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. 2025 Jan;11(1):e70194.
doi: 10.1002/vms3.70194.

Detection and Whole Genome Amplification of the 4d Type of Porcine Hepatitis E Virus in Eastern Tibet, China

Yongzhi Lou  1 , Jiaojiao Xin  1 , Sizhu Suolang  1 , Da Qiong  1 , Zhuoma Dawa  2 , Ga Gong  1
Affiliations

Detection and Whole Genome Amplification of the 4d Type of Porcine Hepatitis E Virus in Eastern Tibet, China

Yongzhi Lou et al. Vet Med Sci. 2025 Jan.

Abstract

Genomic and evolutionary analysis of epidemic porcine hepatitis E virus (HEV) in the Tibetan Plateau was performed. Faecal samples were collected from 216 Tibetan pigs and 78 Tibetan Yorkshire (Large White) and 53 tissue samples from Yorkshire from the Linzhi City slaughterhouse. Total RNA was extracted from faeces and fragments of HEV open reading frame 2 (ORF2) detected by reverse transcription and nested polymerase chain reaction (RT-nPCR) and cloned. Twenty-three samples (23/347; 6.63%) were positive for the virus, including 6.94% (15/216) Tibetan pig and 6.11% (8/131) Yorkshire samples. No tissue samples tested positive for the virus. Cloned sequences were uploaded to GenBank (accession numbers: OR392679-OR392685, OR355817-OR355824 and OR909495-OR909502) and a phylogenetic tree constructed. The entire viral genome was amplified using primers for the 5-month-old Tibetan pig sequence which confirmed that the strain belonged to HEV type 4, subtype d (GenBank accession number: OQ981960) and showed 93.30% homology with Sichuan Tibetan pig sequence, MK410044. Bayesian tree analysis showed that the earliest divergence was in 1999 and evidence of homologous recombination was found. Genomic and evolutionary analysis of HEV in the Tibetan Plateau is presented. The importance of continuous surveillance and genomic analysis of HEV is highlighted, especially in regions like the Tibetan Plateau where new strains may emerge. The findings contribute to our understanding of HEV's genetic diversity, evolutionary history and potential risks to animal and human health.

Keywords: Tibetan pig; detection; genome evolutionary characterisation; hepatitis E virus; whole gene amplification.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Map information for collecting samples of Tibetan pigs in China. The image shows that 347 samples of Tibetan pigs were collected. This includes 78 samples of Yorkshire faeces from Changdu City, 53 samples of Yorkshire liver tissue from a slaughterhouse in Nyingchi City, 129 samples of Tibetan pig faeces from Milin County, and 87 samples of Tibetan pig faeces from Langxian County.
FIGURE 2
FIGURE 2
Results of immunohistochemical analysis. (A) Hepatitis E virus (HEV) negative (20×). (B) HEV positive (20×). Immunohistochemically stained liver cells from York swine using a microscope at 20× magnification revealed that the cell cytoplasm was stained with brown granules.
FIGURE 3
FIGURE 3
Phylogenetic evolutionary tree of hepatitis E virus (HEV) open reading frame 2 (ORF2; 348 bp) fragments. formula imageYorkie pig; ▴ Tibetan native pigs. Phylogenetic trees were constructed using MegA 7 software for 23 positive samples of HEV ORF2 (348 bp) from Tibetan pigs. Phylogenetic analysis showed the 23 strains to belong to the same branch as HEV‐4d Yunnan cow KY233432 isolate. The results revealed that Tibetan pig HEV was type 4d.
FIGURE 4
FIGURE 4
Hepatitis E virus (HEV) whole gene electrophoresis results. Reverse transcription‐polymerase chain reaction (RT‐PCR) successfully amplified nine fragments in the whole genome of Tibetan pig HEV, and the electrophoretic lengths of the nine fragments in DNA were 925 bp, 915 bp, 958 bp, 892 bp, 955 bp, 1007 bp, 1129 bp, 933 bp and 550 bp, respectively.
FIGURE 5
FIGURE 5
Phylogenetic evolutionary tree of hepatitis E virus (HEV) genes. formula image OQ981960; ▴ Type HEV‐4d. A phylogenetic tree was constructed for Tibetan pig OQ981960 using MegA 7 software, related to HEV MK410044 isolated from Tibetan pigs in Sichuan Province in 2018 and HEV KC163335 isolated from human infection in Shandong Province in 2012. The results revealed that Tibetan pig HEV OQ981960 was type 4d.
FIGURE 6
FIGURE 6
Homology analysis of the entire hepatitis E virus (HEV) gene sequence. The homology map of Tibetan pig OQ981960 was generated by Heatmapper and MegAlign.
FIGURE 7
FIGURE 7
Bayesian evolutionary tree of Tibetan pig whole genome isolates. The Bayesian evolutionary tree of Tibetan pig OQ981960 was generated by BEAST.v1.8.4 software package. The earliest divergence time for Tibetan pig sequence, OQ981960, was found to be 1999. Different colours indicate different rates of differentiation, Gansu strain FJ610232, Beijing strain HM152568 and Xinjiang strain AY594199 showed earlier divergence.
FIGURE 8
FIGURE 8
Results of RDP4 reorganisation analysis. Homologous recombination analysis of OQ981960 using RDP4 revealed 5028–6168 bp as the recombination region of OQ981960.
FIGURE 9
FIGURE 9
Results of SimPlot similarity analysis. Homologous recombination analysis of OQ981960 using SimPlot revealed 5028–6168 bp as the recombination region of OQ981960.
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References

    1. Boxman, I. L. A. , Verhoef L., Dop P. Y., Vennema H., Dirks R. A. M., and Opsteegh M.. 2022. “High Prevalence of Acute hepatitis E Virus Infection in Pigs in Dutch Slaughterhouses.” International Journal of Food Microbiology 379: 109830. 10.1016/j.ijfoodmicro.2022.109830. - DOI - PubMed
    1. Chen, Y. , Gong Q. L., Wang Q., et al. 2021. “Prevalence of Hepatitis E Virus Among Swine in China From 2010 to 2019: A Systematic Review and Meta‐Analysis.” Microbial Pathogenesis 150: 104687. 10.1016/j.micpath.2020.104687. - DOI - PubMed
    1. Doceul, V. , Bagdassarian E., Demange A., et al. 2016. “Zoonotic Hepatitis E Virus: Classification, Animal Reservoirs and Transmission Routes.” Viruses 8, no. 10: 270. - PMC - PubMed
    1. Ga, G. , and Sizhu S.. 2020. “Molecular Detection of Hepatitis E Virus in Tibetan Swine.” Pakistan Journal of Zoology 52: 1563–1569.
    1. Geng, Y. , Zhao C., Guo T., et al. 2019. “Detection of Hepatitis E Virus in Raw Pork and Pig Viscera as Food in Hebei Province of China.” Foodborne Pathogens and Disease 16, no. 5: 325–330. 10.1089/fpd.2018.2572. - DOI - PubMed

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