Taxonomy

1983 S. Karger A G. Basel

Intervirology 20: 181-189 (1983) 0300-5526/83/0204-0181 $2.75/0

Coronaviridae1

S. G. Siddell, R. Anderson, D. Cavanagh, K. Fujiwara, H. D. Klenk, M. R. Macnaughton,

M. Pensaert, S.A. Stohlman, L. Sturman, B.A.M. van derZeijst

Key Words. Coronavirus • Viral taxonomy

Summary. The family Coronaviridae comprises a monogeneric group of 11 viruses which

infect vertebrates. The main characteristics of the member viruses are: (i) Morphological: Envel­
oped pleomorphic particles typically 100 nm in diameter (range 60-220 nm), bearing about 20 nm
long club-shaped surface projections, (ii) Structural: A single-stranded infectious molecule of
genomic RNA of about (5-7) x 10° molecular weight. A phosphorylated nucleocapsid protein
[mol.wt. (50-60) x 103] complexed with the genome as a helical ribonucleoprotein; a surface
(peplomer) protein, associated with one or two glycosylated polypeptides [mol.wt. (90-180)
x 103] ; a transmembrane (matrix) protein, associated with one polypeptide which may be glyco­
sylated to different degrees [mol.wt. (20-35) x 103]. (iii) Replicative: Production in infected cells
of multiple 3' coterminal subgenomic mRNAs extending for different lengths in the 5' direction.
Virions bud intracytoplasmically. (iv) Antigenic: 3 major antigens, each corresponding to one
class of virion protein, (v) Biological: Predominantly restricted to infection of natural vertebrate
hosts by horizontal transmission via the fecal/oral route. Responsible mainly for respiratory and
gastrointestinal disorders.

1 Third Report of the Coronavirus Study Group, Since the second report of the Coronavirus
Vertebrate Virus Subcommittee, International Com­ Study Group in 1978 [1], considerable data,
mittee on Taxonomy of Viruses (ICTV).
especially on the structure and replication of
coronaviruses, have been published, and we
Address inquiries to: Dr. S. Siddell, Institute of Vi­
rology, University of Wiirzburg, Versbacher Strasse 7, feel a new report is justified. The Corona­
D-8700 Würzburg (FRG) viridae are a monogeneric family of pleo­
morphic, ether-labile, enveloped viruses. The
virions have a diameter ranging from 60 to
Received: March 21, 1983 220 nm and an average density in sucrose of
182 Siddell/Amlerson/Cavanagh/Fujiwara/Ktenk/Macnaughton/Pensaert/Stohlman/Sturman/van der Zeijst

1.18 g/nil. They characteristically bear club- components of the enzyme have not been iden­
shaped surface projections about 20 nm in tified. Characteristic of coronavirus infection
length from which the group derives its name is the production of 3' coterminal subgenomic
(Latin corona, crown) [1], The genomic RNA RNAs which form a nested set extending in a
is an infectious single-stranded molecule which 5' direction. These RNAs are capped and poly­
is capped and polyadenylated. The molecular adenylated. The replicative structures from
weight is between 5x 106 and 7* 10°, cor­ which they are produced have not been char­
responding to about 15,000-20,000 nucleo­ acterized, but it has been demonstrated that
tides. There is no extensive sequence reiter­ the negative-stranded template from which
ation in the coronavirus genome. Corona- murine hepatitis virus mRNAs are copied is
virions characteristically have three types of of genome length. UV inactivation studies in­
protein: a phosphorylated nucleocapsid pro­ dicate that coronavirus mRNAs are not pro­
tein [mol.wt. (50-60) x 103], complexed with duced by the processing of a larger RNA,
the genome as a helical ribonucleoprotein although extensive sequence homologies have
(RNP); an jV-glycosylated surface peplomer been detected at the 5' ends of ail murine
protein, associated with glycopolypeptides of hepatitis virus-specific subgenomic RNAs. For
(90-180) x 103 molecular weight, which isacyl- murine hepatitis virus, the mRNA function of
ated and is responsible for virus attachment each of the subgenomic viral RNAs has been
and cell-to-cell fusion (this protein may be demonstrated in vitro, and the mRNAs encod­
removed by protease treatment); and a trans­ ing each of the virion proteins, or its precur­
membrane matrix protein, associated with sors, have been identified (fig. 1). Comparing
polypeptides of molecular weight (20-35) x 103 the size of each mRNA with its translation
which have variable degrees of glycosylation. product suggests that the expressed informa­
In the case of murine and bovine corona- tion lies within the 5' sequences of each RNA
viruses this polypeptide bears O-glycosidically which are not found in the next smallest RNA.
linked oligosaccharides, and in the case of For murine hepatitis virus, glycosylation of
avian infectious bronchitis virus it bears N- the peplomer protein is initiated cotranslation-
glycosidically linked oligosaccharides. ally in the rough endoplasmic reticulum,
Most coronaviruses replicate in tissue cul­ whereas the transmembrane protein is glyco­
ture within 12 h at 37°. Infection is often sylated posttranslationally in the Golgi ap­
accompanied by cytopathic changes. There paratus. The infectious bronchitis virus matrix
are conflicting reports as to whether a nuclear protein is glycosylated on the nascent poly­
function is required for coronavirus replica­ peptide. After synthesis, genomic RNA and
tion. There are few data about the early events virion proteins are assembled at the rough
(adsorption, penetration, uncoating, etc.) in­ endoplasmic reticulum and virions bud into
volved in coronavirus replication. It is as­ cisternae, acquiring their lipid membranes
sumed that upon entering the cell the positive- from the cell. The virions are subsequently
stranded genome encodes protein(s) whose transported to and accumulate in Golgi and
function is to replicate the genomic RNA and smooth-walled vesicles. There is an absence of
produce subgenomic mRNA. Recently, there budding from the plasmaiemma. The mecha­
have been reports of virus-specific RNA poly­ nism of virus release has not been character­
merases in coronavirus-infected cells, but the ized.
Coronaviridac 183

RNA P R O T E IN

No. S iz e S iz e D e s ig n a tio n
( x 1 0 ‘ 6) ( x 1 0 " 3)
5' A 3’
1 6.0 200
B
2 3.7 30
3 C
2.9 120 P e p lo m e r
D
4 1.4 1— 1------------ *
1 4 -1 7
5 1.2 H -----------*
F
6 0.9 F -1------- “ 2 3 -2 5 M a tr ix
G
7 0.6 1--------“ 5 5 -60 N u c le o c a p s id

18 16 14 12 10 8 6 4 2 0
1-------- !--------1----

F ig.l. Structure and expression of the MHV ge­ virion nucleocapsid, matrix and peplomer proteins,
nome. The sizeand structural relationships of the MHV respectively. As the size of the translation product for
intracellular mRNAs are shown. No difference be­ each mRNA corresponds approximately to the coding
tween the genome RNA and mRNA 1 has yet been de­ potential of the 5' sequences which are absent from the
scribed. Each mRNA encodes only one protein, and next smallest mRNA, it seems likely that only these
the translation products of mRNAs 7, 6 and 3 have regions are translated into protein.
been identified as the intracellular precursors to the

The relationships between some corona- virus protein. Immunological studies with
viruses have been studied by molecular and monoclonal antibodies, antisera directed
immunological methods, but the data are frag­ against subcomponents prepared from puri­
mentary. Molecular hybridization indicates fied virions, and immune electron microscopy
extensive sequence homology (about 70%) indicate that the antigenic sites responsible for
between murine hepatitis virus strains, in par­ the induction of neutralizing antibodies are
ticular within the gene encoding the nucleo­ associated with the surface peplomer polypep-
capsid protein. This conclusion is supported tide(s). Studies on the antigenic relationships
by oligonucleotide fingerprinting of genomic of coronaviruses present a complex pattern.
RNAs and chymotryptic peptide fingerprint­ Relationships have been determined by a wide
ing of nucleocapsid proteins. Oligonucleotide variety of tests, mainly using polyvalent sera
fingerprinting of the genomic RNA of a num­ from naturally infected or hyperimmunized
ber of infectious bronchitis virus strains in­ animals. These immunological studies indicate
dicates greater sequence divergence both be­ that there are two antigenic groups of mam­
tween and within serotypes of this species. malian coronaviruses and two antigenic groups
Coronaviruses contain 3 major antigens which of avian coronaviruses. One recent porcine
can be distinguished by antibodies against isolate does not appear to fall into either mam­
virion subcomponents. Each antigen corre­ malian group. Many viruses remain to be clas­
sponds to one of the three types of corona- sified.
184 Siddell/Anderson/Cavanagh/Fujiwara/Klenk/Macnaughton/Pensaert/Stohlman/Sturman/van der Zeijst

The geographic distribution of many coro- glutinating encephalomyelitis virus and some
naviruses is known to extend over several con­ murine hepatitis virus strains are associated
tinents and is probably worldwide. A seasonal with encephalomyelitis. Diagnosis of corona­
incidence of infection occurs with some viruses, virus infection is initially clinical, and confir­
namely human coronavirus and transmissible mation is most readily achieved by virus isola­
gastroenteritis virus. Coronaviruses predomi­ tion and propagation in vitro and/or by a
nantly infect their natural vertebrate hosts. variety of immunological procedures [2],
Biological vectors of coronaviruses have not At present the family Coronaviridae is re­
been reported, and the natural hosts form the cognized as 11 species, which are listed below.
major reservoirs for further infection. In most A number of recent isolates meet the morpho­
cases, infection is by the fecal/oral route. Ver­ logical and, to some extent, the molecular cri­
tical intrauterine infection has been reported teria for inclusion in the group, but are as yet
for infectious bronchitis virus and some murine insufficiently characterized to be regarded as
hepatitis virus strains. Transmission from con­ ‘possible’ family members. The question of
taminated clothing and equipment is an im­ speciation and whether the family should
portant source of infection with infectious remain monogeneric will be considered by
bronchitis virus and bovine coronavirus. Co­ the Study Group in the near future. This
ronaviruses are associated with diseases of report does not contain references to primary
economic and clinical importance, predomi­ sources. Extensive bibliographies can be found
nantly respiratory and gastrointestinal dis­ in several recent reviews of coronavirus biol­
orders. Feline infectious peritonitis virus is ogy [3-6]. The acronyms used throughout this
responsible for peritonitis in cats, and hemag- report are defined in section 10.3.
Coronaviridae 185

1 Taxonomy Surface (peplomer) protein: One or

1.1 Family: Coronaviridae two glycosylated polypeptides [mol.
1.1.1 Genus: Coronavirus. wt. (90-180) x IO3]. Location shown
1.1.2 Type species: Avian infectious bron­ for IBV, MHV, HCV, TGEV, BCV,
chitis virus. and HEV. Comparable protein re­
1.2 Taxonomic status: Family with one ported for CCV. Primary sequence
genus. relationship shown for IBV and
2 The virion MHV polypeptides. Acylation shown
2.1 Chemical composition for MHV and BCV polypeptide.
2.1.1 Nucleic acid Transmembrane (matrix) protein:
2.1.1.1 RNA One polypeptide, which may be gly­
2.1.1.2 Single-stranded cosylated to different degrees [(20—
2.1.1.3 Linear 35) x 103 mol.wt.], reported for IBV,
2.1.1.4 Unsegmented MHV, HCV, TGEV, CCV, BCV,
2.1.1.5 Sedimentation coefficient : 50-70S. and HEV. Location shown for IBV,
2.1.1.6 Molecular weight: MHV, HCV, and HEV. Glycosy­
1BV: (5.8-6.9) x 106 lated and nonglycosylated forms in­
MHV : (5.4-6.0) x 10« corporated in MHV and IBV.
HCV : (5.8-6.5)x 106 Others: Glycosylated and nonglyco­
TGEV : 6.8 x 10« sylated envelope proteins reported
BCV : 6.8X106 sporadically for many coronaviruses.
2.1.1.10 Homology studies: High sequence Relationships to other coronavirion
homology among genomes of MHV polypeptides not established.
(molecular hybridization, oligonu­ 2.1.2.5 Enzymes: Protein kinase associated
cleotide fingerprinting). Greater di­ with MHV.
vergence among 1BV genomes (oligo­ 2.1.2.6 Other functional proteins: Hemag­
nucleotide fingerprinting). glutinin found in IBV, HCV-OC43,
2.1.1.11 1nfectivity : Demonstrated for MHV, HEV, BCV, and MHV. Cell fusion
IBV, TGEV. activity associated with MHV pep­
2.1.1.12 Other features: IBV, MHV, TGEV, lomer protein.
BCV and HCV genomic RNA is 2.1.3 Lipids: TGEV contains phospholi­
polyadenylated. MHV genomic pids and glycolipids resembling those
RNA is capped. of the host cell.
2.1.2 Proteins 2.1.3.2 Other features: Fatty acids are cova­
2.1.2.2 Number of polypeptides : lently attached to MHV and BCV
Nucleocapsid protein [mol.wt. (50- peplomer proteins.
6 0 )x l0 3]: Location demonstrated 2.1.4 Carbohydrates: The peplomer pro­
for IBV, MHV, HCV, TGEV, HEV, tein of MHV and IBV is jV-glycosidi-
and BCV. Comparable protein cally linked to complex and high-
shown for CCV. Phosphorylation mannose oligosaccharides. The ma­
demonstrated for IBV, MHV and trix protein of MHV is O-glycosidi-
BCV. cally linked to oligosaccharides. The
186 Siddell/Anderson/Cavanagh/Fujiwara/Klenk/Macnaughton/Pensaert/Stohlman/Sturman/van dcr Zeijst

matrix protein of 1BV is A-glycosidi- 2.4 Morphology

cally linked to complex and high 2.4.1 Overall shape: Pleomorphic, al­
mannose oligosaccharides. though roughly spherical.
2.2 Physicochemical properties 2.4.2 Dimensions: 60-220 nm.
2.2.1 Density: Average density of 1.18 g/ 2.4.3 Surface projections: Usually charac­
ml in sucrose, 1.23-1.24 g/ml in teristic club-shaped projections,
CsCL; HEV has density of 1.17 g/ml about 20 nm long, widely spaced.
in potassium tartrate. IBV, HEV, and MHV can also have
2.2.2 Sedimentation coefficient: HCV, thin cone-shaped projections. BCV
380-400S; IBV, 330S; TGEV, 495S; has two layers of projections.
F1PV, 400S. 2.4.4 Special features in thin sections: In­
2.2.4 Stability of infectivity ner and outer shells, sometimes sepa­
2.2.4.1 pH: Optimal stability of IBV, pH rated by electron-lucent space.
6.0- 6.5; TGEV, pH 6.5; MHV, pH 2.4.5 Other features: Fragile attachment
6.0- 7.0. Conflicting data for more ex­ of projections to surface of virion,
treme conditions. may be removed by protease treat­
2.2.4.2 Heat: Rapid inactivation at 56°, ment. Purified peplomer projections
moderate inactivation at 37°. Mod­ aggregate in aqueous environment.
erately stable at 4° in optimal sus­ Purified transmembrane protein in­
pending medium. teracts specifically with genome RN A
2.2.4.3 Lipid solvents: Chloroform- and at 37°. Inner tongue-shaped mem­
ether-labile. brane in IBV visible by negative
2.2.4.4 Radiation: IBV, MHV, TGEV, and staining.
HEV inactivated by UV radiation 3 Replication
(30,000 erg/mm2). 3.1 Site of accumulation of viral pro­
2.2.4.5 Other agents: Agents capable of in­ teins: Cytoplasm.
activation include SDS, sodium de- 3.2 Nonstructural proteins : Not defined.
oxycholate, formalin, ethanol (70%), Virus-specific nonvirion polypeptides
KMNO-i, (3-propiolactone, hydro- for MHV.
xylamine, and chlorohexidine. 3.3 Mode of nucleic acid replication
2.3 Structure 3.3.1 General account: Genomic RNA as­
2.3.1 Nucleocapsid: Genome and nucleo- sumed to encode enzymes respon­
capsid protein associated as helical sible for amplification of genome and
RNP. production of subgenomic RNA.
2.3.2 Envelope: Lipid-containing envel­ RNA-dependent RNA polymerase
ope (host cell-derived) containing in­ demonstrated in MHV- and TGEV-
tegral and peripheral viral proteins. infected cells, but polypeptide com­
2.3.3 Cores: Electron-dense inner shell vi­ ponents not identified. Subgenomic
sible in thin sections. RNP core den­ RNAs are capped and polyadenyl-
sity: MHV, 1.27-1.28 g/ml in suc­ ated and form a 3' coterminal nested
rose; HCV, 1.31 g/ml in CsCla, sedi­ set. Replicative structures not yet
mentation coefficient 180S. characterized. For MHV, negative-
Coronaviridae 187

stranded template of genome length. HCV suckling mice,

UV inactivation studies on subgen- (some strains) suckling hamsters
omic RNA synthesis argue against IBV suckling mice,
extensive processing. Number of ma­ (some strains) suckling hamsters,
jor subgenomic RNAs: 1BV, 5; suckling rats,
MHV, 6. newborn rabbits
3.3.2 Effect of inhibitors: RNA synthesis MHV rats, hamsters,
is insensitive to actinomycin D, 5- monkeys
iododeoxyuridine, 5-bromodeoxy- TGEV dogs, foxes, cats
uridine, 5-fluorodeoxyuridine, cyto­ HEV suckling mice
sine arabinoside and aminopterin. BCV suckling mice,
Conflicting reports for IBV and rats, hamsters
MHV regarding a-amanitin. RNA CCV pigs
synthesis is sensitive to 6-azauracil F1PV newborn mice,
and virazole. rats, hamsters,
3.4 Site and mechanism of maturation: piglets
Matures in cytoplasm by budding 5.2.2 In vitro: Generally specific to organ
through endoplasmic reticulum. No cultures, primary and secondary cells
budding at plasmalemma. or cell lines derived from species of
3.5 Other features: For MHV, messen­ origin. Also,
ger function of the subgenomic IBV first-passage mon­
RNAs has been demonstrated. key kidney cells:
RNAs 7, 6 and 3 encode the virion VERO, BHK,
nucleocapsid, matrix and peplomer CHO (semiper-
protein(s), or their precursors, re­ missive)
spectively. Involvement of a nucleus CCV CFK, HRT 18
or nuclear function in coronavirus HCV first-passage mon­
replication is equivocal. MHV re­ key kidney cells:
ported to replicate in enucleated cells. BSC1, AGMK,
IBV (Beaudette) reported not to re­ VERO
plicate in enucleated or UV-irradi- MHV L6, HTC, WI38,
ated BHK21 cells. RN-2-2
4 Cooperative interactions: No data. BCV first-passage mon­
4.3 Phenotypic mixing: Reported be­ key kidney cells:
tween MHV and Friend leukemia HRT18, VERO,
virus. PK15, PK3,
5 Host range MA321
5.1 Natural : Generally restricted to na­ TGEV first-passage
tural vertebrate host. canine kidney cells
5.2 Experimental 6 Pathogenicity
5.2.1 In vivo : Generally specific for species 6.1 Association with diseases: IBV - re­
of origin. Also, spiratory disease, nephritis and gona-
188 Siddell/Anderson/Cavanagh/Fujiwara/Klenk/Macnaughton/Pensaert/Stohlman/Sturman/van der Zeijst

dal damage. MHV - acute hepatitis, 9.1 Number of distinct antigenic mole­
encephalomyelitis and infantile diar­ cules in virion : Three for HCV, HEV,
rhea. FIPV - peritonitis and granulo­ IBV, MHV, and TGEV. Each cor­
matous inflammations in many or­ responds to one class of virion pro­
gans. HCV - respiratory diseases. tein.
HEV - vomiting and wasting, ence- 9.2 Antigen involved in neutralization:
phalomyelitis. RCV - pneumonitis, Surface peplomer.
rhinitis. SDAV - sialoadenitis, da- 9.3 Number of distinct nonstructural an­
cryoadenitis. TCV - enteritis. BCV, tigens: Not known.
CCV, TGEV and PEDV associated 9.4 Specificity of different antigens: He­
with diarrhea. magglutinin (IBV, HCV, HEV, BCV,
6.2 Tissue tropism: 1BV - respiratory MHV) associated with peplomer pro­
tract, gonads, kidney. TCV - intes­ tein. Also, virus attachment and cell-
tine, respiratory tract. BCV, CCV, to-cell fusion activity.
PEDV - intestine. FIPV - perito- 9.5 Antigenic properties used for classifi­
neum, lymphoid organs, liver, other cation: Immunological reactivity de­
organs. HCV - respiratory tract. termined by enzyme-linked immuno­
MHV - liver, intestine, CNS, other sorbent assay or radioimmunoassay,
organs. TGEV - intestine, respira­ immunofluorescence, immune elec­
tory tract. HEV - CNS, respiratory tron microscopy, neutralization, he­
tract. RCV - respiratory tract, paro­ magglutinin inhibition, Western blot­
tid gland. SDAV - salivary and lacry- ting.
mal glands. 10 Classification
6.3 Cytopathology: Cellular vacuolation 10.1 Definition of family Coronaviridae:
leading to cell disintegration, some­ Pleomorphic enveloped viruses, aver­
times syncytium formation. aging 100 nm in diameter, bearing
7 Geographical distribution: FIPV, club-shaped projections about 20 nm
HECV, HCV, 1BV, MHV, TGEV, long. The genome is one molecule of
and HEV present over several con­ infectious RNA of about (5-7) x 10«
tinents, probably worldwide. molecular weight. Virions character­
8 Transmission istically contain three (major) struc­
8.1 Vertical: Intrauterine for 1BV, MHV tural proteins: peplomer, matrix and
and FIPV. nucleocapsid. Replication involves
8.2 Horizontal: Probably all coronavi- production of a 3' coterminal nested
ruses. set of subgenomic mRNAs. Virions
8.3 Vectors bud intracellularly. The family is
8.3.1 Biological: None known. monogeneric. Serological relation­
8.3.2 Mechanical: HCV and HEV, air­ ships suggest 2 avian groups, each
borne; IBV and BCV, contaminated with one species (IBV and TCV), and
material; others mainly oral/fecal 2 mammalian groups, comprised of
route. HCV (229E), TGEV, CCV, and
9 Antigenic properties FIPV and of HCV (OC43), MHV,
Coronaviridae 189

SDAV, RCV, HEV, and BCV [5]. determine whether HECV, included
IBV, MHV and HCV have many in the previous report [ I] as a corona­
serotypes. virus, is indeed a coronavirus. The
10.2 Genus Coronavirus problem of the characterization of
10.3 Type species: HECV has been discussed in a recent
IBV avian infectious bronchitis review [7].
virus
Other species:
HCV human Coronavirus References
MHV murine hepatitis virus
1 Tyrrell, D .A .J.; Alexander, D.J.; Almeida, J.D .;
BCV bovine Coronavirus Cunningham, C .H .; Easterday, B.C.; Garwes,
TGEV transmissible gastroenteri­ D .J.; Hierholzer, J.C.; Kapikian, A.; Macnaugh-
tis virus ton, M.R.; McIntosh, K.: Coronaviridae: second
HEV hemagglutinating ence­ report. Intervirology 10: 321-328 (1978).
phalomyelitis virus 2 Bohl, E. H.: Coronaviruses: diagnosis of infections.
Comp. Diagn. viral Dis. IV: 301-328 (1981).
CCV canine coronavirus 3 Siddell, S.G .; Wege, H.; ter Meulcn, V.: The struc­
FIPV feline infectious peritonitis ture and replication of coronaviruses. Curr. Top.
virus Microbiol. Immunol. 99: 131-163 (1982).
Possible species: 4 Siddell, S. G .; Wege, H.; ter Meulen, V.: The biol­
RCV rat (sialodacryoadenitis) ogy of coronaviruses. J. gen. Virol. 64: 761-776
(1983).
coronavirus 5 Sturman, L. S.; Holmes, K. V.: The molecular biol­
TCV turkey coronavirus ogy of coronaviruses. Adv. Virus Res. 28: (in press
PEDV porcine epidemic diar­ 1983).
rhea virus 6 Wege, H .; Siddell, S .; ter Meulen, V.: The biology
and pathogenesis of coronaviruses. Curr. Top.
Microbiol. Immunol. 99: 165-200(1982).
The classification of other isolates re­ 7 Macnaughton, M .R.; Davies, H.A.: Human en­
quires further information. In parti­ teric coronaviruses. Archs Virol. 70 : 301-313
cular, further evidence is required to (1981).