key: cord-0962198-q8g8flo9
authors: Cheley, Steve; Morris, Vincent L.; Cupples, Margaret J.; Anderson, Robert
title: RNA and polypeptide homology among murine coronaviruses
date: 1981-12-31
journal: Virology
DOI: 10.1016/0042-6822(81)90113-6
sha: 6058e813e7b4c4f5c57eba44da2463bd5774574b
doc_id: 962198
cord_uid: q8g8flo9

Abstract Using a 32P complementary DNA (cDNA) prepared against the A59 nucleocapsid protein messenger RNA, we have investigated the extent of homology between A59 and four other strains of murine hepatitis virus (MHV). Analysis by hybridization kinetics of the annealing between A59 [32P]cDNA and infected cell RNA from the other four MHV strains demonstrated 70–80% homology. By gel transfer analysis, the A59 [32P]cDNA was able to detect subgenomic-size virus-specific RNAs in cells infected with all of the five MHV strains. A similar pattern of seven viral RNAs was detected in cells infected with A59, MHV-1, MHV-3, and JHM. In contrast, cells infected with MHV-S contained seven virus-specific RNAs, of which only the two smallest species comigrated with RNAs from the other four strains. The results suggest that as previously shown with A59 (S. Cheley, R. Anderson, M. J. Cupples, E. C. M. Lee Chan, and V. L. Morris (1981) Virology, 112, 596–604), all MHV strains tested encode a nested set of subgenomic RNAs. Analysis of [35S]methionine-labeled viral proteins by SDS-polyacrylamide gel electrophoresis revealed that each strain of MHV specified four major viral polypeptides with apparent molecular weights very similar to those previously reported for the E2, N, El, and PEI polypeptides of A59. The strong degree of interstrain homology among the five MHV strains investigated was confirmed by comparative chymotryptic peptide mapping of the viral N proteins. A majority of the chymotryptic peptides from each of the [35Sknethionine-labeled N proteins was found to coelute by high-performance liquid chromotography. Moreover, this technique of peptide mapping indicated a particularly strong relatedness between MHV-1 and MHV-S and among MHV-3, JHM, and A59.

Coronaviruses include a number of viral pathogens which are responsible for a diverse spectrum of pathological conditions afflicting rodents (Cheever et al, 1949, Gledhill and Andrewes, 1951) , fowl (Beaudette and Hudson, 1937) , humans (Hamre and Procknow, 1966; McIntosh et aL, 1967) as well as several other animal species. Among the coronaviruses pathogenic for rodents, murine hepatitis viruses (MHV) have received considerable attention. Several distinct serotypes of MHV have been recognized (Hierholzer et aL, 19'79; Piazza, 1969) and numerous studies have centered on the pathological effects of MHV infection of mice and rats. Of particular interest is the JHM strain which, unlike other r To whom reprint requests should be sent. MHV strains, shows only a moderate propensity toward visceral tissue, instead, producing a selective infection of the nervous system Cheever et ah, 1949; Weiner, 1973; Lucas et aL, 1977) . Neurological infections by JHM virus can, in some cases, be of a selectively demyelinating nature (Haspel et aL, 1978; Nagashima et aL, 1978; Sorensen et aL, 1980; Weiner, 1973) ; this feature has prompted analogies between JHM disease and human demyelinating conditions of suspected viral etiology . Other strains of MHV produce varying degrees of hepatic disease (Dick et al, 1956; Gledhill and Andrewes, 1951; Manaker et ak, 1961; Nelson, 1952; Rowe et aL, 1963) occasionally with neurological involvement. Apart from serological relationships (Hierholzer et aL, 1979; Piazza, 1969) tides (Anderson et a& 1979; Bond et al, 1979) , very little is presently known about structural similarities or differences among MHV strains at the molecular level. We report here the results of comparative homology determinations on virus-specified RNAs and polypeptides of five selected strains of MHV. In conducting the studies on viral RNAs, we have made use of a complementary DNA probe prepared from nucleocapsid (N) protein messenger RNA isolated from cells infected with the A59 strain of MHV. The specificity of this cDNA including its ability to recognize common sequences in A59specified subgenomic RNAs has been documented elsewhere .

Cell and virus strains. Mouse cell lines, L-2 (Rothfels et aL, 1959) , LM (Merchant and Hellman, 1962) , and 17CL-1 (Sturman and Takemoto, 1972) were used for studies of virus growth. Virus was propagated and plaque assayed (Lucas et ak, 1977) in L-2 cells. Virus strains MHV-1, MHV-3, MHV-S, A59, and JHM were obtained from the American Type Culture Collection.

Assags of virus growth. Confluent monolayer cultures of L-2, LM, and 17CL-1 cells in 25-cm2 tissue culture flasks (Falcon) were inoculated at a multiplicity of infection (m.o.i.) of 0.5 with one of the five MHV strains. After 1 hr adsorption at 37", monolayers were freed from residual inocula by thrice rinsing with minimal essential medium (MEM) and subsequently incubated at 37" with 5 ml of MEM supplemented with 5% fetal calf serum (FCS). Aliquots were removed from the supernatant media at 4-hourly intervals and quantitated for infectious virus by plaque assay.

Isolation of S2P-lubeled A59-infected cell RNA and the preparation of complementav DNA. Complementary DNA (cDNA) was prepared against isolated 0.8 X 106dalton, A59-virus-specific, nucleocapsid protein messenger RNA (mRNA) as previously described .

Hybridization kinetic experiments and analysis using gel electrophoresis and RNA transfer procedures. For the preparation of infected cell RNA, monolayer cultures (approximately 108 cells) of L-2 cells were infected at a m.o.i. of 0.5-l with MHV. Actinomycin D (1 pg/ml) was present in the culture medium from 1 hr postinfection (p.i.) until harvesting (9-10 hr p.i.) at which time the cell monolayers were completely fused. Infected and uninfected cell RNA were isolated as described . For the analysis of hybridization kinetics (Varmus et u& 1973; , the percentage of singlestranded cDNA remaining in aliquots of the annealing mixture was determined with S-l nuclease (Calbiochem) according to the procedure of Leong et al. (1972) . RNA was denatured with glyoxal, subjected to electrophoresis in 1.5% agarose gels, transferred to diazobenzyloxymethyl (DBM)-paper, and hybridized with [%P]-cDNA as previously described (Coulter-Mackie et a& 1980; .

[35SjMethionine labeling of viral polgpep tides. Confluent cultures of L-2 cells in 60mm petri dishes were inoculated at an m.o.i. of 5 each with one of the five MHV strains. Cultures were incubated at 37" for 6-9 hr until virus-induced fusion was evident in virtually all cells; cultures were subsequently incubated for 30 min with [%S]methionine (100 &i/ml) in 1 ml methionine-free MEM. Cells were harvested by scraping, spun into pellets at 650~ for 10 min, and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) as previously described .

Peptide mapping & high-pfmance liquid chromatography (HPLC). Each of the five [36S]methionine-labeled nucleocapsid (N) proteins were excised as slices from dried SDS-polyacrylamide gels after visualization by autoradiography. Proteins were eluted from gel slices with (1 ml) immunoprecipitation (I.P.) buffer (Nusse et aL, 1978, but containing 0.2% instead of 0.1% SDS) for 8 hr at room temperature with periodic mixing. Solubilized protein was freed from gel debris by centrifugation at 60,000~ for 1 hr in a Beckman SW56 rotor. Supernatants were diluted with 1 vol of I.P. buffer lacking SDS in order to attain a final SDS concentration of 0.1% and mixed with 50 ~1 of anti-MHV antiserum. Immune precipitates were pelleted at 60,OOOg and digested for 24 hr at room temperature with 1 ml of 0.05 M ammonium bicarbonate containing chymotrypsin (25 pg/ml; Worthington).

The resulting peptides were lyophilized, dissolved in application buffer (pyridine:acetic acid:water, 1:1:31, and stored at -20" under a nitrogen atmosphere prior to analysis. For peptide mapping, a Waters Associates (Milford, Mass.) liquid chromatograph equipped with a Model U6K injector and a Model 6000A solvent delivery system was used. Peptides were eluted from a C1sp Bondapak reversed phase column (10 pm, 4 mm X 30 cm) using a phosphoric acid-acetonitrile solvent system (Fullmer and Wasserman, 1979) . A linear gradient of 14-36s acetonitrile in 0.1% orthophosphoric acid was found to provide optimal resolution of peptides. Constant flow gradient elution (2 ml/min) was controlled with a Waters Model 720 solvent programmer.

Column effluent was fraction collected in volumes of 0.8 ml and radioactivities quantitated by liquid scintillation spectrometry after the addition of Atomlight (New England Nuclear).

Differences in virus replication and virus-induced cytopathic effect (cpe) have been observed in various cell lines infected with MHV (Lucas et aL, 1977 (Lucas et aL, , 1978 . In order to systematically compare the growth characteristics of different strains of MHV, parallel cultures of L-2 (Rothfels et ah, 1959) , LM (Merchant and Hellman, 1962) , and 17CL-1 (Sturman and Takemoto, 1972 ) cells were each inoculated at an m.o.i. of 0.5 with one of the five strains: MHV-1, MHV-3, MHV-S, A59, or JHM. In all cases, infected L-2 cultures showed the most rapid progression of cpe, in the form of cell-cell fusion which resulted in formation of a virtually complete syncytial monolayer by 12 hr p.i. The light-micro-scopic appearance of individual L-2 cell syncytia produced by infection with either MHV-1, MHV-3, A59, or JHM was identical to that previously observed in JHMinfected RN2 cells (Lucas et d, 1977) . However, L-2 cultures infected with MHV-S produced syncytial foci which tended to contain a large number of predominantly spherical, multinucleated cells which detached from the monolayer more readily than the more flattened syncytia produced by the other four virus strains.

In contrast to infected L-2 cultures, cultures of 17CL-1 cells inoculated with any of the five MHV strains did not show significant cpe until approximately 16 hr pi., at which time a few isolated syncytia were detectable. By 20 hr p.i. approximately 5-10% of the cell monolayer, as estimated by light microscopic observation, consisted of syncytia. Infected LM cell cultures showed the least cpe of any of the three cell lines tested; by 20 hr p.i. only a very few isolated syncytia were visible in each flask.

Virus replication, as determined by plaque assay of supernatant media at 4hourly intervals, is shown in Fig. 1 . In both L-2 and 17CL-1 cell cultures, maximum titers were reached between 12 and 16 hr p.i. for all five MHV strains tested. Growth of virus in LM cells in contrast was markedly reduced and the growth curves more erratic than those observed in L-2 or 17CL-1 cells. Longer term culturing of LM cells infected with all five MHV strains (unpublished) has shown that virus continues to be produced over several weeks in a similar fashion to that in a number of other cell lines previously shown to support a persistent infection of MHV (Lucas et al, 1978) . It is thus interesting that all five MHV strains show the ability to undergo either a productivity lytic (in L-2 cells) or a mildly eytopathic, chronic (in LM cells) type of infection depending on host cell. tein; this cDNA is specific for MHV sequences and shows no significant annealing with uninfected L-2 cell RNA (Cheley et ak, 1981) . It has been demonstrated (Cheley et a& 1981) that this cDNA anneals with six A59-specified RNAs with estimated molecular weights of 0.8 X 106, 1.1 X 106, 1.4 X 106, 1.6 X 106, ca. 3 X 106, and ca. 4 X lo6 daltons. We have now identified an additional high-molecular-weight virus-specific RNA band which migrates more slowly than the ca. 4 X 106-dalton band (Fig. 2) and which would be in the molecular weight range that has been reported for virion RNA (Lai and Stohlman, 1978; .

Using this A59 cDNA as probe for homologous sequences in an RNA transfer procedure, we compared the RNAs from cells infected with the five MHV strains.

In each case, the A59 cDNA annealed with several RNA bands, indicating significant homology between A59 nucleocapsid protein mRNA and the corresponding sequences in the other strains of MHV. The overall similarity in RNA patterns (Fig.  2) is most striking for strains A59, MHV-1, MHV-3, and JHM; for MHV-S a slightly different pattern was observed in which only the two smallest RNA species comigrated with corresponding RNA species seen in the other four samples. In order to enhance the observed migrational differences betwen the RNAs of MHV-S and those of the four other murine coronaviruses (as typified by A59), prolonged times of electrophoresis, were used (Fig. 3) . The 1.4 X 106-and 1.6 X 106-dalton species were missing from the MHV-S pattern, and a 1.2 X 106-dalton virus-specific RNA species .

was present in MHV-S but was absent in RNA species (in 3-4 X lo"-dalton ral 1s) A59 (Fig. 3) . In addition, MHV-S had three1 while A59 only had two viral RNA spe ties higher-molecular-weight virus-specific in this-region of the gel. However, the two smallest RNA species (0.8 X lo6 and 1.1 X 106) of MHV-S comigrated with the corresponding RNA species for A59 (Fig.  3) . In contrast, the higher-molecularweight RNA species for MHV-S did not coelectrophorese with any of the A59 RNA species. Thus, while cells infected with MHV-S and the other MHV strains all contained the 0.8 X 106-and 1.1 X lo'-dalton viral RNA species, the remaining RNA species of MHV-S differed in their electrophoretic mobility from those of the other MHV strains (Figs. 3 and 4) .

We used the cDNA prepared from A59 nucleocapsid mRNA to investigate the homology among the nucleocapsid-encoding sequences of the five different strains of murine Coronavirus.

In agreement with our previous results, it was found that the A59 cDNA annealed essentially to completion with A59 infected cell RNA (Cheley et aL, 1981; Fig. 4 ). The other four murine Coronaviruses that were tested annealed to a maximum extent of '70-80% with the A59 cDNA ( Fig. 4A-D) . The Crtl,e values varied from 0.8 X 10-l to 1.6 X 10-l; this difference could be due to small discrepancies in the initial multiplicity of infection. All of these C,t1,z values correspond to greater than 1000 genome equivalents per infected cell using the calculation method of Varmus et al. (1973) . Thus all five murine hepatitis virus strains produced high levels of virus-specific RNA when infecting L-2 cells. As with our previous work , the cDNA annealed to less than 8% with uninfected L-2 cell RNA or yeast RNA. previously shown for MHV-3 (Anderson et aL, 1979; , JHM (Anderson et aL, 1979) , and A59 these four polypeptides constitute three major size classes of approximately 180,000, 50,000-56,000, and 20,000-25,000 daltons. The striking similarity in electrophoretic mobilities among the five strains of each of the four major polypeptides is evident from Fig. 5 . Nevertheless, slight variations in apparent molecular weight are detectable. Thus, for example, the polypeptides corresponding in size to the nucleocapsid N protein described for A59 (Sturman et cd, 1980 ) have the following approximate molecular weights as deduced from Fig. 5 ; MHV-1, 56,000; MHV-3, 56,000; MHV-S, 56,000; JHM, 55,000, and A59, 52,000. Although it remains to be demonstrated that similarsized polypeptides for each MHV strain are functionally and structurally equivalent, peptide mapping data on the N proteins (in the following section) as well as on the remaining viral proteins (unpublished) strongly suggested that this is, in fact, the case. We have, therefore, adopted the nomenclature proposed for A59 by Sturman et al. (1980) to designate the large ca. 180,000-dalton protein as E2 and the small, ca. 24,000-dalton protein as El, for each of the five MHV strains. As previously shown for MHV-3 El is post-translationally derived from PEl. Moreover, preliminary experiments with the other MHV strains suggest that in all cases a precursor-product relationship exists between the corresponding two polypeptides.

It should be noted that several other labeled proteins are present in certain extracts, particularly those of the JHM ( 

These proteins are also present in uninfected cells and reflect differences among the MHV strains in abilities to inhibit host cell protein synthesis.

Peptide Mapping of MHV Nuckocap~ N Proteins

Analysis of the chymotrypsin-derived peptides from each of the MHV nucleo-capsid N proteins revealed strong overall interstrain similarity and suggested even stronger interrelationships between certain members (Fig. 6) . In all five cases, coeluting peptides were evident at fraction numbers 30,50,58,71, and 96. Two of these peptide peaks (fraction numbers 50 and 58) were of much lower relative intensity in both the MHV-1 and MHV-S peptide profiles than with those of the other MHV strains. The reason for this difference in peak intensity is not known. One explanation is that these two peptides from MHV-1 and MHV-S, while chemically similar to corresponding peptides of the other three MHV strains, may be nevertheless somewhat more deficient in methionine, and hence in [35S]-methionine label. Alternatively, steric factors may limit quantitative liberation of these two peptides from MHV-1 and MHV-S-derived N proteins. Either of these explanations may also be applicable to the relatively low intensity of the fraction 58 peptide (relative to that at fraction 50) in the case of the MHV-3 N protein. As can be seen in Fig.  6 the peptide elution profiles for the MHV-1 and MHV-S N proteins were virtually identical, suggesting very strong relatedness between these two viral strains. Among the other three viruses, MHV-3, JHM, and A59, a majority of the N-protein-derived peptides comigrated, namely those around fraction numbers 10, 18, 30, 50, 58, 71, and 96. Since the migrational characteristics of a peptide may be altered by a single amino acid substitution (Ingram, 1956; Hunt and Ingram, 1959) , we interpret the above data as indicating a high degree of interstrain homology among nucleocapsid N proteins of murine coronaviruses.

The results of the present study extend our knowledge of murine coronaviruses in the following areas.

1. Our previous finding that cells infected with the A59 strain of MHV contain six or seven viruscoded, subgenomic RNAs, has now been expanded to include four additional strains of MHV. As in the case of A59, these subgenomic RNA species contain sequences in common.

2. All five MHV strains tested show a remarkably similar polypeptide profile with only slight variations in apparent molecular weight. Three major size classes of viral polypeptides are evident in each case, which are analogous to those already described for MHV-A59 (Sturman et ok, 1980; Cheley et d, 1981) .

3. Strains MHV-1, MHV-3, MHV-S, and JHM have a high degree of nucleic acid homology (7080%) with the A59 strain as judged by hybridization with labeled cDNA prepared from A59 nucleocapsid protein-encoding mRNA. A further substantiation of strong homology among MHV strains within this coding region was obtained by comparative peptide mapping of each of the virus-coded nucelocapsid polypeptides.

The difference in the pattern of virus-specified subgenomic RNAs, between MHV-S and the other four MHV strains is intriguing.

Comigration is observed, among all five strains, for the two smallest RNAs (0.8 X lo6 and 1.1 X lo6 daltons); however all higher-molecular-weight RNA species of MHV-S fail to line up with the larger RNAs specified by the other four MHV strains. It is obvious from the present study as well as from our previous work that MHV-coded subgenomic RNAs contain common sequences. It has been demonstrated with the avian coronavirus, infectious bronchitis virus (Stern and Kennedy, 1980) , that virus-specific, subgenomic RNAs may form a nested set with sequence homology at the 3' ends. Assuming a similar situation exists among murine coronaviruses, any alteration of a single subgenomic RNA species would also be present in all larger subgenomic RNA species. Thus, the atypical subgenomic RNA pattern exhibited by MHV-S may reflect genetic alteration at the third smallest subgenomic RNA (i.e., the smallest RNA species which shows altered electrophoretic mobility from the corresponding RNA of the other four MHV strains). It is interesting to note, nevertheless, that any such alteration is likely to have occurred within the structural protein-encoding regions of MHV-S RNA, since all three major structural MHV-S proteins show very little deviation in apparent molecular weight from those of the other MHV strains (Fig. 5) .

Homology comparisons of different virus strains have been performed on several virus genera including orthomyxoviruses (Erickson and Kilbourne, 1980, Laver and Downie, 1976; Shild et cd, 1979; Scholtissek et d, 1976) , rhabdoviruses (Burge and Huang, 1979; Doe1 and Brown, 1978; Repik et aL, 1974) , and retroviruses (Albino et a& 1979; Barbacid et cd, 1977; Gautsch et c& 1978; Oroszlan et d, 1975) . From a survey of such studies, it is evident that genetic variation between related strains can involve changes at any sub-genomic site. There may, however, be a relatively greater degree of conservation within regions encoding internal virion polypeptides than within those coding for surface polypeptides. In rationalizing this observation it has been suggested (Doe1 and Brown, 1978; Laver and Downie, 1976) that sequence conservation among internal virion polypeptides may reflect stringent structural constraints imposed on such polypeptides by the precise nature of their interactions during virus assembly. Thus, in the case of MI-IV, in which the major internal protein is the nucleocapsid polypeptide (Struman et al, 1980) , one might expect that this polypeptide be fairly conserved among various strains of MHV. Indeed, the results of the present study demonstrate strong relatedness of all five MHV isolates in both the N polypeptide as well as its corresponding RNA coding sequence. Studies are presently in progress to determine homology relationships of the remaining two major virion structural polypeptides El and E2.

A logical extension of the present study is to attempt to precisely define the sites of genetic variability among the currently recognized strains of MHV. To date considerable differences in virulence and tissue tropism have been demonstrated. JHM, alone among the five MHV strains used in the present work, has been shown to produce a highly selective infection of the central nervous system (Bailey et al, 1979; Cheever et d, 1949; Weiner, 1973) . Of the other four MHV strains, which generally produce liver disease with occasional neurological involvement, strains MHV-1 (Dick et d, 1956; Gledhill and Andrewes, 1951) and MHV-S (Rowe et ak, 1963; Taguchi et d, 1980) appear to be of relatively low pathogenicity.

While disease outcome may be a function of host strain (Bang and Warwick, 1960) , host age (Biggers et al, 1964 , Dick et &, 1956 Weiner, 1973) , antiviral factors (Virelizier and Gresser, 1978) , and passage history of the virus (Rowe et al, 1963; Weiner, 1973) , the underlying differences in biological virulence of varying MHV strains have been shown to reflect primarily virus-specified determinants (Dick et d, 1956; Lucas et aL, 1977; Haspel et al, 1978) . The results of the present study suggest a high degree of relatedness among different MHV strains and thus provide hope that such determinants may eventually be identified.

Tryptic peptide analysis of gag gene proteins of endogenous mouse type C viruses

Comparison of polypeptides of two strains of murine hepatitis virus

A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin

Mouse macrophages as host for the mouse hepatitis virus and the genetic basis of their susceptibility

Evolutionary relationships between gag gene coded proteins of murine and primate endogenous type C RNA viruses

Cultivation of the virus of infectious bronchitis

Lethal intestinal virus infection of mice (LIVIM)

Pathogenic murine coronaviruses: II. Characterization of virus-specific proteins of murine coronaviruses JHMV and A59V

Conserved peptides in the proteins of vesicular stomatitis virus

A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin. I. Isolation and biologic properties of the virus

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Analytical peptide mapping by high performance liquid chromatography

Strain specific markers for the major structural proteins of highly oncogenic murine mammary tumor viruses by tryptic peptide analyses

A hepatitis virus of mice

A new virus isolated from the human respiratory tract

Temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination

New strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice

A terminal peptide sequence of human haemoglobin?

A specific chemical difference between the globins of normal human and sickle cell anaemia haemoglobin

RNA of mouse hepatitis virus

Influenza virus recombination. I. Matrix protein markers and segregation during mixed infections

Virus specific ribonucleic acid in cells producing Rous sarcoma virus: detection and characterization

In tiuo and in vitro models of demyelinating diseases. II. Persistence and host-regulated thermosensitivity in cells of neural derivation infected with mouse hepatitis and measles viruses

In vfvo and in vitro models of demyelinating diseases: Tropism of the JHM strain of murine hepatitis virus for cells of glial origin

A hepatitis virus complicating studies with mouse leukemia

Growth in suckling mouse brain of "IBVlike" viruses from patients with upper respiratory tract disease

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Coronavirus induced subacute demyelinating encephalomyelitis in rats: A morphological analysis

Acute hepatitis associated with mouse leukemia. I. Pathological features and transmission of the disease

Translation of mouse mammary tumor virus RNA: Precursor polypeptides are phosphorylated during processing

Amino acid sequence homology of mammalian type C RNA virus major internal proteins

Detection of homologous RNA sequences among six rhabdovirus genomes

The origin of altered cell lines from mouse, monkey and man as indicated by chromosome and transplantation studies

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Evidence for antigenic variation in influenaa A nucleoprotein

Correlation between RNA fragments of fowl plague virus and their corresponding gene functions. virdogy

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Coronavirus multiplication strategy. II. Mapping the avian infectious bronchitis virus intracellular RNA species to the genome

Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid

Enharmed growth of a murine coronavirus in transformed mouse cells

Resistance to highly virulent mouse hepatitis virus acquired by mice after low-virulence infection: Enhanced antiviral activity of macrophages

Transcription of mouse mammary tumor virus genes in tissues from high and low tumor incidence mouse strains

Role of interferon in the pathogenesis of viral diseases of mice as demonstrated by the use of anti-interferon serum. V. Protective role in mouse hepatitis virus type 3 infection of susceptible and resistant strains of mice

Genomic RNA of the murine coronavirus

Pathogenesis of demyelination induced by a mouse hepatitis virus (JHM virus)

Viral infections and demyelinating diseases

We thank Edwin C. M. Lee Chan for his technical assistance, and Roy Hartwick and Anna Mawrovic for assistance with the photography. We are grateful to T. Morishima for permitting us the use of his HPLC equipment. This work was supported by grants from Medical Research Council of Canada, Grant MA-5941 to R. Anderson and Grant MA-7321 to V. L. Morris.