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}
Efficient production of high-titer rabies virus (RABV) stocks is critical for vaccine development, diagnostic assay design, and virological research. This study employed a Quality by Design (QbD) framework to optimize RABV propagation in Neuro-2a (N2a) cells, targeting infectious titers ≥ 8.0 log₁₀ FFU/mL and cell viability ≥ 85 %. Three genetically distinct RABV strains were evaluated: the laboratory-adapted CVS-11 strain for vaccine production and two field isolates, MN-18 and V-M, relevant to diagnostics and research. A 2 ³ full factorial Design of Experiments (DOE) was implemented to assess the impact of multiplicity of infection (MOI), incubation temperature, and culture medium composition on critical quality attributes (CQAs). Distinct strain-specific responses were observed: CVS-11 and V-M were significantly influenced by MOI (p < 0.05), whereas MN-18 demonstrated a more stable replication profile. Enriched medium significantly enhanced viral titers and cell viability across all strains (p < 0.001). Monte Carlo simulations (n = 10,000) delineated an optimal operational space—MOI 0.1–1.0, temperature 34–35°C, enriched medium—ensuring process robustness with 95 % confidence. This QbD-guided strategy provides a scalable, reproducible platform for generating high-quality RABV stocks, facilitating the manufacture of CVS-11-based vaccines and supporting the diagnostic use of field isolates as biologically standardized reference reagents for diagnostics.
{"title":"Optimization of rabies virus production in neuroblastoma cells using quality by design principles for vaccine and diagnostic applications","authors":"Nayara Ugeda , Orlando Garcia Ribeiro , Elaine Raniero Fernandes , Fernanda Guedes , Sandriana Dos Ramos Silva , Iana Suly Santos Katz","doi":"10.1016/j.jviromet.2025.115289","DOIUrl":"10.1016/j.jviromet.2025.115289","url":null,"abstract":"<div><div>Efficient production of high-titer rabies virus (RABV) stocks is critical for vaccine development, diagnostic assay design, and virological research. This study employed a Quality by Design (QbD) framework to optimize RABV propagation in Neuro-2a (N2a) cells, targeting infectious titers ≥ 8.0 log₁₀ FFU/mL and cell viability ≥ 85 %. Three genetically distinct RABV strains were evaluated: the laboratory-adapted CVS-11 strain for vaccine production and two field isolates, MN-18 and V-M, relevant to diagnostics and research. A 2 ³ full factorial Design of Experiments (DOE) was implemented to assess the impact of multiplicity of infection (MOI), incubation temperature, and culture medium composition on critical quality attributes (CQAs). Distinct strain-specific responses were observed: CVS-11 and V-M were significantly influenced by MOI (p < 0.05), whereas MN-18 demonstrated a more stable replication profile. Enriched medium significantly enhanced viral titers and cell viability across all strains (p < 0.001). Monte Carlo simulations (n = 10,000) delineated an optimal operational space—MOI 0.1–1.0, temperature 34–35°C, enriched medium—ensuring process robustness with 95 % confidence. This QbD-guided strategy provides a scalable, reproducible platform for generating high-quality RABV stocks, facilitating the manufacture of CVS-11-based vaccines and supporting the diagnostic use of field isolates as biologically standardized reference reagents for diagnostics.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115289"},"PeriodicalIF":1.6,"publicationDate":"2025-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145318434","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":"2025-10-15","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}
Pub Date : 2025-10-14DOI: 10.1016/j.jviromet.2025.115279
Jonathan Thibaut , Fabiana Gámbaro , Caspar Geenen , Samuel L. Hong , Joren Raymenants , Sarah Gorissen , Lize Cuypers , Piet Maes , Guy Baele , Simon Dellicour , Emmanuel André
During the COVID-19 pandemic, contact tracing was widely used to limit virus propagation and implement targeted disease control measures. It can however be difficult to assess whether infected cases are actually linked to the traced index case. In this study, we developed and applied an analytical pipeline using genomic data to assess the precision of contact tracing, defined as the proportion of transmission events suggested by contact tracing that are not contradicted by genomic analysis. We exemplify our approach by examining the transmission of SARS-CoV-2 among students at Belgium’s largest university, in Leuven, during the Omicron BA.1 and BA.2 epidemic waves. We analysed 459 case-contact pairs identified through contact tracing. We then aimed to determine whether the pairs, where patients were infected with the same variant, clustered together within a time-scaled phylogeny. Our findings indicate that 34.6 % of transmission events suggested by contact tracing were not invalidated by our combined phylogenetic and single nucleotide polymorphism analysis. Only considering non invalidated transmission events, we estimated serial intervals with a smaller standard deviation than with all case-contact pairs. Our genomic-based pipeline allows us to assess the ability of our contact tracing program to correctly identify transmission chains. While contact tracing is crucial for early outbreak detection and control, monitoring its precision is vital for using this data in public health decisions.
{"title":"A novel methodology for assessing contact tracing precision: Phylogenetic validation of a contact tracing program for COVID-19 in Belgium","authors":"Jonathan Thibaut , Fabiana Gámbaro , Caspar Geenen , Samuel L. Hong , Joren Raymenants , Sarah Gorissen , Lize Cuypers , Piet Maes , Guy Baele , Simon Dellicour , Emmanuel André","doi":"10.1016/j.jviromet.2025.115279","DOIUrl":"10.1016/j.jviromet.2025.115279","url":null,"abstract":"<div><div>During the COVID-19 pandemic, contact tracing was widely used to limit virus propagation and implement targeted disease control measures. It can however be difficult to assess whether infected cases are actually linked to the traced index case. In this study, we developed and applied an analytical pipeline using genomic data to assess the precision of contact tracing, defined as the proportion of transmission events suggested by contact tracing that are not contradicted by genomic analysis. We exemplify our approach by examining the transmission of SARS-CoV-2 among students at Belgium’s largest university, in Leuven, during the Omicron BA.1 and BA.2 epidemic waves. We analysed 459 case-contact pairs identified through contact tracing. We then aimed to determine whether the pairs, where patients were infected with the same variant, clustered together within a time-scaled phylogeny. Our findings indicate that 34.6 % of transmission events suggested by contact tracing were not invalidated by our combined phylogenetic and single nucleotide polymorphism analysis. Only considering non invalidated transmission events, we estimated serial intervals with a smaller standard deviation than with all case-contact pairs. Our genomic-based pipeline allows us to assess the ability of our contact tracing program to correctly identify transmission chains. While contact tracing is crucial for early outbreak detection and control, monitoring its precision is vital for using this data in public health decisions.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115279"},"PeriodicalIF":1.6,"publicationDate":"2025-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145308422","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 : 2025-10-13DOI: 10.1016/j.jviromet.2025.115280
Larissa Baldo Vieira , Taís Fukuta da Cruz , Bruna Lindolfo da Silva , Kayo de Paiva Pereira , João Pessoa Araújo Junior , Angelo José Magro
Porcine circoviruses (PCV) are widespread in the global swine population and is notable for having the smallest known viral genome and structure. Among its species, porcine circovirus 2 (PCV2) is particularly significant due to its association with porcine circovirus diseases (PCVD), a group of syndromes affecting various organs, with the most common symptom being reduced zootechnical performance. Monitoring and preventing this infection are crucial to reducing economic losses and improving animal welfare. In this context, producing synthetic infectious PCV2b particles from a fully designed genome allows insights into the virus's structure and biology. This approach could facilitate the development of engineered viruses for diverse biotechnological applications, such as novel vaccine candidates and diagnostic assay components. The main objective of this study was to standardize the procedures for generating synthetic viral particles in cell culture from a synthetic viral genome. To achieve this, we transfected the synthetic genome into swine testicular (ST) cells and maintained them under optimal conditions, subcultivating the cells to support growth. After transfection, a cell culture infection assay was conducted using the supernatant from the transfected cells. Viral DNA from transfected and infected cultures was quantified by quantitative polymerase chain reaction (qPCR), antigen production was assessed by enzyme-linked immunosorbent assay (ELISA), and the surface structure of the synthetic viral particles was visualized by transmission electron microscopy with negative staining. The results strongly support the successful production of infectious synthetic PCV2b particles, enabling their use in biotechnological applications for this and related species.
{"title":"Experimental production of synthetic infectious porcine circovirus 2b particles in swine testicular cells","authors":"Larissa Baldo Vieira , Taís Fukuta da Cruz , Bruna Lindolfo da Silva , Kayo de Paiva Pereira , João Pessoa Araújo Junior , Angelo José Magro","doi":"10.1016/j.jviromet.2025.115280","DOIUrl":"10.1016/j.jviromet.2025.115280","url":null,"abstract":"<div><div>Porcine circoviruses (PCV) are widespread in the global swine population and is notable for having the smallest known viral genome and structure. Among its species, porcine circovirus 2 (PCV2) is particularly significant due to its association with porcine circovirus diseases (PCVD), a group of syndromes affecting various organs, with the most common symptom being reduced zootechnical performance. Monitoring and preventing this infection are crucial to reducing economic losses and improving animal welfare. In this context, producing synthetic infectious PCV2b particles from a fully designed genome allows insights into the virus's structure and biology. This approach could facilitate the development of engineered viruses for diverse biotechnological applications, such as novel vaccine candidates and diagnostic assay components. The main objective of this study was to standardize the procedures for generating synthetic viral particles in cell culture from a synthetic viral genome. To achieve this, we transfected the synthetic genome into swine testicular (ST) cells and maintained them under optimal conditions, subcultivating the cells to support growth. After transfection, a cell culture infection assay was conducted using the supernatant from the transfected cells. Viral DNA from transfected and infected cultures was quantified by quantitative polymerase chain reaction (qPCR), antigen production was assessed by enzyme-linked immunosorbent assay (ELISA), and the surface structure of the synthetic viral particles was visualized by transmission electron microscopy with negative staining. The results strongly support the successful production of infectious synthetic PCV2b particles, enabling their use in biotechnological applications for this and related species.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115280"},"PeriodicalIF":1.6,"publicationDate":"2025-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145301575","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 : 2025-10-12DOI: 10.1016/j.jviromet.2025.115271
Yumei Chen , Haonan Wang , Jingming Zhou , Chao Liang , Hongliang Liu , Sixuan Wu , Yanhua Qi , Xifang Zhu , Enping Liu , Aiping Wang
Pseudorabies virus (PRV), a zoonotic alphaherpesvirus threatening swine industries and human health, exploits glycoprotein D (gD)-nectin-1 interactions for host cell entry. Here, we report a dual-target neutralizing strategy combining a novel gD-specific monoclonal neutralization antibody (3F7) with nectin-1-blocking mAb. In vitro neutralization assays demonstrated that 3F7 alone achieved potent cross-species inhibition of PRV infection in porcine kidney cells (PK15), human neuroblastoma cells (SK-N-SH), and human embryonic kidney cells (HEK 293 T) (IC50: 0.56–5.39 μg/mL). Co-administration with nectin-1-targeting mAb synergistically enhanced neutralization efficacy by 0.9–25.8 %, revealing a cooperative mechanism between viral glycoprotein blockade and host receptor interference. This study presents the initial evidence indicating that antibodies targeting both the pathogen and its host entry factor, nectin-1, significantly enhance the anti-PRV activity. These findings highlight a novel and effective strategy for mitigating zoonotic PRV transmission, especially among high-risk populations such as swine workers. Moreover, the study provides a promising avenue for the development of next-generation biologics against emerging zoonotic herpesviruses, offering significant implications for both veterinary and human public health.
{"title":"The co-application of nectin-1 and PRV gD monoclonal antibodies: An effective approach to block pseudorabies virus invasion","authors":"Yumei Chen , Haonan Wang , Jingming Zhou , Chao Liang , Hongliang Liu , Sixuan Wu , Yanhua Qi , Xifang Zhu , Enping Liu , Aiping Wang","doi":"10.1016/j.jviromet.2025.115271","DOIUrl":"10.1016/j.jviromet.2025.115271","url":null,"abstract":"<div><div>Pseudorabies virus (PRV), a zoonotic alphaherpesvirus threatening swine industries and human health, exploits glycoprotein D (gD)-nectin-1 interactions for host cell entry. Here, we report a dual-target neutralizing strategy combining a novel gD-specific monoclonal neutralization antibody (3F7) with nectin-1-blocking mAb. In vitro neutralization assays demonstrated that 3F7 alone achieved potent cross-species inhibition of PRV infection in porcine kidney cells (PK15), human neuroblastoma cells (SK-N-SH), and human embryonic kidney cells (HEK 293 T) (IC<sub>50</sub>: 0.56–5.39 μg/mL). Co-administration with nectin-1-targeting mAb synergistically enhanced neutralization efficacy by 0.9–25.8 %, revealing a cooperative mechanism between viral glycoprotein blockade and host receptor interference. This study presents the initial evidence indicating that antibodies targeting both the pathogen and its host entry factor, nectin-1, significantly enhance the anti-PRV activity. These findings highlight a novel and effective strategy for mitigating zoonotic PRV transmission, especially among high-risk populations such as swine workers. Moreover, the study provides a promising avenue for the development of next-generation biologics against emerging zoonotic herpesviruses, offering significant implications for both veterinary and human public health.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115271"},"PeriodicalIF":1.6,"publicationDate":"2025-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145292695","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":"2025-10-11","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 : 2025-10-08DOI: 10.1016/j.jviromet.2025.115277
Obiageli Okafor , Jane Cameron , Andrea Garcia , Charmaine Hinahon , Carmen Salvador-Palomeque , Mitchell Starr , Philip H. Cunningham
Objectives
Monitoring HIV drug resistance is crucial for HIV treatment success. Logistical challenges restrict the use of liquid venous blood specimens for HIV drug resistance (HIVDR) testing, but dried blood spots (DBS) offer a more accessible alternative. This study evaluates the performance of TaqPath™ Seq HIV-1 Genotyping Kit (TaqPath kit) for detecting resistance-associated mutations in the protease (PR), reverse transcriptase (RT), and integrase (INI) regions of HIV-1 pol gene using DBS specimens.
Methods
A total of 79 DBS samples collected from ART-naive and ART-experienced individuals with viral loads ranging from 2830 to 9,095,161 copies/mL and HIV-1 subtypes A, B, C, F, and CRF01_AE were genotyped with TaqPath kit. Performance was assessed using the WHO HIVDR assay validation criteria.
Results
The assay met the amplification sensitivity criterion, achieving 95.7 % for PR/RT and 92.7 % for INI in high viral load samples. However, lower viral load samples between 2000 and 5000 copies/mL demonstrated reduced sensitivity of 50 % and 30 % for the PR/RT and INI regions respectively. Intra- and inter-assay agreement exceeded 99 % and mutation detection showed strong correlation with the reference assay, identifying over 95 % of reference mutations.
Conclusions
The TaqPath kit met the WHO acceptance criteria for HIVDR assay validation, except in the INI region at low viral loads, where amplification sensitivity was below the required threshold. Dried blood spot specimens are compatible with TaqPath kit for baseline and treatment-failure HIVDR genotyping, offering a reliable alternative to plasma in resource-constrained and remote settings.
{"title":"Evaluation of the Applied Biosystems™ TaqPath™ Seq HIV-1 Genotyping Kit for HIV-1 drug resistance testing from dried blood spot specimens","authors":"Obiageli Okafor , Jane Cameron , Andrea Garcia , Charmaine Hinahon , Carmen Salvador-Palomeque , Mitchell Starr , Philip H. Cunningham","doi":"10.1016/j.jviromet.2025.115277","DOIUrl":"10.1016/j.jviromet.2025.115277","url":null,"abstract":"<div><h3>Objectives</h3><div>Monitoring HIV drug resistance is crucial for HIV treatment success. Logistical challenges restrict the use of liquid venous blood specimens for HIV drug resistance (HIVDR) testing, but dried blood spots (DBS) offer a more accessible alternative. This study evaluates the performance of TaqPath™ Seq HIV-1 Genotyping Kit (TaqPath kit) for detecting resistance-associated mutations in the protease (PR), reverse transcriptase (RT), and integrase (INI) regions of HIV-1 pol gene using DBS specimens.</div></div><div><h3>Methods</h3><div>A total of 79 DBS samples collected from ART-naive and ART-experienced individuals with viral loads ranging from 2830 to 9,095,161 copies/mL and HIV-1 subtypes A, B, C, F, and CRF01_AE were genotyped with TaqPath kit. Performance was assessed using the WHO HIVDR assay validation criteria.</div></div><div><h3>Results</h3><div>The assay met the amplification sensitivity criterion, achieving 95.7 % for PR/RT and 92.7 % for INI in high viral load samples. However, lower viral load samples between 2000 and 5000 copies/mL demonstrated reduced sensitivity of 50 % and 30 % for the PR/RT and INI regions respectively. Intra- and inter-assay agreement exceeded 99 % and mutation detection showed strong correlation with the reference assay, identifying over 95 % of reference mutations.</div></div><div><h3>Conclusions</h3><div>The TaqPath kit met the WHO acceptance criteria for HIVDR assay validation, except in the INI region at low viral loads, where amplification sensitivity was below the required threshold. Dried blood spot specimens are compatible with TaqPath kit for baseline and treatment-failure HIVDR genotyping, offering a reliable alternative to plasma in resource-constrained and remote settings.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115277"},"PeriodicalIF":1.6,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145266663","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 : 2025-10-07DOI: 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":"2025-10-07","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}
Feline calicivirus (FCV) is responsible for a highly contagious disease in domestic cats. FCV may cause multiple symptoms and even death to the infected cats. A simple and cost-effective real-time RPA assay was developed for rapid detection of FCV in clinical samples. In this study, specific primers and probe were designed from the genome of FCV that prevalent in south China. The real-time RPA assay was carried out at 39℃ for 20 min before signal analysis by the fluorescence detector. The specificity and sensitivity were thoroughly validated and the results showed that no cross-reaction with irrelevant pathogens were found during the amplification, indicating the good specificity of the new developed real-time RPA assay. RNA standards were constructed and diluted to evaluate the limit of detection. The results showed that the detection limit of the real-time RPA assay could achieve 100 copies/μl, suggesting the high sensitivity of the assay. Additionally, the real-time RPA assay showed excellent performance in clinical sample detection, when compared with a TaqMan qPCR assay. The detection rate of FCV was 38.5 % (57/148) for real-time RPA assay and it was a little higher than 37.2 % (55/148) of the qPCR assay. Taking all together, the real-time RPA assay had potential application of FCV detection in clinical diagnosis. In conclusion, the new developed real-time RPA assay has provided an alternative strategy for rapid and sensitive detection of FCV in laboratories and animal clinics, especially those with limited facilities.
{"title":"Development of a fluorescence isothermal recombinase polymerase amplification assay for rapid detection of feline calicivirus","authors":"Yujun Zhu , Shuzhou Huang , Bihong Huang , Yuexiao Lian , Tongyuan Zhang , Feng Cong , Miaoli Wu","doi":"10.1016/j.jviromet.2025.115275","DOIUrl":"10.1016/j.jviromet.2025.115275","url":null,"abstract":"<div><div>Feline calicivirus (FCV) is responsible for a highly contagious disease in domestic cats. FCV may cause multiple symptoms and even death to the infected cats. A simple and cost-effective real-time RPA assay was developed for rapid detection of FCV in clinical samples. In this study, specific primers and probe were designed from the genome of FCV that prevalent in south China. The real-time RPA assay was carried out at 39℃ for 20 min before signal analysis by the fluorescence detector. The specificity and sensitivity were thoroughly validated and the results showed that no cross-reaction with irrelevant pathogens were found during the amplification, indicating the good specificity of the new developed real-time RPA assay. RNA standards were constructed and diluted to evaluate the limit of detection. The results showed that the detection limit of the real-time RPA assay could achieve 100 copies/μl, suggesting the high sensitivity of the assay. Additionally, the real-time RPA assay showed excellent performance in clinical sample detection, when compared with a TaqMan qPCR assay. The detection rate of FCV was 38.5 % (57/148) for real-time RPA assay and it was a little higher than 37.2 % (55/148) of the qPCR assay. Taking all together, the real-time RPA assay had potential application of FCV detection in clinical diagnosis. In conclusion, the new developed real-time RPA assay has provided an alternative strategy for rapid and sensitive detection of FCV in laboratories and animal clinics, especially those with limited facilities.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115275"},"PeriodicalIF":1.6,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145244609","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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