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Objective To investigate the relationship between HBV -DNA load and serum markers in chronic hepatitis B( CHB) patients in Hohhot,Inner Mongolia,and to explore the mutation of HBV genotype and nucleoside analogue.Methods From January 2015 to December 2017,one hundred and ninety-three CHB patients hospitalized in the People＇s Hospital of Inner Mongolia were selected randomly.The clinical diagnostic criteria for all admitted patients were based on the " Guidelines for the Prevention and Treatment of Chronic Hepatitis B" jointly formulated by the Infectious Diseases Society of 2010. The HBV -DNA load of HBV was detected by real -time quantitative PCR,and the correlation between HBV -DNA load and serum markers was analyzed. Seventy -nine patients were selected from 193 hospitalized patients,PCR-reverse dot blot hybridization was used to analyze HBV genotyping and the drug resistance mutations of different genotypes.Results The differences of HBeAb level and HBV-DNA load between HBeAg positive patients and negative patients were statistically significant(all P<0.001). Of 79 serum specimens of HBV infected people,9 cases(11.4% ) were B genotypes,and 70 cases of C genotype (88.6% ).Of them,25 cases had different loci variation,the rate of variation was 31.6% (25/79),with the unit point rtS213T mutation dominated,accounting for about 24.0% (6/25).Conclusion In Hohhot Inner Mongolia patients with CHB,HBV-DNA load with HBeAg and HBe Ab level are correlated;genotype in patients including B type and C type,which is mainly genotype C;patients with CHB mainly had drug resistance to lamivudine and adefovir dipivoxil, mutations including rtS213T,and hybrid mutation.
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Objective To analyze the VP1 and VP4 genetic region of enterovirus 71 (EV71)isolated from severe cases and mild cases with hanD-foot-mouth disease (HFMD) in Shanghai in 2011.Methods Five EV71 strains isolated from severe cases and five EV71 strains from mild cases in 2011 were included.Reverse transcription-polymerase chain reaction (RT-PCR) method was used to amplify and sequence the VP1 and VP4 genes of EV71,and then the sequencing results were compared with those of A,B,C genotype reference EV71 strains from GenBank by nucleotide alignment,amino acid alignment and phylogenetic tree analyses.Results The homogeneity between EV71 strains from severe cases and mild cases was 96.0％-98.1％ and 93.7％-99.5％ for VP1 and VP4 nucleotide sequences,respectively.The VP1 nucleotide sequences of 5 strains isolated from severe cases and 5 strains from mild cases in Shanghai shared 86.9％-98.2％ and 87.4％ 98.5％ identity with genotype C,respectively,while the homogeneity of VP4 nucleotide sequence was 85.5％-100.0％and 84.5％-99.5％,respectively.In addition,compared with the Fuyang EV71 strains (representative of C4 subtype),the strains from mild and severe cases shared homogeneity of 97.0％-98.2％ and 97.9％-98.5％ for VP1 gene,respectively,96.1％-100.0％ and 97.1％-99.5％ for VP4 gene,respectively.Among 3 strains isolated from severe cases,mutations at the residue 282 in the VP1 protein (N→S) and residue 7 in the VP4 protein (T→A) were discovered simultaneously.Conclusions The 10 EV71 strains isolated from severe and mild cases in Shanghai belong to subgenogroup C4.Among 3 strains isolated from severe cases,mutations at the residue 282 in the VP1 protein (N→S) and residue 7 in the VP4 protein(T→A) are discovered simultaneously.
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Objective To explore the correlation between serum hepatitis B virus (HBV) X antigen/antibody (HBxAg-wild/HBxAb-wild,and HBxAg-mutant/HBxAb-mutant) and the disease progression in patients with chronic HBV infection.Methods A direct enzyme immunosorbent asssay (ELISA) was performed to detect HBxAb using recombinant antigen,and a double antibody sandwich ELISA assay to detect HBxAg using monoclonal antibody and specific rabbit polyclonal antibody.HBxAg-wild/HBxAb-wild and HBxAg-mutant/HBxAb-mutant were tested in sera from cases at different stages of chronic HBV infection.A chi-square test was employed to examine statistical significance.Results The positive rates of HBxAg-wild and HBxAg-mutant in the chronic asymptomatic HBV carriers,chronic hepatitis,hepatitis B-related cirrhosis and liver cancer were 6.2％ (2/32),10.7％ (3/28),28.6％ (6/21),43.6％ (17/39) and 3.1％ (1/32),10.7％ (3/28),33.3％ (7/21),48.7％ (19/39),respectively.The positive rates of HBxAb-wild and HBxAb-mutant in the above mentioned groups were 6.2％ (2/32),21.4％ (6/28),38.1％ (8/21),53.8％ (21/39)and 6.2％ (2/32),25.0％ (7/28),42.9％ (9/21),61.5％ (24/39) respectively.The positive rates of HBxAg-wild and HBxAg-mutant were not significantly different among the above groups (x2 =0.871,0.780,0.565 and 0.317,respectively; all P＞0.05) ; The positive rates of HBxAb-wild and HBxAb-mutant were also similar among all the groups (x2 =0.780,0.709,0.580 and 0.210,respectively; all P＞0.05).The positive rates of HBxAg-wild,HBxAb-wild,HBxAg-mutant,HBxAb-mutant in patients with low viral loads (HBV DNA＜1 × 104 copy/mL) were 36.5％ (23/63),44.4％ (28/63),42.9％ (27/63) and 54.0％ (34/63),respectively,those in patients with high viral loads (HBVDNA≥1×104 copy/mL) were 8.8％ (5/57),15.8％ (9/57),5.3％ (3/57) and 14.0％ (8/57),respectively.The positive rates of HBxAg and HBxAb were significantly higher in cases with low viral loads than those with high viral loads (x2 =12.869,11.522,22.556 and 20.976,respectively; all P＜0.05).The positive rates of HBxAg-wild,HBxAb-wild,HBxAg-mutant,HBxAb-mutant in the HBeAg positive group were 21.7％ (18/83),30.1％ (25/83),22.9％ (19/83) and 32.5％ (27/83),respectively,while those in the HBeAg negative group were 27.0％ (10/37),32.4％ (12/37),29.7％ (11/37) and 40.5％ (15/37),respectively.No significant difference of HBxAg/HBxAb positive rates between HBeAg positive group and HBeAg negative group was noticed (x2 =0.408,0.064,0.638 and 0.722,respectively; all P＞0.05).Conclusions The antigenicity and specificity of HBV X protein remains similar after the occurrence of A1762T/G1764A double mutant in X gene.It is also found that the positive rates of HBxAg and HBxAb increase with disease progression.HBxAg/HBxAb might be promoting factors for tumorigenesis in chronic HBV infection.HBxAg and HBxAb might have negative influence on HBV replication.
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Objective To explore the relevance between sequence variation of human papillomavirus (HPV)16 subtypes E2 gene or long control region (LCR) and cervical lesions.Methods Fifty specimens from HPV16 infected people in Chengdu were collected,including cervical exfoliated cells from 38 HPV carriers,papilloma tissues from 8 cases of genital warts,2 with cervical intraepithelial neoplasia (CIN) Ⅱ and 2 with CIN Ⅲ in this study.Polymerase chain reaction was used to amplify E2 gene and LCR,then an evolutionary tree was constructed.Results In all the 50 specimens,there were 12 mutation sites in E2 gene,among which,C→A existed in one specimen of genital warts,and ≥2 mutation sites existed in all the other 48 specimens.There were 28 mutation sites of LCR sequence of all the specimens.Ten specimens were chosen to construct evolutionary tree and were sequenced.The data showed that 8 specimens were Asian variants,E2 gene mutation existed in all the specimens while the LCR 7867 G→A only existed in the four CIN.Conclusion LCR 7867G→A is a correlative mutation site of cervical lesions in Chengdu.
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ObjectiveTo analyze the Envelope (E) gene of type 1,2,3 dengue virus isolated fromGuangzhouin2010, andtoinvestigatetheinfectionsourceandvirusgenotypes.MethodsEighty-five serum samples were collected from 85 patients in acute phase of dengue fever.Dengue virus was cultured and isolated by C6/36 cells.The whole length of E gene was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and then sequenced.The phylogenetic tree was drawn by neighbor-joining method.The bioinformatics analysis was performed by combining the phylogenetic information and the epidemiologic data.ResultsSix strains of type 1 dengue virus,two strains of type 2 dengue virus and six strains of type 3 dengue virus were isolated from 85 samples.The E gene sequence of these strains was obtained by sequencing.The phylogenetic analysis showed that type 1 and 3 dengue virus belonged to two genotypes (Asian and South Pacific ocean,India subcontinent and Southeast Asia/South Pacific ocean,respectively),and type 2 dengue virus belonged to one genotype (Malaysia/India subcontinent).ConclusionIt's presumed that all strains of type 2 dengue virus are imported,four strains of type 1 dengue virus are imported and four strains of type 3 dengue virus arc imported,the remaining two stains of type 1 and two stains of type 3 dengue virus need mosquito intermediary research further to prove their origins.
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Objective To investigate the genotype distribution of hepatitis C virus (HCV) in Guangdong Province by linear probe assay (LiPA) and core,non-structural protein 5B (NS5B) sequence analysis,and to evaluate the accuracy of LiPA for genotyping.MethodsOne hundred and ten HCV specimens from chronic hepatitis C (CHC) patients in Guangdong Province were genotyped by both core,NS5B sequence analysis and INNO-LiPA 2.0.The data were then analyzed with SPSS 10.0 software.ResultsAmong the 110 specimens,core and NS5B fragment could be amplified from 97 and 62 specimens,respectively,while both fragments were obtained from 57 specimens. The results of genotyping by core sequence analysis were completely consistent with the results of NS5B sequencing.One hundred and two specimens were classified into five subtypes,i.e.genotype 1a,2a,3a,3b,6a,with frequencies of 61.8％ (64),9.8％ (10),3.9％ (4),3.9％ (4),20.6％ (21),respectively.All but genotype 6 strains can be genotyped correctly by using INNO-LiPA 2.0.However,only subtype 1b and 3b could be genotyped accurately.81.5％ genotype 6a strains were genotyped as 1b by mistake. Conclusions Genotype 6a has become the second most prevalent genotype in Guangdong Province.The LiPA cannot distinguish genotype 6a from 1b strains accurately which needs further investigation.
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Objective To determine the pathogen of a local dengue fever outbreak in Shenzhen city in 2010,and to analyze the molecular characteristics of the epidemic dengue virus strain as well as explore the possible origin.Methods The serum samples collected from the suspect dengue fever cases were detected for IgM, IgG by enzyme-linked immunosorbent assay ( ELISA ),immunochromatography and dengue virus nucleic acid by real-time polymerase chain reaction (PCR).Serum samples from patients with early stage dengue fever were used to isolate virus with C6/36 and BHK-21 cell lines.The type of isolated virus strain was determined by RT-semi-nested-PCR and realtime PCR.E gene of isolated virus strain was amplified by RT-PCR and sequenced.Homology and phylogenetic tree of E gene of Shenzhen dengue virus with the strains isolated from other areas were constructed.Results IgM,IgG and RNA of type 1 dengue virus were detected in serum samples from dengue fever suspected patients.Type 1 dengue virus named DEV1-SZ1029 was successfully isolated from the serum sample.The homology of nucleotide sequence of E gene of SZ1029 strain with standard type 1 dengue virus HAWAII 45,Fj231/04,GD14/97 and GD05/99 were 94.8％,99.6％,97.7％ and 98.5 ％,respectively.The phylogenetic tree indicated that SZ1029 had the greatest similarity with the D1/Malaysia/36000/05 strain,SG(EHI)DED142808 strain and Fj231/04 strain and they lied in the same branch of the phylogenetic tree.The isolated dengue virus type 1 belonged to genetype Ⅰ with GZ/80,Taiwan87,All patients lived in a certain construction site in Shenzhen and had no recent travel history outside the area in one month before infection.Conclusions The virological,serological and molecular features all identify that the local dengue fever outbreak in Shenzhen in 2010 is caused by type 1 dengue virus and SZ1029 strain may be transferred from Southeast Asian region,and there may be a plague focus in Shenzhen.
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Objective To construct the eukaryotic vector that expressing hepatitis B virus (HBV) S and the single fragment of variety chain (ScFv) of monoclonal antiboy against cytotoxic Tlymphocyte-associated antigen-4 (CTLA-4) and to analyze the immunological activity of recombinant S-ScFv protein.Methods The oringially constructed pSect2/ScFv4F10 and pSect2/S were double enzyme digested by Sfi I and Hind Ⅲ,respectively.Then the HBV S gene was cloned into the pSect2/ScFv4F10 vector.The pSect2/ScFv4F10 and pSect2/S-ScFv4F10 were expressed in Chinese hamster ovary (CHO) cells,and the expressed proteins were verified through sodium dodecyl sulfatepolyacrylamide gelelectrophoresis(SDS-PAGE)andWesternblotting.Afterultrafiltration concentration and affinity chromatography,the biological affinity of the expressed ScFv4F10 and S-ScFv4F10 proteins were examined by competitive enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) technology.The comparison between groups was done by One-way ANOVA.ResultsThe eukaryotic expression vector of pSect2/S-ScFv4F10was successfully constructed,and relative molecular mass of the expressed protein of S-ScFv4 F10 was about 52 000 that analyzed by SDS-PAGE and Western blotting.With the fixed concentration of 4F10-mAb against CTLA-4,the A570 value of the mixed reaction with purified CTLA-4 antigen gradually increased with the decrease of ScFc fusion protein proportion; when the molar ratio of ScFv,S-ScFv4F10∶4F10=2∶1,the competitiveinhibitionratesagainst 4F10conjugatedantigenwere72.6％and64.5％,respectively.The affinity constants of association kinetics for CTLA-4 mAb,ScFv4F10 and S-ScFv4F10 with CTLA-4 antigen were 7.29 × 108 mol/L,9.52 × 106 mol/L and 2.04 × 106 mol/L,respectively,and the dissociation constants of KD were 1.40 × 10-9 mol/L,1.05 × 10-7 mol/L and 4.91 × 10-7 mol/L,respectively.ConclusionsThe eukaryotic expression vector of pSect2/S-ScFv4F10is successfully constructed,and the recombinant protein of S-ScFv4 F10 has a fairly high affinity with CTLA-4 antigen.
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Objective To study the impact of ribavirin cumulative dose on virological response rates in genotype 1 hepatitis C virus(HCV)infected patients.Methods The medical records of 225 genotype 1 chronic hepatitis C(CHC)patients treated with peginterferon α-2a plus ribavirin were retrospectively analyzed.These patients were divided into four groups according to ribavirin cumulative dose:>97%,80%-97%,60%-79%and97%(65.6%,84/128),80%-97%(60.5%,26/43),60%-79%(58.3%,21/36)(x2=9.538,P=0.023).The relapse rate was 61.5%(8/13)in group of ribavirin cumulative dose97%(20.0%,21/105),80%-97%(23.5% ,8/34),60%-79%(27.6%,8/29)(x2=10.837,P-0.013).Among patients achieved rapid virological response(RVR),SVR in groups of ribavirin cumulative dose>97%,80%-97%,60%-79%and<60 % of standard dose were 92.0%(23/25),88.9%(8/9),85.7%(6/7)and 75.0%(3/4),respectively(x2=1.098,P=0.778).Conclusiom Mlid reduction of ribavirin dose not affect SVR of genotype 1 HCV infected patients.However,the relapse rate is high and SVR is low in patients treated with ribavirin cumulative dose<60% of standard dose.
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ObjectiveTo understand the detections of influenza A (H1N1) in 2009,and haemagglutinin (HA) gene mutations and the comparisons with standard strains.MethodsThe nasopharyngeal swabs from patients with influenza-like illness (ILI) in National Influenza Sentinel Surveillance Hospital and the outbreak epidemic area were collected.The virus typing and A (H1N1) viruses were tested by real time-polymerase chain reaction (RT-PCR).Then the pathogens were isolated with MDCK cells,the virus titer was determined with hemagglutination test and the virus typing was identified with hemagglutination inhibition test (HA1).The RT-PCR products of HA1 gene of virulent strains were sequenced and then analyzed through bioinformatics.Results A total of 996 pharyngeal swab specimens were tested,and nucleic acid positive cases included 337 A (H1N1) subtype,1 seasonal A (H1N1) subtype,67 A (H3N2) subtype,and 12 B type.The positive rate of nucleic acid detection of influenza was 41.87％ and that of A (H1N1) was 33.84％.Thirty-six influenza A (H1N1) virus strains were isolated,and 10 of them were successfully sequenced and several amino acid mutations were identified.There were 6 amino acid mutations found compared with vaccine strain A/California/07/2009 (H1N1),and 1 site was in area B of epitope.Conclusions A (H1N1) is absolute predominant among isolated strains in 2009.HA gene of virulent strains is mutated compared with vaccine strain provided by World Health Organization,which shows that the area B of epitope changes,while the key amino acid position 222 doesn't change.
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Objective To understand epidemic characteristics of human influenza A and the genetic and antigenic variations of H1N1 influenza A isolates in Shanghai area in 2009. Methods Throat swabs were collected from patients with influenza-like illness in the sentinel surveillance clinic in Shanghai area in 2009, then inoculated in Madin-Darby canine kidney (MDCK) cell lines. The types of influenza were identified by direct immunofluorescence assay (DIF) and the subtypes were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Segments of hemagglutinin (HA) and neuraminidase (NA) genes of some 2009 H1N1 influenza A isolates were amplified and sequenced. HA and NA gene mutations of 2009 H1N1 influenza A isolates were analyzed. Results Seasonal H1N1 and H3N2 influenza A viruses co-circulated during the spring of 2009 in Shanghai area. Seasonal H3N2 began to co-circulate with 2009 H1N1 in August (the 32nd week) and finally2009 H1N1 became dominate since the 40th week. The phylogenetic tree of 2009 H1N1 HA segment revealed that the isolates from different regions and months were interspersed with each other, but all were clustered into one branch which closed to strains in Spain, Russia, Denmark and other European countries. Mutations were found in some HA amino acid sites, but none of them was in the antigenic determinant region. No change was observed in the 274 NA amino acid residues which were related to the drug resistance to oseltamivir. PB2 protein analysis showed that the 627 and 701 amino acid residues were glutamic acid and aspartic acid respectively, which were the same encoded amino acid with avian flu PB2 protein. Conclusions Seasonal H1N1 and H3N2 co-circulated in the spring of 2009, then both 2009 H1N1 and seasonal H3N2 were prevalent in Summer and Autumn, and 2009 H1N1 finally became dominate in Autumn. Compared to early 2009 H1N1 strains, variations are detected in H1N1 influenza A viruses, but none of them has epidemiological influence, and viruses still show high affinity with human and low-pathogenic characteristics.
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Objective To understand the structures and functions of enterovirus 71 (EV71)VP1 gene and its encoded protein using bioinformatics method, so as to direct studies of its biological function. Methods VP1 gene and its encoded protein of EV71 2008-GZCH07 strain and other representative EV71 strains were analyzed by online analysis at bioinformatics websites and software packages. Multi-sequence homological alignment and phylogenetic analysis were performed.Physicochemical characteristics, secondary structure, homology modeling of tertiary structure,enzymological characteristics, antigenic epitope of VP1 gene encoded protein were predicted. Results The homology of EV71 2008-GZCH07 strain was highest (97% and 98%) with ZJ001 strain, and lowest with human coxsackievirus A16. The homology of EV71 2008-GZCH07 strain and EV71 types A,B,C was 86%-98%. Phylogenetic analysis demonstrated that 2008-GZCH07 stain was close to ZJ001 and BJ08-Z025-5 stains, which belonged to C4 subtype. In VP1 encoded proteins of EV71 types A,B,C, the relationship between 2008-GZCH07 and EV71-B, EV71-C was closer than EV71-A.The whole length of VP1 gene was 510 bp, with open reading frame (ORF) located at 116-510 bp region,and it encoded 132 amino acids with isoelectric point of 4.39.The protein was rich of a-helix and random coilon without transmembrane regions, and contained 5 high hydrophobic regions and belonged to extracellular protein. The homology modeling of tertiary structure showed that the region was on the surface of protein and formed a binding loop. There was 5 antigen epitopes. And 7 key catalytic sites were located at or close to the loop. Conclusions EV71-VP1 encoded protein contains many phosphorylation sites, with many biological function sites and antigenic epitope regions, which might be a potential target antigen for immunodiagnosis, anti-schistosome drug and vaccine development, and would be basis of further study of diagnosis, treatment and prevention of EV71 infection.
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Objective To detect and analyze the haemagglutinin (HA) gene of the first influenza A-H1N1 viral strain isolated in Guangdong Province during an influenza A pandemic in 2009.Methods A-H1N1 virus strain was isolated from the throat swab of the first patient diagnosed with A-H1N1 virus infection in Guangdong Province in 2009. Viral nucleonic acid was extracted from supernatant of cell culture and amplified using reverse transcriptase-polymerase chain reaction (RT-PCR) with HA gene-specific primers. The product was cloned, sequenced, and the homology was analyzed. Results A 1710 bp HA gene of the first influenza A-H1N1 viral strain in Guangdong Province in 2009 was acquired, which was named as A/GuangzhouSB/01/2009 (H1N1) HA with GenBank access No. GQ268003. The homology of the studied HA gene and the 277 influenza A (H1N1) isolates reported in the epidemic areas was 99.0%-99.8%, and as high as 99.8% when compared with the isolates reported in the United States where the patient had traveled. When the studied HA gene was compared with 25 isolates of Chinese seasonal A-H1N1 virus, the homology was 72.3%-85.6%. Conclusions The homology of the first isolated A-H1N1 viral strain in Guangdong Province in 2009 and epidemic influenza A-H1N1 virus is high, while it is low compared with Chinese seasonal A-H1N1 virus.
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Objective To understand the characteristics of molecular epidemiology of enterovirus 71(EV71) in children with hand, foot and mouth disease (HFMD) in Shanghai area during the first half year of 2009. Methods Seventy-three throat swabs and 38 stool samples were collected from 95 hospitalized children with clinical diagnosis of HFMD in Children's Hospital of Fudan University during April to May 2009. TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR) and nest RT-PCR were used to detect EV71 VP1, followed by gene sequencing analysis. Results Six of the 73 throat swabs were EV71 positive with the detection rate of 8.2%. In the 38 stool samples, 24 were EV71 positive with the detection rate of 63.2%. Twenty-eight nested RT-PCR positive samples were sequenced and the genetic analysis showed that 27 were C4 subtype,which were absolute dominant strain and the other one was C2 subtype. The isolated strain from a fatal case was C4 subtype and there was no obvious mutation found in VP1 region. Conclusions EV71 is an important pathogen in HFMD children in Shanghai area during April to May 2009. C4 subtype strains are absolutely dominant, and accompanied by epidemic strains of subtype C2.
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Objective To understand the nucleotide and amino acid differences of glycoprotein gene (G gene) between isolated rabies viruses in Henan Province and rabies vaccine strains used for human and animals. Methods G gene sequences of nine rabies viruses isolated from dogs in Xinyang city of Henan Province in December 2006 were amplified by reverse transcriptase (RT)-heminestedpolymerase chain reaction (PCR), and then were cloned and sequenced. The phylogenetic trees were constructed for analyzing the genetic characteristics of these rabies viruses. Results The homology of G gene among the nine isolates from Henan Province was 97.6% - 98.9% at nucleotide level and 99.2%-99.8% at amino acid level. The similarities between these isolates and CTN vaccine strain were 85.6%-93.0% and 91.9%-92.9% at nucleotide and amino acid level, respectively, which were higher than those between these isolates and other vaccine strains (80.4% - 83.3% and 87.7% - 92.5% at nucleotide and amino acid level, respectively). The nine isolates had several amino acid substitutions when compared to other genotype 1 rabies virus strains. Conclusions The nine rabies viruses strains isolated from Henan Province all belong to genotype 1. CTN may be an effective vaccine for preventing rabies in Henan Province.
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Objective To sequence and analyze the envelope (E) gene of type Ⅰ dengue virus isolated from Guangzhou in 2009 for tracing the infection source. Methods The serum samples were collected from patients diagnosed with dengue fever in Guangzhou area during 2009. Dengue virus was isolated and cultured in C6/36 cells.The whole length of E gene was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and then sequenced. The phylogenetic tree was drawn by neighbor-joining method. The bioinformatics analysis was performed by combining the phylogenetic information and the epidemiology data. Results Four strains of type Ⅰ dengue virus were isolated from 19 samples. E gene of these strains was amplified and sequenced. The phylogenetic analysis showed that 09/GZ/9104 strain and 09/GZ/9236 strain had identical nucleotide sequence and fell within the American/African group, 09/GZ/11534 stain and 09/GZ/11562 strain had similar sequence homology and fell within the Asian group. Conclusion The typeⅠdengue viruses in Guangzhou area in 2009 are imported, which belong to two genotypes and may come from two independent origins respectively.
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Objective To construct drug-resistant variant recombinant human cytomegalovirus (rHCMV)and identify drug susceptibility by phenotypic and genotypic assays.Methods The UL97 fragments containing Pine I recongnition site and resistant mutation were introduced by site-directed mutagenesis using gene splicing by overlap extension polymerase chain reaction(PCR),and blended with human cytomegalo-virus(HCMV)standard strain ADl 69 genomic DNA proportionally,then the DNA mixture were transfected into MRC-5 fibroblasts by the vector of liposomes.HCMV-PP65 antigen was detected by indirect-immunofluorescent assay to verify rHCMV infection of MRC-5 fibroblasts.When the eytopathic effect(CPE)of homologous recombinant virus reached 100%,the virus was harvested.The purified target virus was screened by plague with different concentrations of ganciclovir(GCV)and the recombinant virus was identified by plague reduction test and DNA sequencing of drug-resistant genes(UL97 and UL54).Results The UL97 fragments containing intended mutations for transfection were constructed successfully.After cotransfected with AD169DNA mixture for 7 days,rHCMV formed cytopathology was obviously visible,which was verified as rHCMV infected focus by HCMV-PP65 antigen test.The UL97 genotypic analysis of recombinant virus obtained by cloning was as expected.No mutation was found by UL54 gene sequencing.The GCV susceptibility of rHCMV positive clone was 15 μmol/L (50% inhibiting concentration),which was 12-fold of standard AD169 strain and was drug-resistant phenotype.Conclusions The rHCMV containing intended mutations is constructed successfully by cotransfeetion into MRC-5 cells and the recombinant virus strain is obtained by GCV screening and plaque purifying.The establishment of this method provides technique platform for identifications of new drug-resistant mutations of HCMV during anti-viral therapy.
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Objective To establish a screening system of efficient small interfering RNA (siRNA) target sites directed against truncated region of human cytomegalovirus (HCMV) UL54 gene (UL54S) with siRNA expression vectors. Methods Two small hairpin RNA (shRNA) expression vectors targeting truncated region of HCMV UL54 gene were constructed based on pAVU6 + 27 vector, and cotransfected into AD293 cells with the fusion protein expression vectors pUL54S-enhanced green fluorescent protein (EGFP). The levels of mRNA and EGFP were evaluated by means of reverse transcription-polymerase chain reaction (RT-PCR), fluorescence microscopy and flow cytometry so as to assess the inhibitory efficiency of siRNA. Analysis of variance was applied to analyze the variance of total fluorescence intensity to screen out the efficient target sites of siRNA.Results shRNA expression vector psiUL54-2 and fusion protein expression vector pUL54S-EGFP were cotransfected into AD293 cells. The EGFP expression level in pUL54S-EGFP/psiUL54-2 cotransfected group was lower than that in pUL54S-EGFP/pAVU6 +27 cotransfected group after 48 h of transfection. Gel analysis showed that the mRNA relative level of UL54S was 19.6 after 48 h of psiUL54-2/pUL54S-EGFP cotransfection, which was significantly lower than those in pUL54S-EGFP/psiUL54-1 group (96.6) and control group (100.0). Cotransfection of psiUL54-1/pUL54S-EGFP for 48h didn't show any effects on the expression of fusion protein UL54S-EGFP (P>0. 05).While psiUL54-1/pUL54S-EGFP cotransfection inhibited the expression of fusion protein UL54S-EGFP(19.43×104±2.29×104vs27.89×104±5.50×104, P<0.01).Conclusion Thescreening system of efficient siRNA targeting truncated region of HCMV UL54S is established successfully. The 1532th-1550th nucleotide acids of UL54S coding sequence are efficient siRNA target sites.
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Objective To study the sequence variation in the env V3-V4 human immunodeficiency virus (HIV)-1 predominant subtype B strains in Hubei Province and to understand the epidemic characteristics and mutations of HIV-1. Methods Epidemiologic survey was done in the HIV-1 carriers in Hubei area. HIV-1 env V3-V4 regions were amplified by nested-polymerase chain reaction (nPCR). The sequences were determined and then phylogenetic analysis was performed. The difference of gene distance were checked by chi square test and the variation of gene distance were descriptively analyzed. Results Four HIV-1 strains or circulating recombinant forms (CRF) were identified in Hubei Province subtype B', B'/C, CRF01_AE and C were 82.69%, 7.69%, 7.69% and 1.92% ,respectively. B' strains were closely related with B. CN. RL 42 from Yunnan Province and B. CN. 02. 02HN from Province Henan, the gene distances were 7.08 ± 2.19 and 7.88 ± 2.28, respectively. Genetic divergence of env of B' strains showed that subtype B' has existed in Hubei area for about 10 years. Amino acid sequence analysis of section env showed V4 was more variable than C3, V3. The top four peptides of V3 loop were GPGR (46.5%), GPGK (30.2%), GPGQ (13.6%) and GQGR (9.3%). Predictions for the potential use of co-receptors on the basis of the critical amino acids within V3 loop disclosed that 16.28% were CCR5-using (R5/NSI), 13.95% were CXCR4-using (X4/SI) while the co-receptor usage of the vast majority (69.77%) could not be predicted. The analysis of glycosylation sites showed that there were 9 sites in env V3-V4 regions of HIV-1 strains in Hubei area and there were deletions in 8 of them, Conclusions Subtype B' is still the main epidemic subtype in Hubei Province and high homologous to the strains from Yunan and Henan Province.
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Objective To develop a clinically useful assay for detecting the mutations of HBV pre-C/BCP based on the pyrosequencing and accuracy, reproducibility and reliability of this assay was evaluated. Methods The pyrosequencing primers for HBV pre-C/BCP mutation were designed through the cluster analysis among one hundred HBV gene sequences. After the amplification of the fragment of pre-C/BCP with the template of pre-C/BCP mutation plasmids, the pyrosequencing method for pre-C/BCP detection was initially set up with this standard sample. The accuracy, reliability and reproducibility of the pre-C/BCP pyrosequencing were confirmed through the pre-C/BCP plasmids as a standard sample when compared with Sanger/genechip sequencing method pre-C/BCP pyrosequencing assay was applied for detecting pre-C/BCP mutation types of 60 chnical serum samples in HBV patients. Results The pre-C/BCP mutation detection assay based on pyrosequencing has been established in our study. The coincidence rate between pyrosequencing and Sanger squencing was 100%. The coincidence rate between the result of pyrosequencing and of genechip method was 91.7%. The reproducibility of this assay was 97. 8%. It indicates the pre-C/BCP pyrosequencing is a high-accurate method with, good-reproducibility and high-reliability. And multi-site detection can be achieved by pyrosequencing one time. A rare mutation T1758C was also detected. Conclusion Pyrosequencing for pre-C/BCP mutations assay is high-throughout method for simultaneous detection of multi-site mutation.