Identification and characterization of endogenous retroviruses upon SARS-CoV-2 infection.

Endogenous retroviruses (ERVs) derived from the long terminal repeat (LTR) family of transposons constitute a significant portion of the mammalian genome, with origins tracing back to ancient viral infections. Despite comprising approximately 8% of the human genome, the specific role of ERVs in the pathogenesis of COVID-19 remains unclear. In this study, we conducted a genome-wide identification of ERVs in human peripheral blood mononuclear cells (hPBMCs) and primary lung epithelial cells from monkeys and mice, both infected and uninfected with SARS-CoV-2. We identified 405, 283, and 206 significantly up-regulated transposable elements (TEs) in hPBMCs, monkeys, and mice, respectively. This included 254, 119, 68, and 28 ERVs found in hPBMCs from severe and mild COVID-19 patients, monkeys, and transgenic mice expressing the human ACE2 receptor (hACE2) and infected with SARS-CoV-2. Furthermore, analysis using the Genomic Regions Enrichment of Annotations Tool (GREAT) revealed certain parental genomic sequences of these up-regulated ERVs in COVID-19 patients may be involved in various biological processes, including histone modification and viral replication. Of particular interest, we identified 210 ERVs specifically up-regulated in the severe COVID-19 group. The genes associated with these differentially expressed ERVs were enriched in processes such as immune response activation and histone modification. HERV1_I-int: ERV1:LTR and LTR7Y: ERV1:LTR were highlighted as potential biomarkers for evaluating the severity of COVID-19. Additionally, validation of our findings using RT-qPCR in Bone Marrow-Derived Macrophages (BMDMs) from mice infected by HSV-1 and VSV provided further support to our results. This study offers insights into the expression patterns and potential roles of ERVs following viral infection, providing a valuable resource for future studies on ERVs and their interaction with SARS-CoV-2.

Persistent and Severe Viral Replication in PBMCs with Moderate Immunosuppression Served an Alternative Novel Pathogenic Mechanism for Canine Morbillivirus.

Measles virus and canine distemper virus (CDV) cause lethal infections in their respective hosts characterized by severe immunosuppression. To furtherly acknowledge the attenuated mechanisms of the regionally ongoing epidemic CDV isolates and provide novel perspectives for designing new vaccines and therapeutic drugs, a recombinant CDV rHBF-vacH was employed with a vaccine hemagglutinin (H) gene replacement by reverse genetics based on an infectious cDNA clone for the CDV wild-type HBF-1 strain. Interestingly, unlike previously published reports that a vaccine H protein completely changed a pathogenic wild-type CDV variant to be avirulent, rHBF-vacH was only partially attenuated by alleviating the degree of viral immunosuppression, and still caused 66.7% lethality in ferrets with a prolonged period of disease. Further comparisons of pathogenic mechanisms proved that the weaker but necessary invasions into peripheral blood mononuclear cells (PBMCs) of rHBF-vacH, and subsequently persistent viral replications in PBMCs and multiple organs, together contributed to its 66.7% mortality. In addition, despite significantly higher titers than the parent viruses, rHBF-vacH would not be a suitable candidate for a live vaccine, with great invasion and infection potentials of PBMCs from 16 tested kinds of host species. Altogether, sustained and severe viral replication in PBMCs with moderate immunosuppression was first proven to be an alternative novel pathogenic mechanism for CDV, which might help us to understand possible reasons for CDV fatal infections among domestic dogs and the highly susceptible wild species during natural transmission. IMPORTANCE Despite widespread vaccine campaigns for domestic dogs, CDV remained an important infectious disease in vaccinated carnivores and wild species. In recent years, the regionally ongoing epidemic CDV isolates have emphasized conservation threats to, and potentially disastrous epidemics in, endangered species worldwide. However, little is known about how to deal with the CDV variants constantly regional epidemic. In this study, we employed a recombinant CDV rHBF-vacH with a vaccine H gene replacement in a CDV wild-type HBF-1 context to attenuate the epidemic CDV variant to design a new vaccine candidate. Interestingly, rHBF-vacH was only partially attenuated by alleviating the degree of viral immunosuppression, and still caused 66.7% lethality in ferrets by weaker but necessary invasions into PBMCs, and subsequently persistent and severe viral replications in PBMCs. Significantly higher virus titers of rHBF-vacH in vitro might indicate the rapid cell-to-cell spreads in vivo that indirectly contribute to fatal infections of rHBF-vacH in ferrets.

Deep Sequencing Analysis of Individual HIV-1 Proviruses Reveals Frequent Asymmetric Long Terminal Repeats.

Effective strategies to eliminate human immunodeficiency virus type 1 (HIV-1) reservoirs are likely to require more thorough characterizations of proviruses that persist on antiretroviral therapy (ART). The rarity of infected CD4+ T-cells and related technical challenges have limited the characterization of integrated proviruses. Current approaches using next-generation sequencing can be inefficient and limited sequencing depth can make it difficult to link proviral sequences to their respective integration sites. Here, we report on an efficient method by which HIV-1 proviruses and their sites of integration are amplified and sequenced. Across five HIV-1-positive individuals on clinically effective ART, a median of 41.2% (n = 88 of 209) of amplifications yielded near-full-length proviruses and their 5'-host-virus junctions containing a median of 430 bp (range, 18 to 1,363 bp) of flanking host sequence. Unexpectedly, 29.5% (n = 26 of 88) of the sequenced proviruses had structural asymmetries between the 5' and 3' long terminal repeats (LTRs), commonly in the form of major 3' deletions. Sequence-intact proviruses were detected in 3 of 5 donors, and infected CD4+ T-cell clones were detected in 4 of 5 donors. The accuracy of the method was validated by amplifying and sequencing full-length proviruses and flanking host sequences directly from peripheral blood mononuclear cell DNA. The individual proviral sequencing assay (IPSA) described here can provide an accurate, in-depth, and longitudinal characterization of HIV-1 proviruses that persist on ART, which is important for targeting proviruses for elimination and assessing the impact of interventions designed to eradicate HIV-1. IMPORTANCE The integration of human immunodeficiency virus type 1 (HIV-1) into chromosomal DNA establishes the long-term persistence of HIV-1 as proviruses despite effective antiretroviral therapy (ART). Characterizing proviruses is difficult because of their rarity in individuals on long-term suppressive ART, their highly polymorphic sequences and genetic structures, and the need for efficient amplification and sequencing of the provirus and its integration site. Here, we describe a novel, integrated, two-step method (individual proviral sequencing assay [IPSA]) that amplifies the host-virus junction and the full-length provirus except for the last 69 bp of the 3' long terminal repeat (LTR). Using this method, we identified the integration sites of proviruses, including those that are sequence intact and replication competent or defective. Importantly, this new method identified previously unreported asymmetries between LTRs that have implications for how proviruses are detected and quantified. The IPSA method reported is unaffected by LTR asymmetries, permitting a more accurate and comprehensive characterization of the proviral landscape.

SeqScreen: accurate and sensitive functional screening of pathogenic sequences via ensemble learning.

The COVID-19 pandemic has emphasized the importance of accurate detection of known and emerging pathogens. However, robust characterization of pathogenic sequences remains an open challenge. To address this need we developed SeqScreen, which accurately characterizes short nucleotide sequences using taxonomic and functional labels and a customized set of curated Functions of Sequences of Concern (FunSoCs) specific to microbial pathogenesis. We show our ensemble machine learning model can label protein-coding sequences with FunSoCs with high recall and precision. SeqScreen is a step towards a novel paradigm of functionally informed synthetic DNA screening and pathogen characterization, available for download at www.gitlab.com/treangenlab/seqscreen .

Changes to the Simian Immunodeficiency Virus (SIV) Reservoir and Enhanced SIV-Specific Responses in a Rhesus Macaque Model of Functional Cure after Serial Rounds of Romidepsin Administrations.

HIV persistence requires lifelong antiretroviral therapy (ART), calling for a cure. The histone deacetylase inhibitor, romidepsin, is used in the "shock and kill" approach with the goal of reactivating virus and subsequently clearing infected cells through cell-mediated immune responses. We tested serial and double infusions of romidepsin in a rhesus macaque (RM) model of SIV functional cure, which controls virus without ART. Off ART, romidepsin reactivated SIV in all RMs. Subsequent infusions resulted in diminished reactivation, and two RMs did not reactivate the virus after the second or third infusions. Therefore, those two RMs received CD8-depleting antibody to assess the replication competence of the residual reservoir. The remaining RMs received double infusions, i.e., two doses separated by 48-h. Double infusions were well tolerated, induced immune activation, and effectively reactivated SIV. Although reactivation was gradually diminished, cell-associated viral DNA was minimally changed, and viral outgrowth occurred in 4/5 RMs. In the RM which did not reactivate after CD8 depletion, viral outgrowth was not detected in peripheral blood mononuclear cells (PBMC)-derived CD4+ cells. The frequency of SIV-specific CD8+ T cells increased after romidepsin administration, and the increased SIV-specific immune responses were associated, although not statistically, with the diminished reactivation. Thus, our data showing sequential decreases in viral reactivation with repeated romidepsin administrations with all RMs and absence of viral reactivation after CD8+ T-cell depletion in one animal suggest that, in the context of healthy immune responses, romidepsin affected the inducible viral reservoir and gradually increased immune-mediated viral control. Given the disparities between the results of romidepsin administration to ART-suppressed SIVmac239-infected RMs and HIV-infected normal progressors compared to our immune-healthy model, our data suggest that improving immune function for greater SIV-specific responses should be the starting point of HIV cure strategies. IMPORTANCE HIV cure is sought after due to the prevalence of comorbidities that occur in persons with HIV. One of the most investigated HIV cure strategies is the "shock and kill" approach. Our study investigated the use of romidepsin, a histone deacetylase (HDAC) inhibitor, in our rhesus macaque model of functional cure, which allows for better resolution of viral reactivation due to the lack of antiretroviral therapy. We found that repeated rounds of romidepsin resulted in gradually diminished viral reactivation. One animal inevitably lacked replication-competent virus in the blood. With the accompanying enhancement of the SIV-specific immune response, our data suggest that there is a reduction of the viral reservoir in one animal by the cell-mediated immune response. With the differences observed between our model and persons living with HIV (PWH) treated with romidepsin, specifically in the context of a healthy immune system in our model, our data thereby indicate the importance of restoring the immune system for cure strategies.

Porcine Epidemic Diarrhea Virus Infection Disrupts the Nasal Endothelial Barrier To Favor Viral Dissemination.

Crossing the endothelium from the entry site and spreading in the bloodstream are crucial but obscure steps in the pathogenesis of many emerging viruses. Previous studies confirmed that porcine epidemic diarrhea virus (PEDV) caused intestinal infection by intranasal inoculation. However, the role of the nasal endothelial barrier in PEDV translocation remains unclear. Here, we demonstrated that PEDV infection causes nasal endothelial dysfunction to favor viral dissemination. Intranasal inoculation with PEDV compromised the integrity of endothelial cells (ECs) in nasal microvessels. The matrix metalloproteinase 7 (MMP-7) released from the PEDV-infected nasal epithelial cells (NECs) contributed to the destruction of endothelial integrity by degrading the tight junctions, rather than direct PEDV infection. Moreover, the proinflammatory cytokines released from PEDV-infected NECs activated ECs to upregulate ICAM-1 expression, which favored peripheral blood mononuclear cells (PBMCs) migration. PEDV could further exploit migrated cells to favor viral dissemination. Together, our results reveal the mechanism by which PEDV manipulates the endothelial dysfunction to favor viral dissemination and provide novel insights into how coronavirus interacts with the endothelium. IMPORTANCE The endothelial barrier is the last but vital defense against systemic viral transmission. Porcine epidemic diarrhea virus (PEDV) can cause severe atrophic enteritis and acute viremia. However, the mechanisms by which the virus crosses the endothelial barrier and causes viremia are poorly understood. In this study, we revealed the mechanisms of endothelial dysfunction in PEDV infection. The viral infection activates NECs and causes the upregulation of MMP-7 and proinflammatory cytokines. Using NECs, ECs, and PBMCs as in vitro models, we determined that the released MMP-7 contributed to the destruction of endothelial barrier, and the released proinflammatory cytokines activated ECs to facilitate PBMCs migration. Moreover, the virus further exploited the migrated cells to promote viral dissemination. Thus, our results provide new insights into the mechanisms underlying endothelial dysfunction induced by coronavirus infection.

Cellular and humoral functional responses after BNT162b2 mRNA vaccination differ longitudinally between naive and subjects recovered from COVID-19.

We have analyzed BNT162b2 vaccine-induced immune responses in naive subjects and individuals recovered from coronavirus disease 2019 (COVID-19), both soon after (14 days) and later after (almost 8 months) vaccination. Plasma spike (S)-specific immunoglobulins peak after one vaccine shot in individuals recovered from COVID-19, while a second dose is needed in naive subjects, although the latter group shows reduced levels all along the analyzed period. Despite how the neutralization capacity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mirrors this behavior early after vaccination, both groups show comparable neutralizing antibodies and S-specific B cell levels late post-vaccination. When studying cellular responses, naive individuals exhibit higher SARS-CoV-2-specific cytokine production, CD4+ T cell activation, and proliferation than do individuals recovered from COVID-19, with patent inverse correlations between humoral and cellular variables early post-vaccination. However, almost 8 months post-vaccination, SARS-CoV-2-specific responses are comparable between both groups. Our data indicate that a previous history of COVID-19 differentially determines the functional T and B cell-mediated responses to BNT162b2 vaccination over time.

Long non-coding RNAs (lncRNAs) NEAT1 and MALAT1 are differentially expressed in severe COVID-19 patients: An integrated single-cell analysis.

Hyperactive and damaging inflammation is a hallmark of severe rather than mild Coronavirus disease 2019 (COVID-19). To uncover key inflammatory differentiators between severe and mild COVID-19, we applied an unbiased single-cell transcriptomic analysis. We integrated two single-cell RNA-seq datasets with COVID-19 patient samples, one that sequenced bronchoalveolar lavage (BAL) cells and one that sequenced peripheral blood mononuclear cells (PBMCs). The combined cell population was then analyzed with a focus on genes associated with disease severity. The immunomodulatory long non-coding RNAs (lncRNAs) NEAT1 and MALAT1 were highly differentially expressed between mild and severe patients in multiple cell types. Within those same cell types, the concurrent detection of other severity-associated genes involved in cellular stress response and apoptosis regulation suggests that the pro-inflammatory functions of these lncRNAs may foster cell stress and damage. Thus, NEAT1 and MALAT1 are potential components of immune dysregulation in COVID-19 that may provide targets for severity related diagnostic measures or therapy.

Recruitment of highly cytotoxic CD8+ T cell receptors in mild SARS-CoV-2 infection.

T cell immunity is crucial for control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been studied widely on a quantitative level. However, the quality of responses, in particular of CD8+ T cells, has only been investigated marginally so far. Here, we isolate T cell receptor (TCR) repertoires specific for immunodominant SARS-CoV-2 epitopes restricted to common human Leukocyte antigen (HLA) class I molecules in convalescent individuals. SARS-CoV-2-specific CD8+ T cells are detected up to 12 months after infection. TCR repertoires are diverse, with heterogeneous functional avidity and cytotoxicity toward virus-infected cells, as demonstrated for TCR-engineered T cells. High TCR functionality correlates with gene signatures that, remarkably, could be retrieved for each epitope:HLA combination analyzed. Overall, our data demonstrate that polyclonal and highly functional CD8+ TCRs-classic features of protective immunity-are recruited upon mild SARS-CoV-2 infection, providing tools to assess the quality of and potentially restore functional CD8+ T cell immunity.

Insufficient immunity led to virologic breakthrough in NAs-treated chronic hepatitis B patients switching to Peg-IFN-ɑ.

BACKGROUND: Virologic breakthrough (VBT) may occur in chronic hepatitis B (CHB) patients after switching from nucleos(t)ide analogues (NAs) to pegylated interferon alpha (Peg-IFN-ɑ). This study aimed to characterize the clinical and immunological features of VBT. METHODS: In NAs-treated patients switching to Peg-IFN-ɑ, innate and adaptive immune cell proportions were examined in peripheral blood and liver biopsy specimens. In vitro effect of IFN-ɑ on the expressions of toll-like receptors 2 (TLR2) and programmed cell death ligand 1 (PDL1) on monocytes, programmed cell death 1 (PD1) on CD8+T cells was examined. Peripheral blood mononuclear cells (PBMCs) were treated with TLR2 agonist and/or PDL1 blockade to evaluate their effect on HBV replication. RESULTS: 33 of 166 patients switching to Peg-IFN-ɑ experienced VBT after NA cessation, with majority being hepatitis B e antigen (HBeAg) positive or having higher hepatitis B core-related antigen (HBcrAg) levels. Patients with VBT exhibited lower proportions of TLR2+monocyte and increased PD1+HBV-specific CD8+T cell during the early phase of Peg-IFN-ɑ therapy after NA cessation in peripheral blood, as well as fewer TLR2+CD68+macrophages but more PDL1+CD68+macrophages and PD1+CD8+T cells in liver tissues. Simultaneous use of TLR2 agonist and PDL1 blockage ex vivo suppressed HBV replication by promoting cytokines production and CD8+T cells cytotoxicity. Upon in vitro IFN-ɑ stimulation, PDL1+monocytes and PD1+CD8+T cells were upregulated, whereas TLR2+monocytes were not increased in PBMC isolated from HBeAg-positive patients, or those with high HBcrAg titers. CONCLUSIONS: In NAs-treated patients, lower TLR2+monocyte and increased PD1+HBV-specific CD8+T cell proportions potentially contribute to VBT after switching to Peg-IFN-ɑ therapy. This insufficient immunity may be associated with the HBeAg status and HBcrAg levels.

HERV-K and W expression in peripheral mononuclear cells of children with Henoch-Schönlein purpura and relation with TLR activation.

BACKGROUND: HERVs expression seems impaired in several diseases, ranging from autoimmune to neoplastic disorders. However, various stimuli may re-activate HERVs transcription. Henoch-Schönlein purpura is the most common cause of vasculitis in children. The role of microbial antigens in the pathogenesis of HSP remains elusive. Toll-like receptors (TLRs) are the first line of defense for the host to initiate an immune and inflammatory response. We aimed to investigate HERV, K and W expression in peripheral mononuclear cells of children with HSP and relation with TLRs activation. METHODS: The study enrolled 63 children affected by HSP. We used RT-PCR to detect GAPDH in the peripheral blood mononuclear cells samples to normalize the data. Subsequently, the viral pol gene HERVs and TLRs transcripts in the same samples were assessed. RESULTS: HERV K and W was expressed in all samples analyzed but the level of expression was much lower in the HSP that in HC P=0.0006 and P=0.0004 for HERV-K and -W respectively. TLR2 was hyper-expressed in 39 vs. 63 (62%) of the HSP, TLR3 in 23 vs. 63 (42%), TLR4 in 42 vs. 63 (66%) and TLR9 in 32 vs. 63 (52%). The different expression was statistically significant only for TLR4 (P=0.0276) no for TLR2,3 and 9 (P=0.1251; 0.3964 and 0.1882 respectively). Spearman's test demonstrated no correlation between the low expression of HERV-K and HERV-W and high expression of TLRs. CONCLUSIONS: The results of the present study demonstrated that mRNA expression of HERV-K and -W and TLRs did not directly correlated.

HLA-A∗02:01 restricted T cell receptors against the highly conserved SARS-CoV-2 polymerase cross-react with human coronaviruses.

Cross-reactivity and direct killing of target cells remain underexplored for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific CD8+ T cells. Isolation of T cell receptors (TCRs) and overexpression in allogeneic cells allows for extensive T cell reactivity profiling. We identify SARS-CoV-2 RNA-dependent RNA polymerase (RdRp/NSP12) as highly conserved, likely due to its critical role in the virus life cycle. We perform single-cell TCRαß sequencing in human leukocyte antigen (HLA)-A∗02:01-restricted, RdRp-specific T cells from SARS-CoV-2-unexposed individuals. Human T cells expressing these TCRαß constructs kill target cell lines engineered to express full-length RdRp. Three TCR constructs recognize homologous epitopes from common cold coronaviruses, indicating CD8+ T cells can recognize evolutionarily diverse coronaviruses. Analysis of individual TCR clones may help define vaccine epitopes that can induce long-term immunity against SARS-CoV-2 and other coronaviruses.

Genetic ancestry effects on the response to viral infection are pervasive but cell type specific.

Humans differ in their susceptibility to infectious disease, partly owing to variation in the immune response after infection. We used single-cell RNA sequencing to quantify variation in the response to influenza infection in peripheral blood mononuclear cells from European- and African-ancestry males. Genetic ancestry effects are common but highly cell type specific. Higher levels of European ancestry are associated with increased type I interferon pathway activity in early infection, which predicts reduced viral titers at later time points. Substantial population-associated variation is explained by cis-expression quantitative trait loci that are differentiated by genetic ancestry. Furthermore, genetic ancestry­associated genes are enriched among genes correlated with COVID-19 disease severity, suggesting that the early immune response contributes to ancestry-associated differences for multiple viral infection outcomes.

Atlas of the HIV-1 Reservoir in Peripheral CD4 T Cells of Individuals on Successful Antiretroviral Therapy.

Knowing the mechanisms that govern the persistence of infected CD4+ subpopulations could help us to design new therapies to cure HIV-1 infection. We evaluated the simultaneous distribution of the HIV-1 reservoir in 13 CD4+ subpopulations from 14 HIV-1-infected individuals on antiretroviral therapy to analyze its relationship with HIV-1 transcription, immune activation, and cell proliferation. A unique large blood donation was used to isolate CD4, CD4 resting (CD4r), CD4 activated (CD4a), T naive (TN), T stem cell memory (TSCM), T central memory (TCM), T transitional memory (TTM), T effector memory (TEM), circulating T follicular helper (cTFH), TCD20, TCD32, and resting memory TCD2high (rmTCD2high) cells. HIV-1 DNA measured by droplet digital PCR ranged from 3,636 copies/106 in TTM to 244 in peripheral blood mononuclear cells (PBMCs), with no subpopulation standing out for provirus enrichment. Importantly, all the subpopulations harbored intact provirus by intact provirus DNA assay (IPDA). TCD32, cTFH, and TTM had the highest levels of HIV-1 transcription measured by fluorescent in situ hybridization with flow cytometry (FISH/flow), but without reaching statistical differences. The subpopulations more enriched in provirus had a memory phenotype, were less activated (measured by CD38+/HLA-DR+), and expressed more programmed cell death 1 (PD-1). Conversely, subpopulations transcribing more HIV-1 RNA were not necessarily enriched in provirus and were more activated (measured by CD38+/HLA-DR+) and more proliferative (measured by Ki-67). In conclusion, the HIV reservoir is composed of a mosaic of subpopulations contributing to the HIV-1 persistence through different mechanisms such as susceptibility to infection, provirus intactness, or transcriptional status. The narrow range of reservoir differences between the different blood cell subsets tested suggests limited efficacy in targeting only specific cell subpopulations during HIV-1 cure strategies. IMPORTANCE The main barrier for HIV-1 cure is the presence of latently infected CD4+ T cells. Although various cell subpopulations have been identified as major HIV-1 reservoir cells, the relative contribution of infected CD4 subpopulations in the HIV-1 reservoir remains largely unknown. Here, we evaluated the simultaneous distribution of the HIV-1 reservoir in 13 CD4+ T-cell subpopulations in peripheral blood from HIV-1-infected individuals under suppressive antiretroviral therapy. We found that the HIV-1 reservoir is composed of a mosaic of cell subpopulations, with heterogeneous proviral DNA, HIV-1 transcription, and activation status. Hence, each cell subpopulation contributes to the HIV-1 persistence through different mechanisms such as susceptibility to infection, rates of intact provirus, transcriptional status or half-life. This research provides new insights into the composition of the HIV-1 reservoir, suggesting that, to be effective, eradication strategies must simultaneously target multiple cell subpopulations.

Garcinia kola and garcinoic acid suppress SARS-CoV-2 spike glycoprotein S1-induced hyper-inflammation in human PBMCs through inhibition of NF-κB activation.

Symptoms and complications associated with severe SARS-CoV-2 infection such as acute respiratory distress syndrome (ARDS) and organ damage have been linked to SARS-CoV-2 spike protein S1-induced increased production of pro-inflammatory cytokines by immune cells. In this study, the effects of an extract of Garcinia kola seeds and garcinoic acid were investigated in SARS-CoV-2 spike protein S1-stimulated human PBMCs. Results of ELISA experiments revealed that Garcinia kola extract (6.25, 12.5, and 25 µg/ml) and garcinoic acid (1.25, 2.5, and 5 µM) significantly reduced SARS-CoV-2 spike protein S1-induced secretion of TNFα, IL-6, IL-1ß, and IL-8 in PBMCs. In-cell western assays showed that pre-treatment with Garcinia kola extract and garcinoic acid reduced expressions of both phospho-p65 and phospho-IκBα proteins, as well as NF-κB DNA binding capacity and NF-κB-driven luciferase expression following stimulation of PBMCs with spike protein S1. Furthermore, pre-treatment of PBMCs with Garcinia kola extract prior to stimulation with SARS-CoV-2 spike protein S1 resulted in reduced damage to adjacent A549 lung epithelial cells. These results suggest that the seed of Garcinia kola and garcinoic acid are natural products which may possess pharmacological/therapeutic benefits in reducing cytokine storm in severe SARS-CoV-2 and other coronavirus infections.

Upregulation of Human Endogenous Retroviruses in Bronchoalveolar Lavage Fluid of COVID-19 Patients.

Severe COVID-19 pneumonia has been associated with the development of intense inflammatory responses during the course of infections with SARS-CoV-2. Given that human endogenous retroviruses (HERVs) are known to be activated during and participate in inflammatory processes, we examined whether HERV dysregulation signatures are present in COVID-19 patients. By comparing transcriptomes of bronchoalveolar lavage fluid (BALF) of COVID-19 patients and healthy controls, and peripheral blood monocytes (PBMCs) from patients and controls, we have shown that HERVs are intensely dysregulated in BALF of COVID-19 patients compared to those in BALF of healthy control patients but not in PBMCs. In particular, upregulation in the expression of specific HERV families was detected in BALF samples of COVID-19 patients, with HERV-FRD being the most highly upregulated family among the families analyzed. In addition, we compared the expression of HERVs in human bronchial epithelial cells (HBECs) without and after senescence induction in an oncogene-induced senescence model in order to quantitatively measure changes in the expression of HERVs in bronchial cells during the process of cellular senescence. This apparent difference of HERV dysregulation between PBMCs and BALF warrants further studies in the involvement of HERVs in inflammatory pathogenetic mechanisms as well as exploration of HERVs as potential biomarkers for disease progression. Furthermore, the increase in the expression of HERVs in senescent HBECs in comparison to that in noninduced HBECs provides a potential link for increased COVID-19 severity and mortality in aged populations. IMPORTANCE SARS-CoV-2 emerged in late 2019 in China, causing a global pandemic. Severe COVID-19 is characterized by intensive inflammatory responses, and older age is an important risk factor for unfavorable outcomes. HERVs are remnants of ancient infections whose expression is upregulated in multiple conditions, including cancer and inflammation, and their expression is increased with increasing age. The significance of this work is that we were able to recognize dysregulated expression of endogenous retroviral elements in BALF samples but not in PBMCs of COVID-19 patients. At the same time, we were able to identify upregulated expression of multiple HERV families in senescence-induced HBECs in comparison to that in noninduced HBECs, a fact that could possibly explain the differences in disease severity among age groups. These results indicate that HERV expression might play a pathophysiological role in local inflammatory pathways in lungs afflicted by SARS-CoV-2 and their expression could be a potential therapeutic target.

Genetic interference exerted by Salmonella-delivered CRISPR/Cas9 significantly reduces the pathological burden caused by Marek's disease virus in chickens.

Efficient in vivo delivery of a CRISPR/Cas9 plasmid is of paramount importance for effective therapy. Here, we investigated the usability of Salmonella as a plasmid carrier for in vivo therapy against virus-induced cancer using Marek's disease virus (MDV) as a model for study in chickens. A green fluorescent protein-expressing CRISPR/Cas9 plasmid encoding the virulence gene pp38 was constructed against Marek's disease virus. Therapeutic plasmids were transformed into Salmonella carrying lon and sifA gene deletions. The animals in 5 groups were intraperitoneally inoculated with phosphate-buffered saline, vector control, or Salmonella before or after MDV infection, or left uninfected as a naïve control. Therapeutic effectiveness was evaluated by observing disease outcomes and the viral copy number in peripheral blood mononuclear cells. The efficacy of plasmid delivery by Salmonella was 13 ± 1.7% in the spleen and 8.0 ± 1.8% in the liver on the 6th day post-infection. The Salmonella-treated groups showed significant resistance to MDV infection. The maximum effect was observed in the group treated with Salmonella before MDV infection. None of the chickens fully recovered; however, the results suggested that timely delivery of Salmonella could be effective for in vivo CRISPR/Cas9-mediated genetic interference against highly pathogenic MDV. The use of Salmonella in CRISPR systems provides a simpler and more efficient platform for in vivo therapy with CRISPR than the use of conventional in vivo gene delivery methods and warrants further development.

PD-1 Blockade Restores the Proliferation of Peripheral Blood Lymphocyte and Inhibits Lymphocyte Apoptosis in a BALB/c Mouse Model of CP BVDV Acute Infection.

Acute infection of bovine viral diarrhea virus (BVDV) is associated with immune dysfunction and can cause peripheral blood lymphopenia and lymphocyte apoptosis. Our previous study has confirmed that programmed death-1 (PD-1) blockade inhibits peripheral blood lymphocytes (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the situation in vivo remains to be further studied and confirmed. Therefore, in this study, we established a BALB/c mouse model of acute BVDV infection with cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain NY-1). Then, we examined the mRNA and protein levels of PD-1 and programmed death-ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC) from BVDV-infected mice and analyzed the effects of PD-1 blockade on the proportions of CD3+, CD4+, and CD8+ T cell subsets, the apoptosis and proliferation of PBL, and the production of IL-2 and IFN-γ. We found that leukopenia, lymphocytopenia, and thrombocytopenia were developed in both CP and NCP BVDV-infected mice at day 7 of post-infection. The mRNA and protein expression of PD-1 and PD-L1 were significantly upregulated in CP and NCP BVDV-infected mice. Moreover, PD-1/PD-L1 upregulation was accompanied by leukopenia and lymphopenia. Additionally, PD-1 blockade inhibited PBL apoptosis and virus replication, restored the proportions of CD3+, CD4+, and CD8+ T cell subsets, and increased IFN-γ production and p-ERK expression in BVDV-infected mice. However, blocking PD-1 did not significantly affect PBL proliferation and IL-2 production in NCP BVDV-infected mice. Our findings further confirmed the immunomodulatory role of PD-1 in peripheral blood lymphocytopenia in vivo and provided a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.

Combined single-cell transcriptional, translational, and genomic profiling reveals HIV-1 reservoir diversity.

Although understanding the diversity of HIV-1 reservoirs is key to achieving a cure, their study at the single-cell level in primary samples remains challenging. We combine flow cytometric multiplexed fluorescent in situ RNA hybridization for different viral genes with HIV-1 p24 protein detection, cell phenotyping, and downstream near-full-length single-cell vDNA sequencing. Stimulation-induced viral RNA-positive (vRNA+) cells from viremic and antiretroviral-therapy (ART)-suppressed individuals differ in their ability to produce p24. In participants on ART, latency-reversing agents (LRAs) induce a wide variety of viral gene transcription and translation patterns with LRA class-specific differences in reactivation potency. Reactivated proviruses, including in p24+ cells, are mostly defective. Although LRAs efficiently induce transcription in all memory cell subsets, we observe induction of translation mostly in effector memory cells, rather than in the long-lived central memory pool. We identify HIV-1 clones with diverse transcriptional and translational patterns between individual cells, and this finding suggests that cell-intrinsic factors influence reservoir persistence and heterogeneity.

Development of a 3-transcript host expression assay to differentiate between viral and bacterial infections in pigs.

Indiscriminate use of antibiotics to treat infections that are of viral origin contributes to unnecessary use which potentially may induce resistance in commensal bacteria. To counteract this a number of host gene transcriptional studies have been conducted to identify genes that are differently expressed during bacterial and viral infections in humans, and thus could be used as a tool to base decisions on the use of antibiotics. In this paper, we aimed to evaluate the potential of a selection of genes that have been considered biomarkers in humans, to differentially diagnose bacterial from viral infections in the pig. First porcine PBMC were induced with six toll-like receptor (TLR) agonists (FliC, LPS, ODN 2216, Pam3CSK4, poly I:C, R848) to mimic host gene expression induced by bacterial or viral pathogens, or exposed to heat-killed Actinobacillus pleuropneumoniae or a split influenza virus. Genes that were differentially expressed between bacterial and viral inducers were further evaluated on clinical material comprising eleven healthy pigs, and six pigs infected with A. pleuropneumoniae. This comprised three virally upregulated genes (IFI44L, MxA, RSAD2) and four bacterially upregulated genes (IL-1ß, IL-8, FAM89A, S100PBP). All six infected pigs could be differentially diagnosed to healthy pigs using a host gene transcription assay based on the geometric average of the bacterially induced genes IL-8 and S100PBP over that of the virally induced gene MxA.