Molecular analysis of the hexon, penton base, and fiber-2 genes of Korean fowl adenovirus serotype 4 isolates from hydropericardium syndrome-affected chickens.

Fowl adenovirus serotype 4 (FAdV-4) is the causative agent of hydropericardium syndrome (HPS), a highly pathogenic disease in poultry. In the present study, hexon, penton base, and fiber-2 genes encoding major capsid proteins were analyzed in four FAdV-4 isolates from HPS-affected chickens in Korea. Nucleotide sequences of the entire hexon (2811 bases), penton base (1578 bases), and fiber-2 (1425 bases) genes from the Korean isolates were 97.5-99.3, 99.1-99.7, and 95.5-99.0 % identical, respectively, to those of foreign FAdV-4 isolates. In the N-terminal tail region of fiber-2, the KRP motif predicted to be the nuclear localization signal was identified in the Korean isolates, whereas KRP/A was detected in other isolates. The VYPF motif in fiber-2, which is known to interact with the penton base, was present in the same region of all FAdV-4 isolates that were compared. Amino acid variations in fiber-2 for HPS and non-HPS isolates revealed that D219 and T300 were conserved among ten HPS isolates from five countries, including Korea. T380 in fiber-2, previously found in HPS isolates, corresponded to A380 in the Korean isolates, indicating that T380 is not relevant for increased virulence. Phylogenetic analysis showed that the four Korean FAdV-4 isolates were more related to MX-SHP95, a Mexican FAdV-4 isolate of HPS origin, than to FAdV-4 isolates of Indian and Chinese origin, suggesting that the genetic relationship among FAdV-4 isolates is independent of geographic distribution. The molecular features of these genes will provide valuable information for vaccine development against HPS in the future.

Abundance of biting midge species (Diptera: Ceratopogonidae, Culicoides spp.) on cattle farms in Korea.

Culicoides biting midges were collected on three cattle farms weekly using light traps overnight from May to October between 2010 and 2011 in the southern part of Korea. The seasonal and geographical abundance of Culicodes spp. were measured. A total of 16,538 biting midges were collected from 2010 to 2011, including seven species of Culicoides, four of which represented 98.42% of the collected specimens. These four species were Culicodes (C.) punctatus (n = 14,413), C. arakawae (n = 1,120), C. oxystoma (n = 427), and C. maculatus (n = 318). C. punctatus was the predominant species (87.15%).

Expression of VP2 gene protein of infectious bursal disease virus detected in Korea.

The VP2 gene DNA (1.4 kb in approximate) of a very virulent infectious bursal disease virus (vvIBDV) Chinju strain detected in Chinju, Korea was cloned into the bacmid, a baculovirus shuttle vector, through transposition of the gene from initially cloned pFastBacHTa plasmid, a baculovirus expression vector, and was subsequently expressed in Spodoptera frugiperda (Sf) cells. Biological properties of the expressed VP2 subunit protein were characterized to aid in the development of genetically engineered diagnostic reagents and vaccines against the vvIVDV. When the VP2 DNA-recombinant bacmid was transfected and propagated in the Sf cells, the cells showed no occlusion formation, which is a positive evidence for the insertion of the VP2 DNA into the polyhedrin gene of the bacmid, whereas the occlusions were observed in the cells infected by the Autographa californica nuclear polyhedrosis virus, a wild baculovirus. The expression of VP2 DNA was identified by strong positive reaction in fluorescent antibody test using chicken anti-IBDV serum. The VP2 protein was determined as a polypeptide band with Mr of 48 kDa by the sodium dodecyl-polyacrylamide gel electrophoresis for the lysate of the Sf cells infected with the recombinant bacmid. The VP2 protein was successfully purified from the cell lysate by Ni-NTA affinity chromatography. The expressed VP2 subunit protein reacted specifically with chicken anti-IBDV serum in Western blotting.

Cloning and nucleotide analysis of segment A gene of infectious bursal disease virus detected in Korea.

A strain of infectious bursal disease virus (IBDV) was detected from bursal tissues of chicks which suffered from infectious bursal disease (IBD) in Chinju, Korea and provisionally named as Chinju strain. A full-length cDNA clone for segment A gene of the virus was constructed, and complete nucleotide sequence of the gene including noncoding region was determined and analyzed by comparison with that of other IBDV strains. The segment A gene of Chinju strain consisted of 3,269 nucleotides including 862 adenine (26.4%), 917 cytosine (28.0%), 854 guanine (26.1%) and 636 thymine (19.5%). There were regions for two open reading frames (ORFs), ORF1 encoding the VP5 with ATG codon at nucleotides 98-100 and ORF2 encoding the polyprotein of VP2, VP4 and VP3 in the nucleotides 132-3,170. In deduced translation the ORF2 encoded 1,012 amino acids. The full nucleotide sequence of segment A gene and amino acid sequence of ORF2 of the Chinju strain showed 98-99% homology with those of the very virulent IBDVs (vvIBDVs) such as HK46, OKYM, UK661, UPM97/ 61, D6948 and BD3/99. In phylogenetic analysis of nucleotide and amino acid sequences, the Chinju strain was also related closely to the vvIBDVs. Hence, it was suggested that the Chinju strain is a vvIBDV. The nucleotide and amino acid sequences of the Chinju strain with pertinent information can be useful for the development of genetically engineered vaccines and diagnostic reagents against vvIBDV.