RT-qPCR for the diagnosis of the vesiculovirus Cocal virus.

Cocal virus (COCV) is one of the causative agents of vesicular stomatitis, presenting clinical signs indistinguishable from those caused by foot-and-mouth disease virus (FMDV). Therefore, the differentiation of these two viruses via laboratory diagnosis is essential. The objective of this study was to develop and validate a real-time quantitative PCR (RT-qPCR) protocol for the diagnosis of COCV directly from epithelial samples. The method developed had 97% accuracy at 3950 pfu and a repeatability error of 1.29%. RT-qPCR was able to distinguish COCV from other viruses that cause vesicular diseases, an important factor because seroneutralization may produce cross-reactivity between COCV and vesicular stomatitis Alagoas virus (VSAV). No epithelial sample originating from vesicular disease outbreaks between 2014 and 2018 in Brazil was positive for COCV.

Porcine parvovirus as a contaminant in cell cultures and laboratory supplies.

Although PPV has been described as a cellular contaminant, few recent studies about the presence of this virus in cell cultures, serum, and trypsin were found in the literature. The purpose of this study was to detect the presence of porcine parvovirus (PPV) by polymerase chain reaction (PCR) in cell cultures, serum, and trypsin used in official public laboratories of educational institutes and research centers. We tested samples of cell cultures (88), batches of trypsin (10), and fetal bovine serum (13) from different manufacturers. The PCR for beta-actin and GAPDH was used to evaluate the efficiency of DNA extraction from samples. The PPV DNA was detected in 52 of 88 (59.1%) cell culture samples. One in ten batches of trypsin tested for PPV DNA was positive. In no sample of fetal bovine serum, amplification of PPV DNA was observed. Positive samples were tested and confirmed by another analyst. In addition, all positive samples were sequenced. Our results indicate that regular PCR testing for PPV in cell cultures and their supplies is important.

Detection of contaminants in cell cultures, sera and trypsin.

The aim of this study was standardization and application of polymerase chain reaction (PCR) for the detection of contaminants in cell cultures, sera and trypsin. Five PCR protocols were standardized to assess the presence of genetic material from mycoplasma, porcine circovirus 1 (PCV1), bovine leukemia virus (BLV) or bovine viral diarrhea virus (BVDV) in cell culture samples. PCR reactions for the genes GAPDH and beta-actin were used to evaluate the efficiency of nucleic acid extraction. The PCR protocols were applied to 88 cell culture samples from eight laboratories. The tests were also used to assess potential contamination in 10 trypsin samples and 13 fetal calf serum samples from different lots from five of the laboratories. The results showed the occurrence of the following as DNA cell culture contaminants: 34.1% for mycoplasma, 35.2% for PCV1, 23.9% for BVDV RNA and 2.3% for BLV. In fetal calf sera and trypsin samples BVDV RNA and PCV1 DNA was detected. The results demonstrated that cell culture, sera and trypsin used by different laboratories show a high rate of contaminants. The results highlight the need for monitoring cell cultures and controlling for biological contaminants in laboratories and cell banks working with these materials.

Phylodynamics of Alagoas vesiculovirus in Brazil.

The vesicular stomatitis virus belongs to the Rhabdoviridae family, genus Vesiculovirus. Four species (New Jersey, Indiana, Cocal, and Alagoas) are responsible for disease outbreaks in Western Hemisphere countries. In Brazil, the Alagoas virus is responsible for the main outbreaks of the disease, mainly in the states of the Northeast, Midwest, and Southeast regions of the country. The present study aimed to perform the genetic characterization of 41 vesicular stomatitis virus samples. RNA was extracted using Trizol and used to amplify part of gene P. Amplicons were sequenced using the Sanger method. The phylogenetic trees generated showed that Alagoas vesiculoviruses were positioned into three groups: group A formed by the first virus isolate; group B by isolates from states in the Northeast region; and group C by isolates from the states of Bahia, Goiás, and Tocantins. Their divergence to date has generated the formation of two genotypes evolving independently in regions that until the present study had little geographic overlap.

Outbreaks of Vesicular Stomatitis in Brazil caused by a distinct lineage of Alagoas vesiculovirus.

This article describes the recurrence of outbreaks of Vesicular Stomatitis in the State of Maranhão, Brazil. The procedures for treating the outbreak of vesicular disease, sample collection, laboratory tests performed, and the results obtained were described. The clinical signs and observed injuries have been described. The sera showed antibodies that cross-react between the Vesiculovirus Indiana, Cocal, and Alagoas. The serological profile shows the presence of high antibody titers for Alagoas vesiculovirus in cattle, swine, and horses. Higher antibody titers indicate the viral serotype present in the outbreak. The genetic sequencing of the isolates confirmed the presence of Alagoas vesiculovirus, which grouped with the virus isolated in 2013 from cattle from the State of Maranhão.

Development and validation of rt-qpcr for vesicular stomatitis virus detection (Alagoas vesiculovirus).

Vesicular stomatitis is an infectious disease that occurs mainly in countries of the Western Hemisphere and affects cattle, swine and horses. The clinical symptoms in cattle and swine are similar to foot-and-mouth disease and include vesicular ulceration of the tongue and mouth. The disease requires a rapid and accurate differential diagnosis, aiming for immediate implementation of control measures. The objective of the present study was to develop and perform validation tests of multiplex RT-qPCR(s) for the detection of RNA from Alagoas vesiculovirus, considering the parameters of sensitivity and analytical specificity, analytical performance (repeatability and reproducibility criteria) and the uncertainty of the measurement. The threshold cycle values obtained in triplicate from each sample were evaluated by considering the variations between days, analysts and equipment in an analysis of variance aimed at determining the variances of repeatability and reproducibility. The results showed that RT-qPCRs had excellent sensitivity and specificity in the detection of RNA of the Alagoas vesiculovirus. The validation parameters showed low coefficients of variation and were equivalent to those found in other validation studies, indicating that the tests presented excellent repeatability and reproducibility.

Molecular epidemiology of Brazilian pseudorabies viral isolates.

Pseudorabies is a disease caused by pseudorabies virus (PRV) and is responsible for considerable economic losses in the swine industry. The objective of this work was to use molecular epidemiology as a tool to facilitate the study of PRV outbreaks in Brazil. The standard PRV strain Shope, the vaccine strain Bartha and isolates from the south and the southeast regions of Brazil, were amplified for gE and gC partial genes by PCR. Results indicated that Brazilian PRV isolates are grouped in two clusters, A and B, except for one isolate that grouped with Bartha and Shope. Most Brazilian PRV isolates belonged to cluster B and diverged from virus isolated from other countries.