Genotype-4 hepatitis E in a human after ingesting roe deer meat in South Korea
A 43-year-old male presented with abdominal discomfort for 3 weeks and jaundice lasting 1 week. He had a past history of diabetes mellitus, which had been diagnosed 3 years prior to admission to our hospital. However, he arbitrarily stopped taking hypoglycemic agents. The patient was a heavy alcohol drinker, with the consumption of 2 to 3 bottles of Soju, distilled liquor, 4 to 5 times a week. He denied any travel outside South Korea in the preceding years. About 6-8 weeks before hospitalization, he ingested raw meat (about 300 g) of a captured wild roe deer inhabiting in Gyeongnam province with his friends, who enjoyed hunting on a regular basis.\nPhysical examination on admission was generally normal, except for jaundice. Mild tenderness was only noted in the epigastric area. Initial laboratory data showed white blood cell count of 4.88×103/mm3 (polymorphonuclear neutrophils, 57.2%; lymphocytes, 36.4%; and eosinophils, 1.5%), elevated serum total bilirubin level of 12.3 mg/dL, serum aspartate aminotransferase (AST) level of 1,637 IU/L, serum alanine aminotransferase (ALT) level of 1,949 IU/L, random glucose level of 220 mg/dL and HbA1c level of 10.8%. Hepatitis B surface (HBs) antigen, immunoglobulin M (IgM) anti-hepatitis B core antigen, anti-hepatitis C virus (HCV), HCV RNA (RT PCR) were all negative with positive anti-HBs. As a result of IgM anti-hepatitis A virus (HAV) was negative with positive immunoglobulin G (IgG) anti-HAV, acute hepatitis A could be excluded. Abdominal computed tomography showed findings compatible with secondary changes in acute hepatitis, and fatty infiltration with splenomegaly, which implied concurrent alcoholic liver disease. Seven days after admission, results of IgM anti-HEV and IgG anti-HEV were both positive, the optical density value of IgM anti-HEV of 3.656 (cut-off value: 0.276) and IgG anti-HEV of 3.384 (cut-off value: 0.375), which confirmed the diagnosis of acute viral hepatitis E. IgM anti-HEV and IgG anti-HEV were measured by a commercial immunoassay (HEV IgM and HEV IgG ELISA, Genelabs Diagnostic Pte. Ltd, Singapore). The serum total bilirubin peaked at 24.3 mg/dL and rapidly decreased. The levels of AST and ALT were highest at the time of admission and then showed a rapid decrease. Twenty-three days after admission, the patient was discharged with a total bilirubin of 8.06 mg/dL, AST of 130 IU/L and ALT of 133 IU/L (). Two months after discharge, IgM anti-HEV and IgG anti-HEV were both still positive with the optical density value of IgM anti-HEV of 3.315 (cut-off value: 0.282) and IgG anti-HEV of 2.753 (cut-off value: 0.375). Diagnosis of hepatitis E was confirmed by the detection of both IgM and IgG anti-HEV in serial samples and by the detection of serum HEV RNA.\nViral RNA was extracted from 140 µL of anti-HEV IgM-positive serum in phosphate-buffered saline using a QIAamp viral RNA mini-kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Purified RNA was used to generate the ORF2 of HEV using One-Step RT-PCR with a PLATINUM Taq Kit (Takara, Shiga, Japan). Briefly, RT-PCR was performed on 5 µL of purified RNA from serum in 50 µL of 2× reaction mix with 0.2 µM each primer (forward: 5' aggttggcgctctgtcgaga-3'; reverse: 5'-acagtcggctcgccattggc-3'). Reverse transcription was performed for 30 min at 50℃ and for 2 min at 94℃ followed by 40 cycles of 94℃ for 30s, 55℃ for 30s and 72℃ for 2 min, and a final extension step of 10 min at 72℃. The second round of PCR was performed under the same conditions as the first-round PCR with 0.2 µM each of the same primers.\nThe relationship between the sequences is shown in the dendrogram. The length of each pair of branches represents the distance between the sequences. Phylogenetic trees were constructed by the neighbor-joining method based on the ORF2 sequence. All strains were separated into four groups according to their genotypes. Sequence analysis was conducted using the software VectorNTI and MEGA 4.1 beta. Bootstrap values are indicated for the major nodes as a percentage of the data obtained from 1000 resamplings.\nWe identified genotype 4 human HEV from the 43-year-old Korean male patient with acute hepatitis, who had never been abroad. He was negative for serum markers of hepatitis A, B and C viruses and positive for anti-hepatitis E virus as IgM class leading to the diagnosis of hepatitis E, whose ORF2 nucleotide sequence from the patient was isolated. The identified Korean HEV strain named 2010-JKSH-CKK (HM769726) belonged to genotype 4, based on comparison with previously reported HEV strains (, ). When comparing the ORF2 region, HM769726 was closely related to the two strains EU849134 and FJ763142 with a nucleotide similarity of 97 and 98%, respectively (). The two human strains by HEV genome from Korean patients, who had never been aboard, were isolated in Gyeonggi-do, Korea. Among swine HEV isolates, HM769726 was closely related to EU332143-45 from Heilongjiang, China ().