Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line.

BACKGROUND: Toll like receptors (TLRs) play an important role in innate immunity and various studies suggest that TLRs play a crucial role in pathogenesis of hepatitis B virus (HBV) infection. The present study aims in looking into the status of crucial host and viral gene expression on inciting TLR7. METHODS: The transcription of TLR7 pathway signaling molecules and HBV DNA viral load were quantified by Real Time-PCR after stimulation of TLR7 with its imiquimod based ligand, R837. Cell cycle analysis was performed using flow-cytometry. Expression of TLR7 and chief cell cycle regulator governing G1/S transition, p53 was also seen in liver biopsysss samples of CHB patients. HBV induced alteration in histone modifications in HepG2 cells and its restoration on TLR7 activation was determined using western blot. RESULTS: The TLR7 expression remains downregulated in HepG2.2.15 cells and in liver biopsy samples from CHB patients. Interestingly HBV DNA viral load showed an inverse relationship with the TLR7 expression in the biopsy samples. We also evaluated the anti-viral activity of R837, an agonist of TLR7. It was observed that there was a suppression of HBV replication and viral protein production upon TLR7 stimulation. R837 triggers the anti-viral action probably through the Jun N-terminal Kinase (JNK) pathway. We also observed a downregulation of histone H3K9Me3 repression mark upon R837 treatment in HBV replicating HepG2.2.15 cells, mimicking that of un-infected HepG2 cells. Additionally, the G1/S cell cycle arrest introduced by HBV in HepG2.2.15 cells was released upon ligand treatment. CONCLUSION: The study thus holds a close insight into the changes in hepatocyte micro-environment on TLR7 stimulation in HBV infection.

Mutation in AIDS restriction gene affecting HIV infection and disease progression in a high risk group from Northeastern India.

BACKGROUND: HIV estimates for 2009 released by National AIDS Control Organization (NACO) reveals that 2.4 million people in India were living with HIV, 39% being female, 4.4% children and 82.4% adult males between the age group of 15 and 49 years. Persons with host genetic polymorphism CCR5Δ32 mutation are known to be partially or fully resistant to HIV infection. Persons with mutation affecting both the alleles (homozygous) are resistant to HIV infection whereas single allele (heterozygous) polymorphism leads to slower progression to AIDS. CCR5Δ32 mutation is commoner in Caucasians but less prevalent amongst Africans and Asians thereby rendering them susceptible to HIV infection. METHOD: 571 HIV serologically naive subjects from a young and homogenous male population hailing from the seven northeastern states; West Bengal and Gorkha people were selected. All the subjects belonged to a special high risk group, sexually active and typically working in difficult and uncongenial terrain involved in frequent moves including overseas missions. Their family lives are severely disrupted. 181 HIV seropositive cases of which 92 cases that were admitted in a large tertiary care hospital were also included. The distribution of CCR5Δ32 polymorphism amongst both HIV seronegative (HSN) and HIV seropositive study cohorts (HSP) using molecular methods was studied. RESULTS AND CONCLUSION: There was failure to detect any CCR5Δ32 amongst this study group suggesting that this population from the northeastern India, West Bengal and Gorkha people are not protected by this specific host polymorphism in respect of acquisition of HIV infection as well as progression to AIDS.

Characterization of antiviral resistance mutations among the Eastern Indian Hepatitis B virus infected population.

BACKGROUND: Antiviral therapy using nucleos(t)ide analogues (NAs) is an effective control measure of chronic hepatitis B virus (HBV) infection; however they need long term treatment. Presence of drug-resistance mutations may get in the way of the efficacy of antiviral therapy. Our study was aimed at defining the prevalence of HBV drug-resistance in HBVrt region in a population of 147 HBsAg positive patients. FINDINGS: HBV/D has shown multiple types of HBVrt mutations both among treatment naïve (65.0%, 13 of 20 HBV/D) and treated patients (56.2%, 9 of 16 HBV/D). In additional, several mutations, with a suggested role in drug resistance, were detected among the treatment naïve as well as the treated patients. The mutations reported to be involved in reduction of drug effectiveness, was common among non-responders to therapy as well as among the naïve patients. Notably, classical antiviral resistance mutations (rtL80I/V-rtI169T-rtV173L-rtL180M-rtA181T/V/S-rtT184A/S/G/C-rtA194T-rtS202C /G/I -rtM204V/I-rtN236T-rtM250V) were not detected. CONCLUSION: The prevalence of putative NAr mutations among non responders to therapy suggests that they might have role in reduced efficacy of currently available antivirals and requires further investigations.

Characterization of the occult hepatitis B virus variants circulating among the blood donors from eastern India.

A previous study from West Bengal documented very high rate of occult HBV infection (OBI) among the HBsAg negative blood donors. This study was aimed to characterize the OBI strains circulating among the blood donors and to estimate the risk associated with the prevailing viral variants/mutants. Blood samples from 2195 voluntary blood donors were included in the study. HBsAg, HBeAg, anti-HBc, and anti-HBs statuses of the samples were done by ELISA based detection. PCR amplification and sequencing were done to determine HBV genotypes, basal core promoter (BCP), and precore (Pre-C) mutations. Among the study samples, 268 were anti-HBc positive/HBsAg negative, among which 65 (24.25%) were HBV DNA positive. Phylogenetic analysis revealed the presence of HBV/D (87.23%), HBV/A (8.51%), and HBV/C (4.26%) (P < 0.0001). HBV/D3 (65.85%) was the significantly prevalent subgenotype over HBV/D2 (26.83%) and HBV/D1 (7.31%) (P = 0.0003). Considerable prevalence of differential BCP (1752C, 1753C, 1762T/1764A, 1753C+1762T/1764A, 1773C, and 1814C) and reverse transcriptase (rt) gene (rtI91L, rtL93P, rtS106C, rtR110G, rtN118T, rtS119T, rtY126H, rtG127W/R, rtC136R, and rtY158H) mutations was identified. Association of specific HBV subgenotypes with OBI was interesting and needs further study. Clinically relevant mutations were prevalent among the OBI strains which are of serious concern.

Molecular evidence for the occurrence of Japanese encephalitis virus genotype I and III infection associated with acute encephalitis in patients of West Bengal, India, 2010.

BACKGROUND: Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is the sole etiologic agent of Japanese Encephalitis (JE); a neurotropic killer disease which is one of the major causes of viral encephalitis worldwide with prime public health concern. JE was first reported in the state of West Bengal, India in 1973. Since then it is being reported every year from different districts of the state, though the vaccination has already been done. Therefore, it indicates that there might be either partial coverage of the vaccine or the emergence of mutated/new strain of JEV. Considering this fact, to understand the JEV genotype distribution, we conducted a molecular epidemiological study on a total of 135 serum/cerebrospinal fluid (CSF) samples referred and/or collected from the clinically suspected patients with Acute encephalitis syndrome (AES), admitted in different district hospitals of West Bengal, India, 2010. FINDINGS: JEV etiology was confirmed in 36/135 (26.6%) and 13/61 (21.3%) 2-15 days' febrile illness samples from AES cases by analyzing Mac-ELISA followed by RT-PCR test respectively. Phylogenetic analysis based on complete envelope gene sequences of 13 isolates showed the emergence of JEV genotype I (GI), co-circulating with genotype III (GIII). CONCLUSION: This study represents the first report of JEV GI with GIII, co-circulating in West Bengal. The efficacy of the vaccine (derived from JEV GIII strain SA-14-14-2) to protect against emerging JEV GI needs careful evaluation. In future, JE outbreak is quite likely in the state, if this vaccine fails to protect sufficiently against GI of JEV.

Full genomic analysis of an influenza A (H1N2) virus identified during 2009 pandemic in Eastern India: evidence of reassortment event between co-circulating A(H1N1)pdm09 and A/Brisbane/10/2007-like H3N2 strains.

BACKGROUND: During the pandemic [Influenza A(H1N1)pdm09] period in 2009-2010, an influenza A (Inf-A) virus with H1N2 subtype (designated as A/Eastern India/N-1289/2009) was detected from a 25 years old male from Mizoram (North-eastern India). OBJECTIVE: To characterize full genome of the H1N2 influenza virus. METHODS: For initial detection of Influenza viruses, amplification of matrix protein (M) gene of Inf-A and B viruses was carried out by real time RT-PCR. Influenza A positive viruses are then further subtyped with HA and NA gene specific primers. Sequencing and the phylogenetic analysis was performed for the H1N2 strain to understand its origin. RESULTS: The outcome of this full genome study revealed a unique reassortment event where the N-1289 virus acquired it's HA gene from a 2009 pandemic H1N1 virus with swine origin and the other genes from H3N2-like viruses of human origin. CONCLUSIONS: This study provides information on possibility of occurrence of reassortment events during influenza season when infectivity is high and two different subtypes of Inf-A viruses co-circulate in same geographical location.

Surveillance in Eastern India (2007-2009) revealed reassortment event involving NS and PB1-F2 gene segments among co-circulating influenza A subtypes.

BACKGROUND: Influenza A virus encodes for eleven proteins, of which HA, NA, NS1 and PB1-F2 have been implicated in viral pathogenicity and virulence. Thus, in addition to the HA and NA gene segments, monitoring diversity of NS1 and PB1-F2 is also important. METHODS: 55 out of 166 circulating influenza A strains (31 H1N1 and 24 H3N2) were randomly picked during 2007-2009 and NS and PB1-F2 genes were sequenced. Phylogenetic analysis was carried out with reference to the prototype strains, concurrent vaccine strains and other reference strains isolated world wide. RESULTS: Comparative analysis of both nucleotide and deduced amino acid sequences, revealed presence of NS gene with A/PR/8/34(H1N1)-like mutations (H4N, Q21R, A22V, K44R, N53D, C59R, V60A, F103S and M106I) in both RNA-binding and effector domain of NS1 protein, and G63E, the HPAI-H5N1-like mutation in NEP/NS2 of five A/H1N1 strains of 2007 and 2009. NS1 of other A/H1N1 strains clustered with concurrent A/H1N1 vaccine strains. Of 31 A/H1N1 strains, five had PB1-F2 similar to the H3N2 strains; six had non-functional PB1-F2 protein (11 amino acids) similar to the 2009 pandemic H1N1 strains and rest 20 strains had 57 amino acids PB1-F2 protein, similar to concurrent A/H1N1 vaccine strain. Interestingly, three A/H1N1 strains with H3N2-like PB1-F2 protein carried primitive PR8-like NS gene. Full gene sequencing of PB1 gene confirmed presence of H3N2-like PB1 gene in these A/H1N1 strains. CONCLUSION: Overall the study highlights reassortment event involving gene segments other than HA and NA in the co-circulating A/H1N1 and A/H3N2 strains and their importance in complexity of influenza virus genetics. In contrast, NS and PB1-F2 genes of all A/H3N2 eastern India strains were highly conserved and homologous to the concurrent A/H3N2 vaccine strains suggesting that these gene segments of H3N2 viruses are evolutionarily more stable compared to H1N1 viruses.

Near full-length genomic characterization of a HIV type 1 BC recombinant strain from Manipur, India.

Genetic complexity of HIV-1 is brought about by recombination between HIV-1 subtypes which leads to the development of epidemiologically significant founder strains. In the present study, the near full-length genome sequence of an HIV-1 isolate from an injecting drug user of Manipur (India) was determined, which evidenced the presence of a novel HIV-1 BC recombinant strain. Near full-length genome was amplified by polymerase chain reaction using primer walking approach. The recombination break points were detected using bootscan and simplot analyses. This isolate exhibited a mosaic structure consisting of subtype C backbone with subtype B insertions at the upstream of pol gene (3026-3259) and the downstream of env gene which spanned till the nef gene (8183-8961). Phylogenetic relationships determined with neighbor-joining trees, revealed that the subtype C sequences clustered with sequences from Indian subtype C HIV-1 strains, and the subtype B sequences clustered with HIV-1 subtype B strains from Thailand. This finding may create a complex scenario of HIV-1 epidemic among the injecting drug users of Manipur in near future.

Variations in the functional domain of basal core promoter of hepatitis B virus among Eastern Indian patients with prevalence of genotypes A, C, and D among the same ethnic population.

Mutations in the basal core promoter (BCP) and precore (PC) regions are associated with persistent and intermittently high hepatitis B virus (HBV) replication in several patients. The variability in the functional domains of BCP and PC region of HBV and their association with disease progression and clinical outcome were assessed in Eastern India, an unique region where three HBV genotypes, A, D, and C are prevalent among the same ethnic group. PCR amplification and direct sequencing of BCP and PC region was done on sera obtained from 130 HBsAg positive subjects with different clinical presentations. Associations of the apparent risk factors with clinical advancement were evaluated by statistical methods including multiple logistic regression analyses (MLR). HBV genotype A was present in 33.08%, C in 25.38%, and D in 41.54% cases. Genotypes A and C were associated with higher rate of T1762/A1764 mutations than the most predominant genotype D. HBeAg negative state was associated with considerably higher rate of C1753 mutation. T1762/A1764 along with C1753 was common among cirrhosis and T1762/A1764 without C1753 was frequent among chronic liver disease cases. No significant association was found between A1896 point mutation and clinical status. Multivariate analysis revealed that T1762/A1764 double mutation, HBV/A, age ≥25 years, C1753 and A1899 were critical factors for clinical advancement while age ≥25 years and C1753 as significant predictor for cirrhosis in comparison with chronic liver disease. In conclusion, the analysis of the BCP variability may help in monitoring the progression towards advanced liver disease in Eastern Indian patients.

Recent pattern of Co-infection amongst HIV seropositive individuals in tertiary care hospital, Kolkata.

BACKGROUND: Opportunistic Infections (OIs) and co-infections are the major cause of deaths amongst HIV infected individuals and this mostly depends upon the risk factors, type of exposure and geographic region. The commonest types of infections reported are tuberculosis, chronic diarrhoea, oral candidiasis, herpes simplex virus-2, cytomegalovirus, hepatitis B virus and hepatitis C virus. Due to the scarcity of OIs data available from this region, we had designed a study to determine the frequency of different OIs amongst HIV seropositive patients. METHODS: Analysis of the different spectrum of OIs/Co-infections were carried out with 204 HIV sero-positive patients (142 males and 62 females) who visited the HIV/AIDS Apex Clinic in a tertiary care hospital from March 2006 to March 2009. The CD4+ count was estimated using FACS Calibur, the routine smear test, serology, nested RT-PCR and DNA sequencing were carried out to determine the different OIs. RESULTS: In this study, HIV seropositive patients were mostly from middle age group (31-40 yrs) with CD4+ counts in majority of symptomatic AIDS patients below 200 cells/mm3. The common co-infections/opportunistic infections were OC (53.43%), CD (47.05%), HSV-2 (36.76%), TB (35.29%), CMV (26.96%), HBV (15.19%) and HCV (7.35%). Dual infections, like HSV-2 & CMV (15.38%), HSV-2 & TB (14.61%), HSV-2 & oral candidiasis (24.61%) and CMV & oral candidiasis (14.61%) were significant in follow-up patients. Triple infections were also common e.g., TB, CD, OC infection occurring frequently in about 14.21% of the study population. Multiple infections like OC, TB, CD amongst the viral co-infected patients with HSV-2, HCV, CMV and HBV are also reported in this study. The genotyping analysis of the HCV co-infected HIV individuals shows that two belonged to HCV genotype 1 and 8 belonged to genotype 3. CONCLUSIONS: A wide spectrum of OIs were observed amongst HIV-infected patients in the HIV/AIDS Apex Clinic. Oral candidiasis, CD, CMV and HSV-2, were the common OIs in those patients. This study aims to provide a clearer picture regarding infections occurring amongst HIV seropositive individuals so that the scientific findings could be translated into sustainable prevention programmes and improved public health policies. TRIAL REGISTRATION: None.

Molecular characterization and comparative analysis of pandemic H1N1/2009 strains with co-circulating seasonal H1N1/2009 strains from eastern India.

During the peak outbreak (July-September 2009), a total 1886 patients were screened in eastern India, of which 139 (7.37%) and 52 (2.76%) were positive for pH1N1 and seasonal H1N1, respectively. Full-length HA1, NA, NS1 and PB1-F2 genes of representative strains were sequenced. Phylogenetic analysis of deduced amino acid sequences of pH1N1 strains revealed HA1 and NS1 to be of North American swine lineage, and the NA gene of Eurasian swine lineage. Consistent with previous reports, the PB1-F2 gene of pH1N1 strains was unique due to a mutation resulting in a truncated protein of 11 aa. The HA, NA and NS1 genes of H1N1/2009 strains clustered with H1N1 strains of 2000-2009, whereas a subset of strains contained a pH1N1-like truncated PB1-F2. The truncated PB1-F2 may confer the advantage of lower pathogenicity but higher replication and infectivity to the human H1N1 strains. This is the first report of seasonal H1N1/2009 strains with a pH1N1/2009-like gene segment.

Anti-hepatitis B core antigen testing with detection and characterization of occult hepatitis B virus by an in-house nucleic acid testing among blood donors in Behrampur, Ganjam, Orissa in southeastern India: implications for transfusion.

BACKGROUND: Occult hepatitis B virus (HBV) infection might transmit viremic units into the public blood supply if only hepatitis B surface antigen (HBsAg) testing is used for donor screening. Our aim was to evaluate the prevalence of occult HBV infection among the HBsAg negative/antiHBc positive donations from a highly HIV prevalent region of India. METHODS: A total of 729 HBsAg negative donor units were included in this study. Surface gene and precore region were amplified by in house nucleic acid test (NAT) for detection of occult HBV infection and surface gene was analyzed after direct sequencing. RESULTS: A total of 220 (30.1%) HBsAg negative donors were antiHBc positive, of them 66 (30%) were HBV DNA positive by NAT. HBV DNA positivity among 164 antiHBc only group, was 27.1% and among 40 antiHBs positive group was 30.0%. HBV/D (93.3%) was predominant and prevalence of both HBV/C and HBV/A was 3.3%. Single or multiple amino acids substitutions were found in 95% samples. CONCLUSION: Thus, a considerable number of HBV infected donors remain undiagnosed, if only HBsAg is used for screening. Addition of antiHBc testing for donor screening, although will lead to rejection of a large number of donor units, will definitely eliminate HBV infected donations and help in reducing HBV transmission with its potential consequences, especially among the immunocompromised population. The HBV genetic diversity found in this donor population are in accordance with other parts of India.

Incidence of multiple Herpesvirus infection in HIV seropositive patients, a big concern for Eastern Indian scenario.

BACKGROUND: Human immunodeficiency virus (HIV) infection is associated with an increased risk for human herpes viruses (HHVs) and their related diseases and they frequently cause disease deterioration and therapeutic failures. Methods for limiting the transmission of HHVs require a better understanding of the incidence and infectivity of oral HHVs in HIV-infected patients. This study was designed to determine the seroprevalence of human herpes viruses (CMV, HSV 2, EBV-1, VZV) antibodies and to evaluate their association with age, sex as well as other demographic and behavioral factors. RESULTS: A study of 200 HIV positive patients from Eastern India attending the Calcutta Medical College Hospital, Kolkata, West Bengal, Apex Clinic, Calcutta Medical College Hospital and ART Center, School of Tropical Medicine, Kolkata, West Bengal was done. Serum samples were screened for antibodies to the respective viruses using the indirect ELISA in triplicates.CytoMegalo virus (CMV), Herpes Simplex virus type 2 (HSV-2), Varicella Zoster virus (VZV), and Epstein Barr virus (EBV-1) were detected in 49%, 47%, 32.5%, and 26% respectively. CONCLUSION: This study has contributed baseline data and provided insights in viral OI and HIV co-infection in Eastern India. This would undoubtedly serve as a basis for further studies on this topic.

Implementation of a multiregion hybridization assay to characterize HIV-1 strains detected among injecting drug users in Manipur, India.

We have implemented the latest technology of a multiregion hybridization assay (MHAbce, version 2) for the molecular characterization of HIV-1 among injecting drug users (IDUs) of Manipur, India. This study provides a more detailed analysis on the basis of probes designed from eight different genomic regions of HIV-1, to achieve a clear picture of HIV-1 genomic diversity in Manipur. Out of 30 samples, 15 were found to be of subtype C, 1 of subtype B, 5 with dual-probe reactivity, 8 with multigenomic recombination pattern and 1 sample showed both dual-probe reactivity and multigenomic variations. In contrast, the heteroduplex mobility assay (HMA) with respect to gag and env genes revealed 21 samples to be of subtype C (gag C/env C), 3 samples of subtype B (gag B/env B) and 6 samples of B/C recombinants (gag C/env B). MHAbce illustrates the occurrence of inter- and intragenomic variants and dual infection in an IDU population from India. It also indicates the possibility of the presence of new circulating recombinant forms of HIV-1 strains, which might have been difficult to trace by HMA alone.

Detection of intersubtype recombinants with respect to env and nef genes of HIV-1 among female sex workers in Calcutta, India.

HIV-1 detected among female sex workers in Calcutta, India was characterized in respect to env and nef genes. A total of 39 HIV-1 seropositive samples were used in the study. Phylogenetic analysis of the nucleotide sequences of respective regions showed that 22 out of 39 samples (56.4%) were infected with subtype C with respect to both env and nef genes; however, 17 samples (43.6%) showed distinct subtype discordance. Simplot analysis of these samples showed the presence of intersubtype recombination between subtypes C and B. Both env C/nef B and env B/nef C recombinants were found to be present; 16 samples were found to be env C/nef B and 1 sample was detected as env B/nef C. This result indicates the emergence of intersubtype recombinants of HIV-1 for the first time in this eastern part of India.

Frequency and significance of hepatitis B virus surface gene variant circulating among 'antiHBc only' individuals in Eastern India.

BACKGROUND: Genetic mutation might account for the presence of hepatitis B virus (HBV) DNA among antiHBc only individuals. The aim of the study was to assess the prevalence and significance of surface gene mutations among antiHBc only cases in our population. METHODS: Three hundred and three antiHBc(+) sera of adults (mean age, 33.7+/-11.0; range 18-65 years) as well as HBsAg(+)/HBV DNA(+) (n=19) controls were included in this study. Surface gene and basal core promoter (BCP)-precore region were amplified and surface gene was analyzed after direct sequencing. RESULTS: One hundred and seventy-eight out of 303 (58.8%) was antiHBc only, 39/171 (22.8%) of them was HBV DNA(+). Genotypes A, C, D were found among both HBsAg(+) and antiHBc(+) samples. Single or multiple amino acids substitutions were found in 82% samples, however, G145R vaccine escape mutation was rare. Individuals having substitutions within as well as outside major hydrophilic loop (MHL) region were detected; some of these mutations were in overlapping RT domain of polymerase (Pol) gene. CONCLUSIONS: The existence of occult HBV infection among antiHBc only individuals could not be explained fully by mutations in the 'a' determinant region of surface gene in our population.

Phylogenetic analysis of env, gag, and tat genes of HIV type 1 detected among the injecting drug users in West Bengal, India.

A recent occurrence of HIV-1 seropositivity among a group of injecting drug users (IDUs) in Darjeeling, a hilly district in northern West Bengal, revealed overall 11.8% HIV seroprevalence. Our study based on env (C2-V3), gag (p24-p7), and tat (exon-1) genomic regions of HIV-1 detected among this population showed that Darjeeling IDU sequences belonged to subtype C. Interestingly, the IDU sequences from Darjeeling were again found to be closer to the C strains from Manipur, a northeastern state in India, which is linked to the Golden Triangle via the Manipur-Myanmar border, rather than the IDU C sequences from Nepal, a neighboring country of India. The outgroup reference strains from different sites of IDU-driven epidemics in the world like Russia, Vietnam, Thailand, and Spain belonged to the nonsubtype C group and formed separate clusters from the subtype C cluster in our analysis. These results indicate a rapid spread of HIV-1 by possible drug trafficking along international boundaries, which might also help in the invasion of HIV-1 among IDUs of Darjeeling through the Manipur-Myanmar border of India.

Determination of gag and env subtypes of HIV-1 detected among injecting drug users (IDUs) in Manipur, India: evidence for intersubtype recombination.

The majority of HIV-1 transmission in Manipur, one of the northeastern states of India, is through the sharing of needles and syringes among the injecting drug users (IDUs). A total of 28 HIV seropositive samples were used to determine the HIV-1 subtypes with respect to both gag and envelope genes. The specific regions within gag and envelope genes were amplified from PBMC DNA by nested PCR using appropriate primers. These amplicons were used in heteroduplex mobility assay followed by DNA sequencing. Phylogenetic analysis of the nucleotide sequences of respective regions showed that 89% of samples (25/28) were infected with subtype C with respect to both gag and envelope genes; however, 11% of the samples (3/28) showed subtype discordance with respect to the envelope (C2-V3) and gag (p24-p7) genomic regions. Simplot analysis of the discordant samples showed the presence of intersubtype recombination between subtype C and Thai B; two samples were found to be subtype C in envelope but Thai B in gag, whereas, one sample was found to be subtype Thai B in envelope and 'C' in gag region.

Determination of gag subtypes of HIV type 1 detected among female sex workers in Calcutta, India.

HIV-1 subtyping is important to study the changing scenario of genetic variation. The gag-based heteroduplex mobility assay (gag-HMA) was developed and evaluated as a powerful and reliable technique for identifying the HIV-1 group M subtypes A to H and the circulating recombinant forms (CRFs). To study the subtype distribution of HIV-1 strains from the eastern part of India, we used the gag-based HMA, followed by the sequencing and phylogenetic analysis. Blood samples from HIV-1-seropositive female sex workers in Calcutta were subjected to gag-HMA. The most prevalent subtype was found to be the C type, among which the C4 subsubtype was prevalent. However, a number of nontypable C strains were found in gag-HMA. Phylogenetic analysis revealed the discrete nature of the C strains and no monophyletic cluster was noticed. This result might indicate a growing tendency of variations among the HIV-1 type C strains circulating in eastern India.

Impact of genetic diversity of HIV-1 on diagnosis, antiretroviral therapy & vaccine development.

HIV-1 strains have diversified extensively through mutation and recombination since their initial transmission to human beings many decades ago in central Africa. The high error rate of HIV reverse transcriptase combined with the estimated in vivo HIV-1 replication rate of ten billion new virions each day leads to extraordinary genetic diversity of HIV. Twenty seven circulating genetic forms of the HIV-1 group M are presently recognized, including 11 subtypes and sub-subtypes, and 16 circulating recombinant forms (CRF). Genotypic analyses have provided a better understanding of the molecular diversity of HIV-1, enabling the detection of emerging HIV-1 variants and improving the tracking of the epidemic worldwide. The rapid evolution of HIV within infected hosts contributes significantly to the elusiveness of this pathogen from host antiviral responses. The complex nature of HIV envelope glycoprotein that is inherently resistant to neutralization, the selective infection, progressive destruction and impaired regeneration of CD4+ T helper cells, generation of cytotoxic T lymphocyte (CTL) escape mutants, together with high genetic diversity with continually evolving HIV variants worldwide, makes design of an effective vaccine a formidable task. Given the rapidity and unpredictability with which HIV-1 genetic forms may propagate in future, a vaccine protective against all major HIV-1 circulating genetic forms is desirable, which could require multivalent formulations. Understanding the kinetics and directions of this continuing adaptation and its impact on viral fitness, immunogenicity and pathogenicity are crucial to the successful design of effective HIV vaccines. In this review, we focus on extensive diversity of HIV-1, emergence of recombinant forms and their impact on diagnosis, antiretroviral therapy, disease progression, transmission, and vaccine development.