Pub Date : 2025-03-05DOI: 10.1016/j.jviromet.2025.115143
Cristina Mendes de Oliveira , Fernanda Yoshika Takahashi , Rodrigo Salazar Silva , Marina Soika , Juliane Nunes Ribeiro , Gabriela Laginestra Francisco , Vanessa Vidotto Frade de Oliveira , Debora Rodrigues de Sousa , José Fernando de Souza , Annelise C.W. Lopes , José Eduardo Levi
Accurate diagnosis of dengue in the acute phase is crucial for proper patient management, reducing mortality rates, and enabling public health authorities to implement effective vector control measures. Our study, conducted during the 2024 dengue epidemic in Brazil, compared the results of a rapid dengue NS1 antigen detection assay with dengue RT-PCR using 300 serum specimens. Ninety-five percent of the patients reported symptoms consistent with dengue infection. We observed substantial agreement between the two tests (kappa = 0.8). The rapid NS1 test demonstrated 87 % sensitivity and 92.7 % specificity, using dengue RT-PCR as the gold standard. Our findings confirm that the rapid NS1 antigen detection test can be reliably used during the acute phase of dengue infection to provide an accurate diagnosis.
{"title":"Evaluation of the performance of a dengue NS1 assay during the 2024 epidemic in Brazil","authors":"Cristina Mendes de Oliveira , Fernanda Yoshika Takahashi , Rodrigo Salazar Silva , Marina Soika , Juliane Nunes Ribeiro , Gabriela Laginestra Francisco , Vanessa Vidotto Frade de Oliveira , Debora Rodrigues de Sousa , José Fernando de Souza , Annelise C.W. Lopes , José Eduardo Levi","doi":"10.1016/j.jviromet.2025.115143","DOIUrl":"10.1016/j.jviromet.2025.115143","url":null,"abstract":"<div><div>Accurate diagnosis of dengue in the acute phase is crucial for proper patient management, reducing mortality rates, and enabling public health authorities to implement effective vector control measures. Our study, conducted during the 2024 dengue epidemic in Brazil, compared the results of a rapid dengue NS1 antigen detection assay with dengue RT-PCR using 300 serum specimens. Ninety-five percent of the patients reported symptoms consistent with dengue infection. We observed substantial agreement between the two tests (kappa = 0.8). The rapid NS1 test demonstrated 87 % sensitivity and 92.7 % specificity, using dengue RT-PCR as the gold standard. Our findings confirm that the rapid NS1 antigen detection test can be reliably used during the acute phase of dengue infection to provide an accurate diagnosis.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"335 ","pages":"Article 115143"},"PeriodicalIF":2.2,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143551531","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 : 2025-02-24DOI: 10.1016/j.jviromet.2025.115131
Federica AM Giardina , Greta Romano , Guglielmo Ferrari , Laura Pellegrinelli , Arlinda Seiti , Cristina Galli , Elena Pariani , Fausto Baldanti , Antonio Piralla
Background
Human enterovirus D68 (EV-D68) has been associated with an increase in mild-to-severe pediatric respiratory diseases worldwide. The rate of circulation of this virus is largely underestimated in the population and genetic evolutionary data are usually available only for partial sequences. To achieve a timely genomic surveillance, a reliable, high-throughput EV-68 sequencing assay is required. Here we report an improved high-throughput EV-D68 whole-genome sequencing assay performed directly on clinical samples that is suitable for short-read sequencing platforms. Between June and December 2022, a total 37 (1.9 %) respiratory samples were EV-D68 positive and together with 52 additional samples with a median cycle of quantification (Cq) of 28.3, ranging from 18 to 36.8 Cq were included in the validation analyses. Overall, all the primers had good performance and no mismatches were detected in more than 85 % of sequences (932 whole-genome dataset). Using a cut-off of Cq < 32 in at least 85.5 % of samples a whole-genome or partial genome was obtained, confirming an acceptable positive sequencing rate for the designed method. A total of 65 whole-genome sequences were obtained and have a mean coverage of 98.4 % across the genome, with a median depth of 6158x (range 2815x-7560x). Based on the obtained data, this method is cost effective resulting in an easy-to-perform protocol helpful for tracing the evolution of EV-D68 in protein different from VP1. EV-D68 could become a significant pathogen for public health in the next future, and thus this protocol for whole genome sequencing could help clinical and molecular virologists to be ready for molecular epidemiology surveillance.
{"title":"A newly developed whole genome sequencing protocol enables early tracking of enterovirus D68 molecular evolution","authors":"Federica AM Giardina , Greta Romano , Guglielmo Ferrari , Laura Pellegrinelli , Arlinda Seiti , Cristina Galli , Elena Pariani , Fausto Baldanti , Antonio Piralla","doi":"10.1016/j.jviromet.2025.115131","DOIUrl":"10.1016/j.jviromet.2025.115131","url":null,"abstract":"<div><h3>Background</h3><div>Human enterovirus D68 (EV-D68) has been associated with an increase in mild-to-severe pediatric respiratory diseases worldwide. The rate of circulation of this virus is largely underestimated in the population and genetic evolutionary data are usually available only for partial sequences. To achieve a timely genomic surveillance, a reliable, high-throughput EV-68 sequencing assay is required. Here we report an improved high-throughput EV-D68 whole-genome sequencing assay performed directly on clinical samples that is suitable for short-read sequencing platforms. Between June and December 2022, a total 37 (1.9 %) respiratory samples were EV-D68 positive and together with 52 additional samples with a median cycle of quantification (Cq) of 28.3, ranging from 18 to 36.8 Cq were included in the validation analyses. Overall, all the primers had good performance and no mismatches were detected in more than 85 % of sequences (932 whole-genome dataset). Using a cut-off of Cq < 32 in at least 85.5 % of samples a whole-genome or partial genome was obtained, confirming an acceptable positive sequencing rate for the designed method. A total of 65 whole-genome sequences were obtained and have a mean coverage of 98.4 % across the genome, with a median depth of 6158x (range 2815x-7560x). Based on the obtained data, this method is cost effective resulting in an easy-to-perform protocol helpful for tracing the evolution of EV-D68 in protein different from VP1. EV-D68 could become a significant pathogen for public health in the next future, and thus this protocol for whole genome sequencing could help clinical and molecular virologists to be ready for molecular epidemiology surveillance.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"335 ","pages":"Article 115131"},"PeriodicalIF":2.2,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143512081","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 : 2025-02-22DOI: 10.1016/j.jviromet.2025.115130
Anne-Marie Lauzier , Émilie Douette , Antoine Labrie , Éric Jubinville , Valérie Goulet-Beaulieu , Fabienne Hamon , Julie Jean
To detect viruses such as hepatitis A virus (HAV) and human norovirus (HuNoV) in foods, RT-qPCR or other molecular methods are used, which cannot distinguish between infectious and non-infectious virions. Samples can be pretreated to limit detection to intact and presumably infectious virions. We compared propidium monoazide (PMA or PMAxx), platinum (IV) chloride (PtCl4), magnetic silica beads and centrifugal filter using HAV or HuNoV inactivated by heat, pulsed light, or sodium hypochlorite (NaOCl). PMAxx completely or nearly eliminated (3.96 ± 1.24 log gc) the RT-qPCR signal of HAV inactivated at 100°C for 10 min. Pretreatments could not reduce significantly RT-qPCR signal of HAV after pulsed light (0.74 ± 0.36 log gc) and NaOCl (0.24 ± 0.14 log gc) inactivation. Enzymatic treatments did not improve the results obtained with PMAxx. The exudate of raspberry, strawberry or oyster used as food matrices needed dilution by at least tenfold for PMAxx to to yield results comparable to virions without a food matrix. Overall, PMAxx shows good potential to discriminate between infectious and non-infectious despite some remaining limitations.
{"title":"Comparison of sample pretreatments used to distinguish between infectious and non-infectious foodborne viruses by RT-qPCR","authors":"Anne-Marie Lauzier , Émilie Douette , Antoine Labrie , Éric Jubinville , Valérie Goulet-Beaulieu , Fabienne Hamon , Julie Jean","doi":"10.1016/j.jviromet.2025.115130","DOIUrl":"10.1016/j.jviromet.2025.115130","url":null,"abstract":"<div><div>To detect viruses such as hepatitis A virus (HAV) and human norovirus (HuNoV) in foods, RT-qPCR or other molecular methods are used, which cannot distinguish between infectious and non-infectious virions. Samples can be pretreated to limit detection to intact and presumably infectious virions. We compared propidium monoazide (PMA or PMAxx), platinum (IV) chloride (PtCl<sub>4</sub>), magnetic silica beads and centrifugal filter using HAV or HuNoV inactivated by heat, pulsed light, or sodium hypochlorite (NaOCl). PMAxx completely or nearly eliminated (3.96 ± 1.24 log gc) the RT-qPCR signal of HAV inactivated at 100°C for 10 min. Pretreatments could not reduce significantly RT-qPCR signal of HAV after pulsed light (0.74 ± 0.36 log gc) and NaOCl (0.24 ± 0.14 log gc) inactivation. Enzymatic treatments did not improve the results obtained with PMAxx. The exudate of raspberry, strawberry or oyster used as food matrices needed dilution by at least tenfold for PMAxx to to yield results comparable to virions without a food matrix. Overall, PMAxx shows good potential to discriminate between infectious and non-infectious despite some remaining limitations.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"335 ","pages":"Article 115130"},"PeriodicalIF":2.2,"publicationDate":"2025-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The Zika virus became a global threat in 2015 due to its association with microcephaly. Preventing its spread depends on developing vaccines, with virus-like particles (VLP) being a promising approach, especially because of their safety profile and high immunogenicity. This study focused on the production of Zika VLP using Sf9 cells and the baculovirus expression system, evaluating cell growth kinetics, nutrient consumption, and metabolite production in Sf-900™ III medium. As a methodology, this study includes bioreactor experiments, cell density and viability quantification, nutrient and metabolite analysis, Dot Blot, Western Blot, and transmission electron microscopy. Among the critical conditions tested are culture medium supplementation with 0.028 mM cholesterol/ 6 nM bovine serum albumin, multiplicity of infection (MOI= 0.2 or 2), and dissolved oxygen tension (DOT= 5 or 30 % air saturation). As a result, in the growth phase, Sf9 cells achieved rapid exponential growth, with doubling times ranging from 22.8 to 35.4 hours and standard nutrient consumption and metabolite generation profiles for this cell line. The infection phase recorded cell death rates between 8200 and 12600 cells mL⁻¹ h⁻¹ , with higher VLP production under low MOI (0.2) and low DOT (5 %). These conditions also reduced protein degradation and nutrient consumption. The produced VLP ranged from 32 to 73 nm in size, with smaller sizes observed under low MOI conditions. Finally, controlling the DOT at 5 % air saturation without cholesterol/albumin supplementation increased VLP production without the need to raise the viral load, highlighting the importance of choosing the appropriate combination of critical parameters (MOI, DOT, and medium supplementation) as key factors in optimizing the upstream process. This finding impacts substantially upstream stage efficiency and economy, which could be useful for future scaling up to the commercial manufacturing scale.
{"title":"Critical parameters on Zika virus-like particles’ generation","authors":"Vinícius Aragão Tejo Dias , Ana Luiza Moraes Octaviano , Júlia Públio Rabello , Fernanda Angela Correia Barrence , Thaissa Consoni Bernardino , Jaci Leme , Soraia Attie Calil Jorge , Eutimio Gustavo Fernández Núñez","doi":"10.1016/j.jviromet.2025.115129","DOIUrl":"10.1016/j.jviromet.2025.115129","url":null,"abstract":"<div><div>The Zika virus became a global threat in 2015 due to its association with microcephaly. Preventing its spread depends on developing vaccines, with virus-like particles (VLP) being a promising approach, especially because of their safety profile and high immunogenicity. This study focused on the production of Zika VLP using Sf9 cells and the baculovirus expression system, evaluating cell growth kinetics, nutrient consumption, and metabolite production in Sf-900™ III medium. As a methodology, this study includes bioreactor experiments, cell density and viability quantification, nutrient and metabolite analysis, Dot Blot, Western Blot, and transmission electron microscopy. Among the critical conditions tested are culture medium supplementation with 0.028 mM cholesterol/ 6 nM bovine serum albumin, multiplicity of infection (MOI= 0.2 or 2), and dissolved oxygen tension (DOT= 5 or 30 % air saturation). As a result, in the growth phase, Sf9 cells achieved rapid exponential growth, with doubling times ranging from 22.8 to 35.4 hours and standard nutrient consumption and metabolite generation profiles for this cell line. The infection phase recorded cell death rates between 8200 and 12600 cells mL⁻¹ h⁻¹ , with higher VLP production under low MOI (0.2) and low DOT (5 %). These conditions also reduced protein degradation and nutrient consumption. The produced VLP ranged from 32 to 73 nm in size, with smaller sizes observed under low MOI conditions. Finally, controlling the DOT at 5 % air saturation without cholesterol/albumin supplementation increased VLP production without the need to raise the viral load, highlighting the importance of choosing the appropriate combination of critical parameters (MOI, DOT, and medium supplementation) as key factors in optimizing the upstream process. This finding impacts substantially upstream stage efficiency and economy, which could be useful for future scaling up to the commercial manufacturing scale.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"334 ","pages":"Article 115129"},"PeriodicalIF":2.2,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143468074","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 : 2025-02-17DOI: 10.1016/j.jviromet.2025.115128
Myoung Gwang Choi , Do-Hyung Kim , Hyoung Jun Kim , Ki Hong Kim
Viral hemorrhagic septicemia virus (VHSV) is a significant pathogen causing mass mortalities in marine and freshwater fish worldwide. Accurate diagnosis through quantitative reverse transcription PCR (RT-qPCR) is essential to prevent its spread, but false negatives can compromise results. In this study, we evaluate the use of heat-inactivated snakehead rhabdovirus (SHRV) as an internal positive control (IPC) for VHSV diagnosis. SHRV's similarity to VHSV in viral structure and genome makes it an ideal IPC. The introduction of SHRV IPC into the RT-qPCR workflow can improve the reliability of diagnostic results by enabling the detection of technical failures during the RNA extraction or amplification process. Our results show that heat-inactivated SHRV preserved RNA integrity for IPC use, and SHRV IPC can provide a useful tool for detecting and interpreting false negatives without affecting assay sensitivity.
{"title":"Evaluation of inactivated snakehead rhabdovirus as an internal positive control for RT-qPCR diagnosis of viral hemorrhagic septicemia virus in fish","authors":"Myoung Gwang Choi , Do-Hyung Kim , Hyoung Jun Kim , Ki Hong Kim","doi":"10.1016/j.jviromet.2025.115128","DOIUrl":"10.1016/j.jviromet.2025.115128","url":null,"abstract":"<div><div>Viral hemorrhagic septicemia virus (VHSV) is a significant pathogen causing mass mortalities in marine and freshwater fish worldwide. Accurate diagnosis through quantitative reverse transcription PCR (RT-qPCR) is essential to prevent its spread, but false negatives can compromise results. In this study, we evaluate the use of heat-inactivated snakehead rhabdovirus (SHRV) as an internal positive control (IPC) for VHSV diagnosis. SHRV's similarity to VHSV in viral structure and genome makes it an ideal IPC. The introduction of SHRV IPC into the RT-qPCR workflow can improve the reliability of diagnostic results by enabling the detection of technical failures during the RNA extraction or amplification process. Our results show that heat-inactivated SHRV preserved RNA integrity for IPC use, and SHRV IPC can provide a useful tool for detecting and interpreting false negatives without affecting assay sensitivity.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"334 ","pages":"Article 115128"},"PeriodicalIF":2.2,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143430148","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 : 2025-02-17DOI: 10.1016/j.jviromet.2025.115126
Juhaeng Heo , Doo-Sung Cheon , Nyun-Ki Chung , Yongrae Kim , Dae-Yong Kim
Sacbrood virus (SBV) is a significant pathogen affecting honeybee health, leading to substantial economic losses in apiculture. Although traditional methods, like reverse transcription quantitative polymerase chain reaction, offer accurate detection and quantification of SBV, they have limitations for use in field settings, such as apiaries. Here, we developed and evaluated the XQ SBV Dx Kit as a diagnostic tool for the XQ Station point-of-care (POC) RT-qPCR device, which integrates nucleic acid extraction, gene amplification, and detection for on-site molecular diagnosis. Diagnostic performance was assessed using clinical samples infected with SBV and was compared with that of standard laboratory-based RT-qPCR. The limit of detection (LOD) for both methods was 102 copies per reaction, with the XQ SBV Dx Kit consistently demonstrating superior sensitivity, detecting 83.3 % of replicates at 101 copies per reaction compared to 58.3 % with RT-qPCR. Specificity testing against 11 other honeybee pathogens confirmed the absence of cross-reactivity, highlighting the diagnostic precision of the XQ SBV Dx Kit. Clinical evaluation revealed 98.4 % sensitivity and 97.0 % specificity, validating its reliability for field applications. Overall, the XQ SBV Dx Kit is an essential advancement in honeybee health management, offering practical and timely solutions for supporting sustainable apicultural practices.
{"title":"Development and validation of a point-of-care molecular assay for sacbrood virus (SBV) diagnosis in apiaries","authors":"Juhaeng Heo , Doo-Sung Cheon , Nyun-Ki Chung , Yongrae Kim , Dae-Yong Kim","doi":"10.1016/j.jviromet.2025.115126","DOIUrl":"10.1016/j.jviromet.2025.115126","url":null,"abstract":"<div><div>Sacbrood virus (SBV) is a significant pathogen affecting honeybee health, leading to substantial economic losses in apiculture. Although traditional methods, like reverse transcription quantitative polymerase chain reaction, offer accurate detection and quantification of SBV, they have limitations for use in field settings, such as apiaries. Here, we developed and evaluated the XQ SBV Dx Kit as a diagnostic tool for the XQ Station point-of-care (POC) RT-qPCR device, which integrates nucleic acid extraction, gene amplification, and detection for on-site molecular diagnosis. Diagnostic performance was assessed using clinical samples infected with SBV and was compared with that of standard laboratory-based RT-qPCR. The limit of detection (LOD) for both methods was 10<sup>2</sup> copies per reaction, with the XQ SBV Dx Kit consistently demonstrating superior sensitivity, detecting 83.3 % of replicates at 10<sup>1</sup> copies per reaction compared to 58.3 % with RT-qPCR. Specificity testing against 11 other honeybee pathogens confirmed the absence of cross-reactivity, highlighting the diagnostic precision of the XQ SBV Dx Kit. Clinical evaluation revealed 98.4 % sensitivity and 97.0 % specificity, validating its reliability for field applications. Overall, the XQ SBV Dx Kit is an essential advancement in honeybee health management, offering practical and timely solutions for supporting sustainable apicultural practices.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"334 ","pages":"Article 115126"},"PeriodicalIF":2.2,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143437877","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 : 2025-02-15DOI: 10.1016/j.jviromet.2025.115127
Sherif Ibrahim, Erica Spackman, David L. Suarez, Iryna V. Goraichuk, Chang-Won Lee
Unprecedented H5N1 highly pathogenic avian influenza (HPAI) outbreaks are occurring around the world and there is growing interest in the use of vaccines in affected regions. Vaccination when properly applied can contribute to HPAI control by significantly reducing virus shedding and breaking the transmission chain, but it requires robust surveillance to ensure that international trade is not affected. Thus, it is imperative to establish a test to differentiate vaccinated only animals from vaccinated and then infected animals (DIVA). In this study, we applied enzyme-linked lectin assay (ELLA) to specifically detect N1 neuraminidase (NA) antibody by inhibition of NA activity and provide a proof-of-concept bench validation using reference and experimental serum samples. We used a wild-type low pathogenic H7N1 virus of North American lineage as the ELLA antigen. The NA inhibition ELLA (NI-ELLA) was evaluated for its specificity and sensitivity using reference and experimental samples. The results demonstrated that the NI-ELLA was highly specific with low background NI activity against influenza-negative sera from different species although varying level of cross-reactivity was observed against sera of different NA subtypes with highest cross-reactivity against N4 subtype sera. Using a conservative positive cut-off threshold of 50 % NI activity, NI-ELLA provides 100 % specificity with all reference sera of 9 different NA subtypes. The relative sensitivity of NI-ELLA was evaluated in detecting H5N1 infection in vaccinated and then challenged birds and NI-ELLA showed higher detection rate of H5N1 infection compared with commercial NP ELISAs and real-time RT-PCR. Overall, the NI-ELLA shows high specificity and sensitivity and has the potential for application in DIVA surveillance with further validation.
{"title":"Evaluation of an N1 NA antibody-specific enzyme-linked lectin assay for detection of H5N1 highly pathogenic avian influenza virus infection in vaccinated birds","authors":"Sherif Ibrahim, Erica Spackman, David L. Suarez, Iryna V. Goraichuk, Chang-Won Lee","doi":"10.1016/j.jviromet.2025.115127","DOIUrl":"10.1016/j.jviromet.2025.115127","url":null,"abstract":"<div><div>Unprecedented H5N1 highly pathogenic avian influenza (HPAI) outbreaks are occurring around the world and there is growing interest in the use of vaccines in affected regions. Vaccination when properly applied can contribute to HPAI control by significantly reducing virus shedding and breaking the transmission chain, but it requires robust surveillance to ensure that international trade is not affected. Thus, it is imperative to establish a test to differentiate vaccinated only animals from vaccinated and then infected animals (DIVA). In this study, we applied enzyme-linked lectin assay (ELLA) to specifically detect N1 neuraminidase (NA) antibody by inhibition of NA activity and provide a proof-of-concept bench validation using reference and experimental serum samples. We used a wild-type low pathogenic H7N1 virus of North American lineage as the ELLA antigen. The NA inhibition ELLA (NI-ELLA) was evaluated for its specificity and sensitivity using reference and experimental samples. The results demonstrated that the NI-ELLA was highly specific with low background NI activity against influenza-negative sera from different species although varying level of cross-reactivity was observed against sera of different NA subtypes with highest cross-reactivity against N4 subtype sera. Using a conservative positive cut-off threshold of 50 % NI activity, NI-ELLA provides 100 % specificity with all reference sera of 9 different NA subtypes. The relative sensitivity of NI-ELLA was evaluated in detecting H5N1 infection in vaccinated and then challenged birds and NI-ELLA showed higher detection rate of H5N1 infection compared with commercial NP ELISAs and real-time RT-PCR. Overall, the NI-ELLA shows high specificity and sensitivity and has the potential for application in DIVA surveillance with further validation.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"334 ","pages":"Article 115127"},"PeriodicalIF":2.2,"publicationDate":"2025-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143433352","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-14DOI: 10.1016/j.jviromet.2025.115116
Bertha Gastelbondo-Pastrana , Luis Flórez , Camilo Guzmán , Karina Torres , Evelin Garay , Jolaime Ballesteros-Villamizar , Rosa Gutierrez , Daniel Echeverri-De la Hoz , Yésica López , Héctor Contreras , Germán Arrieta , Héctor Serrano-Coll , Caty Martínez , Nérlis Pájaro-Castro , Bárbara Arroyo-Salgado , Ricardo Rivero-Herrera , Eliana Hurtado , João Pessoa Araújo Jr. , Salim Mattar
During the COVID-19 pandemic, reagents for SARS-CoV-2 detection were scarce or sold at high prices, particularly in Latin America. In this study, a significant step towards self-sufficiency was achieved through the development of an in-house extraction kit for detecting SARS-CoV-2 from nasopharyngeal swab samples. The purity and concentration of the RNA extracted using the in-house kit were compared to those obtained using the GeneJET RNA Purification Kit (Thermo-Scientific®) as a reference. The applicability of the RNA extracted using the kit was evaluated using four samples positive for SARS-CoV-2 by NGS sequencing with Illumina®. There were no significant differences between the results obtained with the in-house kit and those obtained with the commercial kit. These findings confirm that the in-house protocol demonstrated satisfactory diagnostic accuracy for detecting the virus in patients with COVID-19. The in-house extraction kit works effectively, providing optimal RNA extraction for genomic characterization and lineage assignment of SARS-CoV-2 within the four positive samples analyzed. This phenol-free kit represents a local design and production achievement, offering an effective solution for RNA extraction and detection and sequencing of SARS-CoV-2 from nasopharyngeal swabs. The data highlight the essential contribution of this study to health and biotechnological sovereignty in Colombia.
{"title":"Phenol-free in-house kit for RNA extraction with applicability to SARS-CoV-2 genomic sequencing studies: A contribution to biotechnological sovereignty in Colombia","authors":"Bertha Gastelbondo-Pastrana , Luis Flórez , Camilo Guzmán , Karina Torres , Evelin Garay , Jolaime Ballesteros-Villamizar , Rosa Gutierrez , Daniel Echeverri-De la Hoz , Yésica López , Héctor Contreras , Germán Arrieta , Héctor Serrano-Coll , Caty Martínez , Nérlis Pájaro-Castro , Bárbara Arroyo-Salgado , Ricardo Rivero-Herrera , Eliana Hurtado , João Pessoa Araújo Jr. , Salim Mattar","doi":"10.1016/j.jviromet.2025.115116","DOIUrl":"10.1016/j.jviromet.2025.115116","url":null,"abstract":"<div><div>During the COVID-19 pandemic, reagents for SARS-CoV-2 detection were scarce or sold at high prices, particularly in Latin America. In this study, a significant step towards self-sufficiency was achieved through the development of an in-house extraction kit for detecting SARS-CoV-2 from nasopharyngeal swab samples. The purity and concentration of the RNA extracted using the in-house kit were compared to those obtained using the GeneJET RNA Purification Kit (Thermo-Scientific®) as a reference. The applicability of the RNA extracted using the kit was evaluated using four samples positive for SARS-CoV-2 by NGS sequencing with Illumina®. There were no significant differences between the results obtained with the in-house kit and those obtained with the commercial kit. These findings confirm that the in-house protocol demonstrated satisfactory diagnostic accuracy for detecting the virus in patients with COVID-19. The in-house extraction kit works effectively, providing optimal RNA extraction for genomic characterization and lineage assignment of SARS-CoV-2 within the four positive samples analyzed. This phenol-free kit represents a local design and production achievement, offering an effective solution for RNA extraction and detection and sequencing of SARS-CoV-2 from nasopharyngeal swabs. The data highlight the essential contribution of this study to health and biotechnological sovereignty in Colombia.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"334 ","pages":"Article 115116"},"PeriodicalIF":2.2,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143430147","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Monkeypox was re-emerging in 2022 and spread to more than 100 countries. Two clades of Monkeypox virus (MPXV) result in different lethality rates and varying transmission capabilities. Rapid identification of MPXV and differentiation of its clades and subclades are crucial for effective control of the disease. In this study, we developed an ARMS-Quadruplex-qPCR method to detect MPXV and distinguish clades (Ia, Ib, IIa and IIb). F3L gene was used to detect all clades of MPXV from other orthopoxviruses. A 1953 bp fragment containing the C3L gene was found to be completely absent in clade II. Additionally, a sequence spanning from the 177th to the 1318th position (1142 bp) within the 1953 bp fragment was missing in Ib. Therefore, the 1142 bp sequence was used to distinguish Ia from other subclades, and the sequence with the 1142 bp region missing in Ib was used to discriminate Ib from other subclades. Since subclades IIa and IIb are too close to have large deletions and insertions, a unique single nucleotide polymorphism (SNP) was used to design a primer/probe set for ARMS-qPCR to differentiate clade IIa from IIb. The ARMS-Quadruplex-qPCR system can detect down to 2 copies per reaction of MPXV and effectively differentiate all the four subclades. Altogether, four qPCR primer/probe sets in one tube were deployed to recognize MPXV and differentiate MPXV subclades. The high sensitivity, rapidity and specificity of the developed system make it a promising alternative for the diagnosis of MPXV and the determination of the subclades of the infected MPXV.
{"title":"Development of an ARMS-Quadruplex-qPCR assay for the rapid identification of MPXV and the clades Ia, Ib, IIa and IIb","authors":"Bohan Xu , Dongyan Xiong , Xiaoxu Zhang , Hongping Wei , Junping Yu","doi":"10.1016/j.jviromet.2025.115125","DOIUrl":"10.1016/j.jviromet.2025.115125","url":null,"abstract":"<div><div>Monkeypox was re-emerging in 2022 and spread to more than 100 countries. Two clades of Monkeypox virus (MPXV) result in different lethality rates and varying transmission capabilities. Rapid identification of MPXV and differentiation of its clades and subclades are crucial for effective control of the disease. In this study, we developed an ARMS-Quadruplex-qPCR method to detect MPXV and distinguish clades (Ia, Ib, IIa and IIb). F3L gene was used to detect all clades of MPXV from other orthopoxviruses. A 1953 bp fragment containing the C3L gene was found to be completely absent in clade II. Additionally, a sequence spanning from the 177th to the 1318th position (1142 bp) within the 1953 bp fragment was missing in Ib. Therefore, the 1142 bp sequence was used to distinguish Ia from other subclades, and the sequence with the 1142 bp region missing in Ib was used to discriminate Ib from other subclades. Since subclades IIa and IIb are too close to have large deletions and insertions, a unique single nucleotide polymorphism (SNP) was used to design a primer/probe set for ARMS-qPCR to differentiate clade IIa from IIb. The ARMS-Quadruplex-qPCR system can detect down to 2 copies per reaction of MPXV and effectively differentiate all the four subclades. Altogether, four qPCR primer/probe sets in one tube were deployed to recognize MPXV and differentiate MPXV subclades. The high sensitivity, rapidity and specificity of the developed system make it a promising alternative for the diagnosis of MPXV and the determination of the subclades of the infected MPXV.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"334 ","pages":"Article 115125"},"PeriodicalIF":2.2,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143402598","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 : 2025-02-08DOI: 10.1016/j.jviromet.2025.115114
Andrea Sierra-Mejia , Mohammad Hajizadeh , Habeeb Yinka Atanda , Ioannis E. Tzanetakis
Woody hosts are notoriously resistant to genetic transformation. Traditional methods, such as Agrobacterium-mediated transformation, are often inefficient, and this limitation extends to delivering infectious clones to woody plants. Dodder species (Cuscuta spp.) are holoparasitic plants that can establish direct connections with the vascular tissue of the parasitized plants, allowing them to facilitate virus transmission between unrelated botanical species. We demonstrated that a novel dodder-based approach achieved superior transmission in Rubus spp. compared to direct agroinoculation. The transmission rates for systemic blackberry chlorotic ringspot virus transmission increased from 9 % to 73 %, whereas the transmission of the phloem-limited blackberry yellow vein associated virus rose from 0 % to 46 %. This novel method expands the toolbox available to plant biologists to study virus-host interactions in woody plants.
{"title":"Overcoming the woody barrier: Dodder enables efficient transfer of infectious clones to woody plants","authors":"Andrea Sierra-Mejia , Mohammad Hajizadeh , Habeeb Yinka Atanda , Ioannis E. Tzanetakis","doi":"10.1016/j.jviromet.2025.115114","DOIUrl":"10.1016/j.jviromet.2025.115114","url":null,"abstract":"<div><div>Woody hosts are notoriously resistant to genetic transformation. Traditional methods, such <em>as Agrobacterium-</em>mediated transformation<em>,</em> are often inefficient, and this limitation extends to delivering infectious clones to woody plants. Dodder species (<em>Cuscuta spp.)</em> are holoparasitic plants that can establish direct connections with the vascular tissue of the parasitized plants, allowing them to facilitate virus transmission between unrelated botanical species. We demonstrated that a novel dodder-based approach achieved superior transmission in <em>Rubus</em> spp. compared to direct agroinoculation. The transmission rates for systemic blackberry chlorotic ringspot virus transmission increased from 9 % to 73 %, whereas the transmission of the phloem-limited blackberry yellow vein associated virus rose from 0 % to 46 %. This novel method expands the toolbox available to plant biologists to study virus-host interactions in woody plants.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"334 ","pages":"Article 115114"},"PeriodicalIF":2.2,"publicationDate":"2025-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143377464","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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