The spike (S) protein of a nonfusogenic murine coronavirus, MHV-2, was compared to the S protein of a variant with fusion activity, MHV-2f. Two amino acids differed between the S proteins of these viruses; one was located in the signal sequence and the other was in the putative cleavage site. The amino acid at position 12 in the signal sequence was S in MHV-2 and C in MHV-2f. The amino acid sequence of the cleavage site of MHV-2 was HRARS, while that of MHV-2f was HRARR, showing one amino acid replacement at position 757. In DBT cells infected with MHV-2, the S protein was not cleaved, while the S protein of MHV-2f was cleaved. The S protein of MHV-2f expressed in a transient vaccinia virus expression system was cleaved and was fusogenic in contrast to the nonfusogenic activity of uncleaved MHV-2 S protein. Because the signal sequence is assumed to be removed from the mature S protein soon after synthesis, and because the S protein of MHV-2 was expressed on the cell surface in the same way as the S protein of MHV-2f, the difference in the signal sequence seemed to have had little effect on the transportation and the fusion activity of the S protein. These results showed that MHV-2 does not fuse cells due to the lack of cleavage of its S protein. This conclusion differs from studies on the activity of syncytium formation by the S proteins of fusogenic MHV-JHM and -A59 strains. Possible reasons for these differences in fusion activity are discussed.

The membrane (M) proteins of murine coronavirus (MHV) strains have been reported to contain only O-linked oligosaccharides. The predicted O-glycosylation site consisting of four amino acid residues of Ser-Ser-Thr-Thr is located immediately adjacent to the initiator Met and is well conserved among MHV strains investigated so far. We analyzed the nucleotide sequence of a highly virulent strain MHV-2 M-coding region and demonstrated that MHV-2 had a unique amino acid, Asn, at position 2 at the conserved O-glycosylation site. We also demonstrated that this substitution added N-linked glycans to MHV-2 M protein resulting in increment of molecular mass of MHV-2 M protein compared with JHM strain having only O-linked glycans.

The usefulness of RT-PCR for the detection of MHV in tissues and feces of experimentally infected animals has been reported, but it was unclear whether the method was also applicable for the detection of MHV during a natural outbreak. Enterotropic infection is considered to be the most common form of natural infection among various forms of MHV infection. In this paper, RT-nested PCR was performed to detect MHV excreted in the feces during an outbreak in an immunocompromised A/WySnJ mouse colony. The expected bands were amplified after nested PCR from 20 fecal samples out of 37. These results showed that RT-nested PCR could be applicable for the diagnosis for MHV natural infection.

Abstract:We have isolated the virus from a fecal pellet in the colon of a BALB/c mouse with X-linked immunodeficiency (xid) housed in a room in which there has recently been an epidemic due to mouse hepatitis virus (MHV) and designated it as the MHV-TY strain. Sequence analysis of the MHV-TY strain was performed on major structural, spike (S), membrane (M) and nucleocapsid (N), proteins directly from PCR products. The comparison of nucleotide sequences of MHV-TY with other strains investigated so far revealed that all three structural proteins of the TY strain had some unique amino acid sequences among MHV strains which can be used as markers of this strain. Key words: mouse hepatitis virus, sequence analysis, structural proteins Mouse hepatitis virus (MHV) is one of the most common murine viruses found in laboratory mouse populations [4]. MHV primarily infects the respiratory system or the gastrointestinal tract and causes diverse diseases such as hepatitis, encephalomyelitis and enteritis depending on the strain of the virus and the status of the mice [19,22]. Enterotropic infection is considered to be the most common form of natural infection [3].In one animal room in our facility there has recently been an epidemic of MHV [25]. We isolated an epidemic strain of MHV from a fecal pellet in the colon of a BALB/c mouse with X-linked immunodeficiency (xid) housed in this room, and nucleotide sequences of the major structural proteins of this strain were analyzed.BALB/c xid mice were obtained from the Institute of Medical Science, University of Tokyo and bred in our facility for more than two years. The mice were kept (Received 2 August 1999 / Accepted 18 October 1999 Address corresponding: Y.K. Yamada, Division of Experimental Animal Research, National Institute of Infectious Diseases, Musashimurayama, Japan in negative-pressure ventilated cabinets. All cages and bedding were autoclaved prior to use. A sterile diet and water were provided ad libitum. The animal room was maintained at 23 ± 3°C with a 14-hr light/10-hr dark cycle and 55 ± 5% humidity. We noticed MHV contamination in the room through the results of a routine serological test, although other specific pathogens were negative. Eight 1 to 5-week old mice in the room were exsanguinated after inhalation of chloroform, and tissues of the liver and the brain, and fecal pellets in the colon were taken and frozen at -80°C until use. A piece of the liver or the brain, and a fecal pellet in the colon from each mouse were homogenized in PBS at a concentration of 10%. One hundred µl of ten times diluted homogenate was inoculated on DBT cells which were originally mouse brain tumor cells [10] and routinely used for the propagation and assay of MHV [6].

The spike (S) protein of a non-fusogenic murine coronavirus, MHV-2, was compared to that of a variant, MHV-2f, with fusion activity. Two amino acids differed between the S proteins of these viruses; one was located in the signal sequence (amino acid 12) and the other in the putative cleavage site (amino acid 757). To determine which one of these amino acid changes is important for the alteration of fusogenicity, chimeric S proteins between MHV-2 and-2f were constructed and expressed in DBT cells by a vaccinia virus expression system. The results revealed that one amino acid change (Ser to Arg) at position 757 is responsible for the acquisition of fusogenicity of the MHV-2f S protein. This change also altered the susceptibility to proteolytic cleavage of the MHV-2 S protein which was originally uncleavable. We concluded that the non-fusogenic activity ofMHV-2 results from the lack of cleavage of its S protein.