Phylogenetic Analysis And Clinical Characteristics Of Coxsackievirus A10 In Hand,Foot And Mouth Disease In Qingdao In 2014-2017

Objective:In this study,we analyzed the clinical characteristics of CVA10 in hand-foot-mouth disease in Qingdao from 2014 to 2017,and analyzed the clinical characteristics of CVA10 to understand the clinical characteristics of CVA10 and the possible complications of hand-foot-mouth disease.And the correlation between viral genetic variation and disease epidemics and disease progression,laying the foundation for the study of CVA10 variation and virulence sites,and providing certain judgments for clinical diagnosis and treatment.Methods:During the period of 2014-2017,the routine monitoring of hand,foot and mouth disease cases in Qingdao Women and Children’s Hospital was used as the research object.The diagnostic criteria for hand,foot and mouth disease were collected according to the“Guidelines for Hand,Foot and Mouth Disease Diagnosis and Treatment（2010 Edition）”issued by the Ministry of Health.Swab specimens were collected and clinical data of the subjects were collected by retrospective study;viral nucleic acid extraction and typing were performed on the specimens;and some CVA10 strains in Qingdao were amplified by reverse transcription-polymerase chain reaction（RT-PCR）.Full-length amplification of VP1gene and full-length amplification of CVA10 complete gene,and sequence determination;genetic evolution,alignment and recombination analysis of complete genome sequences using software such as DNAStar,MEGA7.0 and SimPlot3.5.1;clinical analysis of CVA10 Characteristics between severe and mild strains,clinical characteristics of Qingdao’s individual branch strains and other strains in Qingdao in2014,and the clinical characteristics of severe CVA10,EV71 and CVA16 cases were compared and descriptively analyzed.Using t test andχ² test,the difference was statistically significant at P<0.05.Results:In this study,197 strains of CVA10 virus were isolated from Qingdao HFMD in 2014-2017,and32 CVA10 whole gene sequences,108 VP1 sequences and 108 3C region sequences were obtained.The results showed that the nucleotides between Qingdao CVA10 isolates The homology was greater than95.9%,and no significant recombination was found in the genetic recombination analysis.The 62 CVA10gene sequences downloaded from Genebank,together with the whole gene sequence of this study,were used to construct a full-gene phylogenetic tree.The CVA10 strain in Qingdao was in genotype C in the genome-wide phylogenetic tree,Qingdao 91.67%（11/12）CVA10 is severely distributed on the independent branch A of gene C;259 VP1 sequences downloaded from Genebank,and the phylogenetic tree of VP1 sequence of this study,VP1 sequence can be divided into 7 genotypes（A-G）,Qingdao 2014-In 2017,Qingdao strain was divided into five branches in VP1,89.8%of CVA10 strains were located on C4a branch,4 strains were distributed on C4b,and 5 strains were on C4c,only one strain was distributed on C4d,and one strain was distributed in C3.On the branch,in 2014,part of the Qingdao strain formed a separate branch on C4a.The results of mutation site analysis showed that A23V and I283V amino acid mutations occurred in VP1 region,G450A and T693A nucleotide mutations occurred in C4a Qingdao independent branch of VP1 region;A97V amino acid variation occurred in Qingdao branch of 3C region.There were 16 cases of severe CVA10 and 99 cases of mild cases with pathological diagnosis and clinical data.The incidence of fever before severe rash was 68.75%,which was statistically significant（P<0.05）.Laboratory tests showed The mean white blood cell count,neutrophil percentage,hemoglobin and aspartate aminotransferase in the severe group were significantly higher than those in the mild group（P<0.05）.The chest radiograph in the severe group showed an incidence of exudative lesions of 62.5%and abnormal brain CT.There were more mild cases and more statistically significant in the milder group（P<0.001）.In the 3C district in Qingdao,38 cases of the separate branch I of the A97V mutation occurred in Qingdao in 2014 and 55 cases in which no such mutation occurred,the OR value of the A97V amino acid variation in the 3C area caused severe and non-occurrence caused severe symptoms.The OR value was 4.62（95%）.CI:1.139-18.761),the mean number of fever days during hospitalization in the branch I group was 5.86±1.3 days,which was higher than that in the non-branched group I,and the location of the rash in the patients was more scattered,and the difference was statistically significant（P<0.05）.The pathological diagnosis and clinical data were complete in HFMD with severe CVA10 positive in 16 cases,EV71 positive in 50 cases,and CVA16 positive in 24 cases.The CVA10 group had 1.53±0.87days of fever,which was less than the other groups,and the fever was before the rash.The ratio was68.75%,which was higher than the other two groups.The rash was less common in the hands and feet,and there was statistical significance between the two groups(P_EV71/CVA10,P_CVA16/CVA10<0.05).The number of white blood cells,neutrophils and C-reactive protein in CVA10 was higher than that in the other two groups,and there was statistical significance between the two groups(P_EV71/CVA10,P_CVA16/CVA10<0.05).Conclusions:CVA10 may be an important pathogen of severe HFMD.Most of the CVA10 strains in Qingdao in VP1 area are located on the C4a branch in 2014-2017.The independent branch of CVA10 in C4a in Qingdao in 2014 may be an input strain.A97V amino acid variation in the 3C region is a risk factor for severe cases.Fever before the rash,the HFMD first considers the possibility of CVA10 infection.The number of fever days is small and before the rash,the rash is less common in the hands and feet,and the white blood cells,neutrophils,and C-reactive protein are higher.The possibility of CVA10 virus infection is a reference for clinical diagnosis.