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@#Objective To analyze the genetic characteristics of the entire VP1 gene of Coxsackievirus A16(CVA16) strains isolated from the feces of patients with hand,foot and mouth disease(HFMD) in Yunnan Province in 2019.Methods The virus was isolated from human embryonic lung diploid fibroblast(KMB-17) cells and African green monkey kidney(Vero)cells,and the primers for the complete VP1 gene sequence of CVA16 were designed.The target fragment was amplified by RT-PCR and sequenced;the complete VP1 sequence was analyzed by softwares such as MEGA 7.0 and Geneious 9.0.2.Results A total of 26 CVA16 strains were isolated,including eight KMB-17 isolates and 18 Vero isolates.Twenty CVA16isolates were randomly selected for analysis,and three isolates were found to have Bla and 17 B1b genotypes;the nucleotide and amino acid homology of 17 CVA16 B1b isolates were 93.8%—100% and 98.3%—100%,and the nucleotide and amino acid homology with other domestic isolates was 91.1 %—99.2% and 97.3%—99.0%,respectively;the nucleotide and amino acid homology of the three Bla isolates was 98.0%—98.1% and 99.3%,and those with other domestic Bla isolates was 88.7%—98.1% and 98.3%—99.7%,respectively;17 B1b isolates and other three Bla isolates showed the nucleotide and amino acid homology of 87.4%—88.4% and 97.3%—98.7%.Conclusion The CVA16 prevalent in Kunming in 2019 belonged to Bla and B1b genotypes,with B1b as the main strain,and all of them were prevalent strains in the mainland of China.
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@#Objective To express and identify recombinant adenovirus type 5-Norovirus(NoV)GⅡ.4-VP1 virus-like particles(VLPs)in 293T cells. Methods The recombinant adenovirus plasmids pAd5-eGFP and pAd5-NoV-GⅡ.4-VP1 were transfected into 293T cells respectively,and the recombinant adenovirus rAd5-eGFP and rAd5-NoV-GⅡ.4-VP1 were rescued. The rAd5-eGFP was subcultured in 293T cells to verify the function of the vector. The rAd5-NoV-GⅡ.4-VP1 was subcultured in293T cells,expressed and purified,and then NoV-GⅡ.4-VLP was formed by self-assembly,which was detected by Western blot,ELISA and observed by transmission electron microscope. Results The green fluorescence of the recombinant adenovirus rAd5-eGFP of various generations was observed under microscope,and the brightness increased with the increase of generations. NoV-GⅡ.4-VP1 protein was expressed in the harvested solution of recombinant adenovirus rAd5-NoV-GⅡ.4-VP1 of various generations,with a relative molecular mass of about 58 900. NoV-GⅡ.4-VLP showed specific binding to the rabbit anti-NoV-GⅡ.4-VP1 serum;it had similar conformation to natural NoV virus particles and can effectively identify NoV receptors in volunteer saliva samples;microscopic observation showed that the morphology was complete and spherical,with a diameter of 43. 5 — 58. 3 nm,while there were a few protrusions on the surface of the particles,which might be the P domain exposedon the particle surface during self-assembly of VP1. Conclusion The expressed recombinant adenovirus NoV-GⅡ. 4-VLP has complete VLP structure and good specificity,and is expected to be used in the related research of NoV adenovirus vector vaccine.
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Objective To predict and analyze the physicochemical properties, structural characteristics, and antigenic epitopes of viral protein (VP) VP1 of Coxsackievirus A10 (CV-A10) by bioinformatics methods. Methods The physicochemical properties and structural characteristics of CV-A10 VP1 were predicted by ProtParam, SOPMA, SWISS-MODEL, PDBsum, and ProSA-web. The antigenic epitopes of CV-A10 VP1 were predicted and analyzed by DNAstar, ABCpred, Bepipred 2.0, ElliPro, DiscoTope-2.0, NetMHCpan-4.1, NetMHCIIpan-4.0, Consurf, VaxiJen v.2.0, AllerTOP v.2.0, ToxinPred2, and IEDB immunogenicity. Results Bioinformatics analysis showed that CV-A10 VP1 was a basic, unstable, and hydrophilic protein, of which the secondary structure mainly consisted of random coil. The analysis revealed that CV-A10 VP1 had multiple potential B and T cell antigenic epitopes as well as a dominant antigenic epitope based on the potential epitope. Conclusion CV-A10 VP1 has multiple potential sites that induce specific humoral and cellular immunity, providing important support for its experimental identification, molecular epidemiological studies, and vaccine development.
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@#Objective To culture human sapovirus(HuSaV) GⅠ.1 in vitro and prepare polyclonal antibody against the capsid protein VP1.Methods HuSaV GⅠ.1 positive stool specimens preserved in diarrhea department of National Institute for Viral Disease Control and Prevention were inoculated with HuTu-80 cells supplemented with different bile acid salts[glycine chenodeoxycholic acid(GCDCA) and glycine cholic acid(GCA)],and the infection,proliferation and passage of the virus were determined by PCR and RT-qPCR.The VP1 gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-6P-1.The constructed recombinant expression plasmid pGEX-6P-1-VP1 was transformed into E.coli BL21(DE3) and induced by IPTG.Two female New Zealand white rabbits were immunized with the purified recombinant VP1 protein for 4 times.The blood samples were collected 18,28,38 and 48 d after immunization,and the serum titers were detected by ELISA.Results HuTu-80 cells were effectively infected by HuSaV GⅠ.1 in the presence of bile acid salt GCA,and the proliferated virus were stably and continuously transmitted for three generations in HuTu-80 cells.The expressed recom-binant GST-VP1 protein showed a relative molecular mass of about 86 000,and about 60 000 after purification(GST tag excision).The titer of polyclonal antibody against HuSaV VP1 protein was over 1:12 800.Conclusion HuSaV was successfully isolated and cultured in vitro using HuTu-80 cells supplemented with bile acid salt,and polyclonal antibody with high titer against HuSaV VP1 protein was prepared,which laid a foundation of in-depth research of HuSaV identification,infection and pathogenesis.
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Objective:To analyze the non-enterovirus A71 (non-EVA71) and non-coxsackievirus A16 (non-CVA16) enteroviruses causing hand, foot and mouth disease (HFMD) in Kunming and Qujing of Yunnan Province in 2021 by sequencing the VP4/VP2 and VP1 genes and to analyze the phylogenetic characteristics of the VP1 gene of CVA2, aiming to provide reference for the prevention and control of CVA2.Methods:The samples were made and extracted strictly according to the Laboratory Manual for Hand, Foot and Mouth Disease (China Center for Disease Control and Prevention, 2018 Edition). VP4/VP2 junction regions were firstly amplified and sequenced by MD91/OL68-1 primers. These sequences were firstly edited and then "blasted" on the GenBank to determine the virus serotype. To analyze the phylogenetic characteristics of CVA2, the entire VP1 gene sequences were amplified in two segments using enterovirus species A primers. Virus serotype was again confirmed online by "Enterovirus Genotyping Tool Version 0.1". The sequences of the reference virus genotypes/sub-genotypes were downloaded according to the reference. The phylogenetic trees were constructed by Mega5.2 software and the genetic characteristics were analyzed.Results:A total of 749 non-EVA71 and non-CVA16 enteroviruses were detected in the two areas in 2021. Group A enteroviruses were the main pathogens, with CVA16 as the predominant virus, and a small number of group B enteroviruses were reported. Only five strains of CVA2 were detected with a detection rate of 0.67% (5/749), indicating that CVA2 was a rare pathogen for HFMD in the two areas. The sequencing and serotyping results were consistent using the two genomic regions of VP4/VP2 junction region and VP1 region. Phylogenetic analysis showed that three Kunming strains belonged to genotype A, while two Qujing strains belonged to genotype D.Conclusions:The detection rate of CVA2 in Kunming and Qujing was 0.67% in 2021. CVA2 was a rare pathogen for HFMD in the two regions. Phylogenetic analysis showed genotypes A and D spread in Kunming and Qujing, respectively, but had not caused epidemics. To our knowledge, this was the first report of genotype A of CVA2 in China. Strengthening the laboratory surveillance especially molecular epidemiological surveillance is valuable for the monitor and analysis of transmission source for CVA2.
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Background & objectives: BK virus (BKV) is a polyomavirus and cause of a common infection after renal transplantation which could be preceded to BKV-associated nephropathy. It has four main subtypes (I–IV). BKV subtypes II and III are rare, whereas subtype I shows a ubiquitous distribution. The objective of the present study was to investigate the prevailing BKV subtypes and subgroups in renal transplant patients in Sri Lanka. Methods: The presence of BKV in urine was tested through virus load quantification by real-time PCR from 227 renal transplant patients who were suspected to have BKV infection. Of these patients only 41 were found to be BKV infected (>103copies/ml) and those were subjected to conventional PCR amplification of VP1 gene followed by BKV genotyping via phylogenetic analysis based on DNA sequencing data. Results: Persistent BK viral loads varied from 1×103 to 3×108 copies/ml. Of the 41 patient samples, 25 gave positive results for PCR amplification of subtyping region of VP1 gene of BKV. BKV genotyping resulted in detecting subtype I in 18 (72%) and subtype II in seven (28%) patients. BKV subgroups of Ia, Ib-1 and Ib-11, and Ic were identified with frequencies of 6/18 (33.3%), 6/18 (33.3%), 5/18 (27.8%), and 1/18 (5.6%), respectively. Interpretation & conclusions: Findings from this preliminary study showed a high occurrence of subtype I, while the presence of subtype II, which is rare and less prevalent, was a novel finding for this Asian region. This emphasizes the need for further molecular and serological studies to determine the prevalence of different BKV subtypes in Sri Lanka
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Objective:To investigate the predominant types of enteroviruses and the characteristics of the VP1 gene of coxsackievirus A4 (CVA4) causing hand, foot and mouth disease (HFMD) in Yunnan Province from 2018 to 2020.Methods:Throat swab and stool samples were collected from HFMD cases and tested by real-time quantitative PCR for nucleic acid detection. The samples positive for enterovirus nucleic acids were used for viral isolation and sent to the National Center for Disease Control and Prevention. The VP1 gene of the isolated strains was sequenced and analyzed.Results:From 2018 to 2020, a total of 21 757 HFMD samples were collected, 16 457 (75.64%) of which were positive for enteroviruses. Altogether 533 strains were isolated from 4 114 positive samples that were selected for viral isolation, including 89 strains of enterovirus 71 (EVA71, 16.70%), 180 strains of coxsackievirus A16 (CVA16, 33.77%), 76 strains of CVA10 (14.26%), 118 strains of CVA6 (22.14%), 26 strains of CVA4 (4.88%) and 44 strains of other types (8.26%). HFMD occurred mainly in children under five years old with higher incidence in males than in females (1.35∶1). The incidence of HFMD reached the peak in the second and third quarters. In Yunnan Province, CVA4 mainly circulated in Qujing and Kunming, and was sporadically detected in Wenshan and Honghe. The VP1 gene was 915 bp in length. Twenty-six CVA4 strains belonged to C2 subtype, which were genetically far from the prototype strain AY421762-HighPoint. Mutations in the VP1 gene were found at multiple sites including 18, 23, 34, 102, 148, 164, 200, 262, 174, 275, 285 and 303. These strains showed 80.4%-99.0% homology in nucleotide sequence and 95.6%-99.0% in amino acid sequence. Nucleotide mutations were mostly synonymous mutations.Conclusions:CVA16, CVA6, EVA71 and CVA10 were the predominant enteroviruses causing HFMD in Yunnan Province from 2018 to 2020. The prevalence of CVA4 was also worthy of attention. CVA4 isolates in Yunnan Province belonged to C2 subtype, mainly circulating in the east and southeast of Yunnan Province and gradually becoming a cocirculating predominant strain. Long-term dynamic monitoring would be of great public health significance for improving the sensitivity of HFMD early warning.
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Objective To classify and identify the 53 strains of non-polio enterovirus (NPEV) isolated from acute flaccid paralysis (AFP) cases in Chongqing from 2013 to 2020, and to investigate the genotype distribution of the strains. Methods Commercial real-time fluorescence quantitative polymerase chain reaction (real-time PCR) reagents were used for rapid identification of the strains. The nucleotide sequences of VP1 and VP4 regions were used for genotyping. Results Fifty enteroviruses were identified, 33 (66%) in group A and 17 (34%) in group B. Group C and D enteroviruses were not found in these strains，and 3 strains could not be identified. In this study, EV-A71 was the dominant type, with 11 strains (22%), but EV-A71 strain was not isolated since 2016. The sequences of VP4 region and VP1 region were completely consistent in enterovirus grouping. Conclusion When using commercial real-time PCR reagents for enterovirus typing, the identification results of high CT values may be inaccurate. In the genotyping of enterovirus, the nucleotide sequence of VP4 region is first used for grouping, and then the nucleotide sequence of VP1 region is used for genotyping, which could simplify the experimental process. NPEV isolates from AFP cases in Chongqing showed poor genotype diversity. In order to enrich and improve the enterovirus gene database in Chongqing, it is necessary to carry out research on enterovirus transmitted by respiratory tract.
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Objective:To analyze the etiology of hand, foot and mouth disease (HFMD) cases collected from Wenshan prefecture from 2014 to 2018 and the molecular epidemiology of coxsackievirus A6(CV-A6).Methods:Viruses were isolated by RD cells and Hep-2 cells from stool samples collected from HFMD patients in Wenshan prefecture from 2014 to 2018. Virus RNA was extracted and virus VP4/VP2 junction region sequence was firstly amplified and sequenced by MD91 and OL68-1 primer pairs, then the virus serotype was determined. Virus entire VP1 gene sequences were determined by relative primer pairs according to the references. The reference sequences of CV-A6 virus entire VP1 gene were downloaded from the GenBank and the phylogenetic tree was constructed and the genetic characteristics and molecular epidemiology were analyzed.Results:During five years of study period, a total of 581 strains of enteroviruses (EVs) was isolated with an isolation rate of 20.40% (581/2 848). Among 581 strains, 74 strains were CV-A6, accounting for 12.74% (74/581); 124 were CV-A16, accounting for 21.34% (124/581); 374 were EV-A71, accounting for 64.37% (374/581); nine were other EVs, accounting for 1.55% (9/581). The entire VP1 sequences of 74 CV-A6 strains were filtered by constructing a phylogenetic tree and the completely same strains were excluded from analysis. We finally analyzed the phylogenetic characteristics of 22 strains isolated in this study with 52 reference strains. The results showed that all 22 Wenshan strains belonged to D3a sub-genotype, of which 21 strains belonged to cluster 1, and only one strain belonged to cluster 2.Conclusions:From 2014 to 2018, the outbreaks of HFMD in Wenshan prefecture were mainly caused by EV-A71, CV-A16 and CV-A6, accounting for 64.37%, 21.34% and 12.74% respectively. Phylogenetic analysis showed, similar to the situation in China, the sub-genotype D3a of CV-A6 was the predominant virus and the cluster 1 was the main sub-genotype in this outbreak.
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Objective@#To 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.@*Methods@#A 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 CCID @*Results@#The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID @*Conclusion@#This 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.
Sujets)
Humains , Protéines de capside/génétique , Entérovirus humain A/génétique , Entérovirus humain B/génétique , Entérovirus humain C/génétique , Entérovirus humain D/génétique , Épidémiologie moléculaire/méthodes , Typage moléculaire/méthodes , RT-PCR/méthodesRésumé
Aim To investigate the antiviral activity and mechanism of myricetin against enterovirus 71 (E V 7 1) infection. Methods The cytopathic effect (CPE) and plaque assay were used to observe the antiviral effect of myricetin against EV71 in Vero cell. The cells were treated with myricetin at different concentrations combined with crystal violet staining to detectthe cytotoxicity of myricetin. The effect of myricetin on VP1 protein expression was detected by Western blot. The effect of myricetin on VP1 gene expressionwas evaluated byRT-PCR. Results Myricetin pretreatment at 2. 5-20 fimol L-1' significantly inhibitedcell death induced by EV71 infection in a dose-dependent manner with the IC50 value of 5. 6 jxmol • L-1. Compared to virus control group, myricetin could significantly reduce the viral titer at the concentration of 2. 5 ~ 20 u,mol • L-1. The results of Western blot and RT-PCR showed that myricetin could markedlyreduce the gene and protein expression levels of viral capsid protein VP1. Conclusion Myricetin has significant antiEV71 activity in vitro.
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Objective To analyze the genetic characteristics of VP1 3'region of human coxsack-ievirus B2 (CV-B2) strains isolated from Yunnan province. Methods RT-PCR and gene sequencing were performed to analyze the VP1 3'region of 15 CV-B2 strains isolated from acute flaccid paralysis ( AFP) cases during 2005 to 2006, healthy children in 2013 and hand, foot and mouth disease (HFMD) cases in 2014 in Yunnan province. CV-B2 VP1 gene reference sequences were downloaded from the Genbank. Nucleotide (nt) and amino acid (aa) diversities were calculated by MEGA5. 2 software and a phylogenetic tree was constructed. Genetic and molecular epidemiological characteristics of CV-B2 strains circulating in Yunnan province were analyzed. Results A total of 15 CV-B2 strains were isolated, which were one from 232 AFP cases in 2005, one from 240 AFP cases in 2006, 12 from 400 healthy children in 2013 and one from 500 HFMD cases in 2014. Phylogenetic analysis of the 15 CV-B2 strains in Yunnan province and those down-loaded from the GenBank showed that CV-B2 could be genetically divided into five genotypes. The prototype strain Ohio-1 and one strain (01-1) isolated in Taiwan in 1988 belonged to genotype 1. Strains isolated in France in 2006, 2007 and 2010 belonged to genotype 2. Strains isolated in Yunnan, Shandong, Henan, Fu-jian and Taiwan belonged to genotype 3. Strains isolated in Russia, Yunnan AFP cases in 2005 and 2006 and India belonged to genotype 4. Strains isolated in Taiwan, Shandong and New South Wales, Australia be-longed to genotype 5. Different genotypes distributed in different countries/areas with some confined within specific countries/areas. Conclusions The 12 strains isolated from healthy children and one from HFMD cases in Yunnan province belonged to genotype 3, while the two strains isolated from AFP cases belonged to genotype 4. Diversities in nt and aa sequences between the strains isolated from the healthy children and HFMD case were only 0. 76％ and 0. 03％, respectively, indicating that they might come from the same transmission source. However, the nt and aa diversities between the isolates of genotype 3 ( from healthy children and HFMD case) and genotype 4 (from AFP cases in 2005 and 2006) were 15. 11％-15. 22％ and 2. 76％-2. 72％, respectively. Correlation of CV-B2 with AFP and HFMD was worthy of further study.
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Objective To investigate the genetic evolution of VP1 gene of pathogenic coxsackievirus A16 (CV-A16) strain isolated from clinical hand,foot,and mouth disease (HFMD) patients.Methods A total of 160 HFMD cases with CV-A16-positive results were collected from hospitals in Kunming during January 2015 to June 2017.Fecal samples were collected.Real-time polymerase chain reaction (PCR) was used to detect the CV-A16 virus nucleic acid.The VP1 genes of CV-A16-positive samples were amplified by reverse transcription-PCR.The amplified positive products were sequenced and aligned.The homologies were identified and their subgenotypes were determined.The phylogenetic tree was constructed and homology modeling was conducted.Results All the 160 CV-A16 isolates were B2 subtypes.The genetic distance between detected strains of CV-A16 and the strains in Fujian,Beijing,Nanjing was 0.76.The genetic distance to the strains in Malaysia was 0.78,and to the strains in Australia was 1.86.Homologous modeling revealed that the amino acid sequence of the VP1 gene of the strain had a G227R mutation.Conclusions There is no major genetic variation in the CV-A16 strains during 3 years.CV-A16 isolates are close to those of epidemic strains in Beijing,Fujian and Malaysia,but are far fram the strains from Australia.
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Objective@#To analyze the genetic characteristics of Coxsackievirus A4 isolated from Taian, 2017-2018.@*Methods@#Sixty throat swab samples of the children who visited Taian Maternal and Child Health Hospital during the year 2017-2018 and were diagnosed as hand, foot and mouth disease, were collected and aseptically inoculated. Fluorescent quantitative PCR analysis was performed using the universal primer for enteroviruses. The high-throughput sequencing was applied to the enterovirus-positive samples, and the full-length genome sequences of the viruses were obtained. Phylogenetic analysis was performed using Mega5.05 and RaxML respectively, and sequence homology and amino acid mutation sites were also analyzed using Mega5.05.@*Results@#Four whole genome sequences of CV-A4 isolated from infants aged 17-19 months old were obtained. Phylogenetic analysis of the full length CV-A4 genomes showed that apart from MG550920/AA/Henan/2016, the remaining CV-A4 strains from China (97.2%), including the four strains from Taian, fell within Group 3. The VP1 genes could be classified into four genotypes and 98.5% of the Chinese strains belonged to genotype D, and the four strains from Taian belonged to D2. It was notable that the Taian isolate A1/Taian is closely related to two strains C179 and C062 from Australia both in the complete genome and the VP1 gene, as well as one strain YT184R isolated from Yantai in 2016 by us. Compared with the prototype CV-A4 strain High Point, 18 amino acid mutations were found in the P1 region.@*Conclusions@#Both phylogenetic trees estimated using the complete genome and the VP1 gene sequences revealed that the four CV-A4 isolates from Taian fell within the same clade with the majority of CV-A4 strains circulating in China. Compared with the prototype CV-A4 strain, several amino acid variations have occurred in the P1 region, which warrants further investigation.
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Objective@#To detect antibodies to Coxsackievirus B4 (CV-B4), the indirect enzyme-linked immunosorbent assay (ELISA) method was established and optimized using the recombinant VP1 protein expressed in the prokaryote system as the envelope antigen.@*Methods@#The VP1 gene of CV-B4 was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR). It was ligated into the expression vector pET32a(+ ) to obtain the recombinant plasmid pET32(+ )-VP1 and was then transformed into E. coli expression strain Rosetta (DE3). The recombinant VP1 protein was induced by IPTG, which was verified using SDS-PAGE electrophoresis and mass spectrometry. The establishment and optimization of the indirect ELISA reaction system was based on the purified recombinant protein mentioned above as the coating antigen.@*Results@#The CV-B4 VP1 gene was stably expressed in E. coli in the form of inclusion body. The optimal coating concentration of antigen was 7.5 μg per well and the optimal serum dilution was 1∶100. The threshold for determining the negative and positive result of the serum samples was the optical density value of ≥ 0.376 at 450 nm. The purified recombinant protein could be specifically recognized by CV-B4 positive serum without cross-reaction with Coxsackievirus (CV)-A, CV-B1 and CV-B5, indicating that it has good immunogenicity. However, it can cross-react with CV-B3 serum samples.@*Conclusions@#The indirect ELISA detection method based on the CV-B4 VP1 protein could be used in the detection of serum antibody to CV-B4 infection with good sensitivity, specificity and repeatability.
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Objective@#In this study we analyzed the genetic characteristics of echovirus 30 (E-30) VP1 gene sequences from Yunnan province isolated from viral meningitis (VM) cases in 2010-2013.@*Methods@#RT-PCR and VP1 gene sequencing were done for 9 E-30 strains isolated from VM cases in 2010-2013. VP1 gene sequences of E-30 reference strains were downloaded from the GenBank and their nucleotide (nt) and amino acid (aa) diversities were calculated by MEGA 5.1 software, the phylogenetic tree was constructed and the genetic characteristics and molecular epidemiology were analyzed.@*Results@#In 2010-2013, 9 strains of E-30 viruses were detected from 79 VM cases caused by echoviruses, accounting for 11.39%(9/79), the overall positive rate was 1.63%(9/553). Phylogenetic analysis revealed that E-30 strains can be divided into four genotypes (genotype A, B, C and D), and genotype D can be further divided into seven sub-genotypes. Nine Yunnan VM isolates were distributed in D7 sub-genotype, and can be further clustered into 3 branches: 5 strains isolated in 2010 were clustered in branch 1, it is evident that these viruses were responsible for an aseptic meningitis outbreak in Kunming in that year; one 2011 isolate, together with 2013 isolate and one isolate from healthy children in 2010 were clustered in branch 2, these two branches were Yunnan special branches, and two 2011 isolates had the highest homology with 2003 VM outbreaks′ strains isolated from Shandong, Jiangsu, and Zhejiang, showing that these strains may have the same evolutionary sources.@*Conclusions@#Nine Yunnan VM isolates were distributed in D7 sub-genotype, and these strains have different evolutionary sources, showing that at different times E-30 viruses in the same sub-genotypes branch might prevail in different areas.
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Objective@#To detect the enterovirus VP4 and VP1 genes in 510 stool samples collected from hand, foot and mouth disease (HFMD) cases and analyze the phylogenetic characteristics of the entire VP1 genes of coxsackievirus A6 (CV-A6) strains in six prefectures/cities of Yunnan Province in 2018.@*Methods@#Viral RNA was abstracted from the stool samples. VP4 gene sequences were amplified by RT-PCR and sequenced using the MD91/OL68-1 primer pair to identify viral genotypes. Whole VP1 gene sequences were amplified and sequenced using appropriate primer pairs. The whole VP1 gene sequences of CV-A6 reference strains were downloaded from GenBank. MEGA5.2 software was used to analyze the similarity in nucleotide and amino acid sequences between different strains and phylogenetic tree was constructed for analysis of genetic characteristics and molecular epidemiology.@*Results@#VP4 and VP1 gene sequences were obtained from 57 out of 510 stool samples with a positive rate of 11.17% (57/510). There were 43 CV-A6 (8.43%, 43/510), six CV-A10 (1.17%, 6/510), two enterovirus A71 (EV-A71, 0.39%, 2/510) and two CV-A9 (0.39%, 2/510) strains. The other four strains were CV-A4 (0.19%, 1/510), CV-A5 (0.19%, 1/510), CV-B1 (0.19%, 1/510) and E11 (0.19%, 1/510). The phylogenetic analysis showed that all 43 CV-A6 strains belonged to sub-genotype D3.@*Conclusions@#In the 510 HFMD samples, CV-A6 strains were mostly detected with a detection rate of 8.43% and accounted for 75.44% (43/57) of all isolates, followed by CV-A10 (1.17%, 6/510) and EV-A71 (0.39%, 2/510). There was a large HFMD outbreak mainly caused by CV-A6 in Yunnan Province in 2018. The outbreak was caused by CV-A6 of sub-genotype D3, as was the case with pervious outbreaks in China.
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Objective To detect the enterovirus VP4 and VP1 genes in 510 stool samples collect-ed from hand, foot and mouth disease ( HFMD) cases and analyze the phylogenetic characteristics of the en-tire VP1 genes of coxsackievirus A6 (CV-A6) strains in six prefectures/cities of Yunnan Province in 2018. Methods Viral RNA was abstracted from the stool samples. VP4 gene sequences were amplified by RT-PCR and sequenced using the MD91/OL68-1 primer pair to identify viral genotypes. Whole VP1 gene se-quences were amplified and sequenced using appropriate primer pairs. The whole VP1 gene sequences of CV-A6 reference strains were downloaded from GenBank. MEGA5. 2 software was used to analyze the simi-larity in nucleotide and amino acid sequences between different strains and phylogenetic tree was constructed for analysis of genetic characteristics and molecular epidemiology. Results VP4 and VP1 gene sequences were obtained from 57 out of 510 stool samples with a positive rate of 11. 17％ (57/510). There were 43 CV-A6 (8. 43％, 43/510), six CV-A10 (1. 17％, 6/510), two enterovirus A71 (EV-A71, 0. 39％, 2/510) and two CV-A9 (0. 39％, 2/510) strains. The other four strains were CV-A4 (0. 19％, 1/510), CV-A5 (0. 19％, 1/510), CV-B1 (0. 19％, 1/510) and E11 (0. 19％, 1/510). The phylogenetic analy-sis showed that all 43 CV-A6 strains belonged to sub-genotype D3. Conclusions In the 510 HFMD sam-ples, CV-A6 strains were mostly detected with a detection rate of 8. 43％ and accounted for 75. 44％ (43/57) of all isolates, followed by CV-A10 (1. 17％, 6/510) and EV-A71 (0. 39％, 2/510). There was a large HFMD outbreak mainly caused by CV-A6 in Yunnan Province in 2018. The outbreak was caused by CV-A6 of sub-genotype D3, as was the case with pervious outbreaks in China.
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Objective@#To study the molecular epidemiology and genetic variation of coxsackievirus B5 (CV-B5) in certain areas in China.@*Methods@#MEGA 6.0 software was used to analyze the complete VP1 region of CV-B5 isolated strains from certain areas of China by retrieving the GenBank nucleotide database. Besides, the phylogenetic tree was constructed, the homology of nucleotide and amino acids were calculated and the rate of evolution was estimated.@*Results@#A total of 189 Chinese CV-B5 isolated strains were included in this study. Most of Chinese CV-B5 isolated strains belonged to genotype C, accounted for 90.5%. Compared with the genotype A, the homology of nucleotide sequences and amino acid sequences of complete VP1 region of 189 Chinese isolated strains were 79.8%-82.8% and 92.6%-97.9%, respectively; moreover, the nucleotide and the amino acid homology of 189 Chinese CV-B5 isolated strains among themselves ranged from 80.3% to 100.0% and ranged from 91.5% to 100.0%, respectively. The estimated rate of evolution of the CV-B5 was 4×10-3 substitutions/site/year.@*Conclusions@#The majority of CV-B5 isolated strains belonged to genotype C, and subgenotype C1 and C2 were co-circulating together in certain areas of China.
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Human bocaviruses (HBoV) are mainly associated with respiratory and gastroenteric infections. These viruses belong to the family Parvoviridae, genus Bocaparvovirus and are classified in four subtypes (HBoV1-4). Recombination and point mutation have been described as basis of parvovirus evolution. In this study three viral sequences were obtained from positives HBoV sewage samples collected in two Uruguayan cities and were characterised by different methods as recombinant strains. This recombination event was localised in the 5' end of VP1 gene and the parental strains belonged to subtypes 3 and 4. These three Uruguayan strains are identical at the nucleotide sequences in the analysed genome region of the virus. As far as we known, this study represents the first detection of HBoV recombinants strains in the Americas.