Comparative analysis of RNA genomes of mouse hepatitis viruses.

The RNA genomes of various murine hepatitis virus (MHV) strains were studied by T1-oligonucleotide fingerprinting analysis with regard to their structure and sequence relationship. It was found that the MHV particles contained only positive-stranded 60S RNA which had a "cap" structure at its 5' end. No negative-stranded RNA was found. It was also shown that most of the MHV strains had diverged quite extensively in their genetic sequences. However, MHV-3, a hepatotropic strain, and A59, a nonpathogenic strain, were found to have very similar oligonucleotide fingerprinting patterns. Yet, each of them contained two to four specific oligonucleotides. The MHV-3-specific oligonucleotides were conserved in almost all of the hepatotropic MHV strains studied. In contrast, two of the A59-specific oligonucleotides were absent from the genomes of all hepatotropic strains. These findings suggest that these unique oligonucleotides might be localized at the genetic region(s) associated with viral pathogenicity or other biological properties of the virus. Comparison of viral structural proteins also suggests that MHV-3 and A59 are more closely related than other MHV strains. The significance of these findings is discussed.     

The receptor for mouse hepatitis virus in the resistant mouse strain SJL is functional: implications for the requirement of a second factor for viral infection.

The SJL mouse strain is resistant to infection by some strains of the murine coronavirus mouse hepatitis virus (MHV), such as JHM and A59. The block to virus infection has been variously attributed to defects in virus receptors or virus spread. Since the cellular receptors for MHV, mmCGM1 and mmCGM2, have recently been identified as members of the carcinoembryonic antigen family, we reexamined the possible defectiveness of the MHV receptors in SJL mouse strain. Cloning and sequencing of the cDNAs of both mmCGMs RNAs from SJL mice revealed that they were identical in size to those of the susceptible C57BL/6 (B6) mouse. There was some sequence divergence in the N terminus of the mmCGM molecules between the two mouse strains, resulting in a different number of potential glycosylation sites. This was confirmed by in vitro translation of the mmCGM RNAs, which showed that the glycosylated mmCGM2 of SJL was smaller than that of B6 mice. However, transfection of either mmCGM1 or mmCGM2 from SJL mice into MHV-resistant Cos 7 cells rendered the cells susceptible to MHV infection. The ability of the SJL mmCGM molecules to serve as MHV receptors was comparable to that of those from B6. These molecules are expressed in SJL mouse brain and liver in a similar ratio and in amounts equivalent to those in the B6 mouse. Furthermore, we demonstrated that an SJL-derived cell line was susceptible to A59 but resistant to JHM infection. We concluded that the MHV receptor molecules in the SJL mouse are functional and that the resistance of SJL mice to infection by some MHV strains most likely results from some other factor(s) required for virus entry or some other step(s) in virus replication.     

Sequence analysis and molecular detection of mouse hepatitis virus using the polymerase chain reaction.

Sequence analysis of the nucleocapsid protein genes of five strains of mouse hepatitis virus (MHV) disclosed that the 3' region of the nucleocapsid protein gene contains highly conserved sequences unique to MHV. We designed a pair of primers to amplify cDNA from such sequences of MHV by using the polymerase chain reaction (PCR). Six isolates of wild-type MHV, as well as prototype viruses, were amplified successfully and detected in ethidium bromide-stained agarose gels. The sequence identity of PCR products was readily verified by confirming target size and a MflI site within the target. The sensitivity of our PCR assay was estimated to be sufficient to detect a single cell infected with MHV. This new approach may permit more sensitive and rapid detection of MHV in biologic materials than current methods such as virus isolation, the infant mouse bioassay, and the mouse antibody production test.     

Duration and strain-specificity of immunity to enterotropic mouse hepatitis virus.

We examined the duration and strain-specificity of immunity to enterotropic mouse hepatitis virus (MHV). Two strains of enterotropic MHV (MHV-Y and MHV-RI) were determined to be distinct virus strains by serum neutralization and by enzyme immunoassay. BALB/cByJ mice immunized by oral infection with either MHV-Y or MHV-RI developed serum MHV IgG titers that remained stable for more than 6 months. The animals were protected from reinfection with the homologous virus strain at 1 and 6 months after an initial immunizing infection, based on intestinal histology and polymerase chain reaction for a 375-base-pair segment of the membrane glycoprotein gene. Immunity was also fully protective against challenge with the heterologous strain 1 month after initial immunization and partly protective after 6 months. Maternally-derived passive immunity prevented MHV infection in 1-week-old pups challenged with the homologous strain of MHV, and pups challenged with the heterologous virus strain were partially protected.     

Studies on the development of an ELISA kit for microbiological monitoring. 1. Evaluation of the reliability of the prototype kit by field tests

The prototype of an ELISA kit using protein A as the second reaction reagent for mice and anti-rat IgG for rats was prepared for seromonitoring of the Sendai virus and mouse hepatitis virus (MHV)/sialodacryoadenitis virus (SDAV)/Parker's rat coronavirus (PCV) infections. The respective antigen strains and protein concentrations were Sendai virus MN strain, 2 micrograms/ml and MHV Nu-67 strain, 5 micrograms/ml. The reliability of this prototype kit was investigated in two field tests performed on a total of 10,094 mouse and rat sera from 147 institutions. The results indicated that the two types of kits for the two species of animals were highly specific, but it is necessary to increase the detection sensitivity of the MHV antigen for the MHV antibody of mice and SDAV/PCV antibodies of rats.     

High-frequency RNA recombination of murine coronaviruses.

The RNA genome of coronaviruses consists of a single species of nonsegmented RNA. In this communication, we demonstrate that the RNA genomes of different strains of murine coronaviruses recombine during mixed infection at a very high frequency. Susceptible cells were coinfected with a temperature-sensitive mutant of one strain of mouse hepatitis virus (MHV) and a wild-type virus of a different strain. Of 21 randomly isolated viruses released from the coinfected cells at the nonpermissive temperature, 2 were recombinants which differed in the site of recombination. After three serial passages of the original virus pool derived from the mixed infection, the majority of the progeny viruses were recombinants. These recombinant viruses represented at least five different recombination sites between the two parental MHV strains. Such a high-frequency recombination between nonsegmented RNA genomes of MHV suggests that segmented RNA intermediates might be generated during MHV replication. We propose that the RNA replication of MHV proceeds in a discontinuous and nonprocessive manner, thus generating free segmented RNA intermediates, which could be used in RNA recombination via a copy-choice mechanism.     

Interaction of mouse hepatitis virus (MHV) spike glycoprotein with receptor glycoprotein MHVR is required for infection with an MHV strain that expresses the hemagglutinin-esterase glycoprotein.

In addition to the spike (S) glycoprotein that binds to carcinoembryonic antigen-related receptors on the host cell membrane, some strains of mouse coronavirus (mouse hepatitis virus [MHV]) express a hemagglutinin esterase (HE) glycoprotein with hemagglutinating and acetylesterase activity. Virions of strains that do not express HE, such as MHV-A59, can infect mouse fibroblasts in vitro, showing that the HE glycoprotein is not required for infection of these cells. The present work was done to study whether interaction of the HE glycoprotein with carbohydrate moieties could lead to virus entry and infection in the absence of interaction of the S glycoprotein with its receptor glycoprotein, MHVR. The DVIM strain of MHV expresses large amounts of HE glycoprotein, as shown by hemadsorption, acetylesterase activity, and immunoreactivity with antibodies directed against the HE glycoprotein of bovine coronavirus. A monoclonal anti-MHVR antibody, MAb-CC1, blocks binding of virus S glycoprotein to MHVR and blocks infection of MHV strains that do not express HE. MAb-CC1 also prevented MHV-DVIM infection of mouse DBT cells and primary mouse glial cell cultures. Although MDCK-I cells express O-acetylated sialic acid residues on their plasma membranes, these canine cells were resistant to infection with MHV-A59 and MHV-DVIM. Transfection of MDCK-I cells with MHVR cDNA made them susceptible to infection with MHV-A59 and MHV-DVIM. Thus, the HE glycoprotein of an MHV strain did not lead to infection of cultured murine neural cells or of nonmurine cells that express the carbohydrate ligand of the HE glycoprotein. Therefore, interaction of the spike glycoprotein of MHV with its carcinoembryonic antigen-related receptor glycoprotein is required for infectivity of MHV strains whether or not they express the HE glycoprotein.     

Serological examinations on natural infections with mouse pathogens in inbred mouse strains: difference in antibody detection among the strains (author's transl)

Serological surveys on several infections were performed on the inbred mouse strains maintained at the Central Institute for Experimental Animals. In the first survey, 11 strains of mouse, which were 8 weeks of age or older and were kept in separate cages in the same animal room, were tested for antibodies to Salmonella enteritidis, Corynebacterium kutscheri, Tyzzer's organisms, Mycoplasma pulmonis, mouse hepatitis virus (MHV), Sendai virus (HVJ), pneumonia virus of mice (PVM) and minute virus of mice (MVM). Positive results were obtained in MHV, HVJ, PVM and MVM. Positive rates for these viruses except for MVM were different among mouse strains. In the second survey, 5 strains of mouse kept together in the same cage for 4 weeks after weaning were examined for MHV and HVJ antibodies. Positive rates to MHV were different among mouse strains as observed in the first survey. For HVJ antibody, no difference was demonstrated in positive rates unlike in the first survey, but the titers varied between the strains. These results suggest the difference in antibody response to natural infections dependent on mouse strains.     

Enterotropic mouse hepatitis virus infection in nude mice

The cause of emaciation and diarrhea in athymic nude mice was found to be hyperplastic typhlocolitis resulting from infection with enterotropic mouse hepatitis virus (MHV). The disease was reproduced in experimentally-inoculated nude mice using intestinal homogenates from affected mice and cell culture-derived virus. Material derived from an experimental mouse was passed into neonatal Swiss mice and caused acute typhlocolitis. Virus failed to grow in NCTC-1469 cells and 17Cl-1 cells, which are normally permissive for MHV, but grew to low titer in a mouse rectal carcinoma cell line, CMT 93. These results show that an enterotropic strain of MHV can cause chronic enteric disease in athymic nude mice. The pattern of infection differs markedly from the more common MHV wasting syndrome in nude mice caused by non-enteric strains of MHV.     

Differences in antibody production against mouse hepatitis virus (MHV) among mouse strains

Several strains of mice were examined for antibody production after intranasal inoculation with a low virulence strain of mouse hepatitis virus (MHV), MHV-NuU. C57BL/6N mice were shown to be high responders in the production of complement fixing (CF) antibody as compared to C3H/HeN, BALB/c-AnN, DBA/2N mice. F1 hybrids B6C3 and BDF1 from C57BL/6N mice, showed CF antibody responses as high as C57BL/6N, suggesting that high responsiveness is genetically controlled. All these mouse strains were able to produce high titred neutralizing antibody to MHV.     

A role for naturally occurring variation of the murine coronavirus spike protein in stabilizing association with the cellular receptor.

Murine hepatitis virus (MHV), a coronavirus, initiates infection by binding to its cellular receptor (MHVR) via spike (S) proteins projecting from the virion membrane. The structures of these S proteins vary considerably among MHV strains, and this variation is generally considered to be important in determining the strain-specific pathologies of MHV infection, perhaps by affecting the interaction between MHV and the MHVR. To address the relationships between S variation and receptor binding, assays capable of measuring interactions between MHV and MHVR were developed. The assays made use of a novel soluble form of the MHVR, sMHVR-Ig, which comprised the virus-binding immunoglobulin-like domain of MHVR fused to the Fc portion of human immunoglobulin G1. sMHVR-Ig was stably expressed as a disulfide-linked dimer in human 293 EBNA cells and was immobilized to Sepharose-protein G via the Fc domain. The resulting Sepharose beads were used to adsorb radiolabelled MHV particles. At 4 degrees C, the beads specifically adsorbed two prototype MHV strains, MHV JHM (strain 4) and a tissue culture-adapted mutant of MHV JHM, the JHMX strain. A shift to 37 degrees C resulted in elution of JHM but not JHMX. This in vitro observation of JHM (but not JHMX) elution from its receptor at 37 degrees C was paralleled by a corresponding 37 degrees C elution of receptor-associated JHM (but not JHMX) from tissue culture cells. The basis for this difference in maintenance of receptor association was correlated with a large deletion mutation present within the JHMX S protein, as sMHVR-Ig exhibited relatively thermostable binding to vaccinia virus-expressed S proteins containing the deletion. These results indicate that naturally occurring mutations in the coronavirus S protein affect the stability of the initial interaction with the host cell and thus contribute to the likelihood of successful infection by incoming virions. These changes in virus entry features may result in coronaviruses with novel pathogenic properties.     

Bgp2, a new member of the carcinoembryonic antigen-related gene family, encodes an alternative receptor for mouse hepatitis viruses.

Murine coronaviruses such as mouse hepatitis virus (MHV) infect mouse cells via cellular receptors that are isoforms of biliary glycoprotein (Bgp) of the carcinoembryonic antigen gene family (G. S. Dveksler, C. W. Dieffenbach, C. B. Cardellichio, K. McCuaig, M. N. Pensiero, G.-S. Jiang, N. Beauchemin, and K. V. Holmes, J. Virol. 67:1-8, 1993). The Bgp isoforms are generated through alternative splicing of the mouse Bgp1 gene that has two allelic forms called MHVR (or mmCGM1), expressed in MHV-susceptible mouse strains, and mmCGM2, expressed in SJL/J mice, which are resistant to MHV. We here report the cloning and characterization of a new Bgp-related gene designated Bgp2. The Bgp2 cDNA allowed the prediction of a 271-amino-acid glycoprotein with two immunoglobulin domains, a transmembrane, and a putative cytoplasmic tail. There is considerable divergence in the amino acid sequences of the N-terminal domains of the proteins coded by the Bgp1 gene from that of the Bgp2-encoded protein. RNase protection assays and RNA PCR showed that Bgp2 was expressed in BALB/c kidney, colon, and brain tissue, in SJL/J colon and liver tissue, in BALB/c and CD1 spleen tissue, in C3H macrophages, and in mouse rectal carcinoma CMT-93 cells. When Bgp2-transfected hamster cells were challenged with MHV-A59, MHV-JHM, or MHV-3, the Bgp2-encoded protein served as a functional MHV receptor, although with a lower efficiency than that of the MHVR glycoprotein. The Bgp2-mediated virus infection could not be inhibited by monoclonal antibody CC1 that is specific for the N-terminal domain of MHVR. Although CMT-93 cells express both MHVR and Bgp2, infection with the three strains of MHV was blocked by pretreatment with monoclonal antibody CC1, suggesting that MHVR was the only functional receptor in these cells. Thus, a novel murine Bgp gene has been identified that can be coexpressed in inbred mice with the Bgp1 glycoproteins and that can serve as a receptor for MHV strains when expressed in transfected hamster cells.     

A comparison of two antigen strains of each of mouse hepatitis virus and mouse adenovirus for detection of complement fixation antibody in mouse and rat sera

Detection rates of complement fixation antibodies in mice and rats were compared between two antigen strains of each of mouse hepatitis virus (MHV) and mouse adenovirus (MAV). Among 66 and 47 naturally infected MHV-positive sera of mice (18 facilities) and rats (16 facilities) respectively, 17 mouse and 21 rat sera reacted with both Nu-67 and MHV-2 strains, but 49 mouse and 25 rat sera were positive to Nu-67 strain alone. Only one rat serum reacted with MHV-2 strain alone. In comparison with K87 and FL strains of MAV, all 8 positive mouse sera (3 facilities) reacted with K87 strain alone whereas out of 53 positive rat sera (20 facilities), 43, 6 and 4 sera reacted with K87 strain alone, with FL strain alone and with both the two strains, respectively.     

Natural cytotoxicity against mouse hepatitis virus-infected target cells. I. Correlation of cytotoxicity with virus binding to leukocytes

Spleen cells from uninfected control mice selectively lysed BALB/c 3T3 fibroblasts infected with mouse hepatitis virus (MHV), a murine coronavirus. Lysis of infected cells occurred within 3 hr, and histocompatibility between effector and target cells was not required. This natural, cell-mediated, virus-associated cytotoxicity differed from NK cell- and T cell-mediated lysis. Spleen cells from animals infected with MHV were enriched in NK activity and were more cytotoxic to YAC-1 target cells, but did not show enhanced cytotoxicity for MHV-infected target cells. Spleen cells from beige mice, which are deficient in NK cell activity, were able to lyse MHV-infected target cells, as were spleen cells from nude mice, which are deficient in T cell activity. Lysis of MHV-infected target cells could be mediated by cells from the spleen and, to a lesser extent, by cells from the bone marrow, but not by resident peritoneal cells or thymocytes. We suggest the term "virus killer (VK) activity" for this phenomenon. VK activity of splenocytes from different mouse strains correlated with the ability of the splenocytes to bind purified radiolabeled MHV virions. MHV virions caused agglutination of spleen leukocytes from susceptible mouse strains, indicating that leukocyte agglutination or adsorption may provide a useful assay for coronaviruses such as MHV which lack hemagglutinating activity. SJL mouse splenocytes did not bind MHV and did not lyse infected targets. MHV bound relatively well to splenocytes of other mouse strains, but poorly to thymocytes and erythrocytes. Binding of MHV to leukocytes was not influenced by 6 mM EDTA or EGTA, indicating a lack of requirement for Mg++ or Ca++. VK activity was also resistant to EDTA and EGTA, in contrast to NK activity, which was sensitive to those chelating agents. VK activity was also unaffected by actinomycin D, cycloheximide, or puromycin, indicating that new protein synthesis was not required for lysis. Antibody to interferon-alpha/beta did not block lysis, nor was there substantially enhanced lysis mediated by leukocytes from mice infected with virus and thus exposed to high levels of interferon. VK activity was blocked by antibody directed against the peplomeric glycoprotein E2 of MHV. VK activity required infected target cells, because cells with adsorbed MHV virions were not lysed by splenocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of a murine coronavirus defective interfering RNA internal cis-acting replication signal.

The mouse hepatitis virus (MHV) sequences required for replication of the JHM strain of MHV defective interfering (DI) RNA consist of three discontinuous genomic regions: about 0.47 kb from both terminal sequences and a 0.13-kb internal region present at about 0.9 kb from the 5' end of the DI genome. In this study, we investigated the role of the internal 0.13-kb region in MHV RNA replication. Overall sequences of the 0.13-kb regions from various MHV strains were similar to each other, with nucleotide substitutions in some strains; MHV-A59 was exceptional, with three nucleotide deletions. Computer-based secondary-structure analysis of the 0.13-kb region in the positive strand revealed that most of the MHV strains formed the same or a similar main stem-loop structure, whereas only MHV-A59 formed a smaller main stem-loop structure. The RNA secondary structures in the negative strands were much less uniform among the MHV strains. A series of DI RNAs that contained MHV-JHM-derived 5'- and 3'-terminal sequences plus internal 0.13-kb regions derived from various MHV strains were constructed. Most of these DI RNAs replicated in MHV-infected cells, except that MRP-A59, with a 0.13-kb region derived from MHV-A59, failed to replicate. Interestingly, replication of MRP-A59 was temperature dependent; it occurred at 39.5 degrees C but not at 37 or 35 degrees C, whereas a DI RNA with an MHV-JHM-derived 0.13-kb region replicated at all three temperatures. At 37 degrees C, synthesis of MRP-A59 negative-strand RNA was detected in MHV-infected and MRP-A59 RNA-transfected cells. Another DI RNA with the internal 0.13-kb region deleted also synthesized negative-strand RNA in MHV-infected cells. MRP-A59-transfected cells were shifted from 39.5 to 37 degrees C at 5.5 h postinfection, a time when most MHV negative-strand RNAs have already accumulated; after the shift, MRP-A59 positive-strand RNA synthesis ceased. The minimum sequence required for maintenance of the positive-strand major stem-loop structure and biological function of the MHV-JHM 0.13-kb region was about 57 nucleotides. Function was lost in the 50-nucleotide sequence that formed a positive-strand stem-loop structure identical to that of MHV-A59. These studies suggested that the RNA structure made by the internal sequence was important for positive-strand MHV RNA synthesis.     

Selective tropism of a neurotropic coronavirus for ependymal cells, neurons, and meningeal cells.

The ability of a neurotropic virus, mouse hepatitis virus type 3 (MHV3), to invade the central nervous system (CNS) and to recognize cells selectively within the brain was investigated in vivo and in vitro. In vivo, MHV3 induced in C3H mice a genetically controlled infection of meningeal cells, ependymal cells, and neurons. In vitro, purified MHV3 bound to the surface of isolated ependymal cells and cultured cortical neurons but not to oligodendrocytes or cultured astrocytes. MHV3 replicated within cultured cortical neurons and neuroblastoma cells (NIE 115); infected cultured neurons nonetheless survived and matured normally for a 7-day period postinfection. On the other hand, MHV3 had a low affinity for cortical glial cells or glioma cells (C6 line), both of which appear to be morphologically unaltered by viral infection. Finally, MHV3 infected and disrupted cultured meningeal cells. This suggests that differences in the affinity of cells for MHV3 are determinants of the selective vulnerability of cellular subpopulations within the CNS. In vivo, a higher titer of virus was needed for CNS penetration in the genetically resistant (A/Jx) mice than in the susceptible (C57/BL6) mouse strain. However, in spite of viral invasion, no neuropathological lesions developed. In vitro viral binding to adult ependymal cells of susceptible and resistant strains of mice was identical. Genetic resistance to MHV3-CNS infection appeared to be mediated both by a peripheral mechanism limiting viral penetration into the CNS and by intra-CNS mechanisms, presumably at a stage after viral attachment to target cells.     

Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement of an internal and discontiguous sequence for replication.

All of the defective interfering (DI) RNAs of mouse hepatitis virus (MHV) contain both the 5' and 3' ends of the viral genomic RNA, which presumably include the cis sequences required for RNA replication. To define the replication signal of MHV RNA, we have used a vaccinia virus-T7 polymerase-transcribed MHV DI RNA to study the effects of sequence deletion on DI RNA replication. Following infection of susceptible cells with a recombinant vaccinia virus expressing T7 RNA polymerase, various cDNA clones derived from a DI RNA (DIssF) of the JHM strain of MHV, which is a 3.5-kb naturally occurring DI RNA, behind a T7 promoter were transfected. On superinfection with a helper MHV, the ability of various DI RNAs to replicate was determined. Serial deletions from the middle of the RNA toward both the 5' and 3' ends demonstrated that 859 nucleotides from the 5' end and 436 nucleotides from the 3' end of the MHV RNA genome were necessary for RNA replication. Surprisingly, an additional stretch of 135 nucleotides located at 3.1 to 3.3 kb from the 5' end of the genome was also required. This stretch is discontiguous from the 5'-end cis replication signal and is present in all of the naturally occurring DI RNAs studied so far. The requirement for a long stretch of 5'- and 3'-end sequences predicts that the subgenomic MHV mRNAs cannot replicate. The efficiency of RNA replication varied with different cDNA constructs, suggesting possible interaction between different regions of DI RNA. The identification of MHV RNA replication signals allowed the construction of an MHV DI-based expression vector, which can express foreign genes, such as the chloramphenicol acetyltransferase gene.     

Induction of Apoptosis in Murine Coronavirus-Infected Cultured Cells and Demonstration of E Protein as an Apoptosis Inducer

We demonstrated that infection of 17Cl-1 cells with the murine coronavirus mouse hepatitis virus (MHV) induced caspase-dependent apoptosis. MHV-infected DBT cells did not show apoptotic changes, indicating that apoptosis was not a universal mechanism of cell death in MHV-infected cells. Expression of MHV structural proteins by recombinant vaccinia viruses showed that expression of MHV E protein induced apoptosis in DBT cells, whereas expression of other MHV structural proteins, including S protein, M protein, N protein, and hemagglutinin-esterase protein, failed to induce apoptosis. MHV E protein-mediated apoptosis was suppressed by a high level of Bcl-2 oncogene expression. Our data showed that MHV E protein is a multifunctional protein; in addition to its known function in coronavirus envelope formation, it also induces apoptosis.