Ancient recombination events and the origins of hepatitis E virus
© The Author(s). 2016
Received: 29 March 2016
Accepted: 30 September 2016
Published: 12 October 2016
Hepatitis E virus (HEV) is an enteric, single-stranded, positive sense RNA virus and a significant etiological agent of hepatitis, causing sporadic infections and outbreaks globally. Tracing the evolutionary ancestry of HEV has proved difficult since its identification in 1992, it has been reclassified several times, and confusion remains surrounding its origins and ancestry.
To reveal close protein relatives of the Hepeviridae family, similarity searching of the GenBank database was carried out using a complete Orthohepevirus A, HEV genotype I (GI) ORF1 protein sequence and individual proteins. The closest non-Hepeviridae homologues to the HEV ORF1 encoded polyprotein were found to be those from the lepidopteran-infecting Alphatetraviridae family members. A consistent relationship to this was found using a phylogenetic approach; the Hepeviridae RdRp clustered with those of the Alphatetraviridae and Benyviridae families. This puts the Hepeviridae ORF1 region within the “Alpha-like” super-group of viruses. In marked contrast, the HEV GI capsid was found to be most closely related to the chicken astrovirus capsid, with phylogenetic trees clustering the Hepeviridae capsid together with those from the Astroviridae family, and surprisingly within the “Picorna-like” supergroup. These results indicate an ancient recombination event has occurred at the junction of the non-structural and structure encoding regions, which led to the emergence of the entire Hepeviridae family.
The Astroviridae capsid is also closely related to the Tymoviridae family of monopartite, T = 3 icosahedral plant viruses, whilst its non-structural region is related to viruses of the Potyviridae; a large family of plant-infecting viruses with a flexible filamentous rod-shaped virion. Thus, we identified a separate inter-viral family recombination event, again at the non-structural/structural junction, which likely led to the creation of the Astroviridae.
In summary, we have shown that new viral families have been created though recombination at the junction of the genome that encodes non-structural and structural proteins, and such recombination events are implicated in the genesis of important human pathogens; HEV, astrovirus and rubella virus.
Hepatitis E virus (HEV) is a single-stranded RNA, non-enveloped enteric virus and a major etiological agent responsible for acute hepatitis, causing sporadic infections and widespread epidemics globally. Based on seroprevalence, it is estimated that one third of the global population has been infected with HEV at some stage in their lifetime . HEV creates a significant health burden on society, with annual estimates of 20 million clinical infections, over three million reported acute cases, 3,000 stillbirths and 50,000 HEV-related deaths globally [2, 3]. However, it is likely that these figures are an underestimation, given that the virus is often asymptomatic, usually self-limiting and HEV epidemics strike in geographic areas that are the least likely to have effective diagnostic testing available [4, 5].
The Hepeviridae family is currently divided into two genera, Orthohepevirus and Piscihepevirus . These genera include the species: Orthohepevirus A, which infects humans, swine, boar, rabbits, camels, deer and mongooses, Orthohepevirus B, which infects birds, Orthohepevirus C, which infects rodents and ferrets and Orthohepevirus D, which infects bats, whilst Piscihepevirus includes cutthroat trout virus (CTV) . There are currently seven recognized Orthohepevirus A genotypes. Genotype (G) I and GII only infect humans, while GIII and GIV infect a range of animal hosts, as well as humans. GV and GVI infect boars, while GVII infects camelids and humans. Orthohepevirus B has four proposed genotypes, Orthohepevirus C includes two genotypes (HEV-C1 and HEV-C2), while Orthohepevirus D has a single genotype . Additionally there is an unclassified moose HEV, which was recently identified in Sweden .
An initial analysis of the HEV GI non-structural polyprotein in 1992  indicated that the polymerase and helicase sequences were closely related to a plant virus, beet necrotic yellowing vein virus (BNYVV). BNYVV is a member of the newly proposed family Benyviridae and is a causative agent of rhizomania in sugar beet. This virus was first reported in Italy in 1966 , and was subsequently isolated in Japan in 1973 , and found to be transmitted by an obligate intracellular parasite protist vector, Polymyxa betae . HEV and BNYVV both have positive sense single-stranded RNA (ssRNA (+)) genomes, although that of BNYVV is segmented into 4-5 separate RNA fragments , compared to the monopartite genome of HEV. Comparative analysis of the HEV RNA-dependent RNA polymerase (RdRp), methyltransferase and helicase protein sequences with other ssRNA (+) viral sequences also revealed a genetic relationship with rubella virus (RuBV) [8, 9]. These findings led to the initial inclusion of HEV within the “alpha-like” supergroup of viruses, based on similarities in their RdRp, helicase and methyltransferase amino acid sequences and also the presence of a putative papain-like cysteine protease [8, 13].
In 2000, phylogenetic analyses using more sequence data (including four different HEV strains), again analyzing capsid, helicase and RdRp conserved regions, moved HEV out of the Caliciviridae and into a separate, unassigned clade of indeterminate taxonomic position, but closer to the Togaviridae (which includes RuBV) , within the “alpha-like” supergroup. This finally resolved the problem of HEV being both a member of the “Picorna-like” supergroup and the “alpha-like” supergroup. As more and more sequences became available, HEV was seen to be more and more distinct from other viral genera and families, and it was accorded its own genus (Hepevirus) by 2004 and then its own family (Hepeviridae) in 2009 [18, 19]. In 2014, the taxonomy of the Hepeviridae was reviewed again and the family was divided into two new genera: Orthohepevirus and Piscihepevirus, as discussed earlier  and in 2016, Smith and colleagues proposed reference sequences for HEV subtypes within the Orthohepevirus genus . Figure 1 illustrates the timeline of HEV classification through the International Committee on Taxonomy of Viruses (ICTV).
Despite scrutiny of the non-structural protein encoding region, the origin of the HEV capsid remained unresolved, since the Benyviridae are non-enveloped rod-shaped plant viruses , whereas HEV capsids are T = 3 icosahedral in shape, with an estimated 180 copies of the capsid protein [21, 22]. It was not until 2011, that surprisingly the HEV capsid protein was shown to be most structurally related to capsids from members of the Astroviridae family of vertebrate-infecting viruses . Astroviruses, like HEV, also have a T = 3 icosahedral capsid, but which belong with the “Picorna-like” supergroup of viruses. To this day confusion over the ancestry of HEV is still apparent, as the non-structural protein-encoding region is classified within the “alpha-like” supergroup, in contrast to the structural region. Therefore, this study aimed to shed further insight into the evolution and origins of the Hepeviridae family in its entirety. We initially undertook both a phylogenetic approach and an updated analysis of the identity between the individual HEV proteins and other viral proteins. Here we provide evidence that the whole Hepeviridae family likely arose through an ancient recombination event, which could have occurred between plant and insect viruses, with the breakpoint at the junction that separates the non-structural from the structural encoding regions of the genome.
Hepeviridae evolution - Homology searches using the Orthohepevirus A HEV GI ORF1 polyprotein
Individual HEV GI protein viral homologues
aClosest Hepeviridae homologue
1st-ranked Non-HEV viral homologue
2nd-ranked Non-HEV viral homologue
Polyprotein (ORF1) (NP056779)
bHelicoverpa armigera stunt virus (HASV)
1.74 × 10-262.03 × 10-7
26 % 29 %
bDendrolimus punctatus tetravirus
1.28 × 10-21
7.42 × 10-8
7.42 × 10-8
Capsid (ORF2) (NP056788)
2.02 × 10-12
2.74 × 10-12
Phosphoprotein (ORF3) (NP056787)
5.47 × 10-65
5.88 × 10-7
Frangipani mosaic virus
6.50 × 10-6
Y domain (NP056781)
1.0 × 10-96
Hop latent virus
2.0 × 10-18
Blueberry scorch virus
6.0 × 10-18
PCP (Protease) (NP056782)
1.0 × 10-10
Macro domain (X) (NP056784)
1.73 × 10-45
4.61 × 10-8
5.95 × 10-8
7.00 × 10-87
Turnip vein clearing virus
6.04 × 10-13
Maracuja mosaic virus
1.07 × 10-12
3.64 × 10-177
Tick-borne tetravirus-like virus
7.82 × 10-20
Chara australis virus
2.98 × 10-11
The closest non-Hepeviridae homologues to the Orthohepevirus A HEV GI ORF1 polyprotein were found to be from lepidopteran-infecting Alphatetraviridae family members (Fig. 3). Of the Alphatetraviridae, the highest scoring ORF1-encoded homologue by tBlastn or Blastp searching was Helicoverpa armigera (cotton bollworm) stunt virus (HASV) (genus Omegatetravirus), with homology to the MTase and helicase/RdRp regions (EU345431: 26 % and 29 % identity from two fragments comprising 823 amino acids; E-values = 1.74 × 10-26 and 2.03 × 10-07) (Fig. 3 and Table 1). Other Alphatetraviridae viruses with homology to the HEV ORF1 included Dendrolimus punctatus tetravirus (AY594352: 25 %, 25 % and 20 % identity from three fragments comprising 1,116 amino acids, E-values = 1.28 × 10-21 and 7.42 × 10-8), and Nudaurelia capensis beta virus (NP048059: 25 % and 20 % identity from two fragments comprising 1,104 amino acids) (Fig. 3).
Other noteworthy homologies were found, in the RdRp region, with a Sclerotinia sclerotiorum RNA virus , an unassigned dsRNA mycovirus infecting the plant pathogenic fungus S. sclerotiorum, (EU779934: 28 % average identity over 218 amino acids; E-value = 1.17 × 10-9); an unclassified tick-borne tetravirus-like virus  isolated from the ixodid hard tick Dermacentor variabilis (KM048322: 25 % average identity over 348 amino acids; E-value = 3.03 × 10-17) and the unclassified Chara australis virus , an ssRNA(+) virus infecting the charophyte algae C. australis var. nobilis (JF824737: 30 % average identity over 212 amino acids of the RdRp region; E-value = 4.73 ×10-12) (Fig. 3). The previously noted similarities between the HEV RdRp region and Benyviridae and Togaviridae viruses  were also observed in this study (Fig. 3).
Away from the Alphatetraviridae family, numerous homologues (1.0 ×10-9 > E-values > 1.0 ×10-12) were found within the Betaflexiviridae family of plant viruses (Foveavirus, Carlavirus) and Virgaviridae family of plant viruses (Tobamovirus), primarily with the helicase and RdRp regions of human GI HEV (Figs. 3, 5 and 6).
Hepeviridae evolution – Homology searches using the Orthohepevirus A HEV GI individual protein sequences
Out of the Hepeviridae, the closest homologous sequence to the Orthohepevirus A HEV GI methyltransferase protein (MTase; accession number NP056780) was the Orthohepevirus C ferret HEV methyltransferase region (accession number BAO57185; E-value = 5.47 ×10-65, with an overall amino acid identity of 59 % over 182 amino acids) (Table 1). The closest non-Hepeviridae homologue from a tBLASTn search was found to be a methyltransferase protein from the aforementioned HASV (E-value = 5.88×10-7) (Fig. 4, Table 1); a 150 amino acid region with 33 % identity. Other members of the family such as Nudaurelia capensis beta virus (NBetaV, AF102884) were also observed to have weaker homology to the HEV MTase (Fig. 4). Representatives of the Tobamovirus genera (plant virus family Virgaviridae) were also weakly homologous to the HEV MTase (10-6 < E-value <10-4).
The HEV Y domain (accession number NP056781), immediately upstream of the MTase region (Fig. 2) was first described by Koonin et al. , but has no described function. It was found to be well conserved within the Hepeviridae, and the nearest homologue was the corresponding ferret HEV Y domain of the Orthohepevirus C ferret HEV ORF1 polyprotein (BAO57189; E-value = 1.0 × 10-96, 65 % identity over the whole 217 amino acids (Table 1). Outside of the Hepeviridae, no significant identity was observed with the Y domain or any other protein (data not shown) using Blastp or tBlastn searches. However, a Delta-Blast search using a Blosum45 substitution matrix identified a region within the methyltransferase domain of Hop latent virus (NP066258; genus Carlavirus, of the Betaflexiviridae family of plant viruses), as the closest match, with an E-value = 2.0 ×10-18, with 12 % identity over 138 amino acids.
An InterProScan analysis of a concatenated methyltransferase and Y domain sequence showed that when combined, they contain a single region of 348 amino acids, corresponding to the Pfam member “Vmethyltransf” (PF01660, E-value = 7.5 ×10-18), suggestive of a larger contiguous MTase sequence, than that which is represented by NP_056780 alone. Pfam is a protein family database, comprising over 13,000 curated protein families that share significant homology as detected by the profile hidden Markov model method in the HMMER3 suite of programs .
An additional tBlastn search of the GenBank non-redundant viral database was undertaken, this time using the MTase and Y-domain concatenated sequence (Fig. 4). This identified the same hits as for the individual MTase and Y-domain searches above. However, the concatenated sequence search resulted in similar E-values, but with extended regions of homology for some viruses, such as NBetaV (AF102884), which now had a region of 323 amino acids (20 % identity, E-value = 4.0 ×10-5), aligned with the concatenated sequences, compared to 146 amino acids for the MTase alone (24 % identity, E-value = 1.37 ×10-5) (Fig. 4). In addition, with the extended sequence, new homologues were found with negeviruses, a recently described group of insect viruses . The most homologous negeviral sequence was that of the polyprotein of Negevirus Nona 1, with 23 % identity over 313 amino acids (AB972669, E-value = 1.95 ×10-4); the homologous region corresponds to the putative methyltransferase domain  (Fig. 4).
Papain-like cysteine protease
The HEV papain-like cysteine protease domain, of 160 amino acids (PCP; accession number NP056782) is not very well conserved, even within the Hepeviridae, and the nearest non-human Hepeviridae homologue, rat Orthohepevirus C PCP, has homology only at the N-terminal region of the PCP, with 42 % identity, from residues 33 to 78. An alignment of this region with other Hepeviridae family members illustrates the heterogeneity of this small region within the Hepeviridae, with low sequence identity across the family in this region (Additional file 1: Figure S1). Excluding the Hepeviridae from the search and using tBlastn, Blastp or PSI-Blast did not identify any significant homology with any other proteins in the GenBank database.
The Z region comprises amino acids 593-711 inclusive, of the Orthohepevirus A HEV GI ORF1 polyprotein sequence (NP_056779). This region appears to be unique across the Orthohepevirus A, with amino acid sequence identity of >68 % over 119 residues for viruses within the species. However, region Z is highly variable in both sequence and length outside Orthohepevirus A. No significant similarity of the Z region was detected outside the Hepeviridae family.
The polyproline region (PPR) of HEV, or hypervariable region (HVR) as it is also known (accession number NP056783), is an intrinsically disordered region (IDR), whose relaxed structure allows it to bind to multiple ligands, and be involved in the regulation of transcription and translation . As its name suggests, the PPR contains a high level of proline residues, and is extremely variable in its sequence, even between Orthohepevirus A genotypes . Despite significant inter-genotypic variation, within the Orthohepevirus A there is enough intra-genotypic conservation in the PPR to allow the creation of genotype-specific phylogenetic trees (data not shown). Blastp, tBlastn and PSI-Blast searches did not identify any significant homologues with other non-human Hepeviridae viruses, nor any other sequence, viral or non-viral, outside of the Hepeviridae (data not shown).
Macro (X) domain
The macro domain (accession number NP056784) is involved in ADP-ribose metabolism and post-translational modifications. As well as being found in the Hepeviridae, homologues exist in the Coronaviridae and alphaviruses, as well as in bacteria and eukaryotes . Within the Hepeviridae, the closest homologue to the HEV GI was the rat Orthohepevirus C macro domain (E-value = 1.73×10-45; with 56 % identity over 143 amino acids) (Table 1). The closest non-Hepeviridae homology was also seen between HEV and alphaviruses, such as Aura virus and Eastern equine encephalitis virus (EEEV) (AF126284; E-value = 4.61×10-8, with 36 % identity over 111 amino acids) (Table 1). Interestingly, the HEV macro domain also demonstrated similar identity with bacterial macro domains, such as that from Clostridium spiroforme (E-value = 1.62×10-7, with 32 % identity over 116 amino acids). The nearest eukaryotic homologue was the macro domain from the salmon flagellate protist pathogen Spironucleus salmonicida (EST47993; E-value = 5.0×10-6, with 32 % identity over 108 amino acids).
The HEV GI helicase protein (accession number NP056785) is also highly conserved within the Hepeviridae and has homology to non-Hepeviridae and non-viral helicases as well. The strongest non-human Hepeviridae Blast hit was found to be the helicase region of Orthohepevirus C ferret HEV ORF1 polyprotein (E-value = 7.0×10-87, with 62 % identity over 242 amino acids) (Table 1). The non-Hepeviridae viral helicase with the most identity to the HEV helicase was found to be that from the turnip vein clearing virus (Fig. 5), a Tobamovirus of the Virgaviridae family (E-value = 6.04×10-13, with 32 % identity over 250 amino acids). Another close homology was again observed in the helicase region from HASV (EU345431; E-value = 2.43×10-12, with 32 % identity over 250 amino acids) (Fig. 5).
RNA-dependent RNA polymerase
The HEV GI RNA-dependent RNA polymerase (RdRp; accession number NP056786) is highly conserved within the Hepeviridae, with 84-89 % amino acid identity between the human HEV viruses, and 87-50 % within non-human Hepeviridae viruses. The strongest identity was found with the RdRp region of Orthohepevirus C ferret HEV polyprotein (JN998607; E-value = 3.64 ×10-177, with 59 % identity over 471 amino acids) (Table 1). The weakest Hepeviridae homology was with bat Orthohepevirus D and CTV, with 47 % and 34 % amino acid identity, respectively. The HEV RdRp also demonstrated homology to viral RdRps outside of the Hepeviridae family. Of the non-Hepeviridae homologues, the RdRp regions of two unclassified viruses had the highest identity to the human HEV RdRp; tick-borne tetravirus-like virus (KM048322; E-value = 7.82×10-20, with 33 % identity over 224 amino acids), and the algal virus Chara australis virus (JF824737; 2.98×10-11, with 34 % over 335 amino acids) [25, 26] (Fig. 6, Table 1). Other viral families also had RdRps that were highly homologous to HEV RdRp, including Betaflexiviridae and Alphatetraviridae (Fig. 6).
Another unclassified virus, Hemorrhagic kidney syndrome virus (HKSV) , (accession number AF030878), actually had higher homology to HEV RdRp (Fig. 6), than the above-mentioned Tick-borne tetravirus-like virus and Chara australis virus (E-value =1.66×10-23, with 39 % identity over 155 amino acids). HKSV was first identified in salmon in 2000, and originally described as a “togavirus-like” virus . This partial sequence has a recognizable RdRp domain with the characteristic RdRp nucleotide motifs B and catalytic motif C , albeit separated by a stop codon (Fig. 6).
HEV ORF2 (Capsid) protein
The Orthohepevirus A HEV GI capsid protein (encoded by ORF2; accession number NP056788) is well conserved within the Hepeviridae family, with the highest scoring non-human Hepeviridae homologue being the Orthohepevirus C ferret HEV capsid (AB890001; E-value = 0, with 57 % identity over 608 amino acids) (Fig. 7, Table 1). The next closest Orthohepevirus A homologue was that of bat Orthohepevirus D, (JQ001749; E-value = 3.94×10-166, with 55 % identity over 526 amino acids), followed by avian Orthohepevirus B (E-value = 6.55×10-144, with 49 % identity over 536 amino acids) (Fig. 7).
Interestingly, outside of the Hepeviridae, the chicken astrovirus capsid (JN582319; Avastrovirus, family Astroviridae) was found to be the most homologous sequence to the HEV GI capsid (E-value = 1.0 × 10-6, with 25 % identity over 278 amino acids) using a tBlastn search (Blosum62 matrix, word size 2, 3, or 6). Switching to the Blosum45 matrix, for aligning more divergent sequences, resulted in porcine astrovirus capsid being the most homologous sequence identified (JX556690; E-value = 2.02 × 10-12, with 21 % identity over 355 amino acids) (Fig. 7, Table 1). Other astrovirus capsid sequences were also found to be similarly homologous (Fig. 7), with E-values ranging from 10-5 to 10-12, and percentage amino acid identity ranging from 20-26 % (data not shown). In addition, the unclassified virus discussed above, HKSV, had a similar level of homology with the HEV capsid to those of the astroviruses (E-value = 9.95×10-5, 26 % identity over 164 amino acids). Additional PSI-Blast and Delta-Blast searches did not reveal any more significant homology to other capsid sequences from any other viral family or genera. A reciprocal tBlastn and PSI-Blast search using the translated chicken astrovirus capsid sequence JN582319, identified HEV capsid protein as its closest homologue (27 % identity over 278 amino acids; E-value = 3.0 × 10-6).
HEV ORF3 phosphoprotein
The function of the small phosphoprotein encoded by HEV ORF3 (accession number NP056877) is not fully understood, but it plays a role in virion release from infected cells, and has also recently been found to inhibit nuclear factor-κB signaling . It is conserved within the human-infecting Orthohepevirus A genotypes, but not within the Hepeviridae family, even though viruses from the Orthohepevirus B, C and D, and CTV all have small, unrelated ORFs (presumed phosphoproteins) in the same part of their respective genomes. Delta-Blast and PSI-Blast searches did not reveal any other homologous proteins (viral or non-viral).
Hepeviridae evolution – capsid and RdRp phylogeny indicates ancient recombination events
The RdRp tree places the HEV RdRp and CTV RdRp together, forming a small cluster of RdRps with those from the Alphatetraviridae and Benyviridae families. The unclassified tick-borne tetravirus-like virus and Chara australis virus also groups with this cluster (Fig. 8). This small cluster’s next nearest neighbor is RuBV. All of these group together, forming one of the main clusters within the “alpha-like” supergroup of viruses, which can be clearly seen in the unrooted tree (Fig. 9).
In contrast to the RdRp phylogeny, the rooted capsid tree places the HEV capsid within a cluster with the Astroviridae family of viruses (Fig. 8). The Astroviridae capsid is also closely related to the Tymoviridae family of monopartite, T = 3 icosahedral plant viruses. Although the HEV RdRp sequence groups with members of the Benyviridae and RuBV, there is no relationship between the Hepeviridae capsids and capsid sequences from these viruses, which group well away from the Astroviridae cluster (Fig. 8). Indeed, capsids from viruses of the Benyviridae are helical in architecture, not icosahedral. This relationship can also be seen more clearly in the unrooted capsid tree (Fig. 9). In order to reduce the potential effects of saturation  due to the large scale phylogeny of all (+) ssRNA capsids (and their rapid mutation rates), an extended set of capsid sequences was analyzed again, this time including more Hepeviridae and Astroviridae sequences, but with fewer, more closely related families (Additional file 3: Figure S2). This analysis included 223 representatives from the Astroviridae, Benyviridae, Bromoviridae, Caliciviridae, Hepeviridae, Tombusviridae and Tymoviridae families (Additional file 2: Table S3). All of the families form individual clusters, except for the Bromoviridae strains. There is good branch support for several of the intrafamily clusters (Hepeviridae, Tymoviridae, Benyviridae and Caliciviridae), but this is weaker (<60 %) for the other intra- and interfamily clusters (Additional file 3: Figure S2). However, the Astroviridae and Hepeviridae group together, with the Tymoviridae linked to the Hepeviridae after apparent divergence from the Astroviridae cluster (Additional file 3: Figure S2).
Homology searches using the HEV GI proteins
The first part of this study has highlighted four key areas that lead to a greater understanding of the origins and evolution of the Hepeviridae as a whole, with a focus on the prototype virus HEV GI in particular, and these are discussed in more detail below.
MTase, Y-domain, helicase and RdRp
The well-characterized domains with recognizable homology to other viral proteins include the MTase, Y-domain, helicase and RdRp domains. Of these, the Y-domain was originally identified as a distinct domain with weak homology to genomic regions in RuBV and BNYVV . However, no function was ever ascribed to it. Since the preceding methyltransferase domain (NP056780) shares the same Pfam domain classification as the Y-domain, it seems more than likely that the Y-domain is actually just a C-terminal domain of a larger methyltransferase protein. Therefore, the MTase (NP056780) and Y-domain (NP056781) sequences should be considered together as one methyltransferase protein.
Recently, a secondary structure comparison of 50 viral genera identified an “iceberg” region, downstream of the “core” region within viral methyltransferase-guanylyltransferase (MTase-GTase) capping enzymes, to which the HEV Y-domain was also included . Experimental evidence for the iceberg region or Y-domain’s functional role has been demonstrated using C-terminal deletions of the Sindbis virus MTase-GTase . Here, even relatively small C-terminal deletions abolished MTase-GTase activity, demonstrating that the core region alone is insufficient for all capping enzyme activities. This is further evidence that the MTase domain, together with the Y domain, forms either a single protein or single domain.
The individual searches with the Orthohepevirus A HEV ORF1 polyprotein and the individual MTase, Y-domain, helicase and RdRp proteins suggests that the majority of the ORF1 polyprotein of viruses from the Hepeviridae (comprising the MTase-Y-domain, helicase and RdRp domains) share a common ancestor with the Alphatetraviridae family, and that ancestor in turn shares a common ancestor to the Virgaviridae/Benyviridae/Betaflexiviridae plant viral families. Other features of the HEV genome were probably acquired by horizontal gene transfer (polyproline-macro domain) or evolved later as a result of host-virus interactions (polyproline region).
Papain-like cysteine protease, polyproline region, Z region and macro domain
The existence of the HEV protease was first predicted by Koonin et al. , based upon alignments with RuBV and BNYVV viruses. However, the alignment is poor, and subsequent advances in sequence alignment and enlarged databases have not led to the identification of any significant homologues to the HEV protease domain. Indeed, one of the most contentious issues regarding HEV is whether the ORF1 polyprotein is processed at all, either by its own protease or by a host protease. Evidence for in vitro processing of the HEV ORF1 has been reported in E. coli, in human HepG2 and S10-3 cells [38–40], in baculovirus cells  and using vaccinia virus expression systems . However, in all of these studies, the levels of the putative processed proteins are low, and in some cases could be explained by host-cell protease degradation. A recombinant HEV protease purified from E. coli was found to be active on both the HEV capsid protein and the HEV polyprotein, although an inhibitor screen of this protease’s activity led to the conclusion that it was a chymotrypsin-like protease, not a papain-like cysteine protease . However, other studies looking at ORF1 processing in human and hepatic cells reported little or no processing [44, 45]. This data is consistent with the fact that, a specific histidine residue (H590), a key part of the catalytic dyad in papain-like cysteine proteases, is absent in the HEV enzyme . Pertilla et al.  also suggests that the HEV polyprotein is not processed, and functions in a similar manner to the large multifunctional polyproteins of some plant viruses within the “alpha-like” supergroup, consistent with its phylogenetic clustering (Figs. 8 and 9) . Conversely, Parvez and colleagues used in silico modelling of RuBV and HEV proteases to identify a different catalytic dyad in the putative HEV papain-like protease. This study proposed that the catalytic dyad is C434-H443, which is homologous to the C1152-H1273 catalytic dyad in the RuBV protease . Using mutational analyses, the conserved H443 was shown to be is essential for HEV viability . In the current study, the H443 residue was found to be conserved within the Orthohepevirus A, but not across the Hepeviridae family. Irrespective of the functionality (or not) of the protease, its origin remains obscure due to the lack of any significant identity to non-Hepeviridae sequences.
From this study, it is also interesting to note that this “protease” region, which lies between the MTase-GTase and the polyproline region, is approximately the same size across members of the Hepeviridae. Yet the amino acid identity is so low that no genetic relationship is apparent (Additional file 1: Figure S1). This suggests a common functional enzyme does not exist. Instead, perhaps this is a region involved in host-specific interactions. Whether this domain is a degenerate partially functional or non-functional protease domain, or an entirely different domain or protein, remains to be clarified.
One other study has reported deubiquitination activity, a key target for viral infection , associated with a recombinant protein comprising the methyltransferase, Y-domain and protease domain (amino acids 22-592) of HEV ORF1 . A later study showed that a recombinant HEV protease domain, when expressed in HEK293T cells, resulted in reduced IFNβ mRNA production and deubiquitination of RIG-I and TBK-1 . Since cysteine proteases are significantly represented as a subfamily within the larger deubiquitinase (DUB) enzyme family , this could well be the main function, even perhaps the only function, for the protease domain.
The macro domain of HEV is homologous to those found in alphaviruses and bacteria, with slightly less similarity to eukaryotic and archeal macro domains, but the differences in homology are not significant enough to identify unambiguously the origin or common ancestor of this region. It is also unclear whether the HEV macro and protease domains together derive from the same ancestral region, as is the case for the Coronaviridae and alphaviruses , where the macro domain is associated with the protease domain. Macro domains in animal cells have been associated with ADP-ribose metabolism, and post translational protein modification, but few viral families possess macro domains, and in those viral families that do, the macro domains do not appear to be related to any specific feature shared by these viruses (Coronaviridae, alphaviruses, Hepeviridae) . Poly(ADP)-ribosylation is a post-translational modification of proteins in cells, which can be triggered by viral infection, and acts as an apoptosis-inducing signal , and the HEV macro domain has been shown to bind poly(ADP), suggesting a potential role in preventing apoptosis in infected cells. Expression of a recombinant HEV macro domain in HEK293T cells inhibited poly(IC)-induced IRF3 phosphorylation, indicating that one of the HEV macro domain’s function is as an IFN antagonist .
The polyproline region (PPR) was originally described as a protein hinge , but more recent studies have shown it to be the only region within ORF1 under positive selection, and has multiple ligand-binding motifs . The PPR is also involved in viral replication and adaptation, including host-specificity, but not infectivity [54, 55]. An increase in correlated genetic heterogeneity in the macro and polyproline regions has been observed in chronic HEV infection, relative to patients who cleared the virus, suggesting that these two domains are linked in some way in modulating the immune response . Apart from the Hepeviridae, only rubiviruses have a similar arrangement of a PPR upstream of a macro domain, and the rubivirus PPR is also highly diverse genetically, and under positive selection . It is conceivable that ancestral rubiviruses and Hepeviridae viruses at some point acquired or exchanged a PPR-macro domain, which has retained homology in the macro domain, for its shared function in host-virus interactions, but the PPR domain has evolved rapidly due to its host-specificity role.
The Z region of the Orthohepevirus A HEV GI ORF1 is unique across the species and as such, resembles an equivalent alphavirus unique domain (AUD); a region of the alphaviral non-structural protein coding region that has strong sequence identity within alphaviruses, but not outside the genus . Like the AUD, the Z region is highly variable in both sequence and length outside Orthohepevirus A, but is conserved within the Orthohepevirus A group (>68 % identity across 119 residues). It is possible that the AUD-like region in Orthohepevirus A viruses has evolved with changes in host tropism, thus losing any conservation with other members of the Hepeviridae over time. Similar to the Z region in Orthohepevirus A, in alphaviruses a PPR follows the AUD, however unlike HEV both domains are downstream of the macro domain (Fig. 2). In the majority of Orthohepevirus A sequences, the Z region and PPR are both upstream of the macro domain and could have shifted through a translocation event. One exception within Orthohepevirus A is rabbit HEV, and outside Orthohepevirus A exceptions include ferret and rat HEV (Orthohepevirus C) and CTV (Piscihepevirus) which all have a small PPR region at the carboxyl end of the macro domain. This polyproline rich region was originally reported to be an insertion of an inert spacer sequence into the rabbit and rat genomes , but could in fact be a remnant from an incomplete PPR translocation event.
Whereas the majority of the HEV ORF1-encoded polyprotein and those from the Hepeviridae family in general clearly have plant/insect viral ancestry, the HEV capsid sequence appears to have none. Analysis of the capsid spike or projection domain of the human astrovirus serotype 8 (human astrovirus; Yuc8 strain, genus Mamastrovirus) using the DALI protein homology server  identified the HEV capsid P2 domain as the closest structural homologue .
Electron cryomicroscopy also showed that the human astrovirus infectious virions and immature virions were remarkably similar to that of HEV in terms of size, shape and architecture [22, 60]. It is interesting to note that, given the similarity in capsid structural size, the genomes of HEV (~7.2 kb) and astroviruses (6.8-7.1 kb) are also similarly matched in size. A subsequent study  found that the turkey astrovirus spike protein (turkey astrovirus-2; genus Avastrovirus) was a structurally closer match to the HEV capsid, than was found with the previous study involving the human astrovirus capsid spike . The present study however, showed that the porcine astrovirus capsid protein sequence was the closest homologue to the HEV capsid sequence, closely followed by the chicken astrovirus capsid (Fig. 7), and all the homologous Astroviridae capsids sequences had E values between 10-5 – 10-12. Moreover, no other viral family capsid sequences had any significant homology to the HEV capsid. A reciprocal Blast search using a chicken astrovirus capsid sequence identified the HEV capsid as the closest homologue, and no other viral family capsid had any significant identity. Irrespective of which contemporary astrovirus sequence is most closely related to contemporary HEV capsids, it seems that the Hepeviridae and the Astroviridae viruses share a common ancestral capsid sequence.
Surprisingly, it was also found that the Tymoviridae and Hepeviridae capsid sequences have a common ancestor too, which diverged after the Hepeviridae-Astroviridae split (Additional file 3: Figure S2). However, the Hepeviridae and Tymoviridae capsid sequences do not have any significant homology, but are linked phylogenetically, which could be indicative of the (plant-infecting) Tymoviridae capsid sequences evolving faster under different evolutionary pressure(s), than the avian/mammalian-infecting Hepeviridae or Astroviridae capsids.
The absence of any viral homologues to the phosphoprotein encoded by ORF3, even within the Hepeviridae would suggest that ORF3 evolved relatively recently from an overprinting mechanism , utilizing a previously unused ORF within ORF2. ORFs 2 and 3 are bicistronic, with the two start codons closely spaced in different reading frames . ORF3 has evolved to be essential for virulence . Viruses with this type of ORF3 likely occurred after mammalian Orthohepevirus A virus diverged from the common Hepeviridae ancestor and it is possible that ORF3 could have driven host tropism and host restriction . Similar overprinted genes like ORF3, have been identified in numerous other viruses . However, Astroviridae capsid sequences have not been shown to have overlapping proteins expressed from alternative ORFs, and it is seems highly improbable that ORF3 was independently acquired and integrated into the capsid sequence.
Proposed evolutionary scenario for the Hepeviridae
There are few reports in the literature regarding the evolution of contemporary viruses from two or more disparate ancestors. Although there are few examples, one in particular is remarkable; the ssDNA viruses of the Bidnaviridae family (infecting the silkworm Bombyx mori) contain genes derived from Parvoviridae (ssDNA viruses), Reoviridae (dsRNA viruses), Baculoviridae (dsDNA viruses) and polintoviruses . In addition, a recent metagenomics study of viral diversity in a lake has identified a unique hybrid virus, where an ssRNA viral capsid sequence has integrated into a circovirus-like DNA virus (RNA-DNA hybrid virus from Boiling Springs Lake, or “BSL-RDHV”) . The capsid sequence in BSL-RDHV is most closely related to the capsids from multipartite nodaviruses, which mostly infect insects and fish, and to monopartite Tombusviridae capsids (plant viruses), and the remainder of the BSL-RDHV genome resembles circoviruses (infecting birds and mammals) . RNA-RNA recombination in plant viruses and other viruses, and the relevant molecular mechanisms are well-known and well-documented . Some families show high rates of recombination (Picornaviridae), and others low rates (Flaviviridae), or non-existent/undetectable (Leviviridae), nonetheless recombination is vital for generating population variability and maximizing survival .
There are few if any published reports of interfamily capsid exchange in the ssRNA (+) viruses, notwithstanding the fact that such an exchange would in all likelihood require similar sized genomes in order to package properly. The most parsimonious evolutionary scenario, with regard to the exchange, or acquisition of capsid sequences by an ancestral Hepeviridae virus, might be that an ancient ORF1 RNA sequence, from a plant/insect virus ancestral to the Alphatetraviridae/Betaflexiviridae/Virgaviridae/Benyviridae families, underwent recombination with an ancestral Astroviridae capsid RNA sequence, resulting in a recombinant virus that had (fortuitously) similarly sized genome of a plant/insect virus. This virus likely retained capsid sequence packing signals and was able to be subsequently packaged into an astrovirus-like icosahedral capsid. Such a recombination event resulted in the ancestral Hepeviridae hybrid virus, comprising an astroviral capsid from the Picorna-like super-group, and a plant/insect non-structural polyprotein from a virus within the very different alpha-like super-group (Fig. 10). An obvious prerequisite of the above events is that the ancestral viruses involved infected the same host simultaneously; whether this permissive host was a plant, insect or possibly even a vertebrate is at present unclear.
The ancestral Hepeviridae ORF1 probably comprised (from N- to C-terminal) a methyltransferase, “protease”, helicase and RdRp regions. At some time a macro domain was acquired, probably by horizontal transfer, although the putative source is unclear, given the similar, (albeit low) identity to bacterial and alphaviral homologues. Whether this acquisition occurred before, or after the capsid exchange is unclear. This acquisition may have been accompanied by the “protease” region, since these two domains are often closely associated, although no functional link between these domains has yet been demonstrated . The PPR could also have been associated with the macro domain as discussed above. The orientation of the protease and macro domains in Hepeviridae viruses correspond to that seen in alphaviruses, but is the reverse of RuBV, whereas the PPR-macro domain organization in Hepeviridae matches that of rubiviruses, but not alphaviruses in general (which have a much smaller PPR region or lack the PPR altogether) [8, 47] (Fig. 10). Later still, the ORF3 phosphoprotein evolved from an alternate reading frame within the capsid sequence, which could have been present in the capsid region prior to recombination, or evolved afterwards. Subsequently, the evolution of ORF3 could have been influenced by host tropism, leading to the possible evolution of individual species within the Hepeviridae.
Intriguingly, duck astrovirus is unique among the Astroviridae, in causing viral hepatitis, indicating a tropism for hepatocytes, whereas all other astroviruses cause gastroenteritis . It is interesting to speculate that the ancestral virus, from which viruses of the Astroviridae and Hepeviridae obtained their capsids, had a similar tropism. This could help to explain how the descendent capsid of an enteric virus is now used by a virus that causes human hepatitis.
It is also noteworthy that analysis of the Astroviridae capsid and RdRp sequences indicate the possible occurrence of a second ancient inter-viral family recombination event. The astrovirus RdRp sequences are unrelated to those from the Hepeviridae family, but are most closely related to those from viruses of the Potyviridae; a large family of non-enveloped plant-infecting viruses  with a flexible filamentous rod-shaped virion (Figs. 8 and 9). These capsids share no homology whatsoever with the icosahedral astrovirus capsid. This likely indicates that an ancestor of the Potyviridae acquired a distinct Astroviridae-like capsid (i.e., from a different viral family), indicating a recombination event at the non-structural/structural junction, that led to the genesis of the Astroviridae.
Koonin et al.,  first proposed that HEV (and hence subsequently the whole family) had a genetic relationship in the non-structural encoding region of its genome with viruses from the Togaviridae, including RuBV and BNYYV; this is depicted in Fig. 10. In contrast to the Hepeviridae, the Togaviridae (including RuBV), have an alpha-like non-structural genomic region, but a flaviviral-like structural region of their genome (Fig. 10). Given that the two aforementioned recombination predictions likely led to the genesis of the Hepeviridae and the Astroviridae, it is tempting to speculate that the entire Togaviridae family could also have emerged this way. It may have occurred through a recombination event at the non-structural/structural border between viruses from families representing different viral super-groups; flavi-like and alpha-like super-groups.
This study used protein identity searches using the prototype HEV virus ORF1 amino acid sequence to highlight four key areas that lead to a greater understanding of the origins of HEV, the possible functions of its encoded proteins and their relationship to other protein families. This study has also shed additional light on the intriguing evolution and ancestry of several viral families. We show that both the Astroviridae and Hepeviridae, and possibly the Togaviridae, have undergone ancient recombination events at the junction between the non-structural and structural encoding regions of the genome, resulting in their creation. It is likely other families have emerged this way and this could be uncovered with careful analysis of the ever-growing number of new viral sequences being accrued. Finally, based on the results found herein, we hypothesize that viruses from the alpha-like super-group, which usually infect plants and insects, gain an entirely new structure through recombination to enable tropism into new hosts within the animal world.
Hepeviridae genomic sequences
A total of 301 Hepeviridae genomes were identified and downloaded from GenBank (October, 2015). Duplicate sequences were removed using the ElimDupes tool on the HIV sequence database (https://hcv.lanl.gov/content/sequence/ELIMDUPES/elimdupes.html). Orthohepevirus B, C and D genomes, along with the Piscihepevirus CTV genome, were removed. Rabbit HEV (GIII), wild boar HEV (GV), and camel HEV (GVII) sequences were included, but wild boar HEV (GVI) and Moose HEV (unclassified) sequences were omitted. Incomplete sequences, patent sequences of cDNA, recombinant infectious clone constructs and sequences from ex vivo passaged isolates were also excluded, resulting in 228 non-redundant HEV genomes (Additional file 2: Table S1).
Identity searching of Orthohepevirus A HEV GI proteins
To identify homologous Hepeviridae and non-Hepeviridae viral sequences, individual Orthohepevirus A HEV GI protein sequences were used in tBlastn and Blastp searches of the GenBank databases , using the built-in interface in the software package Geneious 9.0.3 . Specifically, protein sequences derived from the prototype HEV Xinjiang strain (accession number NC_001434)  were used, corresponding to the methyltransferase (NP056780), Y-domain (NP056781), papain-like cysteine protease (NP056782), hypervariable region (HVR) or polyproline region (NP056783), X (macro) domain (NP056784), helicase (NP056785) and RNA-dependent RNA polymerase proteins (RdRp, NP056786), shown together in Fig. 2. In addition, searches were also conducted with the complete polyprotein of ORF1 (NP056779), the capsid protein from ORF2 (NP056788) and the phosphoprotein encoded by ORF3 (NP056787) . An “expectation” or “expect value” (E-value) threshold of < 1× 10-4 was used to eliminate weak and random similarities between the query and target sequences .
Homology of HEV GI protein sequences searching by PSI-Blast, Delta-Blast and InterProScan
The individual human HEV GI protein sequences and the HEV ORF1 polyprotein sequences described above (Fig. 2), were also used to screen the non-redundant GenBank protein database for distant homologues, using the position-specific iterated Blast (PSI-BLAST) server at http://blast.ncbi.nlm.nih.gov/ . Initial setup parameters comprised the default settings, using either BLOSUM45 or BLOSUM62 as the initial substitution matrix, word size 2 or 3, up to 5 iterations, and masking of low complexity regions. In addition, searches were made using the same parameters on the Domain Enhanced Lookup Time Accelerated Blast, or Delta-Blast server. This server queries the NCBI conserved domain database (CDD) to construct a scoring matrix prior to searching the protein sequence database . Individual HEV GI protein sequences were also analyzed for protein function against the InterPro protein families’ database, using the InterProScan plugin in Geneious [79, 80].
Multiple sequence alignment and phylogenetic analysis
Capsid and RdRp protein sequences from 104 representative positive sense, single stranded RNA viruses (ssRNA (+)) were collated from GenBank (Additional file 2: Table S2). These sequences were aligned using MAFFT , using the embedded algorithms in Geneious. The best-fit models for amino acid substitution for the phylogenetic analysis were determined by analyzing the capsid and RdRp alignments using ProtTest 3.3 . Phylip files of the alignments were submitted to the ATGC Server at http://www.atgc-montpellier.fr/ to build the phylogeny using PhyML , using the LG substitution model  for RdRp alignments, and the VT substitution model for capsid alignments , with SPR and NNI tree improvements and 100 bootstraps.
The authors wish to thank Associate Professor Mark Tanaka, Dr. John-Sebastian Eden and Professor Eddie Holmes for very useful discussions.
No specific funding was received for this study. NEN was supported by an Australian Postgraduate Award.
Availability of data and material
The datasets analysed during the current study are available from the corresponding author on reasonable request.
AGK, NEN & PAW conceived the study; AGK & PAW designed the study; AGK, NEN & PAW carried out the work, data analysis and drafted the manuscript; AGK, NEN & PAW contributed to interpretation of data; AGK, NEN & PAW critically revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
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