Genetic characteristics of coxsackievirus A16 isolated in Shenzhen from 2016 to 2017 / 中华微生物学和免疫学杂志

Objective@#To investigate the genetic characteristics of VP1 genes carried by coxsackievirus A16 strains isolated from cases of hand foot and mouth disease (HFMD) in Shenzhen during 2016 to 2017.@*Methods@#Fecal and anal swab specimens were collected from patients with mild HFMD in four sentinel hospitals and the Institute of Pathogen Biology, Shenzhen Center for Disease Control and Prevention, China during 2016 to 2017. All specimens were tested for CVA16 viral RNA using real-time RT-PCR. The VP1 genes of 51 randomly selected CVA16 strains were amplified by RT-PCR and then sequenced using TaKaRa Biomedical Technology (Dalian). Bioinformatics software, including Mega6.02, BioEdit and DNAStar, was used for comparison and analysis of the VP1 genes.@*Results@#CVA16 strains in Shenzhen during 2016 to 2017 mainly belonged to B1a and B1b subtypes as well as an emerging subtype B3. The epidemic of B1b subtype was found in both 2016 (28 strains) and 2017 (19 strains), while the B1a subtype (two strains) was only detected in 2017. Two B3 subtype strains were detected in 2017. The strains of B1b subtype were closely related to the strains isolated in Shanghai (JQ314149), Wenzhou (KP289416) and Beijing (KU254598), while the B1a subtype strains were closely related to the strains isolated in Kunming (JQ316639) and Tailand (GQ184139). The B3 subtype strain was an emerging CVA16 epidemic strain in mainland China. Further comparison of the CVA16 epidemic strains in Shenzhen area during 2016 to 2017 with the CVA16 strains causing severe neurological symptoms showed that two amino acid mutations (S14N and M23L) were found in VP1 protein.@*Conclusions@#The epidemic strains of CVA16 were B1b subtype in Shenzhen area in 2016. However, B1a, B1b and the emerging B3 subtype strains were prevalent in 2017. Compared with the CVA16 strains causing severe neurological symptoms, the CVA16 strains circulating in Shenzhen during 2016 to 2017 carried two amino acid mutations inVP1 protein.

Genetic characteristics of coxsackievirus A16 isolated in Shenzhen from 2016 to 2017 / 中华微生物学和免疫学杂志

Objective To investigate the genetic characteristics of VP1 genes carried by coxsack-ievirus A16 strains isolated from cases of hand foot and mouth disease ( HFMD) in Shenzhen during 2016 to 2017. Methods Fecal and anal swab specimens were collected from patients with mild HFMD in four senti-nel hospitals and the Institute of Pathogen Biology, Shenzhen Center for Disease Control and Prevention, China during 2016 to 2017. All specimens were tested for CVA16 viral RNA using real-time RT-PCR. The VP1 genes of 51 randomly selected CVA16 strains were amplified by RT-PCR and then sequenced using TaKaRa Biomedical Technology ( Dalian). Bioinformatics software, including Mega6. 02, BioEdit and DNAStar, was used for comparison and analysis of the VP1 genes. Results CVA16 strains in Shenzhen during 2016 to 2017 mainly belonged to B1a and B1b subtypes as well as an emerging subtype B3. The epi-demic of B1b subtype was found in both 2016 (28 strains) and 2017 (19 strains), while the B1a subtype ( two strains) was only detected in 2017. Two B3 subtype strains were detected in 2017. The strains of B1b subtype were closely related to the strains isolated in Shanghai ( JQ314149 ) , Wenzhou ( KP289416 ) and Beijing (KU254598), while the B1a subtype strains were closely related to the strains isolated in Kunming (JQ316639) and Tailand (GQ184139). The B3 subtype strain was an emerging CVA16 epidemic strain in mainland China. Further comparison of the CVA16 epidemic strains in Shenzhen area during 2016 to 2017 with the CVA16 strains causing severe neurological symptoms showed that two amino acid mutations ( S14N and M23L) were found in VP1 protein. Conclusions The epidemic strains of CVA16 were B1b subtype in Shenzhen area in 2016. However, B1a, B1b and the emerging B3 subtype strains were prevalent in 2017. Compared with the CVA16 strains causing severe neurological symptoms, the CVA16 strains circulating in Shenzhen during 2016 to 2017 carried two amino acid mutations inVP1 protein.

Sequence analysis of VP1-VP4 genes of coxsackievirus A6 strains isolated from children with severe hand, foot, and mouth disease in Shenzhen from 2012 to 2015 / 中华微生物学和免疫学杂志

Objective To analyze the genetic characteristics of VP1-VP4 genes carried by cox-sackievirus A6 (CVA6) strains isolated from severe cases of hand, foot, and mouth disease (HFMD) in Shenzhen during 2012 to 2015. -ethods The VP1-VP4 genes of CVA6 strains isolated from severe HFMD cases in Shenzhen during 2012 to 2015 were amplified and sequenced. Phylogenetic analysis was performed to analyze the VP1-VP4 genes of CVA6 isolates and sequences downloaded from GenBank by using DNASTAR6. 0 and MEGA6. 02 software packages. Results Four cases of severe HFMD were caused by CVA6 in Shenzhen during 2012 to 2015. All of the patients had the symptom of fever, skin rash and aseptic encephalitis. The CVA6 strain causing severe HFMD in 2013 shared 98. 8％-98. 9％ homology in nucleotide sequences and 99. 3％-99. 8％ in amino acid sequences with the strains isolated in 2012. Two amino acid mutations were found in the CVA6 strain isolated in 2013, which were G73E in VP2 region and S13G in VP1 region. However, the CVA6 strain isolated in 2015 only shared 95. 0％ homology in nucleotide sequences and 99. 3％ homology in amino acid sequences with the strain isolated in 2013. Six amino acid mutations were identified including E73G in VP2 region and T5A, S27N, A30V, N137S and V242I in VP1 region. The phylogenetic analysis revealed that the four CVA6 strains belong to D3 sub-genotype. The CVA6 strains causing severe cases in 2012 had the nearest genetic relationship with the strain isolated in Changsha in 2012 (KJ156349). The CVA6 strain isolated in Shenzhen in 2013 had the nearest genetic relationship with the strain isolated in Shanghai in 2013 (KJ612513). The Shenzhen CVA6 isolate in 2015 showed high similarity to Weifang CVA6 isolate in 2014 (KX752785). Conclusions All CVA6 strains causing severe HFMD ca-ses in Shenzhen during 2012 to 2015 belongs to D3 sub-genotype. Mutations of S27N and A30V in the VP1 region of the CVA6 isolate in 2015 are located in the B cell epitopes. In addition, the VP1-V242I mutation in the CVA6 strain isolated in 2015 is located in the binding site of PSGL-1 receptor. These mutations may affect the binding of CVA6 strains to the cellular receptors and their infectivity to people.

Analysis on the epidemiology and etiologic characteristics of first imported severe fever with thrombocytopenia syndrome case in Shenzhen / 中华实验和临床病毒学杂志

Objective@#To study the epidemiology and the etiology characteristics of first imported severe fever with thrombocytopenia syndrome (SFTS) case reported in Shenzhen city in 2017.@*Methods@#Data on descriptive epidemiology was collected to study the characteristics to the epidemic. The serum sample collected from the suspect SFTS case was detected for IgM, IgG by ELISA and severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) nucleic acid by real-time RT-PCR. The samples were further inoculated in Vero cell for virus isolation. The partial fragements of L and S gene were amplified by RT-PCR and sequenced to construct homology comparison and phylogenetic tree with the strains isolated from other areas.@*Results@#The case was laboratory confirmed imported SFTS case in Shenzhen on May 2017. IgM antibody and RNA of SFTSV were detected in the serum sample. SFTSV named GDSZ01/2017/China was successfully isolated from the serum sample. The high nucleotide homology of L and S genome segments were found at 95.3%-98.2% and 93.8%-98.8% with other representative strains from the popular provinces, respectively. The phylogenetic tree indicated that GDSZ01 was most close to SDTA_3 strain, next to strains in Hubei procince. The isolated SFTSV belonged to genotype C3 with HB29, HB154.@*Conclusions@#The virological, serological and molecular features showed that the imported case of SFTS in 2017 was caused by SFTSV C3 genotype.

Role of Toll-like receptor 7 in the production of inflammatory cytokines in EV-A71-infected human Jurkat T cells / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the expression of Toll-like receptor (TLR) mRNA in enterovirus 71(EV-A71) infected human Jurkat T cells and clarify the role of TLRs in the pathogenesis of EV-A71 infection-induced inflammation.</p><p><b>METHODS</b>EV-A71 strains were isolated from feces of children patients with hand, foot and mouth disease in 2014 by Shenzhen Center for Disease Control and Prevention. Human Jurkat T cells were infected with 200 μl EV-A71 at 10(3) cell culture infective dose 50%(CCID50)/ml. The expression of TLR1-TLR10 mRNA in human Jurkat T cells was assessed at different exposure time by RT-PCR. Levels of TLR7 mRNA expression were detected by real-time PCR, and levels of myeloid differentiation factor 88 (MyD88) by western blot. The cytokine secretion of interleukin (IL)-6, IL-8 and Tumor Necrosis Factor α (TNF-α) was analyzed by ELISA assay.</p><p><b>RESULTS</b>The relative expression level of TLR7 mRNA in human Jurkat T cells were 1.26 ± 0.15, 1.75 ± 0.20, 2.26 ± 0.23 and 3.74 ± 0.62 in 6, 12, 24 and 48 h after EV-A71 infection, which the differences were significant with mock-infected group(t values were -2.96, -6.38, -9.57, -7.71; P<0.05). Western blot showed that the protein expression levels of MyD88 had increased 1.34 times and 2.17 times in 24 h and 48 h after EV-A71 infection compared with mock-infected group. After infected for 24 h and 48 h, the levels of IL-6 were (302.86 ± 38.11), (179.70 ± 14.50) pg/ml, which were significantly higher than mock-infected group (176.42 ± 9.60), (179.70 ± 14.50) pg/ml (t values were -5.57, -18.54, P<0.05). The levels of TNF-α in EV-A71 infected group (100.81 ± 9.81) pg/ml was higher than that in mock-infected group (56.19 ± 6.94) pg/ml, and the difference was significant (t=-6.43, P=0.003).</p><p><b>CONCLUSION</b>TLR7 is the main pattern recognition receptor responsible for EV-A71 recognition in immune cells, which then leads to the activation of TLR7 downstream signaling and the production of proinflammatory cytokines.</p>

Sequence analysis of VP1-VP4 genes of enterovirus 71 strains isolated from children with severe or mild hand, foot and mouth disease in Shenzhen, China in 2012 / 中华微生物学和免疫学杂志

Objective To analyze the VP1-VP4 genetic region of enterovirus 71 ( EV71 ) strains isolated from children with severe or mild hand, foot and mouth disease ( HFMD) in Shenzhen in 2012. Methods EV71 strains were isolated from five children with mild HFMD and five children with severe HFMD in Shenzhen in 2012.Reverse transcription-polymerase chain reaction ( RT-PCR) method was used to amplify the sequence of VP1-VP4 genes of EV71 strains.The sequences of the amplified products were analyzed by comparing with those of the EV71 reference strains ( A, B and C genotypes) published in Gen-Bank using nucleotide alignment, amino acid alignment and phylogenetic tree analysis.Results The homo-geneity between the EV71 strains isolated from severe and mild cases was 95.1%-98.2% in nucleotides and 99.2%-100% in amino acids.The VP1-VP4 nucleotide sequences of 5 strains isolated from severe cases and 5 strains from mild cases in Shenzhen shared 87.9%-97.8% homologies in nucleotides and 97.3%-99.9% homologies in amino acids with the genotype C EV71 reference strain.The EV71 strains isolated from children in Shenzhen were highly similar with the EV71 strain (FJ439769) isolated in Fuyang in 2008 and the one isolated in Jingdezhen in 2011 (JQ806378, C4a subtype) in nucleotide sequences.Mutations at the residue 31 in the VP1 region ( N→D ) were detected in 3 strains isolated from children with severe HFMD.Conclusion All of the 10 EV71 strains isolated in Shenzhen in 2012 belonged to the sub-genotype C4a.The mutation ( aa31 N→D) in the VP1 region of EV71 might be related to the different clinical mani-festations of HFMD cases in Shenzhen area.

Analysis of the complete genome sequence of a Shenzhen coxsackievirus A2 strain SHZH13-01 / 中华微生物学和免疫学杂志

Objective To analyze the complete genome sequence of a Shenzhen coxsackievirus A2 strain CVA2-SHZH13-01 and its evolution.Methods RT-PCR was used to amplify the complete genome of CVA2-SHZH13-01 strain.The PCR products were purified and sequenced to analyze their genetic character-istics.Results The complete genome of CVA2-SHZH13-01 strain was 7400 bp in length, encoding 2191 amino acids.CVA2-SHZH13-01 strain was highly similar with the novel recombinant CVA2-HK (431306) strain isolated from Hong Kong sharing the nucleotide homology of 98.3%, 98.8%, 99.0%, 99.2%, 98.8%and 98.9%in 5′UTR, P1 ( VP1 to VP4) , P2, P3, 3′UTR regions and complete genome, respec-tively.CVA2-SHZH13-01 strain was highly identical to the international standard strain CVA2-Fleetwood showing the homology of 81.6% in nucleotide sequences in P1 region, but closely associated with EV71-SHZH03 and EV71-GD2009 strains (82.8%-88.7%) in P2 and P3 regions.The phylogenetic analysis in-dicated that CVA2-SHZH13-01 strain belonged to the CVA2-HK (431306) variant.Data from analysis of amino acid in P1 region showed that there were three amino acid mutations in CVA2-SHZH13-01 strain including aa5L→F, aa666S→G and aa671T→I as compared with CVA2-HK (431306) strain.Conclusion CVA2-SHZH13-01 strain belonged to CVA2-HK (431306) variant.

Analysis of the gene characteristic correspondence on rats with Hantavirus and the human infected Hantavirus in natural epidemic areas of HFRS in Shenzhen / 中华微生物学和免疫学杂志

Objective To compare and contrast the gene characteristic correspondence of hantaviruses(HV) carried by rats in natural epidemic areas of hemorrhagic fever with renal syndrome(HFRS) and infected among HFRS patients in Shenzhen,provide a reasonable scientific basis for controlling of HFRS.Methods We collected the patients serum specimens in acute stage and lung tissues samples of rats.ElISA and direct immunofluorescence were applied to screen the positive samples,respectively.The partial G2 fragments of M segment and S segment were amplified from the representative patients' serum positivesamples and lung tissues positive samples in different areas with reverse transcription-nested-polymerase chain reaction(RT-nested PCR) by the hantaviruses genotype specific primers.The amplified genes were then sequenced,and subjected to genotyping and homology analysis with other known hantaviruses.Results Four hundred and seventy-two rats were trapped in the main epidemic areas,and Hantavirus specific-antigens in lung tissues samples were identified in 47 out of the 472 by direct immunofluorescence.Twelve partial M and S segments were amplified from 60 patients serum specimens positive with specific IgM antibodies against hantavirus with ELISA by RT-nested PCR.The homology of M and S genome segments among 16 strains of Hantaviruses showed more than 95% and 96.5% at nucleotide level,respectively.And the deduced amino acid sequences homology was 98.0% -100% and 98.4%-100%,respectively.The genotype of hantavirus carried by rats and infected among patients were identified to the same subtype-SEO S2.Conclusion The genotype of Hantavirus carried by rats and infected among patients in Shenzhen all belongs to S2 SEOV.The nucleotide homology of SEO type of Hantavirus in the same or nearby areas is higher and the viruses are highly conserved.

Sequence determination and phylogenetic tree analysis of the E gene of dengue virus type 4 isolated from a patient in Shenzhen / 中国人兽共患病学报

To identify the genotype and analyze the molecular characteristics of dengue virus strain SZ0524 isolated from serum samples of patients with early stage of dengue fever in Shenzhen in 2005 so as to explore its possible origin. The C6/36 cell line was cultivated with virus strain SZ0524 and its suspension was harvested. The type of isolated virus strain was determined by RT-semi-nested PCR and fluorescent PCR. E gene of isolated virus strain was amplified by RT-PCR and sequenced. Homology and phylogenetic tree of E gene of this dengue virus with the strains isolated from other areas were constructed. This SZ0524 strain was further identified by fluorescent PCR, and confirmed to be the type 4 virus after obtaining the 392bp band with type 4 specific primers. The homology of nucleotide sequence of E gene of SZ0524 strain with the standard type 4 dengue virus H241 strain were 99.7%, but the homology with the standard dengue virus 1,2,3 in the same fragment were 57.0%, 59.2% and 56.2% respectively. Analysis of the phylogenetic tree indicated that SZ0524 was more close to D4-73NIID and D4-61NIID strain, next to H241 strain, and they lied in the same branch of phylogenetic tree. The isolated dengue virus type 4 belonged to genotype Ⅰand the SZ0524 strain was proved to be dengue virus type 4 in the molecular level. Combined with epidemiology information, it is suggested that this case can be classified as an imported case and the SZ0524 strain may be transferred from the southeast asian region.