Molecular characterization and genogrouping of VP1 of aquatic birnavirus GC1 isolated from rockfish Sebastes schlegeli in Korea

The cDNA nucleotide sequence of genome segment B encoding the VP1 protein was determined for the aquatic birnavirus GC1 isolated from the rockfish Sebastes schlegeli in Korea. The VP1 protein of GC1 contains a 2,538 bp open reading frame, which encodes a protein comprising 846 amino acid residues that has a predicted MW of 94 kDa. The sequence contains 6 potential Asn-X-Ser/Thr motifs. Eight potential Ser phosphorylation sites and 1 potential Tyr phophorylation site were also identified. GC1 contains the Leu-Lys-Asn (LKN) motif instead of the typical Gly-Asp- Asp (GDD) motif found in other aquatic birnaviruses. We also identified the GLPYIGKT motif, the putative GTPbinding site at amino acid position 248. In total, the VP1 regions of 22 birnavirus strains were compared for analyzing the genetic relationship among the family Birnaviridae. Based on the deduced amino acid sequences, GC1 was observed to be more closely related to the infectious pancreatic necrosis virus (IPNV) from the USA, Japan, and Korea than the IPNV from Europe. Further, aquatic birnaviruses containing GC1 and IPNV have genogroups that are distinct from those in the genus Avibirnaviruses and Entomo-birnaviruses. The birnavirusstrains were clustered into 5 genogroups based on their amino acid sequences. The marine aquatic birnaviruses (MABVs) containing GC1 were included in the MABV genogroup; the IPNV strains isolated from Korea, Japan, and the USA were included in genogroup 1 and the IPNV strains isolated primarily from Europe were included in genogroup 2. Avibirnaviruses and entomobirnaviruses were included in genogroup 3 and 4, respectively.

Serosurveillance for Japanese encephalitis, Akabane, and Aino viruses for Thoroughbred horses in Korea

Recent global warming trends may have a significant impact on vector-borne viral diseases, possibly affecting vector population dynamics and disease transmission. This study measured levels of hemagglutination-inhibition (HI) antibodies against Japanese encephalitis virus (JEV) and neutralizing antibodies against Akabane virus (AKAV) and Aino virus (AINV) for Thoroughbred horses in Korea. Blood samples were collected from 989 racehorses in several provinces, between October 2005 and March 2007. Sera were tested using either an HI assay or a virus neutralization test. Approximately half (49.7%; 492/989) of the horses tested were antibody-positive for JEV. The HI titer against JEV was significantly correlated with racehorse age (p < 0.05). Horses with an HI antibody titer of 1: 160 or higher accounted for 3.9% of the animals tested, indicating that vectors transmitting arthropod- borne viruses bit relatively few horses. In contrast, 3.8% (19/497) and 19.5% (97/497) of horse sera collected in March 2007 were positive against AKAV and AINV, respectively. The presence of antibodies against AKAV and AINV may indicate the multiplication of AKAV and AINV in these horses.

Genetic Typing of Bovine Viral Diarrhea Viruses (BVDV) Circulating in Korea

To characterize the genetic diversity of bovine viral diarrhea viruses (BVDV) circulating in Korea, 11 BVDV isolates were obtained from 467 field samples collected during 2005~2006 in Korea. All of the BVDV isolates were identified as non-cytopathic (non-cp) BVDV biotypes. The 5' noncoding region (NCR) genes of the isolates were sequenced and analyzed. In total, ten BVDV isolates were typed as BVDV-1 by comparing the genomic sequences to the 5' NCR. One isolate (05R169) showed 98.6% nucleotide sequence identity with the BVDV-2 reference strain and was therefore typed as BVDV-2. Our results indicate that BVDV-1 is the main genotype circulating in the cattle population of Korea.

Serological Survey of Bovine Coronavirus in Korea

Bovine coronavirus (BCoV) is a causative agent of entero-pathogenic diarrhea in young calves and winter dysentery (WD) in adult cattle. In this study, we conducted a nationwide sero-epidemiological survey of BCoV infection in Korea. In total, 3,029 bovine sera collected between October and December 2005 were screened for the presence of antibodies against BCoV using a hemagglutination inhibition (HI) test. Half (50.0%) of individual cattle tested were positive for BCoV. The regional distribution of the seroprevalence of positive HI antibodies was 55.7% (234/420) in Gyeonggi, 53.0% (316/596) in Jeonra, 51.9% (374/720) in Chungcheong, 48.5% (401/827) in Gyeongsang, 43.9% (79/180) in Jeju, and 38.1% (109/286) in Gangwon Province. Analyzing the distribution of HI titer according to the age of the cattle showed the highest BCoV seropositive rate in 5-year-old cattle, and the incidence of cattle with an HI antibody titer of 1:160 or above was 12.1%.

The seroprevalence of Japanese encephalitis virus in goats raised in Korea

Japanese encephalitis virus (JEV) causes a mosquitoborne viral zoonosis that is becoming increasingly important to public health in east and south Asia. Although JEV is primarily associated with reproductive failure in swine, JEV infection can cause fever and headache in humans and is associated with aseptic meningitis and encephalitis. The exact mode of transmission, including host range and possible source of viral amplification within livestock, is still not completely clear. This study consisted of a serological survey of JEV infection in goats. A total of 804 goat serum samples were collected from 144 farms in Korea between May 2005 and May 2006. The incidence of positive cases was 12.1% (97 out of 804 goats). The seroprevalence of JEV infection in the 144 farms screened was 31.3% (45/144), indicating that JEV infection is frequent in goat farms in Korea. In addition, three districts of Korea (mainly in the southern region) had a higher seroprevalence of JEV compared to other areas. The results suggest that goats could be monitored epidemiologically as a sentinel animal for JEV transmission in Korea.

Alteration in gyrA and parC Gene Associated with Fluoroquinolone Resistance of Enterococcus spp. Isolated from Feces of Chicken

The purpose of this study was to investigate the fluoroquinolone resistance frequency of Enterococcus spp. from normal chicken feces and to analyse mutations of the gyrA and parC gene associated with fluoroquinolone resistance. Among 52 Enterococcus faecalis and 25 E. faecium isolates, 23 (44.2%) E. faecalis and 7 (28.0%) E. faecium were resistant to ciprofloxacin (CIP) by disc diffusion method. Genetic exchange in gyrA and parC gene among 2 CIP intermediate isolates and 15 CIP resistant isolates were found in the amino acid codon of Ser-83 and Asp-87, and Ser-80 and Glu-84, respectively. These mutants contained a change from Ser to Phe, Val, Tyr, Ile, Thr or Pro at codon 83 and from Glu to Gly or Leu at codon 87 in gyrA gene, and a change from Ser to Ile or Thr at codon 80 and from Glu to Asp or Lys at codon 84 in parC gene. The isolates with mutation in gyrA regardless of a mutation in parC showed high resistance (MIC > or =32 microgram/ml) to CIP, enrofloxacin, norfloxacin and ofloxacin. These results suggested that gyrA gene is the primary target for 4 fluoroquinolones resistance in Enterococcus spp.

Antibiotic Resistance and Genotypic Characterization of Escherichia coli Isolated from Pig Farm Environment

The use of antibiotics, including therapeutically in human and veterinary medicine, or as prophylaxis of growth promotion in animal husbandry, ultimately exerts selective pressure favorable for the propagation of antibiotic resistant bacteria. In this study we have determined the resistance for antibiotics of E. coli from pig farm environment, and investigate genetic relatedness by random amplification of polymorphic DNA (RAPD). Six farms were randomly selected in Gyeongsanman-do and Busan provinces for collecting samples from feces, manure and underground water. A total of 88 isolates from feces, 74 isolates from manure and 1 isolate from underground water were analyzed by antibiotic resistance and RAPD. Antibiotic resistance testing was performed by disk diffusion method using 16 antibiotics. The highest percentage of antibiotic resistance of isolates from feces and manure was found to the following antibiotics; tetracycline (100% and 100%), sulfamethoxazole/trimethoprim (60.2% and 62.2%), streptomycin (50.0% and 68.9%), chloramphenicol (56.8% and 56.8%), ampicillin (50.0% and 81.1%) and cephalothin (50.0% and 51.4%). Of isolates from feces and manure, 22.7% and 20.3% showed multiple resistance to 4 and 5 antibiotics, respectively. The isolates from GE pig farm showed six RAPD patterns. A single pattern, RAPD-C, was predominat in feces isolates (50.0%) and manual isolates (46.7%), and the rest of feces isolates showed RADP-A, B and E pattern and manure isolates showed D and E pattern. One isolate from underground water showed F pattern. The appearance of multiresistant in E. coli isolates from pig farms environment is a problem of major concern of public health and RAPD may offer an useful tool of discrimination for the epidemiological investigation.

Development and evaluation of indirect ELISA for the detection of antibodies against Japanese encephalitis virus in swine

The Japanese encephalitis virus (JEV) is one of causative agents of reproductive failure in pregnant sows. An indirect enzyme-linked immunosorbent assay (I-ELISA) was examined for its potential use in the rapid monitoring of the JEV, and the results were compared with those from the hemagglutination inhibition (HI) and serum neutralization (SN) tests. The comparative analysis showed that the results of I-ELISA showed a significant correlation with the conventional HI (r = 0.867) and SN tests (r = 0.804), respectively. When the I-ELISA results were compared with the traditional diagnostic assays, the sensitivity of the I-ELISA was 94.3% with the HI test and 93.7% with the SN test, respectively. The specificity was found to be 81.4% and 80.0% with the HI and SN tests, respectively. To determine the applicability of I-ELISA in the field, the serum samples from 720 pigs were collected from 4 regions in Korea between July and August 2004. The results indicated that 21.7% of screened pigs were seropositive for the JEV. The seropositive rates of JEV in the 4 provinces were 12.6% in Gyeonggi, 45.0% in Gyeongnam, 16.7% in Jeonbuk, and 12.2% in Jeju. The I-ELISA methodology developed in this study was shown to have considerable sensitivity and specificity through a comparison with HI and the SN tests. Therefore, it might be one of convenient methods for screening a large number of samples in various fields.

Immunogenicity of baculovirus expressed recombinant proteins of Japanese encephalitis virus in mice

Genes encoding for the premembrane and envelope (prME), envelope (E) and nonstructural protein (NS1) of Japanese encephalitis virus (JEV) were cloned. Each protein was expressed in baculovirus expression system. Of the three proteins expressed in baculovirus system, only prME had hemagglutination activity. The prME (72 and 54 kDa), E (54 kDa) and NS1 (46 kDa) proteins could be detected by Western blotting in the recombinant virus infected cells. Immunogenicity of the recombinant proteins obtained from infected Spodoptera frugiperda (Sf-9) cells was examined in mice. The 3 week-old ICR mice immunized intraperitoneally with three recombinant proteins three times were challenged with a lethal JEV. A survival rate was increased from about 7.7% in unimmunized mice to 92.3% in E + prME and only E groups. The complete protection was shown in prME and live vaccine inoculated groups, respectively. We also measured neutralizing antibody and three immunoglobulin subtypes of IgG1, IgG2a and IgG2b in the sera of mice before and after challenge. Titers of IgG1 antibodies were approximately two to three times higher than that of IgG2b antibodies in all the immunized groups as compared to the control group. However, IgG2a antibody level somewhat increased after challenge, indicating T-helper type 1 (Th1) cell response. The results of this study can provide useful information for developing efficacious subunit vaccine against JEV.

Establishment of Rapid Detection Methods for Bovine Viral Diarrhea Virus(Pestivirus) in Attenuated Animal Viral Vaccines by RT-PCR and Nested PCR

Bovine viral diarrhea virus (BVDV) of the genus Pestivirus is known as a common contaminant of cell culture-derived vaccines. Hog cholera virus (HCV), which is also of the genus Pestivirus and an important livestock disease in Korea, is recognized as a potential contaminant of vaccines produced in porcine cells. However, it is difficult for the National Biological Assays of korea to adequately detect contamination of these agents in biological products. For these reasons, we established rapid and sensitive methods for the detection of BVDV and HCV contamination in cell cultures and veterinary biologicals by using RT-PCR and nested PCR assays. We designed a Pestivirus primer amplifying 152 bp to detect both BVDV and HCV and a common primer amplifying 237 bp to detect only BVDV. Also, for the differentiation between BVDV type 1 and type 2, nested PCR was conducted using the amplified 237 bp PCR product, to amplify 179 bp in BVDV type 2 genome. The sensitivity of the PCR using common primer for the detection of BVDV was 400 TCID50/ml. All 6 strains of Korean BVDV isolates, 5 vaccines strains and the standard strain NADL could be detected. No reactions were observed when testing 5 types of viruses infecting pigs (HCV, TGEV, PEDV, JEV, PRRSV), 4 types infecting cattle (Akabane virus, BEFV, BCV, BRV) and 4 types infecting cats (FIP, FPL, FCV, FVR). Using this RT-PCR assay, commercial vaccines were tested and, 55 lots from 12 vaccine companies, were negative for BVDV contaminations. Same results were obtained by the immunoflourescence assay. The newly developed PCR or RT-PCR assays can be used as rapid, reliable, sensitive, and simple methods for the detection of BVDV (Pestivirus) in cell cultures, master seeds, and live viral vaccines.

Partial Nucleotide Sequence of Porcine Parvovirus (VRI-1 Strain): Identification of the Putative Defective Genomes

A porcine parvovirus, designated as VRI-1, was isolated from a 30-day-old piglet. Replicative form of viral DNA from ST cells infected with VRI-1 was directly cloned into pUC19. The cloned DNA fragment contained the entire nonstructural and structural protein genes, covering approximately 85% of the viral genome. The nucleotide sequence of VRI-1 showed 99.4~99.5% identity in the nonstructural protein (NS) and 99.0~99.2% identity in the structural protein with previously reported PPV strains, respectively. Among the cloned genes, two types of defective genomes with deletion of 100 and 247 nucleotides at almost similar location of 3' region within NS gene were also identified in this study.

TaqMan reverse transcription polymerase chain reaction for the detection of Japanese encephalitis virus

One step TaqMan reverse transcription polymerase chain reaction (RT-PCR) using TaqMan probe was developed for detection of Japanese encephalitis virus (JEV). Real-time RT-PCR was optimized to quantify JEV using the detection system (Rotor Gene 2000 detector) and dual-labeled fluorogenic probes. The gene specific labeled fluorogenic probe for the 3' non-translated region (3' NTR) was used to detect JEV. When the specificity of the assay using specific JEV primers was evaluated by testing three different JEV strains, other swine viruses and bovine viral diarrhea virus, no cross-reactions were detected with non-JE reference viruses. A single tube TaqMan assay was shown to be 10-fold more sensitive than the conventional two-step RT-PCR method. Detection limits of two step and real-time RT-PCR for JEV were 112 TCID50 /ml and 11.2 TCID50 /ml, respectively. Quantification of JEV was accomplished by a standard curve plotting cycle threshold values (Ct ) versus infectivity titer. Real-time RT-PCR assay using single tube method could be used as a sensitive diagnostic test, and supplied the results in real time for detection and quantification of JEV. We could detect JEV RNA genome in plasma samples of pigs inoculated with KV1899 strain at 2 days post inoculation, but couldn't in 41 fetus samples. This assay was sensitive, specific, rapid and quantitative for the detection of JEV from laboratory and field samples.

Molecular characterization of full-length genome of Japanese encephalitis virus (KV1899) isolated from pigs in Korea

We have determined the complete nucleotide and deduced amino acid sequences of the Japanese encephalitis virus (JEV) strain KV1899, isolated from a fattening pig in Korea. In comparison with 22 fully sequenced JEV genomes currently available, we found that the 10,963-nucleotide RNA genome of KV1899 has a 13-nucelotide deletion in the 3' non-translated variable region and 53 unique nucleotide sequences including 3' non-translated region (NTR). Its single open reading frame has a total of 28 amino acid substitutions. Comparison of the KV1899 genomic sequence with those of the 21 fully sequenced JEV strains in published databases showed nucleotide homology ranging from 97.4% (Ishikawa strain) to 87.0% (CH2195 strain). Amino acid homology with KV1899 strain ranged from 96.4% (K94P05) to 91.0% (GP78). The KV1899 showed the highest nucleotide homology with Ishikawa strain and the highest amino acid homology with K94P05. We performed an extensive E gene based phylogenetic analysis on a selection of 41 JEV isolates available from the GenBank. Compared with Anyang strain, isolated from a pig in 1969, that is current live vaccine strain for swine in Korea, the homology of nucleotide sequence in envelope gene was only 87.1%. The prM gene of the isolate was closely related with those of Ishikawa and K94P05 strains, which were grouped into genotype I of JEV.

Biophysical characterization of Japanese encephalitis virus (KV1899) isolated from pigs in Korea

A virus strain, showing cytopathic effect in Vero cell, was isolated from plasma of a fattening pig in Gyeonggi province, Korea in October 1999. The evaluation of physicochemical/biological properties of the isolate showed that the virus, KV1899, inoculated suckling mouse showed paralysis and died within 7 days post-inoculation, the mouse brain suspension had hemagglutinating activity with goose RBC. Pathogenicity of isolate was carried out by intracranial and intraperitoneal inoculation of 3-4 weeks mice. The mice inoculated with isolate showed 10 4.5 LD50/ 0.03 ml and 10 3.0 LD50/0.5 ml according to the inoculation route. The isolate was identified as RNA and enveloped virus using IUDR and chloroform sensitivity test. The virus particles within the infected Vero cell were measured to be 40-50 nm in size by electron microscopy. The isolate was further characterized by immuno-fluorescence assay using Japanese encephalitis virus (JEV) specific monoclonal antibodies. Reverse transcription polymerase chain reaction (RT-PCR) revealed the presence of JE specific conserved sequences in this isolate. The artificially inoculated pigs had HI titer of 320 to 2,560 against JEV at 14 to 42 days post inoculation. We confirmed this isolate as Japanese encephalitis virus. It was the second isolation of JEV in pigs in Korea.

Glycoprotein g III of aujesky's disease virus espressed in insect cells by a baculovirus

No abstract available.

Expression of neutralizing proteins of pseudorabies virus using recombinant baculovirus

No abstract available.