Rabies, a fatal zoonosis, remains a burden in developing countries. Concerns about indirect environmental transmission and appropriate post‑exposure prophylaxis (PEP) are increasing, yet data on RABV stability under various conditions are limited. In this study, we systematically evaluated the stability of RABV (CVS‑11 strain) on six surface (glass, rubber, plastic, polypropylene surgical mask, fabric, and paper) at 25℃, and its stability in brain tissue, muscle, and simulated saliva under three simulated seasonal conditions: summer (35℃, 75% RH), spring/autumn (15℃, 43% RH), and winter (4℃, 33% RH). Viral titers were determined by fluorescent focus assay, complemented by direct fluorescent antibody staining and qPCR for nucleic acid detection. On surfaces, RABV titers declined from about 10[7] FFU/mL to 10[3] FFU/mL over 72h, with inactivation occurring significantly faster on fabric than on other materials. Under simulated summer conditions, no infectious virus could be recovered from saliva after 12h or from brain and muscle tissues after 24h, but viral RNA remained detectable by qPCR. However, under spring/autumn and winter conditions, the virus still exhibited relatively high titers at 24h, with enhanced stability at 4℃. These findings provide an evidence‑based framework for diagnostic laboratories to interpret nucleic acid tests in degraded specimens and to establish sample acceptance criteria. By clarifying the limited time window of environmental infectivity, they also help alleviate rabies‑related anxiety and support more nuanced post‑exposure prophylaxis decisions.

Accurate and timely sequencing of poliovirus is critical for global eradication efforts, particularly for molecular epidemiology based on the typing region of the genome, viral protein 1 (VP1). While Oxford Nanopore Technologies (ONT) sequencing has expanded capabilities for poliovirus surveillance, the relative performance of different ONT library preparation methods, including ligation-based (Native Barcoding) and transposase-based (Rapid Barcoding) approaches, has not been systematically evaluated. In this study, we compared rapid barcoding and native barcoding workflows for sequencing VP1 amplicons from 17 type 2 poliovirus-positive samples, each processed in triplicate. Native barcoding generated significantly more sequencing output, producing approximately 2.3-fold greater total read yield than rapid barcoding, and demonstrated higher run-to-run reproducibility (R[2] = 0.979-0.998 vs. 0.847-0.929, respectively; p < 0.001). In addition, native barcoding generated 80% of the total yield achieved by rapid barcoding within approximately 7hours, whereas rapid barcoding required approximately 40hours to reach the same output. Despite these differences, both methods produced identical VP1 consensus sequences across all samples, with comparable read quality (median per-base Q-scores of approximately Q17-Q18). Rapid barcoding provided substantial practical advantages, reducing hands-on library preparation time (55 vs. 200minutes) and per-sample cost ($12.82 vs. $16.54), while simplifying workflow and reducing technical complexity. These findings indicate that sequencing yield may not be a determinant of downstream analytical outcomes for poliovirus VP1 ONT sequencing. Rapid barcoding therefore represents a cost-effective and efficient approach for routine poliovirus surveillance, whereas native barcoding remains advantageous in applications requiring rapid data generation or maximal sequencing depth.

Arboviruses such as Usutu virus (USUV) and West Nile virus (WNV) cycle enzootically between competent mosquito vectors and avian reservoir hosts. With USUV established and WNV incidence rising in the Netherlands, detecting antibodies in wild birds captured at ringing sites is critical for surveillance. Currently, no multi-analyte, species-independent serological assays exist for on-site field analysis. Using a recently developed species-independent Lateral flow Microarray ImmunoAssay (LMIA) capable of detecting both USUV non-structural protein 1 (NS1)- and WNV NS1-specific antibodies, a total of 82 wild birds were captured and tested in the field. Of these, 8 birds were USUV LMIA-reactive, all of which were included in a subset of 18 birds that were additionally analysed using gold-standard USUV and WNV 90% Focus Reduction Neutralization Test (FRNT90). Compared to FRNT90 results, the LMIA showed a sensitivity and specificity for USUV of 100% and 89%, respectively. The LMIA also detected seroconversion to USUV in a Eurasian blackbird, which was confirmed by FRNT90. A scoring system based on the WHO's REASSURED criteria was also used as a framework to evaluate the LMIA field performance, receiving 81% of the highest possible score. While minor optimization and broader validation remain, this study demonstrates LMIA feasibility for on-site USUV serological screening. Its rapid, multi-analyte, and equipment-light format supports point-of-capture testing and targeted surveillance in Dutch bird-ringing sites and resource-limited settings.

Positive controls are essential for diagnostics yet access to high quality ones remains a persistent bottleneck for assay validation and quality control. Infected tissues are scarce or even regulated, whereas synthetic DNA fragments lack matrix effects and raise contamination risks. Here, we validate the Virus-Mimicking Artificial Positive Control (ViMAPC) platform for 18 viruses, demonstrating platform scalability across multiple virus taxa. ViMAPCs primarily use genus-matched surrogates and built-in size shifts to easily distinguish natural infections (true positives) from false positives due to contamination. Controls were generated via chimeric primer reverse transcription followed by end-point PCR using peer-reviewed protocols. All targets yielded expected amplicons with designed size differentiation and similar amplification behaviour to naturally infected material, with percent differences in amplicon intensity of ≤ 30%. Independent synthesis and validation in two laboratories achieved 100% success with design-to-use timelines of 4-5 days, demonstrating transferability for routine diagnostic workflows. By providing rapid, low-cost controls that mimic natural infection while remaining clearly identifiable, ViMAPCs enable robust diagnostic validation, contamination tracing, and cross-laboratory comparability, supporting diagnostic laboratories in rapid assay deployment for both newly discovered and quarantine viruses.

Accurate and automated molecular detection of SARS-CoV-2 has played an important role in routine clinical diagnostics and laboratory workflow management. This study evaluated the analytical characteristics of the NeuMoDx™ SARS-CoV-2 assay and compared its clinical detection performance with the BD MAX SARS-CoV-2 system. Analytical performance was assessed using AccuPlex™ SARS-CoV-2 reference materials for dilutional response, reproducibility, and analytical sensitivity evaluation, whereas clinical performance was evaluated using 84 nasopharyngeal swab specimens. The NeuMoDx™ assay demonstrated strong correlations between Ct values and viral RNA concentrations, with R[2] values of 0.9971 and 0.9952 for the N and Nsp2 targets, respectively. Intra-run precision showed low variability, with standard deviations of 0.23 and 0.34 at 500 copies/mL for the N and Nsp2 targets, respectively. Under the experimental conditions of this study, detectable amplification signals were observed at concentrations as low as 50 copies/mL using reference materials. Clinical comparison demonstrated a positive percent agreement (PPA) of 97.3% and a negative percent agreement (NPA) of 100% between the two systems. Positive specimens exhibited Ct values ranging from approximately 20-35. In pooling experiments, detectable amplification signals were observed at a dilution ratio of 1:5 using a representative specimen with a Ct value of approximately 30, although detection consistency decreased at higher dilution ratios. These findings provide additional analytical performance data for an automated SARS-CoV-2 molecular diagnostic platform and contribute to the evaluation of automated PCR-based testing workflows.

One of the most abundant and ubiquitous members of the phytoplankton in the marine ecosystem is the unicellular cyanobacteria of the genus Synechococcus, which contributes to a substantial fraction of the ocean's primary productivity. Nevertheless, like any other organism, Synechococcus is infected by a specific kind of virus: the cyanophages. These viruses, highly abundant and widely distributed throughout the world's oceans, shape the bacterial communities by regulating their abundance, diversity, and physiology. However, the information regarding the occurrence of Synechococcus-infecting phages in the Gulf of California and their impact on host populations in shrimp farming ecosystems is currently unavailable. This is relevant since it is of critical importance for the shrimp's health and growth, as well as for optimizing farm productivity. In this study, a highly specific and sensitive qPCR method to quantitatively estimate the abundance of Synechococcus sp., and the Synechococcus-infecting phages was developed. Moreover, the spatiotemporal abundance patterns of the phage and its host in a shrimp farm on the Gulf of California were estimated, and the ecological impact of this interaction is discussed. Our results contribute to a better comprehension of the virus-host interaction, which could ultimately affect the primary productivity surrounding shrimp farming facilities.

High-throughput sequencing has expanded the known diversity of viruses infecting stone fruits within the family Betaflexiviridae, particularly the subfamily Quinvirinae, complicating routine diagnostics based on species-specific primers. In this study, a universal, degenerate primer set targeting conserved regions of the RNA-dependent RNA polymerase (RdRp) was designed by aligning public sequences from Robigovirus and Foveavirus (4-5 isolates per species where available) and evaluated to enable broad, non-specific detection of multiple Betaflexiviridae species in stone fruits and selected non-Prunus hosts. The assay was validated on (i) reference sources for CNRMV, CGRMV, CRMaV, PVGR, WCaV, ApLV; (ii) locally collected Prunus samples (n = 449); and (iii) non-Prunus hosts, pear (ASPV) and grapevine (GRSPaV). Universal RT-qPCR screening (SYBR Green), followed by conventional RT-PCR and Sanger sequencing (∼280 nt), enabled species attribution and confirmation with species-specific primers. Among 449 Prunus accessions, 45 (10.0%) were qPCR-positive and yielded amplicons of the expected size by RT-PCR. Prevalence was highest in sweet cherry (20.0%, 30/150), followed by apricot (13.3%, 8/60) and peach (5.4%, 7/130); no infection was detected in plum. BLAST analysis identified 21 Robigovirus positives (13 PVGR in cherry; 8 CGRMV in cherry, peach, apricot), 4 Foveavirus (ApLV in apricot and peach; APV2 in apricot and peach), 19 CVA (Capillovirus; 14 in cherry and 5 in apricot/peach), and 1 AVCaV (Prunevirus; apricot). In addition, ASPV was detected in pear and GRSPaV in grapevine. This RdRp-based primer set provides broad detection across Quinvirinae and selected Trivirinae, streamlining large-scale screening and first-line diagnostics for stone fruit health programs. Species-level resolution is readily achieved by Sanger sequencing or secondary species-specific RT-PCR, supporting application in surveillance, certification, and quarantine.

Marek's disease virus (MDV), Chicken anemia virus (CAV), fowl adenovirus (FADV), avian reticuloendotheliosis virus (REV), avian reovirus (ARV), and infectious bursal disease virus (IBDV) are prevalent immunosuppressive diseases affecting broilers, exhibiting a high prevalence rate.To address this issue, a multiplex real-time quantitative PCR(qPCR) method was designed for the detection of all six viruses. The study involved designing primers and probes that specifically target the MDV Meq gene, CAV VP2 gene, FADV penton base gene, REV gp90 gene, ARV S1 gene, and IBDV VP2 gene, and it established a TaqMan probe-based one-step RT-qPCR method.This method is characterized by high efficiency, stability, and sensitivity, achieving a minimum detectable plasmid level of 1 copy/µL for all six viruses. Furthermore, the coefficient of variation for all six viral detection methods is below 3%. The method demonstrates a strong linear relationship (R[2] ≥ 0.998) and an amplification efficiency ranging from 96% to 104%. Testing of 94 clinical liver samples from diseased chickens indicated that CAV exhibited the highest positivity rate at 91.49%. Among the positive samples, the rates of dual, triple, quadruple, and quintuple mixed infections were 30.00%, 37.78%, 13.33%, and 1.11%, respectively.Research indicates that the qPCR method represents a novel approach for detecting six types of immunosuppressive disease viruses in broilers. These viruses demonstrate a high positive rate and co-infection phenomena among chicken flocks in Liaoning Province.

Infectious bursal disease (IBD) is a highly contagious avian disease caused by infectious bursal disease virus (IBDV), mainly affecting the bursa of Fabricius immune organ in young chickens. The virus has heat and acid resistance and can be spread through contaminated environments, feed and insects, leading to immunosuppression and increasing the risk of secondary infections. Affected chickens often show symptoms such as diarrhea, tremors and dehydration. Chickens aged 3-6 weeks are the most susceptible, with an incidence rate of up to 100% and a mortality rate of 5-30%. Therefore, from the perspective of infection control, effective monitoring and prevention of IBDV are of vital importance. The quantum dot fluorescence immunochromatographic strip established in this study can complete the specific detection of infectious bursal disease in chickens within 15 min, with a detection limit of 10[2.5] TCID50/mL for infectious bursal disease virus liquid. It does not cross-react with avian influenza viruses (H9N2, H10N8) and infectious bronchitis virus. This test strip can rapidly and accurately detect infectious bursal disease virus, providing a valuable tool for on-site detection.

Dengue infection remains a major public health concern in Indonesia. Rapid and valid dengue virus (DENV) diagnosis is critical to prompt care and intervention. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), as the gold standard, is challenging to implement in fast-paced settings with limited resources. Therefore, rapid diagnostic tests (RDTs) offer a practical and simpler alternative with differing accuracy. This study aims to compare RDTs' performance with RT-qPCR using specimens of dengue-suspected patients from two hospitals in North Jakarta. A total of 111 archived paired whole blood and plasma specimens were analyzed using the Dengue non-structural protein 1 (NS1) antigen and Dengue Combo NS1 Immunoglobulin (Ig)G/IgM RDT kits, respectively. Diagnostic accuracy and validity parameters were calculated to assess RDT kits' interpretation. The results were further analyzed using the clinical symptoms and blood profiles of patients. Among all the tests, the combination of Combo NS1/IgM results provides the best diagnostic approach to reflect the acute and recent dengue infections in clinical settings, as both combinations showed better sensitivity (79.2%), specificity (79.3%), positive predictive value/PPV (77.8%), and negative predictive value/NPV (80.7%) compared to RT-qPCR. Moreover, Combo NS1/IgM displayed the finest correlation with clinical manifestation according to the World Health Organization (WHO) (OR=12.67, 95% CI=1.57-101.94), though advanced exploration is needed. Meanwhile, IgG showed the lowest performance among all parameters. Dengue RDTs, especially NS1/IgM assays, demonstrated complementary diagnostic value with notable associations to RT-qPCR, clinical features, and hematological parameters. These findings support their integration into routine dengue management while emphasizing its further optimization.

Bovine rhinitis virus (BRV) is an important pathogen causing bovine respiratory disease complex (BRDC). To facilitate the rapid and simultaneous detection of bovine rhinitis A virus (BRAV) and bovine rhinitis B virus (BRBV), specific primers and TaqMan probes targeting the viral 3D gene were designed and synthesized. Following the optimization of primer and probe concentrations and annealing temperatures, as well as comprehensive evaluations of specificity, sensitivity and reproducibility, a dual real-time RT-PCR (RT-qPCR) assay was successfully developed for the reliable differential detection of BRAV and BRBV. The results demonstrated that the developed assay effectively differentiates between BRAV and BRBV. Furthermore, the assay exhibited no cross-reactivity with other common bovine respiratory pathogens, including IDV, BVDV, BCoV, BRSV, BPIV3, IBRV, BAdV-3, M. bovis, M. haemolytica, P. multocida, C. perfringens, and K. pneumoniae, thereby indicating its high specificity. The in vitro transcribed viral 3D gene RNA was used as template, the limit of detection (LOD) for both BRAV and BRBV was 1.0 × 10[2] copies/μL. Standard curves generated by plotting the logarithmic RNA concentrations against the corresponding cycle threshold (Ct) values yielded R[2] values of 0.994 and 0.999, respectively. Finally, both intra- and inter-assay coefficients of variation were less than 3%, demonstrating excellent reproducibility. A total of 790 bovine nasal swab samples were analyzed using the dual RT-qPCR assay, and the results revealed the viral nucleic acid positive rates of 3.79% (30/790) for BRAV and 9.36% (74/790) for BRBV, with a co-infection rate of 1.26% (10/790). The dual RT-qPCR assay developed for the differential detection BRAV and BRBV demonstrated high specificity, sensitivity, and reproducibility, thereby providing a robust technical support for the surveillance, epidemiological investigation, and targeted prevention and control of BRV infection in the cattle industry.

African swine fever (ASF) is a severe disease caused by African swine fever virus (ASFV). In field settings, detection of ASFV can be achieved using pen-side tests, including lateral flow assays (LFAs) targeting virus antigens. In this study, we tested an LFA for the detection of ASFV antigens (an ASFV Ag LFA) in whole blood and serum. As a novel feature, we combined the ASFV Ag LFA with a rapid diagnostic device, the iaX reader, which generated quantitative results for the levels of ASFV antigens. Compared to qPCR, the ASFV Ag LFA in combination with the iaX reader had a diagnostic sensitivity of 100% (1.00, 95% confidence interval 0.92-1.00) and a diagnostic specificity of 91% (0.91, 95% confidence interval 0.72-0.99) for serum. For whole blood, the observed diagnostic sensitivity was lower, 79% (0.79, 95% confidence interval 0.63-0.90), while the diagnostic specificity was comparable to that reported for serum, 95% (0.95, 95% confidence interval 0.77-1.00). When comparing quantitative agreement using intraclass correlation coefficient estimates, the ASFV Ag LFA had a high level of agreement with the ASFV qPCR for serum, while a more moderate agreement was observed for whole blood. Bland-Altman and regression analyses demonstrated that agreement for both types of blood samples was not uniform and was highest when virus levels were high. In summary, the combination of the LFA and the diagnostic reader demonstrated a better performance for the detection of ASFV antigens in serum samples compared to whole blood.

Cytomegalovirus (CMV) is the most common congenital infection and an important cause of childhood disability. Newborn CMV screening and studies of mother-to-child transmission are increasingly common, involving viral detection using various sample types. Here, we describe the analytical evaluation of the AltoStar® CMV PCR Kit 1.5, a commercial diagnostic platform for detection of CMV DNA in diverse sample types relevant for congenital CMV infection. Oral swabs, saliva, dried urine spots (DUS) and dried blood filter paper spots (DBS), umbilical cord blood, amniotic fluid, placental tissue, vaginal swabs, and breast milk from known CMV-negative donors were spiked with known concentrations of viral isolates. Linearity of detection in fluid samples ranged from 0.96 to 1.00. Limits of detection in fluid samples ranged from 2.22 to 2.83 log IU/ml. CMV PCR on dried urine spot samples had greater sensitivity when using off-instrument nucleic acid purification with Quanta Extracta DBS buffer (QuantaBio). This study provides a cross-matrix performance framework for CMV molecular testing in congenital infection research and newborn screening contexts. The AltoStar® CMV PCR platform demonstrated robust performance across clinically relevant specimen types and scalable throughput capacity, supporting its suitability for large-scale epidemiologic and screening applications.