Implications of Local Puumala Hantavirus Genetics and

Implications of Local Puumala Hantavirus Genetics and Epidemiology for Diagnostics and

Vaccine Development

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No 964 – ISSN 0346-6612 - ISBN 91-7305-878-5

From the Department of Clinical Microbiology, Division of Virology Umeå University, Umeå, Sweden

Implications of Local Puumala Hantavirus Genetics and Epidemiology for Diagnostics and

Vaccine Development

Patrik Johansson

Umeå 2005

Copyright © 2005 Patrik Johansson ISBN 91-7305-878-5

Printed in Sweden by

Solfjädern Offset AB, Umeå, 2005

To my family

Abbreviations used in this thesis

a.a. Amino acid bp Base pair cRNA Complementary RNA CTL Cytotoxic T-lymphocyte DNA Deoxyribonucleic acid ELISA Enzyme-linked immunosorbent assay ER Endoplasmic reticulum FRNT Focal reduction neutralization test GC Glycoprotein from the C-terminal half of the polyprotein. (also known as G2) GN Glycoprotein from the N-terminal half of the polyprotein. (also known as G1) GPC Glycoprotein precursor HFRS Hemorrhagic fever with renal syndrome HLA Human leukocyte antigen, see MHC HPS Hantavirus Pulmonary Syndrome IFA Indirect immunofluorescence assay IFN Interferon IgA Immunoglobulin A IgE Immunoglobulin E IgG Immunoglobulin G IgM Immunoglobulin M Kb Kilo base KDa Kilo dalton L The large sized viral genome segment M The medium sized viral genome segment mab Monoclonal antibody MHC Major histocompatibility complex mRNA Messenger RNA N Nucleocapsid protein NE Nephropathia epidemica nt Nucleotide ORF Open reading frame PCR Polymerase chain reaction PTO Phosphorothioate nucleotide RdRp RNA dependent RNA polymerase, the viral polymerase RNA Ribonucleic acid rRNA Ribosomal RNA RT-PCR Reverse transcriptase PCR S The small sized viral genome segment TNF Tumor necrosis factor VLP Virus-like particle vRNA Viral RNA Abbreviation of virus names can be found in Table 1

vi

Table of contents Abbreviations used in this thesis vi Abstract ix Papers in this thesis xiii Sammanfattning på svenska xv Short introduction to hantaviruses and their diseases 1 The nucleocapsid protein 6 The glycoproteins 12 The RNA dependent RNA polymerase 16 The replication cycle 17 Hantaviruses and their hosts 22 Hantavirus caused diseases 31 Diagnostics of NE 34 The immune system 39 Vaccines 43 Aims of this thesis 54 Results and discussions 55 Paper I 55 Paper II 62 Paper III 65 Paper IV 71 Conclusions 77 Acknowledgements 78 References 79

vii

Patrik Johansson 2005

ignoramus et ignorabimus

viii

Abstract

Abstract

Puumala virus is a member of the hantavirus genus in the Bunyaviridae family.

Hantaviruses are enveloped by a lipid bilayer and possesses a tripartite single

stranded RNA genome with negative polarity. The hantaviruses encode four

proteins: a nucleocapsid protein (N), two membrane spanning glycoproteins (GN

and GC) and a RNA dependent RNA polymerase (RdRp). Most hantaviruses are

borne by rodent hosts, and found in most parts of the world. Hantaviruses cause

two forms of human disease, haemorrhagic fever with renal syndrome (HFRS) on

the Eurasian continents, and hantavirus pulmonary syndrome (HPS) in the

Americas. The majority of human hantavirus infections occur in Europe and

Eastern Asia, and the viruses are mainly transmitted to humans by inhalation of

aerosolized excreta from infected rodents.

Human Puumala virus infection results in nephropathia epidemica (NE), a mild

haemorrhagic disease with a mortality of about 0.1%. NE is the the second most

prevailing serious febrile viral infection in Northern Sweden, after influenza. The

yearly incidence is approximately 20 cases per 100 000 inhabitants in Northern

Sweden, and the seroprevalence, in this region reaches 18% for risk groups such as

forestry workers and farmers. It is thus of importance to have a good

understanding of the epidemiology and genetics of these viruses for the

development of new diagnostic methods and for future vaccine development.

The objectives of this thesis are: to characterize a local Puumala virus isolated from

a human patient; to characterize the genetic variability among local Puumala viruses

and their host, the bank vole, Clethrionomys glareolus; to use this information to

develop and evaluate genetic vaccines and to develop diagnostic and

epidemiological tools.

In this thesis, the complete genetic sequence of Puumala Umeå/hu was established,

and genetic analysis revealed that this human isolate clusteres together with other,

bank vole derived, Puumala viruses from that region. Two nucleotides that

ix


interfere with the double stranded RNA, panhandle structure were found in the S-

segment. These mismatches resulted in structural changes in this region, but did

not affect the biological activity of the virus. The N, GN and GC proteins were

expressed as full-length proteins and as overlapping peptides in mammalian cells.

Mutational mapping of the potential glycosylation sites identified four functional

N- and one O-linked glycosylation sites, in the GN and GC proteins.

Genomic analysis of local Puumala viruses and their individual rodent host,

Clethrionomys glareolus, from six different locations was performed. There were no

changes in the viral sequence over the five years that spanned this study, or over

short distances within one capture location. Interestingly, there was no correlation

between viral sequences and proximity of locations. While there could be

significant variation in viral genome sequences over relatively short distances, it

could also be relatively stable over longer distances. In earlier studies the degree of

such changes in the virus genome has been correlated with a similar degree of

changes in the rodent host. However, the phylogenetic analysis of the bank voles in

this study did not show evidence of this or any clear genetic distinction among

bank voles from different capture locations.

Based on the viral sequence information, PUUV-specific, PCR-generated linear

DNA vaccines were developed and tested in mammalian cells and on Balb/c mice.

We demonstrated that appropriate modifications in the flanking regions of these

linear PUUV-specific DNA constructs could protect these fragments against

degradation and is important for development of rapid and long lasting immune

responses. This is probably effected by prolonged biological half life of the

constructs within the cells. A nuclear localization signal peptide further improved

the immune response.

We also designed and fabricated a cDNA microarray for detection and

identification of hantaviruses. The design consisted of more than 2000 genetic

probes of the S and M-segments from twelve hantavirus isolates of six different

hantaviruses. This cDNA microarray was shown to be able to discriminate among

x

Abstract

different hantaviruses as well as individual strains of Puumala virus. Furthermore, it

was able to handle complex samples that were not possible to analyze using

conventional methods.

xi


xii

Papers in this thesis

Papers in this thesis

The thesis is based on the following papers. I. Johansson P, Olsson M, Lindgren L, Ahlm C, Elgh F, Holmström A, Bucht G. Complete gene sequence of a human Puumala hantavirus isolate, Puumala Umeå/hu: sequence comparison and characterisation of encoded gene products. Virus Res. 2004. 105(2):147-55. II. Johansson P, Olsson G, Ahlm C, Juto P, Bucht G, Elgh F. Genetic variability among Puumala viruses and bank voles in an endemic area (Northern Sweden). Manuscript. III. Johansson P, Lindgren T, Lundström M, Holmström A, Elgh F, Bucht G. PCR-generated linear DNA fragments utilized as a hantavirus DNA vaccine. Vaccine. 2002. 20(27-28):3379-88. IV. Nordström H, Johansson P, Li QG, Lundkvist Å, Nilsson P, Elgh F. Microarray technology for identification and distinction of hantaviruses. J. Med. Virol. 2004. 72(4):646-55.

xiii


xiv

Sammanfattning på svenska


Puumalavirus tillhör en grupp av zoonotiska virus i familjen Bunyaviridae som kallas

hantavirus. Alla virus tillhörande denna familj har en arvsmassa som består av tre

olika RNA segment med negativ polaritet. Denna arvsmassa är bunden till ett

nucleokapsidprotein och är tillsammans med ett viralt RNA-beroende RNA-

polymeras, inneslutna i ett lipidmembran, i vilket det sitter två virala transmembran-

proteiner. De senare binder till en cell som därefter blir infekterad. Hantavirus kan

ge upphov till två olika sjukdomar hos människa. Blödarfeber med njurengagemang

(hemmorhagic fever with renal syndrome, HFRS) i Europa och Asien och

hantavirus lungsyndrom (hantavirus pulmonary syndrome, HPS) i Amerika. De

flesta hantavirus har gnagare som värddjur och människan blir sjuk av att inandas

damm som innehåller virus i intorkat sekret från infekterade gnagare. I Europa och

Asien insjuknar årligen 60 000-150 000 personer i hantavirusinfektioner. Dessa

sjukdomar har en dödlighet som varierar mellan 0.1 till 20%, beroende på vilket

hantavirus man är infekterad av. I Amerika smittas betydligt färre personer, och

HPS ger en hög dödlighet (över 40%).

Värddjuret för Puumalavirus är skogssorken, Cletrionomys glareolus, som återfinns i

större delen Europa. Puumalavirus ger en mild variant av HFRS, kallad

nephropathia epidemica (NE) eller sorkfeber. Denna sjukdom har en dödlighet på

cirka 0.1%. Trots att skogssorken finns i hela landet rapporteras fall av sorkfeber

nästan bara norr om Limes Norrlandicus, den kulturhistoriska norrlandsgränsen, en

S-formad linje som sträcker sig från Gävle längsmed Dalälven, genom Västmanland

och ner till Dalsland. Det finns två kända populationer av skogssork i Sverige som

möter varandra i en kontaktzon norr om Sundsvall. Man tror att detta beror på att

sorkarna återinvandrade från två olika håll när glaciärerna smälte efter den sista

istiden, för 6-10 000 år sedan. De två typerna av skogssork kan särskiljas genom

studier av deras mitokondrie-DNA. De Puumalavirus som de olika

sorkpopulationerna bär skiljer sig också och tros ha följt sorkarna under deras

xv


vandring. I norra Sverige varierar antalet sorkar cykliskt med toppar var 3-4 år.

Insjuknandefrekvensen av sorkfeber följer också dessa cykler. I Sverige rapporteras

årligen cirka 150-500 fall med en årlig incidens av ca 20 fall per 100 000 invånare

för norrlandslänen (1998 var ett toppår med 562 rapporterade fall). Västerbottens

och Norrbottens län, som har flest fall av sorkfeber, har en årlig incidens på ca

35/100 000 invånare (67/100 000 för 1998). Antalet infekterade personer är

mycket högre än det registrerade antalet sjuka eftersom man kan se att bara ett fall

av åtta diagnostiseras och rapporteras. Det är också så att vissa yrkergrupper, som

skogsarbetare och bönder, vilka har en seroprevalens upptill 18%, är extra utsatta

för smitta.

Målet med denna avhandling har varit att i detalj karakterisera ett unikt

Puumalavirus, Puumala Umeå/hu, som isolerats från en patient i Umeå. Andra mål

har varit att undersöka den genetiska variabiliteten bland lokala Puumalavirus och

dess värddjur skogsorken och att använda resulteranade informationen för att

förbättra genetisk diagnostik och öka den epidemiologiska kunskapen om sorkfeber

samt att utveckla och utvärdera nya typer av genetiska vacciner mot sorkfeber.

Den fullständiga genetiska sekvensen för Puumala Umeå/hu har bestämts och en

genetisk analys visade att viruset är mycket nära besläktat med andra Puumalavirus

från näraliggande områden. Virusets tre stukturella proteiner producerades i

cellkultur och två typer av sockerstrukturer på membranproteinerna kartlades.

Analys av lokala Puumalavirus och deras värdjur, skogsorken, från sex områden i

Västerbottens kustland, visar att virusets arvsmassa kan skilja sig markant mellan

två närliggande fångstplatser, medan det kan vara relativt oförändrat över längre

avstånd. Tidigare studier har visat att skillnaderna i virusets arvsmassa har

samvarierat med liknande skillnader i värdjuret. Den genetiska analysen av

skogssorkarna visade dock väldigt få genetiska skillnader mellan djuren. Det var

heller ingen genetisk skillnad hos virusen över de fem år som studien varade eller

över korta avstånd inom en fångslokal.

xvi


Linjära DNA-vacciner ämnade att skydda mot Puumalavirusinfektion, framtagna

med s.k. PCR-teknik, utvecklades och undersöktes, dels i cellkultur och därefter i

möss. Vi visade att ett snabbt och långvarigt immunsvar erhölls när de linjära

DNA-fragmenten hade skyddats i flankerna mot DNA-nedbrytande enzymer. När

en kärnlokaliseringssignal kopplades till dessa DNA-fragment förbättrades DNA-

vaccinets egenskaper ytterligare.

Slutligen utvecklade och skapade vi ett hantavirusspecifikt cDNA-microarraychip

och visade att man med hjälp av cDNA-microarrayteknik kan skilja mellan olika

hantavirus och även mellan olika virus i Puumalavirusgruppen. Microarray-tekniken

kan också hantera komplexa prov som inte kan analyseras med konventionella

metoder.

xvii


xviii

Short introduction to hantaviruses and their diseases


Hantaviruses belong to the family of Bunyaviridae. In nature, hantaviruses are

maintained in persistently infected rodents (the only known exception is the

Thottapalayam virus that is carried by an insectivore). The virus is transmitted to

humans through inhalation of infectious material containing the virus, e.g. rodent

urine, excreta or by direct physical contact with infected animals [1]. Hantaviruses

cause hemorrhagic fever with renal syndrome (HFRS) on the Eurasian continents,

and hantavirus pulmonary syndrome (HPS) on the American continents. Currently

four hantavirus serotypes are associated with HFRS, with varying morbidity and

mortality. HFRS cases infected with Hantaan (HTNV) or Seoul (SEOV) viruses are

mainly found in Asia, whereas Dobrava (DOBV) and Puumala (PUUV) viruses

circulate primarily in Europe. A large variation in the mortality has been observed

(PUUV < 0.1%, SEOV<1%, HTNV=5-10% and DOBV<20% [2-4]) among the

60 000-150 000 yearly HFRS cases, a great majority of which are from China [1, 5,

6]. HPS, which can be divided into two variants [Sin Nombre (SNV) and New

York (NYV) virus (prototype) and Bayou (BAYV), Black Creek Canal (BCCV) and

Andes virus (ANDV) (renal variants)], often have a more severe outcome (both

variants show mortality rates of > 40%) [7, 8], but does not infect as many people,

with only a total of 384 cases reported, in the United States, through 1993-2004 [9].

ANDV, on the other hand causes around 200-300 cases annually in areas of South

America [10]. In Europe three hantavirus serotypes are represented. PUUV which

is endemic in many parts of Northern, Central and Eastern Europe [11], DOBV,

found in many parts of Central and Eastern Europe [12], and SEOV that has

recently been found in European rats [13]. In Scandinavia, the only hantavirus

described so far is the PUUV, which is of great concern in Northern Sweden where

it is second to the influenza virus, the most prevailing serious viral infection. There

are about 20 cases per 100.000 people and year and a seroprevalence of up to 18%

for risk groups such as forestry workers and farmers has been reported [14, 15].

The PUUV is carried by the bank vole, Clethrionomys glareolus, which is distributed in

1


most part of Europe, from Britain to the Urals, and from the far north of

Scandinavia down to the northern parts of the Mediterranean regions [16].

History of the diseases

Medical records from a hospital in Vladivostok, indicate that HFRS could be traced

back to 1913 in Russia [17]. In Sweden, in the early 1930´s a disease that was later

named Nephropathia Epidemica (NE) was described independently by two

Swedish physicians, G. Myhrman in Östersund and S.G. Zetterholm in Skellefteå

[18, 19]. Later, records from the Second World War clearly indicated that Finnish,

Russian, German and Japanese soldiers suffered from HFRS [1, 20-22]. In the hunt

for the mysterious pathogen, some gruesome events took place. Japanese soldiers

conducted experiments on Chinese prisoner “volunteers” while trying to develop

the disease-causing agent into a biological weapon [5, 23], while the Soviets infected

“psychiatric patients” in need of “pyrogenic treatment” with the disease [24, 25].

Although the viruses, and the subsequent diseases that they are responsible for, had

been around for a long time, not much was published until UN troops, during the

Korean War 1950-1953, fell ill from a, then unknown and deadly disease called

Korean Hemorrhagic fever. Both UN, South Korean and North Korean soldiers

suffered from the disease that was later confirmed to be HFRS [22, 24]. The disease

causing agent remained unknown until 1976, when Lee HW and co-workers

managed to immunostain lungs from black-striped field mouse (Apodemus agrarius)

but not lungs from other rodents, with sera from patients that had suffered from

Korean Hemorrhagic fever [26]. In 1978, Lee and co-workers isolated the virus

from an immunofluroescent positive Apodemus agrarius rodent by injecting a

suspension of the lung tissue to uninfected Apodemus agrarius rodents. The virus

they isolated was named Hantaan virus (HTNV) after the Hantaan River that flows

close to the demilitarized zone between South and North Korea. [27]. Later, Lee

and others successfully isolated another hantavirus from rats (Rattus norvegicus and

2


Rattus rattus), and this virus was named Seoul virus (SEOV), after the capital city of

South Korea [28]. In 1981, the HTNV was adapted for growth in cell culture.

Human alveolar epithelial cells were incubated with lung tissues of infected

Apodemus agrarius [29]. By using similar techniques, the causative agent for NE was

identified in the lungs of bank voles captured in or near the Finnish village

Puumala, which also gave its name to the NE causing virus, now called Puumala

virus (PUUV) [30]. In 1984, two groups independently succeeded in adapting

PUUV to cell culture, but this time in kidney epithelial cells of African green

monkey kidney (Vero E6 cells), which until today is the most commonly used cell

line for culturing hantaviruses [31, 32].

In 1992, the Dobrava virus (previously named Belgrade virus) was isolated from

yellow-necked mouse, Apodemus flavicollis, as well as from patients suffering from

HFRS in Yugoslavia [2, 33]. Soon after, the first American hantavirus, the Prospect

Hill virus (PHV), was isolated from the meadow vole, Microtus pennsylvanicus, in

Frederick, Maryland, USA [34]. Yet another virus, the Thailand virus (THAIV) was

isolated in Thailand from the greater bandicoot rat, Bandicota indica [35].

Although hantaviruses had been found in rodents in the USA, human illness caused

by hantaviruses had only been observed on the Eurasian continents in 1993.

During that year, young, fit people from the Four Corners region in the south west

of USA, became seriously ill and died soon after, in what was quickly recognized as

a new hantavirus caused disease [36]. This “new” virus, named Sin Nombre virus

(SNV), affected the victim’s heart and lungs in a much more severe way than

hantaviruses of the old world. This virus has been isolated from the deer mouse,

Peromyscus maniculatus. The illness caused by SNV is now called Hantavirus

Pulmonary Syndrome (HPS) or Hantavirus Cardio Pulmonary Syndrome (HCPS).

The discovery of this new deadly disease became a starting point for a new era of

hantavirus research, especially in the Americas where several new hantaviruses were

found and characterized soon after. However, the most notably viruses of the new

world are the SNV in North America and Andes in South America. ANDV,

3


isolated from the long-tailed pygmy rice rat, Oligoryzomys longicaudatus, is the only

hantavirus for which human to human transmission has been reported [37].

Interestingly, it was not realized until 1985 that the Thottapalayam virus (TPMV)

was the first hantavirus to be isolated. It was isolated from the spleen of an

apparently healthy shrew (Suncus murinus) in India already in 1964 [38]. However, at

that time, it was not possible to identify the virus. The virus was re-discovered in

1985, this time in China, by intra-muscular passaging in Apodemus agrarius rodents

[39-41].

The Bunyaviridae virus family

The Bunyaviridae family is one of the largest groups of animal and plant viruses and

comprises more than 300 recognized viruses. The family, based on antigenic

relationships, is divided into five genera (Bunyavirus, Phlebovirus, Uukuvirus,

Nairovirus and Hantavirus) with the first four genera being typical arthropod borne

viruses. The Hantaviruses are the only viruses in the Bunyaviridae family that are not

spread by arthropods [42].

The Bunyaviridae virion is spherical and the diameter is usually between 70-200 nm,

and with a mean diameter of 122 nm [43, 44]. The variation in size could be

explained by the variation of the number of genome segments enclosed in the

virion [45]. The hantavirus particle contain three genome segments, large (L),

medium (M) and small (S), of negative single stranded RNA. The three RNA

segments are surrounded by subunits of the N-protein, and form circular coils

complexed with the RNA dependent RNA polymerase (RdRp). These structures

are enveloped in a bilipid membrane, derived from the host cell ER-Golgi complex

from which the two heterodimeric viral glycoproteins are projecting out. In

electron microscopy (EM) this is visible as a well-defined, grid-like, arrangement of

6-7 nm long spikes (Fig. 1, 2) [43, 44, 46-48].

4

RdRp

N-proteintrimer

GN

GC

Glycosylations


Figure 1. EM picture of Hantaan virus. Amplification is 135 000 times [49].

Figure 2. Schematic picture of the composition of a hantavirus.

In hantaviruses, the small segment (S) is about 1.8 kb and encodes the nucleocapsid

protein (N), and in some bunyaviruses, also a non-structural protein (NSs). The

medium segment (M) (~3.6 kb) encodes a polyprotein that is processed into the

two glycosylated, membrane spanning proteins GN and GC (also known as G1 and

G2). The large segment (L) (~6.4 kb) encodes the viral RNA dependent RNA

polymerase (RdRp) [50]. The 5’ and 3’ ends of each of the three segments are

5


thought to hybridize with each other and form a panhandle structure, previously

shown also for other Bunyaviridae viruses (Fig 3) [51]. These conserved panhandle

structures vary in length among the three segments, being more elongated for the

longer segments. This could compensate for the lower possibility of the longer

fragments ends to find its complementary end in comparison to the shorter

segments, giving them a similar probability of circularization despite the difference

in length [52].

Figure 3. EM picture of Uukuniemi virus RNA. Notice the circular structures, indicating panhandle formation [51].

The nucleocapsid protein

The approximately 50 kDa hantavirus nucleocapsid protein (N), (429-433 a.a.), is

the most abundant protein, both in the virus infected cell and in the virus particle

where the copy number of the N protein is in the order of 3x107 molecules per cell

[53, 54].

The N protein has been reported to have many different functions. In the virus

particle, the primary role is to associate with the viral RNA (vRNA) and to form

the ribonucleoprotein complex, a stable structure necessary for the RNA to be

6


transcribed by the virus encoded RNA dependent RNA polymerase (RdRp), once

internalized into the cell cytoplasm [55-60]. The N protein is also thought to be

involved in the interactions between the glycoproteins and the ribonucleoprotein,

most likely by binding to a YRTL-motif in the cytoplasmic tail of GN when forming

the virion [61, 62]. Furthermore, the N protein is assumed to facilitate the binding

between the RdRp to the ribonucleoprotein [54].

In the infected cell, the major role of the N protein is to bind to the vRNA and

cRNA, which is needed for the RdRp to be able to perform replication and

transcription. Another important function is to regulate the transcription of the

different forms of viral RNA’s (vRNA to mRNA’s, or to cRNA and back to

vRNA) [63]. Beside that, the N protein has been reported to have a role as a

chaperon during the folding of the genome segments into the typical panhandle

structure [64], and also in the binding of the newly synthesized viral

ribonucleoproteins to actin filaments that might facilitate transportation of the viral

components [65].

It is thought that the N protein is expressed in the cytosol where it can be observed

within two hours after infection. The protein is retained in the cytosol for 12 hours

post infection, possibly involved in viral RNA synthesis and interacting with actin

filaments [66]. Thereafter, the N protein is found to be transported, and localised at

the perinuclear structures surrounding the Golgi [53, 66], where the viral replication

is assumed to take place. The ribonucleoproteins are later assembled into virions in

the Golgi or at the plasma membrane [44, 65].

The main interaction between the N protein and the vRNA is thought to be

mediated by a homo-trimeric form of the molecule, formed through intermolecular

interactions between the amino- and carboxyl-terminal parts of three N monomers,

(Fig. 4) [67], and this trimeric form of the N-protein is more stable than the mono-

and dimeric forms. The trimers has a high specific affinity for the panhandle

structure of the genome segments, while the mono- and dimeric forms show more

unspecific affinities for viral RNA structures in general [68]. It has been shown that

7


the N-protein contains a conserved, central region that preferentially binds to the 5’

terminal HTNV vRNA [63].

Although it has not been shown exactly how, and which molecular form of N-

protein that binds to the vRNA first (Fig 5), there are indications that the trimer

binds initially to the vRNA panhandle structure in an entropy-driven process, and

that the vRNA’s primary sequence and secondary structures are of importance for

the binding. The monomeric form is then recruited by its affinity for the already

bound trimer and the RNA motifs itself [64].

One interesting observation is that in a mixed population, where different

molecular forms of N molecules exist simultaneously, the binding affinity between

the trimer and the RNA is lowered. This phenomena could be explained by a more

competitive binding of N monomer- and dimer-binding to the vRNA structures

compared to the trimer, or perhaps structural of property changes of the trimeric

form effected by the monomers or dimers [68]. These and other observations

suggest that the trimer binding to the panhandle structure of the RNA resulting in

encapsulation is complicated, and perhaps not the only biological function of the

N-protein/RNA interactions.

The N-protein has been reported to bind to the cRNA. The plus-stranded

complementary RNA (cRNA), which also can form similar panhandle structures as

those of the vRNA, is also encapsidated by the N protein [69], however with a

lower affinity, than that observed between the N trimer and the vRNA panhandle

[64]. In contrast to vRNA and cRNA, mRNA is not encapsidated [70]. This is

logical since the vRNA and cRNA is supposed to be handled by the viral

polymerase, while the mRNA is translated by cellular ribosomes.

Other interesting observations are that the N-protein binds to the YRTL-motif in

the cytoplasmic tail of GN [62], and that production of N-protein and the

glycoproteins in a cell is enough for the formation of virus-like particles [61].

Finally, since the N-protein is needed for transcription of both the vRNA and

8


cRNA, it can be assumed that there is interaction between the N-protein and the

RdRp [62].

A number of publications have suggested that the N-proteins have interactions

with a number of non-viral components. One interesting observation is that, the N

protein is modified by the SUMO-1 (Small Ubiquitin Modifier 1 also called sentrin)

protein. The function of sumoylation is not clearly understood, but it has been

suggested to have a role in protein transportation and targeting to, and formation

of, certain subnuclear structures such as nuclear bodies (NBs). Other speculations

are that a variety of processes including the inflammatory response, including the

response to TNF-α, are regulated through sumoylation [62, 71-73]. Intriguingly the

N-protein also bind to another protein associated with the NBs, the DAXX

protein, a Fas mediated apoptosis enhancer [74]. Altogether, these different

specificities of the intensively studied multifunctional N protein suggest that the N-

protein could be interfering with the antiviral countermeasures of the host. Other

interesting features are the apparent targeting of the antiviral Myxovirus protein A

(MxA) to the N-protein, and that the N-protein has been shown to interact with

actin-filaments [75-77]. Finally, the N-protein has been suggested to interact with

cellular membranes localized in perinuclear regions of the cell [53].

9


Figure 4. Computer modelling of the C-terminal regions of the N protein trimer. The C-terminal helix-loop-helix structure of three N protein molecules are shown in yellow, red and blue. The core regions of each monomer are illustrated with the coloured ovals. The arrow points towards the amino-terminus of each monomer. The helix-loop-helix structure is shown as a top view (A) and a side view (B) [67].

10

+ +

5’

3’

5’3’

Monomer Dimer Trimer


Figure 5. Association of N protein subunits with the viral RNA. The monomers and dimers require the 5’ terminus of the vRNA for binding and are sensitive to increased ionic strength. The trimeric form of N bind specifically to the 3’/5’ panhandle structure and is resistant to high salt concentration [68].

Antigenic regions on the N-protein

When sera from PUUV IFA positive humans and polyclonal rabbit sera, raised

against the N-protein, were analysed by ELISA, the IgG and IgM antibody profiles

were almost exclusively directed against the amino-terminus of the N-protein [78].

However, when the B-cell epitopes were mapped by using short (10 a.a.) peptides

(PEPSCAN), the results were more inconsistent. Some studies indicate that the

epitopes are relatively evenly spread out over the N-protein, while others pointed

to the middle portion of the protein, and others the amino-terminal [79-81]. When

the PEPSCAN method were used to pin-point IgA binding sites, the carboxyl-

terminus of the protein was suggested to harbour the major binding epitopes [82].

However, the amino-terminal part has been successfully used for detection of

PUUV specific IgA antibodies [83].

T-helper cell epitopes have been identified in the amino-terminal part of the N

protein, as well as some additional epitopes were identified in the central and

11


carboxyl-terminal parts of this protein [81, 84]. Cytotoxic T-cell (CTL) epitopes

have been described throughout the primary sequence of the N protein [84, 85].

A nonstructural S (NSs) protein is encoded by the S-segment in several viruses of

the Bunyaviridae family, and it has been shown to mediate a variety of functions. In

Phleboviruses the NSs protein acts as an antagonist of interferon (IFN) induction,

possibly by shutting down the host cell transcription [86-88]. In Orthobunya

viruses it has been shown to suppress the translation in the host cell and viral RNA

synthesis [86, 89]. In Californian serogroup of Orthobunya viruses, the NSs also

suppresses host response against double stranded RNA, and induce apoptosis [90,

91]. The existence of a NSs protein has also been proposed for hantaviruses

harboured by Arvicolinae and Sigmodontinae, but not the Murinae, rodent. The

expression of the suggested 290 nucleotide NSs ORF, from both the Tula virus

(TULV) and the PUUV, inhibited promoter activities of IFN-β, NF-κB and IRF-3

in cell culture [92, 93].

The glycoproteins

The two glycosylated envelope proteins GN and GC, (N for amino terminal and C

for carboxy terminal) are encoded within a single ORF of the M-segment. The

mRNA encoded by the M-segment is translated into a polyprotein, cotranslationaly

imported into the ER were it is cleaved into the GN and GC proteins. This cleavage

is thought to occur directly after the conserved WAASA motif by a signal peptidase

present on the lumenal side of the ER [46, 94]. The processing procedure is very

rapid and efficient since no full length precursor form of the polyprotein has yet

been identified in hantavirus infected or cDNA transfected cells [95-98]. In the ER

the folding of GN and GC is most likely catalysed by chaperones, and they are

thought to form heterodimers here as well [48, 99, 100].

The molecular weight of the unglycosylated GN is about 64 kDa, and approximately

655 a.a. long (for PUUV), while the, GC is smaller, about 54 kDa and approximately

12

The glycoproteins

490 a.a. long (for PUUV). When glycosylated their weights are 68 and 57 kDa,

respectively [101]. The 1148 a.a. (for PUUV) polyprotein contain four major

hydrophobic regions, one within the first twenty a.a. residues and one region near

the carboxy terminus of GN, which are thought to act as signal peptides for the GN

and GC proteins respectively. The other two hydrophobic regions are thought to be

the transmembrane regions for GN and GC, respectively [46, 94, 102].

Both proteins are type I integral transmembrane proteins with carboxy-terminal

hydrophobic anchor domains facing the cytoplasm, and the amino-terminus

pointing towards the ER lumen. GC has a short cytoplasmatic region (< 9 a.a.)

while GN has an unusually long tail (~150 a.a.). This tail has been suggested to serve

many different functions such as acting as a substitute for a matrix protein by

interacting with the N-protein via its conserved cytoplasmic YRTL motif [46, 61,

62]. The YRTL motif has also been suggested to interact with a cellular AP-2

clathrin-associated adapter protein complex, which is thought to be involved in

regulating its budding from the Golgi [103-105]. The hantavirus glycoproteins, as

well as those from the other Bunyaviridae viruses, show a high number of conserved

cysteine residues, which indicate that cystine bridge formations in the ER may be

important for keeping the correct 3-dimensional structure and conformation [94].

Whether GN and/or GC are retained in the ER, when expressed individually, has

been debated for some time. Some studies claim that both proteins are trapped in

the ER when expressed individually [106-108], while others report that only GC is

retained and GN is transported to the Golgi [107, 109]. However, once in the Golgi

both proteins are retained on site, although it is unclear whether one or both

proteins have Golgi retention signals [46, 109].

Because GN and GC colocalize with a cis-Golgi matrix protein (GM130), and

undergo a high-mannose type of glycosylation, they are not believed to be

transported any further than to the cis-Golgi compartment [99]. Within this

location they are found mainly in lipid rafts [54]. One interesting observation is that

the Old World hantavirus glycoproteins are proposed to be accumulated in the

13


Golgi, where the complete virion assembles [98, 107], while the glycoproteins of

the New World hantaviruses, are proposed to be transported to the plasma

membrane and where their virions are eventually assembled [109, 110]. There is

however no complete consensus on the topic of plasma membrane assembly, and

the discussion is ongoing [46].

Glycosylation

Protein glycosylation is associated with several functions. For the envelope proteins

of hantaviruses, glycosylation has been shown to be essential for folding, targeting

and intracellular sorting and transport of the hantavirus glycoproteins [98, 99]. It

has also been shown that glycosylation is important and involved in recognition

phenomena, signal transduction after receptor binding, and mediation of

membrane fusion and penetration of cell membranes. Glycosylated proteins are

also involved in stabilizing certain structures, recruiting chaperones and to confer

resistance to proteolysis of stem regions in membrane proteins. Furthermore, they

are thought to be directly involved in virus morphogenesis at the budding site, and

are able to elicit a protective immune response [111-114].

The glycoproteins of hantaviruses have four potential N-glycosylation sites that are

conserved among all hantaviruses, three in GN (142, 357 and 409 in PUUV) and

one in GC (937 in PUUV) [115]. When these and other residues were further

investigated it is observed that GN has three or four functional N-glycosylations but

no O-glycosylation while GC has one N-glycosylation as well as one O-

glycosylation [99], [Paper I].

All HPS causing hantaviruses contain an immunoreceptor tyrosine-based activation

motif (ITAM) signalling element in the cytoplasmatic tail of the GN protein. For

the NY-1 virus, it has been shown to be involved in the binding and activation of

cellular kinases from both the src and syk families. Furthermore, it has been

suggested to dysregulate normal immune and endothelial cell responses. This

14

The glycoproteins

ITAM element is not present in HFRS-causing or non-disease-causing viruses of

the hantavirus genus (with TULV as an exception) [116]. In other viruses, including

HIV and SIV, the ITAM elements have been shown to play an immunoregulatory

role [117-119]. It is also thought that pathways that are regulated by ITAM

elements play a crucial role in endothelial cell function [120]. This ITAM element is

ubiquitinated, making the GN very difficult to express in vitro [46, 121-124], [Paper

I]. Destruction of this site greatly stabilizes the protein [121].

During the initial phase of the infection the virus particles attach to the cell surface

through the glycoproteins’ affinity for the receptor β-3 integrin receptor molecules

and possibly to an unidentified 30 kDa receptor [125-127].

The glycoproteins are suggested to undergo conformational changes at low pH

[128]. It has also been shown that hantavirus infected cells fuse under acidic

conditions [129, 130]. As the hantaviruses are internalized by clathrin coated

vesicles and transported into the acidic environment in the lysosomes [131], these

findings indicate that the glycoproteins could facilitate virus release from the

lysosome into the cytoplasm.

The glycoproteins are of high importance for protection and prophylaxis against

hantaviral infection since they contain the only known structure on the virus

particle that, when targeted against, results in a neutralizing immune response and

protection against hantaviral infection. It has also been shown that passive

immunization with immune sera and monoclonal antibodies with neutralizing

capacity, protects against SEOV, HTNV and PUUV infections [96, 132, 133].

Several neutralizing domains have been identified both in GN and GC. Many

different immunoglobulin classes (IgM, IgG and IgA) have been suggested to be

involved in neutralization of the virus [82, 134-136]. However, it seems like IgM is

the most important isotype for neutralization during the acute phase of PUUV

infection, and most likely for the other hantavirus infections as well [82].

15


Most of the epitopes in the glycoproteins seem to be conformational, since few or

none of the currently available monoclonal antibodies (MAbs) are reactive in

immunoblots carried out on denatured proteins [136].

The GC protein contains MAb epitopes that are shared by several hantaviruses,

whereas the epitopes of GN seem to be more virus type specific [137].

The RNA dependent RNA polymerase

Since the RNA genomes of hantaviruses are of negative polarity, they cannot be

transcribed and/or translated by the machinery of the host cell. To overcome this

problem, the virus encodes its own RNA dependent RNA polymerase (RdRp), a

multifunctional enzyme participating in both transcription and replication. To

execute all these activities, it must possess several different enzymatic activities

such as endonuclease, transcriptase, replicase and possibly also RNA helicase

activity [138]. This 2150 a.a. long protein, with a molecular mass of about 250 kDa,

is encoded by the L-segment, and its role as a RdRp has been known for at least

thirty years [139]. The functions of the RdRp has been described by several groups

[140-143]. The RdRp is the least abundant protein that is encoded by the virus,

with about 104 copies per cell, and it has been shown to be associated to

perinuclear membranes [54].

The N protein and the RdRp are the only proteins that are required for replication

of the hantavirus genomes [58].

The error rate during transcription has been estimated to be 10-3 miss-

incorporations per nucleotide position for the RdRp proteins of both PUUV and

TULV [144].

The flanking regions of both vRNA and cRNA are conserved and the 3’ and 5’ end

have been shown to hybridize with each other to form intra-strand panhandle

structures. It has been suggested that the RNA synthesis and RNA replication are

16

U A G U A G U A G/U Pu C U C C ---

A U C A U C A U C A G A Pu G ---

A U

C

AU

G

U A G U

A U C A

G/U

U

APu

C

C C

Pu G

A

U C

U

G

---

---

The RNA dependent RNA polymerase

most efficient when the panhandle forms cork-screw-like structures (Fig 6) [145,

146], indicating that these structures could be recognition and binding sites for the

RdRp. Since both vRNA and cRNA need to be in complex with the N-protein for

transcription and replication by the RdRp [55-60], it can be assumed that there is

some kind of direct interaction between the RdRp and the N-protein.

Figure 6. Suggested folding of hantavirus S-segment panhandle structure. Modified from [145].

The replication cycle

Virus attachment to the cell

The first step in virus entry into the cell involves an interaction between cell-

surface receptors and the viral attachment proteins, GN and/or GC. For pathogenic

hantaviruses it has been shown that virus entry is mediated through αv β3-integrins

[125, 126], while non-pathogenic hantaviruses (PH, TUL and TPM) probably bind

to αv β1-integrins [147, 148]. However, the virus does not bind to the RGD motif as

observed for many other integrin ligands such as vitronectin and fibronectin.

However, the αv β3-integrin binding vitronectin, and the αv β1-integrin binding

fibronectin block the binding of pathogenic and non-pathogenic hantaviruses,

respectively. The underlying mechanism is probably through steric obstruction

rather than direct blocking of the binding domains [126]. Another, still unidentified,

17


30 kDa cellular protein has been suggested as a putative receptor for HTNV entry

[127]. Finally, it has been shown that BCCV infects polarized epithelial cells more

efficiently from the apical side than through the basolateral membranes [110].

Virus entry into the cell

After viral attachment to the cell surface structures, the virus is taken up through

receptor-mediated endocytosis by clathrin-coated vesicles. These vesicles enter the

endocytotic pathway and the viral glycoproteins are thought to undergo a

conformational change induced by acidification of the endosome compartment,

promoting fusion of the viral and cell membranes [128, 131]. This event could

facilitate the escape of the viral ribonucleoprotein (RNP) from the

endosome/lysosome into the cytoplasm.

Figure 7. The infection cycle. Modified from [158].

18


Transcription

After the entry of viral ribonucleoproteins into the cytoplasm, an initial “burst” of

transcription into positive stranded cRNA and mRNA transcripts starts. For

SEOV, that has been observed as soon as one hour after infection, and the number

of positive-stranded S RNA molecules increases logarithmically up to twelve hour

after infection. On the other hand, the concentration of negative-stranded

transcripts decreases during the first four hours, after which it starts to increase in

number. This initial period corresponds to the production of N-protein [66] which

is essential for further transcription since the RdRp can only transcribe and

replicate RNA in association with N-protein [55-59].

There are two hypothesizes about where transcription takes place. The most

accepted idea is based on the fact that the two proteins needed for transcription

and replication (the RdRp and the N-protein) are located at or near the perinuclear

membranes, and that the replication probably takes place there. [53, 54].

Furthermore, it has been shown for positive stranded RNA viruses that RNA

replication is associated with intracellular membranes [149]. One appealing idea is

that maintaining a high local concentration of the viral components in a specific

compartment or cellular structure would enhance the rate of viral replication and

assembly. This idea is consistent with the first hypothesis described above [150].

The other hypothesis is based the fact that the SEOV nucleoprotein is found in the

cytoplasm early after infection, and retains there for up to 12 hours before being

transported to the Golgi region where the ribonucleoproteins then gradually

accumulates [65, 66]. This argument suggests that transcription and replication

takes place in the cytoplasm, possibly in association with actin filaments.

19

3’ 5’(-) strand vRNA

Replication Transcription

Replication

5’ 3’ 3’5’(+) strand cRNA (+) strand mRNA

(-) strand vRNA

Translation

-NH2 -COOH

Packagening and assembly

} NTR

Cap


Figure 8. Hantavirus transcription and replication strategy. Modified from [138].

Transcription of vRNA into mRNA

The viral mRNA transcription is believed to start with an RdRp associated

endonuclease cleavage of 8-17 bases from the 5’-end of cellular mRNA’s “cap-

snatching”. These short fragments, containing a cap structure at the 5’ end, are then

used as primers for the transcription of the viral RNA (vRNA) into viral mRNA

according to the “prime and realign” principle [151]. The transcription of the three

different mRNA species differs in quantity, with most mRNA produced from the

S-segment and the least for the long L-segment. However the mechanism behind

this regulated transcription is not clearly understood [152].

It has also been suggested that there are different ways to terminate the

transcription of each of the three different segments. The N and L encoding

mRNA transcripts are probably not polyadenylated. Instead a 3’ flanking stem-loop

structure has been predicted to be formed. Interestingly, the mRNA transcribed

from the M-segments have been suggested to have a polyadenylation sequence of

about 70 bases, very similar to those found in other negative stranded RNA viruses,

and this poly-A tail has been shown to be functional [152, 153].

20


Transcription of vRNA into cRNA and then back to vRNA

After the primary transcriptions (by the RdRp), when mRNA is the main transcript

to be produced, the transcription switches into producing cRNA, the template for

making new viral RNA (Fig. 7). It is speculated that the accumulation of N protein

might trigger this switch in direction of transcription [138]. This phenomenon has

also been noticed for other negative stranded RNA viruses [154, 155].

The initiation of transcription for other viral members of the Bunyaviridae family is

very similar to the transcription of mRNA, except that instead of using capped

RNA primers, derived from cellular mRNA, a single GTP is used. Both genomic

RNA (vRNA) and antigenomic cRNAs are thereafter encapsidated, but only vRNA

is packaged into virions [142].

Translation

As soon as the mRNA molecules appear after transcription, the translation of viral

proteins starts. The N-protein and the RdRp remains in the cytoplasm after

translation, while the glycoprotein precursor, which has a localization signal, is

cotranslationaly cleaved into GN and GC during the passage and entry into the ER

compartment [46]. The N-protein can be seen within two hours after infection,

while GN and GC are observed in Golgi only after about eight hours post infection

[66].

Virus assembly and release

Since hantaviruses do not have matrix proteins that facilitate binding between the

N-protein and the glycoproteins, it is speculated that the N-protein binds directly

to the one of the glycoproteins. The most likely candidate is the cytoplasmic tail of

GN (~150 a.a.), since the cytoplasmic tail of GC is deemed too short (9 a.a. long) for

21


this interaction. The idea that the N protein binds to one highly conserved YRTL-

motif in the cytoplasmic tail of GN has also been suggested by Kaukinen [62].

Until recently it was generally believed that all Bunyaviridae viruses bud into Golgi

compartment and subsequently get transported out to the plasma membrane in

vesicles. While this is still true for many viruses, several studies indicate that

Sigmodontinae- borne viruses bud at the plasma membrane [44, 110]. It has also

been suggested that the completed BCCV viral ribonucleoproteins are transported

along actin filaments from the place of synthesis to the plasma membrane before

virus assembly [65].

In cell culture released virus particles have been observed 24 hours after infection

with SEOV [66], but not much is known about the mechanisms involved in the

trafficking of viral components, or the final release of new infectious hantaviruses.

For BCCV, electron microscopy and titration of viral particles have demonstrated

their release from the apical side of polarized Vero kidney epithelial cells [110].

Hantaviruses and their hosts

The actual number of different hantavirus serotypes are being discussed, but the 23

officially recognized serotypes can be divided into three groups defined by both

their rodent host and their phylogenetic relationships: those carried by Murinae (Old

World mice and rats), Arvicolinae (voles and lemming, which are spread all over the

northern hemisphere) and those carried by Sigmodontinae (New World mice and rats)

rodents, all of the order Rodentia and family Muridae [156, 157]. Beside these, there

is also one hantavirus, Thottapalayam, which is carried by a shrew, Suncus murinus,

of the order Insectivora.

Of the four hantaviruses carried by the Murinae rodents, three cause disease in

humans; the HTNV that has the striped field mouse, Apodemus agrarius, as its host;

the SEOV that is carried by the black and the brown rats (Rattus rattus and Rattus

22


norvegicus) and the DOBV that has the yellow-necked mouse, Apodemus flavicollis, or

the striped field mouse, Apodemus agrarius, as its host. It has been suggested that the

DOBV carried by Apodemus agrarius should be classified as a separate virus named

Saaremaa virus (SAAV) [158].

Among the six Arvicolinae borne viruses, only the PUUV causes disease in man. It is

carried by the bank vole, Clethrionomys glareolus.

For the eleven Sigmodontinae borne viruses, at least six cause human disease. The

most prominent are the SNV that is carried by the deer mouse, Peromyscus

maniculatus, and the ANDV by the long-tailed pygmy rice rat, Oligoryzomys

longicaudatus.

All known hantaviruses, with the exception of Thottapalayam, are maintained by

one (or a few closely related) rodent species that are believed to be persistently

infected [159]. Other animals can be infected, but they are all accidental “dead end”

hosts for the hantaviruses [160-164]. Human to human transmission usually does

not occur, although an exception might be the ANDV, for which this has been

described [37].

Like other persistent viruses, hantavirus infections appear to consist of a short

acute phase followed by a prolonged persistent phase [165]. However, very little is

known about the mechanism of this persistence.

It has been observed that during the persistent phase of the infection the virus

disappears from some organs, and comes back in a cyclic fashion [166]. It has also

been suggested that the virus attenuates and becomes less infectious during the

persistent phase [167]. One interesting observation is that deletions in the 3’ ends

of the vRNA occur just before a decline in the vRNA and cRNA concentrations in

persistently SEOV infected cells. One speculation is that these truncated segments

compete with the full size RNA segments and down regulate viral replication.

Other similar observations were noticed in the 3’ L cRNA and 5’ L vRNA, where

deletions also occurred in a cyclic fashion [165]. Furthermore, it has been observed

23


that in SEOV infected rats, only the viral cRNA was found during the persistent

phase of the infection [66].

Virus Abbreviated name

Animal host Source location of reference strain

Disease Ref

Order Rodentia, family Muridae Subfamily Murinae Hantaan HTNV Apodemus agrarius Korea HFRS [27] Seoul SEOV Rattus norvegicus, Rattus rattus Korea HFRS [28] Dobrava-Belgrade DOBV Apodemus flavicollis Slovenia HFRS [33, 168] Thailand THAIV Bandicota indica Thailand Unknown [35] Da Bie Shan* DBSV Niviventer confucianus China Unknown [169] Dobrava-Saaremaa* SAAV Apodemus agrarius Estonia HFRS [170, 171] Amur* AMRV Apodemus peninsulae Russia HFRS [172, 173] Subfamily Arvicolinae Puumala PUUV Clethrionomys glareolus Finland HFRS [30] Tula TULV Microtus arvalis Russia Uncertain [174] Topografov TOPV Lemmus sibiricus Russia Unknown [175] Prospect Hill PHV Microtus pennsylvanicus USA Unknown [34, 176] Isla Vista ISLAV Microtus californicus USA Unknown [177] Bloodland Lake BLLV Microtus ochogaster USA Unknown [177] Hokkaido* HOKV Clethrionomys rufocanus Japan Unknown [178] Khabarovsk KBRV Microtus fortis Russia Unknown [179] Vladivostok* VLAV Microtus fortis Russia Unknown [180] Subfamily Sigmodontinae Andes ANDV Oligoryzomys longicaudatus Argentina HPS [181] Rio Mamore RIOMV Oligoryzomys microtis Bolivia Unknown [182, 183] Laguna Negra LNV Calomys laucha Paraguay HPS [184] Bayou BAYV Oryzomys palustris USA HPS [153] Black Creek Canal BCCV Sigmodon hispidus USA HPS [185, 186] El Moro Canyon ELMCV Reithrodontomys megalotis USA Unknown [187] New York NYV Peromyscus leucopus USA HPS [188, 189] Sin Nombre SNV Peromyscus maniculatus USA HPS [36, 190] Rio Segundo RIOSV Reithrodontomys mexicanus Costa Rica Unknown [191] Muleshoe MULV Sigmodon hispidus USA Unknown [186] Caño Delgadito CADV Sigmodon alstoni Venezuela Unknown [192] Lechiguanas* LECV Oligoryzomys flavescens Argentina HPS [193] Oran* ORNV Oligoryzomys longicaudatus Argentina Unknown [194] Bermejo* BMJV Oligoryzomys chacoensis Argentina Unknown [194] Maciel* MACV Bolomys obscurus Argentina Unknown [194] Pergamino* PGMV Akadon azarae Argentina Unknown [194] Choclo* CHOV Oligoryzomys fulvescence Panama HPS [195] Monongahela* MONV Peromyscus maniculatus USA HPS [189] Blue River* BRV Peromyscus leucopus USA Unknown [196] Limestone Canyon* LCV Peromyscus boylii USA Unknown [197] Order Insectivora, family Soricidae Thottapalayam TPM Suncus murinus India Unknown [38]

Table 1. The hantaviruses and their hosts. Viruses not officially recognized as hantavirus serotypes in the 7th report of ICTV [156] are annotated with an asterisk (*). Adapted from: [198] and [159]

24

?

Presence of hantavirus

ANDV

SNV

BCCVDOBV

PUUV

HTNVTULV

SEOV


Geographic spread

Hantaviruses are found in most parts of the world, with a few exceptions like

Antarctica, Greenland and possibly parts of Oceania and Africa. Since hantaviruses

are limited to the geographical region habited by their specific rodent they cannot

extend further than their host. SEOV is spread to most parts of the world since its

host, the rat, has spread with the help of the shipping industry [199].

Figure 9. Map illustrating the regions affected by hantaviruses. SEOV is probably present in most of the major ports around the world. For a more complete list of hantaviruses see Table 1.

Hantaviruses in North and South America

Hantaviruses currently identified in the Americas are either carried by Arvicolinae or

Sigmodontinae rodents. In North America there are, apart from PHV which is carried

by Microtus pennsylvanicus, several closely related viruses that are carried by other

Microtus rodents.

The Sigmodontinae borne viruses of Northern America can be divided into three

subgroups, reflected by the evolutionary relations between their rodent hosts. The

25


first group, which consists of SNV and SNV-like viruses (MONV, NYV and BRV),

are carried by Peromyscus maniculatus or P. leucopus. The second group consists of

ELMCV and RIOSV that are carried by Reithrodontomys species. The third group

consists of BCCV and MULV that are carried by Sigmodon hispidus and BAYV,

carried by Oryzomys palustris [157].

The Sigmodontine rodents, that carry all hantaviruses of South America, are

believed to have immigrated to South America about 5-10 million years ago, which

is relatively recent on the evolutionary time scale. This has resulted in a fast

diversification among both the rodents and the hantaviruses they brought along. At

least three different groups of Sigmodontine rodents carry hantavirus; the

Phyllontini, Akodontini and Oryzomyini. However, all hantaviruses of South

America are closely related and have a 75-90% sequence identity [157].

Hantaviruses on the Eurasian continent

Hantaviruses in Eurasia are hosted by Arvicolinae, which are found in the northern

and eastern regions of Eurasia as well as in North America, and Murinae rodents

that are found through out both Asia and Europe [200].

Hantaviruses in Asia

The hantaviruses carried by Arvicolinae rodents, can be further divided into PUUV-

like viruses hosted by Clethrionomys rodents (Hokkaido and others, carried by C.

rufocanus and possibly other voles), those carried by microtine rodents (TULV and

KBRV, hosted by Microtus arvalis and M. fortis), and TOPV that is carried by Lemmus

sibiricus.

The Hantaviruses carried by Murinae rodents in Asia are SEOV (carried by Rattus

norvegicus and R. rattus), HTNV (carried by Apodemus agrarius), DBSV (carried by

Niviventer confucianus) and THAIV that is carried by Bandicota indica.

26


The only hantavirus not harboured by a rodent, Thottapalayam virus (TPMV)

which is carried by a shrew, Suncus murinus, is also found in Asia [38].

Of these viruses only HTNV and SEOV are thought to cause human disease.

Hantaviruses in Europe

In Europe the Arvicolinae borne hantaviruses are PUUV and TULV carried by

Clethrionomys glareolus and Microtus arvalis, respectively. The Murinae borne

hantaviruses are DOBV and SAAV, carried by Apodemus flavicollis and A. agrarius,

respectively [157]. SEOV that also is Murinae borne (Rattus Norvegicus and R. rattus)

has been found here, but it is doubtful if it should be considered to be a naturally

European virus [13].

In Europe two major types of disease causing hantaviruses dominate, PUUV,

endemic in many parts of Central, Eastern and Northern Europe, and DOBV, that

can be found in many parts of Central and Eastern Europe [12].

TULV are generally not believed to cause human disease, but at least one case of

HFRS caused by TULV has been described [201]. TULV has been identified at

several locations in Central and Eastern Europe [202-207].

Hantaviruses in Fennoscandia

PUUV is endemic among the bank voles in all of Finland and Norway [7, 167], but

only in the northern two thirds of Sweden. Surprisingly, bank voles in the southern

part of Sweden do not seem to be infected with the virus [208]. The border

between infected and non-infected animals roughly coincides with Limes

Norrlandicus (Fig 9). Further to the south, the virus has been found in some

locations in Denmark. [209, 210]. Why southern Sweden present this gap in the

distribution of PUUV is not known. From investigations of mitochondrial DNA of

bank voles from Fennoscandia it can be seen that there are two different bank vole

27

Sundsvall

Sotkamo

Puumala

Helsinki

Stockholm

Oslo

100 km

Contact zone

”Southern” bank vole population

”Northern” bank vole population

Umeå


populations, one southern and one northern that in turn carry a southern and

northern variant of the PUUV (Fig 10) [211]. The reason for this is unclear, but is

probably related to the repopulation following the deglaciation after the last ice age

that happened 6-10 000 years ago [209]. Today the 50 km contact zone between the

southern and northern population can be found just north of the city of Sundsvall

in Northern Sweden [211]. Similar zones can be found in Finland and in Russia

[157].

Figure 10. Illustration of the distribution of the northern and southern bank vole populations. Black rodent figures represent the southern bank vole population and the white represents the northern. Modified from [157].

Coevolution of the viruses with their host animal

The hantaviruses are thought to have coevolved with their rodent hosts over a long

time, possibly for more than 50 million years [212, 213]. This can be seen in the

congruence of the hantavirus and rodent phylogenetic investigations [214].

Exceptions are seen and are thought to be caused by host switching. An example is

28


the KBRV and TOPV that are closely related while their hosts the reed vole

(Microtus fortis) and the Siberian lemmings (Lemmus sibiricus), respectively, are only

distantly related [215]. An other example of possible host switching is found for the

DOBV that can be found in both Apodemus agrarius and Apodemus flavicollis [216].

Classification of hantaviruses

The genus hantavirus can be divided into at least 23 serotypes (Table 1). A serotype

is defined as fourfold or greater two-way difference in a virus neutralization assay,

between the virus in question and previously recognised related virus serotypes

[157]. This is not always enough, since a single amino acid change could lead to loss

of neutralization [136]. Therefore other criteria have been suggested. These include:

1) a unique ecological niche for each virus species (a clear association of a new

hantavirus with a different primary rodent host species or subspecies); 2) at least

7% difference in amino acid sequence from previously identified hantaviruses (on

comparison of complete glycoprotein precursor and N-protein sequences); 3) at

least fourfold difference in cross neutralization tests; and 4) absence of genetic

reassortment with other hantaviruses in nature [157].

Evolution of Hantaviruses

The major part of hantavirus evolution is caused by genetic drift, where nucleotide

changes, insertions and deletions are caused by errors made by the RdRp. The error

rate for RNA viruses usually varies between 10-3 and 10-6 mutations per site, and

for hantaviruses has been shown to be 10-3 [144, 217]. This high mutational rate

results in the formation of complex quasispecies populations within a single host

animal, that can evolve rapidly when a mutant becomes more fit than its “parent”

virus [217]. However, when the virus has adapted to its host over a long time, as

for the hantaviruses, it becomes increasingly difficult to outmatch the adapted

master genotype. The evolution for hantaviruses is now relatively slow, with only

29


0.7x10-7 to 2.2x10-6 nucleotide substitutions per site and year for the PUUV. This

rate is comparable with the estimated rate for the host rodents themselves (1.71-

7.31x10-7) and for other stable RNA viruses. This indicates that it might be the

evolution of the hosts that drives the evolution of the hantaviruses [213].

In addition to genetic drift, there are two other possible ways of evolution, which

are in more dramatic: recombination and segments reassortment.

Recombination, where parts of gene segments from two different viruses

recombine and for a chimeric segment, has been show for several RNA virus [218],

among them TULV [205]. Natural recombination events have been suggested for

PUUV also, and the possibility has been demonstrated in vitro [213, 219].

Segment reassortment, which involves a switch of complete virus genome

segments between viruses, has also been evident in nature [220-222], [Paper I].

Both recombination and reassortment events occur in host that are infected by two

different viruses simultaneously. Since the rodents can be persistently infected by

hantaviruses, the situation of co-infection can be readily achieved by a second

infection with a different virus than the primary infection.

Phenotypical changes in the virus induces by changes in the host

When PUUV was forced to change host, e.g. from bank vole to Vero 6 cells, there

were no changes in the amino acid sequence of the virus proteins, but changes in

the untranslated regions of the S-segment was observed. The cell culture adapted

viruses were also less infectious in their natural host, the bank vole [223]. In the

case of TULV, forced adaptation to cell culture led to the loss of the viral putative

NSs-ORF [93].

30

Hantavirus caused diseases


The severe symptoms of a hantavirus infection is assumed to be the result of

immunopathogenesis due to the host immune response induced by the virus

infection and probably not caused directly by the virus [84, 224]. One indication of

this is that T-cell deficient mice and severe combined immunodeficiency (SCID)

mice survive HTNV or SEOV infection better than immunocompetent control

mice [225, 226]. These experiments, and others, indicate that there might be a

connection between T-cell response and the obtained clinical manifestations.

Furthermore, human leukocyte antigen (HLA)-B8-DR3 and DQ2 alleles have been

associated with a more severe form of NE and HIV in humans [227, 228], while

HLA-B27 has been associated with more benign variants of NE and HIV [228,

229].

Both HFRS and HPS are associated with acute thrombocytopenia and changes in

vascular permeability [230].

The main effects of HFRS causing viruses are seen in the kidneys and the

hemostatic balance, while the symptoms of HPS are mainly correlated to the lungs

and heart. These general features are not absolute, as patients with HPS may also

have symptoms associated to the renal function [10, 231, 232], and about half of

NE patients have pulmonary involvement [233, 234]. However, HFRS patients do

not show any cardiac involvement [235].

Hemorrhagic Fever with renal Syndrome (HFRS)

HTNV, SEOV and PUUV cause 60 000-150 000 cases of HFRS each year, and the

great majority of these cases are found in China [5, 6]. The mortality of HFRS

varies from about 0.1% for PUUV to 20% for DOBV and about 1% for SEOV

and 5-10% for HTNV [2-4]. In Northern Sweden infections caused by PUUV

(NE) is the second most prevailing serious viral infection after influenza virus, with

31


about 20 cases per 100.000 people and year [14]. However, the number of sero-

converted people is higher. In a randomized and stratified study performed in

Northern Sweden, the prevalence of people having PUUV-specific IgGs was found

to be 5.4% among adult humans. When compared with the number of cases

reported, it indicates that only one of eight infected people have been diagnosed

and reported [236].

The incubation period of HFRS is usually 2-4 weeks (1-8 weeks), after which the

clinical progress of HFRS can be divided into five different phases based on the

clinical characteristics (Fig. 11). The febrile phase usually lasts 5-6 days, and is

characterized by high fever, chills with headache, myalgia and nausea. Flushing of

the face, erythematosus rashes, conjunctival injections and abdominal and/or back

pain represents other characteristic findings during this phase. The hypotensive

phase is characterized by a sudden drop in blood pressure and thrombocytopenia

that might contribute to a gradual appearance of bleeding manifestations. In the

most severe cases, shock could also occur during this phase. The oliguric phase has

a duration of 3–7 days associated with nausea, vomiting, and acute renal failure,

often in combination with hypertension due to simultaneous hypervolemia.

Approximately 50% of the lethal HFRS cases appear during this phase. The

polyuric phase is the first stage of recovery, and it is characterized by diuresis and

electrolytic imbalance. The convalescence phase can last for weeks to months while

the renal function gradually recovers [7, 237-239].

Since the virus is believed to show high virus titer only for a couple of days after

the symptoms appear, the treatment of HFRS is mostly based on relieving the

symptoms of the disease, and supportive care. However, trials have been

performed with the antiviral drug Ribavirin which seemed to be relatively

successful in the treatment of HFRS in China [240]. Treatment with NE specific

immune sera has also been tested, however with little effect [241]. In severe cases

dialysis is performed [242]. Plasmapheresis, where the part of the patient’s plasma is

32


exchanged for isotonic solution or albumin, managed to shorten the duration of the

illness in a study consisting of 54 patients [243].

Figure 11. Clinical characteristics of HFRS. The duration of the subsequent phases are shown as they would commonly occur in patients suffering from NE. The percentage shows the proportion of patients showing each symptom or finding in NE/KHF. Modified from [244].

Hantavirus Pulmonary Syndrome (HPS)

In North America about 30 cases of HPS are reported per year [245]. According to

the Pan American Health Organization (PAHO) ANDV causes 200-300 cases of

HPS annually in South America [246]. HPS, both in North and South America,

shows mortality rates of more than 40% [7].

HPS usually has an incubation period of 2-3 weeks (1-5 weeks) [247, 248], after

which the clinical features of HPS can be divided into four phases. A febrile phase

(usually 3-5 days, but varies from 1-12 days), characterized by fever, myalgia and

malaise. Other symptoms include headache, dizziness, anorexia, nausea, vomiting

and diarrhea, and in some patients abdominal pain. The cardiopulmonary phase is

characterized by shock and pulmonary edema. Bleeding manifestations are not the

33


major concern for the North American variant of HPS, however that has been

described in South America types of HPS [10]. The diuretic phase is characterized

by clearance of pulmonary edema and recovery from fever and shock. This phase is

followed by a convalescent phase. The recovery after HPS can take up to two

months and a slight pulmonary dysfunction can be observed for some time [248,

249].

For HPS, supporting treatments with oxygen treatment and in more severe cases,

mechanical ventilation has been conducted [10]. Antiviral treatment with Ribavirin

has been investigated but, in contrast to HFRS, it had limited effect [250].

Diagnostics of NE

Clinical diagnostics

In endemic areas, the most severe cases are diagnosed by physicians by

interpretation of the clinical findings, as those described above. However, for

milder or suspected cases, PUUV-specific laboratory tests are needed for

confirmation or verification before the diagnosis is given. Laboratory findings are

usually low platelet count, hematuria and proteinuria. Renal function is impaired

and the serum creatinine level is elevated.

Specific tests for suspected NE

The incubation period for a hantavirus infection is normally between 2-4 weeks,

and the appearance of symptoms is often accompanied by the development of a

significant immune response, with high levels of hantavirus specific antibodies

[224]. In most cases a single IgM test (IFA or ELISA) is sufficient to confirm a

hantavirus infection. However, during the early stages of the disease, the level of

IgM response might be below the detection limit. Therefore, a negative IgM result

can only exclude hantavirus infection six days or later after the onset of symptoms

34

Diagnostics of NE

[251]. During these early time-points, direct detection of viral RNA, usually RT-

PCR, could be undertaken. Nevertheless, most NE patients would have developed

high enough titers of IgG and IgM antibodies in the first serum sample taken [224].

Serological methods

Indirect immunofluorescence assay (IFA) is a common serological method for the

detection of virus specific antibodies. The patient serum is allowed to react with

virus-infected cells prepared on glass slides. These cells are from either an infected

tissue sample (most often rodent lung), or more commonly virus infected cell

cultures (Vero E6 cells are the most commonly used cell line). Fluorescent labelled

anti-human antibodies are then applied to detect anti-hantavirus antibodies (if

present) that has bound to the virus infected cells. A fluorescence microscope is

used for visualizing the result [26, 29]. The major advantage of IFA is that it utilizes

virus infected cells containing all the viral antigens which are in their natural

conformation, in contrast to purified antigens. However, the IFA analysis requires

an experienced operator to interpret the results which could sometimes be difficult

to evaluate. Furthermore, the production of virus infected cells is complicated since

hantaviruses are risk group 3 pathogens, that require the use of a biosafety level 3

laboratory (BSL3). On top of this, hantaviruses propagate slowly and give low virus

titers in cell culture [252]. However, despite these obstacles, the sensitivity offered

by IFA has led to its popularity in hospitals world-wide.

Enzyme-linked immunosorbent assay (ELISA) is another commonly used

serological method. This method is based on the immobilization of viral protein

antigens (most commonly the N-protein) to ELISA-plate wells. Patient sera are

then applied and if the serum samples contain antigen specific antibodies, they are

detected by the addition of enzyme labelled antibodies specifically directed against

the patient’s antibodies. The enzyme that is conjugated to the secondary anti-

human antibody in-turn breaks down an added substrate reagent, leading to a

35


change in colour. The change in colour can be quantitated by a specialised

spectrophotometer (ELISA reader). There are several variants of the ELISA-

method, and the ELISA as a method has the great advantage of having a high

throughput possibility. It is also sensitive, quantitative, relatively fast and does not

rely on any subjective analysis from the operator [253, 254].

Plaque reduction neutralization test (PRNT) and focal reduction neutralization test

(FRNT) are widely used methods for a more precise identification of the hantaviral

strains, viruses and serotype. These neutralization tests are performed by incubating

hantavirus containing samples with patient sera at various serial dilutions. After a

specified period of incubation, this mixture is seeded on susceptible cells (most

often Vero E6). The neutralizing capacity of the patient’s serum is then analyzed as

the end-point dilution needed to neutralize the virus in the tube, thus inhibiting

viral infection of the tissue culture cells. Since hantaviruses show cytopathic effect

(CPE) only under certain circumstances, and the commonly recognized sign of

cytopathocity, plaques or foci are difficult to be distinguished, techniques have

been developed to improve the visibility of such effect. These often involves

specific staining by fluorescent or enzymatically labelled specific antibodies [255-

257]. To facilitate high throughput assays, these methods have also been adapted to

the 96-well cell culture plate format [179, 258].

In lateral flow immunodiffusion immunochromatic assay, the sample, which could

be a drop of whole blood, is allowed to flow along a filter paper. When the sample

crosses a line pre-loaded with immobilized antigen (the N-protein is currently used)

the specific IgM antibodies, if present in the sample, binds to this antigen to give a

coloured line appearing on the test kit [259-261]. This method is very convenient,

rapid and does not require trained operator to handle. However, this method is less

sensitive in comparison with the local in-house methods, such as IFA and ELISA,

used in Northern Sweden [262].

Examples of other serological methods that have been used for hantavirus

diagnostics are: Hemagglutination-inhibition assay (HI) [263, 264]; Immune

36

Diagnostics of NE

adherence Hemagglutination assay (IAHA) [265, 266], and Complement fixation

test (CFT) [267, 268].

Virus isolation

The best way, and the classical way, of proving that a virus infection has taken

place is by isolating the responsible virus in an appropriate cell line. However, due

to the immunopathogenic characteristics of the disease, it has proven to be very

difficult to isolate viable hantavirus particles from infected patients, since the onset

of symptoms is an indication of an immune response active in neutralizing the

viruses and eliminating virus infected cells. Even when successful, it takes weeks of

blind passaging of the viruses before the virus can be detected [269]. Hantaviruses,

particularly PUUV, are usually very difficult to isolate in cell culture [167].

Electron microscopy (EM)

Electron microscopy has been used successfully for diagnosis of a number of viral

diseases. However, this is not the case for the hantaviruses, mainly because of the

low concentration of virus in the patients, and secondly because hantavirus particle

morphology is not distinctive enough to provide a straightforward morphological

diagnosis [270].

37


Detection of viral antigen

Methods that detect the actual viral antigens have been described as diagnostic

methods for other viral diseases [271], but since the amount of virus particles is

usually very low it is not used for diagnosis of hantaviruses.

Genetic assay

Reverse transcription polymerase chain reaction (RT-PCR) is one genetic method

developed to target the existence of viral RNA (vRNA) in a variety of samples

where the presence of virus particles is suspected. Viral RNA is converted to

complementary DNA by the use of a reverse transcriptase enzyme. This cDNA is

thereafter amplified by PCR and analyzed by agarose gel-electrophoresis. Real time

PCR that relies on fluorescence generated by the presence of PCR product has also

become available. Even though this method is extremely sensitive it is only useful

during early infection, since the virus particles are often eliminated within days after

the appearance of the first symptoms. One disadvantage of this method is the

reliance on specific primer sequences that could lead to false negative due to the

large sequence variability among the different hantavirus species or even among

isolates of the same serotype. Therefore it is of great importance to know the

genetic variability in the local hantavirus population, information of such will

enable good design of specific primers that are useful for the PCR-reaction in

question [272, 273].

Genetic microarray

An interesting method for further analysis of material amplified with PCR is the

microarray technique, where specific DNA probes are spotted onto a glass chip.

The number of individual probes, that can be spotted onto a single chip, range

from 10 000 to more than 100 000. The PCR amplified DNA is labelled with a

fluorescent dye, and hybridized to the DNA probes on the chip. Spots where

38

Diagnostics of NE

labelled sample DNA has hybridized will fluoresce when analyzed in a microscopic

microarray reader. The multiple probes render high specificity by eliminating false

positives due to non-specific PCR amplification. Good design of probes can also

provide detailed information with regards to the genotype of the virus sample [274,

275], [Paper IV].

DNA sequencing

Nucleotide sequencing of amplified DNA provides exact nucleotide sequence, thus

allowing identification of the precise genotype of the virus [Paper I, II]. Several new

methods for sequencing, such as oligonucleotide microarray, mass spectrometry,

and pyrosequencing are now emerging [276-278].

The immune system

The immune response is the mechanism by which the body recognizes and defends

itself against microorganisms, viruses and substances recognized as foreign and

potentially harmful to the body. The response against an infection can be divided

into two major parts; innate and acquired immunity. The innate immunity forms

the first line of defence in the immune response. It is non-specific and recognizes

general patterns of different pathogens, such as double stranded RNA and

unmethylated DNA CpG-motifs. The innate part of the immune system acts soon

after an infection, and the main components of the innate immune system are

granulocytes, mast cells, monocytes, and natural killer cells. Other components are

parts of the complement system and cytokines.

The development of adaptive immunity is delayed when compared with innate

immunity. However, when developed, it becomes very efficient against specific

pathogens, and, due to its memory function, it could respond more rapidly when

39


encountered with the same pathogen subsequently. The adaptive immunity can be

divided into two parts, the humoral (B-lymphocytes) and the cellular (T-

lymphocytes).

B-lymphocytes produce antibodies that bind to a variety of structures on the

pathogens, either in solution or directly on the surface of a pathogen. Antibodies

can neutralize viruses through agglutination or by blocking receptor molecules. It

can also mediate killing of cellular pathogens and infected cells.

The cellular immune response is dependent on T-lymphocytes that circulate in the

body, scanning major histocompability complex (MHC) molecules at the cell

surface. There are two types of MHC molecules, MHC I and MHC II. MHC I is

mostly involved in presenting degraded intracellular proteins while MHC II present

degraded, external, proteins that the cell has phagocytized. The T-lymphocytes will

recognize MHC molecules in complex with foreign proteins. There are two main

types of T-lymphocytes, the CD4+ T-helper (Th) cells that have an

immunoregulatory role, and the CD8+ cytotoxic T-lymphocytes (CTLs) whose role

is to kill infected cells.

Immunity to hantavirus infection

Infection with a hantavirus in believed to result in a life long acquired immunity,

and no reinfection has yet been reported [279, 280]. However, the immune

response is believed to play a significant role in the pathogenesis of hantavirus

diseases (see “Hantavirus caused diseases”). Neutralizing antibodies are thought to

be exclusively directed against the viral glycoproteins, and have been shown to be

able to protect against the disease by inactivating any circulating virus [96]. The N-

protein is thought to induce a cellular response, and it has been shown that

antibodies against the N-protein can protect against hantavirus infection as well

[96, 281, 282].

40

The immune system

Innate Immunity

TNF-α and interleukin (IL)-6 levels are both increased in all, and IL-10 in most

patients, during an infection with PUUV. While IL-6 and IL-10 levels return to

normal, the TNF-α values are still increased one week after the onset of disease.

This is of interest since TNF-α has been associated with symptoms like those

experienced for HFRS [224].

PUUV, as well as the other hantaviruses or other human pathogens of the

Bunyaviridae family are sensitive to interferon-α, and MXA, an interferon induced

dynamin-like GTPase, which is induced by interferon- α [75-77].

Acquired immunity

B-cell response

Hantavirus infections in humans initially induce high levels of virus IgM antibodies

directed against the viral N-protein and two envelope glycoproteins. This response

is soon followed by IgA, IgE and IgG antibodies [83, 280, 283]. The major

response of the IgM, A and G antibodies seems to be directed against the amino-

terminal part of the N-protein [78, 281].

The IgA antibody, which is a crucial part of the mucosal immunity, is detected

during the acute phase of the HFRS and HPS infections. However, the level of IgA

drop after the acute phase in SNV infected patients [284]. Interestingly, neutralizing

levels of IgA antibodies specific for PUUV are still found in sera from NE-patients

2 and 10 years after infection [81].

An IgE response is presumably initiated in HFRS before clinical symptoms are

found, and it has been speculated that they could be involved in the pathogenesis

of the disease. However, no correlation between IgE levels and clinical severity of

HFRS has been described [283].

41


IgG, which is the most abundant antibody type, appears after IgM, during the acute

phase of HFRS and HPS, and these antibodies are mainly directed against the N-

protein, while IgG antibodies directed against the GN and GC appear later, in the

beginning of the convalescence phase [230]. High levels of IgG are found in NE

patients more than 50 years after the recovery from the disease [279].

IgM antibodies are the most important antibody class during the early phase of

infection [280]. GN specific IgM antibodies have been observed before those

directed against the N-protein [285].

Neutralizing antibodies are found soon after the symptoms of NE appear, and

these, early, antibodies have been described as highly cross-reactive IgM and IgA1

antibodies reactive against many hantavirus serotypes [286].

Cross-reactivity

The serological cross-reactivity between different hantaviruses is high, especially

among viruses carried by related rodents (the Murinae, Arvicolinae and Sigmodontinae

rodent groups). This is expected since at the amino acid sequence level, the

nucleocapsid protein displayed more than 79% homology among viruses of the

same rodent group; and displayed between 60-77% homology among viruses from

the different groups [286]. The high degree of cross-reactivity has resulted in

difficulties in serotyping different viruses by ELISA, especially for the

discrimination of SEOV, HTNV and DOBV. This problem has more recently

been was solved by modification of the antigens used [287].

It has also been suggested that the complement system might be involved in the

pathogenesis of NE, and possibly in the damage of tubili and/or functional

changes in glomeruli of the kidneys [288]. The effect of the complement system

could possibly also explain the protective antibody mediated effect observed after

immunization with the N-proteins [289].

42

The immune system

T-cell response

T-cell activation occurs very early in HFRS and is associated with an increase in the

amount of neutrophils, monocytes, B- and T-cells [230, 290]. The T-cell response is

long-lived after a hantavirus infection, with a virus specific CTL responses in

peripheral blood mononuclear cells (PBMCs) of most patients, 6-15 years after

being infected with the virus [124, 291].

Immunogenetics

It has been observed that the severity of the NE disease can be coupled to the

MHC haplotype of the patients, where HLA-B8-DR3 gives a more severe outcome

and a higher viremia [227, 292], and HLA-B27 is associated with milder form of the

disease [229]. In SNV infection, HLA-B*3501 is associated with severe HPS [293].

Vaccines

History of vaccination and prevention

Long before vaccination was described and established as a method to enhance the

immune response and to prevent people from acquiring specific diseases, there

were variolation. People were given a mild infection of a disease, which would then

protect them from a more serious infection in the future. The first written account

of variolation tells about a Buddhist nun that practiced this around 1022 to 1063

AD. She blew powdered scabs of smallpox into the nose of non-immune people

[294]. By the 16th century variolation against smallpox was common practice in

China, India and Turkey, and at that time it was also introduced in Europe and

North America. Although variolation with smallpox is hazardous, the mortality

could be as low as one in two thousand [295].

43


Smallpox was not the only disease that people tried to prevent by variolation. Other

examples are: syphilis, measles, scarlet and yellow fever, muco-cutaneous

leishmaniasis, bovine pleuropneumonia, ruminant anthrax and bovine plague [295].

Vaccination

In the rural parts of the United Kingdom, and perhaps elsewhere too, it was widely

believed that cowpox gave protection against smallpox and vice versa. People that

had survived smallpox were sought for milking herds of cows, since they did not

get infected with, or spread cowpox to other animals. Inoculation of cowpox was

carried out at that time with varying results but no one had yet tried to challenge

any cowpox inoculated patients with smallpox, before Jenner. In 1796 he

inoculated an eight year old boy with lymph from cowpox lesions of an infected

milkmaid. He then challenged the boy with smallpox, and the boy did not develop

the disease. Jenner’s vaccination technique was to transfer lymph from a pustule of

a vaccinated person to a non-immunized person. This was called arm to arm

vaccination. However, sometimes this inoculum could get contaminated with other

diseases, e.g. syphilis. Later vaccines were produced directly from young cows

which resulted in a better and safer vaccine and with a greater availability.

The next step in vaccination did not occur until the end of the 19th century when

Pasteur and co-workers in 1880 managed to permanently attenuate Pasteurella

multocida, that causes fowl cholera, and in 1881 developed a vaccine against Bacillus

anthracis, the cause of anthrax. He later also developed a vaccine against rabies that

consisted of dried spinal cord of rabies infected rabbits. The advantage of the

rabies vaccine was its effectiveness even when given after being infected with

rabies.

Further advances in vaccine development was made in 1886 by Salmon and Smith,

who showed that heat killed salmonella bacteria could be used for immunization.

44

Vaccines

During this period of time vaccines against several infectious diseases were

developed: e.g. Vibrio cholerae, Salmonella typhi, Salmonella paratyphi and Yersina pestis.

Glenny and Hopkins discovered that formalin could be used to detoxify bacterial

toxins. However, the efficiency of the immunization was rather low. In 1926,

Glenny discovered that potassium and aluminium hydroxide, which is still used,

acts as an adjuvant to increase the potency of the immunization.

Other important vaccine discoveries are the BCG vaccine against tuberculosis,

developed in 1921 by Guérin and Calmette, the vaccine against influenza by Francis

and Salk in 1944, the dead Salk-vaccine (1954) and the live Sabin-vaccine (1959)

against polio [295].

The tremendous success of the vaccines in preventing diseases is demonstrated by

the eradication of smallpox and the great reduction in tuberculosis, polio and

measles cases. The latter two could be the next diseases to be eradicated [296].

Short introduction to the immune response to different vaccines

There are two main routes of antigen presentation to the immune system. One is

the MHC class I restricted antigen processing pathway when the antigens are

produced by intracellular parasites, such as viral antigens. Within the cell these

proteins are tagged by ubiquitin, which is a signal for degradation, and then

proteolytically digested by the 26S proteasome. The resulting 8-10 a.a. peptides are

then transported into ER by the TAP-transporter system, where they are bound to

MHC class I molecules. After this binding, the peptide/MHC I complex is

transported to the plasma membrane and presented to the immune systems CD8+

T-cells [297, 298]. This pathway of antigen degradation and presentation results in a

mostly cellular immune response with TNF-α as the main cytokine mediator.

The other route is the MHC class II restricted antigen processing pathway that

processes and presents antigens that are internalized from the exterior of the cell by

endocytosis. While the antigens are degraded to peptides, vesicles containing MHC

45


class II molecules are fused to the endosome/lysosome. The MHC II molecules

binds to the peptides and the complex is thereafter transported to cell surface

where the antigen fragments are presented to CD4+ T helper (Th) cells. The MHC

I pathway results in a essentially cell mediated immune response while the MHC II

pathway response is mainly a humoral response with IFN-γ being the main

cytokine mediator [299].

Vaccines against viral pathogens can be divided into either of the following

categories: live but attenuated pathogens, whole killed pathogens, chimeric viruses,

component vaccines that consist of one or several components of the pathogen, or

genetic vaccines where an antigen coding DNA or RNA molecules are used for

immunization.

The live but attenuated vaccines are the oldest and most tested. Examples from this

category are the vaccinia virus against smallpox, the Sabin polio vaccine, and the

new FluMist live influenza vaccine. These vaccines are supposed to give an

immune response that is similar to a natural infection, which is favourable if the

pathogens themselves induce a long immunity. However, that is not the case for all

diseases, e.g. human hepatitis C and rhinoviruses [300, 301]. A problem with these

vaccines are that although they are attenuated, they might still cause disease, (e.g.

the vaccinia virus that can cause severe infections in some patients) [302]. This is a

great concern for people with lowered immune capacity, such as transplanted

patients on immunosuppressive drugs or people suffering from HIV [303].

Another problem with the use of attenuated pathogens is the possibility of

mutations that might revert an attenuated organism back into a pathogenic strain

again [304].

Attenuated viral RNA genomes (infectious clones) have been suggested for use in

vaccination. This procedure results in an infection with a live virus where the

attenuation can be directly controlled. The attenuating factor is highly defined, in

contrast to most attenuated live vaccine pathogens [305].

46

Vaccines

Whole killed (inactivated) pathogen vaccines are used successfully against several

diseases, e.g. Japanese encephalitis, hepatitis A, rabies and polio. These vaccines are

proven and safe, however batches of incompletely killed vaccines have been

produced and distributed [306]. Another disadvantage of such an approach is that

the killed pathogens are not taken up by the host cells, or presented to the immune

system, by the same route as the corresponding live pathogens. This results in a less

protective immunity that usually requires compensation to some extent by the use

of an adjuvant to boost the immune response. The only adjuvant licensed for

human use is alum (aluminium hydroxide) which has a bias for MHC class II

antigen presentation, leading to a mainly humoral immune response and as a

consequence a lower cellular immune response [307].

Component vaccines, where one or a few selected components of a pathogen

antigens are used for inoculation, have been successfully used against several

diseases, such as tetanus, diphtheria, hemophilus influenzae and hepatitis B [308]. One

disadvantage with component vaccines is that many, especially neutralizing

antigenic epitopes of the pathogen are conformational, and a correct folding of the

antigen is of importance to obtain the required immune response. These

conformational structures are often dependent on the local environment and

modifications of the protein, such as protein-protein interactions, insertions into

lipid membranes or correctly glycosylated amino acid residues. Many of these

structures are difficult to mimic when using recombinantly produced antigens. A

second drawback is that these molecules are usually restricted to the MHC II

presentation pathway [308].

A variant of component vaccines are the recombinant fusion proteins where one

part of the protein self assembles to form virus like particles (VLPs) and the other

part of the protein contain the epitopes of interest. These chimeric VLPs, as

carriers of foreign epitopes and peptides, can help minimise or overcome the

conformational problem of the component vaccines, by giving them a basic

structure, and at the same time act as the antigenic partner (adjuvant). One example

47


is the hepatitis B core (HBc) proteins, that in itself forms VLPs, fused with the

antigenic protein epitopes [309]. One advantage of using HBc particles is that,

besides the humoral response, they also induce cellular immune responses [310].

Another interesting approach for component vaccines are the “edible” vaccines,

where the antigenic component is produced, often in a plant [311, 312]. For

hantavirus this technique is not yet functional [313-315].

Genetic vaccines

When performing genetic vaccination, DNA or RNA molecules are inoculated

directly into the cells of the vaccinees by different techniques, and the encoded

antigens are produced inside the vaccinees’s own cells. This is similar to how the

viral antigens are produced during an actual infection, and a MHC I type of antigen

presentation is favoured which would facilitate cell mediated killing of antigen

producing or virus infected cells. Another advantage of using genetic vaccines is

that they can be used on newborn children. Other types of vaccines are often

neutralized by maternal antibodies obtained before delivery or from breast milk.

Since genetic vaccine antigens are produced in the vaccinee’s own tissue, maternal

antibodies are not a problem [316].

RNA vaccines, where the mRNA coding for the antigen of choice is inoculated,

has the advantage, in comparison to DNA vaccines, of having little or no risk of

integration into the vaccinee’s genome. These mRNAs molecules are produced by

in vitro transcription and capping. However, the production of mRNA, in

comparison to plasmid DNA, is relatively cumbersome, and RNA is not as stable

as DNA.

48

Vaccines

DNA vaccines

The concept of DNA-vaccination has its root in the discovery that injected plasmid

DNA can induce long-term protein expression in muscle tissue [317]. It was soon

after shown that a plasmid that encoded an antigen could induce an immune

response when injected by the same way [318].

DNA vaccines have been shown to give protective immunity against several

infectious diseases, including hantavirus infections (see below) [319].

The main advantages with DNA-vaccines are, apart from the mechanism of their

function and level of protection, that they are cheap and easy to produce. DNA

preparations are done in large scale, and the purification is easy, giving a very high

yield and pure final preparations. This is in contrast to vaccines that are based on

whole pathogens or proteins. Furthermore, DNA is very stable and does not need

to be kept cool, which is a big advantage for a vaccine that needs to be stored and

transported.

The use of DNA vaccines has also been suggested, beside infectious, for the

treatment of cancer, allergies and autoimmune diseases [320-324].

Most DNA vaccines consist of plasmid DNA where the ORF of the antigen is

inserted after a strong mammalian promoter, usually the immediate-early promoter

of human cytomegalovirus (hCMV), and before a polyadenylation/termination

sequence, needed for efficient protein production in cells. Furthermore, these

plasmids usually contain an origin of replication and an antibiotic resistance gene,

which are needed for production of the plasmid it self.

A major concern with DNA vaccines is the risk of integration of the DNA

construct into the vaccinee’s genome, and thereby possibly disrupt or up/down

regulate a gene function that might lead to cancer. Though, this has not been seen

for the more common types of inoculation [325-327], it has been reported for

DNA inoculations followed by electroporation [328]. Another concern is the use of

an antibiotic resistance gene that is part of the plasmid back-bone.

49


Many different ways of inoculation and types of DNA constructs have been used,

and the types of DNA constructs can be divided into different groups, such as: if

the plasmid can replicate in the host cell or not, if there are any immunostimulatory

properties encoded in the DNA construct, if they encode one or more antigens or

ability induce apoptosis of the host cell.

Several different formulations of DNA-vaccines have been investigated. The most

important of these are pure DNA in solution, DNA bound to gold particles for use

with a gene gun, DNA in complex with cationic lipids or biodegradable polymers,

DNA contained in virus like particles or carried by virulence-attenuated infectious

bacteria such as Salmonella typhimurium or Listeria monocytogenes.

Many different routes of DNA administration have been used such as intradermal,

subcutaneous, intramuscular, mucosal (gut, vagina and oral) and intranasal [307].

However, the most frequently used techniques for DNA inoculation are

intramuscular injections of pure plasmid DNA and intradermal delivery with a gene

gun, where the DNA is coated on small gold particles and shot into the epidermis.

Part of the immune response elicited by DNA vaccines are attributed to

immunostimulatory non-methylated CpG motifs in the primary sequence of the

DNA that is being used for inoculation. CpG-motifs are abundant in bacterial

DNA, but not in mammalian DNA, and motifs stimulates the innate part of the

immune system in mammals [329].

Vaccines against hantaviruses

Currently only one vaccine against hantavirus infection is commercially available,

but several groups around the world are investigating both inactivated whole

viruses, recombinant and genetic vaccines.

The commercially available Hantavax™ consists of formalin inactivated HTNV

isolated from suckling mouse brain. It has been used for more than ten years

50

Vaccines

without any serious side effects, suggesting that it is safe to use. It has shown good

seroconversion with 100% seroconversion after 3 immunizations. However only

50% of the vaccinees possessed measurable neutralizing antibodies [230]. In a study

when South Korean soldier were vaccinated with Hantavax™, the vaccine showed

an approximately 75% protection after three immunizations [330]. A similar

vaccine was developed in North Korea where 1.2 million people were reported to

have been immunized in a field trial, with a protective rate of 88-100% [331].

Several vaccines that are based on inactivated HTNV and/or SEOV isolated from

cell culture have been developed in China. These vaccines have shown to have

fewer side effects and a more effective immune response with high levels of

neutralizing antibodies, when compared to rodent brain based vaccines [230].

HTNV N- , GN and GC-proteins, produced and purified from the baculovirus

system, protected hamsters against challenge with the live virus after inoculation

with the N-protein or a combined inoculation with both GN and GC. GN or GC

inoculations did not confer protection when used separately [96]. Baculovirus

derived PUUV N-protein has also been shown to give protection against challenge

in bank voles [281]. Similar results were seen when using E. coli or yeast derived N-

protein [281, 282, 332].

Recombinant vaccinia virus encoding the HTNV N, GN, GC or GN combined with

GC, where explored as a vaccine in hamsters and gerbils. It could be seen that only

the combined GN/GC variant resulted in neutralizing antibodies [96, 97]. Similar

results were obtained when using the SEOV N and GN/GC in a analogous fashion

[333]. When challenged with live virus it was seen that only the combination of

HTNV GN/GC gave full protection, while GN, GC and N-protein by themselves

only gave partial protection [96]. However, for SEOV both the GC/GN

combination and N-protein gave full protection [333]. When a vaccinia vaccine

expressing a combination of both the HTNV GC/GN and N-protein was tested on

humans in phases I and II clinical safety trials it was seen that an earlier immunity

to vaccinia greatly influenced the otherwise promising immunization. 100% of

51


vaccinia negative vaccinees seroconverted after two immunizations and 72%

showed neutralizing antibodies while only 50% of the vaccinia positive vaccinees

seroconverted after two immunizations and only 26% showed neutralizing

antibodies [334].

The use of packaged Sindbis virus replicons has also been evaluated. Here the

structural genes of the positive stranded RNA Sindbis virus was replaced by the

SEOV N-protein or GN/GC coding ORF. The Sindbis virus RNA could replicate

in the host cell, but since the structural genes were missing it could not produce

new viral particles. Hamsters immunized with the N-protein encoding Sindbis

replicon developed non-neutralizing antibodies, and obtained no protection against

the live virus. Most of the hamsters immunized with the GN/GC version developed

neutralizing antibodies and developed partial protection against the virus [335].

Genetic vaccines against hantaviruses

Several groups around the world have studied DNA vaccines against different

hantaviruses. Many vaccines encoding for the N-protein and the two glycoproteins,

independently or in combination, have been investigated. Genetic vaccination with

the gene encoding the N-protein have been shown to give a similar antibody

response as developed against live hantavirus infection [336]. In general, the N-

protein encoding vaccines does not produce neutralizing antibodies, but partial

protection against infection has been described [337, 338]. Constructs encoding

fragments of the SNV glycoproteins have also been investigated. Interestingly,

while none of the constructs resulted in significant levels of neutralizing antibodies,

several gave high level of protection against challenge with the virus [338]. The

most promising results have been obtained when constructs expressing the two

glycoproteins as a pair were used. Vaccines encoding HTN, SEO or AND virus

glycoproteins have all been shown to produce high titers of neutralizing antibodies,

and almost complete protection against challenge with the homologous virus. It

52

Vaccines

was further shown that the HTN based vaccine gave good cross protection against

challenge with both SEO and DOB virus, and that the sera obtained from rhesus

macaques immunized against ANDV or HTNV could be used for passive

immunization of hamsters, giving good protection against challenge against the

respective virus [123, 339, 340].

Animal models for hantavirus infection

For a long time, no adequate animal models for HFRS and HPS infection studies

have been available. One model based on bank voles, the host rodent for PUUV,

has successfully been used in challenge studies after vaccination [281]. However,

bank voles do not develop any illness after infection. To have a more human like

animal model, Groen et al. developed a Cynomolgus macaque model where the

symptoms of NE could be studied after infection with a cell culture adapted PUUV

strain [341]. This model was further improved by Klingström et al. who recorded

human like symptoms after infection with PUUV derived directly from infected

bank voles [342]. An excellent model for ANDV causing HPS is the Syrian

hamsters model developed by Hooper et al. in 2001 [343].

53


Aims of this thesis

The objectives of this thesis are: to characterize a local Puumala virus isolated from

a human patient; to characterize the genetic variability among local Puumala viruses

and their host, the bank vole, Clethrionomys glareolus; to use this information to

develop and evaluate genetic vaccines and to develop diagnostic and

epidemiological tools.

54

Paper I

Results and discussions

A unique PUUV strain, named PUUV Umeå/hu, was previously isolated from a

NE patient [269]. This thesis reports on the further investigations performed on

this isolate. The complete sequence of its genome has been determined and

analysed. Whole and parts of the encoded structural proteins have been expressed

in mammalian cells and their glycosylation patterns and expression profiles have

been determined. We have also studied the phylogenetic relationships of PUU

viruses and their host, the bank vole, from six different locations along the

coastland of the county of Västerbotten, Sweden. The viral sequences were used to

develop a novel microarray method for genetic identification and for future virus

diagnosis. A novel way of genetic immunization was also explored. Important

features of the genetic vaccine include: i) PUUV-specific PCR-produced linear

DNA fragments; ii) partial (5’-3’) exonuclease-protection; and iii) tagging with a

nuclear localization peptide. The approaches and results of the above investigations

are described in three publications and one manuscript summarised below.

Paper I

This paper describes the characterization of the PUUV Umeå/hu isolate. The virus

genome sequence was established and it represents the first complete sequence of a

Puumala virus isolated from a human. Sequence comparisons were made with

other hanta- and PUU-viruses; the N, GN and GC proteins and overlapping

peptides from these proteins were expressed, and the glycosylation patterns of the

GN and GC were investigated.

Comparison of PUUV Umeå/hu and the prototype strain PUUV Sotkamo

revealed that the overall sequence diversity over all three segments were

approximately 19%. However it was as high as 33% in the 3’ non-translated regions

55


of the S-and M-segments (Fig 12), and 5.5%, 8.4% and 6.6% for the N-protein, G-

proteins and polymerase protein amino acid sequences, respectively.

Figure 12. Similarity plot (Stuart Ray’s SimPlot 2.5) of full-length, antigenomic S-segment sequences of PUUV. The plot was calculated by using the PUUV Umeå/hu strain as query and the prototype strain PUUV Sotkamo, and two local PUUV strains, Vindeln and Hällnäs as references. The curves show nucleotide identity comparisons within a sliding window of 200 bases with a step size of 20 bases. The bases between the dotted lines indicate the open reading frame of the N-protein.

When comparing the 3’ and 5’ terminal sequences, which are believed to hybridize

with each other to form the panhandle structure, intrastrand base-pairing seems to

be impaired at distinct positions. There are two adjacent mismatches in position 9

from the 3’ end, and the 10th base from the 5’ end of the genomic sequence. When

sequences from different viral stocks were compared, the two nucleotides were

consistently an A or a C. Furthermore, the complementary nucleotides, in the

proposed panhandle structure, to the A or C were always uracils (U), suggesting

that the presence or absence of this bulge due to nucleotide mismatch, does not

interfere with the biological activity, as shown earlier for the Uukuniemi virus [145].

Another mismatch in position 15 of the S-segment was more consistent.

Phylogenetic trees were constructed from full-length sequences of 11 S and 4 M

full length PUUV sequences. As expected, PUUV Umeå/hu clusters together with

56

Paper I

the other PUUV of Northern Sweden (Fig 13 A). When comparing the

phylogenetic trees of three local PUUV S-segments and that of their M-segments, it

was observed that they where incongruent (Fig 13 B), suggesting that an S- and M

segment exchange might have occurred.

Figure 13. Phylogenetic trees based on complete genome segment sequences of PUUV strains from Northern Europe. (A) Radial tree constructed from PUUV S-segment sequences. (B) Phylograms of the S- and M-segments.

cDNA from the S ORF was expressed in mammalian cells, as the full-length N

protein and as four overlapping peptides (N1–N4). Western blot analysis with anti-

PUUV N serum revealed that all five gene products were present in samples from

transfected COS cells, albeit with different expression levels (Fig 14). The peptides

N1 and N3 and full-length N were detected in high levels both in the total cell

extract and in the tissue culture medium, in contrast to the peptides N2 and N4

that were exclusively detected in total cell extract (Fig 14 A), and in the tissue

culture medium, respectively (Fig 14 B).

57


Figure 14. Western blot analysis of N-peptide expression in COS cells: (A) total cell extract; and (B) tissue culture medium. Cells were transfected with cDNA encoding the full-length nucleocapsid protein (N) or peptides thereof. Neg: “empty” vector; N1: amino acids (a.a.) 1–120; N2: a.a. 105–229; N3: a.a. 215–339; N4: a.a. 324–433, N a.a. 1-433. The N1 and full-length N samples were diluted 50 times for analysis of total cell extracts and 100 times for analysis of the tissue culture medium. The proteins and peptides were detected and visualized with anti-PUUV N antiserum.

The ORF of the M-segment was cloned and expressed as the full-length mature

proteins (GN and GC) (Fig 15 A) and as four overlapping peptides (G1–G4) derived

from GN, and three overlapping peptides (G5–G7) of GC (Fig 15 B). Western blot

of transfected cells revealed that all proteins and peptides were readily detected in

the total cell extract, and that G1, and to some extent G6, could be recovered from

the tissue culture medium as well (Fig 15).

As for the N1–N4 peptides, a difference in the amount of G-proteins/peptides in

samples from COS cells were noticed. In general, peptides derived from GC

exhibited higher expression levels than peptides derived from GN, and the full-

length GN and the carboxy terminal part thereof (G4) were found in low amounts,

possibly because of a postulated ubiquitin recognitions site in GN C-terminus, that

destines the protein for ubiquitination and degradation [121].

58

Paper I

Figure 15. Western blot analysis of COS cells expressing N, GN, GC and peptides thereof: (A) full-length N: a.a. 1-433; GN: a.a. 1-655 and GC: a.a. 656-1148; (B) G1–G7 peptides in cell extracts; and (C) G1–G7 peptides in tissue culture medium. Neg: “empty vector”; G1: a.a. 1–199; G2: a.a. 181–369; G3: a.a. 352–524; G4: a.a. 503–655; G5: a.a. 656–799; G6: a.a. 782–949; G7: a.a. 932–1148. The proteins and peptides were detected and visualized with anti-FLAGTM antibody.

Glycosylation patterns

The two main types of glycosylation (N- and O-linked) of viral proteins, serve

many purposes important for the virus. They are important for receptor binding

and protection from proteolysis. Furthermore they induce protein conformation

and mediate membrane fusion. They are also important for the immune response

to the virus [112, 130, 344, 345].

Of the six N-linked possible glycosylation sites (NXT/S-sequence, where N is

glycosylated), the software NetNGlyc 1.0 [346] predicted three not to be

glycosylated. One of these did not fulfil the general NXT/S rule since X was a

proline (Fig 16 A).

59


We investigated all the conserved N-glycosylation sites in peptides of PUUV

Umeå/hu GN and GC by substituting the specific asparagines (N) with serines (S).

In total, six single amino acid substitutions and one double mutant were generated

in the G1–G7 peptides (Fig 16 A).

Figure 16. Prediction and gel-shift analysis of possible N- and O-glycosylated residues. (A) Schematic representation of the poly-protein precursor of the glycoproteins GN and GC (indicated by bars of different colour). Positions where N- or O-linked glycosylations were predicted are indicated by the amino acids and the number of the residue. (B) Gel-shift analysis of glycoprotein (G) peptides, and (C) postulated O-glycosylation sites. Loss of N- or O-linked glycosylation was observed when mutated peptides migrated significantly faster than the corresponding wild-type peptide, indicated in panel A by underlining the modified amino acids. The proteins and peptides were detected and visualized with anti-FLAG™ antibody.

For O-linked glycosylation, there is no simple consensus sequence pattern. By

using the prediction software NetOGlyc 2.0 [112, 347, 348] three threonines (T):

were suggested to be O-glycosylated (Fig 16 A). Therefore, additional single amino

acid substitutions were made so that these three threonines were substituted with

alanines (A).

60

Paper I

When analyzing cell extracts from transfected cells, using Western blot, of (Fig 16

B), four of the six peptides, mutated for N-glycosylation, migrated significantly

faster than the corresponding wild-type peptides, indicating a loss of an N-linked

glycosylation site (underlined in Fig 16 A). Of the O-mutant peptides, only one

peptide displayed gel-shift in comparison to the corresponding wild-type peptide

(Fig 16 C). This was surprising since this site had been predicted with the lowest

probability of O-linked glycosylation among the postulated sites.

Here we have shown that PUUV Umeå/hu phylogenetically clusters with the other

PUUVs from Northern Sweden (Fig 13), and genetically differ significantly from

the PUU prototype virus Sotkamo. We have also shown that there is some

flexibility allowed in the panhandle structure, allowing both the presence and

absence of a small bulge while keeping it biological function. When peptides and

full length N- and G-proteins were expressed in mammalian cells, there are large

differences in expression levels and localization, for the different proteins and

peptides. (Fig. 14 and 15). Gel-shift analysis of the G1–G7 peptides revealed that

four of the six conserved N-glycosylation, and one of three O-glycosylation sites

were functional.

The presented characterization and use of endemic Puumala virus strains will

improve diagnostics and make it possible to perform the analyses with information

and reagents based on local, or closely related, strains of PUUV. This knowledge

combined with results from other investigations [337], [Paper III, IV], constitute

valuable information for future genetic vaccination studies.

61


Paper II

We wanted to investigate the genetic variability of Puumala viruses in local bank

vole populations to get a better understanding of it in general and particularly for

the requirements in the development of diagnostic methods and future vaccines.

Since the majority of NE patients in Västerbotten County, are believed to have

been infected in the coastal area, we wanted to see if there were any differences

among the local viruses in this area. PCR was run on cDNA from bank voles

collected from six different locations in the Västerbotten County, with primers

specific for the PUUV S-segment, as well as on DNA directly extracted from the

same bank voles using primers specific for the cytochrome B/D-loop region of the

bank vole mitochondrial genome. These PCR-products were then sequenced and

the phylogenetic trees for the two organisms were calculated. While investigating

the viral S-segment sequences it was seen that the phylogenetic relationships in the

northern part of Sweden consists of two branches, which from here on will be

called the southern branch and the northern branch, respectively. The southern

branch consists of PUUV sequences from the Bussjö location and two previously

described sequences from a location (Mellansel) to the south west of the study area.

The northern branch consists of the other five study locations as well as with all

previously described PUUV sequences from the region (with the exception of

Mellansel) (Fig 17). Interestingly the previously described human PUUV isolate

[Paper I], clustered tightly with other, bank vole derived, sequences from Bussjö,

where the patient lived, and probably acquired the disease.

62

P98-2

P98-3

Tavelsjö

Hällnäs

Vindeln

B95-3

B99-2B98-5

B98-4Umea/hu

B95-1

B98-3B98-1

B98-7B98-6B98-1

B99-1B98-2

Mellansel Cg49

Mellansel Cg47

G98-1

G98-4

G98-2

S98-3S98-1

S98-2 G98-3

G98-5

N98-2N98-1

P98-4

Hundberget

D98-1D98-2

D98-4

D98-3D98-5

P98-1N98-5

N98-4

N98-6N98-7

N98-3

B95-2

Bussjö

Gumboda/Skäran

Djäkneböle

Norum/Pålböle

Pålböle

Norum/Pålböle

Paper II

Figure 17. Phylogenetic tree of PUUVs of Västerbotten County. The tree is rooted to the other European PUUVs.

The sequences of the northern branch were very homogenous, even over a distance

of 80 km (Djäkneböle to Skäran). However, we could also see that large variation

(7%) could occur over a short distance (10 km). This was seen for Bussjö and

Djäkneböle, which are the closest locations between the northern and the southern

branch. This, for this short distance, rather dramatic difference in genetic sequence,

could indicate a contact zone. There is one previously described contact zone for

PUUV in Sweden. This contact zone is located approximately 250 km south of the

present study area, and correlates with the contact zone for the southern and

northern subspecies of bank voles.

To investigate if there was a similar difference between the bank voles in the study

area, as was seen for the PUU viruses they where carrying, a part of their

mitochondrial DNA was analyzed phylogenetically. Surprisingly, while there were

large and systematic differences in the viral genomes, there were no systematic

differences in the bank vole mitochondrial DNA sequences. Neither could we find

63


any differences in the virus sequence from viruses caught over a five year period,

nor over short distances (~300 meters).

The differences seen an the a.a. sequence of the N-protein for the previously

described PUUV and those presented in this study show very little variation, and

should not influence serological diagnostics or have any impact on a future vaccine.

However, the differences to the PUUV reference strain, PUUV Sotkamo, and to

the previously described PUUVs of Southern Sweden are more significant. The

impact of these differences in the N-protein, which is used for clinical diagnostics,

should be investigated further. Furthermore, the protein variation of the

glycoprotein encoding M-segment also needs further characterization to develop

better diagnostic methods, and for the development of vaccines.

For genetic diagnostics and epidemiology, the understanding of the local genetic

variation of PUUV is of great importance. As can be seen from this study, there

can be large variation over short distances, at the same time as there can be low

variation over longer distances. This variation has to be considered, when

developing new genetic diagnostic tools.

64

Paper III

Paper III

In this study, we investigated the use of linear DNA for genetic immunization

against the PUUV N-protein.

Earlier studies have shown that PCR-amplified DNA containing the promoter,

ORF and terminator sequence elicits an immune response when administered to

animals [349, 350].

Most genetic vaccinations, to date, involve immunization with circular plasmid

DNA containing genetic material from selected pathogens. In comparison to

plasmids, linear DNA fragments (LDF) containing only eukaryotic regulatory

elements, antigen encoding DNA and a poly-adenylation sequence could have

several advantages. Linear DNA fragments are smaller, more flexible and may,

therefore pass more easily through the nucleopores [351, 352]. In contrast to LDF,

plasmids usually contain other characteristics that are not desirable in DNA

vaccines (e.g. antibiotic resistance genes). Furthermore, plasmids are propagated in

bacteria and might therefore contain bacterial remnants such as lipopolysaccharides

or toxins that could cause unwanted side effects in the vaccinee. Such problems

would be avoided if PCR could be used to generate genetic vaccines.

However, in comparison to circular DNA, one major drawback of using linear

DNA is that they are more rapidly degraded, as their free ends are susceptible to

exonucleases. Earlier studies have shown that phosphorothioate (PTO)-

modifications in the backbone of synthetic oligonucleotides [353], or ligation of

closed loops of DNA to the free ends of linear DNA fragments [350] increases the

biological half-life by protecting the DNA against nuclease degradation.

Several studies have shown that nuclear localization signal peptides (NLS), when

conjugated to e.g. DNA, facilitate the transport of the macromolecules into the

nucleus, [350, 354, 355] which could be beneficial for the DNA half life and

expression.

65


We created PTO-protected DNA fragments with a single NLS-peptide from the

SV40 large T antigen, linked to its 3’-end, by the use of a single NLS-peptide linked

to the 3’ (PTO-modified) primer together with an unlinked PTO modified 5’

primer, during PCR-amplification.

As a template for the PCR an ORF encoding the gene of interest (GOI) was cloned

into a mammalian expression vector, under the control of a CMV promoter (Fig

18). We then designed primers specific for the region starting just upstream of the

CMV promoter and ending just downstream of the bovine growth hormone

(BGH) polyadenylation site.

Figure 18. Concept for generating linear DNA fragments. (A) A map of the eukaryotic expression vector pRc/CMV that was used as a template for generating linear DNA fragments. Control regions are indicated in grey and coding regions in black, and GOI refers to gene of interest. The arrows point out the directions of the genes. The two primer binding sites are denoted [5’] and [3’], and the resulting PCR product is also indicated. (B) The resulting linear DNA fragments obtained by PCR using the three different primer pairs. The non-modified LDF (top), the LDF containing the phosphorothioate-modified nucleotides at the 5’ ends, marked with the black box (middle), and finally (bottom), the PTO/NLS-modified LDF with the NLS peptide attached at the 3’ end (not in scale).

Three different sets of primer pairs for PCR amplification were used: ordinary

oligonucleotides without any modifications; oligonucleotides were the first five 3’

nucleotides had been replaced by a corresponding phosphorothioate (PTO)

nucleotide, and one pair that had the 3’ PTO modifications but where one of the

primers also had a nuclear localization signal (NLS) peptide attached.

66

Paper III

We first showed that the PTO-modified primer generated fragments were more

stable against 3’-5’ exonucleases, than non-modified (Fig 19). Following this, we

tested the different fragments for biological activity when being transfected into a

mammalian cell line, by measuring the activity of a control gene (acetylcholine

esterase) (Fig 20).

Figure 19. Exonuclease degradation test. The relative resistance of the LDF towards exonuclease degradation in vitro was examined using non-modified and phosphorothioate-modified LDF. The DNA fragments were incubated at 37 ◦C in a reaction buffer containing lambda exonuclease and exonuclease I.

Figure 19. Enzyme activity in LDF transfected HEK293 cells. The cells were transfected with LDF amplified with non-modified primers (white bar), PTO-modified primers (light grey bar) and PTO/NLS-modified primers (dark grey bar), and plasmid DNA encoding AChE (black bar). An empty plasmid was used as negative control. The enzyme activity in the cell extract from cells transfected the non-modified fragment was set to one. The diagram represents the average of four individual experiments using the same DNA construct. The error bars indicate the variation in enzymatic activity between the different transfections as standard deviation.

67


We then made a construct encoding the first 120 amino acids of the PUUV N-

protein, which has been shown to be the dominant region for the humoral immune

response towards the N-protein [78, 281]. Balb/c mice were immunized with the

use of a gene gun and received 3 booster shots with 2 week intervals. When

studying the response over the first 12 days, it was seen that circular plasmid DNA

gave the fastest response but that the NLS-modified fragments also were fast in

giving a significant antibody response. This occurred already on day 10, with the

PTO-only fragments also giving a fast response but not as strong as the NLS-

modified or the plasmid responses (Fig 21). Over a longer time period it could be

seen that the humoral immune response elicited by the PTO-modified fragments

caught up with the response of the NLS-fragments and the plasmid DNA at week

4, while it took another 2 weeks for the response of the unprotected fragments to

catch up (Fig 22). Also, the titers caused by the NLS-PTO fragments were more

long lasting than those from the PTO-only or non-modified fragments (Fig 23).

Figure 20. Kinetics of the early immune response. The figures represented by the bars indicate the ELISA reactivity against the antigen after the primary immunization. Serum samples were taken from two individuals of each group of immunized mice every second day. Different mice (two) were chosen at each occasion. From left—non-modified LDF (white bars), PTO-modified (light grey bars), PTO/NLS-modified (dark grey bars) and plasmid DNA (black bars). The lines indicate the suggested increase in antigen reactivity during the first 12 days after primary immunization.

68

0

0,5

1

1,5

2 A. ELISA

2 Weeks after primary immunization

Neg Neg

Abs

orba

nce

at 4

05 n

m

Non-modified LDFPTO-modified LDFPTO/NLS-modified LDFPlasmid DNA

B Western blot4 Weeks after primary immunization

0 2 4 6 8 Weeks after primary immunization

Paper III

Figure 21. Serological analysis of LDF immunized mice. (A) Mean ELISA values from LDF immunized mice. The ELISA was performed with mouse sera from individual animals of different vaccination groups. Blood samples were taken and prepared at the indicated time-points before each DNA vaccination, and analyzed for reactivity against the N peptide (the antigen). The bars at each time-point are: from left—non-modified LDF (white bars), PTO-modified (light grey bars), PTO/NLS-modified (dark grey bars) and plasmid DNA (black bars). (B) The Western blot analysis was performed with mouse sera from 20 mice (four mice per group) at two time-points (2 and 4 weeks after the primary immunization).

In our hands antigen stimulation of T-cells from N-protein immunized mice was

unsuccessful. However low levels of soluble cytokines (γ-INF and IL-4) were

found in tissue culture media from antigen-stimulated cells. Our findings suggest

that linear DNA fragment encoding the amino terminus of PUUV induces a strong

humoral immune response together with a low T-cell-mediated immune response.

These data were also supported by an isotype ELISA. Virtually, all antigen specific

antibodies were found to belong to IgG1 subspecies, correlating to a Th2 type of

immune response (data not shown).

69


Figure 22. Median serum titers. The titers were determined by serial dilutions of mouse sera from the different time-points after the primary immunization. The bars at each time-point after the primary immunization are: from left—non-modified LDF (white bars), PTO-modified (light grey bars), PTO/NLS-modified (dark grey bars) and plasmid DNA (black bars).

All experiments showed that fragments modified both with PTO and NLS-peptide,

performed better than fragments modified only with PTO, which in turn

performed better than non-modified fragments. This was probably due to their

PTO-enhanced stability and faster entry of the DNA to the nucleus, when linked to

NLS.

DNA vaccines against a variety of pathogens are currently investigated by many

research groups. However, most constructs are plasmid based and contain among

other features a genetic resistance marker. These are not desirable. In this study we

have explored the use of linear DNA vaccines that only encode the desired antigen.

Furthermore, we have examined two ways of increasing expression of this antigen

by protection of the construct against nuclease degradation and by attaching a

nuclear localization signal peptide. Both were successful.

70

Paper IV

Paper IV

The objective of this study was to evaluate the potential of the cDNA microarray

technique to identify and discriminate PCR derived amplicons from genetically

highly similar hantaviruses.

Microarray is a hybridisation technique that permits simultaneous analysis of one

genetic sample against, up to tens of thousands of known probes [356, 357]. By

immobilizing various viral gene sequences on a glass slide, a sample can be assayed

for many different viruses simultaneously. These properties have been utilised for

identification of viral pathogens by two basically different approaches: the

oligonucleotide microarray [274, 358, 359], that provides a highly strain specific

virus identification, and the PCR fragment microarray [275], that gives a better

tolerance for inherent sequence variations.

We designed and printed a hantavirus specific microarray, consisting of DNA

fragments reverse transcribed and amplified from 12 virus isolates of six different

hantaviruses. The S and M-segment were represented by 500 bp overlapping and

250 bp non-overlapping DNA fragments. Each DNA fragment was printed as

duplicates in four subgrids, giving a supergrid. This supergrid was also replicated in

triplicate on the chip, giving 6 redundant spots for each individual DNA fragment

on the chip, giving a total of 2,304 spots per chip.

The overall pair-wise sequence identity among the 12 isolates varied between

54.3% (PHV vs. SEOV SR11) and 85.7% (PUUV Kazan vs. PUUV Cg1820) for

the M-segment, and 51.1% (PUUV Sotkamo vs. SEOV SR11) and 92.5% (PUUV

Kazan vs. PUUV Cg1820) for the S-segment.

After labelling, PCR fragments were then hybridized, either alone or in pairs, to the

microarray chip. It was observed that there were very little ambiguities in the results

for the different viruses (Fig 24, Table 2). However, for virus isolates that were

highly similar (PUUV Cg1820 vs. P360 and K27, and SEOV SR11 vs. SEOV 80-

39, which had nucleotide identities of 99% and 96% respectively), this method was

71


not elucidative enough (Table 2). Since these highly similar viruses gave a diluting

effect on the signals, one from each group was chosen, and the others excluded

from further analysis.

Figure 23. Hybridisation patterns of PUUV Umeå/hu represented by green spots, and HTNV 76-118 represented by red spots, to one supergrid of the hantavirus microarray. Yellow spots signify orientation spots.

Results and discriminative power obtained from analysis of M-segments spots were

similar to that derived from the analysis of both segments. However, when using

only spots corresponding to the S-segment, PUUV Cg1820 could not be

distinguished from PUUV Kazan, probably due to a combination of their genetic

similarity and inconsistencies in the amount of probe-DNA spotted on the chip.

To improve the resolution, we further investigated the use of only one relatively

conserved fragment (from the S-segment) of each of three closely related PUUV

(Umeå/hu, Sotkamo and Kazan). Two different fragment lengths from each strain

72

Paper IV

were used for hybridisation, to determine if fragment length would have an effect

on the outcome of analysis. Hybridization (%) a

Printed probe fragments

PUU

V U

meå

PUU

V S

otka

mo

PUU

V K

azan

PUU

V C

G18

20

HTN

V 7

6-11

8

SEO

V S

R11

DO

BV 3

970-

87

PHV

SNV

Con

vict

Cre

ek

Vira

l Con

trol b

Repl

icate

s c

PUUV Umeå 87 0 0 0 0 0 0 0 0 0 5 PUUV Sotkamo 1 82 2 0 0 0 0 0 0 0 2 PUUV Kazan 6 0 88 5 0 0 0 0 0 0 3 PUUV CG1820 6 3 31 52 0 0 0 0 0 0 2 PUUV P360 0 0 6 88 0 0 0 0 0 0 2 PUUV K27 6 6 16 68 0 0 0 0 0 0 2 HTNV 76-118 1 6 0 1 82 2 0 0 0 0 5 SEOV SR11 0 0 0 0 0 80 0 0 0 0 3 SEOV 80-39 0 0 0 0 0 88 0 0 0 0 4 DOBV 3970-87 0 0 0 0 0 0 89 0 0 0 2 PHV 0 0 0 0 0 0 0 100 0 0 3

Hyb

ridiz

ed ta

rget

s

SNV Convict Creek 1 1 4 1 8 1 0 0 72 0 4

Table 2. Specific Hybridisation Values for M and S Segments. a Calculated percentage values for the signals of the viral target sequence in the sample hybridised to the printed probe fragments. b Viral Control: haemorrhagic fever virus control DNA corresponding to the Ebola Zaire, Ebola Reston, Crimean Congo, and Marburg viruses. c Replicates: number of hybridisations of each target.

For a 456 bp fragment (position 43-499 of the S-segment, where the sequence

identities ranged from 88.4%, for Umeå/hu vs. Kazan, to 91.4% for Sotkamo vs.

Kazan) the different isolates could still be differentiated (Fig 25 A). However when

the shorter 250 bp fragment (position 249-499 of the S-segment, with sequence

identities ranging from 84.4%, for Umeå/hu vs. Kazan, to 89.6% for Sotkamo vs.

Kazan) was used, it was not longer possible to differentiate Sotkamo from Kazan

(Fig 25 B). Even though the percentage of sequence identity for the longer

fragment was lower, it performed better than the shorter fragment.

73


Figure 24. Normalised hybridisation signals for fragments, corresponding to position 43 to 499 (A) and position 249–499 (B) for the S segment of PUUV Umeå, PUUV Sotkamo and PUUV Kazan when hybridising target sequence from the three viruses to the hantavirus microarray. In the histograms, columns of average signal with error bars representing the standard deviation described hybridisation of one type of target sequence to certain fragments. These values were calculated from the same sets of hybridisation data that included both types of fragments.

The strength of a microarray chip like the one that we have designed, is its ability to

give an indication of what sort of virus is being analyzed, even if this specific virus,

for what ever reason, is not present on the chip. We tested this by hybridizing

DNA from a specific virus and then deliberately excluding the virus specific spots

from the analysis, to see what kind of indications the results could give us (Table 3).

As expected, a “missing” PUUV was picked by the other PUUV’s spots. The close

relatives, SEOV, HTNV and DOBV, picked each other up, while SNV was picked

74

Paper IV

up by HTNV and the PUUV. For unknown reasons, Prospect Hill virus did not

cross hybridise with any of the viruses tested.

Hybridization (%) a


PUU

V U

meå

PUU

V S

otka

mo

PUU

V K

azan

PUU

V C

G18

20

HTN

V 7

6-11

8

SEO

V S

R11

DO

BV 3

970-

87

PHV

SNV

Con

vict

Cre

ek

Vira

l con

trol b

Repl

icate

s c

PUUV Umeå + 17 44 22 3 0 0 0 3 0 5 PUUV Sotkamo 17 + 41 25 0 10 0 0 9 0 2 PUUV Kazan 29 14 + 36 0 0 3 0 3 0 3 PUUV CG1820 18 15 56 + 0 0 3 0 0 0 2 HTNV 76-118 13 11 10 11 + 28 4 3 16 0 5 SEOV SR11 8 10 0 0 57 + 0 0 0 0 3 DOBV 3970-87 11 0 4 16 21 35 + 7 0 0 2 PHV 0 0 0 0 0 0 0 + 0 0 3 H

ybrid

ized

targ

ets

SNV Convict Creek 6 4 21 7 40 4 2 3 + 0 4

Table 3. Hybridisation values for removed viruses. a Calculated percentage values for the signals of the viral target sequence in the sample hybridised to the printed probe fragments. b,c Similar to Table 2. + Removed virus specific fragment

The ability to analyze complex samples, such as infected tissue from bank voles, is

also important. To test this we performed PCR on cDNA derived directly from six

bank voles caught in the wild just south of Umeå. When these PCR-reactions were

analyzed on agarose gel, only a smear could be seen, but when labelled and

hybridized to the chip they were all recognized as PUUV, and all but one was

recognized, unambiguously, as PUUV Umeå/hu (Table 4).

75


Hybridization (%) a


Sam

ple

PUU

V U

meå

PUU

V S

otka

mo

PUU

V K

azan

PUU

V C

G18

20

HTN

V 7

6-11

8

SEO

V S

R11

DO

BV 3

970-

87

PHV

SNV

Con

vict

Cre

ek

Vira

l Con

trol b

Repl

icate

s c

1 82 0 10 0 0 0 0 0 0 0 1 2 20 17 10 17 10 0 0 7 9 0 2 3 76 7 0 2 0 0 0 0 0 0 2 4 83 1 0 3 0 0 0 0 0 0 2 5 84 0 0 0 0 0 0 0 0 0 2 6 71 6 5 6 0 0 0 0 0 0 2

Table 4. Hybridisation of six samples from lungs of bank voles wild caught outside of Umeå, Sweden. a, b, c similar to table 2.

Our approach of combining a well designed RT-PCR with a microarray of 500

nucleotide fragments covering large parts of the viral genomes, was able to render

surprisingly high discriminative power for differentiating viruses from complex

samples

The ability to discriminate and identify was accompanied by a tolerance for smaller

sequence differences. This makes the microarray suitable for testing samples

containing unknown viruses.

In spite of problems getting equal amounts of DNA in different spots, we have

demonstrated a good discriminative ability, partly by including many replicate spots

and partly by having an adaptable analysis method.

76

Conclusions

Conclusions

In this thesis, a local PUUV isolated from a human patient and PUUV RNA in

wild bank voles in the coastal region of Västerbotten County in Northern Sweden,

were characterised. We found that the genetic differences among hantaviruses

found in different location is not always gradual, but can show large variation even

when there is no obvious difference in their host genetics. We believe that the

resulting information will be of importance for development of both genetic and

serological diagnostic and epidemiological tools, as well as for future vaccine

development. We have also shown that the use of nuclease resistant, nucleotide

analogues and nuclear localization signal peptides greatly improve DNA vaccines

based on linear DNA, and that they could be an alternative to plasmid DNA for

vaccination purposes. Furthermore, the microarray technique was demonstrated to

be very suitable for differentiation among hantaviruses as well as among different

isolates of a specific hantavirus.

77


Acknowledgements

There are so many people that have helped me during the course of my Ph.D. It is impossible to mention everyone here.

So I would start by saying a big THANK YOU to you who are not mentioned on this page. Next, I would like to thank my two supervisors Fredrik and Göran, for making all this possible and for all the help and support that they have given me on and off work. I am also greatly indebted to: Marlene, for working long hard hours with me all the way from the Katab project. And to Bertil, for bringing food, so we wouldn’t starve; Lena, for helping me at work and at Kaptensgatan; Bosse, Anna, Marie, Henrik, Therese, Margaretha and everyone else, that still is or used to be part of the FOI virology group for helping and supporting me at FOI; Gert, for helping me with the bank voles and all the wilderness stuff; Clas for support and help with any clinical questions; Per, Anci, Göran, Ingrid, Katrine and Lena in the department of virology at Umeå University, for their invaluable help and support in all sort of ways; Oleg, for his deep knowledge and help during the write up; Linda Bladh, Henrik Nordström, Anna Wallin, Li Quan Gen and Åke Lundkvist, SMI, for generous help and support; Gunnar Sandström and Åke Sellström, FOI, for help and support; Department for Biomedical Sciences, for giving me support and shelter during my write up; I am also grateful to the CCD personnel at DSO National Laboratories, Singapore, for putting me up and allowing me to do research and writing in their facilities and helping me with all sorts of things. Thank you Mui Tiang, Michelle, Hwee Teng, , Yian Kim, Gek Kee, Jeffrey, Susie, Susan, Soo Sim, Yvonne, Chee Hwee and last but not least Dr. Lee. Christofer, Pär and Fabian, for making sure I had a life outside that lab as well. Pappa, syster yster and Gunborg, for all the help at home in Bankeryd. And last but not least my family, Lee Ching, Lei and Eigil, who have supported, cheered me up and put up with me during the whole journey. I love You!

78

References

References

1. Lee, H.W. and G. van der Groen, Hemorrhagic fever with renal syndrome. Prog Med Virol,

1989. 36: p. 62-102. 2. Gligic, A., N. Dimkovic, S.Y. Xiao, G.J. Buckle, D. Jovanovic, D. Velimirovic, R.

Stojanovic, et al., Belgrade virus: a new hantavirus causing severe hemorrhagic fever with renal syndrome in Yugoslavia. J Infect Dis, 1992. 166(1): p. 113-20.

3. Clement, J., P. Heyman, P. McKenna, P. Colson, and T. Avsic-Zupanc, The hantaviruses of Europe: from the bedside to the bench. Emerg Infect Dis, 1997. 3(2): p. 205-11.

4. Lee, H.W., Hemorrhagic fever with renal syndrome in Korea. Rev Infect Dis, 1989. 11 Suppl 4: p. S864-76.

5. Lee, H.W., Epidemiology and Pathogenesis of Hemorrhagic Fever with Renal Syndrome, in The Bunyaviridae. 1996, Plenum Press: New York. p. 253-64.

6. Song, G., Epidemiological progresses of hemorrhagic fever with renal syndrome in China. Chin Med J (Engl), 1999. 112(5): p. 472-7.

7. Schmaljohn, C. and B. Hjelle, Hantaviruses: a global disease problem. Emerg Infect Dis, 1997. 3(2): p. 95-104.

8. Papadimitriou, M., Hantavirus nephropathy. Kidney Int, 1995. 48(3): p. 887-902. 9. CDC, http://www.cdc.gov/ncidod/diseases/hanta/hps/noframes/caseinfo.htm. Centers for disease

control and prevention, 2005. 10. Castillo, C., J. Naranjo, A. Sepulveda, G. Ossa, and H. Levy, Hantavirus pulmonary syndrome

due to Andes virus in Temuco, Chile: clinical experience with 16 adults. Chest, 2001. 120(2): p. 548-54.

11. Toivanen, A.L., L. Valanne, and T. Tatlisumak, Acute disseminated encephalomyelitis following nephropathia epidemica. Acta Neurol Scand, 2002. 105(4): p. 333-6.

12. Vapalahti, O., J. Mustonen, Å. Lundkvist, H. Henttonen, A. Plyusnin, and A. Vaheri, Hantavirus infections in Europe. Lancet Infect Dis, 2003. 3(10): p. 653-61.

13. Heyman, P., A. Plyusnina, P. Berny, C. Cochez, M. Artois, M. Zizi, J.P. Pirnay, et al., Seoul hantavirus in Europe: first demonstration of the virus genome in wild Rattus norvegicus captured in France. Eur J Clin Microbiol Infect Dis, 2004. 23(9): p. 711-7.

14. Olsson, G.E., F. Dalerum, B. Hornfeldt, F. Elgh, T.R. Palo, P. Juto, and C. Ahlm, Human hantavirus infections, Sweden. Emerg Infect Dis, 2003. 9(11): p. 1395-401.

15. Ahlm, C., A. Thelin, F. Elgh, P. Juto, E.L. Stiernström, S. Holmberg, and A. Tärnvik, Prevalence of antibodies specific to Puumala virus among farmers in Sweden. Scand J Work Environ Health, 1998. 24(2): p. 104-8.

16. Plyusnin, A., O. Vapalahti, H. Lehvaslaiho, N. Apekina, T. Mikhailova, I. Gavrilovskaya, J. Laakkonen, et al., Genetic variation of wild Puumala viruses within the serotype, local rodent populations and individual animal. Virus Res, 1995. 38(1): p. 25-41.

17. Casals, J., B.E. Henderson, H. Hoogstraal, K.M. Johnson, and A. Shelokov, A review of Soviet viral hemorrhagic fevers, 1969. J Infect Dis, 1970. 122(5): p. 437-53.

18. Zetterholm, S.G., Akuta nefriter simulerande akuta bukfall. Läkartidningen, 1934. 31: p. 425-9.

19. Myrhman, En njursjukdom med egenartad sjukdomsbild. Nordisk medicinsk tidskrift, 1934. 7: p. 793-4.

20. Stuhlfauth, K., Bericht uber ein neues schlammfieberähnliches Krankheitbild bei Deutschen Truppen in Lappland. Deutsche medizinische Wochenschrift, 1943. 69: p. 439.

21. Hortling, En epidemi av fältfeber (?) i finska Lappland. Nordisk medicinsk tidskrift, 1946. 30: p. 1001.

22. Smadel, J.E., Epidemic hemorrhagic fever. Am J Public Health, 1953. 43(10): p. 1327-30.

79

http://www.cdc.gov/ncidod/diseases/hanta/hps/noframes/caseinfo.htm


23. Traub, R. and C.L. Wisseman, Jr., Korean hemorrhagic fever. J Infect Dis, 1978. 138(2): p. 267-72.

24. Gajdusek, D.C., Virus hemorrhagic fevers. Special reference to hemorrhagic fever with renal syndrome (epidemic hemorrhagic fever). J Pediatr, 1962. 60: p. 841-57.

25. Johnson, K.M., Hantaviruses: history and overview. Curr Top Microbiol Immunol, 2001. 256: p. 1-14.

26. Lee, H.W. and P.W. Lee, Korean hemorrahagic fever. I. Demonstration of causative antigen and antibodies. Korean Journal of Internal medicine, 1976. 19: p. 371-83.

27. Lee, H.W., P.W. Lee, and K.M. Johnson, Isolation of the etiologic agent of Korean Hemorrhagic fever. J Infect Dis, 1978. 137(3): p. 298-308.

28. Lee, H.W., L.J. Baek, and K.M. Johnson, Isolation of Hantaan virus, the etiologic agent of Korean hemorrhagic fever, from wild urban rats. J Infect Dis, 1982. 146(5): p. 638-44.

29. French, G.R., R.S. Foulke, O.A. Brand, G.A. Eddy, H.W. Lee, and P.W. Lee, Korean hemorrhagic fever: propagation of the etiologic agent in a cell line of human origin. Science, 1981. 211(4486): p. 1046-8.

30. Brummer-Korvenkontio, M., A. Vaheri, T. Hovi, C.H. von Bonsdorff, J. Vuorimies, T. Manni, K. Penttinen, et al., Nephropathia epidemica: detection of antigen in bank voles and serologic diagnosis of human infection. J Infect Dis, 1980. 141(2): p. 131-4.

31. Niklasson, B. and J. Le Duc, Isolation of the nephropathia epidemica agent in Sweden. Lancet, 1984. 1(8384): p. 1012-3.

32. Yanagihara, R., D. Goldgaber, P.W. Lee, H.L. Amyx, D.C. Gajdusek, C.J. Gibbs, Jr., and A. Svedmyr, Propagation of nephropathia epidemica virus in cell culture. Lancet, 1984. 1(8384): p. 1013.

33. Avsic-Zupanc, T., S.Y. Xiao, R. Stojanovic, A. Gligic, G. van der Groen, and J.W. LeDuc, Characterization of Dobrava virus: a Hantavirus from Slovenia, Yugoslavia. J Med Virol, 1992. 38(2): p. 132-7.

34. Lee, P.W., H.L. Amyx, R. Yanagihara, D.C. Gajdusek, D. Goldgaber, and C.J. Gibbs, Jr., Partial characterization of Prospect Hill virus isolated from meadow voles in the United States. J Infect Dis, 1985. 152(4): p. 826-9.

35. Xiao, S.Y., J.W. Leduc, Y.K. Chu, and C.S. Schmaljohn, Phylogenetic analyses of virus isolates in the genus Hantavirus, family Bunyaviridae. Virology, 1994. 198(1): p. 205-17.

36. Nichol, S.T., C.F. Spiropoulou, S. Morzunov, P.E. Rollin, T.G. Ksiazek, H. Feldmann, A. Sanchez, et al., Genetic identification of a hantavirus associated with an outbreak of acute respiratory illness. Science, 1993. 262(5135): p. 914-7.

37. Wells, R.M., S. Sosa Estani, Z.E. Yadon, D. Enria, P. Padula, N. Pini, J.N. Mills, et al., An unusual hantavirus outbreak in southern Argentina: person-to-person transmission? Hantavirus Pulmonary Syndrome Study Group for Patagonia. Emerg Infect Dis, 1997. 3(2): p. 171-4.

38. Carey, D.E., R. Reuben, K.N. Panicker, R.E. Shope, and R.M. Myers, Thottapalayam virus: a presumptive arbovirus isolated from a shrew in India. Indian J Med Res, 1971. 59(11): p. 1758-60.

39. Tang, Y.W., Z.Y. Xu, Z.Y. Zhu, and T.F. Tsai, Isolation of haemorrhagic fever with renal syndrome virus from Suncus murinus, an insectivore. Lancet, 1985. 1(8427): p. 513-4.

40. Zeller, H.G., N. Karabatsos, C.H. Calisher, J.P. Digoutte, C.B. Cropp, F.A. Murphy, and R.E. Shope, Electron microscopic and antigenic studies of uncharacterized viruses. II. Evidence suggesting the placement of viruses in the family Bunyaviridae. Arch Virol, 1989. 108(3-4): p. 211-27.

41. Arthur, R.R., R.S. Lofts, J. Gomez, G.E. Glass, J.W. Leduc, and J.E. Childs, Grouping of hantaviruses by small (S) genome segment polymerase chain reaction and amplification of viral RNA from wild-caught rats. Am J Trop Med Hyg, 1992. 47(2): p. 210-24.

42. Schmaljohn, C.S., Molecular Biology of Hantaviruses, in The Bunyaviridae, R.M. Elliot, Editor. 1996, Plenum Press: New York and London. p. 63-90.

80

References

43. Hung, T., Z.Y. Chou, T.X. Zhao, S.M. Xia, and C.S. Hang, Morphology and morphogenesis of viruses of hemorrhagic fever with renal syndrome (HFRS). I. Some peculiar aspects of the morphogenesis of various strains of HFRS virus. Intervirology, 1985. 23(2): p. 97-108.

44. Goldsmith, C.S., L.H. Elliott, C.J. Peters, and S.R. Zaki, Ultrastructural characteristics of Sin Nombre virus, causative agent of hantavirus pulmonary syndrome. Arch Virol, 1995. 140(12): p. 2107-22.

45. Rodriguez, L.L., J.H. Owens, C.J. Peters, and S.T. Nichol, Genetic reassortment among viruses causing hantavirus pulmonary syndrome. Virology, 1998. 242(1): p. 99-106.

46. Spiropoulou, C.F., Hantavirus maturation. Curr Top Microbiol Immunol, 2001. 256: p. 33-46.

47. Murphy, F.A., A.K. Harrison, and S.G. Whitfield, Bunyaviridae: morphologic and morphogenetic similarities of Bunyamwera serologic supergroup viruses and several other arthropod-borne viruses. Intervirology, 1973. 1(4): p. 297-316.

48. Pettersson, R.F. and L. Melin, Synthesis, assembly and intracellular transport of Bunyaviridae membrane proteins, in The Bunyaviridae, R.M. Elliot, Editor. 1996, Plenum Press: New York. p. 159-88.

49. Hung, T., J. Zhou, S. Xia, and Z. He, Atlas of hemorrhagic fever with renal syndrome. 1988, Beijing: Science Press.

50. Schmaljohn, C.S., S.E. Hasty, S.A. Harrison, and J.M. Dalrymple, Characterization of Hantaan virions, the prototype virus of hemorrhagic fever with renal syndrome. J Infect Dis, 1983. 148(6): p. 1005-12.

51. Hewlett, M.J., R.F. Pettersson, and D. Baltimore, Circular forms of Uukuniemi virion RNA: an electron microscopic study. J Virol, 1977. 21(3): p. 1085-93.

52. Hacker, D., S. Rochat, and D. Kolakofsky, Anti-mRNAs in La Crosse bunyavirus-infected cells. J Virol, 1990. 64(10): p. 5051-7.

53. Ravkov, E.V. and R.W. Compans, Hantavirus nucleocapsid protein is expressed as a membrane-associated protein in the perinuclear region. J Virol, 2001. 75(4): p. 1808-15.

54. Kukkonen, S.K., A. Vaheri, and A. Plyusnin, Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein. J Gen Virol, 2004. 85(Pt 5): p. 1181-9.

55. Bridgen, A. and R.M. Elliott, Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs. Proc Natl Acad Sci U S A, 1996. 93(26): p. 15400-4.

56. Dunn, E.F., D.C. Pritlove, H. Jin, and R.M. Elliott, Transcription of a recombinant bunyavirus RNA template by transiently expressed bunyavirus proteins. Virology, 1995. 211(1): p. 133-43.

57. Flick, R. and R.F. Pettersson, Reverse genetics system for Uukuniemi virus (Bunyaviridae): RNA polymerase I-catalyzed expression of chimeric viral RNAs. J Virol, 2001. 75(4): p. 1643-55.

58. Flick, K., J.W. Hooper, C.S. Schmaljohn, R.F. Pettersson, H. Feldmann, and R. Flick, Rescue of Hantaan virus minigenomes. Virology, 2003. 306(2): p. 219-24.

59. Blakqori, G., G. Kochs, O. Haller, and F. Weber, Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids. J Gen Virol, 2003. 84(Pt 5): p. 1207-14.

60. Accardi, L., C. Prehaud, P. Di Bonito, S. Mochi, M. Bouloy, and C. Giorgi, Activity of Toscana and Rift Valley fever virus transcription complexes on heterologous templates. J Gen Virol, 2001. 82(Pt 4): p. 781-5.

61. Betenbaugh, M., M. Yu, K. Kuehl, J. White, D. Pennock, K. Spik, and C. Schmaljohn, Nucleocapsid- and virus-like particles assemble in cells infected with recombinant baculoviruses or vaccinia viruses expressing the M and the S segments of Hantaan virus. Virus Res, 1995. 38(2-3): p. 111-24.

62. Kaukinen, P., Tula hantavirus nucleocapsid protein. Helsinki University Biomedical Dissertations. Vol. No. 53. 2004, Helsinki: Helsinki University.

63. Xu, X., W. Severson, N. Villegas, C.S. Schmaljohn, and C.B. Jonsson, The RNA binding domain of the hantaan virus N protein maps to a central, conserved region. J Virol, 2002. 76(7): p. 3301-8.

81


64. Mir, M.A. and A.T. Panganiban, The hantavirus nucleocapsid protein recognizes specific features of the viral RNA panhandle and is altered in conformation upon RNA binding. J Virol, 2005. 79(3): p. 1824-35.

65. Ravkov, E.V., S.T. Nichol, C.J. Peters, and R.W. Compans, Role of actin microfilaments in Black Creek Canal virus morphogenesis. J Virol, 1998. 72(4): p. 2865-70.

66. Kariwa, H., H. Tanabe, T. Mizutani, Y. Kon, K. Lokugamage, N. Lokugamage, M.A. Iwasa, et al., Synthesis of Seoul virus RNA and structural proteins in cultured cells. Arch Virol, 2003. 148(9): p. 1671-85.

67. Kaukinen, P., V. Kumar, K. Tulimaki, P. Engelhardt, A. Vaheri, and A. Plyusnin, Oligomerization of Hantavirus N protein: C-terminal alpha-helices interact to form a shared hydrophobic space. J Virol, 2004. 78(24): p. 13669-77.

68. Mir, M.A. and A.T. Panganiban, Trimeric hantavirus nucleocapsid protein binds specifically to the viral RNA panhandle. J Virol, 2004. 78(15): p. 8281-8.

69. Hacker, D., R. Raju, and D. Kolakofsky, La Crosse virus nucleocapsid protein controls its own synthesis in mosquito cells by encapsidating its mRNA. J Virol, 1989. 63(12): p. 5166-74.

70. Severson, W.E., X. Xu, and C.B. Jonsson, cis-Acting signals in encapsidation of Hantaan virus S-segment viral genomic RNA by its N protein. J Virol, 2001. 75(6): p. 2646-52.

71. Wilson, V.G. and D. Rangasamy, Viral interaction with the host cell sumoylation system. Virus Res, 2001. 81(1-2): p. 17-27.

72. Kaukinen, P., A. Vaheri, and A. Plyusnin, Non-covalent interaction between nucleocapsid protein of Tula hantavirus and small ubiquitin-related modifier-1, SUMO-1. Virus Res, 2003. 92(1): p. 37-45.

73. Maeda, A., B.H. Lee, K. Yoshimatsu, M. Saijo, I. Kurane, J. Arikawa, and S. Morikawa, The intracellular association of the nucleocapsid protein (NP) of hantaan virus (HTNV) with small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9). Virology, 2003. 305(2): p. 288-97.

74. Li, X.D., T.P. Makela, D. Guo, R. Soliymani, V. Koistinen, O. Vapalahti, A. Vaheri, et al., Hantavirus nucleocapsid protein interacts with the Fas-mediated apoptosis enhancer Daxx. J Gen Virol, 2002. 83(Pt 4): p. 759-66.

75. Kochs, G., C. Janzen, H. Hohenberg, and O. Haller, Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes. Proc Natl Acad Sci U S A, 2002. 99(5): p. 3153-8.

76. Kanerva, M., K. Melen, A. Vaheri, and I. Julkunen, Inhibition of puumala and tula hantaviruses in Vero cells by MxA protein. Virology, 1996. 224(1): p. 55-62.

77. Frese, M., G. Kochs, H. Feldmann, C. Hertkorn, and O. Haller, Inhibition of bunyaviruses, phleboviruses, and hantaviruses by human MxA protein. J Virol, 1996. 70(2): p. 915-23.

78. Elgh, F., Å. Lundkvist, O.A. Alexeyev, G. Wadell, and P. Juto, A major antigenic domain for the human humoral response to Puumala virus nucleocapsid protein is located at the amino-terminus. J Virol Methods, 1996. 59(1-2): p. 161-72.

79. Lundkvist, Å., S. Björsten, B. Niklasson, and N. Ahlborg, Mapping of B-cell determinants in the nucleocapsid protein of Puumala virus: definition of epitopes specific for acute immunoglobulin G recognition in humans. Clin Diagn Lab Immunol, 1995. 2(1): p. 82-6.

80. Tischler, N.D., H. Galeno, M. Rosemblatt, and P.D. Valenzuela, Human and rodent humoral immune responses to Andes virus structural proteins. Virology, 2005. 334(2): p. 319-26.

81. de Carvalho Nicacio, C., M. Sallberg, C. Hultgren, and Å. Lundkvist, T-helper and humoral responses to Puumala hantavirus nucleocapsid protein: identification of T-helper epitopes in a mouse model. J Gen Virol, 2001. 82(Pt 1): p. 129-38.

82. de Carvalho Nicacio, C., E. Björling, and Å. Lundkvist, Immunoglobulin A responses to Puumala hantavirus. J Gen Virol, 2000. 81(Pt 6): p. 1453-61.

82

References

83. Elgh, F., M. Linderholm, G. Wadell, A. Tärnvik, and P. Juto, Development of humoral cross-reactivity to the nucleocapsid protein of heterologous hantaviruses in nephropathia epidemica. FEMS Immunol Med Microbiol, 1998. 22(4): p. 309-15.

84. Ennis, F.A., J. Cruz, C.F. Spiropoulou, D. Waite, C.J. Peters, S.T. Nichol, H. Kariwa, et al., Hantavirus pulmonary syndrome: CD8+ and CD4+ cytotoxic T lymphocytes to epitopes on Sin Nombre virus nucleocapsid protein isolated during acute illness. Virology, 1997. 238(2): p. 380-90.

85. Van Epps, H.L., C.S. Schmaljohn, and F.A. Ennis, Human memory cytotoxic T-lymphocyte (CTL) responses to Hantaan virus infection: identification of virus-specific and cross-reactive CD8(+) CTL epitopes on nucleocapsid protein. J Virol, 1999. 73(7): p. 5301-8.

86. Bridgen, A., F. Weber, J.K. Fazakerley, and R.M. Elliott, Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis. Proc Natl Acad Sci U S A, 2001. 98(2): p. 664-9.

87. Bouloy, M., C. Janzen, P. Vialat, H. Khun, J. Pavlovic, M. Huerre, and O. Haller, Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs. J Virol, 2001. 75(3): p. 1371-7.

88. Weber, F., A. Bridgen, J.K. Fazakerley, H. Streitenfeld, N. Kessler, R.E. Randall, and R.M. Elliott, Bunyamwera bunyavirus nonstructural protein NSs counteracts the induction of alpha/beta interferon. J Virol, 2002. 76(16): p. 7949-55.

89. Weber, F., E.F. Dunn, A. Bridgen, and R.M. Elliott, The Bunyamwera virus nonstructural protein NSs inhibits viral RNA synthesis in a minireplicon system. Virology, 2001. 281(1): p. 67-74.

90. Colon-Ramos, D.A., P.M. Irusta, E.C. Gan, M.R. Olson, J. Song, R.I. Morimoto, R.M. Elliott, et al., Inhibition of translation and induction of apoptosis by Bunyaviral nonstructural proteins bearing sequence similarity to reaper. Mol Biol Cell, 2003. 14(10): p. 4162-72.

91. Soldan, S.S., M.L. Plassmeyer, M.K. Matukonis, and F. Gonzalez-Scarano, La Crosse virus nonstructural protein NSs counteracts the effects of short interfering RNA. J Virol, 2005. 79(1): p. 234-44.

92. Tulimäki, K., P. Kaukinen, F. Weber, A. Vaheri, and A. Plyusnin. Tula and Puumala Hantavirus NSs ORFs are functional. in The 6th International Conference on Hemorrhagic Fever with Renal Syndrome (HFRS), Hantavirus Pulmonary Syndrome (HPS) and Hantaviruses. 2004. Seoul, Korea.

93. Plyusnin, A., Genetics of hantaviruses: implications to taxonomy. Arch Virol, 2002. 147(4): p. 665-82.

94. Lober, C., B. Anheier, S. Lindow, H.D. Klenk, and H. Feldmann, The Hantaan virus glycoprotein precursor is cleaved at the conserved pentapeptide WAASA. Virology, 2001. 289(2): p. 224-9.

95. Schmaljohn, C.S., S.E. Hasty, L. Rasmussen, and J.M. Dalrymple, Hantaan virus replication: effects of monensin, tunicamycin and endoglycosidases on the structural glycoproteins. J Gen Virol, 1986. 67 ( Pt 4): p. 707-17.

96. Schmaljohn, C.S., Y.K. Chu, A.L. Schmaljohn, and J.M. Dalrymple, Antigenic subunits of Hantaan virus expressed by baculovirus and vaccinia virus recombinants. J Virol, 1990. 64(7): p. 3162-70.

97. Pensiero, M.N., G.B. Jennings, C.S. Schmaljohn, and J. Hay, Expression of the Hantaan virus M genome segment by using a vaccinia virus recombinant. J Virol, 1988. 62(3): p. 696-702.

98. Ruusala, A., R. Persson, C.S. Schmaljohn, and R.F. Pettersson, Coexpression of the membrane glycoproteins G1 and G2 of Hantaan virus is required for targeting to the Golgi complex. Virology, 1992. 186(1): p. 53-64.

99. Shi, X. and R.M. Elliott, Analysis of N-linked glycosylation of hantaan virus glycoproteins and the role of oligosaccharide side chains in protein folding and intracellular trafficking. J Virol, 2004. 78(10): p. 5414-22.

83


100. Arikawa, J., A.L. Schmaljohn, J.M. Dalrymple, and C.S. Schmaljohn, Characterization of Hantaan virus envelope glycoprotein antigenic determinants defined by monoclonal antibodies. J Gen Virol, 1989. 70 ( Pt 3): p. 615-24.

101. Sheshberadaran, H., B. Niklasson, and E.A. Tkachenko, Antigenic relationship between hantaviruses analysed by immunoprecipitation. J Gen Virol, 1988. 69 ( Pt 10): p. 2645-51.

102. Schmaljohn, C.S., A.L. Schmaljohn, and J.M. Dalrymple, Hantaan virus M RNA: coding strategy, nucleotide sequence, and gene order. Virology, 1987. 157(1): p. 31-9.

103. Puffer, B.A., S.C. Watkins, and R.C. Montelaro, Equine infectious anemia virus Gag polyprotein late domain specifically recruits cellular AP-2 adapter protein complexes during virion assembly. J Virol, 1998. 72(12): p. 10218-21.

104. Ohno, H., J. Stewart, M.C. Fournier, H. Bosshart, I. Rhee, S. Miyatake, T. Saito, et al., Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science, 1995. 269(5232): p. 1872-5.

105. Scales, S.J., M. Gomez, and T.E. Kreis, Coat proteins regulating membrane traffic. Int Rev Cytol, 2000. 195: p. 67-144.

106. Deyde, V.M., A.A. Rizvanov, J. Chase, E.W. Otteson, and S.C. St Jeor, Interactions and trafficking of Andes and Sin Nombre Hantavirus glycoproteins G1 and G2. Virology, 2005. 331(2): p. 307-15.

107. Pensiero, M.N. and J. Hay, The Hantaan virus M-segment glycoproteins G1 and G2 can be expressed independently. J Virol, 1992. 66(4): p. 1907-14.

108. Shi, X. and R.M. Elliott, Golgi localization of Hantaan virus glycoproteins requires coexpression of G1 and G2. Virology, 2002. 300(1): p. 31-8.

109. Spiropoulou, C.F., C.S. Goldsmith, T.R. Shoemaker, C.J. Peters, and R.W. Compans, Sin Nombre virus glycoprotein trafficking. Virology, 2003. 308(1): p. 48-63.

110. Ravkov, E.V., S.T. Nichol, and R.W. Compans, Polarized entry and release in epithelial cells of Black Creek Canal virus, a New World hantavirus. J Virol, 1997. 71(2): p. 1147-54.

111. Helenius, A. and M. Aebi, Intracellular functions of N-linked glycans. Science, 2001. 291(5512): p. 2364-9.

112. Hansen, J.E., O. Lund, K. Rapacki, and S. Brunak, O-GLYCBASE version 2.0: a revised database of O-glycosylated proteins. Nucleic Acids Res, 1997. 25(1): p. 278-82.

113. Gabius, H.J., S. Andre, H. Kaltner, and H.C. Siebert, The sugar code: functional lectinomics. Biochim Biophys Acta, 2002. 1572(2-3): p. 165-77.

114. Opdenakker, G., P.M. Rudd, C.P. Ponting, and R.A. Dwek, Concepts and principles of glycobiology. Faseb J, 1993. 7(14): p. 1330-7.

115. Spiropoulou, C.F., S. Morzunov, H. Feldmann, A. Sanchez, C.J. Peters, and S.T. Nichol, Genome structure and variability of a virus causing hantavirus pulmonary syndrome. Virology, 1994. 200(2): p. 715-23.

116. Geimonen, E., R. LaMonica, K. Springer, Y. Farooqui, I.N. Gavrilovskaya, and E.R. Mackow, Hantavirus pulmonary syndrome-associated hantaviruses contain conserved and functional ITAM signaling elements. J Virol, 2003. 77(2): p. 1638-43.

117. Xu, X.N., B. Laffert, G.R. Screaton, M. Kraft, D. Wolf, W. Kolanus, J. Mongkolsapay, et al., Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain. J Exp Med, 1999. 189(9): p. 1489-96.

118. Dehghani, H., C.R. Brown, R. Plishka, A. Buckler-White, and V.M. Hirsch, The ITAM in Nef influences acute pathogenesis of AIDS-inducing simian immunodeficiency viruses SIVsm and SIVagm without altering kinetics or extent of viremia. J Virol, 2002. 76(9): p. 4379-89.

119. Luo, W. and B.M. Peterlin, Activation of the T-cell receptor signaling pathway by Nef from an aggressive strain of simian immunodeficiency virus. J Virol, 1997. 71(12): p. 9531-7.

120. Inatome, R., S. Yanagi, T. Takano, and H. Yamamura, A critical role for Syk in endothelial cell proliferation and migration. Biochem Biophys Res Commun, 2001. 286(1): p. 195-9.

84

References

121. Geimonen, E., I. Fernandez, I.N. Gavrilovskaya, and E.R. Mackow, Tyrosine residues direct the ubiquitination and degradation of the NY-1 hantavirus G1 cytoplasmic tail. J Virol, 2003. 77(20): p. 10760-868.

122. Bharadwaj, M., C.R. Lyons, I.A. Wortman, and B. Hjelle, Intramuscular inoculation of Sin Nombre hantavirus cDNAs induces cellular and humoral immune responses in BALB/c mice. Vaccine, 1999. 17(22): p. 2836-43.

123. Hooper, J.W., D.M. Custer, E. Thompson, and C.S. Schmaljohn, DNA vaccination with the Hantaan virus M gene protects Hamsters against three of four HFRS hantaviruses and elicits a high-titer neutralizing antibody response in Rhesus monkeys. J Virol, 2001. 75(18): p. 8469-77.

124. Terajima, M., H.L. Van Epps, D. Li, A.M. Leporati, S.E. Juhlin, J. Mustonen, A. Vaheri, et al., Generation of recombinant vaccinia viruses expressing Puumala virus proteins and use in isolating cytotoxic T cells specific for Puumala virus. Virus Res, 2002. 84(1-2): p. 67-77.

125. Gavrilovskaya, I.N., M. Shepley, R. Shaw, M.H. Ginsberg, and E.R. Mackow, beta3 Integrins mediate the cellular entry of hantaviruses that cause respiratory failure. Proc Natl Acad Sci U S A, 1998. 95(12): p. 7074-9.

126. Gavrilovskaya, I.N., E.J. Brown, M.H. Ginsberg, and E.R. Mackow, Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins. J Virol, 1999. 73(5): p. 3951-9.

127. Kim, T.Y., Y. Choi, H.S. Cheong, and J. Choe, Identification of a cell surface 30 kDa protein as a candidate receptor for Hantaan virus. J Gen Virol, 2002. 83(Pt 4): p. 767-73.

128. Pekosz, A. and F. Gonzalez-Scarano, The extracellular domain of La Crosse virus G1 forms oligomers and undergoes pH-dependent conformational changes. Virology, 1996. 225(1): p. 243-7.

129. Arikawa, J., I. Takashima, and N. Hashimoto, Cell fusion by haemorrhagic fever with renal syndrome (HFRS) viruses and its application for titration of virus infectivity and neutralizing antibody. Arch Virol, 1985. 86(3-4): p. 303-13.

130. Ogino, M., K. Yoshimatsu, H. Ebihara, K. Araki, B.H. Lee, M. Okumura, and J. Arikawa, Cell fusion activities of Hantaan virus envelope glycoproteins. J Virol, 2004. 78(19): p. 10776-82.

131. Jin, M., J. Park, S. Lee, B. Park, J. Shin, K.J. Song, T.I. Ahn, et al., Hantaan virus enters cells by clathrin-dependent receptor-mediated endocytosis. Virology, 2002. 294(1): p. 60-9.

132. Zhang, X.K., I. Takashima, and N. Hashimoto, Characteristics of passive immunity against hantavirus infection in rats. Arch Virol, 1989. 105(3-4): p. 235-46.

133. Arikawa, J., J.S. Yao, K. Yoshimatsu, I. Takashima, and N. Hashimoto, Protective role of antigenic sites on the envelope protein of Hantaan virus defined by monoclonal antibodies. Arch Virol, 1992. 126(1-4): p. 271-81.

134. Koch, J., M. Liang, I. Queitsch, A.A. Kraus, and E.K. Bautz, Human recombinant neutralizing antibodies against hantaan virus G2 protein. Virology, 2003. 308(1): p. 64-73.

135. Liang, M., M. Mahler, J. Koch, Y. Ji, D. Li, C. Schmaljohn, and E.K. Bautz, Generation of an HFRS patient-derived neutralizing recombinant antibody to Hantaan virus G1 protein and definition of the neutralizing domain. J Med Virol, 2003. 69(1): p. 99-107.

136. Hörling, J. and Å. Lundkvist, Single amino acid substitutions in Puumala virus envelope glycoproteins G1 and G2 eliminate important neutralization epitopes. Virus Res, 1997. 48(1): p. 89-100.

137. Chu, Y.K., C. Rossi, J.W. Leduc, H.W. Lee, C.S. Schmaljohn, and J.M. Dalrymple, Serological relationships among viruses in the Hantavirus genus, family Bunyaviridae. Virology, 1994. 198(1): p. 196-204.

138. Jonsson, C.B. and C.S. Schmaljohn, Replication of hantaviruses. Curr Top Microbiol Immunol, 2001. 256: p. 15-32.

139. Pettersson, R.F., The structure of Uukuniemi virus, a proposed member of the bunyaviruses. Med Biol, 1975. 53(5): p. 418-24.

140. Jin, H. and R.M. Elliott, Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies. J Gen Virol, 1992. 73 ( Pt 9): p. 2235-44.

85


141. Jin, H. and R.M. Elliott, Expression of functional Bunyamwera virus L protein by recombinant vaccinia viruses. J Virol, 1991. 65(8): p. 4182-9.

142. Jin, H. and R.M. Elliott, Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus. J Virol, 1993. 67(3): p. 1396-404.

143. Schmaljohn, C.S. and J.M. Dalrymple, Analysis of Hantaan virus RNA: evidence for a new genus of bunyaviridae. Virology, 1983. 131(2): p. 482-91.

144. Plyusnin, A., Y. Cheng, H. Lehvaslaiho, and A. Vaheri, Quasispecies in wild-type tula hantavirus populations. J Virol, 1996. 70(12): p. 9060-3.

145. Flick, R., F. Elgh, and R.F. Pettersson, Mutational analysis of the Uukuniemi virus (Bunyaviridae family) promoter reveals two elements of functional importance. J Virol, 2002. 76(21): p. 10849-60.

146. Flick, R., G. Neumann, E. Hoffmann, E. Neumeier, and G. Hobom, Promoter elements in the influenza vRNA terminal structure. Rna, 1996. 2(10): p. 1046-57.

147. Mackow, E.R. and I.N. Gavrilovskaya, Cellular receptors and hantavirus pathogenesis. Curr Top Microbiol Immunol, 2001. 256: p. 91-115.

148. Gavrilovskaya, I.N., T. Peresleni, E. Geimonen, and E.R. Mackow, Pathogenic hantaviruses selectively inhibit beta3 integrin directed endothelial cell migration. Arch Virol, 2002. 147(10): p. 1913-31.

149. den Boon, J.A., J. Chen, and P. Ahlquist, Identification of sequences in Brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes. J Virol, 2001. 75(24): p. 12370-81.

150. Kukkonen, S.K., A. Vaheri, and A. Plyusnin, L protein, the RNA-dependent RNA polymerase of hantaviruses. Arch Virol, 2005. 150(3): p. 533-56.

151. Garcin, D., M. Lezzi, M. Dobbs, R.M. Elliott, C. Schmaljohn, C.Y. Kang, and D. Kolakofsky, The 5' ends of Hantaan virus (Bunyaviridae) RNAs suggest a prime-and-realign mechanism for the initiation of RNA synthesis. J Virol, 1995. 69(9): p. 5754-62.

152. Hutchinson, K.L., C.J. Peters, and S.T. Nichol, Sin Nombre virus mRNA synthesis. Virology, 1996. 224(1): p. 139-49.

153. Morzunov, S.P., H. Feldmann, C.F. Spiropoulou, V.A. Semenova, P.E. Rollin, T.G. Ksiazek, C.J. Peters, et al., A newly recognized virus associated with a fatal case of hantavirus pulmonary syndrome in Louisiana. J Virol, 1995. 69(3): p. 1980-3.

154. Beaton, A.R. and R.M. Krug, Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the absence of a 5' capped end. Proc Natl Acad Sci U S A, 1986. 83(17): p. 6282-6.

155. Patton, J.T., N.L. Davis, and G.W. Wertz, N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus. J Virol, 1984. 49(2): p. 303-9.

156. Elliott, R.M., M. Bouloy, C.H. Calisher, R. Goldbach, J.T. Moyer, S.T. Nichol, R.F. Pettersson, et al., Bunyaviridae, in Virus Taxonomy: VIIth report of the International Committee on Taxonomy of Viruses, M.H.V. van Regenmortel, et al., Editors. 1999, Academic Press: San Diego, California. p. 599-631.

157. Plyusnin, A. and S.P. Morzunov, Virus evolution and genetic diversity of hantaviruses and their rodent hosts. Curr Top Microbiol Immunol, 2001. 256: p. 47-75.

158. Sjölander, K.B., I. Golovljova, V. Vasilenko, A. Plyusnin, and Å. Lundkvist, Serological divergence of Dobrava and Saaremaa hantaviruses: evidence for two distinct serotypes. Epidemiol Infect, 2002. 128(1): p. 99-103.

159. Meyer, B.J. and C.S. Schmaljohn, Persistent hantavirus infections: characteristics and mechanisms. Trends Microbiol, 2000. 8(2): p. 61-7.

160. Slonova, R.A., E.A. Tkachenko, E.L. Kushnarev, T.K. Dzagurova, and T.I. Astakova, Hantavirus isolation from birds. Acta Virol, 1992. 36(5): p. 493.

86

References

161. Malecki, T.M., G.P. Jillson, J.P. Thilsted, J. Elrod, N. Torrez-Martinez, and B. Hjelle, Serologic survey for hantavirus infection in domestic animals and coyotes from New Mexico and northeastern Arizona. J Am Vet Med Assoc, 1998. 212(7): p. 970-3.

162. Ahlm, C., K. Wallin, Å. Lundkvist, F. Elgh, P. Juto, M. Merza, and A. Tärnvik, Serologic evidence of Puumala virus infection in wild moose in northern Sweden. Am J Trop Med Hyg, 2000. 62(1): p. 106-11.

163. Danes, L., M. Pejcoch, K. Bukovjan, J. Veleba, and M. Halackova, [Antibodies against Hantaviruses in game and domestic oxen in the Czech Republic]. Cesk Epidemiol Mikrobiol Imunol, 1992. 41(1): p. 15-8.

164. Escutenaire, S. and P.P. Pastoret, [Hantavirus infection epidemiology in Belgium]. Bull Mem Acad R Med Belg, 2001. 156(1-2): p. 137-44; discussion 144-6.

165. Meyer, B.J. and C. Schmaljohn, Accumulation of terminally deleted RNAs may play a role in Seoul virus persistence. J Virol, 2000. 74(3): p. 1321-31.

166. Hutchinson, K.L., P.E. Rollin, and C.J. Peters, Pathogenesis of a North American hantavirus, Black Creek Canal virus, in experimentally infected Sigmodon hispidus. Am J Trop Med Hyg, 1998. 59(1): p. 58-65.

167. Lundkvist, Å., D. Wiger, J. Hörling, K.B. Sjölander, A. Plyusnina, R. Mehl, A. Vaheri, et al., Isolation and characterization of Puumala hantavirus from Norway: evidence for a distinct phylogenetic sublineage. J Gen Virol, 1998. 79 ( Pt 11): p. 2603-14.

168. Avsic-Zupanc, T., A. Toney, K. Anderson, Y.K. Chu, and C. Schmaljohn, Genetic and antigenic properties of Dobrava virus: a unique member of the Hantavirus genus, family Bunyaviridae. J Gen Virol, 1995. 76 ( Pt 11): p. 2801-8.

169. Wang, H., K. Yoshimatsu, H. Ebihara, M. Ogino, K. Araki, H. Kariwa, Z. Wang, et al., Genetic diversity of hantaviruses isolated in china and characterization of novel hantaviruses isolated from Niviventer confucianus and Rattus rattus. Virology, 2000. 278(2): p. 332-45.

170. Plyusnin, A., O. Vapalahti, V. Vasilenko, H. Henttonen, and A. Vaheri, Dobrava hantavirus in Estonia: does the virus exist throughout Europe? Lancet, 1997. 349(9062): p. 1369-70.

171. Nemirov, K., O. Vapalahti, Å. Lundkvist, V. Vasilenko, I. Golovljova, A. Plyusnina, J. Niemimaa, et al., Isolation and characterization of Dobrava hantavirus carried by the striped field mouse (Apodemus agrarius) in Estonia. J Gen Virol, 1999. 80 ( Pt 2): p. 371-9.

172. Yashina, L.N., N.A. Patrushev, L.I. Ivanov, R.A. Slonova, V.P. Mishin, G.G. Kompanez, N.I. Zdanovskaya, et al., Genetic diversity of hantaviruses associated with hemorrhagic fever with renal syndrome in the far east of Russia. Virus Res, 2000. 70(1-2): p. 31-44.

173. Lokugamage, K., H. Kariwa, D. Hayasaka, B.Z. Cui, T. Iwasaki, N. Lokugamage, L.I. Ivanov, et al., Genetic characterization of hantaviruses transmitted by the Korean field mouse (Apodemus peninsulae), Far East Russia. Emerg Infect Dis, 2002. 8(8): p. 768-76.

174. Plyusnin, A., O. Vapalahti, H. Lankinen, H. Lehvaslaiho, N. Apekina, Y. Myasnikov, H. Kallio-Kokko, et al., Tula virus: a newly detected hantavirus carried by European common voles. J Virol, 1994. 68(12): p. 7833-9.

175. Plyusnin, A., O. Vapalahti, Å. Lundkvist, H. Henttonen, and A. Vaheri, Newly recognised hantavirus in Siberian lemmings. Lancet, 1996. 347(9018): p. 1835.

176. Lee, P.W., H.L. Amyx, D.C. Gajdusek, R.T. Yanagihara, D. Goldgaber, and C.J. Gibbs, Jr., New hemorrhagic fever with renal syndrome-related virus in rodents in the United States. Lancet, 1982. 2(8312): p. 1405.

177. Song, W., N. Torrez-Martinez, W. Irwin, F.J. Harrison, R. Davis, M. Ascher, M. Jay, et al., Isla Vista virus: a genetically novel hantavirus of the California vole Microtus californicus. J Gen Virol, 1995. 76 ( Pt 12): p. 3195-9.

178. Kariwa, H., S. Yoshizumi, J. Arikawa, K. Yoshimatsu, K. Takahashi, I. Takashima, and N. Hashimoto, Evidence for the existence of Puumula-related virus among Clethrionomys rufocanus in Hokkaido, Japan. Am J Trop Med Hyg, 1995. 53(3): p. 222-7.

87


179. Hörling, J., V. Chizhikov, Å. Lundkvist, M. Jonsson, L. Ivanov, A. Dekonenko, B. Niklasson, et al., Khabarovsk virus: a phylogenetically and serologically distinct hantavirus isolated from Microtus fortis trapped in far-east Russia. J Gen Virol, 1996. 77 ( Pt 4): p. 687-94.

180. Kariwa, H., K. Yoshimatsu, J. Sawabe, E. Yokota, J. Arikawa, I. Takashima, H. Fukushima, et al., Genetic diversities of hantaviruses among rodents in Hokkaido, Japan and Far East Russia. Virus Res, 1999. 59(2): p. 219-28.

181. Lopez, N., P. Padula, C. Rossi, M.E. Lazaro, and M.T. Franze-Fernandez, Genetic identification of a new hantavirus causing severe pulmonary syndrome in Argentina. Virology, 1996. 220(1): p. 223-6.

182. Bharadwaj, M., J. Botten, N. Torrez-Martinez, and B. Hjelle, Rio Mamore virus: genetic characterization of a newly recognized hantavirus of the pygmy rice rat, Oligoryzomys microtis, from Bolivia. Am J Trop Med Hyg, 1997. 57(3): p. 368-74.

183. Hjelle, B., J. Krolikowski, N. Torrez-Martinez, F. Chavez-Giles, C. Vanner, and E. Laposata, Phylogenetically distinct hantavirus implicated in a case of hantavirus pulmonary syndrome in the northeastern United States. J Med Virol, 1995. 46(1): p. 21-7.

184. Johnson, A.M., M.D. Bowen, T.G. Ksiazek, R.J. Williams, R.T. Bryan, J.N. Mills, C.J. Peters, et al., Laguna Negra virus associated with HPS in western Paraguay and Bolivia. Virology, 1997. 238(1): p. 115-27.

185. Ravkov, E.V., P.E. Rollin, T.G. Ksiazek, C.J. Peters, and S.T. Nichol, Genetic and serologic analysis of Black Creek Canal virus and its association with human disease and Sigmodon hispidus infection. Virology, 1995. 210(2): p. 482-9.

186. Rawlings, J.A., N. Torrez-Martinez, S.U. Neill, G.M. Moore, B.N. Hicks, S. Pichuantes, A. Nguyen, et al., Cocirculation of multiple hantaviruses in Texas, with characterization of the small (S) genome of a previously undescribed virus of cotton rats (Sigmodon hispidus). Am J Trop Med Hyg, 1996. 55(6): p. 672-9.

187. Hjelle, B., F. Chavez-Giles, N. Torrez-Martinez, T. Yates, J. Sarisky, J. Webb, and M. Ascher, Genetic identification of a novel hantavirus of the harvest mouse Reithrodontomys megalotis. J Virol, 1994. 68(10): p. 6751-4.

188. Hjelle, B., F. Chavez-Giles, N. Torrez-Martinez, T. Yamada, J. Sarisky, M. Ascher, and S. Jenison, Dominant glycoprotein epitope of four corners hantavirus is conserved across a wide geographical area. J Gen Virol, 1994. 75 ( Pt 11): p. 2881-8.

189. Song, J.W., L.J. Baek, J.W. Nagle, D. Schlitter, and R. Yanagihara, Genetic and phylogenetic analyses of hantaviral sequences amplified from archival tissues of deer mice (Peromyscus maniculatus nubiterrae) captured in the eastern United States. Arch Virol, 1996. 141(5): p. 959-67.

190. Elliott, L.H., T.G. Ksiazek, P.E. Rollin, C.F. Spiropoulou, S. Morzunov, M. Monroe, C.S. Goldsmith, et al., Isolation of the causative agent of hantavirus pulmonary syndrome. Am J Trop Med Hyg, 1994. 51(1): p. 102-8.

191. Hjelle, B., B. Anderson, N. Torrez-Martinez, W. Song, W.L. Gannon, and T.L. Yates, Prevalence and geographic genetic variation of hantaviruses of New World harvest mice (Reithrodontomys): identification of a divergent genotype from a Costa Rican Reithrodontomys mexicanus. Virology, 1995. 207(2): p. 452-9.

192. Fulhorst, C.F., M.C. Monroe, R.A. Salas, G. Duno, A. Utrera, T.G. Ksiazek, S.T. Nichol, et al., Isolation, characterization and geographic distribution of Cano Delgadito virus, a newly discovered South American hantavirus (family Bunyaviridae). Virus Res, 1997. 51(2): p. 159-71.

193. Lopez, N., P. Padula, C. Rossi, S. Miguel, A. Edelstein, E. Ramirez, and M.T. Franze-Fernandez, Genetic characterization and phylogeny of Andes virus and variants from Argentina and Chile. Virus Res, 1997. 50(1): p. 77-84.

194. Levis, S., J.E. Rowe, S. Morzunov, D.A. Enria, and S. St Jeor, New hantaviruses causing hantavirus pulmonary syndrome in central Argentina. Lancet, 1997. 349(9057): p. 998-9.

88

References

195. Vincent, M.J., E. Quiroz, F. Gracia, A.J. Sanchez, T.G. Ksiazek, P.T. Kitsutani, L.A. Ruedas, et al., Hantavirus pulmonary syndrome in Panama: identification of novel hantaviruses and their likely reservoirs. Virology, 2000. 277(1): p. 14-9.

196. Morzunov, S.P., J.E. Rowe, T.G. Ksiazek, C.J. Peters, S.C. St Jeor, and S.T. Nichol, Genetic analysis of the diversity and origin of hantaviruses in Peromyscus leucopus mice in North America. J Virol, 1998. 72(1): p. 57-64.

197. Sanchez, A.J., K.D. Abbott, and S.T. Nichol, Genetic identification and characterization of limestone canyon virus, a unique Peromyscus-borne hantavirus. Virology, 2001. 286(2): p. 345-53.

198. Nemirov, K., Genetic Relationships of Saaremaa and Dobrava Hantaviruses, in Department of virology, Haartman Institute. 2003, Univeristy of Helsinki: Helsinki. p. 94.

199. Lednicky, J.A., Hantaviruses. a short review. Arch Pathol Lab Med, 2003. 127(1): p. 30-5. 200. Savela, M., Life. 2004. 201. Schultze, D., Å. Lundkvist, U. Blauenstein, and P. Heyman, Tula virus infection associated

with fever and exanthema after a wild rodent bite. Eur J Clin Microbiol Infect Dis, 2002. 21(4): p. 304-6.

202. Song, J.W., L.J. Baek, K.J. Song, A. Skrok, J. Markowski, J. Bratosiewicz-Wasik, R. Kordek, et al., Characterization of Tula virus from common voles (microtus arvalis) in Poland: evidence for geographic-specific phylogenetic clustering. Virus Genes, 2004. 29(2): p. 239-47.

203. Song, J.W., A. Gligic, and R. Yanagihara, Identification of Tula hantavirus in Pitymys subterraneus captured in the Cacak region of Serbia-Yugoslavia. Int J Infect Dis, 2002. 6(1): p. 31-6.

204. Plyusnin, A., Y. Cheng, O. Vapalahti, M. Pejcoch, J. Unar, Z. Jelinkova, H. Lehvaslaiho, et al., Genetic variation in Tula hantaviruses: sequence analysis of the S and M segments of strains from Central Europe. Virus Res, 1995. 39(2-3): p. 237-50.

205. Sibold, C., H. Meisel, D.H. Krüger, M. Labuda, J. Lysy, O. Kozuch, M. Pejcoch, et al., Recombination in Tula hantavirus evolution: analysis of genetic lineages from Slovakia. J Virol, 1999. 73(1): p. 667-75.

206. Bowen, M.D., W. Gelbmann, T.G. Ksiazek, S.T. Nichol, and N. Nowotny, Puumala virus and two genetic variants of Tula virus are present in Austrian rodents. J Med Virol, 1997. 53(2): p. 174-81.

207. Heyman, P., J. Klingström, F. de Jaegere, G. Leclercq, F. Rozenfeld, S. Escutenaire, C. Vandenvelde, et al., Tula hantavirus in Belgium. Epidemiol Infect, 2002. 128(2): p. 251-6.

208. Niklasson, B. and J.W. LeDuc, Epidemiology of nephropathia epidemica in Sweden. J Infect Dis, 1987. 155(2): p. 269-76.

209. Asikainen, K., T. Hanninen, H. Henttonen, J. Niemimaa, J. Laakkonen, H.K. Andersen, N. Bille, et al., Molecular evolution of puumala hantavirus in Fennoscandia: phylogenetic analysis of strains from two recolonization routes, Karelia and Denmark. J Gen Virol, 2000. 81(Pt 12): p. 2833-41.

210. Sironen, T., A. Plyusnina, H.K. Andersen, J. Lodal, H. Leirs, J. Niemimaa, H. Henttonen, et al., Distribution of Puumala hantavirus in Denmark: analysis of bank voles (Clethrionomys glareolus) from Fyn and Jutland. Vector Borne Zoonotic Dis, 2002. 2(1): p. 37-45.

211. Hörling, J., Å. Lundkvist, M. Jaarola, A. Plyusnin, H. Tegelstrom, K. Persson, H. Lehvaslaiho, et al., Distribution and genetic heterogeneity of Puumala virus in Sweden. J Gen Virol, 1996. 77 (10): p. 2555-62.

212. Hughes, A.L. and R. Friedman, Evolutionary diversification of protein-coding genes of hantaviruses. Mol Biol Evol, 2000. 17(10): p. 1558-68.

213. Sironen, T., A. Vaheri, and A. Plyusnin, Molecular evolution of Puumala hantavirus. J Virol, 2001. 75(23): p. 11803-10.

214. Jackson, A.P. and M.A. Charleston, A cophylogenetic perspective of RNA-virus evolution. Mol Biol Evol, 2004. 21(1): p. 45-57.

89


215. Vapalahti, O., Å. Lundkvist, V. Fedorov, C.J. Conroy, S. Hirvonen, A. Plyusnina, K. Nemirov, et al., Isolation and characterization of a hantavirus from Lemmus sibiricus: evidence for host switch during hantavirus evolution. J Virol, 1999. 73(7): p. 5586-92.

216. Nemirov, K., H. Henttonen, A. Vaheri, and A. Plyusnin, Phylogenetic evidence for host switching in the evolution of hantaviruses carried by Apodemus mice. Virus Res, 2002. 90(1-2): p. 207-15.

217. Holland, J.J., J.C. De La Torre, and D.A. Steinhauer, RNA Virus Populations as Quasispecies, in Genetic Diversity of RNA Viruses. 1992, Springer-Verlag: Berlin. p. 1-20.

218. Worobey, M. and E.C. Holmes, Evolutionary aspects of recombination in RNA viruses. J Gen Virol, 1999. 80 ( Pt 10): p. 2535-43.

219. Plyusnin, A., S.K. Kukkonen, A. Plyusnina, O. Vapalahti, and A. Vaheri, Transfection-mediated generation of functionally competent Tula hantavirus with recombinant S RNA segment. Embo J, 2002. 21(6): p. 1497-503.

220. Henderson, W.W., M.C. Monroe, S.C. St Jeor, W.P. Thayer, J.E. Rowe, C.J. Peters, and S.T. Nichol, Naturally occurring Sin Nombre virus genetic reassortants. Virology, 1995. 214(2): p. 602-10.

221. Klempa, B., H.A. Schmidt, R. Ulrich, S. Kaluz, M. Labuda, H. Meisel, B. Hjelle, et al., Genetic interaction between distinct Dobrava hantavirus subtypes in Apodemus agrarius and A. flavicollis in nature. J Virol, 2003. 77(1): p. 804-9.

222. Li, D., A.L. Schmaljohn, K. Anderson, and C.S. Schmaljohn, Complete nucleotide sequences of the M and S segments of two hantavirus isolates from California: evidence for reassortment in nature among viruses related to hantavirus pulmonary syndrome. Virology, 1995. 206(2): p. 973-83.

223. Lundkvist, Å., Y. Cheng, K.B. Sjölander, B. Niklasson, A. Vaheri, and A. Plyusnin, Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment. J Virol, 1997. 71(12): p. 9515-23.

224. Terajima, M., O. Vapalahti, H.L. Van Epps, A. Vaheri, and F.A. Ennis, Immune responses to Puumala virus infection and the pathogenesis of nephropathia epidemica. Microbes Infect, 2004. 6(2): p. 238-45.

225. Nakamura, T., R. Yanagihara, C.J. Gibbs, Jr., H.L. Amyx, and D.C. Gajdusek, Differential susceptibility and resistance of immunocompetent and immunodeficient mice to fatal Hantaan virus infection. Arch Virol, 1985. 86(1-2): p. 109-20.

226. Yoshimatsu, K., J. Arikawa, S. Ohbora, and C. Itakura, Hantavirus infection in SCID mice. J Vet Med Sci, 1997. 59(10): p. 863-8.

227. Mustonen, J., J. Partanen, M. Kanerva, K. Pietila, O. Vapalahti, A. Pasternack, and A. Vaheri, Genetic susceptibility to severe course of nephropathia epidemica caused by Puumala hantavirus. Kidney Int, 1996. 49(1): p. 217-21.

228. McNeil, A.J., P.L. Yap, S.M. Gore, R.P. Brettle, M. McColl, R. Wyld, S. Davidson, et al., Association of HLA types A1-B8-DR3 and B27 with rapid and slow progression of HIV disease. Qjm, 1996. 89(3): p. 177-85.

229. Mustonen, J., J. Partanen, M. Kanerva, K. Pietila, O. Vapalahti, A. Pasternack, and A. Vaheri, Association of HLA B27 with benign clinical course of nephropathia epidemica caused by Puumala hantavirus. Scand J Immunol, 1998. 47(3): p. 277-9.

230. Maes, P., J. Clement, I. Gavrilovskaya, and M. Van Ranst, Hantaviruses: immunology, treatment, and prevention. Viral Immunol, 2004. 17(4): p. 481-97.

231. Riquelme, R., M. Riquelme, A. Torres, M.L. Rioseco, J.A. Vergara, L. Scholz, and A. Carriel, Hantavirus pulmonary syndrome, southern Chile. Emerg Infect Dis, 2003. 9(11): p. 1438-43.

232. Passaro, D.J., W.J. Shieh, J.K. Hacker, C.L. Fritz, S.R. Hogan, M. Fischer, R.M. Hendry, et al., Predominant kidney involvement in a fatal case of hantavirus pulmonary syndrome caused by Sin Nombre virus. Clin Infect Dis, 2001. 33(2): p. 263-4.

90

References

233. Linderholm, M., A. Billström, B. Settergren, and A. Tärnvik, Pulmonary involvement in nephropathia epidemica as demonstrated by computed tomography. Infection, 1992. 20(5): p. 263-6.

234. Schutt, M., H. Meisel, D.H. Krüger, R. Ulrich, K. Dalhoff, and C. Dodt, Life-threatening Dobrava hantavirus infection with unusually extended pulmonary involvement. Clin Nephrol, 2004. 62(1): p. 54-7.

235. Clement, J., P. Colson, and P. McKenna, Hantavirus pulmonary syndrome in New England and Europe. N Engl J Med, 1994. 331(8): p. 545-6; author reply 547-8.

236. Ahlm, C., M. Linderholm, P. Juto, B. Stegmayr, and B. Settergren, Prevalence of serum IgG antibodies to Puumala virus (haemorrhagic fever with renal syndrome) in northern Sweden. Epidemiol Infect, 1994. 113(1): p. 129-36.

237. Settergren, B., Nephropathia epidemica (hemorrhagic fever with renal syndrome) in Scandinavia. Rev Infect Dis, 1991. 13(4): p. 736-44.

238. Settergren, B., C. Ahlm, O. Alexeyev, J. Billheden, and B. Stegmayr, Pathogenetic and clinical aspects of the renal involvement in hemorrhagic fever with renal syndrome. Ren Fail, 1997. 19(1): p. 1-14.

239. Mustonen, J., M. Brummer-Korvenkontio, K. Hedman, A. Pasternack, K. Pietila, and A. Vaheri, Nephropathia epidemica in Finland: a retrospective study of 126 cases. Scand J Infect Dis, 1994. 26(1): p. 7-13.

240. Huggins, J.W., C.M. Hsiang, T.M. Cosgriff, M.Y. Guang, J.I. Smith, Z.O. Wu, J.W. LeDuc, et al., Prospective, double-blind, concurrent, placebo-controlled clinical trial of intravenous ribavirin therapy of hemorrhagic fever with renal syndrome. J Infect Dis, 1991. 164(6): p. 1119-27.

241. Alexeyev, O., Personal communication. 2005. 242. Ahlm, C., Personal communication. 2005. 243. Sirotin, B.Z., I.M. Davidovich, and Y.L. Fedorchenko. Use of Plasmapheresis in Treatment of

Patients with HFRS. in The 6th International Conferance on Hemrrhagic Fever with Renal Syndrome (HFRS), Hantavirus Pulmonary Syndrome and Hantaviruses. 2004. Seoul: International Society for Hantaviruses.

244. Kanerva, M., J. Mustonen, and A. Vaheri, Pathogenesis of puumala and other hantavirus infections. Rev Med Virol, 1998. 8(2): p. 67-86.

245. Graziano, K.L. and B. Tempest, Hantavirus pulmonary syndrome: a zebra worth knowing. Am Fam Physician, 2002. 66(6): p. 1015-20.

246. PAHO, http://www.paho.org/English/AD/DPC/CD/hanta-americas-93-02.pdf. 1993, Pan American Health Organization.

247. Kitsutani, P.T., R.W. Denton, C.L. Fritz, R.A. Murray, R.L. Todd, W.J. Pape, J. Wyatt Frampton, et al., Acute Sin Nombre hantavirus infection without pulmonary syndrome, United States. Emerg Infect Dis, 1999. 5(5): p. 701-5.

248. Enria, D.A., A.M. Briggiler, N. Pini, and S. Levis, Clinical manifestations of New World hantaviruses. Curr Top Microbiol Immunol, 2001. 256: p. 117-34.

249. Nichol, S.T., T.G. Ksiazek, P.E. Rollin, and C.J. Peters, Hantavirus Pulmonary Syndrome and Newly Described Hantaviruses in the United States, in The Bunyaviridae, R.M. Elliott, Editor. 1996, Plenum Press: New York. p. 269-280.

250. Chapman, L.E., G.J. Mertz, C.J. Peters, H.M. Jolson, A.S. Khan, T.G. Ksiazek, F.T. Koster, et al., Intravenous ribavirin for hantavirus pulmonary syndrome: safety and tolerance during 1 year of open-label experience. Ribavirin Study Group. Antivir Ther, 1999. 4(4): p. 211-9.

251. Kallio-Kokko, H., O. Vapalahti, Å. Lundkvist, and A. Vaheri, Evaluation of Puumala virus IgG and IgM enzyme immunoassays based on recombinant baculovirus-expressed nucleocapsid protein for early nephropathia epidemica diagnosis. Clin Diagn Virol, 1998. 10(1): p. 83-90.

252. Sjölander, K.B., F. Elgh, H. Kallio-Kokko, O. Vapalahti, M. Hägglund, V. Palmcrantz, P. Juto, et al., Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica). J Clin Microbiol, 1997. 35(12): p. 3264-8.

91

http://www.paho.org/English/AD/DPC/CD/hanta-americas-93-02.pdf


253. Elgh, F., M. Linderholm, G. Wadell, and P. Juto, The clinical usefulness of a Puumalavirus recombinant nucleocapsid protein based enzyme-linked immunosorbent assay in the diagnosis of nephropathia epidemica as compared with an immunofluorescence assay. Clinical and Diagnostic Virology, 1996. 6(1): p. 17-26.

254. Ivanov, A., O. Vapalahti, H. Lankinen, E. Tkachenko, A. Vaheri, B. Niklasson, and Å. Lundkvist, Biotin-labeled antigen: a novel approach for detection of Puumala virus-specific IgM. J Virol Methods, 1996. 62(1): p. 87-92.

255. Tanishita, O., Y. Takahashi, Y. Okuno, K. Yamanishi, and M. Takahashi, Evaluation of focus reduction neutralization test with peroxidase-antiperoxidase staining technique for hemorrhagic fever with renal syndrome virus. J Clin Microbiol, 1984. 20(6): p. 1213-5.

256. Niklasson, B., M. Jonsson, Å. Lundkvist, J. Hörling, and E. Tkachenko, Comparison of European isolates of viruses causing hemorrhagic fever with renal syndrome by a neutralization test. Am J Trop Med Hyg, 1991. 45(6): p. 660-5.

257. Heider, H., B. Ziaja, C. Priemer, Å. Lundkvist, J. Neyts, D.H. Krüger, and R. Ulrich, A chemiluminescence detection method of hantaviral antigens in neutralisation assays and inhibitor studies. J Virol Methods, 2001. 96(1): p. 17-23.

258. Hörling, J., Å. Lundkvist, J.W. Huggins, and B. Niklasson, Antibodies to Puumala virus in humans determined by neutralization test. J Virol Methods, 1992. 39(1-2): p. 139-47.

259. Hujakka, H., V. Koistinen, P. Eerikainen, I. Kuronen, A. Laatikainen, J. Kauppinen, A. Vaheri, et al., Comparison of a new immunochromatographic rapid test with a commercial EIA for the detection of Puumala virus specific IgM antibodies. J Clin Virol, 2001. 23(1-2): p. 79-85.

260. Hujakka, H., V. Koistinen, P. Eerikainen, I. Kuronen, I. Mononen, M. Parviainen, Å. Lundkvist, et al., New immunochromatographic rapid test for diagnosis of acute Puumala virus infection. J Clin Microbiol, 2001. 39(6): p. 2146-50.

261. Hujakka, H., V. Koistinen, I. Kuronen, P. Eerikainen, M. Parviainen, Å. Lundkvist, A. Vaheri, et al., Diagnostic rapid tests for acute hantavirus infections: specific tests for Hantaan, Dobrava and Puumala viruses versus a hantavirus combination test. J Virol Methods, 2003. 108(1): p. 117-22.

262. Juto, P., Personal communication. 263. Tsai, T.F., Y.W. Tang, S.L. Hu, K.L. Ye, G.L. Chen, and Z.Y. Xu, Hemagglutination-

inhibiting antibody in hemorrhagic fever with renal syndrome. J Infect Dis, 1984. 150(6): p. 895-8. 264. Lu, Q., Z. Zhu, and J. Weng, Immune responses to inactivated vaccine in people naturally infected

with hantaviruses. J Med Virol, 1996. 49(4): p. 333-5. 265. Inouye, S., S. Matsuno, and R. Kono, Difference in antibody reactivity between complement fixation

and immune adherence hemagglutination tests with virus antigens. J Clin Microbiol, 1981. 14(3): p. 241-6.

266. Tang, Y.W., Y.L. Li, K.L. Ye, Z.Y. Xu, S.L. Ruo, S.P. Fisher-Hoch, and J.B. McCormick, Distribution of hantavirus serotypes Hantaan and Seoul causing hemorrhagic fever with renal syndrome and identification by hemagglutination inhibition assay. J Clin Microbiol, 1991. 29(9): p. 1924-7.

267. Sugiyama, K., Y. Matsuura, C. Morita, S. Shiga, Y. Akao, T. Komatsu, and T. Kitamura, An immune adherence assay for discrimination between etiologic agents of hemorrhagic fever with renal syndrome. J Infect Dis, 1984. 149(1): p. 67-73.

268. Gresikova, M. and M. Sekeyova, A simple method of preparing a complement-fixing antigen from the virus of haemorrhagic fever with renal syndrome (Western type). Acta Virol, 1988. 32(3): p. 272-4.

269. Juto, P., F. Elgh, C. Ahlm, O.A. Alexeyev, K. Edlund, Å. Lundkvist, and G. Wadell, The first human isolate of Puumala virus in Scandinavia as cultured from phytohemagglutinin stimulated leucocytes. J Med Virol, 1997. 53(2): p. 150-6.

270. Biel, S.S. and H.R. Gelderblom, Diagnostic electron microscopy is still a timely and rewarding method. J Clin Virol, 1999. 13(1-2): p. 105-19.

92

References

271. Cao, C., C. Shi, P. Li, Y. Tong, and Q. Ma, Diagnosis of hepatitis C Virus (HCV) infection by antigen-capturing ELISA. Clin Diagn Virol, 1996. 6(2-3): p. 137-45.

272. Garin, D., C. Peyrefitte, J.M. Crance, A. Le Faou, A. Jouan, and M. Bouloy, Highly sensitive Taqman PCR detection of Puumala hantavirus. Microbes Infect, 2001. 3(9): p. 739-45.

273. Aitichou, M., S.S. Saleh, A.K. McElroy, C. Schmaljohn, and M.S. Ibrahim, Identification of Dobrava, Hantaan, Seoul, and Puumala viruses by one-step real-time RT-PCR. J Virol Methods, 2005. 124(1-2): p. 21-6.

274. Wilson, W.J., C.L. Strout, T.Z. DeSantis, J.L. Stilwell, A.V. Carrano, and G.L. Andersen, Sequence-specific identification of 18 pathogenic microorganisms using microarray technology. Mol Cell Probes, 2002. 16(2): p. 119-27.

275. Li, J., S. Chen, and D.H. Evans, Typing and subtyping influenza virus using DNA microarrays and multiplex reverse transcriptase PCR. J Clin Microbiol, 2001. 39(2): p. 696-704.

276. Karaman, M.W., S. Groshen, C.C. Lee, B.L. Pike, and J.G. Hacia, Comparisons of substitution, insertion and deletion probes for resequencing and mutational analysis using oligonucleotide microarrays. Nucleic Acids Res, 2005. 33(3): p. e33.

277. Amexis, G., P. Oeth, K. Abel, A. Ivshina, F. Pelloquin, C.R. Cantor, A. Braun, et al., Quantitative mutant analysis of viral quasispecies by chip-based matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. Proc Natl Acad Sci U S A, 2001. 98(21): p. 12097-102.

278. Ronaghi, M., M. Uhlen, and P. Nyren, A sequencing method based on real-time pyrophosphate. Science, 1998. 281(5375): p. 363, 365.

279. Settergren, B., C. Ahlm, P. Juto, and B. Niklasson, Specific Puumala IgG virus half a century after haemorrhagic fever with renal syndrome. Lancet, 1991. 338(8758): p. 66.

280. Lundkvist, Å., J. Hörling, and B. Niklasson, The humoral response to Puumala virus infection (nephropathia epidemica) investigated by viral protein specific immunoassays. Arch Virol, 1993. 130(1-2): p. 121-30.

281. Lundkvist, Å., H. Kallio-Kokko, K.B. Sjölander, H. Lankinen, B. Niklasson, A. Vaheri, and O. Vapalahti, Characterization of Puumala virus nucleocapsid protein: identification of B-cell epitopes and domains involved in protective immunity. Virology, 1996. 216(2): p. 397-406.

282. Dargeviciute, A., K. Brus Sjolander, K. Sasnauskas, D.H. Krüger, H. Meisel, R. Ulrich, and Å. Lundkvist, Yeast-expressed Puumala hantavirus nucleocapsid protein induces protection in a bank vole model. Vaccine, 2002. 20(29-30): p. 3523-31.

283. Alexeyev, O.A., C. Ahlm, J. Billheden, B. Settergren, G. Wadell, and P. Juto, Elevated levels of total and Puumala virus-specific immunoglobulin E in the Scandinavian type of hemorrhagic fever with renal syndrome. Clin Diagn Lab Immunol, 1994. 1(3): p. 269-72.

284. Bostik, P., J. Winter, T.G. Ksiazek, P.E. Rollin, F. Villinger, S.R. Zaki, C.J. Peters, et al., Sin nombre virus (SNV) Ig isotype antibody response during acute and convalescent phases of hantavirus pulmonary syndrome. Emerg Infect Dis, 2000. 6(2): p. 184-7.

285. Groen, J., J. Dalrymple, S. Fisher-Hoch, J.G. Jordans, J.P. Clement, and A.D. Osterhaus, Serum antibodies to structural proteins of Hantavirus arise at different times after infection. J Med Virol, 1992. 37(4): p. 283-7.

286. Vapalahti, O., Å. Lundkvist, and A. Vaheri, Human immune response, host genetics, and severity of disease. Curr Top Microbiol Immunol, 2001. 256: p. 153-69.

287. Araki, K., K. Yoshimatsu, M. Ogino, H. Ebihara, Å. Lundkvist, H. Kariwa, I. Takashima, et al., Truncated hantavirus nucleocapsid proteins for serotyping Hantaan, Seoul, and Dobrava hantavirus infections. J Clin Microbiol, 2001. 39(7): p. 2397-404.

288. Paakkala, A., J. Mustonen, M. Viander, H. Huhtala, and A. Pasternack, Complement activation in nephropathia epidemica caused by Puumala hantavirus. Clin Nephrol, 2000. 53(6): p. 424-31.

93


289. Yoshimatsu, K., J. Arikawa, M. Tamura, R. Yoshida, Å. Lundkvist, B. Niklasson, H. Kariwa, et al., Characterization of the nucleocapsid protein of Hantaan virus strain 76-118 using monoclonal antibodies. J Gen Virol, 1996. 77 ( Pt 4): p. 695-704.

290. Linderholm, M., L. Bjermer, P. Juto, G. Roos, T. Sandström, B. Settergren, and A. Tärnvik, Local host response in the lower respiratory tract in nephropathia epidemica. Scand J Infect Dis, 1993. 25(5): p. 639-46.

291. Van Epps, H.L., M. Terajima, J. Mustonen, T.P. Arstila, E.A. Corey, A. Vaheri, and F.A. Ennis, Long-lived memory T lymphocyte responses after hantavirus infection. J Exp Med, 2002. 196(5): p. 579-88.

292. Plyusnin, A., J. Hörling, M. Kanerva, J. Mustonen, Y. Cheng, J. Partanen, O. Vapalahti, et al., Puumala hantavirus genome in patients with nephropathia epidemica: correlation of PCR positivity with HLA haplotype and link to viral sequences in local rodents. J Clin Microbiol, 1997. 35(5): p. 1090-6.

293. Kilpatrick, E.D., M. Terajima, F.T. Koster, M.D. Catalina, J. Cruz, and F.A. Ennis, Role of specific CD8+ T cells in the severity of a fulminant zoonotic viral hemorrhagic fever, hantavirus pulmonary syndrome. J Immunol, 2004. 172(5): p. 3297-304.

294. Balasubramanian, A.V., Prevention of smallpox in ancient India. CURRENT SCIENCE, 2002. 83(9-10): p. 1055.

295. Bazin, H., A brief history of the prevention of infectious diseases by immunisations. Comp Immunol Microbiol Infect Dis, 2003. 26(5-6): p. 293-308.

296. Wright, P.F., Global immunization--a medical perspective. Soc Sci Med, 1995. 41(5): p. 609-16. 297. Kloetzel, P.M., The proteasome and MHC class I antigen processing. Biochim Biophys Acta,

2004. 1695(1-3): p. 225-33. 298. Lauvau, G. and N. Glaichenhaus, Mini-review: Presentation of pathogen-derived antigens in vivo.

Eur J Immunol, 2004. 34(4): p. 913-20. 299. Giese, M., DNA-antiviral vaccines: new developments and approaches--a review. Virus Genes, 1998.

17(3): p. 219-32. 300. Spengler, U., M. Lechmann, B. Irrgang, F.L. Dumoulin, and T. Sauerbruch, Immune

responses in hepatitis C virus infection. J Hepatol, 1996. 24(2 Suppl): p. 20-5. 301. Callow, K.A., H.F. Parry, M. Sergeant, and D.A. Tyrrell, The time course of the immune

response to experimental coronavirus infection of man. Epidemiol Infect, 1990. 105(2): p. 435-46. 302. Smallpox vaccine adverse events among civilians--United States, February 25-March 3, 2003. MMWR

Morb Mortal Wkly Rep, 2003. 52(9): p. 180-1, 191. 303. Castelli, F. and A. Patroni, The human immunodeficiency virus-infected traveler. Clin Infect Dis,

2000. 31(6): p. 1403-8. 304. Minor, P.D., G. Dunn, M.E. Ramsay, and D. Brown, Effect of different immunisation schedules

on the excretion and reversion of oral poliovaccine strains. J Med Virol, 2005. 75(1): p. 153-60. 305. Mandl, C.W., J.H. Aberle, S.W. Aberle, H. Holzmann, S.L. Allison, and F.X. Heinz, In

vitro-synthesized infectious RNA as an attenuated live vaccine in a flavivirus model. Nat Med, 1998. 4(12): p. 1438-40.

306. Offit, P.A., The Cutter incident, 50 years later. N Engl J Med, 2005. 352(14): p. 1411-2. 307. Davis, H.L. and M.J. McCluskie, DNA vaccines for viral diseases. Microbes Infect, 1999. 1(1):

p. 7-21. 308. Kaiser, B., Vaccinationer. 1997, Lund: Studentlitteratur. 309. Geldmacher, A., D. Skrastina, I. Petrovskis, G. Borisova, J.A. Berriman, A.M. Roseman,

R.A. Crowther, et al., An amino-terminal segment of hantavirus nucleocapsid protein presented on hepatitis B virus core particles induces a strong and highly cross-reactive antibody response in mice. Virology, 2004. 323(1): p. 108-19.

310. Milich, D.R., D.L. Peterson, J. Zheng, J.L. Hughes, R. Wirtz, and F. Schodel, The hepatitis nucleocapsid as a vaccine carrier moiety. Ann N Y Acad Sci, 1995. 754: p. 187-201.

94

References

311. Yu, J. and W.H. Langridge, A plant-based multicomponent vaccine protects mice from enteric diseases. Nat Biotechnol, 2001. 19(6): p. 548-52.

312. Webster, D.E., M.C. Thomas, Z. Huang, and S.L. Wesselingh, The development of a plant-based vaccine for measles. Vaccine, 2005. 23(15): p. 1859-65.

313. Kehm, R., N.J. Jakob, T.M. Welzel, E. Tobiasch, O. Viczian, S. Jock, K. Geider, et al., Expression of immunogenic Puumala virus nucleocapsid protein in transgenic tobacco and potato plants. Virus Genes, 2001. 22(1): p. 73-83.

314. Khattak, S., G. Darai, S. Sule, and A. Rosen-Wolff, Characterization of expression of Puumala virus nucleocapsid protein in transgenic plants. Intervirology, 2002. 45(4-6): p. 334-9.

315. Khattak, S., G. Darai, and A. Rosen-Wolff, Puumala virus nucleocapsid protein expressed in transgenic plants is not immunogenic after oral administration. Virus Genes, 2004. 29(1): p. 109-16.

316. Bot, A. and C. Bona, Genetic immunization of neonates. Microbes Infect, 2002. 4(4): p. 511-20. 317. Wolff, J.A., R.W. Malone, P. Williams, W. Chong, G. Acsadi, A. Jani, and P.L. Felgner,

Direct gene transfer into mouse muscle in vivo. Science, 1990. 247(4949 Pt 1): p. 1465-8. 318. Tang, D.C., M. DeVit, and S.A. Johnston, Genetic immunization is a simple method for eliciting

an immune response. Nature, 1992. 356(6365): p. 152-4. 319. Robinson, H.L. and C.A. Torres, DNA vaccines. Semin Immunol, 1997. 9(5): p. 271-83. 320. Coon, B., L.L. An, J.L. Whitton, and M.G. von Herrath, DNA immunization to prevent

autoimmune diabetes. J Clin Invest, 1999. 104(2): p. 189-94. 321. von Herrath, M.G. and J.L. Whitton, DNA vaccination to treat autoimmune diabetes. Ann Med,

2000. 32(5): p. 285-92. 322. Mimran, A., F. Mor, P. Carmi, F.J. Quintana, V. Rotter, and I.R. Cohen, DNA vaccination

with CD25 protects rats from adjuvant arthritis and induces an antiergotypic response. J Clin Invest, 2004. 113(6): p. 924-32.

323. Schoen, C., J. Stritzker, W. Goebel, and S. Pilgrim, Bacteria as DNA vaccine carriers for genetic immunization. Int J Med Microbiol, 2004. 294(5): p. 319-35.

324. Hartl, A., R. Weiss, R. Hochreiter, S. Scheiblhofer, and J. Thalhamer, DNA vaccines for allergy treatment. Methods, 2004. 32(3): p. 328-39.

325. Manam, S., B.J. Ledwith, A.B. Barnum, P.J. Troilo, C.J. Pauley, L.B. Harper, T.G. Griffiths, 2nd, et al., Plasmid DNA vaccines: tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA. Intervirology, 2000. 43(4-6): p. 273-81.

326. Kang, K.K., S.M. Choi, J.H. Choi, D.S. Lee, C.Y. Kim, B.O. Ahn, B.M. Kim, et al., Safety evaluation of GX-12, a new HIV therapeutic vaccine: investigation of integration into the host genome and expression in the reproductive organs. Intervirology, 2003. 46(5): p. 270-6.

327. Nichols, W.W., B.J. Ledwith, S.V. Manam, and P.J. Troilo, Potential DNA vaccine integration into host cell genome. Ann N Y Acad Sci, 1995. 772: p. 30-9.

328. Nishikawa, M. and L. Huang, Nonviral vectors in the new millennium: delivery barriers in gene transfer. Hum Gene Ther, 2001. 12(8): p. 861-70.

329. Krieg, A.M. and J.N. Kline, Immune effects and therapeutic applications of CpG motifs in bacterial DNA. Immunopharmacology, 2000. 48(3): p. 303-5.

330. Park, K., C.S. Kim, and K.T. Moon, Protective effectiveness of hantavirus vaccine. Emerg Infect Dis, 2004. 10(12): p. 2218-20.

331. Hooper, J.W. and D. Li, Vaccines against hantaviruses. Curr Top Microbiol Immunol, 2001. 256: p. 171-91.

332. de Carvalho Nicacio, C., M. Gonzalez Della Valle, P. Padula, E. Björling, A. Plyusnin, and Å. Lundkvist, Cross-protection against challenge with Puumala virus after immunization with nucleocapsid proteins from different hantaviruses. J Virol, 2002. 76(13): p. 6669-77.

333. Xu, X., S.L. Ruo, J.B. McCormick, and S.P. Fisher-Hoch, Immunity to Hantavirus challenge in Meriones unguiculatus induced by vaccinia-vectored viral proteins. Am J Trop Med Hyg, 1992. 47(4): p. 397-404.

95


334. McClain, D.J., P.L. Summers, S.A. Harrison, A.L. Schmaljohn, and C.S. Schmaljohn, Clinical evaluation of a vaccinia-vectored Hantaan virus vaccine. J Med Virol, 2000. 60(1): p. 77-85.

335. Kamrud, K.I., J.W. Hooper, F. Elgh, and C.S. Schmaljohn, Comparison of the protective efficacy of naked DNA, DNA-based Sindbis replicon, and packaged Sindbis replicon vectors expressing Hantavirus structural genes in hamsters. Virology, 1999. 263(1): p. 209-19.

336. Koletzki, D., R. Schirmbeck, Å. Lundkvist, H. Meisel, D.H. Krüger, and R. Ulrich, DNA vaccination of mice with a plasmid encoding Puumala hantavirus nucleocapsid protein mimics the B-cell response induced by virus infection. J Biotechnol, 2001. 84(1): p. 73-8.

337. Bucht, G., K.B. Sjolander, S. Eriksson, L. Lindgren, Å. Lundkvist, and F. Elgh, Modifying the cellular transport of DNA-based vaccines alters the immune response to hantavirus nucleocapsid protein. Vaccine, 2001. 19(28-29): p. 3820-9.

338. Bharadwaj, M., K. Mirowsky, C. Ye, J. Botten, B. Masten, J. Yee, C.R. Lyons, et al., Genetic vaccines protect against Sin Nombre hantavirus challenge in the deer mouse (Peromyscus maniculatus). J Gen Virol, 2002. 83(Pt 7): p. 1745-51.

339. Hooper, J.W., K.I. Kamrud, F. Elgh, D. Custer, and C.S. Schmaljohn, DNA vaccination with hantavirus M segment elicits neutralizing antibodies and protects against seoul virus infection. Virology, 1999. 255(2): p. 269-78.

340. Custer, D.M., E. Thompson, C.S. Schmaljohn, T.G. Ksiazek, and J.W. Hooper, Active and passive vaccination against hantavirus pulmonary syndrome with Andes virus M genome segment-based DNA vaccine. J Virol, 2003. 77(18): p. 9894-905.

341. Groen, J., M. Gerding, J.P. Koeman, P.J. Roholl, G. van Amerongen, H.G. Jordans, H.G. Niesters, et al., A macaque model for hantavirus infection. J Infect Dis, 1995. 172(1): p. 38-44.

342. Klingström, J., A. Plyusnin, A. Vaheri, and Å. Lundkvist, Wild-type Puumala hantavirus infection induces cytokines, C-reactive protein, creatinine, and nitric oxide in cynomolgus macaques. J Virol, 2002. 76(1): p. 444-9.

343. Hooper, J.W., T. Larsen, D.M. Custer, and C.S. Schmaljohn, A lethal disease model for hantavirus pulmonary syndrome. Virology, 2001. 289(1): p. 6-14.

344. Jentoft, N., Why are proteins O-glycosylated? Trends Biochem Sci, 1990. 15(8): p. 291-4. 345. Owens, R.J., J.W. Dubay, E. Hunter, and R.W. Compans, Human immunodeficiency virus

envelope protein determines the site of virus release in polarized epithelial cells. Proc Natl Acad Sci U S A, 1991. 88(9): p. 3987-91.

346. Gupta, R., E. Jung, and B. S., Prediction of N-glycosylation sites in human proteins. Manuscript in preparation, 2004. http://www.cbs.dtu.dk/services/NetNGlyc/.

347. Hansen, J.E., O. Lund, J. Engelbrecht, H. Bohr, and J.O. Nielsen, Prediction of O-glycosylation of mammalian proteins: specificity patterns of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase. Biochem J, 1995. 308 ( Pt 3): p. 801-13.

348. Hansen, J.E., O. Lund, J. Nilsson, K. Rapacki, and S. Brunak, O-GLYCBASE Version 3.0: a revised database of O-glycosylated proteins. Nucleic Acids Res, 1998. 26(1): p. 387-9.

349. Sykes, K.F. and S.A. Johnston, Linear expression elements: a rapid, in vivo, method to screen for gene functions. Nat Biotechnol, 1999. 17(4): p. 355-9.

350. Zanta, M.A., P. Belguise-Valladier, and J.P. Behr, Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc Natl Acad Sci U S A, 1999. 96(1): p. 91-6.

351. Allen, T.D., J.M. Cronshaw, S. Bagley, E. Kiseleva, and M.W. Goldberg, The nuclear pore complex: mediator of translocation between nucleus and cytoplasm. J Cell Sci, 2000. 113 ( Pt 10): p. 1651-9.

352. Salman, H., D. Zbaida, Y. Rabin, D. Chatenay, and M. Elbaum, Kinetics and mechanism of DNA uptake into the cell nucleus. Proc Natl Acad Sci U S A, 2001. 98(13): p. 7247-52.

353. Stein, C.A. and J.S. Cohen, Oligodeoxynucleotides as inhibitors of gene expression: a review. Cancer Res, 1988. 48(10): p. 2659-68.

96

http://www.cbs.dtu.dk/services/NetNGlyc/

References

354. Adam, S.A. and L. Gerace, Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell, 1991. 66(5): p. 837-47.

355. Lewin, M., N. Carlesso, C.H. Tung, X.W. Tang, D. Cory, D.T. Scadden, and R. Weissleder, Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol, 2000. 18(4): p. 410-4.

356. Shalon, D., S.J. Smith, and P.O. Brown, A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Res, 1996. 6(7): p. 639-45.

357. Kurian, K.M., C.J. Watson, and A.H. Wyllie, DNA chip technology. J Pathol, 1999. 187(3): p. 267-71.

358. Small, J., D.R. Call, F.J. Brockman, T.M. Straub, and D.P. Chandler, Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays. Appl Environ Microbiol, 2001. 67(10): p. 4708-16.

359. Chizhikov, V., M. Wagner, A. Ivshina, Y. Hoshino, A.Z. Kapikian, and K. Chumakov, Detection and genotyping of human group A rotaviruses by oligonucleotide microarray hybridization. J Clin Microbiol, 2002. 40(7): p. 2398-407.

97

Documents

Implications of Local Puumala Hantavirus Genetics and