Pathogenesis Of H5N1 Influenza Virus In Dogs And The Tentative Exploration Of Humanized Antibody

Since 1997, highly pathogenic avian influenza viruses(HPAI) of the H5N1 subtype have caused numerous outbreaks in poultry in Southeast Asia, accompanied with the infected human case. Remarkably, HAPI H5N1 influenza viruses had changed quietly, it had became more pathogenic in various hosts because of its different property, especially for mammalian species. A fatal infection of HAPI H5N1 influenza virus in a dog was documented in Thailand during the outbreak of AI. The serologic detection of 629 village dogs examined for H5-specific antibodies were reported positive. It demonstrated that these dogs may have been likely infected by influenza virus. However, some other experimental data showed no obvious clinical signs and no pathological lesions in dogs after infecting H5N1 virus, so it was conflicting on H5N1 influenza virus infection in dogs.To elucidate the susceptibility of dogs to H5N1 influenza viurs, dogs were inoculated with A/bar-headed goose/Qinghai/3/05 (BHG/QH/3/05), clade 2.2, which were isolated in Qinghai lake and predominant in China. Twelve dogs divided two groups were inoculated with 106 50% egg-infectious dose(EID50) of AIVs by intranasaly (i.n.) or intratrachealy (i.t.), respectively. The dogs showed flu-like disease symptoms, including anorexic, fever, conjunctivitis, labored breathing and cough, and one i.t. inoculated animal died on day 4 post-infection. Replication of H5N1 virus were detected in the upper and lower respiratory tract. Virus shedding was detected from all 6 animals inoculated i.n. and one inoculated i.t. Virus replication was detected in all animals that were euthanized on day 3 or 5 post-infection and in the animal that died on day 4 post-infection. Our results demonstrate that dogs are highly susceptible to H5N1 AIV and may serve as an intermediate host to transfer this virus to humans.In order to explore the method to neutralize the virus in vivo, we employed the technology of phage display to screen the variable fragments pool with affinity for candidate which could be the backbone of humanized antibody. Firstly, the variable fragment of heavy chain and light chain was amplified from the total mRNA extracted from immunized rhesus macaque lymphocytes and subsequently the single-chain variable fragments(VH-Linker-Vλand VH-Linker-Vκ), which were constructed by splicing overlap-extension (SOE)PCR, were displayed on the phage surface. The phage library was constructed in E.coil TG1. Panning of the library of 2.2×106 clones by ELISA allowed the isolation of L11, a high affinity scFv which was further confirmed by SDS-PAGE, Western blot. Subsequently, its activity was analyzed by the method of Indirect Immunofluorescence Assay (IFA) and competitive inhibition ELISA, Results showed that a band about 32ku could specially to bind the virus. Analysis of IgBlast results revealed the Vλgene sharing 91.5% identity with those of human immunoglobulin germ line genes, while 75% identity in VH gene.In order to express a humanized antibody against H5N1 virus, the eukaryotic vectors of expressing the marine-humanized antibody were constructed. Its variable fragments was obtained from hybridoma cell secreted anti-HA specific antibody. Analysis and alignment with IgBlast showed that the homology of marine heavy and light variable genes compared with marine germline genes reached to 92.2% and 93.6%, respectively. The reconstruction of humanized antibody was launched after identification of CDRs. The marine CDRs were kept and discrepancy of amino acids were replaced with humans and eliminated the immunogenicity for human as much as possible. The whole antibody fragments were synthetic and cloned into the vector of pOptiVEC and pcDNA3.3, and then transfected into CHO-dhfr- cell. The whole antibody was expressed under the pressure of dihydrofolate reductase. Our results demonstrated that the humanized antibody could be expressed with this method, the heavy chain(55kD) was also analyzed by Western blot. In our study, the successful construction and expression of the marine-humanized antibody presented a platform which could be used for producing different humanized antibody against influenza virus in the future.