Easing genomic surveillance: A comprehensive performance evaluation of long-read assemblers across multi-strain mixture data of HIV-1 and Other pathogenic viruses for constructing a user-friendly bioinformatic pipeline.

Background: Determining the appropriate computational requirements and software performance is essential for efficient genomic surveillance. The lack of standardized benchmarking complicates software selection, especially with limited resources. Methods: We developed a containerized benchmarking pipeline to evaluate seven long-read assemblers-Canu, GoldRush, MetaFlye, Strainline, HaploDMF, iGDA, and RVHaplo-for viral haplotype reconstruction, using both simulated and experimental Oxford Nanopore sequencing data of HIV-1 and other viruses. Benchmarking was conducted on three computational systems to assess each assembler's performance, utilizing QUAST and BLASTN for quality assessment. Results: Our findings show that assembler choice significantly impacts assembly time, with CPU and memory usage having minimal effect. Assembler selection also influences the size of the contigs, with a minimum read length of 2,000 nucleotides required for quality assembly. A 4,000-nucleotide read length improves quality further. Canu was efficient among de novo assemblers but not suitable for multi-strain mixtures, while GoldRush produced only consensus assemblies. Strainline and MetaFlye were suitable for metagenomic sequencing data, with Strainline requiring high memory and MetaFlye operable on low-specification machines. Among reference-based assemblers, iGDA had high error rates, RVHaplo showed the best runtime and accuracy but became ineffective with similar sequences, and HaploDMF, utilizing machine learning, had fewer errors with a slightly longer runtime. Conclusions: The HIV-64148 pipeline, containerized using Docker, facilitates easy deployment and offers flexibility to select from a range of assemblers to match computational systems or study requirements. This tool aids in genome assembly and provides valuable information on HIV-1 sequences, enhancing viral evolution monitoring and understanding.

Production of Secreted Antibody in Baculovirus Expression Vector System.

Monoclonal antibodies have widespread applications in disease treatment and antigen detection. They are traditionally produced using mammalian cell expression system, which is not able to satisfy the increasing demand of these proteins at large scale. Baculovirus expression vector system (BEVS) is an attractive alternative platform for the production of biologically active monoclonal antibodies. In this chapter, we demonstrate the production of an HIV-1 broadly neutralizing antibody b12 in BEVS. The processes including transfer vector construction, recombinant baculovirus generation, and antibody production and detection are described.

Differences in neutralizing antibody sensitivities and envelope characteristics indicate distinct antigenic properties of Nigerian HIV-1 subtype G and CRF02_AG.

The magnitude of the HIV-1 epidemic in Nigeria is second only to the subtype C epidemic in South Africa, yet the subtypes prevalent in Nigeria require further characterization. A panel of 50 subtype G and 18 CRF02_AG Nigerian HIV-1 pseudoviruses (PSV) was developed and envelope coreceptor usage, neutralization sensitivity and cross-clade reactivity were characterized. These PSV were neutralized by some antibodies targeting major neutralizing determinants, but potentially important differences were observed in specific sensitivities (eg. to sCD4, MPER and V2/V3 monoclonal antibodies), as well as in properties such as variable loop lengths, number of potential N-linked glycans and charge, demonstrating distinct antigenic characteristics of CRF02_AG and subtype G. There was preferential neutralization of the matched CRF/subtype when PSV from subtype G or CRF02_AG were tested using pooled plasma. These novel Nigerian PSV will be useful to study HIV-1 CRF- or subtype-specific humoral immune responses for subtype G and CRF02_AG.

Post-Integrational DNA Repair of HIV-1 Is Associated with Activation of the DNA-PK and ATM Cellular Protein Kinases and Phosphorylation of Their Targets.

Integration of the DNA copy of HIV-1 genome into the cellular genome results in series of damages, repair of which is critical for successful replication of the virus. We have previously demonstrated that the ATM and DNA-PK kinases, normally responsible for repairing double-strand breaks in the cellular DNA, are required to initiate the HIV-1 DNA postintegrational repair, even though integration does not result in DNA double-strand breaks. In this study, we analyzed changes in phosphorylation status of ATM (pSer1981), DNA-PK (pSer2056), and their related kinase ATR (pSer428), as well as their targets: Chk1 (pSer345), Chk2 (pThr68), H2AX (pSer139), and p53 (pSer15) during the HIV-1 DNA postintegrational repair. We have shown that ATM and DNA-PK, but not ATR, undergo autophosphorylation during postintegrational DNA repair and phosphorylate their target proteins Chk2 and H2AX. These data indicate common signaling mechanisms between the double-strand DNA break repair and postintegrational repair of HIV-1 DNA.

Missed Opportunities for HIV Confirmatory Testing Using an Institutional Testing Algorithm Without Reflex to HIV Nucleic Acid Testing.

ABSTRACT: At our medical center, HIV nucleic acid tests are recommended when the HIV antigen-antibody screening immunoassay and antibody differentiation tests are discordant, but not done reflexively. A retrospective chart review found that 35% of discordant test results did not have HIV nucleic acid test completed as recommended.

Efficacy of first-line ART regimens based on tenofovir in HIV-infected patients with pre-existing A62V mutation in reverse transcriptase.

INTRODUCTION: The amino acid substitution A62V in reverse transcriptase was identified as a mutation correlated with virologic failure in patients on first-line therapy including tenofovir (TDF) and tenofovir alafenamide (TAF). A62V is a typically polymorphic mutation in HIV-1 sub-subtype A6, which is the most widespread virus variant in Russia. MATERIALS AND METHODS: The European EuResist (EIDB) database was queried to form two equivalent groups of patients: group 1 ‒ patients with A62V at baseline treated with TDF or TAF on the first-line therapy, group 2 ‒ patients without A62V at baseline treated with TDF or TAF on the first-line therapy. Each group included 23 patients. RESULTS: There was no statistical difference between the two groups in virologic efficacy in 4, 12, and 24 weeks after the start of antiretroviral therapy (ART) and in the frequency of virologic failures. CONCLUSION: This study has some limitations, and the exact role of A62V in the efficacy of the first-line ART based on tenofovir deserves further investigation.

Safety and Effectiveness From the Cabotegravir and Rilpivirine Implementation Study in European Locations Study: Phase 3b Hybrid Type III Implementation Study Integrating Cabotegravir + Rilpivirine Long-Acting Into European Clinical Settings.

BACKGROUND: Cabotegravir + rilpivirine long-acting (CAB + RPV LA) dosed every 2 months (Q2M) is a complete regimen for the maintenance of HIV-1 virologic suppression. In this study, we report month 12 clinical outcomes in patient study participants (PSPs) in the CAB and RPV Implementation Study in European Locations (CARISEL) study. SETTING: CARISEL is a phase 3b implementation-effectiveness study. METHODS: CARISEL was designed as a 2-arm, unblinded study with centers randomized to either enhanced or standard implementation arms. For PSPs, this study is single arm, unblinded, and interventional; all PSPs switched from daily oral therapy to CAB + RPV LA dosed Q2M. The primary objective was to evaluate the perceived acceptability, appropriateness, and feasibility of CAB + RPV LA implementation for staff participants (presented separately). Clinical secondary endpoints assessed through month 12 included the proportion of PSPs with plasma HIV-1 RNA ≥50 and <50 copies/mL (Snapshot algorithm), incidence of confirmed virologic failure (CVF; 2 consecutive plasma HIV-1 RNA levels ≥200 copies/mL), adherence to injection visit windows, and safety and tolerability. RESULTS: Four hundred thirty PSPs were enrolled and treated; the mean age was 44 years (30% ≥50 years), 25% were women (sex at birth), and 22% were persons of color. At month 12, 87% (n = 373/430) of PSPs maintained HIV-1 RNA <50 copies/mL, with 0.7% (n = 3/430) having HIV-1 RNA ≥50 copies/mL. One PSP had CVF. The safety profile was consistent with previous findings. Overall, the results were similar between implementation arms. CONCLUSION: CAB + RPV LA Q2M was well tolerated and highly effective in maintaining virologic suppression with a low rate of virologic failure.

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.

Real world community-based HIV Rapid Start Antiretroviral with B/F/TAF versus prior models of antiretroviral therapy start - the RoCHaCHa study, a pilot study.

BACKGROUND: The rapid start of antiretroviral therapy (RSA) model initiates antiretroviral therapy (ART) as soon as possible after a new or preliminary diagnosis of HIV, in advance of HIV-1 RNA and other baseline laboratory testing. This observational study aims to determine if RSA with a single tablet regimen of bictegravir, emtricitabine, and tenofovir alafenamide (B/F/TAF) is an effective regimen for achieving viral suppression and accepted by patients at the time of diagnosis. METHODS: Adults newly or preliminarily diagnosed with HIV were enrolled from October 2018 through September 2021. Real world advantage, measured in days between clinical milestones and time to virologic suppression, associated with B/F/TAF RSA was compared to historical controls. RESULTS: All Study RSA participants (n = 45) accepted treatment at their first visit and 43(95.6%) achieved virologic suppression by week 48. Study RSA participants had a significantly shorter time (median 32 days) from diagnosis to ART initiation and virologic suppression, in comparison to historical controls (median 181 days) (n = 42). Qualitative feedback from study RSA participants showed high acceptance positive response to RSA. CONCLUSIONS: RSA is feasible and well accepted by patients in a real-world community-based clinic setting. Promoting RSA in community-based clinics is an important tool in ending the HIV epidemic.

Impact of HIV-1 genetic diversity on disease progression: a prospective cohort study in Guangxi.

The high proportion of AIDS cases and mortality rates in Guangxi underscores the urgency to investigate the influence of HIV-1 genetic diversity on disease progression in this region. Newly diagnosed HIV-1 patients were enrolled from January 2016 to December 2021, and the follow-up work and detection of CD4+T lymphocytes were carried out every six months until December 2022. Multivariate logistic regression was used to analyze the factors affecting pre-treatment CD4+T lymphocyte counts, while local weighted regression models (LOESS) and generalized estimating equation models (GEE) were conducted to assess factors influencing CD4+T Lymphocyte Recovery. Cox regression analysis was utilized to examine the impact of subtypes on survival risk. Additionally, HIV-1 env sequences were utilized for predicting CXCR4 and CCR5 receptors. The study encompassed 1867 individuals with pol sequences and 281 with env sequences. Our findings indicate that age over 30, divorced/widowed, peasant, heterosexual infection, CRF01_AE, long-term infection, and Pre-treatment Viral load >10000 copies/ml were factors associated with higher risk for pre-treatment CD4+T lymphocyte decline. Specifically, male gender, age over 30, heterosexual infection (HETs), long-term infection, CRF01_AE, and Pre-treatment CD4 T cell counts below 350/µL were identified as risk factors impeding CD4+T lymphocyte recovery. Pre-treatment CD4+T lymphocyte counts and recovery in individuals infected with CRF01_AE were lower compared to CRF07_BC and CRF55_01B. Additionally, CRF01_AE and CRF08_BC subtypes exhibited higher mortality rates than CRF07_BC, CRF55_01B, and other subtypes. Notably, CRF01_AE demonstrated the highest percentage of CXCR4 affinity ratios. This research unveils the intricate influence of HIV-1 gene diversity on CD4+T lymphocyte dynamics and clinical outcomes. It highlights the multifaceted nature of HIV infection in Guangxi, providing novel insights into subtype-specific disease progression among HIV-infected individuals in this region.

Sensing of an HIV-1-Derived Single-Stranded RNA-Oligonucleotide Induces Arginase 1-Mediated Tolerance.

Small synthetic oligodeoxynucleotides (ODNs) can mimic microbial nucleic acids by interacting with receptor systems and promoting immunostimulatory activities. Nevertheless, some ODNs can act differently on the plasmacytoid dendritic cell (pDC) subset, shaping their immunoregulatory properties and rendering them suitable immunotherapeutic tools in several clinical settings for treating overwhelming immune responses. We designed HIV-1-derived, DNA- and RNA-based oligonucleotides (gag, pol, and U5 regions) and assessed their activity in conferring a tolerogenic phenotype to pDCs in skin test experiments. RNA-but not DNA-oligonucleotides are capable of inducing tolerogenic features in pDCs. Interestingly, sensing the HIV-1-derived single-stranded RNA-gag oligonucleotide (RNA-gag) requires both TLR3 and TLR7 and the engagement of the TRIF adaptor molecule. Moreover, the induction of a suppressive phenotype in pDCs by RNA-gag is contingent upon the induction and activation of the immunosuppressive enzyme Arginase 1. Thus, our data suggest that sensing of the synthetic RNA-gag oligonucleotide in pDCs can induce a suppressive phenotype in pDCs, a property rendering RNA-gag a potential tool for therapeutic strategies in allergies and autoimmune diseases.

Adult Human Brain Tissue Cultures to Study NeuroHIV.

HIV-associated neurocognitive disorders (HAND) persist under antiretroviral therapy as a complex pathology that has been difficult to study in cellular and animal models. Therefore, we generated an ex vivo human brain slice model of HIV-1 infection from surgically resected adult brain tissue. Brain slice cultures processed for flow cytometry showed >90% viability of dissociated cells within the first three weeks in vitro, with parallel detection of astrocyte, myeloid, and neuronal populations. Neurons within brain slices showed stable dendritic spine density and mature spine morphologies in the first weeks in culture, and they generated detectable activity in multi-electrode arrays. We infected cultured brain slices using patient-matched CD4+ T-cells or monocyte-derived macrophages (MDMs) that were exposed to a GFP-expressing R5-tropic HIV-1 in vitro. Infected slice cultures expressed viral RNA and developed a spreading infection up to 9 days post-infection, which were significantly decreased by antiretrovirals. We also detected infected myeloid cells and astrocytes within slices and observed minimal effect on cellular viability over time. Overall, this human-centered model offers a promising resource to study the cellular mechanisms contributing to HAND (including antiretroviral toxicity, substance use, and aging), infection of resident brain cells, and new neuroprotective therapeutics.

May I Help You with Your Coat? HIV-1 Capsid Uncoating and Reverse Transcription.

The human immunodeficiency virus type 1 (HIV-1) capsid is a protein core formed by multiple copies of the viral capsid (CA) protein. Inside the capsid, HIV-1 harbours all the viral components required for replication, including the genomic RNA and viral enzymes reverse transcriptase (RT) and integrase (IN). Upon infection, the RT transforms the genomic RNA into a double-stranded DNA molecule that is subsequently integrated into the host chromosome by IN. For this to happen, the viral capsid must open and release the viral DNA, in a process known as uncoating. Capsid plays a key role during the initial stages of HIV-1 replication; therefore, its stability is intimately related to infection efficiency, and untimely uncoating results in reverse transcription defects. How and where uncoating takes place and its relationship with reverse transcription is not fully understood, but the recent development of novel biochemical and cellular approaches has provided unprecedented detail on these processes. In this review, we present the latest findings on the intricate link between capsid stability, reverse transcription and uncoating, the different models proposed over the years for capsid uncoating, and the role played by other cellular factors on these processes.

Recapitulation of HIV-1 Neutralization Breadth in Plasma by the Combination of Two Broadly Neutralizing Antibodies from Different Lineages in the Same SHIV-Infected Rhesus Macaque.

Viral infection generally induces polyclonal neutralizing antibody responses. However, how many lineages of antibody responses can fully represent the neutralization activities in sera has not been well studied. Using the newly designed stable HIV-1 Env trimer as hook, we isolated two distinct broadly neutralizing antibodies (bnAbs) from Chinese rhesus macaques infected with SHIV1157ipd3N4 for 5 years. One lineage of neutralizing antibodies (JT15 and JT16) targeted the V2-apex in the Env trimers, similar to the J038 lineage bnAbs identified in our previous study. The other lineage neutralizing antibody (JT18) targeted the V3 crown region in the Env, which strongly competed with human 447-52D. Each lineage antibody neutralized a different set of viruses. Interestingly, when the two neutralizing antibodies from different lineages isolated from the same macaque were combined, the mixture had a neutralization breath very similar to that from the cognate sera. Our study demonstrated that a minimum of two different neutralizing antibodies can fully recapitulate the serum neutralization breadth. This observation can have important implications in AIDS vaccine design.

The origin and spread of HIV-1 CRF59_01B epidemic in China: A molecular network and phylogeographic analysis.

Human immunodeficiency virus type 1 CRF59_01B, identified in China in 2013, has been detected nationwide, exhibiting notably high prevalence in Guangzhou and its vicinity. This study aimed to unravel its origin and migration. A data set was established, incorporating all available CRF59_01B pol gene sequences and their metadata from Guangzhou and the public database. Bayesian phylogeographic analysis demonstrated that CRF59_01B originated in Shenzhen, the neighboring city of Guangzhou, around 1998 with posterior probability of 0.937. Molecular network analysis detected 1131 transmission links and showed a remarkably high clustering rate (78.9%). Substantial inter-city transmissions (26.5%, 300/1131) were observed between Shenzhen and Guangzhou while inter-region transmissions linked Guangzhou with South (46) and Southwest (64) China. The centre of Guangzhou was the hub of CRF59_01B transmission, including the inflow from Shenzhen (3.57 events/year) and outflow to the outskirts of Guangzhou (>2 events/year). The large-scale analysis revealed significant migration from Shenzhen to Guangzhou (5.08 events/year) and North China (0.59 events/year), and spread from Guangzhou to Central (0.47 events/year), East (0.42 events/year), South (0.76 events/year), Southwest China (0.76 events/year) and Shenzhen (1.89 events/year). Shenzhen and Guangzhou served as the origin and the hub of CRF59_01B circulation, emphasizing inter-city cooperation and data sharing to confine its nationwide diffusion.

HIV-1 Vpr combats the PU.1-driven antiviral response in primary human macrophages.

HIV-1 Vpr promotes efficient spread of HIV-1 from macrophages to T cells by transcriptionally downmodulating restriction factors that target HIV-1 Envelope protein (Env). Here we find that Vpr induces broad transcriptomic changes by targeting PU.1, a transcription factor necessary for expression of host innate immune response genes, including those that target Env. Consistent with this, we find silencing PU.1 in infected macrophages lacking Vpr rescues Env. Vpr downmodulates PU.1 through a proteasomal degradation pathway that depends on physical interactions with PU.1 and DCAF1, a component of the Cul4A E3 ubiquitin ligase. The capacity for Vpr to target PU.1 is highly conserved across primate lentiviruses. In addition to impacting infected cells, we find that Vpr suppresses expression of innate immune response genes in uninfected bystander cells, and that virion-associated Vpr can degrade PU.1. Together, we demonstrate Vpr counteracts PU.1 in macrophages to blunt antiviral immune responses and promote viral spread.

Genotypic Analysis of Transmitted and Acquired HIV Drug Resistance in People Living with HIV/AIDS in Surabaya, Indonesia, from 2018 to 2019.

BACKGROUND: Despite the availability of various effective antiretroviral (ARV) drugs, human immunodeficiency virus (HIV) infection has come with HIV drug resistance (HIVDR), which compromises its effectiveness in reducing HIV-related morbidity, mortality, and transmission. The emergence of transmitted (TDR) and acquired HIVDR (ADR) among antiretroviral therapy (ART)-naïve and experienced individuals have been reported in several Indonesian regions. Therefore, continuous HIVDR surveillance is needed in Indonesia, especially in Surabaya, which is identified as having the highest prevalence of HIV infection in East Java; thus, this study aimed to identify the emergence of TDR and ADR among people living with HIV/acquired immune deficiency syndrome (AIDS) (PLWHA). METHODS: Fifty-eight PLWHA infected with HIV type 1 (HIV-1), comprising 21 and 37 ART-naïve and experienced individuals were enrolled in this study, respectively. Blood samples collected from study participants were subjected to genotypic analysis, mainly towards the pol gene encoding protease (PR gene) and reverse transcriptase (RT gene) of HIV-1. RESULTS: Seventeen PR and 21 RT genes were successfully amplified and sequenced from 29 samples. HIV-1 subtyping revealed CRF01_AE as the most dominant subtype (24/29; 82.76%), followed by subtype B (3/29; 10.34%). Uncommon subtypes, including subtype D and a recombinant containing subtypes B and G genomic fragments, were also identified. TDR for PR inhibitors was not detected; however, TDR and ADR for RT inhibitors were identified in 11.11% and 41.67% of samples, respectively. Two amino acid insertions at position 69 of the RT gene (69ins), a previously never-reported mutation in Indonesia, were identified in this study. CONCLUSION: Both TDR and ADR have emerged among PLWHA residing in Surabaya, East Java, Indonesia. Uncommon drug-resistance mutations and subtypes were identified in this study. These situations might hamper ART efficacy and treatment success. Continuous surveillance of HIVDR is necessary to monitor both TDR and ADR in Indonesia.

Modelling Role of Protective and Nonprotective HLA Allele Inducing Different HIV Infection Outcomes.

Human immunodeficiency virus (HIV) infects CD4+ cells and causes progressive immune function failure, and CD8+ cells lyse infected CD4+ cell via recognising peptide presented by human leukocyte antigens (HLA). Variations in HLA allele lead to observed different HIV infection outcomes. Within-host HIV dynamics involves virus replication within infected cells and lysing of infected cells by CD8+ cells, but how variations in HLA alleles determine different infection outcomes was far from clear. Here, we used mathematical modelling and parameter inference with a new analysis of published virus inhibition assay data to estimate CD8+ cell lysing efficiency, and found that lysing efficiency fall in the gap between low bound (0.1-0.2 day-1 (Elemans et al. in PLoS Comput Biol 8(2):e1002381, 2012)) and upper boundary (6.5-8.4 day-1 (Wick et al. in J Virol 79(21):13579-13586, 2005)). Our outcomes indicate that both lysing efficiency and viral inoculum size jointly determine observed different infection outcomes. Low lysing rate associated with non-protective HLA alleles leads to monostable viral kinetic to high viral titre and oscillatory viral kinetics. High lysing rate associated with protective HLA alleles leads monostable viral kinetic to low viral titre and bistable viral kinetics; at a specific interval of CD8+ cell counts, small viral inoculum sizes are inhibited but not large viral inoculum sizes remain infectious. Further, with CD8+ cell recruitment, HIV kinetics always exhibit oscillatory kinetics, but lysing rate is negatively correlated with range of CD8+ cell count. Our finding highlights role of HLA allele determining different infection outcomes, thereby providing a potential mechanistic explanation for observed good and bad HIV infection outcomes induced by protective HLA allele.

HIV1 drug resistance among patients experiencing first-line treatment failure in Ethiopia: protocol for a systematic review and meta-analysis.

BACKGROUND: The emergence of HIV drug resistance presents a substantial challenge. Current antiretroviral treatments, along with current classes, face the danger of becoming partially or entirely inactive. As a result, alternative treatment regimens are limited, and treatment choices are complicated. According to the recommendation of the WHO, nations should consider changing their first-line ART regimen if HIV drug resistance exceeds 10%. In spite of the fact that a number of primary studies have been performed on HIV drug resistance in Ethiopia, their pooled prevalence rate has not been determined in a systematic review and meta-analysis, which may provide stronger evidence. Therefore, the objective of this systematic review and meta-analysis will be to estimate the pooled prevalence rate of HIV1 drug resistance in patients with first-line treatment failure in Ethiopia. METHODS: Primary studies will be identified from PubMed/MEDLINE, Scopus, Embase, Web of Science Core Collection, and Google Scholar. The period of search will be from 01 April to 30 June 2024. Studies identified through the search strategies will first be screened by titles and abstracts. Included studies meeting established criteria will be evaluated for risk of bias using the JBI checklist. Data will be extracted, and the pooled prevalence rate of HIV drug resistance will be computed using STATA 14 software. Random effect models will be used when heterogeneity is suspected. The I2 statistic and its corresponding P value will be checked to distinguish heterogeneity. Additionally, publication bias and heterogeneity will be checked using visual funnel plots, Egger's test, trim-and-fill tests, meta-regression, and subgroup analysis. To present and synthesize the results, narrative synthesis will be performed to describe study characteristics and findings, and forest plots will be used to visually represent effect sizes and confidence intervals from individual studies. DISCUSSION: Estimating the pooled prevalence rate of HIV drug resistance through a systematic review and meta-analysis improves the reliability of the evidence, the availability of effective HIV treatment options, and the ability to assist in making decisions for both clinical practice and public health policy in Ethiopia. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42024533975.