Nonstructural Protein Research Articles

Porcine reproductive and respiratory syndrome virus nonstructural protein 2 promotes the autophagic degradation of adaptor protein SH3KBP1 to antagonize host innate immune responses by enhancing K63-linked polyubiquitination of RIG-I.

Non-structural protein 2 (NSP2) of PRRSV is highly variable and plays crucial roles in the virus's life cycle. To elucidate the function of NSP2 during PRRSV infection, we identified SH3KBP1 as an NSP2-interacting host protein using mass spectrometry. Exogenous SH3KBP1 expression significantly inhibited PRRSV replication by enhancing IFN-I and related ISGs production. Conversely, SH3KBP1 knockdown promoted viral replication by downregulating IFN-I and ISGs levels. In vivo experiments revealed that Sh3kbp1-/- mice were more susceptible to VSV infection, exhibiting reduced serum IFN-β levels. Further investigation showed that SH3KBP1 enhances RIG-I signal transduction by increasing K63-linked polyubiquitination through interaction with the E3 ubiquitin ligase TRIM25. We also found that PRRSV infection and NSP2 overexpression induce the autophagic degradation of SH3KBP1, counteracting the host's innate immune response. A critical interaction site was identified within the third proline-rich motif in NSP2 (453PVPAPR458). Recombinant PRRSV lacking this motif displayed reduced virulence and decreased SH3KBP1 degradation. This study advances our understanding of how PRRSV interferes with the host immune response and offers valuable insights for development novel attenuated vaccines against PRRSV.

Cannabinoid-Inspired Inhibitors of the SARS-CoV-2 Coronavirus 2'-O-Methyltransferase (2'-O-MTase) Non-Structural Protein (Nsp10-16).

The design and synthesis of antiviral compounds were guided by computationally predicted data against highly conserved non-structural proteins (Nsps) of the SARS-CoV-2 coronavirus. Chromenephenylmethanone-1 (CPM-1), a novel biphenylpyran (BPP), was selected from a unique natural product library based on in silico docking scores to coronavirus Nsps with high specificity to the methyltransferase protein (2'-O-MTase, Nsp10-16), which is responsible for viral mRNA maturation and host innate immune response evasion. To target the 2'-O-MTase, CPM-1, along with intermediate BPP regioisomers, tetrahydrophenylmethanones (TPMs), were synthesized and structurally validated via nuclear magnetic resonance (NMR) data and DP4+ structure probability analyses. To investigate the activity of these BPPs, the following in vitro assays were conducted: SARS-CoV-2 inhibition, biochemical target validation, mutagenicity, and cytotoxicity. CPM-1 possessed notable activity against SARS-CoV-2 with 98.9% inhibition at 10 µM and an EC50 of 7.65 µM, as well as inhibition of SARS-CoV-2's 2'-O-MTase (expressed and purified) with an IC50 of 1.5 ± 0.2 µM. In addition, CPM-1 revealed no cytotoxicity (CC50 of >100 µM) or mutagenicity (no frameshift or base-pair mutations). This study demonstrates the potential of computational modeling for the discovery of natural product prototypes followed by the design and synthesis of drug leads to inhibit the SARS-CoV-2 2'-O-MTase.

SARS-CoV-2 NSP6 reduces autophagosome size and affects viral replication via sigma-1 receptor.

We have provided a preliminary explanation of the effects on autophagy of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structure protein 6 from the pre-autophagic and late stages, and also found that sigma-1 receptor is likely to be used as a potential target for SARS-CoV-2 therapy to develop relevant drugs.

A comprehensive and single-use foot-and-mouth disease sero-surveillance prototype employing rationally designed multiple viral antigens

Foot-and-mouth disease (FMD) is a great havoc in agri-business-based countries like Bangladesh, for which existing detection system limits the identification and differentiation of serotypes. In this study, an engineered platform was introduced incorporating serotype-specific FMDV VP1 (structural), serotype-independent VP2 (structural) and 3AB (non-structural) proteins for holistic detection. VP1 sequences were engineered combining sequences of BAN/TA/Dh-301/2016 (serotype O), BAN/CH/Sa-304/2016 (serotype A) and BAN/DH/Sa-318/2016 (serotype Asia1). Consensus 3AB sequence was constructed from the selected prevalent viral genomes. Both VP1 and 3AB along with designed VP2 sequences were optimized for codon usage bias, stable mRNA, secondary and tertiary protein structure. Proteins were synthesized in pET-21a ( +) plasmid vector followed by transformation of Escherichia coli BL21(DE3) and IPTG-induced- expression. The western blot analysis of engineered proteins showed that purified VP1 prominently bound to anti-VP1 antibodies in vaccinated sera, whereas 3AB and VP2 bound anti-3AB and anti-VP2 antibodies, respectively from infected cattle sera, all previously collected during epidemiological investigation. Furthermore, dot blot hybridization confirmed efficient antibody capture ability of the membrane-immobilized proteins. This holistic diagnostic platform justifies a comprehensive prototype diagnostic kit that would be cost-effective and efficient for serotype specific and non-specific FMDV sero-surveillance.

Delineating cysteine-reactive compound modulation of cellular proteostasis processes.

Covalent modulators and covalent degrader molecules have emerged as drug modalities with tremendous therapeutic potential. Toward realizing this potential, mass spectrometry-based chemoproteomic screens have generated proteome-wide maps of potential druggable cysteine residues. However, beyond these direct cysteine-target maps, the full scope of direct and indirect activities of these molecules on cellular processes and how such activities contribute to reported modes of action, such as degrader activity, remains to be fully understood. Using chemoproteomics, we identified a cysteine-reactive small molecule degrader of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 14 (nsp14), which effects degradation through direct modification of cysteines in both nsp14 and in host protein disulfide isomerases. This degrader activity was further potentiated by generalized electrophile-induced global protein ubiquitylation, proteasome activation and widespread aggregation and depletion of host proteins, including the formation of stress granules. Collectively, we delineate the wide-ranging impacts of cysteine-reactive electrophilic compounds on cellular proteostasis processes.

PRRSV non-structural protein 5 inhibits antiviral innate immunity by degrading multiple proteins of RLR signaling pathway through FAM134B-mediated ER-phagy.

Viruses employ various evasion strategies to establish prolonged infection, with evasion of innate immunity being particularly crucial. Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant pathogen in swine industry, characterized by reproductive failures in sows and respiratory distress in pigs of all ages, leading to substantial economic losses globally. In this study, we found that the non-structural protein 5 (Nsp5) of PRRSV antagonizes innate immune responses via inhibiting the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs), which is achieved by degrading multiple proteins of RIG-I-like receptor (RLR) signaling pathway (RIG-I, MDA5, MAVS, TBK1, IRF3, and IRF7). Furthermore, we showed that PRRSV Nsp5 is located in endoplasmic reticulum (ER), where it promotes accumulation of RLR signaling pathway proteins. Further data demonstrated that Nsp5 activates reticulophagy (ER-phagy), which is responsible for the degradation of RLR signaling pathway proteins and IFN-I production. Mechanistically, Nsp5 interacts with one of the ER-phagy receptor family with sequence similarity 134 member B (FAM134B), promoting the oligomerization of FAM134B. These findings elucidate a novel mechanism by which PRRSV utilizes FAM134B-mediated ER-phagy to elude host antiviral immunity.IMPORTANCEInnate immunity is the first line of host defense against viral infections. Therefore, viruses developed numerous mechanisms to evade the host innate immune responses for their own benefit. PRRSV, one of the most important endemic swine viruses, poses a significant threat to the swine industry worldwide. Here, we demonstrate for the first time that PRRSV utilizes its non-structural protein Nsp5 to degrade multiple proteins of RLR signaling pathways, which play important roles in IFN-I production. Moreover, FAM134B-mediated ER-phagy was further proved to be responsible for the protein's degradation. Our study highlights the critical role of ER-phagy in immune evasion of PRRSV to favor replication and provides new insights into the prevention and control of PRRSV.

Exploring You-gui Pill for the Treatment of Diabetic Erectile Dysfunction: Data Mining Analysis, Network Pharmacology and Experiments In Vitro.

The You-gui pill (YGP) is a classical compound used for treating antidiabetic erectile dysfunction (DMED). However, the specific active ingredients responsible for its effects on DMED and their mechanisms remain unclear. In this paper, we used data mining techniques to analyze high-frequency herbs and herb combinations used in Chinese medicine for the treatment of DMED based on existing literature. Using network pharmacology to study the active components and mechanism of action of YGP against DMED, molecular docking was used to analyze the interactions of the active components with major structural proteins, nonstructural proteins, and mutants. Also, the therapeutic effect of YGP on hyperglycemic modelling and its underlying mechanisms were experimentally validated in CCEC cells by analyzing the expression of its relevant target mRNAs. Network pharmacological analysis identified the three core components of YGP as quercetin, kaempferol, and β-sitosterol, and constructed a PPI network map of common targets of YGP and DMED, which included HIF-1α, ALB, Bcl-2, INS, IL-1β, IL-6, TNF-α, CASP3, and TP53. Combined with molecular docking results, these targets had a strong binding affinity between them and the active ingredient compounds, with the highest affinity for HIF-1α and TNF-α. During the in vitro cellular assay validation, the HIF-1α, ALB, Bcl-2, TNF-α, and IL-6 mRNA in CCECs cells showed positive regulation after YGP intervention. The combination of "data mining - network pharmacology - molecular docking - experimental validation" provides a powerful methodological basis for the study of the main active components and mechanism of action of YGP against DMED, as well as the development and application of the drug.

Stearoyl coenzyme A desaturase 1 (SCD1) regulates foot-and-mouth disease virus replication by modulating host cell lipid metabolism and viral protein 2C-mediated replication complex formation.

Many positive-stranded RNA viruses, including foot-and-mouth disease virus (FMDV), alter host membranes and lipid metabolism to create a suitable microenvironment for their survival and replication within host cells. In FMDV-infected cells, the endoplasmic reticulum membrane is remodeled, forming vesicular structures that rely heavily on increased free fatty acids, thereby linking lipid metabolism to the FMDV replication complex. Nonstructural FMDV protein 2C is crucial for this complex, while host cell enzyme stearoyl coenzyme A desaturase 1 (SCD1) is vital for lipid metabolism. We found that FMDV infection alters SCD1 expression in host cells. Inhibiting SCD1 expression or its enzymatic activity markedly decreases FMDV replication, while supplementing oleic acid, a catalytic product of SCD1, regulates FMDV replication. Additionally, SCD1 forms part of the FMDV replication complex and helps recruit 2C to a detergent-resistant membrane. Our study provides insights into the pathogenesis of FMDV and a potential novel drug target against the virus.

Gold-enhanced aptasensors for highly sensitive dengue detection: a cost-effective approach

The reemergence of diseases such as Dengue has spurred the development of efficient biosensing devices. This study aims to establish a rapid, cost-effective, and efficient platform for Dengue detection. The surface of the sensor was constructed by sputtering gold onto fluorine-doped tin oxide thin films, that were previously deposited on glass substrates. Varied sputtering deposition times were used. Results obtained from X-Ray Diffraction analysis showed that the films with 4-minute deposition time had the best gold surface coverage and the best cost-benefit ratio for sensor fabrication. Subsequently, the gold thin films were functionalized with the co-immobilization of a self-organized monolayer of aptamers and 6-mercapto-1-hexanol (MCH). The monolayer was analysed using the electrochemical impedance spectroscopy technique, and the ratio of 1:25 (aptamer: total thiol) was determined for surface optimization. The performance of the aptasensor was tested for the detection of the non-structural protein 1 of Dengue serotype 4 (NS1-S4). A good sensitivity of 40.2 ± 3.7 % per decade and a low detection limit of 1.25 pg/mL were obtained. The development of this platform represents an advance in the fast fabrication of aptasensors for the detection of emerging diseases such as Dengue, contributing also to the miniaturization process of point-of-care devices.

Non-lytic spread of poliovirus requires the nonstructural protein 3CD.

Non-enveloped viruses like poliovirus (PV) have evolved the capacity to spread by non-lytic mechanisms. For PV, this mechanism exploits the host secretory autophagy pathway. Virions are selectively incorporated into autophagosomes, double-membrane vesicles that travel to the plasma membrane, fuse, and release single-membrane vesicles containing virions. Loading of cellular cargo into autophagosomes relies on direct or indirect interactions with microtubule-associated protein 1B-light chain 3 (LC3) that are mediated by motifs referred to as LC3-interaction regions (LIRs). We have identified a PV mutant with a severe defect in non-lytic spread. An F-to-Y substitution in a putative LIR of the nonstructural protein 3CD prevented virion incorporation into LC3-positive autophagosomes and virion trafficking to the plasma membrane for release. Using high-angle annular dark-field scanning transmission electron microscopy to monitor PV-induced autophagosome biogenesis, for the first time, we show that virus-induced autophagic signals yield normal autophagosomes, even in the absence of virions. The F-to-Y derivative of PV 3CD was unable to support normal autophagosome biogenesis. Together, these studies make a compelling case for a direct role of a viral nonstructural protein in the formation and loading of the vesicular carriers used for non-lytic spread that may depend on the proper structure, accessibility, and/or dynamics of its LIR. The studies of PV 3CD protein reported here will hopefully provoke a more deliberate look at the presence and function of LIR motifs in viral proteins of viruses known to use autophagy as the basis for non-lytic spread.

Prevalence of Emerging Arboviral Infections: A Tertiary Care Hospital-Based Study from Kashmir, Northern India.

Background: Arboviruses are becoming a global public health menace. The common diseases worldwide caused by arboviruses are dengue, chikungunya, and Zika. This study aims to determine the prevalence of these three arboviral infections in patients with acute febrile illness at a tertiary care hospital in Kashmir, North India. Materials and Methods: A total of 812 blood samples were collected and tested for anti-dengue, anti-chikungunya immunoglobulin M (IgM), and dengue nonstructural protein 1 antigen by enzyme linked immunosorbent assay. Molecular testing of these samples was also done to detect dengue, chikungunya, and Zika viruses by using the CDC Trioplex real-time PCR assay. Results: The prevalence of diseases found among the studied patients was dengue 105/812 (12.93%) followed by chikungunya 17/812 (2.09%), and 3 cases (0.37%) were positive for both dengue and chikungunya; however, no case of Zika was detected. Interestingly, we found that only individuals that had a history of travel to different destinations within the country were positive for these viruses. Most affected cases were males 105/812 (12.93%) compared with females 20/812 (2.46%). Dengue serotyping results indicate that Dengue virus-1 was the most commonly found serotype. The most common symptoms in patients positive for dengue and chikungunya were fever, intense fatigue, myalgia, arthralgia, retro-orbital pain, anorexia, conjunctivitis, and skin rash. Conclusion: This study showed that dengue, chikungunya, and Zika viruses are not prevalent in the indigenous population of Kashmir. However, screening for these agents is required in people who have recently traveled outside Kashmir and have symptoms of acute febrile illness.

Correction for Wu et al., "Enterovirus A71 Promotes Exosome Secretion by the Nonstructural Protein 3A Interacting with Rab27a".

Correction for Wu et al., "Enterovirus A71 Promotes Exosome Secretion by the Nonstructural Protein 3A Interacting with Rab27a".

Long term T cell response and safety of a tetravalent dengue vaccine in healthy children

As robust cellular responses are important for protection against dengue, this phase 2 study evaluated the kinetics and phenotype of T cell responses induced by TAK-003, a live-attenuated tetravalent dengue vaccine, in 4–16-year-old living in dengue-endemic countries (NCT02948829). Two hundred participants received TAK-003 on Days 1 and 90. Interferon-gamma (IFN-γ) enzyme-linked immunospot assay [ELISPOT] and intracellular cytokine staining were used to analyze T cell response and functionality, using peptide pools representing non-structural (NS) proteins NS3 and NS5 matching DENV-1, -2, -3, and -4 and DENV-2 NS1. One month after the second TAK-003 dose (Day 120), IFN-γ ELISPOT T cell response rates against any peptide pool were 97.1% (95% CI: 93.4% to 99.1%), and similar for baseline dengue seropositive (96.0%) and seronegative (98.6%) participants. IFN-γ ELISPOT T cell response rates at Day 120 were 79.8%, 90.2%, 77.3%, and 74.0%, against DENV-1, -2, -3, and -4, respectively, and remained elevated through 3 years post-vaccination. Multifunctional CD4 and CD8 T cell responses against DENV-2 NS peptides were observed, independent of baseline serostatus: CD8 T cells typically secreted IFN-γ and TNF-α whereas CD4 T cells secreted ≥ 2 of IFN-γ, IL-2 and TNF-α cytokines. NAb titers and seropositivity rates remained substantially elevated through 3 years post-vaccination. Overall, TAK-003 was well tolerated and elicited durable T cell responses against all four DENV serotypes irrespective of baseline serostatus in children and adolescents aged 4–16 years living in dengue-endemic countries. TAK-003-elicited CD4 and CD8 T cells were multifunctional and persisted up to 3 years post-vaccination.

Broad-Spectrum Antiviral Agents against SARS-CoV-2 Variants Inhibit the Conserved Viral Protein Nsp1-RNA Interaction.

The ongoing global threats posed by COVID-19 pandemic, catalyzed by SARS-CoV-2, underscores the pressing need for effective antiviral strategies. The viral non-structural protein 1 (Nsp1) significantly influences pathogenicity by impeding host protein expression and enhancing viral RNA translation through its interaction with the stem-loop 1 (SL1) in the 5' untranslated region (UTR). We have developed a novel dual-luciferase reporter assay, designed to investigate the critical Nsp1-SL1 interaction, and identified P23E02 as a potential inhibitor. Our investigation, combining molecular docking studies and alanine mutagenesis, has unveiled that P23E02 disrupts Nsp1-40S ribosomal subunit interaction, liberating translational inhibition and empowering host antiviral responses. P23E02 exhibits antiviral efficacy against various sarbecoviruses, making it a promising candidate for combatting COVID-19 and related diseases. This study underscores the therapeutic potential of targeting the Nsp1/SL1 axis and lays the foundation for innovative antiviral interventions, ultimately fortifying global preparedness against future viral threats.

Study of non-silent and deletion mutations of SARS-CoV-2 in relation to viral behavior in the infectious process

SARS-CoV-2 is a 30,000-nucleotide single-stranded RNA virus encoding structural, non-structural and accessory proteins. Its origin is uncertain, with hypotheses suggesting evolution from bats or pangolins. Genetic recombination events between strains may have resulted in the emergence of SARS-CoV-2, making it capable of infecting humans and increasing its transmissibility. Its mutability rate is similar to that of other coronaviruses, however, due to the occurrence of the pandemic and the propagation clusters, there was an intensification in the number of mutations generated, which evolutionarily gave rise to lineages, sublineages, variants and strains, consecutively. Such mutations, especially non-synonymous ones, phenotypically reflect a variation in virulence, associated with factors of transmissibility, pathogenicity, multiplication, immunological evasion that differ from their ancestors, which are accumulating and becoming fixed in the lineages of this sarbecovirus. In this way, this study aims to understand such non-silent mutations by correlating changes to the viral infectious process.

A single-dose circular RNA vaccine prevents Zika virus infection without enhancing dengue severity in mice

Antibody-dependent enhancement (ADE) is a potential concern for the development of Zika virus (ZIKV) vaccines. Cross-reactive but poorly neutralizing antibodies, usually targeting viral pre-membrane or envelope (E) proteins, can potentially enhance dengue virus (DENV) infection. Although E domain III (EDIII) contains ZIKV-specific epitopes, its immunogenicity is poor. Here, we show that dimeric EDIII, fused to human IgG1 Fc fragment (EDIII-Fc) and encoded by circular RNA (circRNA), induces better germinal center reactions and higher neutralizing antibodies compared to circRNAs encoding monomeric or trimeric EDIII. Two doses of circRNAs encoding EDIII-Fc and ZIKV nonstructural protein NS1, another protective antigen, prevent lethal ZIKV infection in neonates born to immunized C57BL/6 mice and in interferon-α/β receptor knockout adult C57BL/6 mice. Importantly, a single-dose optimized circRNA vaccine with improved antigen expression confers potent and durable protection without inducing obvious DENV ADE in mice, laying the groundwork for developing flavivirus vaccines based on circRNAs encoding EDIII-Fc and NS1.

Computational identification of Vernonia cinerea-derived phytochemicals as potential inhibitors of nonstructural protein 1 (NSP1) in dengue virus serotype-2.

Dengue virus (DENV) infection, spread by Aedes aegypti mosquitoes, is a significant public health concern in tropical and subtropical regions. Among the four distinct serotypes of DENV (DENV-1 to DENV-4), DENV-2 is associated with the highest number of fatalities worldwide. However, there is no specific treatment available for dengue patients caused by DENV-2. This study aimed to identify inhibitory phytocompounds in silico in Vernonia cinerea (V. cinerea), a widely used traditional medicinal plant, for treating DENV-2 associated illnesses. The chemical structures of 17 compounds from V. cinerea were sourced from the Indian Medicinal Plants, Phytochemistry, and Therapeutics (IMPPAT) database. These compounds underwent geometry optimization, were screened against nonstructural protein 1 (NSP1) of DENV-2, and further validated through molecular dynamics simulations (MDS). Baicalein, an established drug against DENV-2, was used for validation in molecular screening, MDS, and MM-GBSA analyses. Among these compounds, Beta-amyrin, Beta-amyrin acetate, Chrysoeriol, Isoorientin, and Luteolin showed promising potential as inhibitors of the NSP1 of DENV-2, supported by the results of thermodynamic properties, molecular orbitals, electrostatic potentials, spectral data and molecular screening. Besides, these compounds adhered to the Lipinski's "rule of 5", showing no hepatotoxicity/cytotoxicity, with mixed mutagenicity, immunotoxicity, and carcinogenicity. Furthermore, final validation through MDS confirmed their potential, demonstrating stable tendencies with significant inhibitory activities against NSP1 of DENV-2 over the control drug Baicalein. Among the screened compounds, Chrysoeriol emerged as the most promising inhibitor of NSP1 of DENV-2, followed by Luteolin and Isoorientin. Taken together, our results suggest that Chrysoeriol is the best inhibitor of NSP1 of DENV-2, which could be evaluated as a therapeutic agent or a lead compound to treat and manage DENV-2 infections.

A curious case of a negative control line on a dengue duo assay due to interference - A case report.

An elderly patient who presented with 1week of fever and respiratory symptoms was tested for dengue infection with an Abbott Bioline™ Dengue Duo immunochromatographic assay. Unexpectedly, the control line of the dengue non-structural protein 1 (NS1) component was absent, necessitating result invalidation. It remained absent when the test was repeated on the SD Biosensor Standard™ Q Dengue Duo, but present on the Wells Bio careUSTM Dengue Combo and Asan Easy Test® Dengue DUO assays, suggesting potential interference. Dilution, polyethylene glycol (PEG) precipitation and centrifugation with a 100kDa filter were performed to reduce/remove the potential interferent. Sera from other patients that showed a control line, and a test line that was either positive or negative for NS1, were used as controls. Upon dilution with negative control serum, a faint control line emerged. PEG precipitation resulted in disappearance of control and test lines in the positive control. Filtration led to emergence of the control line for the patient's serum but caused the test line for the positive control serum to disappear. Overall, investigations suggested the presence of a high molecular weight (>100kDa) substance which interferes with chicken IgY-anti-chicken IgY binding at the control line of affected assays. Our results highlight two important points: firstly, some commonly used laboratory procedures (e.g. PEG or filtration) may inadvertently remove the target biomarker (e.g. multimeric NS1) and should be interpreted with appropriate controls. Secondly, alternative kits that use a different antigen-antibody combination for the control line can be considered when similar patients are encountered in future.

Evaluation and comparison of performances of six commercial NSP ELISA assays for foot and mouth disease virus in Thailand

ELISA kits that detect antibodies to the non-structural protein (NSP) of the foot-and-mouth disease virus (FMDV), commonly referred to as NSP-ELISA, can distinguish between vaccinated and naturally infected animals. They can play an essential role in demonstrating ‘proof-of-freedom’ during the control of FMD. Although various NSP-ELISA kits are available in Thailand, information regarding their performance is lacking. To select the most appropriate NSP-ELISA kit for our specific purpose, we must compare their performance using carefully characterized sera. This will ensure that we maximize the benefits of our testing. In this study, six NSP-ELISA kits sold in Thailand―Biovet, ID Screen, VDPro, IDEXX, PrioCHECK, and KUcheck-F―were evaluated and compared. A total of 800 serum samples were examined, including samples from 357 cattle and 29 buffaloes in outbreak areas, as well as 14 swine serum samples from the Vaccine Quality Control Unit of the Bureau of Veterinary Biologics, Ministry of Agriculture and Cooperation, Thailand. Four hundred samples were confirmed to originate from animals infected with FMDV through ELISA typing (n = 11, tested as representative samples in each farm) and/or RT-PCR (n = 400, all samples), serving as positive control sera. Additionally, 400 negative control sera were obtained from Japan (97 cattle and 300 pigs) and Australia (3 goats), certified by the World Organisation for Animal Health as ‘free of FMD’. The sensitivity and specificity of the six tests were determined based on the results obtained from two-by-two tables. Cohen’s kappa statistics were calculated for the six tests to assess their concordance, and the diagnostic accuracy of the assays was also determined. For all six NSP-ELISA kits, the sensitivity ranged from 97.75 to 99.50%, and the specificity ranged from 97.25 to 100%. Cohen’s kappa statistics ranged from 0.96 to 1.00, and diagnostic accuracy ranged from 98.13 to 99.75%. The study results indicated that the test kits have statistically similar sensitivity, specificity, concordance, and diagnostic accuracy, suggesting they can be used interchangeably. However, ID Screen demonstrated the highest sensitivity and specificity among all kits tested. Therefore, if a single kit were to be selected from the six evaluated, ID Screen would be the most appropriate choice. These findings can aid in selecting the most suitable test kit. Therefore, it is recommended to consider purchasing a diverse range of effective test kits. Furthermore, these findings can provide guidance for expanding the use of test kits, particularly with the growing availability of NSP-ELISA kits in the market.

Distinct chikungunya virus polymerase palm subdomains contribute to viral protein accumulation and virion production.

Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus has established the importance of the nsP4 residue cysteine 483 in replication. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the coxsackievirus B3 3D polymerase. We rescued each variant in mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to host-specific replication. In C6/36 cells, we found that while the nsP4 variants had replicase function similar to that of wild-type CHIKV, many variants presented changes in protein accumulation and virion production even when viral nonstructural and structural proteins were produced. Finally, we found that WT CHIKV and nsP4 variant replication and protein production could be enhanced in mammalian cells at 28°C, yet growing virus under these conditions led to changes in virus infectivity. Taken together, these studies highlight that distinct nsP4 subdomains are required for proper RNA transcription and translation, having major effects on virion production.