Reverse Transcriptase Sequences Research Articles

Human cells contain myriad excised linear intron RNAs with links to gene regulation and potential utility as biomarkers.

A previous study using Thermostable Group II Intron Reverse Transcriptase sequencing (TGIRT-seq) found human plasma contains short (≤300 nt) structured full-length excised linear intron (FLEXI) RNAs with potential to serve as blood-based biomarkers. Here, TGIRT-seq identified >9,000 different FLEXI RNAs in human cell lines, including relatively abundant FLEXIs with cell-type-specific expression patterns. Analysis of public CLIP-seq datasets identified 126 RNA-binding proteins (RBPs) that have binding sites within the region corresponding to the FLEXI or overlapping FLEXI splice sites in pre-mRNAs, including 53 RBPs with binding sites for ≥30 different FLEXIs. These included splicing factors, transcription factors, a chromatin remodeling protein, cellular growth regulators, and proteins with cytoplasmic functions. Analysis of ENCODE datasets identified subsets of these RBPs whose knockdown impacted FLEXI host gene mRNA levels or proximate alternative splicing, indicating functional interactions. Hierarchical clustering identified six subsets of RBPs whose FLEXI binding sites were co-enriched in six subsets of functionally related host genes: AGO1-4 and DICER, including but not limited to agotrons or mirtron pre-miRNAs; DKC1, NOLC1, SMNDC1, and AATF (Apoptosis Antagonizing Transcription Factor), including but not limited to snoRNA-encoding FLEXIs; two subsets of alternative splicing factors; and two subsets that included RBPs with cytoplasmic functions (e.g., LARP4, PABPC4, METAP2, and ZNF622) together with regulatory proteins. Cell fractionation experiments showed cytoplasmic enrichment of FLEXI RNAs with binding sites for RBPs with cytoplasmic functions. The subsets of host genes encoding FLEXIs with binding sites for different subsets of RBPs were co-enriched with non-FLEXI other short and long introns with binding sites for the same RBPs, suggesting overarching mechanisms for coordinately regulating expression of functionally related genes. Our findings identify FLEXIs as a previously unrecognized large class of cellular RNAs and provide a comprehensive roadmap for further analyzing their biological functions and the relationship of their RBPs to cellular regulatory mechanisms.

Trends and Patterns of HIV Transmitted Drug Resistance in China From 2018 to 2023.

National treatment guidelines of China evolving necessitates population-level surveillance of transmitted drug resistance (TDR) to inform or update HIV treatment strategies. We analyzed the demographic, clinical, and virologic data obtained from people with HIV (PWH) residing in 31 provinces of China who were newly diagnosed between 2018 and 2023. Evidence of TDR was defined by the World Health Organization list for surveillance of drug resistance mutations. Among the 22 124 PWH with protease and reverse transcriptase sequences, 965 (4.36%; 95% CI, 4.1-4.63) had at least 1 TDR mutation. The most frequent TDR mutations were nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations (2.39%; 95% CI, 2.19%-2.59%), followed by nucleoside reverse transcriptase inhibitor mutations(1.35%; 95% CI, 1.2%-1.5%) and protease inhibitor mutations (1.12%; 95% CI, .98%-1.26%). The overall protease and reverse transcriptase TDR increased significantly from 4.05% (95% CI, 3.61%-4.52%) in 2018 to 5.39% (95% CI, 4.33%-6.57%) in 2023. A low level of integrase strand transfer inhibitor TDR was detected in 9 (0.21%; 95% CI, .1%-.38%) of 4205 PWH. Presently, the continued use of NNRTI-based first-line antiretroviral therapy regimen for HIV treatment has been justified.

Polio eradication surveillance in Sri Lanka, 2019-2023

Objective: To evaluate the polio laboratory surveillance carried out from January, 2019 to May, 2023 by the Polio Regional Reference Laboratory, Sri Lanka. Methods: This retrospective study analyzed all stool samples received under the acute flaccid paralysis (AFP) and immunodeficient vaccine-derived poliovirus (VDPV) surveillance at Polio Regional Reference Laboratory, Sri Lanka from January, 2019 to May, 2023. The results of the testing methodologies were extracted from the laboratory data system, i.e., poliovirus virus isolation, intra-typic differentiation/VDPV real time reverse transcriptase polymerase chain reaction (ITD/VDPV rRTPCR) and sequencing, along with the data on timing of reporting results, stool adequacy and socio-demographics. Data was analyzed using descriptive statistics. Results: A total of 2141 stool samples from 1 644 cases were received for AFP surveillance from Sri Lanka (93.61%), Maldives (1.52%), and immunodeficient VDPV (4.86%) surveillance. Both polioviruses (19/1 644, 1.15%) and non-polio enteroviruses (73/1 644, 4.44%) were isolated, while Sabin-like 3 virus was detected in majority (12/19, 63.15%) among the poliovirus isolated. Wild polioviruses or circulating VDPVs were not detected among the cases. During all years of the study, the non-polio AFP detection rate was >1/100 000 in children aged less than 15 years, whereas stool adequacy rate was >80%. All results were reported within 14 days of receipt, ensuring timely reporting as per global guidelines. Conclusions: The Polio Regional Reference Laboratory, Sri Lanka plays a vital role in maintaining the polio-free status in the country through its robust laboratory surveillance, while adhering to the surveillance indicators. Non-detection of wild polioviruses and circulating VDPV during the study period reinforces the polio-free status in the country.

Pharmaco-virological outcomes and genotypic resistance profiles among children and adolescents receiving a DTG-based regimen in Togo.

Few data are available on the real-world efficacy of receiving tenofovir-lamivudine-dolutegravir (DTG) as HIV treatment, particularly among young people in West Africa. Here, we evaluated pharmaco-virological outcomes and resistance profiles among Togolese children and adolescents. A cross-sectional study was conducted in Lomé, Togo, enrolling antiretroviral-treated people with HIV aged from 18 months to 24 years. Plasma HIV-1 viral load and antiretroviral concentrations were measured. Next-Generation Sequencing (NGS) of protease, Reverse Transcriptase (RT) and integrase was performed on all samples with viral load >200 c/mL. Drug resistance mutations (DRMs) were identified and interpreted using the ANRS-MIE algorithm. 264 participants were enrolled (median age=17 years), 226 received a DTG-based regimen for a median of 20.5 months. Among them, virological suppression at the 200 c/mL threshold in 80.0% of the participants. Plasma DTG concentrations were adequate (i.e., >640 ng/mL), suboptimal and below the limit of quantification in 74.1%, 6.7% and 19.2% of participants receiving DTG, respectively. Overall, viruses resistant to any of Nucleoside RT Inhibitors, Non-NRTIs, and protease inhibitors were found in 52%, 66% and 1.6% of participants, respectively. A major integrase inhibitor DRM was observed in 9.4% (n=3/32, R263K, E138A-G140A-Q148R, and N155H) of participants with a viral load >200 c/mL. These first findings in such a large series of adolescents in a low-income country, showed a good virological response of 80% and the presence of an integrase DRM in 9.4% of the virological failures, supporting the need to monitor DTG drug resistance to reduce the risk of resistance acquisition.

Abstract PO4-23-11: Resistance Mechanisms to CDK4/6 Inhibitors and/or Tamoxifen Using Comprehensive Thermostable Group II Intron Reverse Transcriptase Sequencing

Abstract Background Understanding the mechanisms of resistance to CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) is pivotal in exploring new therapeutic strategies for hormone receptor-positive (HR+), HER2-negative metastatic breast cancer. To decipher these resistance mechanisms, we analyzed the alterations in comprehensive RNA-seq (coding and non-coding RNAs) of HR+ HER2-negative BC cell lines via thermostable group II intron reverse transcriptase sequencing (TGIRT-seq). Methods We established Tamoxifen-resistant (TMR), Abemaciclib-resistant (ACR), Palbociclib-resistant (PCR), Tamoxifen/Abemaciclib double-resistant (TMR-ACR), and Tamoxifen/Palbociclib double-resistant (TMR-PCR) BC cell lines from MCF7 and T47D HR+/HER2- BC cell lines through stepwise dose-escalation continuous drug exposure. We performed a TGIRT-seq transcriptomic analysis using a protocol that allows the sequencing of both long and short non-coding and protein-coding RNAs in a single library. All libraries were sequenced using paired-end 150 bp on the Novaseq platform, resulting in an average of 50 million reads per library. For analysis, raw reads underwent adapter trimming, small RNA mapping, whole genome mapping, and the generation of annotated genes' read count. We used raw counts to detect differentially expressed genes (DEGs) with DESeq2 in R using the cut-off (log2 [fold change] > 1, FDR < 1e-3). We then used the selected DEGs for Gene Set Enrichment Analysis (GSEA) and Kaplan-Meier survival analysis from the TCGA database. Results The TGIRT-seq analysis identified 1171 to 3472 DEGs in different drug resistance and cell line combinations. Most DEGs in resistant cells were consistently downregulated compared to parent cells. Seventy-five percent of DEGs were protein-coding genes, with the rest being non-coding RNAs, such as small nucleolar RNAs, microRNAs, long non-coding RNAs, and transposable element RNAs, which conventional RNA-seq poorly detects. In principal component analysis (PCA) plots, replicates per drug resistance clustered clearly in both cell line backgrounds. PCA also indicated distinctive pathways for acquiring drug resistance in MCF7 and T47D cells. The estrogen receptor (ER) expression decreased, while HER2 increased in resistant cell lines. DEGs identified were enriched in ESR1-related pathways in all MCF7 and T47D cell lines resistant models. Several ER-regulating genes like IL-1R1 and RET were upregulated, while ADCY1 was consistently downregulated across different resistance types. A significant overlap with single-resistance DEGs was observed among the DEGs in double-resistance cell lines, but 20-50% of up/down-regulated DEGs in double-resistance cell lines were uniquely altered. Several previously identified DEGs (e.g., SALL4, TOP2A) and 21 novel candidate genes correlated with poor survival outcomes. Conclusion The analysis identified unique DEGs in double resistance cell lines, suggesting that double resistance mechanisms may not merely be a cumulative effect of single resistance mechanisms, necessitating further investigation and validation. CDK4/6i and/or ET-resistant BC cell lines displayed significant transcriptome reprogramming during the development of drug resistance. In previously published research, ESR1-related pathway alterations were proposed in tamoxifen resistance cell lines. Here we find that CDK4/6i-resistant cells also modify these ESR1-related pathways. Additionally, we identified several targetable genes, such as IL-1R1 and RET, involved in ESR1-related pathways that could pave the way for developing new treatment strategies. Citation Format: Toshiaki Iwase, Hengyi Xu, Nakyung Oh, Jangsoon Lee, Alan M Lambowitz, Naoto Ueno. Resistance Mechanisms to CDK4/6 Inhibitors and/or Tamoxifen Using Comprehensive Thermostable Group II Intron Reverse Transcriptase Sequencing [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-11.

Lesions and viral antigen distribution in bald eagles, red-tailed hawks, and great horned owls naturally infected with H5N1 clade 2.3.4.4b highly pathogenic avian influenza virus.

An epidemic of highly pathogenic avian influenza (HPAI) began in North America in the winter of 2021. The introduced Eurasian H5N1 clade 2.3.4.4b virus subsequently reassorted with North American avian influenza strains. This postmortem study describes the lesions and influenza A virus antigen distribution in 3 species of raptors, including bald eagles (Haliaeetus leucocephalus, n = 6), red-tailed hawks (Buteo jamaicensis, n = 9), and great horned owls (Bubo virginianus, n = 8), naturally infected with this virus strain based on positive reverse transcriptase polymerase chain reaction and sequencing results from oropharyngeal swabs. The birds presented with severe neurologic signs and either died or were euthanized because of the severity of their clinical signs and suspected influenza virus infection. Gross lesions were uncommon and included forebrain hemorrhages in 2 eagles, myocarditis in 1 hawk, and multifocal pancreatic necrosis in 3 owls. Histological lesions were common and included encephalitis, myocarditis, multifocal pancreas necrosis, multifocal adrenal necrosis, histiocytic splenitis, and anterior uveitis in decreasing frequency. Influenza A viral antigen was detected in brain, heart, pancreas, adrenal gland, kidney, spleen, liver, and eye. In conclusion, bald eagles, red-tailed hawks, and great horned owls infected with the HPAI clade 2.3.4.4b virus strain and showing neurological signs of illness may develop severe or fatal disease with histologically detectable lesions in the brain that are frequently positive for viral antigen.

Transmitted drug resistance to antiretroviral drugs in Spain during the period 2019-2021.

To evaluate the prevalence of transmitted drug resistance (TDR)to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTI,NNRTI),protease inhibitors (PI),and integrase strand transfer inhibitors (INSTI)in Spain during the period 2019-2021, as well as to evaluate transmitted clinically relevant resistance (TCRR)to antiretroviral drugs. Reverse transcriptase (RT),protease (Pro), and Integrase (IN)sequences from 1824 PLWH (people living with HIV) were studied. To evaluate TDR we investigated the prevalence of surveillance drug resistance mutations (SDRM).To evaluate TCRR (any resistance level ≥ 3), and for HIV subtyping we used the Stanford v.9.4.1 HIVDB Algorithm and an in-depth phylogenetic analysis. The prevalence of NRTI SDRMs was 3.8% (95% CI,2.8%-4.6%), 6.1% (95% CI,5.0%-7.3%) for NNRTI, 0.9% (95% CI,0.5%-1.4%) for PI, and 0.2% (95% CI,0.0%-0.9%) for INSTI. The prevalence of TCRR to NRTI was 2.1% (95% CI,1.5%-2.9%), 11.8% for NNRTI, (95% CI,10.3%-13.5%), 0.2% (95% CI,0.1%-0.6%) for PI, and 2.5% (95% CI,1.5%-4.1%) for INSTI. Most of the patients were infected by subtype B (79.8%), while the majority of non-Bs were CRF02_AG (n = 109, 6%). The prevalence of INSTI and PI resistance in Spain during the period 2019-2021 is low, while NRTI resistance is moderate, and NNRTI resistance is the highest. Our results support the use of integrase inhibitors as first-line treatment in Spain. Our findings highlight the importance of ongoing surveillance of TDR to antiretroviral drugs in PLWHparticularly with regard to first-line antiretroviral therapy.

Transmitted drug resistance and subtype patterns of viruses from reported new HIV diagnoses in Germany, 2017–2020

BackgroundThe transmission of resistant HIV variants jeopardizes the effective use of antiretrovirals for therapy and prophylaxis. Molecular surveillance of new HIV diagnoses with a focus on prevalence and type of resistance associated mutations and the subtype of circulating viruses is mandatory.MethodFrom 2017 to 2020, 11,527 new HIV diagnoses were reported in Germany to the Robert Koch Institute (RKI). Protease (PR) and reverse-transcriptase (RT) sequences were obtained from 4559 (39.6%) cases, and PR, RT and integrase (IN) sequences were obtained from 3097 (26.9%) cases. The sequences were analyzed with data from the national HIV reports.ResultsAmong all cases in the analysis, the proportion of primary resistance was 4.3% for nucleoside reverse-transcriptase inhibitors (NRTIs), 9.2% for non-NRTI (NNRTIs), 3.3% for protease inhibitors (PIs) and 1.4% for integrase inhibitors (INIs). Dual-class resistance was highest for NRTIs/NNRTIs with 1.2%. There was no trend in the proportion of viruses resistant to drug classes. Most individual key mutations associated with relevant resistance had a prevalence below 1% including K65R (0.1%) and M184V (0.6%). A notable exception was K103NS, with a prevalence of 2.9% and a significant increase (pTrend=0.024) during 2017–2020. In this period, diagnoses of infections with HIV-1 subtype B were the most common at 58.7%, but its prevalence was declining (pTrend=0.049) while the frequency of minority subtypes (each < 1%) increased (pTrend=0.007). Subtype B was highest (75.6%) in men who have sex with men (MSM) and lowest in reported heterosexual transmissions (HETs, 22.6%).ConclusionThe percentage of primary resistance was high but at a stable level. A genotypic determination of resistance is therefore still required before the start of therapy. The subtype diversity of circulating HIV-1 is increasing.

Pre-Treatment HIV Drug Resistance and Genetic Diversity in Cameroon: Implications for First-Line Regimens.

The efficacy of first-line antiretroviral therapy (ART) may be hampered by the presence of HIV drug resistance (HIVDR). We described HIV-1 pre-treatment drug resistance (PDR) patterns, effect of viral clades on PDR, and programmatic implications on first-line regimens in Cameroon. A sentinel surveillance of PDR was conducted from 2014 to 2019. Sequencing of HIV-1 protease and reverse transcriptase was performed, and HIVDR was interpreted using Stanford HIVdb.v.9.4. In total, 379 sequences were obtained from participants (62% female, mean age 36 ± 10 years). The overall PDR rate was 15.0% [95% CI: 11.8-19.0] nationwide, with significant disparity between regions (p = 0.03). NNRTI PDR was highest (12.4%), of which 7.9% had DRMs to EFV/NVP. Two regions had EFV/NVP PDR above the 10% critical threshold, namely the Far North (15%) and East (10.9%). Eighteen viral strains were identified, predominated by CRF02_AG (65.4%), with no influence of genetic diversity PDR occurrence. TDF-3TC-DTG predictive efficacy was superior (98.4%) to TDF-3TC-EFV (92%), p < 0.0001. The overall high rate of PDR in Cameroon, not substantially affected by the wide HIV-1 genetic diversity, underscores the poor efficacy of EFV/NVP-based first-line ART nationwide, with major implications in two regions of the country. This supports the need for a rapid transition to NNRTI-sparing regimens, with TDF-3TC-DTG having optimal efficacy at the programmatic level.

Resistant mutations within the hepatitis B virus reverse transcriptase sequence in treatment failure patients with chronic HBV infection in Vietnam

ObjectivesWe conducted this study to describe whether mutations in the gene coding for the enzyme reverse transcriptase (RT) were related to drugs used in the treatment of hepatitis B in Vietnam. MethodsPatients receiving antiretroviral therapy with evidence of treatment failure were included in the study. The RT fragment was cloned using the polymerase chain reaction technique after being extracted from patients’ blood samples. The nucleotide sequences were analysed using Sanger method. The HBV drug resistance database contains mutations associated to resistance to existing HBV therapies. Medical records were accessed to collect information on patient parameters, such as treatment, viral load, biochemistry, and blood count. ResultsResistance mutations to lamivudine, telbivudine, and entecavir were found in the highest proportion (75–91.7%) of HBV samples from patients who had failed antiretroviral therapy. Only 20.8% of HBV strains had mutations exhibiting adefovir resistance, while none had mutations conferring tenofovir resistance. M204I/V, L180M, and L80I are frequent variants linked with resistance to lamivudine, telbivudine, and entecavir. In contrast, the A181L/T/V mutation was detected predominantly in tenofovir-resistant HBV strains. Following the drug resistance mutation test, patients achieved the greatest virological response after 24 weeks of therapy with tenofovir and entecavir at a daily dose of one tablet. ConclusionLamivudine, telbivudine, and entecavir were all highly resistant to the RT enzyme modifications in 24 treatment failure patients, with M204I/V, L180M, and L80I being the most prevalent mutations. Tenofovir resistance mutations have not been found in Vietnam.

Highly Ambiguous HIV-1 Pol Positions Encoding Multiple Amino Acids Usually Result from Antiviral or Immune Selection Pressure.

HIV-1 pol nucleotide ambiguities encoding amino acid mixtures occur commonly during population-based genotypic drug resistance testing. However, few studies have addressed the validity of sequences with fully ambiguous codons (FACs) containing codons translatable to more than four amino acids. We identified 839 published HIV-1 pol sequences with 846 FACs at 131 positions and determined their distribution relative to 215 HLA-associated pol positions (HAPs) and 84 drug-resistance positions. Among HIV-1 reverse transcriptase (RT) and protease sequences from antiretroviral therapy (ART)-naive and -experienced persons, there was a strong correlation between the likelihood a position was a FAC and that it was an HAP (Spearman's correlation coefficient rho >0.40; p < 1e-6). Among HIV-1 RT sequences from ART-experienced persons, there was a correlation between the likelihood that a position was a FAC and that it was a drug-resistance position (rho = 0.2; p = 8e-4). In the context of population-based genotypic resistance testing, FACs usually result from antiviral or immune selection pressure.

Co-circulation of all the four Dengue virus serotypes during 2018-2019: first report from Eastern Uttar Pradesh, India.

Dengue fever is an endemic disease in India, transmitted by an infected mosquito bite. In India, the number of concurrent infections and the circulation of multiple dengue virus (DENV) serotypes has increased in recent decades. Molecular surveillance among the DENV serotype is important to keep track of the circulating serotypes, evolutionary changes, and key mutations that can alter the diagnostics. The current study included patients admitted for dengue in the Eastern Uttar Pradesh (E-UP) region during 2018-2019. The genetic characterization of the circulating DENV was accomplished through partial CprM (511 bp) gene amplification via reverse transcriptase polymerase chain reaction and sequencing. Phylogenetic analysis revealed the circulation of all four DENV1-4 serotypes. DENV-2 was the most abundant serotype (44%, 27/61), followed by DENV-3 (32%, 20/61). DENV-1 had a 16% (10/61) predominance, while DENV-4 (6%, 4/61) was found to be the least abundant serotype. DENV-2 genotypes were distributed among lineages I (7.4%), II (85%) and III (7.4%) of genotype IV, DENV-3 to lineage III of genotype III, DENV-1 to genotype III; DENV-2 to lineage B (75%) and C (25%) of genotype I. This primary report on the co-circulation of DENV1-4 serotypes from the E-UP region highlights the requirement of continuous molecular surveillance for monitoring circulating DENV serotypes.

Transmitted Drug Resistance to Integrase-Based First-Line Human Immunodeficiency Virus Antiretroviral Regimens in Mediterranean Europe.

We evaluated the prevalence of transmitted drug resistance (TDR) to integrase strand-transfer inhibitors (INSTIs) and nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and of clinically relevant resistance (CRR) in newly diagnosed people with human immunodeficiency virus (HIV; PWH) naive to antiretroviral therapy (ART) in Europe. MeditRes is a consortium that includes ART-naive PWH newly diagnosed in France, Greece, Italy, Portugal, and Spain during 2018-2021. Reverse transcriptase and INSTI sequences were provided by participating centers. To evaluate the prevalence of surveillance drug resistance mutations (SDRM), we used the calibrated population resistance tools from the Stanford HIV website. To evaluate CRR, defined as any resistance level ≥3, we used the Stanford HIV Drug Resistance Database v.9.1 algorithm. We included 2705 PWH, 72% men, median age of 37 years (interquartile range, 30-48); 43.7% were infected by non-B subtypes. The prevalence of INSTI-SDRMs was 0.30% (T66I, T66A, E92Q, E138T, E138K, Y143R, S147G, R263K; all n=1) and the prevalence of NRTI-SDRMs was 5.77% (M184V: 0.85%; M184I: 0.18%; K65R/N: 0.11%; K70E: 0.07%; L74V/I: 0.18%; any thymidine analog mutations: 4.36%). INSTI-CRR was 2.33% (0.15% dolutegravir/bictegravir, 2.29% raltegravir/elvitegravir) and 1.74% to first-line NRTIs (0.89% tenofovir/tenofovir alafenamide, 1.74% abacavir, 1.07% lamivudine/emtricitabine). We present the most recent data on TDR to integrase-based first-line regimens in Europe. Given the low prevalence of CRR to second-generation integrase inhibitors and to first-line NRTIs during 2018-2021, it is unlikely that newly diagnosed PWH in MeditRes countries would present with baseline resistance to a first-line regimen based on second-generation integrase inhibitors.

Frequency of genotypic factors possibly associated with cabotegravir/rilpivirine failure in antiretroviral treatment-naïve and -experienced HIV-1- infected population.

The long-acting injectable (LAI) cabotegravir (CAB) and rilpivirine (RPV) treatment offers important advantages over oral ART (antiretroviral therapy), however baseline factors possibly contributing to the CAB/RPV treatment failure were identified. The purpose of this study was to describe the frequency of virologic factors previously influencing efficacy of this treatment, namely RPV and CAB resistance-associated mutations (RAMs) and A1/A6 subtype among naïve and treatment-experienced HIV-1-infected patients from Poland. The following datasets of HIV-1 sequences were analysed: 4809 protease and reverse transcriptase (PR/RT) sequences obtained from 4649 Polish Caucasian patients (4122 naive and 687 non-naïve) supplemented with integrase (PR/RT/INT) sequences in 1217 cases (942 naïve and 275 non-naïve). Sub-subtypes A were assigned by phylogenetic methods. Major and minor CAB and RPV RAMs were determined according to the IAS-USA 2019 list, while minor RAMs were additionally defined based on the Stanford database algorithm. Subtype A1/A6 frequency ranged from 6.11% in ART failing cases with PR/RT sequences only, to 15.92% for the PR/RT/INT treatment-naïve dataset, while RPV RAMs were found in up to 5.89% of treatment-naïve and 14.56% of ART failing cases. Regardless treatment history, only <1% sequences had combination of two factors (RPV RAMs and A1/A6 subtype). Furthermore, CAB RAMs were found in 1.27% of treatment-naïve and 14.54% of experienced patients. Despite notable frequency of subtype A1/A6 or CAB/RPV RAMs analysed separately, combination of at least two factors previously associated with failure or this treatment is rare. As subtype A1/A6 becomes more common across real-life cohorts continued subtyping and RAM screening will remain of key importance for LAI treatment implementation. Sequence data from this article have been deposited in GenBank under accession numbers: GU906860, GU906864, GU906871-GU906874, JQ305750-JQ305791, KC409134-KC409222, KM057341-KM057362, KM283892-KM284490, KT340108-KT340205, MZ468643-MZ468894, MZ671788-MZ671823, OP298017-OP302727.

Diversity Chromosome Evolution of Ty1-copia Retrotransposons in Pennisetum purpureum Revealed by FISH

Pennisetum purpureum is a potential species for biofuel production. Characterization and chromosomal distribution of retrotransposons could enhance the comprehension of the role and dynamics of the repetitive elements in plants. In this study, a phylogenetic tree was constructed according to the conserved reverse transcriptase sequences and revealed that these Ty1-copia retrotransposons had a typical structure. Analysis showed that the total Ty1-copia retrotransposons had a significant component, as high as 5.12 × 103 copy numbers in P. purpureum. Then, the chromosomal pattern of four known lineages were also analyzed with the Pennisetum glaucum genome, which suggested that the Sire/Maximus lineage had the highest copy number and followed by Tork/Angela, Tork/TAR, Retrofit/Ale. Additionally, the chromosomal distribution of total Ty1-copia retrotransposons was detected by fluorescence in situ hybridization (FISH) to be a dispersed pattern with weak clustering, mostly near the centromeric regions of P. purpureum chromosomes; interestingly, there were four obvious signals in the subterminal chromosomes. These results suggested that there occurred differential dynamic evolution directions of Ty1-copia retrotransposons within P. purpureum. Furthermore, co-localization of Ty1-copia, 5S rDNA, and 35S rDNA indicated that two chromosome 2 and four chromosome 4 were identified. Concurrently, subterminal signals of Ty1-copia-type retrotransposons were located on four other homologous chromosomes. Altogether, these results shed light on the diversification of Ty1-copia retrotransposons and have the significance for generation of valid chromosomal markers in retrotransposon families.

Non-canonical nematode endogenous retroviruses resulting from RNA virus glycoprotein gene capture by a metavirus.

Reverse-transcribing retroviruses exist as horizontally transmitted infectious agents or vertically transmitted endogenous retroviruses (ERVs) resident in eukaryotic genomes, and they are phylogenetically related to the long terminal repeat (LTR) class of retrotransposons. ERVs and retrotransposons are often distinguished only by the presence or absence of a gene encoding the envelope glycoprotein (env). Endogenous elements of the virus family Metaviridae include the insect-restricted Errantivirus genus of ERVs, for which some members possess env, and the pan-eukaryotic Metavirus genus that lacks an envelope glycoprotein gene. Here we report a novel Nematoda endogenous retrovirus (NERV) clade with core retroviral genes arranged uniquely as a continuous gag-env-pro-pol ORF. Reverse transcriptase sequences were phylogenetically related to metaviruses, but envelope glycoprotein sequences resembled those of the Nyamiviridae and Chrysoviridae RNA virus families, suggesting env gene capture during host cell infection by an RNA virus. NERVs were monophyletic, restricted to the nematode subclass Chromadoria, and included additional ORFs for a small hypothetical protein or a large Upf1-like RNA-dependent AAA-ATPase/helicase indicative of viral transduction of a host gene. Provirus LTR identity, low copy number, ORF integrity and segregation of three loci in Meloidogyne incognita, taken together with detection of NERV transcriptional activity, support potential infectivity of NERVs, along with their recent emergence and integration. Altogether, NERVs constitute a new and distinct Metaviridae lineage demonstrating retroviral evolution through sequential heterologous gene capture events.

Pretreatment Human Immunodeficiency Virus (HIV) Drug Resistance Among Treatment-Naive Infants Newly Diagnosed With HIV in 2016 in Namibia: Results of a Nationally Representative Study.

BackgroundThe World Health Organization (WHO) recommends routine surveillance of pretreatment human immunodeficiency virus (HIV) drug resistance (HIVDR) in children <18 months of age diagnosed with HIV through early infant diagnosis (EID). In 2016, 262 children <18 months of age were diagnosed with HIV in Namibia through EID. Levels of HIVDR in this population are unknown.MethodsIn 2016, Namibia surveyed pretreatment HIVDR among children aged <18 months following WHO guidance. Reverse transcriptase, protease, and integrase regions of HIV-1 were genotyped from remnant dried blood spot specimens from all infants diagnosed with HIV in Namibia in 2016. HIVDR was predicted using the Stanford HIVdb algorithm.ResultsOf 262 specimens genotyped, 198 HIV-1 protease and reverse transcriptase sequences and 118 HIV-1 integrase sequences were successfully amplified and analyzed. The prevalence of efavirenz/nevirapine (EFV/NVP), abacavir (ABC), zidovudine, lamivudine/emtricitabine (3TC/FTC), and tenofovir (TDF) resistance was 62.6%, 17.7%, 5.6%, 15.7%, and 10.1%, respectively. No integrase inhibitor resistance was detected.ConclusionsThe high level of EFV/NVP resistance is unsurprising; however, levels of ABC and TDF resistance are among the highest observed to date in infants in sub-Saharan Africa. The absence of resistance to dolutegravir (DTG) is reassuring but underscores the need to further study the impact of ABC and 3TC/FTC resistance on pediatric protease inhibitor– and DTG-based regimens and accelerate access to other antiretroviral drugs. Results underscore the need for antiretroviral therapy optimization and prompt management of high viral loads in infants and pregnant and breastfeeding women.

Pre-treatment and acquired antiretroviral drug resistance among people living with HIV in Tianjin, China.

This study investigated the prevalence and patterns of pre-treatment and acquired HIV drug resistance mutations among people living with HIV (PLWH) on antiretroviral therapy (ART) for 12 (±3) months in Tianjin, China. From Jan 2018 to Dec 2020, PLWH with HIV-1 RNA greater than 1000 copies/mL visiting the ART clinic in the Tianjin Second People's Hospital were enrolled. Viral RNA isolated from blood samples were taken for genotypic resistance testing using an in-house method. Major drug resistance mutations were analyzed for reverse transcriptase and protease Sanger sequences using the Stanford University HIV Drug Resistance Database. Multivariable Poisson regressions were used to evaluate the factors associated with drug resistance mutations. HIV drug resistance testing was successfully performed on 584 ART-naive and 71 ART-experienced participants. Pre-treatment drug resistance mutation prevalence was 13.5% (79/584) to any antiretroviral drug, 12.5% (73/584) to non-nucleoside reverse transcriptase inhibitors (NNRTIs), 1.5% (9/584) to nucleoside reverse-transcriptase inhibitors (NRTIs), and 0.3% (2/584) to protease inhibitors (PIs). Acquired drug resistance to any antiretroviral drug among PLWH on ART with viral load >1000 copies/mL was 88.7% (63/71). The prevalence of mutation for NNRTIs, NRTIs, and PIs were 93.7% (59/63), 82.5% (52/63), and 3.2% (2/63), respectively. Pre-treatment and acquired drug resistance mutations were highly prevalent among PLWH in Tianjin; therefore, routine baseline genotypic resistance testing and adequate intervals of viral load surveillance are urgently needed for the long-term treatment success. Our findings provide important evidence for first- and second-line regimen drugs for PLWH, especially in China.

Abstract P5-07-03: Disease classification modeling of inflammatory breast cancer based on simultaneous profiling of coding and non-coding RNAs in tumor and blood samples by TGIRT-sequencing

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal breast cancer subtype but lags in disease-specific RNA biomarkers due in part to its paucity of large discrete tumors. A strategy to overcome this challenge is to identify blood-based RNA biomarkers that are minimally invasive and reflect the state of both the diseased breast tissue and the patient's immune response. Here, we identified IBC-specific RNA biomarkers by thermostable group II intron reverse transcriptase sequencing (TGIRT-seq), a recently developed comprehensive RNA-seq technology that enables simultaneous profiling of all RNA biotypes from small amounts of starting material. We used these biomarkers to develop novel disease classification models for IBC based on coding and non-coding RNAs from FFPE tumor slices, PBMCs, and plasma. Methods: We obtained biological samples including FFPE, PBMC, and plasma from a cohort of ten patients with IBC and compared them to samples from six patients with non-IBC and sixteen healthy donors using TGIRT-seq technology. Results: TGIRT-seq of FFPE tumor slices identified differentially expressed mRNAs and miRNAs found previously to distinguish IBC from non-IBC tumors, as well as numerous additional differentially expressed mRNAs and small non-coding RNAs characteristic of IBC. Surprisingly, TGIRT-seq revealed that the differentially expressed protein-coding gene transcripts fall into two categories: mature mRNAs with reads confined to exons, and pre-mRNAs-derived transcripts with reads distributed across exons and introns, to our knowledge, a distinction not made previously for any cancer type. Differentially expressed miRNAs included both mature miRNAs and other transcripts of miRNA loci. IBC PBMCs showed a characteristic inflammatory response not seen in PBMCs from non-IBC patients, as well as differentially expressed tRNAs, snoRNAs, and other sncRNAs, while plasma samples, although of variable quality, included coding and non-coding RNAs distinctive of IBC. Classification models using panels consisting of sets of 50 selected biomarkers profiled by TGIRT-seq achieved a high degree of accuracy under cross-validation, with models based on PBMCs and plasma RNAs correlating with those based on tumor RNAs, and models using both coding and non-coding RNA biomarkers outperforming those based on either alone. Conclusions: Our findings are the first to define a distinct IBC profile across three different tissue types and advance TGIRT-seq as a promising method for high-resolution RNA biomarker profiling of both primary tumors and liquid biopsies with potentially broad utility for diagnosing and defining treatment response in IBC and other cancers. COI: Thermostable group II intron reverse transcriptase (TGIRT) enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas to InGex, LLC. A.M.L., some former and present members of the Lambowitz laboratory, and the University of Texas are minority equity holders in InGex, and receive royalty payments from the sale of TGIRT enzymes and kits and from sublicensing of intellectual property to other companies. Citation Format: Dennis C. Wylie, Xiaoping Wang, Jun Yao, Hengyi Xu, Toshiaki Iwase, Savitri Krishnamurthy, Naoto T. Ueno, Alan M. Lambowitz. Disease classification modeling of inflammatory breast cancer based on simultaneous profiling of coding and non-coding RNAs in tumor and blood samples by TGIRT-sequencing [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-07-03.

Baseline HIV drug-resistance testing: 12 US jurisdictions, 2014-2019.

To understand recent patterns in reported baseline HIV drug-resistance testing over time in the United States. Data from the National HIV Surveillance System for persons who were aged at least 13 years at the time of HIV diagnosis during 2014-2019 and resided in one of 12 US jurisdictions with high levels of reporting in 2014 and 2015. Among persons included in the analysis, we calculated the total proportion of HIV diagnoses occurring during 2014-2019 with a reported baseline sequence by year of diagnosis and sequence type. A baseline sequence was defined as any protease/ reverse transcriptase (PR/RT) or integrase sequence generated from a specimen collected 90 days or less after diagnosis. During 2014-2019, reported levels of baseline PR/RT (with or without integrase) testing varied by year from 46.9% to 51.8% without any clear pattern over time. PR/RT with integrase testing increased (8.3-19.4%) and integrase-only testing remained low (1.9-1.3%). While reported levels of baseline PR/RT (with or without integrase) testing have remained sufficiently high for the purposes of molecular cluster detection, higher levels would strengthen jurisdictions' and the Centers for Disease Control and Prevention's ability to monitor trends in HIV drug-resistance and detect and respond to HIV molecular clusters. Efforts to increase levels of reported baseline testing likely need to address both gaps in testing as well as reporting.