Research On The Adaptive Evolution Of Important Subtypes Of Avian Influenza Virus Based On Sample Frequency

Avian influenza viruses were only prevalent in avian hosts previously,but in recent years,avian influenza viruses have continued to infect humans across host barriers.The world's first case of human infection with the H5N1 virus occurred in Hong Kong in1997.Since 2003,the highly pathogenic H5N1 virus has been reported in humans around the world,and more than 900 people have been infected.The H7N9 virus has been circulating in the population since it first caused infection in 2013.By 2017,it has caused five epidemics in mainland China.In order to cope with the threat posed by the continuous evolution of the avian influenza viruses,it is particularly important to monitor their genetic variation.Whether their virulence,replication ability,ability to evade immunity,or its potential to cross the host barrier,the avian influenza viruses are closely related to the mutations occurred during the adaptive evolution of the viruses.However,the existing researches on viral mutations were carried out for one or several sites.Although the specificity is strong,they lacked the overall control of the mutation rule in the process of adaptive evolution of the viruses.In the first part of this work,we have established a comprehensive and rapid method to search for all important mutations that occurred during the evolution of influenza viruses.The core of the method is to mathematically model the information inherent in the virus.We define and calculate the “strain frequency” and “clade frequency”,and then calculate all the mutations that occurred on the evolution path based on a backtracking method.The biggest advantage of this method is its universality,completely independent of the type of viruses and their features.The only requests for the viruses are that they have complete sequence information and their own inherent information,and they are successive according to the year.At the same time,we also established a mutation evaluation system to evaluate the mutations we calculated,mainly based on the frequency variation and fixation rate in each year of amino acids after mutation.In the second part of the work,we selected the H5N1 virus HA segment as the research object,and used our established method to study the mutation rules in the process of adaptive evolution.A total of 435 mutations were calculated,and a table of amino acid fixation and frequency variation table was obtained.Among them,79 were effective switch,occurring at 36 sites,20 of which were on the epitopes.Combined with the amino acid frequency map,we propose that the sites effective switch emerged are positive selection sites and this method of judging the positive selection sites is more accurate than the methods before.At the same time,we also found 29 high-frequencymutation sites,14 of which were on the HA epitopes and 20 were positive selection sites.Therefore,we believe that high-frequency mutations are closely related to the adaptive evolution of viruses,and it is more convincing to combine high-frequency mutations with effective switches to evaluate mutations.In the third part of this paper,we applied the established method to study the mutation rules of the eight segments of the H7N9 virus genome.After calculation,we finally got 46 mutations on HA,42 mutations on NA,38 on PB1,38 on PB2,31 on PA,20 on NP,31 on NS1,and 1 on NS2,4 on M1 and 18 on M2.In contrast to many existing studies,we found that the mutations we found were very accurate,proving that the method we had built was accurate and reliable.At the same time,we also found that M245I/L and L235 Q on the HA segment are likely to be related to the emergence of the fifth wave of epidemic and highly pathogenic strains.The emergence of L186 I improves the adaptability of the virus;I16T and Y171 H on the NA segment both occur twice in the evolution process of NA,and they are all at the key points in the tree,indicating that they are closely related to the improvement of virus adaptability.There are many mutations in the internal segments which are closely related to the fluctuation of each wave.Studying their function is of great significance for us to explore the causes of each outbreak.Whether the mutation searching method we established or the preliminary mutation evaluation system,it provides a good guiding value for the experimenters.For the study of mutations of influenza,it is not necessary to stick to one or some of the sites observed by the naked eye,but to select the most significant mutations in all the important mutations that are efficiently obtained.It saves time and it can points out the initial direction of the experiment.