Inhibition of foot-and-mouth disease virus replication by small interfering RNA

Ronen Kahana1, Larisa Kuznetzova1, Arie Rogel1, Mordechai Shemesh2, Dalia Hai1, Hagai Yadin1 and Yehuda Stram1

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Foot-and-mouth disease, caused by foot-and-mouth disease virus (FMDV), is one of the most dangerous diseases of cloven-hoofed animals and is a constant threat to the dairy and beef industries in the Middle East and other regions of the world, despite intensive vaccination programmes. In this work, the ability of specific small interfering (si)RNAs to inhibit virus replication in BHK-21 cells was examined. By using bioinformatic computer programs, all FMDV sequences in public-domain databases were analysed. The analysis revealed three regions of at least 22 bp with 100 % identity in all FMDV entries. From these sequences, three specific siRNA molecules were prepared and used to test the ability of siRNAs to inhibit virus replication. By using real-time quantitative PCR to measure the amount of viral RNA in infected cells, it was shown that virus replication was inhibited in cells that were transfected with siRNAs. When viral titres were examined, 100 % inhibition of growth could be demonstrated in cells transfected with a mixture of all three anti-FMDV siRNAs, compared with control cells transfected with anti-LacZ siRNA.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Foot-and-mouth disease virus (FMDV) is one of the most dangerous viruses that affects cloven-hoofed animals. The spreading capacity of the virus and its ability to change its antigenic identity make it a real threat to the beef and dairy industries in many countries. One of the foot-and-mouth disease (FMD)-endemic regions of the world is the Middle East, where several FMD outbreaks occur each year (Stram et al., 1995).

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Computer analysis.
Local homology analysis was performed by using the PILEUP and PRETTY programs in the GCG package. FASTA (Pearson & Lipman, 1988) was used for homology searches in GenBank. From the homology analysis results, three siRNAs were designed and synthesized by Orbigen. Primers and probes for real-time PCR were designed by using PrimerExpress (Applied Biosystems).

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 (70–80 %) 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 (20–50 ng µl–1), 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 4969–4986 and reverse, RC7 5'-CAACAGATGGCTACTGTCTTCCC-3' at nt 5039–5061, 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 4997–5012; 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Anti-FMDV siRNA design
Using FASTA, all FMDV sequences in GenBank were aligned and searched by using the PILEUP and PRETTY programs to find three regions with sequences of at least 22 bp with 100 % identity in all FMDV entries in the database.

Three such regions were found with the sequences: (i) 5'-CCTGTCGCTTTGAAAGTGAAAGC-3' at nt 4900–4922, located in the 3B region; (ii) 5'-GAGATTCCAAGCTACAGATCACTTTACCTGCGTTGGGTGAACGCCGTGTGCGGTGACGC-3' at nt 6934–6992, located in the 3D region; and (iii) 5'-GACGAGTACCGGCGTCTCTTTGAGCC-3' at nt 6892–6917, 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.


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Table 1. Sequences of siRNAs used in this work

siRNAs were synthesized according to the FMDV sequences described in Results.

 
BHK-21 cells were transfected for 18 h with each siRNA or with a mixture of all three. At the end of the transfection period, cells were inoculated with 103 p.f.u. FMDV O1 (G). At designated time points, samples were taken and RNA was extracted. This served as a template for real-time RT-qPCR. As can be seen in Fig. 1, at 24 post-infection (p.i.), relative amounts of viral RNA in cells treated with Cons 7, Cons 8 or Cons 9 (Table 1) or a mixture of all three were 4230, 1708, 2819 or 337, respectively, compared with a value of 20 929 in control-experiment cells, which were transfected with anti-LacZ siRNA. This equated to 80, 92 and 87 % inhibition of viral RNA replication in cells transfected with Cons 7, Cons 8 and Cons 9, respectively. The most effective inhibition by far was found in cells that were transfected with the siRNA mixture, which showed >98 % inhibition compared with the levels of viral RNA in cells transfected with the control anti-LacZ siRNA (Fig. 1).



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Fig. 1. Relative amounts of viral RNA in siRNA-transfected BHK-21 cells. BHK-21 cells in 96-well plates were transfected with 150 ng of each of the siRNAs ({blacklozenge}, Cons 7; {square}, Cons 8; {blacktriangleup}, Cons 9; {blacksquare}, mix; {bullet}, LacZ) for at least 18 h and then inoculated with 103 p.f.u. FMDV serotype O1 (G). At the designated times p.i., cells were frozen at –80 °C. Half of each of the samples was used for RNA extraction to determine the relative amounts of viral RNA by real-time RT-qPCR. The standard used was 10-fold dilutions of 107 p.f.u. O1 (G) virus ml–1. Error bars indicate SD.

 
To show direct inhibition of viral RNA replication, the amount of negative-sense RNA, an essential element of the viral RF, was measured. cDNA representing only the replicative negative-sense RNA was synthesized by using the forward primer FC7 and the amount of viral RNA was measured by qPCR. The amounts of the negative-sense viral RNA in cells transfected with Cons 7, Cons 8 and Cons 9 were 14·12, 17·87 and 25·87, respectively, at 24 h p.i., compared with 92·35 in cells that had been transfected with anti-LacZ siRNA (Fig. 2), representing 85, 81 and 72 % inhibition, respectively. A more substantial reduction in virus replication was observed in cells that had been treated with the mixture of all three siRNAs (Fig. 2). The amount of viral RF at 24 h p.i. was 9·26, compared with 92·35 for the control, representing >90 % inhibition of virus replication.



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Fig. 2. Relative amounts of viral RF RNA in siRNA-transfected BHK-21 cells. BHK-21 cells were transfected with siRNA and infected with FMDV as described in Fig. 1 ({blacklozenge}, Cons 7; {square}, Cons 8; {blacktriangleup}, Cons 9; {blacksquare}, mix; {bullet}, LacZ), except that the reaction was performed in two steps wherein the RT step was carried out with the forward primer to synthesize cDNA representing the negative-sense RF viral RNA. Error bars indicate SD.

 
To show that the presence of the three siRNAs did not have an adverse effect on cellular mRNA, we tested the amount of GAPDH mRNA in cells at 12 h p.i., i.e. 30 h post-transfection, before the beginning of massive viral RNA replication. The relative amount of GAPDH mRNA was in the range of 76·5–217·8 in transfected and infected BHK-21 cells, compared with a value of 97·7 in untreated BHK-21 cells, indicating that anti-FMDV siRNA did not affect the amount of GAPDH mRNA in cells (Table 2).


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Table 2. Relative amounts of GAPDH mRNA in transfected and infected BHK-21 cells

Relative amounts of GAPDH mRNA were determined by real-time RT-qPCR in siRNA-transfected and FMDV-infected cells. A standard curve was established by using serial dilutions of total RNA extracted from BHK-21 cells.

 
Reduction in viral titre.
To corroborate the results with the viral RNA, the same samples that were used for RT-qPCR experiments were used for virus titration. A high correlation was found between RT-qPCR results and the results of virus titration. Viral titres in cells transfected with Cons 7, Cons 8 and Cons 9 at 24 h p.i. were 3·2x10–1, 3·2x10–1 and 6·3x10–1 p.f.u. per cell, respectively (Table 3), compared with 6·3x100 p.f.u. per cell in cells transfected with anti-LacZ siRNA, representing >90 % inhibition of virus yield. As with the RT-qPCR results, virus replication in cells transfected with a mixture of the three siRNAs gave the most significant effect, silencing virus replication completely: the titre at 24 p.i. was 1·2x10–3 p.f.u. per cell, the same as at time zero (Table 3).


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Table 3. Viral titres at 0 and 24 h p.i.

At designated times, samples were taken and viral titres (p.f.u. per cell; ±SD) were determined as described in Methods. ND, Not determined.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This work presents evidence that the use of siRNAs corresponding to FMDV sequences can act as an effective mechanism for inhibition of virus replication. It was demonstrated that RNA levels in cells treated with a single siRNA were at least 80 % lower than in control cells treated with anti-LacZ siRNA. Only when cells were transfected with a mixture of the three siRNAs was there >98 % reduction in the amount of viral RNA. Similar results were obtained when the amount of viral RF was measured. Titration experiments showed that each siRNA could silence virus replication by approximately 90 %. However, most importantly, titration results showed that 100 % silencing could be achieved by the use of all three siRNAs. These results indicated that, to attain the highest levels of virus replication silencing or complete inhibition of virus replication, more than one siRNA targeted to different sites needs to be used.

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 4–5 % of the total viral RNA.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 25 March 2004; accepted 27 July 2004.