Analysis of leader and trailer sequence of genotype III, VIb and VIId Newcastle disease virus determined by modified rapid amplification of cDNA ends (RACE) strategy

Abstract:

Abstract：[Objective] The purpose of this research is to establish a simple rapid amplification of cDNA ends (RACE) strategy for direct mapping of the 3’ end and 5’ end of the genomic RNA of Newcastle disease virus (NDV), and to analyze the leader and trailer sequence of NDV strains belonging to different genotypes. [Methods] Classic RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) was specifically modified for mapping both ends of the NDV genome. 3’-RACE was carried out by genomic RNA ligation with 5’ end phosphated adaptor CL+, and the 5’ end was obtained by first strand cDNA with adaptor CL+. [Results] A modified RLM-RACE strategy was established in this paper, which proved simple, low-cost, repetitive and could be specifically used to map genome ends of NDV. By using this method, the leader and trailer sequence of 5 NDV strains, termed JS/5/05/Go, JS/07/04/Pi, JS/07/16/Pi, JS/7/05/Ch and JS/9/05/Go, belonging to genotype III, VI and VII was determined, respectively. [Conclusion] The initial 8nt at the 3’ and 5’ ends of the genome of genotype I-VI NDV strains were complementary, whereas, the complementary sequences of strain JS/5/05/Go were up to 9 nt due to a mutation from T to C at the 9th nt in the 5’ end. The 3’ end of NDV genomic and anti-genomic RNA was predicted to form a potential hairpin structure. The U→C（T→C）mutation was located in the circle part of the hairpin in the 5’ end of anti-genomic RNA, and had no visible influence on the formation of RNA secondary structure. However, the sequence of the circle part of the hairpin was changed from 3’-UUUC-5’ to 3’-UCUC-5’, more similar to the 3’-UCUUA-5’ in the hairpin of genomic RNA.