東京大学
THE UNIVERSITY OF TOKYO
Udayana University
Amplifications of Terminal Ends of Newcastle Disease Virus Genome by Rapid
Amplification of Complementary Deoxyribonucleic Acid Ends
ANAK AGUNG AYU MIRAH ADI1, NYOMAN MANTIK ASTAWA2, YASUNOBU MATSUMOTO3
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,
1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan.
Corresponding author:mirah638@yahoo.co.id
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-polydATP-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 613-618
Lamb RA, Kolakofsky D, 2001. 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. 2005. 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. 2010. Genomic characterization of the first class I Newcastle disease virus isolated from the mainland of China. Vir. Genes 40:365-371.
Wang LF, Chua KB, Yu M, Eaton BT. 2003. Genome diversity of emerging paramyxoviruses. Curr. Genomics 4:109-121.
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.
