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Introduction: Corona virus disease (COVID-19) is an infectious disease caused by a newly discovered corona virus. Healthcare workers are at risk of developing psychological distress and other mental health related symptoms. This study was intended to evaluate the prevalence and predictors of depression, anxiety, stress, and fear during COVID-19 pandemic among healthcare workers of Nepal. Methods: A cross-sectional survey was conducted between June and July 2020 with 167 healthcare workers. Mental health outcome, specially, fear was estimated using fear of COVID-19 scale (FCV-19S) whereas depression, anxiety and stress were assessed using DASS-21 survey questionnaire. Statistical analysis used: Chi-square test was performed to observe the association between mental health outcomes variables among health workers and the significance level was considered to be a p-value less than 0.05. Results: There were 15.0%, 24.0%, and 5.4% of the respondents who had severe and extremely severe level of depression, anxiety, and stress respectively and 44.3% of the respondents had fear during COVID-19 pandemic. The gender and job position were statistically significant with depression. The gender, job position, and respondents who had any health problems were statistically significant with anxiety. The respondents who had any health problems were statistically significant with stress. The gender, education, and job position were statistically significant with fear. Conclusion: During the COVID-19 outbreak, the frontline healthcare workers have experienced a varying level of depression, anxiety, stress, and fear. Specific counseling, support system, training on handling COVID-19 patient, and assigning normal working hour as government protocol to all healthcare workers are needed to enhance their psychological wellbeing and strengthen the healthcare systems capacity during pandemic.

The COVID-19 pandemic has persisted for more than a year, and post-COVID-19 sequelae of neurological complications, including direct and indirect effects on the central nervous system (CNS), have been recognized. There is a plethora of evidence for neurological, cognitive, and emotional deficits in COVID-19 patients. Acute neurological symptoms like neuroinflammation, cognitive impairment, loss of smell, and brain stroke are common direct effects among SARS-CoV-2 infected individuals. Work-associated stress, lockdowns, social distancing, and quarantine in response to contain SARS-CoV-2 have also affected the mental health of large populations, regardless of age. Public health emergencies have affected individuals and communities, resulting in emotional reactions and unhealthy behaviors. Although vaccines have been widely distributed and administered among large populations, vaccine hesitancy still exists and may be due to apprehension about vaccine efficacy, preliminary trials, and associated side effects. This review highlights the impact of COVID-19 on the CNS by outlining direct and indirect effects and factors contributing to the decline in people's mental health throughout the COVID-19 pandemic both during and after vaccine administration. Furthermore, we also discuss reasons for vaccine hesitancy and why some groups of people are deprived of vaccines. Finally, we touched upon the social determinants of mental health and their impact on disadvantaged populations during times of crisis which may help policymakers set up some action plans to mitigate the COVID-19 mental health turmoil during this ongoing pandemic.

Emerging new variants of SARS-CoV-2 and inevitable acquired drug resistance call for the continued search of new pharmacological targets to fight the potentially fatal infection. Here, we describe the mechanisms by which the E protein of SARS-CoV-2 hijacks the human transcriptional regulator BRD4. We found that SARS-CoV-2 E is acetylated in vivo and co-immunoprecipitates with BRD4 in human cells. Bromodomains (BDs) of BRD4 bind to the C-terminus of the E protein, acetylated by human acetyltransferase p300, whereas the ET domain of BRD4 recognizes the unmodified motif of the E protein. Inhibitors of BRD4 BDs, JQ1 or OTX015, decrease SARS-CoV-2 infectivity in lung bronchial epithelial cells, indicating that the acetyllysine binding function of BDs is necessary for the virus fitness and that BRD4 represents a potential anti-COVID-19 target. Our findings provide insight into molecular mechanisms that contribute to SARS-CoV-2 pathogenesis and shed light on a new strategy to block SARS-CoV-2 infection.

Background Commonly used opioids, such as morphine have been implicated in augmented SIV/HIV persistence within the Central Nervous System (CNS). However, the extent of myeloid cell polarization and viral persistence in different brain regions with diverse environmental niches remains unclear. Additionally, the additive effects of morphine on SIV/HIV dysregulation of gut-brain crosstalk remain underexplored. Therefore, studies focused on understanding how drugs of abuse such as morphine affect immune dynamics, viral persistence and gut brain interrelationships are warranted. MethodsFor a total of 9 weeks, rhesus macaques were ramped-up, and a single daily injection of either morphine (n=4) or saline (n=4) administered. This was later followed with infection with SHIVAD8EO variants. At necropsy, mononuclear cells were isolated from diverse brain (frontal lobe, cerebellum, medulla, putamen, hippocampus (HIP) and subventricular zone (SVZ)) and gut (lamina propria (LP) and muscularis (MUSC) of ascending colon, duodenum, and ileum) regions. Multiparametric flow cytometry was used to were profile for myeloid cell polarity/ activation and results corroborated with indirect immunofluorescence assays. SHIV DNA levels were measured with aid of the digital droplet PCR assay. Luminex assays was used to evaluate soluble plasma/ CSF biomarker levels. Finally, changes in the fecal microbiome were evaluated using the Illumina NovaSeq platform. ResultsFlow Cytometry-based semi-supervised analysis revealed that morphine exposure led to exacerbated M1(CD14/ CD16)/ M2 (CD163/ CD206) polarization in activated microglia that spanned across diverse brain regions. This was accompanied by elevated SHIV DNA within the sites of neurogenesis - hippocampus (HIP) and subventricular zone (SVZ). HIP/SVZ CD16+ activated microglia positively correlated with SHIV DNA levels in the brain (r = 0.548, p = 0.042). Simultaneously, morphine dependence depleted butyrate-producing bacteria, including Ruminococcus (p = 0.05), Lachnospira (p = 0.068) genera and Roseburia_sp._831b (p = 0.068). Finally, morphine also altered the regulation of CNS inflammation by reducing the levels of IL1 Receptor antagonist (IL1Ra). ConclusionsThese findings are suggestive that morphine can promote CNS inflammation by altering receptor modulation, increasing myeloid brain activation, distorting gut-brain crosstalk, and causing selective enhancement of SHIV persistence in sites of neurogenesis.

The current vaccine development strategies for the COVID-19 pandemic utilize whole inactive or attenuated viruses, virus-like particles, recombinant proteins, and antigen-coding DNA and mRNA with various delivery strategies. While highly effective, these vaccine development strategies are time-consuming and often do not provide reliable protection for immunocompromised individuals, young children, and pregnant women. Here, we propose a novel modular vaccine platform to address these shortcomings using chemically synthesized peptides and identified based on the validated bioinformatic data about the target. The vaccine is based on the rational design of an immunogen containing two defined B-cell epitopes from the spike protein of SARS-Co-V2 and a universal T-helper epitope PADRE assembled on the DNA scaffold. The results demonstrate that this assembly is immunogenic and generates neutralizing antibodies against SARS-CoV-2 wild type and its variants of concerns (VOC). This newly designed peptide nanoarray scaffold vaccine is useful in controlling virus transmission in immunocompromised individuals, as well as individuals who are prone to vaccine-induced adverse reactions. Given that the immunogen is modular, epitopes or immunomodulatory ligands can be easily introduced in order to tailor the vaccine to the recipient. This also allows the already developed vaccine to be modified rapidly according to the identified mutations of the virus.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 19 (COVID-19) pandemic. Despite unprecedented research and developmental efforts, SARS-CoV-2-specific antivirals are still unavailable for the treatment of COVID-19. In most instances, SARS-CoV-2 infection initiates with the binding of Spike glycoprotein to the host cell ACE2 receptor. Utilizing the crystal structure of the ACE2/Spike receptor-binding domain (S-RBD) complex (PDB file 6M0J) in a computer-aided drug design approach, we identified and validated five potential inhibitors of S-RBD and ACE-2 interaction. Two of the five compounds, MU-UNMC-1 and MU-UNMC-2, blocked the entry of pseudovirus particles expressing SARS-CoV-2 Spike glycoprotein. In live SARS-CoV-2 infection assays, both compounds showed antiviral activity with IC50 values in the micromolar range (MU-UNMC-1: IC50 = 0.67 μM and MU-UNMC-2: IC50 = 1.72 μM) in human bronchial epithelial cells. Furthermore, MU-UNMC-1 and MU-UNMC-2 effectively blocked the replication of rapidly transmitting variants of concern: South African variant B.1.351 (IC50 = 9.27 and 3.00 μM) and Scotland variant B.1.222 (IC50 = 2.64 and 1.39 μM), respectively. Following these assays, we conducted “induced-fit (flexible) docking” to understand the binding mode of MU-UNMC-1/MU-UNMC-2 at the S-RBD/ACE2 interface. Our data showed that mutation N501Y (present in B.1.351 variant) alters the binding mode of MU-UNMC-2 such that it is partially exposed to the solvent and has reduced polar contacts. Finally, MU-UNMC-2 displayed high synergy with remdesivir, the only approved drug for treating hospitalized COVID-19 patients. IMPORTANCE The ongoing coronavirus infectious disease 2019 (COVID-19) pandemic is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More than 207 million people have been infected globally, and 4.3 million have died due to this viral outbreak. While a few vaccines have been deployed, a SARS-CoV-2-specific antiviral for the treatment of COVID-19 is yet to be approved. As the interaction of SARS-CoV-2 Spike protein with ACE2 is critical for cellular entry, using a combination of a computer-aided drug design (CADD) approach and cell-based in vitro assays, we report the identification of five potential SARS-CoV-2 entry inhibitors. Out of the five, two compounds (MU-UNMC-1 and MU-UNMC-2) have antiviral activity against ancestral SARS-CoV-2 and emerging variants from South Africa and Scotland. Furthermore, MU-UNMC-2 acts synergistically with remdesivir (RDV), suggesting that RDV and MU-UNMC-2 can be developed as a combination therapy to treat COVID-19 patients.

The current vaccine development strategies for the COVID-19 pandemic utilize whole inactive or attenuated viruses, virus-like particles, recombinant proteins, and antigen-coding DNA and mRNA with various delivery strategies. While highly effective, these vaccine development strategies are time-consuming and often do not provide reliable protection for immunocompromised individuals, young children, and pregnant women. Here, we propose a novel modular vaccine platform to address these shortcomings using chemically synthesized peptides identified based on the validated bioinformatic data about the target. The vaccine is based on the rational design of an immunogen containing two defined B-cell epitopes from the spike glycoprotein of SARS-CoV-2 and the universal T-helper epitope PADRE. The epitopes were conjugated to short DNA probes and combined with a complementary scaffold strand, resulting in sequence-specific self-assembly. The immunogens were then formulated by conjugation to gold nanoparticles by three methods or by co-crystallization with epsilon inulin. BALB/C mice were immunized with each formulation, and the IgG immune responses and virus neutralizing titers were compared. The results demonstrate that this assembly is immunogenic and generates neutralizing antibodies against wildtype SARS-CoV-2 and the Delta variant.

Host innate immune response follows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and it is the driver of the acute respiratory distress syndrome (ARDS) amongst other inflammatory end-organ morbidities. Such life-threatening coronavirus disease 2019 (COVID-19) is heralded by virus-induced activation of mononuclear phagocytes (MPs; monocytes, macrophages, and dendritic cells). MPs play substantial roles in aberrant immune secretory activities affecting profound systemic inflammation and end-organ malfunctions. All follow the presence of persistent viral components and virions without evidence of viral replication. To elucidate SARS-CoV-2-MP interactions we investigated transcriptomic and proteomic profiles of human monocyte-derived macrophages. While expression of the SARS-CoV-2 receptor, the angiotensin-converting enzyme 2, paralleled monocyte-macrophage differentiation, it failed to affect productive viral infection. In contrast, simple macrophage viral exposure led to robust pro-inflammatory cytokine and chemokine expression but attenuated type I interferon (IFN) activity. Both paralleled dysregulation of innate immune signaling pathways, specifically those linked to IFN. We conclude that the SARS-CoV-2-infected host mounts a robust innate immune response characterized by a pro-inflammatory storm heralding end-organ tissue damage.

Respiratory epithelial cells are the primary target for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We investigated the 3D human airway tissue model to evaluate innate epithelial cell responses to SARS-CoV-2 infection. A SARS-CoV-2 clinical isolate productively infected the 3D-airway model with a time-dependent increase in viral load (VL) and concurrent upregulation of airway immunomodulatory factors (IL-6, ICAM-1, and SCGB1A1) and respiratory mucins (MUC5AC, MUC5B, MUC2, and MUC4), and differential modulation of select long noncoding RNAs (lncRNAs i.e., LASI, TOSL, NEAT1, and MALAT1). Next, we examined these immunomodulators in the COVID-19 patient nasopharyngeal swab samples collected from subjects with high- or low-VLs (~100-fold difference). As compared to low-VL, high-VL patients had prominent mucoinflammatory signature with elevated expression of IL-6, ICAM-1, SCGB1A1, SPDEF, MUC5AC, MUC5B, and MUC4. Interestingly, LASI, TOSL, and NEAT1 lncRNA expressions were also markedly elevated in high-VL patients with no change in MALAT1 expression. In addition, dual-staining of LASI and SARS-CoV-2 nucleocapsid N1 RNA showed predominantly nuclear/perinuclear localization at 24 hpi in 3D-airway model as well as in high-VL COVID-19 patient nasopharyngeal cells, which exhibited high MUC5AC immunopositivity. Collectively, these findings suggest SARS-CoV-2 induced lncRNAs may play a role in acute mucoinflammatory response observed in symptomatic COVID-19 patients.

SARS-CoV-2 infection initiates with the attachment of spike protein to the ACE2 receptor. While vaccines have been developed, no SARS-CoV-2 specific small molecule inhibitors have been approved. Herein, utilizing the crystal structure of the ACE2/Spike receptor binding domain (S-RBD) complex in computer-aided drug design (CADD) approach, we docked ∼8 million compounds within the pockets residing at S-RBD/ACE2 interface. Five best hits depending on the docking score, were selected and tested for their in vitro efficacy to block SARS-CoV-2 replication. Of these, two compounds (MU-UNMC-1 and MU-UNMC-2) blocked SARS-CoV-2 replication at sub-micromolar IC50 in human bronchial epithelial cells (UNCN1T) and Vero cells. Furthermore, MU-UNMC-2 was highly potent in blocking the virus entry by using pseudoviral particles expressing SARS-CoV-2 spike. Finally, we found that MU-UNMC-2 is highly synergistic with remdesivir (RDV), suggesting that minimal amounts are needed when used in combination with RDV, and has the potential to develop as a potential entry inhibitor for COVID-19.

Pathogenic viruses like SARS-CoV-2 and HIV hijack the host molecular machinery to establish infection and survival in infected cells. This has led the scientific community to explore the molecular mechanisms by which SARS-CoV-2 infects host cells, establishes productive infection, and causes life-threatening pathophysiology. Very few targeted therapeutics for COVID-19 currently exist, such as remdesivir. Recently, a proteomic approach explored the interactions of 26 of 29 SARS-CoV-2 proteins with cellular targets in human cells and identified 67 interactions as potential targets for drug development. Two of the critical targets, the bromodomain and extra-terminal domain proteins (BETs): BRD2/BRD4 and mTOR, are inhibited by the dual inhibitory small molecule SF2523 at nanomolar potency. SF2523 is the only known mTOR PI3K-α/(BRD2/BRD4) inhibitor with potential to block two orthogonal pathways necessary for SARS-CoV-2 pathogenesis in human cells. Our results demonstrate that SF2523 effectively blocks SARS-CoV-2 replication in lung bronchial epithelial cells in vitro, showing an IC50 value of 1.5 µM, comparable to IC50 value of remdesivir (1.1 µM). Further, we demonstrated that the combination of doses of SF2523 and remdesivir is highly synergistic: it allows for the reduction of doses of SF2523 and remdesivir by 25-fold and 4-fold, respectively, to achieve the same potency observed for a single inhibitor. Because SF2523 inhibits two SARS-CoV-2 driven pathogenesis mechanisms involving BRD2/BRD4 and mTOR signaling, our data suggest that SF2523 alone or in combination with remdesivir could be a novel and efficient therapeutic strategy to block SARS-CoV-2 infection and hence be beneficial in preventing severe COVID-19 disease evolution.One Sentence SummaryEvidence of in silico designed chemotype (SF2523) targeting PI3K-α/mTOR/BRD4 inhibits SARS-CoV-2 infection and is highly synergistic with remdesivir.

The emergence and rapid spread of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused over 180 million confirmed cases resulting in over 4 million deaths worldwide with no clear end in sight for the coronavirus disease 19 (COVID-19) pandemic. Most SARS-CoV-2 exposed individuals experience mild to moderate symptoms, including fever, cough, fatigue, and loss of smell and taste. However, many individuals develop pneumonia, acute respiratory distress syndrome, septic shock, and multiorgan dysfunction. In addition to these primarily respiratory symptoms, SARS-CoV-2 can also infiltrate the central nervous system, which may damage the blood-brain barrier and the neuron's synapses. Resultant inflammation and neurodegeneration in the brain stem can further prevent efferent signaling to cranial nerves, leading to the loss of anti-inflammatory signaling and normal respiratory and gastrointestinal functions. Additionally, SARS-CoV-2 can infect enterocytes resulting in gut damage followed by microbial dysbiosis and translocation of bacteria and their byproducts across the damaged epithelial barrier. As a result, this exacerbates pro-inflammatory responses both locally and systemically, resulting in impaired clinical outcomes. Recent evidence has highlighted the complex interactions that mutually modulate respiratory, neurological, and gastrointestinal function. In this review, we discuss the ways SARS-CoV-2 potentially disrupts the gut-brain-lung axis. We further highlight targeting specific responses to SARS-CoV-2 for the development of novel, urgently needed therapeutic interventions. Finally, we propose a prospective related to the individuals from Low- and Middle-Income countries. Here, the underlying propensity for heightened gut damage/microbial translocation is likely to result in worse clinical outcomes during this COVID-19 pandemic.

HIV persists in cellular reservoirs despite effective combined antiretroviral therapy (cART) and there is viremia flare up upon therapy interruption. Opioids modulate the immune system and suppress antiviral gene responses, which significantly impact people living with HIV (PLWH). However, the effect of opioids on viral reservoir dynamics remain elusive. Herein, we developed a morphine dependent SIVmac251 infected Rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. RMs on a morphine (or saline control) regimen were infected with SIVmac251. The cART was initiated in approximately half the animals five weeks post-infection, and morphine/saline administration continued until the end of the study. Among the untreated RM, we did not find any difference in plasma/CSF or in cell-associated DNA/RNA viral load in anatomical tissues. On the other hand, within the cART suppressed macaques, there was a reduction in cell-associated DNA load, intact proviral DNA levels, and in inducible SIV reservoir in lymph nodes (LNs) of morphine administered RMs. In distinction to LNs, in the CNS, the size of latent SIV reservoirs was higher in the CD11b+ microglia/macrophages in morphine dependent RMs. These results suggest that in the proposed model, morphine plays a differential role in SIV reservoirs by reducing the CD4+ T-cell reservoir in lymphoid tissues, while increasing the microglia/reservoir size in CNS tissue. The findings from this pre-clinical model will serve as a tool for screening therapeutic strategies to reduce/eliminate HIV reservoirs in opioid dependent PLWH.IMPORTANCE Identification and clearance of HIV reservoirs is a major challenge in achieving a cure for HIV. This is further complicated by co-morbidities that may alter the size of the reservoirs. There is an overlap between the risk factors for HIV and opioid abuse. Opiates have been recognized as prominent co-morbidities in HIV-infected populations. People infected with HIV also abusing opioids have immune modulatory effects and more severe neurological disease. However, the impact of opioid abuse on HIV reservoirs remains unclear. In this study, we used morphine dependent SIVmac251 infected rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. Our studies suggested that people with HIV who abuse opioids had higher reservoirs in CNS than the lymphoid system. Extrapolating the macaque findings in humans suggests that such differential modulation of HIV reservoirs among people living with HIV abusing opioids could be considered for future HIV cure research efforts.

Identification and clearance of human immunodeficiency virus (HIV) reservoirs is a major challenge in achieving a cure for HIV. This is further complicated by comorbidities that may alter the size of the reservoirs., Human immunodeficiency virus (HIV) persists in cellular reservoirs despite effective combined antiretroviral therapy (cART), and there is viremia flare-up upon therapy interruption. Opioids modulate the immune system and suppress antiviral gene responses, which significantly impact people living with HIV (PLWH). However, the effect of opioids on viral reservoir dynamics remains elusive. Here, we developed a morphine-dependent simian immunodeficiency virus SIVmac251-infected rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. RMs on a morphine (or saline control) regimen were infected with SIVmac251. cART was initiated in approximately half the animals 5 weeks postinfection, and morphine/saline administration continued until the end of the study. Among the untreated RMs, we did not find any difference in plasma/cerebrospinal fluid (CSF) viral loads or in cell-associated DNA/RNA viral loads in anatomical tissues. On the other hand, within the cART-suppressed macaques, there was a reduction in cell-associated DNA load, intact proviral DNA levels, and in inducible SIV reservoirs in lymph nodes (LNs) of morphine-administered RMs. In distinction to those in LNs, in the central nervous system (CNS), the size of latent SIV reservoirs was larger in the CD11b+ microglia/macrophages in morphine-dependent RMs. These results suggest that in the proposed model, morphine plays a differential role in SIV reservoirs by reducing the CD4+ T-cell reservoir in lymphoid tissues while increasing the microglia/macrophage reservoir size in CNS tissue. The findings from this preclinical model will serve as a tool for screening therapeutic strategies to reduce/eliminate HIV reservoirs in opioid-dependent PLWH. IMPORTANCE Identification and clearance of human immunodeficiency virus (HIV) reservoirs is a major challenge in achieving a cure for HIV. This is further complicated by comorbidities that may alter the size of the reservoirs. There is an overlap between the risk factors for HIV and opioid abuse. Opiate abuse has been recognized as a prominent comorbidity in HIV-infected populations. People infected with HIV also abusing opioids have immune modulatory effects and more severe neurological disease. However, the impact of opioid abuse on HIV reservoirs remains unclear. In this study, we used a morphine-dependent simian immunodeficiency virus SIVmac251-infected rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. Our studies suggested that people with HIV who abuse opioids have higher reservoirs in central nervous system (CNS) than in the lymphoid system. Extrapolating the macaque findings to humans suggests that such differential modulation of HIV reservoirs among people living with HIV abusing opioids could be considered for future HIV cure research efforts.

Emerging and re-emerging viral diseases can create devastating effects on human lives and may also lead to economic crises. The ongoing COVID-19 pandemic due to the novel coronavirus (nCoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which originated in Wuhan, China, has caused a global public health emergency. To date, the molecular mechanism of transmission of SARS-CoV-2, its clinical manifestations and pathogenesis is not completely understood. The global scientific community has intensified its efforts in understanding the biology of SARS-CoV-2 for development of vaccines and therapeutic interventions to prevent the rapid spread of the virus and to control mortality and morbidity associated with COVID-19. To understand the pathophysiology of SARS-CoV-2, appropriate animal models that mimic the biology of human SARS-CoV-2 infection are urgently needed. In this review, we outline animal models that have been used to study previous human coronaviruses (HCoVs), including severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV). Importantly, we discuss models that are appropriate for SARS-CoV-2 as well as the advantages and disadvantages of various available methods.

HIV persists in cellular reservoirs despite effective anti-retroviral therapy with rebound of viremia upon therapy interruption. Opioids modulate the immune system and suppress antiviral gene responses, which significantly impact people living with HIV (PLWH). However, the effects of opioids on viral reservoir remains elusive. Herein, we describe a morphine dependent SIVmac251 infected Rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. RMs were ramped up with morphine (n=10) or saline (n=9) for two weeks to a final dosage of 5mg/kg administered twice daily, which was maintained for seven weeks, and then infected with SIVmac251. Combined anti-retroviral therapy (cART) was initiated in approximately half the animals in each group five weeks post-infection and morphine/saline administration continued for 10 months. Among drug naïve macaques, there were no differences in plasma/CSF viral load nor in cell-associated DNA/RNA loads. However, within the cART-suppressed macaques, there was a reduction in cell-associated DNA load, intact proviral DNA copy numbers, and inducible SIV reservoir in both peripheral blood and lymph nodes (LNs) of morphine-administered RMs compared to saline controls. Further, PBMCs of morphine administered RMs, a reduction in Th1 polarized CD4+ T cells and in LNs there was a reduction in the total Tfh and Th1 like Tfh cells were observed, indicating probably have impact on reduction of viral reservoirs. In distinction to PBMC and LNs, within the CNS size of latent SIV reservoirs was higher in the CD11b+ microglia/macrophages of morphine-dependent RMs. These data suggest that morphine plays a role in modulating SIV reservoirs, reduces the CD4+ T-cell reservoir in both peripheral blood and LNs, and increases microglia/macrophage reservoirs in CNS. These findings will aid in understanding of molecular mechanism(s) of opioid-mediated differential modulation of viral reservoirs and evaluation of therapeutic strategies to reduce/eliminate HIV reservoirs in opioid-dependent PLWH.Author summaryOpioids are commonly used as well as abused by HIV infected individuals, are known to suppress immune responses. However, their effects on modulating viral reservoir dynamics is not known. Here we developed a morphine dependent SIVmac251 infected rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs and immune cells. We found that there was no difference in viral loads or cell-associated DNA/RNA loads among morphine dependent vs. saline treated control macaques. On the other hand, when macaques were treated with cART, there was a reduction in SIV reservoirs both in periphery and lymphoid tissues in morphine administered RMs, and the size of latent SIV reservoir was higher in the CNS CD11b+ microglia/macrophages as compared to control macaques. Therefore, these new data form macaque models suggest that PLWH who suffering from opioid use disorders have higher reservoirs in CNS as compared to lymphoid system. Thus, through understanding these reservoirs among PLWH who uses opioids are critical for better designing HIV cure strategies.

Highlights • Knockdown of G3BP1 significantly increases PEDV replication. • Overexpression of G3BP1 lowers virus replication. • Increased pro-inflammatory cytokines in PEDV-infected G3BP1 depleted cells. • PEDV induces stress granules in infected Vero cells., Porcine epidemic diarrhoea virus (PEDV) is a single-stranded, positive-sense RNA virus that belongs to the Coronaviridae. PEDV causes severe diarrhoea and dehydration in nursing piglets, which leads to significant economic losses to the swine industry worldwide. Stress granules (SGs) are sites of mRNA storage that are formed under various stress conditions including viral infections. Increasing evidence suggests that SGs function in antiviral innate immunity of host cells to limit virus replication. Ras-GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) is a key stress granule-resident protein that nucleates stress granule assembly. Depletion of G3BP1 inhibits SGs formation and overexpression of G3BP1 nucleates SGs assembly. We observed that knockdown of G3BP1 by silencing RNA significantly increased PEDV replication. Overexpression of exogenous G3BP1, on the other hand, lowered virus replication by 100-fold compared to vector control. An increase in the levels of mRNAs of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) was also observed in PEDV-infected G3BP1 depleted cells compared to PEDV-infected control cells. Taken together, our results suggest that G3BP1 plays an antiviral role and impairs PEDV replication.