Phylogenic Analysis And Biological Characterization Of H5 Subtype Avian Influenza Virus In Part Of China From 2017 To 2018

Avian influenza(AI)is a viral infection caused by avian influenza viruses in the type A influenza viruses genus of the Orthomyxovirus family,which occurs to various poultry and wild fowl.The disease broke out in Italian flocks firstly in 1978 and is prevalent in many countries around the world.Since the H5N1 highly pathogenic avian influenza had occurred in Guangdong Province in 1996,the virus infected a wider range of hosts,including many birds,mammals and even humans.In addition to the H5N1 subtype,H5subtype highly pathogenic avian influenza virus also has a variety of other NA subtype viruses.At present,H5 subtype highly pathogenic avian influenza presents the characteristics of serosubtype strains such as H5N1,H5N2,H5N6 and H5N8,which are prevalent in flocks simultaneously in China.From 2017 to 2018,this study collected samples of poultry in the live poultry market in some parts of Guangdong,and collected clinical samples from different provinces(Shandong,Hunan,Guangxi,Jiangsu,Hebei,etc.)for virus isolation and identification.39 strains were selected for genetic evolution analysis by whole genome and study on biological characteristics,and 14 of strains were selected for antigenic analysis.Four strains were studied for the pathogenic characteristics in 4-week-old SPF chickens and 4-week-old SPF BALB/c mice.In order to study the molecular characteristics of the H5 subtype strain,the genetic analysis of the HA genes sequence of 39 strains of H5 subtype avian influenza virus revealed that the HA genes of the three strains belonged to Clade 2.3.2 and the remaining 36 strains belonged to Clade 2.3.4.4.There are several consecutive basic amino acids in the HA protein cleavage site of 39 strains of H5 subtype avian influenza viruses,which are consistent with the typical molecular characteristics of highly pathogenic avian influenza virus.One of strains has double sites combination mutation in S137A and T192I.It is speculated that the strain has enhanced binding ability to sialic acid?2-6 galactose receptor.In order to study the antigenicity of H5 subtype strains,14 representative strains were selected to prepare inactivated vaccines and antiserum,and HI cross-matching test was performed to compare antigenic differences between strains.17771 and other 13 strains had similar HA antigens of 87.8%?88.4%,antigen-related values(R)R<0.5,and antigenic differences were significant;17771 and vaccine strain rFJ56-7R values were 0.629836657,with small differences.A value of 1 with the vaccine H5-1806R indicates that they have same antigenicity.The HA antigen similarity in the remaining 13 strains was 96.6%?99.8%,and the antigen-related value(R)was 0.651338947 except for the antigen 18012and the anti-serum 17710,indicating that there was no significant difference.In order to study the pathogenicity of H5 subtype avian influenza virus isolates,four H5 subtype representative strains were selected for challenging in SPF chickens and BALB/c mice in this study.Four-week-old SPF chickens were infected by dropping eye and nasal.The dose of challenging each SPF chicken was 10⁶ID₅₀,which caused 100% death of SPF chicken.Heart,liver,spleen,lung,kidney,and brain all have different degrees of lesions in pathological sections.Anesthetized with isoflurane until the mice were in the form of abdominal breathing.Four-week-old SPF mice were infected by dropping nasal.The dose of challenging each SPF mice was 10⁶EID₅₀.It was found that except the strain18231 did not cause mice death.The mice in this group showed weight loss on the 5th day after challenge and the weight began to rise on the 9th day.The other three strains caused the mice lose weight and die,among which strain 17771 and 18012 mice were 100%dead and 18217 mice had a mortality rate of 75%.In the dead mice,the virus could be detected in the organ.The highest virus titer was detected in the lungs and brain.The strain 8231 did not cause mouse death,but the virus was also detected in the lungs,and no virus replication was detected in the brain.Heart,liver,spleen,lung,kidney and brain were observed in pathological sections with different degrees of lesions.