題名: Determination of genomic sequence of Watermelon bud necrosis virus and quick identification of tospoviruses using degenerate primer pairs 作者: Yeh Yi-Chun
貢獻者: Department of Biotechnology
關鍵詞: Tospovirus;Watermelon bud necrosis virus;degenerate primer pairs 日期: 2009
上傳時間: 2009-11-06T06:11:54Z 出版者: Asia University
摘要: The genus Tospovirus of the family Bunyaviridae has a large genome that consists of three RNA segments, denoted S, M and L RNAs, to code six gene products. The homology of nucleocapsid protein (NP), encoded from the S RNA, is a key criterion for classification of Tospovirus. Based on the NP, 19 virus species within this genus have been characterized.
However, except the NP, other coding sequences are still incompletely determined in Tospovirus. In this investigation, the entire genomic sequence of Watermelon bud necrosis virus (WBNV) was determined.
The S RNA of WBNV is 3,402 nucleotides (nts) in length, encoding 439 amino acids (aa) of NSs protein in the viral (v) sense and a 275 aa of NP in the viral complementary (vc) strand. The M RNA is 4,794 nts in length encoding a 307aa of NSm protein in the v strand and a 1,121 aa of precursor of Gn and Gc glycoproteins (GPs) in the vc strand. The L RNA has 8,914 nts, is of negative polarity, that codes a 2,878 aa of RNA- dependent RNA polymerase in the vc sense. Sequence analyses revealed that WBNV is closely related to Peanut bud necrosis virus (PBNV), Watermelon silver mottle virus (WSMoV) and Capsicum chlorosis virus (CaCV). On the other hand, degenerate primer pairs were designed from the comparison of the determined tospoviral sequences. The previously reported degenerate primer pairs were also used for virus detection in this study. Thirteen distinct tospovirus species, WBNV, PBNV, WSMoV, CaCV, Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV), Tomato spotted wilt virus (TSWV), Tomato chlorotic spot virus (TCSV), Calla lily chlorotic spot virus (CCSV), Iris yellow spot virus (IYSV), Melon yellow spot virus (MYSV), Peanut
chlorotic fan-spot virus (PCFV) and Tomato yellow ring virus (TYRV), were tested by reverse transcription-polymerase chain reaction (RT-PCR). All analyzed tospoviruses can be detected by primer pairs gLl3637/gL4510c and M1/tNSm400c. The primer pairs tNSm410/tNSm870 and
t2740/t3920 can be used to detect 12 tospoviruses, except PCFV.
WSMoV, CaCV, CCSV, MYSV, WBNV, PBNV, IYSV and TYRV, belonging to both WSMoV and IYSV serogroups, can be detected by the primer pairs WG1/WG960 and Gp2150/Gp2730c. Here, we developed a quick method for detection of tospoviruses with RT-PCR using degenerate primer pairs.
Moreover, this method can be applied for determining unknown tospoviral sequences.
