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. 2024 Jan 18;341:199321. doi: 10.1016/j.virusres.2024.199321

Genomic characterization of Rocahepevirus ratti hepatitis E virus genotype C1 in Yunnan province of China

Han Wu a, Bingzhe Li a, Bowen Yu b, Linjie Hu a, Lu Zhou a, Jiaxiang Yin c,⁎, Yihan Lu a,⁎
PMCID: PMC10831724  PMID: 38242291

Highlights

  • •

    The detection rate of Rocahepevirus was 12.95 % (36/278) in animals of order rodentia.

  • •

    All Rocahepevirus sequences amplified in this study belong to HEV-C1.

  • •

    HEV-C1 was isolated in one Niviventer Fulvescens for the first time.

  • •

    The HEV-C1 isolated in this study remained phylogenetically distant from HEV-C1 in North America and Europe.

Keywords: Hepatitis E virus, Rocahepevirus ratti, HEV-C1, Genomic characterization, Rodent, China

Abstract

The Rocahepevirus ratti hepatitis E virus genotype C1 (HEV-C1) has been documented to infect humans. However, the understanding of HEV-C1 remains constrained. This study aims to determine the prevalence and genomic characteristics of HEV-C1 in small animals in Yunnan province of southwestern China. A total of 444 liver tissues were collected from animals covering the orders Rodentia, Soricomorpha, Scandentia and Erinaceomorpha in three regions in Yunnan. Then Paslahepevirus balayani and Rocahepevirus were examined using RT-qPCR. The detection rate of Rocahepevirus was 12.95 % (36/278) in animals of order Rodentia, with 14.77 % (35/237) in Rattus tanezumi and 33.33 % (1/3) in Niviventer fulvescens. No Paslahepevirus balayani was detected. Additionally, two full-length Rocahepevirus sequences (MSE-17 and LHK-54) and thirty-three partial ORF1 sequences were amplified and determined to be HEV-C1. MSE-17 and LHK-54 shared moderate nucleotide identity (78.9 %-80.3 %) with HEV-C1 isolated in rats and humans. The HEV-C1 isolated from Niviventer fulvescens demonstrated a 100 % nucleotide identity with that from Rattus tanezumi. The rat HEV-C1 sequences isolated in our study and other Asian HEV-C1 sequences were phylogenetically distant from those isolated in North America and Europe. Furthermore, the two full-length sequences isolated in our study had less amino acid substitutions in the motifs of RNA-dependent RNA polymerase domain (F204L and L238F), compared with other Asian sequences. In summary, HEV-C1 commonly spreads in rats in Yunnan province of China. Our findings suggest a spatially associated phylogeny, and potential cross-species transmission of HEV-C1.

Graphical abstract

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1. Introduction

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide (Kamar et al., 2012). A recent systematic review and meta-analysis showed that 12.47 % of global population that is approximately 939 million has experienced past infection of HEV, and 15–110 million have recent or ongoing HEV infection (Li et al., 2020). HEV belongs to the family Hepeviridae, and has a positive-sense, single-stranded, 6.8–7.2 kb, RNA genome, which is capped at the 5′ termini and polyadenylated at the 3′ termini (Guerra et al., 2017; Hoofnagle et al., 2012). HEV genome harbors three open reading frames (ORFs), ORF1, ORF2 and ORF3. ORF1 encodes the non-structural polyprotein containing methyltransferase (Met), Y domain, papain-like cysteine protease (PCP), hypervariable region (HVR), X domain, helicase (Hel) and RNA-dependent RNA polymerase (RdRp), which is essential for viral RNA replication and virus infectivity (Koonin et al., 1992; Parvez, 2017). ORF2 encodes the capsid protein (Guerra et al., 2017). ORF3 encodes the multifunctional phosphoprotein (Guerra et al., 2017). Further studies have identified ORF4, enhancing virus replication by interacting with multiple viral and host proteins in HEV-1 (Nair et al., 2016). Additionally, a putative ORF4 has also been recognized in rats and ferrets (Li et al., 2014; Tanggis et al., 2018).

The subfamily Orthohepevirinae comprises four genera, Paslahepevirus (species Paslahepevirus balayani was originally named as Orthohepevirus A), Avihepevirus (Orthohepevirus B), Rocahepevirus (Orthohepevirus C) and Chirohepevirus (Orthohepevirus D), according to the latest report of the International Committee on Taxonomy of Viruses (ICTV) (Purdy et al., 2022). Species Paslahepevirus balayani includes eight genotypes (HEV-1 to HEV-8). HEV-1 and HEV-2 exclusively infect humans. HEV-3 and HEV-4 are zoonotic, with a wide range of hosts including human, swine, wild boar, deer, mongoose and rabbit (Kenney, 2019). HEV-5 and HEV-6 were isolated in wild boars, and HEV-7 and HEV-8 were discovered in dromedary camels and bactrian camels, respectively (Kenney, 2019). Species Rocahepevirus ratti have been isolated in rodent, shrew, ferret, mink and human (Sridhar et al., 2018; Wang et al., 2020). To date, HEV-3 and HEV-4 have been the main sources of zoonotic HEV infection. Additionally, HEV-7 was identified as causing chronic infection in a patient with a liver transplant (Lee et al., 2016), while both HEV-5 and HEV-8 were observed to experimentally infect cynomolgus monkeys (Li et al., 2019; Wang et al., 2019). This amplifies the concern regarding potential zoonotic HEV transmission.

Within the genus Rocahepevirus, two species, namely Rocahepevirus ratti and Rocahepevirus eothenomi, have been recognized by ICTV (Purdy et al., 2022). Species Rocahepevirus ratti has been classified into two genotypes, HEV-C1 and HEV-C2, with hosts in the orders Rodentia and Soricomorpha, and the order Carnivora, respectively (Wang et al., 2020). Besides, the putative HEV-C3 and HEV-C4 have been suggested within the genus Rocahepevirus (Wang et al., 2018; Zhu et al., 2022). Furthermore, HEV-C1 has been proven to infect humans. Multiple human cases with HEV-C1 infection have been reported in Hong Kong of China, particularly among individuals who were immunocompromised due to liver transplantation, cancer therapy, or HIV infection (Sridhar et al., 2021, 2018). Later, one immunocompetent Canadian case was found to be presumably infected with HEV-C1 in central Africa (Andonov et al., 2019). The recent Spanish and French studies discovered three cases of acute hepatitis and one case of persistent hepatitis, respectively, testing positive for HEV-C1 (Rivero-Juarez et al., 2022; Rodriguez et al., 2023).

The zoonotic concern related to HEV-C1 warrants the constant monitoring on the circulation of HEV in rodents and other animals. In China, a study documented a HEV-C1 detection rate of 1.66 % in rodents and shrews in Shenzhen, a city in southern China (Wang et al., 2017). Another study reported an approximate detection rate of 20 % in rodents across various cities in China (He et al., 2018). However, genomic characterization of HEV-C1 remained limited. Thus, we conducted an investigation on HEV-C1 in animals from Yunnan province, a region where HEV-C1 was frequently isolated in China (He et al., 2018; Qian et al., 2022). Subsequently, we performed a comprehensive molecular analysis of isolated HEV-C1 sequences to obtain new insights into the genomic characterization, virus evolution and cross-species transmission.

2. Materials and methods

2.1. Sampling and RNA extraction

Between July and August 2019, we captured a total of 444 animals by cage traps in Lianghe (Hexi village and Mangdong town), Mangshi (Manghai and Zhefang towns), and Mile (Xinshao town) counties, Yunnan province of China (Fig. 1). We set up cage traps in the evening and checked the captures the next morning. All captured animals were alive, except for one berylmys bowersi. The habitats at the sampling sites includes cultivated land, woodland, mountainous terrain, shrubbery, grasslands, indoor spaces and areas near human residences. The species of the trapped animals were determined by morphological identification and sequencing of the cytochrome B (cytB) gene (He et al., 2018) (Supplementary Materials). A total of sixteen species of animals within orders Rodentia, Soricomorpha, Scandentia and Erinaceomorpha were determined (Table 1). Liver tissues were collected in the laboratory immediately. RNA extraction was then performed in the liver tissues using High Pure Viral RNA Kit (TQ-BG-001–96B, Shanghai BioGerm Medical Biotechnology, Shanghai, China) according to the manufacturer's instructions. The extracted RNA was stored at −80 °C until use.

Fig. 1.

Fig. 1

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Geographical map representing the sampling sites in Yunnan province. The detailed sample information were provided in Table 1.

Table 1.

Detection rates of Rocahepevirus among small animals captured in Yunnan province of China.

Orders Species Lianghe Mangshi Mile
Rodentia (n = 278) Rattus tanezumi 19.44 % (21/108) 14.14 % (14/99) 0 (0/30)
Rattus sladeni 0 (0/7) 0 (0/4) 0 (0/3)
Mus pahari 0 (0/5) 0 (0/1) 0 (0/3)
Rattus nitidus 0 (0/2) – 0 (0/4)
Niviventer niviventer 0 (0/4) – –
Niviventer fulvescens – 33.33 % (1/3) –
Mus caroli – – 0 (0/1)
Berylmys bowersi – 0 (0/1) –
Dremomys rufigenis – 0 (0/1) –
Tamiops swinhoei – 0 (0/1) –
Sciurus vulgaris – 0 (0/1) –
Soricomorpha (n = 143) Suncus murinus 0 (0/70) 0 (0/67) –
Crocidura attenuata 0 (0/5) – –
Crocidura horsfieldii 0 (0/1) – –
Scandentia (n = 12) Tupaia belangeri 0 (0/5) 0 (0/3) 0 (0/4)
Erinaceomorpha (n = 11) Hylomys suillus 0 (0/11) – –
Total (444) 9.63 % (21/218) 8.29 % (15/181) 0 (0/45)
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All strains isolated in this study were determined as HEV-C1.

2.2. Detection of HEV RNA

We conducted testing for both Paslahepevirus balayani and Rocahepevirus in the liver specimens. For Paslahepevirus balayani, we directly used a commercial RT-qPCR kit (Shanghai BioGerm Medical Biotechnology Co., LTD, Shanghai, China) (Chen et al., 2019). For Rocahepevirus, we designed the primers and probes targeting Rocahepevirus ORF1 as described elsewhere (Sridhar et al., 2018) and revised the 3′ end quencher of primers to Black Hole Quencher 1 (BHQ1) (Supplementary Materials Table S1). We used diethylpyrocarbonate-treated water as a negative control, and prepared a positive control (Supplementary Materials).

A RT-qPCR was performed using the One Step RT-qPCR Kit (Yongke Biotechnology Co., LTD, Shanghai, China) on a BIO-RAD CFX Connect™ PCR instrument. The reaction system was consisted of 5 μL DNA template, 10 μL 2xNT qPCR SuperMix, 0.8 μL Primer-Foward, 0.8μL Primer-Reverse, 0.4 μL probe, and 8 μL enzyme-free water, following thermal profiles: a reverse transcription step at 50 °C for 10 min, a pre-denaturation step at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, and annealing and extension at 55 °C for 40 s. Compared with internal validation with a positive control, the specimens were considered negative if the cycle threshold value was >38, uncertain if 35–38 which need repeated examination, and positive if ≤35.

2.3. HEV-C sequencing and genotyping

All specimens positive for Rocahepevirus by RT-qPCR were further used for amplification of a partial fragment within ORF1 RdRp domain (ranging from nucleotide positions 4094 to 4414 correspond to the reference sequence GU345042) using the nested broad-spectrum RT-PCR (Supplementary Materials Table S2), as described elsewhere (Johne et al., 2010b). The amplified products were subsequently subjected to Sanger dideoxy sequencing. Moreover, we selected two specimens positive for Rocahepevirus with lowest cycle threshold values in RT-qPCR and then performed the full-length sequencing by next generation sequencing (Supplementary Materials).

All full-length Rocahepevirus genomes available in the GenBank (https://www.ncbi.nlm.nih.gov/GenBank/) were collected (Supplementary Materials Table S3) and aligned to the sequenced fragments using bbmap alignment to identify the Rocahepevirus sequences. The sequences were then spliced using Megahit v1.2.9 and SPAdes v3.14.1 (parameters were provided in Supplementary Materials). Contigs with sequence lengths less than 150 bp were then filtered out. Finally, the contigs were assembled using software CAP3 (parameters were provided in Supplementary Materials) to obtain the genome sequences. The relevant metrics were integrated into the Supplementary Materials Table S4. HEV ORFs were identified by EditSeq 7.1.0 in DNASTAR (https://www.dnastar.com/software/). Furthermore, the schematic diagrams of Rocahepevirus genomes were presented by IBS 1.0.

In this study, two full-length Rocahepevirus genomes (MSE-17 and LHK-54) and thirty-three partial ORF1 sequences were isolated, except one positive specimen failed in sequencing due to insufficient RNA. All sequences belong to HEV-C1 by phylogenetic alignment. These sequences have been deposited in the GenBank (accession numbers ON887282 and ON887283 for the two full-length sequences and OP921725 - OP921757 for the partial ORF1 sequences).

2.4. HEV sequence dataset

Other full-length HEV sequences were retrieved from the GenBank (https://www.ncbi.nlm.nih.gov/genbank/) through 11 September 2023, including thirty-nine reference sequences of Paslahepevirus balayani proposed by the ICTV (Smith et al., 2020), twelve Avihepevirus sequences, forty-nine Rocahepevirus sequences (including thirty-three HEV-C1 sequences), and two Chirohepevirus sequences. Besides, the three partial human HEV-C1 sequences isolated in Spain were also included for phylogenetic analysis (Supplementary Materials Table S3).

2.5. Calculation of sequence identity

We compared the nucleotide and amino acid identities of MSE-17 and LHK-54 with those full-length HEV genomes. Additionally, we generated a nucleotide identity matrix based on the partial ORF1 sequences isolated in this study. Sequence alignment was conducted by Clustal Omega 1.2.4 with default parameters (Sievers et al., 2011). Identity calculation between groups were conducted by MEGA X (Kumar et al., 2018) using p-distance method with default parameters. Standard error (SE) of sequence identities (ID) was estimated using bootstrap method with 1000 replicates (Supplementary Materials Figure S1). The 95 % confidence intervals (CI) of ID was calculated as: 95 % CI (ID) = ID ± Zα/2 * SE, where α represented the probability threshold for statistical significance, and Z represented the critical value that corresponds with α value (in this case, α = 0.05, Z = 1.96).

2.6. Recombination analysis

The potential recombination of the full-length HEV-C1 sequences isolated in this study were investigated employing the RDP software (version 4.101) (Martin et al., 2015), with other full-length Rocahepevirus genomes utilized as references. Seven algorithms (RDP, GENECONV, BootScan, MaxChi, Chimaera, Siscan and 3Seq) with default parameters were used to identify potential recombination. A sliding window of 200-bp and a step size of 20 bp were utilized. The P value was adjusted to 0.01. The recombination identified by three or more algorithms was considered as real recombination.

2.7. Phylogenetic analysis

The RdRp domain of HEV exhibites significant conservation and was frequently employed in the reconstruction of phylogenetic trees. Firstly, the thirty-five HEV-C1 sequences isolated in our study and thirty-six reference HEV-C1 sequences in the HEV sequence dataset were aligned by Clustal Omega 1.2.4 with default parameters, and then the aligned sequences were trimmed to 297 bp by MEGA X. Secondly, the trimmed sequences (within the ORF1 RdRp domain) were used to determine the optimal substitution models by ModelFinder with default parameters according to the Bayesian Information Criterion (BIC) (Kalyaanamoorthy et al., 2017). One HEV-C2 sequence (GenBank accession number AB890001) was included as the outgroup. Finally, the maximum-likelihood (ML) tree was reconstructed based on sequences of 297 bp by IQ-TREE2 (Minh et al., 2020) with ultrafast bootstrap test of 1000 replicates (Hoang et al., 2018), and visualized using the R package “ggtree” (Yu et al., 2017). Additionally, The ML tree based on the full-length sequences was reconstructed using MSE-17 and LHK-54 together with other full-length reference sequences in the HEV sequence dataset.

2.8. Determination of amino acid substitutions

We further included MSE-17, LHK-54, Rocahepevirus prototype sequence GU345042, and representative full-length HEV-C1 sequences (GenBank accession number MN450851, MN450853, MK050105, OP610066, JX120573, LC225389, AB847305, AB847306, LC573546, KM516906, ON644869, MW149254, LC549185, LC549186, LC549186, MZ542728; these sequences were isolated from different regions, and distributed in multiple clades in the phylogenetic tree) to predict the Met, Hel and RdRp domains in ORF1, using pairwise alignment with HEV-1 prototype sequence (GenBank accession number M73218, UniProtKB ID P29324) at amino acid level (Johne et al., 2010a; Koonin et al., 1992) by EMBOSS needle 6.6.0 with default parameters. The predicted functional domains were used to perform multiple sequence alignment using Clustal Omega 1.2.4 with default parameters. Amino acid substitutions in the conserved motifs of these domains were determined, compared with Rocahepevirus prototype sequence (GU345042, Germany, 2009). Data visualization was achieved by Jalview 2.0 (Waterhouse et al., 2009). The detailed information of sequence alignment was provided in Supplementary Materials Figure S2.

2.9. Prediction of RdRp protein structure

We predicted the protein structure of RdRp for MSE-17, LHK-54, and other six representative HEV-C1 sequences used in the amino acid substitution analysis by I-TASSER (Yang and Zhang, 2015). The models were assessed with C-scores and TM scores. A higher C-score signifies a model with a high confidence, and a TM-score >0.5 indicates a model of correct topology (Yang and Zhang, 2015). Based on the protein structure prediction, ligand binding sites and enzyme active sites were further predicted by COFACTOR and COACH (Yang et al., 2013; Zhang et al., 2017). The predicted protein structures and function sites were visualized using VMD 1.9.4a57 (Humphrey et al., 1996). Amino acid substitutions in the conserved motifs of RdRp, compared with Rocahepevirus prototype sequence GU345042, were mapped into the predicted protein structure. The detailed information on prediction of RdRp protein structure, ligand binding sites and enzyme active sites were provided in Supplementary Materials.

3. Results

3.1. Detection rate of Rocahepevirus

Totally, 8.11 % (36/444) of the specimens tested positive for Rocahepevirus, between July to August 2019. The detection rates in Lianghe county and Mangshi county were 9.63 % (21/218) and 8.29 % (15/181), respectively. In contrast, no Rocahepevirus was detected in the specimens collected in Mile county (Table 1). Additionally, no specimen was positive for Paslahepevirus balayani. Two RT-qPCR amplification curves and one chromatogram were provided in Supplementary Materials. Furthermore, all animals positive for Rocahepevirus were rodents. The detection rate in rodents was 12.95 % (36/278), with 14.77 % (35/237) in Rattus tanezumi and 33.33 % (1/3) in Niviventer fulvescens.

3.2. Amplification of Rocahepevirus sequences

Among the thirty-six specimens positive for Rocahepevirus, we obtained thirty-five sequences by Sanger sequencing and next generation sequencing. These included two full-length sequences (MSE-17 and LHK-54) and thirty-three partial ORF1 sequences (297–321 nucleotides), all identified as HEV-C1. MSE-17 was isolated in Mangshi county and LHK-54 was isolated in Lianghe county, both sourced from Rattus tanezumi. The genomic structures and localizations of putative ORFs of MSE-17 and LHK-54 were presented in Fig. 2.

Fig. 2.

Fig. 2

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Genomic structures and localizations of putative ORFs of MSE-17 and LHK-54. The Rocahepevirus prototype sequence (GenBank accession number GU345042, HEV-C1) was presented as a reference.

3.3. Nucleotide and amino acid identity

MSE-17 was 84.3 % (95 %CI: 83.7 %−85.3 %) identical to LHK-54 at nucleotide level. These two sequences shared 78.9 %−80.3 % nucleotide identity with other full-length HEV-C1 genomes. Additionally, MSE-17 and LHK-54 shared similar nucleotide and amino acid identities with the rat HEV-C1, as opposed to the human HEV-C1, across the full-length HEV, ORF1, ORF2 and ORF3 sequences (Table 2).

Table 2.

Nucleotide and amino acid identity ( %) between the two full-length HEV-C1 sequences and other full-length HEV genomes.

Genotypes Nucleotide identity % (95 % CI) Amino acid identity % (95 % CI)*
Full-length* Rocahepevirus (HEV-C1) 79.6 (78.9 - 80.3) –
Rat origin 80.1 (79.4 - 80.8) –
Human origin 78.7 (77.9 - 79.5) –
Rocahepevirus (HEV-C2, C3 and C4) 66.1 (65.2 - 66.9) –
Paslahepevirus balayani 56.5 (55.5 - 57.5) –
Avihepevirus magniiecur 52.3 (51.1 - 53.4) –
Chirohepevirus 52.7 (51.6 – 53.8) –
ORF1 Rocahepevirus (HEV-C1) 78.7 (77.9 - 79.6) 90.2 (89.1 - 91.3)
Rat origin 79.3 (78.4 - 80.1) 90.7 (89.6 - 91.7)
Human origin 77.8 (76.8- 78.7) 89.5 (88.2 - 90.7)
Rocahepevirus (HEV-C2, C3 and C4) 65.0 (64.0 - 66.0) 69.9 (68.1 - 71.7)
Paslahepevirus balayani 55.4 (54.3- 56.6) 53.6 (51.3 - 56.0)
Avihepevirus magniiecur 52.6 (51.3 - 53.9) 47.0 (44.5 - 49.5)
Chirohepevirus 52.0 (50.8 - 53.3) 47.4 (44.9 - 49.8)
ORF2 Rocahepevirus (HEV-C1) 81.8 (80.5 - 83.0) 93.8 (92.4 - 95.1)
Rat origin 82.2 (81.0 - 83.4) 94.1 (92.8 - 95.4)
Human origin 81.0 (79.7 - 82.3) 93.2 (91.7 - 94.8)
Rocahepevirus (HEV-C2, C3 and C4) 68.6 (67.1 - 70.1) 74.4 (71.8 - 77.1)
Paslahepevirus balayani 60.7 (58.8 - 62.6) 57.4 (53.6 - 61.3)
Avihepevirus magniiecur 52.6 (50.5 - 54.7) 45.8 (41.6 - 50.0)
Chirohepevirus 54.9 (52.9 - 56.9) 50.8 (47.0 - 54.6)
ORF3 Rocahepevirus (HEV-C1) 84.5 (81.5 - 87.4) 73.1 (67.3 - 78.9)
Rat origin 85.7 (83.0 - 88.4) 75.3 (69.9 - 80.7)
Human origin 82.3 (79.0 - 85.5) 70.0 (63.6 - 76.4)
Rocahepevirus (HEV-C2, C3 and C4) 62.2 (58.3 - 66.2) 38.6 (32.5 - 44.8)
Paslahepevirus balayani 54.2 (48.8 - 59.5) 29.6 (21.7 - 37.4)
Avihepevirus magniiecur 49.8 (43.0 - 56.5) 30.5 (20.7 - 40.3)
Chirohepevirus 43.9 (38.1 - 49.8) 20.3 (12.3 - 28.3)
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⁎

Amino acid identity was not calculated as the full-length HEV genomes contain non-coding regions (5′ and 3′ UTR).

Furthermore, these two full-length HEV-C1 sequences demonstrated the identities of 77.9 %−79.6 % (nucleotide) and 89.1 %−91.3 % (amino acid) with other HEV-C1 genomes in ORF1. Correspondingly, they were 80.5 %−83.0 % (nucleotide) and 92.4 %−95.1 % (amino acid) identical to other HEV-C1 genomes in ORF2. However, the identity between these two sequences and other HEV-C1 genomes in ORF3 at nucleotide level (81.5 %−87.4 %) was higher than that at amino acid level (67.3 %−78.9 %) (Table 2). Additionally, the nucleotide identity across the partial ORF1 sequences (aligned to 297 nucleotides) isolated in this study was 79.12 %−100 % (Supplementary Materials Table S5).

3.4. Phylogenetic reconstruction

No robust recombination was detected for the full-length HEV-C1 sequences isolated in this study (Supplementary Materials Table S6). TIM2+F + I + G4 was determined as the optimal substitution model to reconstruct the ML tree, based on partial HEV-C1 ORF1 sequences (aligned to 297 nucleotides). For the rat HEV-C1 isolated in our study, some sequences were distributed in clusters A and cluster C, while the remaining sequences were distributed in other unclassified sporadic clusters. These sequences were phylogenetically distant from sequences isolated in other studies (cluster B and cluster D). The sole HEV-C1 sequence isolated from Niviventer fulvescens (MSI-21) clustered with another sequence isolated from Rattus tanezumi (MSI-26), demonstrating a 100 % nucleotide identity. Notably, the sequences isolated in North America and Europe were all distributed in cluster B, indicating a spatially related phylogenetic relationship. However, other sequences isolated in China (rat HEV-C1, LC549184-LC549187, Guangdong, 2012–2015) and Indonesia (rat HEV-C1, AB847305-AB847309, 2011–2012; LC225388-LC225389, 2010–2015) were distributed in both cluster B and cluster D, exhibiting a phylogenetic diversity. For the human HEV-C1, those sequences isolated in Hong Kong (China) were distributed in cluster D, and formed two phylogenetically distant groups (group one: MG813927, MN450851, MN450852, MN450854; group two: MN450853); those sequences isolated in Spain shared the same clade with the Rocahepevirus prototype sequence GU345042 and were phylogenetically distant from French and Canadian HEV-C1 sequences (Fig. 3A). It suggested diverse genetic evolution of HEV-C1, whether of rat or human origin. The ML tree reconstructed with full-length sequences showed a similar phylogenetic relationship (Fig. 3B).

Fig. 3.

Fig. 3

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Phylogenetic analysis of HEV-C1. (A) Phylogenetic tree was reconstructed based on partial HEV-C1 ORF1 sequences (297 nucleotides). The HEV-C1 sequences isolated in our study were annotated with black arcs. (B) Phylogenetic tree was reconstructed based on full-length HEV-C1 sequences. The HEV-C1 sequences isolated in our study were annotated with pentagrams. The phylogenetic trees were reconstructed using maximum-likelihood method with ultrafast bootstrap of 1000 replicates. The dots at the nodes indicated a bootstrap support greater than 70 %. The HEV-C2 sequence (GenBank accession number AB890001) was used as the outgroup.

3.5. Amino acid substitutions

The amino acid substitutions within the motifs of Met, Hel, and RdRp domains of HEV-C1 sequences were identified. Locations of Met, Hel, and RdRp domains in ORF1 were presented based on the Rocahepevirus prototype sequence GU345042 (Fig. 4A). The motifs of these domains were highly conserved across HEV-C1 sequences. Furthermore, eleven amino acid substitutions within the motifs were identified in both the Met and Hel domains (Supplementary Materials Figure S3A and S3B).

Fig. 4.

Fig. 4

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Multiple sequence alignments of HEV RNA-dependent RNA polymerase (RdRp) domain in ORF1. (A) Localization of Met, Hel, and RdRp domains based on Rocahepevirus prototype sequence GU345042. (B) Multiple sequence alignments of RdRp. The motifs in RdRp domain were indicated by black underlines. Substitution sites within the motifs compared with GU345042 were highlighted with red boxes. In addition, multiple sequence alignments of methyltransferase and helicase domains were provided in Supplementary Materials Figure S3.

In RdRp domain, amino acid substitutions within the motifs were identified as follows: F204L in all sequences; T234V in all sequences except MK050105, LC549186, MZ357113, and two sequences isolated in our study (ON887282, ON887282); K237R in MN450853, LC225389, LC549186, MZ357113; L238F in all sequences except those isolated in North America and Europe (MK050105, OP610066, KM516906, ON644869, MW149254); V339L in ON644869; I348V and L403I in sequences isolated in Asia (MN450851, MN450853, JX120573, LC225389, AB847305, AB847306, LC573546, LC549185, MZ542728); and L400M in MZ542728. The two full-length sequences isolated in our study had less amino acid substitutions in the motifs of RdRp, compared with other Asian sequences. Additionally, The GDD motif (in VI motif, amino acid 344–346) was completely conserved (Fig. 4B).

The predicted protein structures of RdRp for MSE-17, LHK-54, and six HEV-C1 genomes (MN450851, KM516906, OP610066, MW149254, MZ542728, and MZ357113) were credible and had the correct topologies, validated by TM-scores >0.5. The amino acid substitutions in LHK-54 (L238F), MN450851 (T234V, L238F, I348V), KM516906 (T234V), MW149254 (T234V), MZ542728 (I348V), and MZ357113 (K237R, L238F) were all close to the ligand binding sites. However, L238F in MSE-17 and T234V in OP610066 (Human HEV-C1, France, 2022) were distant to ligand binding sites and enzyme active sites (Fig. 5, Figure S4).

Fig. 5.

Fig. 5

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Predicted protein structures of RNA-dependent RNA polymerase (RdRp) of HEV-C1 (A) MSE-17, (B) LHK-54, (C) MN450851 (human HEV-C1 in Hong Kong), and (D) KM516906 (rat HEV-C1 in the United States). Compared with Rocahepevirus prototype sequence GU345042, amino acid substitutions in motifs were highlighted in red (except F204L observed in all sequences). The predicted ligand binding sites and enzyme active sites were colored in yellow and purple, respectively. In addition, the protein structures of MW149254, MZ542728, MZ357113 and OP610066 were shown in Supplementary Materials Figure S4.

4. Discussion

In this study, the detection rate of Rocahepevirus was 12.95 % (all identified as HEV-C1) in rodents (order Rodentia), whereas none in other animals (orders Soricomorpha, Scandentia and Erinaceomorpha). So far, HEV-C1 has been detected in multiple countries, including the United States, Hungary, United Kingdom, Italy, France, Vietnam, and Indonesia (Wang et al., 2020) since the first identification in Germany (Johne et al., 2010a). In China, the detection rates differed by areas and animals, such as in a southern city (2.67 % in rodents and 0.51 % in shrews) in 2013–2016 (Wang et al., 2017) and in five southeastern/southern cities (20.19 % in rodents and 0.57 % in shrews) in 2014–2017 (He et al., 2018). It indicated rats may be a principal reservoir for HEV-C1 with varying detection rates. Furthermore, the HEV-C1 detection rate in Rattus tanezumi was 14.77 % (35/237) in our study, which was lower than another Chinese study (32.81 %, 21/64) (He et al., 2018). It might be attributable to sample size, sampling sites or RT-qPCR method.

Notably, to the best of our knowledge, we identified HEV-C1 in one Niviventer fulvescens for the first time, compared with Rattus norvegicus, Rattus tanezumi, and Rattus rattus documented elsewhere (Wang et al., 2020). The partial HEV-C1 sequence isolated from Niviventer fulvescens (MSI-21) clustered with another sequence isolated from Rattus tanezumi (MSI-26), demonstrating a 100 % nucleotide identity. These two rats shared the same habitat (near human residences), suggesting a potential cross-species transmission between rats of distinct species. Similarly, cross-species transmission from swine to rats is also possible, as HEV-3 was identified in rats in few studies (De Sabato et al., 2020; Lack et al., 2012), and rats could be experimentally infected with HEV-3 or HEV-4 (Doceul et al., 2016).

We found that the two full-length HEV-C1 sequences, MSE-17 and LHK-54, shared moderate nucleotide identity (78.9 %−80.3 %) with HEV-C1 isolated in rats and humans. Notably, the Rocahepevirus prototype sequence GU345042 shared significantly higher nucleotide identity (86.2 %−87.4 %) with rat HEV-C1 in Europe and America (KM516906, the United States, 2003; MW149254, Hungary, 2017), compared with that (78.2 %−79.8 %) with rat HEV-C1 in China (MSE-17 and LHK-54 in our study, and MZ542728, MZ868954, MN450855 and MZ357113, 2016–2021). Furthermore, phylogenetic analysis revealed that the HEV-C1 sequences isolated in our study had a relatively distant phylogenetic relationship with HEV-C1 in North America and Europe. These disparities in the sequence identity and phylogenetic relationship might be explained by a spatial spread. Despite the findings, the spread of HEV-C1 remains poorly understood due to limited sequences.

Regarding amino acid substitutions, MSE-17 and LHK-54 exhibited high conservation within the Met, Hel, and RdRp domains, aligning with previous findings (Drexler et al., 2012; Huang et al., 2004; Johne et al., 2010a). Besides, the GDD motif in RdRp was completely conserved (Kamer and Argos, 1984). However, there were several substitutions in other motifs in RdRp, compared with the Rocahepevirus prototype sequence GU345042. It has been documented that the amino acid substitutions might affect viral replication, viral load, and mortality (Parvez, 2017), and be associated with zoonotic infection (Tan et al., 2021). However, further investigation is required to unveil the specific impacts of amino acid mutations on rat and human HEV-C1. Furthermore, the majority of substitutions in HEV-C1 genomes were close to the predicted ligand binding sites, differing from L238F in MSE-17 and T234V in OP610066. Ligand binding and structural change in the ligand region can affect viral dynamics (Bern and Tobi, 2022). However, it remains unclear if and how the localization of substitutions influences the infection of HEV-C1, which warrants further studies on the mechanisms of HEV-C1 infection in animals and humans.

Limitations of this study need to be discussed. Firstly, we collected the animal specimens in three regions in Yunnan province, which limited the representativeness and generalizability of our findings. Secondly, Rattus tanezumi was the dominant species (237/278) among the captured rodents, which limited further comparative analysis across species. Thirdly, the analytical methods we utilized were in silico; hence, additional in vitro and in vivo experiments are imperative to attain a more profound comprehension of HEV-C1.

Our study also has strengths. Initially, we amplified two full-length HEV-C1 sequences, thereby facilitating a comparative analysis with other HEV-C1 genomes, and furnishing foundational data for subsequent in vitro and in vivo experiments. Secondly, we performed a comprehensive bioinformatic analysis on HEV-C1 genomes, including sequence identity, phylogenetic relations, amino acid substitutions and protein structures. Moreover, we concurrently examined the Rocahepevirus and Paslahepevirus balayani in captured animals. Our findings suggested that the local rats were more susceptible to HEV-C1 of Rocahepevirus in contrast to Paslahepevirus balayani.

5. Conclusion

In this study, HEV-C1 commonly spreads in rats. The majority of positive samples pertains to Rattus tanezumi, with only one originating from Niviventer fulvescens. One sequence isolated from Niviventer fulvescens demonstrated a 100 % nucleotide identity with that from Rattus tanezumi. No Paslahepevirus balayani was detected in all specimens. Additionally, the rat HEV-C1 isolated in our study and other Asian HEV-C1 demonstrated a distant phylogenetic relationship with those isolated in North America and Europe. Furthermore, the two full-length sequences isolated in our study had less amino acid substitutions in the motifs of RdRp, compared with other Asian sequences. These findings suggest a spatially associated phylogeny, and potential cross-species transmission of HEV-C1.

Ethics statement

The study was approved by the Institutional Review Board (IRB) of the Fudan University School of Public Health (IRB 00002408 and FWA 00002399) under IRB #2019-TYSQ-03–5 and #2021–04–0892; the Medical Ethics Committee of Dali University (MECDU) under MECDU-201,901–3. We followed all the institutional and national guidelines for the care and use of wild rodents throughout the study.

Disclosure statement

This study shared part of the animal samples with another study published in Chinese entitled “Hepatitis E virus infection and gene polymorphism in murine-shaped animals of plague foci in Yunnan Province” authored by Dr. Jiaxiang Yin and his team at Dali University in Yunnan province, which focused on the prevalence of HEV-C1 among wild rodents. In contrast, we optimized the experimental conditions, independently performed the examination of Rocahepevirus and Paslahepevirus balayani at Fudan University School of Public Health, and conducted data analysis. In our study, we obtained the two full-length HEV-C1 genomes and thirty-three partial sequences, and further characterized HEV-C1 genomics using comprehensive bioinformatics methods. No other potential conflict of interest was reported by the authors. The more detailed information of comparison between the study published in Chinese and our study was provided in Supplementary Materials.

Funding

This work was supported by the National Natural Science Funds of China [grant number 81973101, 81860565].

Data availability statement

The HEV sequences utilized in this study have been deposited and publicly available in the GenBank.

CRediT authorship contribution statement

Han Wu: Writing – original draft, Visualization, Formal analysis, Data curation. Bingzhe Li: Methodology, Investigation, Data curation. Bowen Yu: Investigation. Linjie Hu: Software. Lu Zhou: Data curation. Jiaxiang Yin: Writing – review & editing, Methodology, Funding acquisition, Conceptualization. Yihan Lu: Writing – review & editing, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank the biologists for animal capture and determination of species in the study.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2024.199321.

Contributor Information

Jiaxiang Yin, Email: chinayjx@hotmail.com.

Yihan Lu, Email: luyihan@fudan.edu.cn.

Appendix. Supplementary materials

mmc1.docx (38.4KB, docx)
mmc2.xlsx (16.5KB, xlsx)
mmc3.docx (3.9MB, docx)

Data availability

  • Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.docx (38.4KB, docx)
mmc2.xlsx (16.5KB, xlsx)
mmc3.docx (3.9MB, docx)

Data Availability Statement

The HEV sequences utilized in this study have been deposited and publicly available in the GenBank.

  • Data will be made available on request.

Articles from Virus Research are provided here courtesy of Elsevier

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