Complete genomic characterization of two tick-borne encephalitis viruses isolated from China.

Tick-borne encephalitis (TBEV) is prevalent over a wide area of the Eurasian continent. TBE viruses cause severe encephalitis in humans, with serious sequelae, and have a significant impact on public health in these endemic regions. To gain insight into genetic evolution of tick-borne encephalitis virus (TBEV) in China, the complete genomic sequences of two TBEV strains Senzhang and MDJ01, which were isolated in 1953 and 2001 respectively, were characterized. The complete genome sequences of two strains were all consist 10,784 nucleotides and there are 364 nucleotides deletion in the 3' nontranslated region. Compared with other TBEV strains, homology range from 85.2% (Zausaev) to 99.6% (MDJ02 and MDJ03) on the level of nucleotide. Phylogenetic trees based on the complete genome, open reading frame and E gene nucleotide sequences all showed that the strains Senzhang and MDJ01 belong to Far-Eastern subtype and cluster with other Chinese TBEV strains. All these implied that TBEVs prevalent in China were highly conservative, other measurement should be taken to improve protective efficacy of present vaccine.

Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus.

Coronaviruses (CoVs) are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals. Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. In this study, we created a MHV-A59 temperature sensitive (ts) mutant Wu"-ts18(cd) using the recombinant vaccinia reverse genetics system. Virus replication assay in 17C1-1 cells showed the plaque phenotype and replication characterization of constructed Wu"-ts18(cd) were indistinguishable from the reported ts mutant Wu"-ts18. Then we cultured the ts mutant Wu"-ts18(cd) at non-permissive temperature 39.5 °C, which "forced" the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis showed most of the revertants had the same single amino acid mutation at Nsp16 position 43. The single amino acid mutation at Nsp16 position 76 or position 130 could also revert the ts mutant Wu"-ts18 (cd) to non-ts phenotype, an additional independent mutation in Nsp13 position 115 played an important role on plaque size. The results provided us with genetic information on the functional determinants of Nsp16. This allowed us to build up a more reasonable model of CoVs replication-transcription complex.

Genetic analysis of murine hepatitis virus non-structural protein 16.

MHV-Wüts18 is an RNA-negative, temperature-sensitive mutant of mouse coronavirus, strain murine hepatitis virus (MHV)-A59. We have previously identified the putative causal mutation of MHV-Wüts18 as a C to U transition at codon 2446 in ORF1b, which results in a substitution of proline 12 with serine in non-structural protein 16. Here, we have used a vaccinia virus-based reverse genetic system to produce a recombinant virus, inf-MHV-Wüts18((AGC)) that encodes nsp16 serine 12 with AGC rather than UCU; a difference that facilitates the isolation of second-site revertants. Sequence analysis of nine inf-MHV-Wüts18((AGC)) revertant viruses suggests that their phenotype is most probably due to the intra-molecular substitution of amino acids in nsp16. However, the revertant viruses displayed different plaque sizes and whole genome sequencing of two revertants showed that they were isogenic apart from a mutation in nsp13. These results are discussed in the context of a model of coronavirus MHV nsp16 structure.

Establishment of the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression.

In order to establish the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression. The recombinant plasmid of pTRE-Tight-SARS-N was constructed by using the plasmid p8S as the PCR template which contains a cDNA clone covering the nucleocapsid gene of SARS-CoV HKU-39449. Restriction enzymes digestion and sequence analysis indicated the recombinant plasmid of pTRE-Tight-SARS-N contained the nucleocapsid gene with the optimized nucleotide sequence which will improve the translation efficiency. Positive cell clones were selected by cotransfecting pTRE-Tight-SARS-N with the linear marker pPUR to BHK-21 Tet-on cells in the presence of puromycin. A set of double-stable eukaryotic cell lines (BHK-Tet-SARS-N) with inducible control of the SARS-CoV neucleocapsid gene expression was identified by using SDS-PAGE and Western-blot analysis. The expression of SARS-CoV nucleocapsid protein was tightly regulated by the varying concentration of doxcycline in the constructed double-stable cell line. The constructed BHK-Tet-SARS-N cell strains will facilitate the rescue of SARS-CoV in vitro and the further reverse genetic research of SARS-CoV.

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus.

A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.

Monoclonal antibody induced with inactived EV71-Hn2 virus protects mice against lethal EV71-Hn2 virus infection.

BACKGROUND: Enterovirus 71 (EV71) is a viral pathogen that belongs to the Picornaviridae family, EV71-infected children can develop severe neurological complications leading to rapid clinical deterioration and death. RESULTS: In this study, several monoclonal antibodies (MAbs) were produced by immunizing mice with the inactived EV71 Henan (Hn2) virus strain. The isolated MAbs were characterised by in vitro neutralizing analysis and peptide ELISA. ELISA assay showed that the neutralizing monoclonal antibody 4E8 specifically reacted with synthetic peptides which contain amino acid 240-250 and 250-260 of EV71 VP1. The in vivo protection assay showed that 4E8 can protect two-day-old BALB/c mice against the lethal challenge of EV71 virus. CONCLUSION: The MAb 4E8 could be a promising candidate to be humanized and used for treatment of EV71 infection.

Sequence analysis of six enterovirus 71 strains with different virulences in humans.

Enterovirus 71 (EV71) infection is the main cause of hand, foot and mouth disease (HFMD) and has been associated with severe neurological diseases resulting in high mortalities. In this study, six EV71 strains isolated from patients with different clinical symptoms were sequenced and analyzed in a mouse model of EV71 infection. In a phylogenetic tree, based on the complete VP1 gene sequence, all six strains grouped into the C4 genotype. The sequence analysis revealed that there are nucleotide changes clustered in the internal ribosome entry site (IRES) element of the 5'-nontranslated region (5'-NTR), as well as amino acid differences clustered in the non-structural proteins. Importantly, we identified a unique amino acid difference (Val(1994)-Ile(1994)) that distinguished the more virulent strains, Anhui1 (Ah1), Henan1 (Hn1) and Henan2 (Hn2) from the less virulent strains, Chongqing1 (Cq1), Chongqing2 (Cq2) and Chongqing3 (Cq3). This amino acid difference is located in the finger domain of the viral RNA-dependent RNA polymerase 3D (3D(pol)). Furthermore, two-day-old Balb/c mice were inoculated with the Ah1, Hn1, Hn2, Cq1, Cq2 and Cq3 isolates by the intracerebral or intraperitoneal routes. All of the mice inoculated with Ah1, Hn1 and Hn2 isolates developed hind-leg paralysis and subsequently died. Mice inoculated with the Cq1, Cq2 or Cq3 isolates survived throughout the 21-day observation period. These results show that clinical isolates of EV71 associated with disease of different severity in humans have characteristic sequence differences and cause different mortality rates when inoculated into mice. These data also provide a rational basis to investigate the molecular determinants of EV71 pathogenesis using a reverse genetic approach.

[Laboratory confirmation of the first influenza A (H1N1) imported case in Mainland China].

The clinical throat swab specimen of an imported suspected case of influenza A (H1N1) was detec ted with real-time PCR, RT-PCR and subsequently confirmed by gene sequencing. The presence of influ enza A (H1N1) virus confirmed the first case with A (H1N1) infection in Mainland China.

[Construction and screening of human phage antibody library against SARS virus].

AIM: To construct human phage antibody library against SARS virus. METHODS: Human Fab genes were amplified by RT-PCR from lymphocytes of a convalescent SARS patient and cloned into vector pComb3 to construct phage antibody library. Antibodies against SARS virus were screened by biopanning with immobilized virus antigen. The binding specificity of phage antibody to SARS virus was detected by ELISA. RESULTS: 13 Ig genes were obtained by PCR. A human phage antibody library consisting of 1.3 x 10(6) clones was constructed by electrotransformation and 60 percent of the clones contained Fab genes. After three rounds of panning with SARS virus antigen, phage antibodies against SARS virus were specifically enriched. ELISA detection verified 10 positive phage antibodies that were highly specific for SARS virus. CONCLUSION: Human phage antibody library against SARS virus has been constructed successfully, from which 10 anti-SARS virus antibodies were obtained.