1. Generation of Gateway clone library of virulence associated genes of zoonotic buffalopox virus: state-of-the-art resource for proteome analysis Veterinary Type Culture Collection, National Research Centre on Equines, Hisar, Haryana, India B.C. Bera, Taruna Anand, Sanjay Barua, R. K. Vaid, Nitin Virmani, Riyesh T. & Praveen Malik

2. Generation of repository of Open Reading Frames (ORFs) clones – ORFeome of zoonotic buffalopox virus in Veterinary Type Culture Collection (VTCC) repository Objective VTCC- National Repository Veterinary pathogens Dairy microbes Genetic material Rumen microbes ICAR created this facility for conservation of microbes of veterinary importance Act as biological resource bank for R&D

3.  Refers to the libraries of complete set of clones of protein-coding open reading frames (ORFs)  Collection of plasmids containing ORFs of a genome  Flexible & versatile library allows transfer of ORFs into different destination vectors

4. Advent of systems biology necessitates the cloning of nearly entire sets of ORFs to allow functional studies of proteomes New challenges in post genomic era:  to understand the function of the many genes predicted  Analysis of all genes at a time  high-throughput preparation of versatile resource for the functional and structural studies of proteins Resources for functional genomics projects Why ORF clones ?

5. Proteins profiles ORFeome Localizome Phenome Transcriptome Interactome Proteome Cloned ORFs Mutational phenotypes Expression profiles Protein interactions 1 2 3 4 5 n DNA InteractomeProtein-DNA interactions Cellular, tissue location ORFeome: Gateway b/w Genomics & Omes

6.  Functional genomics- Proteomic studies  To study host-tropism (molecular pathogenesis)  To develop drugs & vaccines Why ORFeome of animalpox viruses?

7.  Zoonotic infections  Reduction of cohort- immunity against poxviruses in humans  Discontinuation of vaccination against smallpox since 1980  Change of host tropism (inter-species jumping)  BPXV – Human & Cow © VTCC - Hisar7 Buffalopox virus

8. Severe cases BPXV Zoonosis In 2011: Meerut, U.P. In 2013: BPXV Nashik, M.P.

9. One step site specific recombination based cloning technology Not dependent on restriction/ligation Efficiency: 100% - only one recombinant DNA product without byproducts No cloning step needed: no need to assay independent clones Very precise recombination system allowing high fidelity DNA engineering Versatile cloning technology:  Genes can be easily transferred into a range of vector systems  Expression, Gene fusion, RNAi… GATEWAY Recombinational Cloning  Based on the bacteriophage lambda integration & excision system Generation of ORF clones by Recombinational cloning

10. attP attB attL attR phage Bacterial genome Excision Integration phage Lambda phage integration & excision system

11. attB2 partial Viral DNA start stop Partial attB1 PCR1 ORF attB1 partial Designed primers ORFs of virulence associated genes of BPXV ORF Complete attB2 Complete attB1 PCR2 Gateway cloning strategy

12. Genes of BPXVFunctions Vaccinia virus homologue genes: CrmB, CKBP, INFA, IL-18, C7L, C3L, ZFA, N1L, K1L, K2L, K3L, B29R, K7R, A39R, A46R, B5R, VACWR208, L5R, H1L, H2R, H3L, VACWR217, A9L, A17L, A21L, VACWR207, A28L, B1R, N2L, F3L, F10L, F34L, A36L, A38L, A40R, A43R, A44L, A46R, A55R, B4R, B6R, B8R, B12R, B13R, B19R, B25R, C12L, M1L, A56R, B18R Modulation of host immune defense *© VTCC - Hisar12 Targeted BPXV-ORFs

13. PCR amplifications of BPXV-ORFs 1kb M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M 1kb M = 1kb DNA marker, L1-18 & L1-14 = amplicons of ORFs of BPXV

14. PCR amplifications of BPXV-ORFs M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1kb M = 1kb DNA marker, L1-14 & L1-16 = amplicons of ORFs of BPXV

15. Phage lambda integration: Integrase & bacterial IHF ORF attB2 attB1 ccdB attP2 attP1 pDONR221 ORF attL2 attL1 Entry clones ccdB BP reaction By product E. Coli transformation ORF attL1 attL2 PCR verification Sequencing & ORF identification (BLAST) Preservation of validated clones in the repository Construction of Entry clones

16. Generated gateway entry clones 50 gateway entry clones of BPXV-ORFs generated All clones validated by sequencing and BLAST analysis Clones preserved in the VTCC repository:  5 clones (recombinant E.coli) of each ORF stored as glycerol stock at -80 o C  Purified recombinant plasmids stored at -80 o C as ethanol precipitate

17. ccdB attR2 attR1 Destination vector- pDEST17 Amp R vORF attL2 attL1 Entry clones- pDONR221-vORF Kan R LR reaction Phage lambda excision: Integrase, IHF & Exisionase vORF attB2 attB1 Destination vector- pDEST17-vORF Amp R ccdB attP2 attP1 By product Kan R E. Coli transformation Selection of Amp resistant clones Expression of recombinant protein Recombinational cloning into destination vector

18. 170 kDa 130 kDa 95 kDa 72 kDa 55 kDa 43 kDa 34 kDa 26 kDa ~32 kDa rA38L Expression of A39R protein

19. Conclusion  Generated entry clone resource of 50 ORFs of virulence associated genes of buffalopox virus -  Platform for functional genomics  Basic biology: molecular networks, structural & functional analysis  Understanding pathogenesis: virus−host interaction  Identifying vaccine candidates : reverse vaccinology

20. *© VTCC - Hisar20 Thank you for kind attention

21. APPLICATIONS OF RECOMBINATIONAL CLONING Reprinted from: Dupuy et. al., Genome Research 14:2169-2175 (2004)