SARS-CoV-2 variants evolve convergent strategies to remodel the host response.

SARS-CoV-2 variants of concern (VOCs) emerged during the COVID-19 pandemic. Here, we used unbiased systems approaches to study the host-selective forces driving VOC evolution. We discovered that VOCs evolved convergent strategies to remodel the host by modulating viral RNA and protein levels, altering viral and host protein phosphorylation, and rewiring virus-host protein-protein interactions. Integrative computational analyses revealed that although Alpha, Beta, Gamma, and Delta ultimately converged to suppress interferon-stimulated genes (ISGs), Omicron BA.1 did not. ISG suppression correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b, which we mapped to specific mutations. Later Omicron subvariants BA.4 and BA.5 more potently suppressed innate immunity than early subvariant BA.1, which correlated with Orf6 levels, although muted in BA.4 by a mutation that disrupts the Orf6-nuclear pore interaction. Our findings suggest that SARS-CoV-2 convergent evolution overcame human adaptive and innate immune barriers, laying the groundwork to tackle future pandemics.

The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.

Protein Interaction Mapping Identifies RBBP6 as a Negative Regulator of Ebola Virus Replication.

Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.

Evolution of enhanced innate immune evasion by SARS-CoV-2.

The emergence of SARS-CoV-2 variants of concern suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on the characterization of changes in the spike protein in variants of concern, mutations outside of spike are likely to contribute to adaptation. Here, using unbiased abundance proteomics, phosphoproteomics, RNA sequencing and viral replication assays, we show that isolates of the Alpha (B.1.1.7) variant3 suppress innate immune responses in airway epithelial cells more effectively than first-wave isolates. We found that the Alpha variant has markedly increased subgenomic RNA and protein levels of the nucleocapsid protein (N), Orf9b and Orf6-all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein that is required for activation of the RNA-sensing adaptor MAVS. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful transmission of the Alpha variant, and may increase in vivo replication and duration of infection4. The importance of mutations outside the spike coding region in the adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the N and Orf9b regulatory regions of the Delta and Omicron variants.

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, and exert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.

The HMOX1 Pathway as a Promising Target for the Treatment and Prevention of SARS-CoV-2 of 2019 (COVID-19).

The coronavirus disease of 2019 (COVID-19) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global pandemic with increasing incidence and mortality rates. Recent evidence based on the cytokine profiles of severe COVID-19 cases suggests an overstimulation of macrophages and monocytes associated with reduced T-cell abundance (lymphopenia) in patients infected with SARS-CoV-2. The SARS-CoV-2 open reading frame 3 a (ORF3a) protein was found to bind to the human HMOX1 protein at a high confidence through high-throughput screening experiments. The HMOX1 pathway can inhibit platelet aggregation, and can have anti-thrombotic and anti-inflammatory properties, amongst others, all of which are critical medical conditions observed in COVID-19 patients. Here, we review the potential of modulating the HMOX1-ORF3a nexus to regulate the innate immune response for therapeutic benefits in COVID-19 patients. We also review other potential treatment strategies and suggest novel synthetic and natural compounds that may have the potential for future development in clinic.

New Delhi metallo-ß-lactamase - type carbapenemases producing Escherichia coli isolates from hospitalized patients: A pilot study.

BACKGROUND & OBJECTIVES: Resistances to carbapenem group of antimicrobials among Escherichia coli due to production of carbapenemases, especially the New Delhi metallo-ß-lactamase (NDM) types, pose serious challenges in the treatment of infections in healthcare settings. This study was undertaken to detect NDM producing E. coli isolates from hospitalized patients with urinary tract infection (UTI). METHODS: A total of 30 non-repetitive isolates of E. coli from hospitalized patients with clinical suspicion of UTI were subjected to antimicrobial susceptibility testing. Screening for the production of extended-spectrum ß-lactamases (ESBL) was carried out by minimum inhibitory concentration (MIC) test strip ESBL followed by phenotypic confirmation by double-disc synergy test. Phenotypic confirmation of carbapenemase production was carried out by MIC test strip metallo-ß-lactamases. Molecular identification of the blaNDM gene was carried out by polymerase chain reaction (PCR) and sequencing of the amplified fragment. RESULTS: Seventeen of the 30 isolates were detected as ESBL producers, of which three were found to be carbapenemase producers. NDM genes were detected by PCR followed by gene sequencing in all three isolates positive for ESBL as well as carbapenemase. The amino acid sequence of the three isolates showed complete identity to the reference sequences of NDM-1, NDM-4 and NDM-8, respectively. INTERPRETATION & CONCLUSIONS: Our study showed the circulation of NDM variants among the clinical isolates of E. coli that were producers of ESBL as well as carbapenemase.

Role of Iron and Copper in the Pathogenesis of Parkinson's Disease.

Parkinson's disease (PD) is an old age disorder of basal ganglia which involves oligomerization of α-synuclein protein and formation of intercellular inclusions known as "Lewy bodies" in substantia nigra and caudate nuclei in brain which is progressive in nature. It is second most prevalent neurodegenerative disorder characterized by tremor at rest, muscle rigidity, slowness of movement (bradykinesia, akinesia), and changes in posture (instability). Both excess and deficiency in levels of transition metals (especially iron, copper) can be detrimental to the central nervous system. Abnormalities in iron (Fe) and copper (Cu) metabolism have been reported to produce oxidative stress which is one of the major cause in pathogenesis of PD. In the present study 35 PD patients and 33 controls of Northern Indian population were included and serum levels of Fe, Cu and ceruloplasmin (Cp) were measured. Serum Fe (p < 0.01) and Cu (p < 0.01) levels were found to be significantly decreased in PD, whereas there was no significant change in Cp levels in PD patients as compared to controls. These results suggest the existence of a defect in iron which over the time, may hasten the entry of iron into the brain and decrease iron in the extracellular compartment in PD patients.

Ocular Surface Squamous Neoplasia in 200 Patients: A Case-Control Study of Immunosuppression Resulting from Human Immunodeficiency Virus versus Immunocompetency.

PURPOSE: To describe and compare the clinical presentation, treatment outcomes, and histopathologic features of ocular surface squamous neoplasia (OSSN) based on human immunodeficiency virus (HIV) status. DESIGN: Case-control study. PARTICIPANTS: A total of 200 patients with OSSN, of whom 83 (41%) had positive results for HIV and were classified as cases and 117 (59%) had negative results for HIV and were classified as controls. METHODS: Enzyme-linked immunosorbent assay for HIV, conjuntival excision biopsy, extended enucleation, orbital exenteration. MAIN OUTCOME MEASURES: Clinical features, treatment outcomes, and histopathologic characteristics. RESULTS: The mean age at presentation of OSSN in both cases and controls was 40 years (median, 40 years; range, 13-65 years) and in controls was 40 years (median, 38 years; range, 15-80 years). On comparison of cases versus controls with OSSN, HIV-positive individuals had larger (12 vs. 8 mm; P < 0.001) and thicker (3.2 vs. 2.3 mm; P = 0.041) tumors, with a higher incidence of corneal (60% vs. 40%; P = 0.007), scleral (19% vs. 9%; P = 0.044), and orbital (13% vs. 3%; P = 0.019) invasion and a higher need for extended enucleation or exenteration (27% vs. 11%; P < 0.001). The bilateral presentation (11% vs. 4%; P = 0.13), need for lamellar sclerectomy (13% vs. 8%; P = 0.29), and tumor recurrence after primary treatment (30% vs. 20%; P = 0.12) was higher in HIV-positive cases compared with HIV-negative controls. However, these features were not statistically significant. Based on American Joint Committee on Cancer classification, T1 tumor was more common in controls (13% in cases vs. 35% in controls; P = 0.0009), and T4 tumor was more common in cases (13% in cases vs. 4% in controls; P = 0.019). None of the patients demonstrated systemic metastases or died of disease during a mean follow-up period of 10 months (median, 4 months; range, <1-75 months) in cases and 9 months (median, 4 months; range, <1-99 months) in controls. CONCLUSIONS: Ocular surface squamous neoplasia in HIV-positive individuals is aggressive with larger and thicker tumors and with higher incidence of corneal, scleral, and orbital invasion. These patients are associated with poor ocular prognosis with higher need for extended enucleation, exenteration, or both.

Interplay between influenza A virus and host factors: targets for antiviral intervention.

Influenza A viruses (IAVs) pose a major public health threat worldwide. Recent experience with the 2013 H7N9 outbreak in China and the 2009 "swine flu" pandemic have shown that antiviral vaccines and drugs fall short of controlling the spread of disease in a timely and effective manner. Major problems include rapid emergence of drug-resistant influenza virus strains and the slow process of vaccine production. With the threat of a highly pathogenic H5N1 bird-flu pandemic looming large, it is crucial to develop novel ways of combating influenza A viruses. Targeting the host factors critical for influenza A virus replication has shown promise as a strategy to develop novel antiviral molecules with broad-spectrum protection. In this review, we summarize the role of currently identified host factors that play a critical role in the influenza A virus life cycle and discuss the most promising candidates for anti-influenza therapeutics.

Lateral eyelid rotation flap: a novel technique for reconstruction of full thickness eyelid defect.

The purpose of this study was to study anatomical, functional, and cosmetic outcomes of a novel technique, 'Lateral Eyelid Rotation Flap' for reconstruction of full thickness eyelid defect. In this prospective interventional study, 10 patients with full thickness eyelid defect measuring 1/2-2/3rd of eyelid width were included. Eyelid reconstruction was performed by single surgeon, using lateral eyelid rotation flap. Anatomic outcome was assessed by analyzing horizontal and vertical palpebral apertures (HPA and VPA), eyelid contour, and lateral canthus. Functional outcome was assessed by measuring tear film break-up time (TBUT) and Schirmer's test in both the eyes. Cosmetic outcome was evaluated by patients. Median age of patients was 56 years. Nine cases had full thickness defect following the excision of eyelid malignancy. The mean horizontal defect size was 17 ± 4.2 mm. HPA did not change significantly after surgery. VPA was statistically comparable to contralateral eye at 1-month follow-up. Lateral canthus angle recovered by 3rd month after surgery. TBUT and Schirmer's tests were comparable to contralateral eye. Eight patients graded cosmetic outcome as good to excellent. This is a new, single-stage technique for reconstruction of full thickness eyelid defects, with full thickness eyelid tissue including margin.

Foam sclerotherapy for periorbital dermoid cysts.

PURPOSE: To report nonsurgical treatment of periorbital dermoid cysts with foam sclerotherapy using sodium tetradecyl sulfate (STS). METHODS: Single-center, retrospective, interventional case series. Clinical records of all patients diagnosed to have congenital orbital dermoid cyst and treated with STS foam sclerotherapy between 2012 and 2013 were reviewed. The ectodermal contents of the dermoid cyst were aspirated through a stab incision with 18G needle, followed by saline lavage. Intraluminal foam sclerotherapy was then performed using STS (30 mg/ml) in a proportion of 10% of the total aspirate volume. Retrospective data analysis included demographic profile, clinicoradiologic findings, and treatment outcomes of foam sclerotherapy. RESULTS: Four patients were treated in the given period. Average age at presentation was 20.2 years. All cysts were reported to be congenital in nature, and the location was medial angular in 2 cases, lateral angular in 1 case, and lateral orbitotemporal in 1 case. The average aspirate of the pultaceous cyst content was 3.75 ml (range, 2-5 ml). Of the 4 patients, 2 dermoid cysts resolved completely within 8 weeks. Two cysts showed partial response and required a second foam sclerotherapy to achieve complete resolution. At an average follow up of 13.25 months (range, 11-16 months), complete cyst resolution was noted. No sclerotherapy-related complications were observed. CONCLUSIONS: Foam sclerotherapy is successful in obliterating periorbital dermoid cysts and provides a minimally invasive nonsurgical approach to achieve an aesthetic result.

Leveraging Resource Centers for Strengthening Immunization Supply Chain.

The efficacy of immunization programs is critically dependent on robust supply chain management, a complex challenge exacerbated by expanding program scopes and evolving vaccine technologies. This comprehensive review underscores the pivotal role of Resource Centers in fortifying the immunization supply chain, presenting a paradigm shift toward enhanced national and global health outcomes. Through a detailed examination of their key activities, the article elucidates how these centers catalyze improvements across various facets of supply chain management - from the integration of suitable technology technologies and specialized training programs to the development of sustainable models and advocacy for policy prioritization. This further explores the multifaceted challenges these centers confront, including funding constraints, capacity building, and infrastructural gaps, alongside the burgeoning opportunities presented by new vaccine introductions, donor interest in health system strengthening, and the potential for broadened scope beyond immunization. By weaving together examples of existing centers worldwide, the review highlights their contributions towards optimizing vaccine logistics, enhancing data management, and ultimately achieving Sustainable Development Goal 3. The insights provided offer valuable guidance for planning and sustaining resource centers, positioning them as indispensable allies in the global pursuit of universal immunization coverage.

Investigating protein C and S levels in pregnant women with recurrent early pregnancy loss versus normal pregnancy.

Miscarriage in the first and second trimesters of pregnancy is very common, and coagulopathy can be a contributing factor. Protein C and S deficiency are rare, inherited disorders that can increase the risk of thrombophilia. Women with these deficiencies have a higher risk of developing blood clots in the placenta, which can lead to placental insufficiency and, ultimately, to a miscarriage. We aimed to compare the levels of protein C and protein S in pregnant females with recurrent first and second-trimester pregnancy loss and normal pregnant females. We performed a detailed history, examination, and various lab tests on a cohort of 40 females with a history of recurrent first and second-trimester abortions visiting an outpatient clinic at a multi-specialty hospital in Kashmir, India. All the findings were compared with 40 women with normal pregnancies. 10% of the participants had low protein C and S levels (P=0.277), out of whom 75% (p<0.001) had intrauterine growth retardation (IUGR) on ultrasound with 67% (p<0.001) having reduced doppler flow in the umbilical artery. 0.05% of participants had isolated protein S deficiency with no concomitant IUGR seen. Patients with protein C and S deficiencies were treated with heparin and progesterone and followed up for pregnancy outcomes. Screening for protein C and S deficiency is mandatory in all cases of recurrent pregnancy loss. Treatment with low molecular weight heparin and progesterone should be initiated to ensure good fetal outcomes and prevent post-partum/postoperative catastrophic venous thromboembolism events.

Proteomic and genetic analyses of influenza A viruses identify pan-viral host targets.

Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.

Heterogeneous Ribonucleoprotein A1 (hnRNPA1) Interacts with the Nucleoprotein of the Influenza a Virus and Impedes Virus Replication.

Influenza A virus (IAV), like other viruses, depends on the host cellular machinery for replication and production of progeny. The relationship between a virus and a host is complex, shaped by many spatial and temporal interactions between viral and host proteome, ultimately dictating disease outcome. Therefore, it is imperative to identify host-virus interactions as crucial determinants of disease pathogenies. Heterogeneous ribonucleoprotein A1 (hnRNPA1) is an RNA binding protein involved in the life cycle of many DNA and RNA viruses; however, its role in IAV remains undiscovered. Here we report that human hnRNPA1 physically interacts with the nucleoprotein (NP) of IAV in mammalian cells at different time points of the viral replication cycle. Temporal distribution studies identify hnRNPA1 and NP co-localize in the same cellular milieu in both nucleus and mitochondria in NP-transfected and IAV-infected mammalian cells. Interestingly, hnRNPA1 influenced NP gene expression and affected viral replication. Most importantly, hnRNPA1 knockdown caused a significant increase in NP expression and enhanced viral replication (93.82%) in IAV infected A549 cells. Conversely, hnRNPA1 overexpression reduced NP expression at the mRNA and protein levels and impeded virus replication by (60.70%), suggesting antagonistic function. Taken together, results from this study demonstrate that cellular hnRNPA1 plays a protective role in the host hitherto unknown and may hold potential as an antiviral target to develop host-based therapeutics against IAV.

Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.

Evolution of enhanced innate immune evasion by the SARS-CoV-2 B.1.1.7 UK variant.

Emergence of SARS-CoV-2 variants, including the globally successful B.1.1.7 lineage, suggests viral adaptations to host selective pressures resulting in more efficient transmission. Although much effort has focused on Spike adaptation for viral entry and adaptive immune escape, B.1.1.7 mutations outside Spike likely contribute to enhance transmission. Here we used unbiased abundance proteomics, phosphoproteomics, mRNA sequencing and viral replication assays to show that B.1.1.7 isolates more effectively suppress host innate immune responses in airway epithelial cells. We found that B.1.1.7 isolates have dramatically increased subgenomic RNA and protein levels of Orf9b and Orf6, both known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein required for RNA sensing adaptor MAVS activation, and Orf9b binding and activity was regulated via phosphorylation. We conclude that B.1.1.7 has evolved beyond the Spike coding region to more effectively antagonise host innate immune responses through upregulation of specific subgenomic RNA synthesis and increased protein expression of key innate immune antagonists. We propose that more effective innate immune antagonism increases the likelihood of successful B.1.1.7 transmission, and may increase in vivo replication and duration of infection.