Accessory Mutations Research Articles

Integrase strand transfer inhibitors resistance-associated mutations in HIV-infected pregnant women.

To date, no data exist regarding the prevalence of integrase inhibitor (INSTI) resistance-associated mutations (HIVDRM) in HIV-infected pregnant women (HPW) in Latin America. We describe the prevalence and transmissibility of integrase HIVDRM in a historical cohort of INSTI-naïve HPW from Argentina (n=56) with Next Generation Sequencing (NGS). Bioinformatics analysis was performed by HyDRA software for 20%, 10%, 5%, 2%, and 1% sensitivity thresholds. We calculated the mutational viral load for each INSTI-HIVDRM, considering those with >1000 c/mL as of high risk of transmissibility. The predominant HIV subtype was BF (78.5%). Major HIVDRM were not detected with the population sequencing 20% filter. With a 1% threshold, the prevalence increased to 8.9%; Y143C/S, E92G, E138K, and T66I mutations were found. The median (range) mutational load (expressed in c/mL) was: 355 (50.2-11705); with only 1 case >1000 c/mL Accessory mutations (G163R/K, T97A) were detected mostly with a 20% sensitivity threshold with an overall prevalence of 23.2%; the median (IQR) mutational load was: 23929 (4009-63158) c/mL; all of them above 1000 c/mL. Our results show evidence of the presence of major INSTI-HIVDRM as aleatory mutations and a high frequency of accessory mutations with potential transmissibility in HPW.

Lack of Resistance Mutations to the Novel HIV-1 Capsid Inhibitor Lenacapavir Among People Living with HIV in Guangdong, China.

The capsid inhibitor (CAI) lenacapavir (LEN) was approved for use in 2022, yet there are few reports about its drug resistance mutations (DRMs) and sensitivity. To delineate the prevalence of CAI DRMs and drug susceptibility among HIV-1 infected individuals living in Guangdong, China. A total of 1035 individuals with HIV-1 infection, including 660highly Active Anti-Retroviral Therapy (HAART) naive individuals and 375hAART experienced individuals whose protease (PR)/ reverse transcriptase (RT) fragments were amplified successfully during drug resistance surveillance between October 2021 and December 2023, were randomly included in this study. The entire HIV-1 gag gene was amplified from plasma in LEN-naive individuals with or without antiretroviral therapy. The epidemiological and demographic information of the enrolled individuals were collected. The Stanford HIV Drug Resistance Database HIVdb program for Capsid was used to interpret the CAI DRMs and the LEN susceptibility. Among 1035 samples, 805 gag sequences were amplified, sequenced and assembled successfully from 518hAART drugs naive individuals and 287hAART drugs experienced individuals. Among them, 0.50% (4/805) carried at least one CAI DRM, of which 0.19% (1/518) from HAART naive individuals and 1.05% (3/287) from HAART experienced individuals. Among the individuals with CAI DRMs, two patients carried CAI major mutations (Q67H) conferring intermediate resistance to LEN and two patients carried CAI accessory mutation (T107A) conferring low level resistance to LEN. Extremely low prevalence of CAI DRMs was detected among people living with HIV (PLWH) in Guangdong, China. Our observations indicate that LEN application may be promising when used in clinical practice in China. Before the administration of LEN, there is no need to consider detecting CAI mutations in PLWH through DRM examination for the time being.

HIV-1 diversity and pre-treatment drug resistance in the era of integrase inhibitor among newly diagnosed ART-naïve adult patients in Luanda, Angola

The surveillance of drug resistance in the HIV-1 naïve population remains critical to optimizing the effectiveness of antiretroviral therapy (ART), mainly in the era of integrase strand transfer inhibitor (INSTI) regimens. Currently, there is no data regarding resistance to INSTI in Angola since Dolutegravir-DTG was included in the first-line ART regimen. Herein, we investigated the HIV-1 genetic diversity and pretreatment drug resistance (PDR) profile against nucleoside/tide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and INSTIs, using a next-generation sequencing (NGS) approach with MinION, established to track and survey DRMs in Angola. This was a cross-sectional study comprising 48 newly HIV-diagnosed patients from Luanda, Angola, screened between March 2022 and May 2023. PR, RT, and IN fragments were sequenced for drug resistance and molecular transmission cluster analysis. A total of 45 out of the 48 plasma samples were successfully sequenced. Of these, 10/45 (22.2%) presented PDR to PIs/NRTIs/NNRTIs. Major mutations for NRTIs (2.2%), NNRTIs (20%), PIs (2.2%), and accessory mutations against INSTIs (13.3%) were detected. No major mutations against INSTIs were detected. M41L (2%) and I85V (2%) mutations were detected for NRTI and PI, respectively. K103N (7%), Y181C (7%), and K101E (7%) mutations were frequently observed in NNRTI. The L74M (9%) accessory mutation was frequently observed in the INSTI class. HIV-1 pure subtypes C (33%), F1 (17%), G (15%), A1 (10%), H (6%), and D (4%), CRF01_AG (4%) were observed, while about 10% were recombinant strains. About 31% of detected HIV-1C sequences were in clusters, suggesting small-scale local transmission chains. No major mutations against integrase inhibitors were detected, supporting the continued use of INSTI in the country. Further studies assessing the HIV-1 epidemiology in the era of INSTI-based ART regimens are needed in Angola.

Kinetic coevolutionary models predict the temporal emergence of HIV-1 resistance mutations under drug selection pressure

Drug resistance in HIV type 1 (HIV-1) is a pervasive problem that affects the lives of millions of people worldwide. Although records of drug-resistant mutations (DRMs) have been extensively tabulated within public repositories, our understanding of the evolutionary kinetics of DRMs and how they evolve together remains limited. Epistasis, the interaction between a DRM and other residues in HIV-1 protein sequences, is key to the temporal evolution of drug resistance. We use a Potts sequence-covariation statistical-energy model of HIV-1 protein fitness under drug selection pressure, which captures epistatic interactions between all positions, combined with kinetic Monte-Carlo simulations of sequence evolutionary trajectories, to explore the acquisition of DRMs as they arise in an ensemble of drug-naive patient protein sequences. We follow the time course of 52 DRMs in the enzymes protease, RT, and integrase, the primary targets of antiretroviral therapy. The rates at which DRMs emerge are highly correlated with their observed acquisition rates reported in the literature when drug pressure is applied. This result highlights the central role of epistasis in determining the kinetics governing DRM emergence. Whereas rapidly acquired DRMs begin to accumulate as soon as drug pressure is applied, slowly acquired DRMs are contingent on accessory mutations that appear only after prolonged drug pressure. We provide a foundation for using computational methods to determine the temporal evolution of drug resistance using Potts statistical potentials, which can be used to gain mechanistic insights into drug resistance pathways in HIV-1 and other infectious agents.

Prevalence and analysis of acquired and transmitted integrase strand transfer inhibitor-associated HIV-1 drug resistance in Chongqing, China

ABSTRACT In this study, we examined the occurrence of acquired and transmitted drug resistance to integrase strand transfer inhibitor (INSTI) in HIV-1 strains in Chongqing (China) for guiding for the routine testing of INSTI-associated HIV-1 genotype resistance. Plasma samples were obtained from HIV-1 patients at Chongqing Public Health Medical Center from July 2019 to August 2022. Besides, amplification, sequence, and analysis of the portion of the HIV-1 pol gene that encodes the integrase protein were implemented to identify INSTI resistance. Integrase sequence data was harvested for a comprehensive cohort of 1032 patients infected with HIV-1. This cohort consisted of 564 ART-naive patients, 465 ART-treated patients, and 3 patients with an unknown treatment history. Within the study group, we identified INSTI resistance in 21 patients (2.03%, 21/1032), including 17 ART-treated patients (3.66%, 17/465). Among the ART-treated patients, 12 were INSTI-treated (11.76%, 12/102), 5 were INSTI-naive (1.38%, 5/363), and 4 were ART-ineffective patients (0.71%, 4/564). The prevalent major resistance mutation was Q148R (0.48%, 5/1032), while the most prevalent accessory resistance mutation was E157Q (1.65%, 17/1032). In light of the above, it is recommended that the incidence of accessory genotype analysis should be considered before starting any future INSTI-based therapy, especially in patients with drug resistance to NRTIs and NNRTIs and the reduction of INSTI sensitivity should be carefully monitored and investigated. Regular monitoring for resistance should be implemented after the use of INSTIs, and, importantly, ongoing monitoring of the decreasing susceptibility to INSTIs is crucial following the initiation of treatment with INSTIs.

Analysis of spike and accessory 3c genes mutations of less virulent and FIP-associated feline coronaviruses in Beijing, China

Mutations in S and 3c genes of feline coronavirus (FCoV) have been associated with the development of feline infectious peritonitis (FIP). In the present study, FCoV S and 3c genes mutations were analyzed in healthy and FIP cats. M1058L mutation was found in 13.64% (3/22) feces from FIP cats, but not in feces from healthy cats (0/39). The intact 3c gene was found in feces from both healthy cats (19/19) and FIP cats (12/12). All parenteral samples from FIP cats carried one or more of the M1058L mutation, S1060A mutation and mutated 3c gene. FCoV reverse-transcriptase polymerase chain reaction (RT-PCR) of parenteral samples (including ascites, pleural effusions and tissue) is recommended as the gold standard for clinical diagnosis of FIP rather than detection of the M1058L mutation, but when cats have severe gastrointestinal symptoms and lesions, detection of the M1058L mutation in feces may be helpful in diagnosing FIP.

Identification of integrase inhibitor-related drug resistance mutations in newly diagnosed ART-naïve HIV patients

BackgroundIn China, the recommended treatment regimens for HIV-infected individuals were tenofovir in combination with lamivudine or emtricitabine as NRTIs, efavirenz or rilpivirine as NNRTIs, lopinavir/ritonavir as protease inhibitors, and raltegravir or dolutegravir as INSTIs. The development of drug resistance increases the risk of viral rebound, opportunistic infections, and ultimately treatment failure such that the early detection of resistance is ideal. This study was developed to explore primary drug resistance characteristics and genotypic distributions in newly diagnosed antiretroviral therapy (ART)-naïve HIV-1 patients in Nanjing with the goal of establishing a basis for their individualized treatment in the clinic. MethodsSamples of serum were collected from newly diagnosed ART-naïve HIV patients from the Second Hospital of Nanjing between May 2021 and May 2022. The HIV-1 integrase (IN), protease (PR), and reverse transcriptase (RT) gene coding sequences were amplified from these samples, sequenced, and assessed for drug resistance-related mutations. ResultsMajor integrase resistance-related mutations were detected in 4/360 amplified samples, with 5 other patient samples exhibiting accessory resistance mutations. The overall prevalence of PR and RT inhibitor-related transmitted drug resistance mutations (TDRMs) in this patient population was 16.99% (61/359). The most common mutations were non-nucleoside reverse transcriptase inhibitor-related mutations (51/359; 14.21%), followed by those associated with nucleoside reverse transcriptase inhibitors (7/359; 1.95%) and protease inhibitors (7/359; 1.95%). Dual-resistant strains were also observed in a subset of patients. ConclusionsIn summary, this study is the first to have surveyed the prevalence of integrase inhibitor resistance-related mutations and other drug resistance-related mutations among newly diagnosed ART-naïve HIV-positive patients in Nanjing, China. These results highlight the need for further molecular surveillance-based monitoring of the HIV epidemic in Nanjing.

Low level of HIV-1C integrase strand transfer inhibitor resistance mutations among recently diagnosed ART-naive Ethiopians

With the widespread use of Integrase strand transfer inhibitors (INSTIs), surveillance of HIV-1 pretreatment drug resistance is critical in optimizing antiretroviral treatment efficacy. However, despite the introduction of these drugs, data concerning their resistance mutations (RMs) is still limited in Ethiopia. Thus, this study aimed to assess INSTI RMs and polymorphisms at the gene locus coding for Integrase (IN) among viral isolates from ART-naive HIV-1 infected Ethiopian population. This was a cross-sectional study involving isolation of HIV-1 from plasma of 49 newly diagnosed drug-naive HIV-1 infected individuals in Addis-Ababa during the period between June to December 2018. The IN region covering the first 263 codons of blood samples was amplified and sequenced using an in-house assay. INSTIs RMs were examined using calibrated population resistance tool version 8.0 from Stanford HIV drug resistance database while both REGA version 3 online HIV-1 subtyping tool and the jumping profile Hidden Markov Model from GOBICS were used to examine HIV-1 genetic diversity. Among the 49 study participants, 1 (1/49; 2%) harbored a major INSTIs RM (R263K). In addition, blood specimens from 14 (14/49; 28.5%) patients had accessory mutations. Among these, the M50I accessory mutation was observed in a highest frequency (13/49; 28.3%) followed by L74I (1/49; 2%), S119R (1/49; 2%), and S230N (1/49; 2%). Concerning HIV-1 subtype distribution, all the entire study subjects were detected to harbor HIV-1C strain as per the IN gene analysis. This study showed that the level of primary HIV-1 drug resistance to INSTIs is still low in Ethiopia reflecting the cumulative natural occurrence of these mutations in the absence of selective drug pressure and supports the use of INSTIs in the country. However, continues monitoring of drug resistance should be enhanced since the virus potentially develop resistance to this drug classes as time goes by.

Prevalence of HIV-1 Natural Polymorphisms and Integrase-Resistance-Associated Mutations in African Children.

Integrase inhibitors (INIs) are a potent option for HIV treatment. Limited data exist on INI resistance in West Africa, particularly in children living with HIV/AIDS. We determined the prevalence of integrase gene polymorphisms and the frequency of naturally occurring amino acid (aa) substitutions at positions associated with INI resistance. Dried blood spot (DBS) samples were obtained from one hundred and seven (107) HIV-1-infected children aged less than 15 years old in two West African countries, Benin and Mali. All children were naïve to INI treatment, 56 were naïve to anti-retroviral therapy (ART), and 51 had received ART. Genetic sequencing of HIV integrase was successful in 75 samples. The aa changes at integrase positions associated with INI resistance were examined according to the Stanford HIV Genotypic Resistance database. The median ages were 2.6 and 10 years for ART-naïve and -treated children, respectively. The most common subtypes observed were CRF02_AG (74.7%) followed by CRF06_cpx (20%). No major INI-resistance mutations at positions 66, 92, 121, 143, 147, 148, 155, and 263 were detected. The most prevalent INI accessory resistance mutations were: L74I/M (14/75, 18.6%) followed by E157Q (8/75, 10.6%), G163E/N/T/Q (5/75, 6.6%), Q95A/H/P (2/75, 2.6%), and T97A (4/75, 5.3%). Other substitutions observed were M50I/L/P, H51E/P/S/Q, I72V, T112V, V201I, and T206S. Polymorphisms at positions which may influence the genetic barrier and/or drive the selection of specific INI-resistance pathways were detected. However, no transmitted drug resistance (TDR) to INI was detected among samples of INI-naïve patients. These findings support the use of this treatment class for children with HIV-1, particularly in West Africa.

Contingency and Entrenchment of Drug-Resistance Mutations in HIV Viral Proteins.

The ability of HIV-1 to rapidly mutate leads to antiretroviral therapy (ART) failure among infected patients. Drug-resistance mutations (DRMs), which cause a fitness penalty to intrinsic viral fitness, are compensated by accessory mutations with favorable epistatic interactions which cause an evolutionary trapping effect, but the kinetics of this overall process has not been well characterized. Here, using a Potts Hamiltonian model describing epistasis combined with kinetic Monte Carlo simulations of evolutionary trajectories, we explore how epistasis modulates the evolutionary dynamics of HIV DRMs. We show how the occurrence of a drug-resistance mutation is contingent on favorable epistatic interactions with many other residues of the sequence background and that subsequent mutations entrench DRMs. We measure the time-autocorrelation of fluctuations in the likelihood of DRMs due to epistatic coupling with the sequence background, which reveals the presence of two evolutionary processes controlling DRM kinetics with two distinct time scales. Further analysis of waiting times for the evolutionary trapping effect to reverse reveals that the sequences which entrench (trap) a DRM are responsible for the slower time scale. We also quantify the overall strength of epistatic effects on the evolutionary kinetics for different mutations and show these are much larger for DRM positions than polymorphic positions, and we also show that trapping of a DRM is often caused by the collective effect of many accessory mutations, rather than a few strongly coupled ones, suggesting the importance of multiresidue sequence variations in HIV evolution. The analysis presented here provides a framework to explore the kinetic pathways through which viral proteins like HIV evolve under drug-selection pressure.

Drug Resistance to Integrase Strand-Transfer Inhibitors among HIV-1-Infected Adults in Guangdong, China.

Integrase strand-transfer inhibitor (INSTI)-containing regimens have gradually been administered in Guangdong Province, China beginning in 2016, and INSTI-related drug resistance (DR) may occur and should be monitored among HIV-1-infected patients. To investigate the prevalence of INSTI-related resistance among HIV-1-infected individuals in Guangdong and provide evidence for the optimal administration of INSTIs. This study recruited 1208 HIV-1-infected patients (including 404 ART-naive and 804 ART-experienced patients) between June 2021 and April 2022. The entire integrase gene was amplified from blood plasma. Demographic and epidemiological information were collected. INSTI mutations and susceptibility were interpreted using the Stanford HIV Drug Resistance Database HIVdb program. Of the 1208 enrolled individuals, 2.65% (32/1208) carried at least one INSTI major or accessory drug resistance mutation (DRM), with 1.49% (6/404) being from ART-naive individuals and 3.23% (26/804) from ART-experienced individuals. Among them, seven polymorphic major mutations were detected. Although no INSTI drug resistance was found among treatment-naive patients, seven ART-experienced patients (0.87%, 7/804) carried mutations conferring resistance to INSTIs. The overall prevalence of INSTI DRMs and DR was comparatively low among ART-naive and ART-treated populations in Guangdong; however, INSTI-related polymorphic mutations were observed. Surveillance should be reinforced before transfer to INSTI-containing regimens.

HIV-1 genetic diversity and transmitted drug resistance to integrase strand transfer inhibitors among recently diagnosed adults in Porto Alegre, South Brazil.

The HIV-1 genetic diversity and the presence of transmitted drug resistance mutations (TDRMs) against integrase strand transfer inhibitors (INSTIs) were assessed sequencing samples of antiretroviral (ARV)-naive HIV-1-infected individuals from South Brazil. Viral RNA from 42 ART-naive individuals was submitted to complete HIV-1 integrase gene amplification by RT-PCR and sequencing. Viral strains carrying TDRMs against INSTIs were not detected in the present study. However, the polymorphisms L74M and L74I were each observed in 4.8% of the individuals. These accessory mutations have been reported as putative causes of TDRMs in ART with raltegravir, but only when associated with additional major mutations. When submitted to HIV-1 subtyping, 50% were classified as subtype C, 21% as recombinant BC, 19% as subtype B, 4.8% as subtype F1 and 4.8% as recombinant CF1. All 42 ARV-naive individuals were apparently susceptible to INSTIs, included in the Brazilian therapeutic guideline since 2009. To the best of our knowledge, this is the first study to evaluate TDRMs against INSTIs in Brazil. The most prevalent HIV-1 subtypes were subtype C, followed by the recombinant BC and subtype B, which is in agreement with previous studies. However, the presence of subtype F1 and recombinant CF1 reported herein was not observed in previous studies.

Molecular Epidemiology of HIV-1 in Eastern Europe and Russia.

The HIV epidemic in Eastern Europe and Russia is large and not well-controlled. To describe the more recent molecular epidemiology of HIV-1, transmitted drug resistance, and the relationship between the epidemics in this region, we sequenced the protease and reverse transcriptase genes of HIV-1 from 812 people living with HIV from Ukraine (n = 191), Georgia (n = 201), and Russia (n = 420) before the initiation of antiretroviral therapy. In 190 Ukrainian patients, the integrase gene sequence was also determined. The most reported route of transmission was heterosexual contact, followed by intravenous drug use, and men having sex with men (MSM). Several pre-existing drug resistance mutations were found against non-nucleoside reverse transcriptase inhibitors (RTIs) (n = 103), protease inhibitors (n = 11), and nucleoside analogue RTIs (n = 12), mostly polymorphic mutations or revertants. In the integrase gene, four strains with accessory integrase strand transfer inhibitor mutations were identified. Sub-subtype A6 caused most of the infections (713/812; 87.8%) in all three countries, including in MSM. In contrast to earlier studies, no clear clusters related to the route of transmission were identified, indicating that, within the region, the exchange of viruses among the different risk groups may occur more often than earlier reported.

Primary resistance against integrase strand transfer inhibitors in integrase strand transfer inhibitor-naive patients failing first- and second-line ART in Tanzania.

Sub-Saharan African countries are introducing integrase strand transfer inhibitors (INSTIs) in their ART programmes as the preferred first-line regimen, and dolutegravir is the INSTI of choice due to its potency, tolerability and high genetic barrier to resistance. Dolutegravir was introduced into the first-line ART regimen in Tanzania in 2019. However, there is a paucity of data on the occurrence of mutations in HIV lineages circulating in Tanzania. This study aimed to determine the prevalence of INSTI primary resistance mutations in Tanzanian patients exposed to ART but not INSTIs. Plasma samples from 50 INSTI-naive patients failing first- or second-line ART [median (IQR) age: 40 (21.93-46.41) years; 68% women] were subjected to Sanger sequencing of the HIV integrase gene. Participants had been on ART for a median (IQR) duration of 7.32 (4.73-9.29) years, with 80% and 20% failing first- and second-line ART, respectively. No major INSTI mutations were found, but 2 (4%) participants had the accessory mutation T97A. Using the REGA HIV-1 subtyping tool, HIV subtype A1 (53.1%) was found to be dominant, followed by subtypes C (30.6%) and D (16.3%). This study found no current evidence for transmitted resistance against INSTIs among unexposed patients failing ART and supports the scale-up of INSTI-based regimens. However, the presence of accessory mutations calls for the surveillance of INSTI resistance mutations to ensure that the anticipated long-term desired outcomes are achieved.

SARS-CoV-2 variant spike and accessory gene mutations alter pathogenesis

The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the spike protein. Although these differences in spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome that may also affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through interference with innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other nonspike mutations on SARS-CoV-2 pathogenesis, we synthesized both viruses possessing deletions in the accessory genes as well as viruses where the WA-1 spike is replaced by each variant spike gene in a SARS-CoV-2/WA-1 infectious clone. We then characterized the invitro and invivo replication of these viruses and compared them to both WA-1 and the full variant viruses. Our work has revealed that the accessory proteins contribute to SARS-CoV-2 pathogenesis and the nonspike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host. This work suggests that while spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may alter clinical disease presentation.

Development and therapeutic manipulation of the head and neck cancer tumor environment to improve clinical outcomes.

The clinical response to cancer therapies involves the complex interplay between the systemic, tumoral, and stromal immune response as well as the direct impact of treatments on cancer cells. Each individual's immunological and cancer histories are different, and their carcinogen exposures may differ. This means that even though two patients with oral tumors may carry an identical mutation in TP53, they are likely to have different pre-existing immune responses to their tumors. These differences may arise due to their distinct accessory mutations, genetic backgrounds, and may relate to clinical factors including previous chemotherapy exposure and concurrent medical comorbidities. In isolation, their cancer cells may respond similarly to cancer therapy, but due to their baseline variability in pre-existing immune responses, patients can have different responses to identical therapies. In this review we discuss how the immune environment of tumors develops, the critical immune cell populations in advanced cancers, and how immune interventions can manipulate the immune environment of patients with pre-malignancies or advanced cancers to improve therapeutic outcomes.

Temporal changes in the accessory protein mutations of SARS-CoV-2 variants and their predicted structural and functional effects.

Emerging variants enable the continuous spread of SARS‐CoV‐2 in humans. The factors contributing to behavioral differences in variants remain elusive despite associations with several Spike protein mutations. Exploring accessory proteins may provide a wider understanding of these differences since these proteins may affect viral processes that occur beyond infection. Various bioinformatics tools were utilized to identify significant accessory protein mutations and determine their structural and functional effects over time. The ViruClust web application was used to retrieve accessory protein amino acid sequences and determine mutation frequencies in these sequences across time. The structural and functional effects of the mutations were determined using Missense3D and PROVEAN, respectively. The accessory and Spike protein mutations were compared using mutation densities. Q57H and T151I of ORF3a; T21I and W27L of ORF6; G38V, V82A, and T120I of ORF7a; S31P and T40I of ORF7b; and R52I, C61F, and I121L of ORF8 were highly frequent in most variants of concern and were within known functional domains. Thus, these are good candidates for further experimental evaluation. Among the accessory proteins, ORF6 and ORF8 were highlighted because of their strong and weak correlation with Spike protein mutations, respectively.

High-level dolutegravir resistance can emerge rapidly from few variants and spread by recombination: implications for integrase strand transfer inhibitor salvage therapy.

The integrase strand transfer inhibitor (INSTI) dolutegravir is commonly used in combination antiretroviral therapy regimens and retains strong potency even with primary resistance mutations to some other INSTIs. Acquisition of accessory mutations to primary mutations results in significant increases in dolutegravir resistance. Previously, we reported that addition of the secondary mutation T97A can result in rapid treatment failure in individuals with INSTI mutations at positions 140 and 148. Here, we conducted a detailed case study of one of these individuals and find that T97A-containing HIV emerged from a large replicating population from only a few (≤4) viral lineages. When combined with primary INSTI resistance mutations, T97A provides a strong selective advantage; the finding that T97A-containing variants spread by replication and recombination, and persisted for months after discontinuing dolutegravir, has important implications as dolutegravir is rolled out worldwide.

Pre-Treatment Integrase Inhibitor Resistance and Natural Polymorphisms among HIV-1 Subtype C Infected Patients in Ethiopia.

Dolutegravir-based antiretroviral therapy (ART) has been scaled up in many developing countries, including Ethiopia. However, subtype-dependent polymorphic differences might influence the occurrence of HIV-drug-resistance mutations (HIVDRMs). We analyzed the prevalence of pre-treatment integrase strand transfer inhibitor (INSTI) HIVDRMs and naturally occurring polymorphisms (NOPs) of the integrase gene, using plasma samples collected as part of the national HIVDR survey in Ethiopia in 2017. We included a total of 460 HIV-1 integrase gene sequences from INSTI-naïve (n = 373 ART-naïve and n = 87 ART-experienced) patients. No dolutegravir-associated HIVDRMs were detected, regardless of previous exposure to ART. However, we found E92G in one ART-naïve patient specimen and accessory mutations in 20/460 (4.3%) of the specimens. Moreover, among the 288 integrase amino acid positions of the subtype C, 187/288 (64.9%) were conserved (<1.0% variability). Analysis of the genetic barrier showed that the Q148H/K/R dolutegravir resistance pathway was less selected in subtype C. Docking analysis of the dolutegravir showed that protease- and reverse-transcriptase-associated HIVDRMs did not affect the native structure of the HIV-1 integrase. Our results support the implementation of a wide scale-up of dolutegravir-based regimes. However, the detection of polymorphisms contributing to INSTI warrants the continuous surveillance of INSTI resistance.

Analysis of HIV‐1 integrase genotypes and polymorphisms among integrase inhibitors‐based antiretroviral treatment naïve patients in South Sudan

HIV-1 genetic diversity and drug resistance mutations remain public health challenges especially in regions where treatment is limited. The aim of this study was to characterize the HIV-1 integrase (IN) subtype and the possible occurrence of drug-resistance mutations or polymorphisms in resource-poor settings in South Sudan. Dried blood spots from integrase inhibitor treatment (Integrase strand transfer inhibitor [INSTI]) naïve HIV-1 infected patients were subjected to DNA amplification and direct sequencing of integrase genes. The sequences were interpreted for drug resistance according to the Stanford algorithm and the International AIDS Society-USA guidelines. Phylogenetic analysis revealed that HIV-1 subtype D, C, G, A1, and recombinant forms accounted for 40%, 10%, 13.3%, 23.4%, and 13.3%, respectively. Furthermore, inter-subtype recombinants were interspersed within viral strains sampled in other African countries, highlighting complex transmission dynamics within a mobile host population. A total of 78 of 288 (27%) amino acid IN positions presented at least one polymorphism each. Major INSTI resistance mutations were absent, however, polymorphic accessory mutations at positions M50ILR (26.6%) and L74I (3.3%) were detected. Despite the limited size of the study population, our findings underscore the need for monitoring minor and natural polymorphisms that may influence the outcome of treatment regimens.