Emergence of HIV-1 Unique DG Recombinant Form in Pakistan

Human immunodeficiency virus type 1 (HIV-1) global epidemics are predominantly driven by group M subtypes, which comprises nine different subtypes (A, B, C, D, F, G, H, J, and K) and several circulating recombinant forms (CRFs). ¹ New CRFs continue to emerge due to frequent recombination between existing strains, especially in high-risk patients who are continuously exposed to diverse HIV-1 strains. ² These recombination events can also generate complex genetic variants and contribute to immune escape, drug resistance, therapeutic failure, and enhanced transmission. ²

The first incidence of HIV-1 infection in Pakistan was reported in 1987. Ever since, HIV-1 cases have continuously increased, turning from isolated cases to outbreaks to a concentrated epidemic, which has now bridged in low-risk communities. ³ Currently, ⁴ Pakistan is experiencing one of the largest HIV-1 outbreaks in children worldwide in the Larkana district of the Sindh province. ⁵ The HIV epidemic in Pakistan predominantly comprises subtype A, ⁶ followed by 12 other subtypes (B, C, D, G, A1D, A1G, 01G, CG, 01_AE, 02A1, 02_AG, and 35_AD) that are commonly circulating in the country. ¹ Recently, the emergence of new CRFs have been observed in Pakistan, for example, CRF56_cpx that was reported by our group last year; and was found to contain multiple major drug resistance mutations (DRMs). ¹

In this study, we report the first case of unique recombinant form (URF) DG from Pakistan. This URF was sequenced from an HIV-infected 35-year-old male patient suffering from virological failure. This patient was diagnosed with HIV-1 infection in 2005 and was reported to have contracted the infection from a blood transfusion in 2004. The patient consented to participate in the study that was approved by the Aga Khan University Ethical Review Committee. The sequence (pol; nucleotides 954 bp) was submitted to GenBank and assigned with accession no. MK927054 (henceforth referred to as AKUDRM_02). HIV-1 genotyping was performed using the REGA HIV subtyping tool, where this sequence was identified as subtype G recombinant form. The results were confirmed by jpHMM ⁷ and RIP tools. ⁸ These recombination analyses identified the virus as the recombinant form of HIV-1 subtype G with HIV-1 subtype D (Fig. 1A). In the next step, BLAST search was performed using the HIV BLAST tool available at the HIV Los Alamos Database (www.hiv.lanl.gov) to identify database sequences exhibiting sequence similarity with our study sequence. The result of BLAST search found 92% sequence identity of AKUDRM_02 sequence with HIV-1 URF_DG sequence submitted from the United Kingdom in 2013 (accession no. MF109700). The sequence homology was further confirmed by maximum likelihood phylogenetic analysis, where AKUDRM_02 sequence clustered with HIV-1 URF_DG sequence from the United Kingdom in 2013 (accession no. MF109700) with aLRT branch support value 1 (near perfect score; Fig. 1B). Furthermore, the recombination analysis of the URG_DG from United Kingdom revealed the recombination sites between the two URFs (United Kingdom and Pakistan) to be almost similar (Fig. 1C).

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FIG. 1.

Recombination pattern and phylogenetic analysis. (A) The recombinant pattern observed in RIP analysis, with 90% confidence threshold and window size 400 residues. X- and Y-axes represent, respectively, the similarity score and the window of analysis. Light and dark gray lines represent, respectively, the HIV-1 subtype G and D fragments in AKUDRM_02 sequence. (B) Maximum likelihood analysis of the AKUDRM_02 sequence was performed using PhyML tool. The phylogenetic tree was constructed using HIV partial pol sequence from AKUDRM_02 sequence and HIV reference sequences belonging to different subtypes and CRFs obtained from HIV Los Alamos Database. The AKUDRM_02 sequence (identified as URF_DG) is shown with gray font and is pointed out by a black arrow. Gray branches signify an LRT SH-like support of ≥0.9. (C) The recombinant pattern observed in RIP analysis for URF_DG from United Kingdom. Light and dark gray lines represent, respectively, the HIV-1 subtype G and D fragments in URF_DG sequence from the United Kingdom in 2013 (accession no. MF109700). CRFs, circulating recombinants forms; URF, unique recombinant form.

This recombinant form has evolved from recombination between HIV-1 subtype D and G, and to date only one sequence has been submitted from the United Kingdom in 2013. Important to note is that the U.K. sequence was submitted as an unassigned subtype and was later characterized as a unique DG recombinant by the Los Alamos Database.

At the time of diagnosis, the baseline CD4 count of the patient was 62, whereas the viral load was 14,965 copies/mL. The patient was started on the first-line antiretroviral therapy (ART), comprising tenofovir/lamivudine (3TC)/efavirenz (EFV) in 2006, however, switched to second-line therapy in 2009 due to poor response. After a year of second-line therapy regimen, third-line ART was started in 2011, which was able to control the viral load and restore CD4 count for 5 years. In 2017, however, the patient experienced virological failure and his CD4 count dropped. Soon after, his sample was analyzed for ART DRM to reveal the presence of three major (M46I, I54V, and V82S) and one minor (L24I) protease inhibitor DRMs in, and three non-nucleoside reverse transcriptase inhibitor DRMs (D67N, K70Q, and L74V).

The aforementioned DRMs are associated with high-level resistance against atazanavir/r (ATV/r), fosamprenavir/r (FPV/r), indinavir/r (IDV/r), lopinavir/r (LPV/r), nelfinavir (NFV), saquinavir/r (SQV/r), and didanosine (DDI); intermediate-level resistance against tipranavir/r (TPV/r), stavudine (D4T), and abacavir (ABC); and low-level resistance against zidovudine (AZT), emtricitabine (FTC), lamivudine (3TC), and tenofovir (TDF). Drug resistance analysis of database sequence (U.K. sequence, accession no. MF109700) was also performed, to compare its drug resistance profile with Pakistani strain. Interestingly, the database sequence had no DRM in the protease and reverse transcriptase region.

The current report of the recombinant form URF_DG suggests that in addition to prevalent subtypes and CRFs, new unassigned HIV-1 URFs are also circulating in Pakistan, indicating that the current HIV epidemic is far more complex than imagined and possibly comprises overlapping transmission chains allowing possible coinfections with the existing HIV-1 strains, resulting in emergence of new URFs. This report warrants the need for continuous monitoring of genotype and drug resistance profile of HIV-positive patients and suggests the implementation of effective infection control measures to control the HIV epidemic in Pakistan.