The First Documented Detection in Taiwan of The Hepatitis E Virus in Rats


 The authors have withdrawn this preprint due to author disagreement.


Background
Hepatitis E virus (HEV) contains a positive-sense, single-stranded RNA genome and is a member of the family Hepeviridae, genus Orthohepevirus. Within the genus Orthohepevirus, HEV is further classi ed into four species, Orthohepevirus A, B, C and D (HEV-A, B, C and D, respectively). The host ranges of these four species are distinct. HEV-A infects humans and other mammals, including domestic pigs and goats, wild boars, deer, and camels. The other 3 species, HEV-B, HEV-C, and HEV-D, infect avian, rodent, and bat species, respectively [1]. HEV-C was rst identi ed in Norwegian rats (Rattus norvegicus) trapped in the manholes of a sewer system in Germany in 2010 [2]. Moreover, HEV-C is further classi ed into genotype 1 (HEV-C1), which can infect rats and shrews, encompassing genotype 2-4 [3,4]. In taxonomy, rat HEV shares approximately 50% or less sequence identity with human HEV-A [5].
The role of rats in human HEV infections is controversial. Rat HEV was experimentally observed to be unable to infect primates in a rhesus monkey model [6]. However, recent HEV-C1 infections in humans challenged the previously known host range of HEV-C1. HEV-C1 was discovered in plasma, stool and liver tissue from a liver transplant recipient in Hong Kong in 2017, and an additional 10 Hong Kong and 1 Canadian HEV-C1 cases during 2017 ~ 2020 were detected and subsequently reported [7,8,9,10]. In Hong Kong, the discovery of human HEV-C1 infection was borne out by the following HEV-C1 infected patients, although the infections were sporadic, with most cases being reported in immunocompromised people with underlying diseases or who were on immunosuppressants [11]. Hong Kong and Taiwan have a similar environment and culture, so the objective of this study was to assess possible disease occurrence in Taiwan using RT-PCR and serological testing methods.

Samples
Fifty human sera samples stored at -20℃ were retrospectively examined. All sera were collected from suspected HEV patients during the period of acute hepatitis in 2018 ( Figure 1). Additionally, these sera samples were previously identi ed as HEV-A negative. Both commensal rodents R. rattus and R. norvegicus are hosts for HEV-C1 [1]. Fifty total rat sera stored at -20℃ were retrospectively sampled from 3 R. rattus and 47 R. norvegicus individuals between January 2017 and June 2019. All rats were trapped from 11 different international airports or harbors in Taiwan for surveillance of potential zoonosis.

RT-PCR analysis
RNA was manually extracted from human and rat serum samples using the QIAamp Viral RNA Mini Kit (Cat. no. 52906, QIAGEN, Hilden, Germany) following the instructions of the manufacturer. Brie y, 140 µL of human or rat serum was applied to a spin column for RNA to bind onto the column. Ethanol was added to the ow-through and RNA and bound to the membrane when the sample was passed through a spin column. After washing, the RNA was eluted in 50 µL of nuclease-free water. Extracted RNA was further tested for the presence of HEV RNA using a broadly reactive nested RT-PCR assay that detects largely divergent HEV variants, including HEV-C1 [12]. Samples positive for nested RT-PCR were tested again using a hemi-nested RT-PCR (an interior primer of the nested RT-PCR was excluded in the following hemi-nested RT-PCR) to avoid false positivity.
Amplicons of the hemi-nested RT-PCR were directly sequenced on both strands using the ABI 3730 XL DNA Analyzer (Applied Biosystem Inc., Foster City, California, USA). The sequences were aligned with other HEV sequences using CLUSTAL W [13]. A phylogenetic tree was constructed by the neighbor-joining method [14] with the Kimura two-parameter correction model and 1,000 replicates of bootstrap resampling as implemented in MEGA 10 (version 10.0.5) [15].
Serological assays HEV-C serological testing of rat sera was performed using a commercial Qualitative Rat Hepatitis E Virus Antibody (Anti-HEV) ELISA kit (Cat. no. MBS9357409, MyBiosource, California, USA). Interpretation of the results was performed according to the manufacturer's instructions. This kit detects both IgG and IgM. Detected immunoglobulin types were not further differentiated.
Results HEV-C RNA was not detected among sera collected from the 50 hepatitis patients or the 3 R. rattus, but viral RNA was observed in sera from 2 R. norvegicus individuals trapped at a harbor on June 6, 2017 (Fig. 1). The two detected partial sequences with a length of 382 base pairs shared 100% genetic identity in the RNA-dependent RNA polymerases gene of HEV. Nucleotide sequences analyzed in this study were submitted to GenBank with accession numbers of MN603692 and MN603693. Phylogenetically, both rat HEV strains were further grouped together with a HEV-C1-G2 variant isolate (HEV species C/genotype 1/genetic group G2, GenBank accession no. LC225389), which was discovered in Indonesia in 2014. The two sequences obtained in this study were also phylogenetically close to isolates of human HEV-C1 infections, including the rst discovered strain from a liver transplant receipt and another strain detected from an immunocompetent adult (Fig. 2). In addition, the seropositive rate of anti-HEV antibodies in the 50 rats was 52% (59%, 42% and 52% in 2017, 2018 and 2019, respectively). The 2 rat sera with HEV-C1 RNA were both negative for anti-HEV antibodies.

Discussion
This study identi ed HEV RNA in a local rat population in Taiwan with a detection rate of 4%. Additionally, anti-HEV antibodies were detected in 52% of trapped wild rats. This RNA result is similar to the Hong Kong study [10]. The detection rates of HEV-C RNA in R. norvegicus were 4.3% and 4.4% in Taiwan and Hong Kong, respectively. Moreover, this nding is consistent with the detection rates of HEV RNA (1.7-22.8%) in previous studies in Europe and Asia in the 2010s [16,17,18]. This nding implies that Taiwan is likely to have a risk of human HEV-C infection, especially given that ongoing sporadic transmission throughout 2017-2020 has already been observed in Hong Kong. The high homology between these 2 rat HEV sequences in this study is not surprising because both rats belonged to the same species, R. norvegicus, and were trapped simultaneously in the same harbor, indicating that HEV-C1 might be circulating and transmitting within rat populations in Taiwan.
Overall, we observed that 52% of wild rats were seropositive for rat HEV in international ports in Taiwan. This prevalence rate is higher than that of early studies, including 4.1-18.1% in Indonesia [19,20], 12.9% in Vietnam [21], 23.3% in China [22], 24.5% in Germany [23], 27.9% in Japan [24], and 31.2% in Lithuania [25]. However, the seropositive rates between studies might be di cult to directly compare due to the lack of identical antigens and detection antibodies used in the ELISA tests. The limited agreement between different ELISA tests for determination of anti-HEV antibodies has also been mentioned previously [26]. Moreover, one report described HEV-positive ELISA results in cattle serum containing HEV neutralizing IgG in the absence of HEV genomes throughout the cattle's life. Antibodies induced by unknown etiological agents that generate cross-reacting antibodies might also increase the di culty of explaining the serological results [27].
Since 2010, HEV-C1 RNA and antibodies have gradually been detected in rats on the Eurasian continent, North American continent, and the Indonesian archipelago [6,19,20,21,22,23,24,25]. This study further documents the rst detection of HEV-C1 in rats in Taiwan, a geographically isolated island.
Phylogenetically, detected rat HEV could also be traced back to the HEV-C1-G2 variant strains discovered in Indonesia in 2014. This possibility indicates that the viruses could have crossed geographic boundaries to achieve transmission. It also re ects the importance of sanitation control of vectors and reservoirs in ships, aircraft and international ports. This study might be insu cient to entirely clarify the prevalence of HEV-C in humans and rats in Taiwan due to the limited sample size. However, the risk for indigenous human infection in Taiwan should not be ignored because HEV-C1 RNA with zoonotic potential has already been detected in the local rodent population.

Conclusions
This study documents the rst detection of HEV-C1 in Taiwan. The high homology between HEV-C1 sequences of rats observed in this study might result from viral circulation and transmission within certain rodent populations. The risk of indigenous human infection in Taiwan should not be ignored given the domestic detection of HEV-C1 RNA.

Declarations
Ethics approval and consent to participate The need for approval was waived by article no. 47 of Infectious Disease Control Act.

Consent for publication
Not applicable.

Availability of data and materials
The datasets generated and analyzed in the current study are available in the GenBank repository, https://www.ncbi.nlm.nih.gov/genbank/.

Competing interest
The authors declare that they have no competing interests.   Figure 1 Sample distribution and ndings of human and rat sera.

Figure 1
Sample distribution and ndings of human and rat sera. Sample distribution and ndings of human and rat sera.