Mutations in the F protein of the live-attenuated respiratory syncytial virus vaccine candidate ΔNS2/Δ1313/I1314L increase the stability of infectivity and content of prefusion F protein.

Respiratory syncytial virus (RSV) is the leading viral cause of bronchiolitis and pneumonia in infants and toddlers, but there currently is no licensed pediatric vaccine. A leading vaccine candidate that has been evaluated for intranasal immunization in a recently completed phase 1/2 clinical trial is an attenuated version of RSV strain A2 called RSV/ΔNS2/Δ1313/I1314L (hereafter called ΔNS2). ΔNS2 is attenuated by deletion of the interferon antagonist NS2 gene and introduction into the L polymerase protein gene of a codon deletion (Δ1313) that confers temperature-sensitivity and is stabilized by a missense mutation (I1314L). Previously, introduction of four amino acid changes derived from a second RSV strain "line 19" (I79M, K191R, T357K, N371Y) into the F protein of strain A2 increased the stability of infectivity and the proportion of F protein in the highly immunogenic pre-fusion (pre-F) conformation. In the present study, these four "line 19" assignments were introduced into the ΔNS2 candidate, creating ΔNS2-L19F-4M. During in vitro growth in Vero cells, ΔNS2-L19F-4M had growth kinetics and peak titer similar to the ΔNS2 parent. ΔNS2-L19F-4M exhibited an enhanced proportion of pre-F protein, with a ratio of pre-F/total F that was 4.5- to 5.0-fold higher than that of the ΔNS2 parent. The stability of infectivity during incubation at 4°C, 25°C, 32°C and 37°C was greater for ΔNS2-L19F-4M; for example, after 28 days at 32°C, its titer was 100-fold greater than ΔNS2. ΔNS2-L19F-4M exhibited similar levels of replication in human airway epithelial (HAE) cells as ΔNS2. The four "line 19" F mutations were genetically stable during 10 rounds of serial passage in Vero cells. In African green monkeys, ΔNS2-L19F-4M and ΔNS2 had similar growth kinetics, peak titer, and immunogenicity. These results suggest that ΔNS2-L19F-4M is an improved live attenuated vaccine candidate whose enhanced stability may simplify its manufacture, storage and distribution, which merits further evaluation in a clinical trial in humans.

In vitro suppression of porcine epidemic diarrhea virus by Panax notoginseng saponins: assessing antiviral potential.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality in neonatal suckling piglets, leading to significant economic losses to the swine industry. Panax notoginseng saponins (PNS) are bioactive extracts derived from the P. notoginseng plant. In this study, we investigated the anti-PEDV effect of PNS by employing various methodologies to assess their impact on PEDV in Vero cells. Using a CCK-8 (Cell Counting Kit-8) assay, we found that PNS had no significant cytotoxicity below the concentration of 128 µg/mL in Vero cells. Using immunofluorescence assays (IFAs), an enzyme-linked immunosorbent assay (ELISA), and plaque formation assays, we observed a dose-dependent inhibition of PEDV infection by PNS within 24-48 hours postinfection. PNS exerts its anti-PEDV activity specifically at the genome replication stage, and mRNA-seq analysis demonstrated that treatment with PNS resulted in increased expression of various genes, including IFIT1 (interferon-induced protein with tetratricopeptide repeats 1), IFIT3 (interferon-induced protein with tetratricopeptide repeats 3), CFH (complement factor H), IGSF10 (immunoglobulin superfamily member 10), ID2 (inhibitor of DNA binding 2), SPP1 (secreted phosphoprotein 1), PLCB4 (phospholipase C beta 4), and FABP4 (fatty acid binding protein 4), but it resulted in decreased expression of IL1A (interleukin 1 alpha), TNFRSF19 (TNF receptor superfamily member 19), CDH8 (cadherin 8), DDIT3 (DNA damage inducible transcript 3), GADD45A (growth arrest and DNA damage inducible alpha), PTPRG (protein tyrosine phosphatase receptor type G), PCK2 (phosphoenolpyruvate carboxykinase 2), and ADGRA2 (adhesion G protein-coupled receptor A2). This study provides insights into the potential mechanisms underlying the antiviral effects of PNS. Taken together, the results suggest that the PNS might effectively regulate the defense response to the virus and have potential to be used in antiviral therapies.

The metabolic footprint of Vero E6 cells highlights the key metabolic routes associated with SARS-CoV-2 infection and response to drug combinations.

SARS-CoV-2 burdens healthcare systems worldwide, yet specific drug-based treatments are still unavailable. Understanding the effects of SARS-CoV-2 on host molecular pathways is critical for providing full descriptions and optimizing therapeutic targets. The present study used Nuclear Magnetic Resonance-based metabolic footprinting to characterize the secreted cellular metabolite levels (exometabolomes) of Vero E6 cells in response to SARS-CoV-2 infection and to two candidate drugs (Remdesivir, RDV, and Azithromycin, AZI), either alone or in combination. SARS-CoV-2 infection appears to force VE6 cells to have increased glucose concentrations from extra-cellular medium and altered energetic metabolism. RDV and AZI, either alone or in combination, can modify the glycolic-gluconeogenesis pathway in the host cell, thus impairing the mitochondrial oxidative damage caused by the SARS-CoV-2 in the primary phase. RDV treatment appears to be associated with a metabolic shift toward the TCA cycle. Our findings reveal a metabolic reprogramming produced by studied pharmacological treatments that protects host cells against virus-induced metabolic damage, with an emphasis on the glycolytic-gluconeogenetic pathway. These findings may help researchers better understand the relevant biological mechanisms involved in viral infection, as well as the creation of mechanistic hypotheses for such candidate drugs, thereby opening up new possibilities for SARS-CoV-2 pharmacological therapy.

Comparative analysis of metacyclogenesis and infection curves in different discrete typing units of Trypanosoma cruzi.

Chagas disease (CD), caused by the complex life cycle parasite Trypanosoma cruzi, is a global health concern and impacts millions globally. T. cruzi's genetic variability is categorized into discrete typing units (DTUs). Despite their widespread presence in the Americas, a comprehensive understanding of their impact on CD is lacking. This study aims to analyze life cycle traits across life cycle stages, unraveling DTU dynamics. Metacyclogenesis curves were generated, inducing nutritional stress in epimastigotes of five DTUs (TcI (MG), TcI (DA), TcII(Y), TcIII, TcIV, and TcVI), resulting in metacyclic trypomastigotes. Infection dynamics in Vero cells from various DTUs were evaluated, exploring factors like amastigotes per cell, cell-derived trypomastigotes, and infection percentage. Statistical analyses, including ANOVA tests, identified significant differences. Varying onset times for metacyclogenesis converged on the 7th day. TcI (MG) exhibited the highest metacyclogenesis potential. TcI (DA) stood out, infecting 80% of cells within 24 h. TcI demonstrated the highest potential in both metacyclogenesis and infection among the strains assessed. Intra-DTU diversity was evident among TcI strains, contributing to a comprehensive understanding of Trypanosoma cruzi dynamics and genetic diversity.

Effect of Melatonin on Herpesvirus Type 1 Replication.

Acute HSV-1 infection is associated with mild symptoms, such as fever and lesions of the mouth, face and skin. This phase is followed by a latency period before reactivation, which is associated with symptoms ranging from ulcers to encephalitis. Despite available anti-HSV-1 drugs, the development of new antiviral agents is sought due to the presence of resistant viruses. Melatonin, a molecule secreted by the pineal gland, has been shown to be an antioxidant, inducer of antioxidant enzymes, and regulator of various biological processes. Clinical trials have explored its therapeutic utility in conditions including infections. This study focuses on melatonin's role in HSV-1 replication and the underlying mechanisms. Melatonin was found to decrease the synthesis of HSV-1 proteins in infected Vero cells measured by immunofluorescence, indicating an inhibition of HSV-1 replication. Additionally, it regulates the activities of antioxidant enzymes and affects proteasome activity. Melatonin activates the unfolded protein response (UPR) and autophagy and suppresses apoptosis in HSV-1-infected cells. In summary, melatonin demonstrates an inhibitory role in HSV-1 replication by modulating various cellular responses, suggesting its potential utility in the treatment of viral infections.

Genetic and virological characteristics of a reassortant avian influenza A H6N1 virus isolated from wild birds at a live-bird market in Egypt.

The first detection of a human infection with avian influenza A/H6N1 virus in Taiwan in 2013 has raised concerns about this virus. During our routine surveillance of avian influenza viruses (AIVs) in live-bird markets in Egypt, an H6N1 virus was isolated from a garganey duck and was characterized. Phylogenetic analysis indicated that the Egyptian H6N1 strain A/Garganey/Egypt/20869C/2022(H6N1) has a unique genomic constellation, with gene segments inherited from different subtypes (H5N1, H3N8, H7N3, H6N1, and H10N1) that have been detected previously in AIVs from Egypt and some Eurasian countries. We examined the replication of kinetics of this virus in different mammalian cell lines (A549, MDCK, and Vero cells) and compared its pathogenicity to that of the ancestral H6N1 virus A/Quail/HK/421/2002(H6N1). The Egyptian H6N1 virus replicated efficiently in C57BL/6 mice without prior adaptation and grew faster and reached higher titers than in A549 cells than the ancestral strain. These results show that reassortant H6 AIVs might pose a potential threat to human health and highlight the need to continue surveillance of H6 AIVs circulating in nature.

Adverse Events Following COVISHIELD and VERO CELL Vaccination Campaigns Against COVID-19.

BACKGROUND: Vaccination against COVID-19 for Nepalese was initiated in January 2021 for various age groups. People were anxious about receiving the vaccines and were concerned about the safety profile of the vaccine they received. In this study, we have tried to observe the Adverse Events Following Immunization of two different vaccines namely COVISHIELD (ChAdOx1 nCOV-19) and VERO CELL (CZ02 strain), used in different phases of vaccination by the government of Nepal. METHODS: We conducted a cross-sectional study among people who received COVID-19 vaccines in this study using a self-administered questionnaire.  Data was cleaned and then exported to IBM SPSS v.20 for analysis, Chi-square test was used to see the association between different variables and a p-value<0.05 was considered statistically significant. RESULTS: Out of 303 respondents, all had received the first and 270 participants had received the second dose of the COVID-19 vaccine, among which, 133 (43.89%) reported at least one side effect after the first dose of vaccination while 58 (21.48%) had self-reported side effects after the second dose of vaccination. Seventeen percent of the respondents had COVID-19 infection within the past 3 months before receiving COVID-19 vaccine. Three percent of participants had re-infection with COVID-19 after receiving the first or the second dose of the COVID-19 vaccine. Among participants who experienced adverse events, 42% and 62.1% of participants experienced mild adverse events following the first dose and second dose of the vaccine, respectively.  Conclusions: The adverse events following immunization for both vaccines after both doses of vaccination were quite low, with 43.89% of participants reporting side effects after the first dose and 21.48% of participants reporting side effects after the second dose. Adverse events were most frequently reported within 24 hours of vaccination and were mostly mild. There was no statistical significance of adverse events between both vaccines.

Whole genome sequencing of recombinant viruses obtained from co-infection and superinfection of Vero cells with modified vaccinia virus ankara vectored influenza vaccine and a naturally occurring cowpox virus.

Modified vaccinia virus Ankara (MVA) has been widely tested in clinical trials as recombinant vector vaccine against infectious diseases and cancers in humans and animals. However, one biosafety concern about the use of MVA vectored vaccine is the potential for MVA to recombine with naturally occurring orthopoxviruses in cells and hosts in which it multiplies poorly and, therefore, producing viruses with mosaic genomes with altered genetic and phenotypic properties. We previously conducted co-infection and superinfection experiments with MVA vectored influenza vaccine (MVA-HANP) and a feline Cowpox virus (CPXV-No-F1) in Vero cells (that were semi-permissive to MVA infection) and showed that recombination occurred in both co-infected and superinfected cells. In this study, we selected the putative recombinant viruses and performed genomic characterization of these viruses. Some putative recombinant viruses displayed plaque morphology distinct of that of the parental viruses. Our analysis demonstrated that they had mosaic genomes of different lengths. The recombinant viruses, with a genome more similar to MVA-HANP (>50%), rescued deleted and/or fragmented genes in MVA and gained new host ranges genes. Our analysis also revealed that some MVA-HANP contained a partially deleted transgene expression cassette and one recombinant virus contained part of the transgene expression cassette similar to that incomplete MVA-HANP. The recombination in co-infected and superinfected Vero cells resulted in recombinant viruses with unpredictable biological and genetic properties as well as recovery of delete/fragmented genes in MVA and transfer of the transgene into replication competent CPXV. These results are relevant to hazard characterization and risk assessment of MVA vectored biologicals.

An in vitro assay for toxicity testing of Clostridium perfringens type C ß-toxin.

Introduction: Veterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the ß-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the ß-toxin. Methods: Development of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for ß-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. Results: The current animal test is estimated to detect 100 - 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the ß-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability. Conclusions: Here, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay's sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.

Promoting the Anticancer Activity with Multidentate Furan-2-Carboxamide Functionalized Aroyl Thiourea Chelation in Binuclear Half-Sandwich Ruthenium(II) Complexes.

A new set of binuclear arene ruthenium complexes [Ru2(p-cymene)2(k4-N2OS)(L1-L3)Cl2] (Ru2L1-Ru2L3) encompassing furan-2-carboxamide-based aroylthiourea derivatives (H2L1-H2L3) was synthesized and characterized by various spectral and analytical techniques. Single-crystal XRD analysis unveils the N^O and N^S mixed monobasic bidentate coordination of the ligands constructing N, S, Cl/N, O, and Cl legged piano stool octahedral geometry. DFT analysis demonstrates the predilection for the formation of stable arene ruthenium complexes. In vitro antiproliferative activity of the complexes was examined against human cervical (HeLa), breast (MCF-7), and lung (A549) cancerous and noncancerous monkey kidney epithelial (Vero) cells. All the complexes are more efficacious against HeLa and MCF-7 cells with low inhibitory doses (3.86-11.02 µM). Specifically, Ru2L3 incorporating p-cymene and -OCH3 fragments exhibits high lipophilicity, significant cytotoxicity against cancer cells, and lower toxicity on noncancerous cells. Staining analysis indicates the apoptosis-associated cell morphological changes expressively in MCF-7 cells. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) analyses reveal that Ru2L3 can raise ROS levels, reduce MMP, and trigger mitochondrial dysfunction-mediated apoptosis. The catalytic oxidation of glutathione (GSH) to its disulfide form (GSSG) by the complexes may simultaneously increase the ROS levels, alluding to their observed cytotoxicity and apoptosis induction. Flow cytometry determined the quantitative classification of late apoptosis and S-phase arrest in MCF-7 and HeLa cells. Western blotting analysis confirmed that the complexes promote apoptosis by upregulating Caspase-3 and Caspase-9 and downregulating BCL-2. Molecular docking studies unfolded the strong binding affinities of the complexes with VEGFR2, an angiogenic signaling receptor, and BCL2, Cyclin D1, and HER2 proteins typically overexpressed on tumor cells.

Langat virus inhibits the gp130/JAK/STAT signaling by reducing the gp130 protein level.

The tick-borne encephalitis virus (TBEV) serocomplex includes several medically important flavivirus members endemic to Europe, Asia, and North America, which can induce severe neuroinvasive or viscerotropic diseases with unclear mechanisms of pathogenesis. Langat virus (LGTV) shares a high sequence identity with TBEV but exhibits lower pathogenic potential in humans and serves as a model for virus-host interactions. In this study, we demonstrated that LGTV infection inhibits the activation of gp130/JAK/STAT (Janus kinases (JAK) and signal transducer and activator of transcription (STAT)) signaling, which plays a pivotal role in numerous biological processes. Our data show that the LGTV-infected cells had significantly lower phosphorylated STAT3 (pSTAT3) protein upon oncostatin M (OSM) stimulation than the mock-infected control. LGTV infection blocked the nuclear translocation of STAT3 without a significant effect on total STAT3 protein level. LGTV inhibited JAK1 activation and reduced gp130 protein expression in infected cells, with the viral NS5 protein mediating this effect. TBEV infection also reduces gp130 level. On the other hand, pretreatment of Vero cells with OSM significantly reduces LGTV replication, and STAT1/STAT2 knockdown had little effect on OSM-mediated antiviral effect, which suggests it is independent of STAT1/STAT2 and, instead, it is potentially mediated by STAT3 signlaing. These findings shed light on the LGTV and TBEV-cell interactions, offering insights for the future development of antiviral therapeutics and improved vaccines.

Cordycepin Inhibits Enterovirus A71 Replication and Protects Host Cell from Virus-Induced Cytotoxicity through Adenosine Action Pathway.

Enterovirus A71 (EV-A71) infection typically causes mild illnesses, such as hand-foot-and-mouth disease (HFMD), but occasionally leads to severe or fatal neurological complications in infants and young children. Currently, there is no specific antiviral treatment available for EV-A71 infection. Thus, the development of an effective anti-EV-A71 drug is required urgently. Cordycepin, a major bioactive compound found in Cordyceps fungus, has been reported to possess antiviral activity. However, its specific activity against EV-A71 is unknown. In this study, the potency and role of cordycepin treatment on EV-A71 infection were investigated. Results demonstrated that cordycepin treatment significantly reduced the viral load and viral ribonucleic acid (RNA) level in EV-A71-infected Vero cells. In addition, EV-A71-mediated cytotoxicity was significantly inhibited in the presence of cordycepin in a dose-dependent manner. The protective effect can also be extended to Caco-2 intestinal cells, as evidenced by the higher median tissue culture infectious dose (TCID50) values in the cordycepin-treated groups. Furthermore, cordycepin inhibited EV-A71 replication by acting on the adenosine pathway at the post-infection stage. Taken together, our findings reveal that cordycepin could be a potential antiviral candidate for the treatment of EV-A71 infection.

Unveiling the Role of Protein Kinase C θ in Porcine Epidemic Diarrhea Virus Replication: Insights from Genome-Wide CRISPR/Cas9 Library Screening.

Porcine epidemic diarrhea virus (PEDV), a member of the Alpha-coronavirus genus in the Coronaviridae family, induces acute diarrhea, vomiting, and dehydration in neonatal piglets. This study aimed to investigate the genetic dependencies of PEDV and identify potential therapeutic targets by using a single-guide RNA (sgRNA) lentiviral library to screen host factors required for PEDV infection. Protein kinase C θ (PKCθ), a calcium-independent member of the PKC family localized in the cell membrane, was found to be a crucial host factor in PEDV infection. The investigation of PEDV infection was limited in Vero and porcine epithelial cell-jejunum 2 (IPEC-J2) due to defective interferon production in Vero and the poor replication of PEDV in IPEC-J2. Therefore, identifying suitable cells for PEDV investigation is crucial. The findings of this study reveal that human embryonic kidney (HEK) 293T and L929 cells, but not Vero and IPEC-J2 cells, were suitable for investigating PEDV infection. PKCθ played a significant role in endocytosis and the replication of PEDV, and PEDV regulated the expression and phosphorylation of PKCθ. Apoptosis was found to be involved in PEDV replication, as the virus activated the PKCθ-B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) axis in HEK293T and L929 cells to increase viral endocytosis and replication via mitochondrial apoptosis. This study demonstrated the suitability of HEK293T and L929 cells for investigating PEDV infection and identified PKCθ as a host factor essential for PEDV infection. These findings provide valuable insights for the development of strategies and drug targets for PEDV infection.

An ascidian Polycarpa aurata-derived pan-inhibitor against coronaviruses targeting Mpro.

Coronaviruses (CoVs) are responsible for a wide range of illnesses in both animals and human. The main protease (Mpro) of CoVs is an attractive drug target, owing its critical and highly conserved role in viral replication. Here, we developed and refined an enzymatic technique to identify putative Mpro inhibitors from 189 marine chemicals and 46 terrestrial natural products. The IC50 values of Polycarpine (1a), a marine natural substance we studied and synthesized, are 30.0 ± 2.5 nM for SARS-CoV-2 Mpro and 0.12 ± 0.05 µM for PEDV Mpro. Our research further demonstrated that pretreatment with Polycarpine (1a) inhibited the betacoronavirus SARS-CoV-2 and alphacoronavirus PEDV multiplication in Vero-E6 cells. As a result, Polycarpine (1a), a pan-inhibitor of Mpro, will function as an effective and promising antiviral option to combat CoVs infection and as a foundation for further therapeutic research.

ALIX and TSG101 are essential for cellular entry and replication of two porcine alphacoronaviruses.

Alphacoronaviruses are the primary coronaviruses responsible for causing severe economic losses in the pig industry with the potential to cause human outbreaks. Currently, extensive studies have reported the essential role of endosomal sorting and transport complexes (ESCRT) in the life cycle of enveloped viruses. However, very little information is available about which ESCRT components are crucial for alphacoronaviruses infection. By using RNA interference in combination with Co-immunoprecipitation, as well as fluorescence and electron microscopy approaches, we have dissected the role of ALIX and TSG101 for two porcine alphacoronavirus cellular entry and replication. Results show that infection by two porcine alphacoronaviruses, including porcine epidemic diarrhea virus (PEDV) and porcine enteric alphacoronavirus (PEAV), is dramatically decreased in ALIX- or TSG101-depleted cells. Furthermore, PEDV entry significantly increases the interaction of ALIX with caveolin-1 (CAV1) and RAB7, which are crucial for viral endocytosis and lysosomal transport, however, does not require TSG101. Interestingly, PEAV not only relies on ALIX to regulate viral endocytosis and lysosomal transport, but also requires TSG101 to regulate macropinocytosis. Besides, ALIX and TSG101 are recruited to the replication sites of PEDV and PEAV where they become localized within the endoplasmic reticulum and virus-induced double-membrane vesicles. PEDV and PEAV replication were significantly inhibited by depletion of ALIX and TSG101 in Vero cells or primary jejunal epithelial cells, indicating that ALIX and TSG101 are crucial for PEDV and PEAV replication. Collectively, these data highlight the dual role of ALIX and TSG101 in the entry and replication of two porcine alphacoronaviruses. Thus, ESCRT proteins could serve as therapeutic targets against two porcine alphacoronaviruses infection.

Developing inhibitory peptides against SARS-CoV-2 envelope protein.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected approximately 800 million people since the start of the Coronavirus Disease 2019 (COVID-19) pandemic. Because of the high rate of mutagenesis in SARS-CoV-2, it is difficult to develop a sustainable approach for prevention and treatment. The Envelope (E) protein is highly conserved among human coronaviruses. Previous studies reported that SARS-CoV-1 E deficiency reduced viral propagation, suggesting that E inhibition might be an effective therapeutic strategy for SARS-CoV-2. Here, we report inhibitory peptides against SARS-CoV-2 E protein named iPep-SARS2-E. Leveraging E-induced alterations in proton homeostasis and NFAT/AP-1 pathway in mammalian cells, we developed screening platforms to design and optimize the peptides that bind and inhibit E protein. Using Vero-E6 cells, human-induced pluripotent stem cell-derived branching lung organoid and mouse models with SARS-CoV-2, we found that iPep-SARS2-E significantly inhibits virus egress and reduces viral cytotoxicity and propagation in vitro and in vivo. Furthermore, the peptide can be customizable for E protein of other human coronaviruses such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The results indicate that E protein can be a potential therapeutic target for human coronaviruses.

Attenuation of carbon tetrachloride-induced nephrotoxicity by gum Arabic extract via modulating cellular redox state, NF-κB pathway, and KIM-1.

The current study investigated the ameliorating impact of GA water extract (GAE) on CCl4-induced nephrotoxicity in renal cells and tissue by comparing its effectiveness with the Ketosteril (Ks) drug in restoring oxidative stress and necroinflammation. The cell morphology, necrosis, and redox state were evaluated in Vero cells. The influence of GAE on CCl4-induced oxidative stress, inflammation, and necrosis was examined in rats. The predicted inhibitory mechanism of GAE phenolic constituents against COX-2 and iNOS was also studied. The results revealed that GAE contains crucial types of phenolic acids, which are associated with its antiradical activities. GAE improved CCl4-induced Vero cell damage and restored renal architecture damage, total antioxidant capacity, ROS, TBARS, NO, GSH, GPX, SOD, and MPO in rats. GAE downregulated the gene expression of renal NF-κB, TNF-α, iNOS, and COX-2, as well as kidney injury molecule-1 (KIM-1) in rats. The GAE improved blood urea, creatinine, cholesterol, and reducing power. The computational analysis revealed the competitive inhibitory mechanism of selected phenolic composites of GAE on COX-2 and iNOS activities. The GAE exhibited higher potency than Ks in most of the studied parameters, as observed by the heatmap plots. Thus, GAE is a promising extract for the treatment of kidney toxicity.

Rickettsia rickettsii virulence determinants RARP2 and RapL mitigate IFN-ß signaling in primary human dermal microvascular endothelial cells.

We compared the growth characteristics of a virulent Rickettsia rickettsii strain (Sheila Smith) to an attenuated R. rickettsii stain (Iowa) and a non-pathogenic species (R. montanensis) in primary human dermal microvascular endothelial cells (HDMEC). All replicated in Vero cells, however, only the Sheila Smith strain productively replicated in HDMECs. The Iowa strain showed minimal replication over a 24-h period, while R. montanensis lost viability and induced lysis of the HDMECs via a rapid programmed cell death response. Both the virulent and attenuated R. rickettsii strains, but not R. montanensis, induced an interferon-1 response, although the response was of lesser magnitude and delayed in the Sheila Smith strain. IFN-ß secretion correlated with increased host cell lysis, and treatment with anti-IFNAR2 antibody decreased lysis from Iowa-infected but not Sheila Smith-infected cells. Both Sheila Smith- and Iowa-infected cells eventually lysed, although the response from Sheila Smith was delayed and showed characteristics of apoptosis. We, therefore, examined whether reconstitution of the Iowa strain with two recently described putative virulence determinants might enhance survival of Iowa within HDMECs. Reconstitution with RARP2, which is inhibitory to anterograde trafficking through the Golgi apparatus, reduced IFN-ß secretion but had no effect on cell lysis. RapL, which proteolytically processes surface exposed autotransporters and enhances replication of Iowa in Guinea pigs, suppressed both IFN-ß production and host cell lysis. These findings suggest distinct mechanisms by which virulent spotted fever group rickettsiae may enhance intracellular survival and replication.IMPORTANCEWe examined a naturally occurring non-pathogenic rickettsial species, R. montanensis, a laboratory-attenuated R. rickettsii strain (Iowa), and a fully virulent R. rickettsii strain (Sheila Smith) for growth in human dermal microvascular endothelial cells. The two avirulent strains replicated poorly or not at all. Only the virulent Sheila Smith strain replicated. IFN-ß production correlated with the inhibition of R. rickettsii Iowa. Reconstitution of Iowa with either of two recently described putative virulence determinants altered the IFN-ß response. A rickettsial ankyrin repeat protein, RARP2, disrupts the trans-Golgi network and inhibits IFN-ß secretion. An autotransporter peptidase, RapL, restores proteolytic maturation of outer membrane autotransporters and diminishes the IFN-ß response to enhance cell survival and permit replication of the recombinant strain. These studies point the way toward discovery of mechanisms for innate immune response avoidance by virulent rickettsia.

Novel silver nanoparticle-antibiotic combinations as promising antibacterial and anti-biofilm candidates against multiple-antibiotic resistant ESKAPE microorganisms.

HYPOTHESIS: The emergence of Multiple Antibiotic Resistance (MAR) in ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens is a global challenge to public health. The inherent antimicrobial nature of silver nanoparticles (AgNPs) makes them promising antimicrobial candidates against antibiotic-resistant pathogens. This study explores the combination of AgNPs with antibiotics (SACs) to create new antimicrobial agents effective against MAR ESKAPE microorganisms. METHODS: AgNPs were synthesized using Streptococcus pneumoniae ATCC 49619 and characterized for structure and surface properties. The SACs were tested against ESKAPE microorganisms using growth kinetics and time-kill curve methods. The effect of SACs on bacterial biofilms and the disruption of cell membranes was determined. The in-vitro cytotoxicity effect of the AgNPs was also studied. FINDINGS: The synthesized AgNPs (spherical, 7.37±4.55 nm diameter) were antimicrobial against MAR ESKAPE microorganisms. The SACs showed synergy with multiple conventional antibiotics, reducing their antibacterial concentrations up to 32-fold. Growth kinetics and time-kill studies confirmed the growth retardation effect and bactericidal activity of SACs. Mechanistic studies suggested that these biofilm-eradicating SACs probably resulted in the loss of bacterial cell membrane integrity, leading to leakage of the cytoplasmic content. The AgNPs were highly cytotoxic against skin melanoma cells but non-cytotoxic to normal Vero cells.