東京大学 THE U NIVERSITY OF T OKYO Udayana University Amplifications of Terminal Ends of Newcastle Disease Virus Genome by Rapid Amplification of Complementary Deoxyribonucleic Acid Ends ANAK AGUNG AYU MIRAH ADI 1, NYOMAN MANTIK ASTAWA 2, YASUNOBU MATSUMOTO 3 1.Pathology Laboratory, 2.Virology Laboratory, Faculty of Veterinary Medicine, Udayana University - Jln. P B Sudirman Denpasar Bali. 3. Laboratory of Global Animal Resource Science, Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences, the University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, Japan. Corresponding Introduction Fragments of viral RNA genome can be amplified by the reaction of reverse transcriptase polymerase chain reaction (RT-PCR). However fragment terminal ends (leader and trailer) of viral RNA genomes could not be amplified by conventional RT-PCR. A methods known as rapid amplification of cDNA Ends (RACE) has developed for this purpose( Bertioli, 2000). Several RACE methods (Bertioli, 2000; Li et al.,2005;Liu et al.,2010)were evaluated in an effort to find a simple and inexpensive method. In this study we attempted to modify an inexpensive RACE method of Li et al. (2005) for amplifying the leader and trailer of NDV/Bali-1/07 genome. Materials and Methods Viral propagation and RNA isolation NDV/Bali-1/07 (acc. number AB426628) was propagated in 9- day-old embryonated SPF chicken eggs. NDV RNA isolation was performed by standard Trizol method. Amplification of termnal ends fragment by 3'- 5' RACE ABSTRACT Leader and trailer (terminal ends) of ribonucleic acid (RNA) sequences is important in characterizing novel Paramyxovirus as they contain important signals for replication and transcription their genomes and therefore important in understanding the process of viral evolution. Conventional polymerase chain reaction (PCR) is unable to amplify these regions. Recently, rapid amplification of cDNA Ends (RACE) is a PCR-based technique which has been developed to amplify these regions in order to determine RNA terminal sequences. In this study, the leader and trailer, of the viral RNA genome of Newcatle disease Virus (NDV)/Bali-/07 were amplified. The leader and trailer sequence of the viral genome was determined using 3'-RACE and 5'-RACE method respectively. With this method both leader and trailer of NDV/Bali-1/07 can be amplified, without using a highly cost RACE kit. Key word:genome, leader, trailer, RACE The genome terminal ends sequences are highly conserved and there is complementary between the 3'- and 5'- termini (Fig 3C). These conserved terminal sequences, especially in the first 12–13-nt, are believed to contain the genome and anti- genome promoters essential for replication and transcription of the virus (Lamb and Kolakofsky, 2001). The sequences are also useful markers for classification of new viruses and for studying virus evolution in the family (Wang et al., 2003). Fig.1. Schematic diagram of 3'- RACE (A ） and 5'-RACE (B). The viral RNA genome (dotted line) is presented in the 3 ' and 5 ' direction. The cDNA is represented in solid line. In 3'-RACE viral RNA was ligated with adaptor DT88, followed by cDNA synthesis. For 5'-RACE, cDNA synthesis was performed first,the cDNA was then polydATP- tailed and polydGTP-tailed. The remaining step PCR and heminested PCR are same for the both 3'-RACE and 5'-RACE, with the exeception of the primers used. References Bertioli D. Rapid amplification cDnA ends. In: Rapley R (ed). The Nucleic Acid Protocols Handbook. Humana Press Inc., Totowa, NJ. p Lamb RA, Kolakofsky D, Paramyxoviridae: the viruses and their replication. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE. (Eds.), Fields Virology, vol.1. Lippincott Williams & Wilkins, Philadelphia, pp. 1305–1340. Li Z, Yu M, Zhang H, Wang HY, Wang LF Improved rapid amplification of cDNA ends (RACE) for mapping both the 5'_ and 3' terminal sequences of paramyxovirus genomes. J. Virol. Methods 130: 154–156. Liu H, Chen F, Zhao Y, Zheng D, Li J, Xu T, Qi L, Wang Z Genomic characterization of the first class I Newcastle disease virus isolated from the mainland of China. Vir. Genes 40: Wang LF, Chua KB, Yu M, Eaton BT Genome diversity of emerging paramyxoviruses. Curr. Genomics 4: Conclusions In this study, modified RACE method was able to amplify the leader and trailer of NDV/Bali-1/07 genome. without using a highly cost RACE kit. The length of 3'-leader and 5'-trailer were 55 and 114 nt, respectively, these length were similar with those of other NDVs strain. Fig.3. Aligntment of the 5' leader and 3' trailer of NDV /Bali-1/07 isolate with several NDVs. The sequences are presented as cDNA in the 5 ' and 3 ' direction. Alignment of the leader (A), the trailer (B). Paired nucleotides at 5' and 3 ' end of Bali-1/07 cDNA are underlined (C). Tab.1. Primers used to generate PCR products. The primers were designed based on the genome sequence of the NDV/LaSota ) and the obtained genome sequence of NDV/Bali-1/07(*). Numbering in superscript indicates the positions on NDV/LaSota genomic sequence Result and discussion By modifying RACE method of Li et al., （ 2005) especially in the viral specific primers both leader and trailer of NDV/Bali- 1/07 could then be amplified(Fig.2A-B). The length of 3'-leader and 5'-trailer were 55 and 114 nt (Fig.3. A-B). Figure 2. Amplification Result of 3'- RACE (A) and 5'-RACE(B) ;(2A).Adaptor ligated cDNA was amplified using Bali-1/07 NP gene specific primers;Line 1 DNA marker 100 bp.Line 2.First PCR using primers DT89 andF1rBali-1. Line 3.Heminested using primers DT 89 and F1r Bali2. (2B).PolydATP/dGTP cDNA was amplified using primer specific L gene of LaSota strain and Bali-1/07 strain. Line 1. Marker 100 bp,line 2.Bali_1 TdT/dA/F29S- oligo dT, line 3.no template/ F29S- oligo dT, line 4. Bali_1 TdT/dG/ F29S- oligo dC, line5.no template/ F29S- oligo dC, line 6. Bali_1 TdT/dA/F30SBali １－ oligo dT, line 7. Bali_1 TdT/dG/ F30SBali- oligo dC, line 8. Bali_1 TdT/dA/F30SBali2- oligo dT, line9. Bali_1 TdT/dG/ F30SBali2- oligo dC,l ine 10.no template/ F30SBali2- oligo dC