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Epstein-Barr virus (EBV), an oncogenic herpesvirus, is predominantly found in the latent infection form and is highly associated with many human malignancies, which mainly have poor prognoses and no effective treatments. Here, we obtained thirteen compounds from small-molecule libraries for specific inhibition of EBV-latently infected cell growth in vitro by high-throughput screening. Among them, cetrimonium bromide (CetB) was identified to selectively inhibit the growth of different EBV-infected B lymphoma cell lines. Importantly, CetB reduced EBNA1 protein stability, activated G1 arrest and early apoptosis of EBV-latently infected cells without viral lytic reactivation, which leads to dramatically inhibit colony formation and tumor growth of EBV-infected cells in vitro and in vivo, and significantly prolong the survival of tumor-bearing mice. Overall, these findings demonstrate that CetB acts as a highly selective inhibitor of the growth of EBV-infected cells and has the potential for further development of effective therapeutic strategies specific against EBV-associated cancers.

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In this study, we obtained the whole genome sequence of GCRV-DY197 and investigated the localization, post-translational modifications, and host interactions of the 11 viral proteins encoded by GCRV-DY197 in grass carp ovary (GCO) cells. The whole genome sequence is 24,704 kb and contains 11 segments (S1-S11). Subcellular localization showed that the VP1, VP2, VP3, VP5, VP56, and VP35 proteins were localized in both cytoplasm and nucleus, whereas the NS79, VP4, VP41, VP6, and NS38 proteins were localized in the cytoplasm. The NS79 and NS38 proteins were phosphorylated, and the ubiquitination modification sites were identified in VP41 and NS38. An interaction network containing 9 viral proteins and 140 host proteins was also constructed. These results offer a theoretical basis for an in-depth understanding of the biochemical characteristics and pathogenic mechanism of GCRV-II.

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Lassa fever continues to be a major public health burden in West Africa, yet effective therapies or vaccines are lacking. The isolation of protective neutralizing antibodies against the Lassa virus glycoprotein complex (GPC) justifies the development of vaccines that can elicit strong neutralizing antibody responses. However, Lassa vaccine candidates have generally been unsuccessful at doing so, and the associated antibody responses to these vaccines remain poorly characterized. Here, we establish an electron microscopy-based epitope mapping workflow that enables high-resolution structural characterization of polyclonal antibodies to the GPC. By applying this method to rabbits vaccinated with a recombinant GPC vaccine and a GPC-derived virus-like particle, we reveal determinants of neutralization that involve epitopes of the GPC-A competition cluster. Furthermore, by identifying undescribed immunogenic off-target epitopes, we expose the challenges that recombinant GPC vaccines face. By enabling detailed polyclonal antibody characterization, our work ushers in a next generation of more rational Lassa vaccine design.

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Infections caused by multidrug resistant (MDR) pathogenic bacteria are a global health threat. Bacteriophages ("phage") are increasingly used as alternative or last-resort therapeutics to treat patients infected by MDR bacteria. However, the therapeutic outcomes of phage therapy may be limited by the emergence of phage resistance during treatment and/or by physical constraints that impede phage-bacteria interactions in vivo. In this work, we evaluate the role of lung spatial structure on the efficacy of phage therapy for Pseudomonas aeruginosa infections. To do so, we developed a spatially structured metapopulation network model based on the geometry of the bronchial tree, including host innate immune responses and the emergence of phage-resistant bacterial mutants. We model the ecological interactions between bacteria, phage, and the host innate immune system at the airway (node) level. The model predicts the synergistic elimination of a P. aeruginosa infection due to the combined effects of phage and neutrophils, given the sufficient innate immune activity and efficient phage-induced lysis. The metapopulation model simulations also predict that MDR bacteria are cleared faster at distal nodes of the bronchial tree. Notably, image analysis of lung tissue time series from wild-type and lymphocyte-depleted mice revealed a concordant, statistically significant pattern: infection intensity cleared in the bottom before the top of the lungs. Overall, the combined use of simulations and image analysis of in vivo experiments further supports the use of phage therapy for treating acute lung infections caused by P. aeruginosa, while highlighting potential limits to therapy in a spatially structured environment given impaired innate immune responses and/or inefficient phage-induced lysis. IMPORTANCE: Phage therapy is increasingly employed as a compassionate treatment for severe infections caused by multidrug-resistant (MDR) bacteria. However, the mixed outcomes observed in larger clinical studies highlight a gap in understanding when phage therapy succeeds or fails. Previous research from our team, using in vivo experiments and single-compartment mathematical models, demonstrated the synergistic clearance of acute P. aeruginosa pneumonia by phage and neutrophils despite the emergence of phage-resistant bacteria. In fact, the lung environment is highly structured, prompting the question of whether immunophage synergy explains the curative treatment of P. aeruginosa when incorporating realistic physical connectivity. To address this, we developed a metapopulation network model mimicking the lung branching structure to assess phage therapy efficacy for MDR P. aeruginosa pneumonia. The model predicts the synergistic elimination of P. aeruginosa by phage and neutrophils but emphasizes potential challenges in spatially structured environments, suggesting that higher innate immune levels may be required for successful bacterial clearance. Model simulations reveal a spatial pattern in pathogen clearance where P. aeruginosa are cleared faster at distal nodes of the bronchial tree than in primary nodes. Interestingly, image analysis of infected mice reveals a concordant and statistically significant pattern: infection intensity clears in the bottom before the top of the lungs. The combined use of modeling and image analysis supports the application of phage therapy for acute P. aeruginosa pneumonia while emphasizing potential challenges to curative success in spatially structured in vivo environments, including impaired innate immune responses and reduced phage efficacy.

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Drug abuse continues to pose a significant challenge in HIV control efforts. In our investigation, we discovered that cocaine not only upregulates the expression of DNA-dependent protein kinase (DNA-PK) but also augments DNA-PK activation by enhancing its phosphorylation at S2056. Moreover, DNA-PK phosphorylation triggers the translocation of DNA-PK into the nucleus. The finding that cocaine promotes nuclear translocation of DNA-PK further validates our observation of enhanced DNA-PK recruitment at the HIV long terminal repeat (LTR) following cocaine exposure. By activating and facilitating the nuclear translocation of DNA-PK, cocaine effectively orchestrates multiple stages of HIV transcription, thereby promoting HIV replication. Additionally, our study indicates that cocaine-induced DNA-PK promotes hyper-phosphorylation of RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) at Ser5 and Ser2 sites, enhancing both initiation and elongation phases, respectively, of HIV transcription. Cocaine's enhancement of transcription initiation and elongation is further supported by its activation of cyclin-dependent kinase 7 (CDK7) and subsequent phosphorylation of CDK9, thereby promoting positive transcriptional elongation factor b (P-TEFb) activity. We demonstrate for the first time that cocaine, through DNA-PK activation, promotes the specific phosphorylation of TRIM28 at Serine 824 (p-TRIM28, S824). This modification converts TRIM28 from a transcriptional inhibitor to a transactivator for HIV transcription. Additionally, we observe that phosphorylation of TRIM28 (p-TRIM28, S824) promotes the transition from the pausing phase to the elongation phase of HIV transcription, thereby facilitating the production of full-length HIV genomic transcripts. This finding corroborates the observed enhanced RNAP II CTD phosphorylation at Ser2, a marker of transcriptional elongation, following cocaine exposure. Accordingly, upon cocaine treatment, we observed elevated recruitment of p-TRIM28-(S824) at the HIV LTR. Overall, our results have unraveled the intricate molecular mechanisms underlying cocaine-induced HIV transcription and gene expression. These findings hold promise for the development of highly targeted therapeutics aimed at mitigating the detrimental effects of cocaine in individuals living with HIV.

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The mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-week supplementation of antiretroviral therapy (ART) with metformin, an indirect mTOR inhibitor used in type-2 diabetes treatment, reduced mTOR activation and HIV transcription in colon-infiltrating CD4+ T cells, together with systemic inflammation in nondiabetic people with HIV-1 (PWH). Herein, we investigated the antiviral mechanisms of metformin. In a viral outgrowth assay performed with CD4+ T cells from ART-treated PWH, and upon infection in vitro with replication-competent and VSV-G-pseudotyped HIV-1, metformin decreased virion release, but increased the frequency of productively infected CD4lowHIV-p24+ T cells. These observations coincided with increased BST2/tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression, and improved recognition of productively infected T cells by HIV-1 envelope antibodies. Thus, metformin exerts pleiotropic effects on post-integration steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH.

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In humans, lymph nodes are the primary site of measles virus (MeV) replication. To understand the immunological events that occur at this site, we infected human lymphoid tissue explants using a pathogenic strain of MeV that expresses GFP. We found that MeV infected 5%-15% of cells across donors. Using single-cell RNA-Seq and flow cytometry, we found that while most of the 29 cell populations identified in the lymphoid culture were susceptible to MeV, there was a broad preferential infection of B cells and reduced infection of T cells. Further subsetting of T cells revealed that this reduction may be driven by the decreased infection of naive T cells. Transcriptional changes in infected B cells were dominated by an interferon-stimulated gene (ISG) signature. To determine which of these ISGs were most substantial, we evaluated the proteome of MeV-infected Raji cells by mass spectrometry. We found that IFIT1, IFIT2, IFIT3, ISG15, CXCL10, MX2, and XAF1 proteins were the most highly induced and positively correlated with their expression in the transcriptome. These data provide insight into the immunological events that occur in lymph nodes during infection and may lead to the development of therapeutic interventions.

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First isolated from neotropical fruit bats in Trinidad in 1956, Tacaribe virus (TCRV) has rarely been detected since. We searched for New World arenavirus reads in roughly 5.7 million sequencing runs available on public databases using Serratus. We recovered a complete genome of a divergent TCRV in metatranscriptomic data derived from heart and eye tissue of an adult male Jamaican fruit-eating bat sampled in the Dominican Republic, 2014. In total, 2,733 reads were mapped resulting in mean coverages of 7.4-fold for the L and 10.2-fold for the S segment. Re-testing original bat specimens showed the highest viral loads in liver tissue (245 copies/mg). Sanger sequencing of PCR amplicons from liver confirmed correctness of and completed the genome recovered from metatranscriptomic data, revealing conserved arenavirus genomic organization, length, intergenic regions, and genome termini. The newly found TCRV strain tentatively named DOM2014 clustered in a basal sister relationship to all other known TCRV strains with which it shared between 83.3%-86.0% genomic and 91.8%-93.7% translated amino acid sequence identity across protein-coding regions. DOM2014 showed a conserved glycine, proline, proline, threonine (GPPT) nucleoprotein motif, which is essential for TCRV interferon ß antagonism. Our data confirm the association of TCRV with the bat genus Artibeus put into question by lethal experimental infections and scarce bat-derived TCRV genomic data. Broad genetic diversity and geographic spread require assessments of TCRV strain-associated pathogenicity, particularly for DOM2014 as a highly divergent TCRV strain. Confirmation of genomic database findings by testing original specimens provides robustness to our findings and supports the usefulness of metatranscriptomic studies. IMPORTANCE: Clade B New World arenaviruses (NWA) include rodent-borne lethal hemorrhagic fever viruses, whereas Tacaribe virus (TCRV) stands out because of its detection in bats and its presumably low zoonotic potential. However, the bat association of TCRV was put into question by lethal experimental neotropical fruit bat infections and rare TCRV detection in bats. Scarce genomic data include near-identical viruses from Caribbean bats and ticks from the US sampled 50 years later. The prototype TCRV isolate used for experimental risk assessments has an extensive passage history in suckling mouse brains. Exploring the true genetic diversity, geographic distribution, and host range of bat-borne NWA is pivotal to assess their zoonotic potential and transmission cycles. We analyzed metatranscriptomic data for evidence of NWA identifying a highly divergent TCRV in bats and confirmed virus detection in original biological materials, supporting the association of TCRV with neotropical bats and warranting investigation of strain-associated TCRV pathogenicity.

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Introduction: This study introduces the Supervised Magnitude-Altitude Scoring (SMAS) methodology, a novel machine learning-based approach for analyzing gene expression data from non-human primates (NHPs) infected with Ebola virus (EBOV). By focusing on host-pathogen interactions, this research aims to enhance the understanding and identification of critical biomarkers for Ebola infection. Methods: We utilized a comprehensive dataset of NanoString gene expression profiles from Ebola-infected NHPs. The SMAS system combines gene selection based on both statistical significance and expression changes. Employing linear classifiers such as logistic regression, the method facilitates precise differentiation between RT-qPCR positive and negative NHP samples. Results: The application of SMAS led to the identification of IFI6 and IFI27 as key biomarkers, which demonstrated perfect predictive performance with 100% accuracy and optimal Area Under the Curve (AUC) metrics in classifying various stages of Ebola infection. Additionally, genes including MX1, OAS1, and ISG15 were significantly upregulated, underscoring their vital roles in the immune response to EBOV. Discussion: Gene Ontology (GO) analysis further elucidated the involvement of these genes in critical biological processes and immune response pathways, reinforcing their significance in Ebola pathogenesis. Our findings highlight the efficacy of the SMAS methodology in revealing complex genetic interactions and response mechanisms, which are essential for advancing the development of diagnostic tools and therapeutic strategies. Conclusion: This study provides valuable insights into EBOV pathogenesis, demonstrating the potential of SMAS to enhance the precision of diagnostics and interventions for Ebola and other viral infections.

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OBJECTIVE: To document the course of neonatal and short-term outcomes in pregnancies after first trimester CMV (cytomegalovirus) seroconversion and negative amniotic fluid (AF) CMV PCR. METHODS: We included 375 patients with a first-trimester CMV seroconversion and amniocentesis at ≥21 weeks. Termination of pregnancy (TOP) was offered in case antenatally severe CMV-related fetopathy was documented either by ultrasound or by MRI. AF CMV PCR-negative fetuses underwent a PCR CMV on neonatal urine (NU). Perinatal and short-term infant outcomes were investigated by a questionnaire, sent to parents. RESULTS: AF CMV PCR was positive in 118/375 cases (31.4%). TOP was performed in 46/118 (38.9%) and fetal demise occurred twice. Questionnaires were sent to 327 patients with an overall response rate of 77%. Three groups were considered: Group 1: the early infected group (AF CMV PCR positive; N=62), group 2: the late infected group (AF CMV PCR negative, NU CMV PCR positive; N=7) and group 3: the control group (AF+NU CMV PCR negative; N=160). Compared with group 3, group 1 was more frequently symptomatic at birth (6.2% vs 19.4%; p=0.006). In short-term follow-up, hearing impairment (23.5%; p<0.001), mild motor deficit - defined as abnormal early motor development or the need for physiotherapy in later life (21.6%; p=0.005) - and subnormal vision (15.7%; p=0.02) were significantly more frequent. Compared with group 3, group 2 showed more often jaundice (57.1%; p=0.04) and petechiae (28.6%; p=0.04) at birth, but other short-term symptoms were lacking. CONCLUSION: Although neonates may screen positive on urine for CMV after an AF CMV negative PCR, they show rarely and only mild sequelae in early life.

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The reduced susceptibility of mRNA vaccines and diminished neutralizing activity of therapeutic monoclonal antibodies against Omicron variants, including BQ.1.1, XBB, and their descendants, highlight the importance of antiviral therapies. Here, we assessed the efficacy of two antivirals, molnupiravir, targeting a viral RNA-dependent RNA polymerase, and nirmatrelvir, targeting a main protease, against BQ.1.1 in hamsters. We found that prophylactic or therapeutic treatment with either drug significantly reduced the viral load in the lungs of infected hamsters. We also evaluated the risk of emergence of drug-resistant viruses in immunocompromised hamsters. Although 13 days of drug treatment reduced viral titers, the immunocompromised hosts could not completely clear the virus. Viruses isolated from drug-treated immunocompromised hamsters did not show reduced susceptibility to the drugs. Molnupiravir and nirmatrelvir remain effective in vivo against variants with reduced susceptibility to monoclonal antibodies and mRNA vaccine-induced antibodies, with limited emergence of drug-resistant variants under the conditions tested.

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Tumor necrosis factor receptor-associated factor 6 (TRAF6) is crucial in flavivirus infections, modulating the host immune response through interactions with viral proteins. Despite its importance, the relationship between TRAF6 and Zika virus (ZIKV) remains poorly understood. Our prior proteomics analysis revealed reduced TRAF6 protein levels in ZIKV-infected human trophoblast cells compared to non-infected controls. Subsequent studies in cell models and murine tissues confirmed a significant reduction in both TRAF6 mRNA and protein levels post-ZIKV infection. Further investigations unveiled that ZIKV induces P62-mediated degradation of TRAF6, with NS1 identified as the primary contributor. Co-localization and interaction studies demonstrated that NS1 promotes the association of P62, a key autophagy mediator, with TRAF6. Notably, our findings revealed TRAF6 enhances ZIKV infection, NS1 ubiquitination, NS1 expression, and the production of inflammatory cytokines and chemokines. These insights highlight the intricate TRAF6-ZIKV relationship, offering potential for drug targeting NS1-TRAF6 interactions to manage ZIKV infections effectively.

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The commercialized genetically modified (GM) papaya cultivars have protected papaya from the devastating disease caused by papaya ringspot virus (PRSV). However, papaya leaf distortion mosaic virus (PLDMV), which causes similar infection symptoms but is serologically distinct from PRSV, was found as a competitive threat to the papaya industry. Our study surveyed the occurrence of PRSV and PLDMV as well as the transgenic markers of the 35S promoter from cauliflower mosaic virus (CaMV 35S) and the neomycin phosphotransferase II (NPT II) gene in feral papaya plants, which were found frequently growing outside of cultivated papaya fields on Hainan Island. In total, 123 feral papayas, comprising 62 (50.4%) GM plants and 61 (49.6%) non-GM ones, were sampled. Among them, 23 (18.7%) were positive for PRSV, 49 (39.8%) were positive for PLDMV, including 5 plants co-infected by PRSV and PLDMV, and 56 (45.5%) plants were free of either virus. In traditional papaya growing regions, we detected fewer PRSV-infected plants (2 in 33, 6%) than in other regions (21 in 90, 23%). But overall, whether transgenic or not made no significance in PRSV incidence (P=0.230), with 9 PRSV-infected plants among 62 GM papayas and 14 among 61 non-GM papayas. Phylogenetic and genetic differentiation analysis showed a clear correlation between PRSV and PLDMV populations and their geographical origins. Negative selection was estimated for the selected gene regions of both viruses. Notably, PLDMV has deviated from neutral evolution and experienced population expansion, exhibiting increased genetic diversity and is becoming the predominant threat to papaya in Hainan.

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BACKGROUND: Natural infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or vaccination triggers antibody production against key viral antigens. However, there is limited evidence on the levels of antibodies produced in naturally infected individuals compared to those vaccinated in Ethiopia. Therefore, we aimed to detect and compare SARS-CoV-2 antibodies produced by naturally infected and vaccinated individuals. MATERIALS AND METHODS: We conducted a multicenter cross-sectional study among a total of 355 naturally infected and 355 vaccinated individuals from November 2022 to April 2023 at 10 selected health facilities in Addis Ababa, Ethiopia. We enrolled the participants consecutively upon their arrival at health facilities until the required sample size was achieved. We used a structured questionnaire to collect data on the demographic and clinical characteristics of the participants. We also collected 3-5 ml of blood samples from all participants and tested for anti-Spike (anti-S) and anti-nucleocapsid (anti-N) antibodies using Cobas 6000. We utilized frequency, mean, or median to describe the data, the Mann-Whitney U test to compare groups, and a generalized linear regression model to assess factors associated with anti-S antibody concentration. We analyzed the data with SPSS version 26, and the level of significance was set at P-value < 0.05. RESULTS: Of the naturally infected participants, 352 (99.5%) had anti-S antibodies and all (100%) had anti-N antibodies, whereas among vaccinated participants, all (100%) had anti-S antibodies, while 323 (91.6%) had anti-N antibodies. Anti-S antibodies produced by vaccinated individuals were significantly (P < 0.001) higher than those produced as a result of natural infection. Being young (P = 0.004), having hypertension (P < 0.001), and having diabetes (P < 0.001) were significantly associated with lower anti-S antibody levels, while being recently vaccinated and having a higher number of vaccine doses were significantly associated with higher anti-S antibody concentrations in vaccinated participants. Having diabetes (P < 0.001) were significantly associated with lower anti-S concentrations in participants who were naturally infected. CONCLUSION: There is a high seropositivity rate in both naturally infected and vaccinated individuals. However, vaccinated individuals had higher levels of SARS-CoV-2 antibodies than those who were naturally infected, which highlights the significant contribution of vaccination in increasing the protection of COVID-19 in Ethiopia.

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OBJECTIVE: To understand parental perspectives regarding universal newborn screening (UNS) for congenital cytomegalovirus (cCMV) in Canada. DESIGN: A qualitative, patient-led study using the Patient and Community Engagement Research approach consisting of online focus groups and in-depth individual interviews to understand parental preferences regarding UNS for cCMV. Data were analysed iteratively using inductive thematic analysis and narrative story analysis. SETTING: Canada-wide study conducted via video conference from October to December 2023. PATIENTS: 12 participants from five Canadian provinces who self-identified as 18 years of age or older and as having parental lived experience with cytomegalovirus (CMV) or cCMV participated in the study. RESULTS: We identified three themes: (1) attitudes about UNS for cCMV, including participants' unanimous support for UNS and confirmation that parental anxiety is not a deterrent for screening, (2) cCMV diagnosis, including the importance of coupling cCMV diagnosis with access to treatment and medical support and (3) awareness of cCMV, where participants shared their frustration about the lack of public and pregnant people's awareness of cCMV. CONCLUSIONS: Parental anxiety is not a deterrent for UNS for cCMV. Children with cCMV and their families deserve every opportunity to attain their best possible outcomes. UNS offers children with cCMV access to early intervention if they need it, and also helps to raise awareness and education to prevent future CMV infections.

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