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.

Sequence analysis of major structural proteins of newly isolated mouse hepatitis virus.

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.

Unique N-linked glycosylation of murine coronavirus MHV-2 membrane protein at the conserved O-linked glycosylation site.

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.

Differential receptor-functionality of the two distinct receptor proteins for mouse hepatitis virus.

We compared the virus-binding activity and receptor-functionality of the receptor proteins isolated from mouse hepatitis virus (MHV)-susceptible BALB/c mice (MHVR1) and MHV-resistant SJL mice (MHVR2). By using a soluble receptor protein which lacked the transmembrane and intracytoplasmic domains, virus overlay protein blot assay and neutralization tests showed that MHVR1 bound to JHM cl-2 virus with 300-500 times higher efficiency than to MHVR2. MHVR1 was revealed to have 10-30 fold higher receptor-functionality than MHVR2 when examined by measuring virus-binding to the receptor expressed on the cell surface. These findings suggested that the differences in susceptibility between BALB/c and SJL mice may depend upon the genotype of the MHV receptor.

Requirement of proteolytic cleavage of the murine coronavirus MHV-2 spike protein for fusion activity.

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 of MHV-2 results from the lack of cleavage of its S protein.

Contamination of mouse-adapted influenza virus with Sendai virus.

In our laboratory animal facility, Sendai virus (HVJ) contamination occurred in a negative flow rack used for experimental infection with 4 strains of mouse-adapted influenza virus (Inf.V). Anti-HVJ antibody (Ab) was detected in 35/42 mice in the rack. To specify the strain of Inf.V contaminated with HVJ, experimental infection was performed by using A, B and D strains of Inf.V in each vinyl isolator. Anti-HVJ Ab was detected in all mice infected with A strain at day 28 post-infection. As a result of experimental infection with A strain of Inf.V which was treated with anti-HVJ mouse serum, the virus suspension was determined not to contain HVJ and allowed for experimental use in our facility, Since then, HVJ contamination has not occurred in our facility.

Application of nested polymerase chain reaction to detection of mouse hepatitis virus in fecal specimens during a natural outbreak in an immunodeficient mouse colony.

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.

Acquired fusion activity of a murine coronavirus MHV-2 variant with mutations in the proteolytic cleavage site and the signal sequence of the S protein.

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.

Difference in virus-binding activity of two distinct receptor proteins for mouse hepatitis virus.

The receptor proteins, MHVR1 (Bgp C or splice variant of mmCGM1 containing two ectodomains) and MHVR2 (mmCGM2) have been reported to be functional receptors for MHV, although there was a significant difference in their virus-binding activity as determined by virus overlay protein blot assay (VOPBA). To compare the receptor function of these proteins, their virus-binding capacities were tested by using soluble forms of the proteins which lacked the transmembrane and intracytoplasmic domains. To estimate the amounts of these proteins expressed, an epitope of influenza HA protein, for which specific monoclonal antibody was available, was used as a tag. Recombinant soluble MHVR1 and MHVR2, expressed in RK 13 cells using recombinant vaccinia virus were secreted into the culture fluids of infected cells expressing these proteins. The inhibitory effect on virus infectivity of MHVR1 was shown to be about 500-fold higher than that of MHVR2. A similar disparity was observed in virus binding by VOPBA. These two proteins worked as functional receptors when they were expressed on resistant BHK-21 cells. However, the efficiency of MHV infection in BHK-21 cells expressing MHVR1 was about 30-fold higher, as compared with those expressing MHVR2. These data show that the receptor function of MHVR1 was significantly higher than that of MHVR2 and suggests that the difference in susceptibility between SJL and BALB/c mice might be due to the specific receptor protein expressed in those animals.

Localization of neutralizing epitopes and receptor-binding site in murine coronavirus spike protein.

To identify the localization of the epitopes recognized by monoclonal antibodies (MAbs) against the S1 subunit of the murine coronavirus JHMV spike protein, we have expressed the S1 proteins with different deletions from the C terminus of the S1. All of MAbs in groups A and B recognized the S1N(330) composed of 330 amino acids (aa) from the N terminus of the S1 and the larger S1 deletion mutants, but failed to react with the S1N(220) composed of 220 aa. MAbs in group C reacted only with the S1utt protein without any deletion. These results indicated that the S1N330 comprised the cluster of epitopes recognized by MAbs in groups A and B. These results together with the fact that all the MAbs in group B retained the high neutralizing activity suggested that the N terminus 330 aa are responsible for binding to the MHV-specific receptors. In pursuit of this possibility, we have expressed the receptor protein and examined the binding of each S1 deletion mutants to the receptor. It was demonstrated that the S1N(330) protein as well as other S1 deletion mutants larger than S1N(330) bound to the receptor. These results indicated that a domain composed of 330 aa at the N terminus of the S1 protein is responsible for binding to the MHV-specific receptor.

Localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein.

To localize the epitopes recognized by monoclonal antibodies (MAbs) specific for the S1 subunit of the murine coronavirus JHMV spike protein, we have expressed S1 proteins with different deletions from the C terminus of S1. S1utt is composed of the entire 769-amino-acid (aa) S1 protein; S1NM, S1N, S1n(330), and S1n(220) are deletion mutants with 594, 453, 330, and 220 aa from the N terminus of the S1 protein. The expressed S1 deletion mutant proteins were examined for reactivities to a panel of MAbs. All MAbs classified in groups A and B, those reactive to most mouse hepatitis virus (MHV) strains and those specific for isolate JHMV, respectively, recognized S1N(330) and the larger S1 deletion mutants but failed to react with S1N(220). MAbs in group C, specific for the larger S protein of JHMV, reacted only with the S1utt protein without any deletion. These results indicated that the domain composed of the N-terminal 330 aa comprised the cluster of conformational epitopes recognized by MAbs in groups A and B. It was also shown that the epitopes of MAbs in group C were not restricted to the region missing in the smaller S protein. These results together with the fact that all MAbs in group B retained high neutralizing activity suggested the possibility that the N-terminal 330 aa are responsible for binding to the MHV-specific receptors. In investigate this possibility, we expressed the receptor protein and examined the binding of each S1 deletion mutant to the receptor. It was demonstrated that the S1N(330) protein as well as other S1 deletion mutants larger than S1N(330) bound to the receptor. These results indicated that a domain composed of 330 aa at the N terminus of the S1 protein is responsible for binding to the MHV-specific receptor.

Detection of mouse hepatitis virus by the polymerase chain reaction and its application to the rapid diagnosis of infection.

Eight different strains of mouse hepatitis virus (MHV) were analyzed by the polymerase chain reaction (PCR) to see whether two sets of oligonucleotides, which were synthesized based on the published nucleotide sequence of MHV-JHM mRNAs 6 and 7, could be used as universal primers for amplification. Total RNA extracted from virus-infected cells or virus-infected culture fluids was transcribed into cDNA by using reverse transcriptase and oligo(dT) as primer, then the cDNA transcripts were amplified by PCR. The MHV-specific fragments of 199-bp and 241-bp were obtained from all eight strains irrespective of nucleotide differences in the primer regions. The same fragments were also amplified from RNA derived from the liver and brain of MHV-JHM-infected mice as soon as day 1 after intraperitoneal injection, even from the liver from which the virus was not detected. Results of PCR amplification from the liver RNA extracts became positive when more than 10(-2)PFU of MHV-JHM was contained in the PCR reaction mixture. In contrast, anti-MHV antibody was not detected by enzyme-linked immunosorbent assay until day 6 after inoculation. These results suggest that PCR is a very sensitive method to identify a variety of MHV infections in laboratory animals, especially at the early phase of infection.

Detection and characterization of mumps virus V protein.

By cDNA cloning, DNA sequencing, and RNAse mapping analyses two distinct mRNA species were shown to be transcribed from the mumps virus P gene; one faithful and the other edited copies. The former was found to direct the synthesis of the V protein (25K), while the latter, after the addition of two nontemplated G residues, was found to direct the synthesis of the P protein (40K-42K). The carboxy terminal of the V protein contained a cysteine-rich region which was similar but not identical to the metal binding domain motif found in several nucleic acid binding proteins. The V protein was detected not only in mumps virus-infected cells but also in the virions by antiserum raised against a synthetic peptide.

Phenotypic characterization of cynomolgus monkey natural killer cells.

Natural killer (NK) activity of cynomolgus monkey peripheral blood lymphocytes (PBL) was determined using B95-8 cells as target cells. Examination for the reactivity of human NK-related monoclonal antibodies (mAbs), anti-Leu-7, anti-Leu-11b, anti-NKH1A, and NC-1, with cynomolgus PBL revealed that Leu-11b (CD16) was the only antigen expressed on cynomolgus PBL. The percentage of Leu-11b-positive (Leu-11b+) cells correlated well with the level of NK activity when PBL taken from 21 monkeys were tested. After depletion of Fc receptor-positive (FcR+) cells, NK activity was lost concomitantly with the disappearance of Leu-11b+ cells. These results show that cynomolgus NK cells are mainly FcR+ which can be detected by mAb directed to Leu-11b. Cynomolgus PBL were separated by Ficoll-Hypaque centrifugation after E rosette formation with 2-aminoethylisothiouronium bromide-treated sheep red blood cells, and NK activities of both E rosette-forming (E+) and nonforming (E-) fractions were determined. The high level of killing was observed in the E- fraction, suggesting that the majority of cynomolgus NK cells was contained in the E- fraction. The separation of PBL by Percoll discontinuous density gradient showed cynomolgus NK cells were enriched in the low density fractions.

Human interferon alpha and gamma production by lymphocytes during the generation of influenza virus-specific cytotoxic T lymphocytes.

We analysed the production of interferons (IFN)-alpha and -gamma during the generation of human influenza-virus specific cytotoxic T lymphocyte (CTL) responses using monoclonal antibodies in a specific radioimmunoassay. The results showed that the peripheral blood mononuclear cells (PBM) of all donors tested produced IFN-gamma and had influenza A virus-specific CTL activity after stimulation. The amount of IFN-gamma produced and the level of CTL activity were significantly correlated. The PBM of some donors also produced IFN-alpha. The level of IFN-gamma produced was low during the first few days and increased subsequently, but IFN-alpha, when it was detected, was produced on day 1. The kinetics of the increase in IFN-gamma correlated with the increase in CTL activity. We also observed an increased percentage of cells bearing interleukin-2 receptors, which may have been a response to the production of IFN-gamma. The T cells active in lysing influenza A virus-infected target cells and in producing IFN-gamma were determined after separating effector cells with monoclonal antibodies. The CTL effector cells were mainly in the T8+ subset, but IFN-gamma-producing cells were found in both T4+ and T8+ subsets. These results suggest that influenza virus-specific T8+ CTL produce IFN-gamma in response to virus, and that T4+ cells which are not CTL effectors also produce IFN-gamma after restimulation with influenza A virus-infected cells.

Influenza virus hemagglutinin-specific cytotoxic T cell response induced by polypeptide produced in Escherichia coli.

We have tested the abilities of various polypeptides of A/PR/8/34 (H1N1) virus, constructed by recombinant DNA techniques, to induce influenza virus-specific secondary cytotoxic T lymphocyte (CTL) responses. A hybrid protein (c13 protein), consisting of the first 81 amino acids of viral nonstructural protein (NS1) and the HA2 subunit of viral hemagglutinin (HA), induced H-2-restricted, influenza virus subtype-specific secondary CTL in vitro, although other peptides did not. Using a recombinant virus, the viral determinant responsible for recognition was mapped to the HA2 portion of c13 protein. Immunization of mice with c13 protein induced the generation of memory CTL in vivo. The CTL precursor frequencies of A/PR/8/34 virus- and c13 protein-immune mice were estimated as one in 8,047 and 50,312, respectively. These results indicate that c13 protein primed recipient mice, even though the level of precursor frequency was below that observed in virus-immune mice.

A study on cadmium-induced nephropathy in rats pretreated with puromycin aminonucleoside.

Nephrotoxicity of cadmium (Cd) was investigated using puromycin aminonucleoside (AN)-pretreated rats. AN pretreatment was performed by iv injection of 100 mg AN/kg body wt 11 days before the initial Cd injection. Since massive proteinuria and focal glomerular deposits were recognized, glomerular permeability is considered to be increased in AN-pretreated rats. AN-pretreated and intact rats were injected sc with 3 mg Cd/kg body wt, 4 times a week. In non-pretreated rats, slight tubular vacuolation was seen after 1-week Cd exposure and severe vacuolation and coagulative necrosis of the tubules was observed after 2-week Cd exposure. On the other hand, AN pretreatment delayed the onset of vacuolation and necrosis for 1 week and made the lesion milder. After 1-week Cd exposure, a larger amount of Cd was excreted into the urine of AN-pretreated rats than of non-pretreated ones, whereas Cd accumulation in the kidney and liver was lower in AN-pretreated rats than in non-pretreated ones. Thereafter, no difference in Cd concentration was recognized between two groups. From these findings, it is suggested that in early stage of Cd administration, Cd was filtered through the glomerular basement membrane modified by AN pretreatment and that this filterable Cd did not have nephrotoxic effects in AN-pretreated rats.

Accumulation and chemical forms of cadmium and its effect on essential metals in rat spleen and pancreas.

Cadmium was injected sc into female Wistar rats at a dose of 3.0 mg Cd/kg body weight, 4 times a week for 1, 2, 3, 4, 5, and 6 wk. Concentrations of cadmium in the spleen and pancreas were determined, together with essential metals, by inductively coupled plasma-atomic emission spectrometry. Cadmium in both tissues increased even after maximum concentration was attained in the liver. Contents of zinc, calcium, and magnesium in the spleen increased with splenomegaly, while content of iron decreased. Concentrations of calcium, magnesium, and iron decreased in the pancreas, while concentration of zinc showed a transitory increase. Cadmium in the spleen and pancreas supernatants was mostly bound to metallothionein, and metallothionein in the pancreas was highly susceptible to oxidation reaction. The spleen and pancreas were histologically less affected by cadmium loading compared to the liver and kidney, and the pancreas showed only slight alterations after injections for 5 and 6 wk.