Ramdas Chatterjee

Human Immunodeficiency Virus Type 1 Env Sequences from Calcutta in Eastern India: Identification of Features That Distinguish Subtype C Sequences in India from Other Subtype C Sequences

ABSTRACT India is experiencing a rapid spread of human immunodeficiency virus type 1 (HIV-1), primarily through heterosexual transmission of subtype C viruses. To delineate the molecular features of HIV-1 circulating in India, we sequenced the V3-V4 region of viralenv from 21 individuals attending an HIV clinic in Calcutta, the most populous city in the eastern part of the country, and analyzed these and the other Indian sequences in the HIV database. Twenty individuals were infected with viruses having a subtype Cenv, and one had viruses with a subtype Aenv. Analyses of 192 subtype C sequences that included one sequence for each subject from this study and from the HIV database revealed that almost all sequences from India, along with a small number from other countries, form a phylogenetically distinct lineage within subtype C, which we designate CIN. Overall, CIN lineage sequences were more closely related to each other (level of diversity, 10.2%) than to subtype C sequences from Botswana, Burundi, South Africa, Tanzania, and Zimbabwe (range, 15.3 to 20.7%). Of the three positions identified as signature amino acid substitution sites for CIN sequences (K340E, K350A, and G429E), 56% of the CIN sequences contained all three amino acids while 87% of the sequences contained at least two of these substitutions. Among the non-CINsequences, all three amino acids were present in 2%, while 22% contained two or more of these amino acids. These results suggest that much of the current Indian epidemic is descended from a single introduction into the country. Identification of conserved signature amino acid positions could assist epidemiologic tracking and has implications for the development of a vaccine against subtype C HIV-1 in India.

Identification of Subtype C Human Immunodeficiency Virus Type 1 by Subtype-Specific PCR and Its Use in the Characterization of Viruses Circulating in the Southern Parts of India

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) subtype C viruses are associated with nearly half of worldwide HIV-1 infections and are most predominant in India and the southern and eastern parts of Africa. Earlier reports from India identified the preponderance of subtype C and a small proportion of subtype A viruses. Subsequent reports identifying multiple subtypes suggest new introductions and/or their detection due to extended screening. The southern parts of India constitute emerging areas of the epidemic, but it is not known whether HIV-1 infection in these areas is associated with subtype C viruses or is due to the potential new introduction of non-subtype C viruses. Here, we describe the development of a specific and sensitive PCR-based strategy to identify subtype C-viruses (C-PCR). The strategy is based on amplifying a region encompassing a long terminal repeat and gag in the first round, followed by two sets of nested primers; one amplifies multiple subtypes, while the other is specific to subtype C. The common HIV and subtype C-specific fragments are distinguishable by length differences in agarose gels and by the difference in the numbers of NF-κB sites encoded in the subtype C-specific fragment. We implemented this method to screen 256 HIV-1-infected individuals from 35 towns and cities in four states in the south and a city in the east. With the exception of single samples of subtypes A and B and a B/C recombinant, we found all to be infected with subtype C viruses, and the subtype assignments were confirmed in a subset by using heteroduplex mobility assays and phylogenetic analysis of sequences. We propose the use of C-PCR to facilitate rapid molecular epidemiologic characterization to aid vaccine and therapeutic strategies.

High incidence and prevalence of HIV-1 infection in high risk population in Calcutta, India

HIV-1 infection in India has been increasing steadily over the last decade. In the absence of potent antiviral therapy, estimates of HIV infection are needed to monitor the epidemic, institute prevention strategies in target populations and determine the suitable populations for vaccine studies. In this report we present the HIV-1 seroprevalence and annual estimates of seroincidence in a high risk population from Calcutta, the most populous city in the eastern part of India. In 1206 high risk subjects tested over two years between February of 1999 and December 2000, we have determined an overall seroprevalence of 40.1% using enzyme-linked immunosorbent assay followed by a confirmatory Western blot testing. Furthermore, using a newly described Standardized Testing Algorithm for Recent HIV-1 Seroconversion (STARHS), we have estimated an annual seroincidence rate of about 7% in this population during this two-year study. Such a high annual seroincidence rate makes this population well suited for studies of HIV-1 prevention, including vaccine trials.

Genetic and functional characterization of the LTR of HIV-1 subtypes A and C circulating in India.

Genetic analysis of HIV-1 sequences circulating in different parts of India have shown that the predominant proportion of HIV-1 subtypes circulating in India is type C and a small fraction are subtypes A, B, E, and CRFs. We sequenced the HIV-1 LTR promoter region of seven subtype C and five subtype A isolates obtained from two major cities in India. Sequence analysis of the complete promoter and TAR regions revealed conserved subtype-specific variability in several major binding sites. Three NF-kappaB sites were present in all subtype C isolates and two isolates contained an insertion in the MFNLP. The transcriptional activity of one of these isolates may have been hindered due to this insertion. Despite the apparent variability between the LTRs we did not observe any significant difference in the transcriptional activity between subtype C and subtype A. To our knowledge, this is the first study characterizing the genetic structure and functional attributes of subtype A LTRs from India.

Immunogenicity of human immunodeficiency virus (HIV) reverse transcriptase: Detection of high levels of antibodies to HIV reverse transcriptase in sera of homosexual men

Immunoglobulin isolated from sera of homosexual men infected with human immunodeficiency virus (HIV) inhibited the reverse transcriptase (RT) activity of HIV. The inhibitory activity was specifically directed against HIV RT, and not against other mammalian retrovirus RT, including human T-lymphotropic virus type I. The relative titer of anti-RT antibody was significantly higher in asymptomatic men than in patients with lymphadenopathy or acquired immune deficiency syndrome (AIDS)-related complex. There was no correlation between the relative titer of anti-RT antibody and the relative titers of antibodies to major virion structural protein as determined by the enzyme-linked immunosorbent assay (ELISA) technique. These data suggest that antibodies to HIV RT may be related to the clinical status and possibly to the different degree of HIV replication in HIV-infected homosexual men.

Genetic characterization of HIV-1 from semen and blood from clade C-infected subjects from India and effect of therapy in these body compartments

Biologic and genetic differences between HIV-1 clade C in India and clade B in US suggest that the effect of anti-viral therapy in various body compartments may differ between these two clades. We examined the effect of therapy on viral loads in semen and blood of HIV-1-clade C infected subjects from India and evaluated whether HIV-1 in the semen is different from that in blood in these subjects. HIV-1 RNA was detected in semen and blood at all stages of the disease. Viral loads in semen and blood were strongly correlated with each other, but not with the CD4+ T cell count. Anti-viral treatment reduced viral load drastically in blood and semen within one month of post therapy. Genetic characterization of HIV-1 in the semen and blood demonstrated that they were highly compartmentalized. These data have important implications of sexual transmission of HIV-1 in clade C HIV-1 infected subjects.

Estimation of the Predictive role of Plasma Viral load on CD4 decline in HIV-1-subtype C infected subjects in India

Background:Plasma viral load has been shown to be a meaningful prognostic marker for disease progression in untreated, HIV-1 subtype B-infected subjects in United States and Western Europe and therefore used as a prognostic marker for disease progression. Because of high expenses of commercially available viral load assays, the role of viral load in disease progression has not been evaluated in HIV-1 subtype C-infected patients in India. Methods:We developed an inexpensive real-time reverse transcriptase-polymerase chain reaction assay to quantify viral load in plasma of HIV-1 subtype C-infected subjects from India and used it in a longitudinal analysis of viral load and CD4 cell number in HIV-infected subjects from Calcutta, India. Results:The real-time reverse transcriptase-polymerase chain reaction assay can quantify plasma viral load with a linear range of detection from 102 to 109 HIV-1 RNA copies per input. Longitudinal analysis of viral load in a cohort of 39 subjects over an average period of approximately 3 years indicates that 1-log increase in HIV-1 RNA level was associated with a decline of 67 CD4 cell count. Furthermore, HIV-1 RNA level between 500 and 50,000 copies per milliliter would predict a 12.9% decrease in CD4 cell count per year, whereas HIV-1 RNA levels above 50,000 copies HIV-1 RNA per milliliter would predict a 25.3% decrease in CD4 cells per year. In addition, we estimated that the mean incubation period of disease development, as defined by the loss of CD4 below 200, is 8.2 years. Conclusion:Our report on the level of viral load on predicting CD4 decline in Indian subjects with HIV-1 provides an additional important tool to the physicians for treating and planning a therapeutic strategy to control HIV-1 infection in India.

Assessment of HIV/AIDS Awareness Level of High School Students in Kolkata

Youths are likely to engage in risky behavior without accurate knowledge about HIV/AIDS. Assessment of their knowledge regarding the disease helps in developing strategies to prevent it. Aim of this study was to evaluate knowledge level of high school students of Kolkata, India, their attitude towards the disease, and enhance their awareness. Effect of previous awareness programs on HIV/AIDS is not well understood. In this cross-sectional study, students (N=2373) were from 38 randomly selected higher secondary schools. Based on answers to questions of a pre-designed, pre-tested, anonymous questionnaire, knowledge level of the students and their attitude towards HIV/AIDS were ascertained using SPSS version 17.0. Approximately one-third (36.5%) of the students knew full form of HIV and AIDS. Many (67%) believed that AIDS can be prevented and 45.9% thought AIDS is curable. Very few (2.6%, 0.6%) identified three major modes of transmission of HIV/AIDS and its three different symptoms. Television was the main source of information about HIV/AIDS for the students. Many students of Kolkata were still deficient in accurate knowledge on HIV/AIDS though they showed positive attitude towards the disease. Frequent awareness programs on HIV/AIDS are required to ensure that students attend at least one program during school curriculum.