LU Gang, YIN Xin, LIU Yan-qin, XIE Jin-xin, LI Shou-jun,

1 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, P.R.China

2 State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150000, P.R.China

3 Bayannur Animal Husbandry Service Center, Bayannur 150800, P.R.China

4 Guangdong Technological Engineering Research Center for Pet, Guangzhou 510642, P.R.China

5 College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, P.R.China

Hepatitis E virus (HEV) infection is associated with epidemic and sporadic viral hepatitis worldwide.It is a small, quasi-enveloped, single-stranded positive-sense RNA virus (Emerson and Purcell 2003).Its genome varies from 6.6 to 7.3 kb in length, containing three open reading frames (ORFs) named ORF1, ORF2, and ORF3.They encode non-structural polyprotein, viral capsid protein, and a small phosphoprotein that is essential for viral release, respectively.As recommended by International Committee on Taxonomy of Viruses (ICTV),HEV (currentlyPaslahepevirusbalayani) is classified into the genusPaslahepevirusin the familyHepeviridae(Smithet al.2014).To date, a total of eight genotypes of HEV(HEV1-8) have been identified inPaslahepevirus balayani(www.ictv.global/report/hepeviridae) and HEV1-4 have been frequently found in human infection.HEV3 and HEV4 could be isolated from pigs and other mammals and are the major causes for zoonotic hepatitis E (Wanget al.2016; Jeonget al.2017).HEV5 and HEV6 have been found in wild boars in Japan (Satoet al.2011), while HEV7 and HEV8 were reported in camels (Wooet al.2014, 2016; Wanget al.2019; Nishizawaet al.2021).

In two molecular epidemiology studies between 2012 and 2013, a total of 203 and 205 fecal samples were collected from adult dromedaries (Camelusdromedarius)in the Middle East and Bactrian camels (Camelusbactrianus) in China respectively for HEV RNA detection(Wooet al.2014, 2016).Intriguingly, the determined viral genome in dromedaries and Bactrian camels differed from all other HEVs by >20% nucleotides.The newly identified viruses were classified into the novelPaslahepevirus balayanigenotypes and named as HEV7 and HEV8 by ICTV.Until now, only one subtype of HEV7 (HEV7a) and one subtype of HEV8 (HEV8a) have been assigned (Smithet al.2020).

Notably, both HEV7 and HEV8 have the potential to cause zoonotic HEV infection as HEV7 RNA has been detected in a patient from the Middle East who underwent liver transplantation and had the habit of consuming camel meat and milk (Leeet al.2016).In addition, animal challenge studies demonstrated monkeys can be infected with both HEV7 and HEV8, posing a high potential zoonotic risk (Liet al.2016; Wanget al.2019; Guoet al.2020).

China is recognized as an endemic region for HEV.A phylogenetic analysis based on 626 HEV isolates from humans and animals in China demonstrated 3 genotypes(HEV1, HEV3, and HEV4) and 11 subtypes of HEV were prevailing in China during 1986-2011 (Liuet al.2012).HEV strains circulating in China had considerable genotype diversity and zoonotic transmission was widely distributed (Liuet al.2012).HEV was firstly recognized in China during a prolonged outbreak in Xinjiang in the late 1980s with ~120 000 people being infected.The causative genotype of this outbreak was HEV1 (Teshaleet al.2010).In addition, HEV was determined being prevalent in farmers with a high seropositive rate of 35.3% in Inner Mongolia, a neighboring region of Xinjiang (Kanget al.2017).

China owns a large number of Bactrian camels, mainly distributed in Xinjiang and Inner Mongolia in northern China.Currently, knowledge on the prevalence and genetic characteristics of HEV in Bactrian camels in China is still lacking.In the present study, we collected fecal samples from Bactrian camels in China, determined the positive rate of HEV RNA in the samples, and performed a genetic characterization on the viral genome.

Between March 2020 and August 2021, a total of 559 fresh fecal samples were individually collected from domestic Bactrian camels in Xinjiang and Inner Mongolia.The presence of HEV RNA in the 559 fecal samples were detected by four nested-PCR (PCR procedure 1 and 3-5)and one heminested-PCR procedures (PCR procedure 2) (Appendix A).The designed primer sequences were targeting HEV1-4, HEV1-6, HEV7, HEV8, and HEV7-8 strains, respectively.Primers for the PCR procedures 3-5(Appendix A) were designed in the present study by aligning the sequences of 9 HEV7 and HEV8 strains retrieved on the NCBI database (accessed on 26 August, 2020).

After gel electrophoresis, PCR products from 17 fecal samples presented the expected bands with a size of 140-338 bp, which were determined by at least one of the 5 PCR procedures.The detailed information on PCR procedures that could successfully detect each of the 17 fecal samples were listed in Appendix B.Then, one PCR product from each positive fecal sample was processed for further Sanger sequencing (BGI, China).

Twelve of these 17 PCR-positive samples were collected from Banner City in Inner Mongolia and the remaining 5 samples were obtained from Hami City and Urumqi City in Xinjiang.After sequencing, the retrieved raw data were used for further analysis by the BLAST tool in the NCBI database (https://www.ncbi.nlm.nih.gov/).The result demonstrated that all the sequencing data were from the 17 PCR-positive samples hit HEV8, with a nucleotide similarity between 88.6 and 94.1% (Appendix B).Accordingly, the positive rate of HEV8 in the tested samples was determined to be 3.0%.

To sequence the viral genome of HEV circulating in camels in China, all the genomic sequences of previously published HEV8 strains were retrieved from the NCBI database and were then aligned by clustalW method using Bioedit 7.0.9.0.Based on the alignment result, a total of 6 primers covering HEV8 strains were designed by Oligo 7.0 and the genome sequences of 3 field HEV8 strains (camel73, camel77, and camel/X-7-19) were sequenced.The primer sequences can be provided upon request.

After sequencing and assembly, the genome sequences of 3 Chinese field HEV8 strains camel73(determined in Urumqi), camel77 (determined in Hami),and camel/X-7-19 (determined in Banner) were obtained and submitted to the GenBank database with accession numbers of ON263152, ON263153, and ON736438,respectively.The genomic sequences of other HEV8 strains determined in the present study were not sequenced owing to low viral RNA contents in fecal samples and/or limited sample amounts.

The genome of camel73, camel77, and camel/X-7-19 included a 15 nt partial 5′UTR, a 7 109 nt viral protein coding region, and a 15 nt partial 3′UTR.The nucleotide similarity of the genome across camel73, camel77, and camel/X-7-19 were calculated.It was demonstrated that camel73 had a high genetic similarity of 99.7% with camel77, and both camel73 and camel77 have a genetic similarity of 93.7% with camel/X-7-19.Sequence alignment result indicated a total of 18 nucleotide differences were determined between genome of camel73 and camel77,and both camel73 and camel77 had 448 nucleotide differences with camel/X-7-19.Further analysis indicated camel73, camel77, and camel/X-7-19 had a higher genetic similarity with HEV8 representative strains compared with HEV1-HEV7 representative strains at the genome, ORF1,ORF2, and ORF3 levels (Appendix C).

No potential recombination events were detected among HEV8 strains or between HEV8 and HEV1-7 strains using Recombination Detection Program (RDP) 4.27.

Four maximum likelihood phylogenetic trees were inferred using MegAlign 7.1.0.to further understand the genetic relationship between the Chinese HEV8 strains determined in the present study (camel73, camel77, and camel/X-7-19) and HEV1-8 representative strains based on their whole genomic, ORF1, ORF2, and ORF3 genetic sequences, respectively (Appendix D).It was noted that all HEV8 strains always had the closest genetic relationship with HEV7 strains when analyzing their whole genomic,ORF1, ORF2, and ORF3 genetic sequences (Fig.1-B).As indicated in all the 4 maximum likelihood phylogenetic trees, camel73, camel77, and camel/X-7-19 were clustered together with each other.In addition, camel73, camel77,and camel/X-7-19 had the closest genetic relationship with BcHEV-GP that was determined in China in 2017 when analyzing their whole genomic, ORF1, and ORF2 genetic sequences.However, ORF3 genetic sequences of camel73, camel77, and camel/X-7-19 had the closest genetic relationship with BcHEV-MNG140 and BcHEVMNG146 that were determined in Mongolia in 2019.This analysis demonstrated ORF3 genetic sequences of camel73, camel77, and camel/X-7-19 presented a distinct evolutionary pattern with ORF1 and ORF2.

According to Smithet al.(2016), strains could be assigned as a new HEV subtype if their genomic sequences were phylogenetically distinct from previously assigned subtypes and at least three complete ORF1 and ORF2 sequences were available that were epidemiologically unrelated (from different studies or localities).The Chinese strain 12XJ (accession number KX387865) was assigned as the reference sequence for subtype 8a (Smithet al.2020).The following study indicated all the 3 strains 12XJ,48XJ, and 62XJ in the first report of HEV8 belonged to subtype 8a (Nishizawaet al.2021).

As indicated in phylogenetic analysis of HEV8 strains based on their genomic sequences, HEV8 strains in China and Mongolia were classified into 4 groups, which contained group 4 (camel73, camel77, and camel/X-7-34), group 2(BcHEV-GP), group 1 (12XJ, 48XJ, and 62XJ), and group 3 (BcHEV-MNG140 and BcHEV-MNG146), respectively(Fig.1).The groups were provisionally designated according to the time when the strains were determined.Group 1 consisted of HEV8 strains 12XJ, 48XJ, and 62XJ of genotype 8a.HEV8 strains of groups 4 were only determined in the present study and were phylogenetically distinct from previously assigned subtype 8a.

As recommended by Smithet al.(2020), though another Chinese strain BcHEV-GP (accession number MH410174) had ap-distance of 0.117 with 12XJ at the genomic level, BcHEV-GP was still regarded as an unclassified strain of genotype 8 as there is only one representative strain by itself.The genetic differences between genomic sequences of camel73, camel77,camel/X-7-34 and other HEV8 strains were calculated(Table 1).Genomic sequences of camel73, camel77,and camel/X-7-34 hadp-distances of 0.119-0.149 with subtype 8a.It was also noted that camel73, camel77, and camel/X-7-34 were determined in three different localities of Urumqi, Hami, and Banner, respectively.

Taken together, camel73, camel77, and camel/X-7-34 were genetically and phylogenetically distinct from previously assigned strains of subtype 8a and were detected in 3 different localities.According to the criteria for HEV subtype assignment proposed by Smithet al.(2016), camel73,camel77, and camel/X-7-34 were assigned as representative strains of a new subtype of HEV8, provisionally designated as HEV8b.RNA-dependent RNA polymerase of members of the familyHepeviridaehas low fidelity and the virus replicates in host cells with high genetic variability (Okamoto 2007).This newly established subtype increases our knowledge of HEV8 heterogeneity and evolution.In addition, it stresses the role of camels as sources for potential novel HEV8 subtypes.

Bactrian camels are very important livestock animals in Xinjiang and Inner Mongolia in northern China.They are important sources for fur, milk, and meat production and also are major draft animals in the local area.Therefore,Bactrian camels have multiple ways to contact human.It was noted that HEV8 was capable of infect cynomolgus macaques without pre-adaption, which finally establish acute and chronic infections and could cause liver and renal injury in infected animals (Wanget al.2019).Accordingly, HEV8 in Bactrian camels is a potential threat to human health in China, such as consuming meat and milk that were obtained from HEV8-infected camels.Onehealth strategy may help reduce the public health risks associated with HEV8.

In summary, we collected 559 fecal samples from Bactrian camels in China and detected HEV RNA in the samples.The result determined the rate of HEV8-RNA positive samples was 3.0%.Further genetic analysis demonstrated they belonged to a new HEV8 subtype.The results of the present study expand our knowledge on the epidemiological characteristics, genetic variability,and evolution of HEV8 in Bactrian camels in China.

Acknowledgements

The authors were supported by the National Natural Science Foundation of China (32172940).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

The authors confirm that the ethical policies of the journal,as noted on the journal’s author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received.The fecal sample collection method was conducted under the guidance of the South China Agricultural University Experimental Animal Welfare Ethics Committee, China.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2023.07.038