1 Virology Division, Kimron Veterinary Institute, PO Box 12, Beit-Dagan 50250, Israel
2 Pathology Division, Kimron Veterinary Institute, PO Box 12, Beit-Dagan 50250, Israel
Correspondence
Yehuda Stram
stramy{at}int.gov.il
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FMDV belongs to the genus Aphthovirus in the family Picornaviridae. The virus genome is an 8·5 kb positive-sense single-stranded RNA that carries a poly(A) tract at its 3' end and a viral genome protein (VPg) at its 5' end (Carroll et al., 1984; Forss et al., 1984
). There exist seven different virus serotypes that do not cross-protect against each other. Each serotype consists of numerous subtypes, about 80 in total (Mason et al., 2003
). This phenomenon is due to the high rate of mutation, especially in the VP1 gene (Dopazo et al., 1988
), which encodes the main viral protein determinant of immunological identity of the virus. The traditional way of protecting against the disease is by vaccination, which greatly reduces its occurrence. Nevertheless, there are hundreds of outbreaks in Asia, Africa, South America and eastern Europe each year, whereas North America and western Europe are considered to be virus-free regions and domestic animals are not vaccinated.
The recent large outbreak in the UK (Samuel & Knowles, 2001), which was previously considered to be free of the disease, emphasizes the need for additional methods to combat this disease. Not only were there massive economic losses to the dairy and meat industry, but large-scale disruption of the entire economy also occurred, due to restrictions on travel and a ban on all agricultural exports. Direct and indirect economical losses as a result of this outbreak were estimated to be in the range of £20 billion.
In this work, we describe the use of RNA interference (RNAi) as a means of inhibiting virus replication. In the last few years, RNAi has been documented in the nematode Caenorhabditis elegans (Fire et al., 1998; Montgomery et al., 1998
; Tabara et al., 1998
), trypanosomes (Ngô et al., 1998
), plants (Waterhouse et al., 1998
) and Drosophila (Kennerdell & Carthew, 1998
), amongst others.
Cleavage of double-stranded (ds)RNA into small, 22 bp fragments is a distinct element of RNAi activity. This small dsRNA serves as a guide sequence that instructs the multi-component, RNA-interfering silencing complex to destroy specific mRNAs (Bass, 2000
). Recently, Bernstein et al. (2001)
identified an enzyme, Dicer, that is responsible for the cleavage of large dsRNAs to produce these small dsRNAs. This enzyme is thought to be conserved in all species, including humans, birds and cattle.
The work of Elbashir et al. (2001) demonstrated that introduction of a 22 bp dsRNA, termed small interfering (si)RNA, into mammalian cells could silence target genes by RNAi. Since then, there have been several publications demonstrating the silencing of various genes by siRNA in mammalian cells (Elbashir et al., 2001
; Sui et al., 2002
). Furthermore, siRNA has been shown to be active in controlling virus replication, including that of human immunodeficiency virus (Capodici et al., 2002
; Park et al., 2002
), poliovirus (Gitlin et al., 2002
) and hepatitis B virus (Giladi et al., 2003
).
In this work, we demonstrated that siRNAs designed from highly conserved regions of the 3B region and the 3D polymerase gene of FMDV could inhibit virus replication. Moreover, these conserved sequences are found in all FMDV serotypes and, thus, these three anti-FMDV siRNAs have the potential to silence all FMDV serotypes.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells, viruses and virus titration.
BHK-21 cells were used for viral infection. Cells were grown in Eagle's : Earl's (45 : 45 %) medium with 5 % fetal calf serum and 0·15 % tryptose phosphate, in 25 cm2 cell-culture flasks (Nunc). The virus used for infection was FMDV serotype O1 Geshur (G).
For virus end-point titration, 5x104 pig kidney cells per well were grown for 24 h in Eagle's : Earl's (45 : 45 %) medium with 5 % fetal calf serum and 0·15 % tryptose phosphate in 96-well plates and inoculated with 10-fold dilutions of each virus sample. Forty-eight hours later, the cytopathic effect of each of the samples was monitored.
siRNA transfection and virus infection.
siRNAs (150 ng) were introduced into almost confluent (7080 %) BHK-21 cells in 96-well plates (Nunc) by lipotransfection for 18 h using an RNAi shuttle (Orbigen), according to the manufacturer's instructions. At the end of the transfection period, cells were washed twice with Earl's medium containing 1 % antibiotic, and 50 µl virus (103 p.f.u.) in Earl's medium with 1 % antibiotic was added to each well. After 45 min absorption at 37 °C, 50 µl medium without serum was added and at designated time points, samples were frozen at 80 °C until further analysis.
RNA extraction.
RNA was extracted by using Tri reagent (Molecular Research Center) according to the manufacturer's instructions. Briefly, 250 µl virus sample was added to 750 µl Tri reagent and 75 µl 1-bromo-3-chloropropane was used as the phase separation reagent. After mixing, the aquatic phase was separated by centrifugation (14 000 r.p.m. for 15 min in an Eppendorf centifuge) at 4 °C and 450 µl aquatic phase was mixed with the same volume of 2-propanol. RNA was precipitated at 14 000 r.p.m. at 4 °C for 15 min and washed once with 75 % ethanol.
Real-time quantitative RT-PCR (RT-qPCR).
The RT-PCR mixture comprised 2 µl RNA (2050 ng µl1), 10 µl RT-qPCR reaction mix (Eurogentec), 0·1 µl Moloney murine leukemia virus RT and RNase inhibitor mix, 100 ng each primer (forward, FC7 5'-CAAAAGATGGTCATGGGC-3' at nt 49694986 and reverse, RC7 5'-CAACAGATGGCTACTGTCTTCCC-3' at nt 50395061, GenBank accession no. AF189157) and 0·8 µl of the minor groove binder (MGB) probe to increase the stability and specificity of hybridization (5'-CCGTCGAACTCATCCT-3' at nt 49975012; Applied Biosystems). Reaction conditions were 48 °C for 30 min and 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All further computations were done using ABI Prism 7000 SDS Software (Applied Biosystems).
To determine the amount of the viral replicative form (RF), cDNA was prepared by using 50 ng RNA, RT buffer (50 mM Tris/HCl, pH 8·3, 50 mM KCl, 10 mM MgCl2, 0·5 mg spermidine and 10 mM DTT), 100 ng forward primer FC7, 12 U avian myeloblastosis virus RT (Chimerx), 10 U RNasin (Promega) and 100 mM each dNTP. Reaction conditions were 42 °C for 30 min and 98 °C for 5 min. For the qPCR, 2 µl RT reaction was used in a reaction containing 12·5 µl reaction mix, SYBR green (Eurogentec) at a final dilution of 1 : 40 000 and 100 ng each primer (FC7 and RC7) in a 25 µl reaction volume. Reaction conditions were 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also analysed as a control by using the TaqMan Rodent GAPDH Control Reagent kit (Applied Biosystems) according to the manufacturer's instructions.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three such regions were found with the sequences: (i) 5'-CCTGTCGCTTTGAAAGTGAAAGC-3' at nt 49004922, located in the 3B region; (ii) 5'-GAGATTCCAAGCTACAGATCACTTTACCTGCGTTGGGTGAACGCCGTGTGCGGTGACGC-3' at nt 69346992, located in the 3D region; and (iii) 5'-GACGAGTACCGGCGTCTCTTTGAGCC-3' at nt 68926917, located in the 3D region. All positions refer to the FMDV serotype O1 (G) sequence (GenBank accession no. AF189157).
Silencing of FMDV serotype O1 (G)
Inhibition of viral RNA replication.
The three sequences identified above were used to design three 21 nt anti-FMDV siRNAs (Table 1) for silencing experiments.
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
From this report, as well as from the results of others (Capodici et al., 2002; Gitlin et al., 2002
; Park et al., 2002
), it is apparent that dsRNA inhibition of viral infection is a very powerful tool for inhibition of virus replication and has a high therapeutic potential.
The use of siRNA as an antiviral agent could lead to a selective pressure on the siRNA target that might result in the appearance of escape variants, due to changes in the target sequence. To address this issue, the chosen virus target sequences were located in conserved regions of the virus genome (Stram & Molad, 1997). Moreover, the siRNA targets chosen had 100 % identity when comparisons were made of all FMDV sequences deposited in GenBank, regardless of their serotype. This level of identity is an indication of a strong selective pressure against mutations, by resisting changes in this sequence in the evolution of the virus. This selective pressure should maintain the siRNA target sequences without changes, ensuring the effective activity of the siRNAs described here.
To further minimize this potentially adverse effect for possible therapeutic use, a mixture of highly conserved sequences needs to be chosen to minimize the opportunity for the appearance of escape variants. In our case, there was an additional benefit to this design, as it also enabled recognition of all different serotypes and subtypes of the virus. With this in mind, siRNAs targeted to three highly conserved sequences were designed that should inhibit all seven viral serotypes. It remains to be seen whether this is the case for the other serotypes.
It has been proposed that post-translational gene silencing (PTGS) is one of the defence mechanisms of plants against pathogens and, in particular, against viruses (Voinnet, 2001; Waterhouse et al., 2001
). The ability of several plant viruses to suppress PTGS (Kasschau & Carrington, 1998
; Voinnet et al., 1999
; Takeda et al., 2002
) suggests that such mechanisms have evolved to enable these viruses to overcome cell-defence mechanisms and to enable them to replicate. Whether PTGS is a defence mechanism that has also evolved in mammalian cells is highly questionable. It should be remembered that inhibition of virus replication or gene silencing by RNAi is only in response to engineered siRNAs and is not a response to large dsRNAs, which induce the interferon chain of reactions, another defence mechanism of vertebrates.
Real-time RT-qPCR proved be an ideal technique for examining the amount of the viral RF. Preparing cDNA with the forward primer resulted in the synthesis of negative-sense cDNA, which served as the template for the synthesis of the positive-sense viral genome strand. Thus, detection of this negative-sense strand is an indication of the potency of virus replication. Our results revealed that the amount of the viral RF was related to the amount of the total viral load and was approximately 45 % of the total viral RNA.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363366.[CrossRef][Medline]
Capodici, J., Karikó, K. & Weissman, D. (2002). Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference. J Immunol 169, 51965201.
Carroll, A. R., Rowlands, D. J. & Clarke, B. E. (1984). The complete nucleotide sequence of the RNA for the primary translation product of foot and mouth disease virus. Nucleic Acids Res 12, 24612472.[Abstract]
Dopazo, J., Sobrino, F., Palma, E. L., Domingo, E. & Moya, A. (1988). Gene encoding capsid protein VP1 of foot-and-mouth disease virus: a quasispecies model of molecular evolution. Proc Natl Acad Sci U S A 85, 68116815.[Abstract]
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. & Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494498.[CrossRef][Medline]
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806811.[CrossRef][Medline]
Forss, S., Strebel, K., Beck, E. & Schaller, H. (1984). Nucleotide sequence and genomic organization of foot-and-mouth disease virus. Nucleic Acids Res 12, 65876601.[Abstract]
Giladi, H., Ketzinel-Gilad, M., Rivkin, L., Felig, Y., Nussbaum, O. & Galun, E. (2003). Small interfering RNA inhibits hepatitis B virus replication in mice. Mol Ther 8, 769776.[CrossRef][Medline]
Gitlin, L., Karelsky, S. & Andino, R. (2002). Short interfering RNA confers intracellular antiviral immunity in human cells. Nature 418, 430434.[CrossRef][Medline]
Kasschau, K. D. & Carrington, J. C. (1998). A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95, 461470.[Medline]
Kennerdell, J. R. & Carthew, R. W. (1998). Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the Wingless pathway. Cell 95, 10171026.[Medline]
Mason, P. W., Grubman, M. J. & Baxt, B. (2003). Molecular basis of pathogenesis of FMDV. Virus Res 91, 932.[CrossRef][Medline]
Montgomery, M. K., Xu, S. & Fire, A. (1998). RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc Natl Acad Sci U S A 95, 1550215507.
Ngô, H., Tschudi, C., Gull, K. & Ullu, E. (1998). Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci U S A 95, 1468714692.
Park, W.-S., Miyano-Kurosaki, N., Hayafune, M., Nakajima, E., Matsuzaki, T., Shimada, F. & Takaku, H. (2002). Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference. Nucleic Acids Res 30, 48304835.
Pearson, W. R. & Lipman, D. J. (1988). Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 85, 24442448.[Abstract]
Samuel, A. R. & Knowles, N. J. (2001). Foot-and-mouth disease virus: cause of the recent crisis for the UK livestock industry. Trends Genet 17, 421424.[CrossRef][Medline]
Stram, Y. & Molad, T. (1997). A ribozyme targeted to cleave the polymerase gene sequences of different foot-and-mouth disease virus (FMDV) serotypes. Virus Genes 15, 3337.[CrossRef][Medline]
Stram, Y., Chai, D., Fawzy, H.-E. & 7 other authors (1995). Molecular epidemiology of foot-and-mouth disease (FMD) in Israel in 1994 and in other Middle-Eastern countries in the years 19921994. Arch Virol 140, 17911797.[Medline]
Sui, G., Soohoo, C., Affar, E. B., Gay, F., Shi, Y., Forrester, W. C. & Shi, Y. (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci U S A 99, 55155520.
Tabara, H., Grishok, A. & Mello, C. C. (1998). RNAi in C. elegans: soaking in the genome sequence. Science 282, 430431.
Takeda, A., Sugiyama, K., Nagano, H., Mori, M., Kaido, M., Mise, K., Tsuda, S. & Okuno, T. (2002). Identification of a novel RNA silencing suppressor, NSs protein of Tomato spotted wilt virus. FEBS Lett 532, 7579.[CrossRef][Medline]
Voinnet, O. (2001). RNA silencing as a plant immune system against viruses. Trends Genet 17, 449459.[CrossRef][Medline]
Voinnet, O., Pinto, Y. M. & Baulcombe, D. C. (1999). Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci U S A 96, 1414714152.
Waterhouse, P. M., Graham, M. W. & Wang, M. B. (1998). Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci U S A 95, 1395913964.
Waterhouse, P. M., Wang, M. B. & Lough, T. (2001). Gene silencing as an adaptive defence against viruses. Nature 411, 834842.[CrossRef][Medline]
Received 25 March 2004;
accepted 27 July 2004.