Study On Interaction Between Nonstructural Protein Of Avain Influenza Virus And Host Protein And Histopathology

Avian influenza(AI) is a fatal syndrome of avian caused by avian influenza virus (AIV),characterized either by the general or respiratory disease,which was defined as the type A infectious disease by OIE.Since the first breakout in Italy in 1887,AIVs have occured worldwide in avian-raising countries.For the past few years,some new characters of AIV have appeared,especially for highly pathogenic avian influenza viruses(HPAIV).Antigen shift or drift has appeared easily for HPAIV,which can make the HPAIV obtain the capability to infection and make the human death,such as in HongKong and Southeast Asia.So HPAIV is more important for the public hygieology.At present,the pathogenic mechanism of AIV in chicken has been still not clear, which maybe the common results of more than one gene.Hemagglutinin(HA),Neuraminidase(NA) and Non-structural protein(NS1)were more important in the all genes of AIV.HA and NA are the key factors for the subtype,antigen variation and pathogenic regulation during influenza virus infection.It has been reported that the NS1 protein,a non-structural protein of the type A influenza virus,can interact with the host cell proteins in vitro via its RNA-binding domain and effector domain,By the protein-protein interaction,NS1 protein can shutoff the host cellular protein synthesis, antagonize the alpha/beta interferon(IFN-α/β) system,induce apoptosis and regulate the viral protein expression.So NS1 protein is an important virulence factor which decides a great damage to host cells on the cell level.Those researches about NS1 Protein were mostly carried out using the human influenza virus and its suitable cell line,but not the avian influenza virus and its natural host(chicken).To elucidate the interaction between NS1 protein and natural host(chicken) protein is not only significant to understand the function of NS1 protein and how to regulate the virulence,but also helpful for preventing and curing the infection of AIV.In present study,the histopathology method and protein-protein interaction technology were applied to study on the pathology of H5NI AIV strain on both cellular and protein level in order to document the reason why the chicken is dead shortly.The results were reported as follow:1.Genetic analysis of the NS1 Genes of H9N2 Influenza VirusesThe nonstructural genes(NS1) of nine H9N2 subtype influenza viruses isolated from diseased chickens on different farms during1998-2005 were amplified by RT-PCR and completely sequenced.The nucleotide and deduced amino acid sequences of NS1 genes of these isolates were analysis and compared.The results showed that NS1 gene of all H9N2 isolates contained 654 bp and encoded 217 amino acids.The homologies of the nucleotide and deduced amino acid of the isolates were 95.4%-99.8%and 93.6%-100%, respectively.Comparison of the amino acid sequences of NS1 proteins of these isolates with other H9N2 viruses demonstrated that NS1 proteins of the nine strains had a deletion of 13 amino acid residues at the carboxy terminus,which may be the molecule mark of the isolates in mainland China.Phylogenetic analysis revealed that the NS1 genes of these isolates fell into the same lineage and belonged to allele A.The other eight isolates belonged to the Ck/SH/F/98-1ike lineage except Ck/HN/A3/98 strain,but all the isolates derived from Ck/BJ/1/94 strain firstly isolated from mainland China in 1994.The result indicated that H9N2 subtype AIV appear differentiation following the time and the viruses belonging to Ck/SH/F/98-like acquired an epidemiological advantage in chicken population in mainland China.2.Study on the apoptosis of SPF chicken challenged with HPAIV(H5N1 subtype)The study on the apoptosis of dead SPF chicken challenged with HPAIV(H5N1 subtype) was carried out using the TdT-mediated X-dUTP nick end labling method (TUNEL) The results documented that HPAIV could induce the different apoptosis in different organs or tissues.The apoptosis in Thymus,Bursa of Fabricius,Pancreas and Kindey was more severe than the other organs or tissues.The apoptosis cells included the vascular endothelial cells,glandular epithelium cartilage corpuscle and lympholeukocyte. According to the researches about the apoptosis,it was deduced that the apoptosis, shortly happened to the chicken incubated with HPAIV,maybe the mechanism which causes the chicken death quickly.3.Study on the distribution of NS1 protein in the different organs of infected chickenThe monoclonal antibody of NS1 protein was applied to develop the immunohistochemistry method(SABC method) in order to detect the NS1 protein.The study on the distribution of NS1 protein was detected in the different organs of chicken infected with HPAIV after 30-31hours.The main organs which can detect the positive signal of NS1 protein was the heart,pancreas,kidney and cerebellum granular layer.The positive signals were stronger than the others.The positive cells were involved in small or microvascular endotheliocyte,myocardial cell,renal tubular epithelial cell,glandular epithelium and epithelium.The results illustrated that the heart,pancreas,kidney cerebellum and microvascular endotheliocyte may be the main target organs in which HPAIV can duplicated effectively and product many effective virion.In this present study,It was presumed that the cardiovascular system was important for the duplication and diffusion of AIV.At the same time,the obvious associativity between the NS1 protein and apotosis was proved. 4.Screening and identification of host protein interacted with NS1 protein using the yeast two hybrid systemIn present study,spleen cDNA library of chicken incubated with HPAIV(H5N1 subtype) was firstly constructed using the switching mechanism at 5′end of RNA transcript technology.Four proteins which can interact with NS1 protein were screened from this cDNA library using the yeast two hybrid system(Y2H).These proteins included the chicken interferon-stimulated gene12-2(ISG12-2),interferon regulatory factor 2(IRF2),Protein phosphatase 1F(PPM1F) and M protein of AIV.The interaction between NS1 protein and ISG12-2 and IRF2 was identified,both in vitro and in vivo,by bacteria two-hybrid system and GST fusion proteins sedimentation technology, respectively.It was deduced that some signal pathway associated with IFN expression may be inhibited by the protein-protein interaction,which would affect the expression of IFN and degress the IFN-mediated antiviral responses.The results would explain the function of NS1 protein and how to regulate the pathogenicity.These studies also will give some reference to the mechanism which HPAIV infects human being.5.Identification of the interaction between NS1 protein and ISG12-2 or IRF2 using the bacterial two hybrid system and GST pull-down testIn this study,the interactons between NS1 protein with ISG12-2 and IRF2 protein were further identified using the bacterial two hybrid system and GST pull-down test,respectively.The results showed that NS1 protein interacted with ISG12-2 and IRF2 protein in vivo and in vitro.It was presumed that some pathways associated with the expressing of interferon were blocked and the expressing of interferon was inhibited by the interactions,which degraded the antivirus immune response and was beneficial for the duplication and diffusion of AIV.