NAME: MUSA MARENA

TOPIC: FILOVIRUS HAEMORRHAGIC FEVER

CONTENTS PAGES

INTRODUCTION 2

HISTORY 3-5

GENOME ABD MORPHOLOGY 6-9

PATHOGENESIS 9-13

VECTOR AND MODE OF TRANSMISSION 14-15

BINDING AND ENTRY 15-16

REPLICATION 17-19

TRANSCRIPTION AND TRANSLATION 20-24

ASSEMBLY AND BUDDING 25-26

CLINICAL FEATURES 26-27

DIAGNOSIS 27

TREATMENT 27

FILOVIRUS HAEMORRHAGIC FEVER

INTRODUCTION

Haemorrhagic fever Is an acute febrile illness characterized by malaise, myalgia, and prostration

dominated by generalized abnormalities of vascular permeability, and regulation. Bleeding

manifestations often occur, particularly in severe cases; they are usually diffuse and reflect

widespread vascular damage rather than life-threatening volume loss.

1

Filoviruses are classic emerging infections. Filoviruses have two main genera and these are Ebola

and Marburg viruses. They belong to the group of viruses called haemorrhagic fever viruses. Four

families of viruses: Arenaviruses (Junin Virus), Bunyaviruses (Nariovirus), Flaviviruses (i.e.

Yellow Fever), Filoviruses (Marburg & Ebola)they are CDC Classification: Biosafety level

(BSL)-4 Agent (cf. HIV is only 2). They are mainly RNA viruses covered in a lipid coating and

are geographically restricted to areas where host species live. Humans are not natural reservoirs

for these viruses.

They are filamentous with a linear ~13-19kb genome. They can infect mice, hamsters, guinea

pigs and monkeys, although the viral reservoir in the wild is not known. Human epidemics seem

to be related to blood-born nosocomial spread (often due to re-use of needles in hospitals;

nosocomial = hospital infection) and to close contact with infected persons (since this is a

hemorrhagic disease, this presumably would involve exposure to large amounts of blood).

Primary infections with Marburg and Ebola are 25-90% fatal. Death is thought to be due to

visceral organ necrosis (e.g., liver) due to viral infection of tissue parenchymal cells. It is

uncertain what role hemorrhage has in death. Wild-caught monkeys are now quarantined before

release to US primate centers. Cloned viruses has help in studying viral genetic and will help the

development of diagnostic serologic tests for infection, and work is also progressing to try to

develop a vaccine for Ebola virus. The first successful vaccination against this virus was reported

in 1998, by Gary Nabel's group at the University of Michigan. In this report, a DNA vaccine

encoding the Ebola virus glycoprotein was able to elicit a T-cell based immune response in

guinea pigs, which was sufficient to protect the animals against infection with a live-Ebola virus

(Xu et al. Nature Medicine 4:37, 1998). Subsequent studies in nonhuman primates have

confirmed that a DNA vaccine can represent an important component of an effective Ebola virus

vaccine. Specifically, a combination of DNA immunization and boosting with adenovirus vectors

encoding viral proteins resulted in the protection of cynomolgus macaques from an otherwise

lethal dose of highly pathogenic, wild-type Ebola Zaire virus (Sullivan et al. Nature 408:2000

HISTORY

Marburg Virus also called (African Hemorrhagic Fever, Green Monkey Disease, and Marburg

Fever). In 1967 simultaneous outbreaks of hemorrhagic fever occurred in Yugoslavia and in

Germany, in lab workers who were processing kidneys from African green monkeys. There were

31 cases and 7 deaths. The virus was first characterized in Marburg, Germany and traced to a

single shipment of Ugandan monkeys. Sporadic additional cases showed up in 1975, 1980, 1982

2

and 1987. Most recently there was the most serious outbreak of Marburg which occurred in

Angola in 2004-2005 had 252 cases with 227 deaths (90%). In 2007 there were 2 cases and 1

death (50%) in Uganda and in 2008 one case who died after 10days illness in Netherlands after

returning from a visit in Uganda

In 1976 there were epidemics of severe hemorrhagic fever in Zaire and Sudan. In Zaire, there

were approximately 300 cases with an 80% fatality rate (due to Ebola-Zaire; ZEBO). In Sudan,

there were a roughly similar number of cases, with a fatality rate of roughly 50% (due to Ebola-

Sudan; SEBO). Ebola virus was originally isolated in Zaire (now Democratic Republic of the

Congo), and it was named after a small river in N.W. Zaire. Ultra-structurally the virus resembled

Marburg virus but it was antigenically (and genetically) distinct. It now appears that at least four

and probably five EBO viruses exist -- EBO-Z (Zaire is 990-1086nm in length), EBO-S (Sudan is

974-1063nm in length), EBO-CI (Côte d’Ivoire), EBO-R (Reston is 1026-1083nm in length) and

more recently EBO-B (Bundinbugyo) from Gulu region Uganda. Figure1. The first two are

known to be highly lethal in humans and are spread via bodily fluids and by close (nonsexual)

contact. The Reston virus appears to be less lethal in humans (0 deaths in 6 cases), although it is

lethal in nonhuman primates. Outbreaks of Ebola occurred in 1995 in the Kikwit area of Zaire

(over 315 cases, with 80% fatality; due to EBO-Z) and in the Gulu region of Uganda in 2000

(over 400 cases, but with roughly 50% fatality; due to EBO-B). It is uncertain how the Kikwit

and Gulu outbreaks started. However, a smaller outbreak in 1996 in Gabon was traced to a group

of 20 young Gabonese who trapped and caught a Chimpanzee that was sick. It is believed that

exposure to Ebola occurred during the preparation of the Chimpanzee, prior to cooking and

consumption of the animal. Interestingly, Ebola was isolated only from meat-eating Chimps, and

not from strictly vegetarian members of the same troupe of animals. Outbreaks of EBO-Reston

have occurred in US primate colonies in the Washington area (Reston, 1989) and in Texas (1990,

1996). These outbreaks were contained by destruction of all animals within the affected area of

the facility. The outbreaks all appear to trace back to shipments of macaques from a single

Philippine exporter. A total of 6 humans have become infected by EBO-Reston, but none had

died. On Wednesday December 10, 2008, the Philippine Department of Agriculture and

the Philippine Department of Health announced that an Ebola virus of the Reston species

has been identified from sick pigs in three locations in the Philippines. This is the first

time globally that domestic pigs are reported to have been infected with an Ebola virus.

High rates of sickness and death in pigs and the pigs were found to be were co-infected

with Porcine Respiratory Reproductive Syndrome one (PRRS) virus and the Ebola

3

Reston virus. Table below show reported and recorded outbreaks excluding the Philippine

Date Species Location # Cases % Fatality1967 Marburg Uganda-Germany 31 231975 Marburg Zimbabwe-South Africa 3 331976 Zaire Ebola DRC (Zaire) 318 881976 Sudan Ebola Sudan 284 531977 Zaire Ebola DRC (Zaire) 1 1001979 Sudan Ebola Sudan 34 651980 Marburg Kenya 2 501987 Marburg Kenya 1 1001989 Reston Ebola USA (VA, TX, PA) 0 01994 Ivory Coast Ebola Ivory Coast 1 01994 Zaire Ebola Gabon 46 651995 Zaire Ebola DRC (Zaire) 315 811995 Ivory Coast Ebola Ivory Coast 1 01996 Zaire Ebola Gabon 97 651998 Marburg DRC 141 822000 Sudan Ebola Uganda 425 532001 Zaire Ebola Gabon 123 792003 Zaire Ebola Republic of Congo 175 862004 Sudan Ebola Sudan 17 422005 Zaire Ebola Republic of Congo 12 752005 Marburg Angola 252 902007 Marburg Uganda 3 1002007 Ebola DRC2007 Ebola Uganda** Uganda2008 Marburg Netherlands ex Uganda 1 100

Filovirus Outbreaks

4

Date Species Location # Cases % Fatality1967 Marburg Uganda-Germany 31 231975 Marburg Zimbabwe-South Africa 3 331976 Zaire Ebola DRC (Zaire) 318 881976 Sudan Ebola Sudan 284 531977 Zaire Ebola DRC (Zaire) 1 1001979 Sudan Ebola Sudan 34 651980 Marburg Kenya 2 501987 Marburg Kenya 1 1001989 Reston Ebola USA (VA, TX, PA) 0 01994 Ivory Coast Ebola Ivory Coast 1 01994 Zaire Ebola Gabon 46 651995 Zaire Ebola DRC (Zaire) 315 811995 Ivory Coast Ebola Ivory Coast 1 01996 Zaire Ebola Gabon 97 651998 Marburg DRC 141 822000 Sudan Ebola Uganda 425 532001 Zaire Ebola Gabon 123 792003 Zaire Ebola Republic of Congo 175 862004 Sudan Ebola Sudan 17 422005 Zaire Ebola Republic of Congo 12 752005 Marburg Angola 252 902007 Marburg Uganda 3 1002007 Ebola DRC2007 Ebola Uganda** Uganda2008 Marburg Netherlands ex Uganda 1 100

Filovirus Outbreaks

5

Figure 1. Geographic locations of Ebola HF outbreaks and phylogenetic relationships of representative filoviruses.

Finally, while the major route of Ebola transmission is clearly close contact with bodily fluids

and blood (e.g., during health care, preparation for burial, etc), it is possible that some Ebola

viruses might be transmissible via an aerosol route in some cases. One piece of evidence to

support this idea is the fact that EBO-Zaire has been shown to infect rhesus monkeys that did not

have direct contact with experimentally inoculated monkeys held in the same room (Jaax et al.

Lancet 346:1669, 1995).

6

GENOME AND MORPHOLOGY

Electron microscopic observation of filoviruses revealed distinctive bacilliform to filamentous

virus particles, and this highly characteristic morphology that inspired their name (Latin filium,

thread). Virion from cultured tissue are pleomorphic, appearing as either U-shaped, six-shaped

(6) or circular (torus) configuration or as elongated filamentous forms. The filamentous forms can

be seen to form branched structure. Figures2 below

The length varied 800-1080nm up to 14,000nm with Marburg virion shorter than Ebola virion

with 860nm and 12,000nm respectively. The virion have a uniform diameter of 80nm, contain a

helical ribonucleoprotein complex or nucleocapsid (NC) roughly 50nm in diameter and have a

central axial space (approx 20nm in diameter) running the length of the particle. Figures3&4. The

NC has a helical periodicity of ~5nm and is surrounded by matrix protein and a closely outer

envelope derived from the host cell plasma membrane. The virion surface is studded with

membrane anchored peplomers projecting ~10nm from the surface figure 3. The virion can appear

ragged or “moth eaten” figures 2D especially in late infection. Virion density has been determined

to be 1.14g/ml by centrifugation in potassium tartan gradient and relative molecular mass Mr 3-

6×108

The virion genome which constitute~1% of virion mass is found on single-stranded, non-

segmented, linear, negative-sense RNA (–ve ssRNA). Filoviruses genome is very similar. Their

organization generally conforms to those of paramyxoviruses and Rhabdoviruses but their

7

figure3

Complexity is more akin to those of paramyxoviruses. Filovirus genome is (~19Kb) long and

making them the largest in the mononegavirales order. They contain seven sequentially arranged

genes in the order 3’-5’ nucleoprotein (NP), viral protein (VP) 35, VP40, glycoprotein (GP),

VP30, VP24 and RNA polymerase L. The genes are delineated by conserved transcriptional

signals and begin close to the 3’ end of genome sequence with a start site and ends with a stop

(polyadenylated) site. Like in other mononegavirale these genes are usually separated by short

intergenic regions (IR) of one or more nucleotides. Unusual feature of gene overlaps are found in

the filovirus genome which have been identify in the genome of some paramyxo and

Rhabdoviruses but does not resemble those of filoviruses. Figure 3

Genes are defined by highly conserved transcriptional start signals at their 3' (3'-

CUNCNUNUAAUU-5'; consensus motif) and termination signals at their 5' ends (3'-

UAAUUCUUUUU-5') Most genes tend to possess long noncoding sequences at their 3' and/or 5'

ends, which contribute to the increased length of the genome

8

The stop site of an upstream gene overlaps the start site of a downstream gene, and overlapping

sequences are limited to the conserved transcriptional signals and are centered on 3’-UAAUU

pentanucleotide sequence common to start and stop sites. Figure4

Marburg genome have one (VP30-VP24) overlap but the characterize Ebola genome have two or

three (VP35-VP40, GP-VP30, VP24-L) overlaps. Filovirus generally have short intergenic

sequence although all have a single lengthy sequence more than 120bases separating GP and

VP30 gene in Marburg (MARV) and VP35 and VP40 on Ebola virus (EBOV). The extragenic

sequence at the 3’ end (Leader) is short in all filovirus genomes ranging in length from 50-

70bases while the length of the 5’ end (Trailer) sequence are variable. These extragenic are

thought to be templates for very small viral, nonpolyadenylated subgenomic RNAs. The extreme

3’ and 5’ ends of the genomes are conserved, show high degree of complementarity and

potentially form stem loops structure. The trailer of Zaire Ebola virus (ZEBOV) is the longest

(677bases), followed Sudan Ebola virus (SEBOV) 381bases MARV 75-95bases and Ebola

Reston REBOV 25bases

9

Phylogenetic analysis of filoviridae family using the coding regions of the glycoprotein gene

indicated EBOV and MARV represent distinct lineages. The five species of EBOV also represent

distinct lineages and this is extraordinary level of genetic stasis within the lineages EBOV.

Nucleotide and amino acid differences between MARV and EBOV are both approx 55% whereas

EBOV species show 37% to 41% difference in nucleotide and amino acid sequence. The same

level of variation are seen when other genes are compared. This is a remarkable genetic stability

within EBOV species indicating that they have reached a high degree of fitness as they have

adapted to their respective niches. MARV have not shown the same degree of variation but two

lineages have been described. The 1987 isolate from Kenya appears to represent a separate

lineage from the three previous isolates but differences of 21-23% amino acid change is not

enough to constitute a separate species. Figure 5

PATHOGENESIS

Viral surface spikes recognize and bind surface receptors of host and Virus enters cell via

endocytosis with Releases of nucleocapsid into cytoplasm.

10

Transcription of viral RNA produces polyadenylated, monocistronic mRNA. Translation and

buildup of viral proteins, primarily NP causes Budding and release of viruses. Host Cell – dies

through intracytoplasmic vesiculation, mitochondrial swelling, organelle breakdown.

Initially, productive infection (that is, that which results in more viral progeny) occurs primarily

in dendritic cells (DCs), monocytes and macrophages. All infected cells can be at least partially

impaired in interferon (IFN) production, but some important differences have been described

between DCs and monocytes: monocytes respond with the production of pro-inflammatory

cytokines, whereas DCs conspicuously lack such a response. Neutrophils are not productively

infected, nor are lymphocytes, but neutrophils are activated by interaction with viral particles

with resultant degranulation and shedding of triggering receptor expressed on myeloid cells 1

(TREM1). Figure6

11

As viral burden increases, lymphocyte apoptosis and a generalized failure of specific immune

responsiveness are observed; we propose these to be rooted in virally induced upregulation of co-

inhibitory molecules (such as B7-H1) on DCs and monocytes, followed by interaction with

programmed death 1 (PD1) receptors on T and B cells. Infection spreads to many cells including

liver hepatocytes, and the increasing release of pro-inflammatory cytokines crosses a threshold

from beneficial to potentially harmful inflammation, also degrading vascular epithelium.

12

Figure7

13

As repeated cycles of viral replication overwhelm and outpace a dysregulated adaptive immune

response, elements of innate immunity and inflammation that are potentially helpful early in the

response only add to the spiraled dysfunction as they collide with high viral burdens.

Dysfunctions in DCs, monocytes and macrophages are particularly important for their secondary

effects on innate and adaptive immune responses, inflammation and vascular integrity.

Figure 8

Marburg virus uses Asialoglycoprotein Receptors (Found exclusively in hepatocytes). These

Recognizes glycoproteins displaying N-linked sugar chains with terminal galactose residues and

binds while Ebola viruses uses Integrins which binds to N-glycosylated transmembrane cell

surface receptors to enter cells. Ebola and Marburg also uses Human folate receptor-α a Co-

factor expressed on cell-surface to enter cells. Figure 7

No Transcriptional RNA Editing causes Over-expression of GP which exhausts Cell Host

Machinery eventual leads to Cell Death. So GP expression and cytotoxicity can be down-

14

regulated by virus through transcriptional RNA editing and sGP expression. sGP inhibits early

activation of neutrophils by Binding to neutrophils via CD16b cell surface receptor. CD16b

activates neutrophils via lateral membrane interaction with CR3. sGP adsorbs neutralizing

antibodies.

Specific region of GP induces cytotoxic effects in endothelial cells. Rapid release of vasoactive

agents from infected cells Induces cell rounding and detachment from extracellular matrices this

Increases cell membrane permeability.

Proteolytic activation of GP0 precursor via cleavage of EBO-Z GP by furin is prerequisite for

fusion between viral envelope and host cell membrane. It Enables virus to replicate in host

leading to systematic infection.

Two sequences contribute to evasion of host immunity. Possible immunosuppressive sequence in

GP2 molecule and or Amino acid sequence at N-terminus suppress lymphocyte mitogen-

stimulated proliferation in vitro

Destruction of the Immune System through Infection of mononuclear phagocytes and fibroblastic

reticular system (associated with lymph nodes), Failure of early T-cell activation Disrupt antigen

trafficking and cytokine production causing Extensive apoptosis of blood leukocytes leading to

Lymphopenia (reduction in lymphocyte number) and severe damage to lymphoid tissue. Infected

Macrophages and circulating monocytes help transmit virus to other tissues

EARLY STAGE: pathology shows Striking lesions usually in liver, spleen and kidney, Necrosis

prominent in liver, lymph tissue, and spleen, little inflammatory response and viral particles

invade phagocytic cells

LATE STAGE: Liver and spleen become enlarged with excess blood, Hemorrhage in the

gastrointestinal tract, pleural, pericardial and peritoneal spaces and into the renal tubules with

deposition of fibrin, Abnormalities in coagulation parameters suggest that disseminated

intravascular coagulation is a terminal event. There is usually also profound leukopenia in

association with bacteremia.

Survived have IgG response against virus’s protein coat early on, cleared circulating antigen and

activated cytotoxic T-cells

Those who end up Dying have No IgG response and barely detectable levels of IgM

15

VECTOR AND MODE OF TRANSMISSION

Ebola Virus has No carrier state (reservoir Unknown). Researchers hypothesize that it is Zoonotic

because some outbreaks are related with contact animal like chimps and bats. Figure 9 Most

animals in Africa have positive antibodies to filoviruses and viruses have been isolated from their

serum. There is an unpopular theory that plant may be the reservoir of the virus. Human-Human

Transmission is through contact with contaminated secretions.

Ebola-Reston which Occurred in the U.S (Reston, VA) in African Green Monkeys imported from

Phillipine. Questions are asked: Why only to non-human primates? (Four scientists found to have

antibody for the disease). There is Circumstantial Evidence of airborne transmission because the

viruses spread within monkey kept within the same and between rooms (national center for

Infectious Diseases). ICEBOV was transmitted through contact with a death chimps carcass.

Figure 9

Marburg Virus maybe transmitted from animal host because fruit bats found in caves have

positive antibodies to the virus and isolates of the virus. It’s not clear whether they can transmit it

16

through contact or suffer from filovirus illness. Human to Human (Close Contact and change of

fluids highly suspect source of transmission)

In short there is no-known vector and it May be A rare species One that usually does not contact

clinical host. If contact is made the virus may not be easily transmitted. Most significant

Hypothesize are those of Bats Plants (fruit bats). Other include rodents, arthropods etc. figure 10

BINDING AND ENTRY

Filoviruses infect variety of cells and this broad tropism is related to large part to the binding

properties of peplomers that populate the surface of virion. Asialoglycoproteins receptor in

17

hepatocytes binds MARV but Ebola virus infects hepatocyte despite its GP lacking sialylated

glycans.

Beta 1 group of integrin are believed to interact with ZEBOV GP on cell surface and during

intracellular trafficking although cell expressing this molecules are not easily infected. Folate

receptors alpha is believe to be a co-factor in viral entry. C-type lectin present on certain

macrophages, dendritic cells and endothelium are capable of binding filovirus peplomers when

N-terminus contains mannose carbohydrate. Antibody binding to peplomers might also enhance.

Infectivity through its interaction with the Fc portion of complement protein C1q bond to the

surface of host cells. Figure 6

Figure 11

18

REPLICATION:

Filoviruses belong to the group of mononegavirale means that the viral genome is of

opposite polarity to mRNA. Thus, the viral genome cannot be used to make proteins until

it has first been transcribed. Figure 12. They include several families like Filoviridae,

Paramyxoviridae and Rhabdoviridae, plus Borna disease virus. These viruses probably

diverged from a single common ancestor as recently as the last ice age. . They are also

transcribed to produce mRNAs.

Figure 12

This has the following implications:

1. Purified virion RNA (without viral proteins) is not infectious (as noted above, it cannot

encode protein)

2. The viruses must bring their own RNA polymerase into the cell in order to make mRNA (ie,

the viral polymerase must be incorporated into the viral particle, or virion). The other key feature

of these viruses is that they make gene-unit length mRNAs (ie, each mRNA encodes only a single

protein). This is achieved by the use of transcriptional stop and start signals, which are located at

the boundaries of all of the viral genes. Stop/start transcription has two major results: Since there

is only a single promoter, located at the 3’ end of the viral genome, the polymerase can only load

onto its RNA template at one site. As it moves along the viral RNA, the polymerase encounters

19

stop/start signals at the boundaries of each of the viral genes. This results in pausing of the

enzyme, which often falls off the template. The result is that more mRNA is made from genes

that are located close to the promoter, and less mRNA is made from genes located far from the

promoter. Figure 13

Figure 13

This means that there is a polarity of transcription (see Figure below). The viruses use

this to regulate the expression of their genes, since highly expressed proteins are encoded

close to the promoter (e.g., structural proteins such as the nucleocapsid protein, N), while

proteins that are needed in only small amounts (e.g., enzymes such as the RNA

polymerase, L) are encoded far away from the promoter.

The other major consequence of stop/start transcription is that it complicates genome

replication. The only way that the complete viral RNA genome can be copied is if the

transcriptional stop/start signals can be ignored or over-ridden. This means that the

critical decision during viral RNA synthesis occurs very early on -- at the first gene

boundary (located between the leader RNA and the N gene). If the stop/start signals here

are obeyed, then only subgenomic mRNAs will be produced. However, if the stop/start

signal here is ignored or over-ridden, then a complete copy of the viral genome can be

made.

3’ end of filovirus genome has a promoter. This derives both the replication and

transcription. It derives the synthesis of full-length complementary/antigenomic RNA

from the encapsidated template. The complementary sequences and stem loop structure at

the ends of the genome are believed to be essential to filovirus replication. The initial

expression of viral genes leads to build up of viral proteins (especially NP) which are

believed to trigger the switch from transcription to replication. This switch leads to

20

synthesis and encapsidation of antigenomic RNA molecule which turn serve as a

template for genomic RNA that is also rapidly encapsidated. Depletion of capsid protein

is believe to cause a return to transcription and eventually an equilibrium is established

wherein transcription and replication are concurrent processes. As replication progress in

infected cells, NC particles containing genomic RNA accumulate and are directed to the

plasma membrane for virion assembly.

Reverse genetic system has shown that NP, VP35 and L proteins are all that is require to

transcribe and replicate MARV mingenomes but EBOV also require VP30

21

TRANSCRIPTION AND TRANSLATION

Upon entry into cell, the non segmented negative sense single stranded RNA genetic dictates that

transcription is the first (and obligatory) viral process which is similar to paramyxo and

Rhabdoviruses.

Once the nucleocapsid is released in the cytoplasm, polyadenylated, monocistronic mRNA are

synthesized from virus genes in 3’ to 5’ direction (with polar attenuated) from the encapsidated

genomic RNA template. Transcription seems to involve a process of starting and stopping as the

polymerase complex encounters conversed start (initiation) and stop

(termination/polyadenylation) sites along the genome. Transcripts are believed to be ‘capped’ at

5’ end by the L protein because it contains conserved motifs associated with this enzymatic

activity. It’s postulated that the leader sequence is transcribed while the intergenic and trailer

sequences are not transcribe. The promoters for initiating RNA synthesis are contained within

156 and 177 nucleotide regions of the genomic and antigenomic RNA 3’ termini respectively

Transcription start sites are 12 or 14 nucleotides in length and end in the consensus sequences 3’-

CUUCUAAUU EBOV and 3’-CUURUAAUU for MARV, while stop sites are 11 or 12

nucleotides long with the conserved sequence 3’-UAAUUC (U)5/6.

Polyadenylation is believed to occur by slippage or stuttering of the polymerase at the five or six

uridines ending the stop sites. Gene overlaps which are short (18-21nucleotides) does not affect

either the polyadenylation of upstream gene or initiation of transcription of downstream gene

because the transcription of VP40 and VP35 genes of ZEBOV is substantial and expression of

VP40 is very strong.

mRNAs have noncoding regions at their 3’ and /or 5’ ends which contribute to the increase length

of genome and may function in the stability of transcripts. 5’ ends of transcripts also have

potential to form stem loop structures that might affect their stability and ribosome binding

capacity/transcription

ZEBOV VP30 has a transcription activation property that is linked to a RNA secondary structure

formed at the 5’ end of the NP gene transcript as it is synthesized. The presence of VP30 is

required for transcription of down stream genes. This property is impaired by phosphorylation at

six serines and one threonine at the N-terminus and can be restored by action of cellular

22

phosphatase. In virion VP30 is partially phosphorylated and it may be the actions of phosphatase

on nucleocapsid required for before transcription proceed efficiently. Corresponding mechanism

has not be found with MARV

Filovirus genes are monocistric (each mRNA is transcribe into one protein products). The

proteins found can be subdivided into those that form nucleocapsid (NC) and those that are

associated with the envelope. The nucleocapsid-associated proteins are involved in transcription

and replication of genome whereas envelope-associated proteins are involved with either viral

assembly or virus entry.

Nucleoproteins: NP and VP30 are the major and minor nucleoprotein respectively and interact

strongly with the genomic RNA molecule to form the viral nucleocapsid. NP and VP30 are

phosphoproteins. NP has a conserved hydrophobic N-terminal half which contains all the cysteine

residues and a divergent hydrophilic C-half which contains most of the proline residues and is

extremely acidic. NP has a predicted mass of 20KDa smaller than the SDS-PAGE migration

attributed to as a result of binding to SDS molecule t negatively charge NP. The central region of

NP is highly conserved region

N-terminal third of VP30 contained a high concentration of basic amino acids (primarily arginine

residues) and may be involve in binding to virus RNA and/or acidic C-terminal of NP. VP30

contain zinc-finger motif ~70-80 residues from the N-terminus that is highly conserved in

filoviruses. ZEBOV VP30 behaves as a transcription activator that is regulated by

phosphorylation but appear absent in MARV.

POLYMERASE COMPLEX PROTEINS:

L and VP35 proteins form the polymerase complex, which acts to transcribe and replicate

filovirus genome. L provides RNA-dependent RNA polymerase activity of the complex, and

motifs linked RNA (template) binding, phosphodiester bonding and a ribonucleotide triphosphate

bonding occurs. It is the largest and least abundant protein

VP35 is a Cofactor in transcription and Replication (Cofactor in polymerase complex). VP35 is

believed to have an essential role as a cofactor that affects the mode of RNA synthesis

(transcription or replication) and acts as a linker between L and NP. VP35 has an antagonistic

effect on the interferon type1 pathway.

MATRIX PROTEIN:

23

VP40 function as matrix protein with VP24 may have a secondary/minor matrix protein function.

VP40 is most abundant protein in the virion while small amount of VP24 are incorporated into

virus particle. VP40 is a Matrix protein necessary for Virus assembly and budding. It forms

hexamers when it contacts the plasma membrane which confers added stability during assembly.

It is the most abundant protein. Both have affinity for membranes and are associated with the

virion envelope. They are easily released from virion by nonionic detergents under low salt

condition. VP40 is critical to the budding process because it initiates and drives the envelopment

of NC by the plasma membrane. VP24 precise role in replication is unclear but in ZEBOV it has

a reported in IFN type1 signaling pathway it has an antagonist activity

GLYCOPROTEIN GP

MARV GP gene encodes a single product GP, in a conventional open reading frame (ORF). It is

the fourth gene in the genome from 3’ to 5’ direction. EBOV GP gene organization and

transcription is quite unusual and distinct from MARV.

EBOV encodes their GP in two reading ORFs (0 and -1). Expression of EBOV GP requires a

transcriptional editing event. Translation of unedited transcript GP gene results in the production

of SGP, a smaller, non structural, secreted glycoprotein, the primary gene product.

24

Figure 14

In the edited transcript, the transcriptional editing event occurs at a series of seven uridines on the

genome RNA template and results in the insertion of an additional adenosine, which connects the

GP open reading frames. This may be due to stuttering of the polymerase on the poly U template

during polyadenylation. Figure 14

Approx 20-25% of the transcript are edited and this single nucleotide editing site occurs with high

degree of fidelity. The nucleotide sequence corresponding to editing site is absent in MARV GP

gene. This results in the production of both a truncated, soluble form of the Ebola virus

glycoprotein (sGP; 50-70 kD in size) and a full-length, transmembrane anchored version of the

same protein (GP; 120-150 kD in size). Ebola virus sGP and GP have different functional

properties, which may be important in disease pathogenesis. The functional subdomains of these

molecules are shown in figure 15. The soluble sGP molecule is secreted as a trimer, and is identical

at its N-terminus to the homologous region of the transmembrane glycoprotein (GP). sGP

interacts with neutrophils through CD16b, the neutrophil-specific form of the Fc © receptor III,

whereas the transmembrane glycoprotein (GP) interacts with endothelial cells but not with

neutrophils (Yang et al. Science 279:1034, 1998). It is possible that interaction of sGP with

25

neutrophils results in the blockade of early events in the activation of these cells, thereby

inhibiting inflammatory responses which might contribute to innate protection against viral

infection. sGP may also act as a "decoy" for antiviral antibodies.

GP The transmembrane glycoprotein is produced as a long precursor, which undergoes cleavage

by a cellular protease (furin), to produce GP1 and GP2. These can be viewed as being somewhat

analogous to HIV-1 gp120 and HIV-1 gp41 (which are produced by cellular proteolytic cleavage

of the gp160 precursor). Ebola virus GP2 remains in the membrane (due to its transmembrane

domain) and is responsible for mediating fusion between the virus and the plasma membrane, via

its fusion domain. The GP1 component is attached to GP2 via a non-covalent linkage, and is

thought to mediate virus attachment to its host cell(s), which include vascular endothelial cells.

Figure 11 Ebola virus GP is also cytotoxic for vascular endothelial cells in vitro, and this is thought

to contribute to the virus’ ability to trigger vascular leakage (hemorrhage) in vivo.

In the Trans-Golgi, the precursor molecule (GP0) is post-translationally cleaved by furin at Furin

cleavage site (Arg-Arg-X-Arg/LYS-Arg) yielding a heterodimer, (GP1-GP2). (Marburg, cleavage

site is more toward N-terminus). Heterodimer is linked together by one disulfide bond, a cysteine

bridge. Figure 11

Figure 16

NON STRUCTURAL GLYCOPROTEIN (sGP)

It’s expressed in EBOV viruses only. The N-terminal ~300 amino acids are identical to the

structural GP but C terminus is unique in the sequence. It is produce from a precursor molecule

cleaved by furin (or furin-like endoprotease) near the C-terminus to release a short peptide that

contain O-linked glycan and name delta peptide. Delta has no identified biological activity. sGP

is structurally distinct from GP and is secreted from infected cells as a homodimer that is likely

form in endoplasmic reticulum. It has a parallel orientation of the dimmer and held together by

disulphide bonds between N- and C terminus cysteine. figure 17 It circulate in large amount in

acutely infected human blood so is likely to contribute to disease progression

26

Figure17

ASSEMBLY AND RELEASE

When sufficient levels of negative-sense nucleocapsids and envelope-associated proteins are

reached, coalescence of these components occurs at the plasma membrane or, to a lesser extent, at

membranes forming intracellular vacuoles. Inclusion bodies forms in infected cells are induced to

form by NP but contain other proteins that form the NC. These structures are facilitated by

expression of VP35 and VP24. NC is believed to interact with VP40 molecules in the budding

process.

27

figure 18: Filovirus replication cycle. Entry: The filovirus replication cycle begins with binding to cellular receptors inducing still undefined cellular uptake mechanisms. Uncoating: Following virus entry into endosomal compartments, acid pH, proteolytic cleavage and an unknown trigger induce fusion of viral and cellular membranes and uncoating of nucleocapsids, which are released into the cytosol. Transcription/translation/replication:Transcription of viralmRNAby the viral polymerase is followed by translation of viral proteins by the host cell. Replication of the viral genome by the viral polymerase is dependent on the presence of viral nucleocapsid proteins which encapsidate the nascent genome. The viral proteins VP35 and VP24 have the capacity to antagonize the interferon pathway of the infected cell. Assembly/Budding: Assembly of viral particles starts with formation of nucleocapsids which accumulate in inclusions in the perinuclear region and are transported to the sites of budding most likely by the activity of the cytoskeleton. Nucleocapsids are cotransported with the matrix protein VP40 to the sites of budding at the plasma membrane. The surface protein GP is transported along the classical secretory pathway and partly recruited to the late endosome tomeet VP40. The late endosome probably plays a key role in the formation of the viral envelope. Budding takes place at long actin-containing protrusions, filopodia, where nucleocapsids are enwrapped with the viral envelope in which GP is incorporated. Abbreviations: CatL/B: Cathepsin L /B, EE: early endosomes,MT: microtubule, LE: late endosomes, Kap a1: Karyopherin alpha 1, STAT1: signal transducer and activator of transcription 1, P: phosphate group, IKK: IkappaB kinase, TBKI: TANK-binding kinase-1, IRF3: interferon regulatory facator 3, NC: nucleocapsids, ER: endoplasmic reticulum,MVB: multivesicular body, ESCRT: endosomal sorting complex required for transport, TACE: tumor necrosis factor-alpha-converting enzyme

28

Membrane/lipid rafts are platforms for the assembly of filovirus virion. They are rigid

microdomains (containing sphingolipids and cholesterol) present in biological membrane and are

isolated from the fluid phospholipids surround them. GP trimers conveyed to the surface

membrane have affinity for these rafts.

Posttranslational processing and intracellular trafficking of VP40 results in deposition of VP40 at

the plasma membrane via the late retrograde endosomal pathway. VP40 ZEBOV is capable of

mediating its own release from mammalian cells to form enveloped virus like particles which are

more efficiently produced what GP and NP are present. VP40 interacts with C-terminus of NP.

VP40 is bond as an oligomeric form and ubiquitinated and subsequently target to endosome by

TSG 101 and VPS 4 (components of vacuoles sorting pathway and is recruited to membrane rafts

through TSG 101 interaction with VP40 and protein raft.. raft-associated VP40 believed to

associate with NCs drawing then tightly to the membrane where they are enveloped and extruded

from host cell as infectious virions

CLINICAL FEATURES

29

Filoviruses infections are generally the most severe of viral haemorrhagic fevers. Abrupt onset

follows an incubation period of 2-21days (Ebola) and 3-9days (Marburg), averaging 4 to 10days.

It’s characterized by flue-like symptoms of fever, chills, malaise, and myalgia.

There is subsequent signs and symptoms of systemic involvement like prostration,

gastrointestinal (anorexia, nausea, vomiting, abdominal pain, diarrhea), respiratory (chest pain,

shortness of breathing, cough), vascular (conjunctival injection, postural hypotension, edema),

and neurologic (headache, confusion, coma) manifestation. Haemorrhagic manifestations develop

during the peak of the illness and include petechiae, ecchymoses, uncontrolled oozing from

venipuncture sites, mucosal hemorrhages and visceral hemorrhagic effusions. The is often a

mucopapular rash associated with varying degrees of erythema at days 5-7 of the illness, this is a

valuable differential diagnostic feature and is usually follow by desquamation in survivors.

Abdominal pain is sometimes associated with hyperamylasemia and true pancreatitis.

In late stages shock, convulsions, severe metabolic disturbances, and, in more than half the cases,

diffuse coagulopathy supervenes.

LABORATORY parameters a less characteristic

Early leucopenia with lymphopenia and subsequent neutropenia, left shift with atypical

lymphocytes, thrombocytopenia, marked elevated serum transaminase level (AST typically

exceeding ALT), hyper proteinemia and proteinuria. Prolonged prothrombin and partial

thromboplastin time, fibrin split products detectable

In later stage, secondary bacteria infection may lead to elevated white blood count. Nonfatal

cases have fever for about 5-9days, and improvement typically occurs around days 7-11days

about the time humoral antibody response. Convalescence is prolonged and sometimes

associated with myelitis, recurrent hepatitis, and psychosis, uveitis. There is an increase risk of

abortion for pregnant women and high death rate in children of infected mothers.

Fatal cases develop clinical signs early during infection and demise typically occurs between days

6 and 16 due to haemoorrhage and hypovolaemic shock

DIAGNOSIS AND PREVENTION

Diagnosis is mainly clinical with high index of suspicion. Filovirus haemorrhagic fever should be

suspected in anyone with clinical signs and symptoms with history of travel to endemic area,

jungles or caves exposure, treatment in local hospital, contact with sick person or wild/domestic

animals like monkey, ape or even pigs. Wide ranges of febrile disease most be considered and

excluded like malaria typhoid etc.

30

LABORATORY DIAGNOSIS.

Clinical microbiology and public healthy labs are ill equip to make diagnosis, so specimens

should be send to national and international reference labs capable of performing require test.

Universal precaution in patient care and sample collections to avoid direct contact with body fluid

and secretions.

Measurement of host specific immune response for detection of IgG and IgM through ELISA.

Viral particles or particle components through RT-PCR, antigens capture ELISA or immunoblot.

Electromicroscope can be use to identify virus in infected cells or through

immunohistochemistry.

Prevention is through avoidance of contact with body fluids, strick barrier nursing with use of

protective clothes and proper disposal of body secretion and fatal caderva.

Vaccine trail have prove effective in primate but it yet to be tested in human as the disease occurs

in sporadic.

TREATMENT

Is mainly supportive directed towards maintenance of effective blood volume, electrolyte

balanced. Management of shock, cerebral edema, renal failure, coagulation disorders and

secondary bacteria infection may be life threatening.

31