Identification and complete genome sequencing of human adenovirus type 55 isolated from a patient with acute hemorrhagic conjunctivitis / 上海预防医学

ObjectiveTo determine the genomic characteristics of a subgenus B human adenovirus strain isolated in Shanghai in 2021. MethodsAn adenovirus type 55 strain was isolated and identified from a patient with acute hemorrhagic conjunctivitis （AHC）. Complete genome of the strain was obtained using the next-generation sequencing （NGS）. Phylogenetic trees were reconstructed based on the sequences of Hexon， Fiber， Penton and complete genome to genomically characterize this strain. ResultsPhylogenetic analysis based on the complete genome classified this strain （MH2021001） into subgenus B， subspecies B2 of HAdV-55. Hexon gene of MH2021001 had close phylogenetic relationship with HAdV-11， while Fiber and Penton genes had close relationship with HAdV-14. The MH2021001 showed high nucleotide identity with currently prevalent HAdV⁃55 strains （>99.90%）. The complete genome had 99.96% nucleotide identity to the 73-GD_CHN_2016 strain isolated in Guangdong. In addition， the amino acid sequence of MH2021001 had several substitutions in regions coding for E1B， L4， E3 and L5. ConclusionThis strain has been classified to HAdV-B55. No recombination event is identified in the complete genome. Due to multiple amino acid substitutions， the biological characteristics of the strain need to be further identified.

Serotype distribution and phylogenetic analysis of virus complete genome from indigenous dengue patients in Guangzhou in 2019 / 中华微生物学和免疫学杂志

Objective:To investigate the serotype distribution and phylogenetic analysis of virus complete genome from indigenous dengue patients in Guangzhou in 2019 and provide evidence for the development of prevention and treatment strategies.Methods:Dengue virus serotypes of indigenous dengue cases in 2019 were detected using serotype specific fluorescent PCR kits. Complete genome in the culture was performed on Illumina platform. Phylogenetic analysis was conducted on complete genomes extracted from ViPR and the isolates from this study with MEGA7.0 software.Results:In 2019, three prevalent serotypes of dengue virus were found in Guangzhou, among which serotype 1 accounted for 80.35%, serotype 2 accounted for 12.97% and serotype 3 accounted for 6.68%. There were no significant differences in gender, age and severity among three serotypes. Phylogenetic analysis of virus complete genome showed that serotype 1 belonged to genotypeⅠand had two origins, which was close to the Cambodian strain; serotype 2 belonged to genotype cosmopolitan, which was close to the epidemic strain in Southeast Asia; serotype 3 belonged to genotypeⅢ, which was in the same branch as the Indian strain.Conclusions:The dengue epidemic was caused by dengue virus serotypes 1, 2 and 3 in Guangzhou in 2019. Each serotype belonged to a genotype.

Multidrogorresistencia de Salmonella infantis en Perú: un estudio mediante secuenciamiento de nueva generación / Multidrug resistance of Salmonella infantis in Peru: a study through next generation sequencing

RESUMEN Objetivos. Describir los patrones fenotípicos y genotípicos de la resistencia antimicrobiana de Salmonella Infantis en Perú. Materiales y Métodos. Se analizaron 297 cepas de Salmonella sp. remitidas al Instituto Nacional de Salud (INS) en el periodo 2014-2016. Las cepas fueron caracterizadas fenotípicamente mediante pruebas microbiológicas, serológicas y de susceptibilidad antimicrobiana. En base a los patrones de resistencia antimicrobiana se seleccionaron 46 cepas que fueron caracterizadas genéticamente mediante secuenciamiento de nueva generación. Resultados. Se identificaron 193/297 (65,0%) cepas de Salmonella Infantis, de la cuales 143 (74,1%) fueron multidrogorresistentes productoras de betalactamasas de espectro extendido (BLEE). Con el secuenciamiento genómico se evidenció un nuevo perfil para Salmonella Infantis, además, se identificó la presencia de 15 diferentes determinantes genéticos de resistencia a los antimicrobianos codificados en cromosoma bacteriano y cinco codificados en un megaplásmido. Los patrones de resistencia fenotípicos y genotípicos coincidieron, a excepción de la ceftazidima. Asimismo, las 46 cepas presentaron resistencia y/o sensibilidad disminuida a las quinolonas. Conclusiones. Salmonella Infantis se ha convertido en una de las serovariedades más frecuentemente referidas al INS, la cual incluye cepas multidrogoresistentes productoras de BLEE con resistencia a las quinolonas. Finalmente, se reafirma la relevancia del secuenciamiento de nueva generación en la caracterización de nuevas variantes de patógenos de importancia para la salud pública y su uso potencial en los sistemas de vigilancia de resistencia antimicrobiana.

ABSTRACT Objectives. To describe the phenotypic and genotypic patterns of the antimicrobial resistance of Salmonella Infantis in Peru. Materials and Methods. Two hundred and ninety-seven strains of Salmonella sp. submitted to the National Institute of Health (INS, in Spanish) during 2014-2016 were analyzed. The strains were phenotypically characterized by microbiological, serological, and antimicrobial susceptibility tests. Based on antimicrobial resistance patterns, 46 strains were selected and genetically characterized by next generation sequencing. Results. 193/297 (65%) strains of Salmonella Infantis were identified, of which 143 (74.1%) were multidrug-resistant producers of extended spectrum beta-lactamases (ESBL). The genomic sequencing evidenced a new profile for Salmonella Infantis; additionally, it identified the presence of 15 different genetic determinants of antimicrobial resistance coded in bacterial chromosome and five coded in a megaplasmid. The phenotypic and genotypic resistance patterns matched, with the exception of ceftazidime. Moreover, the 46 strains presented resistance and/or decreased sensitivity to quinolones. Conclusions. Salmonella Infantis has become one of the sero-varieties most frequently referred to the INS, which includes ESBLproducing multidrug-resistant strains with resistance to quinolones. Finally, the relevance of next generation sequencing is reasserted in the characterization of new variants of pathogens that are important for public health, and their potential use in antimicrobial resistance surveillance systems.

Analysis of genomic characteristics of influenza A (H1N1) pdm09 viruses in Inner Mongolia, 2013-2017 / 中华实验和临床病毒学杂志

Objective@#To evaluate the effect of vaccine and provide scientific evidence for prevention and control of influenza virus, this study aims to analyze the characteristics of genomic variation of influenza A (H1N1) pdm09 viruses in Inner Mongolia.@*Methods@#The 16 viral strains were selected randomly according to the influenza A (H1N1) pdm09 viruses isolated from network laboratories in Inner Mongolia, 2013-2017. The hemagglutinin(HA) and neuraminidase(NA) genomic sequences were obtained by using RT-PCR and sequencing, and genomic characteristics were analyzed via bioinformatics.@*Results@#Compared to the A/California/07/2009 vaccine strain, the relatively obvious variation of antigen of influenza A (H1N1) pdm09 viruses in Inner Mongolia since 2014, and the vaccine provided a poor protection to influenza A (H1N1) pdm09 virus infection, while the A/Michigan/45/2015 vaccine strain recommended by WHO recently has a satisfactory protective effects. Several viral isolates from Inner Mongolia increased the binding force of virus in human upper respiratory tract because of D222N and D222G substitution within HA. E119K and H275Y substitution within NA gene of viral strains, suggesting that the viruses were resistant to NA inhibitors.@*Conclusions@#The influenza A (H1N1) pdm09 viruses had gradual variations as time went on, and the WHO recommended vaccine was relatively lagging. Virulent strains and drug-resistant strains appeared in the population, and the genetic characteristics of influenza virus surveillance should be strengthened to find the new mutants of virus in time, which provide evidence for the prevention and control of influenza.

Successive subculture and genetic stability of hepatitis A virus vaccine strain H2 / 中华实验和临床病毒学杂志

Objective@#To investigate the genetic stability of virus seed H2M20K7 (K7) of live attenuated Hepatitis A virus H2 strain (HAV, H2 strain) for production of hepatitis A (Live) vaccine, lyophilized after continuous passages.@*Methods@#The virus seed K7 of H2 strain was proliferated and passaged in KMB17 cells in cell factories. Viruses of different passages were harvested after continuous passages. Virus RNA was extracted and the complete genomes of different virus passages (K7, K10, K11, K13, K15, K18) were sequenced by using next-generation deep sequencing. The mutation rates of different passages were compared. The infectivity titers of different virus passages of H2 strain were tested by ELISA.@*Results@#The mutation rates of complete genomes of different passages were low after continuous passages of master virus seed. The structure of gene was stable and non-synonymous mutation rate was lower than 0.57%. The mutation rate of 5 ’non-coding regions was lower than 0.1%. There was no significant mutation in VP1/2 A and 2C virulence site. The infectious titers of H2 strains of different passages were within 7.76-8.50 lgCCID50/ml. No statistically significant difference was found in this study.@*Conclusions@#The gene structure of the master virus seed, working seed and different passages of H2M20K7 after subculture was stable and the mutation rate was low. No significant mutation was found in 5’non-coding regions, and the critical virulence sites such as VP1/2 A, 2B and 2C showed attenuated characteristics with low mutation rate. Virulence of the virus did not changed. The H2 strain maintained stable viral infectivity and genetic stability and comply with the requirements as virus seed for vaccine manufacturing.

Complete genome sequence of the aerobically denitrifying thermophilic bacterium Chelatococcus daeguensis TAD1

ABSTRACT Chelatococcus daeguensis TAD1 is a themophilic bacterium isolated from a biotrickling filter used to treat NOx in Ruiming Power Plant, located in Guangzhou, China, which shows an excellent aerobic denitrification activity at high temperature. The complete genome sequence of this strain was reported in the present study. Genes related to the aerobic denitrification were identified through whole genome analysis. This work will facilitate the mechanism of aerobic denitrification and provide evidence for its potential application in the nitrogen removal.

Characterisation of complete high- and low-risk human papillomavirus genomes isolated from cervical specimens in southern Brazil

The classification of human papillomavirus (HPV) intratypic lineages by complete genome sequencing is a determinant in understanding biological differences in association with this disease. In this work, we have characterised complete HPV genomes from southern Brazil. Fifteen cervicovaginal Pap smear negative samples previously categorised as HPV-positive were sequenced using ultradeep sequencing, and 18 complete genomes from 13 different HPV types were assembled. Phylogenetic and genetic distance analyses were performed to classify the HPV genomes into lineages and sublineages. This is the first report describing the distribution of HPV intratype lineages of high and low oncogenic risk in asymptomatic women from southern Brazil.

Complete genome analysis of a sapovirus in a mixed infected patient of hand foot and mouth disease / 中华实验和临床病毒学杂志

Objective@#To understand the potential viral pathogens other than enteroviruses existing in samples of hand foot and mouth disease (HFMD) patient and study their molecular feature and genotype.@*Methods@#The deep sequencing analysis of a fecal specimen collected from HFMD patient was conducted by metagenomics and bioinformatics.@*Results@#Enterovirus A71 and sapovirus mixed infection was found in this case. The nucleic acid of sapovirus was confirmed positive by RT-PCR and the 7 429 bp complete genome sequence of sapovirus was obtained by assembling sequencing reads which consisted of 3 open reading frames. Phylogenetic analysis revealed that this strain of virus should belong to the genotype 1 of sapovirus having a homology of 99.4% with sapovirus Hu/G1/Zhejiang1/China/2014 strain, which is a currently predominant genotype circulating in China.@*Conclusions@#The sapovirus, which is a predominant strain circulating in China, was a mixed infected causative agent existing in HFMD sample identified by deep sequencing. This study will serve as a reference for pathogen detection of HFMD and diarrheal related diseases, as well as provide a sequence reference for molecular feature study of sapovirus in China in the future.

Complete genome sequence of a genogroup I geno type 8 norovirus identified in Huzhou, China / 中国人兽共患病学报

We identified and characterized the full-length genome of a GI.8 norovirus strain CHN/Huzhou/N10 isolated in an outbreak in Huzhou,China.The full-length genome of CHN/Huzhou/N10 was amplified using five pairs of primers which were designed according to the full-length GI norovirus genome sequences published in GenBank database.Multiple alignments were performed using DNAStar,the phylogenetic relationship of CHN/Huzhou/N10 and the representative NoV (Norovirus) strains from each genogroup were assessed using the software MEGA version 6.0.The viral genome of CHN/Huzhou/N10 was 7 740 nucleotides in length,which was consist of three ORFs spanning 5-5 404 nt (ORF1),5 388-7 019 nt(ORF2),and 7 019-7 660 nt (ORF3),respectively.Phylogenetic analysis based on polymerase and capsid sequences VP1 and VP2 region indicated that CHN/Huzhou/N10 belonged to GI.8 genotype.The amino acid sequence analysis of the VP1 region showed that CHN/Huzhou/N10 had 16 mutations compared with the representative strain Boxer/2001/US,12 of these variations were located in the P2 subregion.Moreover,a single amino acid change (T347S) occurred at histo-blood group antigen (HBGA) binding site Ⅱ and another single amino acid change (T397E) occurred at HBGA binding site Ⅲ.In this study,the first full genome of norovirus GI.8 isolated in Huzhou,China was extensively characterized.The data would be helpful not only for the epidemiology study,but also for the diagnostic tool development and effective vaccine design in the future.

Complete genome analysis of influenza A(H1N1) pdm09 virus isolated from one case of pneumonia of unknown etiology (PUE) in Fujian Province, China / 中国人兽共患病学报

To study the biological characteristics and mutations of influenza A(H1N1)pdm09 virus isolated from one case of pneumonia of unknown etiology (PUE),which would provide references for clinical treatment and disease control,the throat swab specimen from the PUE case was isolated in the Madin-Darby Canine Kidney (MDCK) cells,and then the antigenicity,pathogenicity and drug resistance of influenza A (H1N1) pdm09 virus were analyzed after sequencing.As a result,one influenza virus strain was isolated from the specimen and named as A/FujianGulou/SWL64/2016(H1N1).The similarities of nucleotide sequences and amino acids sequences compared with the vaccine strain A/California/07/2009 (H1N1) were 96.9％-98.9％ and 96.7％-99.5％,respectively.Eighteen amino acids had mutated in the HA and 4 mutations,K163Q,S185T,S203T and D222N,were involved in 3 different epitopes,which indicated that the antigenic drift had occurred in the influenza virus.The D222N mutation associated with receptor binding site made the virus infect lower respiratory tract more easily.The virus was still amantadine-resistance and oseltamivir-sensitive.In conclusion,the influenza A (H1N1) pdm09 virus in this study have occurred antigenic drift and has the molecular characterization of causing severe pneumonia,so further surveillance should be performed to prevent and control the influenza epidemic.

Whole genome sequencing for analyzing mutation sites in linezolid-resistant methicillin-resistant Staphylococcus aureus / 中国感染控制杂志

Objective To understand genetic mutation sites in linezolid (LZD)-sensitive and inducible resistant strains of methicillin-resistant Staphylococcus aureus (MRSA) using whole-genome sequencing,and realize mutation sites of LZD-resistant gene.Methods MRSA-MS4 with explicit genotype and whole-genome sequences was induced by LZD of different concentration gradients,LZD-resistant strain MRSA-MS4-LZD100 was obtained,minimum inhibitory concentration(MIC) was detected,domain V of 23S rRNA and ribosomal proteins L3/L4 gene in MRSAMS4-LZD100 were amplified by polymerase chain reaction (PCR),the sequenced products obtained the corresponding mutation site in contrast with the wild-type strain;Illumina PE library was constructed through paired-end sequencing by Illumina HiSeq 2000 technique,and whole genome sequencing was completed based on bioinformatics.Results MRAS-MS4-LZD100 strain was induced after 32 passages,MIC of LZD was 96 μg/mL.Sequencing of PCR products indicated the genetic variations were G2447T mutation in multiple copies of domain V of 23S rRNA gene,and Gly113Val mutation in L3 protein respectively;the whole genome of MRSA-MS4-LZD100 contained 2 744 315 bp,annotation of the whole genome found a total of 2 509 genes,11 tRNA-encoding genes and 2 entire rRNA-encoding operons.The data were submitted to the PubMed,and the GeneBank accession number JXMJ00000000 was assigned;a total of 101 SNPs and 6 Small indels were found,16 of 101SNP mutations occurred in exon,of which the variant proteins with anmino acid sequence alterations included IstB ATP binding domain-containing protein,clumping factor A,IS1272 transposase and so on;3 of 6 Small indel mutations occurred in exon,of which the variant proteins with anmino acid sequence alterations included hypothetical protein,30S ribosomal protein S1,and clumping factor A.Conclusion LZD-resistant strain MRSA-MS4-LZD100 was successfully induced by LZD;beside 23S rRNA V domain and ribosomal L3 protein,the other mutant site exist in this resistant strain,which provide some direction for subsequent study of recessive LZD resistance mechanism.

Research on nucleotide sequence of a newly emerged pandemic norovirus GⅡ.4 genotype / 国际检验医学杂志

Objective Analysis of the complete genome sequence about the newly emerged pandemic norovirus GⅡ.4 genotype, to understand its variation characteristics.Methods 264 patients were collected with diarrhea.The RNA was extracted from 264 fe-cal specimens and cDNA was synthetized.The positive samples were amplified by PCR,the amplified fragments were sequenced. The complete genome sequences of the norovirus was sequenced and analyzed.Results A new norovirus variant strain of Jingzhou GⅡ.4,a pleiston of Sydney GⅡ.4 was isolated.A large variation was found in the new variant subtype,which was mutated inclu-ding in the hypervariable P2 domain of the major capsid protein VP1.Conclusion The result demonstrates the variant strain of Sydney GⅡ.4 was spread to China.VP1 of norovirus GⅡ.4 is evolving rapidly.The spread and evolution situation of the norovirus GⅡ.4 need to be closely monitored in China for the development of effective vaccines and therapeutic monoclonal antibodies.

Origin and Phylogenetic Characteristics of Dengue Virus Isolated from the Large Outbreak of Dengue in Guangdong Province in 2014 / 中山大学学报(医学科学版)

[Objects]To isolate and identify the pathogen of the large outbreak of dengue in Guangdong province in 2014. To understand the origin and the phylogenetic characteristics of the isolates ,and provide scientific foundation for the surveillance and prevention of dengue fever.[Methods]Collected the patient serum samples over all the Guangdong province during the 2014 outbreakperiod,isolated and identified the virus from these samples. Amplified complete E gene and complete genome with certain primers and sequenced all the products. Then the Phylogenetic ,Bayesian phylogeography and mutations analysis were carried.[Results]40 DENV-1 strains were isolated and identified. 40 complete E gene sequences and 6 complete genome sequences of DENV-1 were obtained. Phylogenetic analysis with E gene sequences revealed that the 40 isolates were classified into two genotypes including 16 genotypeⅠ(Asia)and 24 genotypeⅤ(America/Africa). 14 genotypeⅠisolates were clustered closest with isolates from Guangdong province(2013)and Sigapore(2013)which share the nucletide identities of 99.6% ~ 99.9%,other two genotypeⅠisolates were clustered with strains from Malaysia (2013) and both share the nucletide identities of 99.7%;24 genotypeⅤisolates were all classified in one clade with striains from Bangladesh(2009),China(2009)and Bhutan(2013)which share nucletide identities of 99.0%-99.9%. Further analysis with six complete genome sequences showed that five isolates were clustered closest with strains isolated from Guangdong province(2013)share the nucletide identities of 99.6%-99.8% while the sixth stains closest with strains isolated from Myanmar(2002)share the nucletide identities of 98.8%. The isolates have five amino acid mutations compared with strains epidemic in Guangdong province in 2013,three mutations(S88V,E203G,T275R)are in the EⅡdomain and one mutation (S305P)is in the EⅢdomain which associated with virulence.[Conclusions]During the outbreak in Guangdong province in 2014, DENV-1 is the predominant causative serotype,and there are at least two different kinds of genotypes of DENV-1 largely epidemiced in the whole province. Evolution analysis reveals the multiple origins of the isolates which may origin from Guangdong province , Sigapore,Malaysia,Myanmar so that we should enhance the study and surveillance of autochthonous and vectors in order to understand the epidemic way of dengue in Guangdong province. The isolates have had four mutations in the domain associated with virulence which remain further study to know their biological effects.

Genome-wide analyses of HTLV-1aD strains from Cape Verde, Africa

We characterised and reported the first full-length genomes of Human T-cell Lymphotropic Virus Type 1 subgroup HTLV-1aD (CV21 and CV79). This subgroup is one of the major determinants of HTLV-1 infections in North and West Africa, and recombinant strains involving this subgroup have been recently demonstrated. The CV21 and CV79 strains from Cape Verde/Africa were characterised as pure HTLV-1aD genomes, comparative analyses including HTLV-1 subtypes and subgroups revealed HTLV-1aD signatures in the envelope, pol, and pX regions. These genomes provide original information that will contribute to further studies on HTLV-1a epidemiology and evolution.

Genomic evolution characteristics of pathogenicity islands of enteropatho-genic Escherichia coli Deng strain / 中国感染控制杂志

Objective To analyze the genomic evolution characteristics of pathogenicity islands (PAIs)in Deng strain of enteropathogenic Escherichia coli (E.coli,EPEC Deng).Methods EPEC Deng was isolated from infant stool specimen,serotypes were identified and antimicrobial susceptibility testing was performed;whole-genome se-quencing was performed by Illumina 2000 system,the locations of prophages(PPs)in the chromosome were detected using PHAST software,collinearity analysis was performed by MUMmer software,phylogenetic trees of homolo-gous gene were constructed in order to understand the evolutional rule of homology gene.PAIs prediction was per-formed using PAI finder software,the homologous evolutionary rule of PAIs core region(LEE)and core genes were clarified,genetic polymorphism was analyzed.Results The serotype of EPEC Deng strain was O119:H6,the strain was resistant to ciprofloxacin,levofloxacin,and ampicillin,but sensitive to other antimicrobial agents.The complete circular chromosome contained 5 025 482 bp with a GC content of 50.52 %,and the plasmid contained 207 564 bp with a GC content of 49.50%.A total of 17 PPs in the chromosomal genome were discovered,phyloge-netic trees analysis suggested that EPEC Deng strain was highly homologous with O26:H11 and O111 :H strains;PAIs and core genes were highly homologous with RDEC-1 and O26:H413/89-1 strains;genetic diversity analysis showed that the intimin (eae)and its receptor tir had high polymorphism,with the pi (π)value>0.10,the genes in type III secretion system was relatively stable.Conclusion The study clarified the genomic evolution characteris-tics of EPEC Deng genome and it’s PAIs,and is helpful for understanding genetic characteristics of native EPEC.

Full-genome sequences of hepatitis B virus subgenotype D3 isolates from the Brazilian Amazon Region

The Brazilian Amazon Region is a highly endemic area for hepatitis B virus (HBV). However, little is known regarding the genetic variability of the strains circulating in this geographical region. Here, we describe the first full-length genomes of HBV isolated in the Brazilian Amazon Region; these genomes are also the first complete HBV subgenotype D3 genomes reported for Brazil. The genomes of the five Brazilian isolates were all 3,182 base pairs in length and the isolates were classified as belonging to subgenotype D3, subtypes ayw2 (n = 3) and ayw3 (n = 2). Phylogenetic analysis suggested that the Brazilian sequences are not likely to be closely related to European D3 sequences. Such results will contribute to further epidemiological and evolutionary studies of HBV.

Genetic variation and molecular evolution of human bocavirus 1 strains isolated during 2009 to 2014 in Hangzhou, China / 中华微生物学和免疫学杂志

Objective To investigate the genetic variation and molecular evolution of human bo-cavirus 1 (HBoV1) strains isolated during 2009 to 2014 in Hangzhou, China.Methods Throat swab sam-ples were collected from children with acute respiratory tract infections in the Children′s Hospital Affiliated to the Zhejiang University School of Medicine from 2009 to 2014.Real-time PCR was performed for the detec-tion of HBoV1 strains.Fifteen HBoV1 strains with high virus load were screened out for the amplification and sequencing of complete genomes.The complete genomes were submitted to GenBank for further analysis with bioinformatics software.Results A total of 48 nucleotide mutations were detected in the complete genomes of 15 HBoV1 strains, resulting in 11 amino acid mutations with 5 of them located in the active region of phospholipase A2 ( PLA2) .The 15 HBoV1 isolates along with 16 HBoV1 strains in GenBank were classified into three clusters as indicated by the phylogenetic analysis based on their complete coding sequences.All of the 15 strains were belonged to clusterⅠ, the representative strain of which was the Sweden prototype strain ST2.The phylogenetic trees constructed using genes encoding the capsid proteins VP1 and VP2 were highly similar to those based on the complete coding sequences.The estimated mean evolutionary rate of HBoV1 with regard to the complete coding sequence was 3.03×10-4(95%HPD, 2.14×10-4-3.92×10-4 ) substitu-tions per site per year.With regard to each gene, the NS1 gene was considered to the most conserved gene while the NP1 gene showed the highest substitution rate.The dN/dS ratios (ω) of the four genes were all less than 1, indicating that all of them were under negative selection.Moreover, the VP2 gene was under the strongest negative selection, while the NP1 gene was under the weakest negative selection.Conclusion All of the HBoV1 isolates circulating in Hangzhou province during 2009 to 2014 were belonged to ST2 genotype with a relatively high mutation in the area of PLA2.Despite the complete genome was conservative, its evo-lutionary rate was high.Among the four genes, the NP1 gene showed the highest substitution rate.All of the four genes were under negative selection, of which the VP2 gene was under the strongest negative selection.

Concurrent Infection of Torque Teno Sus Virus and Porcine Circovirus Type 2 in a Clinical Case with Post-weaning Multisystemic Wasting Syndrome.

Aims: The goal of this study was to identify possible concurrent infection of torque teno sus virus (TTSuV) and porcine circovirus type 2 (PCV2) in a clinical case with postweaning multisystemic wasting syndrome (PMWS) on certain farm of Shanghai, China. Place and Duration of Study: Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, between June 2009 and June 2010 & Institute of Animal Health, Guangdong Academy of Agricultural Sciences, between September and November, 2013. Methodology: Multiply-primed rolling-circle amplification (MPRCA), a useful molecular tool, was performed to amplify genome sequence of TTSuV and PCV2. For serum sample of SH0822 from a clinical case with PMWS, the products of MPRCA were digested using EcoR I, Xba I, Sma I, Sac I, respectively. Moreover, Clustal W program (DNASTAR software) and MEGA 5.1 software (neighbour-joining method) was used to analysis its nucleotide homology and genetic relationship. Results: Restriction digestion analysis showed one TTSuV genome-size fragment was presented in 1.2 % agarose gel, moreover, another PCV2 genome-size fragment was also presented. Nucleotide sequencing and phylogenetic analysis results suggested that its complete genome were 2823-nucleotide size and 1767-nucleotide size and they were divided into species TTSuV1b and genotype PCV2b, respectively. Conclusion: Concurrent infection of TTSuV and PCV2 in a clinical case with PMWS was identified using MPRCA combining with restriction endonuclease digestion, which indicated that MPRCA was an effective tool to attain simultaneous detection and genome amplification of TTSuV and PCV2.

Genomic characteristics of a coxsackievirus B3 strain KM06/2009 isolated in Kunming,Yunnan,Chi-na / 中华微生物学和免疫学杂志

Objective To analyze the complete genome sequence of a coxsackievirus B 3 (CVB3) strain KM06/2009 and its genetic variation , evolution and cardiovirulence .Methods Eight clones with overlapped gene fragments covering the complete viral genome ( excluding the poly-A tail) were obtained by RT-PCR and sequenced .The nucleotide ( nt ) and amino acid ( aa ) sequences of the eight clones were aligned with sequences of other known CVB 3 clinical strains .Phylogenetic and pairwise alignment analyses based on the genome and complete VP 1 regions were conducted by using Mega 4.1,RDP3 and SimPlot3.5.1 softwares.The RNA secondary structure of CVB3 stem loopⅡ (SLⅡ) was determined by using Mfold web server.Results The complete genome of CVB3 strain KM06/2009 was 7401 nt in length, consisting of 743 nt and 100 nt on 5′untranslated region (UTR) and 3′UTR,respectively.The entire open reading frame contained 6558 nt, encoding a 2185 aa polyprotein.There was no nucleotide deletion or insertion in the coding region,but some changes of amino acid were unique .KM06/2009 strain showed 81.4%and 95.7%identities in nucleotide and amino acid sequences respectively as compared with CVB 3 prototype Nancy strain.It shared 88.4%-98.1%nucleotide and 98.1%-99.4%amino acid homology with the other Chinese clinical strains isolated at the same period .KM06/2009 strain and CVB3 GA strain without cardiovirulence shared 80.7%homology in nucleotide and 96.4% in amino acid.The phylogenetic analysis indicated that the significant spatial and temporal clustering was detected in CVB 3 isolate.CVB3 KM06/2009 strain showed a strong cardiovirulence tendency as indicated by the RNA secondary structure of CVB 3 SL Ⅱ. Conclusion CVB3 KM06/2009 isolate showed a strong cardiovirulence tendency in comparison with other CVB3 clinical isolates based on the bioinformatics analysis .

Characterization of complete genome sequences of human coronavirus NL63 strains derived from Chinese patients / 中华微生物学和免疫学杂志

Objective To sequence and analyze the complete genome of two human coronavirus NL63 (HCoV-NL63) strains collected from Beijing Children Hospital .Methods Eighteen pairs of primers were designed according to the gene sequences of HCoV-NL63 reference strain ( HCoV-NL63_Amsterdam 1) and used to amplify the target fragments covering the complete genome of HCoV-NL63 strains.Rapid ampli-fication of cDNA ends ( RACE) and RT-PCR assays were used to amplify the full length genome of HCoV-NL63 strains.Phylogenetic analysis was conducted by using Mega 5.0 software.Results The complete ge-nome sequences of the two HCoV-NL63 strains were 27 538 bp in length, showing a homology of 99.1%in nucleotide sequences .There were 15 consecutive bases deleted from 1a region.The systematic phylogenetic analysis demonstrated that four genotypes of NL 63 virus including A , B, C and D have been identified , and two domestic strains were belonged to the new genotype D .Conclusion The complete genome sequences of two domestic HCoV-NL63 isolates were identified for the first time .This study provided evidence for further investigation on molecular epidemiology of HCoV-NL63 in China .