Zoonotic transmissions between humans and swine have been highly suspected since partial HEV sequences from both hosts can share more than 99% identity (4
) and since experimental cross-infection of subtype 3a HEV in pigs and primates leads to productive HEV infections (24
). Subtype 3f HEV is the most common subtype in France and Europe (4
) and has been shown to be circulating actively between humans and swine (4
). This subtype was selected to study the effect of an interspecies transmission on the genomic adaptation of HEV in its full-length consensus sequence and its quasispecies.
The oral route mimics natural infection of this enterically transmitted disease but has been shown to be less efficient than intravenous or intrahepatic routes (18
). In the present study, oral exposure of pigs to human subtype 3f HEV led to a productive HEV infection. A previous observation that oral exposure to human genotype 1 did not give rise to infection may thus have been related to the restriction of genotype 1 to humans rather than to the route of inoculation (2
Surprisingly, no nucleotide mutations could be found over the full-length consensus sequence amplified after the interspecies transmission, which demonstrates a clear adaptation of genotype 3 HEV to both humans and swine. Additionally, 29% (12/42) of polymorphic sites of HEV from the human sample were effectively infectious and found to be excreted in the feces of pigs at 16 dpi. As demonstrated in other studies, this spectrum of mutations does not necessarily increase fitness of one virion but might rather lead to increased infectivity and zoonotic potential through the diversity of HEV quasispecies population (7
Conversely, not all polymorphic sites could be transmitted since a large number of mutations were deleterious. At least 2 to 20.7% of HEV quasispecies population have been found to be nonviable. These results represent the lowest range of nonviable sequences since no mutations other than stop codons or proline were considered. Sanjuan et al. estimated that up to 40% of random mutations in RNA viruses are lethal (35
The ratio of transitions/transversions observed in the present study is indicative of whether or not the mutations observed are random. In phylogeny, a bias toward a ratio of 1 is commonly observed since transitions seem favored over transversions, possibly as a result of the underlying chemistry of mutation. In the present study, a ratio closer to 0.5 has been observed, suggesting that mutations seem to occur at random. It is then possible to infer that the development of HEV quasispecies occurs at random, resulting in a high proportion of deleterious mutants, as stated by Sanjuan et al. (35
). HEV quasispecies is then purified of its deleterious mutations, as shown by the negative dN
As Belshaw et al. discussed, mutations and substitutions occur at different tempos and at different biological levels (3
). Substitutions are defined as mutations which are fixed in a population. The present study dealt with nonfixed mutations undetectable at the level of the consensus sequence but observed at the level of the quasispecies and therefore expressed as the average percentage of SNPs. Nonetheless, a correlation in the variation of the mutation rate along the genome observed in this study could be made with a previous report studying the substitution rate along HEV genomes. Variation in the substitution rate along the genome of HEV has been predicted previously as being lower for the region encoding the RdRP (8.4 × 10−4
substitutions per site per year) than for the complete genome (1.51 × 10−3
substitutions per site per year) (30
). In the present study, the mutation rate was also observed to be significantly lower in the RdRP (0.044%) than in other parts of the genome (up to 1.4%), which may be explained by higher functional constraints on this coding region.
In the end, HEV quasispecies resulted in a low-diversity and low-complexity population compared to other human RNA viruses such as HIV or HCV (33
). HEV intrahost nucleotide diversity is closer to what has been found for the zoonotic virus WNV. Indeed, viruses that need to infect diverse hosts to produce a full viral cycle, like arboviruses, are subjected to higher constraints and thus evolve more slowly than other RNA viruses (15
In addition to being a useful tool for discovering new pathogens (5
), HTS is also of great interest in delineating the quasispecies of viruses since the use of specific PCRs to amplify subgenomic regions of the virus, which could introduces bias, is avoided. But great care should be put into the handling of polymorphic data since various biases are reported for HTS techniques (13
Sequence coverage depth is, for example, critical when different samples are compared. Variations of polymorphism observed in this study between the three samples should not be considered as properties of HEV quasispecies in different hosts or sampling points but as a consequence of the differences in nucleotide coverage. A higher sequence coverage contains more information and is therefore more accurate in detecting low-frequency variants. The lower number of SNPs and the smaller intrahost nucleotide diversity observed for HEV from the human sample than from the pig samples are only the results of its lower coverage.
Interestingly, the mutation error rate for Illumina GaIIx calculated in this study as being 0.28% was the same as previously reported (26
). A number of insertions/deletions were found in the HEV sequences, all of which fell under the mutation error rate. The insertion/deletion rate generated by the amplification and high-throughput sequencing processes could be calculated as being 5.7 × 10−7
(data not shown), which is lower than what has been previously established (4 × 10−6
). This insertion/deletion rate was very likely reduced by the second mapping of HEV sequences, which removed all reads containing more than two mismatches.
Here is presented the first report on the use of HTS for the study of full-length genomes of HEV and, more generally, on the use of HTS to analyze viral variability upon interspecies transmission. The observation that the full-length consensus sequence of HEV is conserved in spite of a change of host demonstrates the absence of a species barrier and the clear adaptation of genotype 3f HEV to both hosts. Moreover, this study confirms that HEV exists as a quasispecies in the in vivo setting and that genetic variability extends throughout its genome. Finally, major SNPs were conserved during the interspecies transmission. These results may suggest that transmission of swine HEV to humans would result in the absence of adaptation and in a productive HEV infection.
In conclusion, the transmission of human HEV to pigs did not seem associated with a restriction in genetic diversity, most likely because HEV infection of either host does not impact its viral cycle. According to the typology of zoonosis proposed by Pepin et al. (29
), the transmission of some zoonotic agents can be governed only by ecological drivers. In this case, all viral genotypes circulating in the reservoir are already competent for transmission in the new host. Founder effects or adaptative fine-tuning in the new host could explain the variability of the strains. These results suggest that HEV could belong to this category of viruses.