Establishing Reference Sequences And Carrying Out Mutation Analysis For Each Branch Of SARS-CoV-2

Background:COVID-19 is a serious respiratory disease caused by highly infectious SARS Co V-2.As the COVID-19 continues,the variation of SARS Co V-2 will accumulate.These mutations may not only make the virus spread faster,but also make the current vaccine effect less effective.In this study,we established a reference sequence for each branch defined using the GISAID typing method.The homology analysis of each reference sequence confirmed that the variation rate of SARS-Co V-2was very low.The latest GRY had the lowest homology with other branches(99.89%-99.93%),and the homology between other branches was greater than or equal to99.95%.Variation analysis showed that the earliest S,V and G genes had 2,3 and 3characteristic mutations respectively.In addition,we also found that the geographical distribution of different branches was different.G,GH and GR are very popular in the United States,while GV and GRY are very common in the United Kingdom.Although SARS-Co V-2 genome is more stable than SARS-Co V or MERS-Co V,it has relatively high dynamic mutation rate compared with other RNA viruses.The global spread of COVID-19 provides a lot of opportunities for natural selection of SARS Co V-2 virus mutations.The mutation results in genetic variation,which leads to higher pathogenic adaptability of the virus strain,thus leading to host immune escape and greater infectivity.Therefore,the identification and characterization of important mutations are crucial for the design and development of antiviral vaccines and therapeutic drugs.Our work may help to customize antiviral strategies based on the molecular characteristics of SARS-Co V-2.Methods: In this study,we established the reference sequence of each branch according to the GISAID typing method,and further analyzed the characteristic mutation and high-frequency mutation of each branch.In March 2021,we also downloaded 300 sequences of G,GH,GR,GV and GRY branches of COVID-19 at random from GISAID,and then identified mutations in nucleotide and amino acid levels in G,GH,GR,GV and GRY branches in at least three independent samples compared with the established branch reference sequences.We further analyzed the amino acid mutation in the receptor binding domain(RBD)region,and studied the effect of the mutation on the binding affinity of RBD and angiotensin converting enzyme 2(ACE2)using computer methods.Results: In this study,we downloaded and compared the sequences of several major types of isolates that are popular in the world based on the GISAID typing method,and established the reference sequences of each branch.After comparing with the established reference sequence,we found that the earliest S,V and G motifs had 2,3and 3 characteristic mutations in the whole genome,respectively.There were 5,6 and13 characteristic mutations in GR,GH and GV of G branch,respectively.The latest Clade GRY has 28 characteristic mutations in the genome.In at least three independent samples of G,GH,GR,GV and GRY genes,a total of 348,257,289,241 and 77 mutations in the virus genome were detected,accounting for 212,146,156,123 and 72 mutations at the amino acid level.Mutation number of G branch S＿L452R and N＿D377Y,S of GH＿D80A、S＿E484K and N＿T205I,mutant NSP3 of branch GR＿S37L、NSP3＿K977Q、NSP6＿SGF106-108del、NSP13＿E341D、S＿L18F、S＿T20N、S＿P26S、S＿D138Y、S＿K417T、S＿E484K、S＿N501Y、S＿H655Y、S＿T1027I、S＿V1176F and 3a＿S253P。 In addition,we also found the mutation N in nucleocapsid(N)protein＿D377Y is a characteristic mutation of Delta variant.Electronic docking analysis showed that 6 of the 14 RBD mutations(Q414K,K417 T,L452R,T478 K,E484K,N501Y)had increased binding affinity with ACE2.Conclusions:Firstly,we established the reference sequence of each branch,and found that GH contained the most high-frequency mutations at the nucleotide and amino acid levels,and the number of high-frequency mutations in GH increased with the passage of time.These data indicate that COVID-19 evolves gradually over time and will produce many new variants.Secondly,we downloaded the whole gene sequence of the isolate and compared it with the established reference sequence,and found some important mutations,which have a high frequency and transmission trend in each branch.Moreover,according to the results of molecular-protein docking,these mutations are of high research value.Among them,the N501 Y mutation can greatly deepen the binding of the virus and the ACE2 receptor of the infected person,which leads to the greatly enhanced infectivity of the virus.This study provides an appropriate reference standard for the molecular biology and virology research of SARS Co V-2.Further functional research on these mutations will greatly increase our understanding of the epidemiology and pathogenesis related factors of COVID-19,and facilitate the customization of antiviral strategies.