Multiple Sclerosis and Chlamydia

Although the epidemiological similarity between the two diseases is true, this thesis was not discussed extensively, perhaps because it implied that some children might have been victims of abuse, which sounds false and potentially unfair. We believe that transverse myelitis and MS are the result of an infectious disease, eventually sexually transmitted by chlamydia/gonococcus, which is caused by a subclinical bacterial urethritis/inflamatory pelvic disease (IPD) among adults. In children it is the same disease but caused by common uropathogens/enterobacteria. Both UTI and MS are much more common in girls than in boys [2, 3]. These UTIs would favor herpetic proliferation via toll-like receptors (TLRs), since the virus is endemic and always present, and it is not possible to eradicate it completely. Herpes viral load is counteracted by interferon alpha 1 (IFN alpha-1), present in different cell types, from macrophages to lymphocytes passing through endothelia and fibroblasts. Interferon alpha 1, when interacting with its specific receptors, produces in the intracellular the action of antiviral RNAse and the inhibition of viral protein synthesis

In Vitro Methods to Decipher the Structure of Viral RNA Genomes

RNA viruses encode essential information in their genomes as conserved structural elements that are involved in efficient viral protein synthesis, replication, and encapsidation. These elements can also establish complex networks of RNA-RNA interactions, the so-called RNA interactome, to shape the viral genome and control different events during intracellular infection. In recent years, targeting these conserved structural elements has become a promising strategy for the development of new antiviral tools due to their sequence and structural conservation. In this context, RNA-based specific therapeutic strategies, such as the use of siRNAs have been extensively pursued to target the genome of different viruses. Importantly, siRNA-mediated targeting is not a straightforward approach and its efficiency is highly dependent on the structure of the target region. Therefore, the knowledge of the viral structure is critical for the identification of potentially good target sites. Here, we describe detailed protocols used in our laboratory for the in vitro study of the structure of viral RNA genomes. These protocols include DMS (dimethylsulfate) probing, SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) analysis, and HMX (2′-hydroxyl molecular interference). These methodologies involve the use of high-throughput analysis techniques that provide extensive information about the 3D folding of the RNA under study and the structural tuning derived from the interactome activity. They are therefore a good tool for the development of new RNA-based antiviral compounds.

Pseudorabies virus infection triggers NF-κB activation via the DNA damage response, but actively inhibits NFkB-dependent gene expression

The nuclear factor kappa B (NF-κB) pathway is known to integrate signaling associated with very diverse intra- and extracellular stressors including virus infections, and triggers a powerful (pro-inflammatory) response through the expression of NF-κB-regulated genes. Typically, the NF-κB pathway collects and transduces threatening signals at the cell surface or in the cytoplasm leading to nuclear import of activated NF-κB transcription factors. In the current work, we demonstrate that the swine alphaherpesvirus pseudorabies virus (PRV) induces a peculiar mode of NF-κB activation known as “inside-out” NF-κB activation. We show that PRV triggers the DNA damage response (DDR) and that this DDR response drives NF-κB activation since inhibition of the nuclear ataxia telangiectasia-mutated (ATM) kinase, a chief controller of DDR, abolished PRV-induced NF-κB activation. Initiation of the DDR-NF-κB signaling axis requires viral protein synthesis but occurs before active viral genome replication. In addition, the initiation of the DDR-NF-κB signaling axis is followed by a virus-induced complete shutoff of NF-κB-dependent gene expression that depends on viral DNA replication. In summary, the results presented in this study reveal that PRV infection triggers a non-canonical DDR-NF-κB activation signaling axis and that the virus actively inhibits the (potentially antiviral) consequences of this pathway, by inhibiting NF-κB-dependent gene expression. IMPORTANCE: The NF-κB signaling pathway plays a critical role in coordination of innate immune responses that are of vital importance in the control of infections. The current report generates new insights in the interaction of the alphaherpesvirus pseudorabies virus (PRV) with the NF-κB pathway, as they reveal that (i) PRV infection leads to NF-κB activation via a peculiar “inside-out’ nucleus-to-cytoplasm signal that is triggered via the DNA damage response (DDR), (ii) the DDR-NF-κB signaling axis requires expression of viral proteins but is initiated before active PRV replication, and (iii) late viral factor(s) allow PRV to actively and efficiently inhibit NF-κB-dependent (pro-inflammatory) gene expression. These data suggest that activation of the DDR-NF-κB during PRV infection is host-driven and that its potential antiviral consequences are actively inhibited by the virus.

Ginkgolic Acid Inhibits Coronavirus Strain 229E Infection of Human Epithelial Lung Cells

Since December 2019, the COVID-19 pandemic has affected more than 200 million individuals around the globe and caused millions of deaths. Although there are now multiple vaccines for SARS-CoV-2, their efficacy may be limited by current and future viral mutations. Therefore, effective antiviral compounds are an essential component to win the battle against the family of coronaviruses. Ginkgolic Acid (GA) is a pan-antiviral molecule with proven effective in vitro and in vivo activity. We previously demonstrated that GA inhibits Herpes Simplex Virus 1 (HSV-1) by disrupting viral structure, blocking fusion, and inhibiting viral protein synthesis. Additionally, we reported that GA displays broad-spectrum fusion inhibition encompassing all three classes of fusion proteins, including those of HIV, Ebola, influenza A, and Epstein Barr virus. Here, we report that GA exhibited potent antiviral activity against Human Coronavirus strain 229E (HCoV-229E) infection of human epithelial lung cells (MRC-5). GA significantly reduced progeny virus production, expression of viral proteins, and cytopathic effects (CPE). Furthermore, GA significantly inhibited HCoV-229E even when added post-infection. In light of our findings and the similarities of this family of viruses, GA holds promising potential as an effective antiviral treatment for SARS-CoV-2.

Targeting Stem-loop 1 of the SARS-CoV-2 5’UTR to suppress viral translation and Nsp1 evasion

SARS-CoV-2 is a highly pathogenic virus that evades anti-viral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the mRNA entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the SL1 region of the SARS-CoV-2 5’ UTR is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides (ASOs) inhibits viral translation and makes SARS-CoV-2 5’ UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus’ own virulence mechanism against itself could force a critical trade off between drug resistance and pathogenicity.

At the Crossroads of One Health: Lessons Learned from Foot and Mouth Disease in COVID-19 Pandemic

Foot-and-mouth disease (FMD) and Coronavirus Disease 2019 (COVID-19) are transboundary diseases caused by single-stranded positive-sense RNA viruses with similarities in genome replication and viral protein synthesis. In FMD, asymptomatic infection leads to carrier status and persistently infected animals that threaten the animals vaccinated with a trivalent inactivated whole virus vaccine. Similar information on COVID-19 is not yet available. As COVID-19 vaccination is introduced in January 2021 (since 16 January 2021 in India), its outcome can be assessed by the year-end; and while doing so, the experiences gained in the control of FMD in livestock worldwide can be applied, including monitoring of vaccination response, duration of immunity, level of herd immunity developed, and antigenic matching of the vaccine virus. Antigenic divergence of the virus is a major issue in FMD, and different geographical regions in the world use different virus strains in vaccine preparations to antigenically match circulating virus strains in respective regions for control of the disease. Non-synonymous mutations in the critical antigenic determinants of SARS-CoV-2 have been observed, and there is likely the existence/ development of antigenic variants. Therefore, during the post-COVID-19 vaccination regime, it will be essential to monitor the suitability of the in-use vaccine strain region-wise from time to time, as there could be an eruption of isolated outbreaks in a country arising due to antigenic variation and variants. In the context of the present scenario of COVID-19 around the Globe and multiple ongoing efforts to develop suitable vaccine(s) to control the disease, it is a must to develop NSP-antibody (that differentiate infected from vaccinated) assays to differentiate infected from vaccinated individuals(DIVI; DIVA in veterinary epidemiology). The techniques used and experiences gained in ongoing FMD control programs in the endemic countries can be applied to COVID-19 control in a country; and finally, the Globe. After achieving the control of COVID-19, the aim would be to eradicate the virus, which will be tough even with vaccination, as the disease/ infection may become endemic during the time to come. To achieve this, applying the principles of Progressive Control Pathway for Foot-and-Mouth Disease (PCP-FMD; FAO/OIE) to COVID-19 control will be beneficial in its control. The present review discusses the issue of control of COVID-19.

Cyclopentenone Prostaglandins: Biologically Active Lipid Mediators Targeting Inflammation

Cyclopentenone prostaglandins (cyPGs) are biologically active lipid mediators, including PGA2, PGA1, PGJ2, and its metabolites. cyPGs are essential regulators of inflammation, cell proliferation, apoptosis, angiogenesis, cell migration, and stem cell activity. cyPGs biologically act on multiple cellular targets, including transcription factors and signal transduction pathways. cyPGs regulate the inflammatory response by interfering with NF-κB, AP-1, MAPK, and JAK/STAT signaling pathways via both a group of nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-γ) dependent and PPAR-γ independent mechanisms. cyPGs promote the resolution of chronic inflammation associated with cancers and pathogen (bacterial, viral, and parasitic) infection. cyPGs exhibit potent effects on viral infections by repressing viral protein synthesis, altering viral protein glycosylation, inhibiting virus transmission, and reducing virus-induced inflammation. We summarize their anti-proliferative, pro-apoptotic, cytoprotective, antioxidant, anti-angiogenic, anti-inflammatory, pro-resolution, and anti-metastatic potential. These properties render them unique therapeutic value, especially in resolving inflammation and could be used in adjunct with other existing therapies. We also discuss other α, β -unsaturated carbonyl lipids and cyPGs like isoprostanes (IsoPs) compounds.

The multi-functional reovirus σ3 protein is a virulence factor that suppresses stress granule formation and is associated with myocardial injury

The mammalian orthoreovirus double-stranded (ds) RNA-binding protein σ3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of σ3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of σ3 binding to dsRNA during viral infection is largely unknown. To identify functions of σ3 dsRNA-binding activity during reovirus infection, we engineered a panel of thirteen σ3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of σ3. Two recombinant viruses expressing these σ3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are formed at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not mediate formation of SGs at late times post-infection. These findings indicate that σ3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that σ3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice.

RNase L limits host and viral protein synthesis via inhibition of mRNA export

RNase L is widely thought to limit viral protein synthesis by cleaving host rRNA and viral mRNA, resulting in translation arrest and viral mRNA degradation. Here, we show that the mRNAs of dengue virus and influenza A virus largely escape RNase L–mediated mRNA decay, and this permits viral protein production. However, activation of RNase L arrests nuclear mRNA export, which strongly inhibits influenza A virus protein synthesis and reduces cytokine production. The heterogeneous and temporal nature of the mRNA export block in individual cells permits sufficient production of antiviral cytokines from transcriptionally induced host mRNAs. This defines RNase L–mediated arrest of mRNA export as a key antiviral shutoff and cytokine regulatory pathway.

Structure and Activities of the NS1 Influenza Protein and Progress in the Development of Small-Molecule Drugs

The influenza virus causes human disease on a global scale and significant morbidity and mortality. The existing vaccination regime remains vulnerable to antigenic drift, and more seriously, a small number of viral mutations could lead to drug resistance. Therefore, the development of a new additional therapeutic small molecule-based anti-influenza virus is urgently required. The NS1 influenza gene plays a pivotal role in the suppression of host antiviral responses, especially by inhibiting interferon (IFN) production and the activities of antiviral proteins, such as dsRNA-dependent serine/threonine-protein kinase R (PKR) and 2′-5′-oligoadenylate synthetase (OAS)/RNase L. NS1 also modulates important aspects of viral RNA replication, viral protein synthesis, and virus replication cycle. Taken together, small molecules that target NS1 are believed to offer a means of developing new anti-influenza drugs.