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 In recent years, enterovirus A71 (EV-A71) has been replaced by Coxsackievirus A6 (CV-A6) to become the predominant serotype.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.

Epidemiological and etiological characteristics of viral meningitis for hospitalized pediatric patients in Yunnan, China.

BACKGROUND: Viral infection is the most common cause of aseptic meningitis. The purpose of this study was to identify the viruses responsible for aseptic meningitis to better understand the clinical presentations of this disease. METHOD: Between March 2009 and February 2010, we collected 297 cerebrospinal fluid specimens from children with aseptic meningitis admitted to a pediatric hospital in Yunnan (China). Viruses were detected by using "in house" real-time quantitative polymerase chain reaction or reverse-transcription real-time quantitative polymerase chain reaction from these samples. Phylogenetic analyses were conducted using the Molecular Evolutionary Genetic Analysis version 7.0 software, with the neighbor-joining method. RESULTS: Viral infection was diagnosed in 35 of the 297 children (11.8%). The causative viruses were identified to be enteroviruses in 25 cases (71.4%), varicella-zoster virus in 5 cases (14.3%), herpes simplex virus 1 in 2 cases (5.7%), and herpes simplex virus 2, Epstein-Barr virus, and human herpesvirus 6 in 1 case each (2.9% each). Of the enteroviruses, coxsackievirus B5 was the most frequently detected serotype (10/25 cases; 40.0%) and all coxsackievirus B5 strains belonged to C group. CONCLUSIONS: In the study, a causative virus was only found in the minority of cases, of them, enteroviruses were the most frequently detected viruses in patients with viral meningitis, followed by varicella-zoster virus and herpes simplex virus. Our findings underscore the need for enhanced surveillance and etiological study of aseptic meningitis.

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.

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.

Characterization of a novel echovirus 21 strain isolated from a healthy child in China in 2013.

Echovirus 21 (E21) belongs to the species Enterovirus B, whose members are frequently associated with acute flaccid paralysis. E21 strain 553/YN/CHN/2013 was isolated from a healthy child in Yunnan, China, in 2013. This is the first report of the complete genome sequence of E21 in China. This strain shared 81.7% nucleotide sequence identity and 96.8% amino acid sequence identity with the E21 prototype strain Farina. Although strain 553/YN/CHN/2013 belongs to the E21 serotype, the only similarity to the E21 strain was in the VP1 region, as other genomic regions, including VP2-VP4, were more similar to other EV-B members. Recombination analysis showed evidence of recombination events between E21 and other EV-B viruses. E21 strain 553/YN/CHN/2013 failed to infect suckling mice via intracerebral injection. Surveillance of E21 is very important to help forecast the potential of emerging E21 outbreaks and related diseases.