Pub Date : 2024-05-23DOI: 10.1016/j.jviromet.2024.114959
Rajamanickam Hema Sayee, Madhusudan Hosamani , Narayanan Krishnaswamy, Subramaniyan Shanmuganathan, S.R. Nagasupreeta, Manchikanti Sri Sai Charan, Ganesh Sheshagiri, Vivek Gairola, Suresh H. Basagoudanavar , B.P. Sreenivasa , Veerakyathappa Bhanuprakash
In Foot-and-mouth disease (FMD) enzootic countries, periodic vaccination is the key tool in controlling the disease incidence. Active seromonitoring of the vaccinated population is critical to assess the impact of vaccination. Virus neutralization test (VNT) and enzyme-linked immunosorbent assays (ELISA) are commonly used for antibody detection. Assays like liquid phase blocking ELISA (LPBE) or solid phase competition ELISA (SPCE) are preferred as they do not require handling of live FMDV and are routinely used for seromonitoring or for vaccine potency testing; however, false positives are high in LPBE. Here we report, a monoclonal antibody (mAb) based SPCE as a potential alternate assay for antibody titration. From a panel of 12 mAbs against FMDV serotype A, two mAbs were chosen for the development of SPCE. Based on a set of 453 sera, it was demonstrated that mAb 2C4G11, mAb 6E8D11and polyclonal antibody (pAb) based SPCE had a relative sensitivity of 86.1, 86.1 and 80.3 %; and specificity of 99.6, 99.1 and 99.1 %, respectively. The correlation, repeatability, and level of agreement of the assays were high demonstrating the potential use of mAb in large scale surveillance studies and regular vaccine potency testing.
在口蹄疫(FMD)流行的国家,定期接种疫苗是控制疾病发病率的关键手段。对接种疫苗的人群进行积极的血清监测对于评估疫苗接种的效果至关重要。病毒中和试验(VNT)和酶联免疫吸附试验(ELISA)常用于抗体检测。液相阻断酶联免疫吸附试验(LPBE)或固相竞争酶联免疫吸附试验(SPCE)是首选的检测方法,因为它们不需要处理口蹄疫病毒,可常规用于血清监测或疫苗效力测试;但是,LPBE 的假阳性率很高。在此,我们报告了一种基于单克隆抗体(mAb)的 SPCE,作为抗体滴定的潜在替代检测方法。从 12 种针对 FMDV 血清型 A 的 mAb 中,我们选择了两种 mAb 用于 SPCE 的开发。结果表明,基于 mAb 2C4G11、mAb 6E8D11 和 pAb 的 SPCE 对 453 份血清的相对灵敏度分别为 86.1%、86.1% 和 80.3%,特异性分别为 99.6%、99.1% 和 99.1%。上述检测方法的相关性、可重复性和一致程度都很高,这表明 mAb 可用于大规模监测研究和常规疫苗效力检测。
{"title":"Monoclonal antibody based solid phase competition ELISA to detect FMDV serotype A specific antibodies","authors":"Rajamanickam Hema Sayee, Madhusudan Hosamani , Narayanan Krishnaswamy, Subramaniyan Shanmuganathan, S.R. Nagasupreeta, Manchikanti Sri Sai Charan, Ganesh Sheshagiri, Vivek Gairola, Suresh H. Basagoudanavar , B.P. Sreenivasa , Veerakyathappa Bhanuprakash","doi":"10.1016/j.jviromet.2024.114959","DOIUrl":"10.1016/j.jviromet.2024.114959","url":null,"abstract":"<div><p>In Foot-and-mouth disease (FMD) enzootic countries, periodic vaccination is the key tool in controlling the disease incidence. Active seromonitoring of the vaccinated population is critical to assess the impact of vaccination. Virus neutralization test (VNT) and enzyme-linked immunosorbent assays (ELISA) are commonly used for antibody detection. Assays like liquid phase blocking ELISA (LPBE) or solid phase competition ELISA (SPCE) are preferred as they do not require handling of live FMDV and are routinely used for seromonitoring or for vaccine potency testing; however, false positives are high in LPBE. Here we report, a monoclonal antibody (mAb) based SPCE as a potential alternate assay for antibody titration. From a panel of 12 mAbs against FMDV serotype A, two mAbs were chosen for the development of SPCE. Based on a set of 453 sera, it was demonstrated that mAb 2C4G11, mAb 6E8D11and polyclonal antibody (pAb) based SPCE had a relative sensitivity of 86.1, 86.1 and 80.3 %; and specificity of 99.6, 99.1 and 99.1 %, respectively. The correlation, repeatability, and level of agreement of the assays were high demonstrating the potential use of mAb in large scale surveillance studies and regular vaccine potency testing.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141093770","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 : 2024-05-23DOI: 10.1016/j.jviromet.2024.114968
Tran Thuy Vi , Duong Thi Hue Kien , Vo Thi Long , Le Thi Dui , Vu Thi Tuyet Nhu , Nguyen Thi Giang , Huynh Thi Xuan Trang , Sophie Yacoub , Cameron P. Simmons
Dengue fever, a mosquito-borne viral disease of significant public health concern in tropical and subtropical regions, is caused by any of the four serotypes of the dengue virus (DENV1–4). Cutting-edge technologies like next-generation sequencing (NGS) are revolutionizing virology, enabling in-depth exploration of DENV's genetic diversity. Here, we present an optimized workflow for full-genome sequencing of DENV 1–4 utilizing tiled amplicon multiplex PCR and Illumina sequencing. Our assay, sequenced on the Illumina MiSeq platform, demonstrates its ability to recover the full-length dengue genome across various viral abundances in clinical specimens with high-quality base coverage. This high quality underscores its suitability for precise examination of intra-host diversity, enriching our understanding of viral evolution and holding potential for improved diagnostic and intervention strategies in regions facing dengue outbreaks.
{"title":"A serotype-specific and tiled amplicon multiplex PCR method for whole genome sequencing of dengue virus","authors":"Tran Thuy Vi , Duong Thi Hue Kien , Vo Thi Long , Le Thi Dui , Vu Thi Tuyet Nhu , Nguyen Thi Giang , Huynh Thi Xuan Trang , Sophie Yacoub , Cameron P. Simmons","doi":"10.1016/j.jviromet.2024.114968","DOIUrl":"10.1016/j.jviromet.2024.114968","url":null,"abstract":"<div><p>Dengue fever, a mosquito-borne viral disease of significant public health concern in tropical and subtropical regions, is caused by any of the four serotypes of the dengue virus (DENV1–4). Cutting-edge technologies like next-generation sequencing (NGS) are revolutionizing virology, enabling in-depth exploration of DENV's genetic diversity. Here, we present an optimized workflow for full-genome sequencing of DENV 1–4 utilizing tiled amplicon multiplex PCR and Illumina sequencing. Our assay, sequenced on the Illumina MiSeq platform, demonstrates its ability to recover the full-length dengue genome across various viral abundances in clinical specimens with high-quality base coverage. This high quality underscores its suitability for precise examination of intra-host diversity, enriching our understanding of viral evolution and holding potential for improved diagnostic and intervention strategies in regions facing dengue outbreaks.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141134998","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 : 2024-05-23DOI: 10.1016/j.jviromet.2024.114956
Fabio Morecchiato , Marco Coppi , Claudia Niccolai , Alberto Antonelli , Leandro Di Gloria , Piergiuseppe Calà , Fabrizio Mancuso , Matteo Ramazzotti , Tommaso Lotti , Claudio Lubello , Gian Maria Rossolini
Wastewater-based epidemiology has proved to be a suitable approach for tracking the spread of epidemic agents including SARS-CoV-2 RNA. Different protocols have been developed for quantitative detection of SARS-CoV-2 RNA from wastewater samples, but little is known on their performance. In this study we compared three protocols based on Reverse Transcription Real Time-PCR (RT-PCR) and one based on Droplet Digital PCR (ddPCR) for SARS-CoV-2 RNA detection from 35 wastewater samples. Overall, SARS-CoV-2 RNA was detected by at least one method in 85.7 % of samples, while 51.4 %, 22.8 % and 8.6 % resulted positive with two, three or all four methods, respectively. Protocols based on commercial RT-PCR assays and on Droplet Digital PCR showed an overall higher sensitivity vs. an in-house assay. The use of more than one system, targeting different genes, could be helpful to increase detection sensitivity.
{"title":"Evaluation of different molecular systems for detection and quantification of SARS-CoV-2 RNA from wastewater samples","authors":"Fabio Morecchiato , Marco Coppi , Claudia Niccolai , Alberto Antonelli , Leandro Di Gloria , Piergiuseppe Calà , Fabrizio Mancuso , Matteo Ramazzotti , Tommaso Lotti , Claudio Lubello , Gian Maria Rossolini","doi":"10.1016/j.jviromet.2024.114956","DOIUrl":"10.1016/j.jviromet.2024.114956","url":null,"abstract":"<div><p>Wastewater-based epidemiology has proved to be a suitable approach for tracking the spread of epidemic agents including SARS-CoV-2 RNA. Different protocols have been developed for quantitative detection of SARS-CoV-2 RNA from wastewater samples, but little is known on their performance. In this study we compared three protocols based on Reverse Transcription Real Time-PCR (RT-PCR) and one based on Droplet Digital PCR (ddPCR) for SARS-CoV-2 RNA detection from 35 wastewater samples. Overall, SARS-CoV-2 RNA was detected by at least one method in 85.7 % of samples, while 51.4 %, 22.8 % and 8.6 % resulted positive with two, three or all four methods, respectively. Protocols based on commercial RT-PCR assays and on Droplet Digital PCR showed an overall higher sensitivity <em>vs</em>. an in-house assay. The use of more than one system, targeting different genes, could be helpful to increase detection sensitivity.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0166093424000806/pdfft?md5=36ee29fc35221b9443cef9b1be726584&pid=1-s2.0-S0166093424000806-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141138653","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-22DOI: 10.1016/j.jviromet.2024.114957
Zhangling Fan , Yu Xie , Baoying Huang , Fei Zhao , Yamei Hu , Yu Huang , Shan Mei , Liang Wei , Liming Wang , Lingwa Wang , Zhao Gao , Bin Ai , Jugao Fang , Chen Liang , Fengwen Xu , Wenjie Tan , Fei Guo
Since May 2022, the multi-country outbreak of monkeypox (mpox) has raised a great concern worldwide. Early detection of mpox virus infection is recognized as an efficient way to prevent mpox transmission. Mpox specific detection methods reported up to now are based on the SNPs among mpox virus and other orthopoxviruses. We have therefore developed a real-time PCR based mpox detection method targeting mpox virus specific sequences (N3R and B18Rplus). We have also optimized an orthopoxvirus detection system which targets the highly conserved E9L and D6R genes. The mpox and orthopoxvirus real-time PCR assays have a high sensitivity (1 copy/reaction) and specificity. Mpox viral DNA and clinical samples from mpox patients are detected with the mpox detection system. Furthermore, we have established a multiplex real-time PCR detection system allowing simultaneous and efficient detection of mpox and orthopoxvirus infections.
{"title":"Development of a multiplex real-time PCR assay for the simultaneous detection of mpox virus and orthopoxvirus infections","authors":"Zhangling Fan , Yu Xie , Baoying Huang , Fei Zhao , Yamei Hu , Yu Huang , Shan Mei , Liang Wei , Liming Wang , Lingwa Wang , Zhao Gao , Bin Ai , Jugao Fang , Chen Liang , Fengwen Xu , Wenjie Tan , Fei Guo","doi":"10.1016/j.jviromet.2024.114957","DOIUrl":"10.1016/j.jviromet.2024.114957","url":null,"abstract":"<div><p>Since May 2022, the multi-country outbreak of monkeypox (mpox) has raised a great concern worldwide. Early detection of mpox virus infection is recognized as an efficient way to prevent mpox transmission. Mpox specific detection methods reported up to now are based on the SNPs among mpox virus and other orthopoxviruses. We have therefore developed a real-time PCR based mpox detection method targeting mpox virus specific sequences (N3R and B18Rplus). We have also optimized an orthopoxvirus detection system which targets the highly conserved E9L and D6R genes. The mpox and orthopoxvirus real-time PCR assays have a high sensitivity (1 copy/reaction) and specificity. Mpox viral DNA and clinical samples from mpox patients are detected with the mpox detection system. Furthermore, we have established a multiplex real-time PCR detection system allowing simultaneous and efficient detection of mpox and orthopoxvirus infections.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141093766","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 : 2024-05-18DOI: 10.1016/j.jviromet.2024.114955
Taoni Zhang , Jinwen Tang , Yu Zhang , Yinghao Jin , Zixue Lin , Jiming Chen , Jianni Huang , Meilan Mo
Infectious bronchitis (IB) is an acute, highly contagious contact respiratory disease of chickens caused by infectious bronchitis virus (IBV). IBV is very prone to mutation, which brings great difficulties to the prevention and control of the disease. Therefore, there is a pressing need for a method that is fast, sensitive, specific, and convenient for detecting IBV. In this study, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method was established. Primers and probe were designed based on the conserved regions of the IBV M gene and the reaction concentrations were optimized, then the specificity, sensitivity, and reproducibility of this assay were tested. The results showed that the RF-RAA method could be completed at 39℃ within 20 min, during which the results could be interpreted visually in real-time. The RF-RAA method had good specificity, no cross-reaction with common poultry pathogens, and it detected a minimum concentration of template of 2 copies/μL for IBV. Besides, its reproducibility was stable. A total of 144 clinical samples were tested by RF-RAA and real-time quantitative PCR (qPCR), 132 samples of which were positive and 12 samples were negative, and the coincidence rate of the two methods was 100 %. In conclusion, the developed RF-RAA detection method is rapid, specific, sensitive, reproducible, and convenient, which can be utilized for laboratory detection and clinical diagnosis of IBV.
{"title":"Establishment of a rapid real-time fluorescence-based recombinase-aided amplification method for detection of avian infectious bronchitis virus","authors":"Taoni Zhang , Jinwen Tang , Yu Zhang , Yinghao Jin , Zixue Lin , Jiming Chen , Jianni Huang , Meilan Mo","doi":"10.1016/j.jviromet.2024.114955","DOIUrl":"10.1016/j.jviromet.2024.114955","url":null,"abstract":"<div><p>Infectious bronchitis (IB) is an acute, highly contagious contact respiratory disease of chickens caused by infectious bronchitis virus (IBV). IBV is very prone to mutation, which brings great difficulties to the prevention and control of the disease. Therefore, there is a pressing need for a method that is fast, sensitive, specific, and convenient for detecting IBV. In this study, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method was established. Primers and probe were designed based on the conserved regions of the IBV M gene and the reaction concentrations were optimized, then the specificity, sensitivity, and reproducibility of this assay were tested. The results showed that the RF-RAA method could be completed at 39℃ within 20 min, during which the results could be interpreted visually in real-time. The RF-RAA method had good specificity, no cross-reaction with common poultry pathogens, and it detected a minimum concentration of template of 2 copies/μL for IBV. Besides, its reproducibility was stable. A total of 144 clinical samples were tested by RF-RAA and real-time quantitative PCR (qPCR), 132 samples of which were positive and 12 samples were negative, and the coincidence rate of the two methods was 100 %. In conclusion, the developed RF-RAA detection method is rapid, specific, sensitive, reproducible, and convenient, which can be utilized for laboratory detection and clinical diagnosis of IBV.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141070924","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 : 2024-05-17DOI: 10.1016/j.jviromet.2024.114954
Lele Xu , Zhihao Chen , Haoyang Gong , Xiuxiu Pei , Yiyao Zhu , Yuchen Lu , Yumiao Wang , Shifa Nan , Yupeng Yin , Qin Zhao , Yunpeng Fan , Yani Sun , Shuqi Xiao
Porcine circovirus type 2 (PCV2) is intensely prevalent in global pig farms. The PCV2 vaccine is an important means of preventing and controlling PCV2. The quality control of PCV2 vaccines is predominantly based on detection techniques such as animal testing and neutralizing antibody titration. Measuring the content of effective proteins in vaccines to measure vaccine efficacy is an excellent alternative to traditional methods, which can greatly accelerate the development speed and testing time of vaccines. In this study, we screened a monoclonal antibody (mAb) that can effectively recognize not only the exogenous expression of PCV2 Cap protein but also PCV2 virus. The double antibody sandwich ELISA (DAS-ELISA) was developed using this mAb that specifically recognize PCV2 Cap. The minimum protein content detected by this method is 3.5 ng/mL. This method can be used for the quality control of PCV2 inactivated vaccine and subunit vaccine, and the detection results are consistent with the results of mice animal experiments. This method has the advantages of simple operation, good sensitivity, high specificity and wide application. It can detect the effective antigen Cap protein content of various types of PCV2 vaccines, which not only shorten the vaccine inspection time but also save costs.
猪圆环病毒 2 型 (PCV2) 在全球养猪场的发病率很高。PCV2 疫苗是预防和控制 PCV2 的重要手段。PCV2 疫苗的质量控制主要基于动物试验和中和抗体滴定等检测技术。测定疫苗中有效蛋白的含量来衡量疫苗的效力是传统方法的一种很好的替代方法,可以大大加快疫苗的研发速度和检测时间。本研究筛选了一种不仅能有效识别外源表达的 PCV2 Cap 蛋白,而且能有效识别 PCV2 病毒的单克隆抗体(mAb)。利用这种能特异性识别 PCV2 Cap 的 mAb 开发了双抗体夹心 ELISA(DAS-ELISA)。该方法检测到的最低蛋白质含量为 3.5 纳克/毫升。该方法可用于 PCV2 灭活疫苗和亚单位疫苗的质量控制,检测结果与小鼠动物实验结果一致。该方法具有操作简单、灵敏度高、特异性强、适用范围广等优点。它可以检测各类 PCV2 疫苗的有效抗原帽蛋白含量,不仅缩短了疫苗检验时间,而且节约了成本。
{"title":"Development a high-sensitivity sandwich ELISA for determining antigen content of porcine circovirus type 2 vaccines","authors":"Lele Xu , Zhihao Chen , Haoyang Gong , Xiuxiu Pei , Yiyao Zhu , Yuchen Lu , Yumiao Wang , Shifa Nan , Yupeng Yin , Qin Zhao , Yunpeng Fan , Yani Sun , Shuqi Xiao","doi":"10.1016/j.jviromet.2024.114954","DOIUrl":"10.1016/j.jviromet.2024.114954","url":null,"abstract":"<div><p>Porcine circovirus type 2 (PCV2) is intensely prevalent in global pig farms. The PCV2 vaccine is an important means of preventing and controlling PCV2. The quality control of PCV2 vaccines is predominantly based on detection techniques such as animal testing and neutralizing antibody titration. Measuring the content of effective proteins in vaccines to measure vaccine efficacy is an excellent alternative to traditional methods, which can greatly accelerate the development speed and testing time of vaccines. In this study, we screened a monoclonal antibody (mAb) that can effectively recognize not only the exogenous expression of PCV2 Cap protein but also PCV2 virus. The double antibody sandwich ELISA (DAS-ELISA) was developed using this mAb that specifically recognize PCV2 Cap. The minimum protein content detected by this method is 3.5 ng/mL. This method can be used for the quality control of PCV2 inactivated vaccine and subunit vaccine, and the detection results are consistent with the results of mice animal experiments. This method has the advantages of simple operation, good sensitivity, high specificity and wide application. It can detect the effective antigen Cap protein content of various types of PCV2 vaccines, which not only shorten the vaccine inspection time but also save costs.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141041723","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 : 2024-05-15DOI: 10.1016/j.jviromet.2024.114953
Marley E. Iredale , Galen Cobb , Emily D. Vu , Saptarshi Ghosh , James D. Ellis , Bryony C. Bonning
Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.
{"title":"Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses","authors":"Marley E. Iredale , Galen Cobb , Emily D. Vu , Saptarshi Ghosh , James D. Ellis , Bryony C. Bonning","doi":"10.1016/j.jviromet.2024.114953","DOIUrl":"10.1016/j.jviromet.2024.114953","url":null,"abstract":"<div><p>Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (<em>Apis mellifera</em>) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0166093424000776/pdfft?md5=7988a29a4e469e47a23d51d6d33d6137&pid=1-s2.0-S0166093424000776-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140958439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-14DOI: 10.1016/j.jviromet.2024.114952
Samy Kasem , Ahmed S. Abdel-Moneim , Hideto Fukushi
Primary cell cultures derived from human embryo lung play a crucial role in virology by aiding virus propagation and vaccine development. These cultures exhibit a notable ability to undergo multiple subcultures, often reaching up to 70 passages. However, finding alternative primary cell cultures with similar longevity and usefulness is challenging. In this study, we introduce a novel primary culture cells derived from equine embryo brain (FEB), which cells exhibited remarkable long-term cultivation potential. The FEB was established and maintained using Sumitomo Nerve-Cell Culture System Comparison studies were conducted with fetal equine kidney cell line (FEK-Tc13) to assess growth rates and subculture longevity. Immunological characterization was performed using neuronal markers to confirm the neural nature of FEB cells. Viral growth assessments were conducted using equine herpesviruses (EHV-1 and EHV-4) to evaluate infectivity and cytopathic effects in FEB cells. PCR analysis and real-time PCR assays were employed to detect viral genomic DNA and transcription activity of EHVs in infected FEB cells. FEB cells demonstrated faster growth rates compared to fetal equine kidney cell line (FEK-Tc13 cells) and exhibited sustained subculture capability exceeding 50 passages. Immunostaining confirmed the glial identity of FEB cells. Both equine herpesviruses 1 and 4 EHV-1 and EHV-4 viruses efficiently replicated in FEB cells, resulting in clear cytopathic effects. PCR analysis detected genomic DNA of EHVs in infected FEB cells, indicating successful viral infection. The establishment of FEB cells with extended subculture capability highlights their potential utility as a model system for studying neural cell biology and viral infections.
源自人类胚肺的原代细胞培养物在病毒学中发挥着至关重要的作用,有助于病毒繁殖和疫苗研发。这些培养物具有显著的多次重复培养能力,通常可培养多达 70 次。然而,寻找具有类似寿命和实用性的替代原代细胞培养物是一项挑战。在这项研究中,我们介绍了一种新型原代培养细胞,这种细胞来源于马胚胎脑(FEB),具有显著的长期培养潜力。我们使用住友神经细胞培养系统建立并维持了 FEB,并与胎儿马肾细胞系(FEK-Tc13)进行了比较研究,以评估其生长速度和亚培养寿命。使用神经元标记物进行了免疫学鉴定,以确认 FEB 细胞的神经性质。利用马疱疹病毒(EHV-1 和 EHV-4)进行了病毒生长评估,以评估 FEB 细胞的感染性和细胞病理效应。利用 PCR 分析和实时 PCR 检测法检测病毒基因组 DNA 和受感染 FEB 细胞中 EHV 的转录活性。与胎儿马肾细胞系(FEK-Tc13细胞)相比,FEB细胞的生长速度更快,并表现出超过50次传代的持续亚培养能力。免疫染色证实了 FEB 细胞的神经胶质特性。马疱疹病毒1和4 EHV-1和EHV-4病毒都能在FEB细胞中有效复制,产生明显的细胞病理效应。PCR 分析在感染的 FEB 细胞中检测到了 EHV 的基因组 DNA,表明病毒感染成功。建立具有扩展亚培养能力的 FEB 细胞突显了其作为研究神经细胞生物学和病毒感染模型系统的潜在用途。
{"title":"Establishment of a new equine embryo brain primary cell culture with long-term expansion","authors":"Samy Kasem , Ahmed S. Abdel-Moneim , Hideto Fukushi","doi":"10.1016/j.jviromet.2024.114952","DOIUrl":"10.1016/j.jviromet.2024.114952","url":null,"abstract":"<div><p>Primary cell cultures derived from human embryo lung play a crucial role in virology by aiding virus propagation and vaccine development. These cultures exhibit a notable ability to undergo multiple subcultures, often reaching up to 70 passages. However, finding alternative primary cell cultures with similar longevity and usefulness is challenging. In this study, we introduce a novel primary culture cells derived from equine embryo brain (FEB), which cells exhibited remarkable long-term cultivation potential. The FEB was established and maintained using Sumitomo Nerve-Cell Culture System Comparison studies were conducted with fetal equine kidney cell line (FEK-Tc13) to assess growth rates and subculture longevity. Immunological characterization was performed using neuronal markers to confirm the neural nature of FEB cells. Viral growth assessments were conducted using equine herpesviruses (EHV-1 and EHV-4) to evaluate infectivity and cytopathic effects in FEB cells. PCR analysis and real-time PCR assays were employed to detect viral genomic DNA and transcription activity of EHVs in infected FEB cells. FEB cells demonstrated faster growth rates compared to fetal equine kidney cell line (FEK-Tc13 cells) and exhibited sustained subculture capability exceeding 50 passages. Immunostaining confirmed the glial identity of FEB cells. Both equine herpesviruses 1 and 4 EHV-1 and EHV-4 viruses efficiently replicated in FEB cells, resulting in clear cytopathic effects. PCR analysis detected genomic DNA of EHVs in infected FEB cells, indicating successful viral infection. The establishment of FEB cells with extended subculture capability highlights their potential utility as a model system for studying neural cell biology and viral infections.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140958440","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}
Bacteriophages are viruses that infect bacteria. Researchers use different methods to study the characteristics of bacteriophages. Transmission electron microscope (TEM) is considered the best method to analyze these characteristics. However, the quality of TEM micrographs is significantly influenced by the preparation methods used to prepare the bacteriophages sample. In this study, researchers compared two different methods for preparing the bacteriophage samples. In one method was used SM buffer, while in the other used deionized water. The results were analyzed by TEM and compared with each other. Additionally, the viability of bacteriophage in deionized water and SM buffer at 4°C was determined through plaque assay within 72 hours. TEM micrographs showed that the quality of bacteriophage sample prepared with deionized water is superior to those prepared with SM buffer. Furthermore, the titer of the bacteriophages did not show a significant reduction during 72 hours in both SM and deionized water. In conclusion, the results suggested that preparation method can significantly impact the quality of TEM micrographs. Using sterile deionized water for the preparation of bacteriophages is a simple way to improve the quality of TEM micrographs and it is advisable to send the samples to the laboratory within 72 hours.
噬菌体是感染细菌的病毒。研究人员使用不同的方法来研究噬菌体的特征。透射电子显微镜(TEM)被认为是分析这些特征的最佳方法。然而,噬菌体样本的制备方法对 TEM 显微照片的质量有很大影响。在这项研究中,研究人员比较了两种不同的噬菌体样本制备方法。一种方法使用 SM 缓冲液,另一种方法使用去离子水。研究结果通过 TEM 进行分析并相互比较。此外,还通过斑块检测法测定了去离子水和 SM 缓冲液中的噬菌体在 4°C 温度下 72 小时内的存活率。TEM 显微照片显示,用去离子水制备的噬菌体样品质量优于用 SM 缓冲液制备的样品。此外,在 SM 和去离子水中 72 小时内,噬菌体的滴度都没有明显下降。总之,研究结果表明,制备方法会对 TEM 显微图像的质量产生重大影响。使用无菌去离子水制备噬菌体是提高 TEM 显微图片质量的一个简单方法,建议在 72 小时内将样品送至实验室。
{"title":"A simplified method of bacteriophage preparation for transmission electron microscope","authors":"Sepideh Meidaninikjeh , Parisa Mohammadi , Ameneh Elikaei","doi":"10.1016/j.jviromet.2024.114951","DOIUrl":"10.1016/j.jviromet.2024.114951","url":null,"abstract":"<div><p>Bacteriophages are viruses that infect bacteria. Researchers use different methods to study the characteristics of bacteriophages. Transmission electron microscope (TEM) is considered the best method to analyze these characteristics. However, the quality of TEM micrographs is significantly influenced by the preparation methods used to prepare the bacteriophages sample. In this study, researchers compared two different methods for preparing the bacteriophage samples. In one method was used SM buffer, while in the other used deionized water. The results were analyzed by TEM and compared with each other. Additionally, the viability of bacteriophage in deionized water and SM buffer at 4°C was determined through plaque assay within 72 hours. TEM micrographs showed that the quality of bacteriophage sample prepared with deionized water is superior to those prepared with SM buffer. Furthermore, the titer of the bacteriophages did not show a significant reduction during 72 hours in both SM and deionized water. In conclusion, the results suggested that preparation method can significantly impact the quality of TEM micrographs. Using sterile deionized water for the preparation of bacteriophages is a simple way to improve the quality of TEM micrographs and it is advisable to send the samples to the laboratory within 72 hours.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140944899","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 : 2024-05-10DOI: 10.1016/j.jviromet.2024.114950
Juncong Yan, Joe Tang, Zoila Perez-Egusquiza, Jeremy R. Thompson
The major citrus species include several economically important fruits, such as orange, mandarin, lemon, limes, grapefruit and pomelos. Since the 1980 s, total production and consumption of citrus has grown strongly with the current annual worldwide production at over 105 million tonnes. New Zealand's citrus exports, for instance, had an estimated worth of NZ$ 11.6 million (approx. US$ 7 million) in 2020. Citrus plants are prone to viral diseases, which can lead to substantial economic losses. In New Zealand, the citrus Import Health Standard (IHS) has identified 22 viruses and viroids that are subject to regulation and requires citrus nursery stock to be free of these pathogens. As such, there is a need for reliable, sensitive, and rapid detection methods to screen for these viruses and viroids during post entry quarantine. In this study, we developed TaqMan RT-qPCR assays for the detection of nine of these regulated viruses and viroids, namely citrus leaf rugose virus (CiLRV), citrus leprosis virus C (CiLV-C), citrus leprosis virus C2 (CiLV-C2), citrus leprosis virus N (CiLV-N), citrus psorosis virus (CPsV), citrus yellow mosaic virus (CYMV), citrus bent leaf viroid (CBLVd), citrus viroid V (CVd-V), and citrus viroid VI (CVd-VI). These assays have been validated and found to be highly sensitive, specific, and reliable. The implementation of these assays will facilitate the safe importation of citrus nursery stock, thus safeguarding the country's horticultural and economic interests.
{"title":"Development of TaqMan RT-qPCR for the detection of regulated citrus viruses and viroids in Aotearoa New Zealand","authors":"Juncong Yan, Joe Tang, Zoila Perez-Egusquiza, Jeremy R. Thompson","doi":"10.1016/j.jviromet.2024.114950","DOIUrl":"10.1016/j.jviromet.2024.114950","url":null,"abstract":"<div><p>The major citrus species include several economically important fruits, such as orange, mandarin, lemon, limes, grapefruit and pomelos. Since the 1980 s, total production and consumption of citrus has grown strongly with the current annual worldwide production at over 105 million tonnes. New Zealand's citrus exports, for instance, had an estimated worth of NZ$ 11.6 million (approx. US$ 7 million) in 2020. Citrus plants are prone to viral diseases, which can lead to substantial economic losses. In New Zealand, the citrus Import Health Standard (IHS) has identified 22 viruses and viroids that are subject to regulation and requires citrus nursery stock to be free of these pathogens. As such, there is a need for reliable, sensitive, and rapid detection methods to screen for these viruses and viroids during post entry quarantine. In this study, we developed TaqMan RT-qPCR assays for the detection of nine of these regulated viruses and viroids, namely citrus leaf rugose virus (CiLRV), citrus leprosis virus C (CiLV-C), citrus leprosis virus C2 (CiLV-C2), citrus leprosis virus N (CiLV-N), citrus psorosis virus (CPsV), citrus yellow mosaic virus (CYMV), citrus bent leaf viroid (CBLVd), citrus viroid V (CVd-V), and citrus viroid VI (CVd-VI). These assays have been validated and found to be highly sensitive, specific, and reliable. The implementation of these assays will facilitate the safe importation of citrus nursery stock, thus safeguarding the country's horticultural and economic interests.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140912497","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}