Inter-laboratory comparison of eleven quantitative or digital PCR assays for detection of proviral bovine leukemia virus in blood samples.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts.

Interfering factors in the diagnosis of Senecavirus A.

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.

Phylodynamics of avian influenza A(H5N1) viruses from outbreaks in Brazil.

Our study identified strains of the A/H5N1 virus in analyzed samples of subsistence poultry, wild birds, and mammals, belonging to clade 2.3.4.4b, genotype B3.2, with very high genetic similarity to strains from Chile, Uruguay, and Argentina. This suggests a migratory route for wild birds across the Pacific, explaining the phylogenetic relatedness. The Brazilian samples displayed similarity to strains that had already been previously detected in South America. Phylogeographic analysis suggests transmission of US viruses from Europe and Asia, co-circulating with other lineages in the American continent. As mutations can influence virulence and host specificity, genomic surveillance is essential to detect those changes, especially in critical regions, such as hot spots in the HA, NA, and PB2 sequences. Mutations in the PB2 gene (D701N and Q591K) associated with adaptation and transmission in mammals were detected suggesting a potential zoonotic risk. Nonetheless, resistance to neuraminidase inhibitors (NAIs) was not identified, however, continued surveillance is crucial to detect potential resistance. Our study also mapped the spread of the virus in the Southern hemisphere, identifying possible entry routes and highlighting the importance of surveillance to prevent outbreaks and protect both human and animal populations.

Use of FTA card for the detection of two RNA (CSFV and SV-A) and two DNA viruses (ASFVand SuHV-1) of importance in veterinary medicine.

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.

First report and genetic characterization of the highly pathogenic avian influenza A(H5N1) virus in Cabot's tern (Thalasseus acuflavidus), Brazil.

In 2021, the H5N1 virus lineage 2.3.4.4b spread to the Americas, causing high mortality in wild and domestic avian populations. South American countries along the Pacific migratory route have reported wild bird deaths due to A/H5Nx virus since October 2022. However, limited genomic data resulted in no cases reported in Brazil until May 2023. Brazil reported its first case of highly pathogenic avian influenza virus (HPAI A/H5N1) in May 2023. The virus was detected in Cabot's tern specimen in Marataízes, Espírito Santo. Cases were also found in backyard poultry and other wild birds, but no human or commercial poultry cases occurred. HPAI poses risks to the poultry industry, food security, and public health. Researchers used next-gen sequencing and phylogenetic analysis to study the Brazilian sample. It confirmed its affiliation with the 2.3.4.4b clade and proximity to sequences from Chile and Peru. This sheds light on the spread and evolution of HPAI A/H5N1 in the Americas, emphasizing continuous monitoring to mitigate risks for both avian and human populations. Understanding the virus's genetics and transmission allows implementing effective control measures to protect public health and the poultry industry.

Genetic differentiation of Megalocytivirus by real time PCR and sequencing.

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.

Quantification of in vitro replication kinetics of Alagoas vesiculovirus isolates by digital droplet RT-PCR.

Vesicular stomatitis caused by Alagoas vesiculovirus (VSAV) has generated disease outbreaks in Brazil, mainly in the northeast region. Phylogenetic studies divide the isolates into three distinct genotypes (A, B, and C). However, there is no description of how this genetic divergence reflects on the phenotype of VSAV isolates such as in vitro replication fitness. Therefore, the objective of this work was to evaluate the ability of three distinct genotypes of Brazilian isolates of VSAV to grow in different cell-culture lines (BHK-21, Vero, and NCI-H1299). Quantification of viral RNA was performed using RT-PCR digital droplet from supernatant of cell culture collected every 4 h for a period of 24 h of viral growth in three different cell lines (BHK-21, Vero, and NCI-H1299). It was observed that the genotype C isolate has the lowest replication efficiency among the three analyzed viruses, without major changes in the copies of viral RNA over the entire time of the study.

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.

Detection of megalocytivirus in Oreochromis niloticus and Pseudoplatystoma corruscans in Brazil.

The infectious spleen and kidney necrosis virus (ISKNV) belongs to the genus Megalocytivirus (MCV), a group of double-stranded DNA genome viruses. The aim of this study was to retrospectively analyze samples from suspected foci of MCV infection in freshwater fish in Brazil. Samples were collected from infected fish between 2017 and 2021. Phylogenetic analysis revealed 2 groups of MCV circulating in the country. A genetically homogeneous group formed a clade with ISKNV samples from different parts of the world. Only 2 of the sequences from the state of Goiás showed a small genetic distance when compared to the larger group in the same clade. This study describes the validation of 3 qPCR methods and the presence of MCV in Brazil since 2017, including a genotype not previously described.

Phylodynamics of classical swine fever virus in Brazil.

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.

Evaluation of three different genomic regions for detection of bovine leukemia virus by real-time PCR.

Bovine leukemia virus (BLV) is an oncogenic member of the genus Deltaretrovirus. BLV infects cattle worldwide and is responsible for significant economic losses. The objective of this study was to validate real-time quantitative PCR (qPCR) for the detection of BLV. After identification of the most efficient qPCR, the limits of detection, repeatability, and reproducibility were determined. The results indicate that qPCR can be easily reproduced between laboratories with high sensitivity. The test variation was low in samples from lesions suggestive of bovine leukosis or whole blood.

Study of molecular diagnosis and viremia of bluetongue virus in sheep and cattle.

Bluetongue virus (BTV) is an RNA virus that infects cattle and sheep. The objective of this study was to compare two real-time PCRs for the detection of BTV and to monitor Orbivirus viremia in sheep and cattle for 6 months. The PCR results showed the occurrence of infected animals throughout the experiment without records of clinical signs. The number of positive animals reduced during the experiment, but some animals were positive for BTV RNA during the entire experiment. The performance of the two RT-qPCRs for BTV detection techniques used in this work revealed a kappa index of 0.71 for cattle and 0.75 for sheep.

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.

Evidence for current circulation of an ancient West Nile virus strain (NY99) in Brazil.

INTRODUCTION: In Brazil, West Nile virus (WNV) was first detected, in 2018, in horses with neurological disease. AIM: We report the first case of WNV infection in a horse from Ceará state and the complete genome sequence of an isolate from Espírito Santo state. Both infections occurred in 2019. METHODS: WNV was isolated from the tissues of a horse with neurological signs in Espírito Santo and sequenced by MiSeq. RESULTS: Phylogenetic analysis revealed that the isolate belongs to lineage 1a, clustering with the NY99 strain, a strain that has not circulated in the USA since 2005. CONCLUSIONS: Our findings reinforce the hypothesis that WNV has been silently circulating in Brazil for many years.

Evidence for current circulation of an ancient West Nile virus strain (NY99) in Brazil

Abstract INTRODUCTION: In Brazil, West Nile virus (WNV) was first detected, in 2018, in horses with neurological disease. AIM: We report the first case of WNV infection in a horse from Ceará state and the complete genome sequence of an isolate from Espírito Santo state. Both infections occurred in 2019. METHODS: WNV was isolated from the tissues of a horse with neurological signs in Espírito Santo and sequenced by MiSeq. RESULTS: Phylogenetic analysis revealed that the isolate belongs to lineage 1a, clustering with the NY99 strain, a strain that has not circulated in the USA since 2005. CONCLUSIONS: Our findings reinforce the hypothesis that WNV has been silently circulating in Brazil for many years.

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.

A molecular survey using a validated real-time PCR assay finds no evidence of bovine alphaherpesvirus 2 in samples from animals with suspected vesicular disease in Brazil between 2014 and 2017.

Bovine alphaherpesvirus 2 (BoHV-2) is the etiologic agent of bovine mammillitis (BM) and pseudo-lumpy skin disease. BM is also important because its clinical presentation can be confused with foot-and-mouth disease (FMD), making it necessary to establish differential diagnoses and perform additional laboratory tests. The objective of this work was to use a validated real-time PCR assay to test for the presence of BoHV-2 in samples from cattle and buffalo with suspected vesicular disease in Brazil. The method could detect the virus at a concentration of 0.5 fg/µL and had 99.4% amplification efficiency, a repeatability error of only 4.1%, and good reproducibility with other reagents. No evidence of BoHV-2 causing vesicular disease in cattle and buffalo was found in this work. This study was able to validate a new methodology for detection of BoHV-2 and evaluate its usefulness for investigating outbreaks of vesicular disease Brazil. The importance of BoHV-2 in cases involving other clinical signs should still be studied using the qPCR developed in this work.

Validation of a real-time PCR assay for detection of swinepox virus.

Swine are the only known hosts of swinepox virus (SWPV), the sole member of the genus Suipoxvirus, family Poxviridae. Rapid diagnosis is recommended for appropriate interventions because of the high morbidity associated with this virus. This study describes a real-time quantitative PCR (qPCR) assay for rapid detection and quantification of SWPV. The detection limit, repeatability, reproducibility, and specificity of this assay were determined. The efficiency was 96%, and the R2 value was 0.996. The detection limit was 1 fg or 10-0.5 TCID50/50 µL. Tests showed that the greatest source of error in the SWPV qPCR assay was variation between analysts rather than different qPCR kits or equipment. All nucleic acids from other viruses or samples collected from swine were negative in the specificity test. qPCR for SWPV is a new method with tested variables that allows main sources of error in laboratory diagnosis and viral quantification to be identified.

Quantification of ovine herpesvirus 2 by digital PCR in an outbreak of malignant catarrhal fever.

Infection with ovine gammaherpesvirus 2 (OvHV-2) is generally asymptomatic in sheep; however, when it crosses the species barrier, it causes malignant catarrhal fever (MCF) in cattle. In the present study, we developed a real-time PCR assay and a droplet digital PCR assay and use both methods to study an outbreak caused by OvHV-2. Both PCR methods showed high sensitivity and specificity and were able to detect low copy numbers of OvHV-2 in sheep and cattle. The present study describes the first digital PCR quantification of OvHV-2 genome copies in samples collected from sheep and cattle.

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.