PMC10315653_01
Clinical course of inactive chronic HBV superinfected with HEV in a 63-year-old man at Xiang'an Hospital of Xiamen University before and after treatment. The admission date of the patient in August 2019 was set as day 0. (A) ALT, AST, and GGT levels, anti-HEV and anti-HBV serological status, and HEV RNA and antigen status from October 2019 to January 2021. (B) HEV RNA concentrations in patient serum and stool and HEV antigen levels in patient serum, stool, and urine over time.
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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. 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 ). The patient was diagnosed with chronic hepatitis E as the patient's HEV RNA lasted for more than 6 months ( Figure 1B ). Immunohistochemical staining revealed scattered HBsAg positive cells ( Figure 2D ), whereas HBcAg tests were negative ( Figure S1 ). 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).
Histological and immunohistochemical staining of live tissue sections of a chronic HE patient obtained at day 56 after admission. (A) Liver tissue sections stained with hematoxylin and eosin showing chronic active hepatitis. (B) Reticular fiber staining revealing mild hyperplasia of fibrous tissue. (C, D) Liver tissue section staining with anti-HEV antibody indicating ORF2 antigen (C) and anti-HBV antibody indicating HBsAg (D). Scale bars represent 100 mum.
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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. We also tested the virulence of this HEV strain ( Figure S2 ).
Phylogenetic tree constructed by the neighbor-joining method based on the complete genomic sequence of 12 HEV genotype 3 strains. Genetic distances were calculated using the Kimura two-parameter method. GenBank accession numbers, host, and country of origin are provided. Genome sequences were isolated from chronic HE patient stools at indicated dates.
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Sequencing showed that the three hepatitis E virus strains belonged to genotype 3a, which was consistent at different sampling time ( Figure 3 ). 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 ).
Longitudinal physiological and biochemical tests and cytokine and chemokine profiling of the immune status of an HE patient. (A) Eosinophil counts and (B) serum total IgE before and after patient first admission. Upper limits of the normal eosinophil count (0.52 x 109/L) and percentage (8.0%) in white blood cell (WBC) are indicated as a dashed dotted line and a dotted line, respectively. The admission date in August 2019 was set as day 0. The normal range of serum total IgE (<100 IU/mL) is indicated with a dashed dotted line. (C) Cytokine and chemokine levels before and after antiviral therapy were detected by Bio-Plex assays. The antiviral therapy began on February 4 2020, and this date was set as day 0. Cytokines and chemokines plotted as log10 concentrations over time. Mean concentration of healthy controls is indicated with a dotted line. (D) Anti-HEV IgG titers, IgG avidity, and neutralizing capacity before and after ribavirin and interferon treatment. Neutralization of patient serum was determined by the 50% inhibitory dose (ID50). (E) Anti-HEV specific T-cell responses were detected by IFN-gamma ELISPOT. PBMCs were isolated from this anti-HEV IgG-negative subject and the chronic HE patient and were cultured with HEV 15-mer peptides. IFN-gamma secreting cell numbers were counted after stimulation with peptides.
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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). 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 ). 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 .