A PCR-based protocol for generating West Nile virus replicons.
J Virol Methods
; 148(1-2): 244-52, 2008 Mar.
Article
em En
| MEDLINE
| ID: mdl-18242719
ABSTRACT
A new protocol for the generation of West Nile virus (WNV) replicons was developed. Fragmented cDNAs that covered the entire WNV RNA sequence, except the sequence corresponding to nucleotides 190-2379, were amplified separately by polymerase chain reactions (PCRs) using primer set franking with overlapping sequences of 40-50 bp at the 5'- and the 3'-ends of each fragment. All amplified fragments were mixed together and annealed to each other at the overlapping sequences. The annealed-DNA fragments were elongated by DNA polymerase and amplified by short-cycle PCRs to generate full-sized WNV replicon cDNAs. The WNV replicons were transcribed in vitro using the replicon cDNAs as templates. When the in vitro-transcribed replicon was introduced into mammalian cells, the viral envelope protein and viral positive- and negative-strand RNAs were detected in the replicon-transfected cells. It is noteworthy that the synthesis of the replicon cDNAs and the replicons took just 1 week, and that the use of a high-fidelity DNA polymerase afforded stability to the sequence of the synthetic replicon.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Replicon
/
Vírus do Nilo Ocidental
/
Reação em Cadeia da Polimerase
Limite:
Animals
Idioma:
En
Ano de publicação:
2008
Tipo de documento:
Article