In view of the threat of the potential use of variola virus in a terrorist attack, considerable efforts have been performed to develop new
antiviral strategies against orthopoxviruses. Here we report on the use of RNA interference, either alone or in combination with
cidofovir, as an approach to inhibit orthopoxvirus replication. Two selected small interfering RNAs (siRNAs), named siB1R-2 and siG7L-1, and a previously reported
siRNA, i.e., siD5R-2 (which targets the viral D5R
mRNA), were evaluated for
antiviral activity against vaccinia virus (VACV) by plaque reduction and virus yield assays. siB1R-2 and siG7L-1, administered before or after
viral infection, reduced VACV replication by more than 90%. Also, these two siRNAs decreased monkeypox virus replication by 95% at a concentration of 1 nM. siB1R-2 and siG7L-1 were demonstrated to specifically silence their corresponding transcripts, i.e., B1R and G7L mRNAs, without induction of a
beta interferon response. Strong synergistic effects were observed when siB1R-2, siG7L-1, or siD5R-2 was combined with
cidofovir. In addition, the
antiviral activities of these three siRNAs were evaluated against VACV resistant to
cidofovir and other acyclic
nucleoside phosphonates. siG7L-1 and siD5R-2 remained active against four of five VACV mutants, while siB1R-2 showed activity against only one of the mutants. Our results showed that siRNAs are potent inhibitory agents in vitro, not only against wild-type VACV but also against several
cidofovir-resistant VACV. Furthermore, we showed that a combined
therapy using
siRNA and
cidofovir may be useful in the treatment of
poxvirus infections.