Arch Virol DOI 10.1007/s00705-014-2006-6
BRIEF REPORT
Detection of Aichi virus in South Korea Tae-Hee Han • Sang Hun Park • Eung-Soo Hwang Gabor Reuter • Ju-Young Chung
•
Received: 22 September 2013 / Accepted: 24 January 2014 Ó Springer-Verlag Wien 2014
Abstract Aichi virus (AiV) is considered to be a possible etiologic agent of acute gastroenteritis (GE). We analyzed 1,568 stool samples collected by the Seoul Metropolitan Health Research Center from patients with GE during outbreaks in Seoul, together with 378 archived commonenteric-virus-negative stool samples from children with GE hospitalized at a tertiary hospital in Seoul. AiV was detected in 1.7 % (27/1,568) of the first group but not found in the second group (0 %, 0/378). Genotypes A and B of AiV were both detected in this study. This is the first study confirming the circulation of AiV in Korea. Keywords
Aichi virus Acute gastroenteritis
T.-H. Han Department of Diagnostic Laboratory Medicine, Sanggyepaik Hospital, Inje University College of Medicine, Seoul, South Korea S. H. Park Virus Team, Seoul Health Environment Research Center, Seoul, South Korea E.-S. Hwang Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, South Korea G. Reuter Regional Laboratory of Virology, National Reference ´ NTSZ Regional Institute Laboratory of Gastroenteric Viruses, A of State Public Health Service, Pecs, Hungary J.-Y. Chung (&) Department of Pediatrics, Sanggyepaik Hospital, Inje University College of Medicine, 761-1, Sanggye 7-Dong, Nowon-Gu, Seoul, South Korea e-mail: [email protected]
Aichi virus (AiV), which belongs to the family Picornaviridae, genus Kobuvirus, was first isolated from a patient with acute gastroenteritis (GE) after oyster consumption in Japan [19]. Kobuviruses have a single-stranded, positivesense RNA genome and have a common genome organization: nonstructural protein L (leader), structural capsid proteins (VP0, VP3, and VP1), and nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D) [15]. In the past, AiV was classified into three genotypes with different distributions according to geographical area (A genotype, Japan and Europe; B genotype, Asian countries other than Japan and Brazil; C genotype, Brazil) [1, 12, 17, 20]. However, the genus Kobuvirus was recently reorganized into three species: Aichivirus A (Aichivirus 1, formerly Aichi virus; canine kobuvirus 1; murine kobuvirus 1), Aichivirus B (bovine kobuvirus 1 and sheep kobuvirus 1) and Aichivirus C (formerly porcine kobuvirus 1) [7]. AiV has been suggested to play an etiological role in GE, especially in outbreaks associated with contaminated seafood [1, 12, 19]. Although doubts exist regarding its clinical role because of frequent co-detection (50-80 %) of AiV and other viral agents [1, 9, 14] and low incidence rates (0.41.3 %) in acute GE [8, 9, 11, 14], other studies have recently yielded contrasting results, showing that codetection of AiV with other common enteropathogenic viruses occurred at low rate [21] and that this virus was rarely detected in healthy individuals [2, 3, 21], which supports a possible role of this virus in GE. Klassevirus (KV), which was identified recently and is closely associated with AiV, has been suggested as a possible etiologic agent of GE in Korea, China, and the USA [5, 6, 16]. However, the clinical role and precise epidemiology of klassevirus is unclear because of the lack of data. The purpose of this study was to investigate the presence of AiV in patients with GE in Korea.
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The first retrospective cohort included 1,568 stool samples collected by the Seoul Metropolitan Health Research Center from 1,568 patients with GE during outbreaks in Seoul between January 2010 and March 2011. The second retrospective cohort included 378 archived enteric-virus (rotavirus, astrovirus, adenovirus, and sapovirus)-negative stool samples from 378 children with GE \16 years old, who were hospitalized at Sanggyepaik Hospital, Inje University, between March 2010 and March 2011. The study was approved by the Institutional Review Board of Sanggyepaik Hospital. RNA was extracted from each sample by using a QIAamp Viral RNA Stool Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription polymerase chain reaction (RT-PCR) was performed using 5 lL of each of RNA sample and RT-PCR pre mix (Bioneer, Daejeon, Korea) in 20 lL under the following conditions: 60 min at 42 °C, 5 min at 95 °C, and specific PCR cycling conditions for NoV-GI, NoV-GII, AiV, and KV. For NoV-GI, the first RT-PCR was performed using a primer set for the capsid gene, NV-GIF1 and NV-GIR1, under the following conditions: 40 cycles of 30 s at 94 °C, 30 s at 50 °C, and 60 s at 72 °C. The second round was performed using the primer set NV-GIF2 and NV-GIR1 under the same conditions as in the first round [10]. For NoV-GII, the first RT-PCR was performed using a primer set for the capsid gene, COG2-F and G2-SKR, under the following conditions: 40 cycles of 30 s at 94 °C, 30 s at 50 °C, and 60 s at 72 °C. The second round was performed using the primer set G2F1 and G2-SKR under the same conditions as in the first round [5]. The first RT-PCR reaction for AiV was performed using primer sets for the 5’-NCR (noncoding region), AiV-F274 (CCA GCC TGA CGT ATC ACA GG) and AiV-R1049 (GGA TAG AAC CAG GAT TGG ACA TCA G), under the following conditions: 45 cycles of 30 s at 95 °C, 30 s at 55 °C, and 60 s at 72 °C. The second reaction was carried out for 35 cycles and was performed using modified primer sets AiV-F313 (AAG CTG CTC ACG TGG CAA TTG TG) and AiV-R1039 (CAG GAT TGG ACA TCA GAA TCA TAG AG) under identical conditions, as described previously [3]. RT-PCR for the VP1 gene of AiV was performed using modified primer sets, AiV-F3010 (GCA AAA CCC YGC CCT CIC YTC C) and AiVR3902 (CGT CIG GGG CCA CYT TGC GGA), under the following conditions: 40 cycles of 30 s at 95 °C, 30 s at 60 °C, and 90 s at 72 °C. The second reaction involved 40 cycles performed using AiVF3025 (CTC ICY TCC CAR ACY CTC ACC G) and AiVR3879 (GCC CAG TGG ACG TAG GTG G) under identical conditions. For detection of KV, nested PCR was performed using the following primers: LG0118, LG0117, KL3DF, and KL3DR for the 3D region and LG0119, LG0136, KLVPF, and KLVPR for the gene encoding the viral protein VP0/VP1
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Table 1 Molecular detection of viruses in the study population Virus
No. positive First cohort (n = 1,568)
NoV-GI
47 (2.9 %)
NoV-GII
88 (5.6 %)
Klassevirus AiV
5 (0.3 %) 27 (1.7 %)
Second cohort (n = 387) 2 (0.5 %) 0 (0 %)
under the following conditions: a 30-min RT step at 60 °C and followed by 94 °C for 2 min, and then 40 cycles of 94 °C for 15 s, 56 °C for 30 s, and 68 °C for 90 s [6]. The PCR products were analyzed by 2 % agarose gel electrophoresis with ethidium bromide after extraction using an AccuPrep Gel Purification Kit (Bioneer, Daejeon, Korea). PCR products were cloned into the T-Blunt vector (SolGent, Daejeon, Korea) according to the manufacturer’s instructions. Ligations using the T-Blunt vector (SolGent, Daejeon, Korea) and 69 T-Blunt buffer were carried out at 25 °C for 5 min. Transformation was performed using T-Blunt vector ligation reactions in which the 3- to 6-lL T-Blunt reaction mixture was placed into a tube containing 100 lL of competent E. coli DH5a cells (SolGent, Daejeon, Korea). White colonies were selected and analyzed by plasmid DNA prep (SolGent, Daejeon, Korea). For each PCR product, three or four transfected bacterial colonies were selected, and their plasmids were extracted using an AccuPrep Plasmid Mini Extraction Kit (Bioneer, Daejeon, Korea) for sequencing. The sequences were analyzed using a BigDye Terminator Cycle Sequencing Kit, aligned using BioEdit v7.0, and presented in a topology tree using MEGA 4.1 [18]. The nucleotide sequences identified in this study have been deposited in the GenBank database under the following accession numbers: AiV 50 -NCR (KC167107-34) and VP1 gene (KC167079-KC167106). In the first cohort, NoV-GI was detected in 2.9 % (47/ 1,568), NoV-GII in 5.6 % (88/1,568), AiV in 1.7 % (27/ 1,568), and KV in 0.3 % (5/1,568) of the samples (Table 1). AiV was detected in March 2010 (n = 16) in two outbreaks (14 cases from a freshman orientation at a hotel located in Kangwon Province and two cases from a soccer team of a middle school located in the southern part of Seoul) and then in January 2011 (n = 7) and February 2011 (n = 4). Seventy-four percent of the AiV-positive samples showed mixed infection with other viral agents (NoV-GI in five cases, NoV-GII in 11 cases, NoVGI?NoV-GII in three cases, and NoV-GII?KV in one case). In the second cohort, 378 virus-negative stool samples from hospitalized children with GE were tested using modified primer sets for AiV, but none of the samples were found to be positive for AiV. Otherwise, KV was detected in 0.5 % (2/378) of the second cohort.
Detection of Aichi virus Fig. 1 Phylogenetic analysis of the 50 untranslated region (438 bp) of Aichi virus. The tree was constructed by the neighbor-joining method using the Kimura 2-parameter estimation. The bootstrap values from 1,000 replicates are shown at each branch. The scale bar represents 0.002 substitutions per nucleotide site. The Korean strains (KC167107-KC167134) are indicated in boldface
Phylogenetic analysis of the 50 -NCR gene of AiV showed that 14 Korean strains were clustered into genotype B, close to the Chinese strain Chschc712008, and the other 14 Korean strains were clustered into genotype A, together with the Japanese strain A846/88 and Danish strains D/12535/2004 and BAY/1/2003 (Fig. 1). The number of AiV sequences in the figures (Fig. 1 and Fig. 2) shows a discrepancy with the number (n = 27) of AiV-detected patients because strains belonging to both genotype A (KC167113-50 -NCR gene, KC167085 -VP1 gene) and
genotype B (KC167122-50 -NCR gene, KC167094–VP1 gene) were identified in a university student during the March 2010 outbreak. The VP1 gene of 14 AiV Korean strains formed a distinct phylogenetic cluster with genotype B; 10 strains were most similar to the Chinese strain Chschc712008 (gene similarity, 96.4-96.6 %; amino acid similarity, 97.1-97.8 %), and 4 strains were similar to the Danish strain V12535/2004 (gene similarity, 99-99.5 %; amino acid similarity, 99.2-99.6 %) (Fig. 2). Another 14 Korean strains belonged to genotype A; 10 strains were
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Fig. 2 Phylogenetic analysis of the VP1 (843 bp) of Aichi virus. The tree was constructed using the neighbor-joining method with the Kimura two-parameter estimation. The bootstrap values from 1,000
replicates are shown at each branch. The scale bar represents 0.05 substitutions per nucleotide site. The Korean strains (KC167079KC167106) are indicated in boldface
similar to the Japanese strain J-4432/2002 (gene similarity, 90.6-96.6 %; amino acid similarity, 97.4-97.8 %), and four strains were similar to the Danish strain BAY/1/2003 (gene similarity, 95.5-96.8 %; amino acid similarity, 98.299.2 %) (Fig. 2).
This is the first study to confirm the circulation of AiV in Korea, with the virus detected in 1.7 % of the patients in GE outbreaks between January 2010 and March 2011. In this study, the infection was probably transmitted via consumption of virus-contaminated food, because patients
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in all AiV-positive cases had had a meal at similar places such as a hotel restaurant, school restaurant, or a bistro, although the source of the contaminated food could not be clarified. This result is consistent with the results of previous studies [1, 19] that describe a strong association of AiV infection with consumption of contaminated food, such as oysters. In the present study, co-infection with AiV and other enteric viruses was prevalent (74 %), which is concordant with previous findings [1, 9, 14]. Some previous studies [1, 2, 13, 20–22] showed the geographical distribution of AiV genotypes, whereas this study detected both genotypes A and B, consistent with the results of a recent study [8]. In this study, AiV was not detected in 378 children with GE; this result is concordant with the results of previous studies that showed a low prevalence of this virus (0.21.1 %) [2, 4, 9, 11, 22]. These results suggest that AiV is not an important etiological agent of GE in children. However, some limitations, such as the lack of adequate demographic and clinical data and differences in primer sets and study populations should be considered when interpreting the results. In conclusion, the presence of AiV in adolescents and adults during GE outbreaks was confirmed, but further studies are necessary to determine the exact prevalence of AiV in South Korea. Acknowledgments We would like to thank Dr. Teruo Yamashita for kindly providing the AiV prototype strain A846/88 in 2012. This work was supported by grants from the 2011 Inje University Research Fund.
References 1. Ambert-Bakay K, Lorrot M, Bon F, Giraudon H, Kaplon J, Wolfer M, Lebon P, Gendrel D, Pothier P (2008) Prevalence and genetic diversity of Aichi virus strains in stool samples from community and hospitalized patients. J Clin Microbiol 46:1252–1258 2. Chhabra P, Payne DC, Szilagyi PG, Edwards KM, Staat MA, Shirley SH, Wikswo M, Nix WA, Lu X, Parashar UD, Vinje J (2013) Etiology of viral gastroenteritis infection in children \5 years of age in the United States, 2008–2009. J Infect Dis 208:790–800 3. Drexler JF, Baumgarte S, Luna LK, Eschbach-Budau M, Lukashev AN, Drosten C (2011) Aichi virus shedding in high concentrations in patients with acute diarrhea. Emerg Infect Dis 17:1544–1547 4. Han TH, Kim CH, Chung JY, Park SH, Hwang ES (2010) Klassevirus infection in children, South Korea. Emerg Infect Dis 16:1623–1625 5. Han TH, Kim CH, Chung JY, Park SH, Hwang ES (2011) Emergence of norovirus GII-4/2008 variant and recombinant strains in Seoul, Korea. Arch Virol 156:323–329
6. Holtz LR, Finkbeiner SR, Zhao G, Kirkwood CD, Girones R, Pipas JM, Wang D (2009) Klassevirus 1, a previously undescribed member of the family Picornaviridae, is globally widespread. Virol J 6:86 7. ICTVb—International Committee on Taxonomy of Viruses. Change the names of species Aichi virus and Bovine kobuvirus (family Picornaviridae, genus Kobuvirus) to Aichivirus A and Aichivirus B, respectively. http://ictvonline.org/proposals/2012. 014aV.A.v1.Kobuvirus-Sp,Ren.pdf. Accessed 18 Dec 2013 8. Jonsson N, Wahlstrom K, Svensson L, Serrander L, Lindberg AM (2012) Aichivirus infection in elderly people in Sweden. Arch Virol 157:1365–1369 9. Kaikkonen S, Rasanen S, Ramet M, Vesikari T (2010) Aichi virus infection in children with acute gastroenteritis in Finland. Epidemiol Infect 138:1166–1171 10. Lee JI, Chung JY, Han TH, Song MO, Hwang ES (2007) Detection of human bocavirus in children hospitalized because of acute gastroenteritis. J Infect Dis 196:994–997 11. Nielsen AC, Gyhrs ML, Nielsen LP, Pedersen C, Bottiger B (2013) Gastroenteritis and the novel picornaviruses aichivirus, coasavirus, saffoldvirus, and salivirus in young children. J Clin Virol 57:239–242 12. Oh DY, Silva PA, Hauroeder B, Diedrich S, Cardoso DDP, Schreier E (2006) Molecular characterization of the first Aichi viruses isolated in Europe and in South America. Arch Virol 151:1199–1206 13. Pham NT, Khamrin P, Nguyen T, Kanti DS, Phan TG, Okitsu S, Ushijima H (2007) Isolation and molecular characterization of Aichi viruses from fecal specimens collected in Japan, Bangladesh, Thailand, and Vietnam. J Clin Microbiol 45:2287–2288 14. Rasanen S, Lappalainen S, Kaikkonen S, Hamalainen M, Salminen M, Vesikari T (2010) Mixed viral infections causing acute gastroenteritis in children in a waterborne outbreak. Epidemiol Infection 138:1227–1234 15. Reuter G, Boros A, Pankovics P (2011) Kobuviruses—a comprehensive review. Rev Med Virol 21:32–41 16. Shan T, Wang C, Cui L, Yu Y, Delwart E, Zhao W, Zhu C, Lan D, Dai X, Hua X (2010) Picornavirus salivirus/klassevirus in children with diarrhea, China. Emerg Infect Dis 16:1303–1305 17. Sdiri-Loulizi K, Gharbi-Khelifi H, de Rougemont A, Chouchane S, Sakly N, Ambert-Balay K, Hassine M, Guediche MN, Aouni M, Pothier P (2008) Acute infantile gastroenteritis associated with human enteric viruses in Tunisia. J Clin Microbiol 46:1349–1355 18. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA 4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599 19. Yamashita T, Kobayashi S, Sakae K, Nakata S, Chiba S, Ishihara Y, Isomura S (1991) Isolation of cytopathic small round viruses with BS-C-1 cells from patients with gastroenteritis. J Infect Dis 164:954–957 20. Yamashita T, Sakae K, Ishihara Y, Isomura S, Utagawa E (1993) Prevalence of newly isolated, cytopathic small round viruses (Aichi strain) in Japan. J Clin Microbiol 31:2938–2943 21. Yang S, Zhang W, Shen Q, Yang Z, Zhu J, Cui L, Hua X (2009) Aichi virus strains in children with gastroenteritis, China. Emerg Infect Dis 15:1703–1705 22. Verma H, Chitambar SD, Gopalkkrishna V (2011) Circulation of Aichi virus genotype B strains in children with acute gastroenteritis in India. Epidemiol Infect 139:1687–1691
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BRIEF REPORT
Detection of Aichi virus in South Korea Tae-Hee Han • Sang Hun Park • Eung-Soo Hwang Gabor Reuter • Ju-Young Chung
•
Received: 22 September 2013 / Accepted: 24 January 2014 Ó Springer-Verlag Wien 2014
Abstract Aichi virus (AiV) is considered to be a possible etiologic agent of acute gastroenteritis (GE). We analyzed 1,568 stool samples collected by the Seoul Metropolitan Health Research Center from patients with GE during outbreaks in Seoul, together with 378 archived commonenteric-virus-negative stool samples from children with GE hospitalized at a tertiary hospital in Seoul. AiV was detected in 1.7 % (27/1,568) of the first group but not found in the second group (0 %, 0/378). Genotypes A and B of AiV were both detected in this study. This is the first study confirming the circulation of AiV in Korea. Keywords
Aichi virus Acute gastroenteritis
T.-H. Han Department of Diagnostic Laboratory Medicine, Sanggyepaik Hospital, Inje University College of Medicine, Seoul, South Korea S. H. Park Virus Team, Seoul Health Environment Research Center, Seoul, South Korea E.-S. Hwang Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, South Korea G. Reuter Regional Laboratory of Virology, National Reference ´ NTSZ Regional Institute Laboratory of Gastroenteric Viruses, A of State Public Health Service, Pecs, Hungary J.-Y. Chung (&) Department of Pediatrics, Sanggyepaik Hospital, Inje University College of Medicine, 761-1, Sanggye 7-Dong, Nowon-Gu, Seoul, South Korea e-mail: [email protected]
Aichi virus (AiV), which belongs to the family Picornaviridae, genus Kobuvirus, was first isolated from a patient with acute gastroenteritis (GE) after oyster consumption in Japan [19]. Kobuviruses have a single-stranded, positivesense RNA genome and have a common genome organization: nonstructural protein L (leader), structural capsid proteins (VP0, VP3, and VP1), and nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D) [15]. In the past, AiV was classified into three genotypes with different distributions according to geographical area (A genotype, Japan and Europe; B genotype, Asian countries other than Japan and Brazil; C genotype, Brazil) [1, 12, 17, 20]. However, the genus Kobuvirus was recently reorganized into three species: Aichivirus A (Aichivirus 1, formerly Aichi virus; canine kobuvirus 1; murine kobuvirus 1), Aichivirus B (bovine kobuvirus 1 and sheep kobuvirus 1) and Aichivirus C (formerly porcine kobuvirus 1) [7]. AiV has been suggested to play an etiological role in GE, especially in outbreaks associated with contaminated seafood [1, 12, 19]. Although doubts exist regarding its clinical role because of frequent co-detection (50-80 %) of AiV and other viral agents [1, 9, 14] and low incidence rates (0.41.3 %) in acute GE [8, 9, 11, 14], other studies have recently yielded contrasting results, showing that codetection of AiV with other common enteropathogenic viruses occurred at low rate [21] and that this virus was rarely detected in healthy individuals [2, 3, 21], which supports a possible role of this virus in GE. Klassevirus (KV), which was identified recently and is closely associated with AiV, has been suggested as a possible etiologic agent of GE in Korea, China, and the USA [5, 6, 16]. However, the clinical role and precise epidemiology of klassevirus is unclear because of the lack of data. The purpose of this study was to investigate the presence of AiV in patients with GE in Korea.
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The first retrospective cohort included 1,568 stool samples collected by the Seoul Metropolitan Health Research Center from 1,568 patients with GE during outbreaks in Seoul between January 2010 and March 2011. The second retrospective cohort included 378 archived enteric-virus (rotavirus, astrovirus, adenovirus, and sapovirus)-negative stool samples from 378 children with GE \16 years old, who were hospitalized at Sanggyepaik Hospital, Inje University, between March 2010 and March 2011. The study was approved by the Institutional Review Board of Sanggyepaik Hospital. RNA was extracted from each sample by using a QIAamp Viral RNA Stool Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription polymerase chain reaction (RT-PCR) was performed using 5 lL of each of RNA sample and RT-PCR pre mix (Bioneer, Daejeon, Korea) in 20 lL under the following conditions: 60 min at 42 °C, 5 min at 95 °C, and specific PCR cycling conditions for NoV-GI, NoV-GII, AiV, and KV. For NoV-GI, the first RT-PCR was performed using a primer set for the capsid gene, NV-GIF1 and NV-GIR1, under the following conditions: 40 cycles of 30 s at 94 °C, 30 s at 50 °C, and 60 s at 72 °C. The second round was performed using the primer set NV-GIF2 and NV-GIR1 under the same conditions as in the first round [10]. For NoV-GII, the first RT-PCR was performed using a primer set for the capsid gene, COG2-F and G2-SKR, under the following conditions: 40 cycles of 30 s at 94 °C, 30 s at 50 °C, and 60 s at 72 °C. The second round was performed using the primer set G2F1 and G2-SKR under the same conditions as in the first round [5]. The first RT-PCR reaction for AiV was performed using primer sets for the 5’-NCR (noncoding region), AiV-F274 (CCA GCC TGA CGT ATC ACA GG) and AiV-R1049 (GGA TAG AAC CAG GAT TGG ACA TCA G), under the following conditions: 45 cycles of 30 s at 95 °C, 30 s at 55 °C, and 60 s at 72 °C. The second reaction was carried out for 35 cycles and was performed using modified primer sets AiV-F313 (AAG CTG CTC ACG TGG CAA TTG TG) and AiV-R1039 (CAG GAT TGG ACA TCA GAA TCA TAG AG) under identical conditions, as described previously [3]. RT-PCR for the VP1 gene of AiV was performed using modified primer sets, AiV-F3010 (GCA AAA CCC YGC CCT CIC YTC C) and AiVR3902 (CGT CIG GGG CCA CYT TGC GGA), under the following conditions: 40 cycles of 30 s at 95 °C, 30 s at 60 °C, and 90 s at 72 °C. The second reaction involved 40 cycles performed using AiVF3025 (CTC ICY TCC CAR ACY CTC ACC G) and AiVR3879 (GCC CAG TGG ACG TAG GTG G) under identical conditions. For detection of KV, nested PCR was performed using the following primers: LG0118, LG0117, KL3DF, and KL3DR for the 3D region and LG0119, LG0136, KLVPF, and KLVPR for the gene encoding the viral protein VP0/VP1
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Table 1 Molecular detection of viruses in the study population Virus
No. positive First cohort (n = 1,568)
NoV-GI
47 (2.9 %)
NoV-GII
88 (5.6 %)
Klassevirus AiV
5 (0.3 %) 27 (1.7 %)
Second cohort (n = 387) 2 (0.5 %) 0 (0 %)
under the following conditions: a 30-min RT step at 60 °C and followed by 94 °C for 2 min, and then 40 cycles of 94 °C for 15 s, 56 °C for 30 s, and 68 °C for 90 s [6]. The PCR products were analyzed by 2 % agarose gel electrophoresis with ethidium bromide after extraction using an AccuPrep Gel Purification Kit (Bioneer, Daejeon, Korea). PCR products were cloned into the T-Blunt vector (SolGent, Daejeon, Korea) according to the manufacturer’s instructions. Ligations using the T-Blunt vector (SolGent, Daejeon, Korea) and 69 T-Blunt buffer were carried out at 25 °C for 5 min. Transformation was performed using T-Blunt vector ligation reactions in which the 3- to 6-lL T-Blunt reaction mixture was placed into a tube containing 100 lL of competent E. coli DH5a cells (SolGent, Daejeon, Korea). White colonies were selected and analyzed by plasmid DNA prep (SolGent, Daejeon, Korea). For each PCR product, three or four transfected bacterial colonies were selected, and their plasmids were extracted using an AccuPrep Plasmid Mini Extraction Kit (Bioneer, Daejeon, Korea) for sequencing. The sequences were analyzed using a BigDye Terminator Cycle Sequencing Kit, aligned using BioEdit v7.0, and presented in a topology tree using MEGA 4.1 [18]. The nucleotide sequences identified in this study have been deposited in the GenBank database under the following accession numbers: AiV 50 -NCR (KC167107-34) and VP1 gene (KC167079-KC167106). In the first cohort, NoV-GI was detected in 2.9 % (47/ 1,568), NoV-GII in 5.6 % (88/1,568), AiV in 1.7 % (27/ 1,568), and KV in 0.3 % (5/1,568) of the samples (Table 1). AiV was detected in March 2010 (n = 16) in two outbreaks (14 cases from a freshman orientation at a hotel located in Kangwon Province and two cases from a soccer team of a middle school located in the southern part of Seoul) and then in January 2011 (n = 7) and February 2011 (n = 4). Seventy-four percent of the AiV-positive samples showed mixed infection with other viral agents (NoV-GI in five cases, NoV-GII in 11 cases, NoVGI?NoV-GII in three cases, and NoV-GII?KV in one case). In the second cohort, 378 virus-negative stool samples from hospitalized children with GE were tested using modified primer sets for AiV, but none of the samples were found to be positive for AiV. Otherwise, KV was detected in 0.5 % (2/378) of the second cohort.
Detection of Aichi virus Fig. 1 Phylogenetic analysis of the 50 untranslated region (438 bp) of Aichi virus. The tree was constructed by the neighbor-joining method using the Kimura 2-parameter estimation. The bootstrap values from 1,000 replicates are shown at each branch. The scale bar represents 0.002 substitutions per nucleotide site. The Korean strains (KC167107-KC167134) are indicated in boldface
Phylogenetic analysis of the 50 -NCR gene of AiV showed that 14 Korean strains were clustered into genotype B, close to the Chinese strain Chschc712008, and the other 14 Korean strains were clustered into genotype A, together with the Japanese strain A846/88 and Danish strains D/12535/2004 and BAY/1/2003 (Fig. 1). The number of AiV sequences in the figures (Fig. 1 and Fig. 2) shows a discrepancy with the number (n = 27) of AiV-detected patients because strains belonging to both genotype A (KC167113-50 -NCR gene, KC167085 -VP1 gene) and
genotype B (KC167122-50 -NCR gene, KC167094–VP1 gene) were identified in a university student during the March 2010 outbreak. The VP1 gene of 14 AiV Korean strains formed a distinct phylogenetic cluster with genotype B; 10 strains were most similar to the Chinese strain Chschc712008 (gene similarity, 96.4-96.6 %; amino acid similarity, 97.1-97.8 %), and 4 strains were similar to the Danish strain V12535/2004 (gene similarity, 99-99.5 %; amino acid similarity, 99.2-99.6 %) (Fig. 2). Another 14 Korean strains belonged to genotype A; 10 strains were
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Fig. 2 Phylogenetic analysis of the VP1 (843 bp) of Aichi virus. The tree was constructed using the neighbor-joining method with the Kimura two-parameter estimation. The bootstrap values from 1,000
replicates are shown at each branch. The scale bar represents 0.05 substitutions per nucleotide site. The Korean strains (KC167079KC167106) are indicated in boldface
similar to the Japanese strain J-4432/2002 (gene similarity, 90.6-96.6 %; amino acid similarity, 97.4-97.8 %), and four strains were similar to the Danish strain BAY/1/2003 (gene similarity, 95.5-96.8 %; amino acid similarity, 98.299.2 %) (Fig. 2).
This is the first study to confirm the circulation of AiV in Korea, with the virus detected in 1.7 % of the patients in GE outbreaks between January 2010 and March 2011. In this study, the infection was probably transmitted via consumption of virus-contaminated food, because patients
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Detection of Aichi virus
in all AiV-positive cases had had a meal at similar places such as a hotel restaurant, school restaurant, or a bistro, although the source of the contaminated food could not be clarified. This result is consistent with the results of previous studies [1, 19] that describe a strong association of AiV infection with consumption of contaminated food, such as oysters. In the present study, co-infection with AiV and other enteric viruses was prevalent (74 %), which is concordant with previous findings [1, 9, 14]. Some previous studies [1, 2, 13, 20–22] showed the geographical distribution of AiV genotypes, whereas this study detected both genotypes A and B, consistent with the results of a recent study [8]. In this study, AiV was not detected in 378 children with GE; this result is concordant with the results of previous studies that showed a low prevalence of this virus (0.21.1 %) [2, 4, 9, 11, 22]. These results suggest that AiV is not an important etiological agent of GE in children. However, some limitations, such as the lack of adequate demographic and clinical data and differences in primer sets and study populations should be considered when interpreting the results. In conclusion, the presence of AiV in adolescents and adults during GE outbreaks was confirmed, but further studies are necessary to determine the exact prevalence of AiV in South Korea. Acknowledgments We would like to thank Dr. Teruo Yamashita for kindly providing the AiV prototype strain A846/88 in 2012. This work was supported by grants from the 2011 Inje University Research Fund.
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