Journal of virological methods最新文献

{"title":"Development of a quadruple qPCR assay for simultaneous detection of four common bovine pathogens","authors":"Fuxing Hao ,&nbsp;Chunhao Tao ,&nbsp;Ying Huang ,&nbsp;Ruilong Xiao ,&nbsp;Daoxian Zhu ,&nbsp;Weifeng Yuan ,&nbsp;Zhen Wang ,&nbsp;Yuxin Li ,&nbsp;Hong Jia","doi":"10.1016/j.jviromet.2025.115265","DOIUrl":"10.1016/j.jviromet.2025.115265","url":null,"abstract":"<div><div>Bovine infectious diseases pose a significant threat to cattle health, causing widespread economic losses and profoundly impacting the well-being and productivity of affected herds. Among these, Bovine Herpesvirus 4 (BoHV4), Bovine Ephemeral Fever Virus (BEFV), Bovine Rotavirus (BRV), and Clostridium perfringens (CP) are four common pathogens responsible for a range of clinical manifestations in cattle. Notably, co-infections among these pathogens are relatively prevalent, contributing to the complexity and severity of disease outcomes in affected cattle. To simultaneously detect and differentiate these four pathogens in a single assay, we developed a TaqMan-based multiplex real-time PCR (qPCR) method containing four primer-probe sets, designed to target highly conserved or virulence-associated genes specific to each pathogen. The assay was optimized by adjusting primer-probe concentrations and annealing temperatures. Following optimization, a comprehensive evaluation was conducted to assess the analytical performance, including specificity, sensitivity, repeatability, and clinical applicability. The results demonstrated that the developed method exhibited no cross-reactivity with other bovine pathogens commonly encountered in clinical settings, achieved a detection limit of as few as 5 copies/μL for all four target pathogens, and showed coefficients of variation (CVs) below 2.26 % in repeatability tests. The method was applied to screen 1012 clinical samples collected from two commercial cattle farms in Jiangsu Province. The results revealed a positivity rate of 5.24 % (53/1012) for one or more of the four pathogens, with BRV, CP, BoHV4, and BEFV accounting for 3.66 %, 1.28 %, 0.30 %, and 0 % of the positive cases, respectively. Co-infections involving multiple pathogens were detected in 0.70 % (7/1012) of the samples. In conclusion, this study successfully developed a one-step multiplex qPCR assay for the simultaneous detection and differentiation of four common bovine pathogens. The assay provides a rapid, reliable, and cost-effective tool for bovine infectious disease surveillance and control. Its ability to detect mixed infections, combined with its high sensitivity and specificity, makes it particularly suitable for use in cattle farms, enabling rapid and accurate identification of pathogens to support disease management and control.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115265"},"PeriodicalIF":1.6,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145103075","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":"Evaluation of dead-end hollowfiber ultrafilter options for enumerating somatic and F+ coliphage in ambient waters and wastewater","authors":"Brian R. McMinn,&nbsp;Julie Kelleher,&nbsp;Asja Korajkic","doi":"10.1016/j.jviromet.2025.115267","DOIUrl":"10.1016/j.jviromet.2025.115267","url":null,"abstract":"<div><div>Coliphage are viral indicators of fecal contamination in water while acting as possible proxies for enteric viral pathogens. Depending on contamination levels, coliphage could be present at concentrations necessitating the use of concentrating filters. Hollow-fiber ultrafilters (HFUF) such as Asahi Kasei Rexeed have successfully concentrated coliphage in a dead-end setup (D-HFUF) from environmental waters and are recommended within United States Environmental Protection Agency (USEPA) Method 1642. Asahi Kasei Rexeed are not available within the United States, so replacement filters need to be identified. Additionally, coliphage methods lack recommendations for sample holding times to prevent variability in coliphage concentrations between sample collection and analysis. We compared HFUFs, the Fresenius F160NRE and the Elisio-15H, to the Asahi Kasei Rexeed 15S to determine their efficacy in recovering somatic and F+ coliphage from river, lake, marine, and wastewater. A 2 L volume of each matrix (river, lake, marine, and final effluent [<em>n</em> = 10 each]), were concentrated using D-HFUF for each filter brand with coliphage enumerated using the single agar layer (SAL) assay. There was no significant difference in performance between the three filters regardless of sample matrix (p &gt; 0.05). To establish sample holding times, each water matrix (stored at 4ºC) was analyzed on a weekly basis for endogenous coliphage. In wastewater, significant decay occurred within 48 h of collection (<em>P</em> value range: 0.0175–0.0006), while in other matrices, coliphages were stable ≥ 6 days. In this study, we identified replacement HFUFs and pertinent information regarding sample holding times for coliphage monitoring efforts moving forward.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115267"},"PeriodicalIF":1.6,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145081215","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":"In vitro modeling of human dorsal root ganglion neurons for GCaMP6-based calcium imaging of sensory responses to HSV-1 infection","authors":"Yu-Chih Chen ,&nbsp;Brandon Bullock ,&nbsp;Daniel I. Ogbeh ,&nbsp;Jinglu Ai ,&nbsp;Justin Okoh ,&nbsp;Jia Liu ,&nbsp;Yuhao Qiang ,&nbsp;S. Victor Hsia","doi":"10.1016/j.jviromet.2025.115264","DOIUrl":"10.1016/j.jviromet.2025.115264","url":null,"abstract":"<div><div>Dorsal root ganglion (DRG) neurons play a pivotal role in transmitting sensory information from the periphery to the central nervous system, mediating diverse stimuli such as pain, touch, and temperature. Despite advances, translating findings from rodent models to human applications remains challenging due to species-specific differences, necessitating reliable human DRG neuron models. The immortalized human DRG neuronal cell line HD10.6, derived from embryonic DRG cells and capable of differentiating into functional nociceptive-like neurons, offers a promising in vitro system for studying sensory neuron biology and drug screening. This study explores the utility of GCaMP6s, a genetically encoded calcium indicator, as a molecular tool for imaging sensory activation in HD10.6 cells. To establish HD10.6 as a robust human DRG model, we constructed and characterized adeno-associated virus (AAV9) vectors for efficient GCaMP6s delivery. Differentiated HD10.6 cells were efficiently transduced, and calcium dynamics were validated to assess functional responses to sensory stimuli. The results showed that AAV9 serotype was sufficient to infect HD10.6 and the GCaMP6s was successfully introduced into the cells. The HD10.6-GCaMP6s responded to capsaicin well under the appropriate condition. A series of viral infection studies indicated that herpesvirus HSV-1 triggered robust calcium influx within 5 min after the exposure to the virus. Our findings highlight the potential of GCaMP6s-expressing HD10.6 cells as a high-throughput platform for studying nociception, neuronal signaling, host cell responses to viruses, and therapeutic interventions, bridging the gap between preclinical research and clinical applications.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115264"},"PeriodicalIF":1.6,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145045611","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":"Real-time detection of cucurbit chlorotic yellows virus by RT-LAMP","authors":"Ángela Martínez-Fernández ,&nbsp;Josemaría Delgado-Martín ,&nbsp;Esmeralda Sanahuja-Edo ,&nbsp;Ana Alfaro-Fernández ,&nbsp;José Manuel Estévez ,&nbsp;Isabel Font-San-Ambrosio ,&nbsp;Luis Galipienso ,&nbsp;Luis Rubio","doi":"10.1016/j.jviromet.2025.115262","DOIUrl":"10.1016/j.jviromet.2025.115262","url":null,"abstract":"<div><div>Cucurbit chlorotic yellows virus (CCYV) of the genus <em>Crinivirus</em> and family <em>Closteroviridae</em>, is an emerging infectious agent transmitted by whiteflies that mainly infects cucumber, melon and watermelon plants. Here, we developed a procedure based on reverse transcription (RT) followed by loop-mediated isothermal amplification (LAMP) to detect CCYV in real-time by using a fluorescent dye and incubating at 65 °C. This method detected CCYV in RNA extracts at about 10 min and in nylon membrane-filtered crude extracts between 55 and 95 min. The detection was sensitive, ten times higher than RT followed by polymerase chain reaction (PCR) and specifically detecting different isolates of CCYV without cross-reaction with other viruses of the same genus. This procedure enables the simultaneous analyses of multiple samples, allowing for rapid, sensitive and specific detection of CCYV. By using membrane-filtered crude extracts, RNA purification is unnecessary, so the whole process can be performed in the field with a portable heater and fluorometer, facilitating a rapid response for disease control.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115262"},"PeriodicalIF":1.6,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145040560","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":"Potential of recombinant avian adeno-associated virus as a viral vector for CRISPR/Cas9 delivery to avian cells","authors":"Takumi Terada ,&nbsp;Sodai Fujii ,&nbsp;Nanako Yamanishi ,&nbsp;Ryota Kajihara ,&nbsp;Tenkai Watanabe ,&nbsp;Ryo Ezaki ,&nbsp;Hiroyuki Horiuchi ,&nbsp;Mei Matsuzaki","doi":"10.1016/j.jviromet.2025.115263","DOIUrl":"10.1016/j.jviromet.2025.115263","url":null,"abstract":"<div><div>While genome editing has been established in chickens, where cultured primordial germ cell (PGC) systems are available, the implementation of genome editing remains a major challenge in many other birds due to the lack of robust PGC culture methods. Therefore, the development of reliable and efficient tools can significantly accelerate precision genome modification in avian species. Here, we evaluated the applicability of recombinant avian adeno-associated virus (rA3V) as a delivery vector for a CRISPR/Cas9 construct in avian cells using <em>Staphylococcus aureus</em>-derived Cas9 (SaCas9) and single-guide RNA (sgRNA). Infection with rA3V particles carrying an EGFP expression cassette (rA3V-EGFP) successfully induced EGFP expression in chicken fibroblasts (DF-1) cells, with approximately 80 % EGFP-positive cells at the maximum multiplicity of infection (MOI = 10,000). In plasmid-based transfection experiments, sgRNAs targeting the chicken <em>tyrosinase</em> locus and SaCas9 exhibited DNA cleavage activity in DF-1 cells. Furthermore, infection with rA3V particles encoding these CRISPR components successfully introduced indel mutations into the <em>tyrosinase</em> gene in DF-1 cells, with a calculated indel frequency of approximately 5.4 % at MOI = 40,000 without drug selection. Although EGFP expression was observed in quail fibrosarcoma cells, the percentage of EGFP-positive cells was much lower than that in DF-1 cells. In addition, <em>in vivo</em> infection with rA3V-EGFP of the chicken blastoderm failed to induce EGFP expression in germline cells, even at the highest applicable viral dose. In summary, rA3V can be used as a genome-editing vector in birds, although further investigation of its infectivity and tropism is necessary to expand its applicability to diverse avian species.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115263"},"PeriodicalIF":1.6,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145040574","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":"Curcumin inhibits HIV-1 by modulating FOXP3 and suppressing CCR5 via PI3K/AKT and JAK/STAT pathways","authors":"Mengyuan Shi ,&nbsp;Qing Li ,&nbsp;Wenjin Zheng ,&nbsp;Qingya Li ,&nbsp;Min Jiang ,&nbsp;Jingyi Zhang ,&nbsp;Zhe Wang ,&nbsp;Lu Qiao ,&nbsp;Long Feng","doi":"10.1016/j.jviromet.2025.115261","DOIUrl":"10.1016/j.jviromet.2025.115261","url":null,"abstract":"<div><div>Despite advances in antiretroviral therapy, HIV-1 persistence and immune dysregulation remain unresolved challenges. Here, we demonstrate that curcumin, a low-toxicity natural compound, can inhibit HIV-1 through simultaneous inhibition of the PI3K/AKT and JAK/STAT pathways, leading to downregulation of the viral co-receptor CCR5 and the immune checkpoint transcription factor FOXP3. Using CHIP and EMSA experiments, we found that curcumin disrupts the binding of FOXP3 to the CCR5 promoter, thereby reducing viral entry. Network pharmacology and molecular docking identified STAT3 and AKT1 as key targets. Most importantly, we found that crosstalk between the PI3K/AKT and JAK/STAT pathways is a pharmacological axis for HIV-1 treatment through high-throughput sequencing technology, mass spectrometry and CO-IP experiments. Our findings provide a mechanistic basis for the repurposing of curcumin as an adjuvant to HAART, with implications for therapies targeting viral reservoirs.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115261"},"PeriodicalIF":1.6,"publicationDate":"2025-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145019809","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":"Performance evaluation of a SYBR Green-based real-time quantitative PCR for SARS-CoV-2 detection from animal oropharyngeal samples","authors":"María Emilia Bravi ,&nbsp;Nadia Analía Fuentealba ,&nbsp;Natalia Brasso ,&nbsp;Guillermo Hernan Sguazza ,&nbsp;Marcelo Ricardo Pecoraro ,&nbsp;Carlos Javier Panei","doi":"10.1016/j.jviromet.2025.115259","DOIUrl":"10.1016/j.jviromet.2025.115259","url":null,"abstract":"<div><div>The global emergence of SARS-CoV-2 has highlighted the need for rapid, sensitive, and affordable diagnostic tools, not only for human health but also for animal surveillance within a One Health framework. This study aimed to evaluate the performance of a SYBR Green-based real-time quantitative PCR (qPCR) assay for the detection of SARS-CoV-2 from animal samples, focusing on domestic dogs and cats. A total of 140 oropharyngeal swab samples were collected and analyzed using primers targeting a 139-bp fragment of the <em>N</em> gene of SARS-CoV-2. The assay conditions were optimized through gradient PCR, primer concentration adjustment, and melting curve analysis. Cloning and quantification of the target gene allowed the determination of the limit of detection (LOD), which was estimated at 2.1 × 10² copies/µL. Among the samples tested, 13 were positive for SARS-CoV-2, confirmed by a commercial probe-based qPCR. The assay here evaluated demonstrated high specificity, with no cross-reactivity to canine or feline coronaviruses, and had a highly linear standard curve of 0.977 (R² = 0.997) with a value range of quantification cycle (Cq) from 9.25 to 34.89. In addition, it exhibited a 2-log increase in sensitivity compared to conventional PCR. The intra- and inter-assay coefficients of variation were below 1.1 % and 2 %, respectively, confirming high reproducibility. These results support the use of SYBR Green real-time qPCR as a cost-effective and reliable alternative for SARS-CoV-2 detection from animal samples, particularly in resource-limited settings, serving as a tool for epidemiological control of SARS-CoV-2 infection in animal populations.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115259"},"PeriodicalIF":1.6,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145015801","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":"Evaluation of two IgG-scFv bispecific antibodies for neutralizing Omicron variants of SARS-CoV-2","authors":"Diana Hinojosa-Trujillo ,&nbsp;Freddy Dehesa-Canseco ,&nbsp;Melissa García-Vega ,&nbsp;Verónica Mata-Haro ,&nbsp;Mario Solís-Hernández ,&nbsp;Mónica Reséndiz-Sandoval ,&nbsp;Fanglei Zuo ,&nbsp;Harold Marcotte ,&nbsp;Jesús Hernández","doi":"10.1016/j.jviromet.2025.115258","DOIUrl":"10.1016/j.jviromet.2025.115258","url":null,"abstract":"<div><div>Bispecific antibodies (bsAbs) offer an alternative to monoclonal antibody (mAb) cocktails for addressing the loss of efficacy due to the rapid emergence of SARS-CoV-2 mutants. The structure and specificity of the parental antibodies influence the development of a highly neutralizing bsAb. To design an effective bsAb, the recognition of 44 single-chain fragment variable (scFv) antibodies against variants of SARS-CoV-2 was evaluated, along with an assessment of their ability to competitively bind to the receptor-binding domain (RBD) compared to the most potent neutralizing mAbs. From this analysis, three antibodies − 19n01, 01n21, and 01n27 − were identified for their broad recognition and non-competitive binding behavior. These antibodies were selected as the parental antibodies for the design of two bsAb. The bsAb bis L and bis H were engineered as IgG-scFv constructs, each with the secondary domain oriented differently to introduce new specificities. Both bsAbs retained the neutralizing capabilities of their parental antibodies in live-virus assays, neutralizing the Omicron variants BQ.1.1 and XBB.1.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115258"},"PeriodicalIF":1.6,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145015731","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 dual-target real-time PCR for proactive detection of Mpox variants","authors":"Tracy D. Lee ,&nbsp;Alan O’Dwyer ,&nbsp;Michael Chan ,&nbsp;Branco Cheung ,&nbsp;Frankie Tsang ,&nbsp;John R. Tyson ,&nbsp;Kathleen L. Kolehmainen ,&nbsp;Natalie A. Prystajecky ,&nbsp;Agatha N. Jassem","doi":"10.1016/j.jviromet.2025.115260","DOIUrl":"10.1016/j.jviromet.2025.115260","url":null,"abstract":"<div><div>In 2022, cases of Monkeypox virus (MPXV) in California contained a mutation in the TNF receptor gene (GR2G) that rendered the virus undetectable using a widely adopted public health diagnostic qPCR assay. This underscored the need for a dual-target PCR approach and prompted validation of a second target by the BCCDC Public Health Laboratory. In addition to the GR2G target validated in the original qPCR assay (and duplexed with the endogenous target human β-globin (HBG)), GP113 (OPG128) was identified and validated using both clinical samples and MPXV DNA controls. Mutations in GR2G and GP113 (found in the emerging clade Ib) were also addressed along with the updated target. The new triplex assay (GR2G/GP113/HBG) had 100 % inclusivity and 100 % accuracy with all clinical samples tested and did not cross-react with herpes simplex virus-1 or −2, varicella zoster virus, or enterovirus. It showed &lt; 5 % coefficient of variance between replicates and had a limit-of-detection of 10 copies/μL for GR2G and GP113. The use of two targets presents redundancy against further mutations in MPXV and is recommended for use with all viral qPCR assays moving forward.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115260"},"PeriodicalIF":1.6,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145015721","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":"Cryopreservation of chicken and duck tracheal rings and precision-cut lung slices: A promising tool for the rapid characterization of avian influenza viruses","authors":"Alessandra Napolitan ,&nbsp;Elisa Mazzacan ,&nbsp;Niccolò Fonti ,&nbsp;Sofia Tomasoni ,&nbsp;Erica Melchiotti ,&nbsp;Claudia Zanardello ,&nbsp;Lucrezia Vianello ,&nbsp;Sami Ramzi ,&nbsp;Valentina Panzarin ,&nbsp;Marika Crimaudo ,&nbsp;Ranieri Verin ,&nbsp;Francesco Bonfante ,&nbsp;Eva Mazzetto","doi":"10.1016/j.jviromet.2025.115257","DOIUrl":"10.1016/j.jviromet.2025.115257","url":null,"abstract":"<div><div>Since its emergence in 1996, highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/96 lineage have diversified into multiple clades, culminating in the 2020–2021 global panzootic caused by H5N1 viruses of the clade 2.3.4.4b. Further reassortment events have significantly diversified the phenotypes of these viruses, underscoring the need for continuous monitoring and strain characterization to better adjust control measures and mitigate the impact of the disease in wild birds and poultry. Standardized, ready-to-use <em>ex vivo</em> tissue platforms for rapid phenotyping of avian influenza viruses (AIVs) offer a valid alternative to <em>in vivo</em> models that are financially, ethically and logistically demanding. We optimized explant production and cryopreservation protocols for chicken and duck tracheal organ cultures (cTOCs and dTOCs) and precision-cut lung slices (cPCLS and dPCLS), assessing post-thaw viability, histological integrity, and susceptibility to AIV infection. Trehalose supplementation of cryopreservation solutions based on dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) significantly improved tissue viability. Although cryopreserved tissues were less viable than the fresh explants, viral replication was similar and only a modest reduction in susceptibility to infection was observed. Finally, we used duck and chicken TOCs to assess the ability of cryopreserved explants to discriminate viruses based on their divergent fitness and host preference. These findings underscore the potential of cryopreserved TOCs and PCLS as additional tools for the phenotypic characterisation of emerging AIVs.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115257"},"PeriodicalIF":1.6,"publicationDate":"2025-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144997278","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}