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How severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remains largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected Huh7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a dose-dependent activation of Akt, mTOR, S6K1 and 4E-BP1 at 24 hours post infection (hpi). However, we found a significant inhibition of HIF-1α through 24hpi and 48hpi of the infection, suggesting a crosstalk between the SARS-CoV-2 and the Akt/mTOR/HIF-1 signaling pathways. Inhibition of the mTOR signaling pathway using Akt inhibitor MK-2206 showed a significant reduction in virus production. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe coronavirus disease 2019 (COVID-19) patients.

The ART program in low- and middle-income countries (LMIC) like India, follows a public health approach with a standardized regimen for all people living with HIV (PLHIV). Based on the evidence from high-income countries (HIC), the risk of an enhanced, and accentuated onset of premature-aging or age-related diseases has been observed in PLHIV. However, very limited data is available on residual inflammation and immune activation in the populations who are on first-generation anti-HIV drugs like zidovudine and lamivudine that have more toxic side effects. Therefore, the aim of the present study was to evaluate the levels of systemic inflammation and understand the risk of age-associated diseases in PLHIV on long-term suppressive ART using a large number of biomarkers of inflammation and immune activation. Blood samples were obtained from therapy naïve PLHIV (Pre-ART, = 43), PLHIV on ART for >5 years (ART, = 53), and HIV-negative healthy controls (HIVNC, = 41). Samples were analyzed for 92 markers of inflammation, sCD14, sCD163, and telomere length. Several statistical tests were performed to compare the groups under study. Multivariate linear regression was used to investigate the associations. Despite a median duration of 8 years of successful ART, sCD14 ( < 0.001) and sCD163 ( = 0.04) levels continued to be significantly elevated in ART group as compared to HIVNC. Eleven inflammatory markers, including 4E-BP1, ADA, CCL23, CD5, CD8A, CST5, MMP1, NT3, SLAMF1, TRAIL, and TRANCE, were found to be significantly different ( < 0.05) between the groups. Many of these markers are associated with age-related co-morbidities including cardiovascular disease, neurocognitive decline and some of these markers are being reported for the first time in the context of HIV-induced inflammation. Linear regression analysis showed a significant negative association between HIV-1-positivity and telomere length ( < 0.0001). In ART-group CXCL1 ( = 0.048) and TGF-α ( = 0.026) showed a significant association with the increased telomere length and IL-10RA was significantly associated with decreased telomere length ( = 0.042). This observation warrants further mechanistic studies to generate evidence to highlight the need for enhanced treatment monitoring and special interventions in HIV-infected individuals.

Parechovirus ahumpari 3 (HPeV-3) is among the main agents causing severe neonatal neurological infections such as encephalitis and meningitis. However, the underlying molecular mechanisms and changes to the host cellular landscape leading to neurological disease has been understudied. Through quantitative proteomic analysis of HPeV-3 infected neural organoids, we identified unique metabolic changes following HPeV-3 infection that indicate immunometabolic dysregulation. Protein and pathway analyses showed significant alterations in neurotransmission and potentially, neuronal excitotoxicity. Elevated levels of extracellular glutamate, lactate dehydrogenase (LDH), and neurofilament light (NfL) confirmed glutamate excitotoxicity to be a key mechanism contributing to neuronal toxicity in HPeV-3 infection and can lead to apoptosis induced by caspase signaling. These insights are pivotal in delineating the metabolic landscape following severe HPeV-3 CNS infection and may identify potential host targets for therapeutic interventions.

Severe COVID-19 is characterized by immune-coagulation dysregulation, yet the contribution of related autoantibodies remains poorly understood. We investigated relationships between plasma autoantibody reactivities, whole-blood transcriptomics, plasma proteomics, and clinical laboratory parameters in a cohort of hospitalized COVID-19 patients. Transcriptomic analysis revealed that 42 curated coagulation and complement cascade genes were upregulated in severe cases compared to healthy controls, with 15 genes, including CR1L, ELANE, ITGA2B, ITGB3, VWF, TFPI, PROS1, MMRN1, and SELP (> 1.2 log2 fold-change), also significantly different from mild cases. Autoantibody profiling against eight coagulation-related proteins (ADAMTS13, Factor V, Protein S, SERPINC1, Apo-H, PROC1, Prothrombin, and PF4) showed reactivities below positivity thresholds across all groups. Using an exploratory approach, in severe cases, subthreshold autoantibody candidates (FDR < 0.25) showed negative correlation trends with select gene expressions and inflammatory markers (Factor V with IL-6 and CXCL10), suggesting potential disease-specific immunomodulatory associations. In contrast, while mild cases exhibited stronger gene-protein correlations, they showed limited associations with antigen reactivities or clinical laboratory parameters. Additionally, no correlations were observed between autoantibodies and platelet-counts or Fibrin-D-dimer levels. Age-associated increases in antigen reactivities were noted in severe disease, implying a role for immunosenescence. These findings support further investigation into the role of subthreshold autoantibody candidates in thromboinflammatory COVID-19 pathogenesis.

Picornaviruses are a leading cause of central nervous system (CNS) infections. While genotypes such as parechovirus A3 (PeV-A3) and echovirus 11 (E11) can elicit severe neurological disease, the highly prevalent PeV-A1 is not associated with CNS disease. Here, we expand our current understanding of these differences in PeV-A CNS disease using human brain organoids and clinical isolates of the two PeV-A genotypes. Our data indicate that PeV-A1 and A3 specific differences in neurological disease are not due to infectivity of CNS cells as both viruses productively infect brain organoids with a similar cell tropism. Proteomic analysis shows that PeV-A infection significantly alters the host cell metabolism. The inflammatory response following PeV-A3 (and E11 infection) is significantly more potent than that upon PeV-A1 infection. Collectively, our findings align with clinical observations and suggest a role for neuroinflammation, rather than viral replication, in PeV-A3 (and E11) infection. In comparison to PeV-A1, infection with PeV-A3 is associated with neurological illness in infants. Here, using brain organoids, the authors suggest that the innate inflammatory response as the underlying reason, and not replication kinetics.

Background HIV-1C has been shown to have a greater risk of virological failure and reduced susceptibility towards boosted protease inhibitors (bPIs), a component of second-line combination antiretroviral therapy (cART) in South Africa. This study entailed an evaluation of HIV-1 drug resistance-associated mutations (RAMs) among minor viral populations through high-throughput sequencing genotypic resistance testing (HTS-GRT) in patients on the South African national second-line cART regimen receiving bPIs. Methods During 2017 and 2018, 67 patient samples were sequenced using high-throughput sequencing (HTS), of which 56 samples were included in the final analysis because the patient’s treatment regimen was available at the time of sampling. All patients were receiving bPIs as part of their cART. Viral RNA was extracted, and complete pol genes were amplified and sequenced using Illumina HiSeq2500, followed by bioinformatics analysis to quantify the RAMs according to the Stanford HIV Drug Resistance Database. Results Statistically significantly higher PI RAMs were observed in minor viral quasispecies (25%; 14/56) compared to non-nucleoside reverse transcriptase inhibitors (9%; 5/56; p  = 0.042) and integrase inhibitor RAM (4%; 2/56; p  = 0.002). The majority of the drug resistance mutations in the minor viral quasispecies were observed in the V82A mutation ( n  = 13) in protease and K65R ( n  = 5), K103N ( n  = 7) and M184V (n = 5) in reverse transcriptase. Conclusions HTS-GRT improved the identification of PI and reverse transcriptase inhibitor (RTI) RAMs in second-line cART patients from South Africa compared to the conventional GRT with ≥20% used in Sanger-based sequencing. Several RTI RAMs, such as K65R, M184V or K103N and PI RAM V82A, were identified in < 20% of the population. Deep sequencing could be of greater value in detecting acquired resistance mutations early.

Targeted metabolomics studies reported metabolic abnormalities in both treated and untreated people living with human immunodeficiency virus (HIV) (PLHIV). The present study aimed to understand the plasma metabolomic changes and predicted the risk of accelerated aging in PLHIV on long-term suppressive antiretroviral therapy (ART) in a case-control study setting and its association with the plasma proteomics biomarkers of inflammation and neurological defects. Plasma samples were obtained from PLHIV on successful long-term ART for more than five years (n = 22) and matched HIV-negative healthy individuals (n = 22, HC herein). Untargeted metabolite profiling was carried out using ultra-high-performance liquid chromatography/mass spectrometry/mass spectrometry (UHPLC/MS/MS). Plasma proteomics profiling was performed using proximity extension assay targeting 184 plasma proteins. A total of 250 metabolites differed significantly (p < 0.05, q < 0.1) between PLHIV and HC. Plasma levels of several essential amino acids except for histidine, branched-chain amino acids, and aromatic amino acids (phenylalanine, tyrosine, tryptophan) were significantly lower in PLHIV compared to HC. Machine-learning prediction of metabolite changes indicated a higher risk of inflammatory and neurological diseases in PLHIV. Metabolic abnormalities were observed in amino-acid levels, energetics, and phospholipids and complex lipids, which may reflect known differences in lipoprotein levels in PLHIV that can resemble metabolic syndrome (MetS).

Coronaviruses (CoVs) are positive-stranded RNA viruses that infect humans and animals. Infection by CoVs such as HCoV-229E, -NL63, -OC43 and -HKU1 leads to the common cold, short lasting rhinitis, cough, sore throat and fever. However, CoVs such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the newest SARS-CoV-2 (the causative agent of COVID-19) lead to severe and deadly diseases with mortality rates ranging between ~1 to 35% depending on factors such as age and pre-existing conditions. Despite continuous global health threats to humans, there are no approved vaccines or drugs targeting human CoVs, and the recent outbreak of COVID-19 emphasizes an urgent need for therapeutic interventions. Using computational and bioinformatics tools, here we present the feasibility of reported broad-spectrum RNA polymerase inhibitors as anti- SARS-CoV-2 drugs targeting its main RNA polymerase, suggesting that investigational and approved nucleoside RNA polymerase inhibitors have potential as anti-SARS-CoV-2 drugs. However, we note that it is also possible for SARS-CoV-2 to evolve and acquire drug resistance mutations against these nucleoside inhibitors.

The detection of drug resistance mutations (DRMs) in minor viral populations is of potential clinical importance. However, sophisticated computational infrastructure and competence for analysis of high-throughput sequencing (HTS) data lack at most diagnostic laboratories. Thus, we have proposed a new pipeline, MiDRMpol, to quantify DRM from the HIV-1 pol region. The gag-vpu region of 87 plasma samples from HIV-infected individuals from three cohorts was amplified and sequenced by Illumina HiSeq2500. The sequence reads were adapter-trimmed, followed by analysis using in-house scripts. Samples from Swedish and Ethiopian cohorts were also sequenced by Sanger sequencing. The pipeline was validated against the online tool PASeq (Polymorphism Analysis by Sequencing). Based on an error rate of <1%, a value of >1% was set as reliable to consider a minor variant. Both pipelines detected the mutations in the dominant viral populations, while discrepancies were observed in minor viral populations. In five HIV-1 subtype C samples, minor mutations were detected at the <5% level by MiDRMpol but not by PASeq. MiDRMpol is a computationally as well as labor efficient bioinformatics pipeline for the detection of DRM from HTS data. It identifies minor viral populations (<20%) of DRMs. Our method can be incorporated into large-scale surveillance of HIV-1 DRM.

HIV-1 infection induces a chronic inflammatory environment not restored by suppressive antiretroviral therapy (ART). As of today, the effect of viral suppression and immune reconstitution in people living with HIV-1 (PLWH) has been well described but not completely understood. Herein, we show how PLWH who naturally control the virus (PLWH EC ) have a reduced proportion of CD4 + CCR6 + and CD8 + CCR6 + cells compared to PLWH on suppressive ART (PLWH ART ) and HIV-1 negative controls (HC). Expression of CCR2 was reduced on both CD4 + , CD8 + and classical monocytes in PLWH EC compared to PLWH ART and HC. Longer suppressive therapy, measured in the same patients, decreased number of cells expressing CCR2 on all monocytic cell populations while expression on CD8 + T cells increased. Furthermore, the CD4 + CCR6 + /CCR6 − cells exhibited a unique proteomic profile with a modulated energy metabolism in PLWH EC compared to PLWH ART independent of CCR6 status. The CD4 + CCR6 + cells also showed an enrichment in proteins involved in apoptosis and p53 signalling in PLWH EC compared to PLWH ART , indicative of increased sensitivity towards cell death mechanisms. Collectively, this data shows how PLWH EC have a unique chemokine receptor profile that may aid in facilitating natural control of HIV-1 infection. The expression profiles dynamics of several chemokine receptors are lower for people living with HIV-1 who naturally control the virus compared to those on suppressive antiretroviral therapy and HIV-negative controls, shedding light on the mechanisms of natural control of HIV-1 infection.