Pseudotyped Virus for Avian Influenza Virus and Detection of Swine Influenza Virus by Multi-RT-PCR
|School||Huazhong Agricultural University|
|Course||Preventive Veterinary Medicine|
|Keywords||Influenza Virus Hemagglutinin protein Multi-RT-PCR Pseudotyped virus|
Avian influenza (AI) caused by the AIV (Avian influenza virus) is a common infectious disease of birds. World Organization for Animal Health (OIE) and China defined it as a category A infectious disease. In recent years, AI had new features, especially the highly pathogenic avian influenza virus (HPAIV) through antigen drift and antigen shift, has been received the ability to infect people, even caused the death of people. This gives a more important public health significance. At present, AIV-depth study of pathogenesis, it is still not clear, there are some mechanisms, including protein hemagglutinin (HA) is a major virus invasion of host-related factors, and mediated virus attachment and membrane fusion. HA used cell surface sialic acid residues (SA) as the receptor is the receptor-binding protein, and can stimulate the host immune response, is a good model to study the avian flu virus from entering host cells. Fake virus is a tool to study the relationship between the virus and host cell, compared to a real virus, not only the membrane capsule just has the study glycoprotein, without the interference of other protein, but also it is the replication-deficient virus because nucleic acid molecules encoding capsular protein gene was modified, so that the virus can only be "one cell cycle" of infection, has a strong bio-security. In this study, the false AIV virus expressing HA protein was used to study the mechanism of host cell invasion.1 The establishment of pseudovirus in A/duck/Hubei/W1/2004 (H9N2) strain AIV HA protein of Avian influenza virusIn this study, the hemagglutinin (HA) protein of H9N2 were incorporated into HIV particles, and generated H9 hemagglutinin pseudotyped virus (HIV/H9-HA). HA gene of avian Influenza Virus (AIV) H9N2 subtype was amplified by RT-PCR and cloned into pcDNA3.1(+) vector, then cotransfected with retroviral vectors HIV-1 gag/pol encoded, pCMV△8.2 andthe HIV packagable vector encoding aβ-galactosidase (β-gal) reporter gene, pHR’-CMVLacZ into 293T cells. The HA protein expression could be verified by Western blot .One main bands which is consistent with the expected molecular weight for the full-length HA were seen. Further, Infection activity of HIV/H9-HA were confirmed by LacZ staining and infecting 293Tcells. For hemagglutination test, HIV/H9-HA was detected to make chicken erythrocyte agglutinate, and HA titer≧ 1:8. To test whether the AIV/H9-HA was infectious and displayed the similar host range as AIV, we infected 293T, Vero, PK-15, and MDCK cell strains with HIV/H9-HA. The results showed that HA pseudotyping particles have wide cell tropism. Swine Influenza (SI) caused by influenza virus from pigs (Swine Influenza virus, SIV) is an acute, highly contagious respiratory disease. SI leaded to not only a great loss of pig-raising industry, but also has important public health significance. Virus Isolation and Identification of the traditional methods are time-consuming, can not be fast and accurate diagnosis of SI, therefore in this experiment, the specific HA subtype protein as the target protein was used to establish multiple RT-PCR detection methods for the rapid detection of swine influenza and typing.2.The establishment of Multi-RT-PCR for diagnostic methods in Swine influenza virusThe three subtypes (H1, H3, H5) of gene sequences in swine influenza virus, published in GenBank reference, were applied with Biosystems analysis software selection, designing three subtype-specific primers for swine influenza virus to establish multiple RT-PCR method for diagnostic methods in Swine influenza virus. Porcine reproductive and respiratory syndrome virus amplified in test-specific, classical swine fever virus, porcine pseudorabies virus are HIV negative, and non-specific bands does not appear in cross-test. Its sensitivity has increased after optimization of experimental conditions. The detection of clinical samples, multiple RT-PCR method and virus isolation of chicken embryo in line illustrate the reliablity of the method, which can be applied to clinical.In this study, the establishment of multiple RT-PCR method for rapid and accurate clinical diagnosis of swine flu conditions are provided, establishing the expression of H9 subtype avian influenza HA protein, low-pathogenic avian influenza virus technology model leave in order to further the avian influenza virus receptors, and epitope studies against influenza virus drug screening. A more efficient and convenient diagnostic method, as well as safe and effective vaccine have provided a train of thought and technical support.