Research Collection

Doctoral Thesis

A molecular study of enterotropic murine coronavirus

Author(s): Zhang, Linong

Publication Date: 1996

Permanent Link: https://doi.org/10.3929/ethz-a-001702361

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ETH Library

CIss-ETH e>c.

Dissertation No. 11872

A molecular study of enterotropic murine

coronavirus

A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY

ZURICH

For the degree of Doctor of Natural Science

Presented by Linong Zhang

Master Degree of Medicine from Fujian Medical College, P. R. China

Bom on 25th of October, 1961

Citizen of China

Accepted on the recommendation of

Prof. Dr. F. Wurgler, examiner

Dr. F. R. Homberger, co - examiner

Prof. Dr. P. E. Thomann, co - examiner

ZOrich 1996

ETHICS ETH-BW

00100002920942

1. Introduction

The Coronaviridae were first recognized as a distinct virus family by Tyrrell and

coworkers in 1968 (Tyrrell et a/., 1968). The family comprises two genera,

coronavirus and torovirus, which share similarities in the organization and

expression of their genomes and the structure of the virial gene products (Table 1).

The name "coronavirus" is derived from the solar corona-like ("corona" is Latin for

"crown") appearance of virus particles in negatively stained electron micrographs.

Table 1. Coronaviridae

Natural host virus Acronym

Coronavirus

Chicken

Cattle

Dog

Man

Man

Cat

Mouse

Pig

Pig

Pig

Turkey

Torovirus

Horse

Cattle

Avian infectious bronchitis virus IBV

Bovine coronavirus BCV

Canine coronavirus CCV

Human coronavirus 229E HCV 229E

Human coronavirus OC43 HCV OC43

Feline infectious peritonitis virus FIPV

Murine hepatitis virus MHV

Porcine epidemic diarrhea virus PEDV

Porcine hemagglutinating encephalomyelitis virus HEV

Porcine transmissible gastroenteritis virus TGEV

Turkey coronavirus TCV

Berne virus BEV

Breda virus BRV

The spectrum of diseases in humans and animals caused by coronaviruses

ranges from subclinical infection to rhinitis, enteritis, hepatitis, peritonitis,

encephalomyelitis, and death. One of the best-studied members of the coronavirus

family is mouse hepatitis virus (MHV). MHV is the most prevalent and probably the

most important viral pathogen in contemporary research mouse colonies (Homberger

and Thomann, 1994; Kraft and Meyer, 1990; Lindsey, 1986). Some MHV strains

produce primary respiratory infections, and many MHV strains are enterotropic

(Homberger and Thomann, 1994).

In this chapter a concise overview of the molecular biology of coronaviruses is

presented with the emphasis on MHV.

The virion

Coronaviruses are enveloped viruses, containing a single stranded, positive-

sensed and nonsegmented genome of 27-30 kb in length. A schematic

representation of a coronavirion is shown in Figure 1. The lipid envelope surrounds

a helicoidal nucleocapsid which consists of nucleocapsid phosphoprotein (N) and

the genomic RNA Several glycoproteins are present in the envelope, the membrane

protein (M) which serves as a bridge between the nucleocapsid and the viral

envelope; the spike protein (S) which forms the characteristic peplomers on the

virion surface; the small membrane protein (E); and in some coronaviruses (e.g.

MHV-JHM, BCV, HCV-OC43), the hemaglutinin esterase (HE).

Genome organization

In all coronaviruses the genes encoding the RNA-dependent RNA polymerase

(POL) and the structural proteins are present in the same order. 5' POL - S - sM - M

- N 3' (de Groot et a/., 1989). These genes, each having one or more open reading

frames (ORFs), are separated by conserved stretches of 8 to 10 nucleotides, termed

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FIG. 1. Schematic representation of a coronavirion. Indicated are the RNA genome (RNA), the

nucleocapsid protein (N), the spike protein (S), the membrane protein (M), the small membrane

protein (E) and the hemagglutin-esterase protein (HE) (only present in some coronavirus).

intergenic sequences. So far, three coronavirus genomes have been completely

sequenced, i.e. MHV, HCV-229E and IBV (Boursnell et al., 1987; Bredenbeek et al

1990; Herold etai, 1993; Lee etal., 1991). Genome organizations of these three are

shown in Fig. 2. Some coronaviruses like TGEV, CCV, FIPV and FECV contain one

or two additional ORFs downstream of the N gene (de Groot et al., 1988; Vennema

etal., 1992).

Replication

The first step in the viral replication cycle is the binding of the virus to its cellular

receptor via its S glycoprotein peplomers or HE glycoproteins. This step is believed

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Pol la

Pol la

Pol la

2 HE

MHV

5«L£

S M N

m

A 1113aM5a IBV3b\ \l5b

E

HCV

E N229E

FIG. 2. Genomic organizations of MHV, IBV and HCV 229E. The genomic open reading frames

(ORFs) encoding structural proteins (HE, S, M, N and E, i.e. 5b, 5 and 3c for MHV, HCV and IBV

respectively) are lightly dotted; those encoding nonstructural proteins (ORF 1a and ORF 1b) are filled.

to be one of the major determinants of viral tropism. Following binding of the virus to

the cell, fusion of the viral and cellular membranes takes place and the viral

nucleocapsid enters the host cytosol. The entire replication cycle takes place in the

cytoplasm of the host cell and nuclear functions are unnecessary. Coronaviruses are

positive-stranded RNA viruses and do not have a virion associated polymerase.

After the virion has entered the cell and the genome has been uncoated, gene 1 of

the viral genome is translated to produce the viral RNA-dependent RNA

polymerase(s) (Lai, 1990). Thus, the viral genomic RNA serves as a mRNA for the

RNA polymerase and then as a template for the synthesis of negative-stranded RNA.

From the new negative strands, full-length genomic positive strands as well as, when

transcription starts, the subgenomic mRNAs are synthesized. From these

subgenomic messenger RNAs, secondary translation of the nonstructural and

structural proteins occurs. The N protein is synthesized in the cytoplasm and is

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phosphorylated on serine residues (Stohlman and Lai, 1979). As the only protein

constituent of the nucleocapsid, the N protein binds to the viral genomic RNA. The

M, S, and HE glycoproteins are synthesized on the rough endoplasmic reticulum and

transported to the membrane between the rough endoplasmic reticulum (RER) and

Golgi (reviewed in Spaan et al., 1988). Nucleocapsids are enveloped in the

RER/Golgi via interactions of the N, M and E proteins. Virus particles are released

from cells via exocytosis or cytolysis.

Transcription

The mechanism of coronavirus mRNA synthesis is a matter of considerable

debate. The first RNA believed to be transcribed by the RNA dependent RNA

polymerase is the full-length negative-stranded template (antigenome). The initial

model of coronavirus transcription was based on the observation that only a full-

length copy of the genome was present (Lai et al., 1982). Consequently, this was

believed to be the only template for all positive-stranded RNA species of

coronavirus. The primary transcript in this model was the leader, of 58-90 nt,

encoded at the 3' end of the antigenome (Brown et al., 1984; Lai et al., 1983; Spaan

et al., 1983). The leader/polymerase complex would disassociate from the template

and then realign at a complementary sequence (5'-AAUCUAAAC 3' or a closely

related sequence) in the intergenic regions on the antigenome (Baker and Lai, 1990;

Baric et al., 1983; Budzilowicz et al., 1985; Makino etal., 1986; Spaan etal., 1983).

This complex then primes transcription of the mRNAs. Since the intergenic regions

preceede every transcription unit, in MHV there are 6, this model of leader-primed

transcription thus leads to the nested set of coronavirus RNAs (Cheley et al., 1981;

Lai 1990; Lai etal., 1981; Leibowitz etal., 1981; Spaan etal., 1988). Recently it was

shown that other negative stranded RNAs are made during coronavirus transcription:

full-length copies of each mRNA, containing antileader and poly(U) (Hofman et al.,

1990; Sawicki and Sawicki 1990; Sethna et al., 1989, 1991). It has been

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demonstrated that these subgenomic negative strands are transcriptionally active

(Sawicki and Sawicki, 1990; Schaad and Baric, 1994). Two new models were put

forward to explain these observations. In one, the leader-primed transcription is

considered to occur as thought previously. However, each mRNA that is synthesized

is then a template for minus-strand synthesis, leading to replicons for mRNA

amplification (Sethna etal., 1989). In the second model, subgenomic minus-strands

are believed to be transcribed first from the genomic RNA. The intergenic regions

now serve as leaky transcription termination signals (Sawicki and Sawicki, 1990).

This leads to a nested set of minus stranded RNAs, which is then the template for

the nested set of plus stranded RNAs. Addition of the leader occurs either by leader-

priming, as in the classic model, or by translocation of the minus strand/polymerase

complex to the leader sequence at the extreme end of the genome and continuation

of transcription.

Viral proteins and their genes

The structural proteins S, M and N, and if present, HE are the most abundantly

produced proteins. The S protein is encoded by mRNA 3 in MHV and is a 158 to 200

K glycoprotein (Siddell ef al., 1982). The S protein induces cell fusion in vitro and in

vivo, binds to the cell surface receptor and elicitates neutralizing antibody as well as

cell-mediated immunity (Boyle et al., 1987; Collins et al., 1982; Frana ef al., 1985;

Holmes et al., 1981; Sturman and Holmes 1981; Sturman ef al., 1985). For some

coronaviruses, the mature S protein is cleaved into two 90 KDa subunites S1 and S2

(e.g. MHV and BCV), whereas cleavage does not occur for others (e.g. FIPV, TGEV)

(reviewed in Spaan et al., 1988). The role of S protein cleavage in MHV

pathogenesis is controversial.

The M protein is encoded by mRNA 6 and is a 20 to 30 KDa integral membrane

protein (Siddell ef al., 1982). It is synthesized on the RER/Golgi membranes

(Armstrong et al., 1984; Rottier et al., 1986). The M protein is never transported to

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the plasma membrane of the cell and accumulates in the RER/Golgi where it

interacts with the S protein. Monoclonal antibodies specific for the M protein

neutralize virus infectivity only in the presence of complement (Collins ef al., 1982).

The N protein is encoded by mRNA7 and is a 43 to 60 KDa phosphorylated

protein (Stohlman and Lai, 1979). The N protein probably encapsidates the RNA

genome by interacting with the leader sequence and many other sites of the viral

RNA to form a helical nucleocapside (Stohlman et al., 1988). The nucleocapsid then

interacts with the M protein and buds into the RER/Golgi where it is enveloped. The

interaction between the M protein and the nucleocapsid may determine the site of

virus budding (Machamer and Rose, 1987; Rottier and Rose, 1987; Tooze and

Tooze, 1985; Tooze ef al., 1984). The N protein has been shown to bind to the M

protein and also to the virion RNA in vitro (Baric et al., 1988; Masters et al., 1992;

Robbins etal., 1986; Stohlman etal., 1988; Sturman etal., 1980).

The HE protein is present only in some coronaviruses. It is encoded by mRNA 2-

1 in MHV and is present in the virus envelope as a 120 to 140 KDa disulfide-linked

dimer (King etal., 1985; Shieh etal., 1989; Yokomori etal., 1989). The HE protein is

responsible for hemagglutination by binding to 9-0-acetylated neuraminic acid

residues on red blood cells (Vlasak et al., 1988). The HE protein also has an

esterase activity which cleaves the acetyl group from 9-0-acetylated neuraminic acid

(Pfleiderer ef al., 1991; Schultzeef a/., 1991; Vlasak ef a/., 1988). The HE proteins of

coronaviruses have regions of high homology with the influenza C virus surface

glycoprotein, HEF (Luytjes ef al., 1988). The role of the HE protein in MHV

pathogenesis is unknown.

Recently, a 10 KDa protein encoded by the gene 5b has been determined to be

another envelope protein of MHV (Yu ef al., 1994). This protein has been named

small membrane (sM) or envelope protein (E). It has been shown clearly that E

protein plays an important role in virus budding (Bos etal., 1996).

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RNA recombination

The ability to exchange genetic information may allow RNA viruses to adapt to a

changing environment and to overcome potential deleterious effects caused by the

high error frequency of the RNA polymerase. Viruses with segmented genomes can

undergo RNA reassortment. However, the ability of RNA viruses with nonsegmented

genomes to exchange genetic elements is more limited. Only a few RNA viruses,

including picornaviruses (Cooper 1968,1977; Cooper etal., 1975; King et al., 1982,

1985, 1987; Lake ef al., 1975), coronaviruses (Lai ef al., 1985; ), cowpea chlorotic

mottle virus 3a (Allison ef al., 1990), alphaviruses (Hahn ef al., 1988; Weiss and

Schlesinger, 1991) and bromoviruses (Bujarski and Kaesberg, 1986) have been

reported to undergo RNA-RNA recombination at various efficiencies. During a mixed

infection of two MHV strains, RNA recombination occurs at a high frequency both in

vivo and in vitro (Keck ef al., 1988; Lai ef al., 1985; Makino et al., 1986). RNA

recombination is believed to occur through a copy-choice mechanism in which the

polymerase and partially replicated genome dissociate from the orginal template,

associate with a different viral RNA molecule, and then complete the genome

replication (Fig 3). Based on the analysis of a set of ts-mutants, the frequency of

recombination was found to be very high for coronaviruses (up to 25% over the

whole genome) (Baric ef al., 1990). It is also shown that crossovers can occur more

than once within the genome (Baric et al., 1990). Recombination is almost random

and previously identified hot spots resulted from selection of recombinant viruses

(Banner ef a/., 1991).

Coronaviruses also undergo non-homologous recombination. The original

example of this is the HE pseudogene of MHV-A59 (Luytjes ef al., 1988). This gene

bears significant sequence similarities at the amino acid level with the HEF

sequence of influenza C virus (Luytjes ef al., 1988), and is believed to originate from

a non-homologous recombination event with influenza C HEF RNA. Defective

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interfering (Dl) RNA is also thought to be generated by nonhomologous RNA

ibeen

NcNbNa.

rearrangement and has been detected for most RNA viruses

5'.

i

•N6n

3" Jl » 1 5'

5'—**N._M,_.

5'^-NcNbNa „ „ „

CBA XY 12

FIG. 3. Proposed copy choice model of homologous recombination. Transcription pauses at the site

of a stem-loop structure. The transcription complex is dissociated from the RNA template. The

nascent transcript binds to a different template RNA at an upstream site via complementary

sequences between the nacent RNA and template RNA. If the template RNA has a structure similar to

the original RNA, the transcription complex would bind to the same site and homologous

recombination would result.

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Defective interfering RNAs

The genomes of defective interfering (Dl) particles have lost most of their coding

sequences but have retained the essential cis-acting replication and encapsidation

signals (Huang and Baltimore, 1970). Dl particles can only be replicated and

packaged in the presence of the standard viruses. As a result of the competition for

viral proteins and host factors, Dl viruses interfere with the replication of the

standard viruses (reviewed in Holland, 1991).

Coronavirus Dl RNAs have first been analyzed for MHV strain JHM (Makino ef

al., 1984, 1985). Several Dl RNAs with different sizes were found in virus infected

cells, but only the near-genome length DIssA was packaged into Dl particles (Makino

ef al., 1984). The other smaller Dl RNA, DIssE, apparently lacked a specific RNA

packaging signal since it was packaged inefficiently (Makino and Lai, 1989; Makino

ef al., 1988). So it was considered to be less suitable for molecular genetic studies.

Van der Most ef al. used a natural MHV-A59 Dl RNA (Dl-a) as a template to

construct a Dl cDNA clone, pMIDI. Molecular cloning and sequence analysis showed

that MIDI genome consists of three discontinuous fragments from the viral genome

(Fig. 4) (Van der Most et al., 1991). Dl RNAs transcribed from this clone and from its

derivatives, are replicated upon transfection into MHV-infected cells. The synthetic

Dl RNAs have proven to be excellent tools to investigate viral replication,

transcription, genome packaging and recombination.

Pathogenesis of MHV

MHV strains can be classified into two basic biotypes: Respiratory (polytropic)

and enterotropic (Barthold and Smith, 1984). After oronasal inoculation of infant mice

all of the prototype strains including MHV-1, -3, -A59, -JHM, and others, infect upper

respiratory (nasal) mucosa (Barthold, 1986; Compton ef al., 1993). If the infecting

MHV strain is sufficiently virulent or the mouse is susceptible due to age

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I I MtZTJl AAAAn MHVA 59 genomic RNA2 3 45 6/7 32 KB

J—-P^AAAAn pMIDI RNA

3890 4689 5494 5.5 KB

FIG. 4. A comparison of the structures of pMIDI RNA and the MHV-A59 genomic RNA. Black bars

indicate sequences derived from the 5' end of the genome; the dotted bars represent sequences

derived from ORF1b, and the cross-hatched bars indicate sequences derived from the 3' end.

(preweanling), genotype, or immune status, the virus will disseminate readily to

multiple other organs (Barthold, 1986; Compton ef al., 1993). MHV-JHM has been

used as a model for the respiratory group of viruses because it is polytropic and

moderately virulent. After oronasal inoculation of genetically susceptible BALB/c

mice, virus initiates replication in the nasal epithelium. Virus apparently disseminates

via local lymphatics and blood, with replication in pulmonary vascular endothelium

and secondary viremia to multiple organs (Barthold and Smith, 1992). Virus can be

detected in a variety of tissues including nose, brain, vascular endothelium (lung and

elsewhere), bone marrow, lymphoid tissue (e.g., thymus, lymph node, spleen,

mucosa-associated lymphoid tissues), liver, uterus, placenta, peritoneum, and other

sites. Virus is detectable in intestine but is restricted to gut-associated lymphoid

tissue rather than mucosal epithelium. By contrast, genetically resistant SJL mice

develop significantly less severe infections and clear virus earlier than do BALB/c

mice, with significant virus dissemination and disease occurring only in mice infected

at 1 week of age. Respiratory strains of MHV can infect brain hematogenously during

disseminated infection in immunocompromised or infant mice or directly via olfactory

pathways from the nose to brain, even in the apparent absence of virus

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dissemination to other organs (Barthold, 1988; Barthold ef al., 1986). Several

virulent and avirulent respiratory MHV strains can enter the brain via olfactory

pathways. Encephalitis is common among oronasally inoculated mice but

demyelination and chronic infection are largely experimental phenomena (Barthold,

1988; Barthold ef a/., 1986).

Enterotropic strains of MHV such as MHV-RI, -Y and -DVIM differ significantly

from respiratory MHV strains in their biologic behavior but cannot yet be

differentiated antigenically or genetically. Enterotropic MHV strains replicate

preferentially in intestinal mucosal epithelium and disseminate minimally to other

organs such as liver or brain (Barthold, 1987; Barthold ef al., 1993). This is in

contrast to respiratory strains of MHV which rarely infect intestinal mucosal

epithelium. Mice of all ages are susceptible to active enterotropic MHV infection, but

disease occurs only in very young mice. Oral inoculation of BALB/C and SJL mice at

1, 3, or 6 weeks of age with enterotropic MHV-Y results in nearly equal levels of

virus replication in intestinal mucosa regardless of age or genotype. Disease, in the

form of enteritis, is minimal in mice inoculated at 3 or 6 weeks of age and less severe

in SJL mice. Lesions may be restricted to the presence of a few enterosyncytia in the

surface mucosa of the ascending colon. The ascending colon produces the highest

virus titers and develops the most severe lesions in MHV-Y-infected mice. This also

seems to be true for other enterotropic MHV strains. Mice recover from infection with

no apparent carrier state. Thus, the resistance of SJL mice to respiratory MHV-JHM-

induced disease correlates with low virus titers in target tissues (Barthold and Smith,

1987). In contrast, SJL mice support high titer replication of enterotropic MHV with

only mild disease. Disease and virus titers peak at around 5 days in MHV-JHM-

infected mice, whereas MHV-Y-infected mice develop peak virus titers by 2 days

after inoculation. Enterotropic MHV strains tend not to disseminate from intestine to

other organs, but involvement of mesenteric lymph nodes occurs frequently.

Hepatitis may occur but not to the same extent as with respiratory strains of MHV.

Neonates, less than 48 hours old, infected with enterotropic MHV develop severe

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necrotizing enterocolitis with high mortality within 2 days after inoculation. Epizootics

of enterotropic MHV in naive breeding populations can result in 100% mortality

among neonatal mice (Barthold etal., 1982).

Although the molecular mechanism of the MHV tissue tropism is still poorly

understood, several studies have shown an important role of the S protein in

determining the viral virulence and pathogenesis. The S protein appears to be one of

the most variable components of MHV. In addition to molecular diversity in S from

the various MHV strains, variability in the MHV S protein arises during passage

through rodents, generating antigenic and pathogenic variants (Dalziel ef al., 1986;

Fleming ef a/., 1986; Gallagher ef al., 1990; Haspel etal., 1978; Knobleref a/., 1982;

La Monica ef a/., 1991; Massa ef al., 1988; Matsubara ef a/., 1991; Morris ef al.,

1989; Parker et al., 1989; Robb etal., 1979; Taguchi etal., 1985, Wang etal., 1992).

A study on the MHV strains MHV-1, -3, -S, -JHM, and A59 demonstrated the

presence of five antigenic sites, A through E, among which B is involved with

neurotropism and C with variance (Talbot and Buchmeier, 1985). Such analyses also

uncovered polymorphism in the S protein of MHV-JHM variants isolated after

escaping neutralization by MAb (Buchmeier etal., 1988; Dalziel etal., 1986). Some

isolates obtained in this manner were attenuated and others more virulent when

compared with the parental JHM. Attenuation was manifested through a requirement

for larger inocula to initiate central nervous system disease and a change in

preferential tropism from neurons to oligodendrocytes. Thus, a paralytic,

demyelinating disease rather than an acute encephalitis was elicited (Buchmeier ef

al., 1988).

Aim of the research and scope of the thesis

The goal of this research was to gain insight into the molecular aspects of

enterotropic MHV strains, focusing on the role of S protein in determining tissue

tropism.

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In chapter 2 the S genes of enterotropic strains MHV-RI and -Y were cloned and

sequenced. A high degree of homology was found between the MHV-RI and MHV-

JHM S proteins, whereas the homology that was found between the MHV-Y S protein

and the S proteins of other MHV strains was much lower. These results indicate that

if the spike protein is indeed determining the biotype of a strain only a few amino

acid changes may be responsible for tissue tropism. The fact that the S protein of

MHV-Y is inefficiently cleaved may be due to the presence of several amino acid

changes upstream from the predicted cleavage site.

In chapter 3 the presence and position of open reading frames (ORFs) 4, 5a and

5b of three enterotropic strains were determined. They were found to be very similiar

to those of MHV-JHM, except that due to a frameshift ORF 5a of MHV-Y terminated

after merely 63 nucleotides. The proteins, if present, were found to be highly

conserved among all strains. No biotype-specific pattern could be detected.

In chapter 4 the MHV-RI S gene cDNA was inserted in-frame into pMIDI, a full

length cDNA clone of a MHV-A59 Dl, yielding pDPRIS. RNA in vivo transcribed from

pDPRIS could be replicated and passaged efficiently in MHV-A59 infected L cells. In

the absence of positive selection pressure, a highly specific and sensitive RT-PCR

was used for the detection of recombinant RNAs. Recombinant genomic RNA was

detected in intracellular RNA from pDPRIS transfected and MHV-A59 infected cells

in total lysates as well as in agarose gel purified genomic RNA from these fractions.

Six total RNA PCR products and five virion RNA PCR products were cloned and

sequenced. Crossovers between A59 and Rl RNA were found ranging from 112

nucleotides from the 5' border to 89 nts from the 3' border of sequence homologous

between A59 and Rl S genes. Homologous RNA recombination occurred between

the genomic RNA template and the synthetic Dl RNA template at different locations,

generating a series of MHV recombinant RNAs with chimeric S genes.

In chapter 5 a number of applications of PCR in the study of enterotropic MHV

are described. A universal diagnostic PCR was developed which detected all of 11

different MHV strains. This fast and reliable method was also able to differentiate

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MHV from other non-murine coronaviruses. On the basis of this assay a quantitative

PCR was designed using a mutant template containing a point mutation which

competed for the PCR primers. The amplication and cloning of the structural protein

genes of enterotropic MHV strains in plasmid vectors for subsequent sequencing is

described.

In chapter 6 I describe a strategy which takes advantage of RNA recombination

occurring at high frequency between MHV helper virus and synthetic interfering (Dl)

RNA. A synthesic Dl containing MHV-RI S gene cDNA clone, pDPRIS, was

constructed. RNA in vivo transcribed from the plasmid was passaged in MHV-A59

infected cells. Using a highly specific and sensitive RT-PCR, recombinant genomic

RNA was detected in both intracellular and virion RNA. In addition, the same

intracellular RNA was isolated and separated on a low melting point agarose gel.

The band containing the MHV genomic RNA was excised and the subsequently

eluted RNA yielded a positive signal in the recombinant-specific RT-PCR. Since the

purified genomic RNA did not contain DPRIS RNA, it confirmed that homologous

recombination between MHV-A59 RNA and synthetic Dl RNA had indeed taken

place.

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