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 Yet sen University, Guangzhou
510275, China；
�┆ �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
nucleopolyhedrovirus (SeMNPV, Groups II), the essential
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
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
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
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-
replication and some ORFs including partial
which are unique to SeMNPV. As compared to
MNPV (Groups I), which also generated hypermolar DNA fragments containing the
non-
culture, our results provided
ori
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]