Development of an indirect ELISA against Orf virus using two recombinant antigens, partial B2L and F1L
In this study, an indirect ELISA method for recombinant proteins based on truncated dominant antigenic epitopes of B2L and F1L genes of ORFV was established. A series of conditions and its performance were comprehensively evaluated. The optimized ELISA reaction conditions were: the optimal coating amount of antigen was 0.25μg/mL, 5% skim milk powder was closed for 1hour, the optimal dilution of serum was 1:200, the optimal incubation time of the rabbit anti-goat IgG was 1:8000, the optimal color development time of TMB was 15mins, and the threshold value of negative-positive was 0.358. The method specifically detects anti...
Source: Journal of Virological Methods - February 9, 2024 Category: Virology Authors: Weihao Zheng You Zhang Qinglin Gu Qian Liang Youci Long Qin Wu Simei Xian Source Type: research
The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of infectious hematopoietic necrosis virus (IHNV)
J Virol Methods. 2024 Feb 7;326:114892. doi: 10.1016/j.jviromet.2024.114892. Online ahead of print.ABSTRACTInfectious hematopoietic necrosis virus (IHNV) is an economically important virus causing significant mortalities among wild and cultured salmonid fish worldwide. Rapid and sensitive diagnostic methods of IHNV are crucial for timely controlling infections. For better detection of IHNV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of IHNV in two steps. Following the screening of primer pairs, the re...
Source: Journal of Virological Methods - February 8, 2024 Category: Virology Authors: Feixiang Rong Hongsheng Wang Xiaoqian Tang Jing Xing Xiuzhen Sheng Heng Chi Wenbin Zhan Source Type: research
Diagnostic performance of cross-priming amplification-based lateral flow assay (CPA-LFA) and real-time PCR for koi herpesvirus (KHV) detection
In this study, we developed and validated the diagnostic performance of two molecular diagnostic assays, cross-priming amplification-based lateral flow assay (CPA-LFA) and TaqMan probe-based real-time polymerase chain reaction (PCR). To detect KHV, primers and probe were designed based on the thymidine kinase (TK) genes. The detection limits of developed CPA-LFA and real-time PCR assays were determined to be 675.69 copies/μL and 8.384 copies/μL, respectively. The diagnostic sensitivity and specificity of the developed assay were determined using fish samples (n = 179). CPA-LFA was found to be 93.67% and 100%, respectivel...
Source: Journal of Virological Methods - February 3, 2024 Category: Virology Authors: Guk Hyun Kim Ye Jin Jeong Yu Gyeong Jeon Yun Jung Yang Joon Gyu Min Do-Hyung Kim Kwang Il Kim Source Type: research
Visual, rapid, and cost-effective BK virus detection system for renal transplanted patients using gold nanoparticle coupled loop-mediated isothermal amplification (nanoLAMP)
We report a rapid (two hours from sample collection, processing, and detection), cost-effective (< 2$), highly sensitive and BKV-specific nanoLAMP (loop-mediated isothermal amplification) diagnostic methodology using novel primers and gold nanoparticles complex-based visual detection. The standardized nanoLAMP showed an analytical sensitivity of 25 copies/µl and did not cross-react with closely related JC and SV40 viruses. This nanoLAMP showed diagnostic sensitivity and specificity as 91% and 96%, respectively, taking 50 BK virus-negative (confirmed by qPCR from the plasma of healthy donors) and 57 positive BKV patient...
Source: Journal of Virological Methods - January 30, 2024 Category: Virology Authors: Sunil Kumar Srishty Raman Kishore Sesham Abhishek Gupta Raj Kanwar Yadav Asit Ranjan Mridha Subhash Chandra Yadav Source Type: research
Specific antibody production using recombinant proteins to elucidate seed transmission and nuclear localization of Coguvirus citrulli and Coguvirus henanense in radicles of watermelon crop
J Virol Methods. 2024 Jan 19;325:114886. doi: 10.1016/j.jviromet.2024.114886. Online ahead of print.ABSTRACTWatermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2, both belonging to the genus Coguvirus (family Phenuiviridae), have been identified in watermelon plants in Brazil. To study tissue tropism and the potential for seed transmission of these viruses, we initially planned to produce specific antibodies. However, difficulties in isolating and propagating the virus in host plants hindered the purified virus preparations. To overcome this problem, the nucleocapsid (N) proteins of WCLaV-1 and -2 were produced ...
Source: Journal of Virological Methods - January 21, 2024 Category: Virology Authors: Caterynne M Kauffmann Marina Vendramini Amanda M V Batista Helena B S Mota Ikaro A Andrade Stephanny B S C árdenas Paloma S Queiroz Bruno A Silva Jos é R Correa Tatsuya Nagata Source Type: research
Genome sequencing of the mpox virus 2022 outbreak with amplicon-based Oxford Nanopore MinION sequencing
We present an amplicon-based assay for MinION Nanopore sequencing of mpox virus (MPXV) genomes from clinical specimens, obtaining high-quality results with an average genome coverage of 99% for Ct values of up to 25, and a genome coverage of 97.1% for Ct values from 25 to 30 which are challenging to sequence. This assay is easy to implement in PCR-based workflows and provides accurate genomic data within a short time.PMID:38246565 | DOI:10.1016/j.jviromet.2024.114888 (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - January 21, 2024 Category: Virology Authors: Annika Brinkmann Katharina Pape Steven Uddin Niklas Woelk Sophie F örster Heiko Jessen Janine Michel Claudia Kohl Lars Schaade Andreas Nitsche Source Type: research
Evaluation of long-term preservation methods for viral RNA in mosquitoes at room temperature
J Virol Methods. 2024 Jan 17;325:114887. doi: 10.1016/j.jviromet.2024.114887. Online ahead of print.ABSTRACTMosquitoes are important vectors of various pathogenic viruses. Almost all viruses transmitted by mosquitoes are RNA viruses. Therefore, to detect viral genes, mosquito samples must be kept at low temperatures to prevent RNA degradation. However, prolonged transport from the field to laboratory can pose challenges for temperature control. The aim of this study was to evaluate methods for preserving viral RNA in mosquito bodies at room temperature. Virus-infected mosquito samples were immersed in ethanol, propylene gl...
Source: Journal of Virological Methods - January 18, 2024 Category: Virology Authors: Izumi Kai Daisuke Kobayashi Kentaro Itokawa Chizu Sanjoba Kyo Itoyama Haruhiko Isawa Source Type: research
Establishment and application of an indirect ELISA for Getah virus E2 antibody detection
In this study, we removed specific hydrophobic domains and intracellular structures from E2 proteins and constructed the recombinant plasmid pCold-TF-E2. The recombinant protein was expressed using a prokaryotic expression system, and efficient purification of the rE2 protein was achieved using a nickel affinity column. The purified rE2 protein is suitable for the development of an indirect ELISA (rE2 ELISA). Following the optimization of reaction conditions for the rE2-ELISA, the cut-off value was 0.356. Additionally, the rE2-ELISA method showed a positive rate of 37.1% for IgG antibodies against GETV when testing 986 pig...
Source: Journal of Virological Methods - January 16, 2024 Category: Virology Authors: Dong You Yu-Ling Wang Liang-Peng Ge Yuan-Cheng Zhou Jing Sun Li-Qiao Lang Si-Yuan Lai Yan-Ru Ai Ling Zhu Zhi-Wen Xu Source Type: research
Optimised protocols to generate high titre lentiviral vectors using a novel transfection agent enabling extended HEK293T culture following transient transfection and suspension culture
In this study, we sought to improve lentiviral titre over time by testing two alternative commercially available transfection reagents Fugene® 6 and Genejuice® with the commonly used polycation, polyethyleneimine. Our aim was to identify less cytotoxic transfection reagents that could be used to generate LV particles at high titre past the often used 72 h period when vector is usually collected before producer cell death is caused due to post transfection cytotoxicity. We show that LV could be produced in extended culture using Genejuice® and even by transfected cells in glass flasks in suspension. Because this delivery...
Source: Journal of Virological Methods - January 13, 2024 Category: Virology Authors: Saqlain Suleman Serena Fawaz Terry Roberts Stuart Ellison Brian Bigger Michael Themis Source Type: research
A method for screening CDV microneutralization activity in microvolume samples
CONCLUSION: This study established an economical, stable, and easy-to-operate CDV microneutralizing activity high-throughput screening method, laying a methodological foundation for the development of native CDV neutralizing antibodies based on single B cells.PMID:38195043 | DOI:10.1016/j.jviromet.2024.114883 (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - January 9, 2024 Category: Virology Authors: Xiaoyu Deng Jiazi Su Bo Hu Xue Bai Source Type: research
A method for screening CDV microneutralization activity in microvolume samples
CONCLUSION: This study established an economical, stable, and easy-to-operate CDV microneutralizing activity high-throughput screening method, laying a methodological foundation for the development of native CDV neutralizing antibodies based on single B cells.PMID:38195043 | DOI:10.1016/j.jviromet.2024.114883 (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - January 9, 2024 Category: Virology Authors: Xiaoyu Deng Jiazi Su Bo Hu Xue Bai Source Type: research
Quantification of low-level Cytomegalovirus and Epstein-Barr virus DNAemia by digital PCR
CONCLUSION: dPCR can quantify low-level herpes virus DNAemia with CVs below 8%. Our results indicate that using serum-derived cell-free DNA and droplet-based dPCR is optimal for quantitating low-level viral DNAemia; however, plasma and chip-based approaches are acceptable alternatives and suitable for epidemiologic investigation.PMID:38184072 | DOI:10.1016/j.jviromet.2023.114876 (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - January 6, 2024 Category: Virology Authors: DeVon N Hunter-Schlichting Rachel I Vogel Melissa A Geller Heather H Nelson Source Type: research
Quantification of low-level Cytomegalovirus and Epstein-Barr virus DNAemia by digital PCR
CONCLUSION: dPCR can quantify low-level herpes virus DNAemia with CVs below 8%. Our results indicate that using serum-derived cell-free DNA and droplet-based dPCR is optimal for quantitating low-level viral DNAemia; however, plasma and chip-based approaches are acceptable alternatives and suitable for epidemiologic investigation.PMID:38184072 | DOI:10.1016/j.jviromet.2023.114876 (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - January 6, 2024 Category: Virology Authors: DeVon N Hunter-Schlichting Rachel I Vogel Melissa A Geller Heather H Nelson Source Type: research
Quantification of low-level Cytomegalovirus and Epstein-Barr virus DNAemia by digital PCR
CONCLUSION: dPCR can quantify low-level herpes virus DNAemia with CVs below 8%. Our results indicate that using serum-derived cell-free DNA and droplet-based dPCR is optimal for quantitating low-level viral DNAemia; however, plasma and chip-based approaches are acceptable alternatives and suitable for epidemiologic investigation.PMID:38184072 | DOI:10.1016/j.jviromet.2023.114876 (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - January 6, 2024 Category: Virology Authors: DeVon N Hunter-Schlichting Rachel I Vogel Melissa A Geller Heather H Nelson Source Type: research
Identification of a potent and specific retinoic acid-inducible gene 1 pathway activator as a Hepatitis B Virus antiviral through a novel cell-based reporter assay
J Virol Methods. 2024 Jan 2:114875. doi: 10.1016/j.jviromet.2023.114875. Online ahead of print.ABSTRACTChronic Hepatitis B Virus (HBV) infection remains a global burden. To identify small molecule RIG-I agonists as antivirals against HBV, we developed an HBV-pgRNA-based interferon-β (IFN-β) luciferase reporter assay with high level of assay sensitivity, specificity and robustness. Through HTS screening, lead compound (JJ#1) was identified to activate RIG-I signaling pathway by inducing TBK1 phosphorylation. Knockdown experiments demonstrated that JJ#1-induced retinoic acid-inducible gene 1 (RIG-I) signaling pathway activ...
Source: Journal of Virological Methods - January 4, 2024 Category: Virology Authors: Liping Shi Guangyang Guo Jinying Zhou Zhanling Cheng Ren Zhu George Kukolj Chris Li Source Type: research
