Characterizing enterovirus C96 genome and phylodynamics analysis.

Enterovirus C96 (EV-C96) is a recently discovered serotype belonging to enterovirus C species. It had been isolated from patients with acute flaccid paralysis, hand, foot, and mouth disease, diarrhea, healthy people, or environmental specimens. Despite increasing reports of the virus, the small number of full-length genomes available for EV-C96 has limited molecular epidemiological studies. In this study, newly collected rare EV-C96 strains in China from 1997 to 2020 were combined with sequences available in GenBank for comprehensive analyses. Sequence analysis revealed that the nucleotide sequence similarity of EV-C96 and the prototype strain (BAN00-10488) was 75%-81.8% and the amino acid sequence similarity was 85%-94.9%. EV-C96 had a high degree of genetic variation and could be divided into 15 genogroups. The mean evolutionary rate was 5.16 × 10-3 substitution/site/year, and the most recent common ancestor was dated to 1925. A recombination analysis revealed that EV-C96 may be a recombinant derived from other serotypes in the EV-C group in the nonstructural protein coding region. This comprehensive and integrated analysis of the whole genome sequence of EV-C96 provides valuable data for further studies on the molecular epidemiology of EV-C96 worldwide.

Whole-genome analysis of coxsackievirus B3 reflects its genetic diversity in China and worldwide.

BACKGROUND: Coxsackievirus B3 (CVB3) has emerged as an active pathogen in myocarditis, aseptic meningitis, hand, foot, and mouth disease (HFMD), and pancreatitis, and is a heavy burden on public health. However, CVB3 has not been systematically analyzed with regard to whole-genome diversity and recombination. Therefore, this study was undertaken to systematically examine the genetic characteristics of CVB3 based on its whole genome. METHODS: We combined CVB3 isolates from our national HFMD surveillance and global sequences retrieved from GenBank. Phylogenetic analysis was performed to examine the whole genome variety and recombination forms of CVB3 in China and worldwide. RESULTS: Phylogenetic analysis showed that CVB3 strains isolated worldwide could be classified into clusters A-E based on the sequence of the entire VP1 region. The predominant CVB3 strains in China belonged to cluster D, whereas cluster E CVB3 might be circulated globally compared to other clusters. The average nucleotide substitution rate in the P1 region of CVB3 was 4.82 × 10-3 substitutions/site/year. Myocarditis was more common with cluster A. Clusters C and D presented more cases of acute flaccid paralysis, and cluster D may be more likely to cause HFMD. Multiple recombination events were detected among CVB3 variants, and there were twenty-three recombinant lineages of CVB3 circulating worldwide. CONCLUSIONS: Overall, this study provides full-length genomic sequences of CVB3 isolates with a wide geographic distribution over a long-term time scale in China, which will be helpful for understanding the evolution of this pathogen. Simultaneously, continuous surveillance of CVB3 is indispensable to determine its genetic diversity in China as well as worldwide.

A clinical study of carbon dioxide lattice laser-assisted or microneedle-assisted 5-aminolevulinic acid-based photodynamic therapy for the treatment of hypertrophic acne scars.

OBJECTIVE: To study the clinical efficacy, recurrence rate and safety of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) combined with microneedle or CO2 lattice laser (CO2FL), in comparison with intrascar betamethasone injection in the treatment of hypertrophic acne scar. METHODS: Fifty-two patients with hypertrophic acne scars at the mandibular angle were enrolled and assigned to different therapy groups. Sixteen patients were treated with microneedle-assisted incorporation of ALA. Twenty-eight patients underwent CO2FL-assisted incorporation of ALA. Eight patients received standard therapy with intrascar injection of glucocorticoid. Two dermatologists, blinded to the therapy groups, independently evaluated the scars in all patients using the average value of the Vancouver Scar Scale score, which was treated as an integer variable. RESULTS: After three rounds of treatment, there was no significant difference in therapeutic effective rate among the microneedle, laser and topical glucocorticoid groups (93.75% vs 100% vs 100%, P = .855). One out of 16 patients (6.25%) in the microneedle group, no patient (0%) in the laser group and two out of eight patients (25%) in the topical glucocorticoid group had recurrence. The laser group showed a higher rate of adverse effects, which were usually mild and reversible, except for pigmentation. Adverse reactions could be completely subsided within 3 weeks. CONCLUSIONS: Either CO2FL or microneedle combined ALA-PDT for hypertrophic scar, as to topical glucocorticoid therapy, showed equivalent clinical effects but lower recurrence rate within 6 months of follow-up period.

A deep eutectic solvent magnetic molecularly imprinted polymer for extraction of laminarin from seaweeds.

Magnetic molecular imprinted polymers (MIPs) based on 4-vinylbenzyltrimethylammonium chloride (VBTAC) and 4-vinylbenzoic acid (VBA) deep eutectic solvent as dual functional monomers was successfully synthesized for the specific recognition of laminarin. The MIPs were characterized by transmission electron microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy, thermal gravimetric analysis, and vibrating sample magnetometer analysis. The results showed that the MIPs were spheres of a uniform size, with the surface rich in cavities and excellent superparamagnetism properties. The adsorption experiments showed that MIPs conform to pseudo-second-order kinetics and Langmuir isotherm adsorption. The maximum adsorption capacity under optimal conditions was 322.58 µg·mg-1 and the imprinting factor was 2.13. Under the optimized conditions, the limit of detection (LOD) of the developed material was 6.6 µM. Linearity of the material was obtained within the range 20-800 µM with a coefficient of determination (r2) being better 0.999. Relative standard deviations (RSDs) were less than 3.96%, and satisfactory recoveries were between 94.55 and 97.39%. The actual sample analysis manifested that MIPs could effectively separate laminarin from Laminarin japonica Aiesch.

Coxsackievirus B4: an underestimated pathogen associated with a hand, foot, and mouth disease outbreak.

In order to discover the causes of a coxsackievirus B4 (CV-B4)-associated hand, foot, and mouth disease (HFMD) outbreak and to study the evolutionary characteristics of the virus, we sequenced isolates obtained during an outbreak for comparative analysis with previously sequenced strains. Phylogenetic and evolutionary dynamics analysis was performed to examine the genetic characteristics of CV-B4 in China and worldwide. Phylogenetic analysis showed that CV-B4 originated from a common ancestor in Shandong. CV-B4 strains isolated worldwide could be classified into genotypes A-E based on the sequence of the VP1 region. All CV-B4 strains in China belonged to genotype E. The global population diversity of CV-B4 fluctuated substantially over time, and CV-B4 isolated in China accounted for a significant increase in the diversity of CV-B4. The average nucleotide substitution rate in VP1 of Chinese CV-B4 (5.20 × 10-3 substitutions/site/year) was slightly higher than that of global CV-B4 (4.82 × 10-3 substitutions/site/year). This study is the first to investigate the evolutionary dynamics of CV-B4 and its association with an HFMD outbreak. These findings explain both the 2011 outbreak and the global increase in CV-B4 diversity. In addition to improving our understanding of a major outbreak, these findings provide a basis for the development of surveillance strategies.

Two Coxsackievirus B3 outbreaks associated with hand, foot, and mouth disease in China and the evolutionary history worldwide.

BACKGROUND: Coxsackievirus B3 (CV-B3) is usually associated with aseptic meningitis and myocarditis; however, the association between CV-B3 and hand, foot, and mouth disease (HFMD) has not been clearly demonstrated, and the phylogenetic dynamics and transmission history of CV-B3 have not been well summarized. METHOD: Two HFMD outbreaks caused by CV-B3 were described in Hebei Province in 2012 and in Shandong Province in 2016 in China. To analyze the epidemiological features of two CV-B3 outbreaks, a retrospective analysis was conducted. All clinical specimens from CV-B3 outbreaks were collected and disposed according to the standard procedures supported by the WHO Global Poliovirus Specialized Laboratory. EV genotyping and phylogenetic analysis were performed to illustrate the genetic characteristics of CV-B3 in China and worldwide. RESULTS: Two transmissible lineages (lineage 2 and 3) were observed in Northern China, which acted as an important "reservoir" for the transmission of CV-B3. Sporadic exporting and importing of cases were observed in almost all regions. In addition, the global sequences of CV-B3 showed a tendency of geographic-specific clustering, indicating that geographic-driven adaptation plays a major role in the diversification and evolution of CV-B3. CONCLUSIONS: Overall, our study indicated that CV-B3 is a causative agent of HFMD outbreak and revealed the phylogenetic dynamics of CV-B3 worldwide, as well as provided an insight on CV-B3 outbreaks for effective intervention and countermeasures.

Isolation of an imported subgenotype B5 strain of human enterovirus A71 in Chongqing City, China, 2014.

BACKGROUND: Enterovirus A71 (EV-A71) is the main pathogen responsible for large outbreaks of hand, foot, and mouth disease (HFMD) in mainland China, and the dominant EV-A71 strains belong to subgenotype C4. To date, only one imported subgenotype B5 of EV-A71 has been reported in Xiamen City Fujian Province, 2009. RESULTS: Here, we report on another imported subgenotype B5 of EV-A71 isolated from a HFMD patient in Chongqing City in 2014 (strain CQ2014-86/CQ/CHN/2014, hereafter refer as CQ2014-86). The VP1 coding sequence and the whole genome sequence revealed that strain CQ2014-86 shares the high nucleotide identity with Vietnamese strains isolated in 2011-2013, suggesting that strain CQ2014-86 may have been imported from Vietnam. In the 5'UTR, P2 and P3 regions, recombination events were found between strain CQ2014-86 and other EV-A, such as coxsackievirus A4 (CV-A4), CV-A5, CV-A14 and CV-A16. CONCLUSIONS: This is the second report on importation of subgenotype B5 of EV-A71 in China, implying that we need to pay more attention to the importation of different subgenotypes of EV-A71.

In vitro and in vivo CT imaging using bismuth sulfide modified with a highly biocompatible Pluronic F127.

Probe bismuth sulfide modified with Pluronic F127 (Bi2S3-PF127), which has high biocompatibility and dispersibility, is synthesized using triblock copolymer Pluronic F127 to modify hydrophobic Bi2S3 nanoparticles that are prepared by a hot injection method. TEM results show that most of the probe has a length of about 14.85 ± 1.70 nm and a breadth of about 4.79 ± 0.63 nm. After injected into the tail vein of a mouse, the probe has obvious CT contrast enhancement capability from x-ray CT imaging results. Meanwhile, the probe's in vivo toxicity is also studied. It is found that hematoxylin and eosin stains of major organs have no change. A biochemical analysis (alanine aminotransferase and aspartate aminotransferase) prove the probe has no adverse effects. The results of a blood analysis (white blood cell count, red blood cell count, hemoglobin, and platelet count) are also normal. The biological distribution of Bi by ICP-AES shows that most of nanoparticles are cleaned out after injection 48 h, and the circulation half-life of the probe is 5.0 h, suggesting that Bi2S3-PF127 has a long circulation and indicating that the Bi2S3-PF127 probe has good biocompatibility and safety.

Genotype F of Echovirus 25 with multiple recombination pattern have been persistently and extensively circulating in Chinese mainland.

Echovirus 25 (E25), a member of the Enterovirus B (EV-B) species, can cause aseptic meningitis (AM), viral meningitis (VM), and acute flaccid paralysis (AFP). However, systematic studies on the molecular epidemiology of E25, especially those concerning its evolution and recombination, are lacking. In this study, 18 strains of E25, isolated from seven provinces of China between 2009 and 2018, were collected based on the Chinese hand, foot, and mouth disease (HFMD) surveillance network, and 95 sequences downloaded from GenBank were also screened. Based on the phylogenetic analysis of 113 full-length VP1 sequences worldwide, globally occurring E25 strains were classified into 9 genotypes (A-I), and genotype F was the dominant genotype in the Chinese mainland. The average nucleotide substitution rate of E25 was 6.08 × 10-3 substitutions/site/year, and six important transmission routes were identified worldwide. Seventeen recombination patterns were determined, of which genotype F can be divided into 9 recombination patterns. A positive selector site was found in the capsid protein region of genotype F. Recombination analysis and pressure selection analysis for genotype F showed multiple recombination patterns and evolution characteristics, which may be responsible for it being the dominant genotype in the Chinese mainland. This study provides a theoretical basis for the subsequent prevention and control of E25.

Evolutionary Diversity of Coxsackievirus A6 Causing Severe Hand, Foot, and Mouth Disease - China, 2012-2023.

Introduction: Coxsackievirus A6 (CVA6) has emerged as a significant pathogen responsible for severe cases of hand, foot, and mouth disease (HFMD). This study aims to delineate the demographic characteristics and analyze the viral evolution of severe HFMD associated with CVA6, thereby assisting in its surveillance and management. Methods: In this investigation, 74 strains of CVA6 were isolated from samples collected from severe HFMD cases between 2012 and 2023. The VP1 gene sequences of CVA6 were amplified and analyzed to assess population historical dynamics and evolutionary characteristics using BEAST, DnaSP6, and PopART. Results: A significant portion (94.4%) of severe CVA6-associated HFMD cases (51 out of 54, with 20 lacking age information) were children under 5 years old. Among the 74 CVA6 strains analyzed, 72 belonged to the D3a sub-genotype, while only two strains were D2 sub-genotype. The average genetic distance between VP1 sequences prior to 2015 was 0.027, which increased to 0.051 when compared to sequences post-2015. Historical population dynamics analysis indicated three significant population expansions of severe CVA6-associated HFMD during 2012-2013, 2013-2014, and 2019-2020, resulting in the formation of 65 distinct haplotypes. Consistent with the MCC tree findings, transitioning between regional haplotypes required multiple base substitutions, showcasing an increase in population diversity during the evolutionary process (from 14 haplotypes in 2013 to 55 haplotypes over the subsequent decade). Conclusions: CVA6, associated with severe HFMD, is evolving and presents a risk of outbreak occurrence. Thus, enhanced surveillance of severe HFMD is imperative.

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.

Genomic Epidemiology and Transmission Dynamics of Global Coxsackievirus B4.

The aim of this study was to determine the global genetic diversity and transmission dynamics of coxsackievirus B4 (CVB4) and to propose future directions for disease surveillance. Next-generation sequencing was performed to obtain the complete genome sequence of CVB4, and the genetic diversity and transmission dynamics of CVB4 worldwide were analyzed using bioinformatics methods such as phylogenetic analysis, evolutionary dynamics, and phylogeographic analysis. Forty complete genomes of CVB4 were identified from asymptomatic infected individuals and hand, foot, and mouth disease (HFMD) patients. Frequent recombination between CVB4 and EV-B multiple serotypes in the 3Dpol region was found and formed 12 recombinant patterns (A-L). Among these, the CVB4 isolated from asymptomatic infected persons and HFMD patients belonged to lineages H and I, respectively. Transmission dynamics analysis based on the VP1 region revealed that CVB4 epidemics in countries outside China were dominated by the D genotype, whereas the E genotype was dominant in China, and both genotypes evolved at a rate of > 6.50 × 10-3 substitutions/site/year. CVB4 spreads through the population unseen, with the risk of disease outbreaks persisting as susceptible individuals accumulate. Our findings add to publicly available CVB4 genomic sequence data and deepen our understanding of CVB4 molecular epidemiology.

Molecular Epidemiology and Evolution of Coxsackievirus A14.

As the proportion of non-enterovirus 71 and non-coxsackievirus A16 which proportion of composition in the hand, foot, and mouth pathogenic spectrum gradually increases worldwide, the attention paid to other enteroviruses has increased. As a member of the species enterovirus A, coxsackievirus A14 (CVA14) has been epidemic around the world until now since it has been isolated. However, studies on CVA14 are poor and the effective population size, evolutionary dynamics, and recombination patterns of CVA14 are not well understood. In this study, 15 CVA14 strains were isolated from HFMD patients in mainland China from 2009 to 2019, and the complete sequences of CVA14 in GenBank as research objects were analyzed. CVA14 was divided into seven genotypes A-G based on an average nucleotide difference of the full-length VP1 coding region of more than 15%. Compared with the CVA14 prototype strain, the 15 CVA14 strains showed 84.0-84.7% nucleotide identity in the complete genome and 96.9-97.6% amino acid identity in the encoding region. Phylodynamic analysis based on 15 CVA14 strains and 22 full-length VP1 sequences in GenBank showed a mean substitution rate of 5.35 × 10-3 substitutions/site/year (95% HPD: 4.03-6.89 × 10-3) and the most recent common ancestor (tMRCA) of CVA14 dates back to 1942 (95% HPD: 1930-1950). The Bayesian skyline showed that the effective population size had experienced a decrease-increase-decrease fluctuation since 2004. The phylogeographic analysis indicated two and three possible migration paths in the world and mainland China, respectively. Four recombination patterns with others of species enterovirus A were observed in 15 CVA14 strains, among which coxsackievirus A2 (CVA2), coxsackievirus A4 (CVA4), coxsackievirus A6 (CVA6), coxsackievirus A8 (CVA8), and coxsackievirus A12 (CVA12) may act as recombinant donors in multiple regions. This study has filled the gap in the molecular epidemiological characteristics of CVA14, enriched the global CVA14 sequence database, and laid the epidemiological foundation for the future study of CVA14 worldwide.

Molecular Characteristics and Genetic Evolution of Echovirus 33 in Mainland of China.

Echovirus, a member of the Enterovirus B (EV-B) family, has led to numerous outbreaks and pandemics, causing a broad spectrum of diseases. Based on the national hand, foot, and mouth disease (HFMD) surveillance system, seven strains of echovirus 33 (E33) were isolated from Mainland of China between 2010 and 2018. The whole genomes of these strains were isolated and sequenced, and phylogenetic trees were constructed based on the gene sequences in different regions of the EV-B prototype strains. It was found that E33 may be recombined in the P2 and P3 regions. Five genotypes (A-E) were defined based on the entire VP1 region of E33, of which the C gene subtype was the dominant gene subtype at present. Recombinant analysis showed that genotype C strains likely recombined with EV-B80, EV-B85, E13, and CVA9 in the P2 and P3 regions, while genotype E had the possibility of recombination with CVB3, E3, E6, and E4. Results of Bayesian analysis indicated that E33 may have appeared around 1955 (95% confidence interval: 1945-1959), with a high evolutionary rate of 1.11 × 10-2 substitution/site/year (95% highest posterior density (HPD): 8.17 × 10-3 to 1.4 × 10-2 substitution/site/year). According to spatial transmission route analysis, two significant transmission routes were identified: from Australia to India and from Oman to Thailand, which the E33 strain in Mainland of China likely introduced from Mexico and India. In conclusion, our study fills the gaps in the evolutionary analysis of E33 and can provide important data for enterovirus surveillance.

Molecular Epidemiology Reveals the Co-Circulation of Two Genotypes of Coxsackievirus B5 in China.

Coxsackievirus B5 (CVB5) is an important enterovirus B species (EV-Bs) type. We used the full-length genomic sequences of 53 viral sequences from the national hand, foot, and mouth disease surveillance network in the Chinese mainland (2001-2021). Among them, 69 entire VP1 coding region nucleotide sequences were used for CVB5 genotyping and genetic evolution analysis. Phylogenetic analysis based on a data set of 448 complete VP1 sequences showed that CVB5 could be divided into four genotypes (A-D) worldwide. Sequences from this study belonged to genotypes B and D, which dominated transmission in the Chinese mainland. Two transmission lineages of CVB5 have been discovered in the Chinese mainland, lineage 2 was predominant. Markov chain Monte Carlo analysis indicated that the tMRCA of CVB5 in the Chinese mainland could be traced to 1955, while the global trend could be traced to 1862, 93 years earlier than China. The evolution rate of CVB5 was higher in the Chinese mainland than worldwide. The spatiotemporal dynamics analysis of CVB5 assessed that virus transportation events were relatively active in Central, Northeast, North and Northwest China. Recombination analysis revealed frequent intertypic recombination in the non-structural region of CVB5 genotypes B and D with the other EV-Bs, revealing eight recombination lineages. Our study showed the molecular evolution and phylogeography of CVB5 that could provide valuable information for disease prevention.

Genomic Epidemiology and Phylodynamic Analysis of Enterovirus A71 Reveal Its Transmission Dynamics in Asia.

Enterovirus A71 (EV-A71) is one of the main pathogens causing hand, foot, and mouth disease (HFMD) outbreaks in Asian children under 5 years of age. In severe cases, it can cause neurological complications and be life-threatening. In this study, 200 newly sequenced EV-A71 whole-genome sequences were combined with 772 EV-A71 sequences from GenBank for large-scale analysis to investigate global EV-A71 epidemiology, phylogeny, and Bayesian phylodynamic characteristics. Based on the phylogenetic analysis of the EV-A71 3Dpol region, six new evolutionary lineages (lineages B, J, K, O, P, and Q) were found in this study, and the number of evolutionary lineages was expanded from 11 to 17. Temporal dynamics and recombination breakpoint analyses based on genotype C revealed that recombination of nonstructural protein-coding regions, including 3Dpol, is an important reason for the emergence of new lineages. The EV-A71 epidemic in the Asia-Pacific region is complex, and phylogeographic analysis found that Vietnam played a key role in the spread of subgenotypes B5 and C4. The origin of EV-A71 subgenotype C4 in China is East China, which is closely related to the prevalence of subgenotype C4 in the south and throughout China. Selection pressure analysis revealed that, in addition to VP1 amino acid residues VP1-98 and VP1-145, which are associated with EV-A71 pathogenicity, amino acid residues VP1-184 and VP1-249 were also positively selected, and their functions still need to be determined by biology and immunology. This study aimed to provide a solid theoretical basis for EV-A71-related disease surveillance and prevention, antiviral research, and vaccine development through a comprehensive analysis. IMPORTANCE EV-A71 is one of the most important pathogens causing HFMD outbreaks; however, large-scale studies of EV-A71 genomic epidemiology are currently lacking. In this study, 200 new EV-A71 whole-genome sequences were determined. Combining these with 772 EV-A71 whole-genome sequences in the GenBank database, the evolutionary and transmission characteristics of global and Asian EV-A71 were analyzed. Six new evolutionary lineages were identified in this study. We also found that recombination in nonstructural protein-coding regions, including 3Dpol, is an important cause for the emergence of new lineages. The results provided a solid theoretical basis for EV-A71-related disease surveillance and prevention, antiviral research, and vaccine development.

Molecular evidence of persistent epidemic and evolution of subgenotype B1 coxsackievirus A16-associated hand, foot, and mouth disease in China.

The molecular epidemiology of CVA16 in China between 1999 and 2008 reflects a pattern of endemic cocirculation of clusters B1a and B1b within subgenotype B1 viruses. The annual evolution rate of CVA16 was estimated as approximately 0.91 x 10(-2) substitutions per synonymous nucleotide/year and is slightly lower than that of HEV71.

Retrospective seroepidemiology indicated that human enterovirus 71 and coxsackievirus A16 circulated wildly in central and southern China before large-scale outbreaks from 2008.

BACKGROUND: Large nationwide outbreaks of hand, foot, and mouth disease (HFMD) occurred in China from 2008; most of the cases were in children under 5 years. This study aims to identify the situation of natural human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) infections in children before 2008 in China. RESULTS: Retrospective seroepidemiologic studies of HEV71 and CVA16 were performed with 900 serum samples collected from children ≤5 years of age in 2005. The samples were collected from 6 different geographical areas (Anhui, Guangdong, Hunan, Xinjiang, Yunnan, and Heilongjiang provinces) in mainland China. Of the 900 samples, 288 were positive for HEV71; the total positive rate was 32.0% and the geometric mean titer (GMT) was 1:8.5. Guangdong (43.7% and 1:10.8), Xinjiang (45.4% and 1:11.1), and Yunnan (43.4% and 1:12.0) provinces had relatively high rates of infection, while Heilongjiang province (8.1% and 1:4.9) had the lowest rate of infection. On the other hand, 390 samples were positive for CVA16; the total positive rate was 43.4% and the GMT was 1:9.5. Anhui (62.2% and 1:16.0) and Hunan (61.1% and 1:23.1) had relatively high rates, while Heilongjiang (8.0% and 1:4.6) had the lowest rate. Although there is a geographical difference in HEV71 and CVA16 infections, low neutralizing antibody positive rate and titer of both viruses were found in all 6 provinces. CONCLUSIONS: This report confirmed that HEV71 and CVA16 had wildly circulated in a couple provinces in China before the large-scale outbreaks from 2008. This finding also suggests that public health measures to control the spread of HEV71 and CVA16 should be devised according to the different regional characteristics.

An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand foot and mouth disease in Fuyang city of China.

Hand, foot and mouth disease (HFMD), a common contagious disease that usually affects children, is normally mild but can have life-threatening manifestations. It can be caused by enteroviruses, particularly Coxsackieviruses and human enterovirus 71 (HEV71) with highly variable clinical manifestations. In the spring of 2008, a large, unprecedented HFMD outbreak in Fuyang city of Anhui province in the central part of southeastern China resulted in a high aggregation of fatal cases. In this study, epidemiologic and clinical investigations, laboratory testing, and genetic analyses were performed to identify the causal pathogen of the outbreak. Of the 6,049 cases reported between 1 March and 9 May of 2008, 3023 (50%) were hospitalized, 353 (5.8%) were severe and 22 (0.36%) were fatal. HEV71 was confirmed as the etiological pathogen of the outbreak. Phylogenetic analyses of entire VP1 capsid protein sequence of 45 Fuyang HEV71 isolates showed that they belong to C4a cluster of the C4 subgenotype. In addition, genetic recombinations were found in the 3D region (RNA-dependent RNA polymerase, a major component of the viral replication complex of the genome) between the Fuyang HEV71 strain and Coxsackievirus A16 (CV-A16), resulting in a recombination virus. In conclusion, an emerging recombinant HEV71 was responsible for the HFMD outbreak in Fuyang City of China, 2008.

Genetic Diversity Analysis of Coxsackievirus A8 Circulating in China and Worldwide Reveals a Highly Divergent Genotype.

Coxsackievirus A8 (CV-A8) is one of the pathogens associated with hand, foot and mouth disease (HFMD) and herpangina (HA), occasionally leading to severe neurological disorders such as acute flaccid paralysis (AFP). Only one study aimed at CV-A8 has been published to date, and only 12 whole-genome sequences are publicly available. In this study, complete genome sequences from 11 CV-A8 strains isolated from HFMD patients in extensive regions from China between 2013 and 2018 were determined, and all sequences from GenBank were retrieved. A phylogenetic analysis based on a total of 34 complete VP1 sequences of CV-A8 revealed five genotypes: A, B, C, D and E. The newly emerging genotype E presented a highly phylogenetic divergence compared with the other genotypes and was composed of the majority of the strains sequenced in this study. Markov chain Monte Carlo (MCMC) analysis revealed that genotype E has been evolving for nearly a century and somehow arose in approximately 2010. The Bayesian skyline plot showed that the population size of CV-A8 has experienced three dynamic fluctuations since 2001. Amino acid residues of VP1100N, 103Y, 240T and 241V, which were embedded in the potential capsid loops of genotype E, might enhance genotype E adaption to the human hosts. The CV-A8 whole genomes displayed significant intra-genotypic genetic diversity in the non-capsid region, and a total of six recombinant lineages were detected. The Chinese viruses from genotype E might have emerged recently from recombining with European CV-A6 strains. CV-A8 is a less important HFMD pathogen, and the capsid gene diversity and non-capsid recombination variety observed in CV-A8 strains indicated that the constant generation of deleterious genomes and a constant selection pressure against these deleterious mutations is still ongoing within CV-A8 quasispecies. It is possible that CV-A8 could become an important pathogen in the HFMD spectrum in the future. Further surveillance of CV-A8 is greatly needed.