초록 ▼

○ 연구결과
- 전국 8개 시도에서 소 동절기적리가 발생한 32개 농장 97개 설사분변에서 바이러스 분리를 성공적으로 수행하였음.
- 바이러스 검출을 위해 고전적인 전자현미경법 외에도 국내에서는 사용하지 않았던 ELISA, RT-PCR, nested PCR 기법들을 개발 응용하였음.
- 10개의 소 동절기적리 국내 분리주를 확보함
- 국내에 이미 동절기적리가 만연함을 알 수 있었으며, 구체적인 역학 데이터를 확보함
- 국내에서 분리한 10개의 분리주의 스파이크 당단백질 유전자의 핵산 및 아미노산 염기서열에

○ 연구결과
- 전국 8개 시도에서 소 동절기적리가 발생한 32개 농장 97개 설사분변에서 바이러스 분리를 성공적으로 수행하였음.
- 바이러스 검출을 위해 고전적인 전자현미경법 외에도 국내에서는 사용하지 않았던 ELISA, RT-PCR, nested PCR 기법들을 개발 응용하였음.
- 10개의 소 동절기적리 국내 분리주를 확보함
- 국내에 이미 동절기적리가 만연함을 알 수 있었으며, 구체적인 역학 데이터를 확보함
- 국내에서 분리한 10개의 분리주의 스파이크 당단백질 유전자의 핵산 및 아미노산 염기서열에 대한 분자생물학적 특성을 미국과 캐나다 등에서 보고된 결과와 비교하여 분석함.
- 국내에서 분리한 동절기적리 원인바이러스를 송아지에 재접종하여 기병론에 대한 연구를 성공적으로 수행하였음
- 국내 성우의 코로나 바이러스 항체가 검사를 계절별로 312두분에 대해 수행하여, 연령별, 지역별, 계절병 자료를 확보함.

Abstract ▼

Bovine coronavirus (BoCV), a member of the Coronaviridaefamily, causes severe diarrhea in newborn calves (CD) and is associated with winter dysentery (WD) in adult cattle. WD is characterized by a sudden onset of diarrhea that rapidly affects many adult cattle in the herd and has been reported in ma

Bovine coronavirus (BoCV), a member of the Coronaviridaefamily, causes severe diarrhea in newborn calves (CD) and is associated with winter dysentery (WD) in adult cattle. WD is characterized by a sudden onset of diarrhea that rapidly affects many adult cattle in the herd and has been reported in many parts of the world. WD-affected cattle lose body condition and results in a dramatic decrease in milk production.
BoCV has been incriminated by many researchers around the world as a causative agent of WD. Evidence for BoCV as an etiologic agent of WD is circumstantial, often based on the isolation of the virus from WD-affected cattle or on the detection of BoCV in the fecal samples by electron microscope (EM) or on the presence of high antibody titers against the BoCV in the sera of affected cattle. Experimental reproduction of WD in adult cattle has met with difficulties in the past. Recently, however, experimental WD was successfully reproduced in the BoCV-seronegative adult cattle after they had been exposed to a calf experimentally infected with BoCV.
It has been suspected that WD caused by BoCV does occur and causes enormous economic losses in dairy and beef industry of South Korea. However, the exact epidemiology of WD and the causative agent of WD have never been documented. Therefore, we performed to survey the prevalence of WD by using electron microscopy (EM) as well as antigen-capture ELISA, RT-PCR and nested PCR specific for bovine coronavirus (BoCV) from 97 fecal samples of 32 herds with WD collected from 9 provinces during 2002-2004 and to isolate BoCV from the fecal samples positive for BoCV by ELISA, RT-PCR and nested PCR. The BoCV were consistently detected in all herds with WD collected from 9 provinces. Of other pathogens including bovine viral diarrhea virus, rotavirus, salmonella and coccidian oocyts, only coccidian oocyts were inconsistently but concurrently detected with BoCV. Ten WD BoCV were isolated from the fecal samples and exhibited cytopathic effects on inoculated HRT-18G cells and typical morphology of BoCV on electron microscopy. The isolated BoCV was identified as determined by an agglutination of virus particles by IEM with antiserum to BoCV, positive reaction by immunofluorescent test and ELISA using antisera or monoclonal antibodies specific for BoCV, and amplification of BoCV specific nucleocapsid gene by RT-PCR. From these results, it is concluded that WD caused by BoCV occurred in high frequency and was widespread in Korea. The results provide important epidemiological data for the control and establishment the surveillance system of WD in Kore.
BoCV possesses a single-stranded, enveloped, non-segmented RNA genome of positive polarity (De Vries et al., 1997). The virion contains five major structural proteins; the nucleocapsid (N) protein, the transmem -brane (M) protein, the hemagglutinin/esterase (HE) protein, the spike (S) protein and the small membrane (E) protein. Although both S and HE glycoproteins of BoCV use N-acetyl-9-O acetyl neuraminic acid as receptor determinant to initiate infection, the S glycoprotein requires fewer of these receptors on the surface of erythrocytes for agglutination than the HE protein . Thus the S glycoprotein is the major hemagglutinin of BoCV ,and it is also proposed to be responsible for the primary attachment of BoCV to other cell surface receptors. The variation in host range and tissue tropism of coronaviruses is largely attributable to variations in the S glycoprotein. The S glycoprotein is a type 1 membrane glycoprotein that carries distinct functional domains near the amino (S1) and carboxy (S2) termini. The S1 subunit is peripheral and is associated with receptor binding functions, whereas the S2 subunit is a transmembrane protein mediating fusion of viral and cellular membranes. In general, the S glycoprotein facilitates viral attachment to susceptible cells, causes cell fusion, and induces neutralizing antibodies. Of the two functional subunits, S1 and S2 that contain several antigenic domains, S1 appears to most efficiently elicit monoclonal antibodies (Mabs) with higher neutralizing activity.
The biologic and genetic differences are determined in many viruses by the geographic localities and different time points. Like most RNA viruses, coronaviruses are thought to mutate at a high frequency because of the high error frequencies of RNA polymerases. In addition, a unique feature of coronavirus genetics is a high frequency of RNA recombination. For example, a progenitor of the group II coronaviruses acquired the HE gene from an mRNA of influenza C virus andporcine respiratory coronavirus (PRCV) contained point mutations or deletions within the first 250 amino acids (aa) of transmissible gastroenteritis virus S1 which were associated with reduced enteropathogenicity and loss of hemagglutinating activity. The BoCV causing CD, WD and respiratory infection had point mutations or deletions especially in S1 subunit of S glycoprotein. Besides, molecular analysis of S gene of BoCV has been conducted and compared mainly between American and Canadian isolates and/or strains.
In the present study, we analyzed the S glycoprotein gene to characterize ten winter dysentery (WD) coronavirus isolates circulated in Korea during 2002-2003 and compared the nucleotide (nt) and deduced amino acid (aa) sequences with the other known BoCV. A total of 77 and 52 polymorphic nucleotides were identified in the S1 and S2 subunits of the S glycoprotein of Korean WD isolates compared with the prototype Mebus strain. These polymorphisms led to 46 and 24 aa substitutions at 41 and 20 distinct sites, respectively. The phylogenetic analysis of the entire S glycoprotein gene revealed that the aa sequences of all Korean WD isolates were more homologous to each other and were very closely related to respiratory bovine coronavirus (RBCV) strain OK and enteric bovine coronavirus (EBCV) strain LY-138 but distinct from the other known BoCVs. Based on the phylogenetic analysis of hypervariable region of S1 subunit, all Korean WD isolates clustered with the respiratory strain OK and BCQ3994 and enteric strain LY-138, while the Canadian BCQ calf diarrhea and WD strains, and the American RBCV LSU, French EBCV F15 and avirulent VACC, L9 and Mebus strains clustered on a separate major branch, respectively. Among four aa sites known for RBCV-specific aa substitutions, aa sequences at aa 510, aa 543 and aa 578 were identical in all Korean WD isolates with RBCV specific sites. Seven virulent specific sites in S gene of BoCV were conserved in 9 Korean WD isolates except for a Korean WD isolate KWD9 having aa substitutionat aa 965, leading to increased total charge. Furthermore, all Korean WD isolates had unique aa substitutions at aa 149 and aa 617 of S glycoprotein gene in comparison with all other BoCV. Comparisons of the first hydrophobic region of S2 subunit among virulent and avirulent BoCV represented that virulent BoCVs including all Korean WD isolates had marked increase of hydrophilicity. These data suggest that the WD isolates circulated in Korea had a genetic property of both RBCV and EBCV. In addition, Korean WD isolates as well as virulent BoCVs may have alteration of the fusion activity or pathogenecity due to marked increase of hydrophilicity in the first hydrophobic region.
WD is characterized by huge occurrence in the cattle herds with severe watery to blood diarrhea. Since WD has incriminated by many etiological agents including Campylobacter jejuni, BoCV has been consistently detected or isolated agents by many researchers around the world as a causative agent of WD. However, experimental reproduction of WD in adult cattle has been unsuccessful in the past. Recently, experimental WD was successfully reproduced in the BoCV-seronegative adult cattle after they had been exposed to a calf experimentally infected with BoCV.
The studies on the pathogenicity of BoCV causing calf diarrhea was reported during 1970 decade. However there are no reports about the studies on the pathogenicity of WD BoCV. In addition, the relationships and epidemiology between calf diarrhea and WD BoCV has been still unknown. Therefore, we performed experimental studies in which colostrum-deprived and BoCV-seronegative calves was inoculated with Korean strain of WD BoCV and then sequentially sacrificed at 1, 3, 5, and 8 post-inoculation day (PID), respectively. Each organs and tissues were carefully examined grossly and histopathologically. Fecal virus shedding in each experimental animal was evaluated by antigen-capture ELISA, RT-PCR and nested PCR specific for BoCV. In addition, the change of antibody titer for the BoCV in each experimental animal was checked by indirect antigen-capture ELISA.
Diarrhea was occurred after 1 PID and became more severe as a profuse form after 2 to 3 PID in all experimental animal inoculated with Korean strain of WD BoCV. Grossly, mild atrophy of intestinal wall was only characteristic lesion in the small intestine. Histopathologically, desquamation of villi epithelial cells, atrophy of villi, fusion of villi, increased villi vs crypt ratio, and crypt hyperplasia were detected in the small intestine of BoCV-inoculated experimental animals. Large intestine revealed desquamation of crypt epithelial cells, fusion of each lamina propria, and infiltration of lymphoid cells into lamina propria. At PID 1, the lesions was more severe in small intestines than those in large intestine. Sequentially lesions of large intestine became severe. Finally both small and large intestine had almost equal severe lesions at PID 5 and 8, respectively. Fecal shedding of BoCV was consistently detected in the feces of BoCV inoculated experimental animals from PID 1 to 8 by nested PCR but not in the feces of mock-inoculated animal. From these results, it is concluded that the Korean strain of WD BoCV had a pathogenicity to calves. These results can provide essential data to make protocols of quarantine and epidemiology of WD and calf diarrhea caused by BoCV in Korea.
Although calf diarrhea, WD in adult cattle, and respiratory infection in beef cattle can be caused by infection of BoCV, the exact co-relationship between these diseases are still obscure. At least, we have clarified that the Korean strain of WD BoCV can cause severe diarrhea in the calf. Therefore, these things can provide that calf diarrhea BoCV can induce WD in the adult cattle on the contrary. How does antibody titer of BoCV play the occurrence of WD in the adult cattle? This question should be solved to make a vaccine program to prevent WD in the addult cattle.
In our previous studies, WD caused by BoCV occurred in high frequency and was widespread in Korea. Moreover, the WD strains circulated in Korea had a genetic property of both RBCV and EBCV and were significantly distinct from the ancestral enteric strain. In the veterinary literature as far as we known, there are no reports to survey the antibody titer of BoCV in the adult cattle in Korea. Therefore we performed to survey the antibody titer of BoCV by indirect antigen-capture ELISA in the adult cattle in Korea using the blood specimens sampled seasonally from the 312 cows submitted to the slaughterhouse, respectively.
In the present study, almost all cows had over the 1,600 antibody titer of BoCV. From the seasonal evaluation of antibody titer of BoCV, the blood samples of winter season had higher antibody titer than that of spring and summer. Age distribution of antibody titer of BoCV revealed no differency between the different age population. By the comparison of antibody titer of BoCV among the cattle submitted from Jeonnam, Jeonbuk, and Gwangju, there were no significant differency of antibody titer of BoCV among these districts. However, Sunchang county in Jeonbuk had more 20.3% cattle with high antibody titer than that of Goksung county in Jeonnam. The discrepancy of BoCV antibody titer between beef (Hanwoo) and dairy (Holstein) cattle was not observed. From these results, the adult cattle in Korea had high antibody titer of BoCV, resulting from high occurrence of BoCV infection even of calf diarrhea or WD and meaning widespread of BoCV (calf diarrhea or WD) infection. These results will provide important epidemiological data for the control and establishment of the surveillance system of WD in Kore.
In conclusion, we have developed advanced techniques (indirect antigen-capture ELISA, RT-PCR, and nested PCR) for the detection of WD BoCV as well as calf diarrhea BoCV in the fecal samples and indirect antigen-capture ELISA for the evaluation of BoCV antibody titer in serum. Moreover, we have clarified the molecular characterization of Korean WD strain of BoCV, the pathogenicity of Korean WD BoCV strain to calves, and seroprevalence of BoCV in the adult cattle.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 19
• CONTENTS ... 26
• 목차 ... 32
• 제1장 연구개발 과제의 개요 ... 37
• 제1절 연구개발의 목적과 필요성 ... 37
• 제2절 연구개발의 범위 ... 39
• 제2장 국내외 기술개발 현황 ... 42
• 제3장 연구개발수행 내용 및 결과 ... 43
• 제1절. 소 동절기 적리 코로나바이러스의 진단을 위한 RT-PCR, nested PCR 및 ELISA기법의 개발, 국내 발생 역학조사 및 원인 바이러스 분리·동정 ... 43
• 1. 서 설 ... 45
• 2. 재료 및 방법 ... 46
• 가. 설사분변 ... 46
• 나. 분변의 전자현미경적 관찰 ... 46
• 다. 소 동절기 적리 코로나바이러스 검출을 위한 ELISA 기법 ... 47
• 라. 바이러스 RNA 추출 ... 48
• 마. 소 동절기 적리 코로나바이러스의 검출을 위한 RT-PCR 및 nested PCR용 primer 제작 ... 50
• 바. RT-PCR 기법의 수행 ... 50
• 사. Nested PCR 기법의 수행 ... 51
• 아. 소 동절기 적리 코로나바이러스 분리 ... 52
• 자. 면역형광항체법을 이용한 분리한 바이러스의 동정 ... 52
• 차. 전자현미경 및 면역전자현미경 기법에 의한 분리한 바이러스의 동정 ... 52
• 3. 결 과 ... 56
• 가. 전자현미경을 이용한 바이러스 검출 ... 56
• 나. Antigen-capture ELISA 기법을 이용한 바이러스 검출 ... 58
• 다. RT-PCR 및 nested PCR 기법을 이용한 소 동절기 적리 코로나바이러스 검출 ... 58
• 라. 설사분변 내 세균 및 기생충 검사 ... 60
• 마. RT-PCR 기법을 이용한 설사분변 내 소 바이러스성 설사병 바이러스 및 로타바이러스 검출 ... 60
• 바. HRT-18G 세포주를 이용한 바이러스 분리 ... 60
• 4. 고 찰 ... 65
• 제2절. 동절기 적리 소 코로나바이러스 스파이크 당단백질 유전자의 분자생물학적 특성 ... 68
• 1. 서 설 ... 70
• 2. 재료 및 방법 ... 72
• 가. 바이러스 ... 72
• 나. 바이러스 RNA 추출 ... 72
• 다. RT-PCR 용 primer 제작 ... 73
• 라. 염기서열 분석을 위한 RT-PCR의 수행과 증폭산물의 정제 ... 73
• 마. 염기서열 분석 ... 73
• 3. 결 과 ... 76
• 가. 국내 분리주간의 S 유전자 염기서열의 비교 ... 76
• 나. 국내 분리주와 외국 보고주간의 S 유전자 염기서열의 비교 ... 76
• 다. 국내 분리주와 외국 보고주와의 S2 subunit의 비교 ... 78
• 라. 국내 분리주와 소화기형 및 호흡기형 바이러스의 특이 염기서열 분석 ... 79
• 마. 강독주 관련 특이 염기서열의 분석 ... 79
• 4. 고 찰 ... 85
• 가. 국내 보고주와 외국 보고주간의 계통학적 분석 ... 85
• 나. 국내 분리주와 외국 보고주간의 hypervariable region의 아미노산 치환의 비교 ... 86
• 다. 국내 분리주와 외국 보고주간의 수용체 결합부위 및 S1A immunoreactive region의 비교 ... 87
• 라. S 글라이코단백질 절단부위 주위의 아미노산 차이의 비교 ... 88
• 마. 국내 분리주와 외국 보고주간의 S2 subunit 부위의 염기서열 비교 ... 89
• 바. 국내 분리주와 외국 보고주간의 병원성 특이 염기서열의 비교 ... 89
• 제3절. 국내 성우에서 분리한 소 동절기 적리 코로나바이러스의 송아지에 대한 병원성 ... 90
• 1. 서 설 ... 92
• 2. 재료 및 방법 ... 93
• 가. 바이러스 ... 93
• 나. 공시동물 및 실험설계 ... 93
• 다. 소 동절기 적리 코로나바이러스 검출을 위한 ELISA 기법 ... 94
• 라. 바이러스 RNA 추출 ... 95
• 마. 소 동절기 적리 코로나바이러스의 검출을 위한 RT-PCR 및 nested PCR용 primer 제작 ... 95
• 바. RT-PCR 기법의 수행 ... 96
• 사. Nested PCR 기법의 수행 ... 96
• 아. 소 동절기 적리 코로나바이러스에 대한 항체 검출을 위한 ELISA 기법 ... 97
• 자. 병리조직학적 검사 ... 98
• 3. 결 과 ... 99
• 가. 임상소견 ... 99
• 나. 육안적 소견 ... 99
• 다. 병리조직학적 소견 ... 99
• 라. 분변 내 바이러스 배출 ... 100
• 마. 혈중 항체가 변화 ... 100
• 4. 고 찰 ... 111
• 제4절. 국내 도축장 성우에서 채취한 혈청 내 소 코로나바이러스에 대한 항체가 조사 ... 113
• 1. 서 설 ... 115
• 2. 재료 및 방법 ... 117
• 가. 혈액 재료 ... 117
• 나. 소 동절기 적리 코로나바이러스에 대한 항체 검출을 위한 ELISA 기법 ... 117
• 3. 결 과 ... 121
• 가. 계절별 소 코로나바이러스에 대한 항체가 변동 ... 121
• 나. 연령별 소 코로나바이러스에 대한 항체가 변동 ... 122
• 다. 지역별 소 코로나바이러스에 대한 항체가 변동 ... 125
• 라. 품종별 소 코로나바이러스에 대한 항체가 변동 ... 127
• 4. 고 찰 ... 134
• 제4장 목표달성도 및 관련분야에의 기여도 ... 136
• 1. 연구개발 목표의 달성도 ... 136
• 2. 관련분야에의 기여도 ... 138
• 제5장 연구개발결과의 활용계획 ... 140
• 1. 활용방안 ... 140
• 2. 산업화 추진방안 ... 141
• 제6장 연구개발과정에서 수집한 해외과학 기술정보 ... 142
• 1. 외국분리주와 국내분리주간의 유전자 비교 결과 ... 142
• 2. 외국 보고주와 국내분리주와의 유전자 S2 subunit의 비교 결과 ... 144
• 3. 외국 및 국내 강독주 관련 특이 염기서열의 분석 결과 ... 144
• 제7장 참고문헌 ... 145
• 끝페이지 ... 160