Patent Application
20050164170
The present invention relates to a neuroinvasive and neurovirulent strain of the West Nile virus, named IS-98-ST1, to nucleic acid molecules derived from its genome, to the proteins and peptides encoded by said nucleic acid molecules and to uses thereof.
The present invention also relates to all variants of the viral strain IS-98-ST1 which have at least one mutation in the nucleic acid sequence corresponding to NS5.
The family Flaviviridae includes the viruses of the flavivirus genus which are responsible for serious human pathological conditions such as dengue, yellow fever, tick-borne encephalitis, Japanese encephalitis or West Nile encephalitis and the hepatitis C and G viruses. While flaviviruses are capable of causing considerable morbidity and mortality in humans, the infection is generally asymptomatic and only a fraction of the individuals infected develop a serious disease.
Flaviviruses are small enveloped viruses. Their genome is a single-stranded RNA molecule of positive polarity, approximately 11 000 bases in length. The genomic RNA is associated with several copies of capsid protein C, so as to form the nucleocapsid; it is surrounded by a viral envelope consisting of a double lipid layer derived from the membranes of the endoplasmic reticulum (ER), in which the envelope protein E and the membrane protein M are anchored. The genomic RNA of flaviviruses contains a single open reading frame of approximately 10 500 nucleotides flanked by two short noncoding regions at its 5′ and 3′ ends. The genome is translated into a polyprotein of approximately 3 400 amino acids which is the precursor of the structural proteins C, prM (intracellular precursor of M) and E in its N-terminal portion, and of at least seven nonstructural (NS) proteins, from NS1 to NS5, in its C-terminal portion.
Until very recently, the West Nile virus was recognized as being a relatively nonpathogenic virus responsible for a flu-like syndrome and present in Africa, in southern Europe and in the Middle East; it was isolated during epidemics which occurred, in particular, in Israel in the 1950s and in South Africa in the 1970s.
Very recently, the epidemiology of the West Nile virus became modified and an increasing number of cases of encephalitis was observed during the epidemics occurring in Romania in 1996, in Israel in 1998 and in the USA in 1999.
Pathogenic strains were isolated during these epidemics (Anderson et al., and Lanciotti et al., Science, 1999, 286: 2331-2333, 2333-2337), in particular the strain NY1999 (GenBank No. AF202541, Lanciotti et al., mentioned above), the pathogenicity of which is thought to be correlated with the presence of an NTS glycosylation site in the envelope protein E (Jordan et al., Viral Immunol., 2000, 13, 4: 435-446).
All the poorly identified viral factors may be responsible for the seriousness of the infection, whereas the genetic constitution of the host (human or nonhuman) is thought to contribute to resistance to infection.
However, the data relating to these recently isolated pathogenic strains have not made it possible to determine all the viral factors and host genes involved in sensitivity/resistance to infection with Flaviviridae.
Murine models have made it possible to establish the existence of genetic resistance to infection with flaviviruses. It has been shown that certain mouse lines recently derived from the wild and belonging to the species Mus musculus musculus or Mus spretus (Det, BSVR, BRVR, PRI, CASA/Rk and CAST/Ei) are resistant to infection with flaviviruses, whereas the most common inbred laboratory lines, which derive mainly from the species Mus musculus domesticus, are not resistant to it (Sangster et al., J. Virol., 1993, 67: 340-347).
Resistance is controlled by at least one autosomal locus named Flv, located on chromosome 5, in mice, and three alleles, Flvs, Flvr and Flvmr, confer, respectively, sensitivity, resistance and intermediate resistance to infection with flaviviruses. Using a Murray valley encephalitis flavivirus strain and mice derived from backcrossing the resistant mouse line C3H/RV with the sensitive mouse line C3/He or BALB/c, the Flv locus was located in a 0.9 cM region of chromosome 5, in mice, between the markers D5Mit68 and D5Mit242 (G. R. Shellam et al., Rev. Sci. Tech. Off. Epiz. 1998, 17: 231-248).
The inventors have now isolated a novel strain of the West Nile virus, from samples taken from storks in Israel (in the town of Eilat) in September 1998, which was selected for studying the resistance/sensitivity of a host (human or nonhuman mammal) to infection with viruses of the family Flaviviridae.
In accordance with the invention, said isolated neurovirulent and neuroinvasive strain of the West Nile virus, named IS-98-ST1, is characterized in that its genome consists of the sequence SEQ ID No. 1 which encodes a polyprotein having the sequence SEQ ID No. 2.
The inventors have in particular shown that laboratory mice are extremely sensitive to infection with the strain IS-98-ST1, whereas the mouse lines SEG, WMP, STF and MAI, which derive from wild mice belonging to species which are different although of the same genus Mus, are completely resistant to infection with this strain; an intraperitoneal inoculation of 1 000 FFU (focus-forming units; FFU:LD50=100) is 100% lethal for laboratory mice, whereas the wild mice do not show any symptoms; in addition, the virus replicates in these mice, as shown by the appearance of specific serum antibodies.
A subject of the present invention is also reagents, derived from the strain IS-98-ST1, used for studying and diagnosing infections with Flaviviridae, which reagents are selected from the group consisting of the following reagents:
These various reagents are prepared and used according to the conventional techniques of molecular biology and immunology, according to standard protocols such as those described in Current Protocols in Molcular Biology (Frederick M. AUSUBEL, 2000, Wiley and Son Inc. Library of Congress, USA) and in Current Protocols in Immunology (John E. Coligan, 2000, Wiley and Son Inc. Library of Congress, USA).
The nucleic acid fragments as defined above, in particular those corresponding to the sequences SEQ ID Nos. 3-11, are used, for example, as a probe or as a primer for diagnosing infection with the West Nile virus; the infection is detected, for example, by PCR and/or hybridization, using the nucleic acids extracted from a biological sample taken from an individual liable to be infected or a laboratory animal inoculated with said virus.
According to an advantageous embodiment of said fragments, they comprise at least 15 nucleotides of SED ID No. 1, upstream or downstream of one of the codons at the following position:
Such primers are useful for amplifying fragments containing said codons.
Said primers are preferably located between 10 and 100 nucleotides upstream or downstream of said codons.
According to another advantageous embodiment of said fragments, they consist of the fragments comprising the abovementioned codons, preferably of between 50 and 200 nucleotides, which are amplified using the primers as defined above.
The recombinant vectors as defined above, in particular the expression vectors, and the cells transformed with said expression vectors, are advantageously used for producing the corresponding peptides and proteins.
Said proteins and said peptides, which can be recognized by, and/or can induce the production of, antibodies specific for the West Nile virus, in particular for neurovirulent strains, are useful for diagnosing infection with a West Nile virus; the infection is detected using a suitable technique, in particular EIA, ELISA, RIA or immunofluorescence, using a biological sample taken from an individual liable to be infected or a laboratory animal inoculated with said virus. The proteins and peptides as defined above are also used to investigate cellular partners of these viral proteins which may be involved in the pathogenicity (neurovirulence) of the West Nile virus; these partners are identified using immunoaffinity techniques, for example using immunoaffinity column chromatography.
The antibodies according to the invention are useful for diagnosing infection with a West Nile virus, in particular neurovirulent strains; the infection is detected using a suitable technique, in particular EIA, ELISA, RIA or immunofluorescence, using a biological sample taken from an individual liable to be infected or a laboratory animal inoculated with said virus. Among these antibodies, those produced by immunizing Flvr/Flvr mice with the strain IS-98-ST1 advantageously have a high titer and a very high specificity for the West Nile virus.
The transformed cells according to the invention, in particular neural cells (neurons and endothelial cells) infected with a neurovirulent strain as defined above, are used to identify the genes derived from these cells, the expression of which may be modulated during the viral infection; these genes are detected, for example, using biochip technology according to conventional protocols as described in Atlas Mouse Arrays (#membranes) ATLAS™ NYLON cDNA EXPRESSION ARRAYS (CLONTECH, USA).
A subject of the present invention is also a model for studying sensitivity/resistance to infection with a virus of the family Flaviviridae, characterized in that it comprises at least one neurovirulent strain of the West Nile virus as defined above.
According to an advantageous embodiment of said model, it also comprises a mouse homozygous for the Flvr or Flvr allele.
A subject of the present invention is also a method for detecting Flaviviridae infection, in particular a West Nile virus infection, characterized in that it comprises:
Such a detection may advantageously make it possible to establish a prognosis for the severity of a viral encephalitis caused by the West Nile virus.
The neurovirulent strain of the West Nile virus according to the invention is used to screen cellular genes involved in the resistance of a mammal to infection with a virus of the family Flaviviridae, preferably the hepatitis C virus.
Advantageously, said screening method comprises the following steps:
In accordance with the invention, said detection may comprise establishing the transcript or protein profile using said cells.
A subject of the present invention is also the use of the model as defined above for sorting molecules which are active against a viral infection due to a virus of the family Flaviviridae.
A subject of the present invention is also a method for sorting molecules which are active against infection with a Flavivirus, characterized by:
A subject of the present invention is also a variant of the viral strain as defined above, characterized in that its genome comprises at least one mutation in the nucleotide sequence corresponding to the NS5 protein.
Besides the above arrangements, the invention also comprises other arrangements which will emerge from the following description, which refers to examples of implementation of the subject of the present invention, with references to the attached drawings in which:
FIGS. 4 (A, B and C) represent the kinetics of appearance of the viral antigens in Neuro 2a cells and primary neurons from sensitive mice (BALB/c) infected with the West Nile virus (strain IS-98-ST1),
An isolate of the West Nile (WN) virus was obtained from the central nervous system of a stork exhibiting severe neuropathological problems, in September 1998 in Eilat (Israel). Infection of VERO cells with this isolate is cytolytic and indirect immunofluorescence with an immune mouse ascites specific for the West Nile virus (immune serum of reference WN 8907) is 100% positive. The virus produced on VERO cells was harvested and amplified on AP61 mosquito cells (Després et al., Virol., 1993, 196, 209-219).
Passage 1 (or P1) of the WN virus on AP61 cells was harvested 3. days after infection; it has a titer of 2.5×108 FFU/ml (Focus-Forming Unit) by the technique for titering on AP61 cells described in Despres et al. (mentioned above). The P1 inoculum of the WN virus on AP61 cells was identified as the strain IS-98-ST1.
A P2 was obtained from AP61 cells infected with the strain IS-98-ST1, P1 (titer: 6×107 FFU/ml). The P2 inoculum of IS-98-ST1 is used for the tests for sensitivity to viral infection in adult mice.
A P3 viral inoculum of the strain IS-98-ST1, with a titer of 5×107 FFU/ml, was produced on PA61 cells. A highly purified viral preparation, prepared according to the flavivirion purification protocol described in Deprés et al., 1993) was obtained from twenty 150 cm2 dishes of AP61 cells harvested 3 days after infection with the P3 inoculum of the WN virus strain IS-98-ST1 (multiplicity of infection of 0.4). The strain IS-98-ST1 purified on sucrose gradients has a final titer of 2×1010 FFU/ml. The RNAs extracted from this purified virus are used to amplify the cDNAs corresponding to the C, prM and NS1 viral proteins or to the noncoding sequences at the 5′ and 3′ ends of the viral genome.
The viral genome was extracted from the culture supernatant of the infected VERO cells of example 1 using the “QIAamp Viral RNA” kit (QUIAGEN), according to the manufacturer's instructions. 6 overlapping RT-PCR products were amplified from these RNAs using the primers described by Lanciotti et al. (mentioned above). The 5′ and 3′ ends of the viral genome were amplified, respectively, using the following primers:
The cDNA corresponding to the E viral protein was amplified using the primers 5′GGATGGATGCT(A/T)GG(G/T)AGCAAC 3′ (SEQ ID No. 7) and 5′CCATCCAAGCCTCCACATC 3′ (SEQ ID No. 8), which hybridize, respectively, in the gene of the M protein (positions 889 to 909 of the sequence SEQ ID No. 1) and in the gene of the NS1 protein (positions 2539 to 2557 of the sequence SEQ ID No. 1).
The cDNAs obtained were purified by ion exchange chromatography and precipitated in 2 volumes of isopropanol. The cDNAs were then sequenced on both strands using the “Taq Dye Deoxy Terminator Cycle Sequencing” kit (PERKIN ELMER CORP./APPLIED BIOSYSTEM) and primers 400 base pairs apart on the viral genome (Lanciotti et al., mentioned above). The sequencing was performed with 0.2 pmol of purified cDNA and 30 pmol of primers, according to the protocol recommended by the manufacturer. The sequence alignment is produced using the CLUSTAL W program.
The complete genomic sequence of the strain IS-98-ST1 of the West Nile virus corresponds to the sequence SEQ ID No. 1.
The amino acid sequence alignment for the strain IS-98-ST1 (SEQ ID No. 2) and the strain NY99, given in
However, the differences observed in the strain IS-98-ST1 in the E (A51), NS1 (N17), NS2A (R164), NS2B (G82, E83), NS3 (P496, E521) and NS5 (S54, N280, A372) proteins, respectively, are potentially responsible for the neurovirulence and for the neuroinvasive properties observed with this strain and may be used as a virulence marker for the West Nile virus.
1—The C Protein
The genomic RNA extracted from the IS-98-ST1 virions purified on sucrose gradients described in example 1, using the RNA PLUS 2 solution (Q.BIOGEN), is used as a matrix for amplifying the sequence encoding the C protein (amino acids 1 to 123) using the RT-PCR technique (Titan One Tube RT-PCR kit; Roche Biochemicals #1939 823).
The pair of primers used on the RNA matrix is as follows:
The cDNA of the C protein of the strain IS-98-ST1 (amino acids 1 to 123) of the WN virus was cloned, firstly, between the EcoRI and SmaI sites of the plasmid pCI-neo (Promega #E1841) and, secondly, between the KspI and SmaI sites of the plasmid PIVEX 2.4a (Roche).
The recombined plasmid pCI-C/WN (registered on Jun. 21, 2001, with the Collection Nationale de Culture de Microorganismes [National Collection of Microorganism Cultures] of the Pasteur Institute in Paris, 28, rue du Docteur Roux, 75724, PARIS Cedex 15, under the number I-2688) contains the complete sequence of the C protein gene of the strain IS-98-ST1 of the WN virus, between the T7 and T3 promoters. The transcription, in vitro, of pCI-C/WN linearized with NheI, under the control of the T3 promoter, synthesizes an RNA of approximately 350 bases complementary to the genomic viral sequence. The riboprobe labeled with DIG (digoxigenin) is used to detect the positive-sense viral RNAs in cells infected with the WN virus, using the in situ hybridization technique, according to the protocol described in Després et al., (J. Virol., 1998, 72: 823-829).
The recombinant plasmid pIVEX-C/WN is used for the mass production of the WN virus C protein (amino acids 1 to 123) in bacterial lysate (Roche RTS 500 system). The recombinant C protein produced in vitro has, at its N-terminal end, a [His]6 sequence and the cleavage site recognized by the Xa protease, so as, firstly, to allow it to be purified on an Ni column and, secondly, to allow the histidine residues to be removed. The C protein of the strain IS-98-ST1 of the WN virus thus produced is used for structural studies, to search for cellular partners for this protein on an immunoaffinity column, and to produce monospecific antibodies in rabbits.
2—The M Protein
The cDNAs, of the strain IS-98-ST1 of the WN virus, encoding the M protein (amino acids 215 to 290 of the viral polyprotein) or its 41 amino acid ectodomain (amino acids 215 to 255; acronym ectoM) are cloned:
(1) in phase with the C-terminal end of EGFP, into the plasmid p[95-114]EGFP, derived from the plasmid PEGFP-N1 (Clontech), which comprises residues 95-114 of the C protein of dengue virus type 1 (strain BR/90), fused in phase with the N-terminal sequence of the protein EGFP[215-290]WNV, to give the plasmid p[95-114]EGFP[215-290] WNV,
(2) into the plasmid pIVEX (Roche RTS 500 system), to give the plasmid pIVEX[EGFP][215-255]WNV,
(3) into the retroviral vector TRIPdeltaU3CMV, to give the plasmid TRIPdeltaU3CMV[95-114]EGFP[215-255]WNV.
The plasmid pIVEX[EGFP][215-255]WNV allows the extracellular synthesis and the purification of the chimeric protein EGFP-ectoM WNV, which is used, firstly, to produce monospecific antibodies directed against the M protein of the WN virus and, secondly, to search for cellular partners of the ectoM WNV molecule on an immunoaffinity column.
The plasmid TRIPdeltaU3CMV[95-114]EGFP[215-255]WNV is cotransfected into 293T cells with the plasmids 8.7 and G-VSV, in order to produce the viral particles pseudotyped with the G envelope of the vesicular stomatitis virus (VSV), containing the inner proteins of the acquired immunodeficiency virus (HIV) and chimeric CMV[95-114]EGFP[215-255]WNV RNA molecules. Infection of target cells with the nonreplicative recombined vector allows integration of the CMV[95-114]EGFP(215-255]WNV DNA into the cellular genome and stable expression of the ectodomain of the M-WN protein under the control of the CMV promoter.
3 -The NS1 Protein
The genomic RNA extracted from the purified IS-98-ST1 virions is amplified using the RT-PCR technique (Titan One Tube RT-PCR kit; Roche Biochemicals #1939 823) using the following pair of primers:
The cDNA encoding the NS1 glycoprotein with its signal peptide (amino acids 767 to 1143 of the viral polyprotein) is cloned between the BamH1 and KpnI sites of the retroviral vector TRIPdeltaU3, so as to produce the recombined plasmid TRIPdeltaU3-CMV-NS1-WN (registered on Jan. 9, 2002, with the Collection Nationale de Culture de Microorganismes [National Collection of Microorganism Cultures] of the Pasteur Institute in Paris, 28 rue du Docteur ROUX, 75724, PARIS Cedex 15, under the number I-2770). The plasmid TRIPdeltaU3-CMV-NS1-WN is cotransfected into 293T cells with the plasmids 8.7 and G-VSV, in order to produce viral particles pseudotyped with the G envelope of the vesicular stomatitis virus (VSV), containing the inner proteins of the acquired immunodeficiency virus (HIV) and chimeric CMV-NS1-WN RNA molecules. Infection of target cells with the nonreplicative recombined vector allows integration of the CMV-NS1-WN DNA into the cellular genome and stable expression of the NS1 protein of the WN virus under the control of the CMV promoter. The NS1 protein of the strain IS-98-ST1 of the WN virus thus produced is used for structural studies, to search for cellular partners of this protein on an immunoaffinity column, and to produce monospecific antibodies in rabbits.
1—Sensitive Mouse Lines and Cells
a) Sensitive Mouse Lines
6-week-old mice from Flvs sensitive inbred lines (BALB/c) are inoculated intraperitoneally with 100 FFU of the strain IS-98-ST1 of the West Nile virus (FFU:LD50=10), prepared as described in example 1.
100% of these mice die with a mean mortality time of 9±2 days (
The kinetics of propagation of the strain IS-98-ST1 in the central nervous system of the sensitive mice (BALB/c) was analyzed using brain extracts from the infected mice titered on AP61 cells, according to the technique described in according to the technique described in Despres et al. (J. Virol., 1998, 72, 823-829). The results show that the virusis detected in the murine central nervous system (CNS) on the 5th day of infection and viral production is at a maximum on the 7th day (
Replication of the WN virus in the CNS and the peripheral organs of the mice infected with the strain IS-98-ST1 is also detected by immunohistology, according to conventional protocols as described in Despres et al., 1998 (mentioned above) and by in situ hybridization, according to the protocols described in example 3.
The serum antibodies specifically directed against the proteins of the WN virus are titered by ELISA according to the protocol described in Despres et al., 1993 (mentioned above), using the strain IS-98-ST1 purified on a sucrose gradient as described in example 1, as antigen. The results show that the serum antibodies appear on the 5th day of infection and are significantly detected on the 7th day (
b) Sensitive Cells
b1) Primary Cultures
Primary neurons and astrocytes from the CNS of sensitive mice homozygous for the Flvs allele (Swiss mice, Janvier) are prepared according to conventional protocols. The cells are infected with the strain IS-98-ST1 at a multiplicity of infection of 20 FFU per cell (m.i. of 20). The cytopathic effect is observed by light microscopy, viral production is analyzed by titering on AP61 cells as described previously in example 1, and expression of the viral antigens is analyzed by radioimmunoprecipitation using an anti-West Nile mouse immune serum, according to conventional protocols as described in Duarte Dos Santos et al. (Virology, 2000, 274: 292-308).
The results show that 80% of the neurons in culture produce the viral antigens:
On the other hand, astrocytes of the murine CNS are not permissive to replication of the WN virus strain IS-98-ST1.
b2) Cell Lines
Neuro 2a murine neuroblastoma cells and HepG2 human hepatoma cells, cultured under conventional conditions as described in Marianneau et al. (J. Virol., 1996, 77: 2547-2554), are infected at various multiplicities of infection with the WN virus strain IS-98-ST1, prepared as described in example 1. The cytopathic effect is observed by light microscopy, viral production is analyzed by titering on AP61 cells as previously described in example 1, and expression of the viral antigens is analyzed by radioimmunoprecipitation using an anti-West Nile mouse immune serum, according to conventional protocols as described in Duarte Dos Santos et al., Virol., 2000, 274, 292-308.
The results show that Neuro 2a murine neuroblastoma cells are permissive to replication of the strain IS-98-ST1 of the WN virus. An m.i. of 4 is necessary to infect 80% of the Neuro 2a cells in monolayer. The viral production of 107 FFU/ml (m.i. of 4) after 40 h of infection, and there is massive cell death by necrosis (
The results also show that HepG2 human hepatoma cells are permissive to replication of the strain IS-98-ST1 of the WN virus. At an m.i. of 10, the viral production is [2±1]×106 FFU/ml after 48 h of infection and the CPEs are observed from 72 h.
2—Resistant Mice
The resistant (Flvr) mouse lines which derive from wild mice of the species Mus spretus (SEG/Pas and STF/Pas), Mus musculus musculus (MBT/Pas, MAI/pas) and Mus musculus domesticus (WMP/Pas) are inoculated intraperitoneally with 1 000 FFU (100 LD50) of the strain IS-98-ST1 prepared according to the protocol described in example 1.
Unlike the laboratory mice which are sensitive to infection with the strain IS-98-ST1 and die in about ten days, these mice derived from wild mice are resistant to inoculation with the strain IS-98-ST1 and, nevertheless, permissive to replication of the strain IS-98-ST1. In fact, viral infection of the mice derived from wild mice is asymptomatic, although the virus multiplies, in toto, as demonstrated by the production of anti-WN serum antibodies at high titers; by ELISA, the titers of the sera at a dilution of 1:100, for 106 FFU of purified IS-98-ST1 virion, are greater than 1 O.D. unit at 450 nm.
The mice resistant to viral infection are used to produce immune sera specifically directed against the proteins of the strain IS-98-ST1 of the WN virus. Three weeks after inoculation with the WN virus, the sera taken from resistant mice (0.045 ml per mouse) are mixed, decomplemented for 30 min at 56° C. and then diluted to 1:10 in DPBS* (v/v) supplemented with 0.2% (v/v) of bovine serum albumin (Life Technologies) and 0.05% (w/v) of sodium azide. The diluted sera are divided up into 0.2 ml aliquots and stored at −20° C. The immune sera directed against the strain IS-98-ST1 are used at the final dilutions of 1:500 for indirect immunofluorescence and at 1:1 000 for immunoprecipitation of radiolabeled viral proteins.
1) Methods
a) Model for Analyzing Resistance to Infection with Flaviviridae (
Male mice of the resistant lines MAI/Pas and MBT/Pas are crossed with female mice of the sensitive lines C57BL/6 and BALB/c. The male mice of the F1 generation are backcrossed with female mice of the sensitive lines C57BL/6 and BALB/c, to give a generation of first backcross (BC1) mice.
Five-week-old BC1 mice are inoculated intraperitoneally with the strain IS-98-ST1, prepared according to the protocol described in example 1, under the conditions described in example 2.
The animals are observed every day and the mortality and survival rates are determined 14 days after infection.
b) Genotyping of Flv Alleles
The Flv alleles of the BC1 individuals were mapped by genomic PCR using primers specific for 16 microsatellites of chromosome 5 (Catalogue Research Genetics) surrounding the Flv locus (
2) Results
Analysis of Flv allele distribution in the BC1 mice sensitive and resistant to infection with the strain IS-98ST1 shows that one Flvr allele is sufficient to confer resistance to infection (
The genotyping of the Flv alleles shows that the Flv locus is located in a 0.2 cM region containing the OAS1 gene (
| Number | Date | Country | Kind |
|---|---|---|---|
| 01/04599 | Apr 2001 | FR | national |
| 01/11525 | Sep 2001 | FR | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10474186 | US | |
| Child | 10956085 | Oct 2004 | US |
