ES2401899B1 - USE OF A NON-CODING REGION OF THE VIRUS FEATHER GENOME FOR THE PREPARATION OF AN ANTI-VIRAL MEDICINAL PRODUCT. - Google Patents

Abstract

Use of a non-coding region of the FMD virus genome for the preparation of an antiviral medicament. # The present invention relates to the use of RNAs corresponding to highly structured domains of the non-coding regions of the FMD virus genome. (VFA) and the use of sequences comprising them for the preparation of a pharmaceutical composition for the prophylaxis and / or treatment of diseases caused by interferon-sensitive viruses. Interferon-sensitive viruses are single-stranded RNA viruses, such as foot-and-mouth disease virus, vesicular stomatitis virus and West Nile virus. The invention also relates to the pharmaceutical composition comprising the non-coding regions of the invention and also comprising at least one pharmaceutically acceptable excipient and / or vehicle, as well as the use of said pharmaceutical composition.

Description

Use of a non-coding region of the foot and mouth disease virus genome for the development of an antiviral drug

The present invention relates to the use of a non-coding region of the FMD virus genome for the preparation of a pharmaceutical composition for the prophylaxis and / or treatment of diseases caused by interferon-sensitive viruses.

STATE OF THE PREVIOUS TECHNIQUE

The innate immune response is the first line of defense against pathogen invasion based on various mechanisms and signaling pathways. In the case of a viral infection, one of the mechanisms of the immune response

innate is the production of interferon (IFN) type I (IFN-a and IFN-¡3, or also

denominated IFN-a / ¡3) that has antiviral, antiproliferative effect and presents

immunomodulatory activity The production of type I IFN occurs after the detection of pathogen-associated molecular patterns (PAMPS) present in viral products that are generated in a viral infection.PAMPS include both ribonucleic acid ( Single stranded RNA) as double stranded RNA.

However, interferon-sensitive viruses that evade this defense system have been described. Interferon-sensitive viruses include foot-and-mouth disease virus, vesicular stomatitis virus and Nile virus

Western (Randall RE al. 2008. J Gen Viral 89: 1-47).

Foot and mouth disease virus (VFA, "1001 and moulh disease viruS ', FMDV,) is a member of the P family; cornav; r; dae that is the cause of foot and mouth disease

(glosopeda) that affects humans (Bauer K 1997 Arch Virol (S) 13: 95-97; Hyslop NStG 1976 Bulletin of the Pan American Sanitary Bureau: 375-387) and artiodactylated ungulate mammals, including ruminals and pigs The VFA is

a single stranded RNA virus approximately 8.5 kilobases (Kb) in length that at its thermal ends contains non-coding regions

(NCR, "non-coding regions') containing highly structured domains

forming a double stranded RNA. VFA NCRs contain structures

specific ones involved in the replication control and translation of

viral genome Among the non-coding regions are the so-called

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fragment S, IRES ("internal ribosome entry sile ') and 3'NCR (Witwer CS el al.

2001 Nucleic Acid Res 29: 5079-5089; Escarmis CM al. 1992. Virus Res

26: 113-125; Belsham GJ al. 2009. Virus Res 139: 183-192).

VFA has been shown to be sensitive to IFN-a, IFN-¡3 and IFN -y but is capable

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of developing mechanisms to evade the immune response. Lpro protease

it is the first viral protein that translates and blocks the innate response

host immune, including the INF-mediated response.

Lpro is able to inhibit the induction of messenger RNA (mRNA) of IFN-¡3 and the

expression of genes that stimulate IFN-a / ¡3. This fact represents a

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problem for the control of FMD disease.

Foot and mouth disease is a disease that constitutes a serious problem in the

livestock sector. It is a highly contagious disease that

propagates through infected animals, livestock equipment, clothing or even

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VFA footwear is currently controlled by vaccination and

preventive measures but once infected the animals implies their

sacrifice and therefore results in large economic losses in the sector.

In relation to vaccination against VF A, adaptive immunity appears

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several days after the vaccination of the animals, being of

approximately one week in the case of traditional vaccination. This

It is a problem to be able to expose these animals to the virus

during the window period and be susceptible to infection during that time.

Efforts have been made to cover that susceptibility window

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through the use of replication-defective recombinant adenovirus that

they express IFN-a but said strategy, in addition to involving the use of a vector

viral, it does not cover the VFA susceptibility window in its entirety since

which protects pigs inoculated in this way for 24 hours

Post-infection and protection lasts between 3 and 5 days. It becomes necessary so

therefore, a tool that covers the window of susceptibility to the virus

of foot and mouth disease prior to the development of an effective adaptive immunity in

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which infected animals are susceptible to infection.

The vesicular stomatitis virus (VEV, 'vesicular slomatitis virus', VSV) is a

virus belonging to the family Rhabdoviridae that causes a disease

Acute vesicular in cattle, horses, deer and pigs. Has been found

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serological evidence that infects many other animals and occasionally

It also infects humans. There are two main species, called New

Jersey and Indiana. It is transmitted by insect bite vectors,

mainly sand flies (Lutzomya sp.), black flies (family

Simuliidae) and insects of the genus Culicoides. Its genome is formed by 11

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12 Kb of single-stranded RNA and negative polarity. The VEV is also

able to avoid the host's immune response. In the case of VEV the

infection induces general blockage of gene expression in the cell

host, which affects the production of IFN and other antiviral molecules.

Currently, disease control is performed using measures of

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restriction of movement of infected animals, quarantine, control of

vector insects and vaccination with inactivated virus. An alternative capable of

activating an effective innate immune response against the virus would be very useful

for the control of this disease.

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West Nile virus (WNV, 'West Ni / e Virus', WNV), a transmitted virus

mainly by mosquitoes and whose natural host are birds, is

classified within the family F / aviviridae, along with other important pathogens

animals like the classical swine fever virus, and humans like the virus

hepatitis e, dengue virus, yellow fever virus (Blitvich BJ 2008.

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Anim Health Res Rev 9: 71-86). WNV is a virus with lipid envelope, whose

Virions have icosahedral symmetry and an approximate diameter of 50 nm.

As a genetic material, WNV has a single RNA molecule of

single chain and positive polarity of about 11,000 nucleotides in length. He

WNV is also able to avoid the host's immune response. The WINE

It is a clear example of re-emergent zoonosis, which currently constitutes a serious problem for human and animal health since it causes fatal meningoencephalylises in humans, equidae, bovids, suidae and birds, in which it generally produces subclinical infections, but in which occasionally causes high mortality (Granwehr BP al.

2004. Lancet Infect Dis 4: 547-556, Ringia AM al. 2004 Emerging Infectious Disease 10 (6): 1120-1123; Komar N. al. 2003 Emerging infectious Diseases 9 (3): 311-322; USGS 'National Wildlife Health Center' list of species affected by WNV 2005). In recent years, the number and severity of

cases in humans and horses have increased considerably. In the

currently as tools for the control of it only

It has a commercial vaccine for equids based on the use of inactivated virus and in the case of humans, therapies based on the administration of

interferon (IFN) or antibodies compassionately (Ng al. 2003. Dev Biol 114: 221-227; Davis al. 2006. Ann Neurol 60: 286-300) that are not sufficient for disease control, Therefore, an effective alternative for the prophylaxis and / or treatment of West Nile disease is necessary.

EXPLANATION OF THE INVENTION

The present invention relates to the use of a non-coding region of the foot-and-mouth disease virus genome for the preparation of a pharmaceutical composition for the prophylaxis and / or treatment of diseases caused by interferon-sensitive viruses.

In order to evaluate the antiviral potential of the non-coding regions of the

Foot and mouth disease virus (VFA) genome, in the present invention, non-synthetic RNAs were generated by in vitro transcription

analog encoders of said regions, the ncRNAs of the invention.

Inlerferon-sensitive viruses that have been shown to be

sensitive to the innate immune response induced by the ncRNAs of the present

invention are viruses of the families Picornaviridae, Rhabdoviridae and Flaviviridae.

Within the family Picornaviridae, the FMD virus was chosen in the

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Rhabdoviridae family was chosen vesicular stomatitis virus, while

from the family Flaviviridae the West Nile virus was chosen as the most

representative of these families for the realization of the trials that

demonstrate the activity of the ncRNAs of the invention.

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With the ncRNAs used in the invention, in vitro and in-vitro studies were performed.

live in which the production of messenger RNA (mRNA) of IFN-a or

IFN-¡3 and anliviral response. In addition, trials of

Survival against viral challenge in a nursing mouse model. In the

trials the effect was compared with a known interferon stimulator, the

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poly I: C (Richmond JY al. Proc Natl Acad Sci USA 1969. 64: 81-86) and

demonstrated that the stimulating activity of the innate immune response of

ncRNAs of the invention is superior to this control.

The results obtained with the ncRNAs of the invention show that these

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ncRNAs stimulate the production of I FN in transfected cells. In addition, the

IFN mRNA induction levels correlate with antiviral activity

against vesicular stomatitis virus (VEV). In studies of viral challenge,

ncRNAs of the invention protect against foot-and-mouth disease virus as well as

against the West Nile virus, the IRES sequence of the VFA being the one

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better protection offers in both cases while the S and 3'NCR regions

of said virus offer less protection. Moreover, the production of

IFN occurs in the first hours after transfection and antiviral activity

against VEV is detectable at 12 hours post infection, which converts at

ncRNAs of the invention in an effective tool for the treatment of

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diseases caused by IFN-sensitive viruses since the ncRNAs of the

invention activate the innate immune response quickly. The results

obtained in the present invention show the potential use of the ncRNAs of

the invention for the preparation of an antiviral drug against viruses

IFN sensitive. The ncRNAs of the invention can be very useful in combination with vaccines against diseases caused by IFN-sensitive viruses to cover their susceptibility window.

For all that is described herein, a first aspect of the present invention relates to the use of a non-coding region of the FMD virus genome for the preparation of a pharmaceutical composition.

In the present invention it is understood as "region not codified" (NCR, "noncadíng ruled", "untranslated ruled" or UTR) that region of the foot and mouth disease virus genome located at its terminal ends (5 'and 3') that It contains highly structured domains forming a double stranded RNA that does not encode any protein. Among the non-coding regions are the so-called S fragment, IRES ("internals régosame entry si / e ') at the 5' end, at the so-called 5'NCR and at the 3 'end the 3'NCR region is located.

RNAs obtained by in vitro transcription of the S and IRES regions of the 5'NCR and the 3'NCR region of FMD virus are used in the present invention. Hereinafter we will refer to the S and IRES regions of the 5 'end and the 3'NCR region of the VFA as the "NCRs of the invention" or "non-coding regions of the invention", while we will refer to the RNAs obtained by in vitro transcription of said regions as to the "ncRNAs of the invention" (non-coding RNAs of the invention).

The term "pharmaceutical composition" herein refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases. In the context of the present invention it refers to a composition comprising at least the ncRNAs or NCRs of the invention. The pharmaceutical composition of the invention can be used both alone and in combination with other pharmaceutical compositions, including

vaccines and antivirals. The combination of said pharmaceutical composition with

vaccines or antivirals could make the immune response more effective than

generate, acting as an adjuvant. The term pharmaceutical composition and

Medications are used interchangeably in that invention.

S

In the context of the present invention the term "vaccine" refers to a

antigen preparation used to elicit a system response

Immune to disease caused by a virus. It is an antigen preparation

which, once inside the organism, provokes the response of the system

immune by producing antibodies, and generates memory

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immunological producing permanent or transient immunity.

In the present invention the term "antiviral" refers to any substance

that does not allow virus replication, assembly or release, as per

example interferon, ribavirin, etc.

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The ncRNAs of the invention have proven capable of inducing activity

antiviral against viruses sensitive to INF, such as VFA, VEV, and WNV.

For that reason, a second aspect of the present invention relates to

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use of a non-coding region of the fever virus virus genome

aphthous for the elaboration of a pharmaceutical composition for the treatment

of diseases caused by interferon-sensitive viruses.

"Treatment" refers to both therapeutic and prophylactic treatment or

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preventive measures. Those necessary for treatment include those already

associated with alterations as well as those in which the

disturbance. An "alteration" is any condition that would benefit from

treatment with the composition of the invention, as described in the

present document

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"Interferon sensitive virus" means a virus whose replication is seen

affected or interrupted by interferon secreted by the cells of the

host.

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Interferon is a cyto quina secreted by various cells, including

Immune system cells, which has antiviral effect. Of the groups of

interferons, it is the alpha and beta that are generally induced in

response to viral infection, for that reason in the present invention

"interferon" refers to IFN-a or IFN-¡3.

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The FMD virus (VFA, "1001 and moulh disease virus", FMDV) is the

virus causing foot and mouth disease. It belongs to the family Picornaviridae (genus

Aphlhovirus) and its genome is a single stranded RNA. Seven have been described

different serotypes with multiple subtypes and variants, serotype O, A, C, Asia1,

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SAT1, SAT2 and SAT3. The isolates used in the present invention are the

01 K, belonging to serotype O (the genome of lineage isolates 01, 01 c and

01 K is included in the Genbank access numbers D1 0138 Y

X00871) and the C-S8c1 serotype C isolate (genome in accession number of

Genbank AJ133357). The length of the NCR regions in the different

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isolated varies and the homology is 80% for the S region, 85% for the

5'NCR region comprising IRES in addition to fragments of other regions and

82% for the 3'NCR region, so the non-coding regions of the

VFA would present at least 80% homology between the different serotypes

(Carrillo on al. 2005. J ViroI 79: 6487-6504).

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The ncRNAs used in the invention correspond to the S and IRES regions

from the 5 'end and to the 3'NCR region. The ncRNAs used in the invention

they constitute a non-infectious material of easy production and handling

Biotechnology The results obtained with the ncRNAs used in the

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The present invention demonstrates the potential use of non-coding regions of the

VFA for the preparation of a pharmaceutical composition for treatment

of diseases caused by interferon-sensitive viruses.

In the non-coding region of the 5 '(5'NCR) end of the VFA genome,

find the S fragment. The S fragment encompasses 366 S'-terminal nucleotides

of RNA and its folding is predicted in a secondary structure type

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long and stable hairpin ("hairpin") (included in the access sequence in

GenBank X00871). The S fragment is followed by a poly C region ("heteroplolymeric

poli e tracl ', Cn), several pseudo-knots (pseudoknots, Pk), the element of

ere replication ("cis-acting replication element ') and the internal entry site of

ribosomes (IRES, "internal ribosome entry site ') (Fig. 1). IRES is a region

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of 454 nucleotides structured in multiple domains mediating translation

cap-independent of the viral genome (included in the access sequence

in GenBank AJ133357). On the other hand, at the 3 'end of the viral genome

there is also a non-coding region called 3'NCR. The 3'NCR

includes 90 nucleotides from the termination codon of the reading phase

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open viral RNA and a variable length poly A tail, where said

Length increases with the course of infection (90 nucleotides without poly tail

A are included in the access sequence in GenBank X00871). He

3'NCR of the VFA has been seen to exert a translation activating effect

IRES dependent and that is able to interact with IRES regions and

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S located at the 5 'end by direct RNA-RNA interactions.

In the present invention they have been generated by in vitro transcription

analogs of VFA NCRs. Due to the strategies known for

any subject matter expert derived from biotechnological methods

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used in said in vitro transcription (such as the use of a DNA

complementary inserted into the plasmid used in transcription, cloning

and the use of restriction enzymes), the ncRNAs of the invention contain in

its extra nucleotide terminal ends. The ncRNAs of the invention are

non-coding regions that are functional analogs of the NCRs of the

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invention.

The term "analog" refers to a region of the same function but of different

sequence. In the present invention it is meant that ncRNAs have the

same function as the NCRs regions, comprise the NCR sequence but

contain more nucleotides from the biotechnology strategy

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Used for its synthesis.

The "extra" nucleotides referred to in the present invention are those

Ribonucleotides not present in the VCR NCRs but which are the result of

biotechnological strategies used in the present invention for the

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ncRNAs generation. These extra nucleotides can be incorporated into the

VFA NCRs for the use, for example, of an RNA polymerase, for being

nucleotides present in the plasmid in which the DNA is cloned

Supplementary used for transcription ín viro or to be introduced in the

sequence to generate restriction targets to favor strategies

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Biotechnology

A "restriction target" (or restriction site) is a recognized sequence

by a restriction enzyme, that is, it refers to a site or place that

recognizes an endonuclease that is capable of cutting phosphodiester bonds of

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the nucleotide chain generating a cut in the acid chain

deoxyribonucleic (DNA). In the present invention it refers to a sequence

recognized by a restriction enzyme that is capable of cutting bonds

Complementary ONA phosphodiester used for in vitro transcription. By

example sequences recognized by restriction enzymes Stu I and EcoRV.

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The ncRNA of the 3'NCR region of the VFA (SEQ ID NO: 1) contains 188

ribonucleotides of which the first 15 and the last 16 correspond to

nucleotides not present in the 3'NCR of the VFA and that are the result of

Biotechnological strategies used for its generation. Also contains

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three additional ribonucleotides that have been introduced to generate sites of

restriction that allow its biotechnological manipulation and that do not affect the

secondary structure that is generated in the 3'NCR and therefore to its function. Is

the case of uracil from position 36 that derives from a thymine in the DNA clone

template that generates a restriction target for the enzyme Stu 1 (AGGCCT) and the

case of the guanine of position 108 Y of the uracil of position 110,

derivatives of a guanine and thymine in the template clone that generate a target

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of restriction for EcofN (GATATC). So that the 3'NCR region of the VFA to

that referred to in the present invention is considered as the region

between nucleotide 23 and 114 described in SEO ID NO: 1

(where said nucleotides are also included) in which they are not

nucleotides 36, 108 and 110 are present. On the other hand, the ncRNA of the region

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VFA S (SEO ID NO: 2) contains 404 ribonucleotides of which 20

first and last 18 correspond to nucleotides not present in the

S sequence of VF A and which are the result of biotechnological strategies

used for its generation. So that the S region of the fever virus

aphthous to which the present invention refers is the sequence comprised

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between (and even) nucleotide 21 and 386 described in SEO ID NO: 2. In the

case of the ncRNA of the IRES region of the VFA (SEO ID NO: 3), it contains 476

ribonucleotides of which the first 14 and the last 8 correspond to

nucleotides not present in the IRES of the VFA and that are the result of the strategies

Biotechnology used for its generation. So the IRES region of

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FMD virus referred to in the present invention is the

sequence between (and inclusive) nucleotide 15 and 468 described

in SEO ID NO: 3.

In the present invention the term "region", "sequence" or "sequence

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nucleotide "are used interchangeably in memory.

From the foregoing, a preferred embodiment of the first and second

aspect of the present invention relates to the use of a non-coding region

of the VFA genome where said non-coding region is comprised in the

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nucleotide sequence that is selected from the list comprising: SEO ID

NO: 1, SEO ID NO: 2, SEO ID NO: 3 or any of its combinations.

Where SEO ID NO: 1 comprises the 3'NCR region of the VFA, SEO ID NO: 2

comprises the region or fragment S of the VFA, SEQ ID NO: 3 comprises the IRES region of the VFA

A more preferred embodiment refers to the use where the sequence is SEO ID

NO: 1.

Another more preferred embodiment refers to the use where the sequence is SEO ID

NO: 2.

Another more preferred embodiment refers to the use where the sequence is SEO ID

NO: 3.

The present invention also relates to the use of other NC Rs or ncRNAs of

other virus isolates. For this reason and for the foregoing, the present invention also relates to the use of the non-coding regions of the invention and to the ncRNAs of the invention where the non-coding region refers to a region with a homology of at least 80% with that region.

The present invention also relates to the NCRs or the ncRNAs of the

invention that by biotechnological strategies known by any person skilled in the art have their sequence modified, for example by the generation of restriction targets, but where said modifications do not significantly affect the secondary structure of the RNA

It is known to any person skilled in the art that viruses whose genome is a single chain RNA are more sensitive to IFN than viruses with ONA. Various viruses that are sensitive to interferon have been described, including members of the Picornaviridae, Flaviviridae and Rhabdoviridae families,

for example, the foot and mouth disease virus, West Nile fever virus and vesicular stomatitis virus. Despite presenting sensitivity to INF,

Many viruses are capable of developing mechanisms to avoid this innate immune response, as in the case of VFA, VEV and WNV.

(Randall R E et al. J Viral Gen 2008. 89: 1-47). The present invention demonstrates that the use of the ncRNAs of the invention is useful as an antiviral

even in viruses that present such evasion mechanisms.

As described herein, an even more preferred embodiment refers to the use where the interferon-sensitive virus is a single-stranded RNA virus. Preferably, the virus is a virus of the Picornaviridae family, more

preferably the foot and mouth disease virus. Preferably also the virus is

a virus of the family Rhabdoviridae, more preferably refers to the vesicular stomatitis virus. And also preferably the virus is a virus of the family Ffav; v; ridae, where said virus is preferably the Nile virus

Western.

Another preferred embodiment of the second aspect of the invention relates to the use where said diseases are diseases in non-human mammalian animals. Preferably ungulated animals, more preferably of the

Suidae family (for example from the genus Sus, for example S. scrofa), from the Bovidae family or from the Equidae family. Also the present invention relates to

use in birds, preferably from the families of birds that are selected from the

list comprising: Phasianidae, Corvidae, Paseridae, Fringillidae and Turdidae

(belonging to the Passeriform and Galliform orders), as well as the

Accipitridae and Strigidae families, among others.

Another preferred embodiment of the second aspect of the invention relates to the use where said diseases are diseases in humans. Diseases can be zoonotic diseases.

A third aspect of the invention relates to a pharmaceutical composition.

of the first or second aspects of the invention which further comprises at least one pharmaceutically acceptable excipient and / or vehicle.

The term "excipient" refers to a substance that helps the

Absorption of the pharmaceutical composition comprising the NCR or the ncRNA of the invention, stabilizes or aids in its preparation in the sense of giving it a consistency, shape, flavor or any other specific functional characteristic. Thus, the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.

A "pharmacologically acceptable vehicle" refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants. The carrier can be an inert substance or of analogous action to any of the compounds of the present invention and whose function is to facilitate the incorporation of the drug as well as other compounds, allow a better dosage and administration or give consistency and form to the composition Pharmaceutical When the presentation form is liquid, the vehicle is the diluent. The term "pharmacologically acceptable" refers to the compound referred to being allowed and evaluated so as not to cause damage to the organisms to which it is administered.

As described herein, a preferred embodiment of the third aspect of the invention relates to a pharmaceutical composition that also comprises at least one other active ingredient.

As used herein, the term "active substance" ("active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient") means any component that potentially

provide a pharmacological activity or other effect different in the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect. The pharmaceutical composition or medicament provided by this invention may be provided by any route of administration, for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen. For this reason, a preferred embodiment for this aspect of the invention relates to the pharmaceutical composition wherein said pharmaceutical composition is presented in a form adapted to oral, parenteral or intradermal administration.

The present invention also relates to the use of the pharmaceutical composition of the third aspect of the invention for the preparation of a medicament for the prophylaxis and / or treatment of diseases caused by intelferon-sensitive viruses.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Structural reasons in the NCRs of the VFA genome. Schematic representation of the motifs in the 5'and 3'NCRs of the VFA. NCR,

non-coding region; S, fragment S; IRES, "ribosome entry site"; VPg, "genome linked viral pro / eirl '; Cn, poly C region; Pk, region of" pseudokno / s';

ere, the cis replication element ("cis-acting replication efemen! '); lpro,

protease "Ieade"; 3Dpol, 3D polymerase; SL 1, '' stem-Ioop 1 "; SL2," stem-Ioop 2 "; 3'NCR, 3 '" non coding region "; <VIn, transcribed 3'NCR to which the poly A tail has been removed.

Fig. 2. Induction of IFN- ~ in porcine cells. It shows the production of IFN-p by the ncRNAs of the invention of VFA in porcine cells. SK6 cells were transfected with 20 g / ml of the NCR transcripts or with 10 g / ml of the poly I: C control or the tRNA negative control (transfer RNA). The levels of

induction of IFN-j3 mRNA in cells transfected with respect to cells

transfected with phosphate buffered saline (PBS) at the indicated times were determined by RT-qPCR (quantitative RT-PCR) normalized with respect to

to GAPDH. Error bars show the standard deviation of the mean of

Three independent experiments performed in triplicate. IFN-p, porcine IFN-p messenger RNA; T, time; h, hours

Fig. 3. Analysis of the contribution of VFA 3'NCR sequences

in the induction of IFN-Jl. It shows the production of IFN- ~ by the 3'NCR region of VFA and fragments thereof in porcine cells. RNAs corresponding to SL 1, SL2, 3'NCRóA "as well as 3'NCR RNAs (20 ~ g / ml) treated or not with CIP and poly I: C (10 ~ g / ml) were transfected into SK-6 cells. Induction levels of IFN-p mRNA in transfected cells relative to cells

transfected with PBS at the indicated times were determined by RT

qPCR normalized with respect to GAPDH. Error bars show the

standard deviation of the average between triplicates. IFN-j3, RNA messenger of

Porcine IFN-p; T, time; h, hours; SL 1, "s / em-Ioop 1"; SL2, "stem-Ioop 2"; 3'NCR, 3 '"non coding regiorl'; f) .An, transcribed 3'NCR in which the poly A tail has been removed; 3'NCR + CIP, 3'NCR that has been treated with phosphatases.

Fig. 4. Induction of an antiviral state in cells transfected with the

NCR RNAs. Shows the antiviral effect against vesicular stomatitis virus

in swine cells. After 24 h of transfection with 40 ~ g / ml of the transcripts

corresponding to the NCRs of VFA, or 10 ~ g / ml of tRNA. SK-6 cells were

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infected with VEV (multiplicity of infection (MOI) = 1). Supernatants

of infected cells were collected at 12 and 24 h post-infection and the titers

virals were determined in IBRS-2 cells. The bars indicate the values

means of two independent experiments performed in triplicate plus the

standard deviations Statistical analysis was performed using ANOVA and

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Bonferroni test (O p <0.05.00 p <0.01). T, post infection time; h, hours;

log10pfu / ml, viral titration measured in log10 of the forming units of

plates (pfu, "plaque forming unit", pfu) per milliliter; mock control of

transfection

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Fig. 5. VFA NCRs induce the innate response in a nursing mouse.

It shows the production of IFN-p by the ncRNAs of the invention in mice

infants Groups of 4 to 5 Swiss lactating mice were inoculated

Intraperitonealemnte (i.p.) with 100 ~ g of 3'NCR transcripts. S. IRES or poly I: C.

IFN-a (Al.e IFN- ~ (B) levels in pools of serum extracted at different

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Post-inoculation times for each RNA were determined by ELlSA. The

average values of IFN-a and IFN- ~ for serum pools at O h post inoculation

they were 25 and 6 pg / ml, respectively.

ILLUSTRATIVE EXAMPLES OF THE INVENTION

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The following specific examples provided in this document

The patent serves to illustrate the nature of the present invention. These

Examples are included for illustrative purposes only and should not be

interpreted as limitations to the invention claimed herein. So,

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the examples described below illustrate the invention without limiting the field of

application of it.

.

EXAMPLE 1: VCR genome NCRs contain PAMPs (molecular patterns associated with pathogens) that activate innate response signaling.

The ability to induce IFN-¡3 expression in porcine cells of highly structured elements of the 5 'and 3'NCRs regions of the VFA RNA was initially analyzed. RNAs corresponding to the 5'and 3'NCRs were synthesized by in vitro transcription. and to the S and IRES fragment of the FMD virus (Fig. 1). VFA ncRNAs were used to transfect SK-6 cells (porcine kidney cells) (Fig. 2). IFN-j3 mRNA expression induction was analyzed by quantitative real-time RT-PCR at three different times (Fig, 2) using the sense primer shown in SEO ID NO: 4 and the antisense primer shown In SEO ID NO: 5, notable differences were observed between the different RNAs. At three times the 3'NCR was the most potent IFN-j3 transcriptional inducer of all, reaching levels almost 200 times above the transfection control cells at 9 h posttransfection. The S fragment induced levels 10 times higher than the control followed by the full 5'NCR. For transcrile corresponding to IRES, inductions of 4.5 times were detected, slightly higher than poly I: C. These results suggest that the structures present in the 3'NCR are recognized more efficiently than those of the 5'NCR by the viral sensors present in porcine cells and that the S fragment is the main inducing element in the 5'NCR in these cells.

EXAMPLE 2: Analysis of the characteristics of the 3'NCR of the VFA relevant for the induction of IFN-j3,

The contribution of the different structures present in the 3 'NCR and of the VFA poly A to the induction of the IFN-j3 mRNA was analyzed by Iransfection in SK-6 cells of the corresponding Iranscripts (Fig. 1 and 3). Neither of the two "stem-Ioops" present in the 3'NCR, SL1 (SEO ID NO: 6) and SL2 (SEO ID NO: 7) were able to induce detectable levels of IFN- (3, being minimally active (Fig. 3) However, the 3'NCR with the poly A tail removed (3'NCR 8An, SEQ ID NO: 8) was still a potent activator, reducing only between 2-10 times according to the post-transfection time analyzed (Fig. 3) The activation levels detected did not correlate with

5 the size or molarity of transfected RNAs. The induction role of the 5'-triphosphate end in RNA was analyzed by treatment with alkaline phosphatase (CIP). The effect of this treatment significantly reduced induction levels at 9h post-transfection, although 20-fold inductions were still detected with respect to control cells.

EXAMPLE 3: The "PAMP" elements of the VFA genome stimulate innate immunity and antiviral response in porcine cells

To determine whether the induction of IFN-13 mRNA observed in SK-6 cells

15 transfected with the S, IRES and 3'NCR elements of the VFA corresponded with the activation of an anti-viral state in the cells, the antiviral activity in the transfection supernatants was analyzed, incubating with them IBRS-2 cells (porcine cells of kidney) (Table 1). After 24 hours, the cells were infected with VEV. In all cases antiviral activity was detected (Table

1), except in the controls (cells transfected with poly I: C or tRNA, where the result was the same for both cases), and the levels of inhibition of VEV infection correlated well with the mRNA levels of IFN-13 Induced by each RNA. Treatment of transfection supernatants with specific monoclonal antibodies blocking IFN-a, -13 confirmed

25 that the antiviral activity observed is mainly mediated by IFNs.

Table 1. Antiviral activity in transfected porcine cells.

Antiviral activity

Antibody Antibody Antibody

Supernatant

Monoclonal monoclonal RNA monoclonal

without treating

anti-IFN-a. anti-tFN-p anti-IFN-a. + p

VFA3'NCR

27 <2 6 <2

VFAS

13 3.8

twenty

VFAIRES 9 Poly I: C or tRNA <2 NA NA NA

a SK-6 cells were transfected for 24 h with 40 ~ g / ml of the ncRNAs,

or 10 ~ g / ml of poly I: C or tRNA The antiviral activity is expressed as the

reciprocal of the highest dilution of the transfected SK-6 cell supernatants necessary to reduce the number of VEV plates on IBRS-2 cells by 50%. Data are the average of duplicates of three independent transfection experiments_ In some cases, supernatants were previously incubated for 1 h at 37 Oc with 1 ~ g / ml

of neutralizing monoclonal antibodies against IFN-a, -p or both,

respectively. -, undetermined. NA, not applicable.

On the other hand, the induced antiviral activity on the SK-6 cells transfected with the different RNAs was studied, infecting them with VEV at an MOl of 1. At 12 and 24 h post infection, supernatants were collected and the

viral titer by plating on IBRS-2 cells (Fig. 4 shows

5 results at 12 and 24 hours post infection). In the cells treated with the RNAs the viral yield had been reduced by 3 lag compared to that observed

in the transfected control cells (with tRNA-or with PBS, "mock"). At 12 h post infection the VEV titers showed differences of 5.5 times (although not significant) between the cells treated with the 3 · NCR element with respect to the

10 others, while at 24 hours the viral titles were more similar.

These results suggest that all the NCR RNAs elements used, even

inducing the transcription of IFN-f3 at different levels, they are able to reach the threshold level of induction necessary to trigger in the

15 porcine cells transfected an effective anti-viral response, both autocrine and paracrine.

EXAMPLE 4: Inoculation of the "PAMP" elements of the VFA genome induces an innate immune response in lactating mice

To determine if the "PAMP" elements of the VFA genome were able to

induce an antiviral response in vivo, the transcripts corresponding to the

S, IRES and 3 · NCR regions of the VFA were inoculated intraperitoneally to

Swiss mouse litters 5-7 days old. As a control, poly was inoculated

I: C, a synthetic compound for which an effect on ta

induction of immune response.

5

The levels of tFN-a and IFN-¡3 in serum samples collected at 4, 8, 24 and 48

post-inoculation hours for each ncRNA group were analyzed by ELlSA

(Fig. 5 A and 5 B). All the elements tested proved powerful

IFN-a / ¡3 inducers in lactating mice, even those in cells

pigs in culture had induced low levels of mRNA expression

10

of IFN-¡3.

Serum IFN-a peaks were detected at 8 h after inoculation of the

3'NCR and IRES elements, while for S the IFN-a levels are

they kept high for longer, with a maximum at 24 h post

t5

inoculation (Fig. 5 A). Comparing IFN-a levels at 8 h post-inoculation,

RNA elements of the VFA genome induced 15 to 40 times more than

e l poly I: C, while at 24 h post-inoculation, the levels induced by S

were 90 times higher than those induced by poly I: C. This pattern was repeated.

in the kinetics of IFN-¡3 (Fig. 5 B).

twenty

The biological relevance of these IFN-a / ¡3 levels detected by ELlSA in

serum was checked by a VEV infectivity inhibition assay

in L-929 cells (Table 2), observing a good correlation between the two

essays, both in levels and kinetics.

25

Table 2. Antiviral activity in the serum of lactating mice.

transfected8

h post-inoculation

RNA 4 8 24 48

S

2 128 162 <2

IRES

64 128 8 <2

3'NCR

<2 32 <2 NR

Poly I: C 8 8 <2 <2

Groups of 4-5 mice were inoculated with 100 I-JQ of VFA ncRNAs

or poly I: C. The sera were collected and mixed, at the indicated post-inoculation times, for each RNA. The antiviral activity is expressed as the reciprocal of the highest dilution of serum capable of suppressing the cytopathic effect induced by VEV in L-929 cells in 50% of the wells. No antiviral activity was detected in serum poales collected at O h post-inoculation «2). NR, not done.

Together these results indicate that RNA transcripts

corresponding to the structural elements present in the NCRs of the

3 'and 5' ends of the VFA genome can stimulate an immune response

innate and induce an antiviral state in vivo.

EXAMPLE 5: Survival against the challenge with VFA or with WNV. To determine which of the VFA ncRNAs is the one with the greatest protection

induces against viral challenge with VFA or with WNV, we proceeded to perform

inoculation based protection mice in mice

10 intraperitoneal (i.p.), and intracranial (i.c.) and i.p. in the case of WNV, of the ncRNAs and subsequent infection with different infective doses of the viruses (VFA or WNV) that cause the death of the mice at established doses and times

previously. The protective effect of inoculation with ncRNAs is established

by comparison with the control group of animals inoculated with buffer

15 saline phosphate (PBS). In the case of WNV, control groups were also included

who were inoculated with PBS and challenged with the same amounts of

viruses that had been previously incubated (1: 1 dilution) for 1 hour at

room temperature with a mixture of sera from mice surviving the

infection whose protective capacity against infection with WNV was

20 known (Alonso-Padilla on al. 2011 Vaccine 29: 1830-1835), and untreated groups

that were inoculated I.C. with PBS as a good handling control.

5.1. Protective effect of VFA ncRNAs against infection with VFA

The survival results against the different dilutions tested are shown in Table 3. Briefly, the protective effect of the different

ncRNAs compared to that of poly I: C was gradual. The effect of 3'NCR and poly I: C was similar. From 8 x 10'pfu (10- 'dilution) the poly I: C was better; below that dose (10-4 onwards) were equivalent in

protection. Inoculation of RNA S induced a greater protective effect, with 50% of live animals until day 11 post infection (p.i). of dilution 105 2, same as poly I: C. However, at higher doses of virus, RNA S protected 50% of the animals even against undiluted virus preparation

(8 x 106 pfu), while poly I: e stopped protecting at 10-1 dilution. The best protection results were obtained by inoculating the IRES, with total protection (100%) against VFA until dilution 10-2 and 90% at doses

10 higher to undiluted virus, indicating that even with doses of virus far above physiological levels, VFA is only capable of killing 10% of animals inoculated with this RNA. No animal of the control group

survived from day 1 p.i. with dilution 10-2. The "plateau" effect observed for RNAs with greater protection capacity (S and 15 IRES) is interesting. With these RNAs an antiviral barrier seems to be established that the

virus infedivity is unable to overcome, about 50% for the S and the

10% for eIIRES.

The LD so values (lethal dose-SO / mi) shown in Table 3

20 correspond to the dilution of the viral preparation used necessary to kill 50% of the animals inoculated in each case. For S and IRES, since there is no dilution at which the virus kills a percentage of animals> 50%, it is only indicated that it is <10 °.

25 Table 3. Survival of lactating mice inoculated with ncRNAs at 11

days post-infection with the VFA.

RNA Dilution Percentage of inoculated virus survivors / inoculated survival LDso / mlb

OR

(5/10) fifty

S

-1 -2 (4/8) (7/12) 50 58

-3

(10/11) 90

-4 (12/12) 100 -5 (11/11) 100

O (9/10) 90

-

1 (7/8) 87.5

-

2 (10/10) 100

<10 °

IRES

-

3 (10/10) 100 -4 (10/10) 100 -5 (9/9) 100

-

1 (0/8) O -2 (1/10) 10

3'NCR

-

3 (6/11) 54 9 x 10-3 -4 (5/5) 100 -5 (4/4) 100

O (1/10) 10

-

1 (0/10) OR

-

2 (7/11) 64

7 x 10-2

Polyl: C

-

3 (7/8) 87.5 -4 (12/12) 100 -5 (8/8) 100

-

2 (0/9) O -3 (1/24) 4

PBS

-

4 (7/25) 28 5 x 10-5 -5 (18/27) 67 -6 (9/9) 100

PBS + LP * I -4 (0/6) OR

* Lypofectin; LD, lethal dose. -1, dilution 10- '. -2, dilution 10 -'-- 3, dilution 10- '. 4, dilution 10 -'_ -5, dilution 10- '

5_2_ Dose effect

5 An inoculation experiment was carried out with doses lower than the one commonly used (100 g) with the ncRNA that showed a greater protective capacity, the IRES using a high dose of virus. The animals were inoculated intraperitoneally, as described above, with the RNA and 24 h later with the 10-2 dilution, equivalent to 8 x 10 'pfu (units

plate former) of VFA. The results are shown in Table 4.

Indeed, a dose effect on protection was observed, with a very slight decrease (90%) with 50 ~ g of RNA and greater, 10% with 1 I-JQ. This result confirms the specificity of the protection observed with the ncRNAs and the suitability of the 100 I-Jg dose used in the trials.

Table 4. Survival of inoculated lactating mice with different doses of

IRES of VFA on day 11 post-infection with VFA

IRES dose

survivors / inoculated Survival percentage

50 IJQ

(9/10) 90

10 IJg

(3/10) 30

5IJQ

(4/10) 40

1 1'9

(1/10) 10

10 5.3. Kinetics of protection To assess the time window covering the protection mediated by ncRNAs with respect to the time of infection, dilutions 102 and 10-4 of VF A were inoculated in litters of infants who had previously been inoculated with ellRES (intraperitoneally, 100 ~ g) at different times after

15 RNA inoculation The results are shown in Table 5. The

Total protection against infection with 10-4 dilution of FMD virus was maintained at all times tested from minutes after

inoculation until 72 hours later. For dilution 10. 2 the prolection was complete

after 8 hours of inoculation with the IRES and 60% after 4 hours. However, of

20 almost immediately to inoculation (5-10 min, corresponding to time O) the prolection levels were 100%. On the other hand, 48 hours after

RNA inoculation protection against dilution 10-2 was 50% and at 72 h the animals were totally susceptible to infection.

25 Table 5. Number of survivors per day 10 post-infection / number of

inoculated

RNA pre-inoculadon hours

Challenge with Dil (-2) Dil (-4) virus

-72 8/8 8/8 -48 4/8 8/8 -24 6/7 7/7 -8 lO / lO la / la -4 6/10 9/9 Or 9/9 the

5.4. Effect of lipofectin The effect of lipofectin as a vehicular agent on the protective capacity of ncRNAs was analyzed. The level of protection was studied

5 induced by ellRES with and without lipofectin against dilutions 10-2 and 10-4 of the VFA (Table 6). The deliRES protective effect was reduced by 30% when inoculated without lipofectin against 10-2 dilution and 10% against 10-4 (Tables 1 and 4). No animal inoculated with PBS + lipofectin survived the infection with the 10-2 dilution of the VFA. These results indicate that

10 lipofectin per se does not induce protection although it does enhance the protective effect of ncRNAs probably increasing its stability and / or access to target cells.

Table 6. Survival after infection with VFA at 24 h post-inoculation of 15 IRES of VFA with / without lipofectin (LP)

Survivors Dilution Number Day Day Day Day Day Inocula initial virus of 3 4 5 6 7 animals IRES (+ LP) -2 9 9 9 9 9 9 IRES (-LP) -2 10 9 8 8 7 7 -4 10 10 9 9 9 9 PBS (+ LP) -2 7 1 OOOO

Day 10 9 7 9 W Final% 100 70 90 O

5.5. Protective effect against infection with West Nile virus Survival tests against WNV confirm those obtained against VFA, so that greater protection was obtained with the IRES region of VFA

than with the S region of the VFA, which was also greater than that obtained with the control RNA (poly I: e).

Survival results 15 days post infection against the different

5 dilutions tested are shown in Table 7. Briefly, the protective effect of the different ncRNAs (IRES and S) inoculated 24 hours before viral infection was equal to or better than that of the control (poly I: C). In all conditions of inlection tested (10 ulp / mouse via ic, and 100 ulp / mouse both via jc and ip). The IRES conferred a very high protection, which

10 became complete (100%, 100% and 46.2%). On the other hand, the survival percentages of the S region were also very notable (100%, 96% and 12.5%) compared to those obtained with poly I: C (100%, 33.3% and 0%). The survivals obtained with the ncRNAs of the invention were similar to those obtained when the virus was neutralized with murine sera, whose

The protective capacity was known, prior to inoculation (100%, 100% and 77%). Survival rates of the control groups inoculated with PBS were 12.5%, 40% and 0%, respectively.

Table 7. Survival of lactating mice inoculated with ncRNAs at 15-20 days after intracranial (Le.) Or intraperitoneal (j.p.) postinfection with WNV.

IRES inoculated RNA

Dose virus (pfu / mouse) 10 100 100 Via inoculation (Lp / i.c.) Lc i.p i.c Survivors Inoculated 15/15 12/12 5/13 Survival percentage 100 100 46.2 PS (Chi ') 0.0001 0.0010 0.0266

S

10 100 100 Lc i.p i.c 11/11 16/17 2/14 100 94.1 12.5 0.0002 0.0010 ns

Polyl: C

10 100 100 Lc i.p i.c 12/12 4/12 0/17 100 33.3 O 0.0001 ns ns

2.

PBS

10 .C 1/7 12.5

100

i.p 6/15 40

100

i.c 0/10 OR

Serum

10 You 13/13 100 0.0001

100

i.p 11/11  100 0.0010

100

.C 10/13 77 0.0002

..

Ns, no sglnlflcatlvo

MATERIAL AND METHODS USED

5 Cells and viruses.

The IBRS-2, SK-6 pig kidney cell lines (monkey kidney epithelial cells) of the Animal Health Research Center (CISA-INIA), Valdeolmos, Spain, and murine L-cells have been used 929, of

10 laboratory of Dr. Alcami, of the Severo Ochoa Molecular Biology Center, Madrid, Spain. The cells were grown in DMEM supplemented with 1.0% fetal bovine serum, penicillin / streptomycin and glutamine.

The viruses used in the infection experiments were: the 01K foot-and-mouth disease virus 01K, the vesicular stomatitis virus Indiana and the West Nile virus strain NY -99.

Preparation of RNAs and transfections.

20 RNA transcripts corresponding to the S, 3'NCR fragment and its derivatives SL 1 (3'NCR ~ SL2), SL2 (3'NCR ~ SL1) Y ~ An (with the poly A tail removed) of the VFA 01 genome K (the genome sequence of isolate 01 K is considered collected in Genbank accession numbers D10138 and X00871) were generated by in vitro transcription with T3 RNA polymerase a

25 from plasmids previously described and linearized with Nol I (Serrano PM al. 2006. J Viral Gen 87: 3013-3022). The RNA corresponding to the VFA IRES of the C-S8c1 isolate was obtained from a pGEM-derived clan (Ramos R al. 1999. RNA 5: 1374-1783) assigned by E. Martínez-Salas, after linearizing with Xhol and transcribing in vilro with T7 RNA polymerase (NEB). The RNA corresponding to 5'NCR, including fragment S, IRES and 212

nucleotides of the Lpro coding sequence were synthesized with RNA

SP6 polymerase, using the p01 K clan as a template (Rodríguez-Pulido MF et al. 2009. Journal of Virology 83 (8): 3475-3485) linearized with Accl. Unless otherwise indicated, all transcripts contain a triphosphate group in the

5 'end and the 3'NCR elements of the VFA and its derivatives carry a tail of

58 nucleotide poly A.

After transcription, the RNAs were treated with DNase R01 (1 U / g, Promega), extracted with phenal / chloroform and precipitated with ethanol.

Finally they were resuspended in water and quantified by

spectrophotometry The integrity and size of the RNA were analyzed by

electrophoresis in denaturing gels 6% acrylamide, 7 M urea or agarose gels.

Elimination of the 5 'end phosphate groups in the VFA transcripts

3'NCR was performed by incubating for 90 minutes with 0.5 U / ~ g of phosphalase

alkaline (CIP, New England Biolabs). After this treatment, the RNA will

extracted twice with phenal / chloroform, precipitated with ethanol and treated as indicated above. The integrity of these transcripts was analyzed by electrophoresis in denaturing gels 6% acrylamide, 7 M urea. This

ncRNAS of the invention SEO ID NO: 1, SEO ID NO: 2 and SEO ID NO: 3 were generated.

Before being transfected, the RNAs were heated for 5 minutes at 92 ° C,

They were allowed to cool to room temperature for 10 minutes and kept on ice.

SK-6 cells were transfected using 20 I-Jg / ml of each of the ncRNAs

with Lipofectin (Invitrogen) or with 10 ~ g / ml of poly I: C (Sigma) using

Lipofectamine (Invitrogen).

IFN-j3 expression analysis.

At different post-transfection times, the cells were lysed and the total RNA was

extracted and quantified with the methods known to any expert in the

maleria Retro-transcription (RT) was performed using 500 ng of tolal RNA and the enzyme Transcriptor RT (Roche). The polymerase chain reaction

Quantitative (PCR) was performed from aliquots of the back transcription (RT) reactions (1/10) and the reagents of the kit "LightCyc / er FastStart DNA Master SYBR green l '(Roche) All reactions were performed in triplicate.

Data were analyzed using the ddCt method known to any

skilled. In all assays the expression of the IFN-j3 gene is

normalized with respect to the GAOPH constitutive expression gene, and was expressed as an increase over control transfected cells.

The oligonucleotides used to amplify the mRNA of porcine IFN-j3 were the sequences referred to in SEQ ID NO: 4 (sense primer) and SEQ ID NO: 5 (antisense primer). For GAPOH, previously described oligonucleotides were used (García-Briones MM et al. 2004. Virology 322: 264-75).

IFN bioassay.

The antiviral activity of the supernatants of the transfected SK-6 cells is

determined by means of an inhibition test for the infection of VEV on IBRS-2 cells. Briefly, SK-6 cells were transfected for 24

hours with 40 ~ g / ml of the NCR transcripts, or with 10 ~ g of poly I: C or tRNA (negative control since the transferring RNA is an endogenous RNA and therefore

its transfection should not induce the innate response or the induction of INF). IBRS-2 cells were incubated for 24 hours with different dilutions of the

transfection supernatants, washed and infected with 100 pfu of VEV.

Plates were counted at 24 hours post-infection. In some cases,

Transfection supernatants were previously incubated for 1 hour at 37 ° C with 1 I-Jg of anti-specific neuronizing monoclonal antibodies.

IFN-a porcine (K9, from PBL InterferonSource) or anti-IFN- ~ described previously (Overend C R al. 2007. J Viral Gen 88: 925-31). No effect was observed

inhibitory on the effect of VEV for antibody amounts

monoclonal up to 1 O ~ g. The antiviral activity was expressed as the highest

dilution of supernatant required to reduce the number of plates by 50%.

Mouse experiments.

Litters of Swiss mice (3-7 days old) were inoculated intraperitoneally with 100 ~ I containing 100 ~ g of the transcripts of VFA 3'NCR (SEO ID NO 1), S (SEO ID NO 2), IRES (SEO ID NO: 3) or poly IC, in PBS buffer. For RNA transcripts, 20 g of lipofedin was added

(Invitrogen). At 0, 4, 8, 24 and 48 hours post-inoculation samples were collected from

serum, and samples of 4-5 animals for each RNA were pooled and kept at -80'C until use. The amount of IFN-a and IFN- ~ in serum was determined using ELlSA kits (PBL InterferonSource). To analyze the

antiviral activity in these serum samples, a test of

inhibition of cytopathic effect (Rubinstein SPC al. 1981, J Viral 37: 755-758). Briefly, monolayers of L-929 cells seeded in M multi-well plates

96 were incubated for 24 hours with serial dilutions of the corresponding sera. Subsequently the medium was removed and replaced by

fresh medium containing 100 infective doses-50 (TCIDso) of VEV. At 72

The cytopathic effect was monitored by microscope observation. The antiviral activity was expressed as the reciprocal of the highest serum dilution

It protects against cytotoxicity in 50% of the wells.

Animal management in this study has followed the guidelines at all times.

of the European Community 86/609 / EEC. The protocols were approved by the

INIA Animal Experimentation Ethics Committee (CBS Number Permits

2008/016 for the VFA and 2011/023 for the VNO).

Survival trials Groups of Swiss lactating mice approximately one week old were inoculated intraperitoneally (i.p) with each

RNA (ncRNA) that were subsequently inoculated with different dilutions of

virus: VFA (a litter of 8-12 animals per dilution) 24 hours later by

same route, or WNV (a litter of 9-17 animals per dilution) 24 hours later

well via i.p. or intracranially (i.c.) (both routes of administration were used in different litters). The inoculum had a final volume of 100 1'1 and contained 100 I'g of the RNA to be tested diluted in PBS and 20 I'g of Lipofectin (Invitrogen). Briefly, RNAs synthesized by in vitro transcription

They were heated at 92 'C for 5 min in water. The PBS was then added and allowed to renaturate at room temperature for 10 min. After

Lipofectin was added and after 15 min incubation at room temperature the mixture was injected. Lipofectin is a commercial cationic liposome preparation for routine use in single-stranded RNA transfections. The

VFA RNAs inoculation methodology in mice and pigs has been previously developed. The preparation of the inoculum of poly I: C is also carried out with 100 g of the RNA diluted in PBS but without liposomes since

has described its ability to induce IFN in adult mice after intraperitoneal inoculation as naked RNA. Infection with the virus was performed 24 h

after inoculation with the RNAs for giving the best protection results in previous trials with the 3 · NCR inoculated at 4, 8 and 24 h pre-infection. The inoculum volume of VFA was 100 1.1 diluted in PBS. Dilutions between 10 ° (undiluted) and 10-5 of an infection supernatant in cell culture with a titer of 8x107 plaque forming units (pfu) / ml were tested. In the case of the challenge with the WNV the volume of

inoculation was 10 or 100 1.1, depending on whether the inoculation was Lc. or i.p., diluted in PBS. The challenge was performed with infection supernatant in WNV cell culture strain NY-99 (10 or 100 ufplraton via Lc., And with 100 ufplratón

via i.p) 24 h after inoculation of RNAs.

In the challenge test against WNV, in addition to the control group inoculated with

PBS and virus-challenged (as is the case with VFA) also included control groups that were inoculated with PBS and challenged with the same 5 amounts of virus that had previously been incubated (1: 1 dilution) for 1 hour at room temperature with immune sera from mice whose protective capacity against infection with WNV was known, and groups

untreated that were inoculated i.c. with PBS as a good control

handling.

Claims (23)

1. Use of a non-coding region of the foot and mouth disease virus genome for the preparation of a pharmaceutical composition 2. Use of a non-coding region of the fever virus genome FMD for the development of a pharmaceutical composition for treatment of diseases caused by interferon-sensitive viruses where said viruses are single stranded RNA viruses. 3. Use according to any one of claims 1 to 2 wherein the non-coding region refers to a region with an identity of at least 80% with a nucleotide sequence that is selected from the list that comprises: SEO ID NO: 1, SEO ID NO: 2, SEO ID NO: 3 or any  of their combinations. 4. Use according to any one of claims 1 to 3 wherein said non-coding region consists of the nucleotide sequence that is selected from the list comprising: SEQ ID NO: 1, SEO ID NO: 2, SEO ID NO: 3 or any of its combinations.

5.

Use according to claim 4 wherein the sequence is SEO ID NO: 1.

6.

Use according to claim 4 wherein the sequence is SEO ID NO: 2.

7.

Use according to claim 4 wherein the sequence is SEO ID NO: 3.

8.

Use according to any of claims 2 to 7 wherein the RNA virus

Single chain is a virus of the family Picornaviridae. 9. Use according to claim 8 wherein the virus is the foot and mouth disease virus. 10. Use according to any of claims 2 to 7 where the RNA virus  Single chain is a virus of the family Rhabdoviridae. 11. Use according to claim 10 wherein the virus is vesicular stomatitis virus. 12. Use according to any of claims 2 to 7 where the RNA virus  Single chain is a virus of the Flaviviridae family. 13. Use according to claim 12 wherein the virus is the Nile virus Western.

14.

Use according to any of claims 2 to 13 wherein said diseases are diseases in non-human mammalian animals.

fifteen.

Use according to claim 14 wherein the animals belong to the

Suidae family. 16. Use according to claim 14 wherein the animals belong to the Bovidae family. 17. Use according to any of claims 2 to 7 and 10 to 14 wherein such diseases are diseases in animals that belong to The Equidae family. 18. Use according to any of claims 12 to 13 wherein said  diseases are diseases in birds.

19.

Use according to claim 18 wherein the birds are selected from the families of the list comprising: Phasianidae, Corvidae, Paseridae, Fringillidae, Turdidae, Accipitridae and Strigidae.

twenty.

Use according to any of claims 2 to 13 wherein said diseases are diseases in humans.

twenty-one.

Pharmaceutical composition comprising the non-coding region

5 according to any of claims 1 to 20 which further comprises at least one pharmaceutically acceptable excipient and / or vehicle. 22. Pharmaceutical composition according to claim 21 further comprising at least one other active ingredient. 23. With a pharmaceutical position according to any of claims 21 or 22, wherein said pharmaceutical composition is presented in a form adapted to oral, parenteral or intradermal administration.