Generation
and Analysis of a Repeat DNA Fragment of SeMNPV by Serial Undiluted Passage in
Se301 Insect Cells
YANG Kai, PIJLMAN Gorben1, YU
Mei, VLAK Just, PANG Yi*
( State Key Laboratory for Biocontrol, Sun Yetª²sen University, Guangzhou
510275, China£»ª¤
ª©ªªª¬1 Laboratory of Virology, Wageningen University, Binnenhaven 11,
6709 PD Wageningen, the Netherlands )
Abstract Two major
clades, designated Groups I and II, of nucleopolyhedroviruses (NPVs) from
insect hosts have been previously identified. In order to gain insight into DNA
replication of Spodoptera exigua multicapsid
nucleopolyhedrovirus (SeMNPV, Groups II), the essential cis-acting
DNA segments were studied. A strain, named Se-4, was plaque-purified from
SeMNPV isolate US1. PCR, ELT-PCR and REN showed that Se-4 was genetically
relatively homogeneous and retained the full-length of a hypervariable region
which is usually prone to deletion from SeMNPV genome. To study the stability
of this isolate in vitro, Se-4 was serially passaged in the
Se301 cell line up to 10 times without dilution. Intracellular viral DNA
extracted from every passage was analyzed by REN. A novel 3.5 kb PstI
fragment was observed in passage 7 and the relative intensities of the bands
increased with subsequent passages. In passage 10, the molar ratio of the
fragment was much higher than those of any other viral DNA fragments. This
fragment was thus expected to contain an important cis-acting
element for SeMNPV DNA replication. The fragment was cloned and sequenced and
it was found that it overlapped 3 525 bp with the published SeMNPV genome
sequence (GenBank AF169823), from 81 014 nt to 84 538 nt. The region contained
the SeMNPV non-hr origin (ori) of DNA
replication and some ORFs including partial vlf-1, partial p26,
Se84 as well asSe83, Se85, Se86,
which are unique to SeMNPV. As compared to Autographa californica
MNPV (Groups I), which also generated hypermolar DNA fragments containing the
non-hr ori by serial undiluted passage in IPLB-SF-21 cell
culture, our results provided in vitro evidence that the non-hr
ori may also play an important role in the viral infection cycle of
Groups II NPVs.
Key words Spodoptera exigua multicapsid
nucleopolyhedrovirus; ori; serial undiluted passage; Se301 cell line;
non-hr
*Corresponding author: Tel,
86-20-84113860; Fax, 86-20-84037472; e-mail, [email protected]