Journal of virological methods最新文献

Addressing the need for accessible SARS-CoV-2 testing, carboxy-PEG 12-thiol functionalized gold nanoparticles conjugates were developed for rapid point-of-care (POC) detection against SARS-CoV-2 spike protein, pseudo-SARS-CoV-2, and authentic Beta SARS-CoV-2 virus particles. These conjugates leverage gold nanoparticles (AuNPs) as signal transducers, cross-linked to either angiotensin-converting enzyme 2 (ACE2) or SARS-CoV-2 spike protein receptor-binding domain (RBD) antibodies as bioreceptors and showed a distinct color shift from pink to blue. To assess their POC feasibility, the conjugates were integrated into facemasks and breathalyzers, wherein aerosolized SARS-CoV-2 antigens were successfully detected, producing a color change within 10 and 30 minutes for the breathalyzer and facemask prototypes, respectively. Furthermore, we explored quantitative analysis using varying concentrations of SARS-CoV-2 spike protein. Both conjugates demonstrated a linear relationship between blue color intensity and virus concentration, with linear ranges of 0.08–0.6 ng/mL and 0.04–0.5 ng/mL, respectively. Low limits of detection and quantification were also achieved. They exhibited specificity, responding solely to SARS-CoV-2 even in complex matrices containing diverse proteins, including the SARS-CoV-1 spike protein. Precision tests yielded coefficient of variations below 2 %, showcasing their remarkable reproducibility. This work presents a promising approach for rapid, sensitive, and specific POC detection of SARS-CoV-2 paving the way for improved pandemic response and management.

A method that has rapidly evolved for detection of viral pathogens are loop-mediated isothermal amplification (LAMP) assays. The available LAMP assays usually target the most common viral strains, including enteroviruses, but for the atypical enterovirus D68 strain VR-1197 this method has not yet been developed. Enterovirus D68 are known for severe respiratory distress in children, and atypical strains are less likely to be detected by traditional methods. This study targets the atypical EVD68 strain VR-1197 and have developed a rapid detection method saving time when differentiating enterovirus strains. This study present method development and review the sensitivity and specificity compared to traditional RT-qPCR, and wet lab cross reactivity with other airway pathogens. The EVD68 VR-1197 assay can be a rapid POC (Point of care) test for atypical EVD68 VR-1197 and have the potential as reliable detection method with minimal technological requirements.

Due to shared routes of transmission, including sexual contact and vertical transmission, HIV-HBV co-infection is common, particularly in sub-Saharan Africa. Measurement of viral load (VL), for both HIV and HBV, plays a critical role for determining their infectious phase and monitoring response to antiviral therapy. Implementation of viral load testing in clinical settings is a significant challenge in resource-limited countries, notably because of cost and availability issues. We designed HIV and HBV primers for conserved regions of the HIV and HBV genomes that were specifically adapted to viral strains circulating in West Africa that are HIV-1 subtype CRF02AG and HBV genotype E. We first validated two monoplex qPCR assays for individual quantification and, then developed a multiplex qPCR for simultaneous quantification of both viruses. HIV RNA and HBV DNA amplification was performed in a single tube using a one-step reverse transcription-PCR reaction with primers and probes targeting both viruses. Performance characteristics such as the quantification range, sensitivity, and specificity of this multiplex qPCR assay were compared to reference qPCR tests for both HIV and HBV viral load quantification. The multiplex assay was validated using clinical samples from co- or mono-infected patients and gave comparable viral load quantification to the HIV and HBV reference test respectively. The multiplex qPCR demonstrated an overall sensitivity of 71.25 % [68.16–74.3] for HBV and 82 % [78.09–85.90] for HIV and an overall specificity of 100 % [94.95–100] for both viruses. Although the overall sensitivities of the HIV and HBV assays were lower than the commercial comparator assays, the sensitivity in the clinical decision range of >1000 copies/mL for HIV was 80 % [71.26–88.73] and >1000 IU/mL for HBV was 100 % [95.51–100] which indicates the test results can be used to guide treatment decisions. This in-house developed multiplex qPCR assay represents a useful diagnostic tool as it can be performed on affordable "open" real-time PCR platforms currently used for HIV or SARS-Cov-2 infection surveillance in Mali.

This study describes production of polyclonal antibodies against recently reported novel potyvirid infecting alfalfa (Medicago sativa L.). The virus was first found in alfalfa seed material and later identified in plant samples collected from commercial alfalfa fields in Arizona, USA. It was classified as a novel species related to the members of the genus Ipomovirus and potentially representing a new genus in the family Potyviridae (Nemchinov et al., 2023b). Polyclonal antibodies were produced against the predicted viral coat protein expressed in bacterial cells and used in different types of immunoassays for specific detection of this emerging virus. They could be helpful in plant virus certification programs, screening of alfalfa germplasm, research on pathogenicity, biology, and geographic distribution of this emerging virus.

Five simplex and a multiplex-RT-PCR (m-RT-PCR) protocols were developed for detection and differentiation of bean pod mottle virus (BPMV), cherry leaf roll virus (CLRV), raspberry ringspot virus (RpRSV), soybean mosaic virus (SMV) and tomato ringspot virus (ToRSV) infecting soybean. The simplex RT-PCR protocols produced virus-specific amplicons of 538 bp for BPMV, 139 bp for CLRV, 298 bp for RpRSV, 403 bp for SMV, and 282 bp for ToRSV, with sensitivity down to 10⁻⁴ diluted cDNA. Further, to detect all the five viruses simultaneously in a single tube a quintuplex RT-PCR protocol was optimized with as low as 10⁻³ diluted cDNA and 0.05 µM primer. To validate the reliability of the simplex RT-PCR protocol, imported soybean samples were tested by ELISA as well as RT-PCR. The results revealed that the developed protocol could detect the viruses in imported soybean, and found to be efficient than ELISA in resolving ambiguity in detection of seed borne viruses. The developed simplex and quintuplex RT-PCR protocol will be quite helpful for the diagnosis of soybean germplasm co-infected with viruses during the quarantine processing for ensuring virus free long term seed conservation in the National Gene Bank as well as for quarantine certification.

The cross-species transmissibility of SARS-CoV-2 infection has necessitated development of specific reagents for detecting infection in various animal species. The spike glycoprotein of SARS-CoV-2, which is involved in viral entry, is a highly immunogenic protein. To develop assays targeting this protein, we generated eight monoclonal antibodies (mAbs) against the S1 and seven against the S1/S2 protein (ectodomain) of SARS CoV-2. Based on neutralization capability and reactivity profile observed in ELISA, the mAbs generated against the S1/S2 antigen exhibited a broader spectrum of epitope specificity than those produced against the S1 domain alone. The full-length ectodomain induced antibodies that could neutralize the two most important variants of the virus encountered during the pandemic, namely Delta and Omicron. The availability of these reagents could greatly enhance the development of precise diagnostics for detecting COVID-19 infections in various host species and contribute to the advancement of mAb-based therapeutics.

China has the largest aquaculture eel production in the world. High-density cultivation pattern often results in an outbreak of epidemic diseases. Since the 1990s, eel “mucus sloughing and hemorrhagic septicemia disease” was often broke out in China, and brought huge economic losses to eel breeders. Anguillid herpesvirus 1 (AngHV) was detected and isolated from the diseased eel, and proved to be the pathogen of the disease. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid, sensitive, and specific detection of AngHV. A set of six primers targeting the ORF51 gene of AngHV was designed, which could effectively detect purified AngHV virions, AngHV-infected cells, or eel tissue samples. The suitable reaction temperature is 63℃, and the reaction time is 40 min. There was no cross-reaction with eel and other fish viruses, including Infectious pancreatic necrosis virus (IPNV), Marine birnavirus (MABV), Rana grylio virus (RGV), Cyprinid herpesvirus 3 (CyHV-3), and Eel iridovirus (EIV). The lower detection limit of the AngHV LAMP assay is 10 copies of AngHV genome DNA, which is at least 100 times more sensitive than conventional PCR in detecting AngHV. The assay could effectively detect AngHV from collected samples with typical clinical symptoms of AngHV infection. It suggested that the LAMP assay could be used in specific detection of AngHV and has great potential for early diagnosis of AngHV infection in the farm.

Human enteric viruses, as adenovirus (HAdV), norovirus (HuNoV) and rotavirus (RVA) are significant causes of gastroenteritis associated with consumption of contaminated water worldwide. Various methods have been described for their detection and monitoring in water. The aim of this study was to compare the performance of four conditions for concentrating HAdV, HuNoV and RVA from water matrices, in order to develop a single protocol that could simultaneously concentrate all target viruses from tap water. The tested conditions were based on the adsorption-elution using electronegative filters, in which we evaluated cation-coated filtration by MgCl₂ with or without acid rinse by H₂SO₄ and two elution buffers, namely NaOH and tris-glycine-beef extract. Genomic material was extracted and amplified by real-time PCR and real-time RT-PCR using commercial kits. Based on the statistical analysis of amplification results (cycles of quantification), the condition involving cation-coated filtration by MgCl₂ using electronegative filters with acid rinse by H₂SO₄ combined with NaOH elution allowed efficient recovery of both HAdV, HuNoV and RVA from tap water compared to the other conditions. These findings confirm the effectiveness of the approach used to monitor three major enteric viruses in tap water.

Largemouth bass virus (LMBV) and infectious spleen and kidney necrosis virus (ISKNV) are both belong to Iridoviridae that cause considerable economic losses in the fish industry. There is no reported literature that can detect these two viruses simultaneously. In this study, we established a multiplex quantitative polymerase chain reaction (qPCR) assay that can specifically and simultaneously detect both LMBV and ISKNV in fish samples. The specificity experiment showed that the method only amplified LMBV and ISKNV but not the other 10 common fish viruses. The slope (m), efficiency (E) and linearity (R²) determined from the generated standard curve were all within the optimal range of qPCR values. The detection limit of the multiplex qPCR assay was as low as 4 copies/μL for LMBV DNA and 7 copies/μL for ISKNV DNA, respectively. The established method exhibited adequate repeatability and reproducibility, and the intra- and inter-assay coefficients of variation were both less than 3 %. The accuracy of the multiplex qPCR method was validated using 229 fish samples and was more precise than that of the conventional PCR assay. In summary, the established multiplex qPCR assay can simultaneously detect LMBV and ISKNV to monitor the risk of infection LMBV and ISKNV and control the disease early.

In the employment of serodiagnostic methods for the detection of orthoflavivirus infections, neutralization tests are known to be more accurate than measurements of antibody binding properties employing enzyme-linked immunosorbent assays. However, neutralization tests require infectious virus and laboratories with an appropriate level of biosafety. Single-round infectious particles (SRIPs), which encode a reporter gene instead of the viral structural protein genes, are replication incompetent and represent a safe and reliable alternative to the diagnosis of pathogenic viruses in neutralization tests. The orthoflavivirus SRIPs are produced by co-transfection of plasmids expressing virus-like particles and replicons into mammalian cell lines preferably with high transfection efficacy, such as HEK293T cells. However, certain orthoflavivirus SRIPs have limitations in their efficient expression at 37°C, which is the optimal temperature for mammalian cell growth, resulting in insufficient yields for neutralization tests. Here, we demonstrate that the production of orthoflavivirus SRIPs increases at the lower temperature of 28°C compared to 37°C. Moreover, infections with 28°C-cultured SRIPs in microneutralization tests were specifically inhibited in the presence of serum from mice infected with homologous viruses, suggesting that these SRIPs preserved their neutralizing epitopes for antibodies. Our method to produce high titer SRIPs is anticipated to promote efficient and safe SRIPs neutralization tests as a general serodiagnostic method for detecting virus-specific neutralizing antibodies against orthoflaviviruses.