Human Papillomavirus Virus-Like Particles (HPV-VLPs) are a highly effective vaccine to prevent cervical cancer. Current production and purification processes for HPV-VLPs suffer from poor yield and suboptimal process economics. The current study presents a purification strategy based multi-modal cation exchange chromatography (Capto™ MMC) for the purification of HPV-VLPs produced in Pichia pastoris. Single step purification of disassembled VLPs offered a superior product recovery (> 80 %) and purity (> 70 %) compared to traditional VLP purification platforms that comprise anion exchange and cation exchange chromatography (yield: 32 %, purity: 52 %). Furthermore, it was observed that disassembling the intact VLPs to capsomere subunits before purification provided an improved dynamic binding capacity of up to 18.1 mg/mL (at 2 min residence time), 4 times higher than that with intact HPV-VLPs.
{"title":"Disassembly mediated multimodal chromatography based purification of HPV-VLPs produced in Pichia pastoris","authors":"Rashmi Sharma , Pragya Prakash , Lukas Gerstweiler , Anurag S. Rathore","doi":"10.1016/j.jviromet.2025.115168","DOIUrl":"10.1016/j.jviromet.2025.115168","url":null,"abstract":"<div><div>Human Papillomavirus Virus-Like Particles (HPV-VLPs) are a highly effective vaccine to prevent cervical cancer. Current production and purification processes for HPV-VLPs suffer from poor yield and suboptimal process economics. The current study presents a purification strategy based multi-modal cation exchange chromatography (Capto™ MMC) for the purification of HPV-VLPs produced in <em>Pichia pastoris</em>. Single step purification of disassembled VLPs offered a superior product recovery (> 80 %) and purity (> 70 %) compared to traditional VLP purification platforms that comprise anion exchange and cation exchange chromatography (yield: 32 %, purity: 52 %). Furthermore, it was observed that disassembling the intact VLPs to capsomere subunits before purification provided an improved dynamic binding capacity of up to 18.1 mg/mL (at 2 min residence time), 4 times higher than that with intact HPV-VLPs.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"336 ","pages":"Article 115168"},"PeriodicalIF":2.2,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143847700","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 : 2025-04-14DOI: 10.1016/j.jviromet.2025.115166
Joh-Sin Wu , Ju-Ying Kan , Young-Sheng Chang , Uyen Nguyen Phuong Le , Wen-Chi Su , Hsueh-Chou Lai , Cheng-Wen Lin
To address the human ACE2 dependence for SARS-CoV-2 infection, this study presents a novel strategy for generating ZIKV-hACE2 single-round infectious particles (SRIPs) by incorporating the hACE2 gene into a Zika virus (ZIKV) mini-replicon. SARS-CoV-2 SRIP infection was significantly enhanced in HEK293T cells pre-infected with ZIKV-hACE2, as evidenced by increased cytopathic effects and elevated mRNA and protein levels of the SARS-CoV-2 nucleocapsid (N) protein. A mouse model was also developed with this approach to investigate SARS-CoV-2 infection. Immunohistochemical and real-time RT-PCR analyses confirmed the presence of the SARS-CoV-2 N protein in the lungs of mice injected with ZIKV-hACE2 SRIPs, indicating successful infection. The mouse model displayed COVID-19-like pathological changes, including increased macrophages in BALF, severe lung damage, and elevated pro-inflammatory cytokines (IL-6 and IL-1β). These features mimic severe COVID-19 cases in humans. Additionally, treatment with nirmatrelvir resulted in a 6.2-fold reduction in viral load and a marked decrease in N protein levels. Overall, this ZIKV mini-replicon-mediated hACE2 expression model, both in vitro and in vivo, is a valuable tool for studying SARS-CoV-2 infection and evaluating therapeutic interventions. The mouse model’s pathological features further underscore its relevance for in vivo research on SARS-CoV-2.
{"title":"Developing Zika virus-transduced hACE2 expression models for severe acute respiratory syndrome coronavirus 2 infection in vitro and in vivo","authors":"Joh-Sin Wu , Ju-Ying Kan , Young-Sheng Chang , Uyen Nguyen Phuong Le , Wen-Chi Su , Hsueh-Chou Lai , Cheng-Wen Lin","doi":"10.1016/j.jviromet.2025.115166","DOIUrl":"10.1016/j.jviromet.2025.115166","url":null,"abstract":"<div><div>To address the human ACE2 dependence for SARS-CoV-2 infection, this study presents a novel strategy for generating ZIKV-hACE2 single-round infectious particles (SRIPs) by incorporating the hACE2 gene into a Zika virus (ZIKV) mini-replicon. SARS-CoV-2 SRIP infection was significantly enhanced in HEK293T cells pre-infected with ZIKV-hACE2, as evidenced by increased cytopathic effects and elevated mRNA and protein levels of the SARS-CoV-2 nucleocapsid (N) protein. A mouse model was also developed with this approach to investigate SARS-CoV-2 infection. Immunohistochemical and real-time RT-PCR analyses confirmed the presence of the SARS-CoV-2 N protein in the lungs of mice injected with ZIKV-hACE2 SRIPs, indicating successful infection. The mouse model displayed COVID-19-like pathological changes, including increased macrophages in BALF, severe lung damage, and elevated pro-inflammatory cytokines (IL-6 and IL-1β). These features mimic severe COVID-19 cases in humans. Additionally, treatment with nirmatrelvir resulted in a 6.2-fold reduction in viral load and a marked decrease in N protein levels. Overall, this ZIKV mini-replicon-mediated hACE2 expression model, both in vitro and in vivo, is a valuable tool for studying SARS-CoV-2 infection and evaluating therapeutic interventions. The mouse model’s pathological features further underscore its relevance for in vivo research on SARS-CoV-2.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"336 ","pages":"Article 115166"},"PeriodicalIF":2.2,"publicationDate":"2025-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143828544","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}
This study focuses on three viruses affecting farmed shrimp, including White Spot Syndrome Virus (WSSV), Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), and Taura Syndrome Virus (TSV). Specific primers and probes were designed by their respective conserved gene fragments to establish a triple-RPA-LFS detection method that simultaneously detects WSSV, IHHNV, and TSV. Seven pathogens and healthy shrimp tissues were collected to conduct specificity tests. This method can specifically amplify the gene fragments of WSSV, IHHNV, and TSV, while no fragments were amplified from the muscle tissues of healthy shrimp or other pathogens, indicating strong specificity. The reaction system was optimized, and specificity and sensitivity were validated. Sensitivity tests were conducted using a continuous dilution plasmid method, determining that the detection sensitivity of this method is 101 copies/reaction. Compared with the sensitivity of the qPCR detection method recommended by the World Organization for Animal Health (WOAH, formerly OIE), the triple-RPA-LFS method established in this study is faster and simpler to operate. When applied to test 110 samples simultaneously with the laboratory standard testing method, the results of the qPCR detection matched the results of the laboratory standard method with a 100 % concordance rate. These experimental results indicate that the triple-RPA-LFS detection method established in this study has the characteristics of high specificity, high sensitivity, short detection time, and high accuracy. It can be used for rapid on-site detection and diagnosis of the three pathogens: WSSV, IHHNV, and TSV.
{"title":"Establishment of triple-RPA-LFS detection method for three common shrimp viruses","authors":"Quanling Mu, Cunbao Ding, Ying Xie, Xi Zhen, Jiaming Zhang, Yakun Yu","doi":"10.1016/j.jviromet.2025.115156","DOIUrl":"10.1016/j.jviromet.2025.115156","url":null,"abstract":"<div><div>This study focuses on three viruses affecting farmed shrimp, including <em>White Spot Syndrome Virus</em> (WSSV), <em>Infectious Hypodermal and Hematopoietic Necrosis Virus</em> (IHHNV), and <em>Taura Syndrome Virus</em> (TSV). Specific primers and probes were designed by their respective conserved gene fragments to establish a triple-RPA-LFS detection method that simultaneously detects WSSV, IHHNV, and TSV. Seven pathogens and healthy shrimp tissues were collected to conduct specificity tests. This method can specifically amplify the gene fragments of WSSV, IHHNV, and TSV, while no fragments were amplified from the muscle tissues of healthy shrimp or other pathogens, indicating strong specificity. The reaction system was optimized, and specificity and sensitivity were validated. Sensitivity tests were conducted using a continuous dilution plasmid method, determining that the detection sensitivity of this method is 10<sup>1</sup> copies/reaction. Compared with the sensitivity of the qPCR detection method recommended by the World Organization for Animal Health (WOAH, formerly OIE), the triple-RPA-LFS method established in this study is faster and simpler to operate. When applied to test 110 samples simultaneously with the laboratory standard testing method, the results of the qPCR detection matched the results of the laboratory standard method with a 100 % concordance rate. These experimental results indicate that the triple-RPA-LFS detection method established in this study has the characteristics of high specificity, high sensitivity, short detection time, and high accuracy. It can be used for rapid on-site detection and diagnosis of the three pathogens: WSSV, IHHNV, and TSV.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"336 ","pages":"Article 115156"},"PeriodicalIF":2.2,"publicationDate":"2025-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143852330","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 : 2025-04-10DOI: 10.1016/j.jviromet.2025.115165
Tomo Suzuki , Takehiro Ohki
Potatoes are susceptible to viral diseases, and widespread viral infections lead to reduced production yields. Potato leaf roll virus, potato virus S, potato virus X, and potato virus Y are important viruses predominant in potato fields and cause significant economic damage in Japan. In this study, to reduce the labor and cost of virus testing in seed certification program, a simple molecular diagnostic assay for these potato viruses was developed. This method consists of template preparation by heat-treating a mixture of leaf homogenate and a newly designed primer set, followed by one-step conventional multiplex RT-PCR (mRT-PCR). In particular, to simplify RNA extraction, a procedure was adopted in which leaf homogenates in PBST were diluted 100-fold in RNase-free water, heat-treated with primers for 5 min at 70°C and subjected to one-step mRT-PCR. Interestingly, heat treatment of a mixture of the homogenate and primers improved the detection sensitivity for the virus. This new method’s detection sensitivity of each virus was 10–100 times higher than that of ELISA using commercial kits and 1–10 times higher than that of one-step mRT-PCR with filter paper-based RNA extraction. The heat-treated homogenate with primers was also applicable for detecting eight other potato viruses by one-step conventional mRT-PCR and four potato viruses by real-time mRT-PCR which our research group previously developed. New direct RT-PCR method for potato viruses can be applied to seed certification programs, where a large number of samples need to be tested.
{"title":"A simple detection method for potato viruses combining template preparation by heat treatment of homogenate with primers and one-step multiplex RT-PCR","authors":"Tomo Suzuki , Takehiro Ohki","doi":"10.1016/j.jviromet.2025.115165","DOIUrl":"10.1016/j.jviromet.2025.115165","url":null,"abstract":"<div><div>Potatoes are susceptible to viral diseases, and widespread viral infections lead to reduced production yields. Potato leaf roll virus, potato virus S, potato virus X, and potato virus Y are important viruses predominant in potato fields and cause significant economic damage in Japan. In this study, to reduce the labor and cost of virus testing in seed certification program, a simple molecular diagnostic assay for these potato viruses was developed. This method consists of template preparation by heat-treating a mixture of leaf homogenate and a newly designed primer set, followed by one-step conventional multiplex RT-PCR (mRT-PCR). In particular, to simplify RNA extraction, a procedure was adopted in which leaf homogenates in PBST were diluted 100-fold in RNase-free water, heat-treated with primers for 5 min at 70°C and subjected to one-step mRT-PCR. Interestingly, heat treatment of a mixture of the homogenate and primers improved the detection sensitivity for the virus. This new method’s detection sensitivity of each virus was 10–100 times higher than that of ELISA using commercial kits and 1–10 times higher than that of one-step mRT-PCR with filter paper-based RNA extraction. The heat-treated homogenate with primers was also applicable for detecting eight other potato viruses by one-step conventional mRT-PCR and four potato viruses by real-time mRT-PCR which our research group previously developed. New direct RT-PCR method for potato viruses can be applied to seed certification programs, where a large number of samples need to be tested.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"336 ","pages":"Article 115165"},"PeriodicalIF":2.2,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143847699","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 “National Epidemiological Surveillance of Vaccine-Preventable Diseases” is conducted annually in Japan for various infectious diseases. A susceptibility survey for Japanese encephalitis was conducted by measuring antibody titers using a focus-forming assay. However, this method is complicated to operate and hard to count small focuses; therefore, the development of new measurement methods is required. In this study, we developed and evaluated a single-round infectious particles (SRIPs) assay method and a cytopathic effect (CPE) assay method. The SRIPs assay showed a strong correlation (R = 0.94) with the focus-forming assay. The SRIPs assay method is not only simple and can be expected to improve accuracy, but also has the advantages of extremely low pathogenicity and safety for the operator. The CPE assay also showed a strong correlation (R = 0.80) with the focus-forming assay. It has the advantage of requiring almost no reagents and is easy to interpret. Both SRIPs and CPE assay methods can be used as alternatives to the focus-forming assay method, but further characterization of each method is necessary before one is selected for routine use.
{"title":"Development of neutralization tests using single-round infectious particles and cytopathic effect as an alternative method for measuring antibody titers against Japanese encephalitis virus in national epidemiological surveillance program of vaccine-preventable diseases in Japan","authors":"Shunsuke Yazawa , Yumiko Saga , Mami Matsuda , Ryosuke Suzuki , Shigeru Tajima , Chang-Kweng Lim , Hideki Tani","doi":"10.1016/j.jviromet.2025.115163","DOIUrl":"10.1016/j.jviromet.2025.115163","url":null,"abstract":"<div><div>The “National Epidemiological Surveillance of Vaccine-Preventable Diseases” is conducted annually in Japan for various infectious diseases. A susceptibility survey for Japanese encephalitis was conducted by measuring antibody titers using a focus-forming assay. However, this method is complicated to operate and hard to count small focuses; therefore, the development of new measurement methods is required. In this study, we developed and evaluated a single-round infectious particles (SRIPs) assay method and a cytopathic effect (CPE) assay method. The SRIPs assay showed a strong correlation (R = 0.94) with the focus-forming assay. The SRIPs assay method is not only simple and can be expected to improve accuracy, but also has the advantages of extremely low pathogenicity and safety for the operator. The CPE assay also showed a strong correlation (R = 0.80) with the focus-forming assay. It has the advantage of requiring almost no reagents and is easy to interpret. Both SRIPs and CPE assay methods can be used as alternatives to the focus-forming assay method, but further characterization of each method is necessary before one is selected for routine use.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"336 ","pages":"Article 115163"},"PeriodicalIF":2.2,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143807649","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 : 2025-04-08DOI: 10.1016/j.jviromet.2025.115155
Tanner Grudda , Chenkai Jiang , Che-Min Lo , Nicole E. Skinner , Mark S. Sulkowski , Ashwin Balagopal , Chloe L. Thio
Hepatitis B virus (HBV) affects approximately 2 billion individuals; 254 million have chronic infection. The roles of spliced HBV (spHBV) RNAs in human infection are poorly understood partly due to limitations in quantitative methods. We designed and multiplexed long amplicon (0.3–2kbp) ddPCR assays that simultaneously quantify the most frequently observed spHBV isoforms (sp1, sp2, sp3, sp8, sp9) alongside unspliced pregenomic RNA (pgRNA). Our innovations include the use of ddPCR, long amplicons, and high-dimensional multiplexing. We tested primers and probes using qPCR on synthetic DNA targets to identify a high-fidelity combination recognizing HBV genotype A viruses. Using common forward and reverse primers we accounted for PCR efficiency differences between amplicons of differing size. We altered denaturation and annealing temperatures to enrich spHBV, the minority population of total HBV RNA. To achieve higher-order multiplexing, we increased the number of cycles to 80, added a post-reverse transcription cleanup step, and tested > 360 oligo concentration combinations. We performed spatial transcriptomic sequencing on liver tissue to confirm splice junctions measured by ddPCR. We successfully quantified spHBV and pgRNA in serum, liver, and HBV-infected HepG2NTCP cells. Our assay enables direct, sensitive quantification of spHBV and full-length pgRNA from serum, liver, and tissue culture samples, which will facilitate studies on the impact of spHBV on HBV infection outcomes in people. This technique has potential applications in HBV research and therapy development, with broader implications in virology and oncology.
{"title":"High-dimensional droplet digital PCR of multiple hepatitis B splice variants in serum, liver, and tissue culture","authors":"Tanner Grudda , Chenkai Jiang , Che-Min Lo , Nicole E. Skinner , Mark S. Sulkowski , Ashwin Balagopal , Chloe L. Thio","doi":"10.1016/j.jviromet.2025.115155","DOIUrl":"10.1016/j.jviromet.2025.115155","url":null,"abstract":"<div><div>Hepatitis B virus (HBV) affects approximately 2 billion individuals; 254 million have chronic infection. The roles of spliced HBV (spHBV) RNAs in human infection are poorly understood partly due to limitations in quantitative methods. We designed and multiplexed long amplicon (0.3–2kbp) ddPCR assays that simultaneously quantify the most frequently observed spHBV isoforms (sp1, sp2, sp3, sp8, sp9) alongside unspliced pregenomic RNA (pgRNA). Our innovations include the use of ddPCR, long amplicons, and high-dimensional multiplexing. We tested primers and probes using qPCR on synthetic DNA targets to identify a high-fidelity combination recognizing HBV genotype A viruses. Using common forward and reverse primers we accounted for PCR efficiency differences between amplicons of differing size. We altered denaturation and annealing temperatures to enrich spHBV, the minority population of total HBV RNA. To achieve higher-order multiplexing, we increased the number of cycles to 80, added a post-reverse transcription cleanup step, and tested > 360 oligo concentration combinations. We performed spatial transcriptomic sequencing on liver tissue to confirm splice junctions measured by ddPCR. We successfully quantified spHBV and pgRNA in serum, liver, and HBV-infected HepG2<sup>NTCP</sup> cells. Our assay enables direct, sensitive quantification of spHBV and full-length pgRNA from serum, liver, and tissue culture samples, which will facilitate studies on the impact of spHBV on HBV infection outcomes in people. This technique has potential applications in HBV research and therapy development, with broader implications in virology and oncology.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"336 ","pages":"Article 115155"},"PeriodicalIF":2.2,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143859103","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 : 2025-04-05DOI: 10.1016/j.jviromet.2025.115153
Nick Laurenz Kaiser, Martin H. Groschup, Balal Sadeghi
Nanopore sequencing has proven to be a promising technique in virus surveillance efforts, especially due to the portability of its sequencers. In order to process the long, error-prone reads generated, specialised bioinformatic programs are required. These can be run automatically within pipelines so as to effectively provide decision makers with all relevant information about the molecular characteristics of a virus. The purpose of this systematic assessment was to identify pipelines that are suitable for virus surveillance programs using nanopore sequencing. Promising candidates were then compared in terms of their functional scope. Of 239 initial papers, 22 pipelines were tested, of which six were included in the final assessment. The four pipelines that were exclusively available offline were each missing individual downstream analysis steps considered in our assessment. The other two executed all steps. One of these was only available online and subject to a charge, while the other was freely available both online and offline. While we were able to identify two pipelines that are broadly suitable for virus surveillance using nanopore sequencing, we discovered two major shortcomings in this domain. None of the pipelines integrated basecalling, the initial step of data processing. In addition, there was no pipeline that was easy to install and provided all relevant analysis results with a single program call. We therefore see a need for the development of a pipeline that incorporates both aspects.
{"title":"Identification of bioinformatic pipelines for virus monitoring using nanopore sequence data: A systematic assessment","authors":"Nick Laurenz Kaiser, Martin H. Groschup, Balal Sadeghi","doi":"10.1016/j.jviromet.2025.115153","DOIUrl":"10.1016/j.jviromet.2025.115153","url":null,"abstract":"<div><div>Nanopore sequencing has proven to be a promising technique in virus surveillance efforts, especially due to the portability of its sequencers. In order to process the long, error-prone reads generated, specialised bioinformatic programs are required. These can be run automatically within pipelines so as to effectively provide decision makers with all relevant information about the molecular characteristics of a virus. The purpose of this systematic assessment was to identify pipelines that are suitable for virus surveillance programs using nanopore sequencing. Promising candidates were then compared in terms of their functional scope. Of 239 initial papers, 22 pipelines were tested, of which six were included in the final assessment. The four pipelines that were exclusively available offline were each missing individual downstream analysis steps considered in our assessment. The other two executed all steps. One of these was only available online and subject to a charge, while the other was freely available both online and offline. While we were able to identify two pipelines that are broadly suitable for virus surveillance using nanopore sequencing, we discovered two major shortcomings in this domain. None of the pipelines integrated basecalling, the initial step of data processing. In addition, there was no pipeline that was easy to install and provided all relevant analysis results with a single program call. We therefore see a need for the development of a pipeline that incorporates both aspects.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"336 ","pages":"Article 115153"},"PeriodicalIF":2.2,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143803722","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}
Varicellovirus bovinealpha1 (BoAHV1) is an important causal agent of various pathological conditions, such as respiratory and reproductive diseases, in cattle. An intranasal vaccine containing a temperature-sensitive mutant RLB 106 strain is used to control BoAHV1-related diseases. To monitor the invasion of BoAHV1 wild-type viruses in vaccinated populations, it is essential to develop a simple method for differentiating between the RLB 106 strain and BoAHV1 wild-type viruses. In this study, we developed an allele-specific quantitative polymerase chain reaction (AS-qPCR) assay that targets the point mutation G23136A located in UL40 for detecting the RLB 106 strain. We calculated the difference in Cq values (ΔCq) obtained from a paired qPCR using each point mutation site-targeting primer set and established the cutoff ΔCq for differentiation. The accuracy of differentiation was confirmed by the DNA partial sequencing of UL40. Results demonstrated that differentiation by AS-qPCR was consistent with the results obtained by DNA sequencing, and the field isolates classified as the RLB 106 strain exhibited a temperature-sensitive phenotype. Hence, the AS-qPCR assay developed in this study could be a promising method for the simple differentiation between the RLB 106 strain and wild-type viruses in a single-step reaction.
{"title":"Development of an allele-specific quantitative polymerase chain reaction assay for differentiating the RLB 106 strain from the wild-type viruses of Varicellovirus bovinealpha1","authors":"Keigo Ikeda , Yuto Suda , Hisayuki Tomochi , Shinichi Hatama , Yoshifumi Iwamaru","doi":"10.1016/j.jviromet.2025.115148","DOIUrl":"10.1016/j.jviromet.2025.115148","url":null,"abstract":"<div><div><em>Varicellovirus bovinealpha1</em> (BoAHV1) is an important causal agent of various pathological conditions, such as respiratory and reproductive diseases, in cattle. An intranasal vaccine containing a temperature-sensitive mutant RLB 106 strain is used to control BoAHV1-related diseases. To monitor the invasion of BoAHV1 wild-type viruses in vaccinated populations, it is essential to develop a simple method for differentiating between the RLB 106 strain and BoAHV1 wild-type viruses. In this study, we developed an allele-specific quantitative polymerase chain reaction (AS-qPCR) assay that targets the point mutation G23136A located in UL40 for detecting the RLB 106 strain. We calculated the difference in Cq values (ΔCq) obtained from a paired qPCR using each point mutation site-targeting primer set and established the cutoff ΔCq for differentiation. The accuracy of differentiation was confirmed by the DNA partial sequencing of UL40. Results demonstrated that differentiation by AS-qPCR was consistent with the results obtained by DNA sequencing, and the field isolates classified as the RLB 106 strain exhibited a temperature-sensitive phenotype. Hence, the AS-qPCR assay developed in this study could be a promising method for the simple differentiation between the RLB 106 strain and wild-type viruses in a single-step reaction.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"336 ","pages":"Article 115148"},"PeriodicalIF":2.2,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143803721","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 : 2025-04-05DOI: 10.1016/j.jviromet.2025.115154
Sydney Merritt , Megan Halbrook , Jean Paul Kompany , Prabha Chandrasekaran , Olivia A. Smith , Nicole A. Hoff , Merly Tambu , Skylar A. Martin , Teri Ann Wong , Amie Jarra , Angelica L. Barrall , Kamy Musene , Michael Beya , Robert Orr , Todd Myers , Tracy MacGill , Lisa E. Hensley , Jean-Jacques Muyembe-Tamfum , Didine Kaba , Irina Maljkovic Berry , Anne W. Rimoin
Ebola virus (EBOV) is a highly infectious pathogen, and its long-term consequences continue to be investigated. With its high fatality rate and potential for reinfection or latent infection, continued development of research tools is of utmost importance. Using a cohort (n = 503) of existing bio-banked specimens from the Democratic Republic of the Congo (DRC) two EBOV glycoprotein (GP) immunoglobulin G (IgG) antibody-detection assays were compared: the gold-standard Filovirus Animal Non-Clinical Group (FANG) and a Multiplex bead-based Immunoassay (MIA) with seven pan-filoviral targets. As not all immunoassays have been shown to detect a vaccine-induced immune response, and previous EBOV serosurveillance has been primarily conducted with singleplex technology, this MIA was assessed as an additional resource. Among the cohort, as sample seroreactivity increased, assay correlation increased (r2=0.80). Correlation was sustained among sub-populations of the cohort—in detecting natural immunity among survivors and vaccine-derived responses. Additionally, when results were binarized by seroreactivity, there was high correlation between the two assays (kappa=0.70) with 71 serodiscordant samples. These data indicate that the MIA is an apt alternative to the singleplex FANG assay in detecting relative seroreactivity and can be used as a potential tool for widespread pan-filovirus serosurveillance in the DRC and similar contexts.
{"title":"Comparison of EBOV GP IgG antibody reactivity: Results from two immunoassays in the Democratic Republic of the Congo","authors":"Sydney Merritt , Megan Halbrook , Jean Paul Kompany , Prabha Chandrasekaran , Olivia A. Smith , Nicole A. Hoff , Merly Tambu , Skylar A. Martin , Teri Ann Wong , Amie Jarra , Angelica L. Barrall , Kamy Musene , Michael Beya , Robert Orr , Todd Myers , Tracy MacGill , Lisa E. Hensley , Jean-Jacques Muyembe-Tamfum , Didine Kaba , Irina Maljkovic Berry , Anne W. Rimoin","doi":"10.1016/j.jviromet.2025.115154","DOIUrl":"10.1016/j.jviromet.2025.115154","url":null,"abstract":"<div><div>Ebola virus (EBOV) is a highly infectious pathogen, and its long-term consequences continue to be investigated. With its high fatality rate and potential for reinfection or latent infection, continued development of research tools is of utmost importance. Using a cohort (n = 503) of existing bio-banked specimens from the Democratic Republic of the Congo (DRC) two EBOV glycoprotein (GP) immunoglobulin G (IgG) antibody-detection assays were compared: the gold-standard Filovirus Animal Non-Clinical Group (FANG) and a Multiplex bead-based Immunoassay (MIA) with seven pan-filoviral targets. As not all immunoassays have been shown to detect a vaccine-induced immune response, and previous EBOV serosurveillance has been primarily conducted with singleplex technology, this MIA was assessed as an additional resource. Among the cohort, as sample seroreactivity increased, assay correlation increased (r<sup>2</sup>=0.80). Correlation was sustained among sub-populations of the cohort—in detecting natural immunity among survivors and vaccine-derived responses. Additionally, when results were binarized by seroreactivity, there was high correlation between the two assays (kappa=0.70) with 71 serodiscordant samples. These data indicate that the MIA is an apt alternative to the singleplex FANG assay in detecting relative seroreactivity and can be used as a potential tool for widespread pan-filovirus serosurveillance in the DRC and similar contexts.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"336 ","pages":"Article 115154"},"PeriodicalIF":2.2,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143803720","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}
Reverse genetics (rg) systems are indispensable tools for investigating the pathogenesis of RNA viruses, facilitating vaccine design, and advancing antiviral therapeutic strategies. In this study, we optimized the Infectious Subgenomic Amplicons (ISA) method for generating synthetic r-wt SARS-CoV-2 Wuhan-Hu-1. This system was validated by demonstrating the successful rescue of infectious viral particles from overlapping DNA fragments and their propagation in vitro. Sequencing confirmed 100 % identity of the recovered virus with the Wuhan-Hu-1 reference genome. Importantly, in vivo experiments using K18-hACE2 mice revealed that the r-wt SARS-CoV-2 Wuhan-Hu-1 strain caused clinical symptoms, weight loss, and mortality comparable to those induced by a virulent SARS-CoV-2 field variant. This ISA rg method offers a rapid and reproducible approach to generating synthetic coronaviruses, with potential applications in pathogenesis studies, antiviral testing, and vaccine development.
{"title":"Optimization of an infectious subgenomic amplicons reverse genetics protocol for the rescue of synthetic coronaviruses","authors":"Ilaria Puglia , Marialuigia Caporale , Giovanni Di Teodoro , Massimo Spedicato , Francesca Profeta , Maurilia Marcacci , Chiara Di Pancrazio , Fabrizia Valleriani , Emanuela Rossi , Heidi Auerswald , Alessio Lorusso","doi":"10.1016/j.jviromet.2025.115152","DOIUrl":"10.1016/j.jviromet.2025.115152","url":null,"abstract":"<div><div>Reverse genetics (rg) systems are indispensable tools for investigating the pathogenesis of RNA viruses, facilitating vaccine design, and advancing antiviral therapeutic strategies. In this study, we optimized the Infectious Subgenomic Amplicons (ISA) method for generating synthetic r-wt SARS-CoV-2 Wuhan-Hu-1. This system was validated by demonstrating the successful rescue of infectious viral particles from overlapping DNA fragments and their propagation <em>in vitro</em>. Sequencing confirmed 100 % identity of the recovered virus with the Wuhan-Hu-1 reference genome. Importantly, <em>in vivo</em> experiments using K18-hACE2 mice revealed that the r-wt SARS-CoV-2 Wuhan-Hu-1 strain caused clinical symptoms, weight loss, and mortality comparable to those induced by a virulent SARS-CoV-2 field variant. This ISA rg method offers a rapid and reproducible approach to generating synthetic coronaviruses, with potential applications in pathogenesis studies, antiviral testing, and vaccine development.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"336 ","pages":"Article 115152"},"PeriodicalIF":2.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143795729","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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