Pub Date : 2025-12-16DOI: 10.1016/j.jviromet.2025.115324
D.E.V. Villamor, D. Stainton, I.E. Tzanetakis
Blueberry scorch virus (BlScV) and blueberry virus S (BVS) are closely related carlaviruses with the latter being only recently characterized. Most available PCR assays for BlScV fail to detect some isolates of the virus, as well as BVS. Here, we describe PCR assays designed based on all available virus sequences in public databases that can discriminate BlScV from BVS. Initially, a triplex endpoint PCR assay was developed that yields different amplicon sizes for each virus and incorporates an internal control which assesses the quality of the nucleic acids and the efficiency of the enzymatic reactions. Subsequently, a duplex quantitative PCR was designed employing primers targeting both viruses and discriminatory probes specific to each. Comparison between end-point and qPCR assays revealed near parallel sensitivity. The availability of the two PCR formats reliably improves the detection assays of the viruses in diagnostic facilities, quarantine, and clean plant programs.
{"title":"Development of end-point and quantitative polymerase chain reaction assays to detect and discriminate between blueberry scorch virus and blueberry virus S","authors":"D.E.V. Villamor, D. Stainton, I.E. Tzanetakis","doi":"10.1016/j.jviromet.2025.115324","DOIUrl":"10.1016/j.jviromet.2025.115324","url":null,"abstract":"<div><div>Blueberry scorch virus (BlScV) and blueberry virus S (BVS) are closely related carlaviruses with the latter being only recently characterized. Most available PCR assays for BlScV fail to detect some isolates of the virus, as well as BVS. Here, we describe PCR assays designed based on all available virus sequences in public databases that can discriminate BlScV from BVS. Initially, a triplex endpoint PCR assay was developed that yields different amplicon sizes for each virus and incorporates an internal control which assesses the quality of the nucleic acids and the efficiency of the enzymatic reactions. Subsequently, a duplex quantitative PCR was designed employing primers targeting both viruses and discriminatory probes specific to each. Comparison between end-point and qPCR assays revealed near parallel sensitivity. The availability of the two PCR formats reliably improves the detection assays of the viruses in diagnostic facilities, quarantine, and clean plant programs.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"341 ","pages":"Article 115324"},"PeriodicalIF":1.6,"publicationDate":"2025-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145781285","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 : 2025-12-11DOI: 10.1016/j.jviromet.2025.115326
Sara W.F. Geffert , Soya S. Sam , Karen Chandran , Curt G. Beckwith , Rami Kantor , Angela M. Caliendo
Tasso T20 (T20) is a finger stick-free, self-sampling device capable of at-home blood sample collection. We optimized HIV-1 RNA elution from T20, evaluated its analytical performance, compared its performance to Whatman dried blood spots (DBS), and assessed HIV-1 RNA stability. We used mock viral samples to optimize HIV-1 RNA elution using viral concentrations 10,000 and 3000 copies/mL. Limit of detection (LOD) and limit of quantification (LOQ) were determined, and linearity and reproducibility were verified. Analytical agreement between T20 and DBS was assessed using eight concentrations, from 100,000 to 1000 copies/mL. T20 HIV-1 RNA stability was assessed over time. All samples were tested using the m2000 RealTime HIV-1 DBS assay. The optimized elution conditions selected were 30 min of sonication followed by 30 min of incubation at 55 °C. The 95 % LOD and LOQ were determined to be 3.56 and 3.70 log10 copies/mL, respectively. Good linearity was observed with a strong correlation (R2=0.98) and excellent reproducibility. Analytical agreement analysis demonstrated a mean difference of −0.36 log10 copies/mL (SD ± 0.21 log10 copies/mL). ANOVA indicated all samples remained stable (p value=0.33, F=1.22). The Tasso T20 is capable of quantifying HIV-1 viral loads, with results comparable to DBS.
{"title":"Evaluation of Tasso T20 to collect dried blood for the quantification of HIV-1 RNA","authors":"Sara W.F. Geffert , Soya S. Sam , Karen Chandran , Curt G. Beckwith , Rami Kantor , Angela M. Caliendo","doi":"10.1016/j.jviromet.2025.115326","DOIUrl":"10.1016/j.jviromet.2025.115326","url":null,"abstract":"<div><div>Tasso T20 (T20) is a finger stick-free, self-sampling device capable of at-home blood sample collection. We optimized HIV-1 RNA elution from T20, evaluated its analytical performance, compared its performance to Whatman dried blood spots (DBS), and assessed HIV-1 RNA stability. We used mock viral samples to optimize HIV-1 RNA elution using viral concentrations 10,000 and 3000 copies/mL. Limit of detection (LOD) and limit of quantification (LOQ) were determined, and linearity and reproducibility were verified. Analytical agreement between T20 and DBS was assessed using eight concentrations, from 100,000 to 1000 copies/mL. T20 HIV-1 RNA stability was assessed over time. All samples were tested using the m2000 RealTi<em>m</em>e HIV-1 DBS assay. The optimized elution conditions selected were 30 min of sonication followed by 30 min of incubation at 55 °C. The 95 % LOD and LOQ were determined to be 3.56 and 3.70 log<sub>10</sub> copies/mL, respectively. Good linearity was observed with a strong correlation (R<sup>2</sup>=0.98) and excellent reproducibility. Analytical agreement analysis demonstrated a mean difference of −0.36 log<sub>10</sub> copies/mL (SD ± 0.21 log<sub>10</sub> copies/mL). ANOVA indicated all samples remained stable (p value=0.33, F=1.22). The Tasso T20 is capable of quantifying HIV-1 viral loads, with results comparable to DBS.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"341 ","pages":"Article 115326"},"PeriodicalIF":1.6,"publicationDate":"2025-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145743229","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 : 2025-12-09DOI: 10.1016/j.jviromet.2025.115325
Clara C.P. Mankowski , Matthew W. Hopken , Terry R. Spraker , Amy T. Gilbert
Enhanced rabies surveillance (ERS) of wildlife populations complements passive public health surveillance through active field-based sampling. The combined data augment rabies management decision-making through tracking virus distribution and movement, rapidly identifying potential outbreaks, and informing containment and elimination strategies at regional and landscape scales. Laboratory diagnostic methods are critical to ERS and several gold standard methods are recommended for detection of Lyssavirus rabies (RV); each validated using central nervous system (CNS) tissue which requires necropsy and cold storage, posing challenges and risks in remote field settings. Alternative sample types to CNS tissue may improve ERS efficiency and field personnel safety, provided they offer robust diagnostic specificity and sensitivity. We evaluated multiple sample types opportunistically collected postmortem from naturally infected striped skunks (Mephitis mephitis) and used real-time reverse transcriptase PCR (rtRT-PCR) to evaluate diagnostic sensitivity and specificity of each sample type compared to CNS tissue. The detection rate of RV RNA was 97 % (95 % CI 81–100) for oral swabs and 96 % (95 % CI 80–100) for eye swabs; when swab types were combined, the detection rate was 100 % (95 % CI 85–100) and comparable to detection from CNS. Other sample types demonstrated compromised diagnostic sensitivities (33–76 %). All sample types were 100 % specific for RV diagnosis. The combination of eye and oral swabs as ERS samples may expand sampling opportunities that improve efficiency, mitigate sample collection and storage challenges, and decrease RV exposure risk among field staff while enhancing the information provided to estimate impacts of RV management on target wildlife populations.
加强野生动物种群的狂犬病监测（ERS）通过主动实地抽样补充了被动的公共卫生监测。这些综合数据通过跟踪病毒分布和移动、快速识别潜在疫情以及在区域和景观尺度上通报遏制和消除战略，增强了狂犬病管理决策。实验室诊断方法对ERS至关重要，推荐几种金标准方法用于检测狂犬溶血病毒（RV）；每种方法都使用中枢神经系统（CNS）组织进行验证，这需要尸检和冷藏，这在偏远地区带来了挑战和风险。替代中枢神经系统组织的样本类型可以提高ERS的效率和现场人员的安全，前提是它们具有强大的诊断特异性和敏感性。我们评估了从自然感染的条纹臭鼬（Mephitis Mephitis）死后收集的多种样本类型，并使用实时逆转录酶PCR （rtRT-PCR）来评估每种样本类型与中枢神经系统组织相比的诊断敏感性和特异性。口腔拭子RV RNA检出率为97% (95% CI 81 ~ 100)，眼拭子检出率为96% (95% CI 80 ~ 100)；当结合拭子类型时，检出率为100% (95% CI 85-100)，与中枢神经系统的检出率相当。其他样本类型表现出较差的诊断敏感性（76%至33%）。所有样本类型对RV诊断的特异性均为100%。将眼拭子和口腔拭子结合作为ERS样本可以增加采样机会，从而提高效率，减轻样本收集和储存的挑战，降低现场工作人员暴露于RV的风险，同时增强提供的信息，以估计RV管理对目标野生动物种群的影响。
{"title":"Alternatives to central nervous system samples for enhanced rabies surveillance: Exploring natural infections of Lyssavirus rabies in striped skunks (Mephitis mephitis) in Northern Colorado","authors":"Clara C.P. Mankowski , Matthew W. Hopken , Terry R. Spraker , Amy T. Gilbert","doi":"10.1016/j.jviromet.2025.115325","DOIUrl":"10.1016/j.jviromet.2025.115325","url":null,"abstract":"<div><div>Enhanced rabies surveillance (ERS) of wildlife populations complements passive public health surveillance through active field-based sampling. The combined data augment rabies management decision-making through tracking virus distribution and movement, rapidly identifying potential outbreaks, and informing containment and elimination strategies at regional and landscape scales. Laboratory diagnostic methods are critical to ERS and several gold standard methods are recommended for detection of <em>Lyssavirus rabies</em> (RV); each validated using central nervous system (CNS) tissue which requires necropsy and cold storage, posing challenges and risks in remote field settings. Alternative sample types to CNS tissue may improve ERS efficiency and field personnel safety, provided they offer robust diagnostic specificity and sensitivity. We evaluated multiple sample types opportunistically collected postmortem from naturally infected striped skunks (<em>Mephitis mephitis</em>) and used real-time reverse transcriptase PCR (rtRT-PCR) to evaluate diagnostic sensitivity and specificity of each sample type compared to CNS tissue. The detection rate of RV RNA was 97 % (95 % CI 81–100) for oral swabs and 96 % (95 % CI 80–100) for eye swabs; when swab types were combined, the detection rate was 100 % (95 % CI 85–100) and comparable to detection from CNS. Other sample types demonstrated compromised diagnostic sensitivities (33–76 %). All sample types were 100 % specific for RV diagnosis. The combination of eye and oral swabs as ERS samples may expand sampling opportunities that improve efficiency, mitigate sample collection and storage challenges, and decrease RV exposure risk among field staff while enhancing the information provided to estimate impacts of RV management on target wildlife populations.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"341 ","pages":"Article 115325"},"PeriodicalIF":1.6,"publicationDate":"2025-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145724124","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 : 2025-12-08DOI: 10.1016/j.jviromet.2025.115323
Zheng Niu , Lichen Nie , Jin Yan , Xinru Xu , Xinfeng Hou , Yong Huang , Qian Du , Dewen Tong
Calf diarrhea is a common clinical disease, causing seriously economic losses to the cattle breeding industry. Bovine coronavirus (BCoV), Bovine rotavirus (BRV), Bovine viral diarrhea virus (BVDV), Escherichia coli (E. coli), Salmonella and Clostridium perfringens (C. perfringens) are common pathogens causing calf diarrhea, Diarrhea in calves caused by these six pathogens is clinically similar and most of them have conditions such as mixed or secondary infections. However, the commonly used method of detecting these six pathogens one by one at present is time-consuming and laborious, which seriously restricts the timely and effective prevention and control of calf diarrhea in production practice. This study established a rapid, efficient, and sensitive multiplex fluorescence quantitative PCR detection method, comprising two independent triplex reactions, for the six pathogens causing calf diarrhea. The results show that the drawn standard curve has a good linear relationship, and R2 is greater than 0.990. There is no amplification of other common pathogens in calves. The coefficient of variation (CV) of intra-group repetition and inter-group repetition ranged from 0.17 % to 0.70 %. By testing 932 clinical rectal swab samples of diarrheal calves and comparing them with commercial kits, the results showed that the multiplex fluorescence quantitative PCR detection method established in this study had the characteristics of high specificity and high sensitivity. In conclusion, the detection method established in this study can be used for large-scale detection of clinical samples and is of great applied value for the prevention and treatment of calf diarrhea.
{"title":"Establishment and application of six-plex fluorescent quantitative PCR assay for the detection of calf diarrhea pathogens","authors":"Zheng Niu , Lichen Nie , Jin Yan , Xinru Xu , Xinfeng Hou , Yong Huang , Qian Du , Dewen Tong","doi":"10.1016/j.jviromet.2025.115323","DOIUrl":"10.1016/j.jviromet.2025.115323","url":null,"abstract":"<div><div>Calf diarrhea is a common clinical disease, causing seriously economic losses to the cattle breeding industry. Bovine coronavirus (BCoV), Bovine rotavirus (BRV), Bovine viral diarrhea virus (BVDV), <em>Escherichia coli</em> (<em>E. coli</em>), <em>Salmonella</em> and <em>Clostridium perfringens</em> (<em>C. perfringens</em>) are common pathogens causing calf diarrhea, Diarrhea in calves caused by these six pathogens is clinically similar and most of them have conditions such as mixed or secondary infections. However, the commonly used method of detecting these six pathogens one by one at present is time-consuming and laborious, which seriously restricts the timely and effective prevention and control of calf diarrhea in production practice. This study established a rapid, efficient, and sensitive multiplex fluorescence quantitative PCR detection method, comprising two independent triplex reactions, for the six pathogens causing calf diarrhea. The results show that the drawn standard curve has a good linear relationship, and R<sup>2</sup> is greater than 0.990. There is no amplification of other common pathogens in calves. The coefficient of variation (CV) of intra-group repetition and inter-group repetition ranged from 0.17 % to 0.70 %. By testing 932 clinical rectal swab samples of diarrheal calves and comparing them with commercial kits, the results showed that the multiplex fluorescence quantitative PCR detection method established in this study had the characteristics of high specificity and high sensitivity. In conclusion, the detection method established in this study can be used for large-scale detection of clinical samples and is of great applied value for the prevention and treatment of calf diarrhea.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"341 ","pages":"Article 115323"},"PeriodicalIF":1.6,"publicationDate":"2025-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145724192","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 : 2025-12-08DOI: 10.1016/j.jviromet.2025.115319
Jianhui Nie , Wenbo Wang , Zhiheng Wen , Aijing Song , Kunxue Hong , Shan Lu , Ping Zhong , Jianqing Xu , Wei Kong , Jingyun Li , Hong Shang , Hong Ling , Li Ruan , Youchun Wang
{"title":"Corrigendum to ‘optimization and proficiency testing of a pseudovirus-based assay for detection of HIV-1 neutralizing antibody in China’[journal of virological methods 185 (2012) 267– 275]","authors":"Jianhui Nie , Wenbo Wang , Zhiheng Wen , Aijing Song , Kunxue Hong , Shan Lu , Ping Zhong , Jianqing Xu , Wei Kong , Jingyun Li , Hong Shang , Hong Ling , Li Ruan , Youchun Wang","doi":"10.1016/j.jviromet.2025.115319","DOIUrl":"10.1016/j.jviromet.2025.115319","url":null,"abstract":"","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"341 ","pages":"Article 115319"},"PeriodicalIF":1.6,"publicationDate":"2025-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145714896","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 : 2025-12-05DOI: 10.1016/j.jviromet.2025.115322
Giasemi C. Eptaminitaki, Alexandros Zafiropoulos, George Sourvinos
The collection and storage of clinical specimens are critical determinants of reliable and accurate diagnostic outcomes. Swabs are commonly utilized for specimen collection aimed at the detection and identification of microorganisms. In this study, the ∑-MM™ Molecular Medium (Medical Wire and Equipment), which rapidly inactivates microorganisms, including bacteria, mycobacteria, and viruses, was evaluated for its capacity to preserve the stability of SARS-CoV-2 for up to 90 days under various temperature conditions. A total of 42 newly collected SARS-CoV-2 positive samples were selected to evaluate the performance of the ∑-MM™. Aliquots were stored under different conditions: at 4°C for 7 days, at room temperature for 7 days, at −80°C for one month, at room temperature for one month, at −80°C for three months, and at room temperature for three months. Samples were subsequently tested for SARS-CoV-2 RNA by Real-Time PCR. No statistically significant differences were observed across all storage conditions, when compared to the baseline measurements. The ∑-MM™ effectively preserves SARS-CoV-2 for up to 90 days at ambient temperatures, without statistically significant changes in CT values compared to baseline. These results validate ∑-MM™ as a reliable transport medium that enables specimen storage and transport under ambient conditions without compromising diagnostic accuracy. Given the demonstrated stability, future studies will explore the performance of ∑-MM™ over extended storage durations.
{"title":"Evaluation of ∑-MM™ molecular medium for the SARS-CoV-2 RNA stability over 90 days under different temperature conditions","authors":"Giasemi C. Eptaminitaki, Alexandros Zafiropoulos, George Sourvinos","doi":"10.1016/j.jviromet.2025.115322","DOIUrl":"10.1016/j.jviromet.2025.115322","url":null,"abstract":"<div><div>The collection and storage of clinical specimens are critical determinants of reliable and accurate diagnostic outcomes. Swabs are commonly utilized for specimen collection aimed at the detection and identification of microorganisms. In this study, the ∑-MM™ Molecular Medium (Medical Wire and Equipment), which rapidly inactivates microorganisms, including bacteria, mycobacteria, and viruses, was evaluated for its capacity to preserve the stability of SARS-CoV-2 for up to 90 days under various temperature conditions. A total of 42 newly collected SARS-CoV-2 positive samples were selected to evaluate the performance of the ∑-MM™. Aliquots were stored under different conditions: at 4°C for 7 days, at room temperature for 7 days, at −80°C for one month, at room temperature for one month, at −80°C for three months, and at room temperature for three months. Samples were subsequently tested for SARS-CoV-2 RNA by Real-Time PCR. No statistically significant differences were observed across all storage conditions, when compared to the baseline measurements. The ∑-MM™ effectively preserves SARS-CoV-2 for up to 90 days at ambient temperatures, without statistically significant changes in CT values compared to baseline. These results validate ∑-MM™ as a reliable transport medium that enables specimen storage and transport under ambient conditions without compromising diagnostic accuracy. Given the demonstrated stability, future studies will explore the performance of ∑-MM™ over extended storage durations.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"341 ","pages":"Article 115322"},"PeriodicalIF":1.6,"publicationDate":"2025-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145701404","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}
Hemorrhagic cystitis (HC) following hematopoietic stem cell transplantation (HSCT) is a significant event that can lengthen hospital stays and the need for care. The causes of HC can be multiple, but BK Polyomavirus (BKPyV) is the main protagonist in frequency. Currently, the clinical tool widely used to detect the presence of the virus in urine is PCR-based viral genome testing. We have developed a new rapid test for the antigenic detection of BKPyV in urine. The aim of this diagnostic study was to retrospectively evaluate the performance of this new assay as compared to BKPyV PCR on urine from HSCT patients with suspected HC. 49 samples from 49 different patients were evaluated, 20 of whom presented with HC. Of these 20 samples, 19 (95 %) were positive by the rapid antigen test. Of the 37 BKPyV PCR-positive samples, 31 were above 7 log10 copies/mL, including the 20 patients with HC. Thus, the overall performance of the rapid test for HC is greatly improved compared with PCR, with specificity rising from 62.1 % to 86.2 % and positive predictive value from 64.5 % to 82.6 %. In conclusion, this new tool could be implemented as a point-of-care test to rapidly confirm or rule out a suspicion of BKPyV-HC after HSCT.
{"title":"A rapid immunochromatographic assay for the detection of BK Polyomavirus in urine samples from hematopoietic stem cell transplant recipients","authors":"Etienne Brochot , Etienne Paubelle , Ophélie Fourdinier , Baptiste Demey , Aurélien Aubry , Virginie Morel , Antoine Touzé , Sandrine Castelain , François Helle","doi":"10.1016/j.jviromet.2025.115321","DOIUrl":"10.1016/j.jviromet.2025.115321","url":null,"abstract":"<div><div>Hemorrhagic cystitis (HC) following hematopoietic stem cell transplantation (HSCT) is a significant event that can lengthen hospital stays and the need for care. The causes of HC can be multiple, but BK Polyomavirus (BKPyV) is the main protagonist in frequency. Currently, the clinical tool widely used to detect the presence of the virus in urine is PCR-based viral genome testing. We have developed a new rapid test for the antigenic detection of BKPyV in urine. The aim of this diagnostic study was to retrospectively evaluate the performance of this new assay as compared to BKPyV PCR on urine from HSCT patients with suspected HC. 49 samples from 49 different patients were evaluated, 20 of whom presented with HC. Of these 20 samples, 19 (95 %) were positive by the rapid antigen test. Of the 37 BKPyV PCR-positive samples, 31 were above 7 log<sub>10</sub> copies/mL, including the 20 patients with HC. Thus, the overall performance of the rapid test for HC is greatly improved compared with PCR, with specificity rising from 62.1 % to 86.2 % and positive predictive value from 64.5 % to 82.6 %. In conclusion, this new tool could be implemented as a point-of-care test to rapidly confirm or rule out a suspicion of BKPyV-HC after HSCT.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"341 ","pages":"Article 115321"},"PeriodicalIF":1.6,"publicationDate":"2025-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145701370","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 : 2025-12-04DOI: 10.1016/j.jviromet.2025.115320
Qi Yang , Ziqiang Cheng , Tianxing Yan , Guihua Wang
Gyrovirus homsa 1 (GyH1) is an established causative agent of transmissible viral proventriculitis (TVP) in chickens. This study developed a colloidal gold immunochromatography assay (GICA) for GyH1 detection. Polyclonal and monoclonal antibodies against recombinant VP1 protein were generated. Colloidal gold nanoparticles were synthesized using the trisodium citrate reduction method, yielding uniform particle. Monoclonal antibodies were conjugated to colloidal gold particles, while polyclonal antibodies served as capture reagents immobilized on the membrane. Optimal labeling conditions were determined as pH 8.8 with a monoclonal concentration of 7.2 μg/mL. The assay demonstrated specific recognition of GyH1with a detection limit of 103 copies/μL. Batch-to-batch reproducibility was confirmed across all test strip. In summary, the GyH1 GICA exhibits high specificity, sensitivity, and reproducibility, providing an effective and low-cost tool for monitoring GyH1 early-stage infection status and technical support for field detection applications.
{"title":"Development of a colloidal gold-based immunochromatographic strip assay for rapid detection of Gyrovirus homsa 1 (GyH1)","authors":"Qi Yang , Ziqiang Cheng , Tianxing Yan , Guihua Wang","doi":"10.1016/j.jviromet.2025.115320","DOIUrl":"10.1016/j.jviromet.2025.115320","url":null,"abstract":"<div><div>Gyrovirus homsa 1 (GyH1) is an established causative agent of transmissible viral proventriculitis (TVP) in chickens. This study developed a colloidal gold immunochromatography assay (GICA) for GyH1 detection. Polyclonal and monoclonal antibodies against recombinant VP1 protein were generated. Colloidal gold nanoparticles were synthesized using the trisodium citrate reduction method, yielding uniform particle. Monoclonal antibodies were conjugated to colloidal gold particles, while polyclonal antibodies served as capture reagents immobilized on the membrane. Optimal labeling conditions were determined as pH 8.8 with a monoclonal concentration of 7.2 μg/mL. The assay demonstrated specific recognition of GyH1with a detection limit of 10<sup>3</sup> copies/μL. Batch-to-batch reproducibility was confirmed across all test strip. In summary, the GyH1 GICA exhibits high specificity, sensitivity, and reproducibility, providing an effective and low-cost tool for monitoring GyH1 early-stage infection status and technical support for field detection applications.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"341 ","pages":"Article 115320"},"PeriodicalIF":1.6,"publicationDate":"2025-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145693123","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}
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}
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