Journal of virological methods最新文献

Background and aims

Advances in health, especially in prevention, diagnosis, and treatment, have significantly impacted the way of facing emerging infectious diseases. Yet, events such as the COVID-19 pandemic have shown that there is still a long way to go. Therefore, an urgent need exists for portable and easily deployable point-of-care (POC) detection tools. Biosensors at the POC remain in the laboratory in an analytical characterization step and are not yet mature enough to reach the market massively. In this context, it is necessary to progress in validating these devices to demonstrate their relevance in detecting different disease biomarkers. This work reports on the clinical validation of an electrochemical immunosensor for detecting SARS-CoV-2.

Methods

A monocentric retrospective cohort study was conducted with 150 random nasopharyngeal swabs or tracheal aspiration samples tested by RT-PCR. The immunosensor based on magnetic beads and chronoamperometry detected SARS-CoV-2 through the spike-angiotensin-converting protein (ACE2) immunocomplex.

Results

This biosensor demonstrated 96.04 % clinical sensitivity and 87.75 % clinical specificity in detecting SARS-CoV-2 in the samples, highly correlated with the RT-PCR gold standard.

Conclusions

It demonstrates the potential of electrochemical biosensors to be implemented as highly sensitive and easily deployable detection strategies even in remote locations.

Emerging infectious diseases are a threat that contributes to the decline of global chelonian species. Herpesviruses are among the most impactful pathogens described in chelonians and are frequently associated with a range of presentations across hosts with the potential for severe morbidity and mortality. Trachemys herpesvirus 1 (TrHV1) has been reported in red-eared and yellow-bellied sliders (Trachemys scripta elegans and Trachemys scripta scripta, respectively) but is largely understudied. Invasive red-eared sliders may serve as a reservoir for transmission to sympatric native species. This study aimed to develop a sensitive and specific quantitative real-time PCR (qPCR) assay for the detection of TrHV1 DNA to aid in the characterization of the epidemiology of this virus in aquatic turtles. Two TaqMan-MGB FAM-dye labeled primer-probe sets were designed and evaluated using plasmid dilutions. The higher performing assay was specific for TrHV1 DNA and had a linear dynamic range of 1.0 × 10⁷ to 1.0 × 10¹ copies per reaction with an R² of 0.999, slope of −3.386, and efficiency of 97.39%. The limit of detection was 10¹ copies per reaction, and there was no loss of reaction efficiency in the presence of TrHV1-negative chelonian oral-cloacal DNA. Overall, the Trachemys herpesvirus 1 assay meets established criteria for acceptable qPCR assays and will be a valuable tool in characterizing the epidemiology of Trachemys herpesvirus 1 in chelonians.

Background

Advances in high-throughput sequencing (HTS) technologies and reductions in sequencing costs have revolutionised the study of genomics and molecular biology by making whole-genome sequencing (WGS) accessible to many laboratories. However, the analysis of WGS data requires significant computational effort, which is the major drawback in implementing WGS as a routine laboratory technique.

Objective

Automated pipelines have been developed to overcome this issue, but they do not exist for all organisms. This is the case for human respiratory syncytial virus (RSV), which is a leading cause of lower respiratory tract infections in infants, the elderly, and immunocompromised adults.

Results

We present RSV-GenoScan, a fast and easy-to-use pipeline for WGS analysis of RSV generated by HTS on Illumina or Nanopore platforms. RSV-GenoScan automates the WGS analysis steps directly from the raw sequence data. The pipeline filters the sequence data, maps the reads to the RSV reference genomes, generates a consensus sequence, identifies the RSV subgroup, and lists amino acid mutations, insertions and deletions in the F and G viral genes. This enables the rapid identification of mutations in these coding genes that are known to confer resistance to monoclonal antibodies.

Availability

RSV-GenoScan is freely available at https://github.com/AlexandreD-bio/RSV-GenoScan.

A Tn7-transposition approach was utilized for site-specific insertion of foreign genes into the genome of simian varicella virus (SVV), the causative agent of simian varicella in nonhuman primates. The severe acute respiratory syndrome coronavirus (SARS-CoV-2) nucleocapsid (N) gene and receptor binding domain (RBD) of the spike gene were inserted into the ORF 14 region of the SVV genome cloned into a bacterial artificial chromosome and then transfected into Vero cells to generate the infectious recombinant SVV (rSVV). The rSVV replicated efficiently in infected Vero cells and expressed the N and RBD antigens as indicated by immunoblot and immunofluorescence assays. Tn7-mediated transposition provides a rapid and efficient method for constructing rSVVs which may be evaluated as live-attenuated vaccines.

Senecavirus A (SVA) is a newly identified picornavirus associated with swine vesicular disease and neonatal mortality. The development of an SVA incorporating an exogenous reporter gene provides a powerful tool for viral research. In this study, we successfully constructed a recombinant SVA expressing Gaussia Luciferase (Gluc), termed rSVA-Gluc. The growth kinetics of rSVA-Gluc in BHK-21 cells were found to be comparable to those of the parental virus, and Gluc activity paralleled the virus growth curve. Genetic analysis revealed stable inheritance of the inserted reporter protein genes for at least six generations. We evaluated the utility of rSVA-Gluc in antiviral drug screening, and the results highlighted its potential as an effective tool for such purposes against SVA.

Data availability statement

The data that support the findings of this study are available on request from the corresponding author.

Baculovirus has been widely used for foreign protein expression in biomedical studies, and budded virus (BV) surface display has developed into an important research tool for heterogenous membrane protein studies. The basic strategy of surface display is to construct a recombinant virus where the target gene is fused with a complete or partial gp64 gene. In this study, we further investigate and develop this BV surface displaying strategy. We constructed stable insect cell lines to express the target protein flanking with different regions of signal peptide (SP) and GP64 transmembrane domain (TMD). Subsequently, recombinant BmNPV was used to infect the cell, and the integration of heterogeneous protein into BV was detected. The results indicated that deletion of the n-region of SP (SP^Δn) decreased the incorporation rate more than that of the full-length SP. However, the incorporation rate of the protein fused with h and c-region deletion of SP (SP^Δh-c) was significantly enhanced by 35–40 times compare to full-length SP. Moreover, the foreign protein without SP and TMD failed to display on the BV, while the integration of foreign proteins with GP64 TMD fusion at the c-terminal was significantly enhanced by 12–26 times compared to the control. Thus, these new strategies developed the BV surface display system further.

Introduction

We evaluated the performance of the automated Altostar HEV RNA platform for detecting HEV RNA.

Methods and results

Clinical performance was determined by testing 81 plasma samples and 10 fecal samples manually quantified previously with the Realstar RT-PCR assay using the Magnapure instrument for extraction. The assays were concordant for 79/81 plasma samples (97.5%) and 10/10 (100%) fecal samples. The two plasma samples that tested negative with the Altostar assay had a very low HEV RNA concentration (1.6 and 1.4 log₁₀ IU/ml). Quantitative results obtained with the automated platform and the manual workflow were highly correlated (ρ= 0.98, p<0.01). The intra-run and inter-run standard deviation were 0.09 IU/ml and 0.13 IU/ml respectively. The assay was linear from 2 to 6 log IU/ml. The limit of detection determined by Probit analysis with the WHO HEV RNA standard was 7.6 [95% CI: 4.4–52.5] IU/ml.

Conclusions

The Altostar platform enables highly accurate testing for the detection of HEV RNA in stool and the quantification of HEV RNA in plasma. This allowed us to shorten turnaround times and to save time for the technical staff.

Tomato, an extensively cultivated vegetable crop produces miRNAs in response to infection with Groundnut bud necrosis orthotospovirus, a viral pathogen causing significant economic losses. High-throughput miRNA sequencing was performed on tomato leaves inoculated with GBNV and mock-inoculated leaves as controls. Analysis revealed 73 known miRNAs belonging to 24 miRNA families, with variable expression levels. Interestingly, 39 miRNAs were upregulated, and 34 were downregulated in response to GBNV infection. Stem-loop quantitative reverse transcription PCR validated the differential expression of selected miRNAs. Additionally, 30 miRNA encoded proteins were identified to be involved in disease resistance and susceptibility. The miRNA-target interactions were found to play significant roles in cellular and metabolic activities, as well as modulating signaling pathways during the plant-virus interaction. The findings shed light on the intricate regulatory network of miRNAs in tomato response to viral infection and may contribute to developing strategies for improving crop protection against viral diseases.

This study describes the development and preliminary validation of a new serological assay using MERS-CoV S1 protein in an indirect enzyme-linked immunosorbent assay (ELISA) format. This assay has the advantage of being able to test MERS-CoV serum samples in a PC2 laboratory without the need for a high-level biocontainment laboratory (PC3 or PC4), which requires highly trained and skilled staff and a high level of resources and equipment. Furthermore, this MERS-CoV S1 ELISA enables a larger number of samples to be tested quickly, with results obtained in approximately five hours. The MERS-CoV S1 ELISA demonstrated high analytical specificity, with no cross-reactivity observed in serum of animals infected with other viruses, including different coronaviruses. We tested 166 positive and 40 negative camel serum samples and have estimated the diagnostic sensitivity (DSe) to be 99.4% (95% CI: 96.7 – 100.0%) and diagnostic specificity (DSp) to be 100% (95% CI: 97.2%-100.0%) relative to the assigned serology results (ppNT and VNT) using a S/P ratio cut-off value of >0.58. The findings of this study showed that our MERS-CoV S1 ELISA was more sensitive than the commercial EUROIMMUN ELISA (Se 99.4% vs 84.9%) and comparable to the ppNT assay, and therefore could be used as a diagnostic aid in countries in the Middle East where MERS-CoV is endemic in dromedary camels. The assay reagents and protocol were easily adapted and transferred from an Australian laboratory to a laboratory in the University of Hong Kong. Thus, the results described here show that the MERS-CoV S1 ELISA represents a cheap, rapid, robust, and reliable assay to support surveillance of MERS-CoV in camels in endemic regions.

A 2D primary gill cell culture system of the sevenband grouper (Hyporthodus septemfasciatus) was established to validate the pathogenesis of nervous necrosis virus (NNV) as observed in previous studies. This system, developed using the double-seeded insert (DSI) technique, yielded confluent cell layers. Upon challenge with NNV in a setup containing both autoclaved salt water and L15 media in the apical compartment, viral replication akin to that anticipated based on previous studies was observed. Consequently, we advocate for the utilization of primary gill cell culture as a viable alternative to conventional methodologies for investigating host pathogen interactions.