The Molecular Basis Of Antigenic Variation And Crossing Host Barrier To Infect Mammalian Model Of H9N2 Avian Influenza Viruses

H9N2 subtype avian influenza viruses(AIV) have widely circulated in the world since its first detection from turkeys in Wisconsin in 1966.And have made huge economic losses for poultry industries and damage for human being's health.H9N2 avian influenza virus was firstly isolated from chickens in Guangdong province in 1994,and then the viruses were widely spread in Mainland China.An inactivated vaccine derived from an early isolated, A/Chicken/Shandong/6/96(CK/SD/6/96),has been used in layers and breeders in chicken farms since 1998.However,virus infection still occurred,sometimes even in the vaccinated flockes.In this study,we chose some representative strains of H9N2 viruses that isolated from chickens and ducks from different provinces in China from 1996 to 2002,used reverse genetics system to try to answer the questions:1) the molecular basis of antigenic drift of H9N2 avian influenza viruses 2) the molecular basis of H9N2 avian influenza viruses crossing the host species barrier to infect mice.The ability of influenza virus to constantly alter its antigenic properties has made it very difficult to control this highly contagious illness.The hemagglutinin(HA) and neuraminidase(NA) surface glycoproteins are the most important antigens for inducing protective antibody responses.Accumulating mutations of the HA and NA genes,a process called antigenic drift,enable viruses to escape from immunity induced by prior infection or vaccination resulting in recurring annual epidemics of influenza.The avian influenza virus A/Chicken/Shandong/6/96(H9N2)(CK/SD/6/96),once as a vaccine strain used in Chinese Mainland,could not provide effective protection to the current circulating viruses. A/Chicken/Guangxi/10/99(H9N2)(CK/GD/10/99) was isolated in southern China in 1999, its antigenicity was different obviously from CK/SD/6/96.To understand the molecular mechanism of antigenic drift of H9N2 AIV,reverse genetics was used to determine the minimal amino acid changes that were responsible for causing antigenic drift from CK/GX/10/99 to CK/SD/6/96.Analysis the gene sequence suggested that CK/GX/10/99 differed from CK/SD/6/96 by 16 amino acids in HA1 molecular.After substitution of 2 of the 16 amino acids in the HA1 198,200(the H3 number is 190,192),the CK/GX/10/99 antigenic property similar to CK/SD/6/96 by hemagglutinin inhibition and microneutralization assay using two viruses strains post-infection antisera.Further research showed that the 198 and 200 amino acids are one of the most important antigenic sites of B epitope of HA molecule.The mutation of two amino acids may be change the conformation of B epitope.Otherwise,the 198 amino acid residue is one of the most important of receptor binding sites(RBS) of influenza viruses,mutation 198 amino acid residues may also alter the receptor binding property and block the attachment viruses to cells.The research result indicated that the two amino acid changes at positions 198 and 200 in the HA1 are responsible for the antigenic variation between CK/SD/6/96 and CK/GX/10/99.We also used a panel of H9N2 monoclonal antibody to determinate the antigenicity variation between CK/GX/10/99 and CK/SD/6/96.The result showed that CK/SD/6/96 could react well with the C/B3 MAb,while CK/GX/10/99 could not be recognized by the MAb at all.Using reverse genetics and PCR site-directed mutagenesis,we found that the position 145 amino acid is responsible for antigenicity variation.Deduced from the HA1 gene sequence,the 145-147 amino acid residues is a potential glycosylation site.The SDS-PAGE electrophoresis mobility assay confirmed the oligosaccharide chain located at 145 was expected a glycosylation site in HA glycoprotein.The glycosylation located at A antigenic region,masking the antigenic site A,could block the recognized by the MAb. Similar glycosylation property had been found in other H9N2 avian influenza strains that we isolated in mainland China,such as A/Chicken/Hebei/31/00 and A/Chicken/Guangxi/9/99,which also have a glycosylation site in 145 position of HA₁ glycoprotein,could not be recognized by C/B3 Mab.We may conclude that only one amino acid changed at position 145 in the HA₁ molecular would alter the H9N2 viruses reaction with the MAb.Therefore,monitoring and confirming the antigenic drift stains can be very convenient by using the H9N2 C/B3 MAb in future.Wild aquatic birds are the natural reservoirs of all known 16 HA subtypes influenza A viruses,but only a few of them can cause mild disease in domestic poultry,or severe systemic disease.Most viruses exhibit a relatively restricted host range,with efficient viral replication occurring in the natural host and complete or partial restriction of viral replication occurring in other host species.Many avian influenza A viruses exhibit a host range phenotype characterized by restricted replication only in the respiratory tract of domestic poultry.However,some H9N2 avian influenza viruses,which isolated from domestic poultry in China mainland,could cross the avian-mammalian host species barrier and caused the virus replication in the lung organ of mouse model.We characterized two H9N2 subtype avian influenza viruses that were isolated from chicken, A/Chicken/Shandong/6/96(CK/SD/6/96) and A/Chicken/Guangdong/5/97(CK/GD/5/97). These two viruses are similar to mild pathogenicity for chicken,but differ pathogenicity in mice model.We use reverse genetics to create a series of single-gene segments from CK/SD/6/96 and the remaining seven gene segment from CK/GD/5/97.We found that the PB2 gene of CK/SD/6/96 could attenuate CK/GD/5/97 virus to some extent.And an Asn-to-Asp substitution at position 448 of PB2 gene plays a key role in this function. Conversely,of the recombinant viruses in the CK/SD/6/96 background,only the one that contains the PB2 gene of CK/GD/5/97 was able to replicate in mice.A single amino acid substitution(Asp to Asn) at position 448 of PB2 enabled CK/SD/6/96 to infect in mice.The results demonstrate that amino acid Asn 448 is one of the important determinants for avian influenza virus to cross the host species barrier and infect mice,though the replication of H9N2 influenza viruses involve multiple genes and result from a constellation of gene.Our findings may help us to explain of the host range of replication H9N2 influenza viruses to mouse model.In summary,using reverse genetics,we first demonstrated that the mutation of 145,198, 200 amino acids residues in HA molecule can are responsible for the antigenic variation of H9N2 avian influenza viruses.In addition,we also demonstrated that the difference in PB2 gene are contribute to the H9N2 avian influenza viruses replicated in mouse model,further research showed that a single amino acid at position 448 of PB2 gene is determinant for the different pathogenicity in mice.