Partial pol Sequences from Drug Naive HIV-2 Infected Individuals from Maharashtra,India

Infection with HIV-2 is associated with slower disease progression, low plasma viral loads, and little or no signs or symptoms. Although epicenter of HIV-2 epidemic is believed to be in West Africa, over the years it has spread to other parts of Africa, Europe, United States, as well as India. In India, the prevalence of HIV-2 ranges between 0.29% and 0.8% ^{1 –3} of about 2.1 million HIV infected individuals with estimated 11,500 HIV-2 infections. ⁴ Antiretroviral therapy (ART) differs for HIV-2 infection as the virus possesses natural resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs). ⁵ Most studies on drug resistance are focused mainly on HIV-1 while limited information exists with regard to HIV-2 due to its low prevalence. Hence, it is difficult to monitor the effectiveness of treatment in HIV-2 infected individuals.

The Indian ART guidelines for HIV-2 include two nucleoside reverse transcriptase inhibitors (NRTIs), tenofovir (TDF) and lamivudine (3TC), along with protease inhibitors (PIs), lopinavir boosted ritonavir (LPV/r) in first line, and raltegravir (RAL) with boosted darunavir (DRV) in second line. These limited treatment options make it imperative to know the drug resistance before initiating the ART. Use of PIs and NRTIs makes it important to study the pol gene of HIV-2. Understanding the drug resistance in treatment naive individuals serves important criteria to be considered while determining the treatment policies. Data on the primary drug resistance in HIV-2 infected individuals are not available from India. Hence, in this study, we assessed the presence of drug resistance to NRTIs and PIs in ART naive HIV-2 infected individuals through sequencing the pol gene of HIV-2. The sequence analysis of pol gene showed presence of two unique residues in the protease gene that are specific for Indian HIV-2 viruses. Furthermore, the phylogenetic analysis of all samples confirmed that they belong to HIV-2 subtype A.

The study was carried out on frozen whole blood specimens (in ethylenediaminetetraacetic acid [EDTA]) collected (during January 2014 to August 2014) after obtaining the written informed consent from 32 individuals who were diagnosed with HIV-2 infection in the regional Integrated Counseling and Testing Centers from 13 districts of Maharashtra state. HIV-2 infection was confirmed by Western blot (NEW LAV BLOT II; BioRad, France). The ethics committee of National AIDS Research Institute, Pune, approved the study and use of stored samples. All study participants were ART naive adults with median age of 47 years (range 18–66 years) including 23 males and 9 females.

Total nucleic acids were extracted from 300 μL whole blood using automated NucliSENS^® easyMAG (BioMérieux, France) platform. The pol gene was amplified by nested reverse transcription polymerase chain reaction and sequenced using primers and protocol described previously ⁶ with slight modifications. The primers used for amplification of pol gene covered 801 nucleotides spanning complete PR gene of 99 amino acids and partial RT gene of 168 amino acids. The details of primers and thermal cycling conditions are given in Table 1. Amplified products were sequenced by Sanger sequencing using BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystem, CA). Sequences were compiled using SeqScape v2.7. The phylogenetic analysis of 32 sequences performed with 57 previously reported sequences in Los Alamos HIV database (https://www.hiv.lanl.gov) showed that the study sequences clustered together with previously reported HIV-2 subtype A isolates from Guinea Bissau, Gambia, and two Indian isolates (Fig. 1a). ^{7 –10} The online HIV subtyping tool, REGA (http://bioafrica.mrc.ac.za/rega-genotype/html/subtypinghiv.html), also confirmed HIV-2 subtype A for all the samples.

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FIG. 1.

Phylogenetic relationship between HIV-2 isolates and signature amino acids. (a) Shows maximum likelihood tree constructed using general time reversible model of substitution plus gamma distribution with invariant sites (GTR+G+I) in MEGA 7.0 software. ML tree represents 89 sequences and this includes 32 study sequences (denoted with red dot), other HIV-2 sequences (labeled in brown text), HIV-2 subtype A sequences reported globally (labeled in green text), and sequences from SIV (labeled in blue text). The tree was constructed with 1000 bootstrap replicates; (b) VESPA displaying the signature amino acids in study sequences compared with reference HIV-2 subtype A sequences. ML, maximum likelihood; SIV, simian immunodeficiency virus; VESPA, Viral Epidemiology Signature Pattern Analysis. Color images are available online.

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Table 1.

Details of Primers and Thermal Cycling Conditions for Amplification and Sequencing

Step	Primers	Cycling conditions
Reverse transcription	—	50°C for 30 min followed by 95°C for 15 min
First round PCR	JA218 f 5′-GAAAGAAGCCCCGCAACTTCC-3′ JA221 r 5′-GCTCTGCTTCTGCTAATTCTGTCCA-3′	25 cycles of 94°C for 20 s 56°C for 20 s 72°C for 2 min Final extension at 72°C for 10 min 4°C for ∞
Second round PCR	JA219 f 5′-AGGGGCTA/GACACCAACAGCAC-3′ JA220 r 5′-GTCTTTATT/CCCTGGGTAGATTTGTG-3′	30 cycles of 95°C for 15 s 58°C for 45 s 72°C for 2 min Final extension at 72°C for 5 min 4°C for ∞
Sequencing PCR	JA219 f 5′-AGGGGCTA/GACACCAACAGCAC-3′ JA220 r 5′-GTCTTTATT/CCCTGGGTAGATTTGTG-3′ JA222 f 5′-ACCTCCAACTAATCCTTATAATACC-3′ JA223 r 5′-ACTGAATTTCTGTGAAATCTTGAGT-3′	96°C for 1 min 96°C for 10 s 50°C for 5 s 60°C for 4 min 4°C for ∞

PCR, polymerase chain reaction.

The Viral Epidemiology Signature Pattern Analysis ¹¹ of 32 study sequences against 87 HIV-2 subtype A sequences spanning pol region 2638–3650 (BEN coordinates; HIV-2 isolate [accession no: M30502]) from various countries excluding India (www.hiv.lanl.gov) showed two signature amino acid residues in the protease region of study sequences. Compared with background reference sequences, the most common amino acid at position 14 in protease of study sequences was tyrosine (Y), whereas the common amino acid at this position in reference sequences was histidine (H). Another signature amino acid in study sequences was lysine (K) at position 70 in protease while the same position of reference sequences had arginine (R) as the most common amino acid (Fig. 1b).

Drug resistance mutations conferring resistance to PIs and NRTIs were analyzed using earlier reported online tool “HIV-2EU” (www.hiv-grade.de/HIV2EU/deployed/grade.pl?program=hivalg). ¹² As reported worldwide, mutations Y181I and Y188L known to confer natural resistance against NNRTIs were observed in all 32 sequences. ^13,14 Three out of 32 (9.37%) sequences showed presence of mutations associated with resistance against NRTIs and PIs (Table 2). The resistance against NRTIs only was seen in two samples, whereas one sample had mutations associated with NRTIs as well as PIs. M184V mutation associated with resistance to 3TC was present in all three sequences. Presence of this mutation indicates high-level drug resistance to 3TC. Mutations V111I and K65R associated with resistance to NRTIs were seen in two and one sample, respectively. The mutation V111I has been identified as a novel mutation in the polymerase domain that does not strongly affect drug susceptibility but increases the virus fitness in presence of mutations K65R and Q151M. ¹⁵ Mutations K65R and Q151M have been observed along with V111I in two samples. Mutation V111I in presence of Q151M indicates resistance to TDF, ¹² which is an integral part of the first-line ART for HIV-2 infection in India. As the samples were from the treatment naive individuals, presence of drug resistance mutations indicates transmission of resistant virus.

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Table 2.

Drug Resistance Mutations Associated with Protease Inhibitors and Nucleoside Reverse Transcriptase Inhibitors Resistance

Sample ID	Mutations associated with DR against PIs	Mutations associated with DR against NRTIs	Resistance against ARVs	Intermediate resistance against ARVs
NARI_DR_HIV_2_pol_8	None	M184IM	3TC, FTC	—
NARI_DR_HIV_2_pol_12	None	V111I, Q151M, M184V	3TC, ABC, AZT, D4T, DDI, FTC, TDF/TAF	—
NARI_DR_HIV_2_pol_30	I50L, I82F	K65R, N69S, V111I, M184V	3TC, ABC, DDI, FTC, TDF/TAF ATV/r, IDV/r	D4T, LPV/r

3TC, lamivudine; ABC, abacavir; ATV, atazanavir; ATV/r, atazanavir boosted ritonavir; AZT, ziduvudine; D4T, stavudine; DDI, didanosine; FTC, emticitabine; IDV, indinavir; LPV/r, lopinavir boosted ritonavir; NRTIs, nucleoside reverse transcriptase inhibitors; PI, protease inhibitor; TDF/TAF, tenofovir alafenamide.

Information on ART drug resistance is crucial to achieve the primary goals to contain the spread of the virus. However, to our knowledge, no information on pretreatment drug resistance in HIV-2 infected individuals is available from India. The pretreatment drug resistance of 9.37% is slightly closer to the expected prevalence (10%) among the ART initiators. ¹⁶ Hence, findings from this study highlight the need for country-wide drug resistance analysis on larger number of samples to get a clear idea about HIV-2 drug resistance in the country, which will help the policy makers for deciding ART guidelines for HIV-2 infected individuals.

Sequence Data

The 32 pol sequences have been submitted to GenBank with accession numbers MG460633 to MG460664.