Pub Date : 2025-12-19DOI: 10.1016/j.jviromet.2025.115329
Wenfa Zhang , Xiao Yuan Chen , Sheng-Xiang Lin
Papain-like protease (PLpro) is a key protease encoded by SARS-CoV-2, essential for viral polyprotein processing, replicase-transcriptase complex assembly, and interference with host immune responses. New COVID-19 cases continue to emerge, and WHO has identified coronaviruses with similar PLpro structures as potential pandemic threats. Using molecular modeling, docking, and protease activity assays, we identified four potent inhibitors of SARS-CoV-2 /PLpro with IC₅₀ values of 6.96–20.21 µM. Their binding affinities, determined by fluorescence titration, yielded dissociation constants (KD) of 4.18–13.06, supporting their inhibitory activity. In silico toxicity studies suggest that most inhibitors have LD₅₀ values comparable to GRL0617, a known PLpro inhibitor. Structural analysis revealed that all inhibitors interact with key residues Asp164, Pro248, Tyr264, Tyr268, and Gln269 of PLpro, but differ in specific hydrogen bonding and hydrophobic interactions. Notably, compound P636–0664 exhibits the most extensive interactions, forming 2 hydrogen bonds and 13 hydrophobic contacts. These findings provide a structural basis for the optimization of inhibitors targeting the SARS-CoV-2 PLpro, contributing directly to anti-coronavirus drug discovery.
{"title":"Search and prove of efficient inhibitors against papain-like protease from SARS-CoV-2","authors":"Wenfa Zhang , Xiao Yuan Chen , Sheng-Xiang Lin","doi":"10.1016/j.jviromet.2025.115329","DOIUrl":"10.1016/j.jviromet.2025.115329","url":null,"abstract":"<div><div>Papain-like protease (PLpro) is a key protease encoded by SARS-CoV-2, essential for viral polyprotein processing, replicase-transcriptase complex assembly, and interference with host immune responses. New COVID-19 cases continue to emerge, and WHO has identified coronaviruses with similar PLpro structures as potential pandemic threats. Using molecular modeling, docking, and protease activity assays, we identified four potent inhibitors of SARS-CoV-2 /PLpro with IC₅₀ values of 6.96–20.21 µM. Their binding affinities, determined by fluorescence titration, yielded dissociation constants (KD) of 4.18–13.06, supporting their inhibitory activity. In silico toxicity studies suggest that most inhibitors have LD₅₀ values comparable to GRL0617, a known PLpro inhibitor. Structural analysis revealed that all inhibitors interact with key residues Asp<sup>164</sup>, Pro<sup>248</sup>, Tyr<sup>264</sup>, Tyr<sup>268</sup>, and Gln<sup>269</sup> of PLpro, but differ in specific hydrogen bonding and hydrophobic interactions. Notably, compound P636–0664 exhibits the most extensive interactions, forming 2 hydrogen bonds and 13 hydrophobic contacts. These findings provide a structural basis for the optimization of inhibitors targeting the SARS-CoV-2 PLpro, contributing directly to anti-coronavirus drug discovery.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"341 ","pages":"Article 115329"},"PeriodicalIF":1.6,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145804807","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 : 2025-12-17DOI: 10.1016/j.jviromet.2025.115328
Chiara Filizzolo , Mariangela Pizzo , Marika Munacò , Giovanni M. Giammanco , Gianvito Lanave , Vito Martella , Floriana Bonura , Simona De Grazia
Acute gastroenteritis (AGE) remains a global health concern, particularly affecting children under five. While mortality rates have declined in industrialized countries, AGE continues to pose a significant burden on healthcare systems and remains a major threat in developing regions. This study evaluates the diagnostic performance of two commercial molecular assays for enteric viruses, GastroIntestinal (GI) Viral PLUS ELITe MGB® and GI Norovirus PLUS ELITe MGB®, for the detection of a broad panel of viral genotypes associated with AGE. A total of 222 archived stool samples, previously characterized by reference biomolecular methods, were analyzed. These included 50 rotavirus (RVA)-positive, 57 norovirus (NoV)-positive, 39 adenovirus (AdV)-positive, 45 astrovirus (HAstV)-positive samples, and 10 co-infections. Both assays showed excellent agreement with reference methods (κ ≥ 0.95), with sensitivity ranging from 99.4 % to 100 % and specificity from 98.1 % to 100 %, confirming their ability to detect a broad spectrum of circulating viral genotypes. Minor discrepancies were observed in NoV GII.6 strains, misclassified as Genogroup I (GI), likely due to overlapping annealing profiles. These findings support the use of GI Viral PLUS and GI Norovirus PLUS ELITe MGB® assays as reliable tools for rapid and accurate genotypic diagnosis of viral AGE. Their implementation may enhance patient management, reduce inappropriate antimicrobial use, and support timely outbreak containment, especially in healthcare settings.
{"title":"Evaluation of two commercial multiplex RT-PCR tests for broad genotypic detection of viral gastroenteritis","authors":"Chiara Filizzolo , Mariangela Pizzo , Marika Munacò , Giovanni M. Giammanco , Gianvito Lanave , Vito Martella , Floriana Bonura , Simona De Grazia","doi":"10.1016/j.jviromet.2025.115328","DOIUrl":"10.1016/j.jviromet.2025.115328","url":null,"abstract":"<div><div>Acute gastroenteritis (AGE) remains a global health concern, particularly affecting children under five. While mortality rates have declined in industrialized countries, AGE continues to pose a significant burden on healthcare systems and remains a major threat in developing regions. This study evaluates the diagnostic performance of two commercial molecular assays for enteric viruses, GastroIntestinal (GI) Viral PLUS ELITe MGB® and GI Norovirus PLUS ELITe MGB®, for the detection of a broad panel of viral genotypes associated with AGE. A total of 222 archived stool samples, previously characterized by reference biomolecular methods, were analyzed. These included 50 rotavirus (RVA)-positive, 57 norovirus (NoV)-positive, 39 adenovirus (AdV)-positive, 45 astrovirus (HAstV)-positive samples, and 10 co-infections. Both assays showed excellent agreement with reference methods (κ ≥ 0.95), with sensitivity ranging from 99.4 % to 100 % and specificity from 98.1 % to 100 %, confirming their ability to detect a broad spectrum of circulating viral genotypes. Minor discrepancies were observed in NoV GII.6 strains, misclassified as Genogroup I (<em>GI</em>), likely due to overlapping annealing profiles. These findings support the use of GI Viral PLUS and GI Norovirus PLUS ELITe MGB® assays as reliable tools for rapid and accurate genotypic diagnosis of viral AGE. Their implementation may enhance patient management, reduce inappropriate antimicrobial use, and support timely outbreak containment, especially in healthcare settings.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"341 ","pages":"Article 115328"},"PeriodicalIF":1.6,"publicationDate":"2025-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145794348","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 : 2025-12-17DOI: 10.1016/j.jviromet.2025.115330
Mariane Izabella Abreu de Melo, Alessandra Nunes Duarte Miranda, Antero Silva Ribeiro de Andrade
Yellow Fever Virus (YFV), a mosquito-borne flavivirus, remains a significant public health concern despite the availability of effective vaccines. Accurate differential diagnosis continues to be challenging due to the high antigenic similarity among flaviviruses such as dengue and Zika, which compromises the specificity of current serological assays. Aptamers have emerged as promising alternatives to antibodies for diagnostic applications because of their high specificity, thermal stability, and ease of synthesis. In this study, we developed a DNA aptamer (Flav5) targeting the nonstructural protein 1 (NS1) of YFV using capillary electrophoresis–based SELEX (CE-SELEX). After three selection rounds, aptamer pools were sequenced and analyzed. Specificity assays demonstrated minimal cross-reactivity of Flav5 with NS1 proteins from dengue virus (serotypes 1–4) and Zika virus. The aptamer exhibited a high binding affinity for YFV-NS1, with a dissociation constant (Kd) of 34.5 ± 8.2 nM, as determined by quantitative PCR. The three-dimensional structure of Flav5 was modeled and docked to the tetrameric YFV-NS1 using the HDOCK server, revealing a stable binding interface within the inter-subunit cavity of NS1, supported by high confidence scores (>0.95). The Flav5 aptamer demonstrates strong potential for incorporation into YFV-specific diagnostic platforms, particularly in regions where multiple clinically relevant flaviviruses co-circulate. Its combination of high affinity, specificity, and favorable molecular docking characteristics, position it as a promising candidate for point-of-care diagnostic tools.
{"title":"Targeting Yellow-Fever Virus: Development of a specific aptamer to NS1 protein","authors":"Mariane Izabella Abreu de Melo, Alessandra Nunes Duarte Miranda, Antero Silva Ribeiro de Andrade","doi":"10.1016/j.jviromet.2025.115330","DOIUrl":"10.1016/j.jviromet.2025.115330","url":null,"abstract":"<div><div>Yellow Fever Virus (YFV), a mosquito-borne flavivirus, remains a significant public health concern despite the availability of effective vaccines. Accurate differential diagnosis continues to be challenging due to the high antigenic similarity among flaviviruses such as dengue and Zika, which compromises the specificity of current serological assays. Aptamers have emerged as promising alternatives to antibodies for diagnostic applications because of their high specificity, thermal stability, and ease of synthesis. In this study, we developed a DNA aptamer (Flav5) targeting the nonstructural protein 1 (NS1) of YFV using capillary electrophoresis–based SELEX (CE-SELEX). After three selection rounds, aptamer pools were sequenced and analyzed. Specificity assays demonstrated minimal cross-reactivity of Flav5 with NS1 proteins from dengue virus (serotypes 1–4) and Zika virus. The aptamer exhibited a high binding affinity for YFV-NS1, with a dissociation constant (Kd) of 34.5 ± 8.2 nM, as determined by quantitative PCR. The three-dimensional structure of Flav5 was modeled and docked to the tetrameric YFV-NS1 using the HDOCK server, revealing a stable binding interface within the inter-subunit cavity of NS1, supported by high confidence scores (>0.95). The Flav5 aptamer demonstrates strong potential for incorporation into YFV-specific diagnostic platforms, particularly in regions where multiple clinically relevant flaviviruses co-circulate. Its combination of high affinity, specificity, and favorable molecular docking characteristics, position it as a promising candidate for point-of-care diagnostic tools.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"341 ","pages":"Article 115330"},"PeriodicalIF":1.6,"publicationDate":"2025-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145794314","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 : 2025-12-16DOI: 10.1016/j.jviromet.2025.115327
Mohamed Samy Abousenna, Heba A. Khafagy, Amal Abd El Moneim Mohamed, Sara El Sawy Ahmed, Fady Abd El Mohsen Shasha, Nermeen Gouda Shafik
Lumpy skin disease (LSD) is a transboundary viral disease of cattle caused by lumpy skin disease virus (LSDV), resulting in substantial economic losses. Control of the disease relies primarily on live attenuated vaccines, making accurate potency assessment essential to ensure protective efficacy. Quantitative real-time PCR (qPCR) has emerged as a rapid and sensitive method for viral detection and quantification. In this study, ten commercial batches of live attenuated LSDV vaccines were evaluated using qPCR, and the molecular titers were compared with conventional tissue culture titration and in vivo protection in a calf challenge model. Tissue culture titration was performed on MDBK cells, qPCR quantified viral genome copy numbers, and in vivo challenge testing involved 56 calves. Statistical analyses, including Pearson and Spearman correlations and linear regression, were used to assess the relationships between qPCR-derived titers, tissue culture results, and protection outcomes. Eight of the ten vaccine batches met the WOAH-recommended potency range (3–4 log₁₀ TCID₅₀/mL), while two batches were suboptimal. qPCR-derived titers showed a strong correlation with tissue culture titers (Pearson’s r = −0.994; p < 0.0001) r = −0.994; p < 0.0001) and with protection outcomes following virulent challenge (r = 0.777; p = 0.008), with lower molecular titers corresponding to reduced protection. These results demonstrate that qPCR is a rapid, sensitive, and reliable surrogate for conventional potency testing, offering biosafe and time-efficient evaluation. Integrating molecular quantification into routine vaccine assessment can improve quality control, reduce reliance on in vivo challenge, facilitate harmonized vaccine deployment, and ultimately enhance field protection against LSD.
肿块性皮肤病（LSD）是由肿块性皮肤病病毒（LSDV）引起的跨界牛病毒性疾病，造成重大经济损失。该病的控制主要依靠减毒活疫苗，因此准确的效力评估对于确保保护效果至关重要。实时荧光定量PCR （qPCR）是一种快速、灵敏的病毒检测和定量方法。本研究采用qPCR技术对10批LSDV减毒活疫苗进行了评价，并与传统的组织培养滴定法和小牛攻毒模型的体内保护作用进行了分子效价比较。对MDBK细胞进行组织培养滴定，qPCR量化病毒基因组拷贝数，并对56头小牛进行体内攻毒试验。统计分析，包括Pearson和Spearman相关性和线性回归，用于评估qpcr衍生滴度、组织培养结果和保护结果之间的关系。十个疫苗批次中有八个达到了世界卫生组织推荐的效力范围（3-4log₁₀TCID₅₀/mL），而两个批次则不理想。qpcr衍生的滴度与组织培养滴度（Pearson’s r = -0.994; p < 0.0001）和毒力攻击后的保护结果（r = 0.777; p = 0.008）有很强的相关性，较低的分子滴度对应于较低的保护。这些结果表明，qPCR是一种快速、灵敏、可靠的替代传统效价检测的方法，具有生物安全性和时效性。将分子定量纳入常规疫苗评估可以改善质量控制，减少对体内挑战的依赖，促进疫苗的协调部署，并最终增强对LSD的野外保护。
{"title":"Real time PCR-based evaluation of live attenuated lumpy skin disease virus vaccines for immunogenicity and efficacy","authors":"Mohamed Samy Abousenna, Heba A. Khafagy, Amal Abd El Moneim Mohamed, Sara El Sawy Ahmed, Fady Abd El Mohsen Shasha, Nermeen Gouda Shafik","doi":"10.1016/j.jviromet.2025.115327","DOIUrl":"10.1016/j.jviromet.2025.115327","url":null,"abstract":"<div><div>Lumpy skin disease (LSD) is a transboundary viral disease of cattle caused by lumpy skin disease virus (LSDV), resulting in substantial economic losses. Control of the disease relies primarily on live attenuated vaccines, making accurate potency assessment essential to ensure protective efficacy. Quantitative real-time PCR (qPCR) has emerged as a rapid and sensitive method for viral detection and quantification. In this study, ten commercial batches of live attenuated LSDV vaccines were evaluated using qPCR, and the molecular titers were compared with conventional tissue culture titration and in vivo protection in a calf challenge model. Tissue culture titration was performed on MDBK cells, qPCR quantified viral genome copy numbers, and in vivo challenge testing involved 56 calves. Statistical analyses, including Pearson and Spearman correlations and linear regression, were used to assess the relationships between qPCR-derived titers, tissue culture results, and protection outcomes. Eight of the ten vaccine batches met the WOAH-recommended potency range (3–4 log₁₀ TCID₅₀/mL), while two batches were suboptimal. qPCR-derived titers showed a strong correlation with tissue culture titers (Pearson’s r = −0.994; p < 0.0001) r = −0.994; p < 0.0001) and with protection outcomes following virulent challenge (r = 0.777; p = 0.008), with lower molecular titers corresponding to reduced protection. These results demonstrate that qPCR is a rapid, sensitive, and reliable surrogate for conventional potency testing, offering biosafe and time-efficient evaluation. Integrating molecular quantification into routine vaccine assessment can improve quality control, reduce reliance on in vivo challenge, facilitate harmonized vaccine deployment, and ultimately enhance field protection against LSD.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"341 ","pages":"Article 115327"},"PeriodicalIF":1.6,"publicationDate":"2025-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145781486","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 : 2025-12-16DOI: 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}
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