Journal of virological methods最新文献

{"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":"https://doi.org/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 50µL 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 (2,000 × g for 30min), 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 / 3hours 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 / 3hours 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 / 3hours 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":"2025-07-30","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}

{"title":"Characterization of Goatpox virus major intracellular mature virion proteins A27L and P32 expressed by baculovirus system: Superior to Escherichia coli","authors":"Anand Kushwaha,&nbsp;Poulinlu Golmei,&nbsp;Amit Kumar,&nbsp;B. Mondal,&nbsp;Gnanavel Venkatesan","doi":"10.1016/j.jviromet.2025.115234","DOIUrl":"10.1016/j.jviromet.2025.115234","url":null,"abstract":"<div><div>Baculovirus expression system has become the most widely used eukaryotic system for recombinant protein production due to its ability to perform post-translational modifications and produce proteins with native-like conformation and antigenicity. In this study, two major immunodominant intracellular mature virion genes of Goatpox virus were amplified into three constructs: A27L, full-length P32, and truncated P32 (tr-P32), respectively. The constructs were cloned into the pFastBac HTA vector and expressed in <em>Trichoplusiani</em> (TN5) insect cells using the baculovirus expression system. Recombinant A27L was purified under native conditions, however full-length P32 and tr-P32 required denaturing conditions. tr-P32 showed markedly higher expression and solubility than full-length P32. All recombinant proteins were immunoreactive with hyperimmune GTPV sera, as confirmed by western blotting. Native PAGE analysis of rA27L revealed concentration-dependent oligomerization into dimers, trimers and tetramers. Furthermore, in indirect ELISA, both A27L and P32 exhibited strong reactivity with sera from GTPV and SPPV infected or vaccinated animals at 1:50 dilution and did not show any cross-reactivity with related viruses. These results quantitatively demonstrated that baculovirus-expressed A27L and P32 proteins are superior diagnostic antigens, providing higher sensitivity and specificity for <em>Capripoxviruses</em> serodiagnosis and offering potential as improved candidates for both serological and prophylactic applications.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"338 ","pages":"Article 115234"},"PeriodicalIF":1.6,"publicationDate":"2025-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144756864","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}

{"title":"Chimeric snakehead rhabdoviruses and zebrafish as a model system for investigating the role of fish rhabdoviral glycoproteins in in vivo virulence","authors":"Jae Young Kim,&nbsp;Mariem Bessaid,&nbsp;Kyung Min Lee,&nbsp;Ki Hong Kim","doi":"10.1016/j.jviromet.2025.115232","DOIUrl":"10.1016/j.jviromet.2025.115232","url":null,"abstract":"<div><div>Fish rhabdoviruses pose significant threats to aquaculture due to their pathogenicity and economic impact. This study explores the role of viral glycoprotein (G) and non-virion (NV) genes in determining virulence by generating a panel of chimeric snakehead rhabdoviruses (SHRVs) that express heterologous G or NV genes from pathogenic fish rhabdoviruses. Zebrafish (<em>Danio rerio</em>), which shows susceptibility to multiple rhabdoviruses and low mortality upon SHRV infection, was employed as an <em>in vivo</em> model to evaluate virulence. All chimeric SHRVs replicated efficiently in ZF-4 cells, indicating functional compatibility of the heterologous glycoproteins with the SHRV backbone. However, in the <em>in vivo</em> virulence analysis, G gene substitutions alone, even from highly virulent parental viruses (e.g., VHSV Ia, SVCV), did not significantly enhance mortality, suggesting that the G protein is insufficient by itself to confer high virulence in zebrafish. In contrast, a chimeric SHRV harboring the NV gene from VHSV Ia demonstrated elevated mortality and viral replication <em>in vivo</em> compared to wild-type SHRV, indicating the NV gene's role as a virulence determinant. These findings underscore the complexity of rhabdoviral virulence mechanisms and support the utility of the zebrafish–SHRV system for dissecting gene-specific contributions to pathogenicity in aquatic hosts.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"338 ","pages":"Article 115232"},"PeriodicalIF":1.6,"publicationDate":"2025-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144723737","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}

{"title":"Beyond the limits of conventional ‘endpoint’ ELISA and rescuing the signal with lag k-ELISA","authors":"Ksenia Katsanovskaja ,&nbsp;Federica Marchesin ,&nbsp;Jacquie Ujetz ,&nbsp;Samreen Ijaz ,&nbsp;Richard Tedder ,&nbsp;Myra McClure","doi":"10.1016/j.jviromet.2025.115231","DOIUrl":"10.1016/j.jviromet.2025.115231","url":null,"abstract":"<div><div>Conventional colorimetric e-ELISA assays are widely used for quantifying antibodies in serum but are often labour-intensive, costly, and require serial dilutions for high-titre samples to ensure accuracy. To address these limitations, we propose the lag k-ELISA, a novel method for assessing antibody concentrations against SARS-CoV-2 in blood samples. This technique simplifies workflows while reducing time and labor compared to traditional ELISA. In this study, lag k-ELISA was applied to 169 sera for anti-S1 IgG and 79 samples for anti-S1 IgM assessment. Distinct analytical workflows were developed to process kinetic signal data, allowing for the description of Ab content. Key signal metrics—including time at OD minima, k-rates, and signal kinetics—proved effective in distinguishing samples by dilution factor or unitage. The assay’s performance was systematically evaluated under different operating conditions, such as stirring, readout time, and temperature, which significantly impacted assay outcomes and needed to be optimised to enhance the resolving power of lag k-ELISA. Our findings demonstrate that lag k-ELISA is a robust, efficient complement to traditional ELISA methods, offering reliable results with reduced labour, time, and costs. This technique is particularly advantageous for high-titre samples, streamlining antibody quantification workflows without compromising accuracy.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"338 ","pages":"Article 115231"},"PeriodicalIF":2.2,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144711023","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}

{"title":"Targeted enrichment of Elephant Endotheliotropic Herpesvirus for complete genome sequencing in elephants","authors":"Kirtika Sharma ,&nbsp;K.G. Sai Balaji ,&nbsp;Gaurav Kumar Sharma ,&nbsp;Abhijit M. Pawde ,&nbsp;Sonalika Mahajan ,&nbsp;Ravikant Agrawal ,&nbsp;Pracheta Janmeda ,&nbsp;Karikalan Mathesh","doi":"10.1016/j.jviromet.2025.115230","DOIUrl":"10.1016/j.jviromet.2025.115230","url":null,"abstract":"<div><div>Elephant Endotheliotropic Herpesvirus (EEHV) poses a critical threat to young Asian (<em>Elephas maximus</em>) and African elephants (<em>Loxodonta africana</em>), with high mortality rates due to acute haemorrhagic symptoms from vascular endothelial damage. EEHV remains dormant in adult elephants, reactivating under stress or immune suppression. There are multiple genotypes of EEHV have been reported based on genetic heterogeneity. The diversity among EEHV subtypes, such as EEHV1A and EEHV1B, influences disease severity and host susceptibility. EEHV genomes are large and encode over 115 open reading frames, contributing to viral replication and immune evasion. Hence, generation of complete genome is essential to determine the genotypes of EEHV. However, the inability to culture EEHV <em>in</em>-<em>vitro</em> limits the study of its life cycle and pathogenesis, necessitating molecular techniques for direct analysis. Additionally, variable viral load in the infected tissues limits the success of DNA sequencing using direct sequencing. Hence, in this study several methods of viral or DNA enrichment were considered, and ultracentrifugation method was adopted to concentrate viral copies for improved genomic analysis. Ultracentrifugation demonstrates effectiveness in isolating EEHV, significantly enriching viral DNA for detection and characterization. This technique facilitates advancements in diagnostics, therapeutic development, and vaccine research, addressing the challenges of low viral loads and host DNA contamination in clinical samples.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"338 ","pages":"Article 115230"},"PeriodicalIF":2.2,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144711024","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}

{"title":"Genomic and molecular epidemiological characterization of a novel piscine poxvirus in the red seabream, Pagrus major","authors":"Naritoyo Ishibashi ,&nbsp;Yuri Akase ,&nbsp;Shuntaro Watanabe ,&nbsp;Hiroshi Yokoyama ,&nbsp;Tohru Mekata","doi":"10.1016/j.jviromet.2025.115229","DOIUrl":"10.1016/j.jviromet.2025.115229","url":null,"abstract":"<div><div>Since 2020, unexplained mortality has been recorded in a red seabream hatchery. Affected juveniles exhibit skin darkening and sleep-like symptoms, which occasionally cause mass mortality. In this study, we detected a novel poxvirus genome in diseased fish via next-generation sequencing. Transcriptome analysis of moribund individuals revealed multiple contigs with sequence homology to the salmon gill poxvirus, suggesting the presence of a related viral agent. Subsequent metagenomic analysis led to the assembly of a 308-kbp viral genome of a novel piscine poxvirus, designated Japanese seabream poxvirus (JSPV), containing 338 predicted open reading frames. DNA polymerase of JSPV shared 62 and 44 % amino acid identity with those of the salmon gill poxvirus and carp edema virus, respectively. Comparative genomic analysis of piscine poxviruses revealed that genome synteny was highly conserved in the central region. Subsequently, a PCR method was developed and <em>I7L</em>, which encodes the virion core cysteine protease, was successfully detected in all JSPV genogroups. Based on the partial sequence of <em>I7L</em>, JSPV was classified into two major genogroups: I and II. Genogroup I was further subdivided into genogroups Ia and Ib.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"338 ","pages":"Article 115229"},"PeriodicalIF":2.2,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144702814","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}

{"title":"Establishment and application of a SYBR Green-based absolute real-time quantitative PCR assay for chilli yellow ringspot virus","authors":"Yu Li ,&nbsp;Kuo Wu ,&nbsp;Xia Du,&nbsp;Yongdui Chen,&nbsp;Jie Zhang,&nbsp;Zhongkai Zhang","doi":"10.1016/j.jviromet.2025.115227","DOIUrl":"10.1016/j.jviromet.2025.115227","url":null,"abstract":"<div><div>Chilli yellow ringspot virus (CYRSV), a new emerging pathogen, was first detected in Yunnan Province, China. This virus is a novel agent that causes severe disease in vegetable crops and has a tendency to spread widely. CYRSV infection manifests as yellowing, ringspot and necrosis of the leaves and fruits of tomato plants. Consequently, the development of a sensitive, rapid, and accurate detection system for virus identification and epidemiological surveillance is imperative. In this study, we developed a quantitative PCR (qPCR) assay using SYBR Green for the detection of CYRSV accumulation. To achieve sensitive and rapid detection of CYRSV in tomato, specific primers were designed based on the coding sequence of the CYRSV nucleocapsid protein (N) gene. These primers demonstrated excellent specificity, sensitivity, and reproducibility. After inoculation with CYRSV in a greenhouse, the amount of virus accumulated in tomato leaves was detected. A total of 62 tomato fruit samples were collected from Yuanmou city, Yunnan Province, China, between May and December 2024. Of these, 75.81 % (47/62) tested positive for CYRSV via this qPCR method. These results indicated that the established SYBR Green-based qPCR assay could accurately detect CYRSV in tomato leaves and fruits. In conclusion, our study provides a valuable diagnostic tool for CYRSV detection and epidemiological investigation.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"338 ","pages":"Article 115227"},"PeriodicalIF":2.2,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144711022","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}

{"title":"Detecting rubella virus-specific antibodies by cytopathic effect and immunoenzymatic microneutralization tests","authors":"Iris Medits-Weiss,&nbsp;Heidemarie Holzmann,&nbsp;Lukas Weseslindtner,&nbsp;Karin Stiasny","doi":"10.1016/j.jviromet.2025.115228","DOIUrl":"10.1016/j.jviromet.2025.115228","url":null,"abstract":"<div><h3>Background</h3><div>Rubella in early pregnancy can cause severe congenital malformations, but can be prevented by vaccination. Immunity to rubella is usually assessed by the detection of rubella virus-specific antibodies. Neutralization tests (NTs) are of highest diagnostic significance, because they measure the effectiveness of antibodies to block viral infection.</div></div><div><h3>Objectives</h3><div>The ability of rubella virus to cause a cytopathic effect (CPE) is limited. Therefore, most NT formats are based on immunoenzymatic techniques detecting viral proteins in infected cells. As Vero cells have been described to be susceptible to rubella virus infection with a more pronounced CPE, we aimed to develop an NT with these cells.</div></div><div><h3>Methods</h3><div>We compared rubella NTs with different infectivity readout methods (immunoenzymatic vs. CPE) using 42 human polyclonal samples with a known IgG ELISA concentration (IU/ml).</div></div><div><h3>Results</h3><div>We found a strong positive correlation between rubella virus-specific ELISA IgG units and neutralization titers determined with the two different NTs as well as between the two NT formats.</div></div><div><h3>Conclusion</h3><div>NT titer determination using CPE as a readout for infectivity is a reliable and promising method for rubella serology.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"338 ","pages":"Article 115228"},"PeriodicalIF":2.2,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144696607","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}

{"title":"Corrigendum to \"A high-sensitivity qPCR method for detecting residual vero cell DNA in rabies vaccine production\" [J. Virol. Methods 338 (2025) 115217].","authors":"MaríaP Almario, Jorge Rivera, Catalina Páramo, Valentina Jaramillo, Yanira Chaparro Zulma Rocío Suárez-Moreno","doi":"10.1016/j.jviromet.2025.115226","DOIUrl":"https://doi.org/10.1016/j.jviromet.2025.115226","url":null,"abstract":"","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":" ","pages":"115226"},"PeriodicalIF":2.2,"publicationDate":"2025-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667943","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}

{"title":"A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid colorimetric detection of pepper mild mottle virus (PMMoV)","authors":"Anthony J. Gross ,&nbsp;Salvador Lopez Jr. ,&nbsp;Alexandra Rogers ,&nbsp;Scott Adkins ,&nbsp;Mya Breitbart","doi":"10.1016/j.jviromet.2025.115225","DOIUrl":"10.1016/j.jviromet.2025.115225","url":null,"abstract":"<div><div>Pepper mild mottle virus (<em>Tobamovirus capsici</em>, PMMoV) is a plant virus in the genus <em>Tobamovirus</em> that infects peppers and other members of the family <em>Solanaceae</em>. The virus is transmitted mechanically, poses a significant threat to crops globally, and is one of the most abundant viruses found in human feces and wastewater. Two colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed to detect PMMoV, one targeting the RNA-dependent RNA-polymerase (PMMoV_RdRp) and the other targeting the coat protein (PMMoV_CP). Synthetic gBlock positive controls were used to determine the detection limit of each assay. PMMoV_RdRp detected PMMoV at concentrations greater than or equal to 100 copies/µL, the same sensitivity as the published RT-qPCR assay for this gene. In contrast, the detection limit of the PMMoV_CP RT-LAMP assay was an order of magnitude greater. Both assays were specific to PMMoV and did not amplify plant host tissue or related tobamoviruses. Since these RT-LAMP assays do not require specialized laboratory equipment and yield positive results within 20–30 min, they are advantageous for point-of-use testing. Overall, the RT-LAMP assays described here are sensitive, specific, and more rapid than existing methods for PMMoV detection and quantification and thus have potential widespread applications for agriculture, wastewater treatment assessment, recreational water quality testing, and food safety.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"338 ","pages":"Article 115225"},"PeriodicalIF":2.2,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667942","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}