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.