Coxsackievirus B: The important agent of hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) is a common pediatric infectious illness caused by enteroviruses (EVs). EV-A serotypes are the main pathogens associated with HFMD. In this study, 213 stool samples from 213 children with severe HFMD in Yunnan, China in 2013, 2015, and 2016 were further analyzed retrospectively for EV-B infection. A total of 70.0% of the specimens tested positive for EV.20 EV serotypes were detected. The predominant serotype was enterovirus A71 (EV-A71, 27.7%), followed by coxsackievirus B4 (CV-B4, 16.4%), CV-A16 (9.9%), CV-B5 (6.6%), and Echovirus 9 (E-9,4.7%). EV-A and EV-B accounted for 45.1% and 41.3%, respectively. Among the positive specimens, 28.6% were CV-Bs. Co-infection was present in 19.3% of these cases. In the study, CV-B5 and the majority of CV-B4 isolates belonged to genotypes VI and C3, respectively. This result indicates that EV-B, especially CV-Bs, might be the important agents associated with HFMD and this knowledge will contribute to the prevention and treatment of the disease.

Molecular characterization of a novel clade echovirus 3 isolated from patients with hand-foot-and-mouth disease in southwest China.

Echovirus 3 (E3) belongs to the species Enterovirus B. Currently, three nearly whole-genome sequences of E3 are available in GenBank in China. In this study, we determined the whole genomic sequences of six E3 strains isolated from the stools of patients with hand-foot-and-mouth disease in Southwest China in 2022. Their nucleotide and amino acid sequences shared 82.1%-86.4% and 96.6%-97.2% identity with the prototype Morrisey strain, respectively, and showed 87.1% and 97.2% mutual identity. The six E3 strains are not clustered with other Chinese strains and formed a novel subgenotype (C6) with the recent American and British strains. Recombination analyses revealed that intertype recombination had occurred in the 2 C and 3D regions of the six E3 strains with coxsackieviruses B5 and B4, respectively. This study augments the nearly whole-genome sequences of E3 in the GenBank database and extends the molecular characterization of this virus in China.

Isolation and characterization of a novel clade of coxsackievirus B2 associated with hand, foot, and mouth disease in Southwest China.

Coxsackievirus B2 (CVB2) is an enterovirus B (EV-B) species and can cause aseptic meningitis, myocarditis and hand, foot, and mouth disease (HFMD). We characterized a novel CVB2 (YN31V3) associated with HFMD in Yunnan, Southwest China, in 2019. Although YN31V3 and other Mainland China epidemic strains mainly belonged to genotype C, YN31V3 formed an independent branch. The genome sequence of the strain YN31V3 from this study showed a 12.91% nucleotide difference to its closest strain RW41-2/YN/CHN/2012. Recombination analyses showed that the newly isolated YN31V3 was probably a recombinant, which was closely related to CVB2 strains in the genomic P1 region and other EV-B strains in the P2 and P3 regions, respectively. YN31V3 strain had a temperature-sensitive phenotype. The challenge of suckling BALB/c mice with YN31V3 could cause symptoms of disease and severe pathological lesions.

Characterization of coxsackievirus A10 strains isolated from children with hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) is a contagious disease that threatens the health of children under 5 years of age. Coxsackievirus A10 (CV-A10) is one of the main pathogens of HFMD. Currently, preventive vaccines and specific therapeutic drugs are not available for CV-A10. In this study, a total of 327 stool specimens were collected from pediatric patients from 2009 to 2017 during HFMD surveillance, among which 14 CV-A10 strains could only be isolated from rhabdomyosarcoma cells, but not from KMB17 and Vero cells. Through adaptive culture, 2 and 11 CV-A10 strains were recovered from Vero and KMB17 cell cultures, respectively. The growth of CV-A10 strains in Vero cells was better than that in KMB17 cells. The 14 CV-A10 strains belonged to the F genotype, and the nucleotides and amino acids of their complete genomes shared 92.6%-96.3% and 98.4%-98.9% identities, respectively. The different CV-A10 strains exhibited varying virulence in vivo, but had similar effects on tissue injury, with the hind limb muscles, kidneys, and lungs being severely affected. Additionally, the hind limb muscles had the highest viral loads. CV-A10 was found to exhibit a strong tropism to muscle tissue. The results of this study are critical to developing vaccines against CV-A10 infections.

Co-infection and enterovirus B: post EV-A71 mass vaccination scenario in China.

BACKGROUND: Hand, foot, and mouth disease (HFMD) is a common child infectious disease caused by more than 20 enterovirus (EV) serotypes. In recent years, enterovirus A71 (EV-A71) has been replaced by Coxsackievirus A6 (CV-A6) to become the predominant serotype. Multiple EV serotypes co-circulate in HFMD epidemics, and this study aimed to investigate the etiological epidemic characteristics of an HFMD outbreak in Kunming, China in 2019. METHODS: The clinical samples of 459 EV-associated HFMD patients in 2019 were used to amplify the VP1 gene region by the three sets of primers and identify serotypes using the molecular biology method. Phylogenetic analyses were performed based on the VP1 gene. RESULTS: Three hundred and forty-eight cases out of 459 HFMD patients were confirmed as EV infection. Of these 191 (41.61%) were single EV infections and 34.20% had co-infections. The EVs were assigned to 18 EV serotypes, of which CV-A6 was predominant (11.33%), followed by CV-B1 (8.93%), CV-A4 (5.23%), CV-A9 (4.58%), CV-A 16 (3.49%) and CV-A10 and CVA5 both 1.96%. Co-infection of CV-A6 with other EVs was present in 15.25% of these cases, followed by co-infection with CV-A16 and other EVs. The VP1 sequences used in the phylogenetic analyses showed that the CV-A6, CV-B1 and CV-A4 sequences belonged to the sub-genogroup D3 and genogroups F and E, respectively. CONCLUSION: Co-circulation and co-infection of multiple serotypes were the etiological characteristic of the HFMD epidemic in Kunming China in 2019 with CV-A-6, CV-B1 and CV-A4 as the predominant serotypes. This is the first report of CV-B1 as a predominant serotype in China and may provide valuable information for the diagnosis, prevention and control of HFMD.

Screening of a new candidate coxsackievirus B1 vaccine strain based on its biological characteristics.

Coxsackievirus B1 (CVB1) is one of the significant pathogens causing viral myocarditis, hand, foot, and mouth disease (HFMD), and aseptic meningitis, and it has been associated with type 1 diabetes (T1DM). No effective antiviral drugs against CVB1 infection or preventive vaccines are available. Due to the success of two inactivated vaccines against enterovirus 71 and poliovirus, an inactivated Vero cell-based CVB1 vaccine could be developed. In this study, we isolated a high-growth CVB1 virus strain KM7 in Vero cells and developed a Vero-adapted vaccine candidate strain KM7-X29 via three rounds of plaque purification and serial passages. The KM7-X29 strain was grouped into the GII sub-genotype, which belonged to the Chinese epidemic strain and grew to a titer of more than 107 CCID50/ml in Vero cells. The inactivated CVB1 vaccine produced by the KM7-X29 strain induced an effective neutralizing antibody response in BALB/c mice, and maternal antibodies were able to provide a 100% protective effect against lethal challenges with a CVB1 strain in suckling BALB/c mice. Thus, the KM7-X29 strain might be used as a new candidate coxsackievirus B1 vaccine strain. The neonatal murine model of CVB1 infection will contribute to the development of the CVB1 vaccine.

Molecular characterization of echovirus 9 strains isolated from hand-foot-and-mouth disease in Kunming, Yunnan Province, China.

Echovirus 9 (E9) belongs to the species Enterovirus B. So far, 12 whole genome sequences of E9 are available in GenBank. In this study, we determined the whole genomic sequences of five E9 strains isolated from the stools of patients with hand-foot-and-mouth disease in Kunming, Yunnan Province, China, in 2019. Their nucleotide and amino acid sequences shared 80.8-80.9% and 96.4-96.8% identity with the prototype Hill strain, respectively, and shared 99.3-99.9% and 99.1-99.8% mutual identity, respectively. Recombination analyses revealed that intertype recombination had occurred in the 2C and 3D regions of the five Yunnan E9 strains with coxsackieviruses B5 and B4, respectively. This study augmented the whole genome sequences of E9 in the GenBank database and extended the molecular characterization of this virus in China.

Characterization of Coxsackievirus A6 Strains Isolated From Children With Hand, Foot, and Mouth Disease.

Coxsackievirus A6 (CVA6) is a key pathogen causing hand, foot and mouth disease (HFMD). However, there are currently no specific antiviral drugs or vaccines for treating infections caused by CVA6. In this study, human rhabdomyosarcoma (RD), African green monkey kidney (Vero), and human embryonic lung diploid fibroblast (KMB17) cells were used to isolate CVA6 from 327 anal swab and fecal samples obtained during HFMD monitoring between 2009 and 2017. The VP1 genes of the isolates were sequenced and genotyped, and the biological characteristics of the representative CVA6 strains were analyzed. A total of 37 CVA6 strains of the D3 gene subtypes were isolated from RD cells, all of which belonged to the epidemic strains in mainland China. Using the adaptive culture method, 10 KMB17 cell-adapted strains were obtained; however, no Vero cell-adapted strains were acquired. Among the KMB17 cell-adapted strains, only KYN-A1205 caused disease or partial death in suckling mice, and its virulence was stronger than its RD cell-adapted strain. The pathogenic KYN-A1205 strain caused strong tropism to the muscle tissue and led to pathological changes, including muscle necrosis and nuclear fragmentation in the forelimb and hindlimb. Sequence analysis demonstrated that the KYN-A1205 strain exhibited multiple amino acid mutations after KMB17 cell adaptation. Moreover, it showed strong pathogenicity, good immunogenicity and genetic stability, and could be used as an experimental CVA6 vaccine candidate.