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INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information C om pany 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9307744 Molecular cloning, nucleotide sequencing and genome replication of bovine viral diarrhea virus Deng, Ruitang, Ph.D. The Ohio State University, 1992 UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 MOLECULAR CLONING, NUCLEOTIDE SEQUENCING AND GENOME REPLICATION OF BOVINE VIRAL DIARRHEA VIRUS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ruitang Deng, B.S., M.S. ***** The Ohio State University 1992 Dissertation Committee: Kenny V . Brock Approved by Daral J. Jackwood 1/- Linda J. Saif v Adviser Department of Veterinary David E. Swayne Preventive Medicine To my wife and parents ii ACKNOWLEDGMENTS I express sincere appreciation to my advisor Dr. Kenny V. Brock for his guidance, support and help throughout the research. Thanks go to the other members of my advisory committee, Drs. Daral J. Jackwood, Linda J. Saif and David E. Swayne for their suggestions and comments. The technical assistance of Sylva Riblet and Jerry Meitzler is gratefully acknowledged. To my wife, Fengmin Tian, I express sincere thanks for your encouragement, support, understanding and sacrifice for me. To my little daughter, Stella, I thank you for understanding my frequent absence. iii VITA August 29, 1963 ................ Born-Zhejiang, P. R. of China 1983 ........................... B.S., Zhejiang Agricultural University, Zhejiang, P. R. of China 1986 ........................... M.S., Nanjing Agricultural University, Nanjing, P. R. of China 1986-1989... ................... Research Assistant, Animal Science and Veterinary Medicine Institute, Shanghai, P. R. of China 1989-present .................. Research Associate, Ohio State University, Ohio, USA PUBLICATIONS 1. Ruitang Deng and Kenny V. Brock (1992). Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1. Virology 191, 867-879. 2. Kenny V. Brock, Ruitang Deng and Riblet Sylva (1992). Nucleotide seguencing of 5' and 3' termini of bovine viral diarrhea virus by RNA ligation and PCR. J. Virol. Methods 38, 39-46. 3. Ruitang Deng, Yafen Xu and Naixun Yu (1990). Studies of antibody-forming cells and antibody-secreting cells at the early stage (1-5 days) after immunization of rabbit hemorrhagic disease virus vaccine. J. Shanghai Agricultural Acta 1, 123-128. 4. Naixun Yu, Ruitang Deng and Yafen Xu (1990). Studies on the development of monoclonal antibodies against surface antigen and intracellular antigen of Toxoplasma gondii. J. Shanghai Agricultural Acta 4, 36-41. iv 5. Ruitang Deng, Yafen Xu and Naixun Yu (1989) . The kinetics of rabbit thymus lymphocyte antigen (RTLA) and rabbit bursal eguivalent lymphocyte antigen (RABELA) cells at the early stage (1-5 days) after immunization of rabbit hemorrhagic disease viral vaccine. J. Shanghai Animal Science and Veterinary Medicine 5, 84-90. 6. Ruitang Deng, Weiyan Xu, Nianxing, Xicai Yang and Shixuan Wu (1987). Characteristics of rabbit hemorrhagic disease virus. J. Nanjing Agricultural University 2(2), 110-114. 7. Funan Xu, Shengjiang Liu and Ruitang Deng (1985). Pathological studies on rabbits infected with rabbit hemorrhagic disease virus. Animal Science and Veterinary Medicine 17(6), 244-245. FIELDS OF STUDY Major Field: Veterinary Preventive Medicine Studies in Microbiology, Virology, Immunology, Molecular Biology and Biochemistry. v TABLE OF CONTENTS DEDICATION ................................................ ii ACKNOWLEDGMENTS ........ iii VITA ....................................................... iv LIST OF TABLES ............................................. viii LIST OF FIGURES ........................................... ix INTRODUCTION .............................................. 1 CHAPTER I. LITERATURE REVIEW .............................. 10 1. Persistent infection ....................... 11 2. Mucosal disease ............................. 15 3. Pestivirus genome .......................... 21 4. Viral proteins and genome organization .... 27 5. Antigenic relationship and epitope m a p p i n g ..................................... 35 6. Laboratory diagnosis and vaccination ...... 46 7. Comparison of CP BVDV with NCP B V D V ........ 49 II. NUCLEOTIDE SEQUENCING OF 5' AND 3' TERMINI OF BOVINE VIRAL DIARRHEA VIRUS BY RNA LIGATION AND P C R ............................... 56 Introduction ................................... 56 Materials and Methods ......................... 57 R e s u l t s ......................................... 74 D iscussion..................................... 76 R e f erences..................................... 79 III. MOLECULAR CLONING AND NUCLEOTIDE SEQUENCE OF A PESTIVIRUS GENOME, NONCYTOPATHIC BOVINE VIRAL DIARRHEA VIRUS STRAIN SD - 1 .............. 86 Introduction ................................... 86 Materials and Methods ......................... 88 R e s u l t s ......................................... 96 Discussion..................................... 103 vi References 111 IV. 5' AND 3 ' UNTRANSLATED REGIONS OF PESTIVIRUS GENOME: PRIMARY AND SECONDARY STRUCTURE ANALYSES ..................... 133 Introduction ................................... 13 3 Materials and Methods .......................... 135 R e s u l t s ........................................ 13 6 Discussion..................................... 145 References..................................... 152 V. BOVINE VIRAL DIARRHEA VIRUS REPLICATION: ASYMMETRIC SYNTHESIS OF POSITIVE AND NEGATIVE STRAND VIRAL R N A ............................... 173 Introduction ................................... 173 Materials and Methods .......................... 175 R e s u l t s ......................................... 178 Discussion..................................... 182 References..................................... 186 GENERAL DISCUSSION ....................................... 193 APPENDIX list of NCP BVDV strain SD-1 cDNA clones and subclones.................................... 2 04 LIST OF REF E R E N C E S....................................... 214 vii LIST OF TABLES TABLE PAGE 1. BVDV-specific polypeptides ........................ 54 2. Cloning efficiencies of PCR products with different methods ............................. 76 3. Primary structural features of the 5'-UTR of pestiviruses and other related viruses ........... 160 4. Temporal release of virus from infected cells ..................................... 192 viii LIST OF FIGURES FIGURE PAGE 1 . The genomic organization of bovine viral diarrhea v i r u s ............................................... 55 2 . 5 ' and 3 ' ligation of BVDV genomic RNA and PCR amplification ...................................... 82 3. Cloning of the 5' terminal sequence of BVDV genome by oligo(A)-tailing and PCR amplification ........ 83 4. Cloning of PCR products by G/C-tailing method .... 84 5. Homology of the nucleotide sequence of the 5' and 3' ligation clone of CP BVDV NADL with the published NADL sequence............................ 85 6 . A sample of subcloning for the original SD-1 cDNA clones ........................................ 117 7. Distribution of cDNA clones of NCP BVDV strain SD-1 along the genome of 12308 nucleotides ....... 118 8 . The complete genomic nucleotide sequence of the genomic RNA of NCP BVDV strain S D - 1 .............. 119 9 . Distribution of open reading frames in 6 phases of 2 orientations of NCP BVDV SD-1 cDNA sequence .... 128 1 0 . Comparison of nucleotide and amino acid sequences of NCP BVDV SD-1 with that of other pestiviruses.........................................129 1 1 . Comparison of the predicted amino acid sequence of SD-1 in the four conserved within the region of viral structural proteins and nonstructural protein p54 with that of other pestiviruses..... 130 12. Comparison of the predicted amino acid sequence of SD-1 in the three variable domains within the region of viral structural proteins and nonstructural protein p54 with that of other pestiviruses.........................................131 ix 13.