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Phylogenetic Analyses of Lymphocystis Disease Virus of Fish using Blast and Clustal X

Phylogenetic Analyses of Lymphocystis Disease Virus of Fish using Blast and Clustal X. Dr. Md. Mosharrof Hossain Associate Professor Department of Zoology University of Rajshahi , Bangladesh. mshzool@yahoo.com. Briefly LCDV research History. What is LCDV ?. Family: Iridoviridae

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Phylogenetic Analyses of Lymphocystis Disease Virus of Fish using Blast and Clustal X

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  1. Phylogenetic Analyses of Lymphocystis Disease Virus of Fish using Blast and Clustal X Dr. Md. MosharrofHossain Associate Professor Department of Zoology University of Rajshahi, Bangladesh. mshzool@yahoo.com

  2. Briefly LCDV research History

  3. What is LCDV ? Family:Iridoviridae Genus:Lymphocystivirus Strains/Species: LCDV-1 (Paralichthys flexus) LCDV-C (Paralichthys olivaceus) LCDV-2 (Limada limanda) LCDV-RF (Sebastes schlegeli )

  4. A B C D LCDV virus infection

  5. Objective of Research: To know the biology of LCDV (a) In Vivo, (b) In vitro 2. To find the epitope of infection site 3. To know LCDV taxonomic position

  6. Materials and Methods

  7. Experiment-1. PCR The ExicyclerTM is a Real-Time qPCR system developed by Bioneer. The ExicyclerTM is equipped with an optical system that fits above the thermal cycler. It readily utilizes most fluorescent dyes and thus provide wide choices of excitation / emission wavelengths. It can also be used as a standard thermal cycler for general PCR reactions.

  8. Principle of PCR

  9. Heating 95℃ Cooling 55℃ Target DNA Principle of PCR Basics of PCR 1 Cycle Extension 72℃ Cycling Polymerase Primer Cycling

  10. Primer design and PCRcondition The PCR primers were designed according Kitamura et al. (2006) Forward primer LCC-F 5´-CAA GTG TTA CTA GCG CTT T-3´ Reverse primer LCC-R 5´-ATC CCA TTG AAC CGT TCT-3´ Total PCR reaction mixture was 20 ㎕ PCR Condition: Denaturing- 94℃-1 min Annealing- 54℃- 1 min Extension- 72℃-1 min A total 30 cycles

  11. DNA transformation , cloning and sequencing: Purified DNA Expression in E. coli Send for sequencing

  12. Sequences of DNA for analyses

  13. DNA Sequencing analysis

  14. LCCR-AGCATCTTTATAACCAGAAGTATTTCCACCATTACCACCTGCTGTTATCACTGCTATTGGAGACGTCTTCAATTTAATACTGACATTGGACAAACGACCATAATTGGTAGATCCCATTGGATCTACATCCATCATATTGAGTGAATAGCAATACATGTGATAACCGGTGTCTACAGGAATAGAGCCTCCAAAATAATAAGGTTGAACCAAAGAATAATATTCACTACCCATTTCATTAAGACGAGCACTATTTTCATAAACCAAAGTAACATTTGAAATAGGATCAGCAGCAATACCCGGTAAATCGCTAGCAATTCCACCGTCAAAGATTACAGGAGAAGAACTGGTGTAATTGGATTGTATAGCTTGATAGGTAACATTACGCACACCGAAAAAAAGGATTTTAATGGCATGAGAAAATCTGATGTCAAAATTAGGACTTGGAATAGTTAGAGGTTGAAATACATGTTTAGGTGCTGTTTGTACCTGTTCTACCAAGATGTCTCTAGGTACTGTACCCATTAAACGACGTTCCTCATTGGTTACTACTACATTAGTAATCCATACTTGCACATCCTTTAAATCAGGTTTACCCCAGTCTAAATCGCCTGCTGTCAAAGGCATGATGGTAGAGTCGTTTTTATTTTGAAAGATCAATAATTCAGTCCAATCTCTCAGATGAAAAGTTAATCTTATTTCATTATAAGGCAAAGCAGCGCTGGGTAAAGCCATACCGCTATCTCGAGAAAAGAAATAAGGTAAAGGAAGTATTAACACTTTTTCAGGTAATTGACCATTGGAATCAACGGGTTLCCR-AGCATCTTTATAACCAGAAGTATTTCCACCATTACCACCTGCTGTTATCACTGCTATTGGAGACGTCTTCAATTTAATACTGACATTGGACAAACGACCATAATTGGTAGATCCCATTGGATCTACATCCATCATATTGAGTGAATAGCAATACATGTGATAACCGGTGTCTACAGGAATAGAGCCTCCAAAATAATAAGGTTGAACCAAAGAATAATATTCACTACCCATTTCATTAAGACGAGCACTATTTTCATAAACCAAAGTAACATTTGAAATAGGATCAGCAGCAATACCCGGTAAATCGCTAGCAATTCCACCGTCAAAGATTACAGGAGAAGAACTGGTGTAATTGGATTGTATAGCTTGATAGGTAACATTACGCACACCGAAAAAAAGGATTTTAATGGCATGAGAAAATCTGATGTCAAAATTAGGACTTGGAATAGTTAGAGGTTGAAATACATGTTTAGGTGCTGTTTGTACCTGTTCTACCAAGATGTCTCTAGGTACTGTACCCATTAAACGACGTTCCTCATTGGTTACTACTACATTAGTAATCCATACTTGCACATCCTTTAAATCAGGTTTACCCCAGTCTAAATCGCCTGCTGTCAAAGGCATGATGGTAGAGTCGTTTTTATTTTGAAAGATCAATAATTCAGTCCAATCTCTCAGATGAAAAGTTAATCTTATTTCATTATAAGGCAAAGCAGCGCTGGGTAAAGCCATACCGCTATCTCGAGAAAAGAAATAAGGTAAAGGAAGTATTAACACTTTTTCAGGTAATTGACCATTGGAATCAACGGGTT LCCF- GCTGTAGCTTATTTTGTACGAGAAACTAAACAATGTACCTGGTTCAGTAAATTACCAGTACTTTTAACACGTTGTTCTGGAACACCTAATTTTGATCAAGAATTTTCTGTCAATGTTTCTCGTGGTGGAGATTATGTACTTAATGCTTGGATGACGGTGCGTATTCCTGCTGTTAAATTGAAAACCAATAATCGTATGAACGCCAATGGTACTATCAGATGGTGTAAAAATTTATTTCATAATTTAGTTAAACAAACTTCTGTTCAATTTAATGATTTAGTTGCTCAAAAATTTGAGAGCTACTTTCTTGATTTTTGGTCCTCTTTTGGTATGTGTGGATCTAAACGTATAGGTTATGATAACATGATAGGTAATACTATTGATATGACACAACCCGTTGATTCCAATGGTCAATTACCTGAAAAAGTGTTAATACTTCCTTTACCTTATTTCTTTTCTCGAGATAGCGGTATGGCTTTACCCAGCGCTGCTTTGCCTTATAATGAAATAAGATTAACTTTTCATCTGAGAGATTGGACTGAATTATTGATCTTTCAAAATAAAAACGACTCTACCATCATGCCTTTGACAGCAGGCGATTTAGACTGGGGTAAACCTGATTTAAAGGATGTGCAAGTATGGATTACTAATGTAGTAGTAACCAATGAGGAACGTCGTTTAATGGGTACAGTACCTAGAGACATCTTGGTAGAACAGGTACAAACAGCACCTAAACATGTATTTCAACCTCTAACTATTCCAAGTCCTAATTTTGACATCAGATTTTCTCATGCCATTAAAATCC

  15. Homology of MCP genes

  16. Experiment-2. Cell line infection Cell Line & Virus Inoculation Cells were seeded 1.5×105 cells/㎖ • Cell lines: • FFN • FSP • FHM • CHSE-214 • RTG-2 Overnight confluence Virus injection at 200㎕/wells

  17. 16 10 C P 8 12 M 4 1347bp PCR detection of LCDV

  18. ? Ideal graph 1 Cycle 2 Cycle Real graph 3 Cycle N Cycle Principle of PCR Basics of PCR

  19. Principle of PCR

  20. FFN Cells Seeded in 6-well round plate at 2 x 104 cells/chamber Virus inoculation in the wells Washing cells with PBS Immunofluorescence Test Protocol MAbs treatment for 1h at 37° Washing with PBS Cells Stained with FITC (conjugate) for 1h at 37℃ Washing cells with PBS Mounted with glycerol Microscopy observation

  21. Results and Discussion

  22. LCDV In vivo infection

  23. LCDV in vivo infection in changing temperature

  24. LCDV infection in changing temperature and DNA copies

  25. LCDV in vitro infection in cell lines

  26. Fig.4b. Cytopathic effect morphology at the indicated times (upper panel, A,B,C,D) and immunofluorescence of lymphocystis disease virus infected FFN cells (lower panel, E,F,G,H)(×200, Scale bar 50µm). The infected cells showing strong antigen specific fluorescence (arrows) stained with MAbs and secondary antibody FITC goat anti-mouse IgG (Sigma, USA).

  27. LCDV genotyping and Homology of MCP gene

  28. Molecular phylogenetic tree for the relationship among 63 isolates of lymphocystiviruses and other iridoviruses based on the nucleotide sequence of the MCP gene. Bootstrap value 1000.

  29. Conclusions: • 1. LCDV is an opportunistic pathogen that persistently exists in the flounder epidermis at low temperature and outbreaks at suitable temperature. • 2. LCDV multiply in the optimum temperature at 20℃ when the fish have healthy condition for virus persistence. • FFN is susceptible to LCDV, and LCDV is organ- specific both in in vivo & in vitro infections and multiply in fibroblast cells. • 4. LCDV isolates from different habitats has the specific viral protein expression patterns and common antigenecity. These antigenic proteins enzymatic activity may help to find an epitope for vaccine preparation. These research published in 1. Hossain M. et al. 2008. Journal of Fish Diseases 31(6): 473–479, doi: 10.1111/j.1365-2761.2008.00917.x (IF: 1.697 ) 2. Hossain M. et al. 2009. Journal of Fish Diseases 32(8): 699–703, doi: 10.1111/j.1365-2761.2009.01048.x (IF: 1.697) 3. Hossain M. et al. 2011. Journal of Fish Pathology, 24(2): 47-51.

  30. Thank you for your kind patience

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