Complete VP1 Sequences Research Articles

Phylogenetic characteristics and recombination analysis of echovirus 5 associated with severe acute respiratory infection in China.

Echovirus 5 (E5) belongs to the enterovirus B (EV-B) species, which is one of the pathogens causing respiratory diseases. Only two whole genome sequences (WGSs) of E5 are available in GenBank, and few studies on E5 have been reported worldwide. In this study, human rhabdomyosarcoma (RD) cells were used to isolate E5 from respiratory samples associated with severe acute respiratory infection (SARI). The nucleotide sequence of the VP1 region and the WGSs of two E5 strains were sequenced using Sanger sequencing and high-throughput sequencing technology, respectively. Together with 31 E5 VP1 sequences and 2 WGSs downloaded from GenBank, a total of 33 complete VP1 sequences and 4 WGSs were used for genetic characterization and evolutionary analysis. Phylogenetic analysis revealed five genotypes (A-E) based on complete VP1 sequences. Evolutionary analysis showed that the time to the most recent common ancestor of E5 was approximately 1952 (95% highest posterior density [HPD] range, 1948-1953]), with a mean substitution rate of 7.74 × 10-3 subs/site/year (95% HPD range, 6.44-9.11 × 10-3). In addition, the WGS analyses showed that the nucleotide and amino acid similarities of the coding regions of the two strains obtained in this study with the prototype of the E5 strain Noyce were 80.2% and 97.2%, respectively. Recombination analysis showed that the two E5 strains may have intraspecific recombination with E6, EV-B85, and CVA9 serotypes in the nonstructural region. This study reports the isolation of E5 from SARI cases for the first time, and genetic characterization and evolutionary analysis provide valuable information on global E5 molecular epidemiology.IMPORTANCEThis study is the first report of echovirus 5 (E5) associated with severe acute respiratory infection and obtained the first E5 whole-genome sequence in China. Combined with the sequences available in the GenBank database, the first genotyping, phylogenetic characteristics, recombination, and genetic evolutionary analysis of E5 was performed in this study. Our findings providing valuable information on global E5 molecular epidemiology.

Epidemiology and genetic diversity of norovirus GII genogroups among children in Hubei, China, 2017–2019

Norovirus (NoV) is an important cause of viral acute gastroenteritis (AGE). To gain insights into the epidemiological characteristics and genetic diversity of NoV among children in Hubei, 1216 stool samples from children (≤ 5 years) obtained under AGE surveillance from January 2017 to December 2019 were analyzed. The results showed that NoV was responsible for 14.64% of AGE cases, with the highest detection rate in children aged 7–12 months (19.76%). Statistically significant differences were found between male and female infection rates (χ2 ​= ​8.108, P ​= ​0.004). Genetic analysis of RdRp and VP1 sequences showed that NoV GII genotypes were GII.4 Sydney [P31] (34.35%), GII.3 [P12] (25.95%), GII.2 [P16] (22.90%), GII.4 Sydney [P16] (12.98%), GII.17 [P17] (2.29%), GII.6 [P7] and GII.3 [P16] (each at 0.76%). GII.17 [P17] variants were divided into the Kawasaki323-like lineage and the Kawasaki308-like lineage. A unique recombination event was detected between strains of GII.4 Sydney 2012 and GII.4 Sydney 2016. Significantly, all GII.P16 sequences associated with GII.4/GII.2 obtained in Hubei were correlated with novel GII.2 [P16] variants that re-emerged in Germany in 2016. Antigenic site analysis of complete VP1 sequences from all GII.4 variants from Hubei identified notable variable residues of antibody epitopes. Genotyping under continuous AGE surveillance and observation of the antigenic sites of VP1 are important monitoring strategies for emerging NoV strains.

Identification of two proposed novel human rhinovirus types: Bpat107 and Cpat58.

Human rhinovirus (RV) is an important viral pathogen associated with severe acute respiratory tract infection. The present study retrospectively identified RV types in hospitalized patients with severe acute respiratory infection (SARI) from October 2017 to June 2019 in Henan Province, China. Real-time PCR was used to screen pharyngeal swab samples for RV. Then, the VP1 gene sequences of RV-positive samples were amplified and sequenced with nested primer PCR; subsequently, analyses of the molecular epidemiology and genetic diversity characteristics of the RV types were performed.Seventy-three out of 1015 respiratory samples were identified as RV-positive, from which 65 complete VP1 sequences were successfully sequenced. These RVs were classified into 41 different types, including 26 RV-A types, 2 RV-B types, and 13 RV-C types. The RVs showed an obvious seasonal distribution, with peaks in summer and autumn. The epidemic peak of RV-C was later than that of RV-A. In addition, two new types of species, B and C, were proposed, Bpat107 and Cpat58, respectively. Compared with other types in the same RV species, the pairwise nucleotide p-distances of the two novel RV types were 0.262~0.402 and 0.251~0.508, respectively. This study analyzed the seasonal and genetic characteristics of RV associated with SARI cases in Henan Province, China. Two novel RV types were proposed.

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.

Genetic characteristics of archival noroviruses detected from the 1970s to the 1990s.

The genetic characterization of archival specimens is important for evaluating the evolutionary processes of noroviruses. Complete viral genome sequences, GVIII.1[GII.P28] and GIX.1[GII.P15], were determined from two archival specimens collected in Tokyo, Japan, in 1986 and 1995. In addition, complete VP1 and partial RdRp sequences of four samples collected between 1975 and 1983 were determined. Two viruses were classified as GI.5[P5] and GI.9[P9]; however, the viruses from the other two samples could not be assigned to any known genotypes using norovirus typing tools and phylogenetic analysis, suggesting that they might be untypable genotypes. Further evolutionary analysis of these viruses is warranted.

Epidemiological Evidence for Fecal-Oral Transmission of Murine Kobuvirus.

BackgroundMurine Kobuvirus (MuKV) is a novel picornavirus of the genus Kobuvirus, and was first identified in the feces of murine rodents in the USA in 2011. There is limited information on the transmission route of MuKV. Thus, we conducted a study to investigate virus detection rates in fecal, serum, throat, and lung tissue samples from murine rodents.ResultsA total of 413 fecal samples, 385 lung samples, 269 throat swab samples, and 183 serum samples were collected from 413 murine rodents (Rattus norvegicus, Rattus tanezumi, and Rattus rattus) captured in urban Shenzhen. Kobuviruses were detected via RT-PCR. Only fecal samples were positive, with prevalence rates of 34.9% in Rattus norvegicus and 29.4% in Rattus tanezumi. Phylogenetic analysis based on partial 3D and complete VP1 sequence regions indicated that all of the MuKV sequences obtained belonged to Aichivirus A, and were genetically closely related to other MuKVs reported in China, Hungary, and the USA. Twenty-eight full-length MuKV sequences were acquired. Phylogenetic analysis of two sequences randomly selected from the two species (SZ59 and SZ171) indicated that they shared very high nucleotide and amino acid identity with one another (94.0 and 99.3%, respectively), and comparison with human Kobuvirus revealed amino acid identity values of ~80%. Additionally, a sewage-derived sequence shared high similarity with the rat-derived sequences identified in this study, with respective nucleotide and amino acid identity values from 86.5 and 90.7% to 87.2 and 91.1%.ConclusionThe results of the current study provide evidence that murine Kobuvirus is transmitted via the fecal-oral route.

Molecular epidemiological characteristics of echovirus 6 in mainland China: extensive circulation of genotype F from 2007 to 2018

Echovirus 6 (E6) is associated with various clinical diseases and is frequently detected in environmental sewage. Despite its high prevalence in humans and the environment, little is known about its molecular phylogeography in mainland China. In this study, 114 of 21,539 (0.53%) clinical specimens from hand, foot, and mouth disease (HFMD) cases collected between 2007 and 2018 were positive for E6. The complete VP1 sequences of 87 representative E6 strains, including 24 strains from this study, were used to investigate the evolutionary genetic characteristics and geographical spread of E6 strains. Phylogenetic analysis based on VP1 nucleotide sequence divergence showed that, globally, E6 strains can be grouped into six genotypes, designated A to F. Chinese E6 strains collected between 1988 and 2018 were found to belong to genotypes C, E, and F, with genotype F being predominant from 2007 to 2018. There was no significant difference in the geographical distribution of each genotype. The evolutionary rate of E6 was estimated to be 3.631 × 10-3 substitutions site-1 year-1 (95% highest posterior density [HPD]: 3.2406 × 10-3-4.031 × 10-3 substitutions site-1 year-1) by Bayesian MCMC analysis. The most recent common ancestor of the E6 genotypes was traced back to 1863, whereas their common ancestor in China was traced back to around 1962. A small genetic shift was detected in the Chinese E6 population size in 2009 according to Bayesian skyline analysis, which indicated that there might have been an epidemic around that year.

RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens

ObjectiveTo develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases. MethodsA panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A–C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID50 per μL and copies per μL, and the newly established methods were tested in clinical specimens collected in recent years. ResultsThe sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID50 per μL and 10 virus copies per μL, and for the complete VP1 gene was 1 CCID50 per μL and 100 virus copies per μL, using serially-diluted virus stocks of five serotypes. As a proof-of-concept, 25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons. ConclusionThis RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A–D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

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.

Mosaic genome of Human Coxsackievirus A4 associated with herpangina and HFMD in Yancheng, China, 2016 and 2018

ObjectivesTo better understand the spectrums of pathogens causing herpangina and circulation of Coxsackievirus A4 in Yancheng, China. MethodsStool samples from herpangina and HFMD cases were collected. Real Time PCR Kits was used to identify Enterovirus 71, CV-A16 and CV-A6, and nested reverse transcription PCR (nRT-PCR) to detect the other enterovirus types. Complete VP1 and genome sequence of CV-A4 were amplified by using nRT-PCR. Genetic, phylogenetic and recombination analysis were performed. ResultsCo-circulation of three recombinant CV-A4 groups, including one novel (C2 lineage), was identified in Yancheng, China, 2016 and 2018. One was the major causative agent of herpangina, and another two were responsible for HFMD. Phylogenetic and recombination analysis indicated that the non-structural region of their genome originated from the same ancestry and subsequently adaptation. C2 lineage of CV-A4 group may be introduced from countries outside China and its genome occurred recombination in China. ConclusionNovel recombinant CV-A4 was mainly associated with herpanginain in Yancheng, 2018, China. C2 lineage of CV-A4 group with recombinant non-structural region was also identified in HFMD patients.

Kobuviruses carried by Rattus norvegicus in Guangdong, China

BackgroundMurine kobuviruses (MuKV) are newly recognized picornaviruses first detected in murine rodents in the USA in 2011. Little information on MuKV epidemiology in murine rodents is available. Therefore, we conducted a survey of the prevalence and genomic characteristics of rat kobuvirus in Guangdong, China.ResultsFecal samples from 223 rats (Rattus norvegicus) were collected from Guangdong and kobuviruses were detected in 12.6% (28) of samples. Phylogenetic analysis based on partial 3D and complete VP1 sequence regions showed that rat kobuvirus obtained in this study were genetically closely related to those of rat/mouse kobuvirus reported in other geographical areas. Two near full-length rat kobuvirus genomes (MM33, GZ85) were acquired and phylogenetic analysis of these revealed that they shared very high nucleotide/amino acids identity with one another (95.4%/99.4%) and a sewage-derived sequence (86.9%/93.5% and 87.5%/93.7%, respectively). Comparison with original Aichivirus A strains, such human kobuvirus, revealed amino acid identity values of approximately 80%.ConclusionOur findings indicate that rat kobuvirus have distinctive genetic characteristics from other Aichivirus A viruses. Additionally, rat kobuvirus may spread via sewage.

Epidemiological and genetic characteristics of EV71 strains circulating in Jiangsu Province from 2009 to 2018

Objective To analyze the molecular epidemiology, genetic variations and evolution of enterovirus 71 (EV71) strains isolated in Jiangsu Province from 2009 to 2018. Methods Statistical methods were used to analyze the data about epidemiological characteristics and results of pathogen detection in cases with EV71 infection in Jiangsu Province from 2009 to 2018. The complete VP1 sequences of 80 EV71 strains were amplified and sequenced for analysis of diversity and phylogenesis. Results A total of 41 858 enterovirus-positive hand, foot and mouth disease cases were reported in Jiangsu Province from 2009 to 2018. EV71 was the predominant pathogen, accounting for 36.52%, and responsible for most of the severe cases. However, the percentage of EV71 among all pathogens gradually decreased over time. EV71 infection reached the peak in April to June and mainly occurred in children aged six months to five years old with higher incidence in males than in females. In terms of regional distribution, EV71 infections were characterized by area clustering in Jiangsu Province, mainly detected in Nanjing, Suzhou, Wuxi and Lianyungang. The 80 EV71 isolates belonged to C4a genotype. Nucleotide differences between them and three vaccine strains (H07, FY23 and FY7VP5)were 0.6%-5.5%, 0.8%-5.7% and 1.9%-6.9% and amino acid difference were 0-1.4%, 0.3%-2.0% and 0.3%-2.0%, respectively. Amino acid mutations in the epitopes of the 80 EV71 strains did not marked by years or regions. Conclusions EV71 strains showed obvious epidemiological characteristics in time, population and regional distribution in Jiangsu Province from 2009 to 2018.All of the 80 EV71 isolates belonged to C4a subgenotype. The nucleotide sequences between them and the vaccine strains varied greatly, but the homology of amino acids was relatively high, indicating the existence of some synonymous mutations and no risk of antigenic drift. This study would provide reference for EV71 vaccination in Jiangsu Province. Key words: Hand, foot and mouth disease; Enterovirus 71; Epidemiology; Genetic characteristics

Emerging recombination of the C2 sub-genotype of HFMD-associated CV-A4 is persistently and extensively circulating in China

Sporadic outbreaks caused by coxsackievirus A4 (CV-A4) have been reported worldwide. To further elucidate the detailed genetic characteristics and evolutionary recombination events of CV-A4, virus samples from nationwide hand, foot and mouth disease (HFMD) surveillance, encompassing 27 out of the 31 provinces in China, were investigated. Comprehensive and systematic phylogenetic analyses were performed by using 29 complete genomes, 142 complete CV-A4 VP1 sequences. Four genotypes (A, B, C and D) and five sub-genotypes (C1-C5) were re-identified based on the complete VP1 sequences. C2 is the predominant sub-genotype of CV-A4 associated with HFMD and has evolved into 3 clusters. Cluster 1 is a major cluster that has been persistently and extensively circulating in China since 2006 and has been associated with all severe cases. All the sequences showed high homology with the CV-A4 prototype in the P1 region, while higher identities with CV-A5, CV-14 and CV-16 in the P2 and P3 regions. Recombination analysis revealed that C2 had two specific genetic recombination patterns with other EV-A prototypes in the 5′-UTR and 3D region compared with C5. These recombination patterns might be associated with the increased transmissibility of C2 viruses, which were obtained due to their persistent and extensive circulation in populations.

The resurgence of the norovirus GII.4 variant associated with sporadic gastroenteritis in the post-GII.17 period in South China, 2015 to 2017

BackgroundHuman norovirus is regarded as the leading cause of nonbacterial acute diarrhea in developing and developed countries. Among all genotypes, GII.4 has been the predominant genotype, but in East Asia, it was replaced by the GII.17 in 2014/2015. However, after the prevalence of new GII.17 variant in South China, a sharply increase in the number of norovirus infections associated with sporadic acute diarrhea was detected. In this study, we would investigate the prevalence and genetic diversity of noroviruses in the sporadic acute gastroenteritis cases in the post-GII.17 period in South China.MethodsNorovirus was screened from 217 patients with sporadic acute gastroenteritis from August 2015 to October 2017 by reverse transcription-polymerase chain reaction. Then, two regions including the partial RNA polymerase and the capsid gene of positive samples were amplified and sequenced. Phylogenetic analyses were performed to determine norovirus genotypes. Complete VP1 sequences of GII.4 strains detected in this study were also amplified and subjected into evolutionary tracing analyses.ResultsA total of 43 (19.82%) norovirus samples were confirmed from 217 stool specimens, and it was found that GII.4 resurged as the new predominant variant, accounting for 76.74% (33/43) of positive samples. Only one local strain GZ2015-L550 was clustered with the contemporary GII.P16/GII.4–2012 recombinant variant, and other 32 local strains belonged to the clade with the GII.Pe/GII.4–2012 variant. Other genotypes including GII.17 (n = 4), GII.3 (n = 4), GII.8 (n = 1) and GI. 6 (n = 1) were also detected. Furthermore, all GII.4 strains were phylogenetic analyzed based on their capsid P2 subdomains. Combined with other reported 754 strains, the GII.4–2012 variant could be divided into two clades. Most GII.4 strains collected in 2016 and 2017 in this study (7/8) formed a new cluster A in Clade II with additional 103 contemporaneous strains. In addition, evolutionary tracing of the capsid P2 subdomain of this variant was also analyzed, and one specific amino acid substitutions (N373) was identified for Cluster A.ConclusionIn summary, this study confirmed a norovirus infection peak in the post-GII.17 period in South China, which was caused by the resurgence of the GII.4 variant.

Complete genome sequencing and genetic characterization of porcine sapovirus genogroup (G) X and GXI: GVI, GVII, GX, and GXI sapoviruses share common genomic features and form a unique porcine SaV clade

Sapoviruses (SaVs) are enteric viruses belonging to the family Caliciviridae that infect humans and animals, including pigs. To date, SaVs have been classified into 19 genogroups (G) based on complete VP1 sequences; however, complete genome sequences of some SaV Gs are not yet available. In this study, we determined the full genome sequences of four SaVs (two GX and two GXI SaVs) and analyzed them together with those of other SaVs. The complete genome sequences of GX and GXI SaVs, excluding the poly(A) tails, were 7124, 7142, 7170, and 7179 nucleotides, which were shorter than those of other SaVs, except for porcine GVI and GVII viruses. Genetic characterization revealed that GX SaVs and GXI SaVs shared common features with GVI and GVII viruses, such as the first 10 amino acid residues in the ORF1 coding region, a shorter ORF1 than that of the other genogroups, and the predicted secondary structure of the 5′ end of the genome and the starting region of non-structural protein/structural protein junction. Phylogenetic analyses showed that GX and GXI SaVs branched with porcine GVI, GVII, and GIX SaVs and formed a clade consisting of only porcine SaVs. These findings suggest that porcine GX and GXI SaVs together with porcine GVI, GVII, and possibly GIX SaVs, evolved from a common ancestor in the porcine population.

Molecular detection and genomic characteristics of bovine kobuvirus from dairy calves in China

In this study, 96 diarrheic and 77 non-diarrheic fecal samples from dairy calves were collected from 14 dairy farms in 4 provinces to investigate the molecular prevalence and genomic characteristics of Bovine Kobuvirus (BKoV) in China. The results showed that the BKoV positive rate for the diarrheic feces (35.42%) was significantly higher than that for the non-diarrheic feces (11.69%, p < 0.001). Interestingly, three potential novel VP1 lineages were identified from 15 complete VP1 sequences, and a unique triple nucleotide insertion which can result in an aa insertion, was first observed in the 11/12 VP0 fragments with 660 bp long in this study, compared with known BKoV VP0 sequences. Moreover, the first Chinese BKoV genome was successfully obtained from a diarrheic fecal sample, named CHZ/CHINA. The open reading frame (ORF) of the genome from strain CHZ/China shares 87.4%–88.3% nucleotide (nt) and 93.7%–96.4% amino acid (aa) identity, compared with the three known genomes of BKoV. Interestingly, phylogenetic tree based on aa sequences of these genomes showed that CHZ/CHINA was clustered into an independent branch, suggesting the strain may represent a novel BKoV strain. The findings contribute to better understanding the molecular characteristics and evolution of BKoV.

Molecular characteristic analysis for the VP1 region of coxsackievirus A6 strains isolated in Jiujiang area, China, from 2012 to 2013.

Enterovirus 71 (EV-A71) and Coxsackievirus A16 (CV-A16) are the most common causative agents causing hand, foot, and mouth disease (HFMD). However, coxsackievirus A6 (CV-A6), previously largely ignored, became the predominant pathogen in China in 2012. The objective of this study is to investigate the genetic characteristics and molecular epidemiology of HFMD caused by CV-A6 to guide the diagnosis and treatment of the disease, as well as disease prevention. A total of 138 suspected HFMD cases were enrolled in this study and analyses based on complete VP1 nucleotide sequences were performed to determine the evolutionary trajectory of emerging CV-A6. Among 138 samples in Jiujiang, 125 (90.58%) were positive for enterovirus, the most frequently presented serotypes were CV-A6 (77, 61.60%), CV-A16 (28, 22.40%), EV-A71 (6, 4.80%) and untyped enteroviruses (14, 11.20%). Seventy-seven CV-A6 positive specimens were analyzed for the complete VP1 sequences by sequencing and 36 representative isolates were selected to perform nucleotide sequence similarity analysis. The results showed that 36 strains isolated from HFMD patients were clustered closely to the mainland China and were far from prototype strain CV-A6/Gdula (AY421764) and other international subtypes. Moreover, phylogenetic analysis of the VP1 gene revealed that 36 circulating strains were not significantly concentrated in one branch, but were widely distributed in each branch. Continuous surveillance of HFMD etiological agents other than EV-A71 and CV-A16 is necessary. CV-A6 is emerging as the most common pathogen causing HFMD. Closely monitoring the magnitude and trend of CV-A6 epidemic and the trend of pathogenic spectrum changes can provide scientific basis for this disease prevention and control to the department of disease control.

Genomics Analyses of GIV and GVI Noroviruses Reveal the Distinct Clustering of Human and Animal Viruses.

Noroviruses are highly diverse viruses that are the major viral cause of acute gastroenteritis in humans. Although these viruses can infect multiple mammalian species, their potential for zoonosis is not well understood, especially within Genogroup IV (GIV), which contains viruses that infect humans, canines, and felines. The study of GIV viruses has been, in part, hindered by the limited number of complete genomes. Here, we developed a full-genome amplicon-based platform that facilitated the sequencing of canine noroviruses circulating in the United States. Eight novel nearly full-length canine norovirus genomes and two nearly complete VP1 sequences, including four GIV.2, three GVI.1, and three GVI.2 viruses, were successfully obtained. Only animal strains exhibited GVI/GIV chimeric viruses, demonstrating restrictions in norovirus recombination. Using genomic, phylogenetic, and structural analyses, we show that differences within the major capsid protein and the non-structural proteins of GIV and GVI noroviruses could potentially limit cross-species transmission between humans, canines, and felines.

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.

Detection and molecular characteristics of neboviruses in dairy cows in China.

In this study, 98 diarrhoeic and 70 non-diarrhoeic samples were collected from 13 dairy farms located across 5 provinces in China from April 2017 to May 2018. Approximately 41.8 % (41/98) of diarrhoeic samples and 5.7 % (4/70) of non-diarrhoeic samples were nebovirus-positive based on RT-PCR results, and some diarrhoeic samples were co-infected with bovine rotavirus (73.2 %), bovine coronavirus (36.6 %) and/or bovine viral diarrhoea virus (31.7 %). A phylogenetic analysis of 23 nebovirus RdRp fragments showed that these strains were closely related to Nebraska-like (NB-like) strains but were all located in a unique large branch. Moreover, a phylogenetic analysis of the 18 complete VP1 sequences from this study revealed that 14 strains belonged to lineage 1 and 4 strains belonged to lineage 3. Notably, all four lineage 3 strains shared the same recombination event, with a breakpoint located within the P1A domain. The complete genome of one nebovirus strain, Bo/YLA-2/17/CH, which had a recombination event within the P1A domain of its VP1, was successfully sequenced and was found to be 7453 nt in length, and this may represent a novel nebovirus strain based on the phylogenetic analysis of its complete genome sequence. In conclusion, this study reveals that neboviruses circulate widely in dairy cows in China and exhibit a unique evolution of RdRp. To the best of our knowledge, this is the first reported recombination event located within the P1A domain of nebovirus VP1.