Pathological Characteristics of Echovirus 30 Infection in a Mouse Model.

Echovirus 30 (E30), a member of species B enterovirus, is associated with outbreaks of aseptic meningitis and has become a global health emergency. However, the pathogenesis of E30 remains poorly understood due to the lack of appropriate animal models. In this study, we established a mouse infection model to explore the pathogenicity of E30. The 2-day-old IFNAR-/- mice infected with E30 strain WZ16 showed lethargy and paralysis, and some died. Obvious pathological changes were observed in the skeletal muscle, brain tissue, and other tissues, with the highest viral load in the skeletal muscles. Transcriptome analysis of brain and skeletal muscle tissues from infected mice showed that significant differentially expressed genes were enriched in complement response and neuropathy-related pathways. Using immunofluorescence assay, we found that the viral double-stranded RNA (dsRNA) was detected in the mouse brain region and could infect human glioma (U251) cells. These results indicated that E30 affects the nervous system, and they provide a theoretical basis for understanding its pathogenesis. IMPORTANCE Echovirus 30 (E30) infection causes a wide spectrum of diseases with mild symptoms, such as hand, foot, and mouth disease (HFMD), acute flaccid paralysis, and aseptic meningitis and other diseases, especially one of the most common pathogens causing aseptic meningitis outbreaks. We established a novel mouse model of E30 infection by inoculating neonatal mice with clinical isolates of E30 and observed the pathological changes induced by E30. Using the E30 infection model, we found complement responses and neuropathy-related genes in the mice tissues at the transcriptome level. Moreover, we found that the viral dsRNA localized in the mouse brain and could replicate in human glioma cell line U251 rather than in the neuroblastoma cell line, SK-N-SH.

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.

[Analysis of common viral infection in surveillance cases of febrile respiratory syndrome in 9 provinces of China from 2009 to 2021].

Objective: To understand the common viral infection among the surveillance cases of fever respiratory syndrome (FRS) in nine provinces in China. Methods: The research data were obtained from nine provinces (Anhui, Beijing, Guangdong, Hebei, Hunan, Jilin, Shandong, Shaanxi and Xinjiang) in the "Infectious Disease Surveillance Technology Platform Information Management System" of the Chinese Center for Disease Control and Prevention from January 2009 to June 2021. Finally, 8 243 FRS cases with nucleic acid detection results of eight viruses [human influenza virus (HIFV), human respiratory syncytial virus (HRSV), human adenovirus (HAdV), human parainfluenza virus (HPIV), human rhinovirus (HRV), human metapneumovirus (HMPV), human coronavirus (HCoV) and human Boca virus (HBoV)] were included in the study. The χ2 test/Fisher exact probability method was used to analyze the difference of virus detection rate in different age groups, regions and seasons. Results The M (Q1, Q3) age of 8 243 FRS cases was 4 (1, 18) years old, and 56.56% (4 662 cases) were children under 5 years old. Males accounted for 58.1% (4 792 cases) of all cases. All cases were from outpatient/emergency department (2 043 cases) and inpatient department (6 200 cases). The virus detection rates of FRS cases from high to low were HRSV, HIFV, HPIV, HRV, HAdV, HMPV, HCoV and HBoV. Two or more viruses were detected simultaneously in 524 cases, accounting for 15.66% of virus-positive cases. The difference of the virus detection rate in different age groups was statistically significant (all P values<0.05), and the virus detection rate in children<5 years old was higher (49.96%). The positive rate of any virus in south China was higher than that in north China (P<0.001). The virus-positive FRS cases were detected throughout the year. The detection rate of HRSV was higher in autumn and winter. The detection rate of HIFV was higher in winter. The detection rate of HMPV was higher in winter and spring. The detection rates of HPIV, HRV, HCoV and HBoV were higher in summer and autumn, while there was no significant difference in the detection rate of HAdV in different seasons. Compared with 2009-2019, the detection rate of any virus in 2020-2021 decreased from 41.37% to 37.86%. The detection rate of HIFV decreased sharply from 10.62% to 1.37%. The detection rate of HPIV decreased from 8.24% to 5.88%. The detection rate of HRV and HBoV increased from 5.43% and 1.79% to 9.67% and 3.19%, respectively. Conclusion: HRSV and HIFV infections are more common among FRS cases in nine provinces in China from 2009 to 2021, and the epidemiological characteristics of eight common respiratory viruses vary in different age groups, regions and seasons.

Dental Calculus as a Tool to Study the Evolution of the Mammalian Oral Microbiome.

Dental calculus, the calcified form of the mammalian oral microbial plaque biofilm, is a rich source of oral microbiome, host, and dietary biomolecules and is well preserved in museum and archaeological specimens. Despite its wide presence in mammals, to date, dental calculus has primarily been used to study primate microbiome evolution. We establish dental calculus as a valuable tool for the study of nonhuman host microbiome evolution, by using shotgun metagenomics to characterize the taxonomic and functional composition of the oral microbiome in species as diverse as gorillas, bears, and reindeer. We detect oral pathogens in individuals with evidence of oral disease, assemble near-complete bacterial genomes from historical specimens, characterize antibiotic resistance genes, reconstruct components of the host diet, and recover host genetic profiles. Our work demonstrates that metagenomic analyses of dental calculus can be performed on a diverse range of mammalian species, which will allow the study of oral microbiome and pathogen evolution from a comparative perspective. As dental calculus is readily preserved through time, it can also facilitate the quantification of the impact of anthropogenic changes on wildlife and the environment.

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.

Intranasal immunization with coxsackievirus A16 virus-like particles confers protection against lethal infection in neonatal mice.

Coxsackievirus A16 (CV-A16) is one of the main causative agents of hand, foot and mouth disease (HFMD) in young children and has become prevalent in the Asia-Pacific region in recent years. However, no approved vaccines or drugs are available for CV-A16 infection. CV-A16 virus-like particles (VLPs) are a potential vaccine candidate; however, whether the intranasal route of immunization is suitable for inducing immune responses against CV-A16 infection has not been clarified. In this study, the comprehensive immunogenicity and protective efficacy of the CV-A16 VLP vaccine were evaluated by multiple methods in a mouse model. In mice, a high neutralizing antibody (NTAb) titre could be elicited by intranasal immunization with CV-A16 VLPs, which produced NTAb levels similar to those induced by intranasal immunization with inactivated CV-A16. Passive immunity with NTAbs provided very good protection, as the survival rate of the immunized neonatal mice was 100% after challenges with CV-A16 at a dose of 1000 LD50. Passive protective effects were transferred to the neonates via the mother, thus protecting all the pups against challenges with the homologous or heterologous strains of CV-A16 at a dose of 1000 LD50. In addition, intranasal immunization with CV-A16 VLPs also induced the production of mucosal secretory IgA (s-IgA) antibodies, which may inhibit CV-A16 virus invasion. This study provides valuable supplemental information to facilitate our understanding of the specific protective efficacy of CV-A16 VLPs and has significance for development of the candidate vaccine into a safe and effective vaccine.

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.

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.

Efficacy, safety, and immunogenicity of an enterovirus 71 vaccine in China.

BACKGROUND: Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease or herpangina worldwide. This phase 3 trial was designed to evaluate the efficacy, safety, and immunogenicity of an EV71 vaccine. METHODS: We conducted a randomized, double-blind, placebo-controlled, multicenter trial in which 10,007 healthy infants and young children (6 to 35 months of age) were randomly assigned in a 1:1 ratio to receive two intramuscular doses of either EV71 vaccine or placebo, 28 days apart. The surveillance period was 12 months. The primary end point was the occurrence of EV71-associated hand, foot, and mouth disease or herpangina. RESULTS: During the 12-month surveillance period, EV71-associated disease was identified in 0.3% of vaccine recipients (13 of 5041 children) and 2.1% of placebo recipients (106 of 5028 children) in the intention-to-treat cohort. The vaccine efficacy against EV71-associated hand, foot, and mouth disease or herpangina was 94.8% (95% confidence interval [CI], 87.2 to 97.9; P<0.001) in this cohort. Vaccine efficacies against EV71-associated hospitalization (0 cases vs. 24 cases) and hand, foot, and mouth disease with neurologic complications (0 cases vs. 8 cases) were both 100% (95% CI, 83.7 to 100 and 42.6 to 100, respectively). Serious adverse events occurred in 111 of 5044 children in the vaccine group (2.2%) and 131 of 5033 children in the placebo group (2.6%). In the immunogenicity subgroup (1291 children), an anti-EV71 immune response was elicited by the two-dose vaccine series in 98.8% of participants at day 56. An anti-EV71 neutralizing antibody titer of 1:16 was associated with protection against EV71-associated hand, foot, and mouth disease or herpangina. CONCLUSIONS: The EV71 vaccine provided protection against EV71-associated hand, foot, and mouth disease or herpangina in infants and young children. (Funded by Sinovac Biotech; ClinicalTrials.gov number, NCT01507857.).

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.

Molecular epidemiology of coxsackievirus A6 associated with outbreaks of hand, foot, and mouth disease in Tianjin, China, in 2013.

Since 2008, Mainland China has undergone widespread outbreaks of hand, foot, and mouth disease (HFMD). In order to determine the characteristics of epidemics and enteroviruses (EV) associated with HFMD in Tianjin, in northern China, epidemiological and virological data from routine surveillance were collected and analyzed. In Tianjin, a persistent epidemic of HFMD was demonstrated during 2008-2013, involving 102,705 mild, 179 severe, and 16 fatal cases. Overall, 8234 specimens were collected from 7829 HFMD patients for EV detection during 2008-2013. Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) were the dominant serotypes during 2008-2012, and they were replaced by CV-A6 as the major causative agent in 2013. Phylogenetic analysis based on complete VP1 nucleotide sequences revealed that multiple CV-A6 lineages co-circulated in Tianjin, which grouped together with strains from China and other countries and split into two distinct clusters (clusters 1 and 2). Most Tianjin strains grouped in cluster 1 and were closely related to strains from several eastern and southern provinces of China during 2012 and 2013. Estimates from Bayesian MCMC analysis suggested that multiple lineages had been transmitted silently before the outbreaks at an estimated evolutionary rate of 4.10 × 10(-3) substitutions per site per year without a specific distribution of rate variances among lineages. The sudden outbreak of CV-A6 in Tianjin during 2013 is attributed to indigenous CV-A6 lineages, which were linked to the wide spread of endemic strains around eastern and southern China.

VP2 dominated CD4+ T cell responses against enterovirus 71 and cross-reactivity against coxsackievirus A16 and polioviruses in a healthy population.

Enterovirus 71 (EV71)-associated hand-foot-mouth disease has become a major threat to public health in the Asia-Pacific region. Although T cell immunity is closely correlated with clinical outcomes of EV71 infection, little is known about T cell immunity baseline against EV71 and T cell immunogenecity of EV71 Ags in the population, which has restricted our understanding of immunoprotection mechanisms. In this study, we investigated the cellular immune responses against the four structural Ags of EV71 and determined the immunohierarchy of these Ags in healthy adults. A low frequency of EV71-responsive T cells was detected circulating in peripheral blood, and broad T cell immune responses could be identified in most of the subjects after in vitro expansion. We demonstrated that the VP2 Ag with broad distribution of immunogenic peptides dominates T cell responses against EV71 compared with VP1, VP3, and VP4. Furthermore, the responses were illuminated to be mainly single IFN-γ-secreting CD4(+) T cell dependent, indicating the previous natural acute viral infection of the adult population. Conservancy analysis of the immunogenic peptides revealed that moderately variant peptides were in the majority in coxsackievirus A16 (CV-A16) whereas most of the peptides were highly variant in polioviruses. Less efficient cross-reactivity against CV-A16 might broadly exist among individuals, whereas influences derived from poliovirus vaccination would be limited. Our findings suggest that the significance of VP2 Ag should be addressed in the future EV71-responsive immunological investigations. And the findings concerning the less efficient cross-reactivity against CV-A16 and limited influences from poliovirus vaccination in EV71-contacted population would contribute to a better understanding of immunoprotection mechanisms against enteroviruses.

[Colorimetric detection of coxsackievirus A6 by reverse transcription loop mediated isothermal amplification].

OBJECTIVE: To develop a simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of coxsackievirus A6 (CV-A6) based on the colour chang of hydroxy naphthol blue (HNB). METHODS: The method employed a set of six primers that recognized sequences of VP1 gene for amplification of nucleic acid under isothermal conditions at 63 °C for 50 min. The products were detected through visual inspection of color change by the pre-addition of HNB dye. The specificity was validated by detecting a collection of different human enteroviruses. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of CV-A6 VP1 gene, and compared with real-time RT-PCR (rRT-PCR) in parallel. This assay was evaluated with 92 clinical specimens from patients with hand-foot-mouth disease. RESULTS: A positive color (sky blue) was only observed in the preparation of CV-A6, whereas none of the other 23 kinds of human enteroviruses showed a color change. The HNB based RT-LAMP showed a sensitivity of 100 copies/reaction, which was at the same level as that of the rRT-PCR. The result of RT-LAMP in analysis of 92 clinical specimens was consistent with that of the rRT-PCR. The kappa correlation between the two methods was 1 and both of the sensitivity and specificity of the RT-LAMP assay were 100%. CONCLUSION: The established RT-LAMP assay had good specificity and sensitivity and thus demonstrated to be a promising screening tool for CV-A6 infection. It also has the potential to be used in resource-limited clinical sites and field study.

An outbreak of echovirus 33 in schools in China in 2013.

Echovirus 33 (E33) has been infrequently detected and is less frequently associated with clinical diseases when compared with other types of enteroviruses (EVs) in China. An outbreak of E33 was identified in four schools in Hunan Province, China, in June 2013. For laboratory diagnosis, throat swabs and/or serum specimens were collected from 27 patients. E33 was isolated in cell culture and typed by molecular methods. Complete VP1 gene sequences were determined and analyzed. Specific E33 antibody was measured by virus neutralization testing. From June 3-20, 108 suspected cases were reported, and 19 were confirmed to be associated with E33 by laboratory testing, with seven virologically confirmed and 12 serologically confirmed cases. The suspected cases were in children aged 3-16 years (mean, 11 years), most of whom (94%, 102/108) were ≥6 years old. The majority of cases (98%, 106/108) presented as influenza-like illness (ILI), and two were clinically diagnosed as viral meningitis. Older children aged ≥12 years had a higher hospitalization rate (21%) than younger children (4%). A BLAST query of GenBank with the Hunan E33 strain VP1 gene sequence gave a close match to an E33 isolate from Pakistan, based on a partial VP1 gene sequence. Phylogenetic analyses of the complete E33 VP1 gene sequences from our study revealed an independent cluster with nucleotide sequences that diverge from E33 from other countries by >12%. Due to limited E33 VP1 gene sequence data in GenBank and passive EV surveillance in China and most other parts of the world (excepting hand, foot, and mouth disease surveillance in Asia), the approximate origin of Hunan E33 could not be determined.

Genetic characterization of emerging coxsackievirus A12 associated with hand, foot and mouth disease in Qingdao, China.

To characterize the genetic properties of coxsackievirus A12 (CVA12) strains isolated from hand, foot and mouth disease (HFMD) patients in Qingdao during 2008-2011, the complete genome and VP1 coding region were sequenced and analyzed. Phylogenetic analysis showed that all strains from China clustered into three different branches, suggesting multiple lineages of CVA12 co-circulating in Asia. Sequence analysis indicated a monophyletic group only when the P1 region was examined, indicating possible recombination between CVA12 and other HEV-A serotypes. The emergence of CVA12 involved in an HFMD outbreak in China is a public-health issue.

Emergence, circulation, and spatiotemporal phylogenetic analysis of coxsackievirus a6- and coxsackievirus a10-associated hand, foot, and mouth disease infections from 2008 to 2012 in Shenzhen, China.

Sporadic hand, foot, and mouth disease (HFMD) outbreaks and other infectious diseases in recent years have frequently been associated with certain human enterovirus (HEV) serotypes. This study explored the prevalences and genetic characteristics of non-HEV71 and non-coxsackievirus A16 (CV-A16) human enterovirus-associated HFMD infections in Shenzhen, China. A total of 2,411 clinical stool specimens were collected from hospital-based surveillance for HFMD from 2008 to 2012. The detection of HEV was performed by real-time reverse transcription-PCR (RT-PCR) and RT-seminested PCR, and spatiotemporal phylogenetic analysis was performed based on the VP1 genes. A total of 1,803 (74.8%) strains comprising 28 different serotypes were detected. In the past 5 years, the predominant serotypes were HEV71 (60.0%), followed by CV-A16 (21.2%) and two uncommon serotypes, CV-A6 (13.0%) and CV-A10 (3.3%). However, CV-A6 replaced CV-A16 as the second most common serotype between 2010 and 2012. As an emerging pathogen, CV-A6 became as common a causative agent of HFMD as HEV71 in Shenzhen in 2012. Phylogenetic analysis revealed that little variation occurred in the Chinese HEV71 and CV-A16 strains. The genetic characteristics of the Chinese CV-A6 and CV-A10 strains displayed geographic differences. The CV-A6 and CV-A10 strains circulating in Shenzhen likely originated in Europe. It was found that human enteroviruses have a high mutation rate due to evolutionary pressure and frequent recombination (3.2 × 10(-3) to 6.4 ×10(-3) substitutions per site per year for HEV71, CV-A6, CV-A16, and CV-A10). Since certain serotypes are potential threats to the public health, this study provides further insights into the significance of the epidemiological surveillance of HFMD.

Modified nano zero-valent iron reduce toxicity of polystyrene microplastics to ryegrass (Lolium Perenne L.).

Microplastics pollution in environments has become a major concern and it has been proven to have adverse impacts on plants, so there is an urgent to find approaches to alleviate the detrimental effects of microplastics. In our study, we investigated the influence of polystyrene microplastics (PSMPs) on the growth, photosynthesis, and oxidative defense system changes of ryegrass, as well as the behavior of MPs at roots. Then three types of nanomaterials were applied to alleviate the adverse impact of PSMPs on ryegrass, which were nano zero-valent iron (nZVI), carboxymethylcellulose-modified-nZVI (C-nZVI) and sulfidated nZVI (S-nZVI), respectively. Our results suggested that PSMPs had significant toxicity to ryegrass, leading to decrease of shoot weight, shoot length and root length. Three nanomaterials regained the weight of ryegrass to a varying extent and made more PSMPs aggregate near roots. In addition, C-nZVI and S-nZVI facilitated the entrance of PSMPs into the root and promoted the chlorophyll a and chlorophyll b contents in leaves. Analysis of antioxidant enzymes and malondialdehyde content indicated that ryegrass coped well with the internalization of PSMPs, and all three types of nZVI could alleviate PSMPs-stress in ryegrass. This study elaborates the toxicity of MPs on plants and provides a novel insight into the fixing of MPs by plants and nanomaterials in environments, which needs to be further explored in future research.

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.

Simultaneously typing nine serotypes of enteroviruses associated with hand, foot, and mouth disease by a GeXP analyzer-based multiplex reverse transcription-PCR assay.

Hand, foot, and mouth disease (HFMD) is a contagious enteroviral disease occurring primarily in young children and caused by enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and other serotypes of coxsackievirus and echovirus. In this study, a GeXP analyzer-based multiplex reverse transcription (RT)-PCR assay (GeXP assay) consisting of chimeric primer-based PCR amplification with fluorescent labeling and capillary electrophoresis separation was developed to simultaneously identify nine serotypes of enteroviruses associated with HFMD in China, including EV71, CVA16, CVA4, -5, -9, and -10, and CVB1, -3, and -5. The RNAs extracted from cell cultures of viral isolates and synthetic RNAs via in vitro transcription were used to analyze the specificity and sensitivity of the assay. The GeXP assay detected as little as 0.03 tissue culture infective dose (TCID(50)) of EV71 and CVA16, 10 copies of panenterovirus, EV71, CVA16, CVB1, and CVB5, and 100 copies of 10 (including panenterovirus) premixed RNA templates. A total of 180 stool specimens collected from HFMD patients and persons suspected of having HFMD were used to evaluate the clinical performance of this assay. In comparison with the results of conventional methods, the sensitivities of the GeXP assay for detection of panenterovirus, EV71, and CVA16 were 98.79% (163/165), 91.67% (44/48), and 91.67% (33/36), respectively, and the specificities were 80.00% (12/15), 98.48% (130/132), and 100% (144/144), respectively. The concordance of typing seven other serotypes of enteroviruses with the results of conventional methods was 92.59% (25/27). In conclusion, the GeXP assay is a rapid, cost-effective, and high-throughput method for typing nine serotypes of HFMD-associated enteroviruses.

Myelin protein zero/P0 phosphorylation and function require an adaptor protein linking it to RACK1 and PKC alpha.

Point mutations in the cytoplasmic domain of myelin protein zero (P0; the major myelin protein in the peripheral nervous system) that alter a protein kinase Calpha (PKCalpha) substrate motif (198HRSTK201) or alter serines 199 and/or 204 eliminate P0-mediated adhesion. Mutation in the PKCalpha substrate motif (R198S) also causes a form of inherited peripheral neuropathy (Charcot Marie Tooth disease [CMT] 1B), indicating that PKCalpha-mediated phosphorylation of P0 is important for myelination. We have now identified a 65-kD adaptor protein that links P0 with the receptor for activated C kinase 1 (RACK1). The interaction of p65 with P0 maps to residues 179-197 within the cytoplasmic tail of P0. Mutations or deletions that abolish p65 binding reduce P0 phosphorylation and adhesion, which can be rescued by the substitution of serines 199 and 204 with glutamic acid. A mutation in the p65-binding sequence G184R occurs in two families with CMT, and mutation of this residue results in the loss of both p65 binding and adhesion function.