key: cord-0888071-vpc4xtwl
authors: Lapps, W.; Brian, D. A.
title: Oligonucleotide fingerprints of antigenically related bovine coronavirus and human coronavirus OC43
date: 1985
journal: Arch Virol
DOI: 10.1007/bf01314116
sha: 91cd809ecc303512670cbb5d55d47576e78481d9
doc_id: 888071
cord_uid: vpc4xtwl

Virion RNAs from the bovine enteric coronavirus and the human respiratory coronavirus OC43 were compared by one dimensional gel electrophoresis and by oligonucleotide fingerprinting. For each virus, approximately 55 per cent of the RNA migrated as a 6.8 Md species, 10 per cent as a 0.68 Md species, and 15 per cent as heterogeneous small molecular weight RNA. A sequence homology of greater than 96 per cent was observed between the 6.8 Md species from the two viruses. The 0.68 Md RNA is apparently an intravirion, subgenomic, polyadenylated molecule based on RNAse studies, oligo (dT)-cellulose chromatography, and hybridization to a cDNA clone of the 3′ terminal 1.19 Kb region of the bovine coronavirus genome.

The bovine enteric coronavirus (BCV) causes a severe enteritis in young calves (18) . The human respiratory coronavirus 0C43 (HCV 0C43) causes only a mild upper respiratory disease in humans of all ages (16) . Recent evidence suggests that these viruses are structurally closely related. Both agglutinate erythrocytes from mice, rats, and chickens (10, 15, 19) , and both share antigens as determined by immunofluorescence (20) . No biological or structural data exists to rigorously differentiate between these viruses. Because a significant fraction of the human population (7 to 69 per cent) both in Europe and the United States carry antibodies that are able to neutralize, immunoprecipitate, and indirectly immunofluoresce the bovine coronavirus (22) , the possibility exists that tile bovine corona~drus and the human coronavirus 0C43 are two names for the same virus with zoonotic potential.

In this report we describe studies designed to compare genome sequences between the two viruses. The bovine coronavirus (Mebus strain) and the human respiratory eoronavirus 0C43, when grown on the same human cell line, share a sequence homology of greater than 96 per cent as determined by TI oligonucleotide fingerprinting but each possess unique oligonueleotides and are thus distinctly different viruses. Both viruses incorporated a significant amount of subgenomic 0.68 Md I~NA late in the replication cycle and this may have implications for viral I~NA sequence analyses.

T h e h u m a n rectal a d e n o e a r c i n o m a cell line I-II~T-18 (23) was o b t a i n e d from J. Laporte, France, a n d was g r o w n as m o n o l a y e r s in Dulbeeco Modified Eagle M e d i u m c o n t a i n i n g 50 ~zg g e n t a m i c i n per ml a n d 5 per cent fetal calf serum (Sterile Systems, Logan, U t a h ) h e a t i n a c t i v a t e d a t 56 ° C for 30 minutes. T h e Mebus s t r a i n of B C V (1 l, 19) which h a d u n d e r g o n e a p p r o x i m a t e l y 60 passages in tissue culture was cloned b y four successive isolations from single plaques. T h e h u m a n r e s p i r a t o r y coronavirus OC43 w h i c h h a d u n d e r g o n e seven passages in h u m a n e m b r y o n i c t r a c h e a l o r g a n culture a n d 15 passages in suckling mouse b r a i n (16) was o b t a i n e d from S. Weiss, U n i v e r s i t y of P e n n s y l v a n i a , Philadelphia, P e n n s y l v a n i a , a n d was cloned b y two successive isolations from single plaques. F o r each virus, a clone was passaged twice a t a m u l t i p l i c i t y of < 0.1 P F U per cell, a n d t h e n viral stocks were p r e p a r e d from passages 3 t h r o u g h 6 b y infecting cells a t a m u l t i p l i c i t y of a p p r o x i m a t e l y 0.5 P F U per cell. Viral titers r a n g i n g from 107 P F U p e r ml for OC43 to l0 s P F U per ml for B C V were o b t a i n e d in stock virus p r e p a r a t i o n s .

Confluent m o n o l a y e r s of cells grown in 150 cm 2 flasks were infected w i t h a m u l t i p l i c i t y of 1--5 P F U p e r cell, i~nsed, a n d fed w i t h 20 mls p e r flask of m e d i u m c o n t a i n i n g 10 per cent n o r m a l p h o s p h a t e c o n c e n t r a t i o n , 20 per cent fetal calf s e r u m a n d 50 lxCi of 32Pi per ml. I n f e c t e d cells were i n c u b a t e d 7 2 --9 6 hours a t 37 ° C a n d virus was purified from s u p e r n a t a n t fluids as described previously (2) 

t/,NA was e x t r a c t e d from purified virus using SDS, proteinase K a n d phenol as described previously (2) .

F o r eleetrophoretie analysis virion R N A was d e n a t u r e d in gIyoxa] and d i m e t h y lsulfoxide, a n d electrophoresed on 1 per cent. agarose gels in a vertical slab a p p a r a t u s of 10 e m × 14 cm × 3 m m dimensions using t h e m e t h o d of McMASTER a n d CARMICIIAEL (i7). Gels were d e h y d r a t e d in two successive 30 m i n u t e s b a t h s of 100 p e r cent m e t h a n o l , compressed b y b l o t t i n g to a thickness of < 1 ram, a n d exposed to X -O m a t fihn. 

Experiments in which BCV RNA was labeled with [SH]-uridine for the first 48 hours postinfection and eleetrophoretieally analyzed on formaldehyde-agarose gels revealed only a high molecular weight species that comigrated with porcine transmissible gastroenteritis virus genomic I~NA (2, and d a t a n o t shown). BCV genomie R N A therefore has a molecular weight of a p p r o x i m a t e l y 6.8× 10 s. When virion I~NA from b o t h BCV and 0C43 was labeled in vivo with s2P-orthophosphate for 72 to 96 hours postinfection and a n a l y z e d on denaturing glyoxaLagarose gels, approximately 55 per cent from each virus migrated as a 6.8× 106 M.W. species (Fig. 1) . Approximately 10 per cent migrated as a distinct species with an electrophoretic mobility close to t h a t of 18S ribosomal t~NA and therefore had an a p p a r e n t molecular weight of 0.68 × i06 (Fig. 1 

To measure the degree of relatedness between BCV and HCV 0C43, high molecular weight genomic RNA was isolated for each virus by rate zonal sedimentation in sucrose gradients, fingerprinted separately, and then as a mixture using an equal number of counts (Fig. 2) . Inspection revealed a high degree of similarity between the two viruses consistent with earlier reports of close antigenic relatedness (6, 20) and our own recent work (9) . Of the 27 large resolvable oligonucleotides for BCV, 9 were unique to BCV, and of the 26 large resolvable oligonucleotides for HCV OC43, 8 were unique to I~CV OC4~3. 18 oligonucleotides were in common and represent a comigration of 66--69 per cent of the large oligonucleotides. Assuming that sequence homology demonstrated in the large oligonucleotides is representative of the remaining genome, we conclude there is a common nueleotide sequence of greater than 96 per cent between the two virus genomes based on computer simulation of T1 oligonucleotide mapping (1).

Incubating virus with pancreatic ribonuclease prior to purification, under conditions that completely destroy the integrity of ribosomal t~NA, failed to destroy the 0.68 Md R N A (Fig. 1) . From this we conclude the species is intravirion. The 0.68 Md RNA from BCV annealed to oligo (dT) cellulose indicating that it is polyadenylated (Fig. 1) and annealed toa2P-labeled cloned eDNA representing the 3' terminal 1.19 Kb sequence of BCV genomic I~NA (data not shown) indicating that this I~NA is either an encapsidated subgenomic virus m R N A or possibly a defective genome of the type described for defective interfering particles of other positive-strand viruses.

Our studies demonstrate that BCV (Mebus strain) and HCV OC43 can both be grown on the human rectal adenocarcinoma cell line (HI~T-18). Other strains of BCV, including an isolate from France (13), the LY-138 strain (8) and a primary isolate from the University of Tennessee, College of Veterinary Medicine (our unpublished observations), and HCV OC43-1ike viruses (14) can also grow on these cells without adaptation. Because the HRT-18 cells retain many properties of differentiated tissue cells (23) , these observations are consistent with the notion that there is no rigid species restriction on the growth of these coronaviruses and that they may have zoonotic potential. BCV reportedly caused diarrhea in one investigator and the virus was reisolated from this person (22) .

Our studies further establish that BCV (Mebus strain) and HCV OC43, although similar, are not identical viruses. The cytopathic effects caused by BCV and HCV 0C43 are not identical (data not shown). BCV causes a foamy appearance of the cell cytoplasm and a clumping of infected cells and HCV 0C43 causes a disintegration of the cell into small pieces. Both cause small (i--2 ram) opaque plaques under agar and both can be detected by hemadsorption following agar removal. No difference in size can be detected between the genomes of BCV and ttCV OC43 b y one-dimensional gel electrophoresis of glycosylated RNA. The genome for both viruses therefore measures approximately 6.8 × t06 M.W. since the genome of BCV comigrates with the 6.8× 106 M.W. genome of the porcine transmissible gastroenteritis virus in agarose gels after being denatured with formaldehyde (2 and data not. shown).

The comparative analysis of oligonucleotide fingerprints between the BCV and HCV 0C 43 genome RNAs suggests they have a sequence divergence of 3--4 per cent. The exact degree of divergence cannot be determined using this technique since the large oligonucleotides probably represent less than 10 per cent of the entire genome, but clearly these are not identical viruses. A sequence divergence of 3--4 per cent is consistent with a distinct difference in the behavior of 3 of the 4 homologous proteins we observe between the two viruses (9) .

In preparations of BCV and HCV OC43 labeled with 32p orthophosphate for 72--96 hours we found a 0.68 × 106 M.W. species representing l0 per cent of total virion RNA. For BCV we have shown this to be intravirion, polyadenylated, and subgenomic. Its function is unknown. It was not detected in earlier studies on BCV that, employed 3H-uridine for labeling periods of less than 48 hours (7) . Since our studies here use labeling periods of greater than 48 hours it is possible that the 0.68 × 106 M.W. species becomes incorporated only late in infection and may represent adventitious encapsidation of a viral subgenomic messenger ]~NA of the type described for several coronaviruses (4, 12, 21) . Further studies are needed to confirm this interpretat, ion.

This work was supported by Public H e a l t h Service grant 1~01-AI-14367 from the National Institutes of Health. W. L. is a predoctorM trainee on grant T 32-AI-07123 from the National Institutes of tieal~h.

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