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.