Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Wildlife Diseases

Isolation and Identification of H1N1Subtpye of Panda Influenza a Virus, Sequence Analysis and the Study of of Its Reverse Genetic Technology and Vaccine

Author LiDeSheng
Tutor CuiHengMin
School Sichuan Agricultural University
Course Basic Veterinary Science
Keywords Influenza virus Isolation and Identification Sequence analysis reversegenetic vaccine
CLC S858.9
Type PhD thesis
Year 2012
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Influenza virus is a member of the genus Orthomyxovirus, family Orthomyxoviridae, which divided three types from the difference of protein M and NP, and influenza virus contains eight piece of negative sense single-stranded RNA. Currently, influenza virus circulate and be epidemic in different animal populations throughout of the world countries, and do great damage to the farms, and also threat the human health at all the time. So it is very important to do deeply research in the influenza virus. In order to do that, the author collected some samples of the Pandas and firstly, we isolated and identificated a strain Panda influenza virus which can be cultured stably both in MDCK cells and9-11day eggs by HA, HI and RT-PCR assays from a Sichuan pig samples, and the virus was named A/Panda/Sichuan/0]/20]1(H1N1).Influenza A virus is one of negative sense single-stranded RNA, and its genome is composed of eight solo genes. PB1. PB2and PA are coded by fragments1,2and three genes, and their function is about the virus genome synthesis when the virus infects the cells. HA and NA protein are the most important surface protein, and concerned with the infecting cells and the antigencity of the virus. NP protein is the cod of the genome, and composed of vRNPs with the PB2, PB1and PA protein. M1and M2protein are the peplos of the genome, and it is one of the evidence of the type of the virus. NS protein is the non-construction protein of the virus, and also, it is relation to the early synthesis, but now, human are not very clear about it’function. The each gene5’end and3’end of the virus is very conservative, so it give us a chance to design the primers at the end of the3’end and the5’end, according to the reference strain reported in NCBI, we design11pairs of primers for the virus’genome, and amplified the genome with RT-PCR assays, linked the8fragments to vector pMD18-T, and transfer to the DH5a cells. After the identification of the PCR of plasmids, we sent the recombinant plasmids to shanghai shenggong to determine the sequences of the virus genome. We have constructed the homology and the phylogenetic trees to analyses of H1N1Panda influenza a virus isolates from the world. As the result, A/Panda/Sichuan/01/2011(H1N1) is composed of13595bp and5’end and3’end is very conservative. Based on the phylogenetic analyses of H1N1Panda influenza virus, we found the virus genome is both closed to2009H1N1influenza virus strain.The future of reverse genetics to generate vaccine candidates is lactiferous. The influenza reverses genetics on plasmid DNA include two system12plasmids and8plasmids. The8plasmids system is a bi-directional two-expression construct that contains cDNA virus gene flanked by an RNA polymerase1(pol1) promoter for vRNA synthesis and an RNA polymerase Π (pol Π)promoter for mRNA synthesis. The key point is to screen influenza virus strain that is high yield on MDCK, and clone its whole genome. In this study, we amplified the virus genome cDNA and cloned the eight fragments to pMD18-T vectors, after digestion of BspQl, the fragments was sub-cloned into the vector pBD, then the eight plasmids based on the pBD vectors was constructed, then rescue the A/Panda/Sichuan/01/2011(H1N1).strain which have a high-growth property and the antigen characteristic of the circulating strain. Used HA and HI assays, the recombinant virus was detected, and the viruses also be detected in the electron microscope,The plasmid pM2HA construct expressing a fusion protein of M2joined to the N terminus of the HA could transcripe and translate. The special antibody of M2e of mouse immunized pM2HA show remarkable higher titer than pCI-M2immunized group, and almost no difference to pCl-HA immunized group in HI antibody but a slightly higher neutralization antibody but not significant. After challenged by lethal dose virus, pM2HA immunized group show a satisfied effectiveness according to body weight change and survival rate(100%) and own a good heterologous protecting capacity; After challenged by moderate dose virus, pM2HA group could alleviate lesion of lung compare to control group. It is that fusion HA and M2e provied a new method and ideal to research-develop a new type SI vaccine.

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