University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 9-9-2016 Development of an Improved Live Attenuated Antigenic Marker CSF Vaccine Strain Candidate with an Increased Genetic Stability lauren Gayle Holinka-Patterson [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Holinka-Patterson, lauren Gayle, "Development of an Improved Live Attenuated Antigenic Marker CSF Vaccine Strain Candidate with an Increased Genetic Stability" (2016). Doctoral Dissertations. 1252. https://opencommons.uconn.edu/dissertations/1252 Development of an Improved Live Attenuated Antigenic Marker CSF Vaccine Strain Candidate with an Increased Genetic Stability Lauren Holinka-Patterson, PhD University of Connecticut, 2016 Abstract Classical Swine Fever Virus (CSFV) is an extremely contagious, hemorrhagic and often fatal disease of pigs that causes serious socioeconomic impact on countries which experience outbreaks or are endemically infected. Current control measures utilized for CSFV are contingent upon the epidemiological status of the inflicted area and entail either prophylactic vaccination or non-vaccination, “stamping out”, protocol with the elimination of infected herds and culling of animals on neighboring farms. This latter practice results in less than satisfactory consequences, although once thought to be the answer to eradication of CSFV from a region, “stamping out” results in tremendous economic losses as well as the ethical objection to the potential killing of millions of healthy pigs. Marker vaccines also referred to as DIVA vaccines because they allow for differentiation between naturally infected and vaccinated animals (DIVA), could complement or replace the “stamping out” strategy. The use of a DIVA vaccine with an accompanying diagnostic test would make mass vaccination of susceptible pigs during and outbreak possible thereby preventing the rapid spread of the virus while allowing for identification of CSFV infected animal through serological surveillance. With the advent of reverse genetic technology, it is now possible to rationally design a CSFV vaccine that contains such markers. Recently, we reported the development of a live attenuated CSFV strain with two antigenic markers, named Flag T4v. During the vaccine assessment process, Flag T4v showed evidence of reverting back to the virulent phenotype of wild type CSFV. Analysis of the genome sequence from the recovered revertant virus revealed the presence of four non synonymous substitutions and a deletion of one of the antigenic epitopes compared to the parental Flag T4v genome. In order to improve the genetic stability of Flag T4v, the nucleotide codon sequence in these regions was modified as much as possible from its original sequence without compromising the wild type amino acid sequence by taking advantage of codon redundancy in an attempt to make viral reversion more difficult while maintaining both viral attenuation and reactivity of the antigenic markers. The newly developed virus, Flag T4Gv, was shown to be stably attenuated when assessed in a reversion to virulence test. In addition, Flag T4Gv was also shown to possess similar efficacy of Flag T4v in terms of protecting swine at early and late times post vaccination. Development of an Improved Live Attenuated Antigenic Marker CSF Vaccine Strain Candidate with an Increased Genetic Stability Lauren Holinka-Patterson B.A., University of Connecticut, 2001 M.A., University of Quinnipiac, 2007 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2016 i Copyright by Lauren Holinka-Patterson 2016 ii APPROVAL PAGE Doctor of Philosophy Dissertation Development of an Improved Live Attenuated Antigenic Marker CSF Vaccine Strain Candidate with an Increased Genetic Stability Presented by Lauren Holinka-Patterson, B.A., M.A. Major Advisor____________________________________________________________ Guillermo Risatti Associate Advisor_________________________________________________________ Manuel Borca Associate Advisor_________________________________________________________ Paulo Verardi University of Connecticut 2016 iii ACKNOWLEDGEMENTS Over the past fourteen years it has been my great pleasure to be a member of the Foreign Animal Disease Research Unit (FADRU) at Plum Island Animal Disease Center (PIADC). My supervisor and committee member, Dr. Manuel Borca, has been a great inspiration and exceptional mentor to me. His innovative thinking has made him a leader in the study of CSFV, ASFV and FMDV worldwide. I am grateful for the guidance, support and encouragement he has provided me over the course of this work and throughout my career. I would also like to thank my major advisor, Dr. Guillermo Risatti for sharing his tremendous wealth of knowledge in the areas of microbiology, virology, and pathobiology. I especially would like to thank Dr. Risatti for his ability to explain things clearly and simply when I needed it most. I am also grateful to committee member, Dr. Paulo Verardi for his support and advice along the way. I am also very appreciative for all the assistance that I received from Dr. Zhiqiang Lu with the sequencing of the mutant DNA plasmids and rescued viruses. Lastly, I would like to acknowledge and extend my gratitude to the animal handlers at PIADC for the outstanding service that they provide our scientific staff on a daily basis. Finally, I would like to express my gratitude to my family and friends for their unwavering love and support throughout this work. iv TABLE OF CONTENTS Page INTRODUCTION . 1 Literature Review . 4 Genome . 4 5’UTR . 5 3’UTR . 5 Npro . 6 Core . 8 Erns . 13 E1 . 17 E2 . 20 p7 . 27 NS2 . 29 NS2-3 . 30 NS3 . 31 NS4A . 33 NS4B . 33 NS5A . 36 NS5B . 37 Viral Life Cycle . 38 Translation and Protein Processing . 39 RNA Replication. 41 v Clinical Signs. .42 Pathogenesis. 45 Viral Transmission. 47 Epidemiology. 47 CSFV Reintroduction Pathways into the US . 49 Intentional Release . 49 Unintentional Release . 50 CSF Control Strategies. 52 Live Viral Vector Vaccine Candidates. 55 Non-replicating Subunit Vaccine Candidate . 57 Recombinant Pestivirus Vaccine Candidates . 59 CP7_E2alf Marker Vaccine Candidate . 71 Flag T4v Marker Vaccine Candidate . 76 Thesis Research . 78 MATERIALS AND METHODS . 81 RESULTS. 97 DISCUSSION. 110 REFERENCES. 118 vi LIST OF FIGURES Page Figure 1. Schematic representation of CSFV polyprotein . 138 Figure 2. Schematic representation CSFV Virion Structure. 139 Figure 3. Global distribution of CSFV in 2014 . 140 Figure 4. Schematic representation CSFV RB-22v and C22-Flagv . 141 Figure 5. Schematic representation of CSFV T4v polyprotein . 142 Figure 6. Schematic representation of CSFV Flag T4v polyprotein . 143 Figure 7. Daily temperature of animal groups in minimum protective dose study . 144 Figure 8. Depiction illustrating altered codon usage for Flag T4G . 145 Figure 9. Growth Curve of Flag T4Gv compared with Flag T4v and BICv . 146 Figure 10. Antigenic profile of Flag T4Gv . 147 Figure 11. Viremia Flag T4Gv protection assessment experiment . 148 Figure 12. Viremia of Flag T4GV Early Protection Study . .149 Figure 13. Cytokine Quantification in Serum. 150 Figure 14. qRT-PCR Cytokine Expression . 151 vii LIST OF TABLES Page Table 1. Amino acid and nucleotide changes observed in revertant Flag T4SPv. 152 Table 2. Nucleotide sequence of primers used for sequencing cDNA IC. 153 Table 3. Nucleotide sequence of primers used for evaluation of Flag T4SPv . 156 Table 4. Nucleotide sequence of primers for Flag T4SPv PCR amplification . 157 Table 5. Determination of minimum protective dose experiment . 158 Table 6. Survival and fever response in pigs infected with CSFV chimeric viruses representing mutations in Flag T4SPv . 159 Table 7. Nucleotide sequence of primers used in production of Flag T4Gv . 160 Table 8. Swine survival and fever response in Flag T4Gv infected animals after challenge with parental virulent BICv . ..