Genetic Evolution Analysis And Pathogenicity Study Of H7N9 Influenza Virus In Guangdong,During 2015-2017

Since the first wave of H7N9 broke out in March,2013 in Shanghai,Anhui,and Zhejiang,five waves of the H7N9 influenza virus have been reported since the first half of2017,which seriously threatening China's public health security.Compared with the first four waves,there were mutant strains in the fifth wave.Which had a high lethal rate for poultry and caused significant losses to the poultry industry.Therefore,it is very important to study on the genetic evolution and pathogenicity of the H7N9 influenza virus.In order to keep abreast of the epidemic situation of H7N9 influenza virus and the genetic evolution,pathogenic characteristics in this epidemic wave,the laboratory isolated H7N9 strains from samples which collected in Guangdong Province from 2015 to 2017.According to the principle of distribution at different times and in different regions,50H7N9 strains of Guangdong Province were selected as research objects.In order to compare the Guangdong strains with the strains in the north,16138 strain isolated from the fourth wave and 17528 strain isolated from the fifth wave were selected.After the whole genome sequence analysis of 52 strains,the result show that 13 of 20 isolates from the fifth wave were mutated.Four motify patterns at the cleavage sites in HA protein of mutant strains,is PEVPKRKRTAR ? GLF,PEVPKRRRTAR ? GLF,and PEVPKGKRTAR ? GLF,with several specific amino acids which is a typical feature of high-pathogenic avian influenza virus.The result showed that there were high virulence strains in the fifth wave in Guangdong province,and they were also prevalent with the low pathogenic strains at the same time.The phylogenetic analysis of 32 virus isolated from wave 4 and 20 virus isolated from wave 5 revealed that strains from the fifth wave evolved from the fourth wave's.The HAgene can be divided into the Yangtze River Delta and the Pearl River Delta.the HA and NA of the high-pathogenic strain were all the Yangtze River Delta.It was particularly noteworthy that 3 strains of low pathogenic virulence?15567?15575 and 17064?whose HA is the Yangtze River Delta branch and NA is the Pearl River Delta branch,suggested that the HA and NA genes of low-pathogenic strains are recombinated between the Yangtze River Delta and the Pearl River Delta.In addition,the 6 internal genes?except for HA and NA?of the 15567 and 15575 strains were the closest to the internal genes of A/Duck/Hong Kong/Y439/97?H9N2?,suggested that H9N2 subtype influenza virus was involved in internal gene recombination.Thus,according to eight different genes in different lineages,52 strains were invided to 35 genotypes?G1^G35?.G25 and G28 strains from the fifth wave were mostly,while G4 and G18 strains were mostly in the fourth wave.The dominated genetypes of strains in fifth wave were G25 and G28,and the fourth wave's were G4 and G17,respectively.Through the analysis of key amino acid sites,the results showed that some key sites are different,such as 226 site of the HA protein,strains in the fourth wave were all L,with double mutations at 186 site,while the fifth wave strains were Q.19 of 52 strains were V at 588 site of PB2 protein,27 of 52 strains were A,and 6 strains were T.Just 15567,15575,17527 and 17528 strains at 368 site of PB1 protein were I,while the others were V.Five viruses were screened with 10⁶EID₅₀/200 ?L doses for SPF chickens and BALB/c mice with artificial infection.The results showed that 17390 and 17528 strains isolated from the fifth wave had an increased ability to transmit pathogenicity to chickens and mice than 15709,15567 and 16138 strains from the fourth wave.Four-week-old SPF chickens infected with 17390 and 17528 strains showed the clinical features of high-pathogenic avian influenza,acute death within a week with 100% mortality rate,serious bleeding,and mainly caused by lung and brain lesions.Observed under a microscope,HE stained sections showed pulmonary hemorrhage,inflammatory exudates,interstitial pneumonia and viral encephalitis.In addition,different strains isolated in the same period have different pathogenicity in mice.The mice infected with the Guangdong strains?17392?caused100% mortality rate of mice,while the Hebei strains?17528?caused the mice to lose weight and recover gradually.The virus mainly replicated in lung,turbinate,and brain.