WO2005049815A1 - Flavivirus replication - Google Patents

Abstract

The present invention relates to the modification of the 3'UTR of a vector-borne flavivirus, more particularly the modification of one or more nucleotides within the 3'LSH of the 3'UTR, more particularly modifying one or more nucleotides within pentanucleotide sequence 5'-CAC(A/C)G-3' in the 3' UTR of such a virus in order to reduce the replication of a virus in a vector or vector cell. The present invention also relates to a modified nucleotide sequences and modified viruses comprising a functional 3'UTR of a flavivirus and characterized by one or more modified nucleotides within the 3'LSH of the 3'UTR, more particularly by one or more modified nucleotides within the CAC(A/C)G pentanucleotide motif in the 3' UTR sequence. This invention is applicable for the creation of a vaccine where there is no risk for transmitting the virus particles from one organism to another through a vector.

Description

FLAVIVIRUS REPLICATION-

FIELD OF THE INVENTION The present invention relates to a method for inhibiting the replication of viruses and to a method for inhibiting the transmission of viruses from one organism to another, such as from a vector to its host organism (human, animal) and vice versa. This method can be applied in vaccination strategies to inhibit the spreading of a virus from a vaccinated organism (human, animal) ^"to another through a vector (e.g. mosquito or tick). The present invention also relates to nucleotide sequences, vaccines using the nucleotide sequences, modified virus and pharmaceutical compositions comprising the modified virus.

BACKGROUND OF THE INVENTION RNA viruses are divided in several families including the Arenaviridae, the Picornaviridae, Retro viridae, Coronaviridae, Mosaic Viruses, Orthomyxoviridae and the Flaviviridae among others. The family of the Flaviviridae consists of nearly 80 viruses and can be subdivided into three genera: Flavivirus, Pestivirus and Hepacivirus. The Flavivirus genus contains several human pathogens including yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV). The Pestivirus genus is home to bovine viral diarrhea, classical swine fever virus and border disease virus of sheep. The genus Hepacivirus consists, among others, of hepatitis C virus. Hepatitis G virus (also known as GB vims C) is classified within the family of the Flaviviridae but has not (yet) been assigned to .a genus. Members of the Flavivirus genus are, based on their vector, classified into three groups: (i) flavivimses that are transmitted by mosquitoes (mosquito-bome), (ii) flaviviruses that are transmitted by ticks (tick-borne) (iii) viruses with no known vector (NKV) (Chambers et al, 1990a, Annu. Rev. Microbiol. 44, 649-688). All flaviviruses of human importance belong to the arthropod-borne (mosquito- and tick-bome) group and cause a variety of diseases including encephalitis and (hemorrhagic) fevers. One of the most important flavivimses causing human disease is dengue vims (DENV). It is estimated that there are worldwide annually as many as 50 to 100 million cases of dengue fever and several hundred thousand cases of dengue hemorrhagic fever (DHF), the latter with an overall case fatality rate of -5% (Gubler, 1997, D.J. Gubler and G. Kuno (ed.), Dengue and dengue hemorrhagic fever, CAB International, Wallingford, United Kingdom, 1-23; Monath, 1994, Proc. Natl. Acad. Sci. U.S.A. 91, 2395-2400). In the book "Vaccines for the 21^st Century", National Academy of Sciences, 2000, it is stated that dengue hemorrhagic fever does not figure as important for the U.S. public health burden although it goes on to say that analysis of international disease burden would likely lead to results that would be more favourable. Despite the availability of a very efficacious vaccine, yellow fever (YF) is still a major public health problem. Other important viruses of the genus include Japanese encephalitis (JE), a mosquito-borne arboviral infection and the leading cause of viral encephalitis in Asia (Hennessy et al, 1996, Lancet 347, 1583-1586). Tick-bome encephalitis virus (TBEV) is believed to cause annually at least 11,000 human cases of encephalitis in Russia and about 3000 cases in the rest of Europe. Related viruses within the same group, Louping ill virus (LIV), Langat vims (LGTV) and Powassan vims (POWV), also cause human encephalitis. Three other viruses within the same group, Omsk hemorrhagic fever vims (OHFV), Kyasanur Forest disease vims (KFDV) and Alkhurma virus (ALKV) tend to cause fatal hemorrhagic fevers rather than encephalitis (Gritsun et al, 2003, Antiviral Res. 57, 129-146). Other viruses are Murray Valley encephalitis virus (MVEV), St. Louis encephalitis virus (SLEV) and West Nile virus (WNV). In 1999, the WNV disease appeared for the first time in the northeastern United States and has continued to date to spread across the United States and Canada. The NKV-group holds a few viruses which have been isolated from mice or bats and for which no arthropod-borne or natural route of transmission has (yet) been demonstrated (Kuno et al, 1998, J. Virol. 72, 73-83). To theNKV viruses belong the Modoc vims, Rio Bravo virus, Apoi vims and Montana Myotis leukoencephalitis virus (Charlier et al., J. Gen. Virol. 83, 1887-1896 and J. Gen. Virol. 83, 1875-1885), among others. No information is available about the factors that determine whether a flavivirus is able or not to infect a vector (or vector cells) like mosquitoes or ticks. Differences between the viruses can be found in virulence, replication and transmission efficiency, severeness of pathology and other properties. The genome of the Flavivirus genus (Fig. 1) consists of a linear, positive- sense, single-stranded RNA molecule of about 11 kilobases (kb) in length. This RNA contains a methylated nucleotide cap (type I: m G5'ppp5'A) at the 5' end and lacks a 3' polyadenylate tail. Surrounding the single open reading frame (ORF) are 5' and 3' untranslated regions (UTRs) of about 100 nucleotides and 400 to 700 nucleotides, respectively. These regions contain conserved sequences and predicted RNA structures that are likely to serve as czs-acting elements in the processes of genome amplification, translation or packaging. Translation of the genome results in synthesis of several structural (like membrane and envelope proteins) and non-structural proteins (like a protease and a polymerase). In recent years, the genomic sequences of an increasing number of flaviviruses have been determined. The flaviviruses of which the genome sequence is available, share several overall characteristics, such as the organization of the genome, the presence of homologous protease cleavage sites and conserved motifs in those genes that are believed to be interesting antiviral targets (helicase, polymerase). Also differences between the three flavivirus groups have been described. For example, the folding of the 3' UTR of flavivimses has revealed structural elements that are preserved (i) among members of the arthropod-borne group, (ii) between members of the NKV-group and the tick- or mosquito-bome group and (iii) in all three groups, as well as structural elements that distinguish each group from another. The pentanucleotide motif CACAG located approximately 45-61 nucleotides from the 3' terminus, in the 3' LSH of the 3' UTR of the flavivirus genome, has been put forward to designate a vims as either an NKV or a vector-borne flavivirus. This motif is predicted to be located on a side-loop of a conserved 3 '-terminal secondary stmcture, which plays a role in the formation of a circular RNA molecule (Chambers et al, 1990b; Khromykh et al, 2001, J Virol 75, 6719-6728). It was found that the second position of this pentanucleotide in the 3' UTR of NKV flavivimses (MMLV, MODV, RBV and APOIV) was either U or C, instead of A in the vector-borne flaviviruses. APOIV has, in addition, a C to U change at position 3 of the pentanucleotide motif (table 1) (Charlier, N. et al. J. Gen. Virol. 2002, 83, 1875-1885. At each end of the genome, two teiminal nucleotides, i.e. 5'-AG and CU-3', are conserved among members of the whole Flavivirus genus (vector-bσme and NKV). Strain Sequence alignment S Position TBEV 263 CCCAGA ^« AUAGUCUGACAA -GGA 1 11,092 TBEV^NEU CCCAGA_^ GUAGϋCϋGACAA -GGA 2 11,092 Irøym^pR CCCAGA - _„ AUAGUCϋGACAA -GGA 3 10,786 O 1-BEγv^AS CCCAGA ACAGUCUGACAA -GGA 4 10,878 LGTV CCUAGA , - . .AUAGUCUGAAAA -GGA 5 10,894 POWV UCCAGG" «, ^JAUAGCCUGACAA -GGA 6 10,790 YFV 17D-213 GUGAG- ^* H ϋUUGCOCAAGAA -UAA 7 10,811 YFV 17DD GUGAG-^" >. ^UUUGCUCAAGAA -UAA 8 10,811 YFV neurotropic GUGAG- _ '' ^•> ΛJUUGCUCAAGAA -UAA 9 10,811 o

-α YFV viscerotropic GUGAG- '«■ UUUGCUCAAGAA -UAA 10 10,811 YFV Tαnidad GUGAG-'' , * UUUGCUCAAGAA -UAA 11 10,709

I YFV 85-82H GUGAG- < ^ CUUUGCUCAAGAA -UAA 12 10,811 DENV-1 UCCAGG^ f-- „AACGCCAGAAAA UGGAAU- 13 10,689 DENV-2 UCCAGGV" TAACGCCAGAAAA UGGAAU- 14 10,677 DENV-3 ϋCCAGG^ ^ ^ jAACGCCAGAAAA UGGAAU- 15 10,650 DENV-4 UCCAGG ^"N ^* -lAGCGCCGCAAGA UGGAUU- 16 10,603 LIV CCCAGA_, - ΑUAGUCUGACAA -GGA 1? 10,822 WNV CACGG-¹' ,*"-< 'UGCGCC-GACAϋ AGGUG — 18 10,916 KUNV CACGG-* lUGCGCC-GACAA UGGUG — 19 11,345 JEV ACORGG'f \ _" ~ lAGCGCCGAAGUA UGUA 20 10,928 MVEV -CAAGG _j C -lAGCGCCGAACAC UGUG 21 10,966 CFAV -CCUCC '^• C UUAG-GGAGUU UUGA 22 10,651

RBV U G- C< * AUUGC AUGC UGG 24 NA MODV UCAAG- U^r- " ^AUUG-CUUACUA UGUA 25 10,552

* APOIV GUAAUC CU ^ GUUGGAUOAAAA UUAUCCU 26 NA Table 1: The conserved pentanucleotide-sequence (5 -CACAG-3') in the last 61 nucleotides of the 3' UTR of mosquito- and tick-bome flaviviruses is a C(C/U)(C/U)AG sequence for NKV flaviviruses. (The sequences correspond to the sequences listed in the sequence listing as sequences 1-26)

Since there is no specific treatment for flavivirus infections, and management of patients with the disease is often extremely problematic, much emphasis has been placed on preventive vaccination. Vaccination is currently possible against YFV (live-attenuated), JEV (inactivated and live-attenuated, i.e. JEV-Vax) and TBEV0 (mactivated). Different vaccine strategies exist like the use of empirically derived and cDNA-derived live attenuated vimses (non-lethal deletion or point mutation), recombinant subunit vaccines, inactivated vims vaccines and DNA vaccines. Recent introduction of the so-called "infectious clone technology", which is based on a cDNA copy of an RNA virus genome that can be stably propagated in bacterial plasmid vectors and from which RNA can be transcribed that is infectious following transfection in the appropriate cells, has opened new opportunities in flavivirus vaccine research. The "chimeric " approach for vaccine development is based on the recent observations that the structural genes of one flavivirus can be replaced with homologous genes from another flavivirus (i.e. ChimeriVax vaccines). In this way properties of flaviviruses (such as vimlence, the envelope and replication) can be modified in a chimeric virus by combining genes of different vi ses. Several determinants of specific properties of these flavivimses have already been identified.

However, in the process of developing new vaccines, several factors, such as immunogenicity, vimlence, reactivity and neuroinvasiveness, are still problematic and have to be controlled. For example a vaccine against dengue virus should induce equally high levels of neutralizing antibodies against all four serotypes to prevent the occurrence of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Another problem encountered in the vaccination strategy is the prevention of the spread of vimses from a vaccinated person to a non-vaccinated person by a vector such as mosquitoes.

The major flavivimses (Dengue, Japanese encephalitis, tick-bome encephalitis, yellow fever, and West Nile viruses) cause substantial morbidity and mortality each year. Due to modem transportation and the relaxation of mosquito-control measures there has been a substantial increase of disease caused by flavivimses. Without effective antiviral drugs, vaccination offers the best chance of decreasing the incidence of these diseases, and live virus vaccines are the most promising and cost effective. However, flavivimses can recombine, which raises the possibility of recombination between a vaccine strain and wild-type virus resulting in a new vims with potentially undesirable properties (Seligman SJ and Gould EA, Lancet. 2004 Jun 19;363(9426):2073-5). In view of the importance of treating or preventing of pathologies induced by vimses, there is a need for therapies and/or vaccination strategies with a reduced or no risk for spreading the virus.

SUMMARY OF THE INVENTION

The present invention relates to the modification of nucleotide sequence involved in the formation of the 3 'LSH in the 3 'UTR of vector-bome flavivirasses, more particularly to the modification of one or more nucleotides within or 3' adjacent to the pentanucleotide sequence 5'-CAC(A/C)G-3' in the 3' UTR of vimses in order to reduce the replication of a virus in an organism or a cell, more particularly in a vector, and to reduce or inhibit the transmission of said vims through such a vector. The present invention also relates to a method to reduce the replication of a virus in an organism, more particularly in a vector, comprising modifying the nucleotide sequence involved in the formation of the 3 'LSH in the 3 'UTR of vector-bome flaviviruses, more particularly modifying one or more nucleotides within the CAC(A/C)G pentanucleotide motif in the 3' UTR sequence. The invention further relates to a modified virus comprising a modified 3 'UTR region, more particularly a modified CAC(A/C)G pentanucleotide motif in the 3 ' UTR sequence, its use and pharmaceutical compositions comprising such a virus. The present invention furthermore relates to a vaccine comprising a nucleotide sequence comprising at least the part of the 3' UTR of a flavivirus comprising the conserved pentanucleotide, characterised in that said 3 ' UTR nucleotide sequence comprises a modification in the sequence involved in formation of the 3 'LSH stmcture, more particularly comprising a modified CAC(A/C)G sequence or wherein the pentanucleotide is completely deleted. The present invention relates also to the use of the 3' UTR nucleotide sequence of a flavivirus or a part thereof, modified in sequence involved in the formation of the 3' LSH stmcture, more particularly, modified in the conserved CAC(A/C)G pentanucleotide sequence to reduce the replication of a vims in an organism, more particularly in a vector in the preparation of a vaccine.

The present invention relates to the reduction of the replication of viruses in a vector. In a particular embodiment the viruses are RNA vimses, yet more in particular they are member of the family of the Flaviviridae, still more in particular the viruses are selected from the Flavivirus genus. In a more particular embodiment, the vimses are selected from the vector-bome virasses, such as, but not limited to YFV, Dengue vims or WNV.

The invention further relates to a modified virus (sequence) comprising a modified 3 'UTR, more particularly a modified conserved CAC(A/C)G pentanucleotide motif within the 3' UTR sequence. In a particular embodiment the modified vims is a vector mediated flavivirus such as YFV, DENV and WNV. In another embodiment the modified virus is a chimeric virus constructed by combining genes of at least two viruses of which at least one is a flavivirus.

The present invention relates also to the use of said modified vimses in a pharmaceutical composition, such as a vaccine or for the preparation of a pharmaceutical composition, such as a vaccine.

The present invention furthermore relates to a vaccine comprising a nucleotide sequence comprising at least the part of the 3 ' UTR of a flavivirus comprising the conserved pentanucleotide, characterised in that said 3' UTR nucleotide sequence comprises a modified CAC(A/C)G sequence. In a particular embodiment, the vaccine comprises a chimeric virus with a modified CAC(A/C)G pentanucleotide sequence. According to the present invention, the chimeric vims thus comprises a 3 'UTR of a vector-bome flavivirus which is modified according to the present invention. More particularly, the chimeric vims for use as a vaccine comprises a modified CAC(A/C)G sequence.

In another embodiment, the vaccine further comprises other vimses, chimeric vimses or parts thereof.

DELAILED DESCRIPTION OF THE INVENTION

All flavivimses that cause diseases in humans either belong to the mosquito- or the tick-bome cluster. The third cluster, i.e. the NKV group, consists of viruses for which no arthropod-borne route of transmission has (yet) been demonstrated (Kuno et al, 1998, /. Virol. 12, 73-83). The factor(s) in the flavivirus genome that determine whether a flavivirus is able or not to replicate in vector (cells) have until now not been determined.

The present invention is based on the realization that insight into the determinants that are responsible for vector specificity is important for the development of vaccines against flavivimses. It is observed that the state of the art chimeric vaccine vimses based on the YFV (or DENV) backbone and containing the 3' UTR - and thus the pentanucleotide motif CACAG of vector-bome flavivimses - can still replicate in insect cells (see our observations described below) and can thus theoretically be transmitted by mosquitoes from a vaccinated person to a non- vaccinated person.

The present invention relates to the determinants of the flaviviral replication and transmission and the use of these determinants in the creation of vaccines or in therapy.

The present invention is thus also based on the observation that the viral envelope proteins (prM+E), which are responsible for the initial interaction (i.e. binding and fusion) of flavivimses with the host cell, are only partly responsible for whether a flavivirus is or is not infectious to a vector. This was established by investigating whether the MODV/YFV chimeric vims, which contains the prM and E genes of the NKV flavivirus Modoc vims (MODV) in the genome of a mosquito-borne flavivirus YFV 17D, was infectious to mosquito cells. Since the MODV/YFV chimeric virus contains the envelope proteins of a NKV flavivirus, it was expected that the chimeric vims would not be infectious to mosquito cells. However, the chimeric MODV/YFV replicated as efficiently in mosquito cells as YFV 17D did. This thus proves that the envelope proteins of the NKV flavivimses are not (solely) responsible for the fact that NKV vimses do not replicate in mosquito (vector) cells. Based on these observations it was concluded that other determinants must be responsible for the fact that NKV flavivimses (such as MODV) do not replicate in mosquito cells and, conversely, that vector-borne flaviviruses do replicate in mosquito or tick (cells). In the prior art, a comparative description of the 3' UTR folding of flavivimses can be found. Strong support was found for the presence of four different RNA regions (designated I, II, III and IV) in the 3' UTR of MMLV, MODV and RBV, but not in the 3' UTR of APOIV (Fig. 5). The latter is assumed not to have a region I equivalent. Hairpins, conserved motifs, single stranded parts, Y-shaped structures and pseudoknots are present in the regions. The very 3' terminus of the 3' UTR folds in a manner typical for all flaviviruses, forming the 3' LSH stmcture and a small stem- loop (belonging to region IV and probably coaxially stacking with the long 3' terminal hairpin). The 3' LSH, which preserves its shape despite significant differences in sequence, was calculated to fold in the genome of the four NKV flavivimses with a similar position of the conserved C(C/U)(C/U)AG motif (45-61 nucleotides from the 3' terminus). The present invention demonstrates that one or more modifications in the nucleotides involved in the formation of the 3' LSH, more particularly in the conserved CAC(A/C)G pentanucleotide sequence and/or the stretch of 10 nucleotides 3' thereof, is critical to determining whether or not a vims is capable of replicating in a vector.

The term "vector" as used herein and unless otherwise stated, refers to an organism, which is not a vertebrate, more specifically not a mammal, most particularly not a human, that can carry a virus and transmit it from one organism, particularly a vertebrate, more particularly a mammal, to another. In an aspect of the present invention, a vector is an arthropod, such as but not limited to ticks and mosquitoes.

The term '3' UTR' of a flavivirus as used herein refers to the sequence 3' of the open reading frame, comprising about 400-700 nucleotides. It has been described to contain elements involved in the regulation of essential functions such as translation, replication or encapsidation of the genome (Khromykh et al, 2001, J. Virol. 75, 6719-6728; Proutski et al, 1997a, J Gen. Virol. 78, 1543-1549; 1997b, Nucleic. Acids. Res. 25, 1194-1202). In vector-bome flavivirusses, the 3' UTR comprises a conserved CAC(A/C)G pentanucleotide (sequence) located approximately 45-61 nucleotides from the 3' terminus. A sequence 'corresponding to a functional 3' UTR of a flaviviras' is a sequence which either corresponds to the 3' UTR of a flavivirus or is derived therefrom, while retaining all of the functions normally performed by the 3 'UTR. Thus such a sequence can contain deletions or mutations (other than those described in the present invention) e.g. in non-functional or linker sequences within the 3' UTR.

The very 3' terminus of the 3' UTP_^ folds in a manner typical for all flavivimses, forming the 3 ' long stable hairpin (LSH) stmcture and a small stem-loop. Thus the term '3 'LSH stmcture' as used herein refers to a secondary structure formed by the 3' terminus of the 3 'UTR of flavivimsses. The formation of the 3' LSH stmcture involves a discrete number of nucleotides, which varies between the different flavivimsses. For instance, for YFV strain YFV-17D.204, the 3 'LSH stmcture has been demonstrated to involve nucleotides 68-20 upstream of the 3' UTR terminus (Proutski V. et al. Journal of General Virology (1997), 78, 1543-1549). Without being limited to theory, it is proposed that the 3 'LSH structure ensures specific functions within the 3 'UTR (such as e.g. interaction with intracellular proteins). Modification of one or more nucleotides within the sequence comprised in the 3 'LSH will lead to a modification of said functions.

The term 'conserved CAC(A/C)G pentanucleotide (sequence)' as used herein refers to the pentanucleotide sequence 5'-CACAG-3' or 5'-CACCG-3' located approximately 45-61 nucleotides from the 3' terminus of vector-bome flavivimsses (as described by Wengler & Castle, 1986, J Gen. Virol 61, 1183-1188; see also Table 1 of introduction). The conserved CAC(A/C)G pentanucleotide is part of the 3 'LSH stmcture of the 3 'UTR of such vector-borne flavivimses.

A "modified" CAC(A/C)G pentanucleotide sequence as used herein refers to the CAC(A/C)G nucleotide sequence, but modified by (i) changing or mutating nucleotides into other nucleotides or (ii) deleting or inserting nucleotides. The nucleotides that are referred to can be natural or unnatural (synthetic) nucleotides.

An isolated nucleotide sequence as used herein refers to a DNA or RNA polynucleotide as present outside its natural environment, i.e. as such or as part of a cloning vector or any other recombinant genetic construct. A nucleotide sequence can comprise the complete genome of a vims (or a sequence corresponding thereto) or parts thereof. Sequences encoding structural genes (such as envelope proteins) are also referred to as structural regions, while sequences encoding non-structural proteins (such as helicases, replicases, etc;) are also referred to as non-structural regions. Non-coding regions can include both the 5' and the 3' UTR.

£^• J A 'vaccine' as used herein refers to a composition which, upon introduction into a vertebrate, is capable of directly or indirectly generating a protective immune response to one or more viruses in a vertebrate, without inducing all of the disease symptoms associated with infection of the vims or vimses in a vertebrate. A vaccine0 can comprise one or more (DNA/RNA) nucleotide sequences encoding an immunogenic protein or peptide and/or one or more immunogenic proteins or peptides. According to the present invention, the vaccine is either a live attenuated vims or a nucleotide encoding a live attenuated vims. In a particular embodiment of the present invention, the vaccine comprises a mutated vims or chimeric vims.5 An 'immunogenic composition' refers to a composition which directly induces an immune response when injected into a vertebrate. In the context of the present invention an immunogenic composition comprises one or more parts of the (virally encoded) subunits of the envelope of the virus. The envelope of flavivimses is0 derived from the host cell membrane and comprises virally-encoded transmembrane (M) and envelope (E) proteins.

According to a first aspect, the present invention relates to a nucleotide sequence for use in the preparation of a vaccine against one or more flaviviruses. More5 particularly, the invention relates to a sequence comprising a functional 3 ' UTR of a vector-bome flavivirus or a part thereof comprising the conserved pentanucleotide CAC(A/C)G, wherein one or more nucleotides comprised in the 3' LSH in the 3' terminus of the UTR has been modified. According to a specific embodiment, a nucleotide within the conserved pentanucleotide and/or one or more nucleotides0 within a sequence of 10 nucleotides 3' of the pentanucleotide is modified. According to the present invention, such a modification ensures that the virus is to a much lesser extent or no longer able to replicate in a vector. According to another aspect of the invention, modified vimses are provided comprising the modified nucleotide sequence described above, which have a decreased capacity for replicating within a vector, more particularly have a reduced replication within an arthropod, such as, but not limited to a tick or a mosquito. Thus a vector-borne virus can be modified into a vims which is essentially no longer vector-bome, while maintaining all of its other features, more particularly its envelope proteins, which are critical for eliciting a specific immune response in the context of vaccination. Moreover, according to a particular embodiment of the invention, the replication of the virus in vertebrates is essentially unmodified, which is of interest for the efficiency of the vaccine. According to a particular embodiment of the invention the nucleotide sequence encodes a chimeric vims, i.e. a virus which comprises stmctural and/or non- structural proteins of more than one virus, more particularly more than one flavivirus. According to a more particular embodiment, the nucleotide sequence encodes a chimeric vims as based on the Chimerivax⁷ technology, whereby a live attenuated recombinant virus is constructed from yellow fever virus (YFV) 17D, or another flavivirus such as dengue virus (DENV) in which the envelope protein genes (prM+E) of the parent genes of YFV17D are replaced by the corresponding genes of another flavivirus (for example, but not limited to JEV, WNV, DENV), as is the case with the ChimeriVax ^τ vaccines. Further examples of chimeric vims vaccines, more particularly chimeric flavivirus vaccines, are described in the art and include the live, attenuated chimeric virus vaccine against tick-borne encephalitis virus comprising the preM and E stmctural genes of the tick-bome encephalitis Langat vims and the non- structural genes of the mosquito-borne dengue vims (described in US 6,497,884). Thus, according to a particular embodiment of the invention, the nucleotide sequence and the virus of the present invention correspond to a chimeric virus comprising at least the 3'UTR of a vector-borne flavivirus, more particularly the non-coding regions of the vector-borne flaviras, most particularly the non-coding as well as the coding regions of a vector-borne flavivirus, with the exception of one or more genes encoding structural proteins, whereby one or more nucleotides comprised in the 3'LSH of the UTR, more particularly, one or more nucleotides within the conserved CAC(A/C)G pentanucleotide have been modified. By reducing the ability of the 'backbone' virus (i.e. the virus providing the backbone sequence in which the structural genes of the virus of interest are introduced) to replicate in a vector, transmission of the vims through a vector is prevented. The present invention relates thus to a method for reducing the replication of a virus in an organism or a cell, more particularly in a vector or vector-cell, comprising modifying the 3 'UTR, more particularly modifying the sequence which is part of the 3 'LSH stmcture within the 3 'UTR, most particularly modifying the conserved CAC(A/C)G sequences therein. Envisaged modifications include those that inhibit hybridisation between complementary nucleotides (A-U/T and C-G) potentially required for the fom ation of the secondary stmcture). Most particularly, the modification involves one or more nucleotides within the conserved CAC(A/C)G pentanucleotide motif in the 3' UTR sequence and/or the sequence of 10 nucleotides 3' thereof, more particularly those present in the 3 'LSH stmcture and responsible for the interaction of the 3 'LSH stmcture with intracellular proteins. The pentanucleotide can be modified in different ways. Nucleotides can be changed or mutated into other nucleotides, nucleotides can be deleted or nucleotides can be added in the pentanucleotide sequence. In a particular embodiment, the positions 2 and 3 of the pentanucleotide starting from the 5 '-end are modified, more in particular are changed or mutated into another nucleotide. Therefore, the pentanucleotide can be modified into C(U/T/G/C)(A/T/G/U)AG, or using synthetic nucleotides. and any combination thereof. In another particular embodiment, the pentanucleotide is changed or mutated into 5'-CUCAG-3'. Additionally or alternatively, and more particularly for YFV, the modification involves one or more of the U nucleotides 3' of and adjacent to the conserved pentanucleotide. Most particularly, the modification involves the third nucleotide, 3' of the conserved pentanucleotide. Besides the substitutions with naturally occuring nucleotides, modification or replacement by synthetic nucleotides is also envisaged within the context of the present invention. Several modifications of oligonucleotides have been described in order to increase their stability. Examples of such modifications are oligonucleotides containing modified backbones such as phosphorothioate backbones and oligonucleosides with heteroatom backbones, or non-natural intemucleoside linlcages. Oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linlcages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. Alternatively, both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units can be replaced with novel groups, such as peptide nucleic acids (PNA - Nielsen et al. 1991, Science 254: 1497-1500). Oligonucleotides may also include naturally occurring or synthetic modifications of the "natural" purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

According to the present invention, the 3 'UTR of vector-bome flavivimses is modified, more particularly the natural function of the 3 'LSH structure therein is interfered with, most paiticularly by modification of the conserved CAC(A/C)G pentanucleotide, so as to reduce replication in a vector. In a particular embodiment, the replication of the vims in vector cells is reduced for 100 % and thereby completely inhibited. The reduction of the replication of the vims in vector cells can however be between 95 and 100%, can be higher than 95%, than 90%, 80%, 70%, 60% or 50%. In a particular embodiment, the replication of the virus in a vector is reduced with at least 20 %. Reduction of replication in cell cultures can be established in different ways, based on the production of viral RNA or protein (such as, but not limited to vims-specific real-time quantitative RT-PCR, as described herein, or virus-specific ELISA) or based on cellular features (such as vims-induced cytopathic effects). In order to establish reduction of replication a comparison can be made with the virus or nucleotide not modified according to the present invention. According ^"to a particular emodiment of the present invention, replication of a vector-bome virus within a vector or cells thereof is reduced. The vector in which the replication is reduced can be an anthropod or cells thereof, more in particular an anthropod known as a vector for certain viruses, such as mosquitoes or ticks. Additionally, according to a more particular embodiment of the present invention, replication of the vector-bome vims in cells of the host, e.g. the vertebrate, more particularly the mammal is not significantly reduced as a result of the modification of the present invention, thus allowing replication of the virus in a vertebrate. This is of interest, e.g., for the propagation of the vaccine witliin the vaccinated host. Optimally, according to the present invention, replication of at least 5-10%, more particularly at least 10-20%, most particularly at least 20-50% is retained in vertebrate, more particularly mammal cells. The nucleotides sequences and viruses of the invention modified in the 3'

UTR, more particularly modified in one or more nucleotides present in the 3 'LSH structure therein, most particularly modified in one or more nucleotides within the conserved pentanucleotide sequence CAC(A C)G can be used for the preparation of a pharmaceutical composition, like a vaccine or can be used in a vaccine. In order for the virus or a part thereof to be used as a vaccine, further modifications may be required to ensure its suitability as a vaccine, i.e. sufficient immunogenecity, while not inducing all physical symptoms of the disease normally associated with the introduction of the virus into the body. More particularly, for YFV or JEV it is desirable that the attenuated vims (as such or as produced in vivo upon vaccination with a nucleotide sequence) is less (not) neuroinvasive and less (not) neuropathogenic. Methods for reducing the pathogenicity of a vims, resulting in an 'attenuated' virus are known the to skilled person and include but are not limited to mutations in the coding and non-coding regions. In a naturally attenuated tick-borne flavivirus, Langat (LGT) strain TP21, recovered from ticks in Malaysia, increase in attenuation was associated with three arnino acid substitutions, two located in the stmctural protein E and one in nonstructural protein NS4B (Pletnev AG, Virology. 2001 Apr 10;282(2):288-300). The Dengue virus type 4-, attenuated versions were obtained by deletions in 5' non- coding (NC) region ( i.e., 5'd (82-87), 5'd (73-77), and 5'd (76-81)). Introduction of mutations into the non-structural genes or 3' untranslated region of an attenuated dengue vims type 4 was found to further decrease replication in rhesus monkeys while retaining protective immunity (Hanley KA et al, Vaccine. 2004 Sep 3;22(25- 26):3440-8). rDENdelta30 is a dengue vims vaccine candidate which comprises a 30 nucleotide deletion in the 3' untranslated region about 100 nucleotides upstream of the 3 'LSH. Alternatively, a non-infectuous RNA vaccine has been described based on genetic modifications in the region encoding the capsid protein which simultaneously prevents the assembly of infectious vims particles and promotes the secretion of noninfectious subviral particles that elicit neutralizing antibodies (Kofier RM, Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1951-6. Epub 2004). Alternatively, a chimeric virus is provided, i.e. a combination of e.g. the stmctural genes of the virus of interest (against which a protective immune response is desired) and the non-structural genes of another vims). The ChimeriVax™ system approach replaces the E gene of the 1 D yellow fever vaccine with the analogous gene of the vaccine-targeted flavivirus, and has been used to obtain JE, DEN and West Nile vaccines. Such chimeric viruses have been demonstrated to show lower neurovirulence than the parent vims while inducing a dose-dependent vims- neutralizing antibody response (Lai CJ, Monath TP, Adv Vims Res. 2003;61:469- 509). Antigenic chimeric viruses in which the stmctural genes (the capsid, membrane precursor, and envelope (CME) or the membrane precursor and envelope (ME) gene regions) of dengue virus type 4 (DEN4) have been replaced with the corresponding genes of dengue vims type 2 (DEN2) have been created to obtain a live attenuated tetravalent dengue virus vaccine (Whitehead SS, Vaccine. 2003 Oct l;21(27-30):4307-16). In a particular embodiment of the present invention, the modification of the 3 'UTR, more particularly the modification of the pentanucleotide and/or one or more of the nucleotides 3' thereof is combined with modifying the envelope or a certain part of the envelope of said virus into the envelope of a NKV. The modified virus can at least be mixed with a pharmaceutically acceptable carrier. Suitable pharmaceutical carriers for this purpose are described for instance in Remington's Pharmaceutical Sciences 16^th ed. (1980) and their formulation is well known to those skilled in the art. They include any and all conventional solvents, dispersion media, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like. Additional ingredients may be included in order to control the duration of action of the active ingredient in the composition. Suitable carriers or excipients known to the skilled man are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies 12 harmful to the individual receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers. The "pharmaceutical composition" may be administered by any suitable method within the knowledge of the skilled man. To immunize subjects against flaviviral infection, the vaccines containing immunologically effective amounts of the vims are administered to the subject in conventional immunization protocols involving, usually, multiple administrations of the vaccine. Administration is typically by injection, typically intramuscular or subcutaneous injection; however, other systemic modes of administration may also be employed. Less frequently used, transmucosal and transdermal formulations are included within the scope of the invention as are effective means of oral administration. The efficacy of these formulations is a function of the development of formulation technology rather than the contribution of the present invention. The preferred route of administration is parenterally. In parental administration, the composition of this invention will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with the pharmaceutically acceptable excipients as defined above. However, the dosage and mode of administration will depend on the individual. In a particular embodiment, it is given as a bolus dose.

The following examples, not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying Figures, incorporated herein by reference, in which:

Figure 1. Genomic organization of the Flaviviruses.

Figure 2. Growth kinetics of YFV (D), MODV/YFV (•) and MODV (A) in Vero and mosquito cells. Quantification of viral RNA in the supernatant was performed in triplicate by real-time quantitative RT-PCR using the probe methodology (standard deviation of triplicate determination below 15%) (PCRU = PCR units: One PCR unit is defined to be the lowest template copy number detectable in three of three replicative reactions as determined by limiting dilution series). Figure 3. Growth kinetics of MODV/YFV(CACAG) (•) and the mutant chimeric virus MODV/YFV(CUCAG) (o) in Vero and mosquito cells. Two indepent experiments were carried out that yielded similar findings. Data from one experiment are shown. Quantification of viral RNA in the supernatant was performed in duplicate by real-time quantitative RT-PCR using the probe methodology (standard deviation of triplicate determination below 15%).

Figure 4. Construction of MODV/YFV chimera by using a variant of the fusion- PCR

Figure 5. Proposed secondary structure of the 3' UTR of four NKV flavivimses. The four regions (labelled I to IV) are delineated by boxes. Conserved motifs are shown in bold and boxed. For MMLV and RBV, the predicted pseudoknot is' shown by connecting boxes. For MMLV and MODV, possible stem-loops are connected by dotted lines.

Figure 6. Growth kinetics of MODV/YFV(CACAG) (--♦--), of wild-type YF (-- ■ --) and the mutant chimeric vimses MODV/YFV(CTCAG) (—A—), MODV/YFV(CCCAG)( — ■ — ) and MODV/YFV(CGCAG)( — •— ) in Vero and mosquito cells ('mug').

Figure 7. Growtli kinetics of MODV/YFV(CACAG) (- ♦ -), of wild-type YF(- ■ -) and the mutant chimeric virus MODV/YFV(CTCAG) ( — A— ) in Vero and mosquito cells ('mug').

Examples

Example 1 : Materials and methods Unless otherwise indicated, all the buffers used for restriction enzymes, ligases, and polymerases were provided by the suppliers and used according to their specifications. Cells, viruses andplasmids

Vero and BHK-21 cells were originally obtained from the ATCC (CCL-81 and CCL- 10 respectively). The clone pACNR-MODV/YFV was constructed as described in hereunder (Charlier et al., 2003, J. Virol. Methods 108, 67-74; ). Mosquito (C6/36, Aedes albopictus) cells and the clone pACNR-FLYF17Da can be obtained from the researchers.

Amplification and cloning of a short YFV f^ragment containing the CACAG pentanucleotide sequence A fragment of 590 bp (nt-position 10,694-11,283 of the YFV 17D genome), containing the CACAG pentanucleotide motif, was amplified in a reaction mixture of 50 μl consisting of 30 ng of pACNR-FLYF17Da plasmid, 2 units of Pfu DNA polymerase (Promega), 5 μl of 10 x buffer supplied by the Manufacturer, 400 μM dNTP, and 1.2 μM of each of the two primers (sense primer 5'- GTAGAAAGACGGGGTCTAGAGGT-3' (SEQ ID NO:27) and antisense primer 5'- GGCACTGATGAGGGTGTCAGTG-3' (SEQ ID NO: 28). The conditions for the amplification reaction were as follows: 30 s at 95°C, 30 s at 60°C and 1 min at 72°C repeated for 25 cycles. Following agarose gel electrophoresis the DNA fragment with the expected length was cloned into a TOPO vector using the TOPO TA Cloning kit (version H) (Invitrogen) and One Shot TOP 10 E. coli cells (Invitrogen) to yield pYFV-CACAG.

Mutation of the pentanucleotide sequence

The adenosine at position two of the pentanucleotide motif CACAG was mutated into a thymidine using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, Texas, USA): 50 ng of the pYFV-CACAG plasmide was added to 5 μl 10 x buffer supplied by the Manufacturer, 400 μM dNTP, 0.22 μM of each of the two primers (sense primer and antisense primer 5'-GAGGTCTGTGAGCTCAGTTTGCTCA AGAATAAGCAG-3' (SEQ ID NO: 29) and 2.5 units of Pfu Turbo DNA polymerase. The conditions for the amplification reactions were 30 s at 95°C, 1 min at 55°C and 9 min at 68°C repeated for 12 cycles. Following amplification, the sample was treated with 10 units of the Dpnl restriction enzyme and transformed in One Shot TOP 10 E. coli cells (Invitrogen) to yield pYFV-CTCAG. To assure that the pentanucleotide sequence was altered correctly and that no other mutations were inserted into the genome, the exchanged fragment was sequenced.

Construction of the MODV/YFV full-length clone containing the CUCAG motif The plasmids pYFV-CTCAG and pACNR-MODV/YFV were digested with the restriction enzymes Xbal and Haell (Promega), and the CTCAG containing fragment was inserted in the full-length clone pACNR-MODV/YFV using T4 DNA ligase (Promega). Following ligation, which yielded the plasmid pACNR- MODV/YFV(CTCAG), the latter was transformed into MC1061 E. coli cells.

RNA transcription and transfection

Recombinant viral RNA was transcribed from 5 μg of Aflll-lmssήzQd pACNR- MODV/YFV(CTCAG) using Sp6 RNA-polymerase (mMessage mMachine Kit; Ambion Ltd., Cambridgeshire, United Kingdom). BHK cells were transfected by electroporation as described (van Dinten et al , 1997, Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):991-6). Cell culture medium was harvested at the time that the transfected cells displayed nearly complete CPE. Medium was cleared from cell debris by centrifugation and subsequently used to prepare layer stocks in Vero cells.

Monitoring viral kinetics

Monolayers of Vero and C6/36 cells were inoculated with 10⁷ pfu of either MODV, YFV, MOD YFV(CACAG) or the MODV/YFV(CUCAG) chimeric vims at 37°C and 28°C in 25 cm² culture flasks. Cell culture medium was harvested every day or every two days (between day 0 and 10), and titrated for infectious vims content on Vero cells. Viral RNA load was determined by real-time quantitative RT-PCR (see below) on RNA extracted from the collected media.

Quantitative RT-PCR of MODV, YFV, MODV /YFV (CACAG) and MODV/YFV (CUCAG) RNA RNA extraction was performed using the Qiagen Viral RNA kit (Qiagen) according to the Manufacturer's instructions. For elution of RNA, the columns were incubated with 50 μl of RNase-free water at 80 °C. For the quantitative determination of MODV, YFV and MODV/YFV RNA in the supernatant of infected Vero and C6/36 cells, the reaction conditions were as following. Primers and probes were designed for MODV/YFV 17D: sense primer 5'-TGGGTTTTGGTCTTCTAGCTTTCA-3' (SEQ ID NO:30), antisense primer 5'-CTTGTTCAGCCAGTCATCAGAGTCT- 3'(SEQ ID NO: 31) and probe 5'-CAGGAGTGATGGGAAATCAAGGATGC-3' (SEQ ID NO: 32); YFV 17D: sense primer 5'- AATCGAGTTGCTAGGCAATAAACAC-3' (SEQ ID NO: 33), antisense primer 5'- TCCCTGAGCTTTACGACCAGA-3' (SEQ ID NO: 34) and probe 5^'- ATCGTTCGTTGAGCGATTAGCAG-3' (SEQ ID NO: 35). Primers and probe for quantitation of MODV RNA and the reaction conditions for MODV/YFV 17D, YFV 17D and MODV were as reported earlier (Leyssen et al, 2001, Virology 279, 27-37). Thermal cycling in a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) involved reverse transcription (RT) at 45 °C for 20 min, denaturation at 95°C for 5 min, followed by 45 cycles of 5 s at 95°C and 35 s at 57°C (for YFV) or 20 s at 60°C (for MODV and MODV/YFV) (Wittwer et al, 1997, Biotechniques 22, 176-181; Drosten et al, 2002, /. Clin. Microbiol. 40, 2323-2330).

Rapid and efficient method to create chimeric viruses (i.e. Construction of the clone pACNR-MODV/YFV)

Cells, vimses and plasmids: Vero and BHK-21 (Baby Hamster Kidney) cells, and MODV were originally obtained from the ATCC (CCL-81, CCL-10 and VR-415 respectively). MODV was grown in Vero cells (Leyssen et al, 2001, Virology 279, 27- 37). The clone pACNR-FLYF17Da contains a full-length cDNA of YFV 17D and is identical to pACNR-FLYF17Dx (Bredenbeek et al, 2003, /. Gen. Virol. 84, 1261- 1268) except for the XJioI transcription run-off site which was changed to an Aflll site in pACNR-FLYF17Da. Plasmid pHYF-5' is a derivative of pHYF-5'3TV (Bredenbeek et al, 2003, above) and contains a Notl-Mlul fragment encompassing the Sp6 promoter fused directly to the 5' 2947 bases of YFV 17D. Amplification and cloning of MODV prM+E cDNA: MODV RNA was extracted from 140 μl of cell culture supernatant using the QIAamp Viral RNA kit (Qiagen) according to the Manufacturer's instractions. The cDNA was synthesized and amplified using the One Step RT-PCR kit (Qiagen): 5 μl MODV RNA was added to 10 μl 5 x RT-PCR buffer, 0.4 mM dNTP, 2 μl enzyme-mix, 95 units of HPRI (Amersham Pharmacia Biotech), 0.6 μM of each primer [sense primer, 5'- AAGGTTTTGGAAGATGACTCCGGC-3' (SEQ ID NO: 36) (nt-position 271-294); antisense primer, 5'-GTTAATGACTGGTATGGGGGGTACA-3' (SEQ ID NO: 37)(nt-position 2444-2468)] and 29 μl RNase-free water in a final volume of 50 μl. The following amplification program was used: an RT at 50°C for 30 min, an initial PCR activation step of 15 min at 95°C followed by 30 cycles of 30 s at 94°C, 30 s at 60°C and 2 min at 72°C and a final extension phase of 10 min at 72°C. The DNA fragment with the expected length of 2.2 kb was cloned into a TOPO vector using the TOPO TA Cloning kit (version H) (Invitrogen) and One Shot TOP 10 E. coli cells (Invitrogen) to yield pMODV-prM+E. Primers: Primers used for the construction of the chimeric region were designed based on the nucleotide sequence of YFV 17D (GenBank accession number X03700) (Rice et al, 1985 Science 229, 726-733) and MODV (GenBank accession number NC_003635) (Leyssen et al, 2002, Virology 293, 125-140). The nucleotide sequences of the primers are listed in Table 2.

Table 2 Primers used for fusion-PCR

Name Polarity Sequence A s 5^,-GGAATGCTGTTGTGACGGGTGGA^CCATATTGTCAA■'TGAGTTGTT-3^, (38) Y/M B AS 5'-GCACTGAGGATCCCAATGGAA-3' (39) M C s 5' -GCAACGCGGCGGCCGCGCTAGCGATGACC-3' (40) Y (Nθtf) D S 5'-CTTTCACCACAGGAGTGATGGGAGATCAAGGATGCGCCATCAACTT-3' (41) MY E AS 5^f -GCCTAAATTCAGTTGACTCC-3' (42) Y F S 5'-TGCCTGTTGGAAAAGGATCGT-3' (43) M

Abbreviations: S: sense; AS: antisense; Y: YFV; M: MODV. (Numbers between brackets refer to sequence listing)

Fusion-PCR: Two short fragments (205 bp and 209 bp, Fig. 4), that were to serve as primers in the subsequent fusion-PCR, were amplified in a reaction mix of 50 μl consisting of 100 ng (pHYF-5' or pMODV-prM+E) plasmid, 2 units of Pfu DNA polymerase (Promega), 5 μl of 10 x buffer supplied by the Manufacturer, 400 μM dNTP, and 1.2 μM of each of the two primers (Table 2, A+B and D+E respectively). The conditions for the amplification reactions were as follows: 30 s at 95°C, 30 s at 50°C and 1 min at 72°C repeated for 25 cycles. For the fusion-PCR, the reaction mix of 50 μl consisted of 50 ng (pHYF-5' or pMODV-prM+E) plasmid, 2 units of Pfu DNA polymerase (Promega), 5 μl of 10 x buffer supplied by the Manufacturer, 400 μM dNTP, and 1.2 μM of each of the two primers (Table 2, C and F respectively). The conditions for the amplification reactions were as follows: 1 min at 95°C, 1 min at 59°C and 2 min at 72°C repeated for 35 cycles. Two fragments of 933 bp and 2051 bp respectively were thus obtained (Fig. 4).

Construction of the recombinant plasmid: The fragment of 2051 bp and the pHYF-5' vector were digested with Sad and Hpal (Promega) (Fig. 4). Ligation of 100 ng vector with 65 ng PCR fragment was carried out using the T4 DNA ligase in a 2 x rapid ligation buffer (New England Biolabs GmbH, Frankfurt am Mainz, Germany). The resulting plasmid pHYF-MOl was digested with Hpal and Notl (Promega) and served as a vector for the insertion of the Hpal-Notl digested 933 bp fragment. The resulting plasmid pHYF-MO2 was digested with Mlul-Notl (Promega) and the DNA fragment [encompassing from 5' to 3' the Sp6 promotor fused to the YFV 5' UTR and C gene, MODV prM and E gene and part of the YFV NS1 gene] was ligated into Notl-Mlul digested pACNR-FLYF17Da to construct pACNR-MODV/YFV (Fig. 4). The recombinant region was sequenced in a cycle sequencing reaction with fluorescent dye terminators (Big Dye Terminator Cycle Sequencing Ready Reaction kit, Applied Biosystems Division) and analyzed using an ABI 373 automatic sequencer (Applied Biosystems Division).

Generation of recombinant viral RNA and transfection of BHK cells: Recombinant viral RNA was transcribed in vitro for 2 h at 37°C using 5 μg Aflll-l eaxized pACNR-MODV/YFV plasmid (Fig. 4) as a template and Sp6 RNA-polymerase (1500 units/ml) and RNase inhibitor (1000 units/ml) using the reaction conditions provided by the supplier. The transcription reaction was spiked with 1 μCi [ H]-UTP (46 Ci/mmol) to determine the yield of the transcription reaction. BHK cells were transfected by electroporation essentially as described (van Dinten et αl, 1997, Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):991-6). The medium was harvested from the transfected cells when the CPE was nearly complete, clarified by centrifugation and subsequently used to infect new Vero cells. At 48 h post infection total cellular RNA was isolated from the infected Vero cells and subsequently used in a RT-PCR to verify the chimeric nature of the virus. Example 2: Replication kinetics of MODV. YFV and MODV/YFV in Vero and mosquito cells

To determine whether prM and E of flaviviruses play a role in the vector specificity, the replication efficacy of MODV, YFV and MODV/YFV was compared in Vero and mosquito cells. The kinetics of replication of the three viruses were assessed by means of vims specific real-time quantitative RT-PCR. All viruses reached a plateau of virus production in Vero cells approximately 6 days post infection (Fig. 2). YFV 17D grew as efficiently in mosquito C6/36 cells as in Vero cells. As expected, the NKV flavivims MODV did not replicate in C6/36 cells. If one assumes that the prM and E proteins of MODV are responsible for the fact the virus does not replicate in mosquito cells, one would expect the chimeric vims MODV/YFV (containing the prM and E of MODV) not to replicate in mosquito cells. However, in contrast to expectations, the MODV/YFV replicated as efficiently as the parental YFV 17D in mosquito C6/36 cells (Fig. 2). The factors that determine whether a flavivirus is able to replicate in mosquito cells, are thus not located in the envelope proteins prM and E.

Example 3: Replication kinetics of MODV/YFVfCACAG and MODV/YFV (CUCAG) in Vero and mosquito cells

The pentanucleotide sequence CACAG in the 3' LSH of the 3' UTR in MODV/YFV was mutated into the pentanucleotide motif that is characteristic for NKV flavivimses. The course of replication of the parental MODV/YFV(CACAG) and mutant chimeric MODV/YFV(CUCAG) in Vero and C6/36 cells was compared in two independent experiments. The Icinetics of replication of the two vimses was assessed by means of real-time quantitative RT-PCR. In Vero cells, the chimeric MODV/YFV that contains the NKV pentanucleotide motif, replicated somewhat slower than the chimeric virus that contains the vector-bome CACAG motif. Eventually, both viruses efficiently produced CPE in these cells. In contrast, the chimeric MODV/YFV virus that contained the NKV specific pentanucleotide motif CUCAG, was not longer able to replicate in mosquito (C6/36) cells (Fig. 3). To study whether the inability of MODV/YFV(CUCAG) to replicate in mosquito cells was solely due to the mutation in the pentanucleotide motif (CACAG — CUCAG), the entire genome of the virus was sequenced. No additional mutation was found.

Example 4: Replication kinetics of MODV/YFVfCACAG and MODV/YFV (C(C/G)CAG in Vero and mosquito cells

Monkey kidney cells (Vero cells) and mosquito cells (C6/36) were infected for two hours (day 0) with a) yellow fever vims, b) the original MOD/YF chimeric virus (with CACAG motif) and mutants thereof having c) the motif CTCAG, d) the motif CCCAG and e) the motif CGCAG, according to the procedures described above.

In a first experiment, samples were assayed on day 0, 3 and 8 (Figure 6). In a second experiment, samples were assayed daily during a period of 8 days (Figure 7). All vimses replicated well in the mammalian monkey kidney cells. In mosquito cells however, no replication occurs with the mutant having the CTCAG motif. [quantitative PCR (Taqman) indicates that replication does occur with the mutants having the CCCAG and the CGCAG motif]

Claims

1. An isolated nucleotide sequence comprising a sequence corresponding to a functional 3' UTR of a vector-borne flavivims or a part thereof comprising the conserved pentanucleotide CAC(A/C)G, characterised in one or more nucleotides present in the 3 'LSH within the 3 'UTR is modified.

2. The isolated nucleotide sequence according to claim 1, characterized in that one or more nucleotides witliin said conserved pentanucleotide and/or 10 nucleotides 3' thereof is modified.

3. The isolated nucleotide sequence according to claim 1 or 2, characterized in that one or more nucleotides within said conserved CAC(A/C)G pentanucleotide is modified.

4. The isolated nucleotide of any one of claims 1 to 3, wherein the pentanucleotide is modified into C(U/T/G/C)(A/T/G/U)AG or synthetically modified versions of said nucleotides and any combination thereof.

5. The isolated nucleotide sequence according to any one of claims 1 to 4, which is an RNA corresponding to the complete genome of a flavivirus or a DNA corresponding thereto.

6. The isolated nucleotide sequence according to any one of claims 1 to 5, comprising a non-structural region or a portion thereof of a flavivims, and a structural region or a portion thereof from a different flavivirus.

7. The isolated nucleotide sequence according to any one of claims 1 to 6, wherein said functional 3 'UTR corresponds to a functional 3' UTR of a YFV, DENV or JEV. .

8. The isolated nucleotide sequence of any one of claims 1 to 7, wherein one or more structural regions within the coding region have been replaced by one or more structural regions of another virus.

9. A vaccine comprising the nucleotide sequence of any one of claims 1 to 8.

10. A vaccine comprising a nucleotide sequence, at least comprising a functional 3' UTR of a vector-bome flavivirus or a part thereof comprising the conserved pentanucleotide CAC(A/C)G, characterised in one or more nucleotides present in the 3 'LSH within the 3 'UTR is modified.

11. The vaccine of clam 9 or 10, wherein said one or more nucleotides within said conserved pentanucleotide is modified.

12. The vaccine of any one of claims 9 to 11, wherein said vaccine is a chimeric vims vaccine.

13. The vaccine of any one of claims 9 to 12, further comprising one or more viruses or parts thereof.

14. The vaccine according to claim 12, comprising one or more non-structural protein or a portion thereof of a flavivims, and one or more stmctural proteins or a portion thereof from a different flavivims.

15. A method to reduce the replication of a vector-bome flavivims in an vector comprising modifying one or more nucleotides in the sequence comprised within the 3 'LSH structure in the 3 'UTR.

16. The method according to claim 15 to reduce the replication of a vims in a vector comprising modifying the conserved CAC(A/C)G pentanucleotide motif in the 3' UTR sequence.

17. The method of claim 15 or 16, wherein the replication is not significantly reduced in cells of the vertebrate host of said virus.

18. The method of claims 15 to 17, wherein the pentanucleotide motif is modified in the second and third position from the 5 '-side on.

19. The method of claims 15 to 18, wherein the pentanucleotide is modified into C(U/T/G/C)(A/T/G/U)AG, or synthetic versions of said nucleotides and any combination thereof.

20. The method of claims 15 to 19, wherein the reduction of replication in a vector is higher than 95%.

21. A modified vims comprising a 3 'UTR which has been modified in one or more nucleotides involved in the formation of the 3 'LSH stmcture.

22. The modified virus according to claim 21, which comprises a modified CAC(A/C)G pentanucleotide sequence in the 3' UTR sequence.

23. The modified virus of claim 21 or 22 wherein said modified virus is a chimeric vims.

24. The modified virus comprising the nucleotide of any one of claims 1 to 8.

25. The use of the modified virus of any one of claims 21-24 in a vaccine.

26. A pharmaceutical composition comprising the modified vims of any one of claims 21-24.

27. The pharmaceutical composition of claim 26, further comprising one or more vimses or parts thereof.

28. The use of the 3' UTR nucleotide sequence of a vector-bome flavivirus or a part thereof, modified in the CAC(A/C)G pentanucleotide sequence to reduce the replication of a virus in an organism.

29. The use of the 3 ' UTR nucleotide sequence of a flavivims or a part thereof, modified in the CAC(A/C)G pentanucleotide sequence in the preparation of a vaccine.