Coxsackievirus A6 was the most common enterovirus serotype causing hand,foot,and mouth disease in Shiyan City,central China

BACKGROUND Hand,foot,and mouth disease(HFMD)has become one of the most common infectious diseases in China.Before 2016,the primary causal serotypes were enterovirus A71(EV-A71)and coxsackievirus A16(CV-A16).Following the introduction of EV-A71 vaccines in China since 2016,the situation could change.CV-A6 has recently replaced EV-A71 and CV-A16 in some areas of China.However,the epidemiological characteristics of central China remain unknown.AIM To investigate the clinical symptoms and pathogen spectrum of HFMD in Shiyan City,central China,in recent years.METHODS The epidemiological,clinical,and laboratory data from HFMD cases reported to the Shiyan Center for Disease Control and Prevention between January 2016 and December 2020 were analyzed.196 throat swab specimens were collected from hospitalized HFMD patients between January 2018 and December 2020.To detect and genotype enteroviruses,real-time reverse transcription-polymerase chain reaction and sequencing of the 5'-untranslated region were used.In Shiyan,168 laboratory-confirmed HFMD cases were studied using a logistic regression model to determine the effect of predominant enterovirus serotypes.Based on the logistic regression model,the least absolute shrinkage and selection operator model was used to analyze the correlation between CV-A6 infection and various clinical characteristics in HFMD patients in Shiyan.RESULTS From 2016 to 2020,35840 HFMD cases were reported in Shiyan.The number of cases decreased by 48.4%from 2016 to 2017.Approximately 1.58-fold increases were found in 2018 and 2019 when compared to the previous year,respectively.In 2020,a decrease of about 85.5%was reported when compared to 2019.The most common serotypes shifted from EV-A71 and CV-A16(about 60%-80%in 2016 and 2018)to others(more than 80.0%in 2017,2019,and 2020).EV-A71 lost its dominance in 2017 in Shiyan.Among 196 confirmed HFMD cases,85.7%tested positive for enterovirus,with CV-A6 being the most common serotype(121/168,72.0%).The positive rates for CV-A16 and CVA10 were 4.8%and 3.0%,respectively.There was no EV-A71 discovered.Infection with CV-A6 was linked to fever,myocardial damage,increased creatine kinase MB isoenzyme,and lactate dehydrogenase levels.CONCLUSION CV-A6 was the most common enterovirus serotype in Shiyan City,replacing EV-A71 and CV-A16 as the HFMD pathogen.Developing vaccines against CV-A6 or multiple pathogens,as well as rising CV-A6 surveillance,will help prevent HFMD in central China.

Bioinformatic Analysis of Non-VP1 Capsid Protein of Coxsackievirus A6

This study bioinformatically analyzed the non-VP1 capsid proteins（VP2-VP4） of Coxasckievirus A6（CVA6）, with an attempt to predict their basic physicochemical properties, structural/functional features and linear B cell eiptopes. The online tools Sub Loc, Target P and the others from Ex PASy Bioinformatics Resource Portal, and SWISS-MODEL（an online protein structure modeling server）, were utilized to analyze the amino acid（AA） sequences of VP2-VP4 proteins of CVA6. Our results showed that the VP proteins of CVA6 were all of hydrophilic nature, contained phosphorylation and glycosylation sites and harbored no signal peptide sequences and acetylation sites. Except VP3, the other proteins did not have transmembrane helix structure and nuclear localization signal sequences. Random coils were the major conformation of the secondary structure of the capsid proteins. Analysis of the linear B cell epitopes by employing Bepipred showed that the average antigenic indices（AI） of individual VP proteins were all greater than 0 and the average AI of VP4 was substantially higher than that of VP2 and VP3. The VP proteins all contained a number of potential B cell epitopes and some eiptopes were located at the internal side of the viral capsid or were buried. We successfully predicted the fundamental physicochemical properties, structural/functional features and the linear B cell eiptopes and found that different VP proteins share some common features and each has its unique attributes. These findings will help us understand the pathogenicity of CVA6 and develop related vaccines and immunodiagnostic reagents.

Preparation and immunoprotective effects of a virus-like particle candidate vaccine of the dominant epidemic D3 genotype coxsackievirus A6 in China

Coxsackievirus A6 of the D3a genotype(CVA6 D3a)is a primary pathogen causingmainland of China's hand,foot,and mouth disease(HFMD).Viral‐like particle(VLP)vaccines represent a potential candidate vaccine to prevent HFMD.This study collected Anti‐CVA6 D3a VLPs serum from BALB/c female mice immunized using CVA6 D3a VLPs.The neutralizing antibody levels were compared against the representative 14‐JX2018(D3a)and N4‐YN2015(D3b)strains between the antisera of different immune pathways.The immunoprotective effect of anti‐CVA6 D3a VLPs against these strains was monitored using pathological sections and immuno-histochemical results of lung and skeletal muscle tissues in seven‐day‐old Institute of Cancer Research(ICR)mice.Immunological protection against different branches of viruses was evaluated in 7‐day‐old(serum passive immune protection)and 14‐day‐old(VLPs active immune protection)neonatal ICR mice models.Serum‐neutralizing antibody levels were positively correlated with the number of immunizations and higher against 14‐JX2018 than against N4‐YN2015.Furthermore,these levels were significantly higher with abdominal injection than intramuscular injection.The immunized serum of 7‐day‐old ICR mice inoculated three times was 100%protected against CVA6 D3a 14‐JX2018(lethal titer:106.25 TCID 50)and CVA6 D3b N4‐YN2015(lethal titer:105.25TCID 50)fatal attacks,respectively.For ICR mice that have completed two active immunizations for 14 days,both CVA6 D3a 14‐JX2015(challenge titer:108.25 TCID 50)and CVA6 D3b N4‐YN2015(challenge titer:107.25 TCID 50)were used for the challenge,and the mice were able to survive.Overall,the CVA6 D3a VLPs prepared in this study are a potential vaccine candidate for CVA6,as it has the optimal protective effect against both CVA6 D3a and D3b evolutionary branches viruses.

Genetic Variation of Multiple Serotypes of Enteroviruses Associated with Hand, Foot and Mouth Disease in Southern China

Enteroviruses(EVs)species A are a major public health issue in the Asia–Pacific region and cause frequent epidemics of hand,foot and mouth disease(HFMD)in China.Mild infections are common in children;however,HFMD can also cause severe illness that affects the central nervous system.To molecularly characterize EVs,a prospective HFMD virological surveillance program was performed in China between 2013 and 2016.Throat swabs,rectal swabs and stool samples were collected from suspected HFMD patients at participating hospitals.EVs were detected using generic real-time and nested reverse transcription-polymerase chain reactions(RT-PCRs).Then,the complete VP1 regions of enterovirus A71(EV-A71),coxsackievirus A16(CVA16)and CVA6 were sequenced to analyze amino acid changes and construct a viral molecular phylogeny.Of the 2836 enrolled HFMD patients,2,517(89%)were EV positive.The most frequently detected EVs were CVA16(32.5%,819),CVA6(31.2%,785),and EV-A71(20.4%,514).The subgenogroups CVA16B1 b,CVA6D3 a and EV-A71C4 a were predominant in China and recombination was not observed in the VP1 region.Sequence analysis revealed amino acid variations at the 30,29 and 44 positions in the VP1 region of EV-A71,CVA16 and CVA6(compared to the respective prototype strains Br Cr,G10 and Gdula),respectively.Furthermore,in 21 of 24(87.5%)identified EV-A71 samples,a known amino acid substitution(D31 N)that may enhance neurovirulence was detected.Our study provides insights about the genetic characteristics of common HFMD-associated EVs.However,the emergence and virulence of the described mutations require further investigation.