Objective: To understand prevalence and transmission of transmitted drug resistance (TDR) among HIV infected men who have sex with men (MSM) in Tianjin from 2014 to 2017. Methods: A total of 225 blood samples were collected from HIV infected MSM in Tianjin from 2014 to 2017. Pol gene fragments were obtained by viral RNA extraction and nested PCR amplification. Phylogenetic and drug resistance analyses were conducted. Results: A total of 205 samples were successfully sequenced and analyzed. Based on pol sequences, 53.2% (109/205), 28.8% (59/205), 10.2% (21/205), 4.9% (10/205) and 2.9% (6/205) of the samples were positive for HIV subtypes CRF01_AE, CRF07_BC, B, CRF55_01B and unique recombinant forms (URFs). Twenty transmission clusters, including 75 sequences, were identified and 62.5% (10/16) of sequences with TDR were in 5 clusters. The prevalence of TDR was 7.8% between 2014 and 2017. The annual prevalence rate increased from 3.9% (2/51) in 2014, 5.7% (3/53) in 2015, 9.6% (5/52) in 2016 to 12.2%(6/49) in 2017, the difference was not significant (χ(2)=2.504, P=0.127). CRF01_AE and B strains had high TDR prevalence (3.4%, 7/205) and (2.9%, 6/205), respectively. The TDR mutation was mainly NNRTIs, the TDR prevalence was 6.3% (13/205). In contract, the TDR prevalence of NRTIs and PIs were 1.5% (3/205) and 1.0% (2/205) respectively. Conclusion: Results from this study suggested that the prevalence of HIV-1 TDR strains in MSM was serious in Tianjin. It is necessary to take effective prevention and control measures.
China ; Drug Resistance, Viral/genetics* ; Genes, pol ; Genotype ; HIV Infections/transmission* ; HIV Reverse Transcriptase/genetics* ; HIV Seropositivity/genetics* ; HIV-1/isolation & purification* ; Homosexuality, Male/statistics & numerical data* ; Humans ; Male ; Mutation ; Phylogeny ; Polymerase Chain Reaction ; Prevalence ; RNA, Viral/genetics* ; pol Gene Products, Human Immunodeficiency Virus/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

In order to investigate the mutation of HBV polymerase gene reverse transcription conserved region (P region) in chronic hepatitis B (CHB) patients, 212 CHB patients who took antiretroviral treatment with nucleotide analogues were chosen. The drug resistance mutations of HBV P region and HBV genotype were detected by Pyrosequencing. Sequence analysis showed that the drug resistance sites of HBV P region located at sites 173; 180; 181; 184; 204; 236 and 250. The main site of HBV P region drug resistance was 204 and 180, accounting for 35.8% and 23.5%, respectively. There were significant differences in the mutation rate of site 180 among different age groups. There were also significant differences in the mutation rate of site 204 among younger than 30 age group, 41 to 50 age group and 51 to 60 age group. (P < 0.05, P < 0.01). The mutation rate of site 180 combined with site 204 was 66.6%. The mutation rate of site 181 combined with site 236 was 23.3%. The age of C genotype infected patients was significantly older than B genotype infected patients (P < 0.01). M204V/I mutation mostly existed in the form of joint L180M mutation, the mutation rate was age-related. The detection of HBV genotypes and drug resistance sites of HBV P region have important clinical implications for the treatment and prognosis of patients with CHB.
Adult ; Aged ; Antiviral Agents ; pharmacology ; China ; Drug Resistance, Viral ; Female ; Gene Products, pol ; genetics ; Genotype ; Hepatitis B virus ; classification ; drug effects ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Young Adult

Hepatitis B virus (HBV) is the prototype of hepatotropic DNA viruses (hepadnaviruses) infecting a wide range of human and non-human hosts. Previous studies with duck hepatitis B virus (DHBV) identified duck carboxypeptidase D (dCPD) as a host specific binding partner for full-length large envelope protein, and p120 as a binding partner for several truncated versions of the large envelope protein. p120 is the P protein of duck glycine decarboxylase (dGLDC) with restricted expression in DHBV infectible tissues. Several lines of evidence suggest the importance of dCPD, and especially p120, in productive DHBV infection, although neither dCPD nor p120 cDNA could confer susceptibility to DHBV infection in any cell line. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a binding partner for the N-terminus of HBV large envelope protein. Importantly, knock down and reconstitution experiments unequivocally demonstrated that NTCP is both necessary and sufficient for in vitro infection by HBV and hepatitis delta virus (HDV), an RNA virus using HBV envelope proteins for its transmission. What remains unclear is whether NTCP is the major HBV receptor in vivo. The fact that some HBV patients are homozygous with an NTCP mutation known to abolish its receptor function suggests the existence of NTCP-independent pathways of HBV entry. Also, NTCP very likely mediates just one step of the HBV entry process, with additional co-factors for productive HBV infection still to be discovered. NTCP offers a novel therapeutic target for the control of chronic HBV infection.
Animals ; Carboxypeptidases/genetics/*metabolism ; Gene Products, pol/genetics/metabolism ; Heparan Sulfate Proteoglycans/metabolism ; Hepatitis B virus/*physiology ; Hepatocytes/metabolism/virology ; Organic Anion Transporters, Sodium-Dependent/antagonists & inhibitors/genetics/metabolism ; RNA Interference ; Symporters/antagonists & inhibitors/genetics/metabolism ; Viral Envelope Proteins/metabolism ; Virus Internalization

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

Hepatitis B virus (HBV) is a major cause of chronic liver disease, and frequently results in hepatitis, cirrhosis, and ultimately hepatocellular carcinoma. HBV polymerase (Pol) is an essential viral protein that is important for HBV replication and might be involved in the development of hepatocellular carcinoma. Protein-protein interactions appears to be crucial for its role. The aim of this study was to screen and identify the proteins that interact with Pol using a co-immunoprecipitation-based LC-MS/MS identification technique. The HBV Pol gene was amplified by polymerase chain reaction (PCR) and cloned into pCDNA3.1(+). The recombinant plasmid pCDNA3. 1(+)-Pol-flag was transfected into HeLa cells. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identified 45 proteins that co-immunoprecipitated with flag-tagged HBV Pol. Eleven of these have previously been reported as proteins that interact with HBV Pol. A proof-of-concept-based Ingenuity Pathway Analysis (IPA, www.ingenuity.com) was used to characterize the functions and pathways of these 45 identified proteins and HBV Pol. Among these proteins, four proteins may play a role in three major molecular cellular networks, and are therefore worthy of further investigation.
Cell Line, Tumor ; Chromatography, Liquid ; methods ; Gene Products, pol ; chemistry ; genetics ; metabolism ; Hepatitis B ; genetics ; metabolism ; virology ; Hepatitis B virus ; chemistry ; enzymology ; genetics ; Humans ; Immunoprecipitation ; methods ; Protein Interaction Maps ; Software ; Tandem Mass Spectrometry ; methods

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

To study the CTL antigen epitopes and drug resistance mutations of HIV-1 gag and pol genes through analyzing gag and pol gene sequences. The HIV-1 gag and pol gene fragments were amplified using nested polymerase chain reaction. A total of 23 PCR sequences, 449 cloned gag sequences and 402 cloned pol sequences were obtained. Sequence analyses showed the 23 samples were subtype B or B'. A total of 4 in 8 CTL antigen epitopes appeared 8 mutations in consensus sequence of subtype B and B'. There were no mutations found in the PCR sequences, whereas a few mutations were found in clone sequences (9.80%) in 5 antigen epitopes in p24 region. Eighteen PIs-related mutations and 24 RTIs-related mutations were found in PCR sequences and clone sequences in pol gene region, in which 17 (94.44%) PIs-related mutations and 15 (62.50%) RTIs-related mutations were found only in the clone sequences, respectively. The results showed that the prevalence of HIV-1 drug resistance strains in this study was at a higher level (17.39%), suggesting that some samples were resistant.to existing antiviral drugs.
Antigens, Viral ; immunology ; DNA Mutational Analysis ; Drug Resistance, Viral ; genetics ; Epitopes ; immunology ; HIV-1 ; classification ; drug effects ; genetics ; immunology ; Human Immunodeficiency Virus Proteins ; genetics ; Mutation ; Phylogeny ; T-Lymphocytes, Cytotoxic ; immunology ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; pol Gene Products, Human Immunodeficiency Virus ; genetics

Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).
Anti-HIV Agents ; pharmacology ; Benzoxazines ; pharmacology ; Bioluminescence Resonance Energy Transfer Techniques ; methods ; Fusion Proteins, gag-pol ; genetics ; metabolism ; HEK293 Cells ; HIV Protease ; metabolism ; physiology ; HIV-1 ; enzymology ; High-Throughput Screening Assays ; methods ; Humans ; Plasmids ; genetics ; Protein Precursors ; metabolism ; physiology ; Pyridazines ; pharmacology ; Transfection ; Virion ; growth & development ; Virus Assembly ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism

OBJECTIVETo analyze mutation patterns in the RT region of hepatitis B virus (HBV) P gene in patients treated with nucleoside or nucleotide analogs.

METHODSViral DNA was extracted from 227 serum samples of chronic hepatitis B patients from September, 2005 to March, 2007. The RT region of HBV P gene was PCR-amplified and sequenced.

RESULTSKnown resistant mutations were found in 111 cases (48.9%) among 227 samples, 75 cases with clear therapy history. Novel mutations, including A222T, L229V and S256C, were also found. The incidence of multi-drug resistance in patients sequentially treated with lamivudine and adefovir was 25% (4/16), and none of the patients treated with lamivudine plus adefovir in combination shown multi-drug resistance.

CONCLUSIONThe patterns of mutation is complex in nucleotide analogue treated patients. Switching to adefovir in lamivudine resistant patients may lead to multi-drug resistance. Novel mutations identified in this study need further investigation.

Adolescent ; Adult ; Aged ; Antiviral Agents ; pharmacology ; therapeutic use ; DNA Mutational Analysis ; DNA, Viral ; genetics ; Drug Resistance, Viral ; Female ; Gene Products, pol ; genetics ; Genes, Viral ; Genetic Variation ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; genetics ; virology ; Humans ; Male ; Middle Aged ; Reverse Transcriptase Inhibitors ; pharmacology ; therapeutic use ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Young Adult