ABSTRACT
In this study, we isolated 12 of Brucella (B.) spp. from cattle, which have been positive in Rose Bangal test and tube agglutination test in Gyeongbuk province in 2009. According to AMOS PCR analysis, isolated 12 strains were identified as B. abortus. Murine derived macrophage, RAW 264.7 cells, were infected with isolated 12 strains or reference strain (B. abortus 544), and bacterial internalization were characterized. According to these results, we divided the isolated strains into the following three groups: class I, lower internalization than that of B. abortus 544; class II, similar internalization to that of that of B. abortus 544; class III, higher internalization than that of B. abortus 544 within RAW 264.7 cells. Furthermore, intracellular growth, bacterial adherent assay, LAMP-1 colocalization, virulence in mice and surface protein pattern were characterized. From these results, representative strains of class III showed lower LAMP-1 colocalization, higher adherent efficiency, higher virulence in mice than those of B. abortus 544, and showed different pattern of surface proteins. These results suggest that B. abortus field strains, isolated from cattle in Korea, possess various virulence properties and higher internalization ability of field strain may have an important role for its virulence expression.
Subject(s)
Animals , Cattle , Mice , Agglutination Tests , Brucella , Brucella abortus , Korea , Macrophages , Membrane Proteins , Phagocytes , Polymerase Chain Reaction , Sprains and StrainsABSTRACT
During 3 years surveillance (January 2001 through December 2003) for acute gastroenteritis in human in Daejeon region, 432 out of 4,869 stool samples were selected as rotavirus-positive specimens by means of antigen-capture enzyme-linked immunosorbent assay (ELISA). The P (VP4) and G (VP7) genotypes for 432 stool samples were investigated by reverse transcription polymerase chain reaction (RT-PCR) and nested multiplex PCR. The most prevalent P subtype was P[8] (44.9%), followed by P[4] (25.7%) and P[6] (17.1%). No cases for P[10] and P[9] subtypes were found through the study. In G subtyping, G1 (53.2%) was the most frequently found G type, followed by G2 (23.1%), G3 (9.5%), G4 (6.7%), and G9 (0.9%). The order of detection rates for G2, G3 and G4 was variable by years. The most common G- and P- type combination found in this study was G1P[8] (33.1%), followed by G2P[4] (20.4%), G1P[6] (10.0%), G3P[8] (7.2%) and G4P[6] (4.2%). The mixed types of G and P were observed most frequently in P[8] (1.4%) and G1 (3.2%), respectively. This is the first molecular epidemiological study for Group A rotavirus in Daejeon region. The results might be useful data for evaluating the epidemiological status of rotaviral diarrhea in the region.
Subject(s)
Humans , Diarrhea , Enzyme-Linked Immunosorbent Assay , Epidemiologic Studies , Gastroenteritis , Genotype , Multiplex Polymerase Chain Reaction , Polymerase Chain Reaction , Reverse Transcription , RotavirusABSTRACT
Recombinant DNA vaccines, based on plasmid vectors expressing an antigen under the control of a strong promotor, have several advantages over traditional vaccines. They have been shown to induce a full spectrum of immune responses for humoral and cellular systems and to secure the higher safety and the simplicity of administration. Thus, establishment of DNA vaccines against Newcastle disease virus (NDV) in poultry has been widely investigated using various virus strains and vector systems. In this study, the F and HN genes of NDV CBP-1 strains isolated from diseased pheasants and attenuated by serial passages in egg embryos were cloned using pSLIA vector and constructed two recombinants of pSLIA-tsF and pSLIA-tsHN. The recombinant plasmids were transfected into COS-7 cell and the expression of HN and F proteins were verified by immunofluorescence, SDS-PAGE and Western blot. The recombinant plasmids were injected intramuscularly and intradermally into C57B/6 mouse and a significant increment of HN and F antibodies was detected by ELISA. According to the results, it was implicative that the recombinant DNA could be utilized for development of recombinant DNA vaccine for NDV.
Subject(s)
Animals , Mice , Antibodies , Blotting, Western , Clone Cells , COS Cells , DNA, Recombinant , Electrophoresis, Polyacrylamide Gel , Embryonic Structures , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Newcastle disease virus , Newcastle Disease , Ovum , Plasmids , Poultry , Serial Passage , Vaccines , Vaccines, DNAABSTRACT
Porcine circovirus type 2 (PCV-2) has been nowadays recognized as a major agent causing postweaning multisystemic wasting syndrome (PMWS) in pigs worldwide. PMWS most commonly affects the weaned piglets, being of increasing importance to the pig industry in Korea. Seven commercial farms affected with PMWS and 2 farms free from PMWS, located in the southern part of Gyeonggi province, were selected for this study. The peripheral mononuclear cells were tested for the presence of ORF2 gene by PCR, and 54 (68.4%) of 79 samples were positive. All of 9 herds tested included the positive cases. The positive rates by herds were 50 to 100% in the PMWS-affected herds and 40 to 62.5% in the PMWS-free herd. The nucleotide and amino acid sequences of ORF2 gene of 6 strains were characterized. Homologies among 6 strains revealed 92.1 to 100% in the nucleotide and 92.3 to 100% in the amino acid. The overall ranges of homologies for 25 strains comprised 2 Korean and 23 foreign strains were 91.1 to 100% in the nucleotide and 89.7 to 100% in the amino acid. Three regions of greater heterogeneity were found in immunorelevant epitopes of the capsid protein, and the sequences between 57 to 80 aa revealed higher mutation than other areas. In the phylogenetic tree analysis, KOR 71 strain was clustered together with Korean strains previously isolated in Korea. The remaining 5 strains were closely clustered with other European and Asian strains. The results will be valuable for improving our understanding of the molecular epidemiology of PCV-2 in Korea and development of preventive measures for PMWS.
Subject(s)
Humans , Amino Acid Sequence , Asian People , Capsid Proteins , Circovirus , Epitopes , Korea , Molecular Epidemiology , Polymerase Chain Reaction , Population Characteristics , Swine , Wasting SyndromeABSTRACT
The hemagglutinin-neuraminidase (HN) gene of a thermostable Newcastle disease virus isolated from the diseased pheasants in Korea was cloned using Baculovirus transfer vector system, constructing pVL-NDHN inserted with HN gene (1.75 kbp). The HN recombinant baculovirus was generated in Sf-9 cells by co-transfection with pVL-NDHN and linearized baculovirus DNA. The Sf-9 cells infected with the recombinant baculovirus showed the hemagglutinating activity for chicken erythrocytes, and specific positive reactions in indirect immunofluorescence and indirect dot immunoassay. By SDS-PAGE and Western blot analysis, the expressed HN protein with the size of 74 kDa was detected in the cells infected with the recombinant baculovirus. To evaluate the immunogenicity of expressed HN protein, the chicken inoculated with the lysates of the Sf-9 cells were examined by hemagglutination inhibition and ELISA tests. The substantial levels of antibody responses were detected in both assays. The HN protein expressed in baculovirus recombinant system could be utilized for the development of diagnostic measures for Newcastle disease in poultry, and these results on HN recombinant baculovirus will expedite the development of recombinant ND vaccines.
Subject(s)
Animals , Antibody Formation , Baculoviridae , Blotting, Western , Chickens , Clone Cells , Cloning, Organism , DNA , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Erythrocytes , Fluorescent Antibody Technique, Indirect , Hemagglutination , HN Protein , Immunoassay , Korea , Newcastle disease virus , Newcastle Disease , Poultry , VaccinesABSTRACT
No abstract available.
Subject(s)
Animals , Baculoviridae , Newcastle disease virus , Newcastle DiseaseABSTRACT
No abstract available.
Subject(s)
Animals , Base Sequence , Classical Swine Fever Virus , Classical Swine Fever , Cloning, Molecular , SwineABSTRACT
An attenuated classical swine fever virus (CSFV), Suri strain, is a va.iant derived from a vaccine virus, LOM strain. This study was performed to elucidate the molecular biologcal properties of CSFV Suri strain, and to obtain the basic data for molecular epidemiological approaches for the disease. The truncated form of gp55 gene without the C-terminal transmembrane domain, in size of 1,023bp, was amplified by RT-PCR and sequenced by dye terminator cyclic sequencing method, and inserted into BamHI site of pAcGP67B baculovirus vector, establishing a cloned pAcHEG plasmid. By the nucleotide sequences determined, 341 amino acid sequences were predicted. As compared the nucleotide and amino acid sequences of gp55 of Suri with the various CSFV, Suri strain showed the high homology over 99.1% with ALD and LOM strains, but comparably the lower homology with Alfort and Brescia. In comparison of amino acid sequence in variable domain of gp55 protein, the similar tendency of homology was observed. In hydrophobicity analysis, all of four CSFV strains revealed the analogous patterns of hydrophobicity. The numbers and locations of N-glycosylation site and cysteine residues in gp55 were analyzed, those of Suri strain being coincident with ALD and LOM strains. The results suggest that gp55 in Suri strain has the high similarity to those in ALD and LOM strains in terms of the nucleotide and amino acid sequences and the functional properties of gp55 protein..
Subject(s)
Animals , Amino Acid Sequence , Baculoviridae , Base Sequence , Classical Swine Fever Virus , Classical Swine Fever , Clone Cells , Cysteine , Hydrophobic and Hydrophilic Interactions , Plasmids , Sequence Analysis , SwineABSTRACT
The gene encoding F protein of CBP-1 strain, a heat-stable Newcastle disease virus (NDV) isolated from the diseased pheasants in Korea, was characterized by reverse transcription-polymerase chain reaction (RT-PCR), nucleotide and amino acid sequences. Virus RNA was prepared from the chorioallatoic fluid infected with NDV CBP-1 virus and cDNA was amplified by RT-PCR, cloned and sequenced to analyze. The PCR was sensitive as to detect the virus titer above 25 hemagglutination unit. 1.7kb (1,707bp) size of the cDNA was amplified and cloned into BamHI site of pVL1393 Baculo transfer vector. The nucleotide sequences for F protein were determined by dye terminator cyclic sequencing using four pairs of primers, and 553 amino acid sequences were predicted. In comparison of the nucleotide sequence of F gene of CBP-1 with those of other NDV strains, the homology revealed 88.8%, 98.5% and 98.7% with Kyojungwon (KJW), Texas GB and Beaudette C strains, respectively. As the deduced 553 amino acid sequences of F protein of CBP-1 were compared with those of other NDV strains, the homology appeared 89.9%, 98.7% and 98.9% with KJW, Texas GB and Beaudette C strains, respectively. The putative protease cleavage site (112-116) was R-R-Q-K-R, indicating that CBP-1 strain is velogenic type. The amino acid sequences include 6 sites of N-asparagine-linked glycosylation and 13 cysteine residues. These data indicate that the genotype of CBP-1 strain is more closely associated with the strains of Texas GB and Beaudette C than KJW strain.
Subject(s)
Animals , Amino Acid Sequence , Base Sequence , Clone Cells , Cloning, Molecular , Cysteine , DNA, Complementary , Genotype , Glycosylation , Hemagglutination , Korea , Newcastle disease virus , Newcastle Disease , Polymerase Chain Reaction , RNA , Texas , Viral LoadABSTRACT
PCR implication using the primers for gag, pol and rev genes in BLV (bovine leukemia virus) proviral DNA and syncytium assay were carried out for the Korean native goats experimentally infected with bovine leukemia virus to investigate pathogenesis of BLV in the goats, and to establish a model animal for BLV infection. The oligonucleotide primers used in PCR revealed very high specificity, The minimal amount of FLK-BLV cellular chromosomal DNA to detect the integrated BLV proviral DNA was 10 ng. The peripheral blood lymphocytes from the goat infected with BLV were examined at regular intervals by PCR amplification and syncytium assay. Pol or gag genes were detected in none of three infected goats at the 1st week post-infection (p.i.). At the 4th week p.i., one of three goats showed the amplified gag gene. Thereafter detection rates for the genes were increased, indicating that the BLV proviral genes were integrated in all of the lymphocytes from three goats, at the 16th weeks p.i., when it was evident in syncytium assay that the lymphocytes from all of three goats were infested with infective BLV. Investigating the tissues from the necropsied goats at the 8th month p.i., the amplified BLV proviral genes and infective BLV were detected in all of the peripheral lymphocytes from three infected-goats. Among various tissues examined, the amplified BLV proviral genes were observed in spleen and superficial cervical, mandibular and retropharyngeal lymph nodes, and the infective BLV, in superficial cervical and mandibular lymph nodes. It was assumed that the Korean native goat was quite susceptible to BLV infection, indicating that the goat could be a good model animal for BLV.
Subject(s)
Animals , Cattle , Deltaretrovirus Infections , DNA Primers , DNA , Enzootic Bovine Leukosis , Genes, gag , Genes, rev , Giant Cells , Goats , Leukemia , Leukemia Virus, Bovine , Lymph Nodes , Lymphocytes , Polymerase Chain Reaction , Sensitivity and Specificity , SpleenABSTRACT
No abstract available.