Pub Date : 2026-02-01Epub Date: 2025-10-17DOI: 10.1016/j.jviromet.2025.115290
Hyun Jung Gye , Toyohiko Nishizawa
Enzyme-linked immunosorbent assay (ELISA) is a quantitative immunoassay used to detect antigens and antibodies. In the field of fish diseases, the usefulness of ELISA has been largely overlooked due to its low reproducibility and high background optical density (OD). Nevertheless, ELISA is indispensable for evaluating specific immunity in vaccinated fishes, tracking infection history and analyzing the conformational structures and functions of the surface proteins of fish pathogens. Therefore, a highly quantitative and reproducible ELISA is important and necessary for fish disease research and diagnosis. In this technical review, we used nervous necrosis virus (NNV) as a model to compile the existing knowledge on improving background OD and increasing the reproducibility of ELISA. The issues associated with this method are primarily caused by non-specific reactions of immunoglobulins (Igs) to viral particles and changes in the aggregation states of viral particles. The countermeasures include methods for in vitro virus culture, purification of virus particles, immobilization of virus particle antigens and fish Igs on ELISA plate wells, consideration of physicochemical properties of virus particles and reaction order of the antigens and antibodies. This information will improve the detection of fish viruses other than NNV and specific antibodies against them using ELISA.
{"title":"Optimization of enzyme-linked immunosorbent assay for analyzing surface structures of nervous necrosis virus and detecting virus-specific antibodies: A technical review","authors":"Hyun Jung Gye , Toyohiko Nishizawa","doi":"10.1016/j.jviromet.2025.115290","DOIUrl":"10.1016/j.jviromet.2025.115290","url":null,"abstract":"<div><div>Enzyme-linked immunosorbent assay (ELISA) is a quantitative immunoassay used to detect antigens and antibodies. In the field of fish diseases, the usefulness of ELISA has been largely overlooked due to its low reproducibility and high background optical density (OD). Nevertheless, ELISA is indispensable for evaluating specific immunity in vaccinated fishes, tracking infection history and analyzing the conformational structures and functions of the surface proteins of fish pathogens. Therefore, a highly quantitative and reproducible ELISA is important and necessary for fish disease research and diagnosis. In this technical review, we used nervous necrosis virus (NNV) as a model to compile the existing knowledge on improving background OD and increasing the reproducibility of ELISA. The issues associated with this method are primarily caused by non-specific reactions of immunoglobulins (Igs) to viral particles and changes in the aggregation states of viral particles. The countermeasures include methods for <em>in vitro</em> virus culture, purification of virus particles, immobilization of virus particle antigens and fish Igs on ELISA plate wells, consideration of physicochemical properties of virus particles and reaction order of the antigens and antibodies. This information will improve the detection of fish viruses other than NNV and specific antibodies against them using ELISA.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115290"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145329441","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-02-01Epub Date: 2025-10-07DOI: 10.1016/j.jviromet.2025.115276
Su Lin , Xiuqin Chen , Xiaoli Zhu , Xiaoxia Cheng , Dangdang Jiang , Shifeng Xiao , Shilong Chen , Meiqing Huang , Xiaofei Lin , Shao Wang , Shaoying Chen
To establish a real-time quantitative PCR method for detecting and differentiating virulent and attenuated strains of Muscovy duck-origin goose parvovirus (MDGPV), a pair of common primers and two specific locked nucleic acid (LNA)-TaqMan probes targeting the conserved VP1 gene region were designed and synthesized based on MDGPV genome sequences from GenBank. By labeling the probes with distinct fluorophores and optimizing the reaction conditions, the optimal primer-probe combination was identified, and an LNA-TaqMan based quantitative PCR differentiation method was developed. The results demonstrated that this assay could specifically detect both virulent and attenuated MDGPV strains without cross-reactivity with other waterfowl viruses. The method exhibited high sensitivity, with a detection limit of 6.1 × 100 copies/μL for both the virulent and attenuated strains. The method showed good reproducibility, with a coefficient of variation of less than 3 %. The detection results for the clinical samples were consistent with the sequencing analysis. These findings indicate that the established duplex LNA-TaqMan real-time quantitative PCR method is suitable for the differential detection of MDGPV virulent and attenuated strains in clinical samples, providing an effective technical tool for the control and eradication of MDGPV.
{"title":"Development of a duplex LNA-TaqMan real-time quantitative PCR for differential detection of virulent and attenuated strains of Muscovy duck-origin goose parvovirus","authors":"Su Lin , Xiuqin Chen , Xiaoli Zhu , Xiaoxia Cheng , Dangdang Jiang , Shifeng Xiao , Shilong Chen , Meiqing Huang , Xiaofei Lin , Shao Wang , Shaoying Chen","doi":"10.1016/j.jviromet.2025.115276","DOIUrl":"10.1016/j.jviromet.2025.115276","url":null,"abstract":"<div><div>To establish a real-time quantitative PCR method for detecting and differentiating virulent and attenuated strains of Muscovy duck-origin goose parvovirus (MDGPV), a pair of common primers and two specific locked nucleic acid (LNA)-TaqMan probes targeting the conserved VP1 gene region were designed and synthesized based on MDGPV genome sequences from GenBank. By labeling the probes with distinct fluorophores and optimizing the reaction conditions, the optimal primer-probe combination was identified, and an LNA-TaqMan based quantitative PCR differentiation method was developed. The results demonstrated that this assay could specifically detect both virulent and attenuated MDGPV strains without cross-reactivity with other waterfowl viruses. The method exhibited high sensitivity, with a detection limit of 6.1 × 10<sup>0</sup> copies/μL for both the virulent and attenuated strains. The method showed good reproducibility, with a coefficient of variation of less than 3 %. The detection results for the clinical samples were consistent with the sequencing analysis. These findings indicate that the established duplex LNA-TaqMan real-time quantitative PCR method is suitable for the differential detection of MDGPV virulent and attenuated strains in clinical samples, providing an effective technical tool for the control and eradication of MDGPV.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115276"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145251553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Despite the familiar use of Bryonia alba (Bry-alb) in COVID-19, the method of accomplishment of this homeopathic medicine is not appropriately explored. Studies have revealed that constituents of Bry-alb can up-regulate the haeme-oxygenase-1 (HMOX-1) gene in diverse circumstances. In this experiment, we were concerned to explore whether a COVID-19-induced cytokine dysregulation could be managed by Bry-alb through this pathway.
Methods
Fourteenth-day-old embryonated Gallus gallus domesticus eggs were divided into six experimental groups, and except for the control, all eggs in other groups were challenged with 100 μL of SARS-CoV-2 spike protein receptor binding domain antigen (Ag) and Bry-alb (30 cH or 200 cH) via the amniotic route. Harvesting of all the eggs was performed after 48 h, and 5–10 ml of allantoic fluid was collected in sterile vials and preserved at −80°C. Later, RNA extraction was done, followed by real-time PCR to detect comparative cytokine and HMOX-1 gene expressions.
Results
Equally IFN-α and IL-10 genes were augmented when antigen was injected and followed by administration of Bry-alb 30CH both in therapeutic and prophylactic dose but this alteration was less significant with Bry-alb 200CH. However, when the antigen was confronted after administration of Bry-alb 30CH, IFN-γ and IL-6 gene expression was markedly increased while other cytokines were decreased. In the case of the HMOX-1 gene, mild up-regulation was seen with administration of Bry-alb30CH but overexpression of the aforesaid enzyme was encountered with Bry-alb 200CH. Dose dependent variation in the expression of HMOX-1 is crucial to understand the action of the drug against SARS-CoV-2.
Conclusion
The study finding suggests that Bry-alb has a protective effect against the SARS-CoV-2 spike protein antigen-induced pathogenesis by modulating HMOX-1 gene expression.
{"title":"Ultra-diluted Bryonia alba extract modulates HMOX-1 gene expression to attenuate the pathogenetic effect of SARS-CoV-2 spike protein RBD antigen","authors":"Pritam Goswami , Debasmita Chatterjee , Sayak Ghosh , Krishnendu Paira , Satadal Das","doi":"10.1016/j.jviromet.2025.115274","DOIUrl":"10.1016/j.jviromet.2025.115274","url":null,"abstract":"<div><h3>Introduction</h3><div>Despite the familiar use of <em>Bryonia alba</em> (Bry-alb) in COVID-19, the method of accomplishment of this homeopathic medicine is not appropriately explored. Studies have revealed that constituents of Bry-alb can up-regulate the haeme-oxygenase-1 (HMOX-1) gene in diverse circumstances. In this experiment, we were concerned to explore whether a COVID-19-induced cytokine dysregulation could be managed by Bry-alb through this pathway.</div></div><div><h3>Methods</h3><div>Fourteenth-day-old embryonated <em>Gallus gallus domesticus</em> eggs were divided into six experimental groups, and except for the control, all eggs in other groups were challenged with 100 μL of SARS-CoV-2 spike protein receptor binding domain antigen (Ag) and Bry-alb (30 cH or 200 cH) via the amniotic route. Harvesting of all the eggs was performed after 48 h, and 5–10 ml of allantoic fluid was collected in sterile vials and preserved at −80°C. Later, RNA extraction was done, followed by real-time PCR to detect comparative cytokine and HMOX-1 gene expressions.</div></div><div><h3>Results</h3><div>Equally IFN-α and IL-10 genes were augmented when antigen was injected and followed by administration of Bry-alb 30CH both in therapeutic and prophylactic dose but this alteration was less significant with Bry-alb 200CH. However, when the antigen was confronted after administration of Bry-alb 30CH, IFN-γ and IL-6 gene expression was markedly increased while other cytokines were decreased. In the case of the HMOX-1 gene, mild up-regulation was seen with administration of Bry-alb30CH but overexpression of the aforesaid enzyme was encountered with Bry-alb 200CH. Dose dependent variation in the expression of HMOX-1 is crucial to understand the action of the drug against SARS-CoV-2.</div></div><div><h3>Conclusion</h3><div>The study finding suggests that Bry-alb has a protective effect against the SARS-CoV-2 spike protein antigen-induced pathogenesis by modulating HMOX-1 gene expression.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115274"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145286439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-02-01Epub Date: 2025-11-27DOI: 10.1016/j.jviromet.2025.115307
Stacy Gellenoncourt , Marie Pellerin , Aïlona Marcadet-Hauss , Roxanne Fouillé , Michel Rivoire , Guillaume Passot , Julie Lucifora , David Durantel , Nicole Pavio , Virginie Doceul
Hepatitis E virus (HEV) causes acute hepatitis that can progress to fulminant or chronic hepatitis. For decades, the lack of a pertinent and robust cell culture system for HEV has delayed our understanding on this hepatotropic virus. HepaRG cells are one of the few hepatocyte-derived cell lines able to replicate HEV. These cells can differentiate (dHepaRG) into hepatocytes and cholangiocytes upon treatment with dimethyl sulfoxyde (DMSO) and are very relevant to study interactions between pathogens and hepatocyte innate immunity. However, the suitability of the HepaRG model to study HEV needs to be further investigated. In this study, we found that HEV can infect proliferating HepaRG cells and that DMSO-induced differentiation is not necessary for HEV infection. Moreover, even if treatment with DMSO is needed to maintain optimal differentiation and polarization of dHepaRG, its presence is detrimental for HEV infection. Overall, this study shows that dHepaRG cells cultured without DMSO is a suitable model to study HEV and its interaction with the hepatocyte innate immune system.
{"title":"An alternative model for HEV infection in the HepaRG cell line","authors":"Stacy Gellenoncourt , Marie Pellerin , Aïlona Marcadet-Hauss , Roxanne Fouillé , Michel Rivoire , Guillaume Passot , Julie Lucifora , David Durantel , Nicole Pavio , Virginie Doceul","doi":"10.1016/j.jviromet.2025.115307","DOIUrl":"10.1016/j.jviromet.2025.115307","url":null,"abstract":"<div><div>Hepatitis E virus (HEV) causes acute hepatitis that can progress to fulminant or chronic hepatitis. For decades, the lack of a pertinent and robust cell culture system for HEV has delayed our understanding on this hepatotropic virus. HepaRG cells are one of the few hepatocyte-derived cell lines able to replicate HEV. These cells can differentiate (dHepaRG) into hepatocytes and cholangiocytes upon treatment with dimethyl sulfoxyde (DMSO) and are very relevant to study interactions between pathogens and hepatocyte innate immunity. However, the suitability of the HepaRG model to study HEV needs to be further investigated. In this study, we found that HEV can infect proliferating HepaRG cells and that DMSO-induced differentiation is not necessary for HEV infection. Moreover, even if treatment with DMSO is needed to maintain optimal differentiation and polarization of dHepaRG, its presence is detrimental for HEV infection. Overall, this study shows that dHepaRG cells cultured without DMSO is a suitable model to study HEV and its interaction with the hepatocyte innate immune system.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115307"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145620440","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-02-01Epub Date: 2025-11-03DOI: 10.1016/j.jviromet.2025.115295
Anu E. Jääskeläinen , Anne Pitkäranta , Anu Haaramo , Johanna Nokso-Koivisto , Enni Sanmark
Accurate and cost-effective testing for SARS-CoV-2 is an ongoing need in public health. Nasopharyngeal swab (NPS) samples have been the benchmark as testing material but there are challenges connected to sample collection. We examined the usability of saliva as sample material for high-throughput laboratory molecular testing for SARS-CoV-2. Saliva samples from 108 individuals with suspected acute SARS-CoV-2 infection were collected and tested with cobas® SARS-CoV-2 and cobas® SARS-CoV-2 Duo tests (both Roche Molecular Diagnostics) to detect SARS-CoV-2 nucleic acids and compared to findings from NPS samples. Both cobas® tests performed well for saliva samples with 96 % positive percent agreement for both tests, 98 % negative percent agreement for cobas® SARS-CoV-2 test, and 100 % negative percent agreement for cobas® SARS-CoV-2 Duo test when compared to NPS samples. Saliva is a potential sample material for detecting SARS-CoV-2 infection in adult outpatients and can be considered as sample material to conserve health care resources.
{"title":"Saliva samples in SARS-Cov-2 virus detection compared to the nasopharyngeal RT-PCR findings in individuals with suspected COVID-19 infection","authors":"Anu E. Jääskeläinen , Anne Pitkäranta , Anu Haaramo , Johanna Nokso-Koivisto , Enni Sanmark","doi":"10.1016/j.jviromet.2025.115295","DOIUrl":"10.1016/j.jviromet.2025.115295","url":null,"abstract":"<div><div>Accurate and cost-effective testing for SARS-CoV-2 is an ongoing need in public health. Nasopharyngeal swab (NPS) samples have been the benchmark as testing material but there are challenges connected to sample collection. We examined the usability of saliva as sample material for high-throughput laboratory molecular testing for SARS-CoV-2. Saliva samples from 108 individuals with suspected acute SARS-CoV-2 infection were collected and tested with cobas® SARS-CoV-2 and cobas® SARS-CoV-2 Duo tests (both Roche Molecular Diagnostics) to detect SARS-CoV-2 nucleic acids and compared to findings from NPS samples. Both cobas® tests performed well for saliva samples with 96 % positive percent agreement for both tests, 98 % negative percent agreement for cobas® SARS-CoV-2 test, and 100 % negative percent agreement for cobas® SARS-CoV-2 Duo test when compared to NPS samples. Saliva is a potential sample material for detecting SARS-CoV-2 infection in adult outpatients and can be considered as sample material to conserve health care resources.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115295"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145452381","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-02-01Epub Date: 2025-10-30DOI: 10.1016/j.jviromet.2025.115294
Thoria Donia , Samar S. Alkafaas , Doha F. Ismail , Eiman Adly , Ashraf A. Tabll , Khaled M. Mekkawy , Karim EA Swede , Mohamed Hessien
Viral entry into the host cell is a limiting step in the viral-related pathogenesis, where many viruses utilize different endocytic pathways, particularly clathrin-mediated endocytosis (CME), in cell invasion. Previously, we reclassified endocytosis inhibitors based on their mode of action, where compounds like phenothiazine derivatives inhibit viral endocytosis through different mechanisms. Also, the multifaceted therapeutic potential of these derivatives, like chlorpromazine (CPZ), is attributed to their endocytosis and non-endocytosis-related effects. Thus, this study was designed to investigate CPZ’s antiviral activity against two RNA viruses and to explore how does it interact with structural and regulatory viral proteins. In addition to in silico studies that included molecular interaction and ADME profiling of CPZ, its antiviral activity against infectious bronchitis virus (IBV) and avian influenza virus (N5H1, AIV5) was evaluated by cytotoxicity assay, development of gross lesions in chicken embryos, hemagglutination (HA) assay, and viral replication in chicken embryos by qRT-PCR. The results demonstrated that CPZ interacts with integral IBV proteins, including spike (S), polymerase, and nucleocapsid proteins. Moreover, it interacts with N5H1’s polymerase complexes, nucleoprotein (NP), neuraminidase, and hemagglutinin. Furthermore, ADME profiling suggested that CPZ has better physicochemical characteristics and higher oral bioavailability than Remdesivir, due to its molecular flexibility and polarity. In parallel, experimental investigations revealed the cytotoxic effect at high doses and antiviral activity against both viruses, which led to stunted growth and reduced weight, attenuated replication, induced growth lesions in chicken embryos, and decreased the hemagglutinin (HA) titer at early developmental stages. In summary, the study repurposed CPZ as an antiviral agent, as the synergistic use of molecular docking, ADME studies, in vitro, and in vivo antiviral assays suggested its broad antiviral activity against IBV and N5H1.
{"title":"Insights into antiviral activity of chlorpromazine against RNA viruses: Molecular docking, ADME profile, and semi-in vivo study","authors":"Thoria Donia , Samar S. Alkafaas , Doha F. Ismail , Eiman Adly , Ashraf A. Tabll , Khaled M. Mekkawy , Karim EA Swede , Mohamed Hessien","doi":"10.1016/j.jviromet.2025.115294","DOIUrl":"10.1016/j.jviromet.2025.115294","url":null,"abstract":"<div><div>Viral entry into the host cell is a limiting step in the viral-related pathogenesis, where many viruses utilize different endocytic pathways, particularly clathrin-mediated endocytosis (CME), in cell invasion. Previously, we reclassified endocytosis inhibitors based on their mode of action, where compounds like phenothiazine derivatives inhibit viral endocytosis through different mechanisms. Also, the multifaceted therapeutic potential of these derivatives, like chlorpromazine (CPZ), is attributed to their endocytosis and non-endocytosis-related effects. Thus, this study was designed to investigate CPZ’s antiviral activity against two RNA viruses and to explore how does it interact with structural and regulatory viral proteins. In addition to <em>in silico</em> studies that included molecular interaction and ADME profiling of CPZ, its antiviral activity against infectious bronchitis virus (IBV) and avian influenza virus (N5H1, AIV5) was evaluated by cytotoxicity assay, development of gross lesions in chicken embryos, <em>hemagglutination</em> (HA) assay, and viral replication in chicken embryos by qRT-PCR. The results demonstrated that CPZ interacts with integral IBV proteins, including spike (S), polymerase, and nucleocapsid proteins. Moreover, it interacts with N5H1’s polymerase complexes, nucleoprotein (NP), neuraminidase, and hemagglutinin. Furthermore, ADME profiling suggested that CPZ has better physicochemical characteristics and higher oral bioavailability than Remdesivir, due to its molecular flexibility and polarity. In parallel, experimental investigations revealed the cytotoxic effect at high doses and antiviral activity against both viruses, which led to <em>stunted growth</em> and reduced weight, attenuated replication, induced growth lesions in chicken embryos, and decreased the hemagglutinin (HA) titer at early developmental stages. In summary, the study repurposed CPZ as an antiviral agent, as the synergistic use of molecular docking, ADME studies, <em>in vitro,</em> and <em>in vivo</em> antiviral assays suggested its broad antiviral activity against IBV and N5H1.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115294"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145421998","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-02-01Epub Date: 2025-10-22DOI: 10.1016/j.jviromet.2025.115293
Jillian Redmond, Manoj Pastey
Respiratory Syncytial Virus (RSV) is a major concern in infants, the elderly, and immunocompromised individuals. RSV is a thermolabile virus posing challenges for vaccine development and laboratory handling. We evaluated whether low concentrations of sucrose could enhance RSV stability under standard and stress-inducing conditions. Using plaque assays on HeLa cells, we tested sucrose at concentrations from 0.1 % to 7 %. A statistically significant increase in RSV titer (∼40 %) was observed only with 0.3 % sucrose (p = 0.0009). Follow-up experiments demonstrated that 0.3 % sucrose significantly protected RSV from thermal degradation at 25°C and 37°C and from viability loss over three freeze–thaw cycles. Compared to previously reported stabilizers such as high-concentration sucrose, trehalose, or gelatin, our low-dose sucrose formulation avoids viscosity and processing issues while still offering practical enhancement. This is particularly relevant to live-attenuated vaccine platforms, which require stable formulations to maintain potency during storage and transport. These findings suggest 0.3 % sucrose provides a cost-effective, scalable strategy for improving RSV viability during handling and formulation.
{"title":"Low-concentration sucrose improves respiratory syncytial virus viability under thermal and Freeze–Thaw stress: An efficient solution for virology and vaccine studies","authors":"Jillian Redmond, Manoj Pastey","doi":"10.1016/j.jviromet.2025.115293","DOIUrl":"10.1016/j.jviromet.2025.115293","url":null,"abstract":"<div><div>Respiratory Syncytial Virus (RSV) is a major concern in infants, the elderly, and immunocompromised individuals. RSV is a thermolabile virus posing challenges for vaccine development and laboratory handling. We evaluated whether low concentrations of sucrose could enhance RSV stability under standard and stress-inducing conditions. Using plaque assays on HeLa cells, we tested sucrose at concentrations from 0.1 % to 7 %. A statistically significant increase in RSV titer (∼40 %) was observed only with 0.3 % sucrose (p = 0.0009). Follow-up experiments demonstrated that 0.3 % sucrose significantly protected RSV from thermal degradation at 25°C and 37°C and from viability loss over three freeze–thaw cycles. Compared to previously reported stabilizers such as high-concentration sucrose, trehalose, or gelatin, our low-dose sucrose formulation avoids viscosity and processing issues while still offering practical enhancement. This is particularly relevant to live-attenuated vaccine platforms, which require stable formulations to maintain potency during storage and transport. These findings suggest 0.3 % sucrose provides a cost-effective, scalable strategy for improving RSV viability during handling and formulation.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115293"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145364826","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) are important transboundary pathogens that cause substantial economic losses in the cattle industry globally. Their early detection and control are critical for preventing disease transmission and minimizing their impact on livestock health and productivity. Therefore, establishing a sensitive and robust diagnostic method capable of simultaneously detecting BLV and BVDV is vital for implementing timely control measures. Here, we developed a multiplex RT-dPCR assay to detect BLV and BVDV in a single-tube reaction using nucleic acid extracted from whole blood of infected cattle. The multiplex RT-dPCR assay successfully detected both BLV and BVDV with high specificity, exhibiting no cross-reactivity with other bovine viruses including Akabane virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, bovine herpesvirus 1, and bovine immunodeficiency virus. The assay demonstrated the ability to detect BVDV-1 and BVDV-2 at minimum titers of 10² and 10 ³ TCID₅₀/mL, respectively. For BLV, the multiplex RT-dPCR assay exhibited a detection limit as low as 18.7 viral copies. Our findings represent a significant advance in the detection of BLV and BVDV from extracted RNA and cDNA, highlighting the potential of assays for achieving improved diagnostic accuracy and early disease intervention.
{"title":"Development of multiplex reverse-transcription digital PCR assay for co-detection of bovine leukemia virus and bovine viral diarrhea virus in cattle","authors":"Shakir Ullah , Kosuke Notsu , Akatsuki Saito , Tamaki Okabayashi , Hirohisa Mekata , Mai Shiokawa , Hiroshi Aoki , Satoshi Sekiguchi","doi":"10.1016/j.jviromet.2025.115298","DOIUrl":"10.1016/j.jviromet.2025.115298","url":null,"abstract":"<div><div>Bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) are important transboundary pathogens that cause substantial economic losses in the cattle industry globally. Their early detection and control are critical for preventing disease transmission and minimizing their impact on livestock health and productivity. Therefore, establishing a sensitive and robust diagnostic method capable of simultaneously detecting BLV and BVDV is vital for implementing timely control measures. Here, we developed a multiplex RT-dPCR assay to detect BLV and BVDV in a single-tube reaction using nucleic acid extracted from whole blood of infected cattle. The multiplex RT-dPCR assay successfully detected both BLV and BVDV with high specificity, exhibiting no cross-reactivity with other bovine viruses including Akabane virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, bovine herpesvirus 1, and bovine immunodeficiency virus. The assay demonstrated the ability to detect BVDV-1 and BVDV-2 at minimum titers of 10² and 10 ³ TCID₅₀/mL, respectively. For BLV, the multiplex RT-dPCR assay exhibited a detection limit as low as 18.7 viral copies. Our findings represent a significant advance in the detection of BLV and BVDV from extracted RNA and cDNA, highlighting the potential of assays for achieving improved diagnostic accuracy and early disease intervention.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115298"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145477027","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-02-01Epub Date: 2025-11-22DOI: 10.1016/j.jviromet.2025.115308
Pablo Piñeyro , Brett Webb , Sheela Ramamoorthy
Torque Teno viruses (TTVs) are ubiquitous, small DNA viruses which are highly epidemiologically associated with respiratory infections, hepatitis, neurological disease and autoimmune disorders in humans and animals. Swine TTVs (TTSuVs) can be considered opportunistic pathogens as they exacerbate clinical signs due to coinfecting agents. While further understanding of how TTVs contribute to disease is crucial, there is a notable lack of animal models and tools to study the in vivo infection patterns of TTV. RNA in situ hybridization (RNA-ISH) with multiple probe amplification has recently gained popularity due to its high levels of specificity and sensitivity and ability to detect agent specific RNA or mRNA. Currently there are no commercial TTSuV1 antibodies that allow viral antigen detection by immunohistochemistry assay that can be used to advance the understanding of TTSuV1 pathogenicity. Therefore, the goal of this study was to develop an RNA-ISH assay for TTSuV1. To generate positive control, PK-15 cells grown in chamber slides were either infected with TTSuV1 or transfected with the TTSuV1 genome. A cocktail of TTSuV1 ORF1-specific RNA probes was hybridized to the cells, and specific binding was successfully visualized using a chromogenic reaction. Liver, kidney, heart, spleen and intestines were collected from mice infected with TTSuV1 at 15- and 30-days post infection. Finally, the RNA-ISH was optimized for TTSuV1 mRNA detection in tissues. TTSuV1-specific signal was detected in the hepatocytes and renal tubular epithelium of infected mice at a detection rate of 33 % 15- and 30-days post infection. In summary, the described RNA ISH assay is a useful tool to visualizeTTSuV1 viral replication in tissues and has potential application to clinical specimens in the future.
{"title":"Detection of Torque Teno Sus Virus1 by an RNA in situ hybridization assay","authors":"Pablo Piñeyro , Brett Webb , Sheela Ramamoorthy","doi":"10.1016/j.jviromet.2025.115308","DOIUrl":"10.1016/j.jviromet.2025.115308","url":null,"abstract":"<div><div>Torque Teno viruses (TTVs) are ubiquitous, small DNA viruses which are highly epidemiologically associated with respiratory infections, hepatitis, neurological disease and autoimmune disorders in humans and animals. Swine TTVs (TTSuVs) can be considered opportunistic pathogens as they exacerbate clinical signs due to coinfecting agents. While further understanding of how TTVs contribute to disease is crucial, there is a notable lack of animal models and tools to study the in vivo infection patterns of TTV. RNA in situ hybridization (RNA-ISH) with multiple probe amplification has recently gained popularity due to its high levels of specificity and sensitivity and ability to detect agent specific RNA or mRNA. Currently there are no commercial TTSuV1 antibodies that allow viral antigen detection by immunohistochemistry assay that can be used to advance the understanding of TTSuV1 pathogenicity. Therefore, the goal of this study was to develop an RNA-ISH assay for TTSuV1. To generate positive control, PK-15 cells grown in chamber slides were either infected with TTSuV1 or transfected with the TTSuV1 genome. A cocktail of TTSuV1 ORF1-specific RNA probes was hybridized to the cells, and specific binding was successfully visualized using a chromogenic reaction. Liver, kidney, heart, spleen and intestines were collected from mice infected with TTSuV1 at 15- and 30-days post infection. Finally, the RNA-ISH was optimized for TTSuV1 mRNA detection in tissues. TTSuV1-specific signal was detected in the hepatocytes and renal tubular epithelium of infected mice at a detection rate of 33 % 15- and 30-days post infection. In summary, the described RNA ISH assay is a useful tool to visualizeTTSuV1 viral replication in tissues and has potential application to clinical specimens in the future.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115308"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145596639","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hepatitis delta virus (HDV) is a defective RNA virus that causes severe liver diseases. Despite its endemicity in a few countries, the worldwide prevalence of HDV is unequal. Therefore, the development of sophisticated diagnostic assays for detecting HDV infection is less attractive and the diversity of commercially available kits is limited.
Methods
Recombinant 23 kDa His-tagged sHDAg protein was produced in E. coli and purified by Ni-NTA chromatography. An ELISA plate was assembled using a 3-Aminopropyl triethoxysilane (APTES) linker to create highly capacitive and organized binding. For analysis, prequalified 460 samples (Positive group: HBsAg and anti-HDV/HDV-RNA positive, n = 220; Negative group: HBsAg positive and anti-HDV/HDV-RNA negative, n = 40, anti-HCV and HBsAg negative, n = 200) were used. Based on the optimized protocol, cut-off values were identified using different methods.
Results
We found that the ELISA assay is ultra-sensitive and can detect up to 10^9 times diluted samples. Three different cut-off values (0.8201, 0.7232, and 0.7285) were obtained using the ROC curve, distribution curve, and standard deviation-based methods. The assay demonstrated stable results, including a specificity of 96.25 %-97.08 %, a sensitivity of 96.36 % and an area under the curve (AUC) was 0.98843 (95 %CI=0.97897–0.99789, SE=0.0048, p < 0.001).
Conclusion
ELISA developed using a chemical crosslinking method can detect HDV infection in highly sensitive and rapid ways and should be introduced in clinical practice.
{"title":"Development and validation of an ultra-sensitive ELISA using APTES-based chemical crosslinking method for detection of anti-HDV in human serum","authors":"Nomin Ariungerel , Badmaarag Munkhjin , Ulziigerel Erdembileg , Saruul Enkhjargal , Zaya Batsuuri , Tuul Tsagaantsooj , Sanjaasuren Enkhtaivan , Byambasuren Ochirsum , Purevjargal Bat-Ulzii , Enkhnomin Ochirbat , Nara Bungert Dashdorj , Odgerel Oidovsambuu","doi":"10.1016/j.jviromet.2025.115288","DOIUrl":"10.1016/j.jviromet.2025.115288","url":null,"abstract":"<div><h3>Background</h3><div>Hepatitis delta virus (HDV) is a defective RNA virus that causes severe liver diseases. Despite its endemicity in a few countries, the worldwide prevalence of HDV is unequal. Therefore, the development of sophisticated diagnostic assays for detecting HDV infection is less attractive and the diversity of commercially available kits is limited.</div></div><div><h3>Methods</h3><div>Recombinant 23 kDa His-tagged sHDAg protein was produced in <em>E. coli</em> and purified by Ni-NTA chromatography. An ELISA plate was assembled using a 3-Aminopropyl triethoxysilane (APTES) linker to create highly capacitive and organized binding. For analysis, prequalified 460 samples (Positive group: HBsAg and anti-HDV/HDV-RNA positive, n = 220; Negative group: HBsAg positive and anti-HDV/HDV-RNA negative, n = 40, anti-HCV and HBsAg negative, n = 200) were used. Based on the optimized protocol, cut-off values were identified using different methods.</div></div><div><h3>Results</h3><div>We found that the ELISA assay is ultra-sensitive and can detect up to 10^9 times diluted samples. Three different cut-off values (0.8201, 0.7232, and 0.7285) were obtained using the ROC curve, distribution curve, and standard deviation-based methods. The assay demonstrated stable results, including a specificity of 96.25 %-97.08 %, a sensitivity of 96.36 % and an area under the curve (AUC) was 0.98843 (95 %CI=0.97897–0.99789, SE=0.0048, p < 0.001).</div></div><div><h3>Conclusion</h3><div>ELISA developed using a chemical crosslinking method can detect HDV infection in highly sensitive and rapid ways and should be introduced in clinical practice.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115288"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145313145","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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