| Avian influenza viruses pose a great threat to not only poultry,but also human beings.In 1957 and1968,a world-wide influenza pandemic broke out.The 1957 and 1968 pandemic strains retained five internal gene segments from the 1918 lineage but took on HA,NA and PB1?in 1957?or HA and PB1?in 1968?segments from avian influenza viruses.Therefore,it was hypothesized that reassortment accelerates virus mutations and thus facilitates the rapid adaptation of reassortant viruses to new hosts,causing an influenza pandemic.To test this hypothesis,the reassortant viruses,including r2093HANAPB2/WSN,r2093HANAPB1/WSN,r2093HANAPA/WSN and r2093HANANP/WSN,were rescued by reverse genetics,each of which contains the HA gene,NA gene,and one of polymerase genes or NP gene from A/Chicken/Shanghai/2093/2009?A2093?,and the other 5 genes from A/WSN/1933?H1N1??WSN?.MDCK cells were infected by the four reassortant viruses and their parental viruses at MOI of 0.001respectively,and then the cell supernatants were collected at 12h,24h,48h and 72h after infection for plotting virus growth kinetics curve by plaque assay.The results showed that the virus titers peaked at24h post infection.The virus titers of r2093HANAPB1/WSN and r2093HANAPA/WSN were significantly higher than those of r2093HANAPB2/WSN and r2093HANANP/WSN.To test the replication in mice,4 reassortant viruses and their parental viruses were inoculated intranasally into mice with 105 PFU respectively.The virus titers in the lungs and nasal turbinates of the mice inoculated with the four reassortant viruses were between those of mice inoculated with A2093 and WSN on the 4days post-inoculation.While the titers in the lungs of mice inoculated with 4 recombinant viruses were similar,the titers in the nasal turbinats of r2093HANAPB1/WSN and r2093HANAPA/WSN were significantly higher than those of the mice inoculated with r2093HANAPB2/WSN and r2093HANANP/WSN.The results indicated that there was a significant difference in the replication capacity of reassortant viruses containing different polymerases or NP genes from the avian flu virus.In order to further test the mutation rate of different reassortant viruses in virus passage,MDCK cells were infected at MOI of 0.001.Cell supernatants were collected at 24 h post-infection and were passaged again to fresh MDCK cells.The HA,M,and NS genes of the viruses with pre-passage,fifth-generation,and tenth-generation were used for RT-PCR amplification and deep sequencing.The results showed that mutation rates of HA gene in the fifth generation of r2093HANAPB1/WSN,r2093HANAPA/WSN,A2093,and WSN were 7.085×10-5,4.535×10-5,4.42×10-5 and 4.94×10-5,respectively.Those of the tenth generation were 4.575×10-5,4.335×10-5,4.665×10-5 and 5.055×10-5,respectively.Similarly,the mutation rates of M and NS gene in the fifth generation were 7.58×10-5and6.245×10-5,5.965×10-55 and 5.515×10-5,7.235×10-5 and 4.285×10-5,8.46×10-55 and 5.295×10-5,and those of 10th generation were 6.23×10-5 and 4.8×10-5,5.415×10-5 and 4.655×10-5,6.425×10-5 and 3.715×10-5,7.02×10-5 and 4.935×10-5 respectively.The average mutation rates of the first five generations of viruses were significantly higher than that of the previous ten generations,indicating that more mutations occurred in the first five generations of virus passage.To test whether the viruses showed similar mutation rates in the mice,the viruses were passaged serially 10 times in mice.RNA was extracted from the lungs of mice of fifth and tenth passage,and the HA,M,and NS genes were amplified by RT-PCR.Deep sequencing showed that the mutation rates of the passage viruses in mice were higher than those of the cell.The analysis of the mutation rates of the first five generations r2093HANAPB1/WSN and WSN showed a significant difference,indicating that the viruses were prone to accumulate mutations in mice.This study primarily demonstrated that influenza virus reassortment affected the virus mutation rates and accelerated virus mutation accordingly.To further understand the mutational spectrum of the HA gene during the passage of different reassortant viruses,the ratios of all adenine?A?mutating to thymine?T?,cytosine?C?,or guanine?G?were calculated.Similarly,the ratios of all T to A,C,or G,all C to A,T,or G,and all G to A,T,or C were calculated.The results showed that the ratios of A to G mutations were higher in MDCK cells and in mice than other mutations.The ratios of A to C,C to A,G to T in fifth-generation r2093HANAPB1/WSN in mice were significantly higher than those of WSN strains?P<0.05?,which was consistent with the fact that the r2093HANAPB1/WSN was more likely to accumulate mutations in mice.The ratios of A to G,C to A,C to T,and G to A in the tenth-generation of A2093 were higher than that of the r2093HANAPB1/WSN strain significantly?P<0.05?.When the viruses were passaged in mice,the adaptive ability to the host was different.While the mutation peak of r2093HANAPB1/WSN occurred in the fifth generation,the mutation peak of A2093 virus occurred in the tenth generation,indicating that the PB1 reassortant virus can be adapt to the host rapidly. |
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