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J Clin Microbiol. Jul 2005; 43(7): 3042–3048.
PMCID: PMC1169173

Identification of Novel Human Hepatitis E Virus (HEV) Isolates and Determination of the Seroprevalence of HEV in Korea

Abstract

Hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis. Recently, HEV isolates were subsequently identified in humans and swine in many countries, including Korea. Also, public concerns regarding HEV as a potential zoonotic agent have been increasing. Therefore, we attempted to identify HEV from Korean sera and compare the nucleotide sequences with those of previously identified HEV isolates from other countries. In our study, viral RNA was purified from 568 human sera collected from different regions of Korea. Nested PCR and reverse transcriptase PCR were developed based on the nucleotide sequences of open reading frame 2 (ORF 2) of U.S. and Japanese HEV isolates from humans and Korean HEV isolates from swine. After amplification of the HEV ORF 2 gene from 14 serum samples that were collected mainly from rural areas (2.64% prevalence of HEV viremia), the gene was cloned and sequenced. The isolates were classified into seven different strains, all of which belonged to genotype III. The human isolates we identified were closely related to three Korean swine isolates, with 99.2 to 92.9% nucleotide sequence homology. Our isolates were also related to the Japanese and U.S. HEV isolates, with 99.6 to 97.9% amino acid sequence homology. Human sera were collected from 361 individuals from community health centers and medical colleges. With respect to seroprevalence, 11.9% of the Korean population had anti-HEV immunoglobulin G (IgG). In individuals ranging in age from 40 to over 60 years, the prevalence of anti-HEV IgG was demonstrated by a seroprevalence of almost 15%, especially among populations in rural areas. This is the first report on the identification of human HEV in Korea. Overall, this study demonstrates that subclinical HEV infections may prevail in human populations in Korea and that there is a strong possibility that HEV is a zoonotic agent.

Since hepatitis E virus (HEV) was first identified in a volunteer study in 1983 (5), the virus has been considered the causative agent of enterically transmitted non-A, non-B hepatitis (42). HEV-mediated hepatitis is a serious public health problem in developing countries of Asia, the Middle East, and Africa, as well as in Mexico (2, 23, 40, 41). HEV is primarily transmitted through a fecal-oral route by the consumption of contaminated water (22, 36). A grave feature of HEV infection is the unusually high rates of mortality (from about 20 to 30%) that are observed with pregnant women as a result of fulminant liver disease (18, 24, 37, 40, 41). Recently, the virus has been reclassified in the genus Hepevirus of the family Hepeviridae.

The HEV genome is a 7.5-kb single-stranded, positive-sense RNA virus composed of three open reading frames (ORF), ORF 1, 2, and 3, which encode the nonstructural proteins, the capsid protein, and a cytoskeleton-associated phosphoprotein, respectively (6, 40, 41, 46).

Recently, HEV isolates from several countries worldwide have been classified into four genotypes or nine groups, which include swine and fowl HEV isolates, based on phylogenetic analysis of the full-length genome (43). Genotypes I, II, III, and IV include the Burmese isolates, Mexican isolates, U.S. isolates, and new Chinese isolates, respectively. Group 1 includes isolates of the virus from several countries of Asia and Africa, group 2 from Mexico and Nigeria, group 3 from the United States, group 4 from Italy and swine from New Zealand, group 5 from Greece and Spain, group 6 from Greece, group 7 from Argentina and Austria, group 8 from China, and group 9 from Taiwan (4, 13, 20, 32, 43, 44, 49, 50, 55).

Generally, HEV is not endemic in industrialized nations, but the prevalences of anti-HEV antibodies in U.S. blood donors and healthy Taiwanese individuals in areas of nonendemicity were as high as 21.3 and 11%, respectively (28, 47). In addition, high levels of anti-HEV antibodies were detected in several animal species, including pigs, cattle, dogs, rodents, and monkeys, which live in both countries of HEV endemicity and those of HEV nonendemicity (1, 3, 7, 9, 14, 34). These facts suggest that animals are an important reservoir for HEV infections in humans.

In 1997, a novel swine HEV strain that was genetically and serologically related to the U.S. human HEV strains was isolated (31). This U.S. swine HEV isolate experimentally infected nonhuman primates, and the US2 strain of human HEV also infected pigs, which raised a concern of cross-species infection by the swine HEV strain (17, 33). Several swine HEV isolates from Taiwan, Japan, New Zealand, the United States, The Netherlands, Canada, and Korea were subsequently identified (8, 15, 19, 21, 38, 48, 52, 54). The phylogenetic analysis of swine HEV isolates showed that they were closely related to human HEV isolates in each of these countries (19, 38, 52). An especially high prevalence of anti-HEV antibodies was observed in pigs in both countries of HEV endemicity and those of HEV nonendemicity (3, 7, 9, 15, 19, 31, 34, 54). More importantly, the anti-HEV antibody prevalence among swine farmers and swine veterinarians was higher than that of normal blood donors (11, 35). Overall, this suggests that swine HEV is a possible zoonotic agent for HEV infection in humans. A recent report on swine HEV infection in Korea and the knowledge presented above raise the possibility of human HEV infection in Korea (8). Also, a case of HEV infection with clinical symptoms has been reported in Korea (25).

The aim of this study was to identify human HEV strains in human sera and to characterize the prevalence of anti-HEV antibodies in humans. In this study, 15 human HEV strains were isolated and phylogenetically analyzed, and the serological prevalence of anti-HEV antibodies in Korean human sera was characterized.

MATERIALS AND METHODS

Serum samples.

A total of 749 serum samples used in this study were randomly collected from community health centers and diagnostic laboratories of medical schools and were stored at −70°C prior to analysis. The samples were obtained in 2003 and 2004 from Korean male and female volunteers aged 10 to 60 years. Five hundred sixty-eight of these serum samples were used to identify HEV. Roughly half of the samples were collected from urban areas and the remainder from rural areas. A total of 361 human sera were examined to detect anti-HEV immunoglobulin G (IgG).

Purification of viral RNA and design of PCR primers.

HEV RNA was purified from 140 μl of human serum by using a QIAamp viral RNA minikit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. The viral RNA was eluted with a total volume of 45 μl elution buffer from the spin column and stored at −70°C until further analysis.

Two sets of primers for reverse transcriptase (RT) PCR and nested PCR based on the highly conserved region of HEV ORF 2 from isolates of the U.S. (GenBank accession no. AF060668 and AF060669), Japanese (AB089824), Burmese (M73218 and D10330), and Korean swine (AF516178, AF516179, and AF527942) strains were synthesized. The external set of primers was designed to produce an 813-bp PCR product. The nucleotide sequences of the external set of primers were as follows: forward primer, 5′-TCC CCG CTT ACA TCA TCT GTT GC-3′; backward primer, 5′-CTT TAC TGT TGG CTC GCC ATT GG-3′. The expected size of the PCR product amplified with the nest set of primers was designed to be 720 bp. Nucleotide sequences of the nest set of primers were as follows: forward primer, 5′-AAC CCT CTC TTG CCT CTT CAG G-3′; backward primer, 5′-AGG GCG GGA GTA AAA CAG TTG-3′.

RT-PCR and nested PCR.

RT-PCR was performed by using a QIAGEN OneStep RT-PCR kit according to the manufacturer's instructions. Briefly, a reaction solution was adjusted to 50 μl of the reaction mixture, including 10 μl 5× QIAGEN OneStep RT-PCR buffer, 10 μl 5× Q-Solution, 2 μl deoxynucleoside triphosphate (dNTP) mixture (containing 10 mM of each dNTP), 2 μl external forward primer (100 pmol/μl), 2 μl external backward primer (100 pmol/μl), 2 μl QIAGEN OneStep RT PCR enzyme mixture, 1 μl RNaseOUT RNA inhibitor (10 U/μl; GIBCO BRL, Gaithersburg, Md.), 10 μl template RNA, and 11 μl RNase-free water. The thermal cycling conditions were as follows: one step of reverse transcription for 30 min at 50°C and an initial PCR activation step for 15 min at 95°C. These steps were followed by 40 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 55°C, and extension for 1 min 30 s at 72°C and a final incubation for 10 min at 72°C. The remaining RNA was removed with 2 μl of RNase H (Invitrogen, Carlsbad, CA) by incubation for 20 min at 37°C. Nested PCR was conducted with the following components: 3 μl RT-PCR product, 5 μl 10× Ex Taq PCR buffer (Mg2+ free), 5 μl MgCl2 (25 mg/ml), 4 μl dNTP mixture (10 mM of each dNTP), 1 μl nested forward primer (100 pmol/μl), 1 μl nested backward primer (100 pmol/μl), 0.5 μl Takara Ex Taq polymerase (5 U/μl), and 30.5 μl triple-distilled H2O. The thermal cycling conditions for the nested PCR included 5 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 45°C, and extension for 1 min 30 s at 72°C. This was followed by 35 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 55°C, and extension for 1 min 30 s at 72°C and a final incubation for 7 min at 72°C.

Cloning of PCR products and analysis of the clones.

The nested-PCR products were analyzed in a 1.0% agarose gel stained with ethidium bromide (10 mg/ml) under a UV transilluminator. The 720-bp DNA band specific for human HEV was excised from the gel and purified with the QIAquick gel extraction kit (QIAGEN). The purified DNA was cloned into a pCR-XL TOPO cloning vector (Invitrogen) by following the manufacturer's instructions. The clones containing the insert DNA were identified by colony PCR and restriction enzyme digestion of the plasmid DNA with EcoRI (Takara). The identity of the insert DNA was verified by automatic dye terminator DNA sequencing (CoreBio Research Institute of Lifescience and Biotechnology, Seoul, Korea).

Analysis of nucleotide sequences.

The nucleotide sequences of the Korean human HEV isolates were aligned with other human or swine isolates by a multiple-alignment algorithm (Clustal method) in the MegAlign package (Windows version 3.12e; DNASTAR, Madison, Wis.). The MEGA program was used to construct a phylogenetic tree of the HEV (24). A bootstrap analysis (500 repeats) was performed with the avian HEV as an outgroup in order to evaluate the topology of the phylogenetic tree. The GenBank nucleotide sequence accession numbers of the human and swine HEV isolates analyzed in this study are as follows: JKN-Sap, AB074918; JMY-Haw, AB074920; HE-JA10, AB089824; JJT-Kan, AB091394; JSN-Sap-FH, AB091395; JRA1, AP003430; US1, AF060668; US2, AF060669; KOR1, AF516178; KOR2, AF516179; KOR3, AF527942; China1, D11092; China2, D11093; China3, M94177; hKOR, AY641398; hKOR-JMA, AY715267; hKOR-HSY, AY714268; hKOR-SGK, AY714269; hKOR-DYL, AY714270; hKOR-NAB, AY714271; hKOR-HJY, AY714272; Burma B1, M73218; Burma B2, D10330; Mexican strain, M74506; US-sw, AF082843; and China variant, AJ272108.

ELISA.

For the seroprevalence study, the presence of anti-HEV IgG in human sera was determined by using a commercial HEV enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Genelabs Diagnostics, Singapore). Briefly, 10 μl of the serum sample was added to each well containing 200 μl of the diluent, and the microplate was incubated for 30 min at 37°C. The microplate was washed six times with 300 μl of a wash solution. Horseradish peroxidase-labeled goat anti-human IgG (Genelabs Diagnostics, Singapore) was used as the conjugate. One hundred microliters of a 1:1,000-diluted conjugate was added to each well, and the microplate was incubated for 30 min at 37°C. The microplate was washed six times with 300 μl of a wash solution. Subsequently, 100 μl of the substrate solution containing hydrogen peroxide and o-phenylenediamine 2HCl was added to each well. The microplate was incubated for 15 min in the dark at room temperature. The color-developing reaction was stopped by adding 50 μl of the stop solution to each well. The absorbance for each well was determined at 492 nm with an EMax microplate reader (Sunnyvale, Calif.). The cutoff value was calculated to be 0.5 plus the mean absorbance value of the nonreactive control, per the manufacturer's instructions. A serum sample showing an absorbance value greater than the cutoff value was determined to be positive.

RESULTS

Identification of HEV isolates in human sera.

The isolates of Korean human HEV were identified in serum samples by a nested RT-PCR technique as the 720-bp PCR product (data not shown). The first PCR was performed with the template cDNA and a set of external forward and reverse primers. The expected size of the product was 813 bp. However, no HEV-specific DNA band was observed by agarose gel electrophoresis analysis after the first PCR of this study (data not shown). Therefore, the PCR product was amplified again with a set of nested forward and reverse primers. This method successfully produced the 720-bp human HEV-specific DNA band in 15 out of the 568 serum samples (2.64%). Most of these bands were identified in sera collected from rural areas (Table (Table1).1). Also, the highest identification rate was observed in sera from individuals in their forties, followed by those 20 to 30 years old (Table (Table11).

TABLE 1.
Detection of swine HEV isolates in sera from humans of different geographic areas and ages

Genetic analysis of Korean human HEV isolates with other human and swine HEV isolates.

The Korean isolates from human HEV were genetically analyzed by comparing the highly conserved 720-bp region of HEV ORF 2 to that region in other human and swine HEV isolates (Table (Table2).2). Although the 15 human isolates could be classified into seven different groups based on nucleotide sequence analysis, the Korean isolates showed very high homology in nucleotide sequence to each other (93.2 to 99.9%). Phylogenetic analysis showed that all of the Korean isolates of human HEV were clustered in genotype III, which represents the typical genotype of the human and swine HEV strains isolated from the United States and Japan and the swine isolates from Korea (Fig. (Fig.1).1). The human isolates from Korea were the most closely related to Korean swine isolates KOR1, KOR2, and KOR3, with 92.9 to 99.2% nucleotide homology (Table (Table2).2). In the comparison of the Korean human isolates with those from other countries, a Japanese strain, HE-JA10, was very similar to a Korean isolate, with 92.5% sequence homology. Also, JKN-Sap and JMY-Haw, isolated from Japanese hepatitis patients, showed close relationships to Korean human isolates, with 90.8 to 91.8% nucleotide homology. The nucleotide homology of other Japanese HEV isolates with the Korean isolates ranged from 78.6 to 88.8%. Nucleotide sequence homologies of Korean human isolates to U.S. human HEV isolates US1 and US2 (95.0 to 99.6%) were higher than those of Korean human isolates to the Japanese isolates, but higher similarities in amino acid sequences were observed for both U.S. and Japanese isolates, with 95.0 to 99.6% homology. The U.S. swine strain also showed a high association with the Korean human isolates, with 89.7 to 91.7% homology. Isolates from China, Burma, and Mexico showed some distance from the Korean isolates, with homologies of 76.7 to 78.6, 77.1 to 79.4, and 75.5 to 76.0%, respectively. The Korean human and swine isolates demonstrated 92.9 to 99.0% identity in their nucleotide sequences. Furthermore, the deduced amino acid sequences showed 99.6% similarity between Korean human and swine isolates (Table (Table2).2). These results bear out that swine and human HEV are exceedingly closely related to each other and raise the possibility that HEV may be a serious zoonotic disease in Korea.

FIG. 1.
Phylogenetic tree analysis of isolates of Korean human HEV by comparison of the conserved 720-bp regions of ORF 2 in swine and human HEV isolates. The HEV genotypes are according to a recent report (18). The GenBank accession numbers of the HEV isolates ...
TABLE 2.
Sequence homologies among ORF 2 of HEV isolates

Prevalence of anti-HEV IgG in the human population in Korea.

The prevalence of anti-HEV antibodies in human sera was evaluated by an ELISA specific for anti-HEV IgG. Human sera were collected from 361 individuals from community health centers and diagnostic laboratories of medical schools in five different areas. Serum samples were grouped according to the age of individuals, ranging from 10 to 60 years of age. The total prevalence of anti-HEV IgG in Korean human sera was 11.9% (Table (Table33 and Fig. Fig.2).2). The highest prevalence (18.1%) was observed among individuals from 40 to 49 years old, followed by those in their 50s and 60s (16%) in rural areas. There was no positive result for anti-HEV IgG in the sera of those under 20 years of age. The difference in seroprevalence by sex was not momentous (data not shown). The 11.9% seroprevalence indicates that the human population in Korea has been subclinically infected by HEV even though Korea has been known as a country where HEV is not endemic.

FIG. 2.
Age-dependent distribution of anti-HEV antibodies in human individuals (each diamond represents one individual). The presence of anti-HEV antibodies was determined by using a commercial HEV IgG ELISA kit (Genelabs Diagnostics, Singapore) according to ...
TABLE 3.
Prevalence of anti-HEV antibodies in Korean human populations

DISCUSSION

Although HEV infection is endemic in Southeast and central Asia, several large outbreaks of hepatitis E have been observed in the Middle East, the northern and western parts of Africa, and Mexico (26). Sporadic hepatitis E occurrences also have been observed in several countries where outbreaks have not been reported, including Egypt, Hong Kong, Senegal, and Turkey (10, 12, 16, 27, 29, 39). It is well known that sporadic cases of hepatitis E in regions where HEV is not endemic are associated with travel to regions of HEV endemicity. However, cases in the United States, Italy, and Greece have had no association with travel (27, 45, 56). Recently, there was a case of hepatitis E in Korea not related to travel to a region of HEV endemicity (25). In this study, 15 human HEV isolates were detected and seven human HEV strains were isolated from humans in Korea for the first time. The isolates were characterized, and the prevalence for anti-HEV IgG in Korea was determined.

Genomic analysis of the HEV isolates indicated that the Korean human isolates were divided mainly into two groups. Almost all of the Japanese and U.S. isolates were members of genotype III. Isolates of Mexican origin can be grouped into genotype II, and isolates from China and Burma can be grouped into genotype I. One each of the Japanese and Taiwanese isolates can be grouped into genotype IV. The fact that human HEV infection may be mediated through contact with infected pigs has been indicated previously by phylogenetic analysis (15, 19, 31, 38, 48), experimental cross-species infections (17, 33, 51), and serological studies (11, 19, 35). This phylogenetic study demonstrates that Korean isolates from humans and swine were very highly associated, showing as much as 99.6% identity.

A commercial ELISA for detecting HEV-specific IgG in human sera was used for this prevalence study. The ELISA used type-common epitopes in ORF 2 and ORF 3 of the Mexican and Burmese HEV strains as coating antigens (28, 53). One study reported a 97% concordance in the anti-HEV prevalence results obtained with human and swine HEV antigens (35), which concurrently supports the presence of cross-species HEV epitopes. Additionally, the ELISA could efficiently detect anti-swine HEV IgG in swine serum samples (8). HEV was detected in 2.64% of the Korean human population, and 11.9% of this population had HEV-specific IgG. The detection rate was similar to that in swine, but the prevalence of anti-HEV IgG in humans from the current study was lower than that in humans from the previous study (8). However, there were similarities regarding age in the prevalence of anti-HEV IgG, even though the overall rate was lower in the current study.

The prevalences of anti-HEV in human populations suggest the possibility of subclinical HEV infections in countries like the United States or China (11, 28, 35, 47). Korea is considered a country where HEV is not endemic. However, this study with randomly collected serum samples demonstrates that approximately 12% of Koreans possessed HEV antibody. A recent study in the United States also showed a similar anti-HEV antibody rate among healthy blood donors (35). Therefore, it can be assumed that Koreans have been exposed to HEV without showing clinical signs of infection.

The prevalence of anti-HEV antibody in human populations living in countries where HEV is not endemic was noticeably high (11, 28, 30, 35, 47). This phenomenon agreed with our study. Furthermore, there was a regional difference in HEV particle detection. These results indicate that hepatitis E exists in Korea but presumably in a subclinical state. Furthermore, hepatitis E viral particles are more prevalent in rural areas, and detection of HEV in samples has some relevance within age groups.

In conclusion, this is the first report of the identification of seven new human HEV isolates in Korean human sera. They fell mainly into two groups of genotype III by phylogenetic analysis and were closely related to swine isolates from Korea and human HEV isolates previously identified in the United States and Japan. The prevalence of HEV-specific antibodies in Korean humans was 11.9%. Furthermore, further studies are needed to develop a control measure, such as a vaccine for persons who have any chance of exposure to HEV and especially for pregnant women.

Acknowledgments

This study was supported by the Korea Food and Drug Administration, Brain Korea 21 (KRF-005-E00077), and the Research Institute for Veterinary Science, Seoul National University, Korea.

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