In Atlantic salmon hatcheries, Salmon gill poxvirus (SGPV) can cause parachute, high mortality that can be alleviated by immediate countermeasures. However, this is hampered by a lack of good rapid diagnostics for SGPV. In this study, we developed and evaluated two rapid non-invasive isothermal amplification assays, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), each coupled with a lateral flow device (LFD) for detection of SGPV in gill mucus or lysates from SGPV-infected salmon. Both assays demonstrated high analytical specificity and strong agreement with the reference real-time PCR assay when purified DNA was tested. The RPA-LFD assay achieved sensitivity comparable to real-time PCR but was consistently inhibited by crude gill mucus, limiting its suitability for direct field testing. In contrast, the LAMP-LFD assay maintained high sensitivity and reproducibility using crude gill mucus without prior nucleic acid extraction, detecting SGPV in 14 of 15 field samples with excellent agreement with real-time PCR. Although analytical sensitivity of LAMP was approximately fourfold lower than real-time PCR, this did not compromise SGPV detection in relevant samples and a final mucus concentration of approximately 4% was optimal for SGPV amplification. Both assays showed no cross-reactivity with common salmon gill pathogens, including Candidatus Branchiomonas cysticola, Desmozoon lepeophtherii, and Paramoeba perurans. The LAMP-LFD workflow provides visual results within 45minutes, representing a viable, rapid, and non-invasive diagnostic platform for field surveillance. These findings demonstrate that LAMP-LFD is well suited for SGPV detection and has strong potential to improve early outbreak detection and enhance the management of SGPV in salmon aquaculture.

Porcine epidemic diarrhoea virus (PEDV) can cause fatal watery diarrhoea in newborn piglets, posing a serious threat to the global pig industry. The spike (S) protein is the main envelope antigen of PEDV and an ideal target for antigen detection. In this study, we immunised rabbits to prepare antibodies directed to the receptor binding domain (RBD) and the S2 domain and used the ExpiCHO system to express five previously produced nanobodies that specifically recognise the PEDV S protein. The best antibody pairing scheme was determined via checkerboard titration, and a double antibody sandwich ELISA (DAS-ELISA) was established, using mixed RBD and S2-specific rabbit pAbs as the capture antibody and HRP-labelled Nb9 nanobody as the detection antibody. The cut-off value was set at 0.250-0.280 based on 66 negative anal swab samples. This method showed no cross-reactivity with other diarrhoea viruses. The detection limit for live virus was 10[2.6] TCID50/mL; when the OD450 values for S protein ranged from 0.0625 μg/mL to 3 μg/mL, there was a good linear relationship with antigen concentration. Both intra- and inter-batch coefficients of variation were below 10%, showing good repeatability. Analysis of 166 clinical faecal samples alongside RT-PCR gave an overall concordance of 95.78% with a κ value of 0.915. ROC curve analysis showed a sensitivity of 90.20% and specificity of 92.65%. In summary, the nanobody DAS-ELISA method established in this study can detect PEDV antigens with high specificity and sensitivity, providing a new approach for on-site PEDV surveillance and quantitative vaccine antigen assessment.

Measles virus remains a significant global health threat, and despite the availability of an effective vaccine, measles cases continue to increase worldwide in recent years. Genomic surveillance has become an essential tool for monitoring virus circulation and investigating outbreaks. Here, we describe a wet laboratory method for whole‑genome sequencing of measles virus using a tiled amplicon approach and Illumina sequencing technology. A previously published Oxford Nanopore based tiled primer scheme was adapted to include both circulating measles genotypes and for use on the Illumina platform. Two Illumina library preparation kits, Illumina DNA Prep (IDP) and Nextera XT (XT), were evaluated for performance. The IDP kit demonstrated more complete genomes and consistent genome coverage compared with XT. Using quantified reference genomes, the limit of detection was determined to be 10,000 genome copies for genotype B3 and D8. Sequence accuracy was evaluated using previously characterized clinical samples and showed high concordance. This method provides a reliable and sensitive approach for measles virus whole genome sequencing using Illumina platforms and is suitable for genomic surveillance applications.

The global resurgence of mpox, caused by monkeypox virus (MPXV), has highlighted the need for effective antiviral strategies, rapid diagnostics, vaccination, and integrated preparedness approaches. This review critically examines recent advances in MPXV biology, pathogenesis, transmission, clinical manifestations, prevention, diagnosis, treatment, and emerging nanomedicine-based strategies within a One Health framework. Particular emphasis is placed on nanoparticle-enabled vaccines, diagnostics, therapeutics, and wastewater surveillance, highlighting their potential to improve antigen delivery, diagnostic sensitivity, targeted drug delivery, and population-level monitoring. The review also compares conventional and nanoscale approaches and evaluates the current evidence supporting their efficacy and translational potential. Importantly, most nano-enabled MPXV interventions remain at the preclinical stage, and their reported performance cannot be directly compared because of differences in nanoparticle composition, target antigens, experimental models, assay conditions, and clinical validation strategies. Major challenges include long-term safety, pharmacokinetics, reproducibility, scalability, storage stability, regulatory approval, and the limited availability of well-designed clinical studies. Within the One Health framework, effective MPXV control also requires coordinated surveillance of human, animal, and environmental reservoirs, including standardized wastewater monitoring and cross-sectoral data sharing. Future research should therefore prioritize rigorous clinical validation, standardized evaluation protocols, diverse MPXV clade testing, long-term safety assessment, scalable manufacturing, and integrated human-animal-environmental surveillance.

Nervous necrosis virus (NNV) is a bipartite positive-sense RNA virus that causes viral nervous necrosis in marine larval fish worldwide. Understanding the intracellular trafficking and spatial organization of viral RNAs is essential for elucidating the NNV replication cycle; however, methods that allow strand-specific and simultaneous visualization of multiple NNV RNAs at the subcellular level are limited. In the present study, we developed a strand-specific RNA-fluorescence in situ hybridization (RNA-FISH) protocol for visualizing the subcellular localization of NNV RNAs during infection. Detection specificity was first validated by Northern blot analysis, which distinguished positive-strand RNA1 and RNA2 from their corresponding negative-strands. The designed probes selectively detected each viral RNA without cross-reactivity, enabling simultaneous visualization of two viral RNA species within a single infected cell. This technique can be combined with immunocytochemistry or organelle trackers staining to examine the spatial relationship between viral RNAs and viral or host proteins. Incorporation of 5-bromouridine (BrU) allowed the detection of newly synthesized viral RNAs and the tracking of their intracellular trafficking. Using this approach, nascent viral RNAs were observed to move out of mitochondrial replication/transcription sites toward vesicles associated with coat protein translation and subsequently to the remodeled microtubule-organizing center (MTOC) for viral assembly. This protocol provides a robust tool for studying the intracellular dynamics of viral RNAs and their coordination with viral and host factors and can be adapted for the investigation of other RNA viruses.

Goose astrovirus genotype 1 (GAstV-1) is a major causative agent of visceral gout in goslings, leading to substantial economic losses across Chinese poultry farms. Currently, no commercial vaccine is available, rendering early serological surveillance essential for disease control. In this study, a sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed using a monoclonal antibody (mAb) targeting the ORF2 capsid protein of GAstV-1. Following optimization of antigen coating concentration, serum dilution, mAb concentration, and incubation time, the assay demonstrated a detection limit at a serum dilution of 1:160 and showed no cross-reactivity with antisera against other avian pathogens, including H9N2 avian influenza virus (H9N2-AIV), avian reovirus (ARV), goose parvovirus (GPV), goose astrovirus genotype 2 (GAstV-2), and Tembusu virus (TMUV). The cutoff percent inhibition (PI) value was established at 25%, based on 40 GAstV-negative sera. Intra- and inter-assay coefficients of variation (CV) ranged from 0.41 to 0.88% and 3.12-6.95%, respectively, confirming excellent reproducibility. Evaluation of 35 clinical serum samples revealed an 88.6% (31/35) concordance with virus neutralization tests (VNT), with a kappa value of 0.77. These findings indicate that the ORF2-based ic-ELISA is a reliable, cost-effective, and high-throughput tool for seroepidemiological investigation and post-vaccination monitoring of GAstV-1 in geese.

Bovine viral diarrhea (BVD) is a highly contagious viral disease that affects cattle, causing economic losses to the livestock industry. An effective diagnostic approach that facilitates timely decision-making is essential for the control of BVD virus (BVDV). Therefore, the development of a rapid, sensitive, and reliable assay is critical to prevent and limit the spread of the virus. Although reverse transcription real-time polymerase chain reaction (rRT-PCR) assays are widely employed for BVDV diagnosis, unavoidable delays due to sample transportation and reagent preparation can create a time window that permits further transmission of the disease. In this study, we developed an rRT-PCR assay to detect BVDV directly in bovine serum, without the need for RNA extraction, using a portable PicoGene PCR 1100 device. The newly developed BVDV assay, using an advanced BVDV detection kit, demonstrated high sensitivity by consistently detecting viral titers as low as 10[2] TCID50/mL in serum across all three independent trials. Our findings represent a significant advancement in BVDV on-site diagnosis, achieved through the integration of the PicoGene device system with the newly developed BVDV detection assay.

Okra (Abelmoschus esculentus L.) is an economically important vegetable crop and an important host of several viral pathogens, including tobacco streak virus (TSV). Reliable molecular diagnostics and gene expression studies require high-quality RNA; however, RNA isolation from okra is particularly challenging due to the abundance of mucilage, polysaccharides, and phenolic compounds that co-extract with nucleic acids and interfere with downstream applications. In the present study, a modified SDS-LiCl RNA extraction method was developed to effectively remove mucilage and obtain high-quality RNA. Its performance was compared with three existing protocols, CTAB-LiCl, TRIzol, and the RNeasy Plant Mini Kit for RNA isolation from five different okra tissues, namely leaf, stem, flower, fruit, and seed. RNA quality and quantity were evaluated using spectrophotometric measurements and agarose gel electrophoresis, while suitability for downstream applications was assessed through RT-PCR and RT-qPCR-based detection of TSV. Among the methods evaluated, the modified SDS-LiCl protocol consistently produced improved RNA yield, purity, and integrity across all tissue types. The modified protocol incorporated higher NaCl and SDS concentrations, PVP and β-mercaptoethanol supplementation, additional chloroform:isoamyl alcohol purification, and selective LiCl-sodium acetate precipitation to effectively remove mucilage, polysaccharides, phenolic compounds, and other PCR inhibitors. Among all the methods modified SDS-LiCl and RNeasy Plant Mini Kit yielded mucilage -free RNA. Principal Component Analysis further identified the modified SDS-LiCl method provided the best balance between RNA quality and recovery. RT-PCR detection of the TSV coat protein gene was 100% for RNA obtained using both the modified SDS-LiCl protocol and the RNeasy Plant Mini Kit, compared with 80% for CTAB-LiCl and 70% for TRIzol methods, respectively. RT-qPCR analysis showed improved amplification performance for SDS-LiCl-extracted RNA, with the lowest Ct value for the endogenous elongation factor gene (15.33). The modified SDS-LiCl protocol exhibited the highest analytical sensitivity, enabling TSV detection up to the 10[-4] dilution, one order of magnitude greater than CTAB-LiCl and two orders of magnitude greater than TRIzol and the RNeasy Plant Mini Kit. Overall, the modified SDS-LiCl protocol provides a reliable, cost-effective, and highly efficient strategy for isolation of high-quality RNA from mucilage-rich okra tissues and offers a valuable tool for virus diagnostics, gene expression studies, and other molecular applications in okra and related crops.

Lumpy skin disease (LSD) is a transboundary poxviral disease of cattle and buffalo that has been economically significant in various parts of Africa, the Middle East, Asia and some parts of Europe and therefore requires the development of reliable, high throughput diagnostic tools to support effective surveillance and control. Conventional serological assays are labor intensive, time consuming and involve handling of live virus, which makes them unsuitable for large-scale surveillance. Recombinant antigen based ELISAs offer a safer, scalable and high-throughput alternative; however, identifying optimal diagnostic antigens remains a challenge. In this study, three representative immunogenic proteins of different structural components of the virion of the lumpy skin disease virus (LSDV) were selected for indirect ELISA to detect antibodies against LSDV: A4L (conserved core protein), A27L (intracellular mature virion) and A36R (extracellular enveloped virion). The corresponding genes were cloned and expressed in E. coli using the pET-33b(+) system. Recombinant proteins were purified using Ni-NTA affinity chromatography, and their expression and immunoreactivity were confirmed by SDS-PAGE and western blotting using anti-His antibodies and anti-LSDV sera. Purified proteins were used to optimize indirect ELISAs, and their diagnostic performances were comparatively evaluated. All three ELISAs demonstrated high analytical specificity, with no detectable cross-reactivity against sera positive for other viral infections. The A4L-based ELISA had the highest diagnostic sensitivity and specificity (93% and 94%, respectively) among the antigens evaluated. The diagnostic sensitivity and specificity of the A27L-based ELISA were 89% and 88%, respectively, while the A36R-based ELISA had a sensitivity of 90% and specificity of 86%. These findings indicate that the highly conserved and abundantly expressed core protein A4L is a more suitable serodiagnostic antigen than the evaluated envelope-associated proteins. Overall, among the evaluated antigens, A4L-based indirect ELISA showed better potential for serodiagnosis and large-scale serosurveillance of LSDV. Further large-scale field validation is required to confirm the diagnostic robustness and applicability of the A4L-based indirect ELISA for routine LSDV serosurveillance.

The Oxford Nanopore Technologies (ONT) MinION MK1b sequencer was found to be a useful low-cost entry device for the detection of plant viruses and viroids, especially when double-stranded RNA (dsRNA) is used as input material. Extractions from either dormant wood or mature leaf tissue are suitable for this purpose. A set of 45 plant samples with various known virus and viroid infections was sequenced with both a MinION Mk1b and an Illumina NextSeq 500, for the purpose of comparing detection rates between sequencing methods. Despite the larger sequencing capacity of the NextSeq, the MinION reproduced the Illumina detection results in nearly all instances (94%); a regression model comparison showed no significant difference in performance (p = 0.44), though a non-inferiority test also did not show significance. The relative sensitivity of the MinION sequencer was compared with that of reverse transcriptase PCR (RT-PCR) gel electrophoresis when testing for six different viruses in serially diluted nucleic acid extractions. Three separate criteria for detecting positive virus signals were applied to the ONT dataset; only the most permissive threshold (the presence of any target reads) resulted in ONT showing a statistically significant higher sensitivity than RT-PCR, based on mixed-effects logistic regression modeling (LRT p-value = 0.043). Using the SQK-PCB109 kit for library prep and the Viroscope service for data analysis, screening 12 libraries on one flow cell over a 72 h sequencing run was sufficient to detect all but the weakest pathogen signals known to be present (91% agreement with the expected detection pattern). Overall, the most obvious limiting factor for the MinION as a detection tool for plant pathogens such as viruses is the amount of data generated per run.

Reliable detection of infectious pancreatic necrosis virus (IPNV) by real-time RT-qPCR requires stable internal reference genes to control for variability in RNA quality, extraction efficiency, and reverse transcription. In salmonid diagnostics, the housekeeping gene elongation factor 1 alpha (EF1α) is widely used for this purpose; however, different RT-qPCR protocols targeting EF1α are often adopted without comparative evaluation of assay performance. In this study, we compared three RT-qPCR assays targeting EF1α (Moore, Bowers, and a newly designed assay, ELF4) using 53 field samples from three salmonid species (Salmo salar, Oncorhynchus mykiss, and O. kisutch). Differences in Ct dispersion were observed among assays, with the Bowers and ELF4 protocols showing significantly lower variability than the Moore assay. The ELF4 assay was subsequently validated as an endogenous sample control for diagnostic applications using two independent datasets: 500 pooled kidney-spleen samples collected during a confirmed IPNV outbreak in 2023 and 81 samples (47 survivors and 34 mortality cases) from a previously published experimental challenge. Across outbreak samples, ELF4 Ct values remained consistent, whereas IPNV VP1 Ct values showed wider dispersion, reflecting variability in viral RNA levels. A moderate positive correlation was observed between ELF4 and VP1 Ct values (Spearman's ρ = 0.40, p < 0.001), suggesting that sample-dependent variation in RNA quality may influence viral RNA detectability under field conditions. These results support the use of the ELF4 EF1α RT-qPCR assay as a robust endogenous sample control for IPNV detection. Incorporation of ELF4 into routine diagnostic workflows may improve RNA quality assessment and enhance the reliability of RT-qPCR-based surveillance in salmonid aquaculture.

Parapoxviruses are known for its unique feature of carrying a number of virulence genes in their large genome and deploy them as a weapon to evade host immune response. One of its unique gene is Poxviral anaphase promoting complex regulator (PACR) from the left terminal of the genome named as ORF014. This PACR protein basically mimics the subunit, APC11 of anaphase promoting complex (APC)/cyclosome of the host cell. Orf virus is the prototypic member of Parapoxvirus genera and causes mostly localized infection in animals as well as humans. Considering the importance of gene in ORFV replication and virulence, and the endemicity of the disease in the country affecting sheep and goats, we designed a study to genetically characterize different ORFV isolates from different geographical parts of India. Interestingly, multiple sequence alignment of the gene revealed that sheep and goat isolates encoded different ORF lengths of gene i.e. 282 bp and 288 bp respectively with an additional two amino acids namely valine and lysine at 7th and 8th position in ORFV isolates of goat origin only. Likewise, the difference was also reflected in phylogenetic tree as the sheep and goat isolates of ORFV were clustered into two groups. Based on these salient findings, the difference in sequences was exploited to design species-specific primers for the development of diagnostic PCR to differentiate sheep and goats ORFV isolates. On evaluation of clinical samples and cell culture isolates of ORFV, PCR clearly distinguished them as sheep and goat isolates. The comparable relative specificity and sensitivity of the ORF014 based diagnostic PCR with in-use PCR also revealed a refine potential for epidemiological insights of parapoxviruses. This study seems to have proposed for the first time a basis for differentiating the ORFV isolates by their origin of species.

Solid phase competitive ELISA (SPCE), a WOAH-accepted serological assay for foot-and-mouth disease (FMD), commonly employs rabbit hyperimmune serum for capturing foot-and-mouth disease virus (FMDV). In accordance with the principles of the 3Rs for animal experimentation, this study evaluated native integrin, the cellular receptor for FMDV, as an alternative coating reagent for SPCE. BHK-21, MDBK, and LFBK cell lines were screened for relative abundance of integrin subunits (αV, α5, β1, β3, β5, β6, and β8) by qRT-PCR and for FMDV reactivity by sandwich ELISA. LFBK cells exhibited the highest integrin expression (β5 > β3 > β8 > αV > β6 > β1 > α5) and strongest ELISA reactivity; therefore, the LFBK was selected for integrin isolation. Optimal cell lysis was achieved using 1 mM Triton X-100 for 30 min. Affinity chromatography was performed using full-length fibronectin, GRGDSP peptide, and recombinant fibronectin type III9-10 (FN-III9-10) domain as ligands. Western blot confirmed that all three ligands captured αV integrin; however, FN-III9-10 was selected due to its scalability. Integrin was eluted using NaCl concentrations of 250, 500, and 1000 mM, with maximum yield obtained at 250 mM NaCl. The eluted integrin retained reactivity with anti-αV antibodies and demonstrated the ability to capture FMDV in ELISA. Coating concentration of 50 ng/well was optimized by ELISA titration. Although integrin-coated wells produced lower absorbance than polyclonal serum, specific binding and dilution-dependent inhibition were observed. Antibody titers were comparable to conventional SPCE (Wilcoxon signed-rank test, p = 0.324). These findings provide proof-of-concept that native integrin can be a potential alternate to polyclonal serum in SPCE.

Cardamom mosaic virus (CdMV), associated with mosaic/katte disease in cardamom, is a highly destructive virus that leads to severe reductions in plant growth and yield. High levels of genetic variability approaching species-level divergence exist among CdMV isolates from different geographical regions. Development of a cluster-specific diagnostic assay can enable reliable detection of locally prevalent virus populations and differentiate them from divergent ones. Therefore, in the present study, a CRISPR-Cas12a-coupled reverse transcription-recombinase polymerase amplification-lateral flow assay (RT-RPA-CRISPR-LFA) was developed for cluster IV-specific detection of Idukki isolates of CdMV. The viral target sequence (coat protein) was first amplified through RT-RPA using virus-specific primers designed from conserved region present in all strains of CdMV and crude plant extract template. The amplified product was subsequently recognized by CRISPR-Cas12a complex guided by a crRNA designed to specifically detect CdMV isolates associated with Idukki region of Kerala, triggering trans-cleavage activity of the complex and subsequent cleavage of the lateral flow reporter (LF reporter). This generated a visually detectable signal by LFA. The optimum assay performance was achieved with an RT-RPA incubation time of 10 min, LF reporter, Cas12a, and crRNA concentrations of 500, 75, and 125nM, respectively, and a CRISPR-Cas12a reaction incubation time of 1h. The assay was specific for CdMV strains representing Idukki region of Kerala and exhibited a detection limit of 1 fg (2.4 × 10[2] copies), which was 10 times less sensitive than qPCR. Practical feasibility and consistency of the assay were verified through validation using different field samples of cardamom.