Nonstructural Protein Research Articles

Structural basis of Zika virus NS1 multimerization and human antibody recognition

Zika virus (ZIKV) belongs to the Flavivirus genus of the Flaviviridae family along with the four serotypes of dengue virus (DENV1–4). The recent global outbreaks of contemporary ZIKV strains demonstrated that infection can lead to neurological sequelae in adults and severe abnormalities in newborns that were previously unreported with ancestral strains. As such, there remains an unmet need for efficacious vaccines and antiviral agents against ZIKV. The non-structural protein 1 (NS1) is secreted from the infected cell and is thought to be associated with disease severity besides its proven usefulness for differential diagnoses. However, its physiologically relevant structure and pathogenesis mechanisms remain unclear. Here, we present high-resolution cryoEM structures of ZIKV recombinant secreted NS1 (rsNS1) and its complexes with three human monoclonal antibodies (AA12, EB9, GB5), as well as evidence for ZIKV infection-derived secreted NS1 (isNS1) binding to High Density Lipoprotein (HDL). We show that ZIKV rsNS1 forms tetramers and filamentous repeats of tetramers. We also observed that antibody binding did not disrupt the ZIKV NS1 tetramers as they bound to the wing and connector subdomain of the β-ladder. Our study reveals new insights into NS1 multimerization, highlights the need to distinguish the polymorphic nature of rsNS1 and isNS1, and expands the mechanistic basis of the protection conferred by antibodies targeting NS1.

The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50-100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1β in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1β. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection.

Picornavirus security proteins promote the release of extracellular vesicle enclosed viruses via the modulation of host kinases.

The discovery that extracellular vesicles (EVs) serve as carriers of virus particles calls for a reevaluation of the release strategies of non-enveloped viruses. Little is currently known about the molecular mechanisms that determine the release and composition of EVs produced by virus-infected cells, as well as conservation of these mechanisms among viruses. We previously described an important role for the Leader protein of the picornavirus encephalomyocarditis virus (EMCV) in the induction of virus-carrying EV subsets with distinct molecular and physical properties. EMCV L acts as a 'viral security protein' by suppressing host antiviral stress and type-I interferon (IFN) responses. Here, we tested the ability of functionally related picornavirus proteins of Theilers murine encephalitis virus (TMEV L), Saffold virus (SAFV L), and coxsackievirus B3 (CVB3 2Apro), to rescue EV and EV-enclosed virus release when introduced in Leader-deficient EMCV. We show that all viral security proteins tested were able to promote virus packaging in EVs, but that only the expression of EMCV L and CVB3 2Apro increased overall EV production. We provide evidence that one of the main antiviral pathways counteracted by this class of picornaviral proteins, i.e. the inhibition of PKR-mediated stress responses, affected EV and EV-enclosed virus release during infection. Moreover, we show that the enhanced capacity of the viral proteins EMCV L and CVB3 2Apro to promote EV-enclosed virus release is linked to their ability to simultaneously promote the activation of the stress kinase P38 MAPK. Taken together, we demonstrate that cellular stress pathways involving the kinases PKR and P38 are modulated by the activity of non-structural viral proteins to increase the release EV-enclosed viruses during picornavirus infections. These data shed new light on the molecular regulation of EV production in response to virus infection.

Innate Immune Evasion of PRRSV nsp11 through Degradation of the HDAC2 by Its Endoribonuclease Activity.

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family, represents a persistent menace to the global pig industry, causing reproductive failure and respiratory disease in pigs. In this study, we delved into the role of histone deacetylases (HDAC2) during PRRSV infection. Our findings revealed that HDAC2 expression is downregulated upon PRRSV infection. Notably, suppressing HDAC2 activity through specific small interfering RNA led to an increase in virus production, whereas overexpressing HDAC2 effectively inhibited PRRSV replication by boosting the expression of IFN-regulated antiviral molecules. Furthermore, we identified the virus's nonstructural protein 11 (nsp11) as a key player in reducing HDAC2 levels. Mutagenic analyses of PRRSV nsp11 revealed that its antagonistic effect on the antiviral activity of HDAC2 is dependent on its endonuclease activity. In summary, our research uncovered a novel immune evasion mechanism employed by PRRSV, providing crucial insights into the pathogenesis of this virus and guiding the development of innovative prevention strategies against PRRSV infection.

Genomic Variation in Dengue Virus Non-Structural Protein 1 (NS1)

To understand in an improved way how the dengue virus (DENV) spreads, presents, and becomes hazardous, researching its genetic makeup is necessary. The positive sense RNA of DENV encodes three structural proteins and seven non-structural proteins. One of the non-structural proteins that aids in the replication of viral RNA is the non-structural protein 1 (NS1). The objective was to identify the most frequently repeated mutations in the NS1 protein in DENV RNA isolated from dengue patients in the province Punjab, Pakistan. Selection of 120 DENV isolates was done from laboratories of tertiary care hospitals of Punjab for analysis of sequencing of the whole genome. Only 23 samples were sequenced after viral isolation, quantification and cDNA synthesis. A total of 133 different types of mutations were detected along the entire length of NS.1. The most common mutations with the highest frequency were, K324R and K347R (n=7), D278N (n=6), K174R, and F178S (n=4), found at c-terminal of NS.1 protein. Mutations K347R, K174R, and F178S are novel. Future DENV vaccination development research will be especially profited by the mutations found in the current study. During each DENV outbreak in different places, studying genomic variations is crucial for strengthening societal health and developing new policies for future outbreaks.

Investigating the Presence of Rotavirus in Wastewater Samples of Bhopal Region, India, by Utilizing Droplet Digital Polymerase Chain Reaction.

Rotavirus-induced viral gastroenteritis outbreaks result in over two million hospitalizations globally yearly. Wastewater-based epidemiology (WBE) has emerged as a crucial tool for detecting and monitoring viral outbreaks. The adoption of WBE has been instrumental in the early detection and surveillance of such viral outbreaks, providing a non-invasive method to assess public health. This study aims to utilize droplet digital polymerase chain reaction (ddPCR) technology to detect and quantify Rotavirus in wastewater samples collected from the Bhopal region of India, thereby contributing to the understanding and management of viral gastroenteritis outbreaks through environmental surveillance. In this study, we used ddPCR to detect and quantify Rotavirus in wastewater samples collected from the Bhopal region of India. We monitored its viral presence in municipal sewage treatment plants bi-weekly using an advanced ddPCR assay. Targeting the rotavirus non-structural protein 3 (NSP-3) region with custom primers and TaqMan probes, we detected virus concentration employing polyethylene glycol (PEG). Following RNA isolation, complementary DNA (cDNA) synthesis, and ddPCR analysis, our novel method eliminated standard curve dependence, propelling virus research and treatment forward. Out of the 42 samples collected, a 16.60% positivity rate was observed, indicating a moderate presence of Rotavirus in Bhopal. The wastewater treatment plants (WWTP) attached to a hospital exhibited a 42.85% positivity rate, indicating the need for targeted monitoring. Leveraging ddPCR, precise quantification of rotavirus concentrations (ranging from 0.75 to 28.9 copies/µL) facilitated understanding and supported effective remediation. This study emphasizes the importance of vigilant wastewater surveillance, especially in WWTPs with higher rotavirus prevalence. The significance of ddPCR in comparison to conventional and real-time PCR lies in its superior sensitivity and specificity in detecting and quantifying positive samples. Furthermore, it can identify positive samples even in the smallest quantities without the need for a standard curve to evaluate. This makes ddPCR a valuable tool for accurate and precise detection and quantification of samples.

A live attenuated influenza B virus vaccine expressing RBD elicits protective immunity against SARS-CoV-2 in mice

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to human health globally. It is crucial to develop a vaccine to reduce the effect of the virus on public health, economy, and society and regulate the transmission of SARS-CoV-2. Influenza B virus (IBV) can be used as a vector that does not rely on the current circulating influenza A strains. In this study, we constructed an IBV-based vector vaccine by inserting a receptor-binding domain (RBD) into a non-structural protein 1 (NS1)-truncated gene (rIBV-NS110-RBD). Subsequently, we assessed its safety, immunogenicity, and protective efficacy against SARS-CoV-2 in mice, and observed that it was safe in a mouse model. Intranasal administration of a recombinant rIBV-NS110-RBD vaccine induced high levels of SARS-CoV-2-specific IgA and IgG antibodies and T cell-mediated immunity in mice. Administering two doses of the intranasal rIBV-NS110-RBD vaccine significantly reduced the viral load and lung damage in mice. This novel IBV-based vaccine offers a novel approach for controlling the SARS-CoV-2 pandemic.

COVID-19: A deadly outbreak on planet earth

Coronavirus may be a large group of viruses and recently identified coronavirus called SARS-CoV-2 or COVID-19 was first detected in Wuhan City, China. After that it spread rapidly to other countries. Some studies show positive, and few others exhibit negative relationship between COVID-19 and sunlight. There is controversy about its intermediate host. Snake, turtles, and pangolins are considered the intermediate hosts, however bat is regarded as the primary host. It belongs to Nidovirales order, family is Coronaviridae, and subfamily is Coronavirinae. The transmission may occur through various ways. The structure of COVID-19 is enveloped and spherical with a positive single strand. Various accessory, structural, and non-structural proteins are present in it. Life cycle involves attachment, entry, replication, translation, assembly, and then its release. B.1.1.7, B.1.351, p.1, and b.1.617 are a few important variants of SARS-CoV-2. An increase of cholesterol level supports the entry of SARS-CoV-2. Statins with lower cholesterol level lessens COVID-19 entry. Fever, cough, fatigue, headache, sputum production, diarrhea, dysphonia, etc. are common symptoms of COVID-19. RT-PCR, RT-LAMP, CT scan, antibodies and antigen are various tests for COVID-19 detection. Various drugs and convalescent plasma are used for its treatment. People are also now vaccinated, and these vaccines also have side effects like fever, fatigue, headache etc. Preventive measures must be carried out to prevent its spread.

The Papain-like Protease Domain of Severe Acute Respiratory Syndrome Coronavirus 2 Conjugated with Human Beta-Defensin 2 and Co1 Induces Mucosal and Systemic Immune Responses against the Virus.

Most of the licensed vaccines against SARS-CoV-2 target spike proteins to induce viral neutralizing antibodies. However, currently prevalent SARS-CoV-2 variants contain many mutations, especially in their spike proteins. The development of vaccine antigens with conserved sequences that cross-react with variants of SARS-CoV-2 is needed to effectively defend against SARS-CoV-2 infection. Given that viral infection is initiated in the respiratory mucosa, strengthening the mucosal immune response would provide effective protection. We constructed a mucosal vaccine antigen using the papain-like protease (PLpro) domain of non-structural protein 3 of SARS-CoV-2. To potentiate the mucosal immune response, PLpro was combined with human beta-defensin 2, an antimicrobial peptide with mucosal immune adjuvant activity, and Co1, an M-cell-targeting ligand. Intranasal administration of the recombinant PLpro antigen conjugate into C57BL/6 and hACE2 knock-in (KI) mice induced antigen-specific T-cell and antibody responses with complement-dependent cytotoxic activity. Viral challenge experiments using the Wuhan and Delta strains of SARS-CoV-2 provided further evidence that immunized hACE2 KI mice were protected against viral challenge infections. Our study shows that PLpro is a useful candidate vaccine antigen against SARS-CoV-2 infection and that the inclusion of human beta-defensin 2 and Co1 in the recombinant construct may enhance the efficacy of the vaccine.

Statine-based peptidomimetic compounds as inhibitors for SARS-CoV-2 main protease (SARS-CoV‑2 Mpro)

COVID-19 is a multisystemic disease caused by the SARS-CoV-2 airborne virus, a member of the Coronaviridae family. It has a positive sense single-stranded RNA genome and encodes two non-structural proteins through viral cysteine-proteases processing. Blocking this step is crucial to control virus replication. In this work, we reported the synthesis of 23 statine-based peptidomimetics to determine their ability to inhibit the main protease (Mpro) activity of SARS-CoV-2. Among the 23 peptidomimetics, 15 compounds effectively inhibited Mpro activity by 50% or more, while three compounds (7d, 8e, and 9g) exhibited maximum inhibition above 70% and IC50 < 1 µM. Compounds 7d, 8e, and 9g inhibited roughly 80% of SARS-CoV-2 replication and proved no cytotoxicity. Molecular docking simulations show putative hydrogen bond and hydrophobic interactions between specific amino acids and these inhibitors. Molecular dynamics simulations further confirmed the stability and persisting interactions in Mpro's subsites, exhibiting favorable free energy binding (ΔGbind) values. These findings suggest the statine-based peptidomimetics as potential therapeutic agents against SARS-CoV-2 by targeting Mpro.

Phosphatidylinositol 4-Kinase III Alpha Governs Cytoskeletal Organization for Invasiveness of Liver Cancer Cells

High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.

In silico design and evaluation of multi-epitope dengue virus vaccines: a promising approach to combat global dengue burden

Dengue virus, a pervasive mosquito-borne pathogen, imposes a substantial global health burden and is responsible for numerous fatalities annually globally, with tropical and sub-tropical regions particularly susceptible to dengue outbreaks. Despite decades of efforts, there has been no effective treatment or prevention for dengue, which makes it a life-threatening disease. Hence, this study proposes an innovative bioinformatics-driven approach to construct a vaccine targeting the dengue virus. The study involved a comprehensive analysis of conserved regions of dengue virus serotypes 1–4's non-structural proteins (NS1, NS3, and NS5) and structural protein (E) to predict the potential B & T-cell epitopes which were linked with appropriate adjuvants and linkers to generate four distinct vaccine candidates. The constructed vaccine models underwent rigorous evaluation, considering physicochemical attributes, structural integrity, population coverage, and immune system response through simulation. The results confirm that these vaccine candidates are non-allergenic, non-toxic, antigenic, and immunogenic. Additionally, they exhibit 99.70% world population coverage and 100% conservation across all dengue strains, which is crucial for vaccine efficacy. A Ramachandran plot showed that 95.6% of the amino acid residues of the candidates belong to the optimal zone, while around 4% are in additional allowed regions. Further, molecular docking and dynamic simulation of interaction with the human toll-like receptor 4, a fundamental component of innate immunity, was carried out to gain more insight into interaction dynamics. As a result of these analyses, the candidates' binding dynamics and structural stability were revealed. Overall, this study presents promising vaccine candidates for addressing dengue's global health burden. Their robust design and demonstrated immunogenicity make them attractive candidates for further experimental testing and development as potential vaccines against current strains and future variants.

Japanese encephalitis virus NS1 and NS1' protein disrupts the blood-brain barrier through macrophage migration inhibitory factor-mediated autophagy.

Flaviviruses in the Japanese encephalitis virus (JEV) serogroup, such as JEV, West Nile virus, and St. Louis encephalitis virus, can cause severe neurological diseases. The nonstructural protein 1 (NS1) is a multifunctional protein of flavivirus that can be secreted by infected cells and circulate in the host bloodstream. NS1' is an additional form of NS1 protein with 52 amino acids extension at its carboxy-terminal and is produced exclusively by flaviviruses in the JEV serogroup. In this study, we demonstrated that the secreted form of both NS1 and NS1' can disrupt the blood-brain barrier (BBB) of mice, with NS1' exhibiting a stronger effect. Using the in vitro BBB model, we found that treatment of soluble recombinant JEV NS1 or NS1' protein increases the permeability of human brain microvascular endothelial cells (hBMECs) and leads to the degradation of tight junction proteins through the autophagy-lysosomal pathway. Consistently, NS1' protein exhibited a more pronounced effect compared to NS1 in these cellular processes. Further research revealed that the increased expression of macrophage migration inhibitory factor (MIF) is responsible for triggering autophagy after NS1 or NS1' treatment in hBMECs. In addition, TLR4 and NF-κB signaling was found to be involved in the activation of MIF transcription. Moreover, administering the MIF inhibitor has been shown to decrease viral loads and mitigate inflammation in the brains of mice infected with JEV. This research offers a novel perspective on the pathogenesis of JEV. In addition, the stronger effect of NS1' on disrupting the BBB compared to NS1 enhances our understanding of the mechanism by which flaviviruses in the JEV serogroup exhibit neurotropism.IMPORTANCEJapanese encephalitis (JE) is a significant viral encephalitis worldwide, caused by the JE virus (JEV). In some patients, the virus cannot be cleared in time, leading to the breach of the blood-brain barrier (BBB) and invasion of the central nervous system. This invasion may result in cognitive impairment, behavioral disturbances, and even death in both humans and animals. However, the mechanism by which JEV crosses the BBB remains unclear. Previous studies have shown that the flavivirus NS1 protein plays an important role in causing endothelial dysfunction. The NS1' protein is an elongated form of NS1 protein that is particularly produced by flaviviruses in the JEV serogroup. This study revealed that both the secreted NS1 and NS1' of JEV can disrupt the BBB by breaking down tight junction proteins through the autophagy-lysosomal pathway, and NS1' is found to have a stronger effect compared to NS1 in this process. In addition, JEV NS1 and NS1' can stimulate the expression of MIF, which triggers autophagy via the ERK signaling pathway, leading to damage to BBB. Our findings reveal a new function of JEV NS1 and NS1' in the disruption of BBB, thereby providing the potential therapeutic target for JE.

Expression of the SARS-CoV-2 receptor-binding domain by live attenuated influenza vaccine virus as a strategy for designing a bivalent vaccine against COVID-19 and influenza

Influenza and SARS-CoV-2 are two major respiratory pathogens that cocirculate in humans and cause serious illness with the potential to exacerbate disease in the event of co-infection. To develop a bivalent vaccine, capable of protecting against both infections, we inserted the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein into hemagglutinin (HA) molecule or into the open reading frame of the truncated nonstructural protein 1 (NS1) of live attenuated influenza vaccine (LAIV) virus and assessed phenotypic characteristics of the rescued LAIV-RBD viruses, as well as their immunogenicity in mouse and Syrian hamster animal models. A panel of 9 recombinant LAIV-RBD viruses was rescued using the A/Leningrad/17 backbone. Notably, only two variants with RBD insertions into the HA molecule could express sufficient quantities of RBD protein in infected MDCK cells. Intranasal immunization of mice induced high levels of anti-influenza antibody responses in all chimeric LAIV-RBD viruses, which was comparable to the LAIV virus vector. The RBD-specific antibody responses were most pronounced in the variant expressing RBD194 fragment as a chimeric HA protein. This candidate was further tested in Syrian hamsters and was shown to be immunogenic and capable of protecting animals against both infections.

An evolutionarily conserved ubiquitin ligase drives infection and transmission of flaviviruses

Mosquito-borne flaviviruses such as dengue (DENV) and Zika (ZIKV) cause hundreds of millions of infections annually. The single-stranded RNA genome of flaviviruses is translated into a polyprotein, which is cleaved equally into individual functional proteins. While structural proteins are packaged into progeny virions and released, most of the nonstructural proteins remain intracellular and could become cytotoxic if accumulated over time. However, the mechanism by which nonstructural proteins are maintained at the levels optimal for cellular fitness and viral replication remains unknown. Here, we identified that the ubiquitin E3 ligase HRD1 is essential for flaviviruses infections in both mammalian hosts and mosquitoes. HRD1 directly interacts with flavivirus NS4A and ubiquitylates a conserved lysine residue for ER-associated degradation. This mechanism avoids excessive accumulation of NS4A, which otherwise interrupts the expression of processed flavivirus proteins in the ER. Furthermore, a small-molecule inhibitor of HRD1 named LS-102 effectively interrupts DENV2 infection in both mice and Aedes aegypti mosquitoes, and significantly disturbs DENV transmission from the infected hosts to mosquitoes owing to reduced viremia. Taken together, this study demonstrates that flaviviruses have evolved a sophisticated mechanism to exploit the ubiquitination system to balance the homeostasis of viral proteins for their own advantage and provides a potential therapeutic target to interrupt flavivirus infection and transmission.

Matricellular proteins: Potential biomarkers in head and neck cancer.

The extracellular matrix (ECM) is a complex network of diverse multidomain macromolecules, including collagen, proteoglycans, and fibronectin, that significantly contribute to the mechanical properties of tissues. Matricellular proteins (MCPs), as a family of non-structural proteins, play a crucial role in regulating various ECM functions. They exert their biological effects by interacting with matrix proteins, cell surface receptors, cytokines, and proteases. These interactions govern essential cellular processes such as differentiation, proliferation, adhesion, migration as well as multiple signal transduction pathways. Consequently, MCPs are pivotal in maintaining tissue homeostasis while orchestrating intricate molecular mechanisms within the ECM framework. The expression level of MCPs in adult steady-state tissues is significantly low; however, under pathological conditions such as inflammation and cancer, there is a substantial increase in their expression. In recent years, an increasing number of studies have focused on elucidating the role and significance of MCPs in the development and progression of head and neck cancer (HNC). During HNC progression, there is a remarkable upregulation in MCP expression. Through their distinctive structure and function, they actively promote tumor growth, invasion, epithelial-mesenchymal transition, and lymphatic metastasis of HNC cells. Moreover, by binding to integrins and modulating various signaling pathways, they effectively execute their biological functions. Furthermore, MCPs also hold potential as prognostic indicators. Although the star proteins of various MCPs have been extensively investigated, there remains a plethora of MCP family members that necessitate further scrutiny. This article comprehensively examines the functionalities of each MCP and highlights the research advancements in the context of HNC, with an aim to identify novel biomarkers for HNC and propose promising avenues for future investigations.

Novel mutation N588 residue in the NS1 protein of feline parvovirus greatly augments viral replication.

Feline parvovirus (FPV) infection is highly fatal in felines. NS1, which is a key nonstructural protein of FPV, can inhibit host innate immunity and promote viral replication, which is the main reason for the severe pathogenicity of FPV. However, the mechanism by which the NS1 protein disrupts host immunity and regulates viral replication is still unclear. Here, we identified an FPV M1 strain that is regulated by the NS1 protein and has more pronounced suppression of innate immunity, resulting in robust replication. We found that the neutralization titer of the FPV M1 strain was significantly lower than that of the other strains. Moreover, FPV M1 had powerful replication ability, and the FPV M1-NS1 protein had heightened efficacy in repressing interferon-stimulated genes (ISGs) expression. Subsequently, we constructed an FPV reverse genetic system, which confirmed that the N588 residue of FPV M1-NS1 protein is a key amino acid that bolsters viral proliferation. Recombinant virus containing N588 also had stronger ability to inhibit ISGs, and lower ISGs levels promoted viral replication and reduced the neutralization titer of the positive control serum. Finally, we confirmed that the difference in viral replication was abolished in type I IFN receptor knockout cell lines. In conclusion, our results demonstrate that the N588 residue of the NS1 protein is a critical amino acid that promotes viral proliferation by increasing the inhibition of ISGs expression. These insights provide a reference for studying the relationship between parvovirus-mediated inhibition of host innate immunity and viral replication while facilitating improved FPV vaccine production.IMPORTANCEFPV infection is a viral infectious disease with the highest mortality rate in felines. A universal feature of parvovirus is its ability to inhibit host innate immunity, and its ability to suppress innate immunity is mainly accomplished by the NS1 protein. In the present study, FPV was used as a viral model to explore the mechanism by which the NS1 protein inhibits innate immunity and regulates viral replication. Studies have shown that the FPV-NS1 protein containing the N588 residue strongly inhibits the expression of host ISGs, thereby increasing the viral proliferation titer. In addition, the presence of the N588 residue can increase the proliferation titer of the strain 5- to 10-fold without affecting its virulence and immunogenicity. In conclusion, our findings provide new insights and guidance for studying the mechanisms by which parvoviruses suppress innate immunity and for developing high-yielding FPV vaccines.

Enteric coronavirus nsp2 is a virulence determinant that recruits NBR1 for autophagic targeting of TBK1 to diminish the innate immune response

ABSTRACT Non-structural protein 2 (nsp2) exists in all coronaviruses (CoVs), while its primary function in viral pathogenicity, is largely unclear. One such enteric CoV, porcine epidemic diarrhea virus (PEDV), causes high mortality in neonatal piglets worldwide. To determine the biological role of nsp2, we generated a PEDV mutant containing a complete nsp2 deletion (rPEDV-Δnsp2) from a highly pathogenic strain by reverse genetics, showing that nsp2 was dispensable for PEDV infection, while its deficiency reduced viral replication in vitro. Intriguingly, rPEDV-Δnsp2 was entirely avirulent in vivo, with significantly increased productions of IFNB (interferon beta) and IFN-stimulated genes (ISGs) in various intestinal tissues of challenged newborn piglets. Notably, nsp2 targets and degrades TBK1 (TANK binding kinase 1), the critical kinase in the innate immune response. Mechanistically, nsp2 induced the macroautophagy/autophagy process and recruited a selective autophagic receptor, NBR1 (NBR1 autophagy cargo receptor). NBR1 subsequently facilitated the K48-linked ubiquitination of TBK1 and delivered it for autophagosome-mediated degradation. Accordingly, the replication of rPEDV-Δnsp2 CoV was restrained by reduced autophagy and excess productions of type I IFNs and ISGs. Our data collectively define enteric CoV nsp2 as a novel virulence determinant, propose a crucial role of nsp2 in diminishing innate antiviral immunity by targeting TBK1 for NBR1-mediated selective autophagy, and pave the way to develop a new type of nsp2-based attenuated PEDV vaccine. The study also provides new insights into the prevention and treatment of other pathogenic CoVs. Abbreviations: 3-MA: 3-methyladenine; Baf A1: bafilomycin A1; CoV: coronavirus; CQ: chloroquine; dpi: days post-inoculation; DMVs: double-membrane vesicles; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; GIGYF2: GRB10 interacting GYF protein 2; hpi: hours post-infection; IFA: immunofluorescence assay; IFIH1: interferon induced with helicase C domain 1; IFIT2: interferon induced protein with tetratricopeptide repeats 2; IFITM1: interferon induced transmembrane protein 1; IFNB: interferon beta; IRF3: interferon regulatory factor 3; ISGs: interferon-stimulated genes; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; NBR1: NBR1 autophagy cargo receptor; nsp2: non-structural protein 2; OAS1: 2’-5’-oligoadenylate synthetase 1; PEDV: porcine epidemic diarrhea virus; PRRs: pattern recognition receptors; RIGI: RNA sensor RIG-I; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious doses; VSV: vesicular stomatitis virus.

Seroprevalence and molecular detection of foot and mouth disease virus in cattle in selected districts of Wolaita Zone, Southern Ethiopia

Foot and mouth disease (FMD) is a highly contagious, endemic, and acute viral cattle ailment that causes major economic damage in Ethiopia. Although several serotypes of the FMD virus have been detected in Ethiopia, there is no documented information about the disease's current serostatus and serotypes circulating in the Wolaita zone. Thus, from March to December 2022, a cross-sectional study was conducted to evaluate FMDV seroprevalence, molecular detection, and serotype identification in three Wolaita Zone sites. A multistage sample procedure was used to choose three peasant associations from each study region, namely Wolaita Sodo, Offa district, and Boloso sore district. A systematic random sampling technique was employed to pick 384 cattle from the population for the seroprevalence research, and 10 epithelial tissue samples were purposefully taken from outbreak individuals for molecular detection of FMDV. The sera were examined using 3ABC FMD NSP Competition ELISA to find antibodies against FMDV non-structural proteins, whereas epithelial tissue samples were analyzed for molecular detection using real-time RT-PCR, and sandwich ELISA was used to determine the circulating serotypes. A multivariable logistic regression model was used to evaluate the associated risk variables. The total seroprevalence of FMD in cattle was 46.88% (95% CI 41.86–51.88), with Wolaita Sodo Town having the highest seroprevalence (63.28%). As a consequence, multivariable logistic regression analysis revealed that animal age, herd size, and interaction with wildlife were all substantially related to FMD seroprevalence (p < 0.05). During molecular detection, only SAT-2 serotypes were found in 10 tissue samples. Thus, investigating FMD outbreaks and identifying serotypes and risk factors for seropositivity are critical steps in developing effective control and prevention strategies based on the kind of circulating serotype. Moreover, further research for animal species other than cattle was encouraged.

SARS-CoV-2 nsp15 endoribonuclease antagonizes dsRNA-induced antiviral signaling

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since its emergence in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a recombinant SARS-CoV-2 (nsp15mut) expressing catalytically inactivated nsp15, which we show promoted increased dsRNA accumulation. Infection with SARS-CoV-2 nsp15mut led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15mut and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI cultures.