Anton Andonov

Immunization with Modified Vaccinia Virus Ankara-Based Recombinant Vaccine against Severe Acute Respiratory Syndrome Is Associated with Enhanced Hepatitis in Ferrets

ABSTRACT Severe acute respiratory syndrome (SARS) caused by a newly identified coronavirus (SARS-CoV) is a serious emerging human infectious disease. In this report, we immunized ferrets (Mustela putorius furo) with recombinant modified vaccinia virus Ankara (rMVA) expressing the SARS-CoV spike (S) protein. Immunized ferrets developed a more rapid and vigorous neutralizing antibody response than control animals after challenge with SARS-CoV; however, they also exhibited strong inflammatory responses in liver tissue. Inflammation in control animals exposed to SARS-CoV was relatively mild. Thus, our data suggest that vaccination with rMVA expressing SARS-CoV S protein is associated with enhanced hepatitis.

Mass Spectrometric Characterization of Proteins from the SARS Virus A Preliminary Report

A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a ∼46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted ∼139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS.

Activation of AP-1 signal transduction pathway by SARS coronavirus nucleocapsid protein

Abstract In March 2003, a novel coronavirus was isolated from patients exhibiting atypical pneumonia and subsequently proven to be the causative agent of the disease now referred to as severe acute respiratory syndrome (SARS). The complete genome of the SARS coronavirus (SARS-CoV) has since been sequenced. The SARS-CoV nucleocapsid (SARS-CoV N) shares little homology with other members of the coronavirus family. To determine if the N protein is involved in the regulation of cellular signal transduction, an ELISA-based assay on transcription factors was used. We found that the amount of transcription factors binding to promoter sequences of c-Fos, ATF2, CREB-1, and FosB was increased by the expression of SARS-CoV N. Since these factors are related to AP-1 signal transduction pathway, we investigated whether the AP-1 pathway was activated by SARS-CoV N protein using the PathDetect system. The results demonstrated that the expression of N protein, not the membrane protein (M), activated AP-1 pathway. We also found that SARS-CoV N protein does not activate NF-κB pathway, demonstrating that activation of important cellular pathways by SAS-CoV N protein is selective. Thus our data for the first time indicate that SARS-CoV has encoded a strategy to regulate cellular signaling process.

Analysis of multimerization of the SARS coronavirus nucleocapsid protein

Abstract Severe Acute Respiratory Syndrome (SARS), an emerging disease characterized by atypical pneumonia, has recently been attributed to a novel coronavirus. The genome of SARS Coronavirus (SARS-CoV) has recently been sequenced, and a number of genes identified, including that of the nucleocapsid protein (N). It is noted, however, that the N protein of SARS-CoV (SARS-CoV N) shares little homology with nucleocapsid proteins of other members of the coronavirus family [Science 300 (2003) 1399; Science 300 (2003) 1394]. N proteins of other coronavirus have been reported to be involved in forming the viral core and also in the packaging and transcription of the viral RNA. As data generated from some viral systems other than coronaviruses suggested that viral N–N self-interactions may be necessary for subsequent formation of the nucleocapsid and assembly of the viral particles, we decided to investigate SARS-CoV N–N interaction. By using mammalian two-hybrid system and sucrose gradient fractionations, a homotypic interaction of N, but not M, was detected by the two-hybrid analysis. The mammalian two-hybrid assay revealed an approximately 50-fold increase in SEAP activity (measurement of protein–protein interaction) in N–N interaction compared to that observed in either M–M or mock transfection. Furthermore, mutational analyses characterized that a serine/arginine-rich motif (SSRSSSRSRGNSR) between amino acids 184 and 196 is crucial for N protein oligomerization, since deletion of this region completely abolished the N protein self-multimerization. Finally, the full-length nucleocapsid protein expressed and purified from baculovirus system was found to form different levels of higher order structures as detected by Western blot analysis of the fractionated proteins. Collectively, these results may aid us in elucidating the mechanism pertaining to formation of viral nucleocapsid core, and designing molecular approaches to intervene SARS-CoV replication.

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

Abstract There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.

Characterization Of Protein-protein Interactions Between The Nucleocapsid Protein And Membrane Protein Of The Sars Coronavirus.

Abstract The human coronavirus, associated with severe acute respiratory syndrome (SARS-CoV), was identified and molecularly characterized in 2003. Sequence analysis of the virus indicates that there is only 20% amino acid (aa) identity with known coronaviruses. Previous studies indicate that protein–protein interactions amongst various coronavirus proteins are critical for viral assembly. Yet, little sequence homology between the newly identified SARS-CoV and those previously studied coronaviruses suggests that determination of protein–protein interaction and identification of amino acid sequences, responsible for such interaction in SARS-CoV, are necessary for the elucidation of the molecular mechanism of SARS-CoV replication and rationalization of anti-SARS therapeutic intervention. In this study, we employed mammalian two-hybrid system to investigate possible interactions between SARS-CoV nucleocapsid (N) and the membrane (M) proteins. We found that interaction of the N and M proteins takes place in vivo and identified that a stretch of amino acids (168–208) in the N protein may be critical for such protein–protein interactions. Importantly, the same region has been found to be required for multimerization of the N protein (He et al., 2004) suggesting this region may be crucial in maintaining correct conformation of the N protein for self-interaction and interaction with the M protein.

Conformational Reorganization of the SARS Coronavirus Spike Following Receptor Binding: Implications for Membrane Fusion

The SARS coronavirus (SARS-CoV) spike is the largest known viral spike molecule, and shares a similar function with all class 1 viral fusion proteins. Previous structural studies of membrane fusion proteins have largely used crystallography of static molecular fragments, in isolation of their transmembrane domains. In this study we have produced purified, irradiated SARS-CoV virions that retain their morphology, and are fusogenic in cell culture. We used cryo-electron microscopy and image processing to investigate conformational changes that occur in the entire spike of intact virions when they bind to the viral receptor, angiotensin-converting enzyme 2 (ACE2). We have shown that ACE2 binding results in structural changes that appear to be the initial step in viral membrane fusion, and precisely localized the receptor-binding and fusion core domains within the entire spike. Furthermore, our results show that receptor binding and subsequent membrane fusion are distinct steps, and that each spike can bind up to three ACE2 molecules. The SARS-CoV spike provides an ideal model system to study receptor binding and membrane fusion in the native state, employing cryo-electron microscopy and single-particle image analysis.

Serological and molecular evidence of a plausible transmission of hepatitis E virus through pooled plasma

Recently, hepatitis E virus has been recognized as a new transfusion‐associated risk; however, its efficiency of transmission through blood products requires further investigation. Asymptomatic viremia of short duration has been observed in blood donors from several European countries to the rate of <1:10 000 and HEV transmission in recipients of blood products has been documented in Japan and Europe. Although HEV RNA was detected in large plasma fractionation pools used for manufacturing of plasma derived products, HEV transmission has not been demonstrated so far. In this study, we investigated the possibility of HEV transmission in patients with thrombotic thrombocytopenic purpura whose treatment included up to 40 l of plasma exchange.

Rapid Genotyping of Hepatitis C Virus by Primer-Specific Extension Analysis

ABSTRACT Quick and accurate genotyping of hepatitis C virus (HCV) is becoming increasingly important for clinical management of chronic infection and as an epidemiological marker. Furthermore, the incidence of HCV infection with mixed genotypes has clinical significance that is not addressed by most genotyping methods. We have developed a fluorescence-based genotyping assay called primer-specific extension analysis (PSEA) for the most prevalent HCV genotypes and have demonstrated the capacity of PSEA-HCV for detecting mixed-genotype HCV infections. PSEA-HCV detects genotype-specific sequence differences in the 5′ untranslated region of HCV in products amplified by the COBAS AMPLICOR HCV Test, v2.0. Simulated mixed HCV infection of plasma with RNase-resistant RNA controls demonstrates that PSEA-HCV can detect as many as five genotypes in one specimen. Furthermore, in dual-genotype simulations, PSEA-HCV can unequivocally detect both genotypes, with one genotype representing only 3.1% of the mixture (313/10,000 IU in starting plasma). Compared to INNO-LiPA HCV II, both assays determined the same genotype for 191/199 (96%) patient specimens (175 subtype and 16 genotype-only identifications). Following the initial evaluation, PSEA-HCV was used routinely to genotype HCV from patient specimens submitted to our laboratory (n = 312). Seventeen (5.4%) mixed infections were identified. The distribution of single-infection HCV genotypes in our population was 60.9% type 1 (n = 190), 12.8% type 2 (n = 40), 20.2% type 3 (n = 63), 0.3% type 4 (n = 1), and 0.3% other (n = 1). In conclusion, PSEA-HCV provides an inexpensive, high-throughput screening tool for rapid genotyping of HCV while reliably identifying mixed HCV infections.

An Outbreak of Human Coronavirus OC43 Infection and Serological Cross-Reactivity with SARS Coronavirus

BACKGROUND In summer 2003, a respiratory outbreak was investigated in British Columbia, during which nucleic acid tests and serology unexpectedly indicated reactivity for severe acute respiratory syndrome coronavirus (SARS-CoV). METHODS Cases at a care facility were epidemiologically characterized and sequentially investigated for conventional agents of respiratory infection, SARS-CoV and other human CoVs. Serological cross-reactivity between SARS-CoV and human CoV-OC43 (HCoV-OC43) was investigated by peptide spot assay. RESULTS Ninety-five of 142 residents (67%) and 53 of 160 staff members (33%) experienced symptoms of respiratory infection. Symptomatic residents experienced cough (66%), fever (21%) and pneumonia (12%). Eight residents died, six with pneumonia. No staff members developed pneumonia. Findings on reverse transcriptase-polymerase chain reaction assays for SARS-CoV at a national reference laboratory were suspected to represent false positives, but this was confounded by concurrent identification of antibody to N protein on serology. Subsequent testing by reverse transcriptase-polymerase chain reaction confirmed HCoV-OC43 infection. Convalescent serology ruled out SARS. Notably, sera demonstrated cross-reactivity against nucleocapsid peptide sequences common to HCoV-OC43 and SARS-CoV. CONCLUSIONS These findings underscore the virulence of human CoV-OC43 in elderly populations and confirm that cross-reactivity to antibody against nucleocapsid proteins from these viruses must be considered when interpreting serological tests for SARS-CoV.