Progress 09/01/13 to 08/31/16
Outputs
Target Audience:The target audience constitutes mainly the BRD as well as the herpesvirus community that will benefit once the genome annotation has been revised. In addition, graduate and undergraduate students associated to these studies obtain training that contributes to their intellectual and professional development. Ultimately the knowledge gained from these studies may be adopted by end-users such as farmers and cattle businesses to make informed decisions about how to reduce the economic impact of BRD. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training is an important portion of academic responsibilities. In addition to my current teaching appointment (70%), both graduate and undergraduate students are being trained with this project, with one of them (Kaley Barber) directly funded through this USDA-NIFA grant. This project has involved a lot of computational analysis (e.g. use of Artemis and other softwares and the generation of .gff files) and has resulted in vast learning opportunities for students in my lab. During the summer of 2015, through an NSF grant (DBI-1262901): Undergraduate Research in Computational Biology at Mississippi State University, we recruited an undergraduate student from Hinds County Community College, Allison Martin. She is a computational biology major and helped work in this project generating the files for genome mapping. In addition a graduate student, Victoria Jefferson, participated during the summer of 2016 with laboratory work, mainly RT-PCR analyses. How have the results been disseminated to communities of interest?Kaley Barber, Joseph Reddy, Mariola Edelmann, Bindu Nanduri, Florencia Meyer (2016) Revision of Bovine Herpesvirus Type 1 Annotation Using Experimental Data. 41St International Herpesvirus Workshop, Madison WI. Poster and Oral presentation. Kaley A. Barber, Hillary C. Daugherty, Stephanie E. Ander, Victoria A. Jefferson, Allen Shack, Bindu Nanduri and Florencia Meyer. Protein composition of the Bovine Herpesvirus type 1.1 virion. South Central Branch of the American Society for Microbiology (ASM) held in Lafayette, LA. Poster. What do you plan to do during the next reporting period to accomplish the goals?In the future we plan to start the characterization of the novel transcripts and protein pairs. The work is currently being prepared for publication.
Impacts
What was accomplished under these goals?
p { margin-bottom: 0.08in; direction: ltr; }p.western { ; }p.cjk { ; }p.ctl { ; }a:link { } Our study makes use of proteomic data derived from infected cells to revise the current annotation of the viral genome. The genome of BHV-1 was originally sequenced more than 15 years ago using a combination of 5 viral strains. In 2013 a recent sequencing effort of the BHV-1.1 Cooper strain of has certainly provided clarity to regions of the genome that corresponded to the other 4 strains used in the original sequencing. We now use this sequence (NCBI accession number JX898220) for analysis of our data. Objective 1. Experimentally annotate the BHV-1 genome during productive infection. We infected bovine cells with BHV-1 at multiplicity of infection of 1, and processed whole cell extracts for mass spectrometry. The samples corresponded to 2 hours post infection (hpi), 8 hpi and 16 hpi, in addition to mock infected samples. Three biological replicates of each time point were processed and submitted to the Arizona proteomics Consortium, University of Arizona. Mass spectra and tandem mass spectra were analyzed using our custom proteomics pipeline available at iPlant. Database searches using this pipeline were performed using Xtandem algorithm for peptide identification. The BHV-1 genome (GenBank JX898220) was used to generate a randomized nucleotide sequence database that serves as a decoy for peptide identification analysis and controls for false discovery rates. This randomized database has the same size, base composition and coding potential as the viral genome [1]?. The BHV-1 genome and the decoy database were translated in all 6 reading frames using in house perl scripts, and mass spectra were searched against these two databases to obtain the final list of viral peptides present in each sample. The genomic coordinates for the peptides were obtained by using the proteogenomic mapping tool [2]? and were visualized in Artemis browser. Artemis browser takes the genome sequence and the list of peptides with genome coordinates in .gff format and displays the peptide as a track for easy inspection and evaluation. Viral peptides present at 2, 8 and 16 hours post infection (hpi) were visualized using Artemis. We confirmed the expression of all but 3 of the viral proteins, with an average 26% gene coverage. Approximately 80 unique peptides did not map to any of the currently annotated ORFs. These intergenic peptides may represent novel proteins, 5'/3' extensions of known proteins or novel splicing activity. These intergenic peptides were analyzed separately by scanning up- and downstream genomic regions in search of start and stop codons to predict the potential ORFs. In this way, twenty six novel ORFs have been predicted. Specific primers targeted to these regions were designed to perform strand-specific cDNA synthesis, followed by PCR. We have detected amplification products for ten of the predicted ORFs, suggesting that the transcripts exist. Future experiments will characterize the transcripts to establish their 3' and 5' boundaries. Objective 2. Characterize the virion proteome. With regards to determining the protein composition of the viral particle, we have established a reliable virion purification technique, where we track infectious particles along the purification protocol and assess virion purity with TEM imaging. Imaging at 80,000X magnification revealed clean preparations and a virion morphology typical of herpesviruses with the nucleocapsid measuring 68.55 ± 0.58 nm and enveloped virions 166.0 ± 2.61 nm. Infectious viral titers in these preparations were ~1x109 pfu/mL. Virions were solubilized and trypsinized, and later subjected to LC-MS/MS analysis. To control for cellular debris that may co-purify in the centrifugation process, a negative control consisted of the same volume of uninfected and concentrated culture supernatants. We detected 40 viral proteins that compose the virion. Expectedly we identified most of the glycoproteins with the exception of two, as well as the components of the nucleocapsid UL19, UL38, UL18 and scaffolding proteins UL26 and UL26.5. We detected most of the known tegument components and a number of viral regulatory proteins. Amongst the host proteins found in the virion preparations were histones, tubulin and ribosomal proteins. Other host proteins such as actin, annexins and heat shock proteins also appeared, but these were also present in the uninfected control, suggesting they may be a common co-purifying host protein. This work has been submitted for publication. 1. Kunec D, Nanduri B, Burgess SC (2009) Experimental annotation of channel catfish virus by probabilistic proteogenomic mapping. Proteomics 9:2634-47. doi: 10.1002/pmic.200800397 2. Sanders WS, Wang N, Bridges SM, et al. (2011) The proteogenomic mapping tool. BMC Bioinformatics 12:115. doi: 10.1186/1471-2105-12-115
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
Protein composition of the Bovine Herpesvirus 1.1 virion (2016)
Kaley A. Barber, Hillary C. Daugherty, Stephanie E. Ander, Victoria A. Jefferson, Allen Shak, Bindu Nanduri and Florencia Meyer 1.
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Progress 09/01/14 to 08/31/15
Outputs
Target Audience:The target audience constitutes mainly the BRDC as well as the herpesvirus community that will benefit once the genome annotation has been revised. In addition, graduate and undergraduate students associated to these studies also benefit as they obtain direct training that contributes to their intellectual and professional development. Changes/Problems:I have requested a no-cost extension for this project, which has been granted. Expiration date is now August 31, 2016. What opportunities for training and professional development has the project provided?Training is an important portion of academic responsibilities. In addition to my current teaching appointment (70%), both graduate and undergraduate students are being trained with this project, with one of them (Kaley Barber) directly funded through this USDA-NIFA grant. This project has involved a lot of computational analysis (e.g. use of Artemis and other softwares and the generation of .gff files) and has resulted in vast learning opportunities for students in my lab. During the summer of 2015, through an NSF grant (DBI-1262901): Undergraduate Research in Computational Biology at Mississippi State University, we recruited an undergraduate student from Hinds County Community College, Allison Martin. She is a computational biology major and helped produce the gff files for genome mapping. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We are currently preparing samples for another proteomics experiment using a different protease to digest the infected cell proteins. This is known to improve protein coverage by providing a different array of peptides and increases the chances of identifying more viral proteins. In addition we are also initiating RT-PCR experiments to determine whether there are transcripts that correspond to some of the intergenic peptides that we have encountered. As far as objective number 2, we are completing the 2 additional biological replicates that would complete the virion proteomic experiments. Data analysis and writing of the manuscripts will also be accomplished during the coming year.
Impacts
What was accomplished under these goals?
We are using proteomic data derived from infected cells to revise the current annotation of the viral genome, which was done more than 15 years ago using a composite of 5 viral strains. A recent sequencing effort of the BHV-1.1 Cooper strain of has certainly provided clarity to regions of the genome that corresponded to the other 4 strains used in the original sequencing. We now use this sequence (NCBI accession number JX898220) for analysis of our data. We infected bovine cells with BHV-1 at multiplicity of infection of 1, and processed whole cell extracts for mass spectrometry. The samples corresponded to 2 hours post infection (hpi), 8 hpi and 16 hpi, in addition to mock infected samples. Three biological replicates of each time point were processed and submitted to the Arizona proteomics Consortium, University of Arizona. Mass spectra and tandem mass spectra were analyzed using our open source, 'custom proteomics pipeline' available at iPlant. Database searches using this pipeline were performed using Xtandem algorithm for peptide identification. The BHV-1 genome (GenBank JX898220) was used to generate a randomized nucleotide sequence database that serves as a decoy for peptide identification analysis and controls for false discovery rates. This randomized database has the same size, base composition and coding potential as the viral genome [1]. The BHV-1 genome and the decoy database were translated in all 6 reading frames using in house perl scripts, and mass spectra were searched against these two databases to obtain the final list of viral peptides present in each sample. The genomic coordinates for the peptides were obtained by using the proteogenomic mapping tool [2] and were visualized in Artemis browser. Artemis browser takes the genome sequence and the list of peptides with genome coordinates in .gff format and displays the peptide as a track for easy inspection and evaluation. The retrieved list of peptides contained 170 viral peptides at 8hpi and 401 peptides at 16 hpi, representing a total of 46 viral proteins. We found no viral peptides at 2 hpi. All the proteins detected at 8hpi were also detected at 16 hpi with more peptides representing each protein. The % coverage of detected proteins ranged from 1%-50%, with a median of ranged from In addition, 55 peptides did not map to currently annotated sequences and could constitute novel proteins or 5' or 3' extensions of existing proteins, if mapping to the same reading frame. Some of these peptides map closely together on a given reading frame, supporting the notion that they may derive from a yet undescribed protein. 52 of the intergenic peptides map to predicted ORFs using the FGENESV0 ORF predictor for viral genomes. For example, glycoprotein H was well represented in our data (about 10 peptides), and 2 peptides also mapped to the thymidine kinase gene. However, no peptides were found mapping to UL20 envelope protein. In the case of tegument protein UL21, three peptides represent it, but a 4th peptide maps downstream of annotated coding sequences (on the same reading frame), suggesting that this protein may be longer than originally predicted. With regards to determining the protein composition of the viral particle, we have established a reliable virion purification technique, where we track infectious particles along the purification protocol and assess virion purity with TEM imaging. The purification yiended an infectious viral titer of 1x109 pfu/mL. Virions were treated with 4% SDS to ensure that all virion proteins (including capsid components) were being completely solubilized, and this was followed by tryptic digestion and mass spectrometry analysis. The purification of virions from infected culture supernatants was repeated with the same volume of uninfected culture supernatants to control for cellular debris that may co-purify in the centrifugation process. Preliminary results (one biological replicate) detected 37 virion proteins, including all but one of the glycoproteins and most capsid proteins and more than half of the tegumetn proteins. Our mock-infected control supernatants only retrieved 10 host proteins, while our virion preparation only had 8 host proteins. Two proteins were found in both lists (serum albumin and fetuin). These results indicated that the virion preparation is very clean. In addition, aliquots visualized by TEM also reinforce the clean nature of the preparation. We have two additional biological replicates virion preps, as well as mock-infected concentrated supernatants. Joint analyses of all three replicates will provide a more accurate picture of the virion composition. 1. Kunec D, Nanduri B, Burgess SC (2009) Experimental annotation of channel catfish virus by probabilistic proteogenomic mapping. Proteomics 9:2634-47. doi: 10.1002/pmic.200800397 2. Sanders WS, Wang N, Bridges SM, et al. (2011) The proteogenomic mapping tool. BMC Bioinformatics 12:115. doi: 10.1186/1471-2105-12-115
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
K. Barber, B. Nanduri, M. Edelmann, J. Reddy, A. Martin and F. Meyer. Improving the Annotation of Bovine Herpesvirus-1 Genome Using Experimental Data. South Central Branch of the American Society for Microbiology (ASM) held in Hattiesburg, MS.
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Progress 09/01/13 to 08/31/14
Outputs
Target Audience: The target audience constitutes mainly the BRDC as well as the herpesvirus community that will benefit once the genome annotation has been revised. In addition, graduate and undergraduate students associated to these studies obtain training that contributes to their intellectual and professional development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Training is an important portion of academic responsibilities.In addition tomy currentteaching appointment(70%), Ihavementoredanundergraduate studentunder this project:Kaley Barber's work isdirectlyfunded throughthisUSDA-NIFAgrant.A new PhD student has recently joined the lab and will be working in this project as well. In addition during this period a Master's studentwhograduated in August 2014and an undergraduate student have helped in the early stages of virus concentration and purification.I also mentorseveral undergraduate students and more recently a high school student highly interested in research. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? We plan to submit infected cell samples to the proteomic facility soon. In addition, purified virions will also be submitted very soon. Once the proteomic data I retrieved we will devote to data analysis and annotation efforts. As as part of the personnel in this project we have a computer analyst, Mr. Tony Arick, that will be instrumental with data analysis and genome mapping.
Impacts
What was accomplished under these goals?
IMPACTS: Our research focuses on the study of Bovine herpesvirus type 1 (BHV-1), an agriculturally important pathogen responsible, in part, for the initiation of a complex disease in cattle termed Bovine Respiratory Disease Complex (BRDC). The incidence of BRDC on cattle can account for billion-dollar losses to the beef and dairy cattle industry in the US, and this industry is also an important economic activity in Mississippi. A complete understanding of the virus and its genomic elements is fundamental to advance research to help reduce its overall incidence in the field and to maintaining animal health. Because the information about the genomic arquitecture of this virus is outdated, our studies constitute an effort in re-defining some gene boundaries and in potentially discovering new genomic elements. These in turn may prove to be critical for viral infection and spread. Our studies might ultimately lead to novel therapeutic or practices to prevent the cattle from developing respiratory disease during shipping or stressful conditions. In turn, this will alleviate the cattle industry from large monetary losses. ACCOMPLISHMENTS Our lab is interested in understanding how the virus interacts with the host cells to cause pathogenesis. We believe that a very good first step is to revise the current annotation of the viral genome, which was done more than 15 years ago. A recent effort has sequenced completely the Cooper strain of BHV-1.1 which has certainly contributed clarity to the annotation. We will add further detail to the current annotation by layering experimental proteomic data, that is, characterizing all the viral proteins that are produced in the infected cell, then mapping those peptides back onto the genome. Proteomic data will confirm and revise coding sequence boundaries and may potentially discover new genomic elements. In addition, we will fully characterize the proteome of whole extracellular virions. This project has has had a slow start due to administrative issues [it was awarded on 1/13/2014, and not deposited until mid February 2014]. We have made significant progress in the virion purification technique, tracking infectious particles along the purification protocol and substantiating various steps with TEM imaging. Accordingly we have optimized several of the steps and we believe that we are now close to obtaining high purity virion preparations. Both graduate and undergraduate students are being trained in these efforts. In addition, we are submitting the first set of proteomic samples to work towards the first objective. Meanwhile, the involved parties are being trained in the tools that will be needed for future data analysis, such as the usage of Artemis and other softwares or generation of gff files. This will guarantee that once the proteomic data is retrieved the analysis phase will proceed smoothly.
Publications
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