Lymphoid tissues contribute to plasma viral clonotypes early after antiretroviral therapy interruption in SIV-infected rhesus macaques.

The rebound-competent viral reservoir, composed of a virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after ART interruption (ATI), remains the biggest obstacle to treating HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop therapeutic strategies for reducing the rebound-competent viral reservoir. In this study, barcoded simian immunodeficiency virus (SIV), SIVmac239M, was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood and tissues from secondary lymphoid organs (spleen, mesenteric lymph nodes, and inguinal lymph nodes) and from the colon, ileum, lung, liver, and brain were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX and RNAscope in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy, although plasma viral RNA remained below 22 copies per milliliter. Among the tissues studied, mesenteric lymph nodes, inguinal lymph nodes, and spleen contained viral barcodes detected in plasma. CD4+ T cells were the main cell type harboring viral RNA after ATI. Furthermore, T cell zones in lymphoid tissues showed higher viral RNA abundance than B cell zones for most animals. These findings are consistent with lymphoid tissues contributing to the virus present in plasma early after ATI.

Toll-like Receptor 2 Mediated Immune Regulation in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Toll-like receptors (TLRs) are crucial to the innate immune response. They regulate inflammatory reactions by initiating the production of pro-inflammatory cytokines and chemokines. TLRs also play a role in shaping the adaptive immune responses. While this protective response is important for eliminating infectious pathogens, persistent activation of TLRs may result in chronic immune activation, leading to detrimental effects. The role of TLR2 in regulating HIV-1 infection in vivo has yet to be well described. In this study, we used an SIV-infected rhesus macaque model to simulate HIV infection in humans. We evaluated the plasma of the macaques longitudinally and found a significant increase in the soluble TLR2 (sTLR2) level after SIV infection. We also observed an increase in membrane-bound TLR2 (mb-TLR2) in cytotoxic T cells, B cells, and NK cells in PBMC and NK cells in the gut after infection. Our results suggest that sTLR2 regulates the production of various cytokines and chemokines, including IL-18, IL-1RA, IL-15, IL-13, IL-9, TPO, FLT3L, and IL-17F, as well as chemokines, including IP-10, MCP-1, MCP-2, ENA-78, GRO-α, I-TAC, Fractalkine, SDF-1α, and MIP-3α. Interestingly, these cytokines and chemokines were also upregulated after the infection. The positive correlation between SIV copy number and sTLR2 in the plasma indicated the involvement of TLR2 in the regulation of viral replication. These cytokines and chemokines could directly or indirectly regulate viral replication through the TLR2 signaling pathways. When we stimulated PBMC with the TLR2 agonist in vitro, we observed a direct induction of various cytokines and chemokines. Some of these cytokines and chemokines, such as IL-1RA, IL-9, IL-15, GRO-α, and ENA-78, were positively correlated with sTLR2 in vivo, highlighting the direct involvement of TLR2 in the regulation of the production of these factors. Our findings suggest that TLR2 expression may be a target for developing new therapeutic strategies to combat HIV infection.

Lymphoid tissues contribute to viral clonotypes present in plasma at early post-ATI in SIV-infected rhesus macaques.

The rebound-competent viral reservoir (RCVR), comprised of virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after antiretroviral therapy interruption (ATI), remains the biggest obstacle to the eradication of HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop targeted therapeutic strategies for reducing the RCVR. In this study, barcoded SIVmac239M was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood, lymphoid tissues (LTs, spleen, mesenteric and inguinal lymph nodes), and non-lymphoid tissues (NLTs, colon, ileum, lung, liver, and brain) were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX/RNAscope/ in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy although plasma viral RNA remained < 22 copies/mL. Among the tissues studied, mesenteric and inguinal lymph nodes, and spleen contained viral barcodes detected in plasma, and trended to have higher cell-associated viral loads, higher intact provirus levels, and greater diversity of viral barcodes. CD4+ T cells were the main cell type harboring viral RNA (vRNA) after ATI. Further, T cell zones in LTs showed higher vRNA levels than B cell zones for most animals. These findings are consistent with LTs contributing to virus present in plasma early after ATI. One Sentence Summary: The reemerging of SIV clonotypes at early post-ATI are likely from the secondary lymphoid tissues.

Divergent Cytokine and Chemokine Responses at Early Acute Simian Immunodeficiency Virus Infection Correlated with Virus Replication and CD4 T Cell Loss in a Rhesus Macaque Model.

Cytokine and chemokine levels remain one of the significant predictive factors of HIV pathogenesis and disease outcome. Understanding the impact of cytokines and chemokines during early acute infection will help to recognize critical changes during HIV pathogenesis and might assist in establishing improved HIV treatment and prevention methods. Sixty-one cytokines and chemokines were evaluated in the plasma of an SIV-infected rhesus macaque model. A substantial change in 11 cytokines/growth factors and 9 chemokines were observed during acute infection. Almost all the cytokines/chemokines were below the baseline values for an initial couple of days of infection. We detected six important cytokines/chemokines, such as IL-18, IP-10, FLT3L, MCP-1, MCP-2, and MIP-3ß, that can be used as biomarkers to predict the peripheral CD4+ T cell loss and increased viral replication during the acute SIV/HIV infection. Hence, regulating IL-18, IP-10, FLT3L, MCP-1, MCP-2, and MIP-3ß expression might provide an antiviral response to combat acute SIV/HIV infection.

Simian Immunodeficiency Virus Infection Mediated Changes in Jejunum and Peripheral SARS-CoV-2 Receptor ACE2 and Associated Proteins or Genes in Rhesus Macaques.

Angiotensin converting enzyme-2 (ACE2) and associated proteins play a pivotal role in various physiological and pathological events, such as immune activation, inflammation, gut barrier maintenance, intestinal stem cell proliferation, and apoptosis. Although many of these clinical events are quite significant in SIV/HIV infection, expression profiling of these proteins has not been well reported. Considering the different pathological consequences in the gut after HIV infection, we hypothesized that the expression of ACE2 and associated proteins of the Renin-angiotensin system (RAS) could be compromised after SIV/HIV infection. We quantified the gene expression of ACE2 as well as AGTR1/2, ADAM17, and TMPRSS2, and compared between SIV infected and uninfected rhesus macaques (Macaca mulatta; hereafter abbreviated RMs). The gene expression analysis revealed significant downregulation of ACE2 and upregulation of AGTR2 and inflammatory cytokine IL-6 in the gut of infected RMs. Protein expression profiling also revealed significant upregulation of AGTR2 after infection. The expression of ACE2 in protein level was also decreased, but not significantly, after infection. To understand the entirety of the process in newly regenerated epithelial cells, a global transcriptomic study of enteroids raised from intestinal stem cells was performed. Interestingly, most of the genes associated with the RAS, such as DPP4, MME, ANPEP, ACE2, ENPEP, were found to be downregulated in SIV infection. HNFA1 was found to be a key regulator of ACE2 and related protein expression. Jejunum CD4+ T cell depletion and increased IL-6 mRNA, MCP-1 and AGTR2 expression may signal inflammation, monocyte/macrophage accumulation and epithelial apoptosis in accelerating SIV pathogenesis. Overall, the findings in the study suggested a possible impact of SIV/HIV infection on expression of ACE2 and RAS-associated proteins resulting in the loss of gut homeostasis. In the context of the current COVID-19 pandemic, the outcome of SARS-CoV-2 and HIV co-infection remains uncertain and needs further investigation as the significance profile of ACE2, a viral entry receptor for SARS-CoV-2, and its expression in mRNA and protein varied in the current study. There is a concern of aggravated SARS-CoV-2 outcomes due to possible serious pathological events in the gut resulting from compromised expression of RAS- associated proteins in SIV/HIV infection.

Microcapillary LAMP for rapid and sensitive detection of pathogen in bovine semen.

A microcapillary-based loop-mediated isothermal amplification (µcLAMP) has been described for specific detection of infectious reproductive pathogens in semen samples of cattle without sophisticated instrumentation. Brucella abortus, Leptospira interrogans serovar Pomona and bovine herpesvirus 1 (BoHV-1) cultures were mixed in bovine semen samples. The µcLAMP assay is portable, user-friendly, cost-effective, and suitable to be performed as a POC diagnostic test. We have demonstrated high sensitivity and specificity of µcLAMP for detection of Brucella, Leptospira, and BoHV-1 in bovine semen samples comparable to PCR and qPCR assays. Thus, µcLAMP would be a promising field-based test for monitoring various infectious pathogens in biological samples.HighlightsDetect infectious organism in bovines semenReduction in carryover contamination is an important attribute, which may reduce the false-positive reaction.µcLAMP is a miniaturized form, which could be performed with a minimum volume of reagents.The µcLAMP assay is portable, user-friendly, and suitable to be performed as a POC diagnostic test.

Identification, Characterization, and Transcriptional Reprogramming of Epithelial Stem Cells and Intestinal Enteroids in Simian Immunodeficiency Virus Infected Rhesus Macaques.

Epithelial cell injury and impaired epithelial regeneration are considered key features in HIV pathogenesis and contribute to HIV-induced generalized immune activation. Understanding the molecular mechanisms underlying the disrupted epithelial regeneration might provide an alternative approach for the treatment of HIV-mediated enteropathy and immune activation. We have observed a significant increased presence of α defensin5+ (HD5) Paneth cells and proliferating Ki67+ epithelial cells as well as decreased expression of E-cadherin expression in epithelial cells during SIV infection. SIV infection did not significantly influence the frequency of LGR5+ stem cells, but the frequency of HD5+ cells was significantly higher compared to uninfected controls in jejunum. Our global transcriptomics analysis of enteroids provided novel information about highly significant changes in several important pathways like metabolic, TCA cycle, and oxidative phosphorylation, where the majority of the differentially expressed genes were downregulated in enteroids grown from chronically SIV-infected macaques compared to the SIV-uninfected controls. Despite the lack of significant reduction in LGR5+ stem cell population, the dysregulation of several intestinal stem cell niche factors including Notch, mTOR, AMPK and Wnt pathways as well as persistence of inflammatory cytokines and chemokines and loss of epithelial barrier function in enteroids further supports that SIV infection impacts on epithelial cell proliferation and intestinal homeostasis.

Enhanced Intestinal TGF-ß/SMAD-Dependent Signaling in Simian Immunodeficiency Virus Infected Rhesus Macaques.

Transforming growth factor-ß signaling (TGF-ß) maintains a balanced physiological function including cell growth, differentiation, and proliferation and regulation of immune system by modulating either SMAD2/3 and SMAD7 (SMAD-dependent) or SMAD-independent signaling pathways under normal conditions. Increased production of TGF-ß promotes immunosuppression in Human Immunodeficiency Virus (HIV)/Simian Immunodeficiency Virus (SIV) infection. However, the cellular source and downstream events of increased TGF-ß production that attributes to its pathological manifestations remain unknown. Here, we have shown increased production of TGF-ß in a majority of intestinal CD3-CD20-CD68+ cells from acute and chronically SIV infected rhesus macaques, which negatively correlated with the frequency of jejunum CD4+ T cells. No significant changes in intestinal TGF-ß receptor II expression were observed but increased production of the pSMAD2/3 protein and SMAD3 gene expression in jejunum tissues that were accompanied by a downregulation of SMAD7 protein and gene expression. Enhanced TGF-ß production by intestinal CD3-CD20-CD68+ cells and increased TGF-ß/SMAD-dependent signaling might be due to a disruption of a negative feedback loop mediated by SMAD7. This suggests that SIV infection impacts the SMAD-dependent signaling pathway of TGF-ß and provides a potential framework for further study to understand the role of viral factor(s) in modulating TGF-ß production and downregulating SMAD7 expression in SIV. Regulation of mucosal TGF-ß expression by therapeutic TGF-ß blockers may help to create effective antiviral mucosal immune responses.

Paper-based loop-mediated isothermal amplification and lateral flow (LAMP-LF) assay for identification of tissues of cattle origin.

The present study was made with the objectives of development and standardization of cattle specific paper-based loop-mediated isothermal amplification cum lateral flow assay (LAMP-LFA), as a Point-of-care test (POCT) for identification of tissue of cattle origin. The components of standardized LAMP reaction utilizing cattle specific primer sets were lyophilized over paper buttons, identified best as the carrier of LAMP reagents. Based on probable LAMP amplicon, a pair of probes was designed, tagged and its hybridization with the amplified product of paper LAMP reaction was optimized. The components of lateral flow assay for detection of probe hybridized LAMP products were standardized. Analysis of successful amplification was made by using HNB dye, LAMP-LFA strip, and also by the typical ladder-like pattern on gel electrophoresis. The assay was found highly specific for cattle with an analytical sensitivity of 0.1 pg of absolute DNA. Laboratory validation carried out on samples from different individuals of cattle, coded samples, binary meat admixture, and heat-processed cattle tissues substantiated the accuracy of the assay. Comparison with pre-standardized species-specific PCR assay taken as gold standards revealed 100% conformity. The field utility of the developed assay was further established by its compatibility with the commercial kit eliminating the lengthy DNA extraction step and storage stability of LAMP reagent carrier buttons for 4 months under refrigeration. Thus, the developed assay capable of the result within 3 h in resource-limited settings can be used as POCT for identification of tissue of cattle origin.

Detection of multiple organisms based on the distance-dependent optical properties of gold nanoparticle and dark-field microscopy.

Owing to their unique physical and chemical properties like stability, non-toxic, biocompatibility and feasible to modification with various biomolecules, gold nanoparticle has become a versatile nanomaterial in the field of therapeutic, diagnostic and analytical studies. Various surface plasmon resonance based pathogen detection systems, relying on change in colour, have been proposed. However, all the approaches developed so far were designed for the detection of a single pathogen. In the present study, we have designed a new colorimetric approach based on distant-dependent properties of gold nanoparticle for the detection of multiple targets. A modified multiplex asymmetric PCR in which a universal primer amplifies the multiple targets with the same efficiency was performed. The Limit of detection (LOD) of the designed visual assay is 10 pg of Brucella and Leptospira target DNA and 100 pg of Bovine herpes virus-1 (BoHV-1) target DNA. LOD of 0.5 pg, 0.7 pg and 3.8 pg for Brucella, Leptospira and BoHV-1 respectively was obtained spectrophotometrically. A study on dark field microscopy as a qualitative supporting detection system has also been presented in this study. The designed assay has advantages over earlier reports in terms of multiple organisms detection, specificity and sensitivity of the test.

Rapid Veterinary Diagnosis of Bovine Reproductive Infectious Diseases from Semen Using Paper-Origami DNA Microfluidics.

The health and well-being of cattle is an important issue in maintaining and increasing global agricultural output. In dairy production within low and middle income countries (LMICs), there is a significant biosensing challenge in detecting sexually transmitted infection (STI) pathogens during animal husbandry, due in part to difficulties associated with the limited infrastructure for veterinary medicine. Here we demonstrate low-cost, multiplexed, and sample-to-answer paper-origami tests for the detection of three bovine infectious reproductive diseases in semen samples, collected at a test site in rural India. Pathogen DNA from one viral pathogen, bovine herpes virus-1 (BoHV-1), and two bacteria (Brucella and Leptospira) was extracted, amplified (using loop-mediated isothermal amplification, LAMP), and detected fluorescently, enabling <1 pg (∼ from 115 to 274 copies per reaction) of target genomic DNA to be measured. Data was collected as a fluorescence signal either visually, using a low-cost hand-held torch, or digitally with a mobile-phone camera. Limits of detection and sensitivities of the paper-origami device for the three pathogens were also evaluated using pathogen-inoculated semen samples and were as few as 50 Leptospira organisms, 50 CFU Brucella, and 1 TCID50 BoHV-1. Semen samples from elite bulls at a germplasm center were also tested in double-blind tests, as a demonstrator for a low-cost, user-friendly point-of-care sensing platform, for in-the-field resource-limited regions. The sensors showed excellent levels of sensitivity and specificity, and for the first time a demonstrated ability of the application of paper microfluidics devices for the diagnosis multiple infectious diseases from semen samples.

Rapid and visual detection of Leptospira in urine by LigB-LAMP assay with pre-addition of dye.

Leptospirosis is considered to be the most widespread zoonotic disease caused by pathogenic species of Leptospira. The present study reports a novel set of primers targeting LigB gene for visual detection of pathogenic Leptospira in urine samples through Loop-mediated isothermal amplification (LAMP). The results were recorded by using Hydroxyl napthol blue (HNB), SYBR GREEN I and calcein. Analytical sensitivity of LAMP was as few as 10 leptospiral organisms in spiked urine samples from cattle and dog. LigB gene based LAMP, termed as LigB-LAMP, was found 10 times more sensitive than conventional PCR. The diagnostic specificity of LAMP was 100% when compared to SYBR green qPCR for detection of Leptospira in urine samples. Though qPCR was found more sensitive, the rapidity and simplicity in setting LAMP test followed by visual detection of Leptospira infection in clinical samples makes LigB-LAMP an alternative and favourable diagnostic tool in resource poor setting.

Visual detection of Brucella in bovine biological samples using DNA-activated gold nanoparticles.

Brucellosis is a bacterial disease, which, although affecting cattle primarily, has been associated with human infections, making its detection an important challenge. The existing gold standard diagnosis relies on the culture of bacteria which is a lengthy and costly process, taking up to 45 days. New technologies based on molecular diagnosis have been proposed, either through dip-stick, immunological assays, which have limited specificity, or using nucleic acid tests, which enable to identify the pathogen, but are impractical for use in the field, where most of the reservoir cases are located. Here we demonstrate a new test based on hybridization assays with metal nanoparticles, which, upon detection of a specific pathogen-derived DNA sequence, yield a visual colour change. We characterise the components used in the assay with a range of analytical techniques and show sensitivities down to 1000 cfu/ml for the detection of Brucella. Finally, we demonstrate that the assay works in a range of bovine samples including semen, milk and urine, opening up the potential for its use in the field, in low-resource settings.