Isolation,Identification,Whole Genome Sequence Analysis Of H9 Subtype Avian Influenza Virus From Wild Birds And Establishment Of Duplex RT-LAMP Detection Method In Guangxi

Avian influenza virus(AIV)are the member of the family Orthomyxoviridae.Many members of the family are associated with economic loss for the poultry industries.When affected,AIV subtype H9 causes severe respiratory symptoms including a sharp fall in egg production and decline,slow growth.In addition,H9 subtype of AIV can also affect wild birds and humans.It can also provide partial or the whole cassette of internal genes to other AIVs to generate novel recombinant viruses such as H5N6,H7N9,H10N8,and others,seriously endangering human public health safety.Guangxi has a special geographical location,so it is of great significance to strengthen the research on the ecological evolution of H9 subtype of IV in this region for the effective prevention and control of avian influenza.To know H9 subtype AIV genetic evolution in Guangxi,from 2017 to 2019,we collected throat and cloacal swabs of turtledove,dove,pheasant and other wild birds from different live poultry market for isolation of virus,their detection by RT-PCR followed by sequencing and whole genome analysis for detail genetic evolution analysis.In this study total 15 strains of H9 subtype of AIV were successfully isolated and identified,According to the amino acid sequences analysis,all these 15 strains of H9 subtype of AIV were identified as Low pathogenic influenza virus.In addition,genetic and evolutionary analysis also revealed that their internal gene source was complex,with three different gene combinations.In this study we have established a new loop-mediated isothermal isothermal amplification(LAMP)method,a fast,sensitive and economical technique for the rapid detection of avian influenza viruses.Different HA and NA gene sequences from NCBI were downloaded and analysed by Primer Explorer V5 online software(http://primerexplorer.jp/lampv5e/)to design HA(H5 and H9)and NA(N2)LAMP specific primers.In total four groups with different fluorescence labeled probe(HA-H9-probe and NA-N2-probe combination,H9-probe and H5-probe combination were designed,The newly established fluorescence-based duplex RT-LAMP successfully detected H9 and N2 subtype AIV and H9,H5 subtype of AIV.