Overcoming barriers to establishing autopsy procurement programs in pediatric patients with central nervous system tumors: a call to develop regional centers.

BACKGROUND: While autopsy-repository programs with a variety of pediatric central nervous system (CNS) tumor types are a critical resource for preclinical neuro-oncology research, few exist and there is no published guidance on how to develop one. The goal of this prospective Pediatric Brain Tumor Repository (PBTR) study was to develop such a program at Cincinnati Children's Hospital Medical Center (CCHMC) and then publish the quantitative and experiential data as a guide to support the development of similar programs. METHODS: Protocols and infrastructure were established-to educate oncologists and families, establish eligibility, obtain consent, address pre- and post-autopsy logistics (e.g., patient and tissue transportation), process and authenticate tissue samples, and collect and analyze data. RESULTS: Of the 129 pediatric CNS tumor patients at CCHMC who died between 2013 and 2018, 109 were eligible for our study. Of these, 74% (81 of 109) were approached for PBTR donation, and 68% (55 of 81) consented. In the final year of the study, approach and consent rates were 93% and 85%, respectively. Median time from death to autopsy (postmortem interval, PMI) was 10 h (range, 1.5-30). In the outpatient setting, PMI increased with distance (from the hospice/home where the patient died to CCHMC). In all patients, PMI appeared to be lower, when consent was obtained more than 24 h before death. CONCLUSIONS: Procurement of autopsy specimens need not be a barrier in neuro-oncology research. Regional centers, strict timing-of-consent, patient education, and dedicated staff are all needed to minimize PMI and, thereby, increase the value of the procured tissue for an array of basic and translational research applications.

Evidence of distinct populations of hepatitis C virus in the liver and plasma of patients co-infected with HIV and HCV.

Viral diversity is an important predictor of hepatitis C virus (HCV) treatment response and may influence viral pathogenesis. HIV influences HCV variability in the plasma; however, limited data on viral variability are available from distinct tissue/cell compartments in patients co-infected with HIV and HCV. Thus, this exploratory study evaluated diversity of the hypervariable region 1 (HVR1) of HCV in the plasma and liver for 14 patients co-infected with HIV and HCV. Median intra-patient genetic distances and entropy values were similar in the plasma and liver compartments. Positive immune selection pressure was observed in the plasma for five individuals and in the liver for three individuals. Statistical evidence supporting viral compartmentalization was found in five individuals. Linear regression identified ALT (P = 0.0104) and AST (P = 0.0130) as predictors of viral compartmentalization. A total of 12 signature amino acids that distinguish liver from plasma E1/HVR1 were identified. One signature amino acid was shared by at least two individuals. These findings suggest that HCV compartmentalization is relatively common among patients co-infected with HIV and HCV. These data also imply that evaluating viral diversity, including drug resistance patterns, in the serum/plasma only may not adequately represent viruses replicating with in the liver and, thus, deserves careful consideration in future studies.

Limited infection with occult hepatitis B virus in drug users in the USA.

AIM: Occult HBV infection (O-HBV) is defined as low level HBV replication in the absence of detectable circulating HBV surface antigen. O-HBV has been implicated in HBV reactivation, advanced liver fibrosis and cirrhosis, reduced interferon response rates, elevated liver enzyme levels, and the development of hepatocellular carcinoma. However, the prevalence of O-HBV has not been clearly established in certain at-risk populations, such as injection drug users. METHODS: Therefore, the current pilot study examined the prevalence of O-HBV in a prospective cohort designed to assess the role of injection and non-injection drug use (IDU) on HIV-associated comorbidities. RESULTS: Utilizing two distinct real-time polymerase chain reaction assays, HBV DNA was not detected in 99 participants examined. CONCLUSION: This finding is in contrast to other data from US IDU cohorts and suggests that the prevalence of O-HBV infection is very specific to the cohort studied, is sensitive to other confounding variables such as hepatitis C virus and/or HIV serostatus, and should not be generalized across risk groups or distinct cohorts.

Alternative Lengthening of Telomeres in Pediatric High-Grade Glioma and Therapeutic Implications.

Pediatric high-grade gliomas (pHGGs), including diffuse intrinsic pontine glioma (DIPG), are highly aggressive tumors with dismal prognoses despite multimodal therapy including surgery, radiation therapy, and chemotherapy. To achieve cellular immortality cancer cells must overcome replicative senescence and apoptosis by activating telomere maintenance mechanisms (TMMs) through the reactivation of telomerase activity or using alternative lengthening of telomere (ALT) pathways. Although the ALT phenotype is more prevalent in pHGGs compared to adult HGGs, the molecular pathway and the prognostic significance of ALT activation are not well understood in pHGGs. Here, we report the heterogeneity of TMM in pHGGs and their association with genetic alterations. Additionally, we show that sensitivity to the protein kinase ataxia telangiectasia- and RAD3-related protein (ATR) inhibitor and the ATR downstream target CHK1 is not specific to pHGG ALT-positive cells. Together, these findings underscore the need for novel therapeutic strategies to target ALT in pHGG tumors.

Limitations of radiosensitization by direct telomerase inhibition to treat high-risk medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Previous studies have elucidated the genomic landscape of MB leading to the recognition of four core molecular subgroups (WNT, SHH, group 3 and group 4) with distinct clinical outcomes. Group 3 has the worst prognosis of all MB. Radiotherapy (RT) remains a major component in the treatment of poor prognosis MB but is rarely curative alone and is associated with acute and long-term toxicities. A hallmark of cancer cells is their unlimited proliferative potential which correlates closely with telomere length. The vast majority of malignant tumors activate telomerase to maintain telomere length, whereas this activity is barely detectable in most normal human somatic tissues, making telomerase inhibition a rational therapeutic target in the setting of cancer recurrence and therapy resistance. We and others have previously shown that short telomeres confer sensitivity to ionizing radiation (IR) suggesting that telomerase inhibition mediated telomere shortening will improve the efficacy of RT while minimizing its side effects. Here, we investigated the efficacy of the combination of IR with IMT, a potent telomerase inhibitor, in an in vivo model of group 3 MB. Our results indicate that although IMT inhibited MB telomerase activity resulting in telomere shortening and delayed tumor growth, the combination with IR did not prevent tumor recurrence and did not improve survival compared to the treatment with IR alone. Together, these findings suggest that the radiosensitization by direct telomerase inhibition is not an effective approach to treat high-risk pediatric brain tumors.

OLIG2 maintenance is not essential for diffuse intrinsic pontine glioma cell line growth but regulates tumor phenotypes.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a pediatric lethal high-grade brainstem glioma with no effective therapies. OLIG2 (oligodendrocyte transcription factor 2) was reported to be critical for the growth of a DIPG cell line CCHMC-DIPG-1. Surprisingly, we found that the CCHMC-DIPG-1 cells express little OLIG2 and exhibit a mesenchymal phenotype, which raised a question regarding the role of OLIG2 in the growth of DIPG cells. METHODS: We evaluated the function of OLIG2 in different DIPG cell lines through molecular and genetic approaches and performed transcriptomic and genomic landscape profiling including whole-genome bisulfite sequencing, RNA-seq, ATAC-seq, and ChIP-seq. shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout approaches were utilized to assess OLIG2 functions in DIPG cell growth. RESULTS: We found that DIPG cells are phenotypically heterogeneous and exhibit the characteristics of distinct malignant gliomas including proneural, classical, and mesenchymal subtypes. OLIG2 knockdown did not impact the growth of CCHMC-DIPG-1 cells, wherein OLIG2 is epigenetically silenced. Moreover, OLIG2 deletion did not substantially impair OLIG2-expressing proneural-like DIPG growth but led to an upregulation of HIPPO-YAP1 and epidermal growth factor receptor (EGFR) signaling and a tumor phenotype shift. Targeting HIPPO-YAP1 and EGFR signaling in OLIG2-deficient DIPG cells inhibited tumor cell growth. CONCLUSIONS: Our data indicate that OLIG2 is dispensable for DIPG growth but regulates the phenotypic switch of DIPG tumor cells. OLIG2 downregulation leads to deregulation of adaptive YAP1 and EGFR signaling. Targeting YAP1 and EGFR pathways inhibits the growth of OLIG2-deficient DIPG cells, pointing to a therapeutic potential by targeting adaptive signaling to treat DIPG tumors with nominal OLIG2 expression.

Distinct Neutrophil Populations in the Spleen During PICS.

While mortality after acute sepsis has decreased, the long-term recovery for survivors is still poor, particularly those developing persistent inflammation, immunosuppression, and catabolism syndrome (PICS). While previously thought that activated neutrophils responding to the acute phase of sepsis migrate to the spleen to undergo cell death and contribute to immunosuppression, our data show a significant accumulation of distinct, yet functional, neutrophil populations in the spleen in a murine model of PICS. The exact role and function of neutrophils in this response is still unclear. The objective of our study was to better define the immune function of splenic neutrophils to determine if this could give insight into the pathogenesis of PICS. Using a murine model of cecal ligation and puncture (CLP), which demonstrates all characteristics of PICS by 8 days, spleens were harvested, and neutrophils were identified by Ly6G and CD11b expression via flow cytometry. Nearly all splenic neutrophils expressed CD54, but there were distinct CD54hi and CD54lo cells, with the majority being CD54lo cells during PICS. The CD54hi population showed traditional, proinflammatory properties, but a relatively decreased chemotactic response, while CD54lo cells had significantly higher chemotaxis, yet significantly decreased proinflammatory functions. Using 5-ethynyl-2'-deoxyuridine (EdU) incorporation, we found that the CD54hi population on day 2 after CLP may be participating in emergency myelopoiesis. However, the vast majority of the CD54lo population were paused in the G1 phase at this time point and not proliferating. By day 8 after CLP, most of the CD54hi cells in the spleen were no longer proliferating, while the CD54lo cells were, indicating that CD54lo dominate in extramedullary myelopoiesis at later time points. Almost none of the neutrophils produced arginase or inducible nitric oxide synthase (iNOS), indicating that these are not suppressor cells. Overall, our data demonstrate that neutrophil accumulation in the spleen during PICS is related to extramedullary myelopoiesis, leading to the production of immature neutrophils. While not suppressor cells, the majority have greater chemotactic function but less inflammatory responsiveness, which may contribute to the immunosuppression seen in PICS. Attention to these distinct neutrophil populations after septic or other systemic inflammatory responses is therefore critical to understanding the mechanisms of PICS.

Diffuse Intrinsic Pontine Glioma Cells Are Vulnerable to Mitotic Abnormalities Associated with BMI-1 Modulation.

Diffuse intrinsic pontine glioma (DIPG) is a poor-prognosis pediatric brain tumor with a median survival of less than 1 year. No effective therapy is currently available, and no therapeutic advances have been made in several decades. We have previously identified BMI-1 as a potential therapeutic target in DIPG and have shown that BMI-1 is highly expressed in DIPG tumors regardless of histone 3 subtype. In the present study, we show that the modulation of BMI-1 leads to DNA damage, M phase cell-cycle arrest, chromosome scattering, and cell death. Interestingly, EZH2 inhibition did not alter these effects. Furthermore, modulation of BMI-1 sensitizes DIPG patient-derived stem-like cells to ionizing radiation (IR). Treatment of DIPG stem-like cells with PTC596, a BMI-1 modulator, and IR impairs the kinetics of DNA damage response (DDR). Both DDR foci formation and resolution were delayed, resulting in further reduction in cell viability compared with either treatment alone. In vivo, treatment of mice bearing DIPG xenografts with PTC596 leads to decreased tumor volume and growth kinetics, increased intratumoral apoptosis, and sustained animal survival benefit. Gene expression analysis indicates that BMI-1 expression correlates positively with DIPG stemness and BMI-1 signature. At the single-cell level, the analysis reveals that BMI-1 pathway is upregulated in undifferentiated cells and positively correlates with stemness in DIPG tumors. IMPLICATIONS: Together, our findings indicate that BMI-1 modulation is associated with mitotic abnormalities, impaired DDR, and cell death, supporting the combination of BMI-1 modulation and radiation as a promising novel therapy for children with DIPG.

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.

Induced Telomere Damage to Treat Telomerase Expressing Therapy-Resistant Pediatric Brain Tumors.

Brain tumors remain the leading cause of cancer-related deaths in children and often are associated with long-term sequelae among survivors of current therapies. Hence, there is an urgent need to identify actionable targets and to develop more effective therapies. Telomerase and telomeres play important roles in cancer, representing attractive therapeutic targets to treat children with poor-prognosis brain tumors such as diffuse intrinsic pontine glioma (DIPG), high-grade glioma (HGG), and high-risk medulloblastoma. We have previously shown that DIPG, HGG, and medulloblastoma frequently express telomerase activity. Here, we show that the telomerase-dependent incorporation of 6-thio-2'deoxyguanosine (6-thio-dG), a telomerase substrate precursor analogue, into telomeres leads to telomere dysfunction-induced foci (TIF) along with extensive genomic DNA damage, cell growth inhibition, and cell death of primary stem-like cells derived from patients with DIPG, HGG, and medulloblastoma. Importantly, the effect of 6-thio-dG is persistent even after drug withdrawal. Treatment with 6-thio-dG elicits a sequential activation of ATR and ATM pathways and induces G2-M arrest. In vivo treatment of mice bearing medulloblastoma xenografts with 6-thio-dG delays tumor growth and increases in-tumor TIFs and apoptosis. Furthermore, 6-thio-dG crosses the blood-brain barrier and specifically targets tumor cells in an orthotopic mouse model of DIPG. Together, our findings suggest that 6-thio-dG is a promising novel approach to treat therapy-resistant telomerase-positive pediatric brain tumors. Mol Cancer Ther; 17(7); 1504-14. ©2018 AACR.

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.

BMI-1 is a potential therapeutic target in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a poor-prognosis pediatric brain tumor. No effective curative therapy is currently available and no therapeutic advances have been made in several decades. BMI-1 is a member of the multimeric protein complex Polycomb repressor complex 1. It is highly expressed in a number of diseases and malignancies and has been implicated in self-renewal of normal and cancer cells, and in DNA damage signaling. The role of BMI-1 in DIPG is largely unknown. Here, we show that BMI-1 is highly expressed in tumor tissue samples of DIPG patients and in patient-derived cancer stem-like cells. BMI-1 downregulation leads to the inhibition of DIPG patient-derived neurosphere cell proliferation, cell cycle signaling, self-renewal, telomerase expression and activity, and suppresses DIPG cell migration. Moreover, targeted inhibition of BMI-1 sensitizes DIPG cells to radiomimetic drug-induced DNA damage. Together, our data validate BMI-1 as a potential therapeutic target to treat children with DIPG.

The transcription factor Olig2 is important for the biology of diffuse intrinsic pontine gliomas.

Background: Diffuse intrinsic pontine glioma (DIPG) is a high-grade brainstem glioma of children with dismal prognosis. There is no single unifying model about the cell of origin of DIPGs. Proliferating cells in the developing human and mouse pons, the site of DIPGs, express neural stem/progenitor cell (NPC) markers, including Sox2, nestin, vimentin, Olig2, and glial fibrillary acidic protein, in an overlapping and non-overlapping manner, suggesting progenitor cell heterogeneity in the pons. It is thought that during a restricted window of postnatal pons development, a differentiation block caused by genetic/epigenetic changes leads to unrestrained progenitor proliferation and DIPG development. Nearly 80% of DIPGs harbor a mutation in the H3F3A or the related HIST1H3B gene. Supporting the impaired differentiation model, NPCs derived from human induced pluripotent stem cells expressing the H3F3A mutation showed complete differentiation block. However, the mechanisms regulating an altered differentiation program in DIPG are unknown. Methods: We established syngeneic serum-dependent and independent primary DIPG lines, performed molecular characterization of DIPG lines in vitro and in an orthotopic xenograft model, and used small hairpin RNA to examine Olig2 function in DIPG. Results: The transcription factor Olig2 is highly expressed in 70%-80% of DIPGs. Here we report that Olig2 expression and DIPG differentiation are mutually exclusive events in vitro, and only DIPG cells that retained Olig2 in vitro formed robust Olig2-positive brainstem glioma with 100% penetrance in a xenograft model. Conclusion: Our results indicate Olig2 as an onco-requisite factor in DIPG and propose investigation of Olig2 target genes as novel candidates in DIPG therapy.

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.

Effects of HCV on basal and tat-induced HIV LTR activation.

Hepatitis C virus (HCV) co-infection occurs in ∼30-40% of the HIV-infected population in the US. While a significant body of research suggests an adverse effect of HIV on HCV replication and disease progression, the impact of HCV on HIV infection has not been well studied. Increasing data suggest that hepatocytes and other liver cell populations can serve as reservoirs for HIV replication. Therefore, to gain insight into the impact of HCV on HIV, the effects of the HCV Core protein and infectious hepatitis C virions were evaluated on basal and Tat-induced activation of the HIV long terminal repeat (LTR) in hepatocytes. The HIV LTR was highly induced by the HIV transactivator protein Tat in hepatocytes. Activation varied according to the number of NF-kB binding sites present in the LTRs from different HIV subtypes. Involvement of the NF-kB binding pathway in LTR activation was demonstrated using an NF-kB inhibitor and deletion of the NF-kB binding sites. TNFα, a pro-inflammatory cytokine that plays an important role in HIV pathogenesis, also induced LTR activity in hepatocytes. However, HIV LTR activity was suppressed in hepatocytes in the presence of HCV Core protein, and the suppressive effect persisted in the presence of TNFα. In contrast, infectious hepatitis C virions upregulated HIV LTR activation and gene transcription. Core-mediated suppression remained unaltered in the presence of HCV NS3/4A protein, suggesting the involvement of other viral/cellular factors. These findings have significant clinical implications as they imply that HCV could accelerate HIV disease progression in HIV/HCV co-infected patients. Such analyses are important to elucidate the mechanisms by which these viruses interact and could facilitate the development of more effective therapies to treat HIV/HCV co-infection.

Genetic Characterization of HIV-1 Strains Among the Injecting Drug Users in Nagaland, India.

Global HIV-1 surveillance has led to the detection of its new recombinant forms. This study was carried out for the first time to elucidate the genetic characterization and evolutionary relationship of HIV-1 strains among injecting drug users of Nagaland, northeastern India. A total of 156 injecting drug users participated in this study voluntarily. Among them 18 were seropositive for HIV-1 (11.5%).The Heteroduplex Mobility Assay (HMA) of HIV-1 based on p24-p7 region of gag gene and C2-V3 region of env gene revealed 11 samples to be subtype C (gag/env), 1 sample as subtype B (gag/env) and 6 samples to be recombinants between subtype C and B. Also, the sequencing and phylogenetic analysis of gag (p24-p7) and env (C2-V3) genes from eighteen samples of Nagaland IDUs with different global HIV-1 strains showed the presence of Indian, African, Thai and their recombinant forms. However, more recombinant strains based on different genomic regions of HIV-1 were detected using Multiregional Hybridization Assay (MHA) where 8 out of 18 samples were found to be recombinants between subtype C and B. Thus, multiregional hybridization assay along with heteroduplex mobility assay can serve as an efficient tool in the characterization of recombination pattern among the newly emerging HIV-1 recombinants.

Molecular characterization of tat gene and long terminal repeat region of human immunodeficiency virus type-1 detected among the injecting drug users (IDUs) of Manipur, India: identification of BC recombinants.

The tat gene of human immunodeficiency virus type-1 (HIV-1) is responsible for the initiation and elongation of viral transcription through the LTR (long terminal repeat) transactivation process. Our study included structural and functional analyses of the tat gene and LTR region of 35 injecting drug users (IDUs) from Manipur (a north-eastern state in India and a potential source of HIV-1 recombinants) in order to search for the recombinants and variation in the transactivation process if any due to recombination. Analysis showed prevalence of subtype C with few BC recombinants for the tat gene showing identical recombination breakpoints. Phylogenetic analysis of the LTR region of those IDU strains showed strong resemblance to Indian subtype C forming a completely separate cluster from the other global C LTR sequences. The TAR element (transactivator response region) in all the LTR sequences was fairly conserved. Further study of the transactivation rate of the C and BC tat for the Manipur C LTR showed almost equal transactivity in both the cases. This is the first report of characterisation of tat gene and LTR region of HIV-1 samples among IDUs from north-eastern India.

Phylogenetic analysis of the p24-p7 region of the human immunodeficiency virus type 1 gag gene to determine subtype distribution among female sex workers in Calcutta, India.

Human immunodeficiency virus type 1 (HIV-1) subtype C, based on the envelope region, has been reported to be predominant in India. We sequenced the p24-p7 gag region from 51 HIV-1 seropositive female sex workers in Calcutta, India, for more-detailed molecular characterization. Subtype C was found to be prevalent, although no strong monophyletic cluster was observed.