Zubair Akmal, Zeb Adnan, Shahid Syed Ahsan, Javed Hasnain, Ali Muhammad
Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan.
Natural and Medical Sciences Research Center, University of Nizwa, Birkat al Mouz, 616, Nizwa, Oman.
Virus Genes. 2025 Jun;61(3):324-341. doi: 10.1007/s11262-025-02149-z. Epub 2025 Mar 27.
This study aimed to determine sub-subtypes and circulating recombinant forms (CRFs) in Pakistan using the pol gene, identification of amino acid substitutions, structural changes, and drug resistance evaluation in the p66 region of reverse transcriptase. A total of 50 HIV-positive blood samples were collected from Lahore Pakistan, confirmed by Real Time-Polymerase Chain Reaction. The samples were further processed for pol gene amplification followed by nucleotide sequencing through the Sanger method. Out of 50 samples, 26 samples were amplified, and 14 sequences were obtained. The sequences were aligned with reference sequences to determine subtyping and phylogenetic analysis. Moreover, amino acid substitutions and drug resistance patterns were also determined in the RTp66 region. Phylogenetic analysis showed that 8 sequences of our isolates were closely related to circulating recombinant form (CRF43_02 G), and 3 sequences were of CRF30_026 (CRF02_AG) subtypes while the remaining 3 sequences were related to CRF35_A1D, CRF95_02B (CRF02_AG) and Subtype G of HIV-1. Several amino acid substitutions were identified in our isolates which show no impact on the structure of protein. Furthermore, the isolate QAU-AZ2 (OR086936) proposes variable degree of resistance to nevirapine (NVP), etravirine (ETR), rilpivirine (RPV), efavirenz (EFV), Doravirine (DOR); while no resistance against NNTRI and NTRI was observed in the remaining isolates. Further studies are required to (i) examine the function of identified amino acid substitutions through molecular docking, and (ii) sequence the complete pol gene of the viral isolates from Pakistani patients to determine possible drug resistance associated with amino acid substitutions.
本研究旨在利用pol基因确定巴基斯坦的HIV-1亚亚型和流行重组型（CRF），鉴定逆转录酶p66区域的氨基酸替换、结构变化及耐药性评估。从巴基斯坦拉合尔共收集了50份HIV阳性血液样本，通过实时聚合酶链反应进行确认。对样本进一步处理以扩增pol基因，随后采用桑格法进行核苷酸测序。50份样本中，26份样本成功扩增，获得了14条序列。将这些序列与参考序列进行比对以确定亚型和系统发育分析。此外，还在RTp66区域确定了氨基酸替换和耐药模式。系统发育分析表明，我们分离株的8条序列与流行重组型（CRF43_02G）密切相关，3条序列属于CRF30_026（CRF02_AG）亚型，其余3条序列与CRF35_A1D、CRF95_02B（CRF02_AG）及HIV-其亚型G相关。在我们的分离株中鉴定出了几种氨基酸替换，这些替换对蛋白质结构无影响。此外，分离株QAU-AZ2（OR086936）对奈韦拉平（NVP）、依曲韦林（ETR）、利匹韦林（RPV）、依非韦伦（EFV）、多拉韦林（DOR）呈现不同程度的耐药性；而其余分离株未观察到对NNTRI和NTRI的耐药性。需要进一步开展研究以：（i）通过分子对接研究已鉴定的氨基酸替换的功能；（ii）对巴基斯坦患者病毒分离株的完整pol基因进行测序，以确定与氨基酸替换相关的可能耐药性。
