key: cord-337976-c2auspti
authors: Weiss, Susan R.
title: Coronaviruses SD and SK share extensive nucleotide homology with murine coronavirus MHV-A59, more than that shared between human and murine coronaviruses
date: 1983-04-30
journal: Virology
DOI: 10.1016/s0042-6822(83)80022-1
sha: 
doc_id: 337976
cord_uid: c2auspti

A cDNA probe representing the genome of mouse hepatitis virus (MHV) strain A59 (MHV-A59) was used to measure nucleotide sequence homologies among murine and human coronaviruses and the SD and SK coronaviruses isolated by Burks et al. Since SD and SK were isolated by inoculation of multiple sclerosis (MS) central nervous system (CNS) tissue into mice or cultured mouse cells, it is important to determine their relationships to other murine and human coronavirus isolates. Our results indicate that SD and SK share almost complete nucleotide homology (approximately 90%) with MHV-A59 and generate subgenomic RNAs of the same sizes as MHV-A59. The human coronavirus (HCV) strains tested show less homology with MHV-A59. The immunologically unrelated HCV-229 E shows no nucleotide homology with MHV-A59. The immunologically crossreactive HCV-OC43 shows nucleotide homology with MHV-A59 by blot hybridization but not when hybridized in solution and assayed by S1 nuclease digestion.

Coronaviruses have been associated with acute and chronic neurological diseases in many species of animals (McIntosh, 1974) . Infection of rodents with the murine coronavirus, mouse hepatitis virus MHV strain JHM, has been used as a model system to study virus-induced demyelination (Weiner, 1973; Nagashima et al., 1978; Stohlman and Weiner, 1981) . After initial panencephalitis caused by MHV-JHM, this virus produces a persistent infection with primary demyelination with some evidence for remyelination (Weiner, 1973) . Thus persistent MHV-JHM infection of rodents has been cited as a model to study the human demyelinating disease multiple sclerosis (MS).

Human coronaviruses (HCV) are ubiquitious in nature with a large portion of the human population possessing neutralizing antibodies (McIntosh, 1974) . These viruses were isolated usually as respiratory, and occasionally as enteric viruses.

To whom reprint requests should be addressed.

They are estimated to be responsible for 15% of common colds (McIntosh, 1974) . There are no reports thus far of involvement of human coronaviruses with persistent neurological disease. Some strains of HCV such as 0C43, are antigenically related to murine coronaviruses such as MHV strain JHM (McIntosh, 1974; Gerdes et al., 1981a, b) and may be grown in the brains of suckling mice (McIntosh et al., 1967) . Others such as HCV-229E are unrelated antigenically to MHV or HCV-0C43 (McIntosh, 1974; Pederson et al., 1978) .

Because (1) murine coronaviruses are associated with chronic demyelinating disease in rodents (Weiner 1973; Nagashima et al., 1978) , (2) antibody against HCV is very common in the human population (McIntosh, 1974) , and (3) there is evidence suggesting that MS may be caused by a virus, various workers have undertaken comparisons of human and murine coronaviruses and have started to search for coronaviruses in central nervous system (CNS) tissue from MS patients. There is one report of particles with coronavirus-like morphology seen by electron microscopy in brain tissue from an MS patient (Tanaka et al., 1976) . More recently Burks and co-workers have isolated two coronaviruses, designated SD and SK, by intracerebral inoculation of unfrozen MS CNS autopsy tissue either into weanling mice or into 17CL-1 mouse cells in culture (Burks et al., 1980) . Gerdes et al. (1981a, b) and we (this manuscript) have examined the relationship between these viruses and other known murine and human coronaviruses. Gerdes et al. (1981a, b) showed that SD and SK are antigenically related to MHV-A59 and to HCV-OC43 but not HCV-229E. They were inconclusive about which strains their isolates were more related to. We have further compared murine and human coronaviruses and SD and SK by using molecular hybridization of virus-specific RNA with cDNA probes. Our results show extensive nucleotide homology between SD and SK and MHV-A59, more than that between the human viruses and MHV-A59.

Viruses and cells. MHV-A59 was grown in 17CL-1 cells as previously described (Weiss and Leibowitz, 1983) . SD and SK viruses (Burks et al., 1980) were obtained from Dr. J. Gerdes and were also grown in 17CL-1 cells. HCV-229E was obtained from the American Type Culture Collection (ATCC) and grown in human embryonic lung (L-132) cells also obtained from the ATCC. These viruses were plaque purifled two times and grown in Dulbecco's medium with 10% fetal calf serum (Robb and Bond, 1979) .

HCV-OC43 was obtained as a 20% suckling mouse brain suspension from Dr. J. Hierholzer at the Center for Disease Control (CDC), Atlanta. It was inoculated intracerebrally into C57BL/6 suckling mice, harvested 2 days later, and a 10% brain suspension was made in phosphate-buffered saline (PBS) containing 0.75% bovine serum albumen. The mothers of the suckling mice were obtained from Jackson Labs as MHV-free animals. All were negative for antibodies against MHV-A59 (as de-termined by an enzyme-linked immunosorbent assay) and HCV-OC43 (as shown by a lack of 0C43 hemagglutination inhibiting activity in the sera of these animals (Hierholzer et al., 1969) ) and thus were considered uninfected by these coronaviruses. Virus in brain homogenates was assayed by hemagglutination of chicken red blood cells at room temperature (Hierholzer et al., 1969) . Virus was further verified as HCV-OC43 since hemagglutination was inhibited by an anti-0C43 reference antisera obtained from CDC. HCV-OC43 was also grown in human rectal tumor (HRT) cells (Laporte et al., 1980) obtained from Dr. David Brian. Infected mouse brain homogenates were adsorbed onto monolayers of HRT cells for 1 hr at room temperature. The cells were extensively washed, medium added, and the cells incubated at 33 ~ Virus in the supernatant was titered at various times postinfection by hemagglutination (Hierholzer et al., 1969) . Mock-infected cells were adsorbed with a brain homogenate from uninfected suckling mice.

cDNA probes, cDNAs were synthesized using purified genome RNA as template, oligomers of calf thymus DNA as primers, and reverse transcriptase (Taylor et al., 1976) . cDNAs were labeled with [32P]dCTP to specific activity of approximately l0 s cpm/#g. When used for liquid hybridization, cDNA was synthesized in the presence of actinomycin D and was >95% single stranded. Such cDNAs were validated to be highly virus specific and to represent the majority of the genome RNA as previously described in detail Leibowitz, 1981, 1983) .

RNA extraction. MHV-A59, SD, SK, and HCV-229E virus infections were carried out with a multiplicity of infection of between 0.1 and 1 plaque-forming units per cell. RNA was extracted 18 hr after infection with A59, SD, and SK viruses, when massive syncytia were present. 229E-infected cells were labeled with [3H]uridine in the presence of 10 ttg/ml actinomycin D from 18 to 24 hr postinfection when RNA was extracted. RNA was extracted from OC43-infected HRT cells at 3 days post-infection. RNA was extracted from the cytoplasm of infected cells by SDS-proteinase K treatment followed by phenol extraction as previously described (Weiss and Leibowitz, 1983) . RNA was extracted from suckling mouse brain homogenates by SDS-proteinase K treatment followed by phenol extraction (Weiss, Varmus and Bishop, 1977) .

RNA analysis.

(1) Gel electrophoresis. RNA was electrophoresed in 1% agarose gels, in the presence of methyl mercury hydroxide (Bailey and Davidson, 1976) or formaldehyde (Lehrach et al., 1977) as denaturant. Gels were either fluorographed with sodium salicylate (Chamberlain, 1979) or blotted onto nitrocellulose (Thomas, 1980) . (2) Blots. Dot blots. RNA was adsorbed onto nitrocellulose filters in various amounts (as designated in figure legends), dried, and the filters were baked and hybridized with cDNA (Thomas, 1980) . Northern blots: RNA was electrophoresed in gels, blotted onto nitrocellulose filters, and hybridized with cDNA (Alwine et al., 1977; Thomas, 1980) . (3) Hybridization in solution was carried out at 68 ~ 0.6 MNaC1, and assayed by $1 nuclease digestion as previously described (Leong et aL, 1972) . In Crt curves, increasing amounts of RNA were hybridized with a fixed amount of cDNA to achieve increasing Crt values where Crt = concentration of RNA • time of hybridization.

HCV-OC43 has been difficult to grow and assay in cell culture and this has impaired the study of viral nucleic acids. This virus is usually grown in the brains of suckling mice and titered either by infection of suckling mice or by hemagglutination (Hierholzer et al., 1969) . Schmidt et al. (1979) have reported growing and plaquing HCV-OC43 on human rhabdomyosarcoma (RD) cells. Although we had difficulty with growing the virus in RD cells, we have had some success with growth in human rectal tumor (HRT) cells (Laporte et al., 1980) . We have used hemagglutination to detect and quantitate HCV-OC43 in both infected suckling mouse brain homogenates and HRT cell supernatants. As shown in Table  1 brain homogenates from infected mice contained 40,000 HAU/ml of 0C43 and homogenates from control mock-infected animals had none. This activity could be specifically inhibited by anti-OC43 reference antisera but not by A59 antisera or preimmune sera (data not shown).

Also shown in Table 1 after infection of HRT cells with OC43-infected mouse brain homogenates (800 hemagglutinating units/ 106 cells), hemagglutinating activity could be measured in the medium. As expected pretreatment of brain homogenates with antisera directed against OC43 prevented Hemagglutinating units/ml of OC43. One HAU is defined as the amount of virus present in 0.05 ml of the highest dilution of brain homogenate or supernatant capable of agglutinating 0.05 ml of 0.5% chicken erythrocytes in the standard assay described by Hierholzer et al. (1969) .

b Mock-infected mice or cells are mock-infected with a 10% homogenate or uninfected suckling mouse brains.

c OC43-infected suckling mouse brain homogenate was incubated with anti-OC43 reference antiserum for 1 hr at room temperature before infecting cells. 1981b), the reciprocal e x p e r i m e n t using HCV-229E cDNA was also c a r r i e d out. As i l l u s t r a t e d in Fig. 2 , c D N A r e p r e s e n t i n g the HCV-229E g e n o m e h y b r i d i z e d only to its homologous R N A a n d not to HCV-OC43 R N A or to MHV-A59 RNA.

The blot e x p e r i m e n t s i l l u s t r a t e d in the above sections show the h o m o l o g y between the nucleic acids of c o r o n a v i r u s s t r a i n s MHV-A59 and SD, SK, a n d HCV-0C43 a n d the lack of homology w i t h HCV-229E. These techniques, however, do not q u a n t i t a t e the p e r c e n t a g e of the g e n o m e sequences t h a t are homologous. To be more q u a n t i t a RNA from infected or mockinfected cells or mouse brain homogenates was spotted onto nitrocellulose filters. In the case of intracellular RNAs, 5.0, 0.5, and 0.05 ttg amounts were used. In the case of purified genome RNA, 0.1, 0.01, and 0.001 #g were used. Filters were hybridized to 106 cpm (108 cpm/#g) of A59 [sUP]cDNA and autoradiographed (Alwine et aL, 1977; Thomas, 1980) . Similar hybridizations were carried out with RNAs from cells infected with the other viruses and the plateau values for the percentage cDNA hybridized are summarized in Table 2 . SD and SK are almost completely homologous to MHV-A59, more so t h a n another MHV strain, JHM. HCV-229E showed no homology with A59 cDNA as predicted from the blot experiments. HCV-OC43 showed no homology with the cDNA by this assay. This is probably due to stringency of hybridization and S1 nuclease assay for hybridization (see Discussion). 

To f u r t h e r compare the RNA of the murine and h u m a n strains the intracellular subgenomic RNAs were examined by gel electrophoresis. As illustrated in Fig. 4 , cells infected with MHV-A59 contain six m a j o r subgenomic RNAs as well as genome-sized RNA (band 1) (Cheley et al., 1981a; Leibowitz et aL, 1981; Spaan et al., 1981) . These range in molecular weight from 0.63 • 106 daltons for RNA 7 to 6.1 • 106 daltons for R N A 1. Intraeellular RNAs extracted from eells infected with SD or SK, were eleetrophoresed in parallel with MHV-A59 RNA, blotted onto nitrocellulose and the virusspecific species detected by hybridization with MHV-A59 eDNA. Seven RNA bands were observed, all comigrating with the m a j o r MHV-A59 RNAs. RNAs 2, 4, 5, and, in the ease of SK, RNA 3, are less abundant than with MHV-A59 and the extra band between RNAs 2 and 3 is more prominent. The extra bands between RNAs 3 and 4 are also more p r o m i n e n t in the SD RNA.

The intraeellular RNAs of HCV-229E were also compared to those of MHV. (In this experiment MHV-3 RNA was used instead of MHV-A59. The genome of MHV-3 is 95% homologous to MHV-A59 and MHV-3 generates the same size intraeellular RNAs as MHV-A59 (Cheley et al., 1981b, Weiss and ). Since HCV-229E RNA does not cross-hybridize FIG. 2, Dot blot hybridizations of HCV-229E eDNA with coronavirus RNAs. RNA from infected or mockinfected cells or brain homogenates or from purified virions was spotted onto a nitrocellulose filter. In the case of intracellular RNA, 5.0, 0.5, and 0.05 ~g were used and in the case of purified genome RNA 0.1, 0.01, and 0.001 ug were used. The filter was hybridized with 106 cpm (10 s cpm/#g) of 229E [32P]cDNA and autoradiographed (Alwine et aL, 1977; Thomas, 1980) . with A59 eDNA, 229E intracellular RNA was labeled with [3H]uridine in the presence of actinomycin D (to inhibit host DNA-dependent R N A synthesis). As shown in Fig. 5 , 229E also generates six subgenomic RNAs, but they are of different sizes from MHV-3 and hence from SD and SK. (Genome RNA was difficult to observe in this experiment probably due to some RNA degradation).

M a m m a l i a n coronaviruses have been divided into two antigenic groups (Pederson et aL, 1978) . One includes MHV, HCV-OC43, a RNA from virions, infected cells, or brain homogenates was hybridized with 2000 cpm (10 s cpm/ t~g) of A59 [aZP]cDNA to completion, the plateau portion of a Crt curve. Hybridization was assayed by $1 nuclease digestion. These values have been normalized to 100% hybridization of A59 cDNA with its homologous A59 RNA. The actual values of hybridization of A59 cDNA with A59 RNA ranged from 85 to 100%. gastroenteritis virus of swine, and canine coronavirus. Gerdes et al. (1981a, b) have shown that the SD and SK viruses fall into the first group. They showed that all A59 intracellular proteins are immunoprecipitable with antisera directed against SD, SK, or HCV-OC43. From these experiments, however, it was impossible to determine whether SD and SK were more related to human (0C43) or murine (A59) viruses. This is important in determining the origin of SD and SK and the possible link to MS.

We have used nucleic acid hybridization with cDNA to further explore the relationship among these viruses. Our cDNAs are representative of most if not all of the genome RNA sequences Leibowitz, 1981, 1983) and thus are appropriate reagents for quantitating sequence homologies. The relationship between SD and SK and MHV-A59 as determined by molecular hybridization experiments basically agrees with immunological studies. The SD and SK viruses show extensive homology in nucleotide sequence (approximately 90%) to the A59 strain of MHV even when assayed by the stringent $1 nu-clease assay. This is more homology than that shared between MHV-A59 and another MHV strain, JHM Leibowitz, 1981, 1983) . Furthermore, the pattern of intracellular RNAs generated by SK and SD is very similar to that of MHV-A59. This is not surprising, considering that Gerdes et al., (1981a, b) showed that cells infected by SD or SK have patterns of viral proteins similar to A59-infected cells (Gerdes et al., 1981a, b) . An extra polypeptide of 42,000 daltons molecular weight was observed in , or SK (lane c) were electrophoresed in a 1% agarose gel containing formaldehyde as a denaturant (Lehrach et aL, 1977) . After electrophoresis the gel was stained with ethidium bromide to locate ribosomal RNAs, blotted onto nitrocellulose (Thomas, 1980) , hybridized with l0 s cpm of A59 [32p]cDNA, and autoradiographed (Alwine et al., 1977) . Arrows designate the position of 18 S and 28 S ribosomal RNAs. Viral RNAs are numbered according to (Bailey and Davidson, 1976) . After electrophoresis the gel was stained with ethidium bromide to locate ribosomal RNAs and fluorographed (Chamberlain et aL, 1979) . The positions of 18 S and 28 S ribosomal RNAs are designated by arrows.

There is no extra major RNA band seen in the SD and SK samples. The molar ratios of some of the RNAs are different in SD-and SK-infected cells, specifically, RNAs 2 and 5 are less prominent and the extra RNA band between 2 and 3 is quite prominent. The extra bands between RNAs 3 and 4 are also more prominent in the SD sample. However, there is similar variation among the MHV strains (Cheley et al., 1981b; Leibowitz et al., 1981; Weiss and Leibowitz, 1981 ) and we do not understand its significance. Perhaps this is due to a small amofint of degradation in the extraction of these very large RNAs.

Comparison of the human coronaviruses OC43 and 229E with MHV-A59 reveals less homology than between MHV-A59 and SK and SD. RNA extracted either from brain homogenates of OC43-infected suckling mice or from HRT cells infected with OC43 shows homology with A59 cDNA when assayed by blot hybridization. This homology is not detected when liquid hybridization followed by the more stringent S1 nuclease assay is used. There is precedence for this in at least two other systems. Homology between murine and human papovaviruses (Howley et al., 1979) and murine and human rotaviruses (Schroeder et aL, 1982) is detected only under relaxed hybridization and assay conditions. These viruses are antigenically related; this immunological cross-reactivity is detected without difficulty as is the immunological cross-reactivity between MHV-A59 and HCV-OC43.

There is no homology detected between HCV-229E and MHV-A59 or HCV-OC43 in cross-hybridizations using dot blots and cDNA representing either virus. It is unlikely that there is homology betwen HCV-229E and SD and SK since the latter viruses are so closely related to MHV-A59. This is not surprising considering these viruses fall into different antigenic groups (Pederson et al., 1978) . However, there are reports of immunological cross-reactivity between the nucleocapsid proteins of these viruses (Gerdes et al., 1981b) . These sequences if related, are probably too diverged to be detected by our assay. Perhaps under less stringent blotting conditions, homology could be detected.

These results show that SD and SK are more closely related to MHV-A59 than are the human viruses 0C43 and 229E. Because SD and SK are almost completely homologous (90%) to MHV-A59 it is unlikely that they share much homology with either HCV-OC43 or HCV-229E. It is possible that the 10% of SD and SK sequences that are not homologous to MHV-A59 are homologous to either HCu or HCV-229E. However, if that were the case, it would still suggest that these isolates are more related to murine than human coronaviruses. This, in addition to the fact that SD and SK grow only in murine and not human cells (Gerdes et al., 1981b) , suggests that these viruses are not of human, but murine origin.

Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl paper and hybridization with DNA probes

Methylmercury as a reversible denaturing agent for agarose-gel electrophoresis

Two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients

Fluorographic detection of radioactivity in polyacrylamide gels with the water soluble fluor, sodium salicylate

Intracellular murine hepatitis virus-specific RNAs contain common sequences

RNA and polypeptide homology among murine coronaviruses

Antigenic relationships of coronaviruses detectable by plaque neutralization, competitive enzyme linked immunosorbent assay, and immunoprecipitation

Coronavirus isolates SK and SD from multiple sclerosis patients are serologically related to murine coronaviruses A59 and JHM and human coronavirus OC43 but not to human coronavirus 229E

Standardized viral hemagglutination and hemagglutination-inhibition tests TI. Description and statistical evaluation

A rapid method for detecting and mapping homology between heterologous DNAs

Some characteristics of hemagglutination of certain strains of "IBV-like" virus

Mouse hepatitis virus A59: mRNA structure and genetic localization of the sequence diversion from hepatropic strain MHV-3

Comparative analysis of RNA genomes of mouse hepatitis virus

Une lign6e cellulaire particuli~rement sensible a la r6p-lication du coronavirus ent6ritique: Les cellules HRT 18

RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination

The virus-specific intracellular RNA species of two murine coronaviruses: MHV-A59 and MHV-JHM

Virus-specific ribenucleic acid in cells producing Rous sarcoma virus: Detection and characterization

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

Coronaviruses: A comparative review

Coronavirus induced subacute demyelinating encephalomyelitis in rats: A morphological analysis

Antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species

Pathogenic murine coronaviruses. L Characterization of biological behavior in vitro and virus specific intracellular RNA of strongly neurotropic JHMV and weakly neurotropic A59V viruses

Plaque assay and improved yield of human coronaviruses in a human rhabdomyosarcoma cell line

Sequence relationship between the genome segments of human and animal rotavirus strains

Isolation and identification of virus-specific mRNAs in cells infected with mouse hepatitis virus (MHV-A59)

Chronic central nervous system demyelination in mice after JHM virus infection

Intracisternal virus-like particles in the brain of a multiple sclerosis patient

Efficient transcription of RNA into DNA by avian sarcoma virus polymerase

Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Prec

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

Comparison of the RNAs of murine and human coronaviruses

Characterization of murine coronavirus RNA by hybridization with virus-specific cDNA probes

The size and genetic composition of virus-specific RNAs in the cytoplasm of cells producing avian sarcoma-leukosis viruses

This research was supported by NIH Grant A117418 and Grant RG 1421 from the Multiple Sclerosis Society. I thank Maureen Highkin and David Petcu for help with some of the experiments and Roseann Femia for typing the manuscript.