Search Results (97)

Background: Porcine epidemic diarrhoea virus (PEDV) is a highly contagious pathogen causing severe diarrhoea and high mortality in neonatal piglets. Methods: In this study, consensus sequences encoding the N-terminal domain of spike subunit 1 (S1-NTD) and nucleocapsid (N) protein of PEDV were cloned into a eukaryotic expression vector pJHL204 and transformed into an attenuated Salmonella Typhimurium strain JOL2500. Antigen expression was confirmed by Western blot and immunofluorescence analyses. The recombinant strains were evaluated in vivo for safety, persistence, and immunogenicity. Immunogenicity was characterised by measuring antibody response, virus neutralising assays, cytokine profiling, and flow cytometric analysis of T cell subpopulation. Protective efficacy against salmonellosis in dams and passive transfer of neutralising antibodies to suckling mice were evaluated. Results: Vaccinated mice exhibited no adverse effects or bacterial persistence in major organs, confirming the vaccine’s safety. Immunisation elicited robust PEDV- and Salmonella-specific humoral and cell-mediated immune responses. Upon Salmonella challenge, vaccinated mice showed significantly reduced bacterial loads in splenic tissues. Furthermore, vaccinated dams and their offspring induced detectable anti-PEDV neutralising antibodies, indicating successful passive antibody transfer. Conclusion: Our findings indicate that the designed vaccine constructs provide a promising platform for inducing multifaceted immuno-protectivity against PEDV and salmonellosis. Full article

Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), is an acute and highly contagious intestinal disease that inflicts substantial economic losses on the global swine industry. The nucleocapsid (N) protein of PEDV plays a critical role during viral infection and replication. In this study, the full-length N gene was cloned and expressed using the prokaryotic expression vector pET-32a (+). The purified recombinant N protein was used to immunize BALB/c mice. Subsequently, splenocytes from the immunized mice were fused with SP2/0 cells, and hybridoma cell lines secreting monoclonal antibodies (mAbs) against N protein were screened via indirect ELISA. The linear B-cell epitopes recognized by the mAbs were mapped using truncated N protein fragments. Results showed that three stable hybridoma cell lines (1A3, 1G1 and 1A10) secreting N protein-specific mAbs were obtained. Epitope mapping revealed that mAbs 1A3 and 1G1 recognized the epitope ⁷¹SNWHF⁷⁵, whereas mAb 1A10 recognized ⁶⁶RIEQP⁷⁰. Bioinformatics analysis indicated that these epitopes are highly conserved among the analyzed PEDV strains and show no cross-reactivity with the N proteins of other coronaviruses. These findings could provide valuable experimental materials for further investigation of the N protein’s structure and function and support the development of diagnostic assays and subunit antigen vaccine for PEDV. Full article

Lateral flow assays (LFAs) are among the most successful technologies for point-of-care and at-home testing, but further advances are needed to reduce costs and accelerate development. Alpaca-derived nanobodies (Nbs), single-domain antibody fragments, are promising immunoassay reagents across diverse applications. Their small size and ease of recombinant production make them particularly well suited for diagnostics. Here, we present a paper-based LFA targeting the SARS-CoV-2 nucleocapsid (N) protein that exclusively uses Nbs for direct antigen detection. We also demonstrate in-house synthesis of Nb-coated gold nanoparticles, enabling instrument-free visual readout and detection of N protein down to 40 ng/mL. This design avoids components that require mammalian cell culture and can be produced entirely from in-house reagents, simplifying manufacturing and lowering component costs. Because the assay is read visually without an external reader, it is well suited for deployment in resource-limited settings. Together, these results highlight the speed and practicality of developing Nb-based LFAs and suggest a broadly applicable strategy for detecting other clinically important disease biomarkers. Full article

Climate warming has facilitated the expansion of insect vectors and plant viral pathogens, leading to increased incidence of viral diseases in crops. Cucurbit crops, including cucumber (Cucumis sativus), melon (Cucumis melo), squash (Cucurbita pepo), and watermelon (Citrullus lanatus), are of major economic importance worldwide, but their production is severely threatened by viral infections. Among the most damaging viruses are zucchini yellow mosaic virus (ZYMV; genus Potyvirus), transmitted by aphids, and melon yellow spot virus (MYSV; genus Orthotospovirus), transmitted by thrips, both of which cause significant yield losses in Asia, including Taiwan. Previously, an attenuated ZYMV mutant, ZAC, was shown to confer effective cross-protection against ZYMV in several cucurbit species. In the present study, we engineered a recombinant virus, ZAC-MYnp, by inserting the nucleocapsid protein (NP) open reading frame of MYSV into the ZAC genome. ZAC-MYnp retained the attenuated phenotype of ZAC and remained effective in protecting against ZYMV infection, with protection rates of 70.4% and 87.0% in zucchini and muskmelon plants, respectively. In addition, under both mechanical and thrips-mediated challenge conditions, ZAC-MYnp significantly reduced MYSV symptom severity in muskmelon, with a protection rate of 66.7% and a protective efficacy of 79.0%, respectively. These results demonstrate that ZAC-derived recombinant viruses can function as a bivalent viral vaccine, offering dual protection against an aphid-borne potyvirus and a thrips-borne orthotospovirus. Our study highlights the feasibility of using a bivalent recombinant vaccine to manage two distinct insect-borne viruses in cucurbit crops. Full article

Background/Objectives: Notwithstanding the declaration by the World Health Organization in May 2023 regarding the conclusion of the COVID-19 pandemic, new cases of this potentially lethal infection continue to be documented globally, exerting a sustained influence on the worldwide economy and social structures. Contemporary SARS-CoV-2 variants, while associated with a reduced propensity for severe acute pathology, retain the capacity to induce long-term post-COVID syndrome, including in ambulatory patient populations. This clinical phenomenon may be attributable to potential autoimmune reactions hypothetically triggered by antiviral antibodies, thereby underscoring the need for developing novel, universal vaccines against COVID-19. The nucleocapsid protein (N), being one of its most conserved and highly immunogenic components of SARS-CoV-2, presents a promising target for such investigative efforts. However, the protective role of anti-N antibodies, generated during natural infection or through immunization with N-based vaccines, alongside the potential adverse effects associated with their production, remains to be fully elucidated. In the present study, we aim to identify potential sites of homology in structures or sequences between the SARS-CoV-2 N protein and human antigens detected using hyperimmune sera against N protein obtained from mice, rabbits, and hamsters. Methods: We employed Western blot analysis of lysates from human cell lines (MCF7, HEK293T, THP-1, CaCo2, Hep2, T98G, A549) coupled with mass spectrometric identification to assess the cross-reactivity of polyclonal and monoclonal antibodies generated against recombinant SARS-CoV-2 N protein with human self-antigens. Results: We showed that anti-N antibodies developed in mice and rabbits exhibit pronounced immunoreactivity towards specific components of the human proteome. In contrast, anti-N immunoglobulins from hamsters showed no non-specific cross-reactivity with either hamster or human proteomic extracts because of the lack of autoreactivity or immunogenicity differences. Subsequent mass spectrometric analysis of the immunoreactive bands identified principal autoantigenic targets, which were predominantly heat shock proteins (including HSP90-beta, HSP70, mitochondrial HSP60, and HSPA8), histones (H2B, H3.1–3), and key metabolic enzymes (G6PD, GP3, PKM, members of the 1st family of aldo-keto reductases). Conclusions: The results obtained herein highlight the differences in the development of anti-N humoral responses in humans and in the Syrian hamster model. These data provide a foundational basis for formulating clinical recommendations to predict possible autoimmune consequences in COVID-19 convalescents and are of critical importance for the rational design of future N protein-based, cross-protective vaccine candidates against novel coronavirus infections. Full article

Porcine deltacoronavirus (PDCoV) is a newly discovered porcine intestinal coronavirus that can pose a significant threat to the global commercial swine industry. We established an enzyme-linked immunosorbent assay (ELISA) detection method for the detection of PDCoV antibodies, based on the recombinant nucleocapsid (N) protein expressed using a baculovirus system. The assay was validated using positive and negative serum samples obtained from experimentally immunized rabbits and demonstrated an absence of cross-reactivity with either transmissible gastroenteritis virus (TGEV) or porcine epidemic diarrhea virus (PEDV). The recombinant PDCoV N protein antigen dilution (0.8 μg/mL), sample serum (1:400), and the enzyme-labeled secondary antibody (1:50) were used in this assay. The cut-off value was 0.355, without cross-reactivity including TGEV and PEDV. The ELISA method shows good sensitivity (96.67%), specificity (85.51%), and reproductivity (CV < 10%). We utilized the method to detect PDCoV antibodies in 600 pig serums collected from Zhejiang Province in the last four years (2021–2024). The results showed significant differences in antibody levels between regions and considerable fluctuation in positivity rates across the four-year period. As shown in the results, we developed a sensitive and specific ELISA method for detecting anti-PDCoV N antibodies, which provides a rapid and reliable diagnostic tool for PDCoV surveillance and control. This assay demonstrates significant potential for both epidemiological investigations and commercial applications in swine disease management. Full article

Background: Rapid and high-throughput diagnostic methods are essential for controlling the spread of infectious diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Lateral flow immunoassay (LFIA) strips provide a cost-effective and user-friendly platform for point-of-care testing. However, the sensitivity of conventional LFIA kits is often limited by the performance of their detection probes. This study reports a highly sensitive LFIA strip for detecting the SARS-CoV-2 nucleocapsid (NP) protein using platinum-decorated poly(2-vinylpyridine) nanoparticles (Pt-P2VPs) as probes. Methods: Monoclonal antibodies against SARS-CoV-2 NP were conjugated with Pt-P2VPs and incorporated into LFIA strips. The test line was coated with anti–SARS-CoV-2 NP monoclonal antibody, and the control line with goat anti-mouse IgG. Recombinant proteins, viral strains, and nasopharyngeal swab specimens from patients were used to evaluate assay performance, with reverse transcription polymerase chain reaction (RT-PCR) as the reference standard. Diagnostic accuracy was assessed using nonparametric statistical tests. Results: Pt-P2VP-based LFIA strips enabled sensitive detection of recombinant NP and inactivated SARS-CoV-2, with minimal cross-reactivity. In 200 clinical specimens (100 PCR-negative and 100 PCR-positive), the assay achieved 74% sensitivity and 100% specificity, with strong correlation to viral RNA load. Compared with conventional LFIA kits, Pt-P2VP strips demonstrated superior sensitivity at lower viral loads. Conclusions: Pt-P2VPs represent a promising probe material for enhancing LFIA performance and may facilitate the development of rapid, sensitive, and scalable immunoassays for infectious disease diagnostics in biomedical applications. Full article

Background: With the emergence of SARS-CoV-2 Omicron sub-variants exhibiting increased transmissibility and immune escape, booster immunization is recommended. Ideally, vaccination across all age groups, including children and adolescents, is critical to control viral spread and reduce variant emergence. The heterologous scheme consisting of two doses of SOBERANA^® 02 followed by a third dose of SOBERANA^® Plus, which are recombinant protein subunit vaccines constructed from the ancestral RBD, has proven safety, immunogenicity, and effectiveness in pediatric populations as primary series. This study evaluated the safety and immunogenicity of a SOBERANA^® Plus booster dose administered six months after primary vaccination in individuals aged 3–18 years. Methods: In this follow-up analysis of a phase I/II trial, 244 participants received the booster. Safety was monitored via active surveillance at 1 h, 24 h, and over 28 days post-vaccination. Humoral responses were assessed 28 days post-booster. Antibody responses to the SARS-CoV-2 nucleocapsid (N) protein were assessed in all collected serum samples. Results: Adverse events occurred in 18% of participants, predominantly local (85.2%) versus systemic (14.8%); no serious or severe adverse events were reported. All humoral response parameters increased significantly post-booster, including neutralizing antibodies against D614G (24.7-fold increase) and Omicron BA.1 (55.9-fold increase), with similar responses in N-negative and N-positive individuals. Importantly, cross-neutralizing activity against recent Omicron sub-variants (XBB.1.5 and EG.5.1) was also detected. Conclusions: A SOBERANA^® Plus booster is safe and significantly enhances cross-neutralizing immunity against evolving Omicron sub-variants in children and adolescents. These results highlight the potential of first-generation RBD-based vaccines to maintain broad immunity despite viral evolution. Full article

Background: Rabies virus (RABV) causes approximately 59,000 human deaths annually. Current pre- and post-exposure vaccination relies on inactivated vaccines (INVs) with limited yield and immunogenicity. We engineered a dual-cationic LNP-based nucleocapsid-like nanostructure (NLS) that co-encapsulates RABV G-mRNA and recombinant RABV-N to engage MHC-I/II pathways and enhance protection. Methods: A pVAX-RABV-G plasmid containing 5′/3′UTRs, Kozak, and poly(A) was transcribed in vitro. RABV-N with an N-terminal 6× His tag was expressed in E. coli BL21(DE3) and purified by Ni-Sepharose affinity chromatography. Dual-cationic LNPs (DHA, DOTAP Cl, mPEG-DTA2K, DOPC) were formulated by microfluidics at a 4:1 (G-mRNA:RABV-N) mass ratio. Vaccine quality was assessed by encapsulation efficiency, DLS, PDI, zeta potential, and TEM. Mice received empty LNPs, INV, G-mRNA, or NLS under varied schedules and doses. ELISA measured RABV-G/N-IgG; RFFIT determined neutralizing antibody (nAb) titers; ELISPOT quantified CTL response; qPCR assessed T-cell activation genes. On day 35 after the first immunization of vaccines, mice were challenged intramuscularly with 25 LD₅₀ of CVS-24. Results: G-mRNA purity was >95% and drove strong RABV-G expression in 293T cells. Purified RABV-N was approximately 52 kDa, >90% pure, and reactive to anti-His and anti-N antibodies. NLS achieved >95% encapsulation, a diameter of 136.9 nm, PDI 0.09, and a +18.7 mV zeta potential. A single dose yielded approximately 10 IU mL⁻¹ nAb by day 7; two doses peaked at approximately 1000 IU mL⁻¹. Mice showed 100% survival and no viral rebound in brain, spinal cord, and sciatic nerve. NLS induced stronger MHC-I/II-linked cellular immunity and higher RABV G/N-specific IFN-γ spot frequencies than G-mRNA or INV. Conclusions: The dual-antigen NLS vaccine co-delivering G-mRNA and RABV-N via dual-cationic LNPs robustly activates MHC-I/II, rapidly generates high-titer nAb (≥10 IU mL⁻¹ within 1 week), and sustains potent CD8⁺ CTL and CD4⁺ Th responses. A two-dose regimen (days 0 and 21) conferred complete protection, supporting the NLS platform as a next-generation rabies vaccine candidate. Full article

A novel skin test for an in vivo assessment of SARS-CoV-2-specific T-cell immunity was developed using CoronaDermPS, a multiepitope recombinant polypeptide encompassing MHC II–binding CD4+ T-cell epitopes of the SARS-CoV-2 structural proteins (S, E, M) and full length nucleocapsid (N). In silico epitope prediction and modeling guided antigen design, which was expressed in Escherichia coli, was purified (>95% purity) and formulated for intradermal administration. Preclinical evaluation in guinea pigs, mice, and rhesus macaques demonstrated a robust delayed type hypersensitivity (DTH) response at optimal doses (10–75 µg), with no acute or chronic toxicity, mutagenicity, or adverse effects on reproductive organs. An integrated clinical analysis included 374 volunteers stratified by vaccination status (EpiVacCorona, Gam-COVID-Vac, CoviVac) prior to COVID-19 infection (Wuhan/Alpha, Delta, Omicron variants), and SARS-CoV-2–naïve controls. Safety assessments across phase I–II trials recorded 477 adverse events, of which >88% were mild and self-limiting; no severe or anaphylactic reactions occurred. DTH responses were measured at 24 h, 72 h, and 144 h post-injection by papule and hyperemia measurements. Overall, 282/374 participants (75.4%) exhibited a positive skin test. Receiver operating characteristic analysis yielded an overall AUC of 0.825 (95% CI: 0.726–0.924), sensitivity 79.5% (95% CI: 75.1–83.3%), and specificity 85.5% (95% CI: 81.8–88.7%), with comparable diagnostic accuracy across vaccine, and variant subgroups (AUC range 0.782–0.870). CoronaDerm-PS–based skin testing offers a simple, reproducible, and low-cost method for qualitative evaluation of T-cell–mediated immunity to SARS-CoV-2, independent of specialized laboratory equipment (Eurasian Patent No. 047119). Its high safety profile and consistent performance across diverse cohorts support its utility for mass screening and monitoring of cellular immune protection following infection or vaccination. Full article

Urine-based immunoassay is a non-invasive method with demonstrated utility in detecting anti-SARS-CoV-2 antibodies in unvaccinated patients with COVID-19. To evaluate urine’s potential for serological surveys in a real-world setting, SARS-CoV-2 serology was performed on urine samples from vaccinated individuals, both with and without prior confirmed COVID-19. (1) Methods: An in-house indirect ELISA was used to measure antibodies against recombinant spike (S) and nucleocapsid (N) proteins of SARS-CoV-2 in urine and paired serum from 149 individuals vaccinated with Janssen AD26.COV2.S, an S protein-based COVID-19 vaccine. (2) Results: Anti-S and anti-N levels were higher in the urine and serum of participants with confirmed prior COVID-19 compared to those without prior infection. Urinary anti-S effectively distinguished vaccinated individuals with (AUC = 0.96) and without (AUC = 0.88) prior infection from negative controls (non-vaccinated, non-previously infected individuals) (p < 0.0001). Among vaccinated participants, urinary anti-S and anti-N identified prior infection, with AUC values of 0.73 (p < 0.0001) and 0.60 (p = 0.03), respectively, being recorded. (3) Conclusions: Findings indicate that urinary anti-SARS-CoV-2 antibodies reflect AD26.COV2.S vaccination and previous COVID-19. To further advance the methodology, studies with larger sample sizes and a greater diversity of COVID-19 vaccines are required. Full article

At least three betacoronaviruses have spilled over from bats to humans and caused severe diseases, highlighting the threat of zoonotic transmission. Thus, it is important to enhance surveillance capabilities by developing tools capable of detecting a broad spectrum of bat-borne betacoronaviruses. Three monoclonal antibodies (mAbs) targeting the nucleocapsid (N) protein were generated using recombinant N proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The cross-reactivities of these mAbs were evaluated against a panel of betacoronaviruses. Sandwich ELISAs (sELISAs) were subsequently developed to detect bat-borne betacoronaviruses that have high zoonotic potential. Among the mAbs, 7A7 demonstrated the broadest cross-reactivity, recognizing betacoronaviruses from the Sarbecovirus, Merbecovirus and Hibecovirus subgenera. The first sELISA, based on mAbs 7A7 and 6G10, successfully detected N protein in all clinical swab samples from COVID-19 patients with cycle threshold (Ct) values < 25, achieving 75% positivity overall (12/16). Using this as a reference, a second sELISA was established by pairing mAb 7A7 with mAb 8E2, which binds to multiple merbecoviruses. This assay detected the N protein of two merbecoviruses, namely the human MERS-CoV and bat-borne HKU5-CoV, at high sensitivity and has a limit of detection (LOD) that is comparable to the first sELISA used successfully to detect COVID-19 infection. These broadly reactive mAbs could be further developed into rapid antigen detection kits for surveillance in high-risk populations with close contact with wild bats to facilitate the early detection of potential zoonotic spillover events. Full article

Merbecovirus, a subgenus of Betacoronavirus, includes MERS-CoV and multiple bat-derived viruses with zoonotic potential. Given the unpredictable emergence of these viruses and their genetic diversity, development of broad-spectrum diagnostic tools is expected. In this study, we established a sandwich ELISA targeting the nucleocapsid (N) protein of merbecoviruses. We generated monoclonal antibodies (mAbs) using recombinant N protein of a bat merbecovirus, VsCoV-1, and selected cross-reactive clones for other merbecoviruses. Three mAbs showed strong reactivities with multiple merbecoviruses but not with SARS-CoV-2 or endemic human coronaviruses. Pairwise ELISA screening identified 1A8/10H6 mAbs as the optimal combination for detection of N protein from six merbecoviruses—VsCoV-1, EjCoV-3, MERS-CoV, NeoCoV, HKU4, and HKU5—with limits of detection (LODs) below 7.81 ng/mL, including 1.25 ng/mL for VsCoV-1. Infectious bat merbecovirus EjCoV-3 was detected at 1.3 × 10³ PFU/mL. No cross-reactivity was observed with non-merbecoviruses, indicating its high specificity. This sandwich ELISA offers a rapid, reproducible, and cost-effective diagnostic platform with potential for high-throughput screening and automation. Moreover, its design is amenable to adaptation into point-of-care formats such as lateral flow assays, highlighting its value for field-based surveillance and pandemic preparedness. Full article

By bringing together different variant combinations, recombination can contribute to adaptation in Coronaviridae species, some of which infect humans, and have given rise to epidemics and a pandemic. Therefore, in this work, the impact of the use of different recombination inference methods and sample sizes is addressed using data from 21 Coronaviridae species, and recombination inferences are further supported using a phylogenetic approach. Recombination patterns are shown not to vary greatly between species. A positive correlation is found between gene position and recombination rates, suggesting intrinsic variation in recombination rates along the genome. Within and between species recombination patterns are shown to differ, the module type being the most prevalent between species except for the Membrane and Nucleocapsid genes, whose products are known to interact and thus must co-evolve, explaining why the two genes are often recombined as one unit. It is also shown that within species, the module type is prevalent for the Spike gene only. Moreover, a positive correlation between recombination and selection is here reported. Therefore, intratypic recombination patterns are also shaped by selection. Recombination may thus be an important source of variability upon which selection can act. Full article

Tomato spotted wilt virus (TSWV) is a highly destructive plant pathogen and transmitted by several thrips including the western flower thrips, Frankliniella occidentalis. A structural N protein encoded in the viral genome represents the nucleocapsid protein by binding to the viral RNA genome. However, it remains unknown how the RNA-binding protein specifically interacts with the viral RNA from host RNAs in the target cells. To study the molecular basis of N function, we produced the protein in Escherichia coli and the resulting purified recombinant protein was used to investigate the protein–RNA interactions. The recombinant N protein migrated on agarose gel to the anode in the electric field due to its high basic isoelectric point. This electrostatic property led N protein to bind to DNA as well as RNA. It also bound to both single-stranded (ssRNA) and double-stranded RNA (dsRNA). However, when the total RNA was extracted from plant tissues collected from TSWV-infected host, the RNA extract using the recombinant N protein was much richer in the TSWV genome compared to that without the protein. To investigate the specificity of N protein to ssRNA, the three-dimensional structure was predicted using the AlphaFold program and showed its trimeric oligomerization with the binding pocket for ssRNA. This was supported by the differential susceptibility of N protein with ssRNA and dsRNA against RNase attack. Furthermore, a thermal shift assay to analyze the RNA and protein interaction showed that ssRNA strongly interacted with N protein compared to dsRNA. In addition, the N gene was expressed along with the multiplication of the viral RNA genome segments from the segment-specific fluorescence in situ hybridization analysis in different tissues during different developmental stages of the virus-infected F. occidentalis. These results suggest that the functional trimeric N proteins bind to the viral RNA to form a basic nucleocapsid structure at a specific virus-replicating compartment within the host cells. Full article