Sang-Ik Park

Molecular epidemiology of bovine noroviruses in South Korea

Abstract Since the prevalence of bovine norovirus (BNoV) and their genetic diversity have only been reported in the USA, England, Germany and The Netherlands, this study examined the prevalence and genetic diversity of BNoVs in diarrheic calves in South Korea using 645 diarrheic fecal specimens from calves by RT-PCR and nested PCR assays. Overall, 9.3% of the diarrheic fecal samples tested positive for BNoVs by either RT-PCR or nested PCR, of which 5.9% samples also tested positive for other enteric pathogens including the bovine coronavirus, bovine viral diarrhea virus, bovine torovirus, bovine groups A, B and C rotaviruses, bovine enteric Nebraska-like calicivirus and Escherichia coli. The genetic diversity was determined by direct sequencing of the partial RdRp region of 12 BNoVs detected from the fecal samples by nested PCR. Among the BNoVs examined, one Korean BNoV strain had the highest nucleotide (86.8%) and amino acid (99.1%) identity with the genotype 1 BNoV (GIII-1) strain, while the remaining 11 Korean BNoVs shared a higher nucleotide (88.0–90.5%) and amino acid (93.5–99.1%) identity with the genotype 2 BNoV (GIII-2) strains. The phylogenetic data for the nucleotide and amino acid sequences also demonstrated that one Korean BNoV strain clustered with GIII-1 but the remaining eleven strains clustered with GIII-2. In conclusion, BNoV infections are endemic and there are two distinct genotypes with GIII-2 being the main genotype circulating in the calf population in South Korea.

Detection and molecular characterization of porcine group C rotaviruses in South Korea.

Abstract Group C rotaviruses (GCRVs) cause acute diarrhea in humans and animals worldwide and the evidence for a possible zoonotic role of GCRVs has been recently provided. However, there is little evidence of porcine GCRV infections or of their genetic diversity in South Korea. We examined 137 diarrheic fecal specimens from 55 farms collected from six provinces. RT-PCR utilizing primer pairs specific for the GCRV VP6 gene detected GCRV-positive reactions in 36 (26.2%) diarrheic fecal samples. Of these, 17 samples (12.4%) tested positive for porcine GCRVs alone and 19 samples (13.8%) were also positive for other pathogens. Other enteric pathogens except for GCRV were detected in 64 feces samples (46.7%) and no enteric pathogens were evident in 37 feces samples (27.0%). Phylogenetic and sequence homology analyses of GCRV partial VP6 gene between 23 Korean and other known porcine GCRVs demonstrated that Korean strains belonged to the porcine lineage. Furthermore, one Korean porcine strain shared the highest nucleotide (89.7–89.0%) and deduced amino acid sequence (92.9–93.9%) identities with bovine GCRV strains and was placed in the bovine GCRV lineage indicative of bovine origin. In conclusion, porcine GCRV infections are widespread in piglets with diarrhea in South Korea. The infecting porcine GCRVs mostly belong to the porcine lineage with the exception of one bovine-like GCRV, which possibly originated from bovine GCRV due to interspecies transmission.

Detection and Characterization of Bovine Coronaviruses in Fecal Specimens of Adult Cattle with Diarrhea during the Warmer Seasons

ABSTRACT Bovine coronavirus (BCoV) is an etiological agent associated with winter dysentery (WD), prevalent in adult cattle during the winter. Although we previously detected, isolated, and characterized BCoV strains from adult cattle with WD (WD-BCoV strains) during the winter in South Korea, the precise epidemiology, as well as the causative agent of diarrhea in adult cattle in the warmer seasons, has not been examined. We examined 184 diarrheic fecal specimens collected from 75 herds of adult cattle from seven provinces during the spring (warm), autumn (warm), and summer (hot) seasons. Bovine coronavirus-positive reactions were detected for 107 (58.2%) diarrheic fecal samples (in 47/75 herds). Of these 107 positive samples, 90 fecal samples from 33 herds tested positive for BCoV alone and 17 fecal samples from 14 herds also tested positive for other pathogens. Biological comparisons between the 9 BCoV strains isolated in this study and the 10 previously isolated WD-BCoV strains revealed that there was no receptor-destroying enzyme (RDE) activity against mouse erythrocytes in the 9 BCoV strains but the 10 WD-BCoV strains had high RDE activity. Phylogenetic analysis of the spike (S) and hemagglutinin/esterase (HE) proteins revealed that all the Korean BCoVs clustered together regardless of season and were distinct from the other known BCoVs, suggesting a distinct evolutionary pathway for the Korean BCoVs. These and previous results revealed a high prevalence and widespread geographical distribution of BCoV, suggesting that this virus is endemic in adult cattle with diarrhea in all seasons in South Korea.

Molecular Characterization of Novel G5 Bovine Rotavirus Strains

ABSTRACT Group A rotaviruses are a major cause of acute gastroenteritis in young children as well as many domestic animals. The rotavirus genome is composed of 11 segments of double-stranded RNA and can undergo genetic reassortment during mixed infections, leading to progeny viruses with novel or atypical phenotypes. The aim of this study was to determine if the bovine group A rotavirus strains KJ44 and KJ75, isolated from clinically infected calves, share genetic features with viruses obtained from heterologous species. All 11 genes sequences of the KJ44 and KJ75 strains were sequenced and analyzed. The KJ44 VP4 had 91.7% to 96.3% deduced amino acid identity to the bovine related P[1] strain, whereas the KJ75 strain was most closely related to the bovine related P[5] strain (91.9% to 96.9% amino acid identity). Both KJ44 and KJ75 strains also contained the bovine related VP3 gene. The remaining 9 segments were closely related to porcine group A rotaviruses. The KJ44 and KJ75 strains showed high amino acid identity to the G5 rotaviruses, sharing 90.4% to 99.0% identity. In addition, these strains belonged to the NSP4 genotype B, which is typical of porcine rotaviruses and subgroup I, with the closest relationship to the porcine JL-94 strain. These results strongly suggest that bovine rotavirus strains with the G5 genotype occur in nature as a novel G genotype in cattle as a result of a natural reassortment between bovine and porcine strains.

Reassortment among bovine, porcine and human rotavirus strains results in G8P[7] and G6P[7] strains isolated from cattle in South Korea

Group A rotaviruses (GARVs) cause severe acute gastroenteritis in children and young animals. Although zoonotic infections with bovine-like G6 and G8 GARVs have been reported in many countries, there is little evidence for reassortment between bovine GARVs and GARVs from heterologous species. The finding of bovine GARVs with the G6 and G8 genotypes in combination with the typical porcine P[7] prompted us to characterize all 11 genes of 30 bovine GARVs isolated from clinically infected calves. By the comparison of the full-length ORF of VP7 and NSP1-5, and the partial VP1-4 and VP6 nucleotide sequences between the 30 Korean and other known strains, three different genome constellations were found. Twenty seven strains showed the G8-P[7]-I5-R1-C1-M2-A1-N1-T1-E1-H1 genotypes, a single strain possessed the G6-P[7]-I2-R2-C1-M2-A1-N2-T1-E2-H1 genotype constellation and 2 strains the G6-P[7]-I2-R2-C2-M2-A3-N2-T6-E2-H3 genotype constellation. The complete genome of a single reference strains for each of these three genotype constellations (KJ25, KJ9-1 and KJ19-2) was determined and analyzed. A detailed phylogenetic analysis revealed a complicated picture, with several reassortments among bovine-like, porcine-like and human-like GARV strains, resulting in several different reassortant strains successfully infecting cattle.

Detection and genotyping of Korean porcine rotaviruses.

Abstract 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.

Full-length genomic analysis of porcine G9P[23] and G9P[7] rotavirus strains isolated from pigs with diarrhea in South Korea.

Group A rotaviruses (RVAs) are agents causing severe gastroenteritis in infants and young animals. G9 RVA strains are believed to have originated from pigs. However, this genotype has emerged as the fifth major human RVA genotype worldwide. To better understand the relationship between human and porcine RVA strains, complete RVA genome data are needed. For human RVA strains, the number of complete genome data have grown exponentially. However, there is still a lack of complete genome data on porcine RVA strains. Recently, G9 RVA strains have been identified as the third most important genotype in diarrheic pigs in South Korea in combinations with P[7] and P[23]. This study is the first report on complete genome analyses of 1 G9P[7] and 3 G9P[23] porcine RVA strains, resulting in the following genotype constellation: G9-P[7]/P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1. By comparisons of these genotype constellations, it was revealed that the Korean G9P[7] and G9P[23] RVA strains possessed a typical porcine RVA backbone, similar to other known porcine RVA strains. However, detailed phylogenetic analyses revealed the presence of intra-genotype reassortments among porcine RVA strains in South Korea. Thus, our data provide genetic information of G9 RVA strains increasingly detected in both humans and pigs, and will help to establish the role of pigs as a source or reservoir for novel human RVA strains.

Salmonella enterica Serovar Gallinarum Requires ppGpp for Internalization and Survival in Animal Cells

To elucidate the pathogenic mechanism of Salmonella enterica serovar Gallinarum, we examined the expression of the genes encoded primarily in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. These genes were found to be induced as cultures entered stationary phase under high- and low-oxygen growth conditions, as also observed for Salmonella serovar Typhimurium. In contrast, Salmonella serovar Gallinarum in the exponential growth phase most efficiently internalized cultured animal cells. Analysis of mutants defective in SPI-1 genes, SPI-2 genes, and others implicated in early stages of infection revealed that SPI-1 genes were not involved in the internalization of animal cells by Salmonella serovar Gallinarum. Following entry, however, Salmonella serovar Gallinarum was found to reside in LAMP1-positive vacuoles in both phagocytic and nonphagocytic cells, although internalization was independent of SPI-1. A mutation that conferred defects in ppGpp synthesis was the only one found to affect animal cell internalization by Salmonella serovar Gallinarum. It was concluded that Salmonella serovar Gallinarum internalizes animal cells by a mechanism independent of SPI-1 genes but dependent on ppGpp. Intracellular growth also required ppGpp for the transcription of genes encoded in SPI-2.

Genetic diversity of porcine sapoviruses.

Abstract Sapoviruses (SaVs) within the Caliciviridae family are an important cause of gastroenteritis in both humans and animals. Although the widespread occurrence of divergent human SaV strains has been reported, there have only been a few studies of porcine SaVs examining their genetic diversity. The aim of this study was to assess the genetic diversity of porcine SaVs in piglets with diarrhea in South Korea. Two hundred and thirty-seven fecal specimens from piglets with diarrhea were examined from 78 farms over a 2-year period from six provinces in South Korea. Overall, 69 (29.1%) of the samples from five provinces tested positive for porcine SaVs by either RT-PCR or nested PCR with the primer pairs specific to porcine SaVs. An analysis of the partial capsid gene (757bp) of 12 porcine SaVs detected from fecal samples showed genetic divergence, not only among the Korean porcine SaVs (67.7–99.1%), but also between Korean and American porcine SaVs (69.1–90.2%). Interestingly, one strain (Po/SaV/JN-MA113/05/Korea), formed a second porcine SaV/GIII genotype, and is tentatively called GIII/2. This strain had a 0.236–0.405 inter-cluster distance with the other strains in the same genogroup, which is comparable to the distances between the established GI and GII SaVs. Furthermore, two potential recombinant porcine SaVs were identified. In conclusion, porcine SaV infections are common in diarrheic piglets in South Korea. The infecting strains are genetically diverse, and include a newly recognized genotype and recombinant viruses within genogroup III.

Molecular detection and characterization of unclassified bovine enteric caliciviruses in South Korea

Abstract The unclassified bovine enteric calicivirus (BEC) is a new bovine enteric calicivirus that is different from bovine norovirus, and causes diarrhea and pathologies in the small intestine of calves. This virus includes Nebraska (NB)- and Newbury agent 1 (NA1)-like strains. The prevalence of this BEC and its genetic characterization has only been reported in the UK and the USA. This study examined the prevalence and genetic diversity of these BECs in diarrheic calves in South Korea. Among a total of 645 diarrheic fecal specimens obtained from 629 cattle herds, these unclassified BECs were detected in 59 (9.1%) diarrheic fecal samples from 57 herds (9.3%) by either RT-PCR or nested PCR. Sequence and phylogenetic analyses of the partial RdRp gene showed that all the Korean BECs clustered together and were closely related to the NB-like viruses (80.9–88.1% nucleotide and 84.5–98.4% amino acid) but not to the NA1-like viruses (75.8–78.4% nucleotide and 79.7–82.8% amino acid). Although these viruses could not be classified into NA1- and NB-like viruses from the sequence and phylogenetic data of the entire capsid gene, all the Korean BECs clustered together on a branch separate from the other known BECs. These results show that these BEC infections are endemic in diarrheic calves in South Korea. The infecting strains are genetically closer to the NB-like viruses but have a distinct evolutionary pathway.