Marian C. Horzinek

Coronaviruses : structure and genome expression

Introduction. Progress in coronavirology is illustrated by the number of workshops convened and reviews written. International meetings have been held in Germany (1980), the Netherlands (1983) and the U.S.A. (1986), and the Fourth Coronavirus Symposium will be organized by one of us (D.C.) in Cambridge, U.K. in July 1989. In addition, reviews have appeared which highlighted particularly interesting characteristics of the family, e.g. the replication strategy (Lai, 1986) and the glycoproteins (Sturman & Holmes, 1985). As the last general accounts were published some 5 years ago (Siddell et al., 1983; Sturman & Holmes, 1983) an update is timely. The present article is based on the large amount of sequence data accumulated in these years and focuses on the viral nucleic acids and proteins and their function. Coronaviruses cause infections in man, other mammals and birds. Most experimental data have been obtained from studies of mouse hepatitis virus (MHV) and infectious bronchitis virus of chickens (IBV).

The Genome Organization of the Nidovirales: Similarities and Differences between Arteri-, Toro-, and Coronaviruses

Abstract Viruses in the families Arteriviridae and Coronaviridae have enveloped virions which contain nonsegmented, positive-stranded RNA, but the constituent genera differ markedly in genetic complexity and virion structure. Nevertheless, there are striking resemblances among the viruses in the organization and expression of their genomes, and sequence conservation among the polymerase polyproteins strongly suggests that they have a common ancestry. On this basis, the International Committee on Taxonomy of Viruses recently established a new order, Nidovirales, to contain the two families. Here, the common traits and distinguishing features of the Nidovirales are reviewed.

Recent advances in pestivirus research

Introduction. The term ‘pestivirus’ was coined in 1973 to group together two antigenically related enveloped RNA viruses: hog cholera virus (HCV) and bovine viral diarrhoea virus (BVDV; Horzinek, 1973). A third animal pathogen, the border disease virus (BDV) of sheep, was found later to be a close relative of BVDV. Pestiviruses are among the smallest enveloped animal RNA viruses (about 40 nm in diameter) and possess a nucleocapsid of non-helical, probably icosahedral symmetry (Horzinek et al., 1967); they share these traits with the numerous flaviviruses, of which the arthropod-borne yellow fever virus is the prototype. The pestiviruses are not arthropod-borne and currently hold generic status in the family Togaviridae. Previously, flaviviruses also held generic status in this family. However, when details of flavivirus molecular structure, replication strategy and gene sequence became known in the early 1980s, the Togaviridae Study Group recognized the fundamental differences and proposed the creation of the new family Flaviviridae with Flavivirus as the only genus (Westaway et al., 1985).

Sequence of mouse hepatitis virus A59 mRNA 2: Indications for RNA recombination between coronaviruses and influenza C virus

Abstract The nucleotide sequence of the unique region of coronavirus MHV-A59 mRNA 2 has been determined. Two open reading frames (ORF) are predicted: ORF1 potentially encodes a protein of 261 amino acids; its amino acid sequence contains elements which indicate nucleotide binding properties. ORF2 predicts a 413 amino acids protein; it lacks a translation initiation codon and is therefore probably a pseudogene. The amino acid sequence of ORF2 shares 30% homology with the HA1 hemagglutinin sequence of influenza C virus. A short stretch of nucleotides immediately upstream of ORF2 shares 83% homology with the MHC class I nucleotide sequences. We discuss the possibilitythat both similarities are the result of recombinations and present a model for the acquisition and the subsequent inactivation of ORF2; the model applies also to MHV-A59-related coronaviruses in which we expect ORF2 to be still functional.

Evidence for a coiled-coil structure in the spike proteins of coronaviruses

Abstract The amino acid sequences of the spike proteins from three distantly related coronaviruses have been deduced from cDNA sequences. In the C-terminal half, an homology of about 30% was found, while there was no detectable sequence conservation in the N-terminal regions. Hydrophobic “heptad” repeat patterns indicated the presence of two α-helices with predicted lengths of 100 and 50 Å, respectively. It is suggested that, in the spike oligomer. these α-helices form a complex coiled-coil, resembling the supersecondary structures in two other elongated membrane proteins, the haemagglutinin of influenza virus and the variable surface glycoprotein of trypanosomes.

Primary structure of the glycoprotein E2 of coronavirus MHV-A59 and identification of the trypsin cleavage site

Abstract The nucleotide sequence of the peplomer (E2) gene of MHV-A59 was determined from a set of overlapping cDNA clones. The E2 gene encodes a protein of 1324 amino acids including a hydrophobic signal peptide. A second large hydrophobic domain is found near the COOH terminus and probably represents the membrane anchor. Twenty glycosylation sites are predicted. Cleavage of the E2 protein results in two different 90K species, 90A and 90B (L. S. Sturman, C. S. Ricard, and K. V. Holmes (1985) J. Virol. 56, 904–911), and activates cell fusion. Protein sequencing of the trypsin-generated N-terminus revealed the position of the cleavage site. 90A and 90B could be identified as the C-terminal and the N-terminal parts, respectively. Amino acid sequence comparison of the A59 and 1HM E2 proteins showed extensive homology and revealed a stretch of 89 amino acids in the 90B region of the A59 E2 protein that is absent in JHM.

Feline Herpesvirus Infection: ABCD Guidelines on Prevention and Management:

Overview Feline viral rhinotracheitis, caused by feline herpesvirus (FHV), is an upper respiratory tract disease that is often associated with feline calicivirus and bacteria. In most cats, FHV remains latent after recovery, and they become lifelong virus carriers. Stress or corticosteroid treatment may lead to virus reactivation and shedding in oronasal and conjunctival secretions. Infection Sick cats shed FHV in oral, nasal and conjunctival secretions; shedding may last for 3 weeks. Infection requires direct contact with a shedding cat. Disease signs Feline herpesvirus infections cause acute rhinitis and conjunctivitis, usually accompanied by fever, depression and anorexia. Affected cats may also develop typical ulcerative, dendritic keratitis. Diagnosis Samples consist of conjunctival, corneal or oropharyngeal swabs, corneal scrapings or biopsies. It is not recommended that cats recently vaccinated with a modified-live virus vaccine are sampled. Positive PCR results should be interpreted with caution, as they may be produced by low-level shedding or viral latency. Disease management ‘Tender loving care’ from the owner, supportive therapy and good nursing are essential. Anorexic cats should be fed blended, highly palatable food - warmed up if required. Mucolytic drugs (eg, bromhexine) or nebulisation with saline may offer relief. Broad-spectrum antibiotics should be given to prevent secondary bacterial infections. Topical antiviral drugs may be used for the treatment of acute FHV ocular disease. The virus is labile and susceptible to most disinfectants, antiseptics and detergents.

Phylogeny of antigenic variants of avian coronavirus IBV

Abstract The sequences of the peplomeric S1 protein of four serologically distinct strains of the infectious bronchitis virus (IBV), an avian coronavirus, have been determined. The S1 protein is thought to contain the serotype-specific neutralization epitopes and to be the main target of antigenic variation. An alignment with sequences of three strains published previously showed that from the 545 amino acid residues only 243 have been conserved. Clustering of substitutions suggests that most serotype determinants are located within the first 300 amino acid residues of S1. A phylogenetic tree of the S1 sequences showed very variable rates of divergence. Differences in topology with a tree based on RNAse-T1 fingerprint data indicate that some of the IBV strains have arisen by genetic recombination.

PERSISTENCE AND EVOLUTION OF FELINE CORONAVIRUS IN A CLOSED CAT-BREEDING COLONY

Abstract Feline coronavirus (FCoV) persistence and evolution were studied in a closed cat-breeding facility with an endemic serotype I FCoV infection. Viral RNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in the feces and/or plasma of 36 of 42 cats (86%) tested. Of 5 cats, identified as FCoV shedders during the initial survey, 4 had detectable viral RNA in the feces when tested 111 days later. To determine whether this was due to continuous reinfection or to viral persistence, 2 cats were placed in strict isolation and virus shedding in the feces was monitored every 2–4 days. In 1 of the cats, virus shedding continued for up to 7 months. The other animal was sacrificed after 124 days of continuous virus shedding in order to identify the sites of viral replication. Viral mRNA was detected only in the ileum, colon, and rectum. Also in these tissues, FCoV-infected cells were identified by immunohistochemistry. These findings provide the first formal evidence that FCoV causes chronic enteric infections. To assess FCoV heterogeneity in the breeding facility and to study viral evolution during chronic infection, FCoV quasispecies sampled from individual cats were characterized by RT-PCR amplification of selected regions of the viral genome followed by sequence analysis. Phylogenetic comparison of nucleotides 7–146 of ORF7b to corresponding sequences obtained for independent European and American isolates indicated that the viruses in the breeding facility form a clade and are likely to have originated from a single founder infection. Comparative consensus sequence analysis of the more variable region formed by residues 79–478 of the S gene revealed that each cat harbored a distinct FCoV quasispecies. Moreover, FCoV appeared to be subject to immune selection during chronic infection. The combined data support a model in which the endemic infection is maintained by chronically infected carriers. Virtually every cat born to the breeding facility becomes infected, indicating that FCoV is spread very efficiently. FCoV-infected cats, however, appear to resist superinfection by closely related FCoVs.

Feline immunodeficiency. ABCD guidelines on prevention and management.

Overview Feline immunodeficiency virus (FIV) is a retrovirus closely related to human immunodeficiency virus. Most felids are susceptible to FIV, but humans are not. Feline immunodeficiency virus is endemic in domestic cat populations worldwide. The virus loses infectivity quickly outside the host and is susceptible to all disinfectants. Infection Feline immunodeficiency virus is transmitted via bites. The risk of transmission is low in households with socially well-adapted cats. Transmission from mother to kittens may occur, especially if the queen is undergoing an acute infection. Cats with FIV are persistently infected in spite of their ability to mount antibody and cell-mediated immune responses. Disease signs Infected cats generally remain free of clinical signs for several years, and some cats never develop disease, depending on the infecting isolate. Most clinical signs are the consequence of immunodeficiency and secondary infection. Typical manifestations are chronic gingivostomatitis, chronic rhinitis, lymphadenopathy, weight loss and immune-mediated glomerulonephritis. Diagnosis Positive in-practice ELISA results obtained in a low-prevalence or low-risk population should always be confirmed by a laboratory. Western blot is the ‘gold standard’ laboratory test for FIV serology. PCR-based assays vary in performance. Disease management Cats should never be euthanased solely on the basis of an FIV-positive test result. Cats infected with FIV may live as long as uninfected cats, with appropriate management. Asymptomatic FIV-infected cats should be neutered to avoid fighting and virus transmission. Infected cats should receive regular veterinary health checks. They can be housed in the same ward as other patients, but should be kept in individual cages.