Hyung Kwan Jang

Induction of protective immunity in chickens vaccinated with infectious bronchitis virus S1 glycoprotein expressed by a recombinant baculovirus

A recombinant baculovirus containing the S1 glycoprotein gene of the virulent nephropathogenic KM91 strain of infectious bronchitis virus (IBV) was constructed in order to investigate protective immunity in vaccinated chickens. Results from the protection test were evaluated by re-isolation of virus from the kidneys and tracheas of vaccinated chickens after challenge with strain KM91. After three immunizations, the recombinant S1 (rS1) glycoprotein induced 50% protection of the kidney, whilst inactivated KM91 induced 88% and 50% protection of the kidney and trachea, respectively. In chickens primed with the attenuated H120 vaccine strain, which is heterologous to KM91, the rS1 glycoprotein induced 83% protection of the kidney after two immunizations. Haemagglutination-inhibition titres were also increased in chickens immunized with the rS1 glycoprotein after three immunizations, and significantly higher titres were detected after challenge. These data indicate that the expressed rS1 glycoprotein alone can induce a protective immune response as well as an antibody response.

Identification and transcriptional analysis of the homologues of the herpes simplex virus type 1 UL30 to UL40 genes in the genome of nononcogenic Marek's disease virus serotype 2

Studies on Mareks disease virus serotype 2 (MDV2) are important for understanding the natural nononcogenic phenotype of MDV. This study reports a 27,535 bp nucleotide sequence of part of the MDV2 genome located in the central unique long (U(L)) region. The analysis revealed 11 complete ORFs with high amino acid sequence identities to the products of other alphaherpesviruses. The MDV2 ORFs were arranged collinearly with the prototype sequence of herpes simplex virus type 1, ranging from the UL30 to UL40 genes. Sequences that were particularly well conserved among alphaherpesviruses were the putative functional domain of the DNA polymerase (UL30) and the ribonucleotide reductase large and small subunits (UL39 and UL40). On the other hand, in contrast to oncogenic MDV1, MDV2 did not contain the conserved proline-repeat region in the UL36 homologue. All the genes identified were confirmed to be transcribed as 3-coterminal mRNAs and/or unique transcripts in virus-infected cells.

The genetic organization and transcriptional analysis of the short unique region in the genome of nononcogenic Marek's disease virus serotype 2

Studies on the Mareks disease virus (MDV) serotype 2 (MDV2) genome may be important for understanding the naturally nononcogenic nature of the virus. To determine the complete DNA sequence of MDV2 unique short (Us) region, genomic BamHI fragments F, M1 and R were sequenced. The MDV2 Us region is 12109 bp long and contains 12 potential open reading frames (ORFs) likely to encode for proteins. Seven of them exhibit homologies to herpes simplex virus type 1 (HSV-1) US1 (ICP22), US2, US3 (protein kinase), US6 (gD), US7 (gI), US8 (gE) and US10 genes. These ORFs are conserved in a similar arrangement with those of HSV-1, except for US10 which is transposed in the Us regions of all three MDV serotypes. The predicted amino acid sequence of MDV2 ORF6 is homologous to SORF3 of the other serotypes of MDV serotype 1 (MDV1) and herpesvirus of turkeys (HVT) and to infectious laryngotracheitis virus SR1. In addition, four ORFs, which have been identified around the Us and inverted repeat junction regions, have no apparent relation to any other known herpesvirus genes. The identified ORFs in the MDV2 Us region were more colinear with their previously reported locations of MDV1 than with those of HVT and other alphaherpesviruses. Ten of the 12 ORFs in the MDV2 Us region were expressed and transcribed with 3-coterminal transcripts and/or a unique transcript in the virus-infected cells. Compared to other MDV serotypes, the MDV2 Us-encoded proteins showed 46-70% and 33-59% identities with equivalent of MDV1 and HVT at the amino acid level, respectively. Our present data will be useful to understand the different pathogenicity among serotypes of MDV and to allow precise manipulation of the genes for a possible use in genetically engineered vaccines.

Detection and classification of infectious bronchitis viruses isolated in Korea by dot-immunoblotting assay using monoclonal antibodies

Dot-immunoblotting assay (DIA) using five monoclonal antibodies (MAbs) to infectious bronchitis virus (IBV) was used to detect and classify the viruses propagated in embryonated chicken eggs. Using a group-specific MAb 3F5, 10 reference strains and 12 Korean isolates of IBV were successfully detected by DIA, and the lowest virus titer of IBV detected by DIA was approximately less than 10(3.8) mean embryo infective dose/ml. For evaluating the diagnostic efficiency, DIA was compared with the conventional infectious bronchitis (IB) diagnostic method. IBV antigens in allantoic fluid from embryonated eggs inoculated with IB-suspected field samples were specifically detected by DIA within only one or two egg passages, whereas the conventional embryonated egg inoculation method required four to seven egg passages for confirming IBV infection. These results indicated that DIA could significantly reduce time and cost for IB diagnosis. For examining the possibility of classifying IBV by DIA, four strain-specific MAbs, 3A4, 2A3, 6F7, and 2C6, were used. According to the MAb reacting patterns to the IBV antigens, the 10 IBV reference strains were classified into six groups; seven strains belonged to three different groups, and the other three strains each belonged to an individual group. In the case of 12 Korean isolates of IBV, they were classified in six groups. Among the six groups, the MAb reacting patterns of three groups matched those of the IBV reference strains, but the others did not. These data suggest that at least three variant serotypes of IBV exist in Korea.

Gene arrangement and RNA transcription of the BamHI fragments K and M2 within the non-oncogenic Marek's disease virus serotype 2 unique long genome region.

We determined the nucleotide sequence of a 6593 bp fragment of the Mareks disease virus serotype 2 (MDV2) unique long region located in the right part of genomic BamHI-M2 and the adjacent part of BamHI-K fragments. Within this region five complete open reading frames (ORFs) were identified whose deduced amino acid sequences exhibited homology to the UL53 (glycoprotein K), UL54 (immediate early regulatory protein ICP27), and UL55 gene products of herpes simplex virus type 1 (HSV-1). Homologue to the HSV-1 UL56 was not detected. However, we identified a gene between the MDV2 UL54 and UL55 genes with homology to the first ORF (ORF-1) of equine herpesvirus type 1 and corresponding gene identified in pseudorabies virus. Two adjacent ORFs contained in the BamHI-K fragment, ORF 873s and ORF 873, were found by computer analysis to have the properties of an intron encoding a glycoprotein: ORF 873s encodes a 84 amino acid polypeptide with a stretch of a hydrophobic signal sequence in the C-terminus, and ORF 873 encodes a 873 amino acid polypeptide with a transmembrane domain and putative three N-linked glycosylation sites. All the identified genes were confirmed to be transcribed with 3-coterminal transcripts and/or a unique transcript in the virus-infected cells. Especially, 3.5 kb mRNA of ORF 873s and ORF 873 are transcribed from a potential promoter region of ORF 873s, and splice donor and acceptor sites are used to splice the mRNA after cleavage of a 113 bp-nucleotide sequence.

Transcriptional analyses of the region of the equine herpesvirus type 4 genome encoding glycoproteins I and E

SummaryTo map the transcripts encoding the equine herpesvirus type 4 (EHV-4) glycoproteins I (gI) and E (gE), transcriptional analyses were performed at the right part of the unique short segment of EHV-4 genome. The results revealed that the gI gene is encoded by a 1.6-kb transcript which is 3′ coterminal with a 3.0-kb gD mRNA while the gE gene is encoded by two transcripts of 3.5- and 2.4-kb in size. The transcriptional patterns described in this study for the EHV-4 gI and gE are similar to those found in the equivalent region of herpes simplex virus type 1 and feline herpesvirus type 1. Characterization of EHV-4 gI and gE glycoprotein genes may facilitate future studies to define their roles in the EHV-4 infection.