Biological features of novel avian influenza A (H7N9) virus.

Human infection associated with a novel reassortant avian influenza H7N9 virus has recently been identified in China. A total of 132 confirmed cases and 39 deaths have been reported. Most patients presented with severe pneumonia and acute respiratory distress syndrome. Although the first epidemic has subsided, the presence of a natural reservoir and the disease severity highlight the need to evaluate its risk on human public health and to understand the possible pathogenesis mechanism. Here we show that the emerging H7N9 avian influenza virus poses a potentially high risk to humans. We discover that the H7N9 virus can bind to both avian-type (α2,3-linked sialic acid) and human-type (α2,6-linked sialic acid) receptors. It can invade epithelial cells in the human lower respiratory tract and type II pneumonocytes in alveoli, and replicated efficiently in ex vivo lung and trachea explant culture and several mammalian cell lines. In acute serum samples of H7N9-infected patients, increased levels of the chemokines and cytokines IP-10, MIG, MIP-1ß, MCP-1, IL-6, IL-8 and IFN-α were detected. We note that the human population is naive to the H7N9 virus, and current seasonal vaccination could not provide protection.

[The proliferation of H1N1 subtype influenza viruses in A549 and BEAS-2B cells].

OBJECTIVE: Analyze the proliferation of different host H1N1 subtype influenza viruses in A549 and BEAS-2B cells. METHODS: Human, avain and swine three hosts of the H1N1 influenza viruses infected A549 and BEAS-2B cells and analyze the characteristics of different periods after inocubation. Determine the receptor binding specificity of influenza virus by hemagglutination (HA) test with RBCs with two types of receptor. And the receptors on surfaces of A549 and BEAS-2B cells were tested by flow cytometry. RESULTS: The Cell Pathologic Effect (CPE) is obvious after 24 h inoculation in A549 cells by all the H1N1 influenza viruses, moreover, the peak hemagglutinin (HA) and 50% tissue culture cell infected dose (TCID50) titers was observed after 36 h of culturing in A549 cells. Otherwise, the CPE is not typical from 24 h-120 h inoculated by the same viruses and the HA, TCID50 titers were keep low all the periods in the BEAS-2B cell after inoculation. The receptor-binding preference of H1N1 viruses used in the study was screened by HA assay and some were found with 2-6-receptor binding affinity. Both SA a-2, 3Gal and SA a-2, 6Gal receptors were detected on A549 and BEAS-2B, furthermore, receptor density on A549 cells was significantly higher than that of BEAS-2B cells. CONCLUSION: A549 cells were susceptible to human, avian and swine H1N1 influenza viruses infection and permissively for viral replication. However, BEASE-2B cells with similar receptor pattern and epithelium-derived propriety as A549 cells were unsusceptible to their infection and replication. Possible host factors involved in effective viral infection and replication were needed further study.

[Expression and purification of avian influenza virus H5N1 NP protein, and screening interaction proteins of host cells in vitro].

OBJECTIVE: To express and purify H5N1 influenza virus (A/Anhui/1/2005) NP in prokaryotic system and to explore the NP-interacting proteins of human bronchial epithelial cells BEAS-2B in vitro. METHODS: The full length H5N1 NP gene fragment was amplified by PCR, inserted into prokaryotic expression vector (pET30a) to generate NP expression plasmid pET30a-NP. After transforming pET30a-NP into E. coli (BL21), the expression of soluble NP protein was induced by IPTG. The expressed NP protein was purified by two steps with metal chelation chromatography and ion exchange chromatography. Then the total proteins of BEAS-2B cells was extracted for screening the components which have protein-protein interaction with purified NP by pull-down and LC-MS/MS methods. RESULTS: The expression of H5N1 NP protein could be induced by IPTG in bacterial system using expression plasmid pET30a-NP. The soluble NP was purified. Twenty proteins were found by pull-down and LC-MS/MS, the further experiments may be needed to prove protein-protein interaction between them. CONCLUSION: The soluble H5N1 NP fusion protein with high purity was obtained and twenty proteins were found which could interact with it by pull-down and LC-MS/MS.

[Do pigs play a role in human infection with avian influenza A H9N2 viruses].

OBJECTIVE: To understand whether pigs play a role in human infection with avian influenza A H9N2 viruses. METHODS: The target gene was amplified by RT-PCR, and the PCR product was linked to PGEM-T Vector (Promega, USA) at 4 degrees C, the recombined plasmid was transferred into dH5a bacteria, and the positive colonies were selected and identified with restriction endonuclease. Afterwards, they were sent to Liu He Tong Company in Beijing for nucleotide sequencing. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseq (Version 3.69) software. RESULTS: The genomic characterizations of A/Swine/Shandong /5/2002(H9N2) and A/Swine/Shandong/10/2002(H9N2) viruses were different from those of H9N2 viruses which were isolated either from men or from chickens. The genomic characteristics of H9N2 viruses isolated from humans in China mainland were similar to those of H9N2 viruses isolated from chickens. Whereas, the genomes of H9N2 viruses isolated from men in Hong Kong, China were closely related to those of H9N2 viruses isolated from quails. Avian influenza A H9N2 viruses not only have wide range of host, but their genomes are also diverse. CONCLUSION: Avian influenza A H9N2 viruses can directly infect human. Avian influenza A H9N2 viruses did not require to pass through the pigs as mixing vessels prior to infecting man.

[Sequencing of adenovirus type 7 vaccine strain fragment and characterization of the hexon encoding gene].

OBJECTIVE: To complete the full-length sequencing of the human adenovirus type 7 vaccine strain (Ad7v) for novel vector constructing. METHODS: The Ad7v DNA was digested with SalI and the 17.5-68.0 map unit (mu) fragment was cloned and sequenced. The homology of encoding sequence of Ad7v hexon to those of group A,C,D,E,F and other numbers of group B was accomplished with the software CLUSTAL.V. The three-dimensional structure of the Ad7v hexon was predicted with the RasMo12.71. RESULTS: The fragment contains 17,596 bp, part of E2 and late gene L1, L2 and L3 were encoded by this region. Polypeptide encoded by hexon gene lies in L3 region, which is composed of 934 amino acids. Multiple sequence alignment with the other nine known hexon protein sequences suggested that the variable sequences are mainly concentrated on seven regions, namely hypervariable regions (HVRs). The seven HVRs are related to type-specificity and group-specificity. The three-dimensional structure of the Ad7v hexon revealed that the variable regions are located in the I1 and I2 loops of the molecule mostly on the tower of the hexon. CONCLUSION: The full-length genome sequencing of Ad7v was accomplished at last. Since the deduced amino acid sequence of Ad7v hexon was quite different from other adenoviral vectors such as Ad5 and Ad2, this virus can be potentially used for the construction of novel gene delivery vectors to counterpart the immunity to the vectors widely used at present.

[Expression of the main structural antigen VP6 of human rotavirus by recombinant adenovirus and immune responses induced in vivo].

BACKGROUND: Constructing replication defective recombinant adenovirus vector expressing the group specific antigen VP6 of human rotavirus and studying the immune responses induced in vivo. METHODS: The cDNA of full length VP6 was inserted into the adenovirus vector pShuttle-CMV, and recombinant adenovirus genome DNA was obtained through homological recombination in E.coli,then the recombinant adenovirus was gained after transfecting 293 cell line with the genome DNA. Gene integration of VP6 in resultant adenovirus was confirmed by PCR and Southern blot, respectively gene expression was confirmed in 293 cells by Western blot. BALB/c mice were immunized intranasally(inl)and orally(ora), respectively, to test the immunization effects of the adenovirus. RESULTS: Recombinant adenovirus named rvAd-VP6 was obtained. The cDNA of VP6 was integrated in the adenovirus and was able to be expressed in 293 cells stably. The systemic immune responses to rotavirus VP6 could be induced effectively in both oral and intranasal group, the titer of serum IgG antibody in the two group of mice were 1?1 000 and 1?10 000-1?100 000, respectively. In addition to IgG, the serum IgA specific to VP6 could also be detected at a titer of 1?10-1?100. Secretory IgA(sIgA) was detected in both lung lavage fluid and intestinal homogenate when administered intranasally to BALB/c mice, whereas only found in intestinal homogenate in the oral group. The results indicated that the immunization efficacy of intranasal inoculation was superior to that of oral inoculation. CONCLUSIONS: The recombinant adenovirus vector expressing human rotavirus VP6 was successfully constructed, its ability to induce immune responses has laid a solid foundation for the development of rotavirus genetically engineering vaccine against rotavirus infection.