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.

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 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.

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.

Validation of molecular diagnostics for the detection of pseudocowpox virus.

The pseudocowpox virus (PCPV) is recognized for causing exanthematic lesions in cattle and humans. The diagnosis is important because it is a zoonosis and its clinical signs can be confused with foot-and-mouth disease, a high-impact bovine disease in livestock. The objective of this work is to validate a SYBR Green qPCR and a conventional PCR for virus detection in bovine samples. Detection limit tests, repeatability, reproducibility, sensitivity, and specificity were compared. When two analysts were compared, results demonstrated that training and pipetting influence the repeatability. The qPCR was more sensitive than conventional PCR but showed nonspecific reactions distinguishable by the melting curve. Both showed high repeatability and reproducibility.

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.

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.

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.

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.

A common vaginal microbiota composition among breeds of Bos taurus indicus (Gyr and Nellore).

Describing the bovine vaginal microbiota is essential to better understand its physiology and its impact on health maintenance. Despite the economic importance of reproduction of these animals, bovine vaginal microbial community is still poorly described in comparison with rumen microbiome. Previous studies of our group described the vaginal microbiota of Nellore, an important Bos taurus indicus breed, using metagenomics. In order to better understand this microbiota, the present work aims to investigate another important breed, Gyr. Results have shown bacterial dominance over Archaea and Fungi was observed, with the most abundant bacterial phylum (Firmicutes) representing 40-50% of bacterial population, followed by Bacteroidetes, Proteobacteria, and Actinobacteria. The Fungi kingdom had the Mycosphaerella genus as its main representative, followed by Cladosporium. Archaea were observed at a very low abundance in all animals, with a high relative abundance of Methanobrevibacter genus. These results demonstrate a high microbial diversity on vaginal tract of Gyr, as demonstrated for Nellore and different from the previously described for other species. Our results indicate a great similarity between vaginal microbiota of Nellore and Gyr despite the differences in animal handling and genetic improvement. As observed for both breeds, individual variation is the largest source of microbial diversity between animals.

Development of a droplet digital RT-PCR for the quantification of foot-and-mouth virus RNA.

Foot-and-mouth-disease (FMD) is a highly contagious disease of domestic animals which can result in substantial economic losses, caused by the FMD virus (FMDV). The aim of this study was to develop and standardize a novel reverse transcriptase droplet digital PCR (RT-ddPCR) assay for the quantification of FMDV RNA. This assay was based upon an OIE-recognized real-time RT-PCR that detects the 3D-encoding region of FMDV. The limit of detection at 101.4 TCID50/mL and 26.5 copies was determined using FMDV-A24-Cruzeiro-virus and a plasmid containing the 3D-FMDV sequences, respectively. FMDV O, A and C serotypes and 11 species of non-FMDV were used to confirm the sensitivity and specificity of the assay. The RT-ddPCR was standardized using 60 bovine samples (representing negative and positive samples of epithelium and/or oesophageal-pharyngeal [OP] fluid) from animals suspected of vesicular diseases and previously tested by RT-qPCR. The RT-ddPCR showed robustness, sensitivity, specificity and accuracy, with similar results to the RT-qPCR. Moreover, the new RT-ddPCR diagnostic tool allowed the absolute quantification of FMDV RNA from epithelium and OP-fluid samples, as well as having the advantages of direct quantification by endpoint, eliminating the need for a calibration standard curve required in quantitative real-time RT-PCR.

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.

Spread of poxviruses in livestock in Brazil associated with cases of double and triple infection.

The objective of this work is to describe the distribution of outbreaks of vaccinia virus (VACV), pseudocowpox virus (PCPV), and bovine papular stomatitis virus (BSPV) in Brazil. The Official Laboratory of the Brazilian Ministry of Agriculture received 89 samples from different locations in Brazil in 2015 and 2016 for diagnosis of vesicular and exanthematous disease. Poxvirus coinfections occurred in 11 out of 33 outbreaks, including the first reported triple infection by BPSV, PCPV, and VACV. This occurrence may be associated with the circulation of these viruses in Brazilian cattle.

Detection of pseudocowpox virus in water buffalo (Bubalus bubalis) with vesicular disease in the state of São Paulo, Brazil, in 2016.

BACKGROUND: Parapoxviruses are zoonotic viruses that infect cattle, goats and sheep; there have also been reports of infections in camels, domestic cats and seals. OBJECTIVE: The objective of this report was to describe a case of vesicular disease caused by pseudocowpox virus (PCPV) in water buffalo (Bubalus bubalis) in Brazil. ANIMALS: Sixty buffalo less than 6 months old exhibited ulcers and widespread peeling of the tongue epithelium. There were no cases of vesicular disease in pigs or horses on the same property. METHODS: Samples were analysed by PCR and sequencing. Phylogenetic analysis in MEGA 7.01 was reconstructed using major envelope protein (B2L) by the Tamura three-parameter nucleotide substitution model and the maximum likelihood and neighbor joining models, both with 1000 bootstrap replicates. The genetic distance between the groups was analysed in MEGA using the maximum composite likelihood model. The rate variation among sites was modeled using gamma distribution. RESULTS: The presence of PCPV in the buffalo herd could be demonstrated in epithelium and serum. The minimum genetic distance between the isolated PCPV strain (262-2016) and orf virus and bovine papular stomatitis virus was 6.7% and 18.4%, respectively. The maximum genetic distance calculated was 4.6% when compared with a PCPV detected in a camel. Conclusions/Clinical Importance: The peculiar position of the isolated strain in the phylogenetic trees does not necessarily indicate a different kind of PCPV that infects buffalo. More samples from cattle and buffalo in Brazil must be sequenced and compared to verify if PCPV from buffalo are genetically different from samples derived from cattle.

Molecular epidemiology of senecavirus A associated with vesicular disease in pigs in Brazil.

Senecavirus A (SV-A) may cause vesicular disease and neonatal mortality in pigs, and was first detected in Brazil in 2015. Samples including tissues and serum from pigs with suspected vesicular diseases were collected from January to August in 2015 from farms in the states of Minas Gerais, Santa Catarina, Goiás and Rio Grande do Sul, Brazil, and tested for the presence of SV-A by reverse transcriptase PCR. All samples were negative for foot and mouth disease virus, as well as 13 other infectious agents associated with vesicular diseases in pigs. SV-A was detected by PCR in 65/265 (24.5%) specimens. A 530 base pair fragment sequenced from the VP1 protein coding region indicated a high genetic distance from SV-A in other countries, but a common origin among the Brazilian isolates.

Estudo comparativo e validação de três técnicas de PCR em tempo real (qPCR) para diagnóstico de Peste Suína Africana / Comparative study and validation of three quantitative PCR (qPCR) techniques for the diagnosis of African Swine Fever

Este estudo verificou o desempenho de três técnicas de PCR quantitativa (Real-Time) para o diagnóstico de Peste Suína Africana, uma doença exótica no Brasil, a partir de amostras de tecidos. As três técnicas escolhidas baseiam-se na amplificação de sequências do gene da proteína viral VP72 e são preconizadas, cada uma, por laboratórios oficiais da OIE (PSA-OIE), dos Estados Unidos (PSA-USDA) e da União Europeia (PSA-EU), respectivamente. Oligonucleotídeos iniciadores e sondas de hidrólise marcadas com fluoróforos foram sintetizados conforme a literatura de referência consultada. Sequências-alvo do DNA viral foram inseridos em plasmídeo sintético, os quais serviram de controle positivo para a padronização das técnicas e otimização de reagentes, determinação dos limites de detecção e testes de verificação de desempenho. Para aferição de repetibilidade e reprodutibilidade das técnicas, as técnicas padronizadas foram repetidas em dias diferentes, por um segundo analista, com alteração no mix comercial de reagentes utilizado e em um equipamento diferente, e também por outro laboratório. Realizaram-se, ainda, provas de sensibilidade analítica com amostras de DNA viral de referência e especificidade analítica e diagnóstica, com amostras negativas. As técnicas de PSA-EU e PSA-USDA apresentaram-se mais vantajosas quanto ao consumo de iniciadores. Não houve diferenças significativas nos resultados quantitativos variando-se os dias dos ensaios, os analistas, os equipamentos e o mix de reagentes. As três técnicas apresentaram alta especificidade analítica e diagnóstica e sensibilidade diagnóstica. As três técnicas de qPCR mostraram-se eficazes para serem adotadas por um mesmo laboratório para emissão de diagnósticos oficiais de Peste Suína Africana.(AU)

This study evaluated the performance of three real time PCR techniques (qPCR) for the diagnosis of African Swine Fever in tissue samples. The three chosen techniques are based on amplification of viral protein VP72 gene sequences and are recommended by OIE (PSA-OIE), the United States official laboratories (PSA-USDA) and the European Union (PSA-EU). Target sequences of the viral DNA were inserted into synthetic plasmid, which served as a positive control for the standardization of techniques and optimization of reagents, determination of limits of detection and performance verification testing. To gauge repeatability and reproducibility of techniques, standard procedures were repeated on different days by two analysts and by changing mix reagents and equipment, and also by another laboratory. Analytical sensitivity tests were done with reference samples provided by an OIE reference laboratory and analytical and diagnostic specificity were tested with negative samples. The PSA-EU and PSA-USDA techniques were more advantageous to use because of lower concentration of oligos used. There were no significant differences in quantitative results varying the days of tests, analysts, equipment and the mix of reagents. The three techniques had high analytical and diagnostic specificity and sensitivity. The three qPCR techniques were considered equivalent and effective and can be adopted by any laboratory for issuing official diagnosis of African Swine Fever.(AU)

Detection of multiple viral infections in cattle and buffalo with suspected vesicular disease in Brazil.

Vesicular diseases are of high importance for livestock, primarily because of foot-and-mouth disease (FMD), which is a high-morbidity disease that generates direct losses caused by low milk production, weight loss, and indirect losses because of the need for sanitary barriers. Other vesicular diseases are also of importance for livestock because of direct impacts or because their clinical signs may be confused with those of FMD. We report herein the detection of multiple infections in cattle with suspected vesicular disease in the Brazilian states of Amazonas (AM), Mato Grosso (MT), and Roraima. Thirty-seven epithelial samples from cattle and 1 sample from a buffalo were sent to the laboratory for testing for FMDV and similar disease agents. All samples from MT were positive for parapoxvirus (Pseudocowpox virus and Bovine papular stomatitis virus). In addition, 3 samples were positive for Bluetongue virus, and 5 samples were positive for Bovine herpesvirus 1 Among these samples, 1 was positive for all of these 3 agents. Only 2 samples from AM were negative for parapoxvirus. The molecular tests conducted in this study detected multiple infections, with a high prevalence of parapoxvirus.