MATERIAL AND METHODS
The study was performed on 103 Spanish swine farms with diarrhea outbreaks in which a viral etiology was suspected between 2017-2019. The outbreaks affected nursing piglets (<21 days) (26 farms), postweaning-growing pigs (21-70 days) (11 farms) or fattening pigs (>70 days) (33 farms). The age of affected animals was unknown on 33 farms.
Total RNA was extracted from one pooled fecal sample (2-6 individual samples) per farm using QIAamp Viral RNA Mini Kit (Qiagen) following the manufacturer’s instructions. Then, we performed a reverse transcription PCR (RT-PCR) using a newly designed primer pair, based on the VP6 gene from reference sequences of porcine RVH strains obtained from Genbank (Table 1), and amplifying a 1240 nt fragment. The RT-PCR reactions were carried out with the Verso 1-Step RT-PCR ReddyMix Kit (Thermo Scientific), following the manufacturer’s recommendations, with the following cycling conditions: an initial step of 50ºC for 30 min and 95ºC for 2 min; followed by 45 cycles of 95ºC for 20 s, 50ºC for 30 s and 72ºC for 1 min; with a final extension step at 72ºC for 10 min.
From each positive pooled sample by RT-PCR to RVH, total RNA was extracted using a TRIzol LS reagent (Thermo Scientific) protocol. The total RNA extraction was directly sequenced at the Genomics Bioinformatics Service (SGB) of the Autonomous University of Barcelona (UAB), without using any primer or amplification step. Next Generation Sequencing (NGS) was carried out using an Illumina Miseq Platform. RVH sequences were obtained from NGS outputs applying a tailor-made, virus-specific script developed by us (Cortey et al., 2019). We confirmed each segment identified by BLAST analyses on the assembled sequences using the NCBI GenBank online tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences were aligned using CLUSTALW. The evolutionary relationships among sequences were analyzed with a phylogenetic analysis, using the Maximum-Likelihood method (ML) and the Tamura-Nei substitution model with MEGAX software (Kumar, Stecher, Li, Knyaz, & Tamura, 2018). Sequences were deposited in the NCBI GenBank with the accession numbers MT644949-MT644992.