| H4 subtype of avian influenza virus is a low pathogenic avian influenza virus distributed particularly in the southern parts of China.Surveillance study indicates that H4 subtype of avian influenza viruses infection is gradually increasing and appearing in combination with H1,H3,H5,H6 and H9 subtypes of avian influenza virus due to restructuring and mutations of virus gene segments.Therefore,surveillance to understand relationship of genetic evolution of H4 subtype of avian influenza virus with its pathogenicity in China is of great importance.A surveillance study was conducted at the live poultry market in Guangxi from 2015 to 2016 to monitor H4 subtype of avian influenza virus.Their biological characterization data showed that six isolates belong to low pathogenic avian influenza.They have both SA?2,3 and SA?2,6 receptor affinity.However,they have a higher preferential binding to SA?2,3 receptor.According to genetic evolution analysis,all the eight gene segments of H4 subtype of avian influenza genome belong to the Eurasian lineage.Their HA genes have a high similarity in sequence with that of A/duck/Mongolia/OIE-7438/2011(H4N6)avian influenza virus of Mongolia.The HA genes could be combined with N2 and N8 genes.The N2 geneshave a high similarity in sequence with that of A/duck/Mongolia/154/2015(H1N2)avian influenza virus of Mongolia and that of A/duck/Zhejiang/727042/2014(H6N2)avian influenza virus of Zhejiang province.The N8 genes have a high similarity in sequence with that of A/duck/Vietnam/OIE-2577/2011(H3N8)avian influenza virus of Vietnam.These findings suggest that it is likely that recombined viruses are possibly derived from the neighbouring countries and regions through the migratory birds.To more conveniently detect H4 subtype of avian influenza virus,we developed SYBR Green ? based real-time fluorescence quantitative assay to detect H4 subtype by using ?-actin as a gene of internal control.It is useful to determine the copy numbers of the H4 subtype of avian influenza virus from different tissue samples.To assess the risk of infection by H4 subtype of avian influenza virus,4-week old SPF chickens were infected with 106 EID50 per chicken by six H4 subtype isolates,which were isolated and preserved in our laboratory.The results showed that the adaptation of the six H4 subtype of avian influenza viruses to the chicken body environment is not necessary beforehand.All the six H4 isolates replicated efficiently in the chicken embryos.It was also found that the viruses were discharged to the outside world.A/duck/Guangxi/101D18/2011(H4N6),A/duck/Guangxi/030E93/2015(H4N2)and A/goose/Guangxi/220G30/2016(H4N2)have the ability to transmit horizontally.All the experiments in chickens did not cause systemic infection or death of chickens.Three days post-infection,the dissected sections showed pathological changes such as mild hyperemia in lung and kidney,swelling in spleen,whereas other organs have no indications of anatomical lesions.Pathological slices displayed lymphocytic infiltrates in trachea,lung,liver,heart and kidney.Congestion was observed in the lung and kidney as well.At the same time,11 different organs such as trachea,thymus,brain,liver,spleen,heart,lung,pancreas,kidney,bursa and cecal tonsil were used for virus titration.The virus titration data showed that the virus titers in the trachea and lung virus are higher,and that there is a stronger tissue tropism for the trachea and lung.H4 subtype of avian influenza virus belongs to low pathogenic avian influenza.At present there is no report on H4 subtype viruses that have caused pandemic or prevalence in poultry.However,when mixed infection of H4 subtypes and other AIV subtypes occurs in poultry,gene exchange and reassortment between different AIV subtypes may easily take place,generating novel subtypes of AIV.Therefore,reinforced surveillance of H4 subtype of AIV is of great importance for prevention and control of AIV spreading. |
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