Coronavirus, Infection and Immunity

Encyclopedia of Immunology (Second Edition)
1998, Pages 658-661

HelmutWege

Abstract

The family of Coronaviridae was defined around 1968, primarily by morphologic criteria. The virions are pleomorphic to spherical-shaped particles (120–160 nm diameter), which are surrounded by a fringe of club- or pear-shaped surface projections (12–24 nm long; Figure 1). The virus has a lipid envelope and contains a single-stranded RNA genome of positive polarity. Coronaviruses infect humans, mammals and birds. Based on molecular criteria, the toroviruses represent a second genus within this family. Furthermore, evolutionary relationships to the Arteriviridae are discussed within the concept of a ‘coronavirus-like’ superfamily.

Keywords

Coronaviridae,  virions, coronavirus, Arteriviridae

Cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis

Veterinary Immunology and Immunopathology
Volume 65, Issues 2–4, 23 October 1998, Pages 243-257

A. Kipar, S. Bellmann, J. Kremendahl, K. Köhler, M. Reinacher

Abstract

Twenty-three cats with spontaneous feline infectious peritonitis (FIP) were examined by light microscopy including immunohistology and histochemistry in order to determine the cellular composition and the expression of viral antigen in lesions in FIP. Furthermore, the presence of plasma-cells producing coronavirus-specific antibodies was evaluated in situ. Macrophages and neutrophils were demonstrated by an antibody against calprotectin (leukocyte protein L1, myeloid/histiocyte antigen), neutrophils were recognized due to their chloroacetate esterase activity, and B- and T-lymphocytes were identified by antibodies against the CD3 antigen and the CD45R antigen, respectively. Expression of viral antigen was immunohistologically demonstrated by a monoclonal antibody (mAb) against coronavirus while coronavirus-specific antibodies in situ were identified by the application of feline coronavirus prior to the coronavirus antibody. Lesions were classified as diffuse alterations at serosal surfaces, granulomas with areas of necrosis, granulomas without extended necrosis, focal and perivascular lymphoplasmocytic infiltrates, and granulomatous-necrotizing vasculitis. Diffuse alterations on serosal surfaces were represented either by activated mesothelial cells with single coronavirus antigen-bearing macrophages or by layers of precipitated exudate containing single to numerous granulomas with areas of necrosis. In liver and spleen, the exudate was often underlaid by a small band of subcapsular B-cells with an occasional plasma-cell producing coronavirus-specific antibodies. In other locations, a variably broad band of B-cells and plasma-cells, often infiltrating between underlying muscle fibers, separated the exudate from the unaltered tissue. Some of these plasma-cells were positive for coronavirus-specific antibodies. In granulomas with areas of necrosis, the central necrosis was surrounded by macrophages usually expressing considerable amounts of viral antigen. Few B-cells and plasma-cells were found in the periphery. In granulomas without extended necrosis, the number of macrophages were lower. Only few macrophages expressing low amounts of viral antigen were present. B-cells and plasma-cells formed a broad rim. Few plasma-cells stained positive for coronavirus-specific antibodies. In both types of granulomas, few neutrophils were found between macrophages. Few T-cells were seen scattered throughout the lesions. Focal and perivascular lymphoplasmocytic infiltrates were mainly seen in omentum and leptomeninx. B-cells were the predominant cells; some plasma-cells were positive for coronavirus-specific antibodies. Viral antigen was not readily detected in these alterations. Granulomatous-necrotizing vasculitis was occasionally found in kidneys and leptomeninx. It was dominated by macrophages which often stained strongly positive for coronavirus antigen. Different types of alteration were often seen in the same animal and even the same tissue. There was no obvious correlation between the cat's age, gross pathological changes, and the histological types of alteration. Single plasma-cells positive for coronavirus-specific antibodies were found around blood vessels distant from inflammatory alterations, within the lung parenchyma, as infiltrating cells in the mucosa of the small intestine, and in spleen and mesenteric lymph node. Results show that alterations in FIP are heterogeneous concerning cellular composition and expression of viral antigen. The dominance of B-cells in part of the lesions together with the presence of plasma-cells positive for coronavirus-specific antibodies indicate that these cells may play a role in the maintenance of inflammatory processes in FIP.

Keywords

Feline infectious peritonitis, Immunohistology, Coronavirus antigen, Coronavirus-specific antibodies, B-cells, Plasma-cells

Effects of the common cold on mood and performance

Psychoneuroendocrinology
Volume 23, Issue 7, October 1998, Pages 733-739

Andrew Smith, Marie Thomas, Julie Kent, Karl Nicholson

Abstract

Previous research has shown that both experimentally-induced and naturally occurring upper respiratory tract illnesses (URTIs) influence mood and mental functioning. None of the previous studies of naturally occurring colds has conducted appropriate virological assays to determine the nature of the infecting agent. This is an essential methodological step in studies of malaise associated with URTIs. The aim of this research was to investigate the effects of naturally occurring colds on mood and objective measures of performance. This was done by first conducting a cross-sectional comparison of 37 healthy people and 158 volunteers with colds and then a longitudinal study in which 100 volunteers developed colds and 87 remained healthy. Virological techniques were used to identify infecting agents and comparisons made across the different groups. The results showed that having a cold was associated with reduced alertness and slowed reaction times. These effects were observed both for colds where the infecting virus was identified and those where it was not. Similar effects were obtained for both rhinovirus and coronavirus colds. One may conclude that upper respiratory tract illnesses lead to a reduction in subjective alertness and impaired psychomotor functioning. This was true for both illnesses where the infecting agent was identified and for those clinical illnesses where no virus was detected. It is now important to identify the mechanisms linking infection and illness with the behavioural changes. Similarly, the impact of these effects on real-life activities such as driving needs examining. Finally, methods of treatment need to be developed which not only treat the local symptoms of the illnesses but remove the negative mood and the performance impairments.

Keywords

Common cold, Rhinovirus, Coronavirus, Reaction time, Alertness

Comparison of immunofluorescence with monoclonal antibodies and RT-PCR for the detection of human coronaviruses 229E and OC43 in cell culture

Journal of Virological Methods
Volume 72, Issue 2, June 1998, Pages 145-152

Jacques Sizun, Nathalie Arbour, Pierre J Talbot

Abstract

Human coronaviruses, with two known serogroups named 229E and OC43, cause up to one third of common colds and may be associated with serious diseases such as nosocomial respiratory infections, enterocolitis, pericarditis and neurological disorders. Reliable methods of detection in clinical samples are needed for a better understanding of their role in pathology. As a first step in the design of such diagnostic procedures, the sensitivities and specificities of two viral diagnostic assays were compared in an experimental cell culture model: an indirect immuno-fluorescence assay using monoclonal antibodies and reverse transcriptase-polymerase chain reaction amplification of viral RNA from infected cells. Immunofluorescence detected human coronaviruses in cells infected at a MOI as low as 10−2 (log TCID50/ml=4.25 for HCV-229E and 2.0 for HCV-OC43; log PFU/ml=4.83 for HCV-229E and 1.84 for HCV-OC43) versus 10−3 (HCV-OC43) or 10−4 (HCV-229E) for reverse transcriptase-polymerase chain reaction amplification (log TCID50/ml=1.75 for HCV-229E and 1.5 for HCV-OC43; log PFU/ml=2.3 for HCV-229E and 1.34 for HCV-OC43). There were no false positive signals with other human respiratory pathogens: influenza virus, respiratory syncytial virus and adenovirus. Moreover, each assay was coronavirus serogroup-specific. These results demonstrate the potential usefulness of immunofluorescence with monoclonal antibodies and reverse transcriptase-polymerase chain reaction RNA amplification for the rapid detection of human coronaviruses in infected cell cultures. Both methods could be applied to clinical specimens for the diagnosis of human infections.

Keywords

Coronavirus, 229E, OC43, Immunofluorescence, RT-PCR, Diagnostic

Roles in Cell-to-Cell Fusion of Two Conserved Hydrophobic Regions in the Murine Coronavirus Spike Protein

Virology
Volume 244, Issue 2, 10 May 1998, Pages 483-494

Zongli Luo, Susan R.Weiss

Abstract

The spike (S) protein of coronavirus, mouse hepatitis virus (MHV), mediates attachment and fusion during viral entry and cell-to-cell fusion later in infection. By analogy with other viral proteins that induce cell fusion the MHV S protein would be expected to have a hydrophobic stretch of amino acids that serves as a fusion peptide. Sequence analysis suggests that the S protein falls within the group of fusion proteins having internal rather than N-terminal fusion peptides. Based on the features of known viral fusion peptides, we identified two regions (PEP1 and PEP2) of MHV-A59 S2 as possible fusion peptides. Site-directed mutagenesis and an in vitrocell-to-cell fusion assay were used to evaluate the roles of PEP1 and PEP2, as well as a third previously identified putative fusion domain (PEP3) in membrane fusion. Substitution of bulky hydrophobic residues with charged residues within PEP1 affects the fusion activity of the S protein without affecting processing and surface expression. Similar substitutions within PEP2 result in a fusion-negative phenotype; however, these mutant S proteins also exhibit defects in protein processing and surface expression which likely explain the loss of the ability to induce fusion. Thus PEP1 remains a candidate fusion peptide, while PEP2 may play a significant role in the overall structure or oligomerization of the S protein. PEP3 is an unlikely putative fusion peptide since it is not conserved among coronaviruses and nonconservative amino acid substitutions in PEP3 have minimal effects on cell-to-cell fusion.

Keywords

cell-to-cell, coronavirus, mouse hepatitis virus, protein

Altered Pathogenesis of a Mutant of the Murine Coronavirus MHV-A59 Is Associated with a Q159L Amino Acid Substitution in the Spike Protein

Virology
Volume 239, Issue 1, 8 December 1997, Pages 1-10

Isabelle Leparc-Goffarta, Susan T.Hingley, Ming Ming Chua, Xinhe Jiang, EhudLavi, Susan R.Weiss

Abstract

C12, an attenuated, fusion delayed, very weakly hepatotropic mutant of mouse hepatitis virus strain A59 (MHV-A59) has been further characterized. We have previously shown that C12 has two amino acid substitutions relative to wild type virus in the spike protein, Q159L (within a region of S1 shown to bind to viral receptor in anin vitroassay) and H716D (in the proteolytic cleavage recognition site). We have sequenced the rest of the 31-kb genome of C12 and compared it to wild type virus. Only three additional amino acids substitutions were found, all encoded within the replicase gene. Analysis of C12in vivoin C57Bl/6 mice has shown that despite the fact that this virus replicates in the brain to titers at least as high as wild type and causes acute encephalitis similar to wild type, this virus causes a minimal level of demyelination and only at very high levels of virus inoculation. Thus acute encephalitis is not sufficient for the induction of demyelination by MHV-A59. Analysis of mutants isolated at earlier times from the same persistently infected glial cell culture as C12, as well as mutants isolated from a second independent culture of persistently infected glial cells, suggests that both the weakly demyelinating and the weakly hepatotropic phenotypes of C12 are associated with the Q159L amino acid substitution.

Keywords

pathogenesis, hepatitis virus, murine coronavirus MHV

Infectious Bovine Rhinotracheitis, Parainfluenza-3, and Respiratory Coronavirus

Veterinary Clinics of North America: Food Animal Practice
Volume 13, Issue 3, November 1997, Pages 455-469

Sanjay Kapil DVM, PhD, Randall J.Basaraba DVM, PhD

Abstract

A number of viruses have been proven to be primary respiratory pathogens of cattle. Viruses may play an important role in making cattle susceptible to secondary respiratory bacterial pathogens. Epidemiology, pathogenesis, laboratory diagnosis, and important properties in infectious bovine rhinotracheitis (IBR), parainfluenza-3 (PI-3), and bovine respiratory coronavirus (BRCV) are described in this article.

Keywords

rhinotracheitis, parainfluenza-3, bovine respiratory coronavirus

The Molecular Biology of Coronaviruses

Advances in Virus Research
Volume 48, 1997, Pages 1-100

Michael M.C. Lai, David Cavanagh

Abstract

This chapter discusses the manipulation of clones of coronavirus and of complementary DNAs (cDNAs) of defective-interfering (DI) RNAs to study coronavirus RNA replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors for coronaviruses, and processing of the polymerase. The nature of the coronavirus genome is nonsegmented, single-stranded, and positive-sense RNA. Its size ranges from 27 to 32 kb, which is significantly larger when compared with other RNA viruses. The gene encoding the large surface glycoprotein is up to 4.4 kb, encoding an imposing trimeric, highly glycosylated protein. This soars some 20 nm above the virion envelope, giving the virus the appearance-with a little imagination-of a crown or coronet. Coronavirus research has contributed to the understanding of many aspects of molecular biology in general, such as the mechanism of RNA synthesis, translational control, and protein transport and processing. It remains a treasure capable of generating unexpected insights.

Keywords

Coronavirus, DNAs, RNAs, clones

In vivo study of interferon-alpha-secreting cells in pig foetal lymphohaematopoietic organs following in utero TGEV coronavirus injection

Research in Immunology
Volume 148, Issue 4, May 1997, Pages 247-256

I.Splíchal, Z.Řeháková, M.Šinkora, J.Šinkora, I.Trebichavský, H.Laude, B.Charley

Abstract

Non-infectious UV-inactivated transmissible gastroenteritis virus (TGEV) was previously shown to induce interferon alpha (IFIMα) secretion following in vitro incubation with blood mononuclear cells. In this study, pig foetuses at different stages of gestation were injected in utero with (a) partially UV-inactivated wild TGEV or (b) fully UV-inactivated wild or dm49-4 mutant TGEV Coronavirus. Nucleated cells from foetal liver, bone marrow, spleen and blood were isolated 10 or 20 h after injection and assayed ex vivo for IFNα secretion by ELISPOT and ELISA techniques. The administration of TGEV induced IFNα-secreting cells in foetal lymphohaematopoietic organs at mid-gestation. In contrast, IFNα was not detected in control sham-operated foetuses. A specific point mutation in the amino acid sequence of the viral membrane glycoprotein M of TGEV mutant dm49-4 was associated with lower or absent IFNα in utero inducibility by mutant virus as compared with wild virus. Row cytometry analysis did not show differences in leukocyte surface marker expression between control and TGEV- or between dm49-4 and wild virus-treated foetus cells, with the exception of a reduction in percentages of polymorphonuclear cells in TGEV-treated lymphohaematopoietic tissues, which is probably due to IFNα secretion. The present data provided in vivo evidence of IFNα secretion at the cell level in foetal lymphohaematopoietic organs. Such IFNα-secreting cells in lymphohaematopoietic tissues may be the source of IFNα detected during foetal infections.

Keywords

Coronavirus, Transmissible gastroenteritis virus, IFNα, ELISA, ELISPOT, Foetus, Pig

Bovine coronavirus I protein synthesis follows ribosomal scanning on the bicistronic N mRNA

Virus Research
Volume 48, Issue 1, April 1997, Pages 101-105


Savithra D.Senanayake, David A.Brian

Abstract

The mRNA encoding the 49-kDa nucleocapsid protein (N) of the bovine coronavirus is bicistronic. A 23-kDa protein, termed the I protein for the ‘internal’ open reading frame (ORF), is also synthesized but in the +1 reading frame beginning 61 nt downstream of the N start codon. Sequences flanking the N and I start codons suggest that the I ORF might be accessed by scanning ribosomes passing over the N start codon. Here we test this idea and demonstrate with translation studies both in vitro and in vivo that the I protein is synthesized according to the leaky scanning model for initiation of translation on the subgenomic N mRNA molecule.

Keywords

Coronavirus bicistronic N mRNA, Nucleocapsid protein, Ribosomal scanning

Identification of the polymerase polyprotein products p72 and p65 of the murine coronavirus MHV-JHM

Virus Research
Volume 45, Issue 2, December 1996, Pages 101-109

Hong-QiangGao, Jennifer J.Schiller, Susan C.Baker

Abstract

The RNA polymerase gene of murine coronavirus MHV-JHM encodes a polyprotein of greater than 750 kDa. This polyprotein is proposed to be processed by two papain-like cysteine proteinases, PCP-1 and PCP-2, and a poliovirus 3C-like proteinase domain, 3C-pro, to generate protein products. The amino-terminal product of the MHV polymerase polyprotein, p28, is generated by cleavage of the polyprotein by PCP-1. To identify the viral products downstream of p28, we generated a fusion-protein specific antiserum directed against the region adjacent to p28 and used the antiserum to detect virus-specific proteins from MHV-JHM infected cells. When this antiserum was used to immunoprecipitate radiolabeled proteins from MHV-JHM infected cell lysates, virus-specific proteins of 72 and 65 kDa were detected. Furthermore, pulse and chase experiments demonstrated that p72 is likely a precursor to the mature protein product, p65. To investigate which viral proteinase may be responsible for generating p72 and p65, we expressed the 5′-region of the MHV-JHM RNA polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. We also cloned and expressed the 72 kDa region immediately downstream from p28, and tested the ability of in vitro translated PCP-1 and PCP-2 to cleave p72 to p65 in trans. Our results indicate that neither viral proteinase domain PCP-1 nor PCP-2 is capable of cleavage of p72 to produce p65 in vitro. The role of MHV proteinases in the processing of p72 and p65 is discussed.

Keywords

Coronavirus, Polymerase polyprotein, Proteolytic processing

 

The Common Cold

Primary Care: Clinics in Office Practice
Volume 23, Issue 4, 1 December 1996, Pages 657-675

George L.KirkpatrickMD

Abstract

The common cold has intrigued physicians and the general public for centuries. It has been defined as an acute epidemic respiratory disease characterized by mild coryzal symptoms of rhinorrhea, nasal obstruction, and sneezing. The nasal discharge is usually copious and thin during the first 2 days of illness, then it generally becomes more viscous and purulent.22 The disease is self-limited. Symptoms may persist for 2 days to more than 14 days; however, the cold may abort after only 1 day. Fever, cough, sore throat, or lacrimation may or may not be present. The common cold is of itself harmless, but bacterial invasion frequently follows the initial infection. It is these secondary invaders that may produce disorders of serious consequence.

Keywords

common cold, epidemic, respiratory

Syncytia Formation Induced by Coronavirus Infection Is Associated with Fragmentation and Rearrangement of the Golgi Apparatus

Virology
Volume 221, Issue 2, 15 July 1996, Pages 325-334


EHUD LAVI, QIAN WANG, SUSAN R.WEISS, NICHOLAS K.GONATAS

Abstract

Coronavirus mouse hepatitis virus (MHV) possesses a membrane glycoprotein (M) which is targeted to the Golgi apparatus (GA). We used immunocytochemistry with an organelle-specific antiserum to investigate the morphologic changes of the GA during infection of L2 murine fibroblasts with MHV-A59. Twenty-four hours after infection the GA was fragmented and translocated in the center of syncytia, while the microtubular network was also rearranged displaying radiating elements toward the center of syncytia. Two fusion-defective mutants, which contain an identical amino acid substitution in the cleavage signal sequence of the spike glycoprotein (S), induced fragmentation of the GA. However, the GA migrated only partially to the centers of syncytia during infection with these mutants. Revertant viruses, in which the above mutation was corrected, had fusion properties and GA staining similar to wtMHV-A59. Experiments with brefeldin A (BFA), which induces redistribution of the GA into the rough endoplasmic reticulum (RER), revealed that an intact GA for a period of 4–16 hr postinfection, is required for coronavirus replication and syncytia formation. Thus, during MHV infection, syncytia formation is associated with fragmentation of the GA, followed by a previously undescribed phenomenon of migration of the organelle into the centers of syncytia. The fragmentation of the GA, however, may occur without the formation of syncytia. Therefore, two distinct mechanisms may be responsible for the fragmentation of the GA and its subsequent migration to the center of syncytia.

Keywords

corona virus, mouse hepatitis, golgi apparatus, glycoprotein

Growth characteristics and protein profiles of prototype and wild-type rat coronavirus isolates grown in a cloned subline of mouse fibroblasts (L2p.176 cells)

Virus Research
Volume 41, Issue 1, March 1996, Pages 55-68


Diane J.Gaertner, Susan R.Compton, Deborah F.Winograd, Abigail L.Smith

Abstract

Rat coronaviruses (RCVs) infect laboratory rats and confound biomedical research results. In vitro systems developed so far have limited the growth in knowledge about RCVs by not permitting generation of plaque-cloned virus stocks, reliable isolation of RCVs from rat tissues, or growth of high titered stocks of all isolates. Due to the fact that less than 20% of L2(Percy) cells were becoming infected, sublines were produced and selected for maximal growth of RCVs. Screening of 238 cell sublines yielded L2p.176 cells which were highly susceptible to all RCVs tested; however, susceptibility declined after 30 passages in vitro. Low-passaged L2p.176 cells were used to isolate virus from natural outbreaks and to propagate individual RCV plaques into high titered stocks. Proteins from six RCV isolates were immunoblotted using polyclonal rat and mouse antibodies to sialodacryoadenitis virus and polyclonal monospecific rabbit and goat antibodies against the peplomer (S) and nucleocapsid (N) proteins of mouse hepatitis virus (MHV). Proteins of two prototype, one Japanese and three wild type RCVs were examined and found to be similar to those of MHV, although the exact sizes and ratios of protein forms were unique for most RCV isolats. This study reports the development of a continuous cell line which reliably supports RCVs opening an opportunity for further in vivo studies of the biology of these agents. As a first step in the characterization of RCVs, we have shown that RCV proteins are very similar to those of MHV.

Keywords

Rat coronavirus, Rat, Cell line, Cell culture, Virus strains, Sialodacryoadenitis virus

In situ hybridization technique for the detection of swine enteric and respiratory coronaviruses, TGEV and PRCV, in formalin-fixed paraffin-embedded tissues

Journal of Virological Methods
Volume 56, Issue 2, February 1996, Pages 149-160

Theerapol Sirinarumitr, Prem S. Paul, John P. Kluge, Patrick G. Halbur

Abstract

The in situ hybridization (ISH) technique was developed to detect the swine coronaviruses, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), in cell culture and tissue sections from TGEV-or PRCV-infected pigs. The 35S-labeled RNA probes were generated from two plasmids pPSP.FP1 and pPSP.FP2 containing part of the S gene of TGEV. The procedure was first standardized in cell cultures. The radiolabeled pPSP.FP2 probe detected both TGEV and PRCV in virus-inoculated cell cultures, whereas pPSP.FP1 probe detected TGEV but not PRCV. The probe was then used to detect TGEV or PRCV in tissues of pigs experimentally infected with TGEV or PRCV or naturally infected with TGEV. Again, the probes detected TGEV in intestines of experimentally and naturally infected pigs and PRCV in the lungs of experimentally infected pigs. TGEV RNA was detected mainly within the enterocytes at the tips of villi and, less often, within some crypt epithelial cells. PRCV was shown to replicate mainly in the bronchiolar epithelial cells and in lesser amount in type II pneumocytes, type I pneumocytes, alveolar macrophages and bronchial epithelial cells, respectively. ISH has potential applications as a diagnostic test for the detection and differentiation of TGEV and PRCV in tissues and in studies to gain a better understanding of the mechanism of pathogenesis of enteric and respiratory coronavirus infections.

Keywords

Single stranded RNA probe, In situ hybridization, Transmissible gastroenteritis virus (TGEV), Porcine respiratory coronavirus (PRCV), Coronavirus