초록 ▼

○ 연구결과: 본 연구를 통하여 닭의 마렉병과 백혈병/육종을 감별진단할 수 있는 DNA chip 진단기법을 개발하였다. 본 DNA chip 진단법의 정확도를 확인하기 위하여 표준주 및 야외주 혈청형 1-3 마렉병 바이러스, subgroups A-D, 및 J 백혈병/육종 바이러스들을 배양하여 각각의 바이러스들에 대하여 또한 혼합한 바이러스들에 대하여 DNA chip 진단법을 응용한 결과, 각각의 바이러스 및 혼합된 바이러스들에 특이적인 양성반응을 확인할 수 있었다. 또한 각각의 바이러스들을 단독 혹은 혼합 감염시킨 닭에서 DNA ch

○ 연구결과: 본 연구를 통하여 닭의 마렉병과 백혈병/육종을 감별진단할 수 있는 DNA chip 진단기법을 개발하였다. 본 DNA chip 진단법의 정확도를 확인하기 위하여 표준주 및 야외주 혈청형 1-3 마렉병 바이러스, subgroups A-D, 및 J 백혈병/육종 바이러스들을 배양하여 각각의 바이러스들에 대하여 또한 혼합한 바이러스들에 대하여 DNA chip 진단법을 응용한 결과, 각각의 바이러스 및 혼합된 바이러스들에 특이적인 양성반응을 확인할 수 있었다. 또한 각각의 바이러스들을 단독 혹은 혼합 감염시킨 닭에서 DNA chip 진단법을 응용한 결과, 각각의 바이러스 혹은 혼합 감염된 바이러스들을 검출할 수 있었다. 최종적으로 야외에서 발생한 마렉병 및 백혈병/육종에 대하여 DNA chip 진단법을 응용한 결과 모두 양성의 반응을 검출할 수 있었으며, 이러한 재료를 대상으로 기존에 사용되고 있는 PCR 진단기법과 그 민감도를 비교한 결과, 본 연구에서 개발한 DNA chip 진단법이 더욱 우수함을 알 수 있었다.
DNA chip 진단법에 사용할 야외 마렉병 바이러스의 분리 및 국내 마렉병 바이러스 분리주의 분자적 특성을 규명하기 위하여, 마렉병이 발생한 야외 양계장에서 바이러스를 분리하여, DNA chip 진단법에 사용하였다. 이러한 바이러스들에 대하여 마렉병 바이러스 meq, pp38, vIL-8 유전자에 대하여 분자적 특성을 규명한 결과, 국내에서는 다양한 종류의 바이러스가 존재하며, 더욱이 같은 농장에서도 다양한 바이러스가 존재함을 규명하였다.
국내 육용종계 및 재래 닭에서 외인성 및 내인성 백혈병/육종 그룹 바이러스들의 감염상태를 알아보고, 본 연구의 최종목표인 DNA chip 진단법에 사용하고자, 국내 주요 육용종계 및 재래 닭 종란의 계태아를 사용하여 외인성 및 내인성 백혈병/육종 그룹 바이러스에 대하여 검출을 시도한 결과, 외인성 바이러스는 모두 음성이었지만, 내인성인 EAV, ev, ART-CH, ev/J 바이러스들은 모두 검출되었다. 더욱이 국내에서 검출된 내인성 백혈병/육종 바이러스들은 외국의 표준주와는 계통학적으로 차이가 있음을 규명하였다.
닭 마렉병과 백혈병/육종의 국내 역학조사를 실시한 결과, 닭 마렉병은 닭의 주요 질병임을 규명하였다.
하지만 백혈병/육종의 경우 매우 드물게 발생함을 알 수 있었다. 이러한 닭 백혈병/육종의 산발적 발생은 국내 종계장에서 J 바이러스가 발생한 이후, 종계장에서 닭 백혈병/육종 그룹 바이러스들에 대한 근절대책을 수행한 결과라고 생각된다.

Abstract ▼

Ⅲ. Results and Extents of Research and Development
A. Development of DNA chip diagnostic method for the differential diagnosis of Marek's disease and leukosis/sarcoma of chickens
1. Materials and methods
1) Reference and isolated viruses:
Marek's disease reference viruses used in this st

Ⅲ. Results and Extents of Research and Development
A. Development of DNA chip diagnostic method for the differential diagnosis of Marek's disease and leukosis/sarcoma of chickens
1. Materials and methods
1) Reference and isolated viruses:
Marek's disease reference viruses used in this study included serotype 1 very virulent strain (Md5), virulent strain (JM) and apathogenic strain (CVI988), serotype 2 strain (SB1), and serotype 3 strain (HVT). This study used also the Korean serotype 1 Marek's disease viruses, 03-06-1, 03-06-2, 29-1, 29-2, 29-3, 29-5, 30-7, 31-1 and 46-1 strains.
The ALSVs used in this study were RAV-1 (subgroup A), RAV-2 (subgroup B), RAV-49 (subgroup C) and RAV-50 (subgroup D) strains purchased from the American Type Culture Collection (ATCC, VA, USA), and the HPRS-103 strain (subgroup J) obtained from Dr. K. Venugopal (Viral Oncogenesis Group, Institute for Animal Health, Compton, Berkshire, UK).
Monolayers of the duck embryo fibroblast cell cultures were used to propagate these viruses. The growth medium was M199 supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics (penicillin, dihydrostreptomycin, and mycostatin). The maintenance medium was M199 supplemented with 2% FBS and 1% antibiotics.
2) Experimental inoculation of reference and wild viruses into chickens The one day old chicks were housed in isolation rooms, and given food and water ad libitum. The serotypes 1 (Md5 and 29-1), 2 (HPRS-24) and 3 (HVT) MDV strains were made from duck embryo fibroblast cells.
One-day-old chicks were allotted randomly into 12 groups of 30 birds. The birds in groups 1-4 were inoculated intra-abdominally with 1x104 PFU/0.2 ml of Md/5, 29-1, HPRS-24 and HVT MDVs at 1 week of age, respectively.
The birds in groups 5-8 were inoculated intra-abdominally with 0.2 ml supernatant of ALSV subgroup A, B, C and D viruses at 1 week of age, respectively. The birds in groups 9-12 were inoculated intra-abdominally with 1x104 PFU/0.2 ml of Md/5 and 0.2 ml supernatant of each subgroup A, B, C and D virus, respectively.
The inoculated 3 chicks in each group were euthanized at 1 week intervals. The experiment was terminated when the birds were 10 weeks old.
All of the birds were immediately necropsied upon sacrifice. When the experiment was terminated, all of the surviving birds in all groups were euthanized by cervical dislocation and then exsanguination. At necropsy, almost all organs and tissues were sampled for histological observation, virus detection by PCR and DNA chip application.
3) DNA extraction
The DNA was extracted from each DEF cell infected with each virus, field samples and samples obtained from chickens of each group using a DNA extraction kit. The total DNA recovered was suspended in 50 μl of DNase free water and stored at -80 oC until needed.
4) Polymerase Chain Reaction
The oligonucleotide primers were made for the detection of each serotype of Marek's disease viruses and each subgroup ALSV, and for the amplification of Marek's disease virus and each subgroup ALSV applicable for the DNA chip. The single PCR assay was performed in a 50 μl reaction volume consisting of 5 μl of the template, 5 μl of 10X buffer [100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2, 0.01% gelatin], 5 μl of MgCl2 (25 mM), 1 μl of 10 mM dNTPs, 1 μl of the forward primer (50 pmol), 1 μl of reverse primer (50 pmol), 0.5 μl of Taq polymerase and 31.5 μl of DNase free distilled water. The multiplex PCR assay was almost same with single PCR except the number of primer pairs, in which the amount of DDW was deleted with the amount of primer volume added. The PCR products were visualized on 1.2% agarose gels stained with ethidium bromide.
5) Preparation of probes
The probes specific to viruses were designated between the sequence of amplicons specific for each virus.
6) Preparation of DNA chip
The DNA chip was made by pin microarray method using probes synthesized as above mentioned.
7) Hybridization and detection
The amplified fragments by single or multiplex PCR were hybridized as described previously. Briefly, 10 ㎕ of the amplicons labelled with Cy5-dUTP or Cy5-dCTP were mixed with 10 ㎕ hybridization buffer (50% formamide, 6× SSC, 0.5% SDS, 50 mM Na-phosphate, pH 8.0, 5× Denhard`s). After then, the tube was heated 98℃, 3 min using PCR machine and kept for 3 min in the ice. These final solution were transferred on the DNA chip slides and incubated for 15 hours in the 45℃ humid incubator. The DNA chip slides were washed with 2X SSC, 0.1% SDS for 5 min, 1X SSC for 5 min and 0.5X SSC for 5 min on the shaker and dried in 1500 rpm, 5 min in the centrifuge.
The signal was analyzed by Affymetrix 418 array scanner.
2. Results
1) Confirmation of the presence of reference and wild type viruses by PCR PCR assay with primer pairs specific to each virus was performed with DNA extracted from the cells infected with each virus. As a result, the expected bands for each primer pair were detected in the cells infected with each virus, indicating the presence of each virus in the cells and implying each virus applicable for the development of DNA chip.
2) Clinical and pathological findings of chickens infected experimentally with each virus
The chicks infected experimentally with virulent MDV or concurrently virulent MDV and each ALSV showed depression at 1 day post-inoculation.
However, the chicks infected with serotype 2 and 3, and each ALSV virus did not show any clinical sign. Grossly, any specific lesions were not detected in the chickens experimentally infected with each virus. Histologically, however, multiple lymphoid cell aggregations in the variable size were detected in the chickens infected experimentally with virulent MDV or virulent MDV plus each ALSV but no lesions were observed in the chickens infected with serotype 2 and 3, and each ALSV.
3) Detection of Marek's disease virus and ALSV in the samples obtained from the chickens infected with each virus
The PCR assay was performed to detect each MDV and ALSV in the samples obtained from the chickens infected with each virus. The virulent MDV was continuously detected from the samples obtained from the chickens infected with virulent MDV alone or virulent MDV plus each ALSV from 1 week to 10 weeks post-inoculation. However, positive band was not detected in the samples obtained from the chickens infected with each serotype 2 and 3 MDV. The expected band was present only at 1 week post-inoculation in the samples obtained from the chickens infected with each ALSV or each ALSV plus virulent MDV.
4) Development of DNA chip diagnostic method for the differential diagnosis of Marek's disease and leukosis/sarcoma of chickens In order to confirm if DNA chip can detect reference or wild type MDVs and ALSVs grown in the cells, we applied the DNA chip with the amplicons from each virus strains. The DNA chips showed each specific signal for the amplicons of each virus, indicating that the DNA chip was very specific to detect each virus. Moreover, DNA chip hybridized with amplicons by multiplex PCR revealed positive signals for viruses used, meaning the DNA chip can diagnose the mixed infections.
We applied the DNA chip method to the experimental chickens. The positive signal was detected in the samples obtained from the chickens infected with virulent MDV or virulent MDV plus each ALSV from 1 week post-inoculation to 10 weeks post-inoculation. Moreover, the DNA chip detected positive signal in the samples obtained from the chickens infected with each ALSV or each ALSV plus MDV from 1 week post-inoculation to 2 weeks post-inoculation. From these results, the sensitivity of DNA chip was higher than that of PCR assay.
B. Molecular characterization of Marek's disease viruses isolated in the chickens in South Korea
1. Materials and methods
1) Reference viruses and cell line
Marek's disease reference viruses used in this study included serotype 1 very virulent strain (Md5), virulent strain (JM) and apathogenic strain (CVI988), serotype 2 strain (SB1), and serotype 3 strain (HVT).
Monolayers of the duck embryo fibroblast cell cultures were used to propagate the virus. The growth medium was M199 supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics (penicillin, dihydrostreptomycin, and mycostatin). The maintenance medium was M199 supplemented with 2% FBS and 1% antibiotics.
2) Virus isolation
The MDV isolation was performed from blood and spleens sampled from 35 chickens originated from 5 field layer farms and 3 Korean native chicken farms. These chickens showed emaciation, spastic paralysis of limbs and flaccid paralysis of neck, formed lymphoma in the variable organs and tissues, and tested positive by PCR with primer pairs specific to both MDV meg and pol genes as described below.
The peripheral blood mononuclear cells (PBMC), and single spleen mononuclear and tumor cells were prepared as described elsewhere. The 1x104 cells of each PBMC, single spleen mononuclear cells and tumor cells were mixed with the same number of DEF cells and transferred into 6 well plates containing 3 ml of M199 supplemented with 5% heat-inactivated fetal bovine serum and 1% antibiotics (penicillin, dihydrostreptomycin, and mycostatin).
When monolayers were formed in each well, the growth medium was discarded, replaced with the maintenance medium. The cultures were incubated for 7 days at 37oC in a 5% CO2 atmosphere. When the cytopathic effects including cell rounding and clustering were observed, the isolated virus was passaged 5 times including triple plaque purification prior to characterization. The isolated MDV was confirmed using a PCR, as described below.
3) DNA extraction
The DNA was extracted from each DEF cells infected with each field MDV isolate, using a DNA extraction kit. The total DNA recovered was suspended in 50 μl of DNase free water and stored at -80oC until needed.
4) Polymerase Chain Reaction
The oligonucleotide primers used for sequencing meq, pp38 and vIL-8 genes, and for detecting MDV in the field samples and cultured cells for virus isolation were listed in Table 1. The PCR assay was performed in a 50 μl reaction volume consisting of 5 μl of the template, 5 μl of 10X buffer [100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2, 0.01% gelatin], 5 μl of MgCl2 (25 mM), 1 μl of 10 mM dNTPs, 1 μl of the forward primer (50 pmol), 1 μl of reverse primer (50 pmol), 0.5 μl of Taq polymerase and 31.5 μl of DNase free distilled water. The PCR products were visualized on 1.2% agarose gels stained with ethidium bromide.
5) DNA sequencing and molecular characterization
The PCR products from each gene fragment were purified using a QIAEX II Gel Extraction kit according to the manufacture’s instructions. DNA sequencing was carried out using an automated DNA sequencer. Using the DNA Basic module, each gene sequence were compared with those of the other known MDV strains.
6) Phylogenetic analysis
Phylogenetic analysis based on the nucleotide (nt) and the deduced amino acid (aa) alignments was constructed using the UPGMA method of Molecular Evolutionary Gentetics analysis (MEGA version3.1). A sequence similarity search for the field MDV isolates was performed using the LALIGN Query program of the GENESTREAM network server at the Institut de Génétque Humaine, Montpellier, FRANCE (http://www.eng.uiowa.edu/~tscheetz/sequence-analysis/examples/LALIGN/lalign-guess.html).
2. Results
1) Virus isolation from chicken farms
Of the 35 MDV-positive chickens from 8 farms by PCR, the MDV was isolated from 14 chickens from 5 farms. After the 1st or 2nd passage, infected cultures showed discrete focal plaques, which consisted of clusters of rounded, refractile degenerating cells. The cultures showing characteristic CPE tested positive by PCR with primer pairs specific to MDV pol genes.
2) Detection of MDV meq, pp38 and vIL-8 genes in the field MDV isolates The meq gene is know to be polymorphic [long meq (L-meq), meq, and short meq (S-meq)] in the attenuated serotype 1 MDV and the MDV-transformed cell lines. In order to evaluate the diversity of meq gene in the Korean MDV isolates, PCR assay with primer pair specific to meq gene was performed with 7 Korean MDV isolates, of which 4 isolates (29-1, 29-2, 29-3 and 29-5) were isolated from one farm. The Korean MDV isolates, 29-1 and 46-1 had meq and S-meq but not L-meq. However, 5 Korean MDV isolates (29-2, 29-3, 29-5, 30-7 and 31-1) contained L-meq, meq and S-meq (see chapter 3, section 2, the second year results part, Fig. 7). From this result, it is confirmed that diverse forms of meq gene were present in the field MDV even in the viruses isolated from the same farm. The PCR assays with primer pairs specific to each pp38 and vIL-8 gene detected expected band in the 7 Korean MDV isolates. These 7 isolates were designated the 29-1, 29-2, 29-3, 29-5, 30-7, 31-1 and 46-1 MDV strains.
3) Molecular characterization of meq gene of Korean MDV strains
The genetic diversity of the MDV meg gene was investigated by sequencing 583 nt of the meq gene from 7 Korean MDV strains isolated from the field chickens. Among all strains analyzed, a total of 13 polymorphic nucleotides were identified in the 583 nt of the meq gene, compared with the reference strains. These polymorphisms led to 10 aa changes. Interestingly, proline at 194 aa which was present in the Korean 29-1 and 46-1 MDV strains, and all serotype 1 MDV strains compared was deleted in the Korean MDV strains, 29-2, 29-3, 29-5, 30-7 and 31-1.
Based on the total number of nucleotide substitutions and deletions, a phylogenetic tree of the 583 nt sequences was constructed using the Clustal method with the other known serotype 1 MDV strains. The nt alignment of a part of meq gene sequences suggests that the MDV can be classified into two groups . The Korean MDV strains belonged to group 1. The Korean MDV strains, 46-1 and 29-1 which had only meg and S-meq, located distantly from the other Korean MDV strains which contained L-meq, meq and S-meq. Phylogenetic analysis based on the 193 deduced aa sequences showed almost similar grouping as like that of nt sequences.
4) Phylogenetic analysis of pp38 gene of Korean MDV strains Phylogenetic analysis of the nt and deduced aa sequences of a part (850 bp) of pp38 MDV gene showed that the Korean MDV strains, 29-1 and 46-1 clustered with very virulent (Md5 and Md11) and virulent (GA) MDV strains.
In contrast, the other Korean MDV strains were closer to apathogenic serotype 1 MDV strain, CVI988. Interestingly, a Korean MDV strain, 29-3 was distant from all MDV strains compared.
5) Phylogenetic analysis of vIL-8 gene of Korean MDV strain The MDV vIL-8 gene contained three exons and two introns. Therefore, phylogenetic analysis of nt sequence of MDV vIL-8 gene was conducted.
Phylogenetically, the Korean MDV strains, 46-1 and 29-1 were distantly clustered with the other known MDV strains in order. Interestingly, the other Korean MDV strains clustered with CVI988 strain, apathogenic serotype 1 strain.
C. Detection and molecular characterization of avian
leukosis/sarcoma viruses in fertile Korean chicken eggs
1. Materials and methods
1) Samples
The fertile eggs from three types of broiler chickens [Cobb (18 farms), Ross (1 farm) and Hubbard (1 farm)], which are the most popularly reared chickens in Korea in that order, Korean native chickens (Ogol, Black, Dark-Brown and Light-Brown chickens) and imported White Leghorn chickens, which are used widely for experiments in Korea, were examined. In order to extract the DNA, chicken embryo cells (CEC) were prepared using 11-day-old embryonated chicken eggs using a mincing and trypsin treatment described elsewhere.
2) DNA extraction
The DNA was extracted from 11-day-old CEC of Cobb, Ross and Hubbard meat-type chickens, four lines of Korean native chickens, and White Leghorn chicken, respectively, using a DNA extraction kit. As positive controls of exogenous ALSVs, the DNA was extracted from the RAV-1 (subgroup A), RAV-2 (subgroup B), RAV-49 (subgroup C) and RAV-50 (subgroup D) strains purchased from the American Type Culture Collection (ATCC, VA, USA), and the HPRS-103 strain (subgroup J) obtained from Dr.
K. Venugopal. The total DNA recovered was suspended in 100 μl of DNase free water and stored at -80oC until needed.
3) Polymerase Chain Reaction (PCR)
The endogenous and the exogenous proviruses of the ALSVs in the cells of the embryonated chicken eggs were detected using PCR assays with different primer sets specific to each ALSV. The PCR assay was performed in a 50 μl reaction volume consisting of 5 μl of the template, 5 μl of 10X buffer [100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2, 0.01% gelatin], 5 μl of MgCl2 (25 mM), 1 μl of 10 mM dNTPs, 1 μl of the forward primer (50 pmol), 1 μl of reverse primer (50 pmol), 0.5 μl of Taq polymerase and 31.5 μl of DNase free distilled water. PCR products were visualized on 1.2% agarose gels stained with ethidium bromide.
4) DNA sequencing
The PCR assays with different primer sets specific to a part of the env gene of the ev and the ev/J, the region between the TM and the LTR of the EAV, and the gag-related region of the ART-CH were performed with the DNA extracted from the cells of embryonated chickens eggs. PCR products were purified using a QIAEX Ⅱ gel extraction kit according to the manufacturer’s instructions. The DNA was sequenced using an automated DNA sequencer. The nucleotide (nt) sequences of each ALSV were compared with those of other known endogenous ALSVs using the DNA Basic module.
The deduced amino acid (aa) sequences were then assembled and analyzed on an Amino Acid Basic module. A sequence similarity search was performed for the EAV, ev, ev/J and ART-CH DNA and EAV, ev and ev/J protein using the LALIGN Query program of the GENESTREAM network server at Institut de Génétque Humaine, Montpellier, FRANCE (http://www.eng.uiowa.
edu/~tscheetz/sequence-analysis/examples/LALIGN/lalign-guess.html).
Phylogenetic analyses were conducted using the neighbor-joining and UPGMA methods of Molecular Evolutionary Genetics analysis (MEGA version3.1) with the pairwise distance.
2. Results
1) Detection of exogenous ALSVs by PCR assays
The exogenous ALSVs in the fertile eggs of the meat-type Cobb, Ross and Hubbard chickens, Korean native chickens (Ogol, Black, Dark-Brown and Light-Brown chickens), and imported White Leghorn chickens were detected by PCR assays with the primer pairs specific to each exogenous ALSV on the DNA extracted from each CEC. The amplified fragments specific to each subgroup A, B, C, D and J were detected after amplification with the reference RAV-1 (subgroup A), RAV-2 (subgroup B), RAV-49 (subgroup C), RAV-50 (subgroup D) and HPRS-103 (subgroup J) strains. This suggests that the PCR assays with the primer pairs were specific to each subgroup A, B, C, D and J. In contrast, no positive reaction was detected in the CECs of the meat-type Cobb (18 breeders), Ross (1 breeder) and Hubbard (1 breeder) chickens, four Korean native chickens, and White-Leghorn chickens (data not shown), indicating these ALSVs are not present in the fertile eggs of these chickens.
2) Detection of endogenous ALSVs by PCR assays
The known endogenous ALSVs include EAV, ev, ev/J and ART-CH, which were all detected by PCR assays using the primer pairs specific to each endogenous ALSV in the CECs of Cobb (18 farms), Ross (1 farm) and Hubbard (1 farm) chickens, four Korean native chickens (Ogol, Black, Dark-Brown and Light-Brown), and imported White Leghorn chickens.
3) Molecular analysis of ART-CH, ev, ev/J and EAV ALSV A comparison of the nt sequence of the region between the TM and the LTR of the EAV detected in all Korean native chickens exhibited 100% sequence homology to each other and 99.2-99.6% homology to that of the EAV-0 and other Korean meat-type chickens. However, E-51 had only 73.8-74.6% identity to the four lines of the Korean native chickens, Cobb, Ross and Hubbard and EAV-0, respectively. A comparison of the deduced aa sequences of these regions of the EAV detected in all the Korean broiler chickens showed 100% homology to each other, 98.7% homology with EAV-0, and 70.88% identity with E-51. This indicates that the EAV detected in the Korean meat-type chickens is closely related to EAV-0 but distantly related to E-51. Phylogenetic data based on the total number of nt and deduced aa substitutions in the region between the TM and the LTR of the EAV showed that the EAV can be divided into 4 groups (Fig. 2A and 2B). The first group consists of EAVs detected in the 18 Cobb (EAV-1~18) and 1 Ross (EAV-R) broiler chickens. The second group includes the Korean native chickens (Ogol, Black, Dark-Brown and Light-Brown chickens) and the EAV-0. The third group contains only the EAV detected in the Hubbard broiler chicken. The E51, belonging to the fourth group, is phylogenetically distant from the EAVs of the 1st to 3rd groups.
Because only Brown Leghorn chickens were used to examine the presence of ART-CH in chickens, it is unclear if they are present in the other chicken breeds. In this study, only the variation in the nt sequence could be analyzed because ART-CH does not encode aa due to large and multiple nt deletions.
Paired comparisons of the gag-related nt sequences showed that the Korean meat-type chickens had a high sequence identity (over 98.6%) to each other but slightly low sequence identity with ART-CH clones 5 (94.0-95.8%) and 14 (91.0-91.8%). Based on the total number of nt substitutions and deletions,
the phylogenetic tree of the gag-related sequences was analyzed using the prototype ART-CH elements, clones 5 and 14. Phylogenetically, the gag-related nt sequence of ART-CH indicates that the ART-CHs can be divided into four groups. Furthermore, the first group can be divided into two subgroups. The first subgroup (subgroup 1a) consists mainly of the ART-CHs detected in all the Cobb chickens (ART-CH-C1~18), Hubbard (ART-CH-H) and three Korean native chickens [Black (ART-CH-B), Dark-Brown (ART-CH-D, and Light-Brown chickens (ART-CH-L)], whereas the second subgroup (subgroup 1b) includes only the ART-CH-R detected in the Ross chicken. The second group contains only a single ART-CH element, ART-CH-O, which was detected in the Korean native chicken, Ogol. The ART-CH clones 5 and 14 make the third and fourth groups, respectively, and are clustered distantly from groups 1 and 2.
In the paired comparisons of the ev ALSV detected in the Korean meat-type chickens, the nt sequence substitutions of the ev ALSV indicated the maximum sequence divergence in the Korean native chickens, even in small substitutions (1.2% between Korean native Dark-Brown and Light-Brown). These nt substitutions led to a maximum 2.73% aa changes.
The nt and aa sequence divergences tended to be slightly larger than the other known ev loci, ev-1 (nt; 98.8-99.8% and aa; 97.95-99.09%), ev-3 (98.4-98.9% and 97.49-98.86%) and ev-6 (98.2-98.6% and 96.36-97.50%).
Overall, the nt and aa sequences of a part of the env gene of the ev ALSVs demonstrated a high degree of similarity. The phylogenetic tree of the env gene nt and aa sequences revealed that the ev ALSVs can be divided into two groups. The first group can be further divided into 3 subgroups; the first subgroup (1a) consists of ev ALSVs detected in 18 Cobb (ev-C1~18), 1 Ross (ev-R) and 1 Hubbard (ev-H) chickens, the second subgroup (1b) includes the ev ALSVs detected in the three Korean native chickens [Black (ev-B), Ogol (ev-O) and Light-Brown (ev-L) chickens], and third subgroup consists of the ev-ALSV detected in the Korean native chicken, Dark-Brown (ev-D).
The second group consists of ev-3, forming subgroup 2a, and ev-1 and ev-6 forming subgroup 2b. All the ev-ALSVs were phylogenetically distant from subgroups A, B, C and D ALSVs.
A part of the env gene nt and aa sequences of the ev/J ALSV showed a high level of homology (nt; 97.5-99.5% and aa; 95.84-100%) in all Korean meat-type chickens and the other known ev/J sequences. Moreover, ev/J ALSV including the ev/J viruses detected in all the Korean meat-type chickens and the other known ev/J revealed 96.8-98.1% nt and 94.63-96.98% aa homology to that of subgroup J ALSV. Although all ev/J endogenous viruses showed high nt and aa sequence identity, the ev/J ALSVs can be divided into 3 groups based on the total number of nt and aa substitutions.
The first group consists of subgroups 1a and 1b; subgroup 1a consists of the ev/J viruses detected in the Hubbard (ev/J-H), grey jungle fowl, Cobb (ev/J-C2~5, 7, 8, 13, 14), Ross (ev/J-R) and three Korean native chickens [Black (ev/J-B), Ogol (ev/J-O) and Dark-Brown (ev/J-D) chickens], whereas subgroup 1b consists of ev/J viruses detected in the Cobb chickens (ev/J-C1, 9~12). The second group consists of 2 subgroups; subgroup 2a is an ev/J virus detected in the line 21 chicken, whereas subgroup 2b consists of the ev/J viruses detected in Cobb (ev/J-C15), a Korean native chicken [Light-Brown (ev/J-L)] and the red jungle fowl. Phylogenetically, the group 2 ev/J viruses are most closely related to subgroup J ALSV. The third group can be further divided into four subgroups; subgroup 3a consists of the ev/J viruses detected in the Cobb chickens (ev/J-C6 and 16), subgroup 3b is an ev/J virus detected in the Cobb chicken (ev/J-C17), subgroup 3c is an ev/J virus detected in the line N chicken and subgroup 3d is an ev/J-C18 detected in the Cobb chicken.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 38
• CONTENTS ... 68
• 목 차 ... 71
• 제 1 장 연구개발과제의 개요 ... 73
• 제 1 절 연구개발 목표와 내용 ... 73
• 제 2 절 연구개발의 필요성 ... 73
• 1. 기술적 측면 ... 73
• 2. 경제ㆍ산업적 측면 ... 77
• 3. 사회ㆍ문화적 측면 ... 80
• 제 2 장 국내외 기술개발 현황 ... 82
• 제 3 장 연구개발 수행내용 및 결과 ... 83
• 제 1 절 연구개발 수행내용 ... 83
• 1. 1차년도(2003년도) ... 83
• 가. 제1세부과제: 닭에 종양을 유발하는 바이러스들에 대한 특이 유전자 탐색 ... 83
• 나. 제1협동과제: 국내에서 발생하고 있는 닭의 바이러스성 종양성 질병의 발병조사와 시료채취 ... 85
• 2. 2차년도(2004년도) ... 86
• 가. 제1세부과제: DNA chip의 개발과 국내에서 분리한 바이러스 분리주에 응용 ... 86
• 나. 제1협동과제: 닭 백혈병/육종 그룹 바이러스와 마렉병 바이러스의 분리.동정 및 야외 종양 발생레의 조사 및 재료 확보 ... 88
• 3. 3차년도(2005년도) ... 90
• 가. 제1세부과제: DNA chip의 실험접종례와 야외발생 증례를 이용한 진단법 확립 ... 90
• 나. 제1협동과제: 국내 주요 육용종계 및 재래닭 종란에서 닭 백혈병/육종 바이러스 검출 및 내인성 바이러스의 분자유전학적 분석과 마렉병 바이러스 분리주의 SPF 닭의 실험접종 ... 91
• 제 2 절 연구개발 수행결과 ... 93
• 1. 1차년도(2003년도) ... 93
• 가. 제1세부과제: 닭에 종양을 유발하는 바이러스들에 대한 특이 유전자 탐색 ... 93
• 나. 제1협동과제: 국내에서 발생하고 있는 닭의 바이러스성 종양성 질병의 발병조사와 시료채취 ... 113
• 2. 2차년도(2004년도) ... 122
• 가. 제1세부과제: DNA chip의 개발과 국내에서 분리한 바이러스 분리주에 응용 ... 122
• 나. 제1협동과제: 닭 백혈병/육종 그룹 바이러스와 마렉병 바이러스의 분리.동정 및 야외 종양 발생레의 조사 및 재료 확보 ... 136
• 3. 3차년도(2005년도) ... 153
• 가. 제1세부과제: DNA chip의 실험접종례와 야외발생 증례를 이용한 진단법 확립 ... 153
• 나. 제1협동과제: 국내 주요 육용종계 및 재래닭 종란에서 닭 백혈병/육종 바이러스 검출 및 내인성 바이러스의 분자유전학적 분석과 마렉병 바이러스 분리주의 SPF 닭의 실험접종 ... 168
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 175
• 제 1 절 제1세부과제 연구목표 달성도 ... 177
• 제 2 절 제1협동과제 연구목표 달성도 ... 178
• 제 3 절 관련분야에의 기여도 ... 179
• 제 5 장 연구개발결과의 활용계획 ... 181
• 제 1 절 추가연구의 필요성 및 타연구에의 응용 ... 181
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 183
• 제 7 장 참고문헌 ... 185
• 끝페이지 ... 196