The Screening And Effectiveness Of The Amino Acids Affecting Influenza Virus Polymerase Activity

As an important zoonotic pathogen,the influenza virus can infect wild animals,poultry,and livestock,which also threatens human health.Influenza viruses are affiliated to the family Orthomyxoviridae,divided into the traditional influenza A,influenza B,and influenza C virus and the newly discovered influenza D virus that mainly infect the cattle.Influenza viruses are capable of cross-species transmission from animals to humans by constant mutation,causing seasonal influenza epidemics and even a global influenza pandemic.Both transcription and replication of the influenza virus genome are controlled by influenza virus polymerase protein.Polymerase protein is one of the crucial proteins that determine whether the virus could spread across species and the level of transmission efficiency.The decrease of influenza virus polymerase activity can limit the viral RNA transcription,thereby reducing the viral replication efficiency.The mutations on polymerase protein may improve the influenza viral replication efficiency and the pathogenicity to mammals by enhancing the viral polymerase activity.In this study,we selected influenza A virus subtype H3N2 widely prevalent in wild fowl and poultry,classical influenza A virus subtype H1N1,and the newly discovered influenza D virus for in-depth exploration.For the influenza A virus subtype H3N2 virus,we selected the A/Baikal teal/Shanghai/SH-89/2013(H3N2)virus that widely found in wild birds as our research object.By constructing a random mutation library of H3N2 influenza virus polymerase PB2,we screened and tested which amino acid site mutations may enhance viral polymerase activity,thereby enhancing the viral replication efficiency.From screening experiments,we found that mutations at several amino acid sites in H3N2 influenza virus PB2 could affect the polymerase activity of the virus.The mutation of the H3N2-PB2-D455 L resulted in a 9.42-fold increase in viral polymerase activity.Subsequently,the H3N2-SH89 strain and the H3N2-SH89-D455 L mutant strain were successfully rescued in vitro by reverse genetic manipulation of the influenza virus.The two H3N2 strains were inoculated intranasally in mice.The changes of body weight,in vivo and in vitro replication dynamics were used as indicators to analyze the effect of H3N2 virus mutation on replication efficiency and pathogenicity.It was found that the titer of the SH89-PB2-D455 L mutant virus on MDCK cells was higher than that of the SH89 strain,especially in the early stage of infection.The SH89-PB2-D455 L mutant virus could improve the viral replication ability in mice.Our research can provide molecular markers for the timely detection of newly emerging H3N2-SH89 influenza viruses that may cause influenza epidemics,enhance the surveillance capacity of H3N2 influenza viruses,improve the understanding of H3N2 influenza viruses,and provide a reference for influenza A H3N2 surveillance.For the influenza A virus subtype H1N1 virus,we selected the classic strain A/Puerto Rico/8/1934(H1N1).The PB2 random mutation library of PR8 vaccine backbone strain was constructed,then screening the key amino acid mutation sites that can improve the proliferation capabilities of the H3N2/PR8 vaccine virus in Vero and MDCK cells.From screening experiments,we found that in the PR8 vaccine backbone strain PB2,the PB2-V344 S mutation increased the activity of virus polymerase by6.94-fold,the PB2-S582 P mutation increased the activity of virus polymerase by9.34-fold,the PB2-G590 D mutation increased the activity of virus polymerase by4.55-fold,the PB2-Q350 L mutation increased the activity of virus polymerase by4.73-fold,and the PB2-T530 A mutation increased the activity of virus polymerase by4.65-fold.By employing the virus reverse genetic technology,the AK-PR8 strain and five mutant viruses,AK-PR8-PB2-S582 P,AK-PR8-PB2-G590 D,AK-PR8-PB2-Q350 L,AK-PR8-PB2-T530 A,were successfully rescued in vitro.The replication dynamics of the five mutant strains together with the AK-PR8 strain on Vero and MDCK cells were tested to evaluate whether the mutations could improve the replication capacity of the virus.From the test,we can find that the titer of the three rescued mutant viruses,AK-PR8-PB2-V344 S,AK-PR8-PB2-S582 P,AK-PR8-PB2-T530 A,was higher than that of AK-PR8 strain on both MDCK and Vero cells,among which the PB2-T530 A mutation has the most significant effect on viral replication capabilities.The titer of the AK-PR8-PB2-G590 D strain was the same as the AK-PR8 strain,indicating that this mutation does not affect the viral replication capability.The titer of the AK-PR8-PB2-Q350 L strain was significantly lower than that of the AK-PR8 strain,indicating that the viral polymerase activity increase may enhance the viral replication ability is not absolute.We also tested whether these mutations could improve the replication capacity of viruses in chicken embryos.Our experiments identified three new amino acid mutation sites,PB2-V344 S,PB2-S582 P,and PB2-T530 A,that can increase the polymerase activity of the influenza virus.These three amino acid mutation sites can be used as key sites that modify the backbone of the PR8 vaccine to improve the PR8 vaccine virus replication efficiency in both chicken embryos and cells.The viruses based on the PR8 backbone are often used as the donor strains of the internal genes in vaccine development.Therefore,increasing the titer of the virus in cell line culture can significantly increase the yield of the influenza vaccine and reduce the cost of vaccine production.The PB2-T530 A mutation of our research is expected to be introduced into the PR8 vaccine backbone strain to develop new influenza vaccines based on cell lines and provide more ideal options for the preparation of influenza vaccine strains.For the newly discovered influenza D virus,we first introduced mutations at sites627 and 701 on polymerase PB2,which are associated with enhancing the pathogenicity of the influenza A virus in mammals,into the influenza D virus.Furthermore,we test whether these two sites could also significantly affect the replication efficiency of the influenza D virus.The results of the Dual-Luciferase Reporter(DLR)assay system showed that the PB2-627 K mutation could not enhance the polymerase activity of the influenza D virus;in contrast,the PB2-701 D mutation could increase the polymerase activity of the influenza D virus by 1.47-fold,and the PB2-701 N mutation could increase the polymerase activity of the influenza D virus by 1.20-fold.We created a random mutation library of PB2 fragments of the influenza D virus.The screening experiments showed that several sites could affect viral polymerase activity in the influenza D virus.The two mutations with more obvious effects are the PB2-D533 S mutation and the PB2-G603 Y mutation,which could increase the polymerase activity by 9.92 times and 8.22 times separately.The combined mutation of PB2-D533 S and PB2-G603 Y could also increase viral polymerase activity by 1.46 times,much lower than that of two separate mutations.It may be due to some unknown changes in the structure of the viral polymerase when the two sites are mutated simultaneously,resulting in a down-regulation of the efficiency of enhancing the viral polymerase activity.We also carried out a large number of experiments to rescue the influenza D virus,including constructing an eleven-plasmids reverse genetic operating system and a seven-plasmids reverse genetic operating system by molecular cloning and other means.We found that rescuing the influenza D virus was a challenging task.When the rescued viruses infected cell lines,except replicate in ST cells for the first generation,they could not replicate more generations in MDCK and ST cells.Acording to high-throughput sequencing analysis,we doubt that the Y320 C mutation of the PB1 and the T452 A mutation of the P3 may be the key sites that affect the ability of the influenza D virus to replicate in cells.Subsequent work will strengthen the experimental analysis of this hypothesis and continue the rescue of the influenza D virus.The key amino acid sites identified in this research can provide molecular markers for the timely detection of emerging highly pathogenic influenza viruses,enhance the surveillance capacity,and improve the understanding of the pathogenic molecular basis.By the method we established,the screening of key sites that can improve the replication efficiency of vaccine virus strains will provide important guidance for the solution of potential influenza pandemics and the production of inactivated influenza vaccines.