Arlene R. Collins

In Vitro Detection of Apoptosis in Monocytes/Macrophages Infected with Human Coronavirus

ABSTRACT Human coronavirus (HCoV) strain 229E infection, but not HCoV strain OC43 infection, of monocytes/macrophages from healthy donors and patients with multiple sclerosis in remission resulted in increased apoptosis, as measured by DNA changes and annexin V staining. Apoptosis correlated with the differential release of infectious virus. HCoV strain 229E titers were 103.5 to 106 50% tissue culture-infective doses (TCID50)/ml, and HCoV strain OC43 titers were only 101.2 to 102.7 TCID50/ml.

SARS Coronavirus Spike Protein-Induced Innate Immune Response occurs via Activation of the NF-κB pathway in Human Monocyte Macrophages in vitro

Abstract A purified recombinant spike (S) protein was studied for its effect on stimulating human peripheral blood monocyte macrophages (PBMC). We examined inflammatory gene mRNA abundances found in S protein-treated PBMC using gene arrays. We identified differential mRNA abundances of genes with functional properties associated with antiviral (CXCL10) and inflammatory (IL-6 and IL-8) responses. We confirmed cytokine mRNA increases by real-time quantitative(q) RT-PCR or ELISA. We further analyzed the sensitivity and specificity of the prominent IL-8 response. By real-time qRT-PCR, S protein was shown to stimulate IL-8 mRNA accumulation in a dose dependent manner while treatment with E protein did not. Also, titration of S protein-specific production and secretion of IL-8 by ELISA showed that the dose of 5.6nM of S produced a significant increase in IL-8 (p =0.003) compared to mock-treated controls. The increase in IL-8 stimulated by a concentration of 5.6nM of S was comparable to concentrations seen for S protein binding to ACE2 or to neutralizing monoclonal antibody suggesting a physiological relevance. An NF-κB inhibitor, TPCK (N-Tosyl-L-Phenylalanine Chloromethyl Ketone) could suppress IL-8 production and secretion in response to S protein in PBMC and THP-1 cells and in HCoV-229E virus-infected PBMC. Activation and translocation of NF-κB was shown to occur rapidly following exposure of PBMC or THP-1 cells to S protein using a highly sensitive assay for active nuclear NF-κB p65 transcription factor. The results further suggested that released or secreted S protein could activate blood monocytes through recognition by toll-like receptor (TLR)2 ligand.

HLA class I antigen serves as a receptor for human coronavirus OC43

Human coronaviruses are associated with acute respiratory and enteric disease in man as such their target cells are probably the epithelial cells lining the respiratory and enteric tract. Attachment of virus to specific receptors on the cell surface is a major determinant of virus tropism in pathogenesis (1). Recently, aminopeptidase-N was identified as a cell receptor for the 229e coronavirus (2). Cell receptor(s) for OC43 coronavirus have not been identified. However, it is of pathologic significance that OC43 virus shares DNA sequence homology with the two coronavirus isolates, SK and SD, from the brain of patients with multiple sclerosis (MS) (3). Probing MS and control brain with probes specific for human, murine, porcine and bovine coronavirus by in situ hybridization resulted in the detection of coronavirus RNA in 12 of 22 MS brain samples; five of which were positive with the OC43 probe (4). A study of virus-ligand interactions of OC43 with human rhabdomyosarcoma (RD) cells, which are highly susceptible to virus infection, was undertaken to identify possible cell receptors. The binding of virus collected from the supernatant of infected cells to cell proteins immobilized on nitrocellulose paper was used to screen for virus-ligand interactions. The next step was the identification or development of antibodies to each of the ligands, and to test their ability to blockade receptor activity by culturing infected cells in medium containing the ligand antibodies and measuring the effect on virus yield. The preliminary experiments reported here reveal an interesting observation of strong affinity of OC43 virus for the HLA class I antigen.

Regulation of viral persistence in human glioblastoma and rhabdomyosarcoma cells infected with coronavirus OC43

Abstract Cultures of human rhabdomyosarcoma (RD) and human glioblastoma (U87-MG) were compared for their ability to sustain a persistent infection with coronavirus OC43. Within 28 days, infectious virus and hemagglutinin were being produced at high levels in both types of cells. Temperature sensitive plaque variants were recovered at 31 °C. In both cell types, the virus caused increased antigen synthesis and cell death, if the temperature was lowered to 31 °C. Infectious virus was lost if cells were treated with antiserum to whole virus or if the temperature was raised to 39.5 °C. Probing the cured cells with OC43-specific 32P-cDNA showed that cured cells contained no detectable viral RNA. The relative ease of establishment and cure of these persistent infectious makes them attractive as models to study coronavirus regulatory processes.

Human Macrophages are Susceptible to Coronavirus OC43

Adherent adult and cord blood macrophages were infected with human coronavirus OC43 at a multiplicity of 1-1.5 and washed twice to remove unbound virus. Virus progeny was detected in the supernatant on day 1 and peaked at 2-3 days at an average titer of 5 +/- 3.9 x 10(6) pfu/ml from seven samples. Viral RNA was detected by nested set RT-PCR in infected macrophages incubated for 48 hr. Intracellular viral nucleocapsid was detected in 15% of the cells and surface staining for viral spike antigen was observed using monoclonal antibodies. Amplification of infectious virus and detection viral RNA and antigen synthesis in macrophages in vitro indicates susceptibility to OC43 virus.

Human Coronavirus OC43 Interacts with Major Histocompatibility Complex Class I Molecules at the Cell Surface to Establish Infection

Human coronaviruses have been associated with common colds, diarrhea and enterocolitis, and have been implicated in multiple sclerosis. HLA class I molecules may play a critical role as receptor for OC43 because monoclonal antibody (mAb)W6/32 to HLA-A, -B and -C specificities completely blocks infectivity in human rhabdomyosarcoma (RD) cells. The role of HLA class 1 antigen as the virus receptor was examined using HLA-A3.1 stably transfected human plasma cells and untransfected HMY.C1R cells which do not express HLA-A and -B molecules. When the cells (5 x 10(6)) were infected at a multiplicity of one, the HLA.A3 transfected cells produced 10(8) PFU of virus whereas no replication occurred in the HMY.C1R cells mAb W6/32 reduced the virus yield by 99.9%. Cell membranes from HMY.C1R, HMY.A3 cells and chicken erythrocytes were biotinylated as live cells. Immunoprecipitation with polyclonal antiviral antibody to detect binding of biotinylated cell membranes to virus revealed that biotinylated HMY.A3 membranes coprecipitated with virus-antibody complexes when the immunoprecipitates were electrophoresed on SDS-PAGE gel, electroblotted and stained with Avidin-horseradish peroxidase. The results provide direct evidence that OC43 virus can recognize HLA class I as receptor on the cell surface.

Interferon γ Potentiates Human Coronavirus OC43 Infection of Neuronal Cells by Modulation of HLA Class I Expression

HCN-1A, a human cerebral cortical neuron cell line, was examined for its susceptibility to human coronaviruses. The 229e strain replicated efficiently, but the OC43 strain did not replicate well, if at all. Treatment of the cells with interferon gamma at 20U/ml for 48 hr markedly increased the susceptibility of the cells to infection with OC43 virus as shown by a 100-fold increase in secretion of infectious virus over a four day period as compared to untreated controls. The increased susceptibility was shown to be due to membrane expression of HLA class I by receptor-blockade with a monoclonal antibody specific for HLA molecules.

Interferon production and response to exogenous interferon in two cell lines of mouse brain origin persistently infected with Sendai virus.

SummaryTwo persistently infected cell lines established from C3H mouse brain cells infectedin vivo with Sendai virus were shown to differ with respect to interferon (IF) production and response to exogenous IF. MB/Sen carrier cells contained 1–5 per cent antigen positive cells when examined by immunofluorescence, and virus was occasionally recovered from the culture medium. MB/SenAS carrier cells were maintained with 0.16 per cent Sendai antiserum in the supernatant medium. All MB/SenAS cells contained viral antigen and infectious virus was present in the culture medium.MB/Sen released IF spontaneously into the culture medium. Further IF production could be stimulated in MB/Sen by superinfection with Newcastle disease virus (NDV) or vesicular stomatitis virus (VSV). Exogenous IF provided good protection against VSV challenge.In contrast, MB/SenAS produced no IF spontaneously but could be stimulated by NDV and VSV to produce IF. Exogenous IF failed to reduce the amount of VSV released into the supernatant fluid. Replication of VSV was restricted in MB/SenAS as shown by a 2.3 log10 lower virus yield compared to MB/Sen.

Induction of Apoptosis in MRC-5, Diploid Human Fetal Lung Cells after Infection with Human Coronavirus OC43

Human coronaviruses (HCoV) cause upper respiratory tract infections manifested by symptoms of the common cold including rhinitis, tussis, fever, headache and myalgia (Hruskova et al 1990). HCoV respiratory illness is primarily virus-mediated; immunologic events have not been postulated to play a role (Makela et al 1998). Evidence showing the presence of viral genome in spinal fluid and brain suggests that HCoV may play an etiologic role in multiple sclerosis (Stewart et al 1992, Cristallo et al 1997). However, the pathogenesis of HCoV infections is poorly understood. In human embryonic tracheal organ cultures, HCoV causes a slow patchy destruction of the ciliated epithelial cells and in respiratory epithelial tissue cultures, the cytopathic effect is subtle, evident only by vacuolization and spindling.

Comparison of the Replication of Distinct Strains of Human Coronavirus OC43 in Organotypic Human Colon Cells (Caco-2) and Mouse Intestine

Three strains of human coronavirus (HCV) OC43 were compared for their ability to cause enteric infections and to induce interferon alpha (IFN alpha) using the Caco-2 human colon carcinoma cell line which exhibits spontaneous epithelial differentiation in vitro. MRC-5 cell culture grown stocks were prepared from: 1. CV Paris, a strain of OC43 recovered from an outbreak of necrotizing enterocolitis in newborns. 2. CV Mb, a neurotropic strain of OC43 which exhibits strict neuronal specificity in murine neuronal cell cultures. 3. CV Rd, a strain of OC43 which grows to a high titer in human rhabdomyosarcoma (RD) cells. Immunofluorescent staining for nucleocapsid antigen and plaque assay in MRC-5 cells was used to detect viral replication. BG-9 (human foreskin) cells challenged with vesicular stomatitis virus were used to detect IFN alpha production by human peripheral blood monocytes (PBMC) stimulated by virus infected Caco-2 cells. Caco-2 cells infected with virus at a multiplicity of infection of 0.5 yielded 10(4.6) and 10(4.4) plaque forming units/ml (pfu/ml) with CV Rd and CV Paris respectively, while CV Mb yielded only 10(3) pfu/ml. Caco-2 cells infected with CV Rd induced 64 IU/ml of IFN alpha in PBMC while these cells infected with CV Paris induced less than 2 IU/ml IFN alpha. In cells infected with CV Mb 4 IU/ml IFN alpha was detected. The results suggest that a lack of IFN alpha induction by CV Paris may be an indicator of its enteropathogenic potential.(ABSTRACT TRUNCATED AT 250 WORDS)