Evolution Of H5N1 Influenza Virus From Tigers And Lions And Construction Of Recombinant Canine Adenovirus Type 2 Expressing Hemagglutinin Gene

Advisor:Xia Xian-zhu,Membership of Chinese Academy of EngineeringSince late 2003, H5N1 virus has reached epizootic levels in domestic fowl in anumber of Asian countries. More recently, the H5N1 virus spread to poultry in 45countries in Asia, Europe, and Africa. Moreover, H5N1 virus was reported to be ableto transmit to humans with a high mortality rate. Of 192 laboratory-confirmed cases,109 patients died of H5N1 virus infection as of 6 April 2006. These events have led tosignificant global concern about the potential for this virus to evolve to pandemicproportions with the capacity to cause millions of deaths. The first outbreak of H5N1influenza virus infected tigers was reported in China in 2002. This report is the first ofH5N1 virus infection causing death in nonhuman mammals.Twenty-one specimens of lung of tigers and lions, died of high fever, anorexia andtwitch, were collected from Beijing, Harbin, Taiyuan, Yichang, Guilin, Datong,Zhengzhou, Shanghai, Nanjing, and Tangshan from 2002~2005. All of specimensdetected by RT-PCR with H5N1 virus special primers, 18 specimens were positive. 15isolates of influenza-like virus were obtained from the positive specimens. The isolateswere confirmed to be H5N1 subtype influenza A virus by characterization of theirvirology. Two hundreds and seventy-five sera from 1998~2005 were tested forevidence of exposure to H5N1 virus by hemagglutination inhibition test. Among thosefrom 2002~2005, 98 were HI antibody positive. These results indicated that H5N1influenza rapidly spread to capture tigers and lions from south to north in China in ashort time.To test the pathogenicity of the Tiger H5N1 influenza viruses(TIV), the cell andanimal infection experiments were carried out. TIV infected PK-15, Vero, F81 andBHK-21 cell with obvious cytopathic effect(CPE). The amount of IVPI in chickens isto 2.23, hence the TIVs could be determined to be strains of HPAI subtype. Theaverage half mouse lethal dose (MLD50) of TIVs was from10-6.8 to 10-7.1/50μl. Threecats infected with TIVs showed clinical signs, including anorexia, depression andraised body temperature. One survived in the infection and the other two died. Onnecropsy, dull red focus of infection was found on the lung of dead cats. Histologiclesions observed in the infected cats showed diffusing alveolar damage and a numberof macrophage infiltrating and a few of protein serosity effusions in alveolus.Influenza A virus antigen was demonstrated in endochylema of bronchia epithelialcells and sporadic macrophage by the method of immunohistochemistry. H5N1 TIVswere recovered from the lung, brain, heart and kidney tissues. The virus titers in thelung were the highest, ranged from10-5.9 to 10-6.8 /g.To shed light on the evolution and genetic backgrounds of these TIVs, 9 of 15isolates were selected for whole genome analysis. Homology analysis showed that 8gene segments of TIVs had high homology with the gene segments of H5N1 influenzaA virus (96%~100%). Phylogenetic analysis showed that the TIVs from2002~2004 isolates clustered within the lineage of the 1996~2004 H5N1 avianand human isolates from china, the TIVs from 2005 isolates clustered within thelineage of the 2003-2005 H5N1 avian and human isolates from China, Japan,South Korea, and Russia. Genetic analysis revealed that the HA proteolyticcleavage site of TIVs harbor multiple basic amino acids(RRRKKR) , indicatives ofhigh virulence. All 9 isolates of TIVs had a glutamine at position 222 and a glycine atposition 224 of HA protein,which are related to receptor binding sites specific foravian species. All 9 isolates of TIVs contained a 20 amino acid deletion in the NAstalk (49~68), and contained a 5 amino acid deletion at positions 80~84 of NS1protein. There is no mutations related to amantadine resistance properties in M2protein of TIVs and therefore, the TIVs were presumably sensitive to amantadines.To find out the molecular determination of the H5N1 virus infected tigers, thereceptor binding specificity of all the TIVs and the type of influenza virus receptor intiger trachea were studied. All of the sialic acid was removed from chicken red bloodcells (CRBCs) using Vibrio cholerae neuraminidase (VCNA). Subsequently,resialylation was performed using α2,3-(N)-sialyltransferase orα2,6-(N)-sialyltransferase and 1.5 mM CMP-sialic acid. To assay the receptor bindingspecificity of tiger isolates, the Hemagglutination assay was used with treated CRBCs.All TIVs binds the α2,3 sialic acid(Avian) cellular receptor. To identify the type ofreceptor in tiger trachea, tiger trachea sections were incubated withdigoxigenin-labeled SNA and MAA lectin, respectively. Subsequently the sectionswere incubated with FITC-conjugated anti-DIG antibody. The results showed thatthere are abundant α2,3 sialic acid linked in tiger trachea.To develop a new type of vaccine for Felidae influenza prevention, recombinantreplication-competent canine adenovirus Type 2 expressing hemagglutinin gene ofH5N1 subtype tiger influenza virus was constructed. A/tiger/Harbin/01/2003(H5N1)HA gene was cloned into PVAX1. The HA expression cassette which included CMVand HA and PolyA was ligated into the E3 deletion region of pVAXE. Therecombinant plasmid was named pEHA. The pEHA and the pPoly2-CAV2 weredigested with Nru I/Sal I, respectively. The purified Nru I/Sal I DNA fragmentcontaining the HA expression cassette was cloned into pPoly2-CAV2 to generate therecombinant plasmid pCAV-2/HA. The recombinant genome was released frompCAV-2-HA, and was transfected into MDCK cells by Lipofectamine. Therecombinant virus named CAV-2-HA was obtained. Anti-H5N1influenza virus HIantibody (1:8~1:16) could be detected in cats immunized with CAV-2-HA.