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Emergence of Novel Unique Recombinant Forms and Multiple Subtypes in Gag-Pol Region of HIV-1 in Punjab, Pakistan

PJZ_54_5_2353-2363

Emergence of Novel Unique Recombinant Forms and Multiple Subtypes in Gag-Pol Region of HIV-1 in Punjab, Pakistan

Amreen Zahra1*, Mushtaq A. Saleem2, Hasnain Javed3,

Muhammad Azmat Ullah Khan4 and Abdul Rauf Shakoori4

1Department of Biochemistry, University of Central Punjab, Lahore 54782, Pakistan

2Ghazi National Institute of Engineering and Sciences (GNIES), Taunsa by Pass Road, Pakistan Chowk DG Khan, Pakistan

3Advanced Diagnostic Laboratory, Punjab AIDS Control Programme, Government of Punjab, Lahore 54000, Pakistan

4Department of Biochemistry and Molecular biology, University of Gujrat, Gujrat 50700, Pakistan

5School of Biological Sciences, University of the Punjab, Quaid-I-Azam Campus, Lahore 54590, Pakistan

ABSTRACT

The current era faces a major health challenge of drug resistance and viral escape mutants of human immunodeficiency virus (HIV-1) worldwide. Therefore, a deep molecular insight unveiling the genetic characteristics of the prevailing strains is the need of the hour for successful disease control interventions in endemic countries like Pakistan. Here we present, a detailed molecular and computational analysis of the major genetic constituents, Gag-Pol region of HIV-1 for the very first time in Punjab-Pakistan including three regions of outbreak i.e. Kot Imrana Sargodha, Faisalabad and Gujrat. Drug resistance mutations such as T12A, I13A, K14R, I15V, K20I, T31S, E35D, M36I, N37D, R41K, K43X, R57K, Q61H, H69K, K70R and L89M were observed across several sites across the whole length of PR region among the isolates. A significant accessory resistance mutation V32I conferring a low level of resistance against non-nucleoside reverse transcriptase inhibitors was observed among two isolates. This kind of genotypic pattern demonstrated a striking evidence of multiple transmission routes and co-circulating viruses due to increased genetic diversity in Pakistani isolates. However, SNAP analyses demonstrated higher values for Subtype A1 and Subtype C denoting a positive selection pressure and sequence conservation among the HIV-1 isolates in Pakistan. Our phylogenetic analyses revealed that HIV subtype A1 was found to be the most predominant subtype found in Pakistani population with 30% ratio among the isolates, 10% was of CRF02_AG, Subtype C a potential recombinant had 3.33% and unique recombinant form URFs accounted for 13.3% in the HIV Gag sequences. All Pol (URFs) demonstrated 40% of ratio among the isolates, and Subtype D with 6.66%. The present study shows that multiple subtypes of HIV-1 are present in the affected population. Hence, this study highlights the demand of continuous future studies on disease surveillance coupled with responsible evaluation of drug resistance in HIV-1 at higher resolution. This study emphasizes on the sudden emergence of circulating HIV-1 subtypes and its deadly drug resistant variants in Pakistan in the recent years. However, such molecular approaches in developing nations like Pakistan, will pave favourable strategies for advanced drug design, diagnostic assays and antiretroviral regimen therapies.


Article Information

Received 24 August 2021

Revised 29 September 2021

Accepted 07 October 2021

Available online 13 December 2021

(early access)

Published 08 July 2022

Authors’ Contribution

AZ conceived, designed and wrote the research article. MAS proofread the article. HJ confirmed the data and sources. MAUK and ARS helped in writing of the article and analysis of data.

Key words

Gag-Pol, Unique recombinant forms (URFs), Protease inhibitors (PI), Mutations, Drug resistance

DOI: https://dx.doi.org/10.17582/journal.pjz/20210824080807

* Corresponding author: amreenzara1@gmail.com

0030-9923/2022/0005-2353 $ 9.00/0

Copyright 2022 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).



INTRODUCTION

Acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV) which is a lentivirus and belongs to the family retroviridae. It is an RNA virus with a 9.2-kb genome that encodes structural and regulatory proteins in the order 5’-gag-pol-vif-vpr-tat-rev-vpuenv-3’ (Girard et al., 2006). HIV-1 probably originated from Africa from non-human pri­mates and was later transferred to humans early in the 20th century (Worobey et al., 2008). HIV-1 and HIV-2 are the two main types which initiate the onset of infection. HIV-1 having its dwelling roots from Southern Camer­oon was transferred to humans from wild chimpanzees during the twentieth century (Gao et al., 1999; Keele et al., 2006). Its evolution was from a Simian immunode­ficiency virus. On the other hand, HIV-2 seems to have its roots in Sooty Mangabey (Cercocebusa­tys), Gabon, and Cameroon (Reeves and Doms, 2002). HIV-1 possesses a high genetic variation and the deficiency of proof reading in the viral transcriptase provokes an increased rate of recombination and genetic mutations frequently. These mutations have given rise to emerging escape mutants with the passage of time (Abram et al., 2010). However, the increasing number of mutations in HIV-1 in the current situation has evolved to multiple genetic recombinants mainly such as circulating recombinant forms (CRFs) and unique recombinant forms (URFs) (Tongo et al., 2016). The universal Los Alamos HIV database does however, displays many CRFs circulating around the world. Asian countries have a predominancy of Subtypes A, B, C, D and G with multiple CRFs (Junqueira et al., 2016; Sanders-Buell et al., 2010; Sarker et al., 2008). In a developing country like Pakistan with a high burden of socio-economic conditions, HIV poses a significant health concern with the increasing number of cases and outbreaks. The trend has shifted from low to high rise in HIV infection in the country. Like any other developing country due to resource limited settings, AIDS is a major public health burden in Pakistan due to increase in HIV-1 positive cases in the near past. Recent reports of NACP ranked Punjab with the highest number of HIV cases of 75000, Sindh with 60,000, Balochistan as 5275 followed by Khyber Pakhtunkhwa with 16322 cases (Khanani et al., 1988). Urban development, migration from smaller cities for education, employment and medical facilities contributed to population rise mainly in the Punjab region. Thus migrational influx however could be one of the possible reasons of the increased number of HIV-1 cases. In low income nations like Pakistan, monitoring of HIV drug resistance and population-based surveillances are highly the need of the hour, to ensure the efficacy of continuing the treatment regimen for HIV-1 (Bennett et al., 2006; Jordan et al., 2008; Gilks et al., 2006). Increased urbanization in Punjab contributes as a major facilitating factor for exploration of HIV-1 subtypes, drug resistance mutations and genetic variations. Pakistan, however, demonstrates paucity in molecular and computational HIV related studies to date. This study focusses on investigating the drug-resistance mechanisms of the circulating HIV-1 subtypes in Punjab and highlighting the phylogenetic relationship among the isolates. The recombinant strains and URFs discussed in this study are however reported for the first time in both Gag and Pol genes in HIV-1 patients in Punjab, Pakistan. The data analyzed in the current study, unveils for the first time the major targeted areas of outbreak in Punjab for both Gag and Pol genes molecularly and computationally. Hence, such studies on this deadly virus will provoke necessary health implementations for treatment and control of HIV-1 in Pakistan in the near future.

MATERIALS AND METHODS

HIV-1 positive samples were directed from different treatment centers, to Punjab AIDS Control Programme (PACP) Lahore between October 2018 to March 2020. Among them only 122 HIV-1 positive samples were selected with increased viral load for the current study. Only 30 HIV-1 (0.1%) positive isolates fulfilled the quality processing criteria for genotype analysis. Informed consent was taken from all the selected subjects. RNA was extracted from 200 μl of plasma by QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA, United States) according to the manufacturer’s instructions. cDNA was prepared by the Revert Aid First Strand method. Both Gag (HXB2:781–1861) and Pol fragments were amplified (HXB2: 2147–3462) in a nested polymerase chain reaction (Chen et al., 2014). The successfully amplified samples for HIV-1 Gag and Pol were used for genotyping and phylogenetic analysis with their mentioned sociodemographics (Table I). Amplified samples were sequenced by First Base Laboratories Sdn Bhd, Seri Kembangan, and Selangor, Malaysia on an ABI 3730xl automated DNA analyzer (Applied Biosystems, Foster City, CA). Edited sequences were evaluated for drug resistance mutations using the surveillance drug resistance mutations (SDRM) software and the Stanford University Algorithm (http://hivdb.stanford.edu). REGA HIV subtyping tool was used for subtyping of Gag and Pol (http://www.bioafrica.net/subtypetool/html/). Intersubtype mosaicism in the potential sequences was investigated by the Recombination Identification Program (RIP version 3.5.1). The neighbor-joining method using MEGA (Molecular Evolutionary Genetics Analysis, version 5.1) was used for phylogenetic analyses (Tamura et al., 2007). For non synonymous–synonymous substitution (dn–ds) analysis of sequences SNAP programme was used. DNA sequences were aligned using CLUSTAL W (Huang et al., 2012; Wilkinson et al., 2014). The HIV-1 isolates of pol gene, were analyzed for RT and PR regions by Calibrated Population Resistance (CPR) Tool (Version 6.0) (Chen et al., 2012). HIV-1 sequences were then submitted to GenBank under the following accession numbers: MT462197-MT462205, MT436255-MT436257, MT489302-MT489309 and MT561289- MT561297. The Gag and Pol sequences were edited using Bio-Edit 7.0 software.

RESULTS

Demographic characteristics of study isolates

The isolates selected in the current study were located in the districts of Lahore (n =54), Faisalabad (n= 38), Jalalpur Jattan-Gujrat (n= 32), Kot Imrana-Sargodha (n= 42), Sheikhupura (n=13 ), Kasur (n=21 ), Multan (n =29), Bhawalpur (n=18), Rawalpindi (n=31), Jhelum (n =13 ), Layyah (n=17) and Rahim Yar Khan (n =11) in Punjab. Table I shows the demographic characteristics of the 30 selected subjects in the Punjab for this study. The ratio of males to females was 63.3% in males and 26.6% in females among the 30 isolates with mean age as ± 32 years. Lahore being the major suburb had a ratio of 16.6% followed by 10% for Sargodha, 6.66% Multan, 6.66% Rawalpindi,10% Bhawalpur, 6.66% Sheikhupura, 3.33% Jhelum, 6.66% Rahim Yar Khan, 10% Faisalabad, 10% Layyah, 6.66% Kasur and 13.3% in Gujrat. Among these regions we

 

Table I. Socio-demographic features and genotypes of the study subjects.

Isolate ID

Age

Gender

Transmission route

City

Gene

HIV subtype

CD4 count

Drug resistant mutations

MBBL01

26

Male

IDU

Lahore

Gag

Subtype A1

383

MBBL02

31

Male

IDU

Lahore

Gag

Subtype A1

506

MBBL03

44

Male

IDU

Lahore

Gag

Subtype A1

149

MBBL04

29

Male

IDU

Sargodha

Gag

CRF02_AG

225

MBBL05

55

Male

Immigrant worker

Multan

Gag

CRF02_AG

280

MBBL06

47

Male

Quack treatment

Rawalpindi

Gag

Subtype A1

422

MBBL07

42

Female

Quack treatment

Bhawalpur

Gag

Subtype A1

137

MBBL08

36

Female

Surgeries

Sheikhupura

Gag

Subtype C/ Potential recombinant

335

MBBL09

33

Female

FSW

Rawalpindi

Gag

Subtype A1

196

MBBL10

29

Transgender

Sex

Multan

Gag

CRF02_AG

882

MBBL11

41

Transgender

Sex

Jhelum

Pol

URF/A1+G

341

MBBL12

50

Male

Immigrant worker

Rawalpindi

Gag

Subtype A1/URF

400

MBBL13

38

Male

IDU

Kot Momin-Sargodha

Gag

Subtype A1

475

MBBL14

26

Male

IDU

Kot Momin- Sargodha

Gag

Subtype A1

687

MBBL15

37

Female

Spouse HIV positive

Sargodha

Pol

URF

474

NRTI/PI

MBBL16

32

Male

IDU

Rahim Yar Khan

Pol

URF

124

MBBL17

44

Male

IDU

Lahore

Pol

URF

225

PI/PI SDRM

MBBL18

23

Female

FSW

Multan

Pol

URF

109

NRTI

MBBL19

33

Male

Immigrant worker

Bhawalpur

Pol

URF

256

NRTI

MBBL20

27

Female

FSW

Faisalabad

Pol

URF

338

NNRTI

MBBL21

22

Male

Quack treatment

Faisalabad

Pol

URF

117

NRTI/PI

MBBL22

34

Male

IDU

Jalalpur Jattan-Gujrat

Pol

URF

90

MBBL23

25

Male

IDU

Gujrat

Pol

URF

678

NRTI/PI

MBBL24

28

Male

MSM

Jhelum

Pol

URF

567

NRTI/NNRTI

MBBL25

31

Male

MSM

Layyah

Pol

URF

347

NNRTI

MBBL26

22

Female

Spouse HIV positive

Jalalpur Jattan-Gujrat

Pol

Subtype A1/ URF

109

NNRTI

MBBL27

30

Male

Immigrant worker

Faisalabad

Gag

CRF02_AG

415

MBBL28

25

Transgender

Sex

Lahore

Gag

Subtype A1

223

MBBL29

29

Male

IDU

Kasur

Gag

Subtype A1

446

MBBL30

39

Female

Spouse HIV positive

Kot Momin -Sargodha

Gag

Subtype A1/ URF

258

NA

 

For abbreviation see Table I.

 

present for the very first time molecular and computational analysis of Gag-Pol from Punjab region including three regions of outbreak Kot Imrana Sargodha, Faisalabad and Gujrat. The major transmission routes among the detected subjects were heterosexual transmission accounting of 16.6% as Immigrant workers, 10% as FSW, 6.66% as infection through spouse and 6.66% as transgenders. Intravenous drug transmission ranked the second highest route with 26.6% while the other transmission routes i.e. 10% as homosexual transmission and unknown routes accounted for 6.66% of infections (Table I).

HIV genotyping analysis

Prediction of genetic variability reveals all the possible viral evolutionary information. Therefore, HIV-1 Gag and Pol sequences were analyzed by REGA HIV subtyping tool for subtype determination (Jeannot et al., 2009). HIV subtype A1 was found to be the most predominant subtype found in Pakistani population with 30% ratio among the isolates. CRF02_AG accounted for 10%, Subtype C a potential recombinant had 3.33% and the Unique Recombinant forms (URFs) accounted for 13.3% in the HIV Gag sequences (Table I). Boot scanning analysis of the Gag sequences however revealed four unique recombinant forms as shown in Table I. All the isolate sequences were subjected with bootscan cluster support 1.0 and 0.87 with a confidence level of 99.9%. The prevalence of URFs 13.3 % and CRF02_AG 10% was synchronous with each other which strongly strikes towards the notion that there might have been frequent recombination among the subtype A1 main strains itself (Table I). The subtype A1 in Pakistani population was significant among the IDUs. Isolate MBBL03, MBBL09, MBBL12 and MBBL30, demonstrated isolate MBBL03, MBBL09, MBBL12 and MBBL30, demonstrated HIV Subtype A1as shown in Figure 1. Among the pol sequences, bootscanning analysis revealed that maximum number of subtype determination was accounted for unique recombinant forms (URFs) among the 13 isolates (Table II, Fig. 3). Intersubtype recombination was demonstrated strongly among the pol genotyped isolates. All Pol URFs demonstrated 40% of ratio among the isolates, and subtype D with 6.66% (Table II). The subtype genetic variability and diversity varied with different routes of transmission in infection (Table II). HIV infection caused due to quack treatment also demonstrated subtype A1 prevalence as it is caused by common syringe usage. Some IDUs and 2 isolates MBBL06 and MBBL07 were quack infected and demonstrated genotype A1 (Table I). However, two isolates MBBL05 and MBBL27 did show the genotype CRF02_AG which does suggest the notion that there might be a strong heterosexual transmission route from expats returning from abroad depicting this genotype (Fig. 2A-C). This kind of genotypic pattern shows an association with multiple transmission routes (Tables I and II). The proportion of URFs among the pol genotyped isolates was significantly higher than Gag. This increased prevalence strikes towards an increased vulnerable hypervirulency in the HIV virus (Zahra et al., 2021).

 

Recombination and substitution analysis

Genetic diversity initiates intersubtype mosaicism among the candidate genes. There was a high rate of recombination among the isolates identified with Gag gene in our study (Supplementary Fig. 1). Window size= 200, Confidence threshold = 0.9, Gap option= 3, and Multistate characters = yes, were used for the recombination analysis of the isolates. The RIP anlaysis of these four isolates MBBL01, MBBL05, MBBL14 and MBBL10 demonstrated a similarity score of 0.87 (Supplementary Fig. 1A), 0.91 (Supplementary Fig. 1B), 0.92 (Supplementary Fig. 1C), and 0.89 (Supplementary Fig. 1D). This increased level of mosaicism however denotes to a high rate of intersubtype mosaicism occurring among the region. However previous studies in Pakistan did demonstrate this fact about co-circulating viruses due to increased genetic diversity (Abidi et al., 2014). To measure the rate of natural selection, 30 isolate sequences were subjected to SNAP programme for computing dn/ds ratio. The SNAP results however revealed higher values for Subtype A1 and Subtype C denoting a positive selection pressure and sequence conservation within the Pakistani population. Cumulative behavior by SNAP analysis was shown in Supplementary Figure S2 and Supplementary Table SI.

Genotypic analysis of drug resistance

Anti HIV drugs such as nucleoside reverse transcriptase inhibitors, protease inhibitors and non-nucleoside analog reverse transcriptase inhibitors are generally the recommended combination of drugs used by HIV-1 infected individuals in Punjab, Pakistan as provided at PACP treatment centers. Substitution mutations with a high degree of drug resistance poses a challenge on the treatment and efficacy of HIV-1. Hence, to combat the hinderances evaluation of the RT and PR region in Pol gene would pave pathways in exploring the associated drug resistance in circulating HIV-1 subtypes. Our analysis demonstrated an accessory resistance mutation V32I only in one isolate MBBL17 originating from Lahore. These findings however exhibit a synchronization with previous studies done in Sindh-Pakistan where a single primary mutation (L90M) was detected in a patient with PI usage history (Shah et al., 2011). The drug resistance analysis in our study neither showed major nor minor mutations significantly suggesting the fact that HIV-1 strains are sensitive to NNRTIS and NRTIS being used in Pakistan. Our investigations also shed light on the notion that the circulating viruses reflect sensitivity especially towards protease inhibitors like lopinavir, darunavir and atazanavir. Some non-significant substitutions were observed in the whole length of the RT region. The substitutions were: V35T, D121Y, V60I, K49R, I135T, K122E, I142L, K166R, Q207G, R211K, A272P, K275R, K277R, Q278H,

 

Table II. Demographic and genetic characteristics of HIV-1 isolates with URFs in the Pol gene.

Isolate ID

Gender

Age

Route of trans-mission

City

CD4 count

Gene

Subtype

Drug resistant mutations

NRTI

NNRTI

PI

PI SDRM

MBBL15

Female

37

Spouse HIV positive

Sar-godha

474

Pol

URF

Q58P, Q61X

M66I, 147 V,D60G, 189 M

MBBL17

Male

44

IDU

Lahore

225

Pol

URF

E21X, D29X, D3C

V32I

MBBL18

Female

23

FSW

Multan

109

Pol

URF

K43X

MBBL19

Male

33

Immigrant Worker

Bha-walpur

256

Pol

URF

K43X

MBBL20

Female

27

FSW

Faisal-abad

338

Pol

URF

Y188NY, H198HP, W229RW

MBBL21

Male

22

Quack Treatment

Faisal-abad

117

Pol

URF

R78RT, R172E, D1DE

L5V, R8L, P9A, A62AG, N54NY, D186DE

MBBL23

Male

25

IDU

Gujrat

678

Pol

URF

R78RT, R172E, D1DE

L5V, R8L, P9A, A62G, I167N

MBBL24

Male

28

MSM

Jhelum

567

Pol

URF

Q222R, E194X, L234P

Y181D, Y188N, E194X, H198L, K220R, G231R, W229X, Y232N, W239C

MBBL25

Male

31

MSM

Layyah

347

Pol

URF

Y188N, H198X, K201Q, P217X, W229

MBBL26

Female

22

Spouse HIV positive

Jalapur Jattan-Gujrat

109

Pol

Subtype D/URF

Y188NY, H198HP, W229RW

 

Table Legend: IDU, intravenous drug users; FSW, female sex workers; MSM, males who have sex with males; URFs, Unique recombinant forms.

 

 

L282C, L283I and R248X in the isolates MBBL15, MBBL16, MBBL17, MBBL18, MBBL19 and MBBL20. The RT region exhibited minor mutations leaving an insignificant impact on drug resistance to date (Rhee et al., 2003; Lai et al., 2014; Gatanaga et al., 2010). The current combination of drugs being used for HIV-1 so far in Pakistan showed low prevalence for drug resistance thus satisfying the continuation of these drugs for HIV-1 patients in Pakistan. However, non-significant substitutions such as T12A, I13A, K14R, I15V, K20I, T31S, E35D, M36I, N37D, R41K, K43X, R57K, Q61H, H69K, K70R and L89M were observed in the study subjects (MBBL18, MBBL19, MBBL20, MBBL21, MBBL23, MBBL24, MBBL25 and MBBL26) at several sites across the whole length of PR region. Q58P, Q61X, K43X, R78RT, R172E, D186DE, Q222R, E194X and L234P were identified as unique NRTI mutations. Among the NNRTI mutations Y188NY, H198HP, W229RW, Y181D, Y188N, E194X, H198L, K220R, G231R, W229X, Y232N, W239C, Y188N, H198X, K201Q, P217X, W229, Y1888NY, H198HP and W229RW were identified among the isolates (Table II). M66I, 147V, D60G,189M, E21X, D29X, D30C, L5V, R8L, P9A, A62AG, N54NY, D186DE, L5V, R8L, P9A and I167N were the detected PIs. Only one isolate MBBL17 from Lahore had an accessory mutation V32I. Similarly, absence of substantial changes in the RT region denoted a conferred resistance to reverse transcriptase inhibitors (RTIs). Only one isolate had a mutation V32I which conferred a low level resistance to NNRTIs (Table II). Medium and low resistance levels to six PIs were detected against atazanavir (ATV), indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), saquinavir (SQV) and tipranavir (TPV). High-level and moderate resistances were also revealed among six NRTIs such as lamivudine (3TC), zidovudine (AZT), abacavir (ABC), stavudine (D4T), didanosine (DDI) and entricitabine (FTC)) and four NNRTIs [efavirenz (EFV), etravirine (ETR), nevirapine (NVP) and rilpivirine (RPV)].

Phylogenetic analysis

Phylogenetic analysis demonstrated that 17 Gag sequences from this study clustered into three different clades. Among the isolates, 10 clustered with subtype A

originating from Pakistan, Kenya, Uganda and Rawanda. Of these, 3 clustered with sequences corresponding sub-subtype 02_AG, having its descent from Kenya, Ghana and South Africa. Three isolates demonstrated URFs and 1 sequence clustered within a clade representing subtype A and G clustered within a clade representing sub-type G from Kenya, Cameroon and Ghana (Fig. 4A, B), showing an evolutionary origin from African countries. The isolate’s travel histories were insufficient to have reasonably connected probable ancestral links. As a result, clustering of subtypes 02 AG, A, and G in African countries, therefore brought to evidence an evolutionary origin of HIV-1 from Africa (Huet et al., 1990). However, 13 Pol

 

sequences were identified in our study, among them the highest ratio was of URFs. Forty percent of the sequences clustered with unique recombinant forms and only one isolate MBBL26 clustered with subtype D. This nature of clustering isolates with a wide range of reported databases therefore, indicates the genetic diversity, heterogeneity and potential evolutionary dynamics of HIV-1. These analyses give the notion of presence of many subtypes and inter-subtype recombinant forms at the population level (Song et al., 2018).

DISCUSSION

Investigations based on HIV-1 Gag and Pol analysis among the sequences targeting the circulating genotypes in the Punjab Province was the main aim of the current study. The sociodemographic analysis of this study however, revealed that Lahore due to its urbanization and economic development does have the highest number of HIV-1 patients in the Punjab including three major regions of outbreak i.e., Kot Imrana (Sargodha), Gujrat (Jalalpur Jattan) and Faisalabad. In the regions of outbreak common syringe usage by the local quacks, and rural medical facilities was the main culprit of the outbreak (Zaid and Afzal 2018; Wahid, 2019). These findings do support the fact that indirect facilitation and fast population mobility could possibly be the major routes in spreading of HIV-1 and transmitting the viruses to the community as well. In this study, HIV-1 genotypes prevailing predominantly in the heterosexually infected population in Punjab were investigated based on the analysis of HIV-1 Gag and Pol sequences. Lahore being the capital of Punjab province could possibly lead to increased genetic diversity of HIV-1 due to its growing population. Lack of proper biosafety measures in hospitals, frequent migration in the suburb from nearby regions for education and health has contributed to the rise in infection (Rana and Bhatti, 2018). To add to the fact, previously reported researches however have brought to light the phenomenon of HIV-1 infection among people migrating frequently resulting in emergence of novel subtypes, inter-subtype recombinants, and CRFs (Zhong et al., 2007; Kalichman et al., 2017; Lebedev et al., 2019). The subtype, SNAP and RIP analysis of this study however reveals major molecular revelations in HIV-1 sequences from Punjab, Pakistan. Nonetheless, phylogenetic and genotypic analyses of drug resistance in HIV-1 sequences brings to light the major significant and non-significant mutations especially in the Pol region. Such novel molecular analysis from Punjab, Pakistan, reported in our study could possibly pave pathways in understanding of the underlying mechanisms for HIV-1 in resource limited countries like Pakistan. HIV-1 subtype distribution appears to be quite diverse in other Asian countries too. In Iran previous studies have reported HIV-1 subtypes A and B mainly in the IDUs (Sarrami-Forooshani et al., 2006). Similarly, studies reported from countries like Uzbekistan, Kazakhstan and Yemen also demonstrated a prevalence of subtypes A, B and CRF02_AG which share synchrony with the present data indicating the commonly circulating prevalent subtypes in Asia (Kurbanov et al., 2003; Carr et al., 2005; Bobkov et al., 2004; Saad et al., 2005). HIV molecular epidemiological research in Pakistan is still in the infant stages, and yet well affirmed HIV prevention policies need to be designed. Previously reported data in Pakistan in HIV-1 genotypes evidently demonstrates genetic diversity with existing co- circulating genotypes such as: CRF_ AG, A, B and CRF_AE which are synchronous with our findings also (Mujeeb and Altaf, 2003; Altaf and Mujeeb, 2002; Hyder and Khan, 1998). However, our data demonstrates that apart from Subtype A1, the URFs were becoming more prevalent than the previously reported subtypes A, G and CRF_02 AG (Khan et al., 2006; Yaqub et al., 2019). This changing trend of introduction of novel and new recombinant subtypes is because of returning migrant workers and expatriates in Pakistan. This unchecked migrational influx of expatriates, ultimately provokes increased incidence of HIV-1 infection. It has also contributed in altering the pattern of different HIV-1 subtypes and increased drug resistances turning it to a more challengeable situation for the concerned health authorities. Absence of major resistance-associated substitutions in our findings likewise agree with another study conducted in Sindh, Pakistan, where no major resistances were recognized (Baqi et al., 1998; Khanani et al., 2011; Khan et al., 2018). Our study does come up with the notion that circulating viruses are highly sensitive to all protease inhibitors, implicating lopinavir, atazanavir and darunavir. The 30 isolates in this investigation had no significant major or minor mutations in the RT area that conferred resistance to reverse transcriptase inhibitors (RTIs). This strongly denotes the fact, that the prevalent HIV-1 strains in Pakistan are sensitive to the NRTIs and NNRTIs. PR and RT regions display down regulation in prevalence of drug-resistance associated mutations in the infected individuals. Combination drug regimens of PIs in combination with RTIs like lopinavir, ritonavir, nevirapine, lamivudine, tenofovir and zidovudine, could be continued to treat patients. Furthermore, continuation of these studies pave ways for essential modifications in drug regimens which could further be applicable for effective therapeutics and subsequent control in the future. Hence, the current study unveils novel information on circulating subtypes and drug resistance mutations in Gag-Pol genes for HIV-1 patients for the first time in Pakistan’s Punjab province. The phylogenetic and computational revelations made by this study will be extremely beneficial for clinicians, physicians and researchers in determining the best possible therapeutic strategies for controlling the transmission of this virus. It also uncovers primitive information about the evolutionary links and drug resistance patterns of the recent existing HIV strains in a developing country like Pakistan. Future studies are therefore highly the need of the hour to control the spread of this virus and monitor drug resistance mechanisms for implementation of appropriate therapeutic options.

ACKNOWLEDGMENTS

We are highly grateful to Dr. Asim Altaf and Dr. Munir Ahmad Malik, Punjab AIDS Control Programme for providing assistance and facilities at PACP. We are also very grateful to the laboratory staff at PACP for providing the opportunity to collect the samples and experimental work as recommended by WHO. This study was undertaken in collaboration and support by the Punjab AIDS Control Programme (PACP) and Institute of Public Health (IPH) Lahore, Pakistan.

Funding

The research study was funded by the University of Central Punjab Pakistan and Punjab AIDS Control Programme.

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20210824080807

Statement of conflict of interest

The authors have declared no conflict of interest.

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