Given that the focus for viral detection in enterically transmitted non-A, non-B hepatitis historically had been fecal shedding, the first method available for detection of the virus (now known as HEV) responsible for this disease was immune electron microscopy (IEM) (4
), but the detection rate of HEV in fecal samples by IEM was low: the detection limit of HEV by IEM is estimated to be only 106
). Application of RT-PCR methods resulted in a dramatic increase in the detection of HEV RNA among patients with acute hepatitis E (76/166 [45.8%] or 47/67 [70.1%]) (9
). In the present study, although the number of patients studied was small, all 11 patients had detectable HEV RNA in their fecal specimens. The HEV load in fecal supernatant was low, at <100 copies/ml for 5 of the 11 patients, and was only 7.1 × 102
to 5.7 × 104
copies/ml for 5 other patients, indicating that a large proportion of patients were excreting the virus, albeit in small quantities, after the onset of clinical symptoms. To our surprise, high-titer HEV RNA of 2.0 × 107
copies/ml was detected in the stool sample of the remaining patient (patient 1), although the fecal sample was collected on day 22 after the presumed day of onset or 10 days after the day with the highest ALT value during the observation period. This finding encouraged us to investigate whether HEV in the fecal supernatant can replicate in established cell lines. Using the fecal suspension obtained on day 22 (JE03-1760F) from patient 1, we successfully developed an efficient cell culture system for HEV in PRC/PRF/5 and A549 cells in our previous study (55
Except for experimental HEV infection in monkeys (9
), only a few studies had been conducted for detection of HEV RNA in serial fecal samples from patients with acute hepatitis E. In a study of HEV transmission in a single volunteer in which RT-PCR was employed for sequential detection of HEV RNA (8
), HEV RNA was detected in fecal samples from the patient up to day 16 after the onset of symptoms; stools collected on days 4 and 7 transmitted disease to three monkeys. Since subsequent samples were not collected from the patient, it was unknown how long fecal shedding of HEV lasted. Based on data on serial fecal samples, Nanda et al. (36
) reported that four patients with acute hepatitis E in India showed fecal virus shedding up to the 9th, 10th, 12th, and 52nd days of the illness, respectively. In the largest available data set on serial fecal samples, HEV RNA was detectable in 16 (80%) of 20 patients in an outbreak of hepatitis E in India, and the maximum duration after the onset of the first symptom at which a stool sample was positive for HEV RNA was 30 days (2
). Most previous studies on detection of HEV RNA in fecal samples from patients with epidemic or sporadic hepatitis E had been conducted cross-sectionally. In a study of single fecal samples from patients with acute hepatitis E, Clayson et al. (10
) found HEV RNA in 53% (8/15), 77% (10/13), and 50% (1/2) of stool samples collected within the first 3 days of illness, within 8 to 11 days, and within 12 to 15 days, respectively; no stool samples were collected after 15 days. Several other studies of single stool samples from infected individuals indicated that fecal shedding occurs for approximately 2 weeks (1
). Therefore, to summarize the previous studies, prolonged fecal shedding of HEV in humans was shown in a small group of patients, and the longest duration of fecal excretion of HEV thus far reported is 52 days.
In the present study, we were able to obtain serial fecal samples from 11 sporadic cases of domestic or imported acute hepatitis E, and therefore it was possible to investigate the duration of HEV excretion and load of HEV shed into the feces. Nine of the 11 patients studied had short-term virus fecal excretion of 14 to 29 days' duration, corroborating the findings of the previous study in which loss of virus fecal shedding was observed within 30 days of illness for patients with acute HEV infection, except for one patient, who showed fecal shedding up to the 52nd day of illness (36
). One of the most remarkable results of this study was that HEV RNA was detectable in the fecal specimen obtained from a patient (patient 1) even on day 121 after the presumed onset of hepatitis, or 109 days after the day on which the peak ALT level of 620 IU/liter was observed. Recently, prolonged fecal virus shedding (beyond 56 dpi) was observed in only 2 of 15 pigs experimentally infected with genotype 3 HEV (14
) and only one of nine chickens experimentally infected with avian HEV (6
). The observation of prolonged fecal virus shedding from a natural case of human hepatitis E, coupled with similar observations in HEV infections from a very small number of experimentally infected animals, indicates that, indeed, prolonged fecal shedding does occur during HEV infection. This has important implications for understanding HEV pathogenesis and transmission.
The precise reason for the markedly long duration of virus fecal excretion found for a particular patient remains unknown. Unlike the other 10 patients studied, patient 1 had an underlying disease of chronic renal failure and contracted de novo HEV infection just before the initiation of hemodialysis. As patients on maintenance hemodialysis have an impaired immune response to viral protein or to vaccination (12
), they may be unable to raise an adequate immune response to viral protein and to efficiently eliminate an infecting virus. The impaired immune response in hemodialysis patients is thought to be related to low levels of T-cell proliferation upon mitogenic stimuli because of impaired costimulation by accessory cells (16
). Prolonged viremia of HEV also was reported for a patient with T-cell lymphoma during chemotherapy (54
). Therefore, one possible explanation is that protracted fecal shedding of HEV in patient 1 was attributable to impaired host immunity at the initiation of hemodialysis and during maintenance hemodialysis. However, high and persistent excretion of HAV by immunocompetent patients has been reported (60
), and transient, short-term viremia of HEV was observed in three hemodialysis patients who acquired subclinical HEV infection (31
), suggesting that it is important to consider an alternative explanation for the obtained results. HEV in the fecal suspension obtained from patient 1 on day 22 (JE03-1760F) could grow efficiently in our cell culture system, reaching a high titer of up to 108
copies/ml, and HEV progeny released in the culture medium were successfully passaged five times in culture cells (55
). Therefore, it is likely that the JE03-1760F strain has a higher replicative capability than other HEV strains.
For patient 1, fecal shedding of HEV lasted approximately 100 days after normalization of the ALT level and 83 days after the cessation of viremia, suggesting extrahepatic replication of HEV. Of interest, it has been shown that both swine HEV and human HEV replicate in extrahepatic organs of experimentally infected pigs, including the colon and intestines (65
), which may be responsible for the long duration of virus fecal excretion. Whether the JE03-1760F strain is likely to replicate efficiently and for a longer duration in the colon and/or intestines needs further investigation.
It remains unknown what mutations in the HEV genome are associated with the heightened multiplication ability of HEV. Therefore, in the present study, we determined the full-length genomic sequence of the JE03-1760F isolate obtained from the fecal suspension with the highest HEV load of 2.0 × 107 copies/ml of patient 1 and found 29 nucleotide substitutions over the entire genome that are unique to the JE03-1760F isolate and that are not seen in any of the 25 reported HEV isolates of the same genotype (genotype 3). Among the 29 nucleotide substitutions, six substitutions lead to amino acid changes in ORF1, which encodes nonstructural proteins: two substitutions were seen in the poly-proline hinge, one each in the X domain and helicase, and two in the RNA-dependent RNA polymerase. Therefore, it is tempting to speculate that these substitutions could be candidates for mutations associated with high levels of replication activity and long-term fecal shedding of HEV in infected hosts and the capability of efficient replication in a cell culture system; further studies are needed to clarify this important issue. Studies using a mutagenized, infectious cDNA clone of the HEV genome may elucidate the mechanism by which the observed nucleotide substitutions with or without amino acid changes lead to active replication and protracted fecal shedding of HEV.
Viral RNA became detectable again on day 121 but not on day 124 in patient 1. A better explanation as to why fecal virus shedding was positive again on day 121 even after 6 weeks of negative results may be required in future studies. A sequence comparison between the genome of day 121 virus and those of the earlier viruses (such as day 79 virus) may provide important information on the mechanism of prolonged fecal virus shedding. The precise reason why HEV in the fecal specimen on day 121 from patient 1 did not grow as efficiently as that on day 30 with a comparable HEV load remains unknown. One possible explanation is that antibodies against HEV secreted into the gastrointestinal tract have neutralized the virus. However, no significant signals of IgG, IgM, and IgA classes of HEV antibodies were detectable in fecal samples throughout the observation period in patient 1 or in the other 10 patients studied (data not shown). Fecal samples contain large amounts of phenolic and metabolic compounds and polysaccharides and have a very heterogeneous composition (66
), suggesting that the performance of our cell culture system may vary between samples, although we diluted fecal specimens 1:5 in PBS(−) containing 0.2% BSA prior to inoculation. It also is likely that replication-defective HEV genomes or those with lowered replicative activity have appeared at the end stage of long-term HEV infection. In this context, further studies by molecular approaches using the cell culture system for HEV and various clinical samples are needed.
In conclusion, the present study indicates that a delayed protracted virus shedding in feces occurs in a fraction of patients with acute sporadic HEV infection and that HEV shed into the feces at least on day 30 after the onset of hepatitis has the capability of efficiently replicating in PLC/PRF/5 cells, suggesting that careful attention to hygiene and sanitation is necessary even after patients with hepatitis E enter the recovery phase. Therefore, fecal-oral transmission of HEV also should be taken into consideration as a possible transmission route for individuals with no immunity against HEV, even in industrialized countries where the occurrence of domestic HEV infection as a zoonosis currently is emphasized.