WO2002095040A1 - Deletions des replicons de l'arterivirus - Google Patents
Deletions des replicons de l'arterivirus Download PDFInfo
- Publication number
- WO2002095040A1 WO2002095040A1 PCT/NL2002/000314 NL0200314W WO02095040A1 WO 2002095040 A1 WO2002095040 A1 WO 2002095040A1 NL 0200314 W NL0200314 W NL 0200314W WO 02095040 A1 WO02095040 A1 WO 02095040A1
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- rna
- virus
- protein
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- prrsv
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C—CHEMISTRY; METALLURGY
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- C12N2770/00011—Details
- C12N2770/10011—Arteriviridae
- C12N2770/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the invention relates to recombinant Arterivirus replicons.
- Porcine reproductive and respiratory syndrome virus is a positive strand RNA virus that belongs to the Arteriviridae family (reviewed in Snijder and Meulenberg, 1998), together with equine arteritis virus (EAV), lactate dehydrogenase -elevating virus (LDV), and simian hemorrhagic fever virus (SHFV) (Meulenberg et al., 1993b).
- EAV equine arteritis virus
- LDV lactate dehydrogenase -elevating virus
- SHFV simian hemorrhagic fever virus
- PRRSV is the causative agent of respiratory problems in pigs and stillbirths in sows, and accounts for huge economical losses worldwide. PRRSV was first isolated in the Netherlands in 1991 (Wensvoort et al., 1991), and was designated Lelystad virus (LV).
- PRRSV PRRSV genome of PRRSV
- the genome of PRRSV is a 5'-capped and 3'- polyadenylated RNA molecule of 15.1 kb (Meulenberg et al., 1993b).
- the 5' two-third of this RNA is translated into two large polyproteins.
- These are subsequently cleaved by virus-encoded proteases to yield at least 12 non-structural proteins, including the viral RdRp (Snijder et al., 1994; van Dinten et al., 1999; van Dinten et al., 1996; van Marie et al., 1999b; Wassenaar et al., 1997).
- sg mRNAs are produced through a process of discontinuous mRNA transcription.
- These sg mRNAs each contain a leader sequence derived from the 5' UTR fused to a body part derived from the 3' part of the genome (de Vries et al., 1990; Lai, 1990; Meulenberg et al., 1995; Meulenberg et al., 1993a).
- Leader-body fusion occurs at a transcription- regulating sequence (TRS) and results in the production of a 3' nested set of sg mRNAs. They collectively specify the viral structural proteins.
- ORF7 encodes the nucleocapsid protein N, ORF6 the membrane protein M, ORF5 the major envelope glycoprotein GP5, and ORFs2-4 the minor envelope glycoproteins GP2, GP3, and GP4 (Meulenberg et al., 1995).
- E a novel structural protein called E
- the PRRSV genome contains a 5'UTR of 221 nucleotides (Snijder and Meulenberg, 1998), which carries the cap at its 5' end (Meulenberg et al., 1998; Sagripanti et al., 1986), and a 3'UTR of 114 nucleotides to which the poly(A)-tail is attached (Meulenberg et al., 1993b).
- Positive strand RNA viruses replicate in infected cells by a process which is mediated by RNA-dependent RNA-polymerase (RdRp).
- RdRp RNA-dependent RNA-polymerase
- the positive strand genomic RNA serves as a template for the production of negative strand genomic RNA, which is used in turn as a template for the synthesis of new plus strands.
- the process of replication requires the recruitment of the RdRp to specific sequences or structures within the templates, also known as cis-acting elements. These elements are usually located in the non-coding regions at the termini of the viral RNA, where RdRp complexes initiate the synthesis of plus and minus strands (Buck, 1996). Cis-acting elements have been characterized for several viruses and show a wide variety of structures.
- RNAs can be structures with no apparent structure, e.g. the plus strand promoter from a satellite RNA of a carnovirus (Guan et al., 2000); stem-loop structures, e.g. in the 5' untranslated region (UTR) of arterivirus RNAs (Hwang and Brinton, 1998), pseudoknots, e.g.
- cis-acting elements are located within a coding region, e.g. the long-range pseudoknot of bacteriophage Qb RNA (Klovins and van Duin, 1999). This is the only known sequence in the coding region which is involved in a long-range interaction that is essential for RNA replication.
- Cis-acting elements for EAV genome replication, transcription, and packaging have been roughly mapped by using a Defective Interfering (DI) genome (Molenkamp et al, 2000a). So far, it has not been elucidated which sequences in the region of the arterivirus genome encoding the structural proteins are essential for RNA replication and/ or transcription.
- DI Defective Interfering
- the invention provides the insight that an Arterivirus replicon having at least some of its original arteriviral nucleic acid encoding ORF-7 deleted, as provided herein, can still be capable of in vivo RNA replication, even when further comprising nucleic acid derived from at least one heterologous micro-organism, thereby also providing viable Arteriviruses with deletions proximal to the 3'end of the genome.
- the present application describes the requirements for replication and transcription on the RNA/nucleic acid level, and the use for vaccine development or vector systems. Further this application teaches how these biological processes can be influenced. It demonstrates that for producing a replicon, it is essential that a long distance interaction between a (34-)nucleotide stretch in a coding region of the viral genome (which stretch is highly conserved among PRRSV isolates and folds into a putative stem-loop structure) and particularly between a 7-base sequence within the loop of this structure needs be maintained with a sequence present in the 3'noncoding region, which in turn occurs in the loop of a predicted, strongly conserved hairpin structure. However, it is the base-pairing ability, not the sequences per se, that is essential, for example, complementary substitution of a short (3-7, preferably 5-)base sequence in either of the loops still allows the generation of a replicon.
- the invention relates to recombinant Arterivirus replicons and methods to obtain these.
- the invention provides the insight that an Arterivirus replicon having at least some of its original arteriviral nucleic acid (such as encoding a distinct part of ORF-7) deleted, as provided herein, can still be capable of in vivo RNA replication, even when further comprising nucleic acid derived from at least one heterologous micro-organism, thereby also providing viable Arteriviruses with deletions proximal to the 3'end of the genome, provided that said long-distance interaction is kept in place.
- the invention provides a method for generating a replicon wherein a short, approximately 5-base sequence in either of the loops is modified in that it is complementary substituted while maintaining said longdistance interaction, as for example shown in figure 4.
- the invention provides a method for generating a replicon of an Arterivirus, preferably of PRRSV, wherein by mutation the genome of said Arterivirus is altered, but wherein the ability of the two predicted loops to base-pair, (albeit not their primary sequences per se) is functionally kept intact.
- the invention furthermore provides arterivirus replicon having at least some of its original arteriviral nucleic acid (such as that encoding ORF-7) deleted, wherein the primary sequences of said loops are no longer wild-type sequences (as for example known from Genbank datasubmissions NC-001961, AF066183, AF331831, NC-002534, U87392, NC-002533, M96262, AF184212, NC-001639, AF159149, AF046869, and U15146) but wherein the ability of the two loops to base-pair (as for example identified in figure 4) is functionally kept intact.
- This finding thus provides distinct metes and bounds for the production of
- WO 005387 provides an infectious clones of PRRSV eventually supplemented with heterologous genetic material, but does not teach the minimally essential requirement of said long-distance interaction, nor do US 6 110467 (relating to a PRRS vaccine, attenuated or inactivated, obtained by serial passages of defined PRRSV strains belonging to the US genotype) nor WO 96/06619 which relatesto polynucleic acids and proteins originated from PRSV and there use. Nucleic acids are used for encoding one or more PRRSV proteins in expression systems such as baculovirus but no recombinant replicating system based on PRRSV is provided.
- US 5 998601 relates to a DNA sequence which comprises full length or part of VR2332, an US PRRSV isolate, but does not teach the minimally essential requirement of said long-distance interaction either.
- the invention provides a replicon wherein a kissing loop interaction between 3'noncoding and coding sequences is maintained where its primary sequences essential for wild-type Arterivirus RNA replication are modified.
- replication of arteriviruses requires cis-acting elements to initiate the synthesis of genomic negative strands. These cis- acting elements are now known to be located in the 5' and 3' non-coding regions, as well as in sequences from the long open reading frame lab (ORFlab) encoding the nonstructural proteins.
- RNA hairpin in which loop residues are complementary to nucleotides from the loop of another hairpin within the 3'UTR.
- kissing loop interaction is required for RNA replication of PRRSV.
- the invention provides the insight that an Arterivirus replicon having at least some of its original arteriviral nucleic acid encoding ORF-7 deleted, as provided herein, can still be capable of in vivo RNA replication, even when further comprising nucleic acid derived from at least one heterologous micro-organism.
- the invention provides a deletion in the region around the N gene stop codon. Alignment of the N protein sequence and the 3'UTR of different PRRSV strains revealed heterogeneity at the C-terminus of the N protein and at the 5' end of the 3'UTR.
- a deletion analysis of this region was therefore performed using the available infectious cDNA clone (Meulenberg et al., 1998a) of Lelystad virus (LV) to determine the limits of the sequences that can be removed without significantly affecting virus viability, hereby providing the generation of viable arterivirus mutants containing a deletion in the viral genome, which is stably maintained after multiple passages in vitro.
- the thus obtained attenuated hve vaccine candidates of porcine reproductive and respiratory syndrome virus (PRRSV) each comprise one of a series of deletions introduced at the 3'end of the viral genome, for example using the infectious cDNA clone of the Lelystad Virus (LV) isolate.
- RNA transcripts from the full-length cDNA clones were transfected into BHK-21 cells. The culture supernatant of these cells was subsequently used to infect porcine alveolar macrophages to detect the production of progeny virus.
- C-terminal truncation of the nucleocapsid protein N, encoded by ORF7 was tolerated for up to 6 amino acids without blocking the production of infectious virus. Mutants containing larger deletions produced neither virus nor virus-like particles containing viral RNA. Deletion analysis of the 3'UTR immediately downstream of ORF7 showed that infectious virus was still produced after removal of 7 nucleotides behind the stop codon of ORF7.
- PRRSV has a concise genome, like other RNA viruses. Since RNA viruses have evolved to optimal fitness, most of the genetic information is expected to be essential. Second, the ORFs that encode the structural proteins of the virus are partially overlapping. Deletions in overlapping regions would therefore result in the mutation of two structural proteins, which would almost inevitably lead to the production of a nonviable virus.
- the invention furthermore provides the use of a replicon according to the invention for obtaining a vaccine, said vaccine preferably comprising such a replicon; however a killed vaccine or subunit vaccine based on using the replicon to produce the necessary antigenic mass is also provided.
- a vaccine can be used for vaccinating animals, preferably pigs susceptible to PRRSV infections.
- RT-PCR strategy for (A) and results of (B) the detection of genomic positive strand RNA (1) and genomic negative strand RNA (2), sg positive strand mRNA7 (3) and sg negative strand mRNA7 (4).
- BHK-21 cells were electroporated with RNA transcripts from pABV437, pABV668, and pABV696, and cellular RNA was isolated 12 hours after transfection.
- the viral RNA was reverse transcribed and ampHfied by PCR, as outlined in A. Products were analyzed in a 1% agarose gel. Numbers of the constructs from which the amplification products were derived are indicated beneath the lanes. The numbers on the left indicate the marker sizes in kilobases.
- the nucleotide positions of the primers are indicated between brackets beneath the primers.
- FIGURE 4 (A) Schematic representation of the predicted secondary structure in the 34- nucleotide stretch (nucleotides 14653-14686) in ORF7 of LV (GenBank M96262). (B) Predicted secondary structure of a hairpin within the 3'UTR, with 7 nucleotides in its loop complementary to 7 nucleotides in the predicted loop within the 34-nucleotide stretch. Nucleotide differences with other PRRSV strains are indicated alongside. FIGURE 4
- LV and VR2332 are the prototypes of the European and American PRRSV strains, respectively. Underlined is the part of the Pad-site present in the 3'UTR. The conserved residues are indicated beneath the alignments with *.
- PRRSV PRRSV. Deletions were introduced by PCR-mutagenesis, and cloned into the full- length cDNA clone pABV437 (Meulenberg et al., 1998a). The constructs, the deleted nucleotides, the plasmid numbers, the observed expression of M and N protein, and the production of viable virus are indicated. The boxes indicate the present regions, the lines indicate the deleted regions. Expression of the viral proteins M and N was analysed by IPMA 24 hours after transfection of BHK-21 cells using MAb 126.3 and MAb 122.17, respectively. Positive staining is indicated by +; no staining is indicated by -. The bar above the constructs indicates the antigenic domains of the N protein. FIGURE 8
- deletions were introduced into the infectious cDNA clone of LV (Meulenberg et al., 1998) and their RNA transcripts were transfected into BHK-21 cells. Their ability to transiently express the remaining viral structural protein genes was tested by immunoperoxidase monolayer assay
- IPMA IP-associated reverse transcriptase
- RNA transcripts lacking ORF2 through the 5' part of ORF6 induced the expression of the N protein in the transfected BHK-21 cells, indicating that sg mRNAs were still produced and that replication and ⁇ transcription were not affected.
- RNA transcripts lacking the entire ORF7 pABV521) gene did not express any of the remaining structural proteins after transfection into BHK-21 cells.
- the 34-nucleotide region in ORF7 is essential for RNA replication.
- RNA transcripts were produced by pABV668 to generate positive and negative strand genomic and subgenomic viral RNA, as outlined in Figures 2A1 to A4.
- Total RNA isolated from cells transfected with transcripts from pABV668 yielded an amplification product only after testing for positive strand genomic RNA ( Figure 2B1). This product was probably derived from the input RNA, because pABV668 lacks the 3'UTR sequences and is therefore unlikely to yield RNA transcripts that are replication competent.
- RNA isolated from cells that had been transfected with transcripts from pABV437 RT-PCR products of the expected sizes were obtained for both the genomic positive and negative strand ( Figure 2B1 and 2B2) and for the sg mRNA7 positive and negative strand ( Figure 2B3 and 2B4).
- RNA from cells transfected with transcripts from pABV696 we obtained similar results as for pABV668 ( Figure 2B1 to B4).
- the identity of the PCR products was verified by their size and by restriction enzyme analysis (data not shown).
- an immunofluorescence assay IF A was performed using an antiserum against the nonstructural precursor protein nsp2/3 of PRRSV.
- Ns 2/3 is translated from genomic RNA, but the level of nsp2/3 produced from non-replicating transcripts is too low to be detected by the antiserum. Therefore, positive staining of nsp2/3 by the antiserum is dependent on RNA replication.
- transcripts from pABV696 no expression of nsp2/3 was detected, as was the case with transcripts from our negative control pABV668.
- transcripts from our positive control pABV437 we clearly detected the expression of the nsp2/3 precursor protein (data not shown).
- transcripts from pABV696 were impaired in the synthesis of both positive and negative strand genomic and sg mRNAs. More specifically, the 34-nucleotide stretch in ORF7 appears to be essential for genomic minus-strand RNA synthesis.
- the 34-nucleotide stretch is highly conserved in PRRSV isolates and is predicted to form a stem-loop structure
- the position of the 34-nucleotide sequence is important for its function
- RNA transcripts when transfected into BHK-21 cells, showed no detectable expression of the M protein in IPMA. This indicated that the RNA replication and/ or transcription could not be restored by relocation of the 34-nucleotide stretch.
- RNA viruses Signals regulating the replication of RNA viruses are generally located within the terminal non-coding regions of the genome.
- a domain essential for viral replication within a coding region of the porcine arterivirus RNA the most 3'ORF specifying the viral nucleocapsid protein N.
- Deletion of this 34- nucleotide domain from genomic RNA completely abolished negative strand RNA synthesis.
- Theoretical analysis of its sequence predicts it to fold into a stem-loop structure that is highly conserved among porcine arteriviruses.
- a 7- nucleotide sequence within the loop of this structure appeared to be engaged in a kissing loop interaction with a domain located in the 3'UTR.
- the 34-nucleotide domain critical for PRRSV RNA replication is located in the coding region of the N gene.
- the N protein would have any role in viral RNA replication in addition to its functioning in virus assembly, the effects of deletions in this gene might simply be explained by its debilitating consequences on the protein's functioning.
- a role of the arterivirus N protein in replication has indeed been suggested on the basis of its co-localization with the polymerase and helicase proteins in the viral replication complex (Pedersen et al., 1999; van der Meer et al., 1999).
- a similar multifunctional role has also been attributed to the N protein of the related coronaviruses.
- DI RNAs For a number of positive strand RNA viruses, DI RNAs have been used to map cis-acting sequence elements that participate in replication and transcription.
- the only arteriviral DI described so far (Molenkamp et al., 2000a) had lost most of its sequences encoding the non-structural proteins, but retained the entire region encoding the structural proteins.
- Further trimming of this DI genome using a cDNA clone revealed that the 3' terminal 1066 nucleotides were essential either for replication, transcription or packaging. This region included besides the ORF7 gene, the ORF6 gene, as weU as the 3' end of ORF5.
- ORF7 Since ORF7 is located at the 3'end of the viral genome, the 34-nucleotides might have a role in the formation of complexes for initiation of minus-strand synthesis. Therefore, its position, i.e. the relative distance to the 3'UTR and the adjacent nucleotide sequence, might be important for its structure and therefore for its function.
- the negative effect of the relocation of the 34- nucleotide stretch on the RNA replication indeed confirms this.
- At least 10 non- structural viral proteins are involved in RNA replication, and, moreover, host- encoded proteins may take part in the formation of such complex. Protein binding might stabilize the kissing loop interaction, or might prevent or melt the interaction, thereby shutting off minus strand synthesis.
- RNA structure trans-activation response element Tar
- PCBP cellular factor Poly(rC) binding protein
- 3CD represses the translation and promotes negative-strand RNA synthesis
- Proteins are also involved in RNA rephcation (Hwang and Brinton, 1998; Liu et al, 1997; Yu and Leibowitz, 1995) and transcription (Huang and Lai, 1999) of coronaviruses.
- the ways in which they act are not yet elucidated. Potentially, the interaction with specific proteins might regulate whether the genomic RNA is used for RNA replication or sg mRNA transcription of PRRSV.
- BHK-21 cells were grown in BHK-21 medium (Gibco BRL), complemented with 5% FBS, 10% tryptose phosphate broth (Gibco BRL), 20 mM Hepes pH 7.4 (Gibco BRL), 200 mM glutamine, 10 U/ml penicillin, 10 ⁇ g/ml streptomycin, 20 ⁇ g/ml kanamycin, 5 ⁇ g/ml polymixine B, and 0.2 ⁇ g/ml fungizone.
- pABV402 was digested with EcoRI and Ndel, treated with Klenow-enzyme, and self-Hgated, resulting in subclone pABV593.
- This subclone was extended to a fuU-length cDNA clone by insertion of the Pmll-Spel region of pABV399, which comprises the 5' one-third of the viral genome (Meulenberg et al., 1998), generating pABV594.
- ORF7 was deleted from pABV442, a fuU- length cDNA clone containing a Swal-site directly downstream of ORF7 (Meulenberg et al., 1998).
- pABV442 was digested with Hpal and Swal, and self-Hgated, generating pABV521. Third, the region comprising ORF2 through 6, except for the TRS of ORF7, was deleted. Plasmid pABV402 was digested with EcoRI and Hpal, treated with Klenow-enzyme, and self-Hgated, resulting in subclone pABV663. pABV663 was restored into a fuH length cDNA clone by insertion of the Pmll-Spel fragment of pABV399, resulting in pABV664.
- the double-mutant containing these mutations in both loops was constructed by ligation of the Hpal-Pacl fragment from pABV769 into the Hpal-Pacl digested pABV768. This resulted in pABV770.
- the mutations are described in Figure 4.
- the constructed cDNA clones were in vitro transcribed using 1 ⁇ g Hnearized plasmid DNA, and were subsequently treated for 15 minutes with 10 U DNAse at 37°C. BHK- 21 ceHs were transfected with the resulting RNA by electroporation as described (Meulenberg et al., 1998).
- IPMA Immunoperoxidase monolayer assay
- RNA was extracted three times using phenol-chloroform (pH 4.0), once using chloroform, and was then precipitated with isopropanol.
- RNA was reverse transcribed as indicated in Fig. 3A.
- the PCR consisted of 39 cycli, each comprising 30 seconds of denaturation at 94°C, 30 seconds of anneaHng at 62°C, and 2 minutes of elongation at 72°C.
- the PCR products were analyzed in 2% agarose gels.
- RNA secondary structures were predicted with M. Zuker's Mfold server at www.ibc.wustl.edu/ ⁇ zuker/rna/. REFERENCES with example 1
- AH subgenomic mRNAs of equine arteritis virus contain a common leader sequence. Nucleic Acids Res, 18, 3241-3247.
- RNA secondary structure in the 3 ' untranslated region of the murine coronavirus genome J Virol, 74, 6911-6921.
- Coronavirus how a large RNA viral genome is repHcated and transcribed. Infect Agents Dis, 3, 98-105.
- RNAs of Lelystad virus contain a conserved leader-body junction sequence. J Gen Virol, 74,
- (rC) binding protein 2 forms a ternary complex with the 5'-terminal sequences of poHovirus RNA and the viral 3CD proteinase. Rna, 3, 1124-1134.
- the 5'-end sequence of the murine coronavirus genome impHcations for multiple fusion sites in leader-primed transcription. Virology, 156, 321-330.
- BHK-21 ceHs were grown in BHK-21 medium (Gibco BRL) complemented with 5% FBS, 10% tryptose phosphate broth (Gibco BRL), 20 mM Hepes pH 7.4 (Gibco BRL), 200 mM glutamine, 10 U/ml penicillin, 10 ⁇ g/ml streptomycin, 20 ⁇ g/ml kanamycin, 5 ⁇ g/ml polymixine B, and 0.2 ⁇ g/ml fungizone.
- BHK-21 ceHs were grown in BHK-21 medium (Gibco BRL) complemented with 5% FBS, 10% tryptose phosphate broth (Gibco BRL), 20 mM Hepes pH 7.4 (Gibco BRL), 200 mM glutamine, 10 U/ml penicillin, 10 ⁇ g/ml streptomycin, 20 ⁇ g/ml kanamycin, 5 ⁇ g/ml polymixine B, and 0.2 ⁇ g
- Porcine alveolar lung macrophages were maintained in MCA-RPMI-1640 medium containing 10% FBS, 100 ⁇ g/ml kanamycin, 50 U/ml penicillin, 50 ⁇ g/ml streptomycin, 25 ⁇ g/ml polymixine B, and 1 ⁇ g/ml fungizone.
- Serial passage of the recombinant PRRS viruses was performed by inoculation of 500 ⁇ l of the culture supernatant of transfected BHK-21 ceUs onto 1x107 PAMs. The inoculum was removed after 1 hour and 5 ml of fresh medium was added.
- the culture supernatant containing the produced virus was harvested when the first signs of cytopathogenic effect (cpe) were observed, generally around 48 hours after infection.
- the virus was further passaged by repeatedly inoculating 500 ⁇ l of the harvested culture medium of the previous passage onto 1x107 PAMs and again harvesting the culture supernatant after 48 hours.
- Virus titres expressed as 50% tissue culture infective doses [TCID50] per ml) were determined on PAMs by end point dilution (Wensvoort et al., 1986).
- PCR-mutagenesis was used to introduce sequences into the Pad-mutant of the genome-length cDNA clone of LV (pABV437) (Meulenberg et al, 1998a).
- the primers used for PCR-mutagenesis are Hsted in Table 1.
- PCR-fragments generated to introduce deletions into ORF7 were digested with Hpal and Pad, and Hgated into these sites of pABV437.
- PCR- fragments generated to introduce deletions into the 3'UTR were digested with Pad and Xbal, and Hgated into these sites of pABV437.
- Standard cloning procedures were performed essentiaUy as described (Sambrook, 1989). Transformation conditions were maintained as described (Meulenberg et al., 1998a). Sequence analysis was performed to confirm the introduced mutations.
- the constructs are schematicaUy drawn in Fig. 7
- IPMA Immunoperoxidase monolayer assay
- RNA of recombinant viruses Genetic analysis of genomic RNA of recombinant viruses.
- 200 ⁇ l of the culture supernatant or of the fraction was diluted with an equal volume of proteinase K buffer (100 mM Tris-HCl [pH 7.2], 25 mM EDTA, 300 mM NaCl, 2% [wt/vol] sodium dodecyl sulfate), and 0.08 mg proteinase K was added. After incubation for 30 minutes at 37°C, the RNA was extracted with phenol-chloroform and precipitated with ethanol.
- proteinase K buffer 100 mM Tris-HCl [pH 7.2], 25 mM EDTA, 300 mM NaCl, 2% [wt/vol] sodium dodecyl sulfate
- RNA was reverse transcribed with primer LV76, and PCR was performed using primers 119R218R and LV20 flanking the region of the viral genome containing the deletions.
- the ampHfied fragments were analysed in 2% agarose gels, the PCR fragments were excised from the gel and purified with SpinX columns (Costar). Sequence analysis of the fragments was performed using the antisense primer of the PCR.
- Radioimmunoprecipitation (RIP). MetaboHc labelling and immunoprecipitation of proteins expressed in PAMs was performed essentiaUy as described (Meulenberg & Petersen den Besten, 1996). MAb 122.17 was used to immunoprecipitate the N protein. PAMs were infected with passage 5 of the viruses at a multipHcity of infection of 1, and were labeUed for 4 hours with Tran[35-S]-label (Amersham) at 15 hours post infection. Samples were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 14% acrylamide gel.
- SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
- Virus concentration and purification To analyse the production of (noninfectious) virus particles, BHK-21 ceUs were electroporated with RNA transcripts from pABV747 and pABV437, and 15 hours after transfection the cells were metabolically labeUed with 75 ⁇ l (10.5 mCi/ml) Tran[35-S] -label (Amersham) for 24 hours (Meulenberg & Petersen den Besten, 1996). The particles in the supernatant were concentrated by centrifuging the supernatant through a 0.5 M sucrose cushion at 26,000 rpm for 5 hours at 4°C (Meulenberg & Petersen den Besten, 1996).
- PRRSV is an RNA virus with a very concise genome, most of its genetic information is expected to be essential. Therefore, genomic cDNA clones containing deletions -especiaUy in the conserved regions- generally do not produce infectious transcripts (Verheije, M.H., unpublished results). In order to identify regions of heterogeneity, where deletions might be tolerated, sequence comparisons were performed. The ORF7 gene at the 3' end of the LV genome was selected because this ORF does not overlap with other ORFs.
- the amino acid sequence is highly conserved up tiH residue 119 of LV. Downstream of this conserved region, a short stretch without amino acid conservation occurs.
- the N protein of LV is 4 amino acids longer than that of VR2332 (Fig. 6A). It was therefore anticipated that deletions in the heterogeneous C-terminus of the N protein of LV might be tolerated, and this region was selected as a target to introduce deletions.
- LV accepts C-terminal truncations of up to 6 amino acids of the N protein.
- cDNA clones with deletions in the sequence coding for the two (pABV639), four (pABV694), and nine (pABV695) C-terminal amino acids of the N protein were constructed by PCR-mutagenesis and cloning of the PCR-fragments into the infectious cDNA clone of LV containing a Pad-site at the stop codon of ORF7 (Meulenberg et al., 1998a) (Fig. 7).
- the RNA transcripts of these constructs were transfected into BHK-21 ceUs and tested for their ability to repHcate by analysing the expression of the structural proteins in IPMA (Fig. 7).
- LV mutants producing an N protein with a C-terminal deletion of up to 4 produce infectious virus
- mutants producing an N protein with a C-terminal deletion of 9 amino acids do not produce infectious virus at aU.
- stepwise deletions in the region coding for the 5 to 8 most C-terminal amino acids The fragments generated by PCR-mutagenesis were again introduced into pABV437, resulting in pABV745, 746, 747, and 748 coding for N proteins lacking 5, 6, 7, and 8 C-terminal amino acids, respectively (Fig. 7).
- the growth characteristics of the viruses vABV746 and vABV693 were investigated by determining their growth curves and comparing them with that of wUd type vABV437.
- PAMs were infected with viruses from passage 5 at a multiplicity of infection of 0.05, and samples were taken from the culture media at various time points.
- Virus titres were determined by end point dUution on macrophages. As is clear from Fig. 8, no significant differences in growth rates could be observed between recombinant viruses and wild type virus.
- N protein expressed by the recombinant virus vABV746 was analysed by immunoprecipitation.
- PAMs were infected, metaboIicaUy labeUed with 35S-amino acids, and ceU lysates were prepared.
- the N protein in the lysates was precipitated with MAb 122.17, which is directed against the D-domain of the protein and analysed by SDS-PAGE.
- an N protein of wUd type size (15 kDa) was immunoprecipitated from lysates of ceUs transfected with vABV437 (Fig.
- At least 7 nucleotides in this region were dispensable for virus production; removal of 32 nucleotides was, however, fatal.
- Both the virus with a 6- amino acid truncation of the N protein and the virus with the 7 nucleotide deletion in the 3'UTR had in vitro growth characteristics and antigenic profiles simUar to that of wUd type virus. Moreover, these viruses were both geneticaUy stable.
- the dramatic effect of truncation at the 7th residue of the LV N protein was quite surprising, and was not predicted by the sequence.
- the C-terminal 9 residues sequence of the LV N protein is very different from that of the VR2332 isolate except for its high content of hydroxyl amino acids.
- 6 out of 10 residues and 3 out of 6 residues at the very C-terminus, respectively, are serines or threonines. The function of this domain and of these particular residues is unknown.
- two other arteriviruses, LDV and SHFV contain hydroxyl amino acids at the extreme C-terminus of their N protein, namely 3 out of 10 and 4 out of 10 amino acids, respectively.
- hydroxyl amino acids are fuUy lacking in the last 10 amino acids of the EAV N protein.
- WMle coronavirus N proteins generaUy do have a relatively high serine content (7-11%) (Masters & Sturman, 1990), the proportion of serines and threonines at their carboxy terminus is quite insignificant; in these viruses this region is markedly acidic.
- these variable characteristics do not aUow predictions for the role of the C-terminus of the N protein in the viral Hfe cycle.
- the truncated N protein had the same antigenic profile as the wUd type N protein, since it reacted with aU MAbs directed against antigenic domains of the N protein. This is consistent with observations by Meulenberg et al. (1998b), who identified that domain D, the most C-terminal domain of N, is a conformation dependent or discontinuous epitope that involves amino acids 51-67 and 80-90.
- Viral particle production appeared to be blocked after truncation of the LV N protein by 7 amino acids. This strongly indicates a defect at the level of virus assembly.
- For a Canadian PRRSV isolate it has been demonstrated that non-covalent interactions between the C-terminal regions of N proteins are critical for formation of the isometric capsid protein (Wootton & Yoo, 1999). In a system expressing only the N protein, they showed that the last 11 amino acids were involved in these interactions. This might indicate that the C-terminus of PRRSV is essential for nucleocapsid formation. Our study supports this idea.
- N protein has been impHcated in various other processes, such as interaction with the viral RNA ((Dea et al., 2000), for MHV (Cologna & Hogue, 1998, Molenkamp & Spaan, 1997), and interaction with other viral proteins (for MHV (Narayanan et al., 2000).
- infectious virus was stUl not produced from the deletion mutants expressing truncated N proteins lacking 7 amino acids or more after lowering the incubation temperature to 30°C
- extension of the C-terminus of the N protein by a 9 amino acid sequence of the influenza virus HA protein significantly impaired viral growth.
- RNA viruses have at their termini non-coding sequences that play essential roles in RNA rephcation and sg mRNA transcription. Mutations in these domains are likely to affect the virus Hfe cycle. Consistently, when we introduced deletions in the 5' terminal region of the LV 3'UTR we found out that removal of a smaU 7-nucleotides variable sequence was accepted, whUe removal of a somewhat larger, 32-nucleotide stretch was not. From the inability of the RNA transcripts to express the M and N protein, we conclude that the defect likely resides in an effect on RNA rephcation or sg mRNA transcription. This suggests that this region of the 3'UTR probably contains an essential RNA signal.
- viruses obtained were characterised in vitro, and fulfilled the most important requirements, good growth and genetic stabiHty. Because their in vitro growth characteristics on PAMs were identical to'those of wUd type virus, virus production for in vivo studies can easUy be accompHshed. The growth characteristics in vitro do not necessarUy correlate with or predict the behaviour of the virus in vivo. Thus, many currently used vaccines are attenuated in vivo, but show no differences in in vitro propagation (Yang et al., 1998). Therefore, only animal experiments wUl teU how these viruses behave in vivo, whether they are sufficiently attenuated and whether they induce immune responses that wiU protect against infection with virulent PRRSV.
- RNA secondary structure in the 3 ' untranslated region of the murine coronavirus genome Journal of Virology 74, 6911-6921.
- Lelystad virus contain a conserved leader-body junction sequence.
- Lelystad virus the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virology 192, 62-72.
- Equine arteritis virus-infected ceUs contain six polyadenylated virus-specific RNAs.
- Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus are structural proteins of the virion. Journal of Virology 70, 4767-4772. Wensvoort, G., Terpstra, C, Boonstra, J., Bloemraad, M. & Van Zaane, D. (1986).
- PRRSV Porcine Reproductive and Respiratory Syndrome Virus
- PRRSV causes abortion and poor Htter quaHty in third trimester pregnant sows. Moreover, it may cause respiratory disease in young pigs. Infection of late term pregnant sows (80-95 days) with PRRSV can cause profound reproductive faUure, especiaUy due to a high level of mortaUty among the off-spring of these sows at birth and during the first week after birth. PRRSV is a ubiquitous pathogen. Two distinct antigenic types can be distinguished, i.e. the European and the American type. CHnical effects after a PRRSV infection depend on the type of strain involved. Vaccination of pigs with a PRRS vaccine influences the way a PRRSV-chaUenge works out on an animal and a farm level. The level and duration of viraemia, and shedding of the field- virus is reduced by this vaccination.
- vABV707 LDV-PRRS chimeric virus (ectodomain of M exchange)
- vABV741 aa9 deletion of the M-protein of PRRSV
- vABV746 18 nucleotide deletions at the C-terminal part of ORF7
- vABV688 mutations at position 88-95 of ORF2
- vABV437 wUd-type recombinant of Lelystad virus
- vaccinates were separated from these challenged animals for 24 hours and re-united thereafter. 28 days after challenge, aU pigs were removed and destroyed. vABV437 served as a positive control. A chaUenge control was included for 14 days starting at the moment of chaUenge in order to control chaUenge efficacy with LV-tH and SDSU#73, Animals were treated as described for the other animals during the chaUenge phase.
- Table 1 AUocation of pigs to designated groups. Each mutant group consisted of 5 vaccinated pigs and 1 sentinel (*so each PRRSV-mutant had two groups). Groups 11 and 12 served as challenge control groups (**) consisting of 5 animals per group;only two of these pigs were intranasally exposed to LV-tH or SDSU#73. All mutant groups were housed in isolation recombinant facilities, whereas the wiM fr p ⁇ groups were housed in standard isolation facilities.
- the vaccines were administered intramuscularly according to a SOP (2 ml deep intramuscularly in the neck halfway between the shoulder and the right ear; min titer 10 5 TCID ⁇ o/ml). All inoculae were titrated before and after usage and were stored on melting ice at all times.
- All mutant virus groups showed a reduced type 1 and type 2 viremia score as compared to vABV437.
- vABV707 vaccinated pigs showed a reduced type 1 and type 2 viraemia score prior to challenge as compared to the score of the pigs in all other groups.
- At the moment of challenge no animals were shown to be viraemic any more. All sentinels became viraemic and sero-converted, meaning that the viruses shedded from the exposed pigs to the sentinels.
- the studied recombinant mutant PRRS viruses show a reduced virulence as determined by a reduction of viraemia (length and height) as compared to wUd- type (vABv437).
- AU mutants instigate an effective immune response for the protection of pigs against a wUd-type field PRRSV.
- the homologous protection seems to be somewhat more effective than the heterologous one.
- the humoral response is measurable by a commercial ELISA (IDEXX) in aU cases. No adverse reactions are ehcited.
Abstract
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WO2006091824A2 (fr) * | 2005-02-25 | 2006-08-31 | Idexx Laboratories, Inc. | Peptides de detection d'anticorps du virus du syndrome respiratoire reproducteur porcin |
WO2006129139A1 (fr) * | 2005-02-25 | 2006-12-07 | Pfizer Products Inc. | Mutants de la proteine n du virus du syndrome dysgenesique et respiratoire du porc (prrs) |
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