Natural Polyphenols Inhibit the Dimerization of the SARS-CoV-2 Main Protease: The Case of Fortunellin and Its Structural Analogs

3CL-Pro is the SARS-CoV-2 main protease (MPro). It acts as a homodimer to cleave the large polyprotein 1ab transcript into proteins that are necessary for viral growth and replication. 3CL-Pro has been one of the most studied SARS-CoV-2 proteins and a main target of therapeutics. A number of drug candidates have been reported, including natural products. Here, we employ elaborate computational methods to explore the dimerization of the 3CL-Pro protein, and we formulate a computational context to identify potential inhibitors of this process. We report that fortunellin (acacetin 7-O-neohesperidoside), a natural flavonoid O-glycoside, and its structural analogs are potent inhibitors of 3CL-Pro dimerization, inhibiting viral plaque formation in vitro. We thus propose a novel basis for the search of pharmaceuticals as well as dietary supplements in the fight against SARS-CoV-2 and COVID-19.

The novel ORFV protein ORFV113 activates LPA-p38 signaling

Viruses have evolved mechanisms to subvert critical cellular signaling pathways that regulate a wide range of cellular functions, including cell differentiation, proliferation and chemotaxis, and innate immune responses. Here, we describe a novel ORFV protein, ORFV113, that interacts with the G protein-coupled receptor Lysophosphatidic acid receptor 1 (LPA1). Consistent with its interaction with LPA1, ORFV113 enhances p38 kinase phosphorylation in ORFV infected cells in vitro and in vivo, and in cells transiently expressing ORFV113 or treated with soluble ORFV113. Infection of cells with virus lacking ORFV113 (OV-IA82Δ113) significantly decreased p38 phosphorylation and viral plaque size. Infection of cells with ORFV in the presence of a p38 kinase inhibitor markedly diminished ORFV replication, highlighting importance of p38 signaling during ORFV infection. ORFV113 enhancement of p38 activation was prevented in cells in which LPA1 expression was knocked down and in cells treated with LPA1 inhibitor. Infection of sheep with OV-IA82Δ113 led to a strikingly attenuated disease phenotype, indicating that ORFV113 is a major virulence determinant in the natural host. Notably, ORFV113 represents the first viral protein that modulates p38 signaling via interaction with LPA1 receptor.

Efficient Selection of Recombinant Fluorescent Vaccinia Virus Strains and Rapid Virus Titer Determination by Using a Multi-Well Plate Imaging System

Engineered vaccinia virus (VACV) strains are used extensively as vectors for the development of novel cancer vaccines and cancer therapeutics. In this study, we describe for the first time a high-throughput approach for both fluorescent rVACV generation and rapid viral titer measurement with the multi-well plate imaging system, IncuCyte®S3. The isolation of a single, well-defined plaque is critical for the generation of novel recombinant vaccinia virus (rVACV) strains. Unfortunately, current methods of rVACV engineering via plaque isolation are time-consuming and laborious. Here, we present a modified fluorescent viral plaque screening and selection strategy that allows one to generally obtain novel fluorescent rVACV strains in six days, with a minimum of just four days. The standard plaque assay requires chemicals for fixing and staining cells. Manual plaque counting based on visual inspection of the cell culture plates is time-consuming. Here, we developed a fluorescence-based plaque assay for quantifying the vaccinia virus that does not require a cell staining step. This approach is less toxic to researchers and is reproducible; it is thus an improvement over the traditional assay. Lastly, plaque counting by virtue of a fluorescence-based image is very convenient, as it can be performed directly on the computer.

Application of a real-time cell analysis system in the process development and quantification of Rift Valley fever virus clone 13

Conventional cell-culture viral quantification methods, namely viral plaque and 50 % tissue culture infective dose assays, are time-consuming, subjective and are not suitable for routine testing. The viral plaque formation assay is the main method utilized for Rift Valley fever virus (RVFV) clone 13 quantification. The RVFV is a mosquito-borne RNA Phlebovirus belonging to the family Bunyaviridae. The virus comprises a single serotype and causes the zoonotic Rift Valley fever disease. The real-time cell analysis (RTCA) system has been developed for the monitoring of cell growth, cell adhesion, cell viability and mortality using electronic impedance technology. In this study, Vero cell growth kinetics and RVFV clone 13 replication kinetics were investigated in a roller bottle and RTCA systems. In roller bottles, Vero cell growth was measured by cell counts through trypan blue staining, whilst impedance expressed as the cell index (CI) was used for Vero growth measurement in the RTCA system. Similar growth patterns were observed in both roller bottle and RTCA systems. Exponential growth phase was observed between 48 and 100 h, followed by a stationary phase from 100 to 120 h, before cell death was observed. Viral plaque assay quantification of RVFV clone 13 in the roller bottle system and the time required for the CI to decrease 50 % after virus infection (CIT50) in the RTCA system were comparable. The highest RVFV clone 13 titre was obtained at 120 h in both roller bottle and RTCA systems. An increase in time for cytopathic effect (CPE) formation was observed with a decrease in the concentration of the virus used to infect the RTCA plates. A positive correlation was observed between the viral concentration and the time for a CPE and was used to calculate CIT50. A similar correlation was observed between the viral concentration and the time for a CPE in the roller bottle system. This study shows that the RTCA system can be used as an alternative method for conducting cell culture kinetics and viral quantification.

Viral Plaque Assay v1

This is part 3.6 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols. Collection Abstract: MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation. Abstract: Viral plaque assays are used to determine influenza viral titers. A diluted solution of egg-adapted Influenza A viruses/lung-infected tissue homogenates are applied to a six-well tissue culture dish containing a monolayer of Madin-Darby canine kidney (MDCK) cells. The infected MDCK cells grow under a semisolid overlay medium (agar) containing trypsin. A plaque is produced when a virus particle infects a cell, replicates, and then kills the cell. This process can be repeated several times as surrounding cells can be infected by newly replicated virus and killed. When visualized by eye, plaques appear as white spots. The assay is measured in PFU/mL.

Rapid Inactivation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by Tungsten Trioxide-Based (WO3) Photocatalysis

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), is transmitted person-to-person via respiratory droplets and, likely, via smaller droplet nuclei light enough to remain suspended in the air for hours and contaminate surfaces particularly in indoor conditions. Thus, effective measures are needed to prevent SARS-CoV-2 transmission in indoor environments. In this regard, we have investigated whether a system based on a filter combining Tungsten Trioxide-Based (WO3) photocatalysis and an antiviral fabric treated-copper nanocluster could inactivate SARS-CoV-2. To this purpose, an infectious SARS-CoV-2 suspension was introduced in the upper opening of a closed cylinder containing a WO3 filter and a lightbased system that activates WO3 and the antiviral fabric. From the bottom exit, aliquots of fluid were collected every 10 min (up to 60 min) and tested for their infectivity by means of a viral plaque assay in Vero cells whereas, in parallel, the viral RNA content was measured by quantitative PCR (qPCR). As we have previously shown for SARS-CoV, a 1:1,000 ratio of plaque forming units (PFU) vs. viral RNA copies was observed also for SARS-CoV-2. After 10 min, the infectious viral content was already decreased by 98.2% reaching 100% inactivation after 30 min whereas the SARS-CoV-2 RNA load was decreased of 1.5 log10 after 30 min. Thus, in spite of only a partial decrease of viral RNA, SARS-CoV-2 infectivity was completely abolished by the WO3 photocatalysis system by 30 min. These results support the hypothesis that this system could be exploited to achieve SARS-CoV-2 inactivation in indoor environments.

Culex interfor and Culex saltanensis (Diptera: Culicidae) are susceptible and competent to transmit St. Louis encephalitis virus (Flavivirus: Flaviviridae) in central Argentina

Background St. Louis encephalitis virus (SLEV) is endemic and autochthonous on the American continent. Culex pipiens quinquefasciatus is a vector of SLEV; however, Culex interfor and Culex saltanensis have also been found to be naturally infected with SLEV. The aim of this study was to determine the vector competence of C. interfor and C. saltanensis for SLEV from Argentina compared with C. p. quinquefasciatus. Methods Female of the Culex species were orally infected by feeding on viraemic chicks that had been inoculated with SLEV. Abdomens, legs and saliva blood-fed mosquitoes were analysed by viral plaque assay. Results Mosquitoes were susceptible to orally acquired infection, dissemination and transmission of SLEV in the saliva. Conclusions Our results demonstrate that C. saltanensis and C. interfor are susceptible to SLEV and competent for its transmission.

Whole Genomic Analysis and Comparison of Two Canine Papillomavirus Type 9 Strains in Malignant and Benign Skin Lesions

Papillomaviruses (PVs) usually cause benign proliferative lesions in the stratified epithelium of various animal species. However, some high-risk types of PVs have been proven to lead to malignant transformations. In dogs, several canine papillomaviruses (CPVs) have been identified in malignant lesions and are suggested as one of the risk factors for the development of squamous cell carcinomas (SCCs). In the present study, the full genomes of two CPV9 strains from recurrent SCCs of Dog 1 and skin viral papilloma (viral plaque) of Dog 2 were sequenced. Alignment of the two CPV9 sequences with the genome of the reference CPV9 strain (accession no. JF800656.1) derived from a solitary pigmented plaque was performed. Compared with the reference strain, a 27 bp in-frame insertion in the E1 gene was identified in both CPV9 strains in this study. In comparison with the CPV9 strains derived from benign lesions, the CPV9 from the SCCs of Dog 1 exhibited a 328 bp deletion at the 3′ end of the E2 and spacer sequence, which encoded a truncated deduced E2 protein and a chimeric E8^E2 protein. However, there was no difference in the mRNA expression levels of viral oncoproteins of E6 and E7 between the two CPV9 cases, suggesting that the oncogenesis of CPV9 for malignant transformation might be different from that of human papillomaviruses. The roles of E2 and E8^E2 deleted CPV9 in the oncogenesis of benign and malignant lesions should be further investigated.

Prunin suppresses viral IRES activity and is a potential candidate for treating enterovirus A71 infection

Human enterovirus A71 (HEVA71) causes hand, foot, and mouth disease (HFMD) in young children and is considered a major neurotropic pathogen but lacks effective antivirals. To identify potential therapeutic agents against HFMD, we screened a 502-compound flavonoid library for compounds targeting the HEVA71 internal ribosome entry site (IRES) that facilitates translation of the HEVA71 genome and is vital for the production of HEVA71 viral particles. We validated hits using cell viability and viral plaque assays and found that prunin was the most potent inhibitor of HEVA71. Downstream assays affirmed that prunin disrupted viral protein and RNA synthesis and acted as a narrow-spectrum antiviral against enteroviruses A and B, but not enterovirus C, rhinovirus A, herpes simplex 1, or chikungunya virus. Continuous HEVA71 passaging with prunin yielded HEVA71-resistant mutants with five mutations that mapped to the viral IRES. Knockdown studies showed that the mutations allowed HEVA71 to overcome treatment-induced suppression by differentially regulating recruitment of the IRES trans-acting factors Sam68 and hnRNPK without affecting the hnRNPA1-IRES interaction required for IRES translation. Furthermore, prunin effectively reduced HEVA71-associated clinical symptoms and mortality in HEVA71-infected BALB/c mice and suppressed hepatitis C virus at higher concentrations, suggesting a similar mechanism of prunin-mediated IRES inhibition for both viruses. These studies establish prunin as a candidate for further development as a HEVA71 therapeutic agent.