Noninfectious Virus-Like Particle Antigen for Detection of Swine Vesicular Disease Virus Antibodies in Pigs by Enzyme-Linked Immunosorbent Assay

An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits. We developed a blocking ELISA using the VLPs and SVDV-specific neutralizing monoclonal antibody 3H10 (VLP-ELISA) for detection of SVDV serum antibodies in pigs. The VLP-ELISA showed a high specificity of 99.9% when tested with pig sera that are negative for SVDV neutralization (n = 1,041). When tested using sera (n = 186) collected periodically from pigs (n = 19) with experimental infection with each of three different strains of SVDV, the VLP-ELISA detected SVDV serum antibodies as early as 3 days postinfection and continued to detect the antibodies from all infected pigs until termination of the experiments (up to 121 days postinfection). This test performance was similar to that of the gold standard virus neutralization test and indicates that the VLP-ELISA is a highly specific and sensitive method for the detection of SVDV serum antibodies in pigs. This is the first report of the production and diagnostic application of recombinant VLPs of SVDV. Further potential uses of the VLPs are discussed.     

Three clusters of bovine kobuvirus isolated in Korea, 2008–2010

Fecal samples (n = 107) were collected from cattle with ascertained or suspected diarrheal disease on Korean farms during 2008–2010. Of these, 37 samples tested positive for bovine kobuvirus. The 37 positive samples came from 32 cattle that exhibited diarrhea and five cattle that were non-diarrhetic. The majority of the virus-positive feces samples were from calves under 1 month of age (n = 25). Nine of the 37 cattle infected with bovine kobuvirus were confirmed to have a co-infection with other viruses including bovine rotavirus (n = 3), bovine coronavirus (n = 1), bovine viral diarrhea virus (n = 1), and both bovine coronavirus and bovine viral diarrhea virus (n = 4). A neighbor-joining tree grouped 36 of the Korean kobuvirus strains (with the exception of the KB8 strain) into three clusters (G1, G3, and G4), while strains derived from Thailand and Japan (except the U1 strain) were included in the G2 cluster. The results indicated that Korean bovine kobuvirus has diverse lineages regardless of disease status and species.     

Identification of origin of unknown derivative chromosomes by array-based comparative genomic hybridization using pre- and postnatal clinical samples

Microarray-based comparative genomic hybridization (array CGH) is a high-resolution and comprehensive method for detecting both genome-wide and chromosome-specific copy-number imbalance. We have developed an array CGH analysis system (consisting of an array CGH chip plus its exclusive analysis software) for constitutional genetic diagnosis and have evaluated the suitability of our system for molecular diagnosis using pre- and postnatal clinical samples. In a blind study, each of the 264 sample karyotypes identified by array CGH analysis was consistent with that identified by traditional karyotype analysis – with one exception, case (47, XXX) – and we were able to identify origins, such as small supernumerary marker chromosomes, which cannot be determined by conventional cytogenetics. We also acquired very accurate, fast and reliable results using a diminutive amount of clinical samples. Taken together, the array CGH platform developed in this study is a rapid, powerful and sensitive technology for pre- and postnatal diagnosis using a very small amount of clinical sample. J. Choe, J.-K. Kang, C.-J. Bae and D.-S. Lee contributed equally to this work.     

Cross-protective efficacy of the O1 Manisa + O 3039 bivalent vaccine and the O 3039 monovalent vaccine against heterologous challenge with FMDV O/Jincheon/SKR/2014 in pig

After massive foot-and-mouth disease (FMD) outbreaks originated from Jincheon County from Dec. 2014 to Apr. 2015, the effectiveness of the previous FMD vaccine containing only the O1 Manisa as the O antigen, O1 Manisa?+?A Malaysia 97?+?Asia 1 Sharmir trivalent vaccine, was questioned in South Korea, and a change in the O antigen in FMD vaccines was demanded to control the FMD caused by FMDV O/Jincheon/SKR/2014, the O Jincheon strain. Therefore, the efficacies of O1 Manisa?+?O 3039 bivalent vaccine and O 3039 monovalent vaccine were studied for cross-protection against heterologous challenge with the O Jincheon strain. In this study, the efficacy of the O1 Manisa?+?O 3039 bivalent vaccine was better than that of the O 3039 monovalent vaccine, even though the serological relationship (r₁ value) between O Jincheon and O 3039 was matched according to the OIE Terrestrial Manual. According to serological test results from vaccinated specific pathogen free pigs, virus neutralization test titers against Jincheon were good estimates for predicting protection against challenge. A field trial of the O1 Manisa?+?O 3039 bivalent vaccine was performed to estimate the possibility of field application in conventional pig farms, especially due to concerns about the effect of maternally derived antibodies (MDA) in field application of the FMD vaccine. According to the result of the field trial, the O1 Manisa?+?O 3039 bivalent vaccine was considered to overcome MDA. The results of the efficacy and field trials indicated that the O1 Manisa?+?O3039 vaccine could be suitable to replace previous FMD vaccines to control the FMD field situation caused by O Jincheon FMDV.     

Comparison and Analysis of the Complete Nucleotide Sequence of Foot-and-Mouth Disease Viruses from Animals in Korea and Other PanAsia Strains

During the last 3 years, foot-and-mouth disease virus serotype O, named PanAsia, caused two outbreaks in the Republic of Korea. To determine if there was an obvious genetic relationship between the virus isolated in 2002 (O/SKR/2002) and the O/SKR/2000, and to further analyze the epidemiological relationships between the PanAsia viruses and the viruses identified in Korea, the complete nucleotide sequence of the O/SKR/2002 and the O/SKR/2000 were determined by automatic cycling sequencing and primer walking. The nucleotides and the deduced amino acid (aa) sequences of the strains identified in Korea were compared with each other and also those enrolled in the GenBank database. In comparison and analysis of the viruses identified in Korea, any deletions or insertions in the specific fragment gene of both the O/SKR/2002 and O/SKR/2000 were not identified. However, comparison of the aa sequence of the identified virus in 2002 from pigs with those of other PanAsia strains revealed significant substitutions of 4 aa in the VPI region and 8 aa in the 3A region. In phylogenetic analysis based on the translated region, the identified virus in 2002 appeared to be the divergence of approximately 1% degree with other PanAsia viruses. Also, animal experiments indicated that O/SKR/2000 is not host-restricted and develop the clinical signs in the main susceptible livestock species (cattle and pigs). However, O/SKR/2002 did not develop the clinical signs in cattle and showed severe clinical signs only in pigs.These analytic data suggest that 2002 outbreaks in Korea is not re-occurred but re-introduced from nowhere.     

Promoter hypermethylation of the CFTR gene and clinical/pathological features associated with non-small cell lung cancer

Background and objective: The exact role of the cystic fibrosis transmembrane conductance regulator (CFTR) in pathophysiology, and the mechanisms regulating its expression are poorly understood. The CFTR gene is known to be genetically or epigenetically associated with several cancers. In the present study, the methylation status of the promoter region of the CFTR gene and its expression in primary non‐small cell lung cancer (NSCLC) were investigated. Methods: The methylation status of the promoter region of the CFTR gene in NSCLC tissue was assessed by pyrosequencing and methylation‐specific PCR. Expression of the CFTR gene was analysed by real‐time PCR, and CFTR gene reactivation was investigated using 5‐aza‐2′‐deoxycytidine. The correlation between methylation of the CFTR gene and the clinical features of the patients was assessed. Results: Methylation of the CFTR gene in NSCLC was quantitatively high by pyrosequencing analysis and qualitatively frequent by methylation‐specific PCR analysis. Expression of the CFTR gene was significantly lower in NSCLC compared with normal lung tissue. In addition, the demethylating agent 5‐aza‐2′‐deoxycytidine increased CFTR gene expression. Methylation of the CFTR gene was significantly greater in squamous cell carcinomas than in adenocarcinomas. CFTR gene methylation was associated with significantly poorer survival in young patients, but not in elderly patients. Conclusions: These findings suggest that DNA methylation may be important for downregulation of CFTR gene expression in lung cancer. Promoter hypermethylation of the CFTR gene may be an important prognostic factor in younger patients with NSCLC.     

Phylogenetic analysis of bovine astrovirus in Korean cattle

Bovine astrovirus (BAstV) belongs to a genetically divergent lineage within the genus Mamastrovirus. The present study showed that BAstV was associated with the gastroenteric tracts of cattle in nine positive fecal samples from 115 cattle, whereas no positive samples were found in the brain tissues of 14 downer cattle. Interestingly, the positive diarrheal samples were obtained mainly from calves aged 14 days–3 months. Bayesian inference tree analysis of the partial ORF1ab and capsid (ORF2) gene sequences of BAstVs identified four divergent groups. Eleven BAstVs, four porcine astroviruses, and two deer astroviruses (DAstVs; CcAstV-1 and -2) belonged to group 1; group 2 contained two BAstVs (BAstK08–51 and BAstK10–96) with another two in group 3 (BAstK08–2 and BAstK08–53); and group 4 comprised the BAstV-NeuroS1 strain derived from a cattle brain tissue sample and an ovine astrovirus. The same divergent groups were obtained when the pairwise alignments were produced using both amino acid and nucleotide sequences. The Korean BAstVs isolated from infected cattle had a nationwide distribution and they belonged to groups 1, 2, and 3.     

Detection and genetic characterization of feline kobuviruses

In order to survey for feline kobuviruses infection, fecal samples (n = 39) of cats with diarrhea were collected during 2011–2012. Six (14.5 %) of the fecal samples tested were positive for feline kobuviruses. The partial nucleotide sequences of feline kobuviruses based on the RNA-dependent RNA polymerase gene were compared to those of other species. Feline kobuviruses were most closely related to canine kobuvirus in terms of their amino acid and nucleotide levels. In a phylogenetic tree, feline kobuviruses were also closely clustered with canine kobuvirus, Aichi virus (human), and mouse kobuvirus. This is the first report of the detection and genetic characterization of feline kobuviruses.     

Canine kobuvirus infections in Korean dogs

To investigate canine kobuvirus (CaKoV) infection, fecal samples (n = 59) were collected from dogs with or without diarrhea (n = 21 and 38, respectively) in the Republic of Korea (ROK) in 2012. CaKoV infection was detected in four diarrheic samples (19.0 %) and five non-diarrheic samples (13.2 %). All CaKoV-positive dogs with diarrhea were found to be infected in mixed infections with canine distemper virus and canine parvovirus or canine adenovirus. There was no significant difference in the prevalence of CaKoV in dogs with and without diarrhea. By phylogenetic analysis based on partial 3D genes and complete genome sequences, the Korean isolates were found to be closely related to each other regardless of whether they were associated with diarrhea, and to the canine kobuviruses identified in the USA and UK. This study supports the conclusion that CaKoVs from different countries are not restricted geographically and belong to a single lineage.     

Highly pathogenic avian influenza (H5N1) outbreaks in wild birds and poultry, South Korea

Highly pathogenic avian influenza (H5N1) among wild birds emerged simultaneously with outbreaks in domestic poultry in South Korea during November 2010-May 2011. Phylogenetic analysis showed that these viruses belonged to clade 2.3.2, as did viruses found in Mongolia, the People's Republic of China, and Russia in 2009 and 2010.