| Highly pathogenic avian influenza （HPAI） is a worldwide infectious disease with great damaging power. Breakouts of HPAI often destroy local poultry industry. It is a list A diseases by the Office International des Epizooties （OIE） and is on the list of animal infectious diseases by the International Treaty Banning Biological Weapons. Many domestic and wild fowls and birds are susceptible to HPAI virus （HPAIV）. The possible major cause for the worldwide epidemic of HPAI is the carrying of the virus by migrating birds, showing birds, free range fowls, and water fowls. In addition, avian influenza virus has many serological subtypes and high variability related to both antigenic shift and antigenic drift. Large variations are found among viruses of different subtypes, and sometimes among different isolates of the same subtype. Furthermore, AIV has been demonstrated in recently years to be able to break species barrier and infect mammals and sometimes humans directly, which increases the difficulty of controlling avian influenza. In recent years, HPAI has been happening non-stop worldwide, has done great damages and attracted much attention in all countries involved.Based on the epidemic of HPAI, we carried out studies to examine the pathogenic characteristics of HPAIV of local isolates, to clone and perform phylogenetic analysis of the HA and NA genes of HPAIV, and to develop a H5N1 AIV-pseudorabie virus （PRV） recombinant vaccine for prevention and control of avian influenza infection in pigs. Major results are as follows.1. Pathogenic characteristics of H5N1 subtype AIV local isolates Pathogenicity in whole organism level:A local H5N1 isolates from goose （ZFE strain） had EID50 of 10-9.8/0.2ml, IVPI of 2.85 （228/80）, and ICPI of 3.00 （240/80）. Chickens challenged with the H5N1 virus fell ill within 36 h and 100% died from infection, and their clinical symptoms and pathological changes were of typical HPAI, indicating that the isolate had strong toxicity and pathogenicity. Pigs challenged with this and several other local H5N1 AIV primarily showed respiratory symptoms and pathological changes of typical pneumonia. At 45 days after challenge, all the serum samples from infected pigs were positive for AIV as detected by ELISA, demonstrating that pigs are susceptible to AIV H5N1 subtype. Pathogenicity at the cellular level:Typical morphological characteristics of apoptosis were observed in MDCK cells infected with isolated HPAIV using electron microscopy. Changes include shrinking of the cell, condensation of cell nucleus, indentation of cell membrane, formation of apoptosis bodies, and compartmentation of mitochondria region. The dynamics of apoptosis in cultured cells induced by one AIV H5N1 subtype isolate from chicken was studied by observing changes throughout the time course （0, 6, 12, 18, 24, 30 h）. Changes characteristic of early stage apoptosis can be observed at 12 h after infection, as well as DNA ladder which is indication for biochemical changes. At this time, however, no obvious cytopathic effect （CPE） could be observed under inverted light optical microscope, indicating that before cultured cells show apparent pathology, cells have already started apoptosis mechanism.Spleen and brain tissues of AIV H5N1 subtype infected chicken have many brown cells which are strongly positive in the TUNEL assay, suggesting that the AIV has high affinity for spleen cells, and induces their apoptosis. This might has important implication in the high pathogenicity of HPAIV: since the virus directly acts on the lymphoid tissue cells of the host and induces their apoptosis, leading to reduction of lympoid cells, immunosuppression, lymphoid organ damage and lack of host immune resistance to AIV infection.Pathogenicity of HSN1 subtype AIV at the molecular level:Nucleotide sequences of HA gene of the H5N1 AIV isolates were compared. All of the translated amino acid sequences have the R-R-R-K-K-R↓G motif at the supposed cleavage site at position 341-347. The six consecutive basic amino acids are considered one of the molecular characteristics of HPAIV.2. Cloning of the HA and NA gene of H5N1 subtype AIV isolates and their phylogenetic analysisIn this study, HA and NA gene were cloned from five H5N1 AIV strains isolated from local chicken, duck, wild duck, and wild goose during the avian influenza outbreak from the winter of 2003 to the spring of 2004. The cloned HA and NA genes were sequenced. Phylogenetic analysis were performed on their sequence in comparison with reference sequences of H5N1 AIV strains in water fowl, chickens and pigs found in Vietnam, Thai, Indonesia, and China’s Hong Kong, Guanggong, Guangxi, Shanghai, Shandong. The study aimed to understand the molecular epidemiology features and genetic evolutional characteristics of local H5 subtype HPAIV on the gene level, and to provide evidence for forecasting avian influenza outbreak and evolving trend, which is of economic and public health significance.The full length of the HA gene cDNA from all five local H5N1 AIV isolates were 1707 bp, coding for 568 amino acid residues with intact reading frame. The full length of the NA gene cDNA from all five local H5N1 AIV isolates were 1350 bp, coding for 449 amino acid residues with intact reading frame.When HA nucleotide sequences were compared, sequence homology was found highest （99.6%～99.9%） among the 4 2004 Hubei AIV isolates. They are closely related and all evolved from the HA gene of one wild goose AIV isolate., supporting the view that water fowls are major sources for H5N1 AIV in epidemic regions.One AIV Hubei duck isolate is evolutionally distant from others and it is the common ancestor to all strains plotted in the phylogenetic tree in the current study. Nucleotide variations were identified at multiple locations in the HA signal sequence, HA1 and HA2 region, indicating the stain’s importance in epidemiology.HA gene from a Shandong pig and a Guangdong goose H5N1 AIV isolate evolved into Jilin chicken isolate and further evolved to two major branches including Hubei branch and East Asia branch. This provides evidence that in the "Avian-Swine-Human" interspecies transmission chain, pigs serve an important role as intermediate host and mixer. A Vietnam human H5N1 AIV HA gene has evolved via a unique route and reached human in relatively short time, imposing a tremendous threat onto the human species. From HA gene phylogenetic tree, this Vietnam human H5N1 AIV has low homology with 5 Hubei isolates and they also differ in recent evolutionary route, indicating that they are not related directly.From the phylogenetic tree of NA gene, the A/DK/guangxi/53/2002 （H5N1） isolate from our country shares low homology with a 2004 Tailand quail isolate, and shares lowest homology （95.6%） with a 2004 Tailand human isolate. It seems that there is no direct relationship between the Thailand quail influenza, the Thailand avian influenza infection in human and avian influenza in our country.From the genetic phylogenic tree, it appears that AIV NA gene had experienced the evolutional process of "from duck-back to duck". This provides supporting evidence that water fowl not only are giant reservoir for AIV, but also become susceptible to AIV and form important sources for AIV transmission.The NA and HA gene of the four local 2004 H5N1 AIV isolates （JZJ, ZFE, TMJ, XFY） are in the same branch as one H5N1 AIV strain （327DW） we isolated in 2002. They share 99.6～99.9% nucleotide homology and are closely related, suggesting little molecular variation among H5N1 subtype HPAIV circulating in Hubei since 2002.3. Construction of H5N1 subtype AIV-Psudorabies virus recombinant vaccine strainThe aim is to insert the major antigenic gene of H5N1 AIV, HA, into PRV Ea TK-/gE-/gI-/LacZ+ parent strain, to construct H5N1 subtype AIV-PRV recombinant live vectored vaccine. The PRV in the vaccine vector could prevent psudorabies virus infection in pigs, and the insertion of H5N1AIV-HA could induce specific immune response to AIV HA. This could provide technical reserve for preventing H5N1 subtype HPAIV infection in pigs and has important public health implications.The full length H5N1 AIV HA gene was cloned into shuttle vector pIECMV. The plasmid and the genome of PRV TK-/gE-/gI-/LacZ+ were used to co-transfect IBRS-2 cells to obtain recombinant PRV TK-/gE-/gI-/HA+ strain. Cells infected with recombinant virus has higher fluorescent intensity （HA） on their cell membrane than the nucleus, suggesting the primary cellular location of HA protein is the cell membrane, which might be of advantage in inducing strong immune response.The TK-/gE-/gI-/HA+ recombinant vaccine had similar good growth characteristics as the parent strain in IBRS-2 cells. Immunization with the recombinant vaccine induced anti-AIV antibodies. Meanwhile the profile and level of anti-PRV levels were comparable with the parent PRV vaccine, indicating the recombinant PRV-HA vaccine could be potentially useful as vaccine against both PRV and AIV in pigs. More detailed and deeper work regarding the recombinant vaccine, including HA expression in vivo, level of cellular and humoral immunity and protective immunity, will be studied in the future. |
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