H. Wege

The Biology and Pathogenesis of Coronaviruses

The coronavirases were first recognized and morphologically defined as a group by Tyrrell and co-workers (1968, 1975, 1978). Biochemical studies have recently provided additional information which allows better characterization of these agents. Presently, coronaviruses are defined as being particles which are pleomorphic to rounded with a diameter of 60–220 nm, surrounded by a fringe or layer of typical club-shaped spikes. The virion is composed of about four to six proteins and possesses a lipid bilayer. The genome consists of a single-stranded polyadenylated RNA which is infectious and of positive polarity. During maturation these viruses are released by internal budding into vesicles derived from the endoplasmatic reticulum. These viruses are widespread in nature and are associated with a great variety of diseases with an acute, subacute, or subclinical disease process.

The biology of coronaviruses.

Introduction. The Coronaviridae is a monogeneric family comprising 11 viruses which infect vertebrates. Members of the group are responsible for diseases of clinical and economic importance, in particular respiratory and gastrointestinal disorders (Table 1). The group was originally recognized on the basis of a characteristic virion morphology (Tyrrell et al., 1968), but can now be defined by biological and molecular criteria. Various aspects of coronavirus biology have been dealt with in recent reviews (Robb & Bond, 1979; Siddell et al., 1982; Wege et al., 1982). Structure. Morphology. Coronavirions are pleomorphic, although generally spherical, 60 to 220 nm in diameter and bear widely spaced, club-shaped surface projections about 20 nm in length. Complete virions have a density in sucrose of about 1.18 g/ml. In thin sections the virion envelope may be visualized as inner and outer shells separated by a translucent space. In negatively stained preparations of avian infectious bronchitis virus (IBV) an inner tongue-shaped membrane is visible (Bingham & Almeida, 1977; Fig. 1).

Adoptive transfer of EAE-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis

Viruses have been found to induce inflammatory demyelinating lesions in central nervous system (CNS) tissue of both animal and man, either by natural infections or after vaccination1,2. At least two different pathogenic mechanisms have been proposed for these changes, a cytopathic viral infection of oligodendroglia cells with subsequent cell death, and a host immune reaction against virus and brain antigens. We now report the occurrence of cell-mediated immune reactions against basic myelin proteins in the course of coronavirus infections in Lewis rats. Infection of rats with the murine coronavirus JHM leads to demyelinating encephalomyelitis developing several weeks to months post-infection3–7. Lymphocytes from these diseased Lewis rats can be restimulated with basic myelin protein (BMP) and adoptive transfer of these cells leads to lesions resembling those of experimental allergic encephalomyelitis (EAE) in recipients, which can be accompanied by a mild clinical disease. This model demonstrates that a virus infection in CNS tissue is capable of initiating an autoimmune response which may be of pathogenic importance.

The Structure and Replication of Coronaviruses

Coronaviruses were recognized as a group in 1968 primarily on the basis of their characte ristic morphology as seen in the electron microscope (Tyrrell et al. 1968). Since that time our knowledge of the structure and replication of these viruses has increased steadily and has been periodically reviewed (McIntosh 1974; Tyrrell et al. 1978; Robb and Bond 1979a). The basis for this review, which concentrates on the molecular biology of coronaviruses, is principally the new data which has become available in the last 2 years. The pathogeni-city of these viruses, which are associated with many diseases of clinical importance in animals and humans, is the subject of the accompanying article.

Corona virus induced subacute demyelinating encephalomyelitis in rats: a morphological analysis.

SummaryThirty percent of weanling rats infected with JHM murine corona virus developed a subacute demyelinating encephalomyelitis approximately 3 weeks after intracerebral inoculation. Small demyelinating foci were located in the deep cerebral white matter and large, sharply demarcated demyelinating lesions were detectable in optic chiasma, pons and spinal cord. Axons as well as neurons were well preserved in the demyelinating plaques in areas where the lesions extended to the gray matter. Perivascular cuffings, consisting of plasma cells and mononuclear cells, were frequently found.Viral antigen was found mostly in the white matter and in glial cells, leaving neurons unstained. Electron microscopic studies of the early lesions of white matter disclosed two different kinds of cell degeneration which developed prior to the myelin disruption and mononuclear cell infiltration. One was a small pyknotic cell, which is thought to be an oligodendrocyte and the other is a ballooned cell containing abundant microtubules. Virus particles could be demonstrated only in the latter cell type. Discussion about astrocytes as well as oligodendrocytes was made in relation to the initial stage of demyelination caused by virus infection. This animal model may be useful in the analysis of the mechanisms leading to demyelination in subacute or chronic infections.

Coronavirus JHM: Intracellular Protein Synthesis

Coronavirus JHM contained six major proteins, four of which were glycosylated. The proteins were gp170, gp98, gp65, p60, gp25 and p23. Sac(-) cells infected with JHM shut off host cell protein synthesis, and the synthesis of three major (150K, 60K and 23K) and three minor (65K, 30K and 14K) polypeptides was detected by pulse-labelling with 35S-methionine. Antiserum directed against purified virus proteins specifically immunoprecipitated the three major intracellular species and also the 65K minor species. During a chase period, species 150K and 23K were processed and three new immunoprecipitable species, 170K, 98K and 25K appeared. The intracellular species 170K, 98K, 65K, 60K, 25K and 23K co-electrophoresed with virion proteins. Two-dimensional gel electrophoresis of infected cell polypeptides showed that the 60K, 23K, 25K and 14K species were relatively basic polypeptides whilst the 98K and 170K were relatively acidic and heterogeneously charged polypeptides. Additionally, a charge-size modification of the 23K species during processing was detected, which was not apparent using one-dimensional gel analysis.

Demyelinating encephalomyelitis induced by a long-term corona virus infection in rats

SummaryAbout 30% of weanling rats inoculated with JHM virus developed a subacute demyelinating encephalomyelitis (SDE) 3 weeks after inoculation (a.i.). From the remaining animals, 5% displayed overt neurological signs 3,6, and 8 months a.i. Animals with and without clinical signs 6–8 months a.i. were morphologically examined.Fresh demyelinating lesions could be demonstrated in paralyzed animals. Viral antigen was demonstrated and infectious JHM virus could be recovered from one animal which developed clinical signs at 3 months a.i. In one animal with clinical onset of 8 months a.i., completely remyelinated areas as well as recent demyelinating lesions were observed, suggesting a recurrence of the disease process. Remyelinated areas were also found in 40% of clinically silent animals. The morphology of the late onset of the demyelination was similar to that occurring in SDE. Remyelination consisted of both CNS and PNS-type. This animal model offers the possibility to investigate the virus-host relationship which is responsible for the induction of a demyelinating process after a long incubation period.

Structural Polypeptides of the Murine Coronavirus JHM

Analysis by SDS-polyacrylamide gel electrophoresis shows that the purified coronavirus JHM contains six polypeptides. The apparent mol. wt. of the polypeptides (GP1, GP2, GP3, VP4, GP5 and VP6) are 170000; 125000; 97500; 60800; 24800 and 22700, respectively. Four polypeptides are glycosylated (GP1, GP2, GP3 and GP5). The analysis of particles obtained after limited proteolysis with pronase suggests that GP2 and GP3 are protruding from the lipid envelope and, together with GP1, form the spike layer. Protein VP6 and a part of GP5 are located within the lipid bilayer. Protein VP4 is susceptible to digestion at a concentration of pronase which changes the morphology of the virus particles making the interior of the virus accessible. Subviral particles produced after treatment with the detergent Nonidet P40 banded at a higher density than the virus and contained only VP4, GP5 and VP6.

Genomic RNA of the Murine Coronavirus JHM

Genomic RNA extracted from the purified murine coronavirus JHM sediments between 52S and 54S in aqueous sucrose gradients. The RNA is single-stranded and has an apparent mol. wt. of 5.4 to 6.5 X 10(6), as determined by electrophoresis in polyacrylamide agarose gels of different concentrations. The presence of polyadenylate sequences in the RNA is demonstrated by binding to oligo-)dT) cellulose and digestion with ribonucleases A and T1. The purified RNA does not dissociate into subunits at high temperatures or in high concentrations of DMSO and is infectious.

The Peplomer Protein E2 of Coronavirus JHM as a Determinant of Neurovirulence: Definition of Critical Epitopes by Variant Analysis

We selected murine coronavirus JHM variants specifically changed in defined antigenic sites of the peplomer protein E2. Variants were isolated from the supernatants of monoclonal antibody hybridoma cell cultures which continued to secrete neutralizing antibodies after being infected with JHM. Comparative antigenic analysis and biological tests were performed in order to refine an operational epitope map and to characterize functional domains important for pathogenicity. The reaction patterns (neutralization, inhibition of cell fusion, immunofluorescence and binding in ELISA) between the variant viruses and the panel of monoclonal antibodies were very similar. Four groups of variants were characterized each of which revealed distinct changes affecting one defined antigenic site. These observations indicated that at least four independently mutable antigenic sites were associated with domains involved in cell fusion, neutralization and pathogenicity (E2-Aa, -Ab, -Ba and -Bb). JHM variants with alterations in the E2-Aa, -Ab or -Bb sites were similar to wild-type virus. These variants caused acute hepatitis and encephalomyelitis in mice. In contrast, JHM variants with changes in site E2-Ba had a strong propensity to induce chronic disease accompanied by demyelination persisting for several months.