A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood. The technology utilizes special primers and Taqman MGB fluorescence probe to measure amplification products from the gag-pro-pol polyprotein gene of HTLV-I. HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the beta-actin gene, The amplification system was sensitive to detect 5 copy/microL. The standard curve had a good linearity when the quantity for the gene was between 10(3) and 10(7) copy/microL (R2 = 0.999). Good reproducibility was observed in each intra- and inter-assay. We also measured proviral load in peripheral blood in 12 HTLV-I seropositive former blood donors. Proviral load for HTLV-I infected donors ranged from 0.015 to 12.819 copy/cell in WBC with the mean of 3.116 copy/cell.
Gene Products, gag ; genetics ; Gene Products, pol ; genetics ; Human T-lymphotropic virus 1 ; genetics ; isolation & purification ; Humans ; Molecular Probes ; Polymerase Chain Reaction ; methods ; Viral Proteins ; genetics

Alanine Transaminase ; blood ; DNA, Viral ; analysis ; Gene Products, pol ; genetics ; Hepatitis B ; drug therapy ; Humans ; Lamivudine ; therapeutic use ; Mutation

OBJECTIVETo explore the characteristics of mutation in HBV polymerase (P) gene reverse transcriptase region (RT region) in lamivudine-resistant chronic hepatitis B (CHB) patients.

METHODSThis study involved 115 CHB patients who developed clinical resistance to lamivudine. Direct sequencing of the PCR products was used to detect lamivudine genotypic resistance.

RESULTSLamivudine resistant mutation was detected in 103 patients, and the major mutations included rtL180M+rtM204V and rtM204I, accounting for 58.3% and 22.3%, respectively. Other resistant substitutions included rtL80V/I, rtT184S, and rtA200V, and combined mutation of triple resistant substitutions was detected in HBV RT region of 5 patients by direct sequencing.

CONCLUSIONFor lamivudine-treated patients, combined mutation at the sites other than rtL180 and rtM204 in HBV P gene should also be detected for drug resistance evaluation.

Anti-HIV Agents ; therapeutic use ; Drug Resistance, Viral ; genetics ; Gene Products, pol ; genetics ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; therapeutic use ; Mutation

BACKGROUND/AIMS: Lamivudine, a nucleoside analogue has been widely used as an effective antiviral agent for the treatment of patients with chronic hepatitis B infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine very frequently occurs after long-term use of lamivudine. It is well known that the mutation is selected by the lamivudine. We hypothesized that a few mutant strains of YMDD motif are present as quasispacies before the lamivudine treatment, are selected by the treatment, and breakthrough during treatment. We investigated the prevalence of the YMDD motif mutants in patients with chronic hepatitis B infection who had not been treated by antiviral agents before. METHODS: The study included the serums of 40 patients with chronic heptitis B infection, which stored at -70 degrees C. Thirty-four patients had chronic hepatitis and 6 patients had cirrhosis. Thirty-one patients were diagnosed by liver biopsy. The average age and range were 29 years and 13-57 years respectively. None had taken any antiviral agents before. To detect YMDD mutants, YVDD (M552V), and YIDD (M552I), we used direct sequencing and the restriction fragment length polymorphism (RFLP) method. RESULTS: The YMDD mutant was detected by RFLP method in 7.5% (3/40) of the patients with chronic hepatitis B infection, in two patients with chronic hepatitis and one with cirrhosis. All were YMDD+ YIDD mutants. CONCLUSIONS: The YMDD motif mutation occurs spontaneously without antiviral therapy in patients with chronic hepatitis B infection.
Adolescent ; Adult ; Drug Resistance, Viral/genetics ; Female ; Gene Products, pol/*genetics ; Hepatitis B virus/*genetics ; Hepatitis B, Chronic/*virology ; Humans ; Male ; Middle Aged ; *Mutation ; Polymorphism, Restriction Fragment Length

This study aims to compare the influence of different genes to the results of HIV-1 subtype B' phylogenetic analyses. We first split 47 near full-length genome sequences of subtype B' into different regions (gag, pol, vif, vpr, vpu, env, nef), which derived from various risk populations and geographic regions from Thailand, Myanmar and China from published studies. Phylogenetic analyses were performed to each region obtained. The phylogenetic results of different regions were compared to that of the near full-length genome sequences. The pol gene was found to have the lowest diversity and evolutionary rate, and could repeat the phylogenetic results by using near full-length genome sequences. Although the env gene has the highest diversity and evolutionary rate, it could not achieve the similar results. This study compared the influence to the results of HIV-1 subtype B' phylogenetic analyses by using different genes and laid foundation for further molecular survey and analyses of the transmission of subtype B' in China.
Amino Acid Sequence ; China ; epidemiology ; Cluster Analysis ; Genes, Viral ; genetics ; HIV-1 ; classification ; genetics ; Molecular Epidemiology ; Molecular Sequence Data ; Phylogeny ; pol Gene Products, Human Immunodeficiency Virus ; chemistry ; genetics

OBJECTIVETo investigate the distribution and proportion of subtypes of pol gene in HIV-1 epidemic strains in Guangxi Autonomous Region.

METHODS152 HIV-1 patients were enrolled from 11 cities in Guangxi Autonomous Region from 2010 to 2012 by convenient sampling. Inclusion criterias were listed as the fdlowing: HIV-1 infection was confirmed by Western blot, HIV-1 viral load >1 000 copies/ml, > 18 year-old, and without any serious illnesses. 5 ml of peripheral blood samples were obtained from each patient. The viral RNA was isolated from plasma and used for amplification of full-length pol gene by nested RT-PCR. The amplified products were sequenced. After editing and modification, all sequences were characterized for preliminary subtyping by genotyping and confirmed with phylogenetic tree constructed by MEGA 5.03 software. The recombinant identification of 2 unknown recombinant strains was determined by RIP and jpHMM at GOBICS.

RESULTSAmong 152 patients, 137 full-length pol genes were successfully amplified and 127 HIV-1 subtypes were identified. The distribution and proportion of subtypes was summarized as the following 71 cases of CRF01_AE, accounting for 55.9% (71/127), 38 CRF08_BC, 29.9% (38/127), 13 CRF07_BC, 10.2% (13/127), and 3 B (B'), 2.4% (3/127), 2 unknown recombinant strains, 1.6% (2/127). In 11 cites of Guangxi Autonomous Region, subtype CRF01_AE was the dominant strain. Among heterosexual transmitted patients and drug abusers, the proportions of subtype CRF01_AE were 67.4% (58/86) and 34.1% (14/41), respectively. There was a significance different in the distribution of CRF01_AE in different routes of transmission (χ(2)=15.07, P<0.001). In age 21- 35, age 36- 60 and age>60 groups, the proportions of CRF01_AE was 43.6% (17/39), 57.6% (38/66), 77.3% (17/22), and CRF08_BC was 43.6% (17/39), 28.8% (19/66), 9.1% (2/22), respectively, the difference in proportions was significant(χ(2)=8.48, P= 0.014). The patterns of two unknown recombinant strains were found to be CRF01_AE/B (B') and CRF01_AE/C/B(B'), respectively.

CONCLUSIONCRF01_AE was the dominant HIV-1 subtype in Guangxi Autonomous Region from 2010 to 2012, with heterosexual transmission as its main spreading route. The two unknown recombinant strains in Guangxi Autonomous Region were reconstructed by subtype CRF01_AE and CRF_BC.

Blotting, Western ; China ; epidemiology ; Cities ; Drug Users ; Genes, pol ; Genotype ; HIV Infections ; epidemiology ; transmission ; virology ; HIV-1 ; genetics ; Humans ; Phylogeny ; Polymerase Chain Reaction ; RNA, Viral ; blood ; pol Gene Products, Human Immunodeficiency Virus ; genetics

BACKGROUNDConstruction of replication-deficient recombinant adenovirus expressing gag-pol and env genes of human immunodeficiency virus (HIV) in mice.

METHODSgag-polDelta and gp140TM genes were cloned into shuttle vector pAdTrack-CMV respectively, and then the plasmids containing gag-polDelta or gp140TM gene were cotransformed with the backbone of adenovirus into E.coli BJ5183. Transfections of the recombinants were performed to obtain recombinant adenoviruses. Its immunogenicity was evaluated by testing antibody levels of mice primed with DNA vaccines and boosted with recombinant adenoviruses.

RESULTSThe replication-deficient recombinant adenovirus could express Gp140TM, Gag P55 and P24 proteins correctly. The mice primed with DNA vaccines and boosted with recombinant adenoviruses elicited high titer of HIV-1-specific antibody compared with that inoculated with DNA vaccines only.

CONCLUSIONReplication-deficient recombinant adenovirus expressing gag-polDelta and gp140TM can elicit high titer HIV-1-specific antibodies.

AIDS Vaccines ; immunology ; Adenoviridae ; genetics ; Animals ; Female ; Fusion Proteins, gag-pol ; biosynthesis ; genetics ; Gene Products, env ; biosynthesis ; genetics ; HIV-1 ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Recombination, Genetic ; Transfection ; Vaccines, DNA ; immunology ; env Gene Products, Human Immunodeficiency Virus

OBJECTIVETo investigate the primary structure and heterogenenity of P gene region including YMDD motif in hepatitis B virus.

METHODSFrom serum samples collected from 4 patients who had never been treated with anti-viral drugs, DNA fragments of 1057bp long of P gene were amplified and cloned into pUC19. Twenty positive clones were chosen randomly from each sample. The YMDD motif mutation was detected by mismatched PCR-RFLP. Finally last ten positive clones of two samples were sequenced.

RESULTSNucleotide mutation rates among clones of Sample 1 and 2 were 0.3% - 1.1%, 0.4% - 1.7%, respectively. Among 80 clones, the variations from YMDD to YMGD were revealed in two clones.

CONCLUSIONThere are HBV quasispecies in the P gene region including YMDD motif of hepatitis B virus and a novel mutation of YMDD motif in the sera of patients without being therapied by anti-viral drugs.

Adult ; Amino Acid Motifs ; Base Sequence ; Gene Products, pol ; genetics ; Hepatitis B virus ; genetics ; Humans ; Male ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

Clinically being applied treatment against chronic hepatitis has three limitations: low response rates, severe adverse effects and a high rate of drug resistance. Hence, novel targets for antiviral therapy need to be developed so as to provide an armory of different strategies. During the replication of hepatitis B virus, the interaction of viral polymerase (P protein, also called P) and epsilonRNA is indispensable for the initiation of reverse transcription via protein priming and the pregenome RNA (pgRNA) packaging. Three strategies are currently developed for blocking P-epsilon interaction: heat shock protein inhibitors, epsilonaptamers and chemical compounds for blocking formation of P-epsilon complex. Previously, our group has for the first time worldwide in vitro screened several aptamers, which are able to interfere with the P-epsilon interaction. A strong inhibition against HBV was observed in vitro and in vivo experiments, respectively. In conclusion, the so far developed chemicals suppressing the P-epsilon interaction may bypass or overcome the viral resistance problems during clinic treatment and represent a highly attractive option for therapeutic intervention.
Animals ; Gene Expression Regulation, Viral ; Gene Products, pol ; antagonists & inhibitors ; genetics ; metabolism ; Hepatitis B ; therapy ; virology ; Hepatitis B virus ; enzymology ; genetics ; physiology ; Humans ; RNA, Viral ; genetics ; metabolism ; Virus Replication

OBJECTIVETo study the heterogeneity of polymerase gene (P gene) within hepatitis B virus (HBV) genotypes based on a systematic analysis of 202 HBV P genes, providing some useful references for further studies on the relationship among HBV genotypes, P gene mutations, replication and nucleoside analogues drug-resistance.

METHODS202 HBV complete sequences containing P genes were obtained from GenBank and were analysed using computer softwares.

RESULTSThere were some genotype-related characteristics of HBV P genes. As reverse transcriptase domain was concerned, there were more amino acid divergences in genotype C and D compared with these in genotype A. There were also amino acid substitutions in the A-F conserved regions of the reverse transcriptase domain within and between HBV genotypes.

CONCLUSIONSThere are divergences of P genes and amino acids within and between HBV genotypes, which should be considered when amino acid changes are analyzed whether they are proposed to be drug-resistance mutations or the results from quasispecies-selected. Moreover, these divergences may affect the antiviral effect of nucleoside analogues on HBV with different genotypes.

Amino Acid Sequence ; DNA, Viral ; genetics ; DNA-Directed DNA Polymerase ; genetics ; Gene Products, pol ; genetics ; Genetic Heterogeneity ; Genotype ; Hepatitis B virus ; genetics ; physiology ; Humans ; Molecular Sequence Data ; Mutation ; Phylogeny