Molecular Aetiological Characteristics Of Hand-foot-and-mouth Disease CV-A16 In Anhui Province From 2016 To 2020

Background Hand,foot and mouth disease(HFMD)is an acute infectious disease caused by a variety of enteroviruses.Most of the symptoms of hand,foot and mouth disease are mild and recover well,but there are still a few patients in critical condition,even life-threatening.With the vaccination of EV-A71 vaccine,the proportion of HFMD caused by CV-A16 has gradually increased in recent years,but the vaccine for CV-A16 is still under development and research.ObjectiveTo analyze the prevalence of CV-A16 associated with hand,foot and mouth disease in Anhui Province from 2016 to 2020,and to understand the genotype changes and molecular genetic evolution characteristics of CV-A16 enterovirus in Anhui Province during the past five years,so as to provide guidance for prevention and control of hand,foot and mouth disease.MethodFrom January 2016 to December 2020,pharyngeal swab samples of children with hand,foot and mouth disease in Anhui area were collected for etiological detection.Representative 436 samples with positive CV-A16 nucleic acid were selected from 5years,and PCR was used for amplification of gene segments in the VP1 region.It was sent to biotechnology company for sequencing.MEGA 6.0 was used for sequence alignment of the determined sequences.Evolutionary tree construction and mutation site analysis were carried out for the sequences in Anhui Province during 2016-2020.Meg Align software in DNA Star software package was used to analyze the homology of amino acid and nucleotide.Results1.The results of etiological surveillance showed that 16650 laboratory confirmed cases of HFMD were reported in 16 prefecture-level cities in Anhui Province from2016 to 2020,including 1057 in 2016,2955 in 2017,4202 in 2018,3962 in 2019,and4474 in 2020.1296 EV-A71 types(7.78%),3809 CV-A16 types(22.88%),and 11545 other types(69.34%);The annual composition of EV-A71 in enterovirus positive cases was 36.9%(390 cases)in 2016,17.6%(52 cases)in 2017,5.33%(224 cases)in2018,1.74%(69 cases)in 2019,and 2.08%(93 cases)in 2020,respectively.The annual composition of CV-A16 in enterovirus positive cases was 20.72%(219 cases)in 2016,22.91%(677 cases)in 2017,23.89%(1004 cases)in 2018,42.58%(1687cases)in 2019,and 5.0%(222 cases)in 2020,respectively.The annual composition of other viruses in enterovirus positive cases was 42.38%(448 cases)in 2016,59.49%(1758 cases)in 2017,70.78%(2974 cases)in 2018,55.68%(2206 cases)in 2019,and92.96%(4159 cases)in 2020.2.290 VP1 sequences were obtained from 436 CV-A16 positive specimens,all of which belonged to B genotype,including 225 from B1 b and 65 from B1 a.The nucleotide homology of 290 VP1 sequences was 86.8%～100%,and that of G-10 VP1 sequence was 74.1%～76.8%.The nucleotide homology between the VP1 sequence of225 B1 b strains and the reference strains identified as genotype B1 b in Gen Bank database was 88.4%-99.6%.The nucleotide homology between 65 strains VP1 of B1 a and reference strains identified as genotype B1 a in Gen Bank database was93.0%～99.9%.Amino acid variation analysis with the prototype strain G-10 showed that 104 sites of CV-A16 had been changed during the five years.In this study,the B1 b strain in Anhui Province from 2016 to 2020 had mutation at the L23 M site at the23 rd site,and the mutation incidence was 90.2%,205/225.Conclusion1.In this study,436 representative samples of CV-A16 positive nucleic acid from Anhui Province during 2016-2020 were amplified by PCR,and 290 effective sequences were obtained successfully.2.Through evolutionary tree analysis,it can be seen that:(1)B1a and B1 b gene subtype of HFMD CV-A16 strain co-circulated in Anhui from 2016 to 2020,with B1 b being the dominant gene subtype,followed by B1 a.(2)HFMD CV-A16 strains from Anhui region may have co-cyclic evolution characteristics with those from Southeast Asia and southern China.3.The analysis of amino acid variation in the VP1 gene sequence of 290 strains in Anhui Province from 2016 to 2020 showed that,except for 2 strains of B1 b subtype(19BB002-2019013 b and 19BB004-2019015b)in 2019,amino acid was replaced by threonine with serine at position 13.The remaining 288 strains were all replaced by threonine with asparagine at site 13 amino acid.It is suggested that the subtype of CV-A16 B1 b gene in Anhui has evolved into a new clade during cyclic evolution,which needs to be confirmed by further studies.