High Prevalence and Genetic Heterogeneity of Genotype 3 Hepatitis E Virus in Wild Boar in Umbria, Central Italy

Abstract

Hepatitis E virus (HEV) is an important human pathogen and, in developed countries, most human infections are due to a zoonotic cycle, mainly maintained by domestic and wild suids. In European countries several genotype 3 strains have been found to circulate in human population and animal reservoirs, with human infections being related mostly to pork or wild boar meat consumption. In this study, we surveyed HEV circulation in wild boar in Umbria (Italy) during the 2021-2022 hunting seasons, using a stratified sampling. Liver samples were tested for HEV presence by real-time RT-qPCR. Positive samples were characterized by nested RT-PCR followed by sequencing of partial region of the capsid gene. Overall, 78 out of 179 wild boar liver samples tested positive to HEV (43.6%), with viral load ranging between 1.47 and 7.35 log genome copies/g (median 3.20 log). Variations, although not statistically significant, were observed considering geographical, age, weight, and gender factors, in terms of either prevalence or viral load. In particular, in animals younger than 1 year of age, the viral load was 2 log higher (median viral load of 5.50 log genome copies/g of liver tissue) than in older age groups. Sequence analysis characterized the 41 obtained sequences into genotype 3 subtype 3c (n = 4), 3f (n = 11), and 3e (n = 1), while 13 sequences clustered with two genotype 3 genomes (GenBank MF959764 and MK390971) still unassigned to subtypes. Additional, 12 sequences did not cluster with any known subtype or unassigned genome and on phylogenetic analysis segregated into two distinct groups of eight and four sequences, respectively. Interestingly, some wild boar sequences of subtype 3f were intermingled with sequences of HEV strains previously identified in human patients in Central Italy. Sharing of molecular data for HEV in animals is pivotal to decipher the intricate ecology of HEV.

Figure 1

Geographic distribution of the three different hunting districts (ATCs) in the region of Umbria.

Figure 2

Dispersion of HEV quantitative values in liver samples according to category. The box-and-whisker plot displays median values, first and third quartiles (boxes), minimum and maximum values (whiskers), and the intermediate individual values (dots) arranged according to the different categories.

Figure 3

Phylogenetic tree of the partial HEV ORF2 sequences obtained from wild boar (liver and muscle samples). The maximum likelihood phylogenetic tree was constructed using the Tamura–Nei model with a discrete gamma distribution and sites evolutionarily invariable (TN93 + G + I). Codon positions included were 1st + 2nd + 3rd + noncoding. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 73 nucleotide sequences and a total of 410 positions. Bootstrap values >70% are shown next to the branches. Analyses were conducted in MEGA X. Genotype and subtypes are referred according to Smith et al., 2020 [3]; genotype 3 genome sequences unassigned according to current classification (AB290313, KP294371, MF959764, LC260517, MF959765, MK390971) were reporting adding also the uc number adopted in the HEV typing tool.