| dc.description.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. | |
