Journal of Virological Methods

Rotavirus genotyping is useful for surveillance purposes especially in areas where rotavirus vaccination has been or will be implemented. RT-PCR based molecular methods have been applied widely, but quantitative assays targeting a broad spectrum of genotypes have not been developed. Three real time RT-PCR panels were designed to identify G1, G2, G9, G12 (panel GI), G3, G4, G8, G10 (panel GII), and P[4], P[6], P[8], P[10], P[11] (panel P), respectively. An assay targeting NSP3 was included in both G panels as an internal control...

The world-wide HIV epidemic is characterized by increasing genetic diversity with multiple viral subtypes, circulating recombinant forms (CRFs) and unique recombinant forms (URFs). Antiretroviral drug design and basic virology studies have largely focused on HIV-1 subtype B. There have been few direct comparisons by subtype, perhaps due to the lack of uniform and standardized culture systems for the in vitro propagation of diverse HIV-1 subtypes. Although peripheral blood mononuclear cells (PBMCs) are major targets and reservoirs of HIV, PBMCs culturing is relatively difficult and not always reproducible...

Sheeppox is now enzootic in Morocco. The development of a reliable method for rapid diagnosis of the disease is a central part of any control strategy. The aim of this study is to determine the diagnostic value of a variety of clinical samples such as ovine nasal, ocular or rectal swabs for the detection of sheeppox virus (SPPV) by qualitative conventional polymerase chain reaction (PCR), using a single pair of primers targeting the inverted terminal repeats of the SPPV InS-1 strain, a virulent field isolate...

A novel assay, the CSFV DNAChip, was developed to clearly and rapidly discriminate three genotypes of classical swine fever virus (CSFV). Total RNA was extracted from clinical samples and then subjected to a one-step reverse-transcription polymerase chain reaction (RT-PCR) using Cy3-labeled primers from the 5' non-coding region (NCR) of CSFV. Amplicons were hybridized to the CSFV DNAChip and fluorescence scanning was performed for detection of CSFV. A cut-off fluorescence intensity value of 5000 was determined by two-graph receiver operating curve (TG-ROC) analysis...

Due to an intensive use of chemical insecticides, resistance mechanisms to insecticides together with adverse effects on non-target organisms have been largely reported. Improvement in pest control strategy represents an urgent need to optimize efficiency in the control of pest insects. In this context, a novel method based on the use of insect specific virus applied in combination with chemical insecticide, which could lead to sensitization of the insect target to insecticides is described. Insect virus, the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), applied onto Sf9 cells induces an increase of intracellular calcium concentration via extracellular calcium influx...

Dengue fever is the most important arthropod-transmitted viral disease affecting humans. It is a blood-borne disease characterized by persistent fever and joint pain. In the blood, primary peripheral blood mononuclear cells (PBMCs), in particular monocytes, are the main target of the dengue virus (DENV). These cells are poorly permissive for in vitro dengue virus infection and their infectivity varies from donor to donor. To overcome this barrier, an anti-dengue antibody was used to improve the infectivity of DENV-2 clinical isolates to PBMCs, the monocytic leukemia cell line, THP-1 and the granulocyte cell line, KU812...

West Nile virus (WNV) is one of flaviviruses and has emerged recently in the United States as a significant cause of viral encephalitis. Although cellular entry of WNV is important for viral pathogenesis, its mechanisms have not been elucidated fully. To explore the entry mechanisms, a virus-particle tracking system in live cells by using fluorescently labeled subviral particles (SVPs) and time-lapse epifluorescence microscopy was established. This study revealed that, following cellular entry, SVP movements could be divided into two phases: early (slow movement) and late (fast movement) phase...

Hepatitis D virus (HDV) infection is often accompanied by hepatitis B virus (HBV) infection. Co-infection of HDV and HBV may lead to more severe symptoms and even death. Current methods for HDV diagnosis have high false-positive rates and show significant result discrepancies. The Abbott AxSYM AUSAB test, currently a standard test for HDV detection, is too laborious and expensive for routine application. Therefore, new sensitive and cost-efficient methods for HDV diagnosis are urgently needed. In this study, S-HDAg protein was produced in high yield in Escherichia coli...

Development and improvement of quality control tests for live attenuated vaccines are a high priority because of safety concerns. Live attenuated influenza vaccine (LAIV) viruses are 6:2 reassortants containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from circulating influenza viruses to induce protective immune responses, and the six internal gene segments from a cold-adapted Master Donor Virus (MDV). LAIV candidate viruses for the 2012-2013 seasons, A/Victoria/361/2011-CDC-LV1 (LV1) and B/Texas/06/2011-CDC-LV2B (LV2B), were created by classical reassortment of A/Victoria/361/2011 and MDV-A A/Leningrad/134/17/57 (H2N2) or B/Texas/06/2011 and MDV-B B/USSR/60/69...

Heteroduplex mobility (HMA) and tracking assays (HTA) are used to assess genetic relationships between DNA molecules. While distinguishing relationships between clonal or nearly clonal molecules is relatively straightforward, inferring population structures is more complex. To address this issue, HIV-1 quasispecies with varying levels of diversity were studied using both HTA and DNA sequencing. Viral diversity estimates and the temporal features of virus evolution were found to be generally concordant between HTA and DNA sequencing...

The extraction and purification of nucleic acids is a critical step in the molecular detection of enteric viruses from environmental or fecal samples. In the present study, the performance of three commercially available kits was assessed: the MO BIO PowerViral Environmental DNA/RNA Isolation kit, the Qiagen QIAamp Viral RNA Mini kit, and the Zymo ZR Virus DNA/RNA Extraction kit. Viral particles of adenovirus 2 (AdV), murine norovirus (MNV), and poliovirus type 1 (PV1) were spiked in molecular grade water and three different types of sample matrices (i...

The development of rapid and effective methods to detect water- and food-borne enteric viruses is important for the prevention and control of mass infection. This study represents an attempt to develop a reliable cell culture-based detection system and optimize an effective and rapid protocol for the assaying of environmental samples for the presence of infectious enteric viruses. Six enteric viruses were used in this study: poliovirus, Coxsackie virus A9, Coxsackie virus B5, human rotavirus G1, hepatitis A virus, and adenovirus type 41...