key: cord-308953-4mhjtitv
authors: Kim, Hyun-Jeong; Park, Sang-Ik; Ha, Thi Phuong Mai; Jeong, Young-Ju; Kim, Ha-Hyun; Kwon, Hyoung-Jun; Kang, Mun-Il; Cho, Kyoung-Oh; Park, Su-Jin
title: Detection and genotyping of Korean porcine rotaviruses
date: 2010-08-26
journal: Vet Microbiol
DOI: 10.1016/j.vetmic.2010.01.019
sha: 
doc_id: 308953
cord_uid: 4mhjtitv

Porcine group A rotavirus (GARV) is considered to be an important animal pathogen due to their economic impact in the swine industry and its potential to cause heterologous infections in humans. This study examined 475 fecal samples from 143 farms located in 6 provinces across South Korea. RT-PCR and nested PCR utilizing primer pairs specific for the GARV VP6 gene detected GARV-positive reactions in 182 (38.3%) diarrheic fecal samples. A total of 98 porcine GARV strains isolated from the GARV-positive feces were analyzed for G and P genotyping. Based on the sequence and phylogenetic analyses, the most predominant combination of G and P genotypes was G5P[7], found in 63 GARV strains (64.3%). The other combinations of G and P genotypes were G8P[7] (16 strains [16.3%]), G9P[7] (7 strains [7.1%]), G9P[23] (2 strains [2.0%]), and G8P[1] (1 strain [1.0%]). The counterparts of G or P genotypes were not determined in three G5, five P[7], and one P[1] strains. Interestingly, phylogenetic analysis indicated that all Korean G9 strains were more closely related to lineage VI porcine and human viruses than to other lineages (I–V) of GARVs and to Korean human G9 strains (lineage III). These results show that porcine GARV infections are common in diarrheic piglets in South Korea. The infecting strains are genetically diverse, and include homologous (G5P[7]), heterologous (G8P[1]), and reassortant (G8P[7]), as well as emerging G9 GARV strains.

Group A rotavirus (GARV), a member of the Reoviridae family, is one of the major pathogens that cause severe, acute dehydrating diarrhea in young children and in a wide variety of domestic animals (Estes and Kapikian, 2007; Gentsch et al., 2005; Glass et al., 1997) . The rotavirus genome consists of 11 segments of double-stranded (ds) RNA enclosed in a trilaminar capsid and encodes six structural (VP1-VP4, VP6, and VP7) and six nonstructural proteins (NSP1-NSP6) (Estes and Kapikian, 2007; Gentsch et al., 2005; Parashar et al., 2006) . Due to the segmented nature of the genome, GARVs can undergo genetic reassortment if two different GARVs of the same group co-infect one cell (Estes and Kapikian, 2007; Gentsch et al., 2005; Parashar et al., 2006) .

Recently, a new rotavirus classification system was proposed, in which nucleotide percentage identity cut-off values define different genotypes for all the 11 genomic RNA segments (Matthijnssens et al., 2008) . The VP7 and VP4 outer capsid proteins independently elicit neutralizing antibody responses and are used to classify GARVs into G (for glycoprotein) and P (for protease-sensitive) types (Ciarlet and Estes, 2002; Estes and Kapikian, 2007; Glass et al., 1997) . Currently, 23 G and 31 P genotypes have been described for GARVs of humans and animals (Abe et al., 2009; Ursu et al., 2009) . As many more reassortant or new genotypes are predicted to appear, continuous monitoring of circulating rotaviruses is important for improving regional epidemiological information and updating the vaccine strains.

Porcine GARVs can cause enormous economic losses in the swine industry and are a potential source of heterologous GARV infections in humans (Jain et al., 2001; Leite et al., 1996; Martella et al., 2005; Timenetsky et al., 1994; Unicomb et al., 1999) . Thus, molecular epidemiology on porcine GARVs in South Korea is needed to determine the prevalence, as well as the extent of diversity in the circulating strains to improve vaccination programs by updating the vaccine strains. This paper reports the prevalence of porcine GARVs in diarrheic piglets, along with the genetic diversity of the porcine GARVs based on the characterization of the G and P genotypes.

From 2006 to 2007, 475 fecal specimens from diarrheic pigs were obtained from 143 farms across 6 provinces in South Korea during the spring (215 samples/53 farms), summer (86 samples/17 farms), autumn (69 samples/20 farms), and winter (105 samples/53 farms). The ages of the pigs tested from these provinces ranged from 3 to 70 days old. The fecal samples were examined for common bacterial enteric pathogens including Escherichia coli (E. coli) and Salmonella spp. using specific agar media. Brachyspira hyodysenteriae was detected by PCR with the specific primers B.hyo nest3 (5 0 -CTGCTGCCTTCTTCA-TAAAT-3 0 ) and B.hyo nest 5 (5 0 -AAGAATGGGTATTG-TTGCTG-3 0 ) (La et al., 2003) . For the extraction of viral RNA, fecal suspensions of each sample were prepared immediately by diluting the feces 1:10 in 0.01 M phosphate-buffered saline (PBS), pH 7.2. The suspensions were then vortexed for 30 s, centrifuged (1200 Â g for 20 min), and then the supernatants were collected and stored at À80 8C until needed.

The RNA was extracted from a 200 ml starting volume of centrifuged 10% fecal suspensions and from the lysates of GARV-infected fetal rhesus monkey kidney (TF-104) cells using the SV Total RNA Isolation System reagent (Promega Corporation, Madison, WI) according to the manufacturer's instructions. The total RNA recovered was suspended in 50 ml of RNase free water and stored at À80 8C until used.

RT-PCR assays with different primer sets (Table 1) for the detection of porcine groups A-C rotaviruses (GARVs-GCRVs), porcine sapovirus (PSaV), porcine norovirus (PNoV), porcine torovirus (PToV), transmissible gastroenteritis coronavirus (TGEV), and porcine epidemic diarrhea coronavirus (PEDV) were performed using a standard one-step RT-PCR as previously described (Jeong et al., 2007) . In order to increase the sensitivity and specificity of RT-PCR, nested PCR assays with the primer pairs specific to porcine GARV, GCRV and PSaV (Table 1 ) were performed as previously described (Jeong et al., 2007) . The amplification products were analyzed by 1.5 or 2% agarose gel electrophoresis and visualized by UV after ethidium bromide staining.

Monolayers of TF-104 cells (a cloned derivative of MA-104 monkey kidney cells) grown for 3 or 4 days in 6-well plates were used to isolate GARVs, as previously described (Bohl et al., 1984; Park et al., 2006) . The isolated GARVs were confirmed by direct immunofluorescence (IF) tests and RT-PCR (Bohl et al., 1984; Park et al., 2006) .

To obtain genomic data on the G and P genotypes of Korean porcine GARVs, porcine GARVs isolated from the diarrheic fecal samples were subjected to RT-PCR with primer pairs specific to each VP7 and VP4 gene of GARVs (Table 1) . RT-PCR products amplified by each primer pair were selected based on the intensity of the bands shown by agarose gel electrophoresis and ethidium bromide visualization. Before sequencing, the RT-PCR products from each gene fragment were purified using a QIAEX II Gel Extraction kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. DNA sequencing was carried out using an ABI system 3700 automated DNA sequencer (Applied Biosystems, Foster City, CA).

Using the DNA Basic module (DNAsis MAX, Alameda, CA), the nucleotide and deduced amino acid sequences of the partial VP4 gene (834 bp, devoid of primer pair sequences) and VP7 gene (1020 bp, devoid of primer pair sequences) were compared with those selected from other known GARVs (Table 2) . Phylogenetic analysis based on the nucleotide alignments was constructed using the neighbor-joining method and the UPGMA method of Molecular Evolutionary Genetics analysis (MEGA version 4.0) with a pair-wise distance comparison (Tamura et al., 2007) . A sequence similarity search was performed for the GARV VP4 and VP7 genes using the LALIGN Query program of the GENESTREAM network server at Institut de Gé né tique Humaine, Montpellier, France (http://www. eng.uiowa.edu/$tscheetz/sequence-analysis/examples/ LALIGN/lalign-guess.html).

In order to determine the prevalence of porcine GARVs in diarrheic Korean piglets, a total of 475 fecal samples from diarrheic pigs in 143 farms across South Korea were screened by RT-PCR and nested PCR using two sets of primer pairs (Table 1) . By RT-PCR, 106 out of 475 diarrheic fecal samples tested positive for porcine GARVs. In nested PCR, an additional 76 samples were found to be positive for porcine GARVs. Overall, 182 (38.3%) out of 475 diarrheic fecal samples were positive for porcine GARVs (Table 3) .

Of the 182 porcine GARV-positive diarrheic fecal specimens, 58 fecal samples (12.2%) tested positive only for the porcine GARVs, while the other 124 fecal samples (26.1%) were also positive for other enteric pathogens, including GBRV, GCRV, PSaV, PToV, E. coli, Salmonella and B. hyodysenteriae (Table 3 ). In addition, 168 fecal specimens (35.4%) that tested negative for porcine GARVs were positive for other enteric pathogens (Table 3) . No enteric pathogens were detected in the remaining 125 fecal samples (26.3%).

Porcine GARV infections were more prevalent in fecal samples of pigs in summer than in the other seasons: 87 (40.5%) out of 215 fecal samples were positive in spring; 43 (50.0%) out of 86 fecal samples were positive in summer; 17 (24.6%) out of 69 fecal samples were positive in autumn; and 35 (33.3%) out of 105 fecal samples were positive in winter.

Of the 182 porcine GARV-positive fecal samples by RT-PCR or nested PCR, porcine GARVs were isolated from 98 fecal samples. After the second or third passage, cytopathic effect (CPE), characterized by rounded and detached cells in clusters, was observed in the cultures inoculated with each fecal sample from diarrheic piglets at post inoculation days 1-2. No differences in CPEs were observed among the isolates. CPE was not observed in the mock-infected TF-104 cells. The direct IF test detected GARV-specific cytoplasmic fluorescence in the TF-104 cells inoculated with each of these samples at the second or third passage. A specific band was detected after amplification of all isolates using a RT-PCR assay targeting a 308 bp fragment of the VP6 gene of GARVs.

Using RT-PCR to amplify full length sequence (1062 nucleotides in length) of the VP7 gene, amplicons could be achieved for 92 out of 98 strains and could be sequenced. A comparison of the nucleotide and deduced amino acid sequences of the VP7 gene between all Korean porcine GARV strains and the GARV strains representing all 23 G genotypes was performed with a 1020 bp fragment (excluding the primer sequences) (Tables 4 and 5). Sixty-six Korean strains showed high nucleotide (97.6-99.8%) and deduced amino acid (94.5-100%) identities with the G5 strains, which include the porcine OSU and JL94 strains, and the bovine KJ44 strains. On the other hand, these strains had comparatively lower nucleotide (63.4-83.2%) and deduced amino acid (54.2-89.8%) identities with the other G genotypes (data not shown). Phylogenetic analysis also confirmed that the VP7 gene of 66 Korean porcine GARV strains was closely related to the G5 strains and clustered with the porcine G5 strains (Fig. 1A) . Seventeen of the 92 Korean porcine GARV strains had 84.7-97.8% nucleotide and 90.9-98.2% deduced amino acid identities to the G8 GARVs including the bovine BRV16, Sun9, KAG80, and NGRBg8 strains (Table 4) , whereas they showed relatively lower nucleotide (61.8-78.7%) and deduced amino acid (54.4-84.7%) identities with other G genotypes (data not shown). Phylogenetically, these strains are grouped with G8 strains, including the bovine BRV16, Sun9, KAG80, and NGRBg8 strains. The remaining nine Korean porcine GARV strains showed high nucleotide (85.3-97.2%) and deduced amino acid (87.1-99.1%) identities with the G9 strains (Table 5 ). In contrast, these strains shared lower nucleotide (60.7-80.4%) and deduced amino acid (53.9-87.2%) identities with the other G genotypes (data not shown). Phylogenetic analysis showed that all G9 Korean porcine strains clustered with those of lineage VI. In addition, all Korean human G9 strains were found to be grouped with those of lineage III (Fig. 1B) .

A part of the VP4 gene (874 nucleotides in length) was able to be amplified in 95 out of 98 isolated strains. The nucleotide and deduced amino acid sequences encoding 290 amino acids representing VP8* and the amino terminus of VP5* of the 95 strains were compared with GARV strains representing all the 31 P genotypes. Of the 95 Korean strains, 91 had high nucleotide (88.7-99.8%) and deduced amino acid (91.0-99.3%) identities with the P[7] GARVs including the porcine OSU, JL94, and SW20/21 strains, and the bovine PP-1 strain (Table 6 ), but less than 73.8% nucleotide and 79.4% deduced amino acid identities with the other P genotypes (data not shown). Phylogenetic analysis of the VP4 gene provided a molecular basis for their similarity to the P[7] genotype strains (Fig. 2) . The sequences of the Korean strains, 11-1 and 66-1, were most closely related to the bovine BRV033, NCDV, C486, and RF (Table 6 ). In contrast, these strains showed lower nucleotide (53.0-76.1%) and deduced amino acid (43.6-80.4%) identities to the representatives of other P genotypes (data not shown). Phylogenetically, these two strains clustered with those of the P[1] genotype (Fig. 2) . The remaining two strains, 06-52-1 and 06-285, shared high nucleotide (84.5-89.7%) and deduced amino acid (94.1-95.1%) identities to the P[23] strains (A34, JP32-4, and Hokkaido-14 strains) ( Table 6 ), but less than 73.9% nucleotide and 82.1% deduced amino acid identities compared to representatives of the other P genotypes (data not shown). Phylogenetic analysis of the VP4 gene showed that these strains were grouped with those of the P[23] genotype (Fig. 2) .

Based on the sequence and phylogenetic analyses of 98 Korean porcine GARVs, G and P genotype combinations were determined in the Korean porcine GARVs (Table 7) . The most common combination of G and P genotypes was G5P[7], which was detected in 63 GARVs. Sixteen GARVs had the G8P[7] combination, while G9P[7] GARVs were detected in 7 strains. Two GARVs showed the G9P[23] combination, and one strain had the G8P[1] combination. In addition, the counterparts of G and P genotypes were not determined in three G5, five P[7], and one P[1] GARV strains (Table 7) .

Epidemiological information related to the prevalence and genotype specificities of porcine GARVs are beneficial for the development of effective vaccines (Rosen et al., 1994) . Therefore, we investigated the prevalence of porcine GARV infections as well as their genotype diversities in South Korea. The fecal prevalence of porcine GARV infections in diarrheic piglets has been reported to be 3.3% in Argentina (Parra et al., 2008) , 4% in Southern Germany (Wieler et al., 2001) , 9.2% in Canada (Morin et al., (Khamrin et al., 2007) and 35.3% in Brazil (Rá cz et al., 2000) . In this study, porcine GARV infections in South Korea were found widespread and highly prevalent at 38.3%, similar to Brazil at 35.3% (Rá cz et al., 2000) . This suggests that porcine GARV infections are epidemic in diarrheic piglets in South Korea. This is the first large-scale, epidemiological study on the prevalence of porcine GARV infections in diarrheic piglets in South Korea.

Epidemiological studies have demonstrated that five G genotypes (G3, G4, G5, G9, and G11) and P[26] ) are the most frequent VP7 and VP4 types associated with porcine GARV infections (Kobayashi et al., 2007) . In this study, two-thirds of the VP7 and VP4 genotypes were comprised of G5 and P[7] genotypes, respectively. The other G and P genotypes including G8, G9, P[1], and P[23] were a minority of the VP7 and VP4 genotypes. However, G3, G4, G11, P[6], P[13], P[19], and P[26] genotypes, which were known to be common, were not detected in this study. It is unclear whether the data in this study exactly reflected the true prevalence of G and P genotypes in the field farms due to the difficulty in cultivating some rotaviruses in cell culture (Zaberezhny et al., 1994) . For example, P[6] porcine GARVs are quite Fig. 1. (A) Phylogenetic tree of the complete VP7 genes of the sixty-six G5, seventeen G8, and nine G9 strains of Korean porcine GARVs indicating their genetic relationships with other G genotypes. Black triangles contain rotavirus G5, G8, and G9 strains. (B) A detailed phylogenetic tree of the complete VP7 genes of the nine Korean porcine G9 strains with other known G9 strains indicating their genetic relationships with other known VI lineages of G9 genotype. Reference sequences used in the analysis (A and B) were obtained from the GenBank database (Table 2) . Reference sequences used in the analysis were obtained from the GenBank database (Table 2) . common in nature, but are not usually cultivatable (Martella et al., 2006; Zaberezhny et al., 1994) . Since we analyzed only cell culture cultivated porcine GARV strains, future studies should use the fecal samples for G and P genotyping of porcine GARVs to generate a more accurate picture of GARV genotypes in South Korea (Zaberezhny et al., 1994) .

In this study, we isolated 17 G8 GARVs (17.3%) in combination with P[1] and P[7] across South Korea, which were ranked the second most frequently detected G and P types, indicating that these strains may be prevalent throughout South Korea. The discovery of these G8 GARVs is important to the swine industry, veterinary practitioners, and GARV vaccine producers in South Korea. It should be noted that the serotype G8 is one of the major bovine serotypes in combination with P[5] and P[1] genotypes (Alfieri et al., 2004; Chang et al., 1996; Fukai et al., 2004; Gentsch et al., 1992) . In addition, G8 GAVR serotype has been detected in rare cases in humans (Adah et al., 2001; Cunliffe et al., 1999; Fischer et al., 2003; Matthijnssens et al., 2006; Palombo et al., 2000; Steele et al., 1999) , and pigs . Among the G8 GARVs, one strain contained bovine-like P[1]-VP4 gene, indicating bovine-like G8P[1] strains can infect heterologous species in nature, such as pigs. The remaining 16 G8 strains contained the porcine-like P[7]-VP4 gene. This result implies that reassortant events between porcine and bovine GARVs occur in nature. In previous reports (Ha et al., 2009; Park et al., unpublished data) , we demonstrated that reassortant GARVs between bovine and porcine, and heterologous GARVs whose 11 genome segments are of pig origin infect calves and induce diarrhea. Therefore, interspecies transmission of GARVs between bovine and porcine, either as whole virions or by gene segment reassortment, appear to occur in nature at a relatively high frequency in South Korea.

Since G9 GARV was first detected in a child with gastroenteritis in the United States in 1983 (Clark et al., 1987) and subsequently in other countries (Das et al., 1993; Nakagomi et al., 1990; Urasawa et al., 1992; Zizdić et al., 1992) , G9 GARVs have not been reported in humans for a decade. From mid-1990s, G9 GARVs reemerged and efficiently spread throughout the world as the fifth globally important serotype (Ramachandran et al., 2000; Santos and Hoshino, 2005) . Recently, G9 GARVs have been classified into I-VI lineages, with I-II consisting of strains isolated in the 1980s, and III-VI composing of strains isolated from the mid-1990s (Phan et al., 2007) . Of these, lineages III and VI were found in both humans and pigs (Phan et al., 2007) . In the present study, G9 GARVs were isolated and identified as the third most important genotype in the diarrheic pigs. All Korean strains were clustered in lineage VI of known porcine and human G9 GARVs. Thus, continuous genotypic characterization of the GARVs and cautions against the increase of the G9 is necessary in South Korea. Moreover, human G9 GARV infections belonging to lineage III have been emerging in South Korea since 2002 (Kang et al., 2005) , meaning that Korean porcine G9 GARVs are different from Korean human G9 GARVs.

Human GARVs showed a striking seasonal pattern of infection in developed countries, with epidemic peaks occurring in the cooler months of each year (Estes and Kapikian, 2007) . This may be related to the influence of low relative humidity as a factor facilitating the survival of GARVs on surfaces (Brandt et al., 1982) . Studies describing the seasonal pattern of porcine GARVs in diarrheic piglets have rarely been published, and those published data varied widely Svensmark et al., 1989) . In one of the few comparable studies, the seasonal curves of porcine GARV infections were highest in winter and the slightly higher in late summer and early autumn in Iowa, USA . In contrast, Danish porcine GARV infections showed a slight increase during the autumn (Svensmark et al., 1989) . In this study, however, porcine GARVs occurred throughout the year with the highest prevalence during the summer months. The reason for the seasonal pattern variations around the world is not yet known. Therefore, more intensified epidemiological studies throughout the world will be needed to fully understand the seasonal pattern of porcine GARV infections and to establish porcine GARV surveillance programs to prevent infections.

In summary, this study demonstrates that porcine GARV infections are epidemic and widespread in diarrheic piglets in South Korea. The infecting strains are genetically diverse, and include homologous (G5P[7]), heterologous (G8P[1]), and reassortant (G8P[7]), as well as emerging G9 GARV strains.

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This study was supported by the National Veterinary Research and Quarantine Services (NVRQS), Ministry of Agriculture and Forestry, the Korea Science and Engineering Foundation (KOSEF) grant (2009-0081752), and the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy (MOCIE), Republic of Korea. The authors would like to acknowledge a graduate fellowship from the Korean Ministry of Education and Human Resources Development through the Brain Korea 21 project.