1. 1 Amplification and cloning of infectious bursal disease virus genomic RNA segments by long and accurate PCR Aydemir Akin, Ching Ching Wu*, Tsang long Lin Department of Veterinary Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette Received in revised form 17 May 1999; accepted 18 May 1999

2. 2 Abstract RNA denaturation by heating RTase lacking RNase-H activity RNase-H digestion Long and accurate PCR(LA-PCR) Utility for any IBDV strains or isolates

3. 3 Table of contents 1. Introdction 2. Material and methods –2.1. Virus –2.2. Extraction of viral RNA –2.3 Oligodeoxynuclotide primers –2.4. Reverse transcription and LA-PCR –2.5. Cloning of cDNA amplicons of small and large IBDV genome segments –2.6 nucleotide sequencing 3. Results and discussion –3.1. Extraction of viral dsRNA and synthesis of cDNA –3.2. LA-PCR amplification

4. 4 Introduction Birnaviridae –Aquabirnavirus (e.g. infectious pancreatic necrosis virus) –Entomobirnavirus (e.g. drosophila-X virus) –Avibirnavirus (e.g. IBDV) Immunosuppression Two serotype Nonenveloped and icosohedral shaped virion Two double-stranded RNA segment

5. 5 Pathogenesis Duodenum Jejunum caecum Liver bursa of Fabricius 64-72 hrs Clinical disease and death

6. 6 Old method Whole genome separated into smaller overlapping fragments Screened and sequenced

7. 7 Resembled by RE digestion Subcloning into plasmid

8. 8 Method detail PCR TA-cloning approach by Taq But the disadvantage is 2 kb limitation

9. 9 Improvement Up to 42 kb Additions of proof reading activity (3'-5' exonuclease) enzyme into PCR mixture RNA Digestion of RNA-DNA hybrid by RNase-H Use RTase lacking RNase-H activity

10. 10 Viral RNA preparation 3-week-old chickens orally infected Bursae homogenization in PBS Freeze-thawed three times Proteinase K, 1% SDS, DEPC KOAc, absolute ethanol, 14000 X g Lithium chloride(2 and 4M) Concentration measurement by spectrophotometry (260 nm)

11. 11 primers

12. 12 RT 1.dsRNA denaturation by heating 2.Superscript II system (MMLV RTase, 45 ℃, 60 min) 3.RNase-H digestion 4.Inactivation

13. 13 LA-PCR 1.Amount of cDNA 2.Removal of RNA:cDNA hybrids 3.Magnesium concentration 4.Duration of denaturation during PCR amplification 5.Different polymerase mixtures

14. 14 Cloning dATP + Taq for addition of A on 3' end Screening with T7N and Sp6N primers, or segment-specific primers Orientation and identity were confirmed with internal primerinternal primer

15. 15 Sequencing Li-Cor long-read sequencing reaction kit

16. 16 Result and discussion 1.Extraction of viral dsRNA and synthesis of cDNA 2.LA-PCR amplification

17. 17 Result of preparation Produced 1.98 mg of very good quality ( 260/280  1.8) Only MMLV produced long cDNA in both specifically and non-specifically primed cDNA.

18. 18 Discussion of preparation Random hexamers better than virus specific primers RTase lacking RNase-H activity enables the production of long cDNA transcripts. RNase-H digestion; In PCR, RNA may be serve as template (It will reduce efficiency) Denaturation by heating without adding dimethyl-sulphoxide(DEPC) or methymercury hydroxide

19. 19 Result of LA-PCR Taq : Pfu = 64:1 (v/v) These sequences obtained from IBDV strain E have the identity of IBDV. 3182 2777

20. 20 Q&A