The helicase genes and hypervariable regions (HVRs) of three avian hepatitis E viruses (HEVs) detected at three different farms were sequenced and characterized. Two isolates (DW-L and GI-B2) were classified as genotype 2 and one isolate (GR-B) was classified as genotype 1. A phylogenetic tree, based on the helicase gene and HVR nucleotide sequences, revealed the newly detected viruses and other avian HEVs were classified similarly. Unlike previously reported avian HEVs, the DW-L isolate detected in broiler breeders with characteristic lesions of avian HEV had no prolinerich motif in its HVR, suggesting that the proline-rich motif is non-essential for viral replication and infection.

To evaluate avian hepatitis E virus (aHEV) as an RNA vaccine platform, open reading frame 2 (ORF2) of aHEV was replaced by heterologous genes, such as enhanced green fluorescent protein (eGFP) and hemagglutinin (HA)-tag, in aHEV infectious cDNA clones. eGFP and HA-tag replicons were expressed in leghorn male hepatoma (LMH) cells. To confirm expression of the heterologous protein, ORF2 was replaced with the antigenic S1 gene of infectious bronchitis virus (IBV). The IBVS1 replicon was expressed in LMH cells. To our knowledge, this is the first investigation showing potential as a RNA vaccine platform using an aHEV. In the future, it may be used in the development of RNA vaccines against various pathogens.

To evaluate avian hepatitis E virus (aHEV) as an RNA vaccine platform, open reading frame 2 (ORF2) of aHEV was replaced by heterologous genes, such as enhanced green fluorescent protein (eGFP) and hemagglutinin (HA)-tag, in aHEV infectious cDNA clones. eGFP and HA-tag replicons were expressed in leghorn male hepatoma (LMH) cells. To confirm expression of the heterologous protein, ORF2 was replaced with the antigenic S1 gene of infectious bronchitis virus (IBV). The IBVS1 replicon was expressed in LMH cells. To our knowledge, this is the first investigation showing potential as a RNA vaccine platform using an aHEV. In the future, it may be used in the development of RNA vaccines against various pathogens.

The helicase genes and hypervariable regions (HVRs) of three avian hepatitis E viruses (HEVs) detected at three different farms were sequenced and characterized. Two isolates (DW-L and GI-B2) were classified as genotype 2 and one isolate (GR-B) was classified as genotype 1. A phylogenetic tree, based on the helicase gene and HVR nucleotide sequences, revealed the newly detected viruses and other avian HEVs were classified similarly. Unlike previously reported avian HEVs, the DW-L isolate detected in broiler breeders with characteristic lesions of avian HEV had no prolinerich motif in its HVR, suggesting that the proline-rich motif is non-essential for viral replication and infection.
Agriculture ; Base Sequence ; Genotype ; Hepevirus ; Korea ; Trees

Two infectious bronchitis virus (IBV) K046-12 and K047-12 strains were isolated and the nearly complete genomes of them were sequenced. Sequence comparisons showed that the K046-12 genome was most similar to Korean IBV strains, and the K047-12 genome was most similar to QX-like IBV strains. Phylogenetic analysis showed that nearly all K046-12 and most K046-12 genes were placed in the same cluster as Korean IBV isolates, but the S1 region was placed in the same cluster as Mass-type IBVs. For K047-12, nearly all K047-12 and most K047-12 genes were located in the same cluster as QX-like IBVs, but the M region was located in the same cluster as Korean IBV isolates with K047-12. Recombination analysis confirmed that K046-12 is a recombinant strain with the primary parental sequence derived from Korean IBVs and minor parental sequence derived from Mass-type IBV, and K047-12 is a recombinant strain with the major parental sequence derived from QX-IBV and minor parental sequence derived from Korean IBVs. This study showed that new IBV recombinants are constantly generated among various IBVs, including those used for vaccination. Therefore, genetic analysis of new virus isolates should be performed for effective infectious bronchitis control and appropriate vaccine development.
Bronchitis ; Genome ; Humans ; Infectious bronchitis virus ; Korea ; Parents ; Recombination, Genetic ; Vaccination

Infectious bursal disease virus (IBDV) is a member of the Avibirnavirus genus of the Birnaviridae family which genome consists of two segments (A and B) of double stranded RNA. Segment A gene of KNU08010 isolate, which was isolated from a 15-day-old chicken flock in 2008, was sequenced and compared with other IBDV isolates including SH/92 strain, the first Korean very virulent (vv) IBDV isolate. The amino acid sequences of segment A gene showed that KNU08010 had 99.2% homology with SH92 strain. KNU08010 isolate had specific amino acids A222, I242, I256, I294 and S299 which are highly conserved among vvIBDV strains. Phylogenetic analysis based on the nucleotide sequences of variable region of the VP2 gene of 18 IBDV strains revealed that KNU08010 was grouped with vvIBDVs and was closely related to Korean vvIBDVs isolated from wild birds.
Amino Acid Sequence ; Amino Acids ; Avibirnavirus ; Base Sequence ; Birds ; Birnaviridae ; Chickens ; Genes, vif ; Genome ; Humans ; Infectious bursal disease virus ; Korea ; RNA, Double-Stranded ; Sequence Analysis ; Sprains and Strains

This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken interferon-gamma (ChIFN-gamma) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-atilde was constructed. Twice at 2-week intervals, twoweek-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines plus either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-gamma were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-gamma was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-gamma groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-gamma had no significant effect.
Adjuvants, Immunologic ; Animals ; Antibodies, Viral/blood ; Birnaviridae Infections/*immunology/*prevention & control/virology ; Bursa of Fabricius/immunology/virology ; Cell Proliferation ; Chickens ; CpG Islands/immunology ; Enzyme-Linked Immunosorbent Assay/veterinary ; Immunization/methods/*veterinary ; Infectious bursal disease virus/*immunology ; Interferon-gamma/immunology/therapeutic use ; Lymphocytes/cytology/immunology ; Oligonucleotides/immunology ; Poultry Diseases/immunology/*prevention & control/*virology ; Specific Pathogen-Free Organisms ; Vaccines, DNA/immunology/therapeutic use ; Viral Vaccines/*immunology/therapeutic use

The isolation rate of Escherichia (E.) coli in poultry litter was investigated at 44 broiler farms, 20 that used fresh litter and 24 that used recycled litter. The patterns of resistance to antibiotics of the E. coli isolates were compared. In litter sampled before the rearing period, the isolation rate of E. coli was higher at farms that used fresh litter; E. coli was present in the litter in 94.5% (35 out of 37 flocks tested) of the farms that used fresh litter vs. 51.2% (21 out of 41 flocks) of the farms that used recycled litter. The susceptibility of the 93 isolates of E. coli to 13 antibiotics was studied. Before the rearing period, E. coli isolates from the farms that recycled litter showed higher resistance rates than isolates from farms that replaced litter with fresh litter. Comparing the antibiotic resistance patterns of isolates from litter sampled before and at the end of the rearing period, the antibiotic resistance rates at the end of the rearing period increased dramatically compared with rates before the rearing period.
Agriculture ; Anti-Bacterial Agents ; Drug Resistance, Microbial* ; Escherichia coli* ; Escherichia* ; Poultry*

Two subgroup J avian leukosis viurses (ALVs) were isolated from broiler breeder flocks, in which myeloid leukosis had occurred. The isolates could be classified as subgroup J ALV. by the positive reaction in polymerase chain reaction (PCR) with primers specific for subgroup J ALV. Two isolates replicated in chicken embryo fibroblast (CEF) cells from the alv6 chicken line in which cells are resistant to subgroup A and E ALVs. In in vitro serum neutralization tests with other subgroup ALVs including ADOL-Hc1, the prototype of subgroup J ALVs isolated in the United States of America, two isolates were partially neutralized by antibody to ADOL-Hc1, indicating that Korean isolates and ADOL-Hc1 may be antigenically related, but not identical. When the PCR was done with a primer pair designed to amplify genes of E element and long terminal repeat of proviral DNA, the PCR product size of one isolate (KOAL-PET) was smaller than that of ADOL-Hc1, suggesting that some sequences in these regions are deleted.
Animals ; Antibodies, Viral/immunology ; Antigens, Viral/immunology ; Avian Leukosis/virology ; Avian leukosis virus/*classification/genetics/immunology/*isolation & purification ; Cell Line ; Chick Embryo ; Chickens/*virology ; Korea ; Neutralization Tests ; Polymerase Chain Reaction ; Poultry Diseases/virology

Twelve Korean infectious bronchitis viruses (IBVs) were isolated in the field from chickens suspected of being carriers of infectious bronchitis between 2001 and 2003. The S1 glycoprotein genes of these IBV isolates were amplified by reverse transcriptase-polymerase chain reaction (RTPCR) and analyzed by restriction fragment length polymorphism (RFLP) analysis. These Korean IBV isolates were classified into three groups according to their RFLP patterns obtained using the restriction enzyme HaeIII. Half of the twelve isolates were similar to the KM91 RFLP pattern, which is a common pattern in Korea. Three more isolates were related to the Arkansas strain pattern, but with some unique variations. The other three viruses showed variant RFLP patterns. For a comparison with the published sequences for non-Korean IBV strains, amplified PCR products from the twelve isolates were cloned and sequenced. The Korean IBV field isolates had 71.2-99.7% nucleotide sequence homology with each other and 45.9-80.7% nucleotide sequence homology with non-Korean IBV strains. With respect to the deduced amino acid sequence, the Korean IBV isolates had 71.5-99.3% similarity with each other and 44.9-80.3% similarity with non-Korean IBV strains. Phylogenetic tree analysis revealed that some of the IBV isolates appear to belong to a new group, different from the non-Korean IBV strains or from previously isolated Korean IBV strains. Specifically, the new Korean IBV isolates K10217-03, K3-3 and K1255-03 represented a separate group. These findings suggest that the Korean IBVs appear to be continuously evolving.
Amino Acid Sequence ; Animals ; Coronavirus Infections/*veterinary/virology ; Glycoproteins/chemistry/*genetics ; Infectious bronchitis virus/*classification/*genetics/isolation & purification ; Molecular Sequence Data ; *Phylogeny ; Polymorphism, Restriction Fragment Length ; Poultry ; Poultry Diseases/*virology ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Sequence Alignment/veterinary ; Sequence Analysis ; Sequence Homology, Amino Acid ; Viral Proteins/chemistry/genetics