Uncovering neglected subtypes and zoonotic transmission of Hepatitis E virus (HEV) in Brazil

We were able to sequence two complete genomic sequences (PRsw1 and PEsw1) (Table 1) and, four nearly complete porcine-derived genomic sequence (RJsw1, RJsw2, RJsw3 ad RJsw4), and a fragment of approximately 1000 nucleotides of the first recorded human-derived sequence in Brazil (RJh1). (Fig. 1a). The two complete genomic sequences exhibited the typical features of an HEV genome and were 7215 and 7233 nucleotides length, respectively. Both HEV-3 genomes contain three partially overlapping open reading frames, with ORF3 overlapping ORF1 and ORF2 (Fig. 1b).

Because we were not able to obtain complete genomic sequences of all positive samples and considering the epidemiological importance of the genetic information, we performed further analyses with multiple datasets to compare with HEV subtype reference sequences. Accordingly, we compared the two complete genomic sequences obtained, the nearly complete genomic sequences and the complete capsid sequence of all six sequences from pigs to a set of HEV-3 reference sequences (Fig. 2a and b). Our analysis suggests that full genome sequences from South (PRsw1) and Southeastern (RJsw1), previously classified as HEV-3 subtype i, likely represent a new subtype with nucleotide divergence of 15.5% from subtype ‘m’ together with two other sequences found in Uruguay (MW596896 and MZ969073) (Fig. 3; Supplementary Table 2). Moreover, the nucleotide divergence of PEsw1 from the northeast region, previously classified as subtype 3f in our previous study [20], differed from the HEV-3f reference sequence at 12.0%, but remained in the same subtype classification. Interestingly, both nearly-complete-genome- and capsid-based analysis suggests that strain RJsw2 (from Southern region) clustered between the subtype 3i and a group of unassigned sequences represented by MF959764 (UN-V). Strains RJsw3 and RJsw4, from the same region, are related to subtype 3b (Fig. 2b). On the one hand, the robust results on the capsid-based phylogeny corroborate that ORF2 sequences can be alternatively used in the absence of full genomic sequences (Fig. 2c), as previously suggested [11, 20, 26], including with subgenomic regions of ORF1 and ORF2 optimized for robust phylogenetic inference and subtyping [26]. By contrast, this illustrates the high genetic variability of the HEV strains circulating in Brazil and upholds the need to increase the number of complete genomic sequences from different samples from Latin America. Overall, the consistent topology derived from multiple inferences supports the robustness of the new proposed subtype.

To investigate the zoonotic origin of the first autochthonous human case of acute hepatitis E reported in Brazil, we compared the partial fragments of the capsid and the ORF1. Phylogenetic analyses using the ORF2 fragment, as well as the previously published ORF1 fragment, indicated that the sequence from the human patient (RJh1) clusters among those from pigs from southeastern region (RJsw3 and RJsw4) with nucleotide identities of 99.5 and 99.6%, respectively (Fig. 2d; Supplementary Fig. 1 and Supplementary Table 2). In the previous study [18], only small fragments of ORF1 and ORF2 were available, and by providing a larger fragment of ORF2, our data support the robustness of the evolutionary inferences and the evidence of zoonotic transmission. Therefore, regardless of whether strains RJsw3 and RJsw4 represent a new subtype or not, our results show strong evidence that the first reported case of HEV in Brazil is likely of zoonotic origin.

Noteworthy, our study is limited by the lack of success in obtaining larger fragments of four sequences which were probably related to the volume availability and conservation status of the samples. However, except for the human sample, the study provided a good amount of retrospective information that allowed robust analysis. Although our findings and those previously published support that the capsid-based analyses are robust, as recommended by the ICTV Hepeviridae Study Group, it would be necessary to obtain full genomic sequence before proposing a new subtype [12].

Finally, the genetic variability of HEV in Brazil, along with the occurrence of zoonotic transmission, illustrates the challenges and lack of knowledge of HEV epidemiology in South America. Therefore, further studies, including more representative sequences, are needed to investigate the genetic diversity of HEV in humans, animal reservoirs, food, and environmental samples to obtain an accurate picture of the genetic variability of HEV in South America and its implication in zoonotic transmission and pathogenicity.