A 63-year-old man was admitted to Xiang'an Hospital of Xiamen University, Xiamen, China, for hepatitis on August 17, 2019. Five months before presentation, his physical examination yielded abnormally elevated transaminase levels ( Figure 1A ); yet, the patient reported no fatigue, anorexia, jaundice, or abdominal pain. Afterward, the patient received oral bicyclol tablets and compound glutathione for hepatoprotective treatment, but his transaminase levels continued to fluctuate above the normal level. The patient visited Xiang'an Hospital of Xiamen University for further diagnosis and treatment at this point.
The patient underwent surgical treatment for tuberculosis of the lumbar transverse process in 1984. After that, he received regular anti-tuberculosis treatment for 1 year and healed well afterward. In 2015, he underwent laparoscopic cholecystectomy due to cholecystitis. Hepatitis B was diagnosed in the same year, with normal transaminase levels, and the patient was diagnosed as being anti-HBe positive at this time ( Figure 1A ,  Table S1 ), indicating an inactive phase of chronic hepatitis B (CHB) infection. The patient was not treated with antiviral therapy and was followed up regularly. Anti-HBs seroconversion and clearance of serum HBsAg occurred during follow-up. HBV DNA tests were negative when abnormal liver function was found upon physical examination five months before admission(March 2019). The patient had no history of autoimmune disease and denied receiving immunosuppressive drugs. The patient, however, had traveled abroad several times, including the Philippines sixteen months before admission (April 2018), Singapore and Malaysia eight months before admission (November 2018),and Nepal three months before admission (May 2019).
After being admitted to Xiang'an hospital, the patient tested positive for anti-HEV IgM, and both serum and stool samples were positive for HEV RNA. The patient was diagnosed as being in the inactive phase of CHB and being superinfected with hepatitis E. Diagnostic test results for infectious diseases including hepatitis A, C, D and G, HIV, liver fluke, and plasmodium were all negative ( Tables S2 ,  S3 ). Diagnostic test results for liver-related autoantibodies, including anti-nuclear antibody, anti-mitochondrial antibody, and anti-smooth muscle antibody, were negative, and ceruloplasmin was within the normal range ( Table S2 ). His routine blood tests at admission showed a normal white blood cell count (WBC, 8.75 x 109/L), neutrophilic granulocyte count (NEU, 3.23 x 109/L), lymphocyte count (LYM, 3.87 x 109/L), and monocyte count (MON, 0.51 x 109/L) and a slightly increased basophilic granulocyte count (EOS, 1.07 x 109/L;  Tables S2 ,  S4 ). The serum total IgM, IgG, IgA, and complement (C3 and C4) were all within the normal ranges ( Table S2 ). His CD3+ T cell count (1343/microL), CD4+ T cell count (555/microL), CD8+ T cell count (591/microL), and CD4/CD8 ratio (0.94) were also within the normal ranges, indicating that the patient showed no evidence for immune deficiency ( Table S2 ).
After the patient was admitted to Xiang'an hospital, his serum, feces, and urine samples were continuously collected for serological testing by ELISA and qRT-PCR ( Figure 1B ). Liver enzymes, including alanine aminotransferase (ALT), aspartate aminotransferases (AST), and glutamyl transpeptidase (GGT), fluctuated more than the normal range after admission, while the patient exhibited normal liver function four years (October 2015) and one year (July 2018) before admission ( Figure 1A ), indicating that his hepatitis was derived from HEV infection. HEV RNA was detected in the serum and feces of the patient at a titer above 105 IU/mL and 107 IU/mL, respectively. The patient was diagnosed with chronic hepatitis E as the patient's HEV RNA lasted for more than 6 months ( Figure 1B ). Therefore, treatment with 900 mg/d ribavirin was initiated on 170th day after admission (February 4, 2020). Subsequently, 600 mg/d of ribavirin combined with 180 microg/w of PEG-IFN was initiated three months later (April 27,2020). The transaminase and GGT levels returned to normal after 2 and 4 weeks of ribavirin treatment, respectively, indicating the recovery of the patient's liver function. Serum HEV RNA decreased below the detectable limit after 6 weeks of ribavirin treatment, while fecal HEV shedding persisted for more than 12 weeks after the start of ribavirin therapy. HEV antigen (Ag) levels in the serum and urine decreased after this treatment, consistent with the declining trend of viral RNA.
Liver puncture biopsy was performed two months after being admitted (October 2019), and this revealed the chronic active hepatitis with mild inflammation (METAVIR A1) and moderate fibrosis (METAVIR F1) ( Figure 2 ). Regional watery degeneration of liver cells, fatty degeneration of partial liver cells, scattered focal necrosis, and apoptotic bodies were all observed. ( Figure 2A ). An incomplete liver plate in the portal area was observed, with the reticular fibers proliferating and extending to the lobules, suggesting mild hyperplasia of fibrous tissue ( Figure 2B ). Immunohistochemical staining revealed scattered HBsAg positive cells ( Figure 2D ), whereas HBcAg tests were negative ( Figure S1 ). Widely distributed HEV Ag was detectable in the liver using a mouse anti-HEV capsid protein antibody (mAb #4,  Figure 2C ), indicating chronic HBV superinfected with HEV.
Complete genome sequencing of HEV isolates from the patient's stool specimens at several distinct dates was performed, and three genome sequences (Genbank accession OQ801358, OQ801359, OQ801360) were successfully identified. Sequencing showed that the three hepatitis E virus strains belonged to genotype 3a, which was consistent at different sampling time ( Figure 3 ). Sequence similarity analysis of the HEV whole genome showed that these isolates were the most identical to the variant KT447526, with 97.0% similarity. Zhu et al. described that the HEV genotype 3a National University Hospital (NUH) clade represented by KT447526 and KT227528 was an endemic local lineage of HEV in Singapore. Given the fact that the patient had traveled to Singapore and that his ALT and AST levels were normal a few months before going abroad thirteen months before presentation (July 2018) ( Figure 1A ), we proposed that the patient was infected with HEV in Singapore in eight months before admission into Xiang'an hospital (November 2018).
We also tested the virulence of this HEV strain ( Figure S2 ). This strain exhibited stronger infectivity to cells in vitro than the virus isolated from an acute patient but had comparable infectivity to a strain isolated from another chronic patient. It is possible that the strain isolated from the chronic patient had replicated in the patient's body and produced adaptive mutations as we found ( Figure S3 ).
We analyzed the levels of 48 cytokines and chemokines in the patient's plasma. The patient showed type-2 (anti-helminths) immune responses, with an elevation of the type-2 antibody isotype IgE and the hallmark type-2 cytokine IL-5, which is produced by Th2 cells and ILC2s and is the most potent activator of eosinophils ( Figures 4 ,  S4 ). To test whether the patient was infected with helminths, we performed eukaryotic 18S ribosome amplicon and metagenome sequencing with blood samples and intestinal samples, but no parasitic pathogen was found (data not shown). Biomarkers for atopic dermatitis, including CTACK (strong correlation), IL-18 (strong correlation), and IgE (moderate correlation), were also increased, although the patient had no obvious clinical manifestations of atopic dermatitis. Moreover, the patient showed high serum total IgE at admission. Sustained higher eosinophil levels in the blood of the patient were observed as early as 2015 ( Figure 4A ); then, elevated eosinophils were confirmed by bone marrow examination four months after presentation (December, 2019) ( Table S5 ).
The patient showed decreasing serum anti-HEV IgG titer over time from over 30 WHO unit per mL (Wu/mL) before treatment to around 10 Wu/mL, along with reducing viral shedding, after four months of antiviral therapy ( Figure 4D ). The patient also exhibited a notably higher anti-HEV IgG level compared to individuals who were vaccinated with Hecolin  and other acute HE patients and a medium range of IgG avidity ( Figure S5 ). The patient showed remarkably high neutralizing capacity with 50% inhibitory dose (ID50) values up to 16,634, in comparison to acute HE patients ( Figure S5C ). The patient's serum neutralizing activity also decreased over time, which was consistent with the patient's anti-HEV IgG level. In order to detect which epitopes antibody recognized were induced after chronic infection of hepatitis E virus, we employed competitive ELISA using a panel of murine mAbs that targeting diverse antigenic clusters on the capsid protein as previously described. The patient serum HEV specific antibodies predominantly recognize 8G12-like epitope ( Figure S5D ). When compared to acute hepatitis E patients, antibodies recognizing 8C11-like epitope and 8E1-epitope were relatively less dominant in chronic patient serum. These results are consistent with the predominant type of epitopes recognized by antibodies in sera from acutely infected individuals. To test for the generation of anti-HEV specific T-cell responses following infection, samples of the patient's PBMCs were collected. We stimulated PBMCs from the patient and two anti-HEV IgG negative controls with peptides derived from ORF1, ORF2, and ORF3 of HEV ( Table S6 ). HEV specific T-cell responses were detected in the patient's PBMCs, and these presented a slightly decreasing tendency but were significantly higher compared to HEV-negative controls ( Figure 4E ), indicating a mild to moderate T cell responses to HEV in the chronic HE patient. The T cell response was consistent with the patient's HEV RNA titer and IgG levels. The overview of the case's diagnosis, treatment, and follow-up process and timelines of major clinical events are summarized in  Figure S6 .