A Pilot Study on Hepatitis E Virus (HEV) Epidemiology in Animal Reservoirs in Bulgaria


 Information on hepatitis E virus (HEV) epidemiology in animal reservoirs in Bulgaria is still lacking. Herein, by screening HEV seropositive sera obtained from Bulgarian swine and wild boars, viral RNA was detected at high prevalence rate (28.2%) in industrial pigs. Sequence analysis of the partial polymerase (RdRp) region revealed the highest genetic correlation with HEVs of genotype (Gt) 3 identified in French and Dutch patients. For three such strains a 700-bp fragment of the ORF 2 gene was generated. On phylogenetic analysis, the Bulgarian strains clustered tightly (93.8-98.3% nt) with human and animal HEVs classified within the Gt3 subtype c.

molecular epidemiology and on the genetic features of the circulating strains has not yet been collected, thus hindering to depict a complete portrait on the impact that animal reservoirs may play in the transmission of infection to humans. In this study, we report the results of a preliminary molecular survey performed on serum samples collected between January 2018 and February 2020 from a selection of 39 HEV seropositive pigs and wild boars in different districts of Bulgaria [12,14,15]. In detail, ten sera (collection SNB: Swine Northern Bulgaria) were obtained from fattening pigs bred in an industrial farrowto-nish farm in Vidin district (Northern Bulgaria), ten (collection SSB: Swine Southern Bulgaria) were from fattening pigs farmed in an industrial Southern Bulgaria herd located in the Yambol district, and an additional nine group of sera (collection SEB: Swine Eastern Bulgaria) was collected from fattening East Balkan swine, an aboriginal pig breed, from Burgas district (Eastern Bulgaria). Furthermore, 10 sera were collected from wild boars during the o cial hunting season (from November 2019 to February 2020) in  [17] into the Topo TA cloning vector (Invitrogen, Ltd, Milan, Italy). Tenfold serial dilutions of the plasmid ranging from 10 0 to 10 9 copies per reaction were used in each PCR run. A standard curve was generated from the copy number and corresponding cycle threshold (Ct) value. The rst WHO international standard for HEV RNA (code 6329/10) was used to standardize the system. On molecular screening by qRT-PCR of 39 serum samples, HEV RNA was detected in a total of 11 sera (28.2%; 11/39) with viral loads ranging from 7.4 x 100 to 3.5 x 102 RNA copies/5 μl of template (mean 1.3 x 102 RNA copies) ( Table 1). Out of 11 positive specimens, 10 (90.9%; 10/11) were identi ed in pigs from Northern Bulgaria (collection SNB) with an overall prevalence of 100% (10/10) and only one (10.0%; 1/10) was detected in a pig from the collection SSB. None of the sera from East Balkan swine and from wild boars resulted positive for HEV RNA. When testing the sera with pan-hepeviruses primer pairs [18], nine samples all of collection SNB (90.0%; 9/10), yielded a band of expected size (Table 1). Partial RdRp sequences were determined and deposited in GenBank under the accession numbers MZ519907-MZ519915. Upon BLAST (www.ncbi.nlm.nih.gov/blast) and FASTA (www.ebi.ac.uk/fasta33) analyses, the amplicons shared 99.4-100% nucleotide (nt) identity to each other and displayed the closest relatedness (96.4-98.8%) to human HEV Gt3 strains previously detected in serum samples from French patients (GenBank accession no. MW355218, MW355367, MW355268, MW355295) [19] and in solid organ transplant recipients in the Netherlands (GenBank accession no. JQ015416 and JQ015424) [20].
In order to achieve a more clear characterization of swine HEVs detected, all of the positive samples were further tested by RT-PCR and nested PCR targeting respectively regions of 755 bp and 348 bp of the ORF2 gene [21]. The sequence of ~ 700 nt was obtained for the strains SNB2, SNB6 and SNB8 (GenBank accession no. MZ555941-42 and MZ555944), whilst one additional specimen, HEV SNB7 (MZ555943), resulted positive after second-round ampli cation in nested PCR ( Table 1). The partial ORF2 genes (~ 700 nt) were compared to reference and prototype sequences of known HEV Gt3 subtypes [22] and used to generate a Maximum Likelihood tree [23]. In the partial capsid-based tree (Fig. 1), the Bulgarian strains segregated within the clade 1 (abchkijlm) [22,24,25] displaying the highest identity (93.8-98.3%) to animal and human HEVs assigned to subtype c. Identities to the other Gt3 subtypes were from 81.6% (Gt3ra) to 88.9% (Gt3i).
We next compared the ORF2 sequences of the porcine strains detected in this study with the sequences of 63 Bulgarian human Gt3 HEVs [10] available on the database, since the sequences overlapped by ã 240 nt portion. The overall nucleotide identity ranged from 80.1% to 93.9%, with the best match (92.0-93.9%) to the strains ISS2/Sof2013 and ISS76/Dob/2014 (GenBank accession no. MH203165 and MH203204) classi ed as subtype c ( Table 2).
In the last few years, improved surveillance activities in Bulgaria have shown that zoonotic HEV infection is emerging [8][9][10]. Herein, to provide insights into the epidemiology of HEV, we screened by a multitarget gene approach a collection of sera from Bulgarian swine and wild boars previously found positive for HEV IgG antibodies. HEV RNA was detected in 28.2% (11/39) of the animals tested. This high molecular rate seems to con rm previous suspicious on a lively viral spread in the geographical area analysed. Serosurveys performed in Bulgaria report prevalences ranging from 40.0% to 60.3% in domestic swine [11][12][13], to 40.8% in wild boars [15] and to 82.5% in East Balkan swine (autochthonous pig breed) [14].
Out of the four collections assessed in this study, the highest prevalence (100%; 10/10) was found in pig sera obtained from a closed-cycle industrial farm in North Bulgaria (collection SNB), in which speci c HEV antibodies were detected with an overall rate of 63.3% (19/30) (data unpublished). Accordingly, it could be hypothesized that at the sampling time point most of the pigs were seropositive and viremic. Also, sequence analysis of the RdRp fragment (338 nt) generated for 9 strains revealed a clonal origin (99.4-100% identity) that may be compatible with the circulation of the same variant within the herd investigated.
Only one strain (10.0%) from collection SSB with an estimated seroprevalence of 23.3% [12], resulted positive by quantitative RT-PCR, but attempts made to obtain sequence data failed. All serum samples collected from East Balkan swine (seroprevalence rate of 76.7%) [14] and wild boars (seroprevalence rate of 73.3%) [15] resulted negative either when tested with quantitative or conventional PCR. It is likely that these results rather than mirroring the actual geographical distribution of HEV in Bulgaria, are merely linked to other factors such as the absence of viraemic status at the time of sampling and/or the limited number of animals analysed.
Data on the diffusion and distribution of HEV Gt3 subtypes circulating in Bulgaria in human population are still limited. Phylogenetic and coalescence analyses based on a 355-nt partial ORF2 region of 63 HEV Gt3 strains identi ed in patients with hepatitis E [10,26] provided evidence that subtypes 3e (39/63, 62%), 3f (15/63, 24%) and 3c (8/63, 13%) are the most common. In our study, sequence analysis of a 700-nt portion of ORF2 allowed a subtyping classi cation of the swine Bulgarian strains SNB2, SNB6 and SNB8, revealing the highest genetic correlation to HEV Gt3 subtype c. This cluster comprises HEV strains of human and animal origin frequently signalled in Europe [25]. Accordingly, the spread of this subtype in Bulgaria could be accounted for by geographical changes in the circulation of HEV strains, likely due to trading of HEV-infected animals from other European countries. We retrieved from GenBank database all of the Bulgarian HEV human sequences [10] and compared with the porcine sequences generated in this study. On pairwise alignment of a 240-nt ORF2 overlapping fragment, two human sequences (ISS2/Sof2013 and ISS76/Dob/2014) were genetically similar (92.0-93.9%) to the HEV strains detected in swine, suggesting that similar HEVs are circulating in humans and animals in the same geographical setting.
In summary, our study con rmed molecularly the presence of HEV in pigs in Bulgaria. A high prevalence of HEV RNA was found in an industrial pig farm from Northern Bulgaria. Large-scale molecular surveillance at environmental/animal/food levels could be useful to improve the knowledge on HEV ecology in order to establish local adequate control measures. RT-PCR ORF2 [21] Nested PCR ORF2 [21] SNB (Swine Northern Bulgaria) Phylogenetic tree constructed on the partial ORF2 gene of three Bulgarian HEV swine strains (GenBank accession no. MZ555941, MZ555942 and MZ555944). Tree was generated using Maximum Likelihood method based on the Tamura-Nei model and supplying statistical support with bootstrapping of 1000 replicates. The scale bar indicates nucleotide substitutions per site. The reference strains representative