CRISPR editing of CCR5 and HIV-1 facilitates viral elimination in antiretroviral drug-suppressed virus-infected humanized mice.

Treatment of HIV-1ADA-infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) and HIV-1 proviral DNA gene editing. This led to sequential viral suppression, restoration of absolute human CD4+ T cell numbers, then elimination of replication-competent virus in 58% of infected mice. Dual CRISPR therapies enabled the excision of integrated proviral DNA in infected human cells contained within live infected animals. Highly sensitive nucleic acid nested and droplet digital PCR, RNAscope, and viral outgrowth assays affirmed viral elimination. HIV-1 was not detected in the blood, spleen, lung, kidney, liver, gut, bone marrow, and brain of virus-free animals. Progeny virus from adoptively transferred and CRISPR-treated virus-free mice was neither detected nor recovered. Residual HIV-1 DNA fragments were easily seen in untreated and viral-rebounded animals. No evidence of off-target toxicities was recorded in any of the treated animals. Importantly, the dual CRISPR therapy demonstrated statistically significant improvements in HIV-1 cure percentages compared to single treatments. Taken together, these observations underscore a pivotal role of combinatorial CRISPR gene editing in achieving the elimination of HIV-1 infection.

Autologous neutralizing antibodies increase with early antiretroviral therapy and shape HIV rebound after treatment interruption.

Early initiation of antiretroviral therapy (ART) alters viral rebound kinetics after analytic treatment interruption (ATI) and may play a role in promoting HIV-1 remission. Autologous neutralizing antibodies (aNAbs) represent a key adaptive immune response in people living with HIV-1. We aimed to investigate the role of aNAbs in shaping post-ATI HIV-1 rebound variants. We performed single-genome amplification of HIV-1 env from pre-ART and post-ATI plasma samples of 12 individuals who initiated ART early after infection. aNAb activity was quantified using pseudoviruses derived from the most common plasma variant, and the serum dilution that inhibited 50% of viral infections was determined. aNAb responses matured while participants were on suppressive ART, because on-ART plasma and purified immunoglobulin G (IgG) demonstrated improved neutralizing activity against pre-ART HIV-1 strains when compared with pre-ART plasma or purified IgG. Post-ATI aNAb responses exerted selective pressure on the rebounding viruses, because the post-ATI HIV-1 strains were more resistant to post-ATI plasma neutralization compared with the pre-ART virus. Several pre-ATI features distinguished post-treatment controllers from noncontrollers, including an infecting HIV-1 sequence that was more similar to consensus HIV-1 subtype B, more restricted proviral diversity, and a stronger aNAb response. Post-treatment control was also associated with the evolution of distinct N-glycosylation profiles in the HIV-1 envelope. In summary, aNAb responses appeared to mature after early initiation of ART and applied selective pressure on rebounding viruses. The combination of aNAb activity with select HIV-1 sequence and reservoir features identified individuals with a greater chance of post-treatment control.

Differential effect of histone H3.3 depletion on retroviral repression in embryonic stem cells.

BACKGROUND: Integration of retroviruses into the host genome can impair the genomic and epigenomic integrity of the cell. As a defense mechanism, epigenetic modifications on the proviral DNA repress retroviral sequences in mouse embryonic stem cells (ESC). Here, we focus on the histone 3 variant H3.3, which is abundant in active transcription zones, as well as centromeres and heterochromatinized repeat elements, e.g., endogenous retroviruses (ERV). RESULTS: To understand the involvement of H3.3 in the epigenetic silencing of retroviral sequences in ESC, we depleted the H3.3 genes in ESC and transduced the cells with GFP-labeled MLV pseudovirus. This led to altered retroviral repression and reduced Trim28 recruitment, which consequently led to a loss of heterochromatinization in proviral sequences. Interestingly, we show that H3.3 depletion has a differential effect depending on which of the two genes coding for H3.3, H3f3a or H3f3b, are knocked out. Depletion of H3f3a resulted in a transient upregulation of incoming retroviral expression and ERVs, while the depletion of H3f3b did not have the same effect and repression was maintained. However, the depletion of both genes resulted in a stable activation of the retroviral promoter. These findings suggest that H3.3 is important for regulating retroviral gene expression in mouse ESC and provide evidence for a distinct function of the two H3.3 genes in this regulation. Furthermore, we show that Trim28 is needed for depositing H3.3 in retroviral sequences, suggesting a functional interaction between Trim28 recruitment and H3.3 loading. CONCLUSIONS: Identifying the molecular mechanisms by which H3.3 and Trim28 interact and regulate retroviral gene expression could provide a deeper understanding of the fundamental processes involved in retroviral silencing and the general regulation of gene expression, thus providing new answers to a central question of stem cell biology.

Intact Proviral DNA Analysis of the Brain Viral Reservoir and Relationship to Neuroinflammation in People with HIV on Suppressive Antiretroviral Therapy.

HIV establishes a persistent viral reservoir in the brain despite viral suppression in blood to undetectable levels on antiretroviral therapy (ART). The brain viral reservoir in virally suppressed HIV+ individuals is not well-characterized. In this study, intact, defective, and total HIV proviral genomes were measured in frontal lobe white matter from 28 virally suppressed individuals on ART using the intact proviral DNA assay (IPDA). HIV gag DNA/RNA levels were measured using single-copy assays and expression of 78 genes related to inflammation and white matter integrity was measured using the NanoString platform. Intact proviral DNA was detected in brain tissues of 18 of 28 (64%) individuals on suppressive ART. The median proviral genome copy numbers in brain tissue as measured by the IPDA were: intact, 10 (IQR 1-92); 3' defective, 509 (225-858); 5' defective, 519 (273-906); and total proviruses, 1063 (501-2074) copies/106 cells. Intact proviral genomes accounted for less than 10% (median 8.3%) of total proviral genomes in the brain, while 3' and 5' defective genomes accounted for 44% and 49%, respectively. There was no significant difference in median copy number of intact, defective, or total proviruses between groups stratified by neurocognitive impairment (NCI) vs. no NCI. In contrast, there was an increasing trend in intact proviruses in brains with vs. without neuroinflammatory pathology (56 vs. 5 copies/106 cells, p = 0.1), but no significant differences in defective or total proviruses. Genes related to inflammation, stress responses, and white matter integrity were differentially expressed in brain tissues with >5 vs. +5 intact proviruses/106 cells. These findings suggest that intact HIV proviral genomes persist in the brain at levels comparable to those reported in blood and lymphoid tissues and increase CNS inflammation/immune activation despite suppressive ART, indicating the importance of targeting the CNS reservoir to achieve HIV cure.

Combination Antiretroviral Therapy and Immunophenotype of Feline Immunodeficiency Virus.

Feline Immunodeficiency Virus (FIV) causes progressive immune dysfunction in cats similar to human immunodeficiency virus (HIV) in humans. Although combination antiretroviral therapy (cART) is effective against HIV, there is no definitive therapy to improve clinical outcomes in cats with FIV. This study therefore evaluated pharmacokinetics and clinical outcomes of cART (2.5 mg/kg Dolutegravir; 20 mg/kg Tenofovir; 40 mg/kg Emtricitabine) in FIV-infected domestic cats. Specific pathogen free cats were experimentally infected with FIV and administered either cART or placebo treatments (n = 6 each) for 18 weeks, while n = 6 naïve uninfected cats served as controls. Blood, saliva, and fine needle aspirates from mandibular lymph nodes were collected to quantify viral and proviral loads via digital droplet PCR and to assess lymphocyte immunophenotypes by flow cytometry. cART improved blood dyscrasias in FIV-infected cats, which normalized by week 16, while placebo cats remained neutropenic, although no significant difference in viremia was observed in the blood or saliva. cART-treated cats exhibited a Th2 immunophenotype with increasing proportions of CD4+CCR4+ cells compared to placebo cats, and cART restored Th17 cells compared to placebo-treated cats. Of the cART drugs, dolutegravir was the most stable and long-lasting. These findings provide a critical insight into novel cART formulations in FIV-infected cats and highlight their role as a potential animal model to evaluate the impact of cART on lentiviral infection and immune dysregulation.

HIV Reservoir: How to Measure It?

PURPOSEOF REVIEW: In the current quest for a complete cure for HIV/AIDS, the persistence of a long-lived reservoir of cells carrying replication-competent proviruses is the major challenge. Here, we describe the main elements and characteristics of several widely used assays of HIV latent reservoir detection. RECENT FINDINGS: To date, researchers have developed several different HIV latent reservoir detection assays. Among them, the in vitro quantitative viral outgrowth assay (QVOA) has been the gold standard for assessing latent HIV-1 viral load. The intact proviral DNA assay (IPDA) based on PCR also demonstrated the predominance of defective viruses. However, these assays all have some drawbacks and may still be inadequate in detecting the presence of ultralow levels of latent virus in many patients who were initially thought to have been cured, but eventually showed viral rebound. An accurate and precise measurement of the HIV reservoir is therefore needed to evaluate curative strategies, aimed to functional cure or sterilizing cure.

Evolutionarily Young African Rhinoceros Gammaretroviruses.

High-throughput sequences were generated from DNA and cDNA from four Southern white rhinoceros (Ceratotherium simum simum) located in the Taronga Western Plain Zoo in Australia. Virome analysis identified reads that were similar to Mus caroli endogenous gammaretrovirus (McERV). Previous analysis of perissodactyl genomes did not recover gammaretroviruses. Our analysis, including the screening of the updated white rhinoceros (Ceratotherium simum) and black rhinoceros (Diceros bicornis) draft genomes identified high-copy orthologous gammaretroviral ERVs. Screening of Asian rhinoceros, extinct rhinoceros, domestic horse, and tapir genomes did not identify related gammaretroviral sequences in these species. The newly identified proviral sequences were designated SimumERV and DicerosERV for the white and black rhinoceros retroviruses, respectively. Two long terminal repeat (LTR) variants (LTR-A and LTR-B) were identified in the black rhinoceros, with different copy numbers associated with each (n = 101 and 373, respectively). Only the LTR-A lineage (n = 467) was found in the white rhinoceros. The African and Asian rhinoceros lineages diverged approximately 16 million years ago. Divergence age estimation of the identified proviruses suggests that the exogenous retroviral ancestor of the African rhinoceros ERVs colonized their genomes within the last 8 million years, a result consistent with the absence of these gammaretroviruses from Asian rhinoceros and other perissodactyls. The black rhinoceros germ line was colonized by two lineages of closely related retroviruses and white rhinoceros by one. Phylogenetic analysis indicates a close evolutionary relationship with ERVs of rodents including sympatric African rats, suggesting a possible African origin of the identified rhinoceros gammaretroviruses. IMPORTANCE Rhinoceros genomes were thought to be devoid of gammaretroviruses, as has been determined for other perissodactyls (horses, tapirs, and rhinoceros). While this may be true of most rhinoceros, the African white and black rhinoceros genomes have been colonized by evolutionarily young gammaretroviruses (SimumERV and DicerosERV for the white and black rhinoceros, respectively). These high-copy endogenous retroviruses (ERVs) may have expanded in multiple waves. The closest relative of SimumERV and DicerosERV is found in rodents, including African endemic species. Restriction of the ERVs to African rhinoceros suggests an African origin for the rhinoceros gammaretroviruses.

Detection of HTLV-1 proviral DNA in cell-free DNA: Potential for non-invasive monitoring of Adult T cell leukaemia/lymphoma using liquid biopsy?

Introduction: Fragmented genomic DNA is constitutively released from dying cells into interstitial fluid in healthy tissue. In cancer, this so-called 'cell-free' DNA (cfDNA) released from dying malignant cells encodes cancer-associated mutations. Thus, minimally invasive sampling of cfDNA in blood plasma can be used to diagnose, characterise and longitudinally monitor solid tumours at remote sites in the body. ~5% of carriers of Human T cell leukaemia virus type 1 (HTLV-1) develop Adult T cell leukaemia/lymphoma (ATL), and a similar percentage develop an inflammatory CNS disease, HTLV-1 associated myelopathy (HAM). In both ATL and HAM, high frequencies of HTLV-1 infected cells are present in the affected tissue: each carrying an integrated DNA copy of the provirus. We hypothesised that turnover of infected cells results in the release of HTLV-1 proviruses in cfDNA, and that analysis of cfDNA from infected cells in HTLV-1 carriers might contain clinically useful information pertaining to inaccessible sites in the body- e.g. for early detection of primary or relapsing localised lymphoma type ATL. To evaluate the feasibility of this approach, we tested for HTLV-1 proviruses in blood plasma cfDNA. Methods: CfDNA (from blood plasma) and genomic DNA (gDNA, from peripheral blood mononuclear cells, PBMC) was isolated from blood from 6 uninfected controls, 24 asymptomatic carriers (AC), 21 patients with HAM and 25 patients with ATL. Proviral (HTLV-1 Tax) and human genomic DNA (the beta globin gene, HBB) targets were quantified by qPCR using primer pairs optimised for fragmented DNA. Results: Pure, high quality cfDNA was successfully extracted from blood plasma of all study participants. When compared with uninfected controls, HTLV-1 carriers had higher concentrations of cfDNA circulating in their blood plasma. Patients with ATL who were not in remission had the highest levels of blood plasma cfDNA in any group studied. HTLV-1 proviral DNA was detected in 60/70 samples obtained from HTLV-1 carriers. The proviral load (percentage of cells carrying proviruses) was approximately tenfold lower in plasma cfDNA than in PBMC genomic DNA, and there was a strong correlation between the proviral load in cfDNA and PBMC genomic DNA in HTLV-1 carriers that did not have ATL. cfDNA samples in which proviruses were undetectable also had very low proviral load in PBMC genomic DNA. Finally, detection of proviruses in cfDNA of patients with ATL was predictive of clinical status: patients with evolving disease had higher than expected total amount of proviruses detectable in plasma cfDNA. Discussion: We demonstrated that (1) HTLV-1 infection is associated with increased levels of blood plasma cfDNA, (2) proviral DNA is released into blood plasma cfDNA in HTLV-1 carriers and (3) proviral burden in cfDNA correlates with clinical status, raising the possibility of developing assays of cfDNA for clinical use in HTLV-1 carriers.

Confounding factors in profiling of locus-specific human endogenous retrovirus (HERV) transcript signatures in primary T cells using multi-study-derived datasets.

BACKGROUND: Human endogenous retroviruses (HERV) are repetitive sequence elements and a substantial part of the human genome. Their role in development has been well documented and there is now mounting evidence that dysregulated HERV expression also contributes to various human diseases. While research on HERV elements has in the past been hampered by their high sequence similarity, advanced sequencing technology and analytical tools have empowered the field. For the first time, we are now able to undertake locus-specific HERV analysis, deciphering expression patterns, regulatory networks and biological functions of these elements. To do so, we inevitable rely on omics datasets available through the public domain. However, technical parameters inevitably differ, making inter-study analysis challenging. We here address the issue of confounding factors for profiling locus-specific HERV transcriptomes using datasets from multiple sources. METHODS: We collected RNAseq datasets of CD4 and CD8 primary T cells and extracted HERV expression profiles for 3220 elements, resembling most intact, near full-length proviruses. Looking at sequencing parameters and batch effects, we compared HERV signatures across datasets and determined permissive features for HERV expression analysis from multiple-source data. RESULTS: We could demonstrate that considering sequencing parameters, sequencing-depth is most influential on HERV signature outcome. Sequencing samples deeper broadens the spectrum of expressed HERV elements. Sequencing mode and read length are secondary parameters. Nevertheless, we find that HERV signatures from smaller RNAseq datasets do reliably reveal most abundantly expressed HERV elements. Overall, HERV signatures between samples and studies overlap substantially, indicating a robust HERV transcript signature in CD4 and CD8 T cells. Moreover, we find that measures of batch effect reduction are critical to uncover genic and HERV expression differences between cell types. After doing so, differences in the HERV transcriptome between ontologically closely related CD4 and CD8 T cells became apparent. CONCLUSION: In our systematic approach to determine sequencing and analysis parameters for detection of locus-specific HERV expression, we provide evidence that analysis of RNAseq datasets from multiple studies can aid confidence of biological findings. When generating de novo HERV expression datasets we recommend increased sequence depth ( > = 100 mio reads) compared to standard genic transcriptome pipelines. Finally, batch effect reduction measures need to be implemented to allow for differential expression analysis.

Immune activation and exhaustion marker expression on T-cell subsets in ART-treated adolescents and young adults with perinatal HIV-1 infection as correlates of viral persistence.

HIV-1 infection in memory CD4+ T cells forms a latent reservoir that is a barrier to cure. Identification of immune biomarkers that correlate with HIV-1 reservoir size may aid with evaluating efficacy of HIV-1 eradication strategies, towards ART-free remission and cure. In adults living with non-perinatal HIV-1, the immune exhaustion marker PD-1 on central memory CD4+ T cells (Tcm) correlates with measures of HIV-1 reservoir size. Immune correlates of HIV-1 are less defined in adolescents and young adults with perinatal HIV-1. With multi-parameter flow cytometry, we examined immune activation (CD69, CD25, HLA-DR), and exhaustion (PD-1, TIGIT, TIM-3 and LAG-3) markers on CD4+ T cell subsets (naïve (Tn), central memory (Tcm), and the combination (Ttem) of transitional (Ttm) and effector memory (Tem) cells, in 10 adolescents and young adults living with perinatal HIV-1 (median age 15.9 years; median duration of virologic suppression 7.0 years), in whom HIV-1 reservoir size was measured with the Intact Proviral HIV-1 DNA Assay (IPDA) and an enhanced Tat/Rev limiting dilution assay (TILDA). RNA-seq was also performed on the unstimulated CD4+ T cells. The median total HIV-1 DNA concentration in memory CD4+ T cells was 211.90 copies per million CD4+ T cells. In the 7 participants with subtype B HIV-1 infection, the median intact proviral DNA load was 7.96 copies per million CD4+ T cells. Levels of HLA-DR and TIGIT on the Ttem were correlated with total HIV-1 DNA (r=0.76, p=0.015) and (r=0.72, p=0.023), respectively, but not with intact proviral load or induced reservoir size. HIV-1 DNA load was also positively correlated with transcriptional clusters associated with HLA-DR expression by RNA-seq. In contrast, PD-1 expression on Tcm was inversely correlated with total HIV-1 DNA (r=-0.67, p=0.039). Reservoir size by IPDA and TILDA were correlated (r=0.81, p=0.036). Thus, in this cohort of youths with long-standing treated perinatal infection, HLA-DR and TIGIT on Ttem were the key correlates of HIV-1 infected cell frequencies by total HIV-1 DNA, and not PD-1. Total HIV-1 DNA was negatively correlated with PD-1 expressing Tcm. These differences in longstanding perinatal HIV-1 infection compared with adult infection requires further study in larger cohorts.

Phylogenetic identification of feline leukemia virus A and B in cats with progressive infection developing into lymphoma and leukemia.

To date, only a few studies have examined the impacts of feline leukemia virus (FeLV) subgroups on disease development in spontaneously infected cats. The present study identified FeLV-A and FeLV-B subgroups in cats with lymphoma and leukemia and explored the phylogenetic relationships of env sequences. Twenty-six cats with lymphoma (n=16) or leukemia (n=10) were selected. FeLV p27 antigen positivity was determined using ELISA, and proviral DNA in blood samples was detected using nested PCR. Positive animals in both tests were classified as cases of FeLV progressive infection and subjected to a second nested PCR for env amplification and subgroup determination. Six samples of FeLV-A and five samples of FeLV-B were sequenced using the Sanger method, and the results were used to build a phylogenetic tree and estimate evolutionary divergence. Among cats with lymphoma, 68.8% carried FeLV-AB and 31.2% FeLV-A. Among cats with leukemia, 70% carried FeLV-AB and 30% FeLV-A. Regarding cat characteristics, 50% were young, 30.8% young adults, and 19.2% adults; 88.5% were mixed-breed and 11.5% pure breed; and 42.3% were males and 57.7% were females. Among lymphomas, 62.5% were mediastinal, 31.3% multicentric, and 6.3% extranodal. Regarding histological classification, lymphoblastic and small non-cleaved-cell lymphomas were the most frequently detected. Among leukemia cases, 30% were acute lymphoid, 30% chronic myeloid, and 40% acute myeloid. Phylogenetic analysis showed that FeLV-A SC sequences were closely related to the Arena, Glasgow-1, and FeLV-FAIDS variants. Meanwhile, FeLV-B SC sequences were divergent from one another but similar to the endogenous FELV env gene (enFeLV). In conclusion, FeLV-AB is prevalent in cats with lymphoma and leukemia, highlighting the genetic diversity involved in the pathogenesis of these neoplasms in Brazil.

A virus-borne DNA damage signaling pathway controls the lysogeny-induction switch in a group of temperate pleolipoviruses.

Many prokaryotic viruses are temperate and their reactivation is tightly regulated. However, except for a few bacterial model systems, the regulatory circuits underlying the exit from lysogeny are poorly understood, especially in archaea. Here, we report a three-gene module which regulates the switch between lysogeny and replicative cycle in a haloarchaeal virus SNJ2 (family Pleolipoviridae). The SNJ2 orf4 encodes a winged helix-turn-helix DNA binding protein which maintains lysogeny through repressing the expression of the viral integrase gene intSNJ2. To switch to the induced state, two other SNJ2-encoded proteins, Orf7 and Orf8, are required. Orf8 is a homolog of cellular AAA+ ATPase Orc1/Cdc6, which is activated upon mitomycin C-induced DNA damage, possibly through posttranslational modification. Activated Orf8 initiates the expression of Orf7 which, in turn, antagonizes the function of Orf4, leading to the transcription of intSNJ2, thereby switching SNJ2 to the induced state. Comparative genomics analysis revealed that the SNJ2-like Orc1/Cdc6-centered three-gene module is common in haloarchaeal genomes, always present in the context of integrated proviruses. Collectively, our results uncover the first DNA damage signaling pathway encoded by a temperate archaeal virus and reveal an unexpected role of the widely distributed virus-encoded Orc1/Cdc6 homologs.

Isolation of an Ecotropic Porcine Endogenous Retrovirus PERV-C from a Yucatan SLAD/D Inbred Miniature Swine.

Xenotransplantation may compensate the limited number of human allografts for transplantation using pigs as organ donors. Porcine endogenous retroviruses inherit infectious potential if pig cells, tissues, or organs were transplanted to immunosuppressed human recipients. Particularly, ecotropic PERV-C that could recombine with PERV-A to highly replication-competent human-tropic PERV-A/C should be excluded from pig breeds designed for xenotransplantation. Because of their low proviral background, SLAD/D (SLA, swine leukocyte antigen) haplotype pigs are potential candidates as organ donors as they do not bear replication-competent PERV-A and -B, even if they carry PERV-C. In this work, we characterized their PERV-C background isolating a full-length PERV-C proviral clone number 561 from a SLAD/D haplotype pig genome displayed in a bacteriophage lambda library. The provirus truncated in env due to cloning in lambda was complemented by PCR, and the recombinants were functionally characterized, confirming an increased infectivity in vitro compared to other PERV-C. Recombinant clone PERV-C(561) was chromosomally mapped by its 5'-proviral flanking sequences. Full-length PCR using 5'-and 3'-flanking primers specific to the PERV-C(561) locus verified that this specific SLAD/D haplotype pig harbors at least one full-length PERV-C provirus. The chromosomal location is different from that of the previously described PERV-C(1312) provirus, which was derived from the porcine cell-line MAX-T. The sequence data presented here provide further knowledge about PERV-C infectivity and contribute to targeted knockout in order to generate PERV-C-free founder animals. IMPORTANCE Yucatan SLAD/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro, the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.

Nonsuppressible viremia during HIV-1 therapy meets molecular virology.

HIV-1 replication can be suppressed with antiretroviral therapy (ART), but individuals who stop taking ART soon become viremic again. Some people experience extended times of detectable viremia despite optimal adherence to ART. In this issue of the JCI, White, Wu, and coauthors elucidate a source of nonsuppressible viremia (NSV) in treatment-adherent patients - clonally expanded T cells harboring HIV-1 proviruses with small deletions or mutations in the 5'-leader, the UTR that includes the major splice donor site of viral RNA. These mutations altered viral RNA-splicing efficiency and RNA dimerization and packaging, yet still allowed production of detectable levels of noninfectious virus particles. These particles lacked the HIV-1 Env surface protein required for cell entry and failed to form the mature capsid cone required for infectivity. These studies improve our understanding of NSV and the regulation of viral functions in the 5'-leader with implications for rationalized care in individuals with NSV.

The FOXP3-924 A/G Single Nucleotide Polymorphism May Be Associated with Predictive Factors for Human T Lymphotropic Virus 1 Associated Myelopathy.

Human T lymphotropic virus 1 (HTLV-1) is a retrovirus associated with inflammatory diseases, including HTLV-1-associated myelopathy (HAM), and host genetic factors may be involved in disease evolution. The forkhead Box P3 (FOXP3) transcription factor is linked to homeostasis of the immune system, and the presence of polymorphisms in the promoter region of the FOXP3 gene should reflect its expression levels and consequent activation of regulatory T cells, which may contribute to severe inflammatory disorders, such as HAM. This study evaluated the rs2232365 polymorphism (-924 A/G) located in the promoter region of the FOXP3 gene and its association with HAM. Forty DNA samples from asymptomatic carriers and 25 samples from HAM patients were used, in addition to 130 control samples. The polymorphism was genotyped by conducting real-time polymerase chain reaction (PCR) (quantitative PCR [qPCR]) on extracted DNA. The proviral loads (PVLs) and CD4+ and CD8+ T lymphocyte counts were determined by qPCR and FACSCalibur flow cytometry, respectively. The PVLs, CD4+ T lymphocyte concentrations, and tumor necrosis factor-α dosages were considered predictive factors of the clinical profiles of HTLV-1 infection, all of which had higher levels in the HAM group. Carriers of the GG genotype for the polymorphism rs2232365 had high PVLs and CD4+ T lymphocyte concentrations.

Antiretroviral therapy reveals triphasic decay of intact SIV genomes and persistence of ancestral variants.

The decay kinetics of HIV-1-infected cells are critical to understand virus persistence. We evaluated the frequency of simian immunodeficiency virus (SIV)-infected cells for 4 years of antiretroviral therapy (ART). The intact proviral DNA assay (IPDA) and an assay for hypermutated proviruses revealed short- and long-term infected cell dynamics in macaques starting ART ∼1 year after infection. Intact SIV genomes in circulating CD4+T cells showed triphasic decay with an initial phase slower than the decay of the plasma virus, a second phase faster than the second phase decay of intact HIV-1, and a stable third phase reached after 1.6-2.9 years. Hypermutated proviruses showed bi- or mono-phasic decay, reflecting different selective pressures. Viruses replicating at ART initiation had mutations conferring antibody escape. With time on ART, viruses with fewer mutations became more prominent, reflecting decay of variants replicating at ART initiation. Collectively, these findings confirm ART efficacy and indicate that cells enter the reservoir throughout untreated infection.

Predictors of intact HIV DNA levels among children in Kenya.

OBJECTIVE: We determined predictors of both intact (estimate of replication-competent) and total (intact and defective) HIV DNA in the reservoir among children with HIV. DESIGN: HIV DNA in the reservoir was quantified longitudinally in children who initiated antiretroviral therapy (ART) at less than 1 year of age using a novel cross-subtype intact proviral DNA assay that measures both intact and total proviruses. Quantitative PCR was used to measure pre-ART cytomegalovirus (CMV) viral load. Linear mixed effects models were used to determine predictors of intact and total HIV DNA levels (log 10 copies/million). RESULTS: Among 65 children, median age at ART initiation was 5 months and median follow-up was 5.2 years; 86% of children had CMV viremia pre-ART. Lower pre-ART CD4 + percentage [adjusted relative risk (aRR): 0.87, 95% confidence intervals (95% CI): 0.79-0.97; P  = 0.009] and higher HIV RNA (aRR: 1.21, 95% CI: 1.06-1.39; P  = 0.004) predicted higher levels of total HIV DNA during ART. Pre-ART CD4 + percentage (aRR: 0.76, 95% CI: 0.65-0.89; P < 0.001), CMV viral load (aRR: 1.16, 95% CI: 1.01-1.34; P  = 0.041), and first-line protease inhibitor-based regimens compared with nonnucleoside reverse transcriptase-based regimens (aRR: 1.36, 95% CI: 1.04-1.77; P  = 0.025) predicted higher levels of intact HIV DNA. CONCLUSION: Pre-ART immunosuppression, first-line ART regimen, and CMV viral load may influence establishment and sustainment of intact HIV DNA in the reservoir.

Development of droplet digital PCR for quantification of bovine leukemia virus proviral load using unpurified genomic DNA.

Bovine leukemia virus (BLV) is the causative agent of a B-cell tumor called enzootic bovine leukosis. Preventing BLV spreading is required to reduce economic loss related to BLV infection of livestock. To quantify proviral load (PVL) more easily and rapidly, we developed a quantification system of PVL using droplet digital PCR (ddPCR). This method uses a multiplex TaqMan assay of the BLV provirus and housekeeping gene RPP30 for the quantification of BLV in BLV-infected cells. Furthermore, we combined ddPCR with DNA purification-free sample preparation (unpurified genomic DNA). The percentage of BLV-infected cells based on unpurified genomic DNA was highly correlated with that based on purified genomic DNA (correlation coefficient: 0.906). Thus, this new technique is a suitable method to quantify PVL of BLV-infected cattle in a large sample number.

Virally Suppressed People Living with HIV Who Use Opioids Have Diminished Latency Reversal.

Of the 12 million people who inject drugs worldwide, 13% live with HIV. Whether opioid use impacts HIV pathogenesis and latency is an outstanding question. To gain insight into whether opioid use influences the proviral landscape and latent HIV reservoir, we performed intact proviral DNA assays (IPDA) on peripheral blood mononuclear cells (PBMCs) from antiretroviral therapy (ART)-suppressed people living with HIV (PWH) with or without current opioid use. No differences were observed between PWH with and without opioid use in the frequency of HIV intact and defective proviral genomes. To evaluate the latent reservoir, we activated PBMCs from ART-suppressed PWH with or without opioid use and assessed the induction of HIV RNA. PWH using opioids had diminished responses to ex vivo HIV reactivation, suggesting a smaller reversible reservoir of HIV-1 latently infected cells. However, in vitro studies using primary CD4+ T cells treated with morphine showed no effect of opioids on HIV-1 infection, replication or latency establishment. The discrepancy in our results from in vitro and clinical samples suggests that while opioids may not directly impact HIV replication, latency and reactivation in CD4+ T cells, opioid use may indirectly shape the HIV reservoir in vivo by modulating general immune functions.

miRNAs in Herpesvirus Infection: Powerful Regulators in Small Packages.

microRNAs are a class of small, single-stranded, noncoding RNAs that regulate gene expression. They can be significantly dysregulated upon exposure to any infection, serving as important biomarkers and therapeutic targets. Numerous human DNA viruses, along with several herpesviruses, have been found to encode and express functional viral microRNAs known as vmiRNAs, which can play a vital role in host-pathogen interactions by controlling the viral life cycle and altering host biological pathways. Viruses have also adopted a variety of strategies to prevent being targeted by cellular miRNAs. Cellular miRNAs can act as anti- or proviral components, and their dysregulation occurs during a wide range of infections, including herpesvirus infection. This demonstrates the significance of miRNAs in host herpesvirus infection. The current state of knowledge regarding microRNAs and their role in the different stages of herpes virus infection are discussed in this review. It also delineates the therapeutic and biomarker potential of these microRNAs in future research directions.