Suppression subtractive hybridization method for the identification of a new strain of murine hepatitis virus from xenografted SCID mice.

During attempts to clone retroviral determinants associated with a mouse model of Langerhans cell histiocytosis (LCH), suppression subtractive hybridization (SSH) was used to identify unique viruses in the liver of severe combined immunodeficiency (SCID) mice transplanted with LCH tissues. A partial genomic sequence of a murine coronavirus was identified, and the whole genome (31428 bp) of the coronavirus was subsequently sequenced using PCR cloning techniques. Nucleotide sequence comparisons revealed that the genome sequence of the new virus was 91-93% identical to those of known murine hepatitis viruses (MHVs). The predicted open reading frame from the nucleotide sequence encoded all known proteins of MHVs. Analysis at the protein level showed that the virus was closely related to the highly virulent MHV-JHM strain. The virus strain was named MHV-MI. No type D retroviruses were found. Degenerate PCR targeting of type D retrovirus and 5'-RACE targeting of other types of retroviruses confirmed the absence of any retroviral association with the LCH xenografted SCID mice.

Mouse hepatitis virus liver pathology is dependent on ADP-ribose-1''-phosphatase, a viral function conserved in the alpha-like supergroup.

Viral infection of the liver can lead to severe tissue damage when high levels of viral replication and spread in the organ are coupled with strong induction of inflammatory responses. Here we report an unexpected correlation between the expression of a functional X domain encoded by the hepatotropic mouse hepatitis virus strain A59 (MHV-A59), the high-level production of inflammatory cytokines, and the induction of acute viral hepatitis in mice. X-domain (also called macro domain) proteins possess poly-ADP-ribose binding and/or ADP-ribose-1''-phosphatase (ADRP) activity. They are conserved in coronaviruses and in members of the "alpha-like supergroup" of phylogenetically related positive-strand RNA viruses that includes viruses of medical importance, such as rubella virus and hepatitis E virus. By using reverse genetics, we constructed a recombinant murine coronavirus MHV-A59 mutant encoding a single-amino-acid substitution of a strictly conserved residue that is essential for coronaviral ADRP activity. We found that the mutant virus replicated to slightly reduced titers in livers but, strikingly, did not induce liver disease. In vitro, the mutant virus induced only low levels of the inflammatory cytokines tumor necrosis factor alpha and interleukin-6 (IL-6). In vivo, we found that IL-6 production, in particular, was reduced in the spleens and livers of mutant virus-infected mice. Collectively, our data demonstrate that the MHV X domain exacerbates MHV-induced liver pathology, most likely through the induction of excessive inflammatory cytokine expression.

Intrahepatic endothelial and Kupffer cells involved in immunosuppressive cytokines and natural killer (NK)/NK T cell disorders in viral acute hepatitis.

During acute viral hepatitis, the intrahepatic tolerance sustained by immunosuppressive cytokines such as interleukin (IL)-4, IL-10, transforming growth factor (TGF)-beta and prostaglandin E2 (PGE2), produced by Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC), natural killer (NK) T cells and natural regulatory T cells may be disturbed. NK cells are recruited normally in the liver and produce interferon (IFN)-gamma to control viral replication. The use of mouse hepatitis virus type 3 (MHV3) attenuated variants showing selected tropisms for KC or LSEC have allowed determining their roles in the disturbances of immune tolerance during viral hepatitis. Groups of C57BL/6 mice were infected with the pathogenic L2-MHV3 (KC+, LSEC+), low attenuated 51.6-MHV3 (KC+, LSEC-) or high attenuated CL12-MHV3 (KC-, LSEC-) variants for the first 3 days. Results showed that IL-10, TGF-beta and PGE2 production in the liver decreased in L2-MHV3-infected mice and increased in 51.6-MHV3- and CL12-MHV3-infected mice. The ratio of IFN-gamma/IL-4 in liver decreased in L2-MHV3-infected mice, while it was not (or low) altered in mice infected with the attenuated MHV3 variant mice. Phenotypic analysis of intrahepatic mononuclear cells revealed that apoptotic NK and NK T cells increased in mice infected with the L2-MHV3, but were minor in 51.6-MHV3- and CL12-MHV3-infected mice. The numbers of CD4+ forkhead box P3+ cells increased in the livers from low pathogenic CL12-MHV3 and YAC-MHV3-infected mice. These results indicate that viral permissivity of KC and LSEC is involved in the decrease of IL-10 and PGE2, while KC may play an additional role in the apoptosis of NK and NK T cells during acute viral hepatitis.

Mouse hepatitis virus does not induce Beta interferon synthesis and does not inhibit its induction by double-stranded RNA.

Mouse hepatitis virus (MHV) does not induce interferon (IFN) production in fibroblasts or bone marrow-derived dendritic cells. In this report, we show that the essential IFN-beta transcription factors NF-kappaB and IFN regulatory factor 3 are not activated for nuclear translocation and gene induction during infection. However, MHV was unable to inhibit the activation of these factors and subsequent IFN-beta production induced by poly(I:C). Further, MHV infection did not inhibit IFN-beta production mediated by known host pattern recognition receptors (PRRs) (RIG-I, Mda-5, and TLR3). These results are consistent with the notion that double-stranded RNA, produced during MHV infection, is not accessible to cellular PRRs.

Murine hepatitis virus strain 1 produces a clinically relevant model of severe acute respiratory syndrome in A/J mice.

Severe acute respiratory syndrome (SARS) is a life-threatening infectious disease which has been difficult to study and treat because of the lack of a readily available animal model. Intranasal infection of A/J mice with the coronavirus murine hepatitis virus strain 1 (MHV-1) produced pulmonary pathological features of SARS. All MHV-1-infected A/J mice developed progressive interstitial pneumonitis, including dense macrophage infiltrates, giant cells, and hyaline membranes, resulting in death of all animals. In contrast, other mouse strains developed only mild transitory disease. Infected A/J mice had significantly higher cytokine levels, particularly macrophage chemoattractant protein 1 (MCP-1/CCL-2), gamma interferon, and tumor necrosis factor alpha. Furthermore, FGL2/fibroleukin mRNA transcripts and protein and fibrin deposits were markedly increased in the lungs of infected A/J mice. These animals developed a less robust type I interferon response to MHV-1 infection than resistant C57BL/6J mice, and treatment with recombinant beta interferon improved survival. This study describes a potentially useful small animal model of human SARS, defines its pathogenesis, and suggests treatment strategies.

Differentiation of mouse hepatitis virus genotypes by restriction fragment length polymorphism analysis.

Reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the nucleocapsid (N) gene of mouse hepatitis virus (MHV) successfully detected 36 strains from the faeces of mice that had been housed in animal facilities in Japan from 2000 to 2003. Subsequent restriction fragment length polymorphism (RFLP) analysis, using the enzymes Acc I, Alu I, EcoR I and Mbo I, demonstrated that these strains could be divided into five distinct subgroups and that the same MHV strains were detected from closely related mouse facilities. Furthermore, strains from the same facility showed the same RFLP pattern, irrespective of the year of detection, but this pattern varied between different locations. This study shows that RFLP analyses are a rapid and useful method for differentiation of MHV strains.

The sialate-4-O-acetylesterases of coronaviruses related to mouse hepatitis virus: a proposal to reorganize group 2 Coronaviridae.

Group 2 coronaviruses are characterized within the order Nidovirales by a unique genome organization. A characteristic feature of group 2 coronaviruses is the presence of a gene encoding the haemagglutinin-esterase (HE) protein, which is absent in coronaviruses of groups 1 and 3. At least three coronavirus strains within group 2 expressed a structural protein with sialate-4-O-acetylesterase activity, distinguishing them from other members of group 2, which encode an enzyme specific for 5-N-acetyl-9-O-acetylneuraminic acid. The esterases of mouse hepatitis virus (MHV) strains S and JHM and puffinosis virus (PV) specifically hydrolysed 5-N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2) as well as the synthetic substrates p-nitrophenyl acetate, 4-methylumbelliferyl acetate and fluorescein diacetate. The K(m) values of the MHV-like esterases for the latter substrates were two- to tenfold lower than those of the sialate-9-O-acetylesterases of influenza C viruses. Another unspecific esterase substrate, alpha-naphthyl acetate, was used for the in situ detection of the dimeric HE proteins in SDS-polyacrylamide gels. MHV-S, MHV-JHM and PV bound to horse serum glycoproteins containing Neu4,5Ac2. De-O-acetylation of the glycoproteins by alkaline treatment or incubation with the viral esterases resulted in a complete loss of recognition, indicating a specific interaction of MHV-like coronaviruses with Neu4,5Ac2. Combined with evidence for distinct phylogenetic lineages of group 2 coronaviruses, subdivision into subgroups 2a (MHV-like viruses) and 2b (bovine coronavirus-like viruses) is suggested.

Sequence analysis of the S gene of recombinant MHV-2/A59 coronaviruses reveals three candidate mutations associated with demyelination and hepatitis.

Coronaviruses, mouse hepatitis virus (MHV) strains, exhibit various degrees of neurotropism and hepatotropism following intracerebral (IC) infection of 4-week-old C57Bl/6 mice. Whereas MHV-A59 produces acute meningitis, encephalitis, hepatitis, and chronic demyelination, a closely related strain, MHV-2, produces only acute meningitis and hepatitis. We previously reported that the spike glycoprotein gene of MHV contains determinants of demyelination and hepatitis. To further investigate the site of demyelination and hepatitis determinants within the S gene, we sequenced the S gene of several nondemyelinating recombinant viruses. We found that three encephalitis-positive, demyelination-negative, hepatitis-negative recombinant viruses have an MHV-A59-derived S gene, which contains three identical point mutations (I375M, L652I, and T1087N). One or more of the sites of these mutations in the MHV-A59 genome are likely to contribute to demyelination and hepatitis.

Virus-neutralizing monoclonal antibody expressed in milk of transgenic mice provides full protection against virus-induced encephalitis.

Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine beta-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis.

Differentiation of mouse hepatitis viruses in animal facilities in Japan by use of nucleotide analysis of the nucleocapsid gene.

The nucleotide sequences of the coding region of the nucleocapsid (N) gene of 12 mouse hepatitis virus (MHV) strains recently found in animal facilities in Japan were analyzed. Nucleotide sequencing was performed directly on polymerase chain reaction (PCR) products amplified by reverse transcription (RT) and polymerase chain reaction (RT-PCR) analysis from fecal samples or isolated viruses. Phylogenetic analysis of these MHV strains along with those reported previously indicated that sequence analysis of the N gene was a useful tool for differentiation of MHV strains,although most MHV strains in Japanese facilities were phylogenetically close. Results suggested that interchange of mice infected with MHV among facilities provided opportunities of introduction of MHV into otherwise MHV-free facilities and that the source of MHV infection could be traced by use of nucleotide analysis of the N gene.

Difference in Bgp-independent fusion activity among mouse hepatitis viruses.

Mouse hepatitis virus (MHV) utilizes a mouse biliary glycoprotein (Bgp) as a receptor. Co-cultivation of MHV-nonpermissive hamster BHK cells devoid of mouse Bgp with mouse DBT cells infected with MHV-A59 or JHMV induces syncytia formation on BHK cells (Bgp-independent fusion). This study shows the difference in Bgp-independent fusion activity among various MHV strains. Under a phase contrast microscopy, JHMV (cl-2, sp-4) induced the Bgp-independent syncytia on BHK cells similar to those observed on DBT cells, while such syncytia were not seen with the infection of other MHV strains (MHV-1, MHV-3, MHV-A59, MHV-S, srr7, srr11 and srr18). Tiny syncytia detectable only by immunofluorescence were produced with the latter MHV strains except for srr7 which failed to produce syncytia. MHVs except for srr7 grew in BHK cells after Bgp-independent infection. The Bgp-independent fusion by JHMV was inhibited either by anti-S1 or anti-S2 antibodies. These results showed that the JHMV spike protein had a remarkably high Bgp-independent fusion activity.

Prevalence of enterotropic and polytropic mouse hepatitis virus in enzootically infected mouse colonies.

Mouse hepatitis virus (MHV) causes the most prevalent viral infection in contemporary laboratory mouse colonies. According to their primary replication site, different strains of MHV segregate into two overlapping biotypes, enterotropic and polytropic. These two groups vary greatly in disease pattern, pathogenicity, immune response, and duration of infection. Historically, the polytropic MHV strains represent the extensively studied prototype strains that have minimal enterotropism, whereas enterotropic MHV strains have been less characterized. Anecdotal reports suggest that most MHV strains encountered today belong to the enterotropic biotype. We have identified 15 isolates of MHV from 19 independent enzootically infected mouse colonies. Sequencing of a variable region of the nucleoprotein (N) gene of each isolate confirmed that all were independent genetic variants. The principal tissue tropism of the new isolates was determined by experimental inoculation of infant mice and examination of intestine, liver, spleen, and brain for lesions. Nine isolates infected only intestine; four isolates infected intestine and liver; one isolate infected intestine, liver, and brain; and one isolate infected liver. These results confirm that the enterotropic MHV biotype is predominant in contemporary laboratory mouse colonies. The MHV biotype features need to be taken into consideration when dealing with MHV infections.

Genetic complementation among three panels of mouse hepatitis virus gene 1 mutants.

Temperature-sensitive (ts) mutant viruses have been useful for the study of replication processes in many viral systems. To determine how our panel of MHV-JHM-derived RNA- ts mutants (Robb et al., 1979) is genetically related to other panels of MHV RNA- ts mutants, we tested our mutants for complementation with representatives from two different sets of MHV-A59 ts mutants (Koolen et al., 1983; Schaad et al., 1990). These three ts mutant panels together comprise eight genetically distinct complementation groups. Considerable genetic similarity was observed among the three mutant panels. Only three complementation classes are unique to their particular mutant panel, and genetically equivalent mutants were not observed within the other two mutant panels. There are two overlapping complementation groups between the mutant sets derived from MHV-A59 and four overlapping complementation classes between the MHV-JHM panel and the MHV-A59 panels. Two complementation groups had representative mutants in all three mutant panels. One of these latter complementation classes demonstrated nonreciprocal complementation patterns consistent with intragenic complementation.

-Entrance of PCR for studying murine coronaviruses-. / Der Einsatz der PCR zum Studium muriner Coronaviren.

This paper describes a number of applications of the Polymerase Chain Reaction (PCR) in the study of enterotropic murine coronaviruses [mouse hepatitis virus (MHV)]. A diagnostic PCR was developed which detected all of 11 different MHV strains. This fast and reliable method was also able to differentiate 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 amplification and cloning of the structural protein genes of enterotropic MHV strains it plasmid vectors for subsequent sequencing is described. In addition an RT PCR was developed which was able to selectivity detect artificially generated recombinant coronavirus.

[Mouse hepatitis virus]. / Mäusehepatitis-Virus.

Mouse hepatitis virus (MHV), the coronavirus of the mouse (mus musculus), is one of the most important viral pathogens in contemporary laboratory mouse colonies. It is a highly mutable virus consisting of numerous antigenically distinct serotypes with different pathology. These can be divided according to their tissue tropism into respiratory and enterotropic strains. The course of an MHV infection is dependent on virus strain and host factors. Generally MHV causes an acute, self limiting infection which is inapparent in adult mice. Neonates are highly susceptible to disease and show high mortality. In an enzootically infected colony however, they are protected by maternally derived passive immunity. MHV's importance in biomedical research on one hand stems from its potential as an interfering agent, mainly in the field of immunology. On the other hand MHV serves as a model for coronaviruses of other species including man in studies on virus replication and tissue tropism. Since MHV infections are usually subclinical, detection depends on serological screening of colonies using Enzyme-linked immunosorbent assay (ELISA) or immunofluorescence. MHV is controlled by culling and rederivation of the affected colony using hysterectomy or embryo transfer or by elimination by cessation of breeding.

Induction of macrophage procoagulant activity by murine hepatitis virus strain 3: role of tyrosine phosphorylation.

The induction of a unique macrophage procoagulant molecule by murine hepatitis virus strain 3 correlates with the severity of viral hepatitis. The role of tyrosine phosphorylation in the signalling pathway leading to procoagulant expression was studied. Murine hepatitis virus strain 3 initiated a rapid increase in phosphotyrosine accumulation. Tyrosine kinase inhibition precluded this increase and abrogated expression of the virus-induced procoagulant mouse fibrinogen-like protein (musfiblp) gene. These findings suggest that manipulation of this signalling pathway in vivo might represent a novel approach to treating this disease.

Pattern of disease after murine hepatitis virus strain 3 infection correlates with macrophage activation and not viral replication.

Murine hepatitis virus strain (MHV-3) produces a strain-dependent pattern of disease which has been used as a model for fulminant viral hepatitis. This study was undertaken to examine whether there was a correlation between macrophage activation and susceptibility or resistance to MHV-3 infection. Peritoneal macrophages were isolated from resistant A/J and susceptible BALB/cJ mice and, following stimulation with MHV-3 or lipopolysaccharide (LPS), analyzed for transcription of mRNA and production of interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), mouse fibrinogen-like protein (musfiblp), tissue factor (TF), leukotriene B4, and prostaglandin E2 (PGE2). Macrophages from BALB/cJ mice produced greater amounts of IL-1, TNF-alpha, TGF-beta, leukotriene B4, and musfiblp following MHV-3 infection than macrophages from resistant A/J mice, whereas in response to LPS, equivalent amounts of IL-1, TNF-alpha, TGF-beta, and TF were produced by macrophages from both strains of mice. Levels of mRNA of IL-1, TNF-alpha, and musfiblp were greater and more persistent in BALB/cJ than in A/J macrophages, whereas the levels and kinetics of IL-1, TNF-alpha, and TF mRNA following LPS stimulation were identical in macrophages from both strains of mice. Levels of production of PGE2 by MHV-3-stimulated macrophages from resistant and susceptible mice were equivalent; however, the time course for induction of PGE2, differed, but the total quantity of PGE2 produced was insufficient to inhibit induction of musfiblp, a procoagulant known to correlate with development of fulminant hepatic necrosis in susceptible mice. These results demonstrate marked differences in production of inflammatory mediators to MHV-3 infection in macrophages from resistant A/J and susceptible BALB/cJ mice, which may explain the marked hepatic necrosis and fibrin deposition and account for the lethality of MHV-3 in susceptible mice.

Genotyping of mouse hepatitis virus strains by restriction endonuclease analysis of amplified nucleocapsid protein genes.

Restriction endonuclease analysis of amplified nucleocapsid protein genes from mouse hepatitis virus (MHV) was used to differentiate 12 strains isolated from mouse liver or transplantable tumors from five facilities, and the restriction patterns of the isolates were compared with those of five well-defined MHV strains, A59, JHM, 2, S and Nu-67. The patterns of 10 isolates from three facilities were the same as that of Nu-67. The remaining two isolates revealed different patterns from the five reference strains. This study showed that reverse transcription and the polymerase chain reaction assay based restriction analysis are feasible for the detection and genotyping of MHV, and the Nu-67 related strain was the most prevalent type found in the clinical samples.

Characterization of the S protein of enterotropic murine coronavirus strain-Y.

The pathogenesis of enterotropic murine coronavirus strain MHV-Y differs extensively from that of prototypic respiratory strains of murine coronaviruses. The S protein of MHV-Y was characterized as a first step towards identifying viral determinants of enterotropism. Immunoblots of MHV-Y virions using anti-S protein specific antiserum revealed that the MHV-Y S protein was inefficiently cleaved. The MHV-Y S gene was cloned and sequenced. It encodes a protein predicted to be 1361 amino acids long. The presence of several amino acids changes within and surrounding the predicted cleavage site of the MHV-Y S protein may contribute to its inefficient cleavage.