Infected Vero Cells Research Articles

Ethnomedicinal Plant Stephania hernandifolia and its Active Fraction 2- Chloroethyl Linoleate Inhibits HSV-2 Infection by Blocking Viral Immediate Early and Early Transcription

Aim: The aim of this study is validation of antiviral activity of Stephania hernandifolia against HSV-2. Background: Ethnomedicinal plant Stephania hernandifolia, traditionally used for the management of skin, digestive and nerve ailments demonstrated significant anti-HSV-1 activity; similar to Stephania cepharantha having neuroinflammatory and anti-HSV activities. Objectives: Thus, the present study aimed to validate the potential of the most active fraction-2 (F-2) of S. hernandifolia against HSV-2 in vitro, along with the underlying mode or mechanism of action. Methods: The standardized F-2 was characterized by GC-MS, 1H-NMR, Mass and FTIR spectroscopy. Cytotoxicity (CC50) and antiviral activity (EC50) were evaluated by MTT and Plaque reduction assay. To determine the mode of action, we have used time-of-addition, virus inactivation, and entry (attachment and penetration) assays, followed by semiquantitative PCR. Furthermore, the protein expression levels of immediate early (IE) and early (E) gene products of drug-treated virions were measured by Western blot. Results: The results showed that HSV-2G and ICMR/VU-2012/20, the clinical isolate of HSV-2, were inhibited by F-2 at EC50 of 20.0 and 20.43 μg/ml respectively, with Selectivity Index (SI) of 12. Timeof- addition assay showed that F-2 significantly inhibited HSV-2 infection in Vero cells at 4-8 h posttreatment. The infectivity of the virion was lost within 1h of exposure to F-2 (EC50 and EC99). Furthermore, semi-Q-PCR and Western blot studies demonstrated significant downregulation of IE and E gene products. The characterization revealed that 2-chloroethyl linoleate is the lead compound in the F-2 fraction. Conclusion: Thus, our results showed that the bioactive fraction F-2 inhibits both IE and E transcription of HSV-2.

Adherence of SARS-CoV-2 Delta variant to a surgical mask and N95 respirators.

The use of facial protection has been adopted globally due to the COVID-19 pandemic. We dissected four respirators and one surgical mask into layers to determine whether or not the virus adheres to them. These individual layers were contaminated with the SARS-CoV-2 Delta variant, and its release by vortexing was performed. Samples were used to infect Vero cells, and a plaque assay was used to evaluate the adherence of the virus to the layers. Results showed that a cumulative log reduction of the layers reduced the load of the virus by at least sixfold. Our study confirms the effectiveness of facial protection in reducing the transmission and/or infection of SARS-CoV-2.

From functional genomics of vero cells to CRISPR-based genomic deletion for improved viral production rates.

Despite their wide use in the vaccine manufacturing field for over 40 years, one of the main limitations to recent efforts to develop Vero cells as high‐throughput vaccine manufacturing platforms is the lack of understanding of virus‐host interactions during infection and cell‐based virus production in Vero cells. To overcome this limitation, this manuscript uses the recently generated reference genome for the Vero cell line to identify the factors at play during influenza A virus (IAV) and recombinant vesicular stomatitis virus (rVSV) infection and replication in Vero host cells. The best antiviral gene candidate for gene editing was selected using Differential Gene Expression analysis, Gene Set Enrichment Analysis and Network Topology‐based Analysis. After selection of the ISG15 gene for targeted CRISPR genomic deletion, the ISG15 genomic sequence was isolated for CRISPR guide RNAs design and the guide RNAs with the highest knockout efficiency score were selected. The CRISPR experiment was then validated by confirmation of genomic deletion via PCR and further assessed via quantification of ISG15 protein levels by western blot. The gene deletion effect was assessed thereafter via quantification of virus production yield in the edited Vero cell line. A 70‐fold and an 87‐fold increase of total viral particles productions in ISG15−/− Vero cells was achieved for, respectively, IAV and rVSV while the ratio of infectious viral particles/total viral particles also significantly increased from 0.0316 to 0.653 for IAV and from 0.0542 to 0.679 for rVSV‐GFP.

Characterization of replication and variations in genome segments of a bat reovirus, BatMRV/B19-02, by RNA-seq in infected Vero-E6 cells.

Mammalian orthoreoviruses (MEVs) that can cause enteric, respiratory, and encephalitic infections have been identified in a wide variety of mammalian species. Here, we report a novel MRV type 1 strain detected in Miniopterus schreibersii that may have resulted from reassortment events. Using next-generation RNA sequencing (RNA-seq), we found that the ratios of the RNA levels of the 10 reovirus segments in infected cells were constant during the late stages of infection. We also discovered that the relative abundance of each segment differed. Notably, the relative abundance of M2 (encoding the µ1 protein) and S4 (encoding the σ3 protein) RNAs was higher than that of the others throughout the infection. Additionally, massive junctions were identified. These results support the hypothesis that defective genome segments are generated and that cross-family recombination occurs. These data may further the study of gene function, viral replication, and virus evolution.

A Preliminary Study on the Anti-dengue Viral Activity of Munronia pinnata

Dengue infection is a major health care problem in tropical and subtropical countries. The recently approved dengue vaccine has limitations, and there is no antiviral drug for treatment at present.For centuries plants and plant extracts have been used in traditional medicine for the treatment of various infections. The whole plant of Munronia pinnata, which has been used for treating fever patients in Sri Lankan traditional medicine, was tested for anti-dengue viral activity.The cytotoxicity assay of M. pinnata on Vero cells using 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) revealed Maximum Nontoxic Dose (MNTD) as 125 μg/ml and 50% cytotoxic concentration (CC50) as 428.9 ± 21.55 μg/ml. Plaque reduction antiviral assay performed on dengue-4 virus infected Vero cells demonstrated half-maximal inhibitory concentration (IC50) as 26.12 ± 0.91 μg/ml. The selectivity index (SI) of Dengue infection is a major health care problem in tropical and subtropical countries. The recently approved dengue vaccine has limitations, and there is no antiviral drug for treatment at present.For centuries plants and plant extracts have been used in traditional medicine for the treatment of various infections. The whole plant of Munronia pinnata, which has been used for treating fever patients in Sri Lankan traditional medicine, was tested for anti-dengue viral activity.The cytotoxicity assay of M. pinnata on Vero cells using 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) revealed Maximum Nontoxic Dose (MNTD) as 125 μg/ml and 50% cytotoxic concentration (CC50) as 428.9 ± 21.55 μg/ml. Plaque reduction antiviral assay performed on dengue-4 virus infected Vero cells demonstrated half-maximal inhibitory concentration (IC50) as 26.12 ± 0.91 μg/ml. The selectivity index (SI) of M. pinnata was 16.42.Based on the selectivity index, Munronia pinnata appears to be a viable candidate for identifying biologically active compounds with anti-dengue viral activity.Keywords: Dengue, Munronia pinnata, Antiviral, Plaque reduction Assay, Vero Cells was 16.42.Based on the selectivity index, Munronia pinnata appears to be a viable candidate for identifying biologically active compounds with anti-dengue viral activity.
 Keywords: Dengue, Munronia pinnata, Antiviral, Plaque reduction Assay, Vero Cells

The Anti-Histamine Azelastine, Identified by Computational Drug Repurposing, Inhibits Infection by Major Variants of SARS-CoV-2 in Cell Cultures and Reconstituted Human Nasal Tissue.

Background and purpose: The COVID-19 pandemic continues to pose challenges, especially with the emergence of new SARS-CoV-2 variants that are associated with higher infectivity and/or compromised protection afforded by the current vaccines. There is a high demand for additional preventive and therapeutic strategies effective against this changing virus. Repurposing of approved or clinically tested drugs can provide an immediate solution. Experimental Approach: We applied a novel computational approach to search among approved and commercially available drugs. Antiviral activity of a predicted drug, azelastine, was tested in vitro in SARS-CoV-2 infection assays with Vero E6 cells, Vero cells stably overexpressing the human TMPRSS2 and ACE2 proteins as well as on reconstituted human nasal tissue using the predominant variant circulating in Europe in summer 2020, B.1.177 (D614G variant), and its emerging variants of concern; B.1.1.7 (alpha), B.1.351 (beta) and B.1.617.2 (delta) variants. The effect of azelastine on viral replication was assessed by quantification of viral genomes by droplet digital PCR or qPCR. Key results: The computational approach identified major drug families, such as anti-infective, anti-inflammatory, anti-hypertensive, antihistamine, and neuroactive drugs. Based on its attractive safety profile and availability in nasal formulation, azelastine, a histamine 1 receptor-blocker was selected for experimental testing. Azelastine reduced the virus-induced cytopathic effect and SARS-CoV-2 copy numbers both in preventive and treatment settings upon infection of Vero cells with an EC50 of 2.2–6.5 µM. Comparable potency was observed with the alpha, beta and delta variants. Furthermore, five-fold dilution (containing 0.02% azelastine) of the commercially available nasal spray formulation was highly potent in inhibiting viral propagation in reconstituted human nasal tissue. Conclusion and Implications: Azelastine, an antihistamine available as nasal sprays developed against allergic rhinitis may be considered as a topical prevention or treatment of nasal colonization by SARS-CoV-2. A Phase 2 efficacy indicator study with azelastine-containing nasal spray that was designed based on the findings reported here has been concluded recently, confirming accelerated viral clearance in SARS-CoV-2 positive subjects.

P-073 SARS-CoV2 infection in human testis and sperm: in vivo and in vitro studies

Study questionCan severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) enter somatic and germinal cells of human testis or ejaculated sperm, thus affecting male reproductive function?Summary answerThis research provides a biological background of the potential route for infection of SARS-CoV-2 and may enable rapid deciphering of COVID-19-induced male-related reproductive disorders.What is known alreadyEpidemiologic studies suggest a significant male sex susceptibility for severe COVID19 symptoms. SARS-CoV-2 is known to affect certain cell types based on their expression of angiotensin-converting enzyme 2 (ACE2) and Transmembrane serine protease2 (TMPRSS2). ACE2 makes available the binding site for the Spike protein of SARS-CoV-2. TMPRSS2 facilitates virus entry by cleaving the S antigen into S1 (the active binding site). Several studies reported the presence of ACE2 in Leydig and Sertoli cells as well as in germ cells from spermatogonia to spermatozoa. These data suggest that the human testis and gametes are a target for SARS-CoV-2.Study design, size, durationTo address this question, we examined the gene expression profile of SARS-CoV-2-associated receptors and proteases (ACE2-TMPRSS2) as well as their protein expression and localization in testicular tissue of males undergoing diagnostic surgery and in sperm of healthy, normozoospermic donors referring to the Unit of Assisted Reproduction, Siena University Hospital, from April 2020 to January 2022.Participants/materials, setting, methodsAssays were performed on tissue biopsies (n = 3) or on freshly ejaculated sperm of men (n = 6) undergoing routine semen analysis after granting informed consent. To this end, tissue biopsies and ejaculated sperm have been co-cultivated for 5-12h with infected VERO E6 cells. Immunoelectron microscopy, in situ hybridization, and highly sensitive digital droplet PCR analysis, have been used to assess if particles containing SARS-CoV- 2 antigens can be detected in testicular tissue or spermatozoa in vitro.Main results and the role of chanceWe carefully investigated the presence of the SARS-CoV-2 virus and the possible effect on male fertility, both at the molecular and ultrastructural levels. To elucidate the mechanisms underlying virus infection in germ cells and male gametes, we analyzed the expression of both ACE2 and TMPRSS2. Both genes are expressed in testes and ejaculated sperm, confirming the possibility that SARS-CoV2 can enter these cells. We also provided evidence of the expression of the corresponding proteins in both testis and sperm by immunofluorescence assays. The immunofluorescence staining of paraffin-embedded slices of testicular tissue with anti-ACE2 antibody revealed a strong signal in Leydig cells. However, ACE2 staining was also present in human Sertoli cells and was concentrated in the adluminal half of the cell, i.e. surrounding spermatocytes and spermatids.The analysis of TRPMSS2 showed similar results. Indeed, this protease is localized mainly in the interstitium, at the level of Leydig cells. This is an intriguing datum, since, while Leydig cells may be considered as a high-risk cells because of the co-expression of TMPRSS2 and ACE2, germ cells may not be at increased risk of ACE2and TMPRSS2-mediated viral entry and spread, given the lack of co-expression in these testicular cell type.Limitations, reasons for cautionThe low number of analyzed samples may limit the statistical power of this study. Whether the SARS-CoV-2 infection change the gene expression profile of other SARS-CoV-2-associated proteases is under investigationWider implications of the findingsThis validated RT-PCR assay allows reliable screening of SARS-CoV-2 in sperm, useful for investigating the presence of the virus in patients undergoing ART, as well as for explaining the molecular and cellular mechanisms related to the gender specificity of the more severe infection-related symptoms.Trial registration numberNot applicable

Identification of clinical candidates against West Nile virus by activity screening in vitro and effect evaluation in vivo.

The West Nile virus (WNV) is a member of the flavivirus and is known to causeencephalitis. There is currently no specific treatment for WNV infection. Repurposing of clinically approved drugs appeared promising for rapidly identifying effective, safe, and readily available candidates for antiviral drugs. Here, we screened the small-molecule compounds with anti-WNV activity from 978 Food Drug Administration-approved drugs. Four compounds, including cilnidipine, mycophenolate mofetil, nitazoxanide, and teriflunomide, were found to efficiently abrogate WNV infection in Vero cells and human neuroblastoma SH-SY5Y cells. The four compounds also exert broad-spectrum antiviral activity against the Zika virus, Japanese encephalitis virus, yellow fever virus, tick-borne encephalitis virus, and chikungunya virus. Furthermore, nitazoxanide (a synthetic benzamide) and teriflunomide (an inhibitor of dihydroorotate dehydrogenase, DHODH) protected 20% and 40% of mice from lethal WNV challenge, respectively. Both drugs, which are orally bioavailable and have been approved clinically for many years, may be promising therapeutics for WNV infection. Moreover, the other two DHODH inhibitors,ML390 and vidofludimus, also displayed potent activity against WNV infection in vitro and in vivo.

Identification of potent inhibitors of arenavirus and SARS-CoV-2 exoribonucleases by fluorescence polarization assay

Viral exoribonucleases are uncommon in the world of RNA viruses. To date, they have only been identified in the Arenaviridae and the Coronaviridae families. The exoribonucleases of these viruses play a crucial role in the pathogenicity and interplay with host innate immune response. Moreover, coronaviruses exoribonuclease is also involved in a proofreading mechanism ensuring the genetic stability of the viral genome. Because of their key roles in virus life cycle, they constitute attractive target for drug design.Here we developed a sensitive, robust and reliable fluorescence polarization assay to measure the exoribonuclease activity and its inhibition in vitro. The effectiveness of the method was validated on three different viral exoribonucleases, including SARS-CoV-2, Lymphocytic Choriomeningitis and Machupo viruses. We performed a screening of a focused library consisting of 113 metal chelators. Hit compounds were recovered with an IC50 at micromolar level. We confirmed 3 hits in SARS-CoV-2 infected Vero-E6 cells.

Natural Polyphenols, 1,2,3,4,6-O-Pentagalloyglucose and Proanthocyanidins, as Broad-Spectrum Anticoronaviral Inhibitors Targeting Mpro and RdRp of SARS-CoV-2.

The natural plant dietary polyphenols 1,2,3,4,6-O-Pentagalloylglucose (PGG) and proanthocyanidin (PAC) have potent antioxidant activity and a variety of pharmacological activities, including antiviral activity. In this study, we examined the inhibitory effect of PGG and PAC on SARS-CoV-2 virus infection, and elucidated its mode of action. PGG and PAC have dose-dependent inhibitory activity against SARS-CoV-2 infection in Vero cells. PGG has a lower IC50 (15.02 ± 0.75 μM) than PAC (25.90 ± 0.81 μM), suggesting that PGG has better inhibitory activity against SARS-CoV-2 than PAC. The PGG and PAC inhibit similar Mpro activities in a protease activity assay, with IC50 values of 25–26 μM. The effects of PGG and PAC on the activity of the other essential SARS-CoV-2 viral protein, RdRp, were analyzed using a cell-based activity assay system. The activity of RdRp is inhibited by PGG and PAC, and PGG has a lower IC50 (5.098 ± 1.089 μM) than PAC (21.022 ± 1.202 μM), which is consistent with their inhibitory capacity of SARS-CoV-2 infection. PGG and PAC also inhibit infection by SARS-CoV and MERS-CoV. These data indicate that PGG and PAC may be candidate broad-spectrum anticoronaviral therapeutic agents, simultaneously targeting the Mpro and RdRp proteins of SARS-CoV-2.

Abiraterone Acetate Attenuates SARS-CoV-2 Replication by Interfering with the Structural Nucleocapsid Protein.

The drug repurposing strategy has been applied to the development of emergency COVID-19 therapeutic medicines. Current drug repurposing approaches have been directed against RNA polymerases and viral proteases. Recently, we found that the inhibition of the interaction between the SARS-CoV-2 structural nucleocapsid (N) and spike (S) proteins decreased viral replication. In this study, drug repurposing candidates were screened by in silico molecular docking simulation with the SARS-CoV-2 structural N protein. In the ChEMBL database, 1994 FDA-approved drugs were selected for the in silico virtual screening against the N terminal domain (NTD) of the SARS-CoV-2 N protein. The tyrosine 109 residue in the NTD of the N protein was used as the center of the ligand binding grid for the docking simulation. In plaque forming assays performed with SARS-CoV-2 infected Vero E6 cells, atovaquone, abiraterone acetate, and digoxin exhibited a tendency to reduce the size of the viral plagues without affecting the plaque numbers. Abiraterone acetate significantly decreased the accumulation of viral particles in the cell culture supernatants in a concentration-dependent manner. In addition, abiraterone acetate significantly decreased the production of N protein and S protein in the SARS-CoV-2-infected Vero E6 cells. In conclusion, abiraterone acetate has therapeutic potential to inhibit the viral replication of SARS-CoV-2.

Formulation of Neem oil-loaded solid lipid nanoparticles and evaluation of its anti-Toxoplasma activity

BackgroundToxoplasmosis is caused by an intracellular zoonotic protozoan, Toxoplasma gondii, which could be lethal in immunocompromised patients. This study aimed to synthesize Neem oil-loaded solid lipid nanoparticles (NeO-SLNs) and to evaluate the anti-Toxoplasma activity of this component.MethodsThe NeO-SLNs were constructed using double emulsification method, and their shape and size distribution were evaluated using transmission electron microscope (TEM) and dynamic light scattering (DLS), respectively. An MTT assay was employed to evaluate the cell toxicity of the component. The anti-Toxoplasma activity of NeO-SLNs was investigated using vital (trypan-blue) staining. Anti-intracellular Toxoplasma activity of NeO-SLNs was evaluated in T. gondii-infected Vero cells.ResultsThe TEM analysis represented round shape NeO-SLNs with clear and stable margins. DLS analysis showed a mean particle size 337.6 nm for SLNs, and most of nanoparticles were in range 30 to 120 nm. The cell toxicity of NeO-SLNs was directly correlated with the concentration of the component (P-value = 0.0013). The concentration of NeO-SLNs, which was toxic for at least 50% of alive T. gondii (cytotoxic concentration (CC50)), was > 10 mg/mL. The ability of NeO-SLNs to kill Toxoplasma was concentration-dependent (P-value < 0.0001), and all concentrations killed at least 70% of alive tachyzoites. Furthermore, the viability of T. gondii- infected Vero cells was inversely correlated with NeO-SLNs concentrations (P-value = 0.0317), and in the concentration 100 μg/mL at least 75% of T. gondii- infected Vero cells remained alive.ConclusionsOverall, our findings demonstrated that the NeO-SLNs was able to kill T. gondii tachyzoites in concentration 100 μg/mL with a cell toxicity lower than 20%. Such results suggest that employing SLNs as carrier for NeO can effectively kill T. gondii tachyzoites with acceptable cell toxicity. Our findings also showed that SLNs capsulation of the NeO can lead to prolonged release of the extract, suggesting that NeO-SLNs could be also employed to clear cyst stages, which should be further investigated in animal models.

Investigation of Virulence Variation of Enterovirus 71 Strains during Thermal Adaptive Evolution

To study the virulence variation of enterovirus 71 (EV71) during thermal adaptive evolution, providing references for the prevention and control of the EV71-related hand, foot and mouth disease. Parental strains and thermal-adapted strains originating from EV71 sibling strains (lineage #100 and #101) were used for plaque assay validation, CCK-8 cytotoxicity experiment, and host proteomics studies after Vero cell infection. Plaque morphology and cell inhibition rate of the viral strains were obtained. Mass spectrometry was used to examine and analyze the functions of proteins that were differential expressed in the host cells. Plaque morphology variation was found only in the heat-adapted strain of lineage #101. Increase in cell inhibition rate was observed in all the thermal-adapted strains, but the amount of increase varied in different strains. According to the results of clustering analysis and principal component analysis, after infection of Vero cells, the host cell protein profile of the heat-adapted strains was similar to that of the parental strains and the host cell protein profile of cold-adapted strains was similar to that of cell-adapted strains. It showed that 500 kinds of proteins presented inter-group difference in their expression, with 239 kinds being up-regulated proteins and 261 being down-regulated. The function of the up-regulated proteins were related to post-translational protein modification, while the functions of the down-regulated proteins were related to SRP-dependent cotranslational protein translocation/targeting to membrane and retrograde protein transport. Virulence variations of enterovirus 71 may accompany thermal adaptive evolution, but its mechanism of action still awaits further investigation.

Anti-SARS-CoV-2 potential of Cissampelos pareira L. identified by connectivity map-based analysis and in vitro studies

BackgroundViral infections have a history of abrupt and severe eruptions through the years in the form of pandemics. And yet, definitive therapies or preventive measures are not present. Herbal medicines have been a source of various antiviral compounds such as Oseltamivir, extracted using shikimic acid from star anise (Illicium verum) and Acyclovir from Carissa edulis are FDA (Food and Drug Administration) approved antiviral drugs. In this study, we dissect the anti-coronavirus infection activity of Cissampelos pareira L (Cipa) extract using an integrative approach.MethodsWe analysed the signature similarities between predicted antiviral agents and Cipa using the connectivity map (https://clue.io/). Next, we tested the anti-SARS-COV-2 activity of Cipa in vitro. Molecular docking analyses of constituents of with key targets of SARS-CoV2 protein viz. spike protein, RNA‑dependent RNA‑polymerase (RdRp) and 3C‑like proteinase. was also performed. A three-way comparative analysis of Cipa transcriptome, COVID-19 BALF transcriptome and CMAP signatures of small compounds was also performed.ResultsSeveral predicted antivirals showed a high positive connectivity score with Cipa such as apcidin, emetine, homoharringtonine etc. We also observed 98% inhibition of SARS-COV-2 replication in infected Vero cell cultures with the whole extract. Some of its prominent pure constituents e.g. pareirarine, cissamine, magnoflorine exhibited 40–80% inhibition. Comparison of genes between BALF and Cipa showed an enrichment of biological processes like transcription regulation and response to lipids, to be downregulated in Cipa while being upregulated in COVID-19. CMAP also showed that Triciribine, torin-1 and VU-0365114–2 had positive connectivity with BALF 1 and 2, and negative connectivity with Cipa. Amongst all the tested compounds, Magnoflorine and Salutaridine exhibited the most potent and consistent strong in silico binding profiles with SARS-CoV2 therapeutic targets.

Intracellular flow cytometry complements RT-qPCR detection of circulating SARS-CoV-2 variants of concern.

Basic and antiviral research on SARS-CoV-2 rely on cellular assays of virus replication in vitro. In addition, accurate detection of virus-infected cells and released virus particles is needed to study virus replication and to profile new candidate antiviral drugs. Here, by flow cytometry, we detect SARS-CoV-2 infection at single cell level and distinguish infected Vero E6 cells from uninfected bystander cells. Furthermore, based on the viral nucleocapsid expression, subpopulations of infected cells that are in an early or late phase of viral replication can be differentiated. Importantly, this flow cytometric technique complements our duplex RT-qPCR detection of viral E and N, and it can be applied to all current SARS-CoV-2 variants of concern, including the highly mutated Omicron variant.

Long-Read RNA Sequencing Identifies Polyadenylation Elongation and Differential Transcript Usage of Host Transcripts During SARS-CoV-2 In Vitro Infection.

Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) using Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analyzed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~101 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.

Differential Transcriptomics Analysis of IPEC-J2 Cells Single or Coinfected With Porcine Epidemic Diarrhea Virus and Transmissible Gastroenteritis Virus.

Porcine epidemic diarrhea (PED) and transmissible gastroenteritis (TGE) caused by porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are two highly contagious intestinal diseases in the swine industry worldwide. Notably, coinfection of TGEV and PEDV is common in piglets with diarrhea-related diseases. In this study, intestinal porcine epithelial cells (IPEC-J2) were single or coinfected with PEDV and/or TGEV, followed by the comparison of differentially expressed genes (DEGs), especially interferon-stimulated genes (ISGs), between different groups via transcriptomics analysis and real-time qPCR. The antiviral activity of swine interferon-induced transmembrane protein 3 (sIFITM3) on PEDV and TGEV infection was also evaluated. The results showed that DEGs can be detected in the cells infected with PEDV, TGEV, and PEDV+TGEV at 12, 24, and 48 hpi, and the number of DEGs was the highest at 24 hpi. The DEGs are mainly annotated to the GO terms of protein binding, immune system process, organelle part, and intracellular organelle part. Furthermore, 90 ISGs were upregulated during PEDV or TGEV infection, 27 of which were associated with antiviral activity, including ISG15, OASL, IFITM1, and IFITM3. Furthermore, sIFITM3 can significantly inhibit PEDV and TGEV infection in porcine IPEC-J2 cells and/or monkey Vero cells. Besides, sIFITM3 can also inhibit vesicular stomatitis virus (VSV) replication in Vero cells. These results indicate that sIFITM3 has broad-spectrum antiviral activity.

Circulation of SARS-CoV-2 Variants among Children from November 2020 to January 2022 in Trieste (Italy).

Introduction: The ongoing coronavirus disease 19 (COVID-19) outbreak involves the pediatric population, but to date, few reports have investigated the circulation of variants among children. Material and Methods: In this retrospective study, non-hospitalized pediatric patients with SARS-CoV-2-positive nasopharyngeal swabs (NPS) were enrolled at the Institute for Maternal and Child Health-IRCCS Burlo Garofolo, Trieste (Italy), from November 2020 to January 2022. SARS-CoV-2 variants were identified by in vitro viral isolation, amplification, automatic sequencing of the receptor binding domain (RBD) of the SARS-CoV-2 spike coding gene, and subsequent next-generation sequencing. The growth curves of the isolated strains were defined in vitro by infecting Vero-E6 cells and quantifying the viral load in the supernatants up to 72 h post-infection by qRT–PCR. The neutralization activity of sera obtained from a COVID-19 vaccinated subject, recovered (2020) patient, vaccinated and recovered (2021) patient, and seronegative subject was assessed by microneutralization assay against the different variants. Results: In total, 32 SARS-CoV-2-positive children, 16 (50%) females, with a median age of 1.4 years (range: 1 day–13 years), were enrolled. The D614G amino acid substitution was detected in all isolated and amplified viral strains. Of the 32 isolates, 4 (12.5%) carried a nonsynonymous nucleotide mutation leading to the N439K (3/4), lineage B.1.258 (∆H69/∆V70), and S477N (1/4) substitution. In 7/32 (21.8%) isolates, amino acid substitutions allowed the identification of a delta variant, lineage B.1.617.2-AY.43, and in 1/32 (3.1%), the Omicron strain (B.1.1.529.BA1) was identified. The growth curves of the B.1, B.1.258 (∆H69/∆V70), B.1.617.2-AY.43, and B.1.1.529.BA1 variants did not show any significant differences. A reduction in the serum neutralizing activity against B.1.258 (∆H69/∆V70) only in a vaccinated subject (1.7-fold difference), against B.1.617.2-AY.43 in a vaccinated subject and in recovered patients (12.7 and ≥2.5-fold differences, respectively), and against B.1.1.529.BA1 variant (57.6- and 1.4-fold differences in vaccinated and in vaccinated and recovered patients) were observed compared to the B.1 variant. Conclusions: SARS-CoV-2 variants carrying the B.1.258 (∆H69/∆V70) and S477N substitutions were reported here in a pediatric population for the first time. Although the growth rates of the isolated strains (B.1.258, B.1.617.2-AY.43, B.1.1.529.BA1) did not differ from the B.1 variant, neutralizing activity of the sera from vaccinated subjects significantly decreased against these variants. Attention should be devoted to the pediatric population to prevent the spread of new SARS-CoV-2 variants in an unvaccinated and predominantly naive population.

The Screen of a Phage Display Library Identifies a Peptide That Binds to the Surface of Trypanosoma cruzi Trypomastigotes and Impairs Their Infection of Mammalian Cells.

BackgroundChagas is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. On the order of seven million people are infected worldwide and current therapies are limited, highlighting the urgent need for new interventions. T. cruzi trypomastigotes can infect a variety of mammalian cells, recognition and adhesion to the host cell being critical for parasite entry. This study focuses on trypomastigote surface ligands involved in cell invasion.MethodsThree selection rounds of a phage peptide display library for isolation of phages that bind to trypomastigotes, resulted in the identification of the N3 dodecapeptide. N3 peptide binding to T. cruzi developmental forms (trypomastigotes, amastigotes and epimastigotes) was evaluated by flow cytometry and immunofluorescence assays. Parasite invasion of Vero cells was assessed by flow cytometry and immunofluorescence assays.ResultsPhage display screening identified the N3 peptide that binds preferentially to the surface of the trypomastigote and amastigote infective forms as opposed to non-infective epimastigotes. Importantly, the N3 peptide, but not a control scrambled peptide, inhibits trypomastigote invasion of Vero cells by 50%.ConclusionThe N3 peptide specifically binds to T. cruzi, and by doing so, inhibits Vero cell infection. Follow-up studies will identify the molecule on the parasite surface to which the N3 peptide binds. This putative T. cruzi ligand may advance chemotherapy design and vaccine development.

Gramicidin S and melittin: potential anti-viral therapeutic peptides to treat SARS-CoV-2 infection

The COVID19 pandemic has led to multipronged approaches for treatment of the disease. Since de novo discovery of drugs is time consuming, repurposing of molecules is now considered as one of the alternative strategies to treat COVID19. Antibacterial peptides are being recognized as attractive candidates for repurposing to treat viral infections. In this study, we describe the anti-SARS-CoV-2 activity of the well-studied antibacterial peptides gramicidin S and melittin obtained from Bacillus brevis and bee venom respectively. The EC50 values for gramicidin S and melittin were 1.571 µg and 0.656 µg respectively based on in vitro antiviral assay. Significant decrease in the viral load as compared to the untreated group with no/very less cytotoxicity was observed. Both the peptides treated to the SARS-CoV-2 infected Vero cells showed viral clearance from 12 h onwards with a maximal viral clearance after 24 h post infection. Proteomics analysis indicated that more than 250 proteins were differentially regulated in the gramicidin S and melittin treated SARS-CoV-2 infected Vero cells against control SARS-CoV-2 infected Vero cells after 24 and 48 h post infection. The identified proteins were found to be associated in the metabolic and mRNA processing of the Vero cells post-treatment and infection. Both these peptides could be attractive candidates for repurposing to treat SARS-CoV-2 infection.