RESULTS AND DISCUSSION
We detected RVH in nine out of the 103 pooled samples (8.7%, Figure 1),
most of them from fattening pigs (6 positive samples out of 33) or
postweaning-growing pigs (2 positive samples out of 11) while only one
positive sample was detected in nursing piglets. A similar result was
reported in commercially raised pigs in the USA (15% of positive fecal
samples) being the odds of RVH positive 5.9 in the >55-day
group as compared with odds for the 4-20-day piglets (Marthaler et al.,
2014). However, in our research, the number of RVH outbreaks did not
differ significantly between age groups when compared using Fisher exact
test (p=0.139). Although no significant differences were demonstrated in
the number of RVH outbreaks between age groups, clearance of maternal
antibodies together with the mix of piglets after weaning may explain a
higher percentage of positive samples in postweaning pigs
(>21 days-old) as compared with suckling piglets (18%
versus 4%).
Whole genome sequencing was attempted in all the strains and we achieved
the complete genome (11 segments) in three of them (SP-VC18, SP-VC29 and
SP-VC36). In another strain (SP-VC19) complete segment sequence was
achieved for VP4, VP6, VP1, NSP1, NSP2, NSP4 and NSP5 while partial for
VP7, VP2, VP3 and NSP3. The nucleotide identity among sequences varied
between 82.0%–100% for VP7, 83.2%–100% for VP4, 86.1%–100% for
VP6, 87.0%–100% for VP1, 84.4%–100% for VP2, 81.6%–100% for
VP3, 84.0%–100% for NSP1, 91.0-%–100% for NSP2, 83.5%–100% for
NSP3, 81.5%–100% for NSP4 and 92.3%–100% for NSP5.
The four sequences of porcine RVH strains recovered from Spanish farms
were compared with those available in Genbank, including partial and
complete genome sequences of porcine RVH isolates from Japan (n=11), USA
(n=2), Brazil (n=3), South Africa (n=1) and Vietnam (n=5), as well as
human (n=3) and bat (n=1) RVH strains.
High pairwise identities at the nucleotide levels (69.4-93.7%) were
observed for all genomic segments of the Spanish porcine RVH strains
when compared with the proposed porcine RVH genotypes (Appendix). In
contrast, Spanish porcine RVH strains were distantly related to human
RVH strains at the nucleotide levels (31.3–71.3%), as well as to the
bat RVH strain (15.9-68.0%). Accordingly, the ML phylogenetic trees
shown that Spanish porcine RVH strains were more closely related to the
porcine RVH strains from Japan, USA, Brazil, South Africa and Vietnam,
and more distantly related to human RVH strains from Bangladesh and
China and were also distinct from a bat RVH strain from Cameroon (Figure
2). Phylogenetic analyses indicate that genomic sequences of RVH
isolates recovered from different host species cluster in clearly
distinct sub-groups (porcine, human and bat), suggesting the lack of
recent interspecies transmission events. However, this observation is
based on a very limited number of fully sequenced RVH strains recovered
until now in these three different host species, particularly in humans
and bats. More global whole genome RVH sequences are needed to
conclusively determine the RVH evolutionary pathways and their zoonotic
potential.
Recently a full genome-base genotyping system for RVH has been proposed
(Suzuki & Inoue, 2018). Based on the recommended cut-off values, the
Spanish porcine RVH strains were classified into one or two different
genotypes for each genomic segment (Table 2 and Figure 2). We proposed a
new genotype for VP7 (G11), since the pairwise sequence identities of
SP-VC29 and SP-VC36 exceeded cut-off values proposed for the genomic
segments (86%) when compared with the rest of the available strains
(Appendix). However, this result should be taken with caution due to
limited number of RVH sequences available. This is particularly obvious
in the VP6 segment (Figure 2), where the ML tree clustering seems to
point to a single – very diverse – group, but according to the
proposed thresholds the isolate SP-VC36 might be considered a new
genotype (Appendix). More RVH sequences should be added to the proposed
genotyping system and cut-offs for each genome segment should be
reviewed based on them.
Herein we report the first complete genome sequence of four porcine RVH
strains from Spain being the first RVH strains identified in Europe. Our
data indicate that RVH is relatively widespread in Spanish swine
population being identified in almost 9% of the investigated diarrhea
outbreaks. In addition, full genome sequencing showed its usefulness in
the characterization the isolates, will facilitate further RVH
surveillance in pigs globally as well as the development of improved
diagnostic methods for RVH detection on swine farms.