The Effect Of PB2 K627E Mutation On The Replication Competence Of Classical H1N1 Subtype Swine Influenza Virus

Swine influenza(SI) is one of the most common diseases caused by swine influenza virus(SIV).The feature of this disease is it spread fast, has a high morbidity, and has a high fatality rate especially to piglets who have weaker immune systems. Though SIV can hardly cause death of pigs, its high morbidity, declining the pigs’ immunity, accompany or secondary infection of other diseases and even transmits to human, and thus has attract humans’ attation, so it’s urgent to research the pathogenicity of SIV.The reverse genetics technology is used to study the influenza viruses’ pathogenicity abroadly. In this study, we chose A/Swine/Guangdong/1/2011(H1N1), the classical SIV as the material to rescue the virus. In order to know the biological characteristics of the rescued strain and the wild one, checks were taken on the MDCK cells. The results showed that: the CPE was same at the same time of the two strains, hemagglutination titers were both 26, the morphology of plaque was same; the growth curve had no difference; the sequence analysis showed that the sequences of the two are same, too. All the above results indicated that the biological characteristics were same of the two strains.The H1N1 subtype avian influenza virus is prevalent in domestic and has caused huge damage to breeding industry. In this study, we chose the avian origin SIV to establish the reverse genetic, by amplifying the eight segments by RT-PCR, and then transfected together into 293 T cells after purification and obtain the rescued strain, and the experiment of coagulation indicated that the rescued viruse had blood clotting activity and successfully got the virus.The PB2 residue 627 is regarded as the virulent determinant of influenza virus and has an effect on the replication ability of human influenza and avian influenza virus. However, the impact of this position on SIV on the replication has not been evaluated popularly. In this study, on the basic of the platform of reverse genetic of classical H1N1 subtype SIV, we changed the lysine(K) into glutamic acid(E) and then studied the replication ability on MDCK cells and mice. The results showed that, compared with the mutant strain and the rescued one in the same case, the degree of CPE of the former was less than the latter; the morphology of plaque was much smaller; the growth curve showed that it’s had a significant distinct difference(P<0.001) in 24h、36h、48h, a very significant difference(P<0.01)was shown 60h、72h. The results of experiments on mice showed that the mutant strain has a low pathogenicity and cannot lead to loss the weight. However, the rescued strain caused the weight loss on the fifth day, caused death on eighth, and until the tenth day, the weight of the mice had recovered slowly, and on the thirteenth day, it recovered to the normal level. The results of virus titer on the 3d’ and 5d’ lung tissues showed that there is a significant distinct difference between the two strains. Compared with the normal mutant strains, the rescued ones focal inflammatory cell infiltrated, and multiple focal can be seen, at the same time, the alveolar walls partial enlargement, which indicated that the mutant strain has low pathogenic than the rescued one. The results above showed that the pathogenicity of the mutant virus, from K to E of PB2 of the classical H1N1 subtype, had become lower in vivo and in vitro. This conclusion demonstrates that it’s not only one of the important virulence marker of PB2 627, but also provided the condition for further clarify its pathogenic mechanism.