First detection of multiple cases related to CV-A16 strain of B1c clade in Beijing in 2022.

Coxsackievirus A16 (CV-A16) is a significant etiologic agent of hand, foot, and mouth disease (HFMD) and herpangina (HA), with the capacity to progress to severe complications, including encephalitis, aseptic meningitis, acute flaccid paralysis, myocarditis, and other critical conditions. Beijing's epidemiological surveillance system, established in 2008, encompasses 29 hospitals and 16 district disease control centers. From 2019 to 2021, the circulation of CV-A16 was characterized by the co-circulation of B1a and B1b clades. Multiple cases of HFMD linked to clade B1c has not been reported in Beijing until 2022. This study enrolled 400 HFMD and 493 HA cases. Employing real-time RT-PCR, 368 enterovirus-positive cases were identified, with 180 selected for sequencing. CV-A16 was detected in 18.89% (34/180) of the cases, second only to CV-A6, identified in 63.33% (114/180). Full-length VP1 gene sequences were successfully amplified and sequenced in 22 cases, revealing the presence of clades B1a, B1b, and B1c in 14, 3, and 5 cases, respectively. A cluster of five B1c clade cases occurred between June 29 and July 17, 2022, within a 7-km diameter region in Shunyi District. Phylogenetic analysis of five complete VP1 gene sequences and two full-genome sequences revealed close clustering with the 2018 Indian strain (GenBank accession: MH780757.1) within the B1c India branch, with NCBI BLAST results showing over 98% similarity. Comparative sequence analysis identified three unique amino acid variations (P3S, V25A, and I235V). The 2022 Shunyi District HFMD cases represent the first instances of spatiotemporally correlated CV-A16 B1c clade infections in Beijing, underscoring the necessity for heightened surveillance of B1c clade CV-A16 in HFMD and HA in this region.

Phylogenetics and phylogeographic characteristics of coxsackievirus A16 in hand foot and mouth disease and herpangina cases collected in Beijing, China from 2019 to 2021.

Coxsackievirus A16 (CV-A16) is a significant pathogen responsible for causing hand foot and mouth disease (HFMD) and herpangina (HA). This study aimed to investigate the recent evolution and spread of CV-A16 by monitoring HFMD and HA cases in 29 hospitals across 16 districts in Beijing from 2019 to 2021. The first five cases of HFMD and the first five cases of HA each month in each hospital were included in the study. Real-time reverse transcription polymerase chain reaction was used to identify CV-A16, CV-A6, and EV-A71. From each district, two to four CV-A16 positive samples with a relatively long sampling time interval every month were selected for sequencing. A total of 3344 HFMD cases and 2704 HA cases were enrolled in this study, with 76.0% (2541/3344) of HFMD and 45.4% (1227/2704) of HA cases confirmed to be infected by enterovirus. Among the EV-positive samples, CV-A16 virus was detected in 33.61% (854/2541) of HFMD cases and 13.4% (165/1227) of HA cases, with the predominant cluster being B1a. Both B1a and B1b had a co-circulation of local and imported strains, with different origin time (1993 vs. 1995), different global distribution (14 countries vs. 10 countries), and different transmission centers but mainly distributed in the southern and eastern regions of Beijing. Strengthening surveillance of HFMD in southern and eastern regions will improve the prevention and control efficiency of enterovirus infections.

Molecular epidemiology and phylodynamic analysis of enterovirus 71 in Beijing, China, 2009-2019.

BACKGROUND: Enterovirus 71(EV71)-associated hand, foot and mouth disease (HFMD) decreased dramatically in Beijing from 2009 to 2019. This study was to investigate the epidemiological characteristics, evolutionary dynamics, geographic diffusion pathway, and other features of EV71 in Beijing, China. METHODS: We conducted a retrospective study of EV71-associated HFMD and its causative agent in Beijing, China, from 2009 to 2019. Phylogenetic and phylogeographic methods based on the EV71 genome were used to determine the evolution features, origin, and spatiotemporal dynamics. Positive selection sites in the VP1 gene were identified and exhibited in the tertiary structure. Bayesian birth-death skyline model was used to estimate the effective reproductive number (Re). RESULTS: EV71-associated HFMD decreased greatly in Beijing. From 2009 to 2019, EV71 strains prevalent in Beijing shared high homology in each gene segment and evolved with a rate of 4.99*10- 3 substitutions per site per year. The genetic diversity of EV71 first increased and peaked in 2012 and then decreased with fluctuations. The time to the most recent common ancestor (TMRCA) of EV71 in Beijing was estimated around 2003 when the EV71 strains were transmitted to Beijing from east China. Beijing played a crucial role in seeding EV71 to central China as well. Two residues (E145Q/G, A293S) under positive selection were detected from both the VP1 dataset and the P1 dataset. They were embedded within the loop of the VP1 capsid and were exposed externally. Mean Re estimate of EV71 in Beijing was about 1.007. CONCLUSION: In recent years, EV71 was not the primary causative agent of HFMD in Beijing. The low Re estimate of EV71 in Beijing implied that strategies for preventing and controlling HFMD were performed effectively. Beijing and east China played a crucial role in disseminating EV71 to other regions in China.

Evolution, recombination and geographic spreading of global Coxsackievirus A6.

BACKGROUND: The increasing incidence of hand, foot, and mouth disease (HFMD) associated with Coxsackievirus A6 (CVA6) has become a very significant public health problem. The aim of this study is to investigate the recombination, geographic transmission, and evolutionary characteristics of the global CVA6. METHODS: From 2019 to 2022, 73 full-length CVA6 sequences were obtained from HFMD patients in China and analyzed in combination with 1032 published whole genome sequences. Based on this dataset, the phylogenetic features, recombinant diversity, Bayesian phylodynamic characteristics, and key amino acid variations in CVA6 were analyzed. RESULTS: The four genotypes of CVA6, A, D, E, and F, are divided into 24 recombinant forms (RFs, RF-A - RF-X) based on differences in the P3 coding region. The eastern China region plays a key role in the dissemination of CVA6 in China. VP1-137 and VP1-138 are located in the DE loop on the surface of the CVA6 VP1 protein, with the former being a highly variable site and the latter having more non-synonymous substitutions. CONCLUSIONS: Based on whole genome sequences, this study contributes to the CVA6 monitoring, early warning, and the pathogenic mechanism by studying recombination diversity, geographical transmission characteristics, and the variation of important amino acid sites.

Biochemical characterization of enterovirus 71 3D RNA polymerase.

An unusual enterovirus 71 (EV71) epidemic has begun in China since 2008. EV71 RNA polymerases (3D(pol)) showed polymerase activity with an Mn(2+). Little activity was detected with Co(2+), and no activity was detected with Mg(2+), Ca(2+), Cu(2+), Ni(2+), Cd(2+), or Zn(2+). It is a primer-dependent polymerase, and the enzyme functioned with both di- and 10-nucleotide RNA primers. DNA primer, dT15, increased primer activity, similar to other enterovirus 3D(pol). However, EV71 3D(pol) initiated de novo transcription with a poly(C) template and genome RNA. Its RNA binding activity was weak. Terminal nucleotidyl transferase and reverse transcriptase activity were not detected. The Km and Vmax for EV71 3D(pol) were calculated from classic Lineweaver-Burk plots. The Km values were 2.35±0.05 (ATP), 5.40±0.93 (CTP), 1.12±0.10 (GTP) and 2.81±0.31 (UTP), and the Vmax values were 0.00078±0.00005/min (ATP), 0.011±0.0017/min (CTP), 0.050±0.0043/min (GTP) and 0.0027±0.0005/min (UTP). The Km of EV71 3D(pol) was similar to that of foot and mouth disease virus and rhinovirus. Polymerase activity of BrCr-TR strain and a strain from a clinical isolate in Beijing, 2008 were similar, indicating the potential for 3D(pol) as an antiviral drug target.

Molecular analysis of virulent determinants of enterovirus 71.

Enterovirus 71 (EV71) is the most important causative agent of hand, foot and mouth disease (HFMD) in children. In most cases, it is a self-limiting illness. However some EV71 infectious cases can develop severe clinical outcomes, such as encephalitis, meningitis, poliomyelitis like paralysis, and even death. To identify the determinants of virulence, the deduced amino acid sequence of polyprotein and nucleotide sequence of 5'-NTR and 3'-NTR in 25 SC-EV71 strains (strains from severe cases) and 31 MC-EV71 strains (strains from mild cases) were analyzed. Results showed four amino acids on two positions (Gly(P710)/Gln(P710)/Arg(P710) and Glu(P729)) on the DE and EF loop of VP1, one (Lys(P930)) on the surface of protease 2A and four nucleotides on three positions (G(P272), U(P488) and A(P700)/U(P700)) in the 5'-NTR region are associated with EV71 virulent phenotype. Predicted secondary structure of RNA using the consensus sequence of 5'-NTR by RNAStructure showed the mutation of nucleotide at position 488 in strain BJ08-Z004-3 (position 491 in prototype strain BrCr) can result in the discrepancy of an additional pair of nucleotides and thus change the stability of the second structure of IRES. Fragment base content analysis showed that in the region 696 to 714 bp at the 5'-NTR, where the A(P700)/U(P700) was located, the nucleotide constitution ratios differed significantly between SC-EV71 and MC-EV71 strains. In conclusion, comparative genomic analysis showed that virulence of EV71 strains are mainly determined by the amino acids on two positions of VP1, one position of protease 2A and the nucleotides on three positions in 5'-NTR.

[Human enterovirus 71 was isolated from specimens of hand foot mouth disease in Beijing in 2007].

OBJECTIVE: Analyzing and identifying the type of enterovirus of human Hand Foot Mouth Disease (HFMD) outbreak in Daxing district in Beijing at the end of May in 2007. METHODS: The liquid of Herpes, throat swab and stool specimen were collected. Using reverse transcription-polymerase chain reaction (RT-PCR) method to amplify the enterovirus specific nucleotide acid fragment from specimens and virus positive cultures, the sensitivity of two methods was compared. Then identifying and typing of the enterovirus of HFMD through analyzing the results of nucleotide sequencing of the virus positive cultures. RESULTS: In 10 specimens from 5 HFMD children patients, enterovirus specific nucleotide acid fragment was detected in 8 patients, the RT-PCR positive ratio (80%) was higher than enterovirus isolation positive ratio (30%). In 5 enteroviruses isolated from 5 patients and 9 close contacts, 4 isolated from 2 patients and 1 close contact were Enterovirus 71.1 isolated from close contact was Coxsackievirus A16. CONCLUSION: Enterovirus 71 was the pathogen of HFMD outbreak in Daxing district at the end of May in 2007.

[Study on the etiology of hand-foot-mouth disease outbreaks in Beijing in 2007].

OBJECTIVE: To identify the etiology of 8 human hand-foot-mouth disease (HFMD) outbreaks in Beijing, during May to July 2007. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) method was used to directly type the specimens including fluid from the herpes and throat swabs from the HFMD patients. Using RD cell lines, the collected stool specimens were cultured followed by typing. Partial VP1 region of selected EV positive specimens and cultures were sequenced and both nucleic acid sequence and predicted amino acid sequence were analyzed. RESULTS: The two HFMD outbreaks in Daxing region in Beijing in 2007 were caused by enterovirus 71 type (EV71), and the others were caused by Coxsackie virus A16 (Cox A16). Two EV71 strains caused epidemics in Daxing region in 2007 belonged to C4 subgenotype but on different branches in VP1 gene phylogenetic tree. The differences on nucleic acid sequence and amino acid sequence were 3.7% and 0.8% between the two EV71 stains, respectively. The Cox A16 strain in Shunyi region and the other strains were on different branches in phylogenetic tree, and the difference on nucleic acid and amino acid sequence were 3.7% and 0% respectively between the two Cox A16 stains. CONCLUSION: The HFMD outbreaks occurred in Beijing in 2007 were caused mainly by EV71 and Cox A16, and there were two individual epidemic virus strains. Cox A16 seemed to spread more widely than EV71 in Beijing, 2007.

[Genetic characteristics of enterovirus 71 isolated in Beijing, 2006-2008].

OBJECTIVE: To sequence and analyze the VP1 region of isolated enterovirus from different sources in Beijing, 2006-2008. METHODS: 9 EV71 were selected from the isolates identified through the specimen of human hand foot mouth disease (HFMD), acute flaccid paralysis (AFP) and healthy children in Beijing, 2006-2008. Reverse transcription-polymerase chain reaction (RT-PCR) method was used to amplify and sequence the whole VP1 gene of enterovirus. Phylogenetic tree was constructed, with the means of nucleotide homology and distance between/within groups analyzed. RESULTS: The 9 selected strains were clustered with C4 subgenotype reference strains in Phylogenetic tree and showed high nucleotide acid identity (92.1%-93.9% ) in nucleotide homology analysis, and had higher homology than C1, C2, C3 subgenotype reference strains (88.8%-89.5%, 89.4%-90.0% and 88.4%-89.3%, respectively). High homologous (95.9%-100.0%) was noticed between the isolated stains from three different sources, but low homologous (93.3% -93.9%, 92.1%-92.9%, respectively) showed between the isolated stains and C4 reference strains isolated in 1998. There appeared larger variations between groups in C4 subgenotype when analyzing the distance between groups means, especially between the reference strains and isolated strains (D = 0.052-0.071). CONCLUSION: The EV71 isolated in Beijing, from 2006 to 2008 also appeared to be C4 subgenotype and there was no significant difference found in the whole sequence of VP1 gene of the strains isolated from different regions, sources, or under different diseases occurred in the same period. There were more nucleotide variations and more chances for the presence of new subgenotype, suggesting that it is necessary to strengthen the surveillance program on EV71 isolates.