Pub Date : 2025-11-07DOI: 10.1016/j.jviromet.2025.115299
Rajamanickam Hema Sayee , Madhusudan Hosamani , Narayanan Krishnaswamy , Subramaniyan Shanmuganathan , Manchikanti Sri Sai Charan , Dasu Ramadoss Vignesh , Veerakyathappa Bhanuprakash
In enzootic regions like India, foot-and-mouth disease (FMD) is managed through vaccination and strict biosecurity. Traditionally, FMD vaccine potency is assessed via in vivo challenge, which raises animal welfare concerns. As an alternative, this study evaluated a serology-based indirect potency test using 87 serum samples derived from calves 28 days post-vaccination. Samples were analyzed via virus neutralization test (VNT) and SPCEs employing polyclonal (pAb) and monoclonal antibodies (mAbs 2C4G11 and 6E8D11). Log₁₀ serum titres (ti) were correlated with protection outcomes from live virus challenge (FMDV serotype A/40/2000) using logistic regression. Four logit models were developed, and ROC analysis determined ti cut-offs for 50 %, 75 %, and 95 % protection probabilities (pi). VNT and mAb SPCEs showed sigmoid logit curves, indicating strong ti–pi correlation. Based on AIC and Somers’ D, mAb 2C4G11 SPCE was the best-fit model. At 75 % pi, ti thresholds for VNT, pAb, mAb 2C4G11, and mAb 6E8D11 were > 1.204, > 1.041, > 1.204, and > 1.204, respectively, with mAb 6E8D11 showing highest accuracy. Thus, mAbs 2C4G11 and 6E8D11 SPCEs are promising tools for predicting homologous protection. Further validation with larger sample sizes is recommended.
{"title":"Development of monoclonal antibody based SPCE logistic regression model to predict the protective status of animals vaccinated against FMD virus type A","authors":"Rajamanickam Hema Sayee , Madhusudan Hosamani , Narayanan Krishnaswamy , Subramaniyan Shanmuganathan , Manchikanti Sri Sai Charan , Dasu Ramadoss Vignesh , Veerakyathappa Bhanuprakash","doi":"10.1016/j.jviromet.2025.115299","DOIUrl":"10.1016/j.jviromet.2025.115299","url":null,"abstract":"<div><div>In enzootic regions like India, foot-and-mouth disease (FMD) is managed through vaccination and strict biosecurity. Traditionally, FMD vaccine potency is assessed via <em>in vivo</em> challenge, which raises animal welfare concerns. As an alternative, this study evaluated a serology-based indirect potency test using 87 serum samples derived from calves 28 days post-vaccination. Samples were analyzed via virus neutralization test (VNT) and SPCEs employing polyclonal (pAb) and monoclonal antibodies (mAbs 2C4G11 and 6E8D11). Log₁₀ serum titres (<em>ti</em>) were correlated with protection outcomes from live virus challenge (FMDV serotype A/40/2000) using logistic regression. Four logit models were developed, and ROC analysis determined <em>ti</em> cut-offs for 50 %, 75 %, and 95 % protection probabilities (<em>pi</em>). VNT and mAb SPCEs showed sigmoid logit curves, indicating strong <em>ti–pi</em> correlation. Based on AIC and Somers’ D, mAb 2C4G11 SPCE was the best-fit model. At 75 % <em>pi</em>, <em>ti</em> thresholds for VNT, pAb, mAb 2C4G11, and mAb 6E8D11 were > 1.204, > 1.041, > 1.204, and > 1.204, respectively, with mAb 6E8D11 showing highest accuracy. Thus, mAbs 2C4G11 and 6E8D11 SPCEs are promising tools for predicting homologous protection. Further validation with larger sample sizes is recommended.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115299"},"PeriodicalIF":1.6,"publicationDate":"2025-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145477071","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":"2025-11-06","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 : 2025-11-05DOI: 10.1016/j.jviromet.2025.115297
Jennifer Kopetzky , Sarah C. Mansour , Rachel Floyd , Liam Byrne , Christine Tchao , Natalie Prystajecky
The ability to monitor the prevalence of respiratory viruses in a community depends on testing infrastructure, surveillance programs, and community engagement. Wastewater-based surveillance has recently evolved as a tool to monitor disease at a population level with reduced costs, diverse surveillance coverage, and inherently passive population participation. This study designed a molecular assay for the detection and quantification of multiple respiratory viruses—SARS-CoV-2, influenza A, influenza B, and Respiratory Syncytial Virus (RSV)—from wastewater samples. The presence of inhibitory substances in the wastewater matrix and multiple molecular targets did not prove to be limiting factors for the performance of the assay. Evaluation of the assay using municipal wastewater treatment plant samples showed a strong concordance with publicly available surveillance and clinical case data in British Columbia, Canada. Existing and future community testing programs for respiratory viruses should integrate wastewater-based surveillance to capture asymptomatic/non-testing populations, expand surveillance coverage, and provide greater insight into the community dynamics of respiratory viruses.
{"title":"Development and evaluation of a molecular assay for the detection and quantification of SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus from municipal wastewater in British Columbia","authors":"Jennifer Kopetzky , Sarah C. Mansour , Rachel Floyd , Liam Byrne , Christine Tchao , Natalie Prystajecky","doi":"10.1016/j.jviromet.2025.115297","DOIUrl":"10.1016/j.jviromet.2025.115297","url":null,"abstract":"<div><div>The ability to monitor the prevalence of respiratory viruses in a community depends on testing infrastructure, surveillance programs, and community engagement. Wastewater-based surveillance has recently evolved as a tool to monitor disease at a population level with reduced costs, diverse surveillance coverage, and inherently passive population participation. This study designed a molecular assay for the detection and quantification of multiple respiratory viruses—SARS-CoV-2, influenza A, influenza B, and Respiratory Syncytial Virus (RSV)—from wastewater samples. The presence of inhibitory substances in the wastewater matrix and multiple molecular targets did not prove to be limiting factors for the performance of the assay. Evaluation of the assay using municipal wastewater treatment plant samples showed a strong concordance with publicly available surveillance and clinical case data in British Columbia, Canada. Existing and future community testing programs for respiratory viruses should integrate wastewater-based surveillance to capture asymptomatic/non-testing populations, expand surveillance coverage, and provide greater insight into the community dynamics of respiratory viruses.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115297"},"PeriodicalIF":1.6,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145471344","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 : 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":"2025-11-03","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 : 2025-11-01DOI: 10.1016/j.jviromet.2025.115296
M. Jane Morwitzer, Ying Yi Zheng, Heather Friberg, Jeffrey R. Currier
The SARS-CoV-2 spike (S) protein plays a central role in viral entry through receptor binding and membrane fusion, making it a key target for therapeutic interventions. While existing assays for studying spike-mediated fusion can be complex and lack real-time monitoring, we present a split GFP-based cell fusion assay that provides a straightforward and adaptable platform for evaluating fusion dynamics. This assay utilizes a split GFP system in non-adherent 293-F cells to detect fusion events, allowing for reliable assessment of spike-targeting monoclonal antibodies and fusion inhibitors. Our study demonstrates the effectiveness of this approach in evaluating the inhibitory potential of therapeutics against multiple SARS-CoV-2 variants. The results highlight the assay’s ability to distinguish variant-specific fusion characteristics and inhibitor responses, particularly in the context of Omicron’s altered entry pathways. By enabling the quantitative, real-time assessment of spike-mediated fusion, this platform provides a valuable tool for therapeutic screening, supporting future efforts to develop antiviral strategies against SARS-CoV-2 and other emerging coronaviruses.
{"title":"A split GFP approach to assay SARS-CoV-2 spike-dependent cell fusion","authors":"M. Jane Morwitzer, Ying Yi Zheng, Heather Friberg, Jeffrey R. Currier","doi":"10.1016/j.jviromet.2025.115296","DOIUrl":"10.1016/j.jviromet.2025.115296","url":null,"abstract":"<div><div>The SARS-CoV-2 spike (S) protein plays a central role in viral entry through receptor binding and membrane fusion, making it a key target for therapeutic interventions. While existing assays for studying spike-mediated fusion can be complex and lack real-time monitoring, we present a split GFP-based cell fusion assay that provides a straightforward and adaptable platform for evaluating fusion dynamics. This assay utilizes a split GFP system in non-adherent 293-F cells to detect fusion events, allowing for reliable assessment of spike-targeting monoclonal antibodies and fusion inhibitors. Our study demonstrates the effectiveness of this approach in evaluating the inhibitory potential of therapeutics against multiple SARS-CoV-2 variants. The results highlight the assay’s ability to distinguish variant-specific fusion characteristics and inhibitor responses, particularly in the context of Omicron’s altered entry pathways. By enabling the quantitative, real-time assessment of spike-mediated fusion, this platform provides a valuable tool for therapeutic screening, supporting future efforts to develop antiviral strategies against SARS-CoV-2 and other emerging coronaviruses.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115296"},"PeriodicalIF":1.6,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145431527","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 : 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":"2025-10-30","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 : 2025-10-27DOI: 10.1016/j.jviromet.2025.115292
Michelle L. Rock , Jessica B. Huskey , Sarah Hernandez , Divya P. Shinde , Thomas H. Oguin III , Sara Ping , Arabella Lewis , Jesse J. Waggoner , M. Anthony Moody , Gregory D. Sempowski , Brook E. Heaton
The Centers for Research in Emerging Infectious Diseases (CREID) was established to enhance pandemic preparedness by studying emerging/reemerging pathogens, especially in resource-limited regions. To overcome infrastructure challenges, a low-cost, field-deployable method for extracting total nucleic acids is essential, eliminating reliance on expensive equipment, power, and cold chain systems used in traditional extraction techniques. To address this challenge, we developed an RNA extraction and storage method (RNAES) that meets these criteria. Herein, we report RNAES inactivation efficacy against nine prototype viruses (Middle Eastern respiratory syndrome coronavirus, Japanese encephalitis virus, West Nile virus, Hantaan virus, measles virus, Heartland virus, enterovirus A71, chikungunya virus, and Western equine encephalitis virus) representing seven pandemic potential virus families. We compare the RNAES method to the Qiagen QIAamp kit across various viral loads and field sample types. The presence of infectious virus in RNA samples was quantified using plaque assays. Successful inactivation of viruses was demonstrated for six enveloped virus families spiked into matrices routinely collected at field sites. The seventh family tested (Picornaviridae) was not completely inactivated, likely due to non-enveloped viruses being differentially susceptible to the lysis chemistry of the RNAES kit. The commercial comparator inactivated all viruses tested. Specialized biosafety facilities, specific detailed permits, and comprehensive logistics are required to ensure safety when handling and shipping potentially infectious samples. Inactivating pathogens at the point of collection reduces risks and simplifies sample transfer for critical outbreak research. Confidently ensuring that an isolated nucleic acid sample is non-infectious using RNAES will enable safer, and more efficient downstream analysis.
{"title":"Validation of prototype virus inactivation from seven virus families of pandemic potential with a novel low-cost, field-deployable RNA extraction and storage method","authors":"Michelle L. Rock , Jessica B. Huskey , Sarah Hernandez , Divya P. Shinde , Thomas H. Oguin III , Sara Ping , Arabella Lewis , Jesse J. Waggoner , M. Anthony Moody , Gregory D. Sempowski , Brook E. Heaton","doi":"10.1016/j.jviromet.2025.115292","DOIUrl":"10.1016/j.jviromet.2025.115292","url":null,"abstract":"<div><div>The Centers for Research in Emerging Infectious Diseases (CREID) was established to enhance pandemic preparedness by studying emerging/reemerging pathogens, especially in resource-limited regions. To overcome infrastructure challenges, a low-cost, field-deployable method for extracting total nucleic acids is essential, eliminating reliance on expensive equipment, power, and cold chain systems used in traditional extraction techniques. To address this challenge, we developed an RNA extraction and storage method (RNAES) that meets these criteria. Herein, we report RNAES inactivation efficacy against nine prototype viruses (Middle Eastern respiratory syndrome coronavirus, Japanese encephalitis virus, West Nile virus, Hantaan virus, measles virus, Heartland virus, enterovirus A71, chikungunya virus, and Western equine encephalitis virus) representing seven pandemic potential virus families. We compare the RNAES method to the Qiagen QIAamp kit across various viral loads and field sample types. The presence of infectious virus in RNA samples was quantified using plaque assays. Successful inactivation of viruses was demonstrated for six enveloped virus families spiked into matrices routinely collected at field sites. The seventh family tested (<em>Picornaviridae</em>) was not completely inactivated, likely due to non-enveloped viruses being differentially susceptible to the lysis chemistry of the RNAES kit. The commercial comparator inactivated all viruses tested. Specialized biosafety facilities, specific detailed permits, and comprehensive logistics are required to ensure safety when handling and shipping potentially infectious samples. Inactivating pathogens at the point of collection reduces risks and simplifies sample transfer for critical outbreak research. Confidently ensuring that an isolated nucleic acid sample is non-infectious using RNAES will enable safer, and more efficient downstream analysis.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115292"},"PeriodicalIF":1.6,"publicationDate":"2025-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145401281","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 : 2025-10-26DOI: 10.1016/j.jviromet.2025.115291
Sahar Mahmood , Saumya S. Thomas , Craig S. Ross , Louise Fothergill , Rowena D.E. Hansen , Rachel Jinks , Ian H. Brown , Marco Falchieri , Joe James , Ashley C. Banyard , Scott M. Reid
Whilst molecular methods which detect viral nucleic acid have replaced more traditional assays such as virus isolation (VI) as frontline diagnostic assays for avian influenza virus (AIV), the ability to isolate live viruses from samples remains critical for all downstream research and virus categorisation activities. VI, when successful, enables detailed functional characterisation of the AIV strain and provides essential reference material for diagnostic testing for animal and public health. However, VI is resource intensive, requiring specialist facilities, reagents, trained staff and time. Therefore, understanding the factors which contribute to successful VI is paramount in targeting efforts. In this study, using a range of clinical samples collected from wild birds during the high pathogenicity AIV 2020–2022 panzootic, we assessed factors which limited successful VI including sample collection time, sample type, subtype, originator species and sample viral RNA level. Although virus stability is highly temperature-sensitive and cold chain failure or prolonged exposure of carcasses or samples to ambient temperatures could negatively impact virus viability despite strong RT-PCR Cq values, we propose an upper RT-PCR Cq value, which increases the likelihood for optimal VI when handling poor quality material. Understanding these factors enables efficient triage for VI to direct resources for maximum success.
{"title":"Understanding limitations to successful avian influenza virus isolation from wild birds","authors":"Sahar Mahmood , Saumya S. Thomas , Craig S. Ross , Louise Fothergill , Rowena D.E. Hansen , Rachel Jinks , Ian H. Brown , Marco Falchieri , Joe James , Ashley C. Banyard , Scott M. Reid","doi":"10.1016/j.jviromet.2025.115291","DOIUrl":"10.1016/j.jviromet.2025.115291","url":null,"abstract":"<div><div>Whilst molecular methods which detect viral nucleic acid have replaced more traditional assays such as virus isolation (VI) as frontline diagnostic assays for avian influenza virus (AIV), the ability to isolate live viruses from samples remains critical for all downstream research and virus categorisation activities. VI, when successful, enables detailed functional characterisation of the AIV strain and provides essential reference material for diagnostic testing for animal and public health. However, VI is resource intensive, requiring specialist facilities, reagents, trained staff and time. Therefore, understanding the factors which contribute to successful VI is paramount in targeting efforts. In this study, using a range of clinical samples collected from wild birds during the high pathogenicity AIV 2020–2022 panzootic, we assessed factors which limited successful VI including sample collection time, sample type, subtype, originator species and sample viral RNA level. Although virus stability is highly temperature-sensitive and cold chain failure or prolonged exposure of carcasses or samples to ambient temperatures could negatively impact virus viability despite strong RT-PCR Cq values, we propose an upper RT-PCR Cq value, which increases the likelihood for optimal VI when handling poor quality material. Understanding these factors enables efficient triage for VI to direct resources for maximum success.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115291"},"PeriodicalIF":1.6,"publicationDate":"2025-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145390457","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 : 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":"2025-10-22","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}
Pub 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":"2025-10-17","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}
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