Identification of Swine Hepatitis E Virus(HEV) and Prevalence of Anti-HEV Antibodies in Swine and Human Populations in Korea
Department of Infectious Diseases, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul 151-742,1 Institute of Intron Biotechnology, Seoul 138-200, Korea2/, http://www.100md.com
Received 9 September 2002/ Returned for modification 23 January 2003/ Accepted 8 June 2003/, http://www.100md.com
ABSTRACT/, http://www.100md.com
The swine hepatitis E virus (HEV) is considered to be a new zoonotic agent due to its close genomic resemblance to the human HEV and its ability to infect nonhuman primates. Hepatitis caused by HEV infection has been a serious public health problem in developing countries. However, recent seroprevalence studies indicate that the HEV also circulates in industrialized countries. In this study, a nested reverse transcription (RT)-PCR was developed to detect a part of the swine HEV open reading frame 2. Three Korean isolates of swine HEV were identified in 128 swine sera (2.3% prevalence) by the nested RT-PCR method. They were isolated from 2- to 3-month old pigs showing an age-specific prevalence of the HEV viremia. A phylogenetic tree analysis with a number of swine and human HEV isolates indicated that all Korean isolates of the swine HEV belong to genotype III. They were closely related to the swine and human HEV isolates that were identified in the United States and Japan. In addition, they formed a distinct branch in genotype III, showing a 92.7 to 99.8% identity at their nucleotide sequences. The overall prevalence of anti-swine HEV antibodies in swine was 15%. Antibodies to the swine HEV were not detected in 1-month-old pigs. However, the anti-swine HEV antibodies appeared in pigs older than 1 month and also showed an age-specific prevalence. The antibody prevalence rates to the swine HEV were 6.0, 10.0, 36.0, and 25.0%, in 2-, 3-, 4-, and 5-to-7-month-old pigs, respectively. In addition, the seroprevalence in sows to the swine HEV was 8.8%. On the other hand, 18% of blood donors in Korea were found to be positive for anti-HEV antibodies. Overall, this study indicates that subclinical HEV infections may prevail in swine and human populations in Korea.
INTRODUCTION7k]](t0, 百拇医药
The hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis (33). HEV-mediated hepatitis is a serious public health problem in developing countries of Asia, Middle East, and Africa and also in Mexico (1). The highest incidence of HEV infections occurs in young adults, and the mortality rate of HEV-infected pregnant women is approximately 20% (1, 4, 12, 17, 20). HEV is mainly transmitted through a fecal-oral route by consuming contaminated water (3, 13). It is a nonenveloped small virus with an approximately 7.5-kb single-stranded, positive-sense RNA genome (9, 40). HEV was previously classified into the family Caliciviridae. However, it is now reclassified into the Hepatitis E-like viruses as an unassigned genus (8, 32). The HEV genome is composed of three open reading frames (ORF). These are ORF 1, 2, and 3, which encode the nonstructural proteins, the capsid protein, and a cytoskeleton-associated phosphoprotein, respectively (9, 40, 51).
HEV isolates are generally classified into four genotypes based on the phylogenetic analysis of the full-length genome (37). Genotypes 1, 2, 3, and 4 are represented by the Burmese isolates, the Mexican isolate, the U.S. isolates, and the new Chinese isolates, respectively (5, 15, 22, 37, 40, 43, 44). Most HEV infections in developed countries were identified in travelers who had visited countries of HEV endemicity (37, 52). However, novel variants of HEV have recently been isolated in patients with acute HEV infections in developed countries where HEV is not endemic who never traveled to areas of HEV endemicity or had contacts with infected individuals (34-36, 38, 46, 52). The prevalence rates of anti-HEV antibodies in American blood donors and healthy Taiwanese individuals in areas of nonendemicity were as high as 21.3 and 11%, respectively (25, 41). In addition, high levels of anti-HEV antibodies were detected in several animal species, including pigs, cattle, dogs, rodents, and monkeys, which lived in both countries of HEV endemicity and countries of nonendemicity (2, 6, 10, 11, 16, 29). These facts suggest that animals are an important reservoir of HEV infections in humans.
In 1997, a novel swine HEV strain was isolated in pigs in the United States (26). The U.S. swine HEV isolate was genetically related to the U.S. human HEV strains and cross-reacted with antibodies against the human HEV capsid protein. The U.S. swine HEV isolate experimentally infected nonhuman primates, and the US-2 strain of human HEV also infected pigs, which raised a concern of cross-species infection by the swine HEV (19, 28). Several swine HEV isolates were subsequently identified in pigs in Taiwan, Japan, New Zealand, The Netherlands, and Canada (18, 21, 31, 42, 47, 50). The phylogenetic analysis of the swine HEV isolates identified in Taiwan and Japan indicated that they were closely related to the human HEV strains isolated in Taiwan and Japan, respectively (21, 31, 47). The swine HEV strains identified in The Netherlands and New Zealand also showed high levels of nucleotide identity with the European HEV strains (18, 42). Especially, a high prevalence of anti-HEV antibodies was observed in pigs in both countries of HEV endemicity and countries of nonendemicity (6, 10, 11, 18, 21, 26, 29, 50). More importantly, the anti-HEV prevalence of swine farmers and swine veterinarians was higher than that of normal blood donors (14, 30). Overall, this suggests that swine HEV is a possible zoonotic agent for HEV infection in humans.
The aim of this study was to identify the swine HEV in pig serum and to measure the prevalence rate of anti-HEV antibodies in pigs and humans in Korea. In this study, three strains of Korean swine HEV were isolated and characterized by phylogenetic analysis. The serological evidence of the HEV infections was observed in both pigs and humans.u*+4t, 百拇医药
MATERIALS AND METHODSu*+4t, 百拇医药
Serum samples. All serum samples used in this study were stored at -70°C prior to analysis. A total of 128 serum samples of 1- to 4-month old pigs from 10 swine farms were used for the isolation of the swine HEV RNA. A total of 264 serum samples of 1- to 7-month old pigs and sows from 13 swine farms were used for the prevalence study of the anti-HEV immunoglobulin G (IgG). The swine serum samples used in this study were randomly selected from the sera that had been submitted to the Department of Infectious Diseases, College of Veterinary Medicine, Seoul National University, Korea, for a routine examination of bacterial and viral diseases. A total of 96 human serum samples that had been obtained in 1995 from 40- to 60-year-old Korean volunteers regardless of their gender were examined to detect the anti-HEV IgG.
Purification of viral RNA and designing of PCR primers. The HEV RNA was purified from 140 µl of swine serum by using a QIAamp viral RNA minikit (Qiagen, Valencia, Calif.) according to the manufacturer's instructions. The viral RNA was eluted from the spin column with 45 µl of the elution buffer and stored at -70°C. Two sets of degenerate primers based on the highly conserved regions of the ORF 2 from the U.S. (AF082843) and Japanese (AB073912) swine HEV isolates were synthesized. The external set of primers was designed to produce a 1,040-bp (nucleotides 5457 to 6496 of the US swine HEV) PCR product. The external set of primers had the following sequences: forward primer, 5'-CTG CCC CCC GTC GTC GAT C-3'; and reverse primer, 5'-ATC CTG AAT AAC CAC ACG GG-3'. The expected size of the PCR product amplified with the nest set of primers was 860 bp (nucleotides 5533 to 6392 of the U.S. swine HEV). The nest set of primers had the following sequences: forward primer, 5'-TGT ACC TGA TGT TGA CTC ACG-3'; and reverse primer, 5'-GCT CGC CAT TGG CCG AGA C-3'.
RT-PCR and development of a nested PCR. Reverse transcriptase PCR (RT-PCR) was performed by using a QIAGEN OneStep RT-PCR kit according to the manufacturer's instructions. Briefly, a reaction tube contained 50 µl of the reaction solutions, including 10 µl of the 5x QIAGEN OneStep RT-PCR buffer, 2 µl of the dNTP mix (containing 10 mM of each dNTP), 10 µl of the 5x Q-Solution, 2 µl of the external forward primer (100 pM/µl), 2 µl of the external reverse primer (100 pM/µl), 2 µl of the QIAGEN OneStep RT-PCR enzyme mix, 1 µl of the RNaseOut RNA inhibitor (10 units/µl; Gibco BRL, Gaithersburg, Md.), 10 µl of the template RNA, and 11 µl of the RNase-free water. The thermal cycling conditions included one step of reverse transcription for 30 min at 50°C and an initial PCR activation step for 15 min at 95°C. This was followed by 40 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 50°C, and extension for 1 min 15 s at 72°C, and a final incubation for 10 min at 72°C. A nested PCR was conducted with the following components: 3 µl of the RT-PCR product, 5 µl of the 10x PCR buffer, 5 µl of MgCl2 (25 mg/ml), 4 µl of the dNTP mix (10 mM of each dNTP), 1 µl of the nested forward primer (100 pM/µl), 1 µl of the nested reverse primer (100 pM/µl), 0.5 µl of Takara Ex Taq polymerase (5 units/µl), and 30.5 µl of the double-distilled H2O. The thermal cycling conditions for the nested PCR included 5 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 45°C, and extension for 1 min 15 s at 72°C. This was followed by 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 53°C, and extension for 1 min 15 s at 72°C, and a final incubation for 7 min at 72°C.
Cloning of PCR products and analysis of clones. The nested PCR products were analyzed in a 1.0% agarose gel stained with ethidium bromide (10 mg/ml) under a UV transmitter. The 860-bp DNA band specific for the swine HEV was excised from the gel and purified with the QIAquick gel extraction kit (Qiagen). The purified DNA was cloned into a TOPO TA Cloning vector (Invitrogen, Carlsbad, Calif.) according to the manufacturer's protocol. The clones containing the insert DNA were identified by restriction enzyme digestion of the plasmid DNA with EcoRI (Promega, Madison, Wis.). The identity of the insert DNA was verified by using an automatic dye terminator DNA sequencing (Takara Korea Biomedicals, Seoul, Korea).1@::9eh, http://www.100md.com
Analysis of nucleotide sequences. The nucleotide sequences of the swine and human HEV isolates were aligned by the 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 accession numbers of the swine and human HEV isolates analyzed in this study are as follows: 01-18934D2, ; 01-19248-3, 01-22807-2, ; swUS1, NLSW15, NLSW20, NLSW22,; NLSW85, ; NLSW99, swJ681, swKOR1, swKOR2, swKOR3, TW32SW, ; TW3SW, UMC13A, ; JKN-Sap, JMY-Haw, US1, US2,JRA1, ; 87, China 5, Mexico, ; Morocco, India 2, ; Burma 1, ; China 1, D Pakistan SAR55, Egypt 93, and Avian HEV,
ELISA. The presence of anti-HEV IgG was determined in human and swine sera by using a commercial HEV enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Genelabs Diagnostics, Singapore). All serum samples were tested in duplicate. 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 250 µl of a wash solution. Horseradish peroxidase-labeled goat anti-human IgG (Genelabs Diagnostics, Singapore) and rabbit anti-pig IgG (Sigma, Steinheim, Germany) were used as the conjugates for the human and pig sera, respectively. 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 250 µl of the wash solution. Subsequently, 100 µl of the substrate solution containing 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 of the nonreactive control according to the manufacturer's instructions. The serum sample showing an absorbance value greater than the cutoff value was determined to be a positive.
Nucleotide sequence accession numbers. The GenBank accession numbers of the swine and human HEV isolates analyzed in this study are as follows: 01-18934D2, ; 01-19248-3, ; 01-22807-2, ; swUS1, ; NLSW15, NLSW20, NLSW22, ; NLSW85, ; NLSW99, ; swJ681, ; swKOR1, ; swKOR2, ; swKOR3, ; TW32SW, ; TW3SW, ; UMC13A, ; JKN-Sap JMY-Haw, US1, US2, ; JRA1, ; 87, ; China 5, Mexico, ; Morocco,India 2, Burma 1, China 1, ; Pakistan SAR55, Egypt 93, and Avian HEV, .i, http://www.100md.com
RESULTSi, http://www.100md.com
Identification of HEV isolates in swine sera. The swine isolate of HEV was identified in swine serum samples by a nested RT-PCR technique as the 860 bp PCR product (Fig. 1). The first PCR was performed with the template cDNA and a set of external forward and reverse primers. The reaction was expected to produce a 1,040-bp PCR product. However, the first PCR conditions used in this study did not produce any HEV-specific DNA band (data not shown). Therefore, the product of PCR was amplified again with a set of nest forward and reverse primers. This method successfully produced the 860-bp swine HEV-specific DNA band (Fig. 1). Three swine HEV strains were identified in a total of 128 pig sera collected from 10 pig herds, which indicated a 2.3% rate of HEV viremia prevalence (Table 1). Their nucleotide sequences were reported to GenBank, and they were given accession numbers for swKOR1, for swKOR2, and for swKOR3. Two strains, swKOR1 and swKOR2, were isolated in two 3-month-old pigs, and swKOR3 was isolated in a 2-month-old pig. These results suggest that most HEV infections in swine occur in young pigs approximately 2 to 3 months old.
fig.ommitted)*g, 百拇医药
FIG. 1. Identification of the Korean isolates of swine HEV in swine sera. HEV RNA was purified from swine serum. A nested RT-PCR was then performed to amplify a region of swine HEV ORF2. The swine HEV-specific 860-bp DNA bands were identified from three pig sera. Lanes: M, standard 100-bp DNA ladder; 1, Korean isolate 1 of swine HEV (swKOR1; GenBank accession number of ); 2, swKOR2 (); 3, swKOR3 ().)*g, 百拇医药
fig.ommitted)*g, 百拇医药
TABLE 1. Detection of swine HEV isolates in pigs of different ages)*g, 百拇医药
Genetic analysis of Korean isolates of swine HEV with other human and swine HEV isolates. Three Korean isolates of the swine HEV were genetically analyzed by comparing the highly conserved 300 bp of ORF2 to those of other human and swine HEV isolates. Phylogenetic analysis showed that all the Korean isolates of swine HEV were clustered in genotype III, which is typically represented by the human and swine HEV strains isolated from the United States and Japan (Fig. 2). They formed a distinct branch in genotype III, and they were most closely related to two human HEV strains, JKN-Sap and JMY-Haw, which were isolated from Japanese hepatitis patients, and one swine HEV strain, 01-19248-3, which was isolated from a pig in the United States. They were also closely related to one Netherlands swine HEV isolate, NLSW22, and other U.S. human and swine HEV isolates, including US1, US2, swUS1, and other recently identified swine isolates (Fig. 2). Among the swine HEV isolates examined in this study, the U.S. isolates, including the prototype swUS1, demonstrated the highest nucleotide identity (89.3 to 92.3%) with the Korean isolates (Table 2). The percent nucleotide identities between Korean and Japanese, Dutch, and Taiwanese swine HEV isolates were 84.7 to 86.7, 81.3 to 91.3, and 73.3 to 84.3%, respectively (Table 2). The three Korean isolates demonstrated a 92.7 to 99.8% identity at their nucleotide sequences (data not shown). However, their deduced 286 amino acids showed a 99.3 to 99.7% identity (data not shown). This suggests that most variations in the swine HEV genome silently occur at the third nucleotides of the amino acid codons.
fig.ommitted3x3h9ur, 百拇医药
FIG. 2. Phylogenetic tree analysis of the Korean isolates of swine HEV by comparison of the conserved 300 bp of the ORF2 in the swine and human HEV isolates. The HEV genotyping was determined according to a recent report (37). The GenBank accession numbers of the HEV isolates used in this study are indicated in Materials and Methods.3x3h9ur, 百拇医药
fig.ommitted3x3h9ur, 百拇医药
TABLE 2. Pairwise comparison of the nucleotide identity (%) in the ORF2 of the swine HEV isolatesa3x3h9ur, 百拇医药
Prevalence of anti-HEV IgG in swine and human populations. The prevalence rates of anti-HEV antibodies in swine and human sera were examined with ELISAs specific for anti-HEV IgG (Table 3 and Fig. 3). The swine sera were collected from 264 pigs in 13 farms and prepared according to the age of the swine, ranging from 1 to >5 months old. The highest prevalence of anti-HEV IgG was observed in 4-month-old pigs, showing a 36% seroprevalence. None of the 1-month-old pigs tested positive for anti-HEV antibodies in their sera. Other age groups of the pigs, including 2-, 3-, and 5- to 7-month-old pigs demonstrated 6, 10, and 25% anti-HEV antibody prevalence, respectively. Sows demonstrated an 8.8% seroprevalence to HEV. These results indicate that the prevalence of anti-HEV IgG in swine is age dependent. The overall prevalence of anti-HEV antibodies in Korean pigs was 14.8%. With respect to herd prevalence of anti-HEV antibodies, 11 out of 13 pig farms had HEV-positive pigs, demonstrating an 84.6% prevalence in the pig herds (data not shown). In the case of humans, a total of 96 serum samples randomly collected from blood donors in Korea in 1995 were tested. A total of 17 individuals (17.7%) were found to be HEV positive (Table 3). These results indicate that the swine and human populations in Korea have been subclinically infected by the HEV even though Korea has been known as a country where HEV is not endemic.
fig.ommitted#x-6n|, 百拇医药
TABLE 3. Prevalence of anti-HEV antibodies in swine and human populations#x-6n|, 百拇医药
fig.ommitted#x-6n|, 百拇医药
FIG. 3. Age-dependent distribution of anti-HEV antibodies in swine sera. The presence of anti-HEV antibodies was examined with a commercial HEV IgG ELISA kit (Genelabs Diagnostics, Singapore) according to the manufacturer's instructions. Samples showing an optical density (OD) value greater than 0.54 were considered to be HEV positive. The cutoff value was determined by a calculation suggested in the manufacturer's instructions.#x-6n|, 百拇医药
DISCUSSION#x-6n|, 百拇医药
It has been recognized that a large portion of pigs are infected with the HEV in the world (6, 10, 11, 18, 21, 23, 26, 29, 42, 50). The experimental cross-species infections (19, 28, 45), phylogenetic studies (18, 21, 26, 31, 42), and seroprevalence data (14, 21, 30) also indicate that HEV infections in humans may be mediated through contacts with infected pigs. Based on these findings, swine HEV is considered to be a zoonotic agent. In this study, three swine HEV strains were isolated from pigs in Korea for the first time, the isolates were characterized, and the prevalence rates of HEV-specific antibodies in pigs and humans were determined.
It has been observed that swine HEV can be detected in the feces at 2 to 6 weeks and in the sera at 4 to 6 weeks after experimental infections (27). Swine HEV RNA is typically isolated from fecal and serum samples obtained from 2- to 4-months old pigs (23, 31, 48). The overall prevalence rates of HEV viremia determined with serum samples were 1.5, 3.0, and 4.6% in pigs raised in Taiwan, Japan, and India, respectively (7, 31, 48). In this study, it was demonstrated that the prevalence of HEV viremia in pigs raised in Korea was 2.3%. Korean isolates of the swine HEV were also detected only in 2- to 3-month-old pigs. However, the prevalence of swine HEV RNA was much higher in the feces than in the sera, as demonstrated in recent studies (23, 42). When swine HEV RNA was detected in the pooled fecal samples from swine farms in The Netherlands, 22% of samples demonstrated the presence of the HEV (42). In the United States, when the presence of HEV RNA was detected by RT-PCR in the feces and sera obtained from 2- to 4-month-old pigs, the prevalence rates of HEV RNA were 40 and 12.5% in the feces and sera, respectively (23). These data indicate that more HEV particles are excreted in the feces than in the serum. And the discrepancy of HEV prevalence rates reported in several studies can also be explained by the different experimental materials they used. In this study, we used only sera as the experimental material for detecting the HEV because we primarily wanted to know the HEV prevalence in swine sera. The HEV prevalence in swine feces will be determined in subsequent study.
Genomic analysis of the HEV isolates indicates that HEV can be grouped into four major genotypes in which they can be further subgrouped into 9 groups (37). Genotype I contains the isolates from Burma and several Asian and African countries. Genotype II is represented by the isolates from Mexico and Nigeria. Genotype III contains the human isolates from the United States, Japan, Italy, Greece, Spain, Argentina, and Austria and the swine isolates from the United States, Japan, and New Zealand (31). Genotype IV contains the human and swine isolates from China and Taiwan (21, 48). The three newly isolated swine HEV strains in Korea are included in genotype III. They are most closely related to two Japanese isolates recently identified from acute hepatitis patients (39). They also have close relations with several swine isolates identified in the United States, Japan, and The Netherlands (23, 31, 42). This suggests that Korean strains of swine HEV might be variants introduced from the United States or other countries. Among the Korean HEV isolates, swKOR1 and swKOR2 were isolated from pigs in the same farm located in the southeastern region of Korea, and they displayed almost identical genomic sequences. In contrast, swKOR3 was isolated from a pig raised in a farm located in the central region of Korea. The identities of the 860-bp nucleotide sequence of swKOR3 with swKOR1 and swKOR2 were lower (92.7 to 92.9%) than those between swKOR1 and swKOR2 (99.8%). In the phylogenetic tree analysis, swKOR3 is divergent from the branch containing swKOR1 and swKOR2. This indicates the genetic variation of swine HEV in different regions.
The prevalence of anti-swine HEV antibodies in swine sera has been studied with an ELISA that uses the capsid antigen of the human Pakistan strain, HEV Sar-55, as a coating antigen because of a high percentage of identity in its amino acid sequence of ORF2 with that of swine HEV (26, 29). In this study, a commercial ELISA kit that was developed for detecting HEV-specific IgG in human sera was used to identify the anti-swine HEV antibodies in swine sera. The ELISA uses type-common epitopes in ORF2 and ORF3 of the Mexican and Burmese HEV strains as coating antigens (25, 49). Surprisingly, the ELISA very efficiently detected the anti-swine HEV IgG in swine serum samples. This indicates that the type-common HEV epitopes found in the Mexican and Burmese strains are also recognized by the anti-swine HEV IgG. A recent report demonstrated a 97% concordance in the anti-HEV prevalence results obtained with the human and swine HEV antigens (30), which also concurrently supports the presence of cross-species HEV epitopes. The highest prevalence of anti-swine HEV antibodies was observed in 4-month-old pigs. The overall anti-swine HEV prevalence rate determined in this study was 15%, whereas other studies have shown rather higher prevalence rates ranging from 30 to 70% (7, 10, 21, 26, 29, 50). The discrepancy in the antibody prevalence rates can be attributed to the different assay systems and to the different calculation methods used to decide a cutoff value.
The seroprevalence rates to HEV in human populations living in countries where HEV is not endemic are considerably high, suggesting the possibility of subclinical HEV infections in those countries (14, 25, 30, 41). Korea has been considered to be a country where HEV is not endemic. However, this study with randomly collected serum samples in Korea demonstrates that approximately 18% of Korean people possess HEV antibodies. A recent study in the United States also shows a similar anti-HEV antibody rate in normal blood donors (30). Therefore, it can be assumed that Korean people have been exposed to HEV without showing clinical signs of infection. Subsequent study will be undertaken to isolate the human HEV strain in Korea. If the human HEV is isolated, comparative analysis can be done to determine the genomic relationship between swine and human HEV isolates.9s, 百拇医药
In conclusion, three new swine HEV isolates were identified in 2- to 3-month-old pigs in Korea. They were grouped into genotype III by phylogenetic tree analysis and were closely related to the swine and human HEV isolates identified in the United States and Japan. The swine showed age-specific antibody prevalence rates to HEV. The prevalence rates of the HEV-specific antibodies in swine and humans were 15 and 18%, respectively.
ACKNOWLEDGMENTS'd4-, 百拇医药
This work was supported by funds from Brain Korea 21 and Research Institute for Veterinary Science, Seoul National University, Korea.'d4-, 百拇医药
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Received 9 September 2002/ Returned for modification 23 January 2003/ Accepted 8 June 2003/, http://www.100md.com
ABSTRACT/, http://www.100md.com
The swine hepatitis E virus (HEV) is considered to be a new zoonotic agent due to its close genomic resemblance to the human HEV and its ability to infect nonhuman primates. Hepatitis caused by HEV infection has been a serious public health problem in developing countries. However, recent seroprevalence studies indicate that the HEV also circulates in industrialized countries. In this study, a nested reverse transcription (RT)-PCR was developed to detect a part of the swine HEV open reading frame 2. Three Korean isolates of swine HEV were identified in 128 swine sera (2.3% prevalence) by the nested RT-PCR method. They were isolated from 2- to 3-month old pigs showing an age-specific prevalence of the HEV viremia. A phylogenetic tree analysis with a number of swine and human HEV isolates indicated that all Korean isolates of the swine HEV belong to genotype III. They were closely related to the swine and human HEV isolates that were identified in the United States and Japan. In addition, they formed a distinct branch in genotype III, showing a 92.7 to 99.8% identity at their nucleotide sequences. The overall prevalence of anti-swine HEV antibodies in swine was 15%. Antibodies to the swine HEV were not detected in 1-month-old pigs. However, the anti-swine HEV antibodies appeared in pigs older than 1 month and also showed an age-specific prevalence. The antibody prevalence rates to the swine HEV were 6.0, 10.0, 36.0, and 25.0%, in 2-, 3-, 4-, and 5-to-7-month-old pigs, respectively. In addition, the seroprevalence in sows to the swine HEV was 8.8%. On the other hand, 18% of blood donors in Korea were found to be positive for anti-HEV antibodies. Overall, this study indicates that subclinical HEV infections may prevail in swine and human populations in Korea.
INTRODUCTION7k]](t0, 百拇医药
The hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis (33). HEV-mediated hepatitis is a serious public health problem in developing countries of Asia, Middle East, and Africa and also in Mexico (1). The highest incidence of HEV infections occurs in young adults, and the mortality rate of HEV-infected pregnant women is approximately 20% (1, 4, 12, 17, 20). HEV is mainly transmitted through a fecal-oral route by consuming contaminated water (3, 13). It is a nonenveloped small virus with an approximately 7.5-kb single-stranded, positive-sense RNA genome (9, 40). HEV was previously classified into the family Caliciviridae. However, it is now reclassified into the Hepatitis E-like viruses as an unassigned genus (8, 32). The HEV genome is composed of three open reading frames (ORF). These are ORF 1, 2, and 3, which encode the nonstructural proteins, the capsid protein, and a cytoskeleton-associated phosphoprotein, respectively (9, 40, 51).
HEV isolates are generally classified into four genotypes based on the phylogenetic analysis of the full-length genome (37). Genotypes 1, 2, 3, and 4 are represented by the Burmese isolates, the Mexican isolate, the U.S. isolates, and the new Chinese isolates, respectively (5, 15, 22, 37, 40, 43, 44). Most HEV infections in developed countries were identified in travelers who had visited countries of HEV endemicity (37, 52). However, novel variants of HEV have recently been isolated in patients with acute HEV infections in developed countries where HEV is not endemic who never traveled to areas of HEV endemicity or had contacts with infected individuals (34-36, 38, 46, 52). The prevalence rates of anti-HEV antibodies in American blood donors and healthy Taiwanese individuals in areas of nonendemicity were as high as 21.3 and 11%, respectively (25, 41). In addition, high levels of anti-HEV antibodies were detected in several animal species, including pigs, cattle, dogs, rodents, and monkeys, which lived in both countries of HEV endemicity and countries of nonendemicity (2, 6, 10, 11, 16, 29). These facts suggest that animals are an important reservoir of HEV infections in humans.
In 1997, a novel swine HEV strain was isolated in pigs in the United States (26). The U.S. swine HEV isolate was genetically related to the U.S. human HEV strains and cross-reacted with antibodies against the human HEV capsid protein. The U.S. swine HEV isolate experimentally infected nonhuman primates, and the US-2 strain of human HEV also infected pigs, which raised a concern of cross-species infection by the swine HEV (19, 28). Several swine HEV isolates were subsequently identified in pigs in Taiwan, Japan, New Zealand, The Netherlands, and Canada (18, 21, 31, 42, 47, 50). The phylogenetic analysis of the swine HEV isolates identified in Taiwan and Japan indicated that they were closely related to the human HEV strains isolated in Taiwan and Japan, respectively (21, 31, 47). The swine HEV strains identified in The Netherlands and New Zealand also showed high levels of nucleotide identity with the European HEV strains (18, 42). Especially, a high prevalence of anti-HEV antibodies was observed in pigs in both countries of HEV endemicity and countries of nonendemicity (6, 10, 11, 18, 21, 26, 29, 50). More importantly, the anti-HEV prevalence of swine farmers and swine veterinarians was higher than that of normal blood donors (14, 30). Overall, this suggests that swine HEV is a possible zoonotic agent for HEV infection in humans.
The aim of this study was to identify the swine HEV in pig serum and to measure the prevalence rate of anti-HEV antibodies in pigs and humans in Korea. In this study, three strains of Korean swine HEV were isolated and characterized by phylogenetic analysis. The serological evidence of the HEV infections was observed in both pigs and humans.u*+4t, 百拇医药
MATERIALS AND METHODSu*+4t, 百拇医药
Serum samples. All serum samples used in this study were stored at -70°C prior to analysis. A total of 128 serum samples of 1- to 4-month old pigs from 10 swine farms were used for the isolation of the swine HEV RNA. A total of 264 serum samples of 1- to 7-month old pigs and sows from 13 swine farms were used for the prevalence study of the anti-HEV immunoglobulin G (IgG). The swine serum samples used in this study were randomly selected from the sera that had been submitted to the Department of Infectious Diseases, College of Veterinary Medicine, Seoul National University, Korea, for a routine examination of bacterial and viral diseases. A total of 96 human serum samples that had been obtained in 1995 from 40- to 60-year-old Korean volunteers regardless of their gender were examined to detect the anti-HEV IgG.
Purification of viral RNA and designing of PCR primers. The HEV RNA was purified from 140 µl of swine serum by using a QIAamp viral RNA minikit (Qiagen, Valencia, Calif.) according to the manufacturer's instructions. The viral RNA was eluted from the spin column with 45 µl of the elution buffer and stored at -70°C. Two sets of degenerate primers based on the highly conserved regions of the ORF 2 from the U.S. (AF082843) and Japanese (AB073912) swine HEV isolates were synthesized. The external set of primers was designed to produce a 1,040-bp (nucleotides 5457 to 6496 of the US swine HEV) PCR product. The external set of primers had the following sequences: forward primer, 5'-CTG CCC CCC GTC GTC GAT C-3'; and reverse primer, 5'-ATC CTG AAT AAC CAC ACG GG-3'. The expected size of the PCR product amplified with the nest set of primers was 860 bp (nucleotides 5533 to 6392 of the U.S. swine HEV). The nest set of primers had the following sequences: forward primer, 5'-TGT ACC TGA TGT TGA CTC ACG-3'; and reverse primer, 5'-GCT CGC CAT TGG CCG AGA C-3'.
RT-PCR and development of a nested PCR. Reverse transcriptase PCR (RT-PCR) was performed by using a QIAGEN OneStep RT-PCR kit according to the manufacturer's instructions. Briefly, a reaction tube contained 50 µl of the reaction solutions, including 10 µl of the 5x QIAGEN OneStep RT-PCR buffer, 2 µl of the dNTP mix (containing 10 mM of each dNTP), 10 µl of the 5x Q-Solution, 2 µl of the external forward primer (100 pM/µl), 2 µl of the external reverse primer (100 pM/µl), 2 µl of the QIAGEN OneStep RT-PCR enzyme mix, 1 µl of the RNaseOut RNA inhibitor (10 units/µl; Gibco BRL, Gaithersburg, Md.), 10 µl of the template RNA, and 11 µl of the RNase-free water. The thermal cycling conditions included one step of reverse transcription for 30 min at 50°C and an initial PCR activation step for 15 min at 95°C. This was followed by 40 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 50°C, and extension for 1 min 15 s at 72°C, and a final incubation for 10 min at 72°C. A nested PCR was conducted with the following components: 3 µl of the RT-PCR product, 5 µl of the 10x PCR buffer, 5 µl of MgCl2 (25 mg/ml), 4 µl of the dNTP mix (10 mM of each dNTP), 1 µl of the nested forward primer (100 pM/µl), 1 µl of the nested reverse primer (100 pM/µl), 0.5 µl of Takara Ex Taq polymerase (5 units/µl), and 30.5 µl of the double-distilled H2O. The thermal cycling conditions for the nested PCR included 5 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 45°C, and extension for 1 min 15 s at 72°C. This was followed by 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 53°C, and extension for 1 min 15 s at 72°C, and a final incubation for 7 min at 72°C.
Cloning of PCR products and analysis of clones. The nested PCR products were analyzed in a 1.0% agarose gel stained with ethidium bromide (10 mg/ml) under a UV transmitter. The 860-bp DNA band specific for the swine HEV was excised from the gel and purified with the QIAquick gel extraction kit (Qiagen). The purified DNA was cloned into a TOPO TA Cloning vector (Invitrogen, Carlsbad, Calif.) according to the manufacturer's protocol. The clones containing the insert DNA were identified by restriction enzyme digestion of the plasmid DNA with EcoRI (Promega, Madison, Wis.). The identity of the insert DNA was verified by using an automatic dye terminator DNA sequencing (Takara Korea Biomedicals, Seoul, Korea).1@::9eh, http://www.100md.com
Analysis of nucleotide sequences. The nucleotide sequences of the swine and human HEV isolates were aligned by the 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 accession numbers of the swine and human HEV isolates analyzed in this study are as follows: 01-18934D2, ; 01-19248-3, 01-22807-2, ; swUS1, NLSW15, NLSW20, NLSW22,; NLSW85, ; NLSW99, swJ681, swKOR1, swKOR2, swKOR3, TW32SW, ; TW3SW, UMC13A, ; JKN-Sap, JMY-Haw, US1, US2,JRA1, ; 87, China 5, Mexico, ; Morocco, India 2, ; Burma 1, ; China 1, D Pakistan SAR55, Egypt 93, and Avian HEV,
ELISA. The presence of anti-HEV IgG was determined in human and swine sera by using a commercial HEV enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Genelabs Diagnostics, Singapore). All serum samples were tested in duplicate. 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 250 µl of a wash solution. Horseradish peroxidase-labeled goat anti-human IgG (Genelabs Diagnostics, Singapore) and rabbit anti-pig IgG (Sigma, Steinheim, Germany) were used as the conjugates for the human and pig sera, respectively. 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 250 µl of the wash solution. Subsequently, 100 µl of the substrate solution containing 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 of the nonreactive control according to the manufacturer's instructions. The serum sample showing an absorbance value greater than the cutoff value was determined to be a positive.
Nucleotide sequence accession numbers. The GenBank accession numbers of the swine and human HEV isolates analyzed in this study are as follows: 01-18934D2, ; 01-19248-3, ; 01-22807-2, ; swUS1, ; NLSW15, NLSW20, NLSW22, ; NLSW85, ; NLSW99, ; swJ681, ; swKOR1, ; swKOR2, ; swKOR3, ; TW32SW, ; TW3SW, ; UMC13A, ; JKN-Sap JMY-Haw, US1, US2, ; JRA1, ; 87, ; China 5, Mexico, ; Morocco,India 2, Burma 1, China 1, ; Pakistan SAR55, Egypt 93, and Avian HEV, .i, http://www.100md.com
RESULTSi, http://www.100md.com
Identification of HEV isolates in swine sera. The swine isolate of HEV was identified in swine serum samples by a nested RT-PCR technique as the 860 bp PCR product (Fig. 1). The first PCR was performed with the template cDNA and a set of external forward and reverse primers. The reaction was expected to produce a 1,040-bp PCR product. However, the first PCR conditions used in this study did not produce any HEV-specific DNA band (data not shown). Therefore, the product of PCR was amplified again with a set of nest forward and reverse primers. This method successfully produced the 860-bp swine HEV-specific DNA band (Fig. 1). Three swine HEV strains were identified in a total of 128 pig sera collected from 10 pig herds, which indicated a 2.3% rate of HEV viremia prevalence (Table 1). Their nucleotide sequences were reported to GenBank, and they were given accession numbers for swKOR1, for swKOR2, and for swKOR3. Two strains, swKOR1 and swKOR2, were isolated in two 3-month-old pigs, and swKOR3 was isolated in a 2-month-old pig. These results suggest that most HEV infections in swine occur in young pigs approximately 2 to 3 months old.
fig.ommitted)*g, 百拇医药
FIG. 1. Identification of the Korean isolates of swine HEV in swine sera. HEV RNA was purified from swine serum. A nested RT-PCR was then performed to amplify a region of swine HEV ORF2. The swine HEV-specific 860-bp DNA bands were identified from three pig sera. Lanes: M, standard 100-bp DNA ladder; 1, Korean isolate 1 of swine HEV (swKOR1; GenBank accession number of ); 2, swKOR2 (); 3, swKOR3 ().)*g, 百拇医药
fig.ommitted)*g, 百拇医药
TABLE 1. Detection of swine HEV isolates in pigs of different ages)*g, 百拇医药
Genetic analysis of Korean isolates of swine HEV with other human and swine HEV isolates. Three Korean isolates of the swine HEV were genetically analyzed by comparing the highly conserved 300 bp of ORF2 to those of other human and swine HEV isolates. Phylogenetic analysis showed that all the Korean isolates of swine HEV were clustered in genotype III, which is typically represented by the human and swine HEV strains isolated from the United States and Japan (Fig. 2). They formed a distinct branch in genotype III, and they were most closely related to two human HEV strains, JKN-Sap and JMY-Haw, which were isolated from Japanese hepatitis patients, and one swine HEV strain, 01-19248-3, which was isolated from a pig in the United States. They were also closely related to one Netherlands swine HEV isolate, NLSW22, and other U.S. human and swine HEV isolates, including US1, US2, swUS1, and other recently identified swine isolates (Fig. 2). Among the swine HEV isolates examined in this study, the U.S. isolates, including the prototype swUS1, demonstrated the highest nucleotide identity (89.3 to 92.3%) with the Korean isolates (Table 2). The percent nucleotide identities between Korean and Japanese, Dutch, and Taiwanese swine HEV isolates were 84.7 to 86.7, 81.3 to 91.3, and 73.3 to 84.3%, respectively (Table 2). The three Korean isolates demonstrated a 92.7 to 99.8% identity at their nucleotide sequences (data not shown). However, their deduced 286 amino acids showed a 99.3 to 99.7% identity (data not shown). This suggests that most variations in the swine HEV genome silently occur at the third nucleotides of the amino acid codons.
fig.ommitted3x3h9ur, 百拇医药
FIG. 2. Phylogenetic tree analysis of the Korean isolates of swine HEV by comparison of the conserved 300 bp of the ORF2 in the swine and human HEV isolates. The HEV genotyping was determined according to a recent report (37). The GenBank accession numbers of the HEV isolates used in this study are indicated in Materials and Methods.3x3h9ur, 百拇医药
fig.ommitted3x3h9ur, 百拇医药
TABLE 2. Pairwise comparison of the nucleotide identity (%) in the ORF2 of the swine HEV isolatesa3x3h9ur, 百拇医药
Prevalence of anti-HEV IgG in swine and human populations. The prevalence rates of anti-HEV antibodies in swine and human sera were examined with ELISAs specific for anti-HEV IgG (Table 3 and Fig. 3). The swine sera were collected from 264 pigs in 13 farms and prepared according to the age of the swine, ranging from 1 to >5 months old. The highest prevalence of anti-HEV IgG was observed in 4-month-old pigs, showing a 36% seroprevalence. None of the 1-month-old pigs tested positive for anti-HEV antibodies in their sera. Other age groups of the pigs, including 2-, 3-, and 5- to 7-month-old pigs demonstrated 6, 10, and 25% anti-HEV antibody prevalence, respectively. Sows demonstrated an 8.8% seroprevalence to HEV. These results indicate that the prevalence of anti-HEV IgG in swine is age dependent. The overall prevalence of anti-HEV antibodies in Korean pigs was 14.8%. With respect to herd prevalence of anti-HEV antibodies, 11 out of 13 pig farms had HEV-positive pigs, demonstrating an 84.6% prevalence in the pig herds (data not shown). In the case of humans, a total of 96 serum samples randomly collected from blood donors in Korea in 1995 were tested. A total of 17 individuals (17.7%) were found to be HEV positive (Table 3). These results indicate that the swine and human populations in Korea have been subclinically infected by the HEV even though Korea has been known as a country where HEV is not endemic.
fig.ommitted#x-6n|, 百拇医药
TABLE 3. Prevalence of anti-HEV antibodies in swine and human populations#x-6n|, 百拇医药
fig.ommitted#x-6n|, 百拇医药
FIG. 3. Age-dependent distribution of anti-HEV antibodies in swine sera. The presence of anti-HEV antibodies was examined with a commercial HEV IgG ELISA kit (Genelabs Diagnostics, Singapore) according to the manufacturer's instructions. Samples showing an optical density (OD) value greater than 0.54 were considered to be HEV positive. The cutoff value was determined by a calculation suggested in the manufacturer's instructions.#x-6n|, 百拇医药
DISCUSSION#x-6n|, 百拇医药
It has been recognized that a large portion of pigs are infected with the HEV in the world (6, 10, 11, 18, 21, 23, 26, 29, 42, 50). The experimental cross-species infections (19, 28, 45), phylogenetic studies (18, 21, 26, 31, 42), and seroprevalence data (14, 21, 30) also indicate that HEV infections in humans may be mediated through contacts with infected pigs. Based on these findings, swine HEV is considered to be a zoonotic agent. In this study, three swine HEV strains were isolated from pigs in Korea for the first time, the isolates were characterized, and the prevalence rates of HEV-specific antibodies in pigs and humans were determined.
It has been observed that swine HEV can be detected in the feces at 2 to 6 weeks and in the sera at 4 to 6 weeks after experimental infections (27). Swine HEV RNA is typically isolated from fecal and serum samples obtained from 2- to 4-months old pigs (23, 31, 48). The overall prevalence rates of HEV viremia determined with serum samples were 1.5, 3.0, and 4.6% in pigs raised in Taiwan, Japan, and India, respectively (7, 31, 48). In this study, it was demonstrated that the prevalence of HEV viremia in pigs raised in Korea was 2.3%. Korean isolates of the swine HEV were also detected only in 2- to 3-month-old pigs. However, the prevalence of swine HEV RNA was much higher in the feces than in the sera, as demonstrated in recent studies (23, 42). When swine HEV RNA was detected in the pooled fecal samples from swine farms in The Netherlands, 22% of samples demonstrated the presence of the HEV (42). In the United States, when the presence of HEV RNA was detected by RT-PCR in the feces and sera obtained from 2- to 4-month-old pigs, the prevalence rates of HEV RNA were 40 and 12.5% in the feces and sera, respectively (23). These data indicate that more HEV particles are excreted in the feces than in the serum. And the discrepancy of HEV prevalence rates reported in several studies can also be explained by the different experimental materials they used. In this study, we used only sera as the experimental material for detecting the HEV because we primarily wanted to know the HEV prevalence in swine sera. The HEV prevalence in swine feces will be determined in subsequent study.
Genomic analysis of the HEV isolates indicates that HEV can be grouped into four major genotypes in which they can be further subgrouped into 9 groups (37). Genotype I contains the isolates from Burma and several Asian and African countries. Genotype II is represented by the isolates from Mexico and Nigeria. Genotype III contains the human isolates from the United States, Japan, Italy, Greece, Spain, Argentina, and Austria and the swine isolates from the United States, Japan, and New Zealand (31). Genotype IV contains the human and swine isolates from China and Taiwan (21, 48). The three newly isolated swine HEV strains in Korea are included in genotype III. They are most closely related to two Japanese isolates recently identified from acute hepatitis patients (39). They also have close relations with several swine isolates identified in the United States, Japan, and The Netherlands (23, 31, 42). This suggests that Korean strains of swine HEV might be variants introduced from the United States or other countries. Among the Korean HEV isolates, swKOR1 and swKOR2 were isolated from pigs in the same farm located in the southeastern region of Korea, and they displayed almost identical genomic sequences. In contrast, swKOR3 was isolated from a pig raised in a farm located in the central region of Korea. The identities of the 860-bp nucleotide sequence of swKOR3 with swKOR1 and swKOR2 were lower (92.7 to 92.9%) than those between swKOR1 and swKOR2 (99.8%). In the phylogenetic tree analysis, swKOR3 is divergent from the branch containing swKOR1 and swKOR2. This indicates the genetic variation of swine HEV in different regions.
The prevalence of anti-swine HEV antibodies in swine sera has been studied with an ELISA that uses the capsid antigen of the human Pakistan strain, HEV Sar-55, as a coating antigen because of a high percentage of identity in its amino acid sequence of ORF2 with that of swine HEV (26, 29). In this study, a commercial ELISA kit that was developed for detecting HEV-specific IgG in human sera was used to identify the anti-swine HEV antibodies in swine sera. The ELISA uses type-common epitopes in ORF2 and ORF3 of the Mexican and Burmese HEV strains as coating antigens (25, 49). Surprisingly, the ELISA very efficiently detected the anti-swine HEV IgG in swine serum samples. This indicates that the type-common HEV epitopes found in the Mexican and Burmese strains are also recognized by the anti-swine HEV IgG. A recent report demonstrated a 97% concordance in the anti-HEV prevalence results obtained with the human and swine HEV antigens (30), which also concurrently supports the presence of cross-species HEV epitopes. The highest prevalence of anti-swine HEV antibodies was observed in 4-month-old pigs. The overall anti-swine HEV prevalence rate determined in this study was 15%, whereas other studies have shown rather higher prevalence rates ranging from 30 to 70% (7, 10, 21, 26, 29, 50). The discrepancy in the antibody prevalence rates can be attributed to the different assay systems and to the different calculation methods used to decide a cutoff value.
The seroprevalence rates to HEV in human populations living in countries where HEV is not endemic are considerably high, suggesting the possibility of subclinical HEV infections in those countries (14, 25, 30, 41). Korea has been considered to be a country where HEV is not endemic. However, this study with randomly collected serum samples in Korea demonstrates that approximately 18% of Korean people possess HEV antibodies. A recent study in the United States also shows a similar anti-HEV antibody rate in normal blood donors (30). Therefore, it can be assumed that Korean people have been exposed to HEV without showing clinical signs of infection. Subsequent study will be undertaken to isolate the human HEV strain in Korea. If the human HEV is isolated, comparative analysis can be done to determine the genomic relationship between swine and human HEV isolates.9s, 百拇医药
In conclusion, three new swine HEV isolates were identified in 2- to 3-month-old pigs in Korea. They were grouped into genotype III by phylogenetic tree analysis and were closely related to the swine and human HEV isolates identified in the United States and Japan. The swine showed age-specific antibody prevalence rates to HEV. The prevalence rates of the HEV-specific antibodies in swine and humans were 15 and 18%, respectively.
ACKNOWLEDGMENTS'd4-, 百拇医药
This work was supported by funds from Brain Korea 21 and Research Institute for Veterinary Science, Seoul National University, Korea.'d4-, 百拇医药
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