Desenvolvimento e padronização de PCR digital e metagenômica viral para avaliação do Senecavirus A em surtos de doenças vesiculares ocorridos em suínos no Brasil

Abstract

In February 2015, there was an outbreak of vesicular diseases on pig farms in different states of Brazil. As a result, swine biological samples were sent to one of the laboratories of Ministry of Agriculture, Livestock and Supply (MAPA), the Laboratório Federal de Defesa Agropecuária de Minas Gerais (LFDA-MG). The only infectious agent found in these samples was Senecavirus A (SVA). This virus is of Senecavirus genera belonging to Picornavirales Order, Picornaviridae family and Riboviria realm. The Picornaviridae family also includes the Aphtovirus genera, whose species type is Foot-and-mouth disease virus (FMDV), which causes the foot-and-mouth disease, a highly notifiable disease under the strict control of MAPA and recommended by the World Organization for Animal Health (OIE). The SVA had never been reported in Brazil, having been officially reported by the LFDA-MG and first published by two Brazilian groups in July 2015. Highlighting the fact that the disease caused by SVA is incipient in the country and the world at large, pointing to the need for basic and applied studies of SVA. The objective of this study was to check the presence of SVA and other viruses in these biological samples, and analyse their association with the vesicular disease outbreaks which occurred on the pig farms of different Brazilian states using the viral metagenomic approach. In addition to techniques requiring no cell culture, viral isolation and characterization of these isolates were also performed, aiming at the standardization of techniques such as viral seroneutralization, among others, for the improvement of SVA diagnosis in official Brazilian laboratories. Digital PCR for FMDV and SVA (RT-ddPCR) were developed and standardized, evaluating parametres such as analytical and diagnostic sensitivity and specificity. Swine samples (epithelium and serum) belonging to the outbreak of vesicular disease of 2015 were extracted and submitted to RT-ddPCR-SVA in order to be selected for viral metagenomics analysis (virome) from the absolute quantification data. Virome was performed from total nucleic acid in six samples (pool of epithelium or serum) with subsequent elimination of DNA. The HiSeq platform was used and data analysis was performed using the MetLab pipeline with the addition of quality parameters. The present work offered two new tools (RT-ddPCR) sensitive and specific for the diagnosis of FMDV and SVA, and the RT-ddPCR-SVA was applied in the samples submitted to virome. In relation to virome analysis, 19 families of RNA and 21 of DNA were initially classified. However, after joint evaluation using the following criteria: filter by size, e-value, identity / similarity, coverage, evaluation of the contig region, these numbers reduced to one family of RNA (Picornaviridae) and four (4) families of DNA (Geminiviridae, Inoviridade, Microviridae and Siphoviridae). Senecavirus A was the only RNA virus confirmed and identified in five (5) of six (6) samples. In addition to this virus, phages and a plant virus were classified and confirmed. The present work contributed to the validation of the viral metagenomic approach and digital PCR in government reference laboratories for use, especially in outbreak situations, as well as in the epidemiological surveillance of viral agents in animal species of importance in the national scenario. In addition, it highlights the importance of the need for strict quality control, taking into account several parameters with regards to the confirmation of viral classifications in order to avoid false positive results and erroneous speculation from massive data.