ABSTRACT
BACKGROUND: Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits. METHODS: Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples. RESULTS: In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values. CONCLUSIONS: Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis.
Subject(s)
Picornaviridae Infections , Picornaviridae , RNA, Viral , Swine Diseases , Animals , Picornaviridae/genetics , Picornaviridae/isolation & purification , Swine , Picornaviridae Infections/diagnosis , Picornaviridae Infections/veterinary , Picornaviridae Infections/virology , RNA, Viral/genetics , Swine Diseases/virology , Swine Diseases/diagnosis , Sensitivity and Specificity , Real-Time Polymerase Chain Reaction/methods , Swine Vesicular Disease/diagnosis , Swine Vesicular Disease/virology , Foot-and-Mouth Disease/diagnosis , Foot-and-Mouth Disease/virology , Brazil , Reproducibility of ResultsABSTRACT
The FTA card has emerged as a promising alternative for nucleic acid extraction. The FTA card is a filter paper impregnated with chemicals that preserve and stabilize the genetic material present in the sample, allowing for its storage and transport at room temperature. The aim of this study was to test the card for the detection of RNA and DNA nucleic acids. Two RNA viruses (Senecavirus A and classical swine fever virus) and two DNA viruses (African swine fever virus and suid alphaherpesvirus 1) were tested, and in all cases, there was a decrease in sensitivity. The methods exhibited good repeatability and demonstrated a rapid and practical use for sample transport and nucleic acid extraction.
Subject(s)
African Swine Fever Virus , Animals , Swine , African Swine Fever Virus/isolation & purification , African Swine Fever Virus/genetics , Classical Swine Fever Virus/genetics , Classical Swine Fever Virus/isolation & purification , Herpesvirus 1, Suid/isolation & purification , Herpesvirus 1, Suid/genetics , RNA, Viral/genetics , RNA, Viral/isolation & purification , Veterinary Medicine/methods , Swine Diseases/virology , Swine Diseases/diagnosis , DNA Viruses/genetics , DNA Viruses/isolation & purification , Picornaviridae/genetics , Picornaviridae/isolation & purification , Picornaviridae/classification , Sensitivity and Specificity , DNA, Viral/genetics , RNA Viruses/genetics , RNA Viruses/isolation & purification , RNA Viruses/classification , DNA Virus Infections/veterinary , DNA Virus Infections/diagnosis , DNA Virus Infections/virology , Specimen Handling/methods , Specimen Handling/instrumentationABSTRACT
BACKGROUND: Megalocytiviruses (MCV) are double-stranded DNA viruses that infect fish. Two species within the genus are epidemiologically important for fish farming: red sea bream iridovirus (RSIV) and infectious spleen and kidney necrosis virus (ISKNV). The objective of this work was to study regions that allow the differentiation and correct diagnosis of RSIV and ISKNV. METHODS: The regions ORF450L, ORF342L, ORF077, and the intergenic region between ORF37 and ORF42R were sequenced and compared with samples from the database. RESULTS: The tree constructed using the sequencing of the PCR product Megalocytivirus. ORF077 separated the three major clades of MCV. RISV genotypes were well divided, but not ISKNV. All qPCRs tests showed acceptable repeatability values, that is, less than 5%. CONCLUSION: Two qPCRs for ISKNV detection and two for RSIV were considered suitable for use in the diagnosis and typing of MCV. The results of this study demonstrate the importance of an accurate evaluation of methodologies for the differentiation of MCV.
Subject(s)
DNA Virus Infections , Fish Diseases , Iridoviridae , Iridovirus , Animals , Iridoviridae/genetics , Real-Time Polymerase Chain Reaction , DNA Virus Infections/genetics , DNA Virus Infections/veterinary , PhylogenyABSTRACT
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.
Subject(s)
Vesicular Stomatitis , Animals , Brazil/epidemiology , Phylogeny , Vesicular stomatitis Indiana virus/genetics , Vesiculovirus/geneticsABSTRACT
The classical swine fever virus is the etiologic agent of one of the diseases with the greatest impact on swine farming worldwide. An extensive area of Brazil is considered free of the disease, but some states in Northeast Brazil have registered outbreaks since 2001. The objective of this study was to analyze the genetic variations of the virus and its spread over time and space. Partial sequences of the viral E2 protein obtained from samples collected during the Brazilian outbreaks were compared with sequences from the GenBank database (NCBI). The results demonstrated the continuous presence of the virus in the state of Ceará, with diffusion to at least two other states. The Brazilian Northeast virus presents specific polymorphisms that separate it from viruses isolated in other countries.
Subject(s)
Classical Swine Fever Virus , Classical Swine Fever , Viruses , Animals , Brazil/epidemiology , Classical Swine Fever/epidemiology , Classical Swine Fever/genetics , Classical Swine Fever Virus/genetics , Disease Outbreaks , Phylogeny , Swine , Viral Proteins/geneticsABSTRACT
OBJECTIVE: The aim of this study was to use partial Ul44 sequences (glycoprotein C) of Suid herpesvirus 1 to examine the evolution and dynamics of the virus in different periods and hosts. METHODS: Phylogenetic trees were constructed using the software MrBayes after analysis in the software jModelTest to evaluate the best phylogenetic models. The software SplitsTree 4.0 was used to create phylogenetic networks, and the BEAST program was used to generate data on phylogeography. Replication kinetics and serum neutralization tests were applied to tree strains from different phylogenetic groups. RESULTS: Ul44 sequences derived from domestic swine and wild swine clustered in different clades and had different selective pressures depending on the host. We found no differences in replication kinetics and serum neutralization tests in the strains tested. Data show that the evolution of herpesviruses is complex, and different genetic groups may be evolving at different rates. Ul44 is an important marker for molecular evolution and epidemiology studies, but it is not useful for biological information.
