The Pathogenic Mechanism Of Reassortants Derived From H5, H6 And H7 AIVs To BALB/c Mice

Influenza A virus is characterized by a genome composed of eight single-stranded, negative sense RNA segments, which allow for reassortment between different strains when they co-infect the same host cell. Reassortment is an important driving force for the evolution of influenza viruses which could modify host range, pathology, and transmission of the influenza A viruses.In this process,the influenza virus strainwithepidemic and/or pandemic potential can be created and pose a constant threat to the health of humans andanimals. Since the late 1990 s, multiple genotype H5, H6, and H9 influenza viruses have been circulating within multiple bird species, which provides opportunities for genetic reassortment.Recent years, the rapid antigenic and genetic change of avian influenza virusesfacilitated the emgergence of a number of different HA and NA pairings.The new HA and NA pairings not only infect poultry and wild birds, but also infect human.Therefore, to study the genetic evolution and pathogenicity of avian influenza viruses and their reassortant follow natural lawtimely is importantto prevent and control avian influenza.To investigate the genetic basis of H5 Nx and H7N9 avian influenza viruses, we performed genome cloning, sequence and phylogenic analysis of 34 viruses isolated during recent years. HA genetic analysis indicated that 30 H5 Nx viruses belonged to clade7.2, clade 2.3.4.4, clade 2.3.4, and clade 2.3.2.1. And the calde 7.2 and clade 2.3.4.4 were the dominant branch. Among them, most of H5N2 avian influenza viruses belonged to clade 7.2, and most of H5N1, H5N6, and H5N8 avian influenza viruses belonged to clade 2.3.4.4.Amino acid analysis of the major antigen districts of HA protein showed that there were some amino acids took mutation compared to Re-1（A/goose/Guangdong/1/1996）, Re-4（A/chicken/Shanxi/1/2006）, Re-5（A/duck/Anhui/1/2005）, Re-6（A/duck/Guangdong/s1322/2010）, Re-7（ A/chicken/Liaoning/s4092/11）, and Re-8（A/chicken/Guizhou/4/2013）vaccine strains used currently in China. The variation rate of theviruses belonged to clade 2.3.4.4 were about 0%～50% compared with Re-8 vaccine strain. The variation rate of the viruses belonged to clade 2.3.2.1 were about 20% compared with Re-6 vaccine strain.The variation rate of the viruses belonged to clade 2.3.4 were about 20% compared with Re-5 vaccine strain. The variation rate of the viruses belonged to clade 7.2 were about 26%～36% and 0%～20% compared with Re-4 and Re-7 vaccine strains, respectively. Phylogenic analysis of the 30 H5 Nx AIVs indicated that multi-reassortant happened amongdifferent geneotypes of the same subtypes avian influenza virus and/or different subtypesavian influenza virus.Phylogenic analysis of H7N9 avian influenza viruses showed that the novel H7N9 avian influenza viruses were multi-reassortant viruses, whose gene segments derived from different avian influenza viruses. The HA gene of them belonged to H7-like/Eurasia lineage, their NA gene clustered into N9-like/Eurasia lineage and their internal genes were from the H9N2-like/Eurasia lineage. Therefore, reassortment events in nature occurred among H5 and H7 avian influenza viruses isolated during recent years.To understand the reassortant in the evolution and molecular determinants of the H5N1, H6N2, and H7N9 viruses to BALB/c mice, we here used reverse genetics to evaluate the reassortant of H5N1, H6N2, and H7N9 viruses. When one gene segement was substituted correspondingly between H5N1 and H6N2 avian influenza viruses,we could successfully rescue the reassortant viruses, whichindicated that reassortant between H5N1 and H6N2 avian influenza viruses may occur.To investigate the pathogenicity of thereassortant viruses in BALB/c mice,we inoculated SPF female BALB/c mice intranasally with 106EID₅₀/50μl these viruses.The results indicated thatthe viruses carried the PB2 and NS genes of the DK65（H6N2） virus were lower pathogenicity than the wild-type CA1（H5N1） virus to the mice. The virus carried the NS gene of the CA1（H5N1） virus had higher replication ability than the wild-type DK65（H6N2） virus in mice. Therefore, the NS gene played a major role in the pathogenicity between the CA1（H5N1） virus and the DK65（H6N2） virus in mice. The viruscarried the NS gene of the DK65（H6N2） virus was lower pathogenicity than the wild-type C381（H5N1） virus to the mice. The virus carried the NS gene of the C381（H5N1） virus had higher replication ability than the wild-type DK65（H6N2） virus in mice. Therefore, the NS gene was the determinative factor of the pathogenicity between the C381（H5N1）virus and the DK65（H6N2） virus in mice. When one gene segment was substituted correspondingly between the CA1（H5N1） virus and the GD134（H7N9） virus, we also successfully rescued the reassortant viruses,which indicated that reassortant between H5N1 and H7N9 avian influenza viruses may occur. When SPF female BALB/c mice inoculated intranasally with 106EID₅₀/50μl these reassortant viruses, we found that the virus carried the PA gene of the GD134（H7N9） virus was lower pathogenicity than the wild-type CA1（H5N1） virus to the mice. The virus carried the PA gene of the CA1（H5N1） virus had higher replication ability than the wild-type GD134（H7N9） virus in mice. Therefore, the PA gene mainly determined the difference of pathogenicity to mice between the CA1（H5N1） virus and the GD134（H7N9） virus.In summary, our results indicated that reassortment events among H5N1, H6N2, and H7N9 avian influenza virus could be naturallyoccurring, and the viruses carried NS gene or PA gene from H5N1 viruses had higher pathogenicity than the wild-type viruses in mice.