Histopathological Evaluation of the Efficacy for Plant-produced E2 Protein Vaccine against Classical Swine Fever Virus (CSFV) in Piglets

Classical swine fever (CSF), previously known as hog cholera, remains one of the most important swine-related contagious diseases worldwide. In order to eradicate classical swine fever virus (CSFV), it is commonly used in LOM-850 strain as a live attenuated CSF vaccine. However, there are symptoms of vaccination, such as the depression of feed intake, and difficulty of differentiation between infected and vaccinated hosts is impossible based on the antibodies induced. Nicotiana benthamiana were considered as an alternative to the production of recombinant vaccines on account of higher yields and levels of soluble protein than other models and crops in protein recombinant products. This study was conducted to evaluate histopathological validation of the plant-produced E2 fusion protein (ppE2) in piglets. The piglets were challenged by an injection of YC11WB strain in 7 days, 11 days and 14 days after one shot of the vaccination. The histopathological examination indicated that ppE2 can protect against lethal CSFV challenge at least 11 days of vaccination in piglets. These data suggest that the ppE2 can be an effective vaccine against CSFV in piglets.

Apolipoprotein E Genotype and Expression Correlated with Hepatitis C Virus Genotype and Infection

The hepatitis C virus (HCV) is a globally prevalent human pathogen that causes persistent liver infections in most infected individuals. Several studies reported that HCV particles are enriched in apolipoprotein E (apoE) and that apoE is required for HCV infectivity and production. However, the relationship between apoE gene polymorphisms and HCV genotypes in patients with HCV is less well understood. The aim of this study was to investigate the association between apoE gene polymorphism and HCV genotypes in patients. The HCV genotypes were identified among the 124 patients infected with HCV, and the genetic characteristics of the HCV genotype were analyzed. In addition, the results of the clinical laboratory test were comparatively analyzed according to the classified genotypes. Both HCV 1b (n=80) and 2a (n=42) patients had higher AFP, AST, ALT, ALP, γ-GTP, apoB, and apoE values compared with the normal control group. In particular, apoB and apoE levels were statistically significantly higher in the HCV 2a patients (P<0.05) and apoE levels were significantly higher in the HCV 1b patients (P<0.000). According to the results the patients with HCV genotype 1b showed higher values of liver damage related indicators and apoB expression than the patients with HCV genotype 2a. The fat related indicators and apoE expression were not different between the two major HCV genotypes (2a and 1b). We anticipate that the apoE ε3 allele is the most common type in HCV genotype 1b (89.2%) and 2a (91.7%). As a result of apoE genotyping, we confirmed an association with HCV infection and the apoE ε3 allele. However, the ratios of the apoE ε3 allele among the patients with genotype 1b and 2a were similar to each other.

Differences in Hematological Characteristics, Including Cholesterol and Apolipoprotein B and E, between Hepatitis B Virus and Hepatitis C Virus Patients in Korea

Hepatitis B virus (HBV) and hepatitis C virus (HCV) chronically cause hepatitis, liver cirrhosis, and hepatocellular carcinoma, and biomarkers related to liver damage are elevated in HBV and HCV patients. However, comparisons of biomarkers between HBV and HCV patients have not previously been reported. The aim of this study was to investigate differences in hematological biomarker in the sera of HBV and HCV patients and to find a key biomarker to differentiate between HBV and HCV infections. HBV (n=115) and HCV (n=128) samples (serum and whole blood) were collected and tested using a biochemical analysis system. The obtained data were analyzed with SPSS 18.0 statistical software. The mean age of the HCV group (60.3±14.1) was much higher than that of the HBV group (51.1±12.4). Male and female rates were 71.3% and 28.7% in the HBV group and 53.9% and 46.1% in the HCV group, respectively (p = 0.005). AST, ALT, and TG values were higher in the HCV group than in the HBV group. Although γ-GTP and LDL levels were higher in the HBV group than in the HCV group, apoB and apoE levels were much higher in HCV group than in HBV group (p < 0.001). There were no significant differences in the other hematological biomarkers between the HBV and HCV groups. In conclusion, HBV rates were higher in male patients, and HCV rates were higher in older patients. In particular, apoE and apoB were more highly expressed in HCV patients, and they might be key markers to differentiate HCV infection.

HMG CoA Reductase Inhibitors Inhibit HCV RNA Replication of HCV Genotype 1b but Not 2a

Replication of hepatitis C virus (HCV) is regulated by statin, one of 3-hydroxy-3-methylglutaryl CoA reducatase (HMG CoA reductase) inhibitors that block mevalonate pathway and cholesterol biosyntheis, which has been used usefully for health improvement and disease control in clinic. In order to know which statin can be used to inhibit HCV replication, we examined the effects of HCV genotype 1b replication by 6 kinds of statins with different structure. We treated six statins to HCV genotype 1b replicon cell. Atorvastatin, simvastatin, fluvastatin, mevastatin, and lovastatin inhibited HCV RNA replication and HCV protein expression in HCV genotype 1b replicon cells, though pravastatin did not affect HCV replication. In order to know whether inhibition of HCV replication by statin is depended on HCV genotype, we treated the statins to HCV genotype 2a producing cells, and investigated HCV RNA replication and HCV protein expression. HCV RNA replication and protein expression was not affected in HCV genotype 2a producing cells by treatment of statins and cholesterol inhibitor. These results suggest that HMG-CoA reductase and cholesterol inhibitors might be used depending on HCV genotype. In addition, inhibition of HCV genotype 1b replication by statins has been depended on structure of various statins which should be seriously selected for HCV clinic. In future, we will study on inhibition of another HCV genotype replication by HMG-CoA reductase and cholesterol inhibitors.

Genospecies Classification of Acinetobacter calcoaceticus-Acinetobacter baumannii Complex by Restriction Fragment Length Polymorphism

Acinetobacter calcoaceticus-Acinetobacter baumannii (A. calcoaceticus-A. baumannii) complex, which includes A. calcoaceticus (genospecies 1), A. baumannii (genospecies 2), Acinetobacter genospecies 3 and 13, has been identified as A. baumannii by automated bacteria identification system. The purpose of this study is to develop rapid genospecies classification of A. calcoaceticus-A. baumannii complex by molecular techniques. Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) were determined for 4 reference strains and 80 isolates of A. calcoaceticus-A. baumannii complex from clinical sources. Four and eleven RAPD patterns were observed among the reference strains and the isolates, respectively. RAPD might be useful for genomic typing but not for genospecies classification of Acinetobacter spp. RFLP of 16S-23S rRNA intergenic spacer gene with three selected restriction enzymes (ApaLI, SwaI, and SalI) showed only four RFLP patterns in the reference and the isolates. Of 80 isolates, 10 of A. calcoaceticus (12.5%), 50 of A. baumannii (62.5%), 11 of A. genospecies 3 (13.75%), and 9 of A. genospecies 13 (11.25%) were classified by RFLP. This result suggests that RFLP of 16S-23S rRNA intergenic spacer gene of A. calcoaceticus-A. baumannii complex might be useful for genospecies classification.

Correlation Between the Prevalence of Superantigenic Toxin Genes and Coagulase Serotypes of Staphylococcus aureus Isolates

A heterogenic group of staphylococcal exotoxins, including staphylococcal superantigenic toxins, enterotoxin (SE), toxic shock syndrome toxin-1 (TSST-1), and coagulase are the most important virulence factors of Staphylococcus aureus. We analyzed the prevalence of genes encoding five enterotoxins and TSST-1 in S. aureus isolated from clinical ear discharges. The genes were identified by multiplex PCR and we compared the results to references of coagulase serotypes. In 102 isolates of S. aureus, 44 of them were methicillin-resistant S. aureus (MRSA) and the others were methicillin-susceptible S. aureus (MSSA). Among both types of S. aureus, 33 strains were positive for sea, 2 for seb, 23 for sec, 26 for see, and 26 for tst. Overall, 59 (57.8%) isolates were positive for one or more superantigenic toxin genes. From these, 71.2% (42/59) strains harbored more than one toxin gene in different combinations. The major combinations of genes were sea and see, and sec and tst. The degree of possession of superantigenic toxic genes was similar in both MRSA and MSSA isolates (56.8% vs 58.6%, respectively), yet significant differences in toxin gene profiles and coagulase serotypes between two isolates were detected. All of 13 positive strains for sec and tst were MRSA and belonged to coagulase serotype II. On the other hand, 80.0% of 20 positive strains for sea and see were MSSA with coagulase serotype IV and VII, whereas 20.0% of them were MRSA with coagulase serotype IV. This data indicates that the profile of superantigenic toxin genes correlates to coagulase serotype and methicillin resistance in S. aureus isolates.

Correlation between Sau1 Restriction and Modification Complex Type and Coagulase Serotype or SCCmec Type of Staphylococcus aureus

Staphylococcus aureus coagulase serotype I to VIII isolated from clinical samples could be classified into two groups, methicillin-sensitive S. aurues (MSSA) and methicilln-resistant S. aurues (MRSA), by antibiotics susceptibility and existence of mecA which is a gene related with methicillin resistance. Coagulase serotype I, VI, and VIII were MSSA which showed different antimicrobial susceptibility. Coagluase serotype II-V and VII were MRSA in which mecA and SCCmec were detected. To analyze Sau1 restriction and modification (R-M) complex types by coagulase type and SCCmec type, sau1hsdR, sau1hsdM and sau1hsdS genes involved in Sau1 R-M complex were detected by PCR, we found five complex types such as M1, R2M2, R2M2, R2M2S1, and R2M2S2. Coagulase serotype I, VI, and VIII of MSSA were M1, R2M2 and R2M2, respectively. SCCmec type II and coagulase serotype II, SCCmec type III and coagulase serotype III, SCCmec type IV and coagulase serotype V, and SCCmec type IV and coagulase serotype IV, VII of MRSA were Sau1 R-M complex type R2M2S1, R2M2, R2M2, and R2M2S2, respectively. Taken together, correlation between Sau1 R-M complex types and coagulase or SCCmec types of S. aureus was found.

Characterization of Vancomycin-Resistant Enterococci Isolated from Stools and Their Acquisition of Vancomycin Resistance

We have isolated 6 vancomycin resistant (VR) Enterococcus faecium and 5 VR-E. gallinarum. Vancomycin resistant enterococcus (VRE) isolates were resistant to multi-drugs, but susceptible to linezolid and quinupristin/dalfopristin. VRE isolates showed 10 VanA phenotypes and 1 VanB phenotype (E. gallinarum). However, all of them showed vanA genotype. vanA gene was detected on both genomic and plasmid DNA from all VRE isolates. Almost of VR-E. faecium had IS1216V which is worldwide type and almost of VR-E. gallinarum had IS1542 which is European type. IS1216V and IS1542 genes were not related with antibiotic types of VRE. Copy numbers of vanA were decreased in VRE with IS1216V or IS1542 but not in VRE with both ISs in broth without vancomycin. The copy numbers of vanA were significantly decreased in VanB phenotype of VRE with IS1542 in broth without vancomycin. Copy numbers of vanA were recovered in the presence of vancomycin. Growth time of reference E. faecium is faster than that of reference E. faecalis when cultured in the broth containing vancomycin. Reference strains cultured in the broth containing vancomycin showed intermediate resistance or resistance to antibiotics without acquisition of van genes. Naturally, multidrug-resistant E. faecium might be fast adapted in the presence of vancomycin compared to E. faecalis. Taken together, VanA phenotype E. gallinarum as well as E. feacium have been increasing in nosocomial infection and showed acquired inducible resistance. E. faecium and E. faecalis showed intermediate resistance in long exposure of vancomycin without acquisition of vanA.

Correlation Between Staphylococcal Cassette Chromosome mec Type and Coagulase Serotype of Methicillin-Resistant Staphylococcus aureus

Staphylococcal cassette chromosome mec (SCCmec) type and coagulase serotype are important epidemiologic factors in methicillin-resistant Staphylococcus aureus (MRSA). To investigate correlation between SCCmec type and coagulase serotype of MRSA, we analyzed SCCmec types of MRSA strains isolated from clinical sources and compared the results to coagulase serotypes and antimicrobial susceptibility patterns. A total of 108 MRSA isolates were classified into four SCCmec types: II (55.6%), IV (21.3%) III (13.0%) and IIIA (8.3%), and five coagulase serotypes: II (54.6%), IV (21.3%), V (18.5%) and VII (2.8%). All of coagulase type II, IV and V strains belonged to SCCmec type II, III/IIIA and IV, respectively. SCCmec types II, III and IIIA were multidrug resistant, whereas SCCmec type IV strains were non-multidrug resistant except beta-lactams and erythromycin. The data provide that there is a significant correlation between SCCmec types and phenotypic characteristic of coagulase serotypes.

Cloning and Expression of Hemagglutinin-Neuraminidase Gene of a Thermostable Isolate of Newcastle Disease Virus by Baculovirus Recombinants

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.

Expression of F Protein Gene of a Thermostable Isolate of Newcastle Disease Virus Using Baculovirus Expression System

No abstract available.

Molecular Cloning and Nucleotide Sequence of the Gene Encoding Gp44 Protein of Suri strain: an Attenuated Classical Swine Fever Virus

No abstract available.

Molecular Cloning and Nucleotide Sequence of the Gene Encoding Fusion(F) Protein of the Thermostable Newcastle Disease Virus Isolated from a Diseased Pheasant

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.

Detection of BLV Proviral DNA in Korean Native Goats Experimentally Infected with Bovine Leukemia Virus by Polymerase Chain Reaction

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.

Infectivity of bovine leukemia virus to Korean native goats I. antibody responses and syncytium assay for Korean native goats experimentally infected with bovine leukemia virus

No abstract available.