Journal of Virological Methods

Dendritic cells (DCs) play a pivotal role in maintaining immune tolerance. Using recombinant adenovirus (rAd) to deliver vectors to immature dendritic cells (imDCs) is an important method for studying the tolerogenic function of DCs. We found that using RPMI medium and a higher MOI during transduction increased the expression of CD80, CD86, and MHC-II on the surface of imDCs. Our data reveal a significant increase in the secretion of the pro-inflammatory cytokine IL-6 in the group showing the most pronounced phenotypic changes...

Human norovirus (HuNoV) is regularly involved in food-borne infections. To detect infectious HuNoV in food, RT-qPCR remains state of the art but also amplifies non-infectious virus. The present study combines pre-treatments, RNase and propidium monoazide, with three molecular analyses, including long-range PCR, to predominantly detect infectious Tulane virus (TuV), a culturable HuNoV surrogate. TuV was exposed to inactivating conditions to assess which molecular method most closely approximates the reduction in infectious virus determined by cell culture (TCID50 )...

Bagaza virus (BAGV) is a mosquito-borne orthoflavivirus known to occur in regions of southern Europe, Africa, India and the Middle East. The virus has been associated with neurological disease and fatalities in various wild bird species. Association with human disease is not confirmed although limited serological evidence has suggested human infection. Surveillance programs for screening mosquitoes for evidence of arbovirus infection play an important role in providing information regarding the circulation and spread of viruses in specific regions...

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne zoonotic orthonairovirus of public health concern and widespread geographic distribution. Several animal species are known to seroconvert after infection with CCHFV without showing clinical symptoms. The commercial availability of a multi-species ELISA has led to an increase in recent serosurveillance studies as well as in the range of species reported to be exposed to CCHFV in the field, including wild boar (Sus scrofa). However, development and validation of confirmatory serological tests for swine based on different CCHFV antigens or test principles are hampered by the lack of defined control sera from infected and non-infected animals...

Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in China. Screening suitable cells for LSDV replication is vital for future research on pathogenic mechanisms and vaccine development. Previous comparative studies have identified that the rodent-derived BHK21 is a highly susceptible cell model to LSDV infection. Using western blot, indirect immune-fluorescence assay, flow cytometry, and transmission electron microscopy methods, this study is the first to identify the murine osteoblastic cell line MC3T3-E1 as a novel permissive cell model for LSDV infection...

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-footed animals. Immunization with inactivated virus vaccine is effective to control the disease. Six-monthly vaccination regimen in endemic regions has proven to be effective. To enable the differentiation of infected animals from those vaccinated, non-structural proteins (NSPs) are excluded during vaccine production. While the antibodies to structural proteins (SPs) could be observed both in vaccinated and infected animals, NSP antibodies are detectable only in natural infection...

Polioviruses (PV), the main causative agent of acute flaccid paralysis (AFP), are positive-sense single-stranded RNA viruses of the family Picornaviridae. As we approach polio eradication, accurate and timely detection of poliovirus in stool from AFP cases becomes vital to success for the eradication efforts. Direct detection of PV from clinical diagnostic samples using nucleic acid (NA) extraction and real-time reverse transcriptase polymerase chain reaction (rRT-PCR) instead of the current standard method of virus isolation in culture, eliminates the long turn-around time to diagnosis and the need for high viral titer amplification in laboratories...

INTRODUCTION: SARS-CoV-2 is usually diagnosed from naso-/oropharyngeal swabs which are uncomfortable and prone to false results. This study investigated a novel diagnostic approach to Covid-19 measuring volatile organic compounds (VOC) from patients' urine. METHODS: Between June 2020 and February 2021, 84 patients with positive RT-PCR for SARS-CoV-2 were recruited as well as 54 symptomatic individuals with negative RT-PCR. Midstream urine samples were obtained for VOC analysis using ion mobility spectrometry (IMS) which detects individual molecular components of a gas sample based on their size, configuration, and charge after ionization...

This study aimed to evaluate diagnostic accuracy of SARS-CoV-2 RNA detection in saliva samples treated with a guanidine-based or guanidine-free inactivator, using nasopharyngeal swab samples (NPS) as referents. Based on the NPS reverse transcription-polymerase chain reaction (RT-PCR) results, participants were classified as with or without COVID-19. Fifty sets of samples comprising NPS, self-collected raw saliva, and saliva with a guanidine-based, and guanidine-free inactivator were collected from each group...

Hepatitis B virus (HBV) infection is a global public health burden and affects approximatively 300 million people around the world. Since, HBV population is represented with genetic diversity, having different viral effects. Development of a new prognosis method play a key role on the efficiency of the different treatment. The HBx protein of HBV has a potential role in Hepatocellular Carcinoma (HCC), which makes it a valuable target for HCC prognosis. In this context, the first quantitative real-time PCR (qRT-PCR) assay in the Mediterranean area was developed and validated...

Optimal sampling, preservation, and culturing of SARS-CoV-2 from COVID-19 patients are critical for successful recovery of virus isolates and to accurately estimate contagiousness of the patient. In this study, we investigated the influence of the type of sampling media, storage time, freezing conditions, sterile filtration, and combinations of these to assess the optimal pre-analytic conditions for virus recovery and estimation of infectious viral load in COVID-19 patients. Further, we investigated the viral shedding kinetics and mucosal antibody response in 38 COVID-19 hospitalized patients...

Quantitative PCR (qPCR) is the gold standard for detecting nucleic acid sequences specific to the target pathogen. For COVID-19 diagnosis, several molecular assays have been developed. In this study, we present an optimization strategy for the measurement of SARS-CoV-2 RNA via multiplex qPCR and digital PCR (dPCR). Compared to qPCR, both droplet and chip-based dPCR, which are known to be more sensitive and accurate, showed a better resilience to suboptimal assay compositions and cycling conditions following the proposed optimizations...

Adenovirus protein VII (pVII) is a highly basic core protein, bearing resemblance to mammalian histones. Despite its diverse functions, a comprehensive understanding of its structural intricacies and the mechanisms underlying its functions remain elusive, primarily due to the complexity of producing a good amount of soluble pVII. This study aimed to optimise the expression and purification of recombinant pVII from four different adenoviruses with a simple vector construct. This study successfully determined the optimal conditions for efficiently purifying pVII across four adenovirus species, revealing the differential preference for bacterial expression systems...

Reverse transcription polymerase chain reaction (RT-PCR) tests are commonly utilized in commercial settings but pose challenges due to labor-intensive procedures and extended response times during peak demand. In contrast, real-time fluorescence and isothermal amplification assays using Crossing Priming Amplification (CPA) offer faster genetic material analysis, eliminate subjectivity, and require less manipulation and personnel training. This study aimed to validate the EasyNAT SARS-CoV-2-Assay, a diagnostic kit based on CPA, using oral and nasopharyngeal samples...

Plant virus detection and identification in crops is a pillar for disease management, import of crop material, production of clean stock plants and basic plant virology studies. In this report, we present a platform for the enrichment and isolation of known or unknown viruses. This platform is based on carbon nanotube arrays inside a microfluidic device that can be a solution for the identification of low titer viruses from plants. Using our microfluidic devices, we achieved enrichment of two economically important viruses, the orthotospovirus, tomato spotted wilt orthotospovirus (TSWV) and the potyvirus, zucchini yellow mosaic virus (ZYMV)...

Fig mosaic virus (FMV) is recognized as the main viral agent associated with the mosaic disease (MD) of fig trees (Ficus carica). Due to its worldwide occurrence, FMV represents the most significant global threat to the production of fig fruit. A disease management strategy against the MD in fig orchards has never been effective; and therefore, expression of recombinant antibody in plant cells could provide an alternative approach to suppress FMV infections. In this study we focused on expressing a specific recombinant antibody, a single-chain variable fragment (scFv), targeting the nucleocapsid protein (NP) of FMV in planta...

Red sea bream iridovirus (RSIV) is a highly contagious viral infection that affects various fish species and poses a significant threat to the global aquaculture industry. Thus, accurate and timely diagnosis is paramount for sustainable management of fish health. This study rigorously evaluated the diagnostic efficacy of various polymerase chain reaction (PCR) assays, focusing on those recommended by the World Organization for Animal Health (WOAH) and the assays newly proposed by WOAH's Aquatic Animals Health Standards Commission...

Many methods have been developed to measure the neutralizing capacity of antibodies to SARS-CoV-2. However, these methods are low throughput and can be difficult to quickly modify in response to emerging variants. Therefore, an experimental system for rapid and easy measurement of the neutralizing capacity of antibodies against various variants is needed. In this study, we developed an experimental system that can efficiently measure the neutralizing capacity of sera by using a GFP-carrying recombinant SARS-CoV-2 with spike proteins of multiple variants (B...

West Nile Virus (WNV) is an arthropod-borne virus that is spread through mosquito vectors. WNV emerged in the US in 1999 and has since become endemic in the US, causing the most domestically acquired arboviral disease in the country. Mosquito surveillance for WNV is useful to monitor arboviral disease burden over time and across different locations. RT-qPCR is the preferred method for WNV surveillance, but these methods are labor-intensive. The Panther Fusion System has an Open Access feature that allows for laboratory-developed tests (LDTs) to run on a fully automated system for nucleic acid extraction, RT-qPCR, and result generation...

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.