The poultry industry is a major global source of animal protein but remains vulnerable to immunosuppressive viral infections that compromise bird health and productivity. This study evaluated five viral purification methods for metagenomic analysis of respiratory samples from broiler chickens in Santa Catarina, Brazil. Tracheal swabs from ten flocks (one per farm) were pooled, and 50 µL of a herpes simplex virus type 2 (HSV-2) and murine norovirus (MNV-1) mix was added as an internal positive control. The sample was centrifuged (2000 × g for 30 min), filtered (0.45 μm), and subjected to five purification methods. The filtrate was subjected to five different purification methods. Method 1 (M1) was based on nucleic acid direct genomic extraction of the supernatant. Method 2 (M2): a pre-treatment with DNase was used, followed by genomic extraction. Method 3 (M3) was performed using ultracentrifugation at 100,000 × g / 3 h at 4 °C, followed by genomic extraction. In Method 4 (M4), the sample was submitted to ultracentrifugation on a 25 % sucrose cushion at 100,000 × g / 3 h at 4 °C, followed by genomic extraction. Finally, in Method 5 (M5), the sample was ultracentrifuged on a 25 % sucrose cushion at 100,000 × g / 3 h at 4 °C, and the pellet was treated with DNase followed by genomic extraction. All genomic extractions were performed using the RNeasy Mini kit. Samples were reverse transcribed into cDNA and sequenced by the MiSeq Sequencing System. The efficiency of M1-5 was evaluated based on the yield of viral genetic material. All methodologies employed demonstrated varying rates of genome recovery from viruses identified in poultry production. Notable viruses included avian gyrovirus 2 (AGV-2), avian leukosis virus (ALV), and the avian endogenous retrovirus EAV-HP found within chicken genomes. However, M5 showed the best performance, recovering 9.32 % of viral sequences, 44 % of HSV-2, as internal viral control, 32 % of EAV-HP, 8 % of ALV, and 7 % of AGV-2. In conclusion, this study successfully evaluated and compared five distinct viral purification methods, contributing significantly to the characterization of avian viromes and enhancing comprehension of viral ecology.
家禽业是全球动物蛋白的主要来源，但仍然容易受到免疫抑制性病毒感染的影响，从而损害鸟类的健康和生产力。本研究评估了巴西圣卡塔琳娜肉鸡呼吸样本宏基因组分析的五种病毒纯化方法。收集10只鸡（每个农场1只）的气管拭子，加入50µL单纯疱疹病毒2型（HSV-2）和小鼠诺如病毒（MNV-1）混合物作为内部阳性对照。样品离心（2000 × g） 30min，过滤（0.45 μm），经过5种纯化方法。滤液采用五种不同的纯化方法。方法1 （M1）是基于核酸直接基因组提取上清的方法。方法2 (M2)：采用dna酶预处理，然后进行基因组提取。方法3 （M3）在4℃下以100,000 × g / 3h超离心，然后进行基因组提取。在方法4 （M4）中，样品在25%的蔗糖缓冲液上在4°C下以100,000 × g / 3小时的速度进行超离心，然后进行基因组提取。最后，在方法5 （M5）中，样品在25%的蔗糖缓冲液上以100,000 × g / 3小时在4°C下进行超离心，用DNase处理，然后进行基因组提取。所有基因组提取均使用RNeasy Mini试剂盒进行。将样品逆转录成cDNA，并通过MiSeq测序系统进行测序。以病毒遗传物质的产量评价M1-5的效率。所采用的所有方法都表明，从家禽生产中发现的病毒中恢复基因组的速度各不相同。在鸡基因组中发现的主要病毒包括禽回旋病毒2 （AGV-2）、禽白血病病毒（ALV）和禽内源性逆转录病毒EAV-HP。其中，M5表现最好，可恢复9.32%的病毒序列、44%的HSV-2、32%的EAV-HP、8%的ALV和7%的AGV-2。总之，本研究成功地评估和比较了五种不同的病毒纯化方法，对禽病毒组的表征和增强对病毒生态学的理解具有重要意义。
{"title":"Protocol for virome characterization in low-volume respiratory samples from broiler chickens.","authors":"Giulia Von Tönnemann Pilati, Henrique Borges da Silva Grisard, Rafael Cadamuro Dorighello, Vilmar Benetti Filho, Mariane Dahmer, Beatriz Pereira Savi, Mariana Alves Elois, Gleidson Biasi Carvalho Salles, Eduardo Correa Muniz, Gislaine Fongaro","doi":"10.1016/j.jviromet.2025.115233","DOIUrl":"10.1016/j.jviromet.2025.115233","url":null,"abstract":"<p><p>The poultry industry is a major global source of animal protein but remains vulnerable to immunosuppressive viral infections that compromise bird health and productivity. This study evaluated five viral purification methods for metagenomic analysis of respiratory samples from broiler chickens in Santa Catarina, Brazil. Tracheal swabs from ten flocks (one per farm) were pooled, and 50 µL of a herpes simplex virus type 2 (HSV-2) and murine norovirus (MNV-1) mix was added as an internal positive control. The sample was centrifuged (2000 × g for 30 min), filtered (0.45 μm), and subjected to five purification methods. The filtrate was subjected to five different purification methods. Method 1 (M1) was based on nucleic acid direct genomic extraction of the supernatant. Method 2 (M2): a pre-treatment with DNase was used, followed by genomic extraction. Method 3 (M3) was performed using ultracentrifugation at 100,000 × g / 3 h at 4 °C, followed by genomic extraction. In Method 4 (M4), the sample was submitted to ultracentrifugation on a 25 % sucrose cushion at 100,000 × g / 3 h at 4 °C, followed by genomic extraction. Finally, in Method 5 (M5), the sample was ultracentrifuged on a 25 % sucrose cushion at 100,000 × g / 3 h at 4 °C, and the pellet was treated with DNase followed by genomic extraction. All genomic extractions were performed using the RNeasy Mini kit. Samples were reverse transcribed into cDNA and sequenced by the MiSeq Sequencing System. The efficiency of M1-5 was evaluated based on the yield of viral genetic material. All methodologies employed demonstrated varying rates of genome recovery from viruses identified in poultry production. Notable viruses included avian gyrovirus 2 (AGV-2), avian leukosis virus (ALV), and the avian endogenous retrovirus EAV-HP found within chicken genomes. However, M5 showed the best performance, recovering 9.32 % of viral sequences, 44 % of HSV-2, as internal viral control, 32 % of EAV-HP, 8 % of ALV, and 7 % of AGV-2. In conclusion, this study successfully evaluated and compared five distinct viral purification methods, contributing significantly to the characterization of avian viromes and enhancing comprehension of viral ecology.</p>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":" ","pages":"115233"},"PeriodicalIF":1.6,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144765052","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 : 2025-11-27DOI: 10.1016/j.jviromet.2025.115307
Stacy Gellenoncourt , Marie Pellerin , Aïlona Marcadet-Hauss , Roxanne Fouillé , Michel Rivoire , Guillaume Passot , Julie Lucifora , David Durantel , Nicole Pavio , Virginie Doceul
Hepatitis E virus (HEV) causes acute hepatitis that can progress to fulminant or chronic hepatitis. For decades, the lack of a pertinent and robust cell culture system for HEV has delayed our understanding on this hepatotropic virus. HepaRG cells are one of the few hepatocyte-derived cell lines able to replicate HEV. These cells can differentiate (dHepaRG) into hepatocytes and cholangiocytes upon treatment with dimethyl sulfoxyde (DMSO) and are very relevant to study interactions between pathogens and hepatocyte innate immunity. However, the suitability of the HepaRG model to study HEV needs to be further investigated. In this study, we found that HEV can infect proliferating HepaRG cells and that DMSO-induced differentiation is not necessary for HEV infection. Moreover, even if treatment with DMSO is needed to maintain optimal differentiation and polarization of dHepaRG, its presence is detrimental for HEV infection. Overall, this study shows that dHepaRG cells cultured without DMSO is a suitable model to study HEV and its interaction with the hepatocyte innate immune system.
{"title":"An alternative model for HEV infection in the HepaRG cell line","authors":"Stacy Gellenoncourt , Marie Pellerin , Aïlona Marcadet-Hauss , Roxanne Fouillé , Michel Rivoire , Guillaume Passot , Julie Lucifora , David Durantel , Nicole Pavio , Virginie Doceul","doi":"10.1016/j.jviromet.2025.115307","DOIUrl":"10.1016/j.jviromet.2025.115307","url":null,"abstract":"<div><div>Hepatitis E virus (HEV) causes acute hepatitis that can progress to fulminant or chronic hepatitis. For decades, the lack of a pertinent and robust cell culture system for HEV has delayed our understanding on this hepatotropic virus. HepaRG cells are one of the few hepatocyte-derived cell lines able to replicate HEV. These cells can differentiate (dHepaRG) into hepatocytes and cholangiocytes upon treatment with dimethyl sulfoxyde (DMSO) and are very relevant to study interactions between pathogens and hepatocyte innate immunity. However, the suitability of the HepaRG model to study HEV needs to be further investigated. In this study, we found that HEV can infect proliferating HepaRG cells and that DMSO-induced differentiation is not necessary for HEV infection. Moreover, even if treatment with DMSO is needed to maintain optimal differentiation and polarization of dHepaRG, its presence is detrimental for HEV infection. Overall, this study shows that dHepaRG cells cultured without DMSO is a suitable model to study HEV and its interaction with the hepatocyte innate immune system.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115307"},"PeriodicalIF":1.6,"publicationDate":"2025-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145620440","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-22DOI: 10.1016/j.jviromet.2025.115308
Pablo Piñeyro , Brett Webb , Sheela Ramamoorthy
Torque Teno viruses (TTVs) are ubiquitous, small DNA viruses which are highly epidemiologically associated with respiratory infections, hepatitis, neurological disease and autoimmune disorders in humans and animals. Swine TTVs (TTSuVs) can be considered opportunistic pathogens as they exacerbate clinical signs due to coinfecting agents. While further understanding of how TTVs contribute to disease is crucial, there is a notable lack of animal models and tools to study the in vivo infection patterns of TTV. RNA in situ hybridization (RNA-ISH) with multiple probe amplification has recently gained popularity due to its high levels of specificity and sensitivity and ability to detect agent specific RNA or mRNA. Currently there are no commercial TTSuV1 antibodies that allow viral antigen detection by immunohistochemistry assay that can be used to advance the understanding of TTSuV1 pathogenicity. Therefore, the goal of this study was to develop an RNA-ISH assay for TTSuV1. To generate positive control, PK-15 cells grown in chamber slides were either infected with TTSuV1 or transfected with the TTSuV1 genome. A cocktail of TTSuV1 ORF1-specific RNA probes was hybridized to the cells, and specific binding was successfully visualized using a chromogenic reaction. Liver, kidney, heart, spleen and intestines were collected from mice infected with TTSuV1 at 15- and 30-days post infection. Finally, the RNA-ISH was optimized for TTSuV1 mRNA detection in tissues. TTSuV1-specific signal was detected in the hepatocytes and renal tubular epithelium of infected mice at a detection rate of 33 % 15- and 30-days post infection. In summary, the described RNA ISH assay is a useful tool to visualizeTTSuV1 viral replication in tissues and has potential application to clinical specimens in the future.
{"title":"Detection of Torque Teno Sus Virus1 by an RNA in situ hybridization assay","authors":"Pablo Piñeyro , Brett Webb , Sheela Ramamoorthy","doi":"10.1016/j.jviromet.2025.115308","DOIUrl":"10.1016/j.jviromet.2025.115308","url":null,"abstract":"<div><div>Torque Teno viruses (TTVs) are ubiquitous, small DNA viruses which are highly epidemiologically associated with respiratory infections, hepatitis, neurological disease and autoimmune disorders in humans and animals. Swine TTVs (TTSuVs) can be considered opportunistic pathogens as they exacerbate clinical signs due to coinfecting agents. While further understanding of how TTVs contribute to disease is crucial, there is a notable lack of animal models and tools to study the in vivo infection patterns of TTV. RNA in situ hybridization (RNA-ISH) with multiple probe amplification has recently gained popularity due to its high levels of specificity and sensitivity and ability to detect agent specific RNA or mRNA. Currently there are no commercial TTSuV1 antibodies that allow viral antigen detection by immunohistochemistry assay that can be used to advance the understanding of TTSuV1 pathogenicity. Therefore, the goal of this study was to develop an RNA-ISH assay for TTSuV1. To generate positive control, PK-15 cells grown in chamber slides were either infected with TTSuV1 or transfected with the TTSuV1 genome. A cocktail of TTSuV1 ORF1-specific RNA probes was hybridized to the cells, and specific binding was successfully visualized using a chromogenic reaction. Liver, kidney, heart, spleen and intestines were collected from mice infected with TTSuV1 at 15- and 30-days post infection. Finally, the RNA-ISH was optimized for TTSuV1 mRNA detection in tissues. TTSuV1-specific signal was detected in the hepatocytes and renal tubular epithelium of infected mice at a detection rate of 33 % 15- and 30-days post infection. In summary, the described RNA ISH assay is a useful tool to visualizeTTSuV1 viral replication in tissues and has potential application to clinical specimens in the future.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115308"},"PeriodicalIF":1.6,"publicationDate":"2025-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145596639","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-22DOI: 10.1016/j.jviromet.2025.115309
Teresa Murphy, Rebecca M. Lynch
In vitro models to study HIV-1 escape from broadly neutralizing antibodies (bNAbs) are highly important for designing in vivo bNAb combination therapy. Frequently, short-term viral escape is studied in cell lines, which do not express physiological levels of receptors or with antigenic libraries that do not allow for the observation of concurrent escape or compensatory mutations. We designed an in vitro viral escape assay to measure the ability of HIV-1 to escape from single bNAbs in a high-throughput manner. We tested the multiplicity of infection (MOI) of virus, cloned and un-cloned virus stocks, and different concentrations of antibody. From these results, we developed a 56-day assay to measure escape from bNAbs by adding multiple concentrations of antibody that is gradually increased over time. In this assay, we observed both common escape mutations previously published, but also novel mutations that could be either escape or compensatory mutations. This in vitro bNAb escape assay will lead to a deeper understanding of viral escape, to better inform the design of highly effective bNAb cocktails targeting multiple conserved sites.
{"title":"Development of In Vitro assay for viral escape from broadly neutralizing antibodies","authors":"Teresa Murphy, Rebecca M. Lynch","doi":"10.1016/j.jviromet.2025.115309","DOIUrl":"10.1016/j.jviromet.2025.115309","url":null,"abstract":"<div><div><em>In vitro</em> models to study HIV-1 escape from broadly neutralizing antibodies (bNAbs) are highly important for designing <em>in vivo</em> bNAb combination therapy. Frequently, short-term viral escape is studied in cell lines, which do not express physiological levels of receptors or with antigenic libraries that do not allow for the observation of concurrent escape or compensatory mutations. We designed an <em>in vitro</em> viral escape assay to measure the ability of HIV-1 to escape from single bNAbs in a high-throughput manner. We tested the multiplicity of infection (MOI) of virus, cloned and un-cloned virus stocks, and different concentrations of antibody. From these results, we developed a 56-day assay to measure escape from bNAbs by adding multiple concentrations of antibody that is gradually increased over time. In this assay, we observed both common escape mutations previously published, but also novel mutations that could be either escape or compensatory mutations. This <em>in vitro</em> bNAb escape assay will lead to a deeper understanding of viral escape, to better inform the design of highly effective bNAb cocktails targeting multiple conserved sites.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115309"},"PeriodicalIF":1.6,"publicationDate":"2025-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145596678","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 : 2025-11-19DOI: 10.1016/j.jviromet.2025.115302
Laiqing Li , Hongrui Lai , Huankun Liang , Guiling Guo , Cuicui Chen , Qiang Jia
Objective
Canine distemper virus (CDV) and canine parvovirus type 2 (CPV-2) are two highly contagious and severely pathogenic viruses commonly found in dogs, particularly lethal to puppies. This study aimed to establish a double-labeling time-resolved fluorescence immunoassay (TRFIA) to test and distinguish CDV and CPV-2 infection.
Methods
A sandwich TRFIA method was established and optimized using europium(III) (Eu3 +)/samarium(III) (Sm3+) chelates dual labeling. Subsequently, it was formulated into a kit and constructed the TRFIA platform, and both its laboratory performance (limit of detection, specificity, accuracy and stability) and its ability to detect target viruses in canine clinical samples were evaluated.
Results
A dual-label TRFIA platform for simultaneous CDV and CPV-2 detection was constructed and rigorously validated. The TRFIA platform exhibited limit of detection of 0.43 ng/mL for CDV and 0.73 ng/mL for CPV-2, with high specificity for both targets. Clinical cut-offs were established at 5.47 ng/mL (CDV) and 6.96 ng/mL (CPV-2). Intra-assay coefficients of variation ranged from 1.11 % to 5.53 %, with spike-recoveries between 104.74 % and 108.50 %. Concordance with PCR was excellent (Pearson’s χ² test, P < 0.001). Clinical validation yielded diagnostic sensitivities/specificities of 90.24 %/97.37 % for CDV and 88.37 %/98.65 % for CPV-2, respectively.
Conclusion
A TRFIA platform for simultaneous detection of CDV and CPV-2 demonstrated robust limit of detection, specificity, accuracy, plus reliable clinical sensitivity and specificity. It offers an additional tool for distinguishing CDV from CPV-2 infections and may enhance future disease-prevention strategies.
{"title":"Development of a dual-label time-resolved fluorescence immunoassay platform for simultaneous detection of canine distemper virus and parvovirus","authors":"Laiqing Li , Hongrui Lai , Huankun Liang , Guiling Guo , Cuicui Chen , Qiang Jia","doi":"10.1016/j.jviromet.2025.115302","DOIUrl":"10.1016/j.jviromet.2025.115302","url":null,"abstract":"<div><h3>Objective</h3><div>Canine distemper virus (CDV) and canine parvovirus type 2 (CPV-2) are two highly contagious and severely pathogenic viruses commonly found in dogs, particularly lethal to puppies. This study aimed to establish a double-labeling time-resolved fluorescence immunoassay (TRFIA) to test and distinguish CDV and CPV-2 infection.</div></div><div><h3>Methods</h3><div>A sandwich TRFIA method was established and optimized using europium(III) (Eu<sup>3 +</sup>)/samarium(III) (Sm<sup>3+</sup>) chelates dual labeling. Subsequently, it was formulated into a kit and constructed the TRFIA platform, and both its laboratory performance (limit of detection, specificity, accuracy and stability) and its ability to detect target viruses in canine clinical samples were evaluated.</div></div><div><h3>Results</h3><div>A dual-label TRFIA platform for simultaneous CDV and CPV-2 detection was constructed and rigorously validated. The TRFIA platform exhibited limit of detection of 0.43 ng/mL for CDV and 0.73 ng/mL for CPV-2, with high specificity for both targets. Clinical cut-offs were established at 5.47 ng/mL (CDV) and 6.96 ng/mL (CPV-2). Intra-assay coefficients of variation ranged from 1.11 % to 5.53 %, with spike-recoveries between 104.74 % and 108.50 %. Concordance with PCR was excellent (Pearson’s χ² test, <em>P</em> < 0.001). Clinical validation yielded diagnostic sensitivities/specificities of 90.24 %/97.37 % for CDV and 88.37 %/98.65 % for CPV-2, respectively.</div></div><div><h3>Conclusion</h3><div>A TRFIA platform for simultaneous detection of CDV and CPV-2 demonstrated robust limit of detection, specificity, accuracy, plus reliable clinical sensitivity and specificity. It offers an additional tool for distinguishing CDV from CPV-2 infections and may enhance future disease-prevention strategies.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115302"},"PeriodicalIF":1.6,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145574029","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}
Gosling gout, caused by goose astrovirus 2 (GAstV-2), poses a significant threat to the goose industry due to its high morbidity and mortality, while effective control measures remain unavailable. The development of specific countermeasures is hindered by a limited characterization of monoclonal antibodies (mAbs) and epitopes against the major ORF2 antigen. To address this, we generated two mAbs (A12G1 and A10D12) against GAstV-2 ORF2 using hybridoma technology. Epitope mapping identified two novel linear B-cell epitopes, 1MADRA5 (A12G1) and 36YKPQKLPMKA45 (A10D12), residing in the conserved S domain. These epitopes were highly conserved among GAstV-2 isolates but demonstrated notable divergence from other avian astroviruses. The specific reactivity of both mAbs with GAstV-2 was confirmed by Western blotting, immunofluorescence, and immunohistochemistry. Furthermore, leveraging mAb A10D12, we established a highly sensitive and specific indirect competitive ELISA (icELISA) for detecting GAstV-2 antibodies in goose serum. The icELISA demonstrated excellent reproducibility, high sensitivity, and no cross-reactivity with antisera against other common waterfowl pathogens. This study not only expands the known epitope repertoire of GAstV-2 ORF2 but also provides valuable mAbs and a robust serological tool for functional studies of ORF2 and for monitoring GAstV-2 infections.
{"title":"Development of monoclonal antibodies against goose astrovirus 2 ORF2 protein and establishment of an indirect competitive ELISA detection method","authors":"Anping Wang, Zhi Wu, Qingkang Zhou, Xiaolu Zhang, Li Liu, Shanyuan Zhu","doi":"10.1016/j.jviromet.2025.115306","DOIUrl":"10.1016/j.jviromet.2025.115306","url":null,"abstract":"<div><div>Gosling gout, caused by goose astrovirus 2 (GAstV-2), poses a significant threat to the goose industry due to its high morbidity and mortality, while effective control measures remain unavailable. The development of specific countermeasures is hindered by a limited characterization of monoclonal antibodies (mAbs) and epitopes against the major ORF2 antigen. To address this, we generated two mAbs (A12G1 and A10D12) against GAstV-2 ORF2 using hybridoma technology. Epitope mapping identified two novel linear B-cell epitopes, <sub>1</sub>MADRA<sub>5</sub> (A12G1) and <sub>36</sub>YKPQKLPMKA<sub>45</sub> (A10D12), residing in the conserved S domain. These epitopes were highly conserved among GAstV-2 isolates but demonstrated notable divergence from other avian astroviruses. The specific reactivity of both mAbs with GAstV-2 was confirmed by Western blotting, immunofluorescence, and immunohistochemistry. Furthermore, leveraging mAb A10D12, we established a highly sensitive and specific indirect competitive ELISA (icELISA) for detecting GAstV-2 antibodies in goose serum. The icELISA demonstrated excellent reproducibility, high sensitivity, and no cross-reactivity with antisera against other common waterfowl pathogens. This study not only expands the known epitope repertoire of GAstV-2 ORF2 but also provides valuable mAbs and a robust serological tool for functional studies of ORF2 and for monitoring GAstV-2 infections.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115306"},"PeriodicalIF":1.6,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145573973","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 : 2025-11-19DOI: 10.1016/j.jviromet.2025.115305
Qiufang Huang , Tingting Zhao , Wenhao Su , Shishi Li , Jingjing Liu , Zihao Ge , Benyao Zhang , Xiuxiu Ren , Xiaohuan Zhang , Jiangbo Wei
The plaque purification is a critical step in the screening of traditional live-attenuated vaccines and recombinant viral vaccines, aiming to acquire vaccine clones with homogeneous characteristics and desirable immunogenicity to address outbreaks of emerging diseases such as monkeypox, chikungunya fever, and dengue fever. The traditional plaque purification process to screen out a vaccine strain with genetically consistent stability from a mixed pool of viral clones generally requires laborsome work. We utilized live-cell imaging technique enabling us to isolate monoclonal vaccine strains to simplify and improve the efficiency of this process. Here, we genetically engineered the vaccinia virus TianTan (VTT) using CRISPR/Cas9 system to generate recombinant VTT viruses (VTT-WS01-EGFP) that expressed enhanced green fluorescent protein (EGFP). Initially, we performed 9 rounds of plaque purification using traditional plaque assay, yielding 50 candidate clones. The Incucyte Live-Cell Imaging and Analysis system was subsequently performed to conduct a rigorous, high-resolution screening of these candidates in a more automated, sensitive and high-throughput way. Through this screening process, we ultimately obtained 31 pure viral clones that were free of parental strain contamination, followed by the analysis of plaque formation, fluorescent plaque size, and plaque morphology, and 11 candidate clones were selected for immunological evaluation. Furthermore, we found that clone 49 induced a relatively high titer of anti-VTT neutralizing antibodies and elicited the production of cross-reactive IgG against monkeypox virus antigens, thereby validating its potential as a candidate strain as a monkeypox virus vaccine. Taken together, our data demonstrates that live-cell imaging technique significantly accelerates the screening process for the isolation of monoclonal viral clones as recombinant viral vaccines, and holds considerable potential in attenuated strain selection as well as investigations into biological characteristics of viruses, including viral replication.
{"title":"Screening of monoclonal vaccine strains based on real-time live-cell imaging technology","authors":"Qiufang Huang , Tingting Zhao , Wenhao Su , Shishi Li , Jingjing Liu , Zihao Ge , Benyao Zhang , Xiuxiu Ren , Xiaohuan Zhang , Jiangbo Wei","doi":"10.1016/j.jviromet.2025.115305","DOIUrl":"10.1016/j.jviromet.2025.115305","url":null,"abstract":"<div><div>The plaque purification is a critical step in the screening of traditional live-attenuated vaccines and recombinant viral vaccines, aiming to acquire vaccine clones with homogeneous characteristics and desirable immunogenicity to address outbreaks of emerging diseases such as monkeypox, chikungunya fever, and dengue fever. The traditional plaque purification process to screen out a vaccine strain with genetically consistent stability from a mixed pool of viral clones generally requires laborsome work. We utilized live-cell imaging technique enabling us to isolate monoclonal vaccine strains to simplify and improve the efficiency of this process. Here, we genetically engineered the vaccinia virus TianTan (VTT) using CRISPR/Cas9 system to generate recombinant VTT viruses (VTT-WS01-EGFP) that expressed enhanced green fluorescent protein (EGFP). Initially, we performed 9 rounds of plaque purification using traditional plaque assay, yielding 50 candidate clones. The Incucyte Live-Cell Imaging and Analysis system was subsequently performed to conduct a rigorous, high-resolution screening of these candidates in a more automated, sensitive and high-throughput way. Through this screening process, we ultimately obtained 31 pure viral clones that were free of parental strain contamination, followed by the analysis of plaque formation, fluorescent plaque size, and plaque morphology, and 11 candidate clones were selected for immunological evaluation. Furthermore, we found that clone 49 induced a relatively high titer of anti-VTT neutralizing antibodies and elicited the production of cross-reactive IgG against monkeypox virus antigens, thereby validating its potential as a candidate strain as a monkeypox virus vaccine. Taken together, our data demonstrates that live-cell imaging technique significantly accelerates the screening process for the isolation of monoclonal viral clones as recombinant viral vaccines, and holds considerable potential in attenuated strain selection as well as investigations into biological characteristics of viruses, including viral replication.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115305"},"PeriodicalIF":1.6,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145573958","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 : 2025-11-17DOI: 10.1016/j.jviromet.2025.115301
Ebrahim Abbasi
Background
Mosquito-borne arboviruses pose a growing threat to public health, particularly in ecologically vulnerable and climatically dynamic regions. This study aimed to investigate the diversity of emerging arboviruses in mosquito populations from high-risk provinces in southern and southeastern Iran using a metagenomic surveillance approach.
Methods
Adult mosquitoes were collected from 36 sites across Hormozgan, Sistan and Baluchestan, and Khuzestan provinces. Specimens were pooled by species and location, followed by RNA extraction and high-throughput sequencing. Bioinformatics analysis was performed to identify viral taxa and assess phylogenetic relationships.
Results
A total of 4275 mosquitoes representing six species were analyzed. Virome analysis revealed 43 viral taxa, including medically important arboviruses such as dengue virus serotype 2 (DENV-2), chikungunya virus (CHIKV), and West Nile virus (WNV). Multiple novel viral sequences were also detected, including putative members of Phenuiviridae and Orthomyxoviridae. Viral diversity was highest in Hormozgan province and positively correlated with ambient temperature.
Conclusion
This study provides the first comprehensive metagenomic insight into mosquito viromes in Iran, revealing both endemic and potentially novel arboviruses. These findings underscore the need for integrated genomic surveillance and regional vector-borne disease preparedness.
{"title":"Metagenomic surveillance of emerging viruses in mosquito populations from high-risk regions of Iran","authors":"Ebrahim Abbasi","doi":"10.1016/j.jviromet.2025.115301","DOIUrl":"10.1016/j.jviromet.2025.115301","url":null,"abstract":"<div><h3>Background</h3><div>Mosquito-borne arboviruses pose a growing threat to public health, particularly in ecologically vulnerable and climatically dynamic regions. This study aimed to investigate the diversity of emerging arboviruses in mosquito populations from high-risk provinces in southern and southeastern Iran using a metagenomic surveillance approach.</div></div><div><h3>Methods</h3><div>Adult mosquitoes were collected from 36 sites across Hormozgan, Sistan and Baluchestan, and Khuzestan provinces. Specimens were pooled by species and location, followed by RNA extraction and high-throughput sequencing. Bioinformatics analysis was performed to identify viral taxa and assess phylogenetic relationships.</div></div><div><h3>Results</h3><div>A total of 4275 mosquitoes representing six species were analyzed. Virome analysis revealed 43 viral taxa, including medically important arboviruses such as dengue virus serotype 2 (DENV-2), chikungunya virus (CHIKV), and West Nile virus (WNV). Multiple novel viral sequences were also detected, including putative members of <em>Phenuiviridae</em> and <em>Orthomyxoviridae</em>. Viral diversity was highest in Hormozgan province and positively correlated with ambient temperature.</div></div><div><h3>Conclusion</h3><div>This study provides the first comprehensive metagenomic insight into mosquito viromes in Iran, revealing both endemic and potentially novel arboviruses. These findings underscore the need for integrated genomic surveillance and regional vector-borne disease preparedness.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115301"},"PeriodicalIF":1.6,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145556892","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 : 2025-11-17DOI: 10.1016/j.jviromet.2025.115304
Zhen Zhong , Guoyan Li , Guanglin Liang , Tongwei Ren , Cuijing Teng , Jingpin Xiong , Guangqiang Ji , Min Zheng , Yan Pan , Yifeng Qin , Kang Ouyang , Yeshi Yin , Ying Chen , Weijian Huang , Zuzhang Wei
This study aimed to develop a rapid and visually interpretable nucleic acid detection assay for Foot-and-Mouth Disease Virus serotype O (FMDV-O) by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Specific RPA primers and CRISPR RNA (crRNA) sequences were designed and optimized based on the conserved 3D gene region of FMDV-O. An assay combining RPA pre-amplification with Cas12a-mediated cleavage was subsequently established. The sensitivity and specificity of the RPA-CRISPR/Cas12a method were systematically evaluated, and its diagnostic utility was further assessed using clinical samples. The results demonstrated that the primer set RPA-F1/R1 paired with crRNA1 constituted the optimal combination, with an ideal reaction system comprising 50 nM Cas12a protein and 200 nM crRNA. This system exhibited a detection limit of 2.60 × 10² copies/μL for target plasmid DNA following a 20-minute incubation at 37°C. Specificity analysis confirmed positive detection exclusively for FMDV-O plasmids, with no cross-reactivity observed with other tested pathogens. When applied to clinical samples, the proposed method demonstrated a superior detection rate relative to conventional PCR. In conclusion, a novel diagnostic platform for FMDV-O was successfully developed based on RPA-CRISPR/Cas12a. This method is characterized by its rapidity, operational simplicity, high sensitivity, and excellent specificity, holding significant promise for application in clinical diagnostics, epidemiological surveillance, and field-based testing.
{"title":"Establishment of a nucleic acid detection method for foot-and-mouth disease virus serotype O utilizing RPA-CRISPR/Cas12a technology","authors":"Zhen Zhong , Guoyan Li , Guanglin Liang , Tongwei Ren , Cuijing Teng , Jingpin Xiong , Guangqiang Ji , Min Zheng , Yan Pan , Yifeng Qin , Kang Ouyang , Yeshi Yin , Ying Chen , Weijian Huang , Zuzhang Wei","doi":"10.1016/j.jviromet.2025.115304","DOIUrl":"10.1016/j.jviromet.2025.115304","url":null,"abstract":"<div><div>This study aimed to develop a rapid and visually interpretable nucleic acid detection assay for Foot-and-Mouth Disease Virus serotype O (FMDV-O) by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Specific RPA primers and CRISPR RNA (crRNA) sequences were designed and optimized based on the conserved 3D gene region of FMDV-O. An assay combining RPA pre-amplification with Cas12a-mediated cleavage was subsequently established. The sensitivity and specificity of the RPA-CRISPR/Cas12a method were systematically evaluated, and its diagnostic utility was further assessed using clinical samples. The results demonstrated that the primer set RPA-F1/R1 paired with crRNA1 constituted the optimal combination, with an ideal reaction system comprising 50 nM Cas12a protein and 200 nM crRNA. This system exhibited a detection limit of 2.60 × 10² copies/μL for target plasmid DNA following a 20-minute incubation at 37°C. Specificity analysis confirmed positive detection exclusively for FMDV-O plasmids, with no cross-reactivity observed with other tested pathogens. When applied to clinical samples, the proposed method demonstrated a superior detection rate relative to conventional PCR. In conclusion, a novel diagnostic platform for FMDV-O was successfully developed based on RPA-CRISPR/Cas12a. This method is characterized by its rapidity, operational simplicity, high sensitivity, and excellent specificity, holding significant promise for application in clinical diagnostics, epidemiological surveillance, and field-based testing.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115304"},"PeriodicalIF":1.6,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145557269","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 : 2025-11-16DOI: 10.1016/j.jviromet.2025.115303
Yousef Douzandegan , Ali Kargar Kheirabad , Sayed‑Hamidreza Mozhgani , Gholamreza Tavoosidana , Abbas Rahimi Foroushani , Mehdi Norouzi
Background
Human T-lymphotropic virus type 1 (HTLV-1) is a delta-retrovirus responsible for severe diseases such as adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Accurate and accessible diagnostic tools are required, especially in endemic regions such as Iran, to manage reasonably and prevent HTLV-1 infections.
Objective
This study aimed to develop and validate an HTLV-1 detection protocol using loop-mediated isothermal amplification (LAMP) method on clinical samples from Iranian patients.
Methods
A LAMP assay was designed using optimized primers targeting the HTLV-1 genome. It was validated with clinical samples, and its sensitivity and specificity were evaluated compared to the polymerase chain reaction (PCR) method. Reaction conditions were optimized for fast and specific amplification, and results were visually analyzed utilizing a Luminator device and a real-time thermocycler.
Results
The LAMP assay revealed a sensitivity of 100 % and specificity of 94.7 % for HTLV-1 detection. Within 15–30 min, the method produced results in isothermal conditions with good performance. The fluorescence-based detection approach is easy to use. It does not need a sophisticated laboratory facility and has a limit of detection of 2.5 copies/µL.
Conclusion
This study demonstrates the feasibility of a LAMP-based method for HTLV-1 detection that is rapid, cost-effective, and accessible compared to PCR. For this reason, this alternative could enhance HTLV-1 diagnosis in both qualitative or quantitative form in endemic and low-resource scenarios. Future research should explore further validation in more diverse populations and integration with new technologies for digital detection.
{"title":"Development of a LAMP-based diagnostic method for HTLV-1 using Iranian clinical samples","authors":"Yousef Douzandegan , Ali Kargar Kheirabad , Sayed‑Hamidreza Mozhgani , Gholamreza Tavoosidana , Abbas Rahimi Foroushani , Mehdi Norouzi","doi":"10.1016/j.jviromet.2025.115303","DOIUrl":"10.1016/j.jviromet.2025.115303","url":null,"abstract":"<div><h3>Background</h3><div>Human T-lymphotropic virus type 1 (HTLV-1) is a delta-retrovirus responsible for severe diseases such as adult<!--> <!-->T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Accurate and<!--> <!-->accessible diagnostic tools are required, especially in endemic regions such as Iran, to manage reasonably and prevent HTLV-1 infections.</div></div><div><h3>Objective</h3><div>This study aimed to develop and validate an HTLV-1 detection protocol using loop-mediated isothermal amplification (LAMP) method on clinical samples from Iranian<!--> <!-->patients.</div></div><div><h3>Methods</h3><div>A LAMP assay was designed using optimized primers targeting the HTLV-1 genome. It was validated with clinical samples, and its sensitivity and specificity were evaluated compared to the polymerase chain reaction (PCR)<!--> <!-->method. Reaction conditions were optimized for fast and specific amplification, and results were visually analyzed utilizing a Luminator device and a real-time thermocycler.</div></div><div><h3>Results</h3><div>The LAMP assay revealed a sensitivity<!--> <!-->of 100 % and specificity of 94.7 % for HTLV-1 detection. Within 15–30 min, the method produced results in isothermal conditions<!--> <!-->with good performance. The fluorescence-based detection approach is easy to<!--> <!-->use. It does not need a sophisticated laboratory facility and has a limit of detection of 2.5 copies/µL.</div></div><div><h3>Conclusion</h3><div>This study demonstrates the feasibility of a LAMP-based method for HTLV-1 detection that is rapid, cost-effective, and accessible compared to PCR. For this reason,<!--> <!-->this alternative could enhance HTLV-1 diagnosis in both qualitative or quantitative form in endemic and low-resource scenarios. Future research should explore further validation in more diverse populations and integration with new<!--> <!-->technologies for digital detection.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115303"},"PeriodicalIF":1.6,"publicationDate":"2025-11-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145549275","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}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号