HIV-1 DNA Testing in Viremic Patients Identifies More Drug Resistance Than HIV-1 RNA Testing.

Background: The Department of Health and Human Services HIV-1 Treatment Guidelines recommend drug resistance testing in HIV-1 RNA to guide the selection of antiretroviral therapy in patients with viremia. However, resistance-associated mutations (RAMs) in HIV-1 RNA may reflect only the patient's current regimen and can be lost during prolonged absence of therapy. We determined if HIV-1 DNA testing can provide drug resistance information beyond that identified in contemporaneous plasma virus. Methods: This was a retrospective database review of results obtained for patients with viremia for whom commercial HIV-1 RNA and HIV-1 DNA drug resistance testing was ordered on the same day. Resistance-associated mutations and drug susceptibility calls were compared between paired tests, and the effect of HIV-1 viral load (VL) on test concordance was assessed using Spearmen's rho correlation. Results: Among 124 paired tests, more RAMs were identified in HIV-1 DNA in 63 (50.8%) cases, and in HIV-1 RNA in 11 (8.87%) cases. HIV-1 DNA testing captured all contemporaneous plasma virus RAMs in 101/117 (86.3%) cases and identified additional RAMs in 63/117 (53.8%) cases. There was a significant positive correlation between the viral load at the time of resistance testing and the percentage of plasma virus RAMs detected in HIV-1 DNA (rs = 0.317; P < .001). In 67 test pairs demonstrating pan-sensitive plasma virus, resistance in HIV-1 DNA was seen in 13 (19.4%) cases. Conclusions: HIV-1 DNA testing identified more resistance than HIV-1 RNA testing in most patients with viremia and may be informative in patients whose plasma virus reverts to wild-type following therapy discontinuation.

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.

High replication fitness and transmission efficiency of HIV-1 subtype C from India: Implications for subtype C predominance.

HIV-1 subtype C has been the predominant subtype throughout the course of the HIV-1 epidemic in India regardless of the geographic region of the country. In an effort to understand the mechanism of subtype C predominance in this country, we have investigated the in vitro replication fitness and transmission efficiency of HIV-1 subtypes A and C from India. Using a dual infection growth competition assay, we found that primary HIV-1 subtype C isolates had higher overall relative fitness in PBMC than subtype A primary isolates. Moreover, in an ex vivo cervical tissue derived organ culture, subtype C isolates displayed higher transmission efficiency across cervical mucosa than subtype A isolates. We found that higher fitness of subtype C was not due to a trans effect exerted by subtype C infected PBMC. A half genome A/C recombinant clone in which the 3' half of the viral genome of subtype A was replaced with the corresponding subtype C3' half, had similar replicative fitness as the parental subtype A. These results suggest that the higher replication fitness and transmission efficiency of subtype C virus compared to subtype A virus from India is most probably not due to the envelope gene alone and may be due to genes present within the 5' half of the viral genome or to a more complex interaction between the genes located within the two halves of the viral genome. These data provide a model to explain the asymmetric distribution of subtype C over other subtypes in India.

Construction and characterization of an infectious molecular clone of HIV-1 subtype A of Indian origin.

India has the second highest number of HIV-1 infected people next to South Africa. The predominant proportion of HIV-1 circulating in India is of subtype C origin, with a small fraction made up of subtypes A and B. In this report, we describe the construction and characterization of the first full-length infectious molecular clone p1579A-1 HIV-1, from an HIV-1 subtype A infected person from India, using long PCR and successive ligation of the amplimers. Phylogenetic analysis of the sequence of the entire proviral DNA and LTR confirmed p1579A-1 to be an HIV-1 subtype A. Analysis of the env gene of p1579A-1 showed a conserved GPGQ motif and the absence of basic amino acids at positions 11 and 25 suggesting CCR5 coreceptor usage. Analysis of env N-linked glycosylation sites revealed fewer sites in the V1 region of envelope compared to other subtype A. Transcription factor binding site analysis of the LTR sequences identified conserved as well as unique transcription factor binding sites (TFBS) in p1579A-1. This infectious clone of HIV-1 can be useful to study the molecular mechanism of dominance of subtype C in India.