Coxsackievirus A16 Strains Research Articles

Pathological and molecular studies on Coxsackie virus A-16 isolated from hand, foot, and mouth disease cases in India: Approach using neonatal mouse model.

The present study highlights pathogenesis and molecular aspects of Coxsackie virus A-16 (CVA-16) strains isolated from hand, foot, and mouth disease (HFMD) cases from India using a neonatal mice model. ICR mice were intraperitoneally inoculated with CVA-16/311 strain isolated from HFMD cases. Mice developed hind and forelimb paralysis on day 3 of post infection. Histopathological observations of hind limb muscles showed necrosis, dissolution of muscle fiber cells, infiltration of inflammatory cells, marked dilated ventricle, hemorrhages, and neuronal degeneration in the brain. Immunohistochemical studies revealed high expression of CVA-16/311-specific viral antigen in limb muscles, brain, heart from day 3 till day 7 of post-infection. VP1 gene-based reverse transcription polymerase chain reaction conducted in RNA samples of different tissue organs of infected mice followed by sequencing of the positive amplimers revealed presence of CVA-16/311-specific viral sequences. Phylogenetic analysis based on the VP1 gene showed the presence of B1c sub genotype of CVA-16/311 strain in targeted tissue organs. Sequence analysis revealed major genetic changes in heart, skeletal muscle tissues at the nucleotide and amino acid levels. Genetic changes occurred in organs of mice might predict some potential targets and might act as markers of virulence for neuronal tropism. Pathogenesis and molecular studies of CVA-16 strains isolated from HFMD cases using neonatal mice model was conducted for the first time from India.

Biological characteristics of coxsackievirus A6 clinical isolates

Objective To analyze the biological characteristics of clinical isolates of coxsackievirus A6 (CVA6), a pathogen of hand, foot and mouth disease (HFMD), and to provide reference for vaccine development. Methods CVA6 strains were isolated from 21 stool and throat swab specimens of patients with HFMD in Yunnan Province and then identified. Their growth characteristics, plaque morphology and virulence to suckling mice were analyzed. Results Five CVA6 strains, named CVA6-129, CVA6-113, CVA6-57, CVA6-94 and CVA6-162, were isolated and all belonged to D3 subtype. Only the CVA6-129 strain could proliferate rapidly in Vero and KMB17 cells and the proliferation peaked 30 h after inoculation. The infectious titer of the CVA6-129 strain was 7.54 lgCCID50 (50% cell culture infective dose)/ml in KMB17 cells. Different morphologies of plaques were formed by the CVA6-129 strain in Vero and KMB17 cells at the same time points, which were small and round with clear edges in Vero cells, and large and irregular with blurry edges in KMB17 cells. Suckling mice were susceptible to CVA6 via intramuscular and intraperitoneal injection. The most common symptoms in infected suckling mice were reduced mobility, hind limb paralysis and quadriplegia. CVA6 infection could result in death in severe cases. Conclusions This study isolated five CVA6 strains from a number of clinical samples of suspected HFMD cases, of which the CVA6-129 strain showed potential as a vaccine candidate. Key words: Coxsackievirus A6 (CVA6); Hand, foot and mouth disease (HFMD); Vaccine; Biological characteristics

Enterovirus type 71-immunized chicken egg yolk immunoglobulin has cross antiviral activity against coxsackievirus A16 in vitro.

To exploit a cross passive immunotherapy for enterovirus-induced hand-foot-and-mouth disease (HFMD), the cross antiviral activity of a neutralizing antibody against enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) was investigated in vitro. White Leghorn specific-pathogen-free chickens were immunized with EV71 antigens and a specific isolated immunoglobulin (IgY) was prepared from the chicken egg yolk. IgY was further purified and characterized by SDS-PAGE, ELISA, western blotting and bidirectional immune agar diffusion testing. The antiviral activity and dose-response of the IgY were determined by assessing the cytopathic effect in rhabdomyosarcoma (RD) cells in vitro. It was indicated that the levels of IgY were increased at day 7, peaked at week 7 and were maintained at a higher level for 4 weeks following immunization when compared with the negative control. The results of western blotting and bidirectional immune agar diffusion testing revealed that the IgY had cross-binding properties in EV71 and CVA16 strains through targeting the envelope proteins (VP0, VP1 and VP3) of EV71 and CVA16. Neutralization assay results indicated that the infectivity of EV71 and CVA16 strains in RD cells was cross-blocked by IgY in a dose-dependent manner. To conclude, these findings indicate that IgY has cross antiviral activity against EV71 and CVA16 in vitro, and could potentially be developed as a passive immunotherapy for EV71- and CVA16-induced HFMD.

Molecular characteristic analysis for the VP1 region of coxsackievirus A6 strains isolated in Jiujiang area, China, from 2012 to 2013.

Enterovirus 71 (EV-A71) and Coxsackievirus A16 (CV-A16) are the most common causative agents causing hand, foot, and mouth disease (HFMD). However, coxsackievirus A6 (CV-A6), previously largely ignored, became the predominant pathogen in China in 2012. The objective of this study is to investigate the genetic characteristics and molecular epidemiology of HFMD caused by CV-A6 to guide the diagnosis and treatment of the disease, as well as disease prevention. A total of 138 suspected HFMD cases were enrolled in this study and analyses based on complete VP1 nucleotide sequences were performed to determine the evolutionary trajectory of emerging CV-A6. Among 138 samples in Jiujiang, 125 (90.58%) were positive for enterovirus, the most frequently presented serotypes were CV-A6 (77, 61.60%), CV-A16 (28, 22.40%), EV-A71 (6, 4.80%) and untyped enteroviruses (14, 11.20%). Seventy-seven CV-A6 positive specimens were analyzed for the complete VP1 sequences by sequencing and 36 representative isolates were selected to perform nucleotide sequence similarity analysis. The results showed that 36 strains isolated from HFMD patients were clustered closely to the mainland China and were far from prototype strain CV-A6/Gdula (AY421764) and other international subtypes. Moreover, phylogenetic analysis of the VP1 gene revealed that 36 circulating strains were not significantly concentrated in one branch, but were widely distributed in each branch. Continuous surveillance of HFMD etiological agents other than EV-A71 and CV-A16 is necessary. CV-A6 is emerging as the most common pathogen causing HFMD. Closely monitoring the magnitude and trend of CV-A6 epidemic and the trend of pathogenic spectrum changes can provide scientific basis for this disease prevention and control to the department of disease control.

Evolution of VP1 gene of coxsackievirus A16 in hand, foot, and mouth disease

Objective To investigate the genetic evolution of VP1 gene of pathogenic coxsackievirus A16 (CV-A16) strain isolated from clinical hand, foot, and mouth disease (HFMD) patients. Methods A total of 160 HFMD cases with CV-A16-positive results were collected from hospitals in Kunming during January 2015 to June 2017. Fecal samples were collected. Real-time polymerase chain reaction (PCR) was used to detect the CV-A16 virus nucleic acid. The VP1 genes of CV-A16-positive samples were amplified by reverse transcription-PCR. The amplified positive products were sequenced and aligned. The homologies were identified and their subgenotypes were determined. The phylogenetic tree was constructed and homology modeling was conducted. Results All the 160 CV-A16 isolates were B2 subtypes. The genetic distance between detected strains of CV-A16 and the strains in Fujian, Beijing, Nanjing was 0.76. The genetic distance to the strains in Malaysia was 0.78, and to the strains in Australia was 1.86. Homologous modeling revealed that the amino acid sequence of the VP1 gene of the strain had a G227R mutation. Conclusions There is no major genetic variation in the CV-A16 strains during 3 years. CV-A16 isolates are close to those of epidemic strains in Beijing, Fujian and Malaysia, but are far fram the strains from Australia. Key words: Hand, foot and mouth disease; Coxsackievirus A16; VP1 gene; Phylogenetic tree; Homology modeling

Recombinant virus-like particle presenting a newly identified coxsackievirus A10 neutralization epitope induces protective immunity in mice

Coxsackievirus A10 (CVA10) has emerged as one of the major pathogens of hand, foot, and mouth disease in recent years. However, there are no approved vaccines or effective drugs against CVA10. Several experimental CVA10 vaccines have been shown to elicit neutralizing antibodies that could confer protection against viral infection. However, neutralizing antigenic sites on CVA10 capsid have not been well characterized. Here, we report the characterization of linear neutralization epitopes of CVA10 and the development of a CVA10 vaccine based on the identified epitopes. We showed that peptide VP2-P28, corresponding to residues 136 to 150 of VP2, were recognized by anti-inactivated CVA10 sera and effectively inhibited anti-CVA10 sera-mediated neutralization, suggesting that this peptide contains neutralizing epitopes. Insertion of VP2-P28 into hepatitis B core antigen (HBc) resulted in a chimeric virus-like particle (VLP; designated HBc-P28) with the CVA10 epitope exposed on the particle surface. HBc-P28 VLP elicited strong antibody responses against VP2-P28 in mice. Anti-HBc-P28 sera could neutralize both CVA10 clinical isolates and prototype strain, consistent with the fact that the VP2-P28 sequence is highly conserved among CVA10 strains. In addition, anti-HBc-P28 sera failed to cross-neutralize other HFMD-causing enteroviruses, indicating that neutralizing antibodies elicited by HBc-P28 VLP were CVA10-specific. Importantly, anti-HBc-P28 sera were able to provide efficient protection against lethal CVA10 infection in recipient mice. Collectively, these data show that peptide VP2-P28 represents a CVA10-specific linear neutralizing antigenic site and chimeric VLP displaying this peptide is a promising epitope-based CVA10 vaccine candidate.

A potential therapeutic neutralization monoclonal antibody specifically against multi-coxsackievirus A16 strains challenge

ABSTRACT Coxsackievirus A16 (CA16) has caused worldwide epidemics of hand, foot and mouth disease (HFMD), particularly in infants and pre-school children. Currently, there are no vaccines or antiviral drugs available for CA16-associated disease. In this study, a CA16-specific monoclonal antibody (MAb) NA11F12 was derived with an epidemic CA16 strain (GenBank no. JX127258). NA11F12 was found to have high cross-neutralization activity against different CA16 subgenotypes but not EV71 using RD cells. The neutralizing titers of NA11F12 ranged from 1:1024 to 1:12288 against A, B1, B2 and C subgenotypes of CA16 and was less than 8 against EV71 strain. In the neonatal mouse model, a single treatment of NA11F12 showed effective protection with a dose- and time-dependent relationship against lethal challenge by CA16 strain (GenBank no. JX481738). At day 1 post-infection, administering more than 0.1 μg/g of NA11F12 could protect 100% newborn mice from mobility and mortality challenged by CA16. With dose of 10 μg/g of NA11F12, a single administration fully protected mice against CA16-associated disease within 4 days post-infection. And there were 80% and 60% mice protected by administering NA11F12 at day 5 post-infection and day 6 post-infection when the control mice had shown clinical symptoms for 1- and 2-day, respectively. Immunohistochemical and histological analysis confirmed that NA11F12 significantly prohibited CA16 VP1 expression in various tissues and prevented CA16-induced necrosis. In conclusion, a CA16-specific MAb NA11F12 with high cross-neutralization activity was identified, which could effectively protect lethal CA16 challenge in mice. It could be a potential therapeutic MAb against CA16 in the future.

Sequence analysis of VP1-VP4 genes of coxsackievirus A6 strains isolated from children with severe hand, foot, and mouth disease in Shenzhen from 2012 to 2015

Objective To analyze the genetic characteristics of VP1-VP4 genes carried by coxsackievirus A6 (CVA6) strains isolated from severe cases of hand, foot, and mouth disease (HFMD) in Shenzhen during 2012 to 2015. Methods The VP1-VP4 genes of CVA6 strains isolated from severe HFMD cases in Shenzhen during 2012 to 2015 were amplified and sequenced. Phylogenetic analysis was performed to analyze the VP1-VP4 genes of CVA6 isolates and sequences downloaded from GenBank by using DNASTAR6.0 and MEGA6.02 software packages. Results Four cases of severe HFMD were caused by CVA6 in Shenzhen during 2012 to 2015. All of the patients had the symptom of fever, skin rash and aseptic encephalitis. The CVA6 strain causing severe HFMD in 2013 shared 98.8%-98.9% homology in nucleotide sequences and 99.3%-99.8% in amino acid sequences with the strains isolated in 2012. Two amino acid mutations were found in the CVA6 strain isolated in 2013, which were G73E in VP2 region and S13G in VP1 region. However, the CVA6 strain isolated in 2015 only shared 95.0% homology in nucleotide sequences and 99.3% homology in amino acid sequences with the strain isolated in 2013. Six amino acid mutations were identified including E73G in VP2 region and T5A, S27N, A30V, N137S and V242I in VP1 region. The phylogenetic analysis revealed that the four CVA6 strains belong to D3 sub-genotype. The CVA6 strains causing severe cases in 2012 had the nearest genetic relationship with the strain isolated in Changsha in 2012 (KJ156349). The CVA6 strain isolated in Shenzhen in 2013 had the nearest genetic relationship with the strain isolated in Shanghai in 2013 (KJ612513). The Shenzhen CVA6 isolate in 2015 showed high similarity to Weifang CVA6 isolate in 2014 (KX752785). Conclusions All CVA6 strains causing severe HFMD cases in Shenzhen during 2012 to 2015 belongs to D3 sub-genotype. Mutations of S27N and A30V in the VP1 region of the CVA6 isolate in 2015 are located in the B cell epitopes. In addition, the VP1-V242I mutation in the CVA6 strain isolated in 2015 is located in the binding site of PSGL-1 receptor. These mutations may affect the binding of CVA6 strains to the cellular receptors and their infectivity to people. Key words: Hand, foot, and mouth disease; Coxsackievirus A6; Severe case; Genotype; Mutation

Phylogenetic and antigenic analyses of coxsackievirus A6 isolates in Yamagata, Japan between 2001 and 2017

Although coxsackievirus A6 (CV-A6) is generally recognized as a causative agent of herpangina in children, CV-A6 infections globally emerged as a new and major cause of epidemic hand-foot-and-mouth-diseases (HFMDs) around 2008. To clarify the longitudinal epidemiology of CV-A6, we carried out sequence and phylogenetic analyses for the VP1 and partially for the VP4-3D regions as well as antigenic analysis using 115 CV-A6 isolates and 105 human sera in Yamagata, Japan between 2001 and 2017. Phylogenetic analysis revealed that CV-A6 isolates were clearly divided into two clusters; strains in circulation between 2001 and 2008 and those between 2010 and 2017. Neutralizing antibody titers of two rabbit antisera, which were immunized with Yamagata isolates in 2001 and 2015, respectively, against 28 Yamagata representative strains as well as the prototype Gdula strain were 1:2560–1:5120 and 1:160–1:640, respectively. The neutralizing antibody titers among residents in Yamagata against the above two strains were similar. Our analyses revealed that there were cross-antigenicities among all analyzed CV-A6 strains, although the newly emerged strains were introduced into Yamagata around 2010 and replaced the previous ones. With regard to control measures, these findings suggest that we can prevent CV-A6 infections through the development of a vaccine that effectively induces neutralizing antibodies against CV-A6, irrespective of genetic cluster.

Longitudinal study on enterovirus A71 and coxsackievirus A16 genotype/subgenotype replacements in hand, foot and mouth disease patients in Thailand, 2000–2017

BackgroundEnterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are the major causative agents of hand, foot and mouth disease (HFMD) worldwide, particularly in the Asia-Pacific region. Several strains have emerged, circulated, and faded out over time in recent decades. This study investigated the EV-A71 and CV-A16 circulating strains and replacement of genotypes/subgenotypes in Thailand during the years 2000–2017. MethodsThe complete VP1 regions of 92 enteroviruses obtained from 90 HFMD patients, one asymptomatic adult contact case, and one encephalitic case were sequenced and investigated for serotypes, genotypes, and subgenotypes using a phylogenetic analysis. ResultsThe 92 enterovirus isolates were identified as 67 (72.8%) EV-A71 strains comprising subgenotypes B4, B5, C1, C2, C4a, C4b and C5, and 25 (27.2%) CV-A16 strains comprising subgenotypes B1a and B1b. Genotypic/subgenotypic replacements were evidenced during the study period. EV-A71 B5 and C4a have been the major circulating strains in Thailand for more than a decade, and CV-A16 B1a has been circulating for almost two decades. ConclusionsThis study provides chronological data on the molecular epidemiology of EV-A71 and CV-A16 subgenotypes in Thailand. Subgenotypic replacement frequently occurred with EV-A71, but not CV-A16. Monitoring for viral genetic and subgenotypic changes is important for molecular diagnosis, vaccine selection, and vaccine development.

An Epidemic of Hand, Foot, and Mouth Disease Caused by Coxsackievirus A6 in Osaka City, Japan, in 2017.

The second largest epidemic of hand, foot, and mouth disease since 1982 occurred in 2017, which involved 6,173 cases in Osaka City, Japan. The main causative agent was coxsackievirus A6 (CV-A6). Phylogenetic analysis revealed that the detected CV-A6 strains belonged to genetic groups A3 and A4 in clade A.

Molecular surveillance of coxsackievirus A16 reveals the emergence of a new clade in mainland China

Coxsackievirus A16 (CV-A16) of the genotypes B1a and B1b have co-circulated in mainland China in the past decades. From 2013 to 2017, a total of 3,008 specimens from 3,008 patients with mild hand, foot, and mouth disease were collected in the present study. Viral RNA was tested for CV-A16 by a real-time RT-PCR method, and complete VP1 sequences and full-length genome sequences of CV-A16 strains from this study were determined by RT-PCR and sequencing. Sequences were analyzed using a series of bioinformatics programs. The detection rate for CV-A16 was 4.1%, 25.9%, 10.6%, 28.1% and 12.9% in 2013, 2014, 2015, 2016 and 2017, respectively. Overall, the detection rate for CV-A16 was 16.5% (497/3008) in this 5-year period in Shenzhen, China. One hundred forty-two (142/155, 91.6%) of the 155 genotype B1 strains in the study belonged to subgenotype B1b, and 13 (13/155, 8.4%) strains belonged to subgenotype B1a. Two strains (CVA16/Shenzhen174/CHN/2017 and CVA16/Shenzhen189/CHN/2017) could not be assigned to a known genotype. Phylogenetic analysis of these two strains and other Chinese CV-A16 strains indicated that these two CV-A16 strains clustered independently in a novel clade whose members differed by 8.4%-11.8%, 8.4%-12.1%, and 14.6%-14.8% in their nucleotide sequences from those of Chinese B1a, B1b, and genotype D strains, respectively. Phylogenetic analysis of global CV-A16 strains further indicated that the two novel CV-A16 strains from this study grouped in a previously uncharacterized clade, which was designated as the subgenogroup B3 in present study. Meanwhile, phylogenetic reconstruction revealed two other new genotypes, B1d and B4, which included a Malaysian strain and two American strains, respectively. The complete genome sequences of the two novel CV-A16 strains showed the highest nucleotide sequence identity of 92.3% to the Malaysian strain PM-15765-00 from 2000. Comparative analysis of amino acid sequences of the two novel CV-A16 strains and their relatives suggested that variations in the nonstructural proteins may play an important role in the evolution of modern CV-A16.

Molecular characterization of Coxsackievirus A16 strains isolated from children with severe hand, foot, and mouth disease in Yunnan, Southwest China, during 2009-2015.

Coxsackievirus A16 (CV-A16) commonly causes mild symptoms, but severe diseases, such as aseptic meningitis, encephalitis, and even fatal cases, have been reported. Thirteen CV-A16 strains were isolated from patients with severe hand, foot, and mouth disease in Yunnan, Southwest China, from 2009 to 2015. Subgenotype B1a and B1b of CV-A16 were predominantly circulating the region with B1b the predominant strain in recent years. The mean rate of nucleotide substitution based on the VP1 gene sequence was 4.545 × 10 -3 substitution per site per year from 2009 to 2015. These results may help in understanding the genetic diversity of CV-A16 and develop a CV-A16 vaccine.

Genetic characteristics of the P1 coding region of Coxsackievirus A16 associated with hand, foot, and mouth disease in China.

Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot, and mouth disease (HFMD) in young children. To investigate the genetic characteristics of the P1 coding region gene of CVA16 associated with HFMD in China, we included the sequences of CVA16 specimens obtained from outbreak investigations and sporadic HFMD cases between 1998 and 2014 in China from GenBank, we genotyped the CVA16 sequences and analyzed P1 coding region sequences that encode structural proteins with bioinformatics software. CVA16 was classified into genotypes A and B1 based on the VP1 gene; the B1b and B1a subgenotypes were the major CVA16 strains and predominated in the coastal areas of China. Four strains were found to show inter- and intra-typic recombination in the P1 region. The amino acid identities of VP1, VP2, VP3, and VP4 proteins in all Chinese CVA16 strains were 88.2-100%, 83.0-100%, 87.6-100%, and 72.4-100%, respectively. A total of 251 amino acid substitution sites were detected in the structural proteins encoded by the P1 coding region gene. The amino acid sequences of the P1 coding region in Chinese CVA16 strains were highly conserved, although some amino acid mutations occurred with high frequency: VP1-T11A (10%), N14S (14%), L23M/V (11%), T98M (16%), V107A (14%), N102D (6.1%), E145V (8.8%), N218D (10%), E241K (22%), T248A/I (6.8%); VP2-I217V (22%), T226A (38%); VP3-N141S/G (5.4%), and N240D (15%). The genetic characteristics of CVA16 in the P1 coding region gene may provide a basis for developing a CVA16 vaccine and preventing and controlling HFMD in China.

A virus-like particle vaccine protects mice against coxsackievirus A10 lethal infection

Coxsackievirus A10 (CVA10) has emerged worldwide as one of the main pathogens of hand, foot, and mouth disease (HFMD) in recent years. However, there is currently no commercial vaccine available to prevent CVA10 infection. Here we report the development of a recombinant virus-like particle (VLP) based candidate vaccine for CVA10. Co-expression of the capsid protein precursor P1 and the protease 3CD of CVA10 in Pichia pastoris resulted in cleavage of P1 into three capsid subunit proteins VP0, VP1, and VP3. These three subunit proteins co-assembled into CVA10 VLPs, which were visualized as spherical particles with a diameter of ∼30 nm under electron microscope. Immunization studies showed that CVA10 VLP could efficiently induce antigen-specific serum antibodies in mice. The anti-VLP sera were able to potently neutralize homologous and heterologous CVA10 strains. Importantly, passively transferred anti-VLP sera fully protected recipient neonatal mice from lethal CVA10 infection. In addition, neonatal mice born to the VLP-immunized dams were also completely protected from CVA10 lethal challenge. Collectively, these data show that CVA10 VLP represents a promising CVA10 vaccine candidate.

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer

ObjectiveKnowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. MethodsIn this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. ResultsWithin the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run. ConclusionMinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.

Complete Genome Sequences of Four Coxsackievirus A16 Strains Isolated from Four Children with Severe Hand, Foot, and Mouth Disease.

ABSTRACTHere, we report the complete genome sequences of four coxsackievirus A16 strains isolated from four children with severe hand, foot, and mouth disease. Three of them were assigned to subgenotype B1b based on phylogenetic analysis of the VP1 gene, and the other one belonged to subgenotype B1a.

Persistent circulation of Coxsackievirus A6 of genotype D3 in mainland of China between 2008 and 2015

A total of 807 entire VP1 sequences of Coxsackievirus A6 (CV-A6) from mainland of China from 1992 to 2015, including 520 in this study and 287 from the GenBank database, were analysed to provide a basic framework of molecular epidemiological characteristics of CV-A6 in China. Sixty-five VP1 sequences including 46 representative CV-A6 isolates from 807 Chinese strains and 19 international strains from GenBank were used for describing the genotypes and sub-genotypes. The results revealed that CV-A6 strains can be categorised into 4 genotypes designated as A, B, C, and D according to previous data and can be further subdivided into B1–B2, C1–C2, and D1–D3 sub-genotypes. D3 is the predominant sub-genotype that circulated in recent years in mainland of China and represents 734 of 807 Chinese isolates. Sixty-six strains belong to D2, whereas B1 and C1 comprise a single strain each, and five AFP strains formed B2. Sub-genotype D3 first circulated in 2008 and has become the predominant sub-genotype since 2009 and then reached a peak in 2013, while D2 was mostly undetectable in the past years. These data revealed different transmission stages of CV-A6 in mainland of China and that sub-genotype D3 may have stronger transmission ability.

Epidemiological characterizations, pathogen spectrum and molecular characteristics of Coxsackievirus A16 from patients with HFMD in Yantai, Shandong, China between 2011 and 2015

ABSTRACTThis study aimed to investigate the epidemiological characterizations and pathogen spectrum of hand, foot, and mouth disease (HFMD) in Yantai City, Shandong Province, China, during 2011–2015, and to study the nucleotide evolution and amino acid variation of coxsackievirus A16 (CV-A16) epidemic strains that caused HFMD. The HFMD epidemic began to rise in March, and became prevalent from May to August, reached its peak in June, and then declined in September every year, children aged one to 5 years-old had the highest incidence rate whereas the incidence in children under 6 months was very low, and there were more males than females. Enterovirus nucleic acid detection using real-time reverse transcription polymerase chain reaction was performed on 2130 clinical specimens collected from patients with HFMD between 2011 and 2015, and 2012 enterovirus positive samples were detected, including 678 CV-A16, 639 EV-A71, and 695 other enteroviruses. In total, 60 CV-A16 isolates were randomly selected each year for virus isolation, of which 33 CV-A16 strains were randomly selected for further characterization because CV-A16 is the predominant serotype that caused HFMD in Yantai City, and a phylogenetic tree based on the VP1 region was constructed. All 33 CV-A16 strains belonged to the Bla and B1b genotypes, with a nucleotide similarity of 87.9–100% and deduced amino acid similarity of 98.6–100%. Compared with the reference strain Tainan/5079/98 (AF177911), amino acid mutations were identified at positions 11, 23, 25, 31, 99, 145, and 289, where differences were observed among 33 strains, indicating a unique mutation map of CV-A16 in Yantai City. Our findings demonstrate the etiologic characteristics of HFMD, provide supporting evidence for the prevention and control of HFMD, and open a promising avenue for vaccine development against HFMD, by targeting CV-A16.

Characterization of genome sequences and clinical features of coxsackievirus A6 strains collected in Hyogo, Japan in 1999-2013.

Coxsackievirus A6 (CV-A6) is an enterovirus, which is known to cause herpangina. However, since 2009 it has frequently been isolated from children with hand, foot, and mouth disease (HFMD). In Japan, CV-A6 has been linked to HFMD outbreaks in 2011 and 2013. In this study, the full-length genome sequencing of CV-A6 strains were analyzed to identify the association with clinical manifestations. Five thousand six hundred and twelve children with suspected enterovirus infection (0-17 years old) between 1999 and 2013 in Hyogo Prefecture, Japan, were enrolled. Enterovirus infection was confirmed with reverse transcriptase-PCR in 753 children (791 samples), 127 of whom (133 samples) were positive for CV-A6 based on the direct sequencing of the VP4 region. The complete genomes of CV-A6 from 22 positive patients with different clinical manifestations were investigated. A phylogenetic analysis divided these 22 strains into two clusters based on the VP1 region; cluster I contained strains collected in 1999-2009 and mostly related to herpangina, and cluster II contained strains collected in 2011-2013 and related to HFMD outbreak. Based on the full-length polyprotein analysis, the amino acid differences between the strains in cluster I and II were 97.7 ± 0.28%. Amino acid differences were detected in 17 positions within the polyprotein. Strains collected in 1999-2009 and those in 2011-2013 were separately clustered by phylogenetic analysis based on 5'UTR and 3Dpol region, as well as VP1 region. In conclusion, HFMD outbreaks by CV-A6 were recently frequent in Japan and the accumulation of genomic change might be associated with the clinical course.