First National Prevalence in Italian Horse Population and Phylogenesis Highlight a Fourth Sub-Type Candidate of Equine Hepacivirus

Abstract

Equine hepacivirus (EqHV, Flaviviridae, hepacivirus) is a small, enveloped RNA virus generally causing sub-clinical hepatitis with occasional fatalities. EqHV is reported in equids worldwide, but for Italy data are limited. To address this, a survey study was set up to estimate prevalence at a national level and among different production categories (equestrian; competition; work and meat; reproduction) and national macro-regions (North, Central, South, and Islands). Data obtained testing 1801 horse serum samples by Real-Time RT PCR were compared within the categories and regions. The NS3 fragment of the PCR-positive samples was sequenced by Sanger protocol for phylogenetic and mutational analysis. The tertiary structure of the NS3 protein was also assessed. The estimated national prevalence was 4.27% [1.97-6.59, 95% CI] and no statistical differences were detected among production categories and macro-regions. The phylogenesis confirmed the distribution in Italy of the three known EqHV subtypes, also suggesting a possible fourth sub-type that, however, requires further confirmation. Mutational profiles that could also affect the NS3 binding affinity to the viral RNA were detected. The present paper demonstrates that EqHV should be included in diagnostic protocols when investigating causes of hepatitis, and in quality control protocols for blood derived products due to its parental transmission.

Keywords: Italy; biomolecular prevalence; equine hepacivirus; horses; phylogenesis.

Figure 1

EqHV PCR-positive samples detected in this study are shown on the map. Red dots represent equestrian (EQU) positive samples, red diamonds competition (COM) positive samples, red triangles work/meat (W/M) positive samples, and red squares reproduction (REP) positive samples. Only regional borders are reported. The green shading represents the numeric density of the Italian equine premises.

Figure 2

Phylogenetic analysis of NS3 partial gene of equine hepacivirus. The figure reports the phylogenetic tree based on 497-nt fragment of NS3 gene of 35 EqHV strains detected in the present study or retrieved from GenBank database, including the reference sequences of sub-types 1 (O and ●), 2 (O and ●), and 3 (O and ●). Molecular evolutionary analyses were performed using Maximum Likelihood method based on the Tamura–Nei model with bootstrap test (n = 1000). Bootstrap values > 50% are shown. In grey dotted ellipses, the potential transmission clusters can be observed (all supported by 99% of bootstrap). Sequence KC411778 [46] of rodent hepacivirus was used as outgroup. Scale bar indicates nt substitutions per site.

Figure 3

The three-dimensional structure of equine hepacivirus NS3 protein constructed by I-Tasser software using the PDB model of the human hepacivirus (HCV 1CU1) as reference. The functional domains are color-coded in the equine hepacivirus NS3 protein as follows: serine protease domain (1–180, green); helicase subdomain I (181–326 cyan); helicase subdomain II (327–481 dodger blue); helicase subdomain III (482–631 navy blue); residues S356, S357, L358, V377, D396, N405, V447, S488, and T490, showing their side chains as spheres, are reported in grey.