Progress 11/15/06 to 11/14/10
Outputs
OUTPUTS: The full-length genomes for 44 coronaviruses were sequenced, assembled, edited and annotated in this project. The data were disseminated to the scientific community in a timely manor by submitting genomes to the NCBI database, GenBank as they were completed. In addition, presentations at scientific meetings were used to update the scientific community on our progress and several publications with significant findings were submitted and published. The research involved students and the broader coronavirus research community in several ways. A series of "Special Problems" laboratory courses at the graduate level, were offered, and datasets from this project were used as raw material for a computer laboratory course (GENE8940) in the Institute for Bioinformatics, UGA. Finally a conference, which includes a section on molecular characterization and evolution of coronaviruses, will be held in Athens, GA on May 15-18, 2011. PARTICIPANTS: Dr. Jackwood, the PI, has been actively involved in sequencing the structural protein genes for a number of group III coronaviruses (sequences available through GenBank) and recently, using a hierarchical sequencing strategy, completed the whole genome sequence for the Massachusetts 41 (Mass 41) strain of infectious bronchitis virus (IBV, GenBank accession # AY851295). Paterson's laboratory has contributed to sequencing and annotation of the rice and chicken genomes, pioneered a reduced-representation approach that is gaining widespread use in plants, and is coordinating the US Department of Energy Joint Genome Institute sequencing of sorghum (http://www.jgi.doe.gov/sequencing/why/CSP2006/sorghum.html). Dr. Kissinger's laboratory is involved with integrating data sets for Trypanosoma cruzi and Apicomplexan pathogens as well as conducting comparative genomics studies for Cryptosporidium. TARGET AUDIENCES: The educational opportunities in this proposal were designed to train both students and the greater coronavirus research community. Jackwood and Kissinger used data from this project in a course (GENE8940) at the University of Georgia which is open to both graduate and undergraduate students. In addition, Dr. Jackwood with help from USDA/ARS (Dr. Patti Miller, SEPRL, Athens GA) organized a conference on molecular virology and comparative genomics of coronaviruses and parpmyxoviruses in the final year of the project to provide a means of disseminating the resources and findings of this work to the broader coronavirus research community. Dr. Jackwood was also responsible for developing and teaching a series of " Special Problems" laboratory type courses at the graduate level, whereby a number of students participated in library preparation, template production, sequencing, closure, assembly, annotation, alignment, phylogenetic analysis, and electronic publishing of data. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
During the course of this project we established a method of full-length genome sequencing of coronavirus that combined a random primed template displacement RT-PCR and specific overlapping primer amplification and sequencing to rapidly sequence each genome at 5 to 10X coverage. Results that extended directly from this project include significant molecular virology findings such as; recombination events can lead directly to emergence of new coronaviruses and coronavirus diseases, extremely rapid mutation and selection occurs in vaccine viruses (after only one infectious cycle) and in cell culture, evolution of coronaviruses occurs at different rates and can be masked by live attenuated vaccine usage, and an enormous amount of recombination occurs among coronaviruses with apparently no biological consequences. We found differences in the S1 sequence within the same vaccine serotype from different manufacturers, differences in S1 sequence between different vaccine serials from the same manufacturer, and intra-vaccine differences. To our knowledge, this is the first report showing selection of a more fit quasispecies as well as genetic mutations associated with replication of modified live IBV vaccine viruses in chickens. Comparative genomics revealed two recombination sites that replaced the spike gene in IBV with an unidentified sequence likely from another coronavirus, resulting in cross-species transmission and a pathogenicity shift. Following emergence in turkeys, TCoV diverged to different serotypes through the accumulation of mutations within spike. This is the first evidence that recombination can directly lead to the emergence of new coronaviruses and new coronaviral diseases. Analysis of the consensus full-length genome for three different IBV serotypes showed that passage in embryonated eggs, to attenuate the viruses for chickens, resulted in 34.75 percent to 43.66 percent of all the amino acid changes occurring in nsp 3 within a virus type. Both attenuated and pathogenic viruses of the same serotype replicated similarly in embryonated eggs suggesting that mutations in nsp 3, which is involved in replication of the virus, might play an important role in the reduced replication observed in chickens leading to the attenuated phenotype. The full-length genomes of eleven avian coronavirus field isolates isolated over a 41, 25 and 8 year period respectively, were sequenced and analyzed to determine the mutation rates and level of polymorphisms across the genome. Lower levels of genetic diversity among the Mass and Conn viruses as well as sequence similarities with vaccine virus genomes suggest that the origin of the Mass and all but one of the Conn viruses was likely vaccine virus that had been circulating in the field for an unknown but apparently short period of time. We observed recombination occurring among coronaviruses across the entire genome. Every sequence included in the analysis was recognized as a potential recipient of horizontally acquired sequences at some point in the viral evolutionary past. Regions involved in the greatest proportion of recombination events are nsp3 and spike.
Publications
- McKinley, E. T., D. A. Hilt, and M. W. Jackwood Avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination. Vaccine 26:1274-1284. 2008.
- McKinley, E. T., M. W. Jackwood, D. A. Hilt, J. C. Kissinger, J. S. Robertson, C. Lemke, A. H. Paterson. Attenuated live vaccine usage affects accurate measures of virus diversity and mutation rates in avian coronavirus infectious bronchitis virus. Submitted, Virus Research. January 2011.
- Phillips, J. E., M. W. Jackwood, E. T. McKinley, S. W. Thor, D. A. Hilt, N. D. Acevedol, S. M. Williams, J. C. Kissinger, A. H. Paterson, J. S. Robertson, C. Lemke. Changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus. Submitted, Virus Research. January 2011.
- Phillips, J. E., M. W. Jackwood, D. A. Hilt, J. C. Kissinger, A. H. Paterson, J. S. Robertson, C. Lemke. Comparative sequence analysis of the complete genomes of Feline Infectious Peritonitis Virus at different tissue culture passage levels and virus replication in cell culture. In preparation February 2011.
- Thor, S. W., M. W. Jackwood, J. E. Phillips, D. A. Hilt, J. C. Kissinger, A. H. Paterson, J. S. Robertson, C. Lemke. Recombination in coronaviruses. In preparation February 2011.
- Jackwood, M. W., T. O. Boynton, D. A. Hilt, E. T. McKinley, J. C. Kissinger, A. H. Paterson, J. Robertson, c. Lemke, A. W. McCall, S. M. Williams, J. W. Jackwood, and L. A. Byrd. Emergence of a Group 3 Coronavirus Through Recombination. Virology, 398:98-108. 2010.
- Jackwood, M. W., D. A. Hilt, S. M. Williams, P. Woolcock, C. Cardona, and R. O Connor. Molecular and serologic characterization, pathogenicity and protection studies with infectious bronchitis virus field isolates form California. Avian Dis. 51:527-533, 2007.
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Progress 11/15/08 to 11/14/09
Outputs
OUTPUTS: We have sequenced, assembled and annotated the full-length genome of 24 coronaviruses. Those sequences were submitted to GenBank. We are in the final stages of annotating full-length genomes for 7 more coronaviruses and are conducting finishing sequencing and assembly of 16 additional coronaviruses. PARTICIPANTS: Mark W. Jackwood, Tye O. Boynton, Deborah A. Hilt, Enid T. McKinley, Amber W. McCall, Susan M. Williams, Joshua W. Jackwood, Lauren A. Byrd Department of Population Health, College of Veterinary Medicine, 953 College Station Road, University of Georgia, Athens, GA, 30602, USA Jessica C. Kissinger, Department of Genetics, Center for Tropical and Emerging Global Diseases, University of Georgia, 500 D. W. Brooks Drive, Athens GA 30602 Andrew H. Paterson, Jon Robertson, Conelia Lemke Plant Genome Mapping Laboratory, Departments of Crop and Soil Sciences, Plant Biology, and Genetics, University of Georgia, 111 Riverbend Road, Athens GA 30602 Undergraduate training and development students: Joshua Jackwood, Lauren Byrd GENE8940 Evolution of RNA viruses was taught to graduate students Sp2009. TARGET AUDIENCES: Information on emergence of coronaviruses was reported to the scientific community via the publication in Virology. In addition, timely uploads of full-length genome sequence to GenBank was made available to the coronavirus scientific community at large. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
To study the rate of IBV evolution, we sequenced the entire genome of eleven IBV field viruses of the Massachusetts (Mass), Connecticut (Conn) and California (Cal) serotypes isolated over a forty-one, twenty-five and eight year period respectively. The Cal viruses, which Poultry producers do not vaccinate against showed evidence of genetic evolution and had a higher mutation and evolutionary rates than the Mass and Conn isolates that are routinely vaccinated against. The Mass and Conn field viruses in our study were likely vaccine viruses that were circulating in the field for a short period of time while the Cal viruses were true field isolates circulating in birds for a long time. This is the first detailed and compressible sequence analysis of coronavirus genomes, namely IBV, that assesses the rate of CoV evolution from viruses isolated over several decades. Turkey coronavirus (TCoV) an enteric disease virus that is highly similar to infectious bronchitis virus (IBV) an upper-respiratory tract disease virus in chickens, were examined to determine the adaptive potential, and genetic changes associated with emergence of this group 3 coronavirus. Comparative genomics revealed two recombination sites that replaced the spike gene in IBV with an unidentified sequence likely from another coronavirus. This change resulted in cross-species transmission and a pathogenicity shift. Then TCoV diverged to different serotypes through the accumulation of mutations within spike. This is the first evidence that recombination can directly lead to the emergence of new coronaviruses and new coronaviral diseases, emphasizing the importance of limiting exposure to reservoirs of coronaviruses that can serve as a source of genetic material for emerging viruses.
Publications
- Jackwood, Mark W., Tye O. Boynton, Deborah A. Hilt, Enid T. McKinley, Jessica C. Kissinger, Andrew H. Paterson, Jon Robertson, Conelia Lemke, Amber W. McCall, Susan M. Williams, Joshua W. Jackwood, Lauren A. Byrd Emergence of a group 3 coronavirus through recombination. Virology Accepted Nov 2009.
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Progress 11/15/06 to 11/14/07
Outputs
OUTPUTS: We are sequencing a total of 54 coronaviruses. To provide a comprehensive database of genetic information for the animal coronaviruses, we will sequence viruses from dogs, cats, pigs, cattle, rats, rabbits, mice, turkeys and chickens. Viruses of the same type (infectious bronchitis virus (IBV), Mass 41 and Conn) isolated over a 40-year period (from 1965 to 2005) are being sequenced to provide information on the rate of genetic changes that can be used to study Darwinian adaptive evolution among coronaviruses. We are also sequencing a temporal series of TCoV, GA98, and Delaware IBV isolates obtained prior to, during, and after the emergence of TCoV and GA98. Phylogenetic analysis of these genomic sequences will reconstruct the changes associated with tissue tropism changes and host shifts that can lead to newly emerging coronaviral diseases. Finally, we are sequencing paired pathogenic and attenuated progeny viruses from cats and chickens, which will provide critical
databases of variation that can be used to identify genetic markers of disease as well as develop unique diagnostic tests and novel vaccines. All of the viruses have been thoroughly characterized and carefully selected to provide biologically significant data. We are still in the process of collecting data, sequence assembly, editing and annotation. We will begin examining the biological significance of the data once we have the first group of related group of viruses completely sequenced. Initially, the sequence will be made public through timely submissions to GenBank (www.ncbi.nlm.nih.gov/Genbank/index.html).
PARTICIPANTS: Three graduate students (two Ph.D. and one M.S. student) in Dr. Jackwood's laboratory are currently working on viral genome amplification and cloning for the project. A course on nucleic acid sequencing and annotation was taught at the University of Georgia (Genetics Department) by Dr. Kissinger with input from Dr. Jackwood. Dr. Paterson's group has been involved with processing clones and generating sequence data. A graduate level special problems course on sequence analysis, annotation, and analysis will be taught (SP2008) at the University of Georgia by Dr. Jackwood.
TARGET AUDIENCES: The genomic sequence data developed herein will provide databases of sequence variation that can be used by the greater coronavirus research community to design sensitive and specific diagnostic tests as well as develop novel vaccine approaches.
PROJECT MODIFICATIONS: None.
Impacts
We are finishing the first year of a 3 year project. The first 2 years are dedicated to collecting and analyzing the data whereas the final year will be dedicated to examining results and describing findings. Thus, there are no findings, results or techniques to report at this time.
Publications
- No publications reported this period
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