Analysis of JHM central nervous system infections in rats.

Intracerebral inoculation of murine coronavirus JHM into 2- to 3-day-old Wistar Furth rats causes an acute encephalomyelitis, while inoculations at 10 days of age usually result in hind leg paralysis. To examine the distribution of viral antigens within this infected central nervous system (CNS) tissue, we used the avidin-biotin-peroxidase method to detect monoclonal and polyclonal antibodies bound to JHM structural proteins; in addition we used the Western blot technique to detect viral proteins. Our study demonstrated the following characteristics: Infected neuronal and glial cells produced viral nucleocapsid and E2 glycoprotein. The synthesis of these viral structural proteins was not restricted to cells in any particular part of the central nervous system. While JHM E2 proteins could be detected in individual cells of JHM-infected CNS tissue, the relative level of detectable E2 protein in the total CNS tissue of infected rats was reduced by more than 13-fold compared with JHM-infected tissue culture cells. Hippocampus neuronal cells provided a sensitive indication of JHM infection. These cells invariably contained antigens in both acutely and chronically infected animals. The distribution of cells containing viral antigens differed markedly for JHM-induced acute encephalitis and chronic demyelinating disease. Acutely infected brains had large lesions containing low levels of viral antigen scattered throughout the brain. One percent to ten percent of histologically normal cells in many parts of the brain contained viral antigens; in addition, more neuronal cells than glial cells were observed to be antigen-positive. The hippocampus appeared normal with hematoxylin-eosin staining; however, a scattered infection of neuronal cells was apparent.(ABSTRACT TRUNCATED AT 250 WORDS)

Western and dot immunoblotting analysis of viral antigens and antibodies: application to murine hepatitis virus.

Viral proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred quantitatively to nitrocellulose by electroblotting in SDS-containing buffer. Monoclonal antibodies directed against previously defined epitopes on the viral proteins were used as probes to detect viral protein synthesis and processing, as well as expression in animal tissues. Circulating polyclonal antibodies were also probed and characterized for their polypeptide specificities. Under appropriate conditions, this Western immunoblotting technique was quantitative. Finally, a highly sensitive dot immunoblotting assay was used to analyze the sensitivity to denaturation of various epitopes on the viral proteins. This assay detected picogram quantities of viral antigens and antibodies.

Activation of glial cells by human coronavirus OC43 infection.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease that could be triggered by a viral infection. Coronaviruses induce an MS-like disease in rodents, are neuroinvasive in humans and can infect primary cultures of human astrocytes and microglia. Infection of the human astrocytic cell line U-373MG by the OC43 strain of human coronavirus caused an upregulation of IL-6, TNF-alpha, and MCP-1 mRNA expression. This virus also modulated the activity of matrix metalloproteinases-2 and -9 and augmented nitric oxide production in both U-373MG cells and the human microglial cell line CHME-5. Thus, a coronaviral infection of glial cells could lead to the production of inflammatory molecules that have been associated with central nervous system pathologies such as MS.

Antigenic variation among murine coronaviruses: evidence for polymorphism on the peplomer glycoprotein, E2.

A panel of 28 monoclonal antibodies (MAb) against the structural proteins of murine hepatitis virus-4, strain JHM (MHV-4) was used in three antigen binding assays to determine the extent of antigenic homology among six strains of murine coronaviruses. The antigenic determinants studied were highly conserved on the E1 glycoproteins and nucleocapsid (N) proteins of all strains tested. In contrast, antigenic polymorphism was observed among the E2 glycoproteins. Of three previously described antigenic determinants against which neutralizing antibodies are directed, only one, termed A(E2), was conserved on all strains. Antigenic site B(E2) was found only on the strongly neurotropic MHV-4 and site C(E2) was present on the virulent MHV-4 and MHV-3 (hepatotropic) strains, but absent on the weakly pathogenic MHV-A59, MHV-1 and MHV-S strains. Four non-neutralizing antibodies against at least one topographically distinct antigenic determinant, which we previously designated D(E2), gave binding patterns consistent with two distinct sites. One of these was present on all MHV strains tested and the other was present on all strains except MHV-S. These non-neutralizing antigenic sites were redesignated E(E2) and D(E2) respectively.

Sequence analysis of human coronavirus 229E mRNAs 4 and 5: evidence for polymorphism and homology with myelin basic protein.

Human coronaviruses (HCV) are important pathogens responsible for respiratory, gastrointestinal and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the nucleotide sequence of the 5'-unique regions of mRNAs 4 and 5 were determined from cloned cDNAs. Sequence analysis of the cDNAs synthesized from mRNA 4 revealed a major difference with previously published results. However, polymerase chain reaction amplification of this region showed that the sequenced cDNAs were produced from minor RNA species, an indication of possible genetic polymorphism in this region of the viral genome. The mutated messenger RNA 4 contains two ORFs: (1) ORF4a consisting of 132 nucleotides which potentially encodes a 44-amino acid polypeptide of 4653 Da; this coding sequence is preceded by a consensus transcriptional initiation sequence, CUAAACU, similar to the ones found upstream of the N and M genes; (2) ORF4b of 249 nucleotides potentially encoding an 83-amino acid basic and leucine-rich polypeptide of 9550 Da. On the other hand, mRNA 5 contains one single ORF of 231 nucleotides which could encode a 77-amino acid basic and leucine-rich polypeptide of 9046 Da. This putative protein presents a significant degree of amino acid homology (33%) with its counterpart found in transmissible gastroenteritis coronavirus (TGEV). The proteins in the two different viruses exhibit similar molecular weights and are extremely hydrophobic. Interestingly, a sequence homology of five amino acids was found between the protein encoded by ORF4b of HCV-229E and an immunologically important region of human myelin basic protein.

Different host-cell shutoff strategies related to the matrix protein lead to persistence of vesicular stomatitis virus mutants on fibroblast cells.

Acute infection of fibroblastic cell lines by the Indiana strain of vesicular stomatitis virus (VSV) usually induces dramatic cytopathic effects and shutoff of cellular gene expression. We have compared a series of independent mutants with differences in shutoff induction and found that M was mutated either in the N-terminus (M(51)R) or C-terminus (V(221)F and S(226)R). Furthermore, only double mutants (M mutation and a ts mutation related or not to M) were able to persist on fibroblast cell lines at 39 degrees C. A more detailed investigation of the infection was performed for the mutants T1026, TP3 and G31, differing in their host shutoff effects related to M protein. Viral activity in persistently infected mouse L-929 and monkey Vero cell lines was followed by viral proteins detection, RNA synthesis throughout infection and finally detection of infectious particles. All three mutants cause extensive CPE followed by emergence of persistently infected cells on Vero cells. The same thing is seen on L-929 cells except for T1026 which causes little CPE. Taken together, the results form a basis of further studies to clarify how various viral and cellular factors interact in the establishment of a persistent infection by VSV mutants.

Characterization of protection against coronavirus infection by noninternal image antiidiotypic antibody.

Previously, we have reported protective vaccination of mice against a coronavirus infection using rabbit polyclonal noninternal image Ab2gamma anti-idiotypic (anti-Id) antibody specific for a virus-neutralizing and protective monoclonal antibody (mAb) 7-10A against the viral surface S glycoprotein. To characterize further the mechanisms involved in the induction of protective immunity by this noninternal image anti-Id, plasma and splenocytes from Ab2gamma-immunized BALB/c mice were passively transferred to naive BALB/c mice, followed by viral challenge. A reproducible significant delay in mortality observed in mice to which plasma was passively transferred, together with the presence of specific in vitro neutralizing antiviral Ab3 identified the humoral immune response as the major element responsible for protection. The activation of specific and cross-reactive T lymphocytes by both virus and anti-Id in immunized mice and the absence of adoptive transfer of protection by splenocytes suggested the participation of T helper activity in the induction of protective virus-neutralizing Ab3. To obtain more defined monoclonal reagents for a better understanding of anti-Id-induced protection, mAb2 were generated against the same mAb1 7-10A and characterized. We report the successful generation of mAb2 of the gamma type. However, unlike the polyclonal Ab2gamma, they were not capable of inducing a protective immune response.

Characterization of phage-displayed recombinant anti-idiotypic antibody fragments against coronavirus-neutralizing monoclonal antibodies.

Murine coronaviruses provide useful animal models for human neurological disorders such as multiple sclerosis. In an effort to better understand the mechanisms involved in protection from coronavirus infection, we are studying the role of the idiotypic network in the modulation of viral infectivity. We have explored the feasibility of using single-chain antibodies displayed on phage surfaces for the isolation of recombinant anti-idiotypic antibodies (anti-Ids) with antigen-mimicking properties, which has proven to be difficult with conventional hybridoma approaches. A phage-display library containing more than 10(8) different antibody specificities was screened for the presence of anti-Ids by successive rounds of panning with three different in vitro neutralizing and in vivo protective antiviral monoclonal antibodies. After five rounds of panning, between 32% and 84% of all individual clones tested showed antibody-binding in an enzyme-linked immunosorbent assay (ELISA). Although several clones showed identical antibody sequences, a number of different clones were identified and further characterized. None of the selected clones induced the production of antiviral or neutralizing antibodies or conferred reproducible protection from viral challenge in BALB/c and C57BL/6 mice. These results demonstrate that anti-Ids can be isolated from a phage-display library, although high-affinity antigen-mimicking phages with antiviral protective capacities were apparently not represented in this library. This argues for the development of more diverse phage-display libraries.

Increased viral titers and enhanced reactivity of antibodies to the spike glycoprotein of murine coronavirus produced by infection at pH 6.

Infection of cell monolayers by murine coronavirus A59 at pH 6 rather than 7 yielded a ten-fold increase in the infectious titer and a remarkable enhancement of the reactivities of monoclonal and polyclonal antibodies against the spike glycoprotein in immunoblotting, immunoprecipitation and enzyme-linked immunosorbent assays. These observations are very useful for detecting antibodies against the S glycoprotein of coronaviruses and enhancing infectious titers.

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

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.

Growth of a murine coronavirus in a microcarrier cell culture system.

The growth of the murine coronavirus MHV-A59 on murine DBT cells adapted to dextran-made Cytodex 1 microcarriers was studied in comparison with cells grown on plastic dishes. With a microcarrier concentration of 5 g/l in spinner flasks, a density of 3 x 10(6) cells/ml was reached in 7 days. Under these conditions, cells supported virus growth to the same extent as when they were grown on the plastic substratum. This was shown by a similar development of virus-induced syncytia, the release of an equivalent number of infectious progeny virions per cell, similar recoveries observed after concentration and purification and an identical appearance of the purified virus under the electron microscope. On the other hand, the technical convenience of microcarriers and the ease of scale-up emphasize their potential for the growth of coronaviruses.

Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay.

The SARS-associated human coronavirus (SARS-HCoV) is a newly described, emerging virus conclusively established as the etiologic agent of the severe acute respiratory syndrome (SARS). This study presents a single-tube RT-PCR assay that can detect with high analytical sensitivity the SARS-HCoV, as well as several other coronaviruses including other known human respiratory coronaviruses (HCoV-OC43 and HCoV-229E). Species identification is provided by sequencing the amplicon, although a rapid screening test by restriction enzyme analysis has proved to be very useful for the analysis of samples obtained during the SARS outbreak in Toronto, Canada.

Survival of human coronaviruses 229E and OC43 in suspension and after drying onsurfaces: a possible source ofhospital-acquired infections.

Strains OC43 and 229E of human coronaviruses (HCoV) cause one-third of common colds and hospital-acquired upper respiratory tract HCoV infections have been reported in premature newborns. To evaluate possible sources of infection, virus survival was studied in aqueous suspensions and on absorptive and non-absorptive surfaces representative of a hospital environment. Virus susceptibility to chemical disinfection with standard products was also characterized. Virus survived in saline solution for as long as six days but less in culture medium, with or without added cells. After drying, HCoV-229E infectivity was still detectable after 3h on various surfaces (aluminum, sterile latex surgical gloves, sterile sponges) but HCoV-OC43 survived 1h or less. Of the various chemical disinfectants tested, Proviodine reduced the virus infectious titre by at least 50%. This study suggests that surfaces and suspensions can be considered as possible sources of contamination that may lead to hospital-acquired infections with HCoV and should be appropriately disinfected.

Coronavirus-related nosocomial viral respiratory infections in a neonatal and paediatric intensive care unit: a prospective study.

The incidence of nosocomial viral respiratory infections (NVRI) in neonates and children hospitalized in paediatric and neonatal intensive care units (PNICU) is unknown. Human coronaviruses (HCoV) have been implicated in NVRI in hospitalized preterm neonates. The objectives of this study were to determine the incidence of HCoV-related NVRI in neonates and children hospitalized in a PNICU and the prevalence of viral respiratory tract infections in staff. All neonates (age< or =28 days) and children (age>28 days) hospitalized between November 1997 and April 1998 were included. Nasal samples were obtained by cytological brush at admission and weekly thereafter. Nasal samples were taken monthly from staff. Virological studies were performed, using indirect immunofluorescence, for HCoV strains 229E and OC43, respiratory syncytial virus (RSV), influenza virus types A and B, paramyxoviruses types 1, 2 and 3 and adenovirus. A total of 120 patients were enrolled (64 neonates and 56 children). Twenty-two samples from 20 patients were positive (incidence 16.7%). In neonates, seven positive samples, all for HCoV, were detected (incidence 11%). Risk factors for NVRI in neonates were: duration of hospitalization, antibiotic treatment and duration of parenteral nutrition (P<0.01). Monthly prevalence of viral infections in staff was between 0% and 10.5%, mainly with HCoV. In children, 15 samples were positive in 13 children at admission (seven RSV, five influenza and three adenovirus) but no NVRI were observed. In spite of a high rate of community-acquired infection in hospitalized children, the incidence of NVRI with common respiratory viruses appears low in neonates, HCoV being the most important pathogen of NRVI in neonates during this study period. Further research is needed to evaluate the long-term impact on pulmonary function.

Protection of mice from lethal coronavirus MHV-A59 infection by monoclonal affinity-purified spike glycoprotein.

Numerous studies have provided indirect evidence that the spike glycoprotein of coronaviruses (E2 or S) bears determinants for pathogenesis and the induction of protective immunity. In order to directly evaluate its immunogenicity, the E2 glycoprotein of the murine hepatitis virus, strain A59, was purified by immunoaffinity chromatography. High titers of neutralizing and fusion inhibiting antibodies were induced in mice vaccinated with purified E2/S in Freund's adjuvant, which were protected from an intracerebral challenge with 10 LD50 of MHV-A59. This study provides a direct demonstration of the importance of the coronavirus spike glycoprotein in the induction of a protective immune response.

Molecular characterization of the 229E strain of human coronavirus.

Human coronaviruses (HCV) cause various respiratory, gastrointestinal and possibly neurological disorders. Very little is known of the molecular biology of these ubiquitous pathogens. We have undertaken the molecular characterization of the prototype 229E strain of HCV. The virus grew to the highest titers on a human embryonic lung cell line (L132) at 33 degrees C and purification was optimal on Renografin-60 gradients. Metabolic labeling with [35S]methionine or [3H]glucosamine or galactose and analysis by SDS-PAGE revealed at least five structural proteins, which could be identified by analogy with murine coronaviruses as follows: the spike glycoprotein (E2/S), in both monomeric (88-97 kDa) and dimeric (190-200) forms, the nucleoprotein (N) at 52-53 kDa and the matrix protein (E1/M), in both glycosylated (25-26 kDa) and non-glycosylated (20-22 kDa) forms. Monomeric, dimeric and multimeric (greater than 200 kDa) forms of E2/S incorporated glucosamine and galactose, whereas only galactose was incorporated into E1/M. Multimers of E1/M, with apparent molecular masses of 44, 74 and 140 kDa, were formed in the absence of a reducing agent.

Induction of a protective immune response to murine coronavirus with non-internal image anti-idiotypic antibodies.

Neurotropic murine coronaviruses (MHV) provide an excellent animal model to study experimental modulation of the immune response to a viral pathogen with anti-idiotypic antibodies. It is known that among the various types of anti-idiotypic antibodies (anti-Id), those designated beta (beta) or internal image can molecularly mimic the antigen and induce biological activities such as anti-viral protection and neutralization. We have recently shown that polyclonal non-internal image anti-idiotypic antibodies of the gamma-type could induce protective anti-coronavirus immunity. In the present study, a polyclonal anti-Id (Ab2) was induced against a neutralizing murine monoclonal antibody (MAb1), designated 5B170.11. Mice immunized with this affinity-purified rabbit Ab2 alpha, a non-internal image antibody, were partially protected against lethal infection by the JHM strain of MHV. However, other polyclonal and monoclonal non-internal image Ab2 induced against another neutralizing Mab1, designated 4-11G.6, were not able to protect mice against lethal infection with the A59 strain of MHV. These results demonstrate that anti-viral protection by altering the idiotypic network with non-internal image-bearing anti-idiotype reagents can be achieved even with some anti-Id of the alpha-type.

Persistent infection of neural cell lines by human coronaviruses.

Human coronaviruses (HCV) have been associated mainly with infections of the respiratory tract. Accumulating evidence from in vitro and in vivo observations is consistent with the neurotropism of these viruses in humans. To verify the possibility of a persistent infection within the central nervous system (CNS), various human cell lines of neural origin were tested for their ability to maintain chronic infection by both known strains of HCV, OC43 and 229E. Production of infectious progeny virions was monitored by an immunoperoxydase assay on a susceptible cell line and viral RNA was observed after RT-PCR. Astrocytic cell lines U-373 MG and U-87 MG did not sustain a persistent HCV-229E infection, even though they were susceptible to an acute infection by this virus. On the other hand, these two cell lines could maintain a persistent infection by HCV-OC43 for as many as 25 cell passages (about 130 days of culture). Relatively stable titers of infectious viral particles, as well as apparently constant amounts of viral RNA were detected throughout the persistent infection of U-87 MG cells. However, persistent infection of U-373 MG cells was accompanied by the detection of infectious viral particles from passage 0 to passage 13 and then from passage 20 to the end of the experiment. This gap in the production of infectious virions was correlated by a drop in the apparent amount of viral RNA detected at passages 15 and 20. These results confirm the ability of HCV-OC43 to persistently infect cells of an astrocytic lineage and, together with our previous observations of HCV infection of primary cultures of human astrocytes and the detection of HCV RNA in human brains, are consistent with the possibility that this human coronavirus could persist in the human CNS by targeting astrocytes.

Characterization of the nonstructural and spike proteins of the human respiratory coronavirus OC43: comparison with bovine enteric coronavirus.

The nucleotide sequence of the region between the spike (S) and the membrane (M) protein genes, and sequences of the S and ns2 genes of the OC43 strain of human coronavirus (HCV-OC43) were determined. The ns2 gene comprises an open reading frame (ORF) encoding a putative nonstructural (ns) protein of 279 amino acids with a predicted molecular mass of 32-kDa. The S gene comprises an ORF encoding a protein of 1353 amino acid residues, with a predicted molecular weight of 149,918. Sequence comparison between HCV-OC43 and the antigenically related bovine coronavirus (BCV) revealed more sequence divergence in the putative bulbous part of the S protein (S1) than in the stem region (S2). The cysteine residues near the transmembrane domain and the internal predicted protease cleavage site are conserved in the HCV-OC43 S protein. Nucleotide sequence analysis of the region between the S and M gene loci revealed the presence of an unexpected intragenomic partial leader sequence and two ORFs encoding potential proteins of 12.9 and 9.5-kDa. These two proteins were identified as nonstructural by comparison with the homologous BCV genes. In vitro translation analyses demonstrated that the HCV-OC43 9.5-kDa protein, like its BCV counterpart, is poorly translated when situated down-stream of the 12.9-kDa ORF, but is expressed in infected cells, as shown by immunofluorescence. Interestingly, two ORFs, potentially encoding 4.9 and 4.8-kDa ns proteins in BCV are absent in HCV-OC43, indicating that they are not essential for viral replication in HRT-18 cells.

A functional model of microvascular thrombosis.

A new model of microvascular thrombosis is presented, with the evaluation of single-dose heparin in the prevention of microvascular thrombosis. The technique, which involves arterial crushing and an arteriotomy with intimal abrasion, was performed on the superficial femoral artery of the rat. The model was applied to a series of 30 consecutive rat superficial femoral arteries. A 100 percent thrombosis rate was seen immediately and at 24 hours in 10 nonheparinized animals. An operator control group of 10 vessels without intimal abrasion had a patency rate of 100 percent immediately and at 24 hours. Ten vessels following single-dose heparin and intimal abrasion were all patent initially, with 7 remaining patent at 24 hours. Reproducibility of the model was documented by a second operator with similar results. Utilizing this model, single-dose heparin was effective in maintaining vessel patency.