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Novel Plant Viral Genome Sequencing and Characterization

Novel Plant Viral Genome Sequencing and Characterization. Jiaxi Quan Bioengineering Center. Hypothesis. The genomic organization of plant virus plays an important role in the interactions of its host with the environment.

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Novel Plant Viral Genome Sequencing and Characterization

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  1. Novel Plant Viral Genome Sequencing and Characterization Jiaxi Quan Bioengineering Center

  2. Hypothesis • The genomic organization of plant virus plays an important role in the interactions of its host with the environment. • Revealing the genomic structure of virus will provide evidence for addressing the questions of virus-host interaction, virus taxonomy and evolutionary relationships. • Closely related viruses are homologous in coat protein genes and this homology determines the serological relationship among these viruses.

  3. Specific Aims • Determine the population of viruses that are present in different distinct regions of Costa Rican rain forest. • Discover and characterize previously unobserved virus via in silico analysis. • Verify the classification of selected novel viruses through serological analysis and ELISA of cloned virus genomes. • Analyze the host effect on virus genetic diversity with both in silico and statistical analysis.

  4. Why study virus? Viruses play a major role of all complex ecosystems Viruses are one of the most understudied taxanomic groups Significance

  5. Backgroundvirus • Viruses are small biological entities that require host for reproduction • Hosts could be animals, plants or bacteria • Composed of nucleic acid and protein coat • Virus genomes may be either DNA or RNA tobacco mosaic virus seen by transmission electron microscopy tobacco mosaic virus cartoon

  6. Backgroundvirus

  7. 1. Sample collection and cDNA libraries construction 2. cDNA sequencing 3. Genome annotation 4. Phylogenetic study 5. Functional study Materials and Methods Outline

  8. BackgroundPlant virus • Most plant virus genomes are single (+)stranded RNA • Compared to animal hosts, the plant host system is advantageous • Plant viruses are ideal models for related studies in virus evolution

  9. Specific Aim 1 • Determine the population of viruses that are present in different distinct regions of Costa Rican rain forest by: • Sequencing approximately 5,000 dsRNA plant virus genomes using massively parallel 454-based pyrosequencing. • The dsRNA first will be converted into cDNA libraries and then multiplexed viral genomic DNA samples will be subjected to 454-based pyrosequencing.

  10. Biotin Specific Aim 1General overview of 454 DNA Sequencing Amplified, Tagged Viral cDNA Linker/Adaptor Ligation Purification of fragments with AB combination using strepavidin beads SS-DNA Isolation (library) Amplification (emPCR) Pyrosequencing (on 454-GS20)

  11. Specific Aim 1Emulsion PCR • Anneal Single- Stranded template to DNA Capture beads • Emulsify beads and PCR reagents in water-in-oil microreactors • - “B” primer is in solution and attached to capture bead • - “A” primer is biotinylated • Emulsion PCR Amplification • Break Emulsion Microreactors • Enrich for DNA positive beads • Load beads into Picotiter Plate Before PCR After PCR

  12. 44 μm Specific Aim 1Load Beads into 454 Plate Load Enzyme Beads Load beads into PicoTiterPlate Centrifugation

  13. Polymerase DNA Bead • Polymerase adds • nucleotide (dNTP) (1) dTTP A A T C G G C A T G C T A A A A G T C A T APS Annealed Primer (2) • Pyrophosphate • is released (PPi) PP Sulfurylase Luciferase ATP (3) i • Sulfurylase creates ATP • from PPi and APS Enzyme Bead (5) luciferin (4) CCD camera detects bursts of light • Luciferase hydrolyses ATP • to oxidize luciferin and • produce light Light + oxy luciferin Specific Aim 1Pyrosequencing

  14. Specific Aim 1Sample Collection and cDNA library construction Plant samples are collected from ACG (Area Conservation Guanacaste in Northwestern Costa Rica) Extraction of dsRNA • dsRNA is a hallmark of plant virus infection

  15. Specific Aim 1Tagged, Random Hexamer RT-PCR Tagged primer design

  16. Specific Aim 1cDNA sequencing on the 454/Roche GS-FLX • The cDNA of twenty samples, each one amplified with its unique tagged primer, • Pooled and loaded on each of the 16 strips of 454 slides • This gives around 7X coverage of each sample

  17. Specific Aim 1Data Processing signals across the wells flowgrams assembly

  18. Specific Aim 2 • Discover and characterize previously unobserved virus through in silico analysis by: • Developing an Oracle database to store viral collection information, DNA sequence results, protein coding regions and database search results that will provide a platform for studying the diversity distribution of these viruses on different plant species in the distinct rain forest climates. • Classify new virus into their corresponding taxonomic groups based on phylogenetic trees, genomic structure and motif information.

  19. Compiled sequences Search against Genbank Deposit into Oracle Database Find novel sequences Find identical sequences Find similar sequence groups Predict ORF/Gene Predict taxonomical Status of novel sequences Predict Gene Function Multi-infection analysis Genomic diversity and distribution Specific Aim 2Data Analysis

  20. Specific Aim 2Genome Annotation Genomic organization of Potyvirus family Genome structure of Kyuri green mottle mosaic virus (KGMMV-C1) Genome organization of CiLV-C

  21. Specific Aim 2Blastp

  22. Specific Aim 2Blastp

  23. Specific Aim 2Blastp …

  24. Specific Aim 2How to deal with blast results? Each blast result Select high similarity hits (E<e-5) yes no Novel viral sequence Complete genome Partial genome Parse selected data i.e. Identities Global alignment Parse data with Perl Known viral sequence Sequences that need further analysis Potentially known virus Store into different tables Parse data with Perl

  25. Specific Aim 2Artemis

  26. Specific Aim 2Database Schema

  27. Specific Aim 2Phylogenetic studies to find the taxonomical grouping of novel viruses ClustalW – make multiple alignment

  28. Specific Aim 2Phylogenetic study • Phylip – draw phylogenetic tree Relationship between coat protein amino acid sequences of selected potyviruses with Phylip

  29. Specific Aim 3 • Verify the classification of selected novel viruses through serological analysis and ELISA of cloned virus genomesby: • Cloning selected viruses and measuring their infectivity after inoculation into plants. • Produce antibodies against the viruses extracted from the infected plants and cross reactions using ELISA • Determine the taxonomy of selected viruses phylogenetically.

  30. Specific Aim 3Serological analysis Convert dsRNA sample into full length cDNA Amplify the cDNA with designed primers, 5’primer contains CaMV 35S promoter sequence Clone the PCR product into vector pUC18 Full length cDNA clone construction:

  31. Specific Aim 3Serological analysis Infectivity assay and morphology detection: • Full length cDNA clone controlled with CaMV 35S promoter is mechanically innoculated into the plants with the aid of carborundum • The viruses of infected plant samples are extracted • Electron microscope detection

  32. Specific Aim 3Serological analysis Obtaining polyclonal antiserum of the virus: • The extracted virus particles will be emulsified in Freund’s complete adjuvant for the first injections followed by two injections in incomplete Friund’s adjuvant • The antiserum will be separated from blood collected after the final immunization and then will be used in ELISA

  33. Specific Aim 3Serological analysis - ELISA • Apply Antigen Coat each well of a microtiter plate by adding virus particles diluted in coating solution. • Block Plate Block wells by adding BSA/PBS and incubate. • React Primary Antibody Add primary antibody diluted in BSA/PBS to each well. • Add Secondary Antibody Solution Add diluted secondary antibody conjugated to horseradish peroxidase to each well. • React Substrate Substrate solution is added into each well, develop at room temperature. The entire plate is placed into a plate reader at interval times.

  34. Specific Aim 3Multiple infection and generic diversity • Plant hosts will be infected with full length cDNA clones • Collected sap samples used to inoculate more hosts up to 10 passages • Viral RNAs from individual plants extracted and used for RT-PCR • PCR product cloned and sequenced with Sanger method, 20 clones selected for each population • Sequences will be aligned and compared to find mutation bias, mutation type, mutation cluster, hot spots and mutation-free zones

  35. Summary • Sequence the plant virus genomes collected from ACG • Data analysis to characterize virus genomes • Classify the novel viruses into their taxonomical status • Develop database to study the virus diversity, incidence, multi-infection • Serological studies based on ELISA to confirm the classification based on phylogenetic analysis • Genetic diversity analysis

  36. Acknowledgment • Dr. Roe • Dr. Nollert, Dr. Harrison, Dr. Li, Dr. Sikavitsas • All the members in the lab • Dr. Roossinck at Noble Foundation • NSF

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