Hongbo Zhang

Characterization of an H10N8 influenza virus isolated from Dongting lake wetland

BackgroundWild birds, especially those in wetlands and aquatic environments, are considered to be natural reservoirs of avian influenza viruses. It is accepted that water is an important component in the transmission cycle of avian influenza virus. Monitoring the water at aggregation and breeding sites of migratory waterfowl, mainly wetland, is very important for early detection of avian influenza virus. The epidemiology investigation of avian influenza virus was performed in Dongting lake wetland which is an international important wetland.ResultsAn H10N8 influenza virus was isolated from Dongting Lake wetland in 2007. Phylogenetic analysis indicated that the virus was generated by multiple gene segment reassortment. The isolate was lowly pathogenic for chickens. However, it replicated efficiently in the mouse lung without prior adaptation, and the virulence to mice increased rapidly during adaptation in mouse lung. Sequence analysis of the genome of viruses from different passages showed that multiple amino acid changes were involved in the adaptation of the isolates to mice.ConclusionsThe water might be an important component in the transmission cycle of avian influenza virus, and other subtypes of avian influenza viruses (other than H5, H7 and H9) might evolve to pose a potential threat to mammals and even humans.

Cross-protection against influenza virus infection by intranasal administration of M2-based vaccine with chitosan as an adjuvant.

Influenza vaccines based on conserved antigens could provide cross-protection against infections by multiple subtypes of influenza A virus. Influenza matrix protein 2 (M2) is highly conserved in all influenza A strains. In this study, we deleted the transmembrane domain of the M2 of the avian influenza virus (AIV) A/Chicken/Jiangsu/7/2002 (H9N2) strain to create an M2 without a transmembrane domain, named sM2, which was efficiently expressed in Escherichia coli. The sM2 protein was administered intranasally to mice in combination with chitosan adjuvant three times at an interval of 3xa0weeks. Three weeks after the last immunization, the mice were challenged with a lethal dose (5xa0×xa0LD50) of A/Chicken/Jiangsu/7/2002 (H9N2) virus, PR8 (H1N1) virus and A/Chicken/Henan/12/2004 (H5N1) virus. The protective immunity of the vaccine was evaluated by determining the survival rates, residual lung virus titers, body weight, and the serum antibody titers of the mice. Nasal administration of 15xa0μg sM2 in combination with chitosan completely protected mice against the homologous virus and protected 90 and 30% of the mice against the heterologous H1N1 and H5N1 viruses, respectively. The study indicated that the sM2 protein was a candidate antigen for a broad-spectrum influenza virus vaccine and that the adjuvant chitosan improved the efficacy of the sM2 vaccine.

Characterization of highly pathogenic H5N1 avian influenza viruses isolated from poultry markets in central China.

H5N1 highly pathogenic avian influenza (HPAI) viruses have seriously affected the Asian poultry industry since their recurrence in 2003. While surveillance in southern China has revealed that H5N1 viruses underwent extensive genetic reassortment to generate many different viral genotype viruses, little is known concerning the genotypes of H5N1 virus that circulated in central China in recent years. In this study, 16 H5N1 influenza viruses were isolated from the poultry market in central China during late 2006 and early 2007, and the genotypes and pathogenicity of the viruses were identified and characterized. All eight segments of each virus were sequenced, and phylogenetic analysis showed that the two surface glycoprotein genes, hemagglutinin (HA) and neuraminidase (NA), of all the viruses were closely related to the H5N1 viruses isolated in poultry in southern China since 2006. Phylogenetic analysis of the internal protein genes indicated that four viral genotypes circulated in poultry markets in central China. The virulence of 7 of the 16 isolates was tested in chickens and mice. The results showed that the 7 isolates were highly pathogenic for SPF chickens, and had a varied virulence in mice. Our results indicate that the H5N1 viruses circulated in central China have diversified characteristics of genotype and virulence.

Characterization of a pathogenic H9N2 influenza A virus isolated from central China in 2007

The entire genome of the A/Chicken/Hubei/C1/2007 (H9N2) virus, isolated from central China in 2007, was completely sequenced and phylogenetically analyzed. Phylogenetic analysis demonstrated that A/Chicken/Hubei/C1/2007 (H9N2) virus represents multiple reassortant lineages, with genes coming from the early mainland China strain (Ck/Bejing/1/94), an H9N2 virus with special genotype (Ck/shanghai/F/98) and other lineages from poultry in Asia. Infection studies indicated that A/Chicken/Hubei/C1/2007 (H9N2) virus replicated efficiently in MDCK cells and in BALB/c mice. The H9N2 virus also replicated to high titers in chicken respiratory tracts and caused overt clinical signs in chickens. Our results suggest that attention should be paid to the natural evolution of H9N2 influenza viruses and to the control of H9N2 influenza viruses in animals.

Characterization of low-pathogenic H6N6 avian influenza viruses in central China

Three strains of H6N6 subtype avian influenza virus (AIV) were isolated from live-poultry markets of central China during 2009-2010. A phylogenetic analysis showed that these isolates originated from gene reassortment among different virus lineages of the H6 subtype. In an experimental infection of animals, the selected isolate was non-pathogenic for chickens and low-pathogenic for mice. The wild-type isolate was capable of replication in mouse lung without prior adaptation, and the virulence to mice increased rapidly during adaption in mouse lung. The genomes of viruses of passage 0 (P0), P4, and P8 were sequenced and compared, and virulence-related amino acid substitutions were found in multiple sites during mouse lung passage.

Induction of cross-protection against influenza A virus by DNA prime-intranasal protein boost strategy based on nucleoprotein.

BackgroundThe highly conserved nucleoprotein (NP) is an internal protein of influenza virus and is capable of inducing cross-protective immunity against different influenza A viruses, making it a main target of universal influenza vaccine. In current study, we characterized the immune response induced by DNA prime-intranasal protein boost strategy based on NP (A/PR/8/34, H1N1) in mouse model, and evaluated its protection ability against a lethal dose challenge of influenza virus.ResultsThe intranasal boost with recombinant NP (rNP) protein could effectively enhance the pre-immune response induced by the NP DNA vaccine in mice. Compared to the vaccination with NP DNA or rNP protein alone, the prime-boost strategy increased the level of NP specific serum antibody, enhanced the T cell immune response, and relatively induced more mucosal IgA antibody. The overall immune response induced by this heterologous prime-boost regimen was Th-1-biased. Furthermore, the immune response in mice induced by this strategy provided not only protection against the homologous virus but also cross-protection against a heterosubtypic H9N2 strain.ConclusionsThe NP DNA prime-intranasal protein boost strategy may provide an effective strategy for universal influenza vaccine development.

Perpetuation of H5N1 and H9N2 avian influenza viruses in natural water bodies.

Water bodies are an important route for the spread and transmission of avian influenza virus (AIV). The determining factor for an AIV to transmit through diffusion in water is the term of viability of the virus in the water body. To better understand the perpetuation of AIV in natural water bodies, and thus the risks of AIV spread and transmission via such bodies, we systematically studied the inactivation dynamics of two AIV strains (H5N1 and H9N2) at different temperatures in water bodies of important migratory bird habitats within China (Dongting Lake, Poyang Lake, the Hubei segment of the Yangtze River and Qinghai Lake). We also studied the impact of water-borne micro-organisms on the perpetuation of AIV. Our findings indicated that water is very likely an important route for the epidemic spread of AIV, especially during the autumn and winter seasons. In addition, water-borne micro-organisms might antagonize the persistence of AIV.

Characterization of H9N2 influenza viruses isolated from Dongting Lake wetland in 2007

In 2007, a total of eight H9N2 influenza viruses were isolated from the water and fowl feces in Dongting Lake wetland, China. The genomes of the eight viruses were sequenced, and all eight gene segments were subjected to phylogenetic analysis. The results showed that all the isolates belonged to the same genotype, in which the HA, NA and NS gene segments were Chicken/Beijing/94-like; the PB2, PB1, PA and NP gene segments were Chicken/Shanghai/F/98-like; and the M gene was Quail/Hong Kong/G1/97-like. Animal experiments showed low pathogenicity of the selected viruses for chickens, although some chickens died after inoculation. The viruses showed no overt clinical signs in mice, but they could replicate in murine lungs prior to adaptation.

Characterization of an H4N2 avian influenza virus isolated from domestic duck in Dongting Lake wetland in 2009

In January 2009, an H4N2 subtype of avian influenza virus [A/duck/Hunan/8-19/2009 (H4N2)] was isolated from domestic ducks in Dongting Lake wetland. The whole genome of the virus was sequenced and the results indicated that multiple gene segments of the virus had a high homology with viruses isolated from wild waterfowl, which indicated that the virus was probably transmitted from wild waterfowl to domestic ducks. Phylogenetic analysis revealed that the each gene belonged to the Eurasian lineage of avian influenza viruses, but genetic reassortment occurs between viruses of different subtypes.

An adjuvanted inactivated murine cytomegalovirus (MCMV) vaccine induces potent and long-term protective immunity against a lethal challenge with virulent MCMV

BackgroundHuman cytomegalovirus (HCMV) is a ubiquitous pathogen that causes serious problems in immunocompromised or immunologically immature hosts. Vaccination is the preferred approach for prevention of HCMV infection, but so far no approved HCMV vaccine is available. In this study, we assessed the immunogenicity and protective immunity of a formalin-inactivated murine cytomegalovirus vaccine (FI-MCMV) in a mouse model in combination with adjuvants MF59, alum, or chitosan.MethodsSpecific-pathogen-free BALB/c mice aged 6–8 weeks were immunized twice, 3xa0weeks apart, with various doses of FI-MCMV (0.25xa0μg, 1xa0μg, 4xa0μg) with or without adjuvant. Mice were challenged with a lethal dose (5u2009×u2009LD50) of a more virulent mouse salivary gland-passaged MCMV 3xa0weeks after the second immunization. The protective immunity of the vaccine was evaluated by determining the survival rates, residual spleen and salivary gland viral loads, body weight changes, and serum anti-MCMV IgG titers.ResultsImmunization with FI-MCMV vaccine induced a high level of specific antibody response. Antigen sparing was achieved by the addition of an adjuvant, which significantly enhanced the humoral response to vaccine antigens with a wide range of doses. The level of live virus detected in the spleen on day 5 and in the salivary glands on day 21 after the lethal challenge was significantly lower in adjuvant-treated groups than in controls. Survival rates in adjuvant-treated groups also increased significantly. Furthermore, these protective immune responses were sustained for at least 6xa0months following immunization.ConclusionsThese results show that inactivated MCMV vaccine is effective, and that the adjuvanted FI-MCMV vaccine provides more effective and longer-term protection than the adjuvant-free vaccine.