The nucleocapsid (N) protein of SARS-CoV-2 is an essential structural protein for viral replication, assembly, and immune modulation. The N protein is more structurally conserved than the spike protein, making it a promising target for antiviral drug development. To identify novel inhibitors, a high-throughput virtual screening of 65,000 antiviral compounds was conducted against both domains of the N protein using AutoDock Vina. Top candidates were prioritized based on binding energy and key molecular interactions, followed by ADMET evaluation via SwissADME. Ten compounds that met these criteria were subjected to 200 ns molecular dynamics simulations using GROMACS 2022.2, revealing four compounds (C4, C7, C8, and C9) with the highest binding affinities and stability. Fluorescence spectroscopy confirmed that the N protein’s tertiary structure remains unchanged upon compound binding. Isothermal titration calorimetry (ITC) measurements indicated binding by hydrogen-bond formation, with compounds 7 and 9 displaying the highest Gibbs free energies ( -8.67*104 and -4.36*103 cal/mol). Surface plasmon resonance (SPR) analysis corroborated the fluorescence spectroscopy and ITC findings, showing that compounds 7, 9, 8, and 4 exhibit the highest binding constants (1.67*10-4, 4.50*10-4, 2.92*10-4, and 7.77*10-4 M, respectively), while compound 9 demonstrates a faster dissociation rate, indicating lower stability. Liquid–liquid phase separation (LLPS) assays revealed that the N protein forms spherical biocondensate in the presence of poly U, and compounds 4, 7, 8, and 9 deform condensate formation. These results, confirmed by fluorescence microscopy and Förster resonance energy transfer, suggest functional inhibition of the N protein, positioning C4, C7 and C8 as promising antiviral candidates. Collectively, these findings support further evaluation of these inhibitors in cell-based and in vivo studies. Advancing these compounds holds significant promise for developing therapeutics targeting the highly conserved N protein, thereby complementing existing vaccine approaches and addressing the urgent challenge posed by neutralization-escape mutants.

Arenavirus mRNAs defy the central dogma of eukaryotic gene expression by achieving stability and efficient translation without the poly(A) tail and with short 5’UTRs, long considered essential for both processes. Despite the growing public health threat posed by arenaviruses like Lassa virus (LASV), the molecular basis for its unconventional stability and translation strategies remains poorly understood. To elucidate the molecular basis of these mechanisms, we applied Nanopore direct RNA sequencing to first define the precise termini of LASV transcripts. These analyses revealed the presence of compact, GC-rich intergenic regions (IGRs) at the 3’ end of LASV mRNAs forming conserved stem-loop structures and short, structured 5′ untranslated regions (UTRs). In addition, we uncovered a strong preference for U-rich cap-snatched sequences in NP and GPC mRNAs. Using luciferase reporters that incorporate these sequence and structural features, we found that the LASV NP 3′ IGR enhances mRNA stability up to six-fold compared to polyadenylated controls, while the native 5′ UTR drives strong, cap-dependent translation even in the absence of a poly(A) tail. Synthetic IGR variants confirmed that GC content and predicted folding energy correlate with increased resistance from 3′→5′ exonuclease decay. Finally, degron-based screens identified essential host translation factors that drive the non-canonical translation of arenavirus mRNAs.

Together, these findings define how LASV mRNAs encode their own stability and translation logic through structured RNA elements independent of canonical poly(A)-dependent interactions. This mechanism expands our understanding of non-canonical gene expression and suggests new design principles for broad antivirals that target the unique features of arenavirus mRNAs.

Pathogen-associated molecular patterns, such as viral dsRNA replication intermediates, activate expression of interferon-stimulated genes (ISGs). We generated mice transgenic for a picornaviral RNA-dependent RNA polymerase (RdRp^tg mice; the RdRp is from Theiler’s murine encephalomyocarditis virus). RdRp^tg mice have elevated tissue levels of dsRNAs (the RdRp templates on host cell RNAs), and the result is constitutive activation of the MDA5/MAVS pathway, which results in markedly elevated, lifelong expression of a broad panel of ISGs in multiple organs, including brain, spinal cord, liver, lung, heart, and kidney. It is a pure MDA5 activation state in the sense that the other main RIG-I-like receptor, RIG-I, is not involved. The ISG upregulations are completely abrogated by MDA5 (or MAVS) gene (Ifih1) knockout. RIG-I gene KO has no effect. This MDA5 pathway confinement mirrors the MDA5-confined response to replicating picornaviruses. The mice are profoundly resistant to diverse RNA and DNA viruses (EMVC, pseudorabies virus, vesicular stomatitis virus, Friend retrovirus, Theiler virus itself). However, in marked contrast to other human and murine chronic MDA5 activation states, RdRp^tg mice are healthy, with normal longevity and a lack of detectable autoimmune or autoinflammatory diseases. To determine whether the distinctive innate immune system of RdRp^tg mice is transferable post-developmentally by hematopoietic elements, we carried out serum transfers, parabiosis, and bone marrow transplantation (BMT). Intravenous serum transfers did not cause ISG upregulation, but parabiosis of RdRp^tg mice to wild-type mice conferred stable upregulation of ISGs in liver but not brain. BMT conferred increased ISG expression in both liver and brain. BMT also transferred robust resistance to a normally lethal challenge with encephalomyocarditis virus. Thus, cellular elements in blood and bone marrow confer a characteristic RdRp^tg mouse innate immune system reconfiguration with nervous system actualization requiring BMT.

RNA virus double-stranded RNAs (dsRNAs) are key pathogen-associated molecular patterns. Their sensing by MDA5 and/or RIG-I triggers transient, antivirally protective interferon-stimulated gene (ISGs) expression. We transgenically expressed the RNA-dependent RNA polymerase (RdRp) of the picornavirus Theiler virus at low levels (mRNA FPKM values ~ 2) in mice and found that RdRp templating on host RNAs generated dsRNAs in mouse tissues. The outcome was remarkable, lifelong, body-wide MDA5-MAVS pathway activation and global ISG upregulation at very high levels. MDA5 knockout abrogated this while RIG-I knockout had no effect. The constitutive MDA5 pathway activation conferred robust protection from RNA and DNA viral diseases (EMVC, pseudorabies virus, vesicular stomatitis virus, Friend retrovirus, Theiler virus itself), but in contrast to other chronic MDA5 hyperactivation states, the mice suffer no autoimmune or other health consequences. They further confound expectations by resisting a strong autoimmunity provocation (BM12 model lupus induction). However, when we knocked out one allele of the RNA editor ADAR1, the autoinflammation-protected state of RdRp^tg mice was broken. The result was a severe disease that resembles interferonopathies caused by MDA5 gain-of-function protein mutations. Adar^+/- mice were healthy but Adar^+/- RdRp^tg mice had shortened lifespan, stunted growth, premature fur graying, poorly developed teeth, skeletal abnormalities, and extreme ISG elevations. These features are similar to human Singleton–Merten syndrome, while differing in some respects such as lack of aortic calcification. A-to-I edits were both abnormally distributed and increased (numbers of genes and sites). These results, with a nucleic acid-triggered and MDA5-wild type model, illuminate the ADAR1-MDA5 axis in the healthy regulation of innate immunity and establish that viral polymerase-sourced dsRNA can drive autoinflammatory disease pathogenesis.

Baculoviruses are a family of entomopathogenic double-stranded DNA viruses enclosed within a protein matrix (occlusion bodies, OBs), widely employed in biological pest control due to their high host-specificity. This family comprises four genera, of which two selectively infect Lepidoptera: Nucleopolyhedrovirus (NPV) and Granulovirus (GV).

Mythimna unipuncta, a phytophagous noctuid moth native from North America, established itself in Europe in the 19^th century and its attacks have become more prominent in recent years in Spain. Its larvae nourish on Gramineae, causing significant losses in maize and forage grasses used for mowing and cattle grazing. Four baculovirus were isolated out of field samples from dead larvae with symptoms compatible with baculovirus infection, two Nucleopolyhedrovirus (MyunNPV-SP1 and MyunNPV-SP2) with similar REN profiles, and two Granulovirus (MyunGV-SP1 and MyunGV-SP2), with significant differences. This study explores the entomopathogenic potential of these viruses in pest control against M. unipuncta.

Phylogenetic analysis of concatenated sequences from the highly preserved regions lef8, lef9, and polyhedrin/granulin confirmed the NPVs are closely related to M. unipuncta’s Kentucky NPV isolate, while MyunGV-SP2 groups with its Hawaiian GV isolate. MyunGV-SP1, however, clusters with GVs from Xestia c-nigrum and Helicoverpa armigera, although its distance with these suggest it may represent a novel GV species.

Bioessays on ten noctuid species revealed NPVs’ host range limits to M. unipuncta, while GVs infect both Trichoplusia ni and Chrysoideixis chalcites, and MyunGV-SP1 C. includens and Spodoptera exigua additionally. Median lethal concentration (CL50) on second instar larva reached values over 10⁴OBs/ml for the NPVs and around 10⁸OBs/ml and 10⁷OBs/ml for GVs in M. unipuncta. Simultaneous infection (coinfection), a natural phenomenon, showed non-antagonistic interaction between the viruses, although NPV production prevails over GVs. Further studies are underway to assess the mean time to mortality, OB production per larva, and the effects of coinfection.

The U.S. Four Corners region, where Arizona, New Mexico, Utah, and Colorado converge, supports diverse ecosystems, high rodent biodiversity, and increasing human–wildlife contact, making it a critical area for monitoring emerging infectious diseases. Over the past century, New Mexico has undergone a tenfold population increase, accompanied by expanding urban and agricultural development. These shifts, coupled with climate-driven changes in vector and reservoir distributions, are thought to underlie the state’s persistently high incidence of hantavirus pulmonary syndrome since national surveillance began in 1993. Recently, Sin Nombre virus (SNV) was detected in several rodent species, including six not previously recognized as hosts. Together, these trends emphasize the need for proactive surveillance strategies to anticipate and prevent zoonotic spillover.

To address this need, we implemented a two-pronged approach integrating serological profiling with genomic detection. We analyzed serum and tissue samples from small mammals collected across New Mexico to assess exposure to a panel of viral and bacterial pathogens and to identify high-priority candidates for next-generation sequencing (NGS). Using a high-throughput MAGPIX multiplex immunoassay, we screened more than 700 serum samples representing over 22 species, revealing widespread exposure to pathogens endemic and non-endemic to this region of the United States. SNV-seropositive cases were further investigated through quantitative PCR to confirm active infection and narrow down top candidates for sequencing. NGS revealed mutations and diversity of circulating variants that may affect epitope structure and influence the performance of existing medical countermeasures.

By coupling broad immunological screening with targeted viral genomics, this work provides a scalable surveillance model to study pathogen exposure and transmission dynamics. Additionally, it can help inform early warning systems and strengthen national preparedness for future zoonotic threats.

Poliomyelitis remains a globally significant paralytic disease; widespread use of oral poliovirus vaccine (OPV) and, to a lesser extent, inactivated poliovirus vaccine (IPV) has driven progress toward eradication, with wild poliovirus serotypes 2 and 3 declared eradicated in 2015 and 2019, respectively, while serotype 1 persists in two endemic countries. Environmental (wastewater) surveillance, long established as a complementary tool to syndromic acute flaccid paralysis (AFP) surveillance, is critical for detecting silent poliovirus circulation and for certifying interruption of transmission. Using ongoing wastewater monitoring in the Barcelona metropolitan area, we detected a circulating vaccine-derived poliovirus type 2 (cVDPV2) in 2024 and Sabin-like type 3 (SL3) strains in 2025; the SL3 genomes carried reversion-to-virulence substitutions T6I and A54V/T. Targeted upstream sewer-catchment analysis enabled localization of the source and, in coordination with clinical surveillance, identification of an index case. Although high vaccination coverage in the affected areas was associated with no observed AFP cases, these detections underscore the necessity of sustained environmental surveillance, rapid genomic characterization, and coordinated public-health responses to limit spread and to protect vulnerable populations, particularly immunocompromised individuals, while supporting global eradication efforts.

Molnupiravir is an oral broad-spectrum nucleotide analog that enhances mutagenesis. Although authorized for COVID-19 treatment, its use raises concerns about a potential sublethal mutagenesis that could accelerate SARS-CoV-2 evolution and generate variants with enhanced pathogenicity, immune escape or antiviral resistance. To address these concerns, we performed cell culture and animal studies to characterize the effects of molnupiravir exposure on SARS-CoV-2. Compared to the original virus (OV), the molnupiravir-exposed virus 1 (MEV1) exhibited increased genetic diversity, with a 24-fold increase in new single-nucleotide polymorphisms (SNPs) and 16-to-290-fold increases in molnupiravir-signature transitions. MEV1 also acquired numerous substitutions across the genome, including in the Nsp12 and Nsp14 proteins involved in molnupiravir antiviral activity. In growth kinetics assays, compared to OV, MEV1 maintained viral fitness in Vero E6 and A549-ACE2 cells but exhibited reduced fitness in Calu-3 cells. High quasispecies diversity was maintained after MEV1 was serially passaged without drug pressure in Vero E6 and in A549-ACE2 cells. Compared to OV, infection with MEV1 caused attenuated disease in Syrian hamsters, characterized by preserved body weight, milder lung pathology, reduced viral loads and a blunted host interferon transcriptional response. MEV1 also exhibited reduced susceptibility to molnupiravir compared to OV, showing a 2-fold increase in EC₅₀ and higher infectivity titers with reduced mutagenesis after molnupiravir re-treatment. Such a phenotype was not mediated by Nsp12 or Nsp14 substitutions. In independent molnupiravir exposure experiments, MEV2 and MEV3 exhibited different mutational landscapes than MEV1 but similar reduced susceptibility to molnupiravir re-treatment. Interestingly, all MEVs maintained their susceptibility and barrier to resistance to remdesivir. In conclusion, molnupiravir-induced sublethal mutagenesis significantly enhanced SARS-CoV-2 quasispecies diversity, which resulted in reduced molnupiravir susceptibility. Importantly, hypermutated SARS-CoV-2 populations decreased virulence in vivo and did not accelerate the emergence of antiviral resistance, supporting the safe and broader use of molnupiravir against RNA viruses.

Newcastle Disease virus (NDV) is a single-stranded, non-segmented RNA virus that encodes six structural and two non-structural proteins. It belongs to the genus Avulavirus of the family Paramyxoviridae and is divided into class I and class II groups. Class I isolates are classified as a single genotype with four sub-genotypes, while class II isolates were found to be much more diverse, divided into 18 genotypes. Several complete whole-genome sequences have been reported from different parts of the world. However, despite attempts to capture the evolutionary dynamics of NDV at regional levels, there is limited research on comparative genomic and evolutionary analysis worldwide. Therefore, this study examined overall nucleotide diversity and genomic characteristics to deduce an evolutionary relationship among strains representing diverse geographical regions worldwide, utilizing various bioinformatics tools. A total of 935 whole-genome NDV sequences from 48 countries were included in this study, comprising 132 class I and 797 class II, with the remaining six classified as unknown. The nucleotide diversity(π) and the haplotype diversity among the whole genome sequences were 0.162 and 0.999, respectively. Based on RDP4 analysis, the highest number of recombination events was found in gene L, followed by genes NP, HN, and P and F, M genes, respectively. Although different algorithmic approaches reveal the number of positive selection sites in each gene, none of them exhibited a mean dN/dS greater than 1. In conclusion, this study enhances our understanding of ongoing phylogenomic and evolutionary dynamics, providing a more profound comprehension of NDVs and informing effective disease control interventions.

The continuous emergence of SARS-CoV-2 variants has intensified the urgency to discover therapeutic agents with novel mechanisms of action. This study focused on validating the antiviral potential of Allium sativum (garlic) derivatives to disrupt the critical initial step of infection: the binding of the viral Spike protein's receptor-binding domain (RBD) to the human ACE2 enzyme. Through an integrated experimental and computational approach, the extraction process was optimized, identifying that the freeze-drying of the Tigre cultivar preserved the highest biological activity. Subsequent bio-guided fractionation led to an aqueous fraction that showed good inhibitory potency in ELISA assays, blocking the RBD-ACE2 interface with 57.26% inhibition at low concentration (0.01 ug/mL). The MS chemical profile of this fraction revealed a mixture rich in polar and sulfur compounds. Molecular docking analysis identified three candidate ligands (L36, L20, and L17) that bind strongly to the RBD-ACE2 interface, with predicted affinities (Gibbs free energy) in a favorable range of -7.5 to -6.9 kcal/mol}. A rigorous in silico pharmacokinetic and toxicological analysis determined that compound L17 is the most promising candidate. L17 is distinguished by exhibiting high gastrointestinal absorption and blood-brain barrier permeability, fully complying with all major drug-likeness rules, and, most importantly, showing no predicted inhibition of Cytochrome P450 isoforms or active risk of mutagenicity or nephrotoxicity.These findings suggest that the chemical scaffold of L17 is a viral entry inhibitor derived from a natural product, with a superior safety profile, prioritizing it for the preclinical development of new therapies against SARS-CoV-2.