CN1406277A - Recombinant attenuation of PRRSV - Google Patents
Recombinant attenuation of PRRSV Download PDFInfo
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- CN1406277A CN1406277A CN01804245A CN01804245A CN1406277A CN 1406277 A CN1406277 A CN 1406277A CN 01804245 A CN01804245 A CN 01804245A CN 01804245 A CN01804245 A CN 01804245A CN 1406277 A CN1406277 A CN 1406277A
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
- C12N7/04—Inactivation or attenuation; Producing viral sub-units
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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Abstract
The present invention relates to live PRRS viruses which are attenuated by amino acid mutations in a specific site of the viral protein coded by the open reading frame (ORF) selected from the group of ORF 1a, ORF 1b and/or ORF 2. The invention also pertains to nucleotide sequences coding said viruses, methods of generating such viruses and their use for the preparation of a pharmaceutical composition for the prophylaxis and treatment of PRRS infections.
Description
Invention field
The present invention relates to by amino acid mutation taking place and the PRRS virus alive of attenuation in viral protein specificity site, wherein said viral protein is by the open reading frame coding that is selected from ORF1a, ORF1b and/or ORF2.The invention still further relates to the nucleotide sequence of the described virus of coding, the method that produces described virus is used for preventing and treating the purposes of the pharmaceutical composition of PRRS infection with them in preparation.
Background of invention
A kind of mysterious pig disease was renamed afterwards and was Porcine Reproductive and Respiratory Syndrome (PRRS), was (E.J.Snijdet, 1998) that the positive chain RNA enveloped virus by the Arteriviridae Viraceae causes.Before about 10-15, at US and European two kinds of different PRRS virus strain appear respectively.This disease now in the North America, the localized epidemics of many product pigs country in Europe and Asia.It is the major cause that causes pig fecundity forfeiture and respiratory disease always, according to estimates should disease at the prevalence rate of the U.S. up to 70%.
This virus by suck, ingest, mating, the approach of biting wound or acupuncture propagate, and duplicate in the scavenger cell of mucous membrane, lung or regional area.
This disease causes and clinical inapparent infection (resolution) or persistent infection.By the animal of persistent infection virus is released in fluid, blood, excrement, urine and the seminal fluid of mouth/pharynx.
The clinical symptom of sow relates to miscarriage or the low little piglet of prematurity of viability, the pig and the self-dissolving fetus of stillbirth.There is higher mortality ratio in infected newborn piglet or catches pneumonia, and pneumonia and concurrent infectation of bacteria make that nursing and the raising to pig is very difficult subsequently, and mortality ratio increases.Boar is easy to have a fever and the variation of sperm morphological takes place.
The same with all arterivirus, the PRRS viral genome is the strand positive chain RNA molecule that is about 15kb.ORF (open reading frame) 1a and 1b coding replicative enzyme, ORF2 to 5 coding supposition glycoprotein (gp1-4), ORF6 coding membranin (M) and ORF7 coding nucleocapsid protein (N).
To the U.S. (isolate ATCC VR-2332, be preserved in American type culture collection on July 18th, 1991, it is at rockville, Maryland, the U.S., Genebank U87392 U00153) and Europe (WO9221375, isolate Lelystad Agent (CDI-NL-2.91), be preserved in Institute Pasteur on June 5th, 1991, Paris, preserving number I-1102) the initial description infected of PRRS differentiated the virus that has genome and serology difference.Comparison shows that they all have common ancestors, these ancestors break up before the later stage eighties 20th century, clinical symptom was described.The full-length gene group sequence of multiple PRRS virus and complete structure (Snijder etc., 1998 of protein-coding region thereof have now been reported; Meulenberg etc., 1993b; Conzelmann etc., 1993; Murtaugh etc., 1995; Kapur etc., 1996).PRRS virus can be so-called CL-2621 and MARC-145 (K.D.Rossow at external pig pulmonary macrophage, monocyte, neurogliocyte and two kinds of MA-104 cell subsets (monkey embryonic kidney cells), reproduction of pig and respiration syndrome, Vet Pathol 35:1-20,1998) duplicate in.The existing at present recombination method (EP0839912A1) that produces the PRRS clone that infectivity is arranged.
Now existing commercially available PRRS attenuated live vaccine (RespPRRS/Ingelvac
PRS MLV, Boehringer Ingelheim) is used to protect pig.
Producing on the immunoreactive effect of all-round protection, even exist under the situation of adjuvant, killed vaccine (totivirus of deactivation) or subunit vaccine (viral protein of conventional purifying or heterogenous expression and purifying) are usually not as living vaccine.Confirm that for PRRS with respect to present available inactivated vaccine, attenuated vaccine causes the immunizing power more lasting and effective (Snijder etc., reference is the same) to disease.Present PRRS vaccine alive is to carry out attenuation according to the method for routine, promptly continuously virus is gone down to posterity up to pathogenic forfeiture (EP0529584B1) in the host cell that is fit to.Present PRRS vaccine alive still needs to improve.The first, they can not prevent to infect again, and the second, they can not make the animal and the animal that infects wild virus difference mutually on serology of immunization.Though but the most important is from the living vaccine perform toxic attenuation of complete microorganism, but may be accompanied by serious safety problem, particularly all the more so for RNA viruses such as PRRS virus, this is because think that RNA viruses is owing to lack the check and correction of rna replicon enzyme and cause the out of true of rna gene group duplicated and caused high mutation rate.
Attenuated live virus potential is replied the serious threat that may cause immune animal.By repeatedly molecule source and still the unknown of genetic stability of the attenuated virus that obtains of propagating method of routine, and the formation of revertant is unpredictable.
Therefore, the technical problem to be solved in the present invention provides a kind of little PRRS virus that may revert back to wild-type virus.
Summary of the invention
The present invention relates to PRRS virus alive, this virus is by attenuation obtains to carrying out amino acid mutation by the specificity site on the viral protein of open reading frame (ORF) coding that is selected from ORF1a, ORF1b and/or ORF2.The invention still further relates to the nucleotide sequence of the described virus of coding, produce method and their application in the pharmaceutical composition that preparation prevents and treatment PRRS infects of this virus.
Description of drawings
The aminoacid sequence of the ORF1a of Figure 1A TTC VR-2332 has wherein marked the preferred attenuation of the present invention site.
The aminoacid sequence of the ORF1b of Fig. 2 ATTC VR-2332 has wherein marked the preferred attenuation of the present invention site.
The aminoacid sequence of the ORF2 of Fig. 3 ATTC VR-2332 has wherein marked the preferred attenuation of the present invention site.
The nucleotide sequence of the ORF1a of Fig. 4 ATTC VR-2332, wherein runic is represented the preferred attenuation of the present invention site, underscore is site most preferably.
The nucleotide sequence of the ORF1b of Fig. 5 ATTC VR-2332, wherein runic is represented the preferred attenuation of the present invention site, underscore is site most preferably.
The nucleotide sequence of the ORF2 of Fig. 6 ATTC VR-2332, wherein runic is represented the preferred attenuation of the present invention site, underscore is site most preferably.
Detailed Description Of The Invention
This specification and the technical scheme that have been defined by the claims feature provide the above-mentioned technology that solves The method of problem.
Before describing technical scheme of the present invention, must be clear that the word " a ", " an " and " the " that use in this paper and the attached claim have comprised the plural meaning, unless know in the text and indicated its implication.Therefore, for example, " a PRRS virus " comprises multiple described PRRS virus, and " cell " refers to one or more cells and equivalent known to those skilled in the art, or the like.Unless otherwise defined, all technology used herein and scientific terminology have the common identical meanings of understanding of those skilled in the art in the invention.Though in practice or detect can use when of the present invention to literary composition in describe similar or the method and the material that are equal to, preferable methods, equipment and material have still been described in the present invention.All publications of mentioning in the literary composition all are incorporated herein by reference, be used for describing and disclose report on these publications can be used for clone of the present invention, carrier and method.Content disclosed by the invention can be owing to openly cause losing the power that ranks forefront at preceding invention this.
The contriver is surprised to find PRRS virus and contains specific gene group site on some open reading frame, and they can stiffly revert back to the amino acid of the initial VR-2332 of street strain on these sites.This locational evolution pressure that is called " virulence specificity site " now or abbreviates " site of the present invention " as or only be called " site " is huge.Might utilize two kinds of RespPRRS to reply strains and confirm that first the amino acid mutation on this virulence specificity site of initial street strain has the rational difference in ground at US and European.Has the defective that to avoid preparing at present attenuated vaccine as the living vaccine of the specific sudden change on attenuation of the present invention basis, another advantage of described attenuation sudden change is on their the known molecular uniqueness that this uniqueness makes them can be used as the remarkable mark of attenuated virus and makes them to come with the wild-type virus difference.
With the amino acid of conventional attenuated virus RespPRRS and comparing of nucleotide sequence and the initial VR-2332 of street strain, in order to identify virulence specificity site, between two kinds of virulence revertants of mentioning and and VR-2332 and RespPRRS between compare respectively.
The feasible like this virulence specificity site that can differentiate on every kind of viral protein relevant with the virulence of PRRS virus.
The result, one aspect of the present invention relates to PRRS virus alive, this virus is not RespPRRS, and a little less than PRRS virus of A TCC VR-2332 toxicity, it is characterized in that it contains a kind of by open reading frame (ORF) encoded protein, this open reading frame is selected from the ORF1a of the described VR-2332 strain of following group: Fig. 1 description, the ORF1b of the described VR-2332 strain of describing among Fig. 2, and/or the ORF2 of the described VR-2332 strain of Fig. 3 description, wherein at least one amino acid in the virulence specificity site of determining is not equal to VR-2332 strain at least one amino acid on described corresponding position.
The numbering of amino acid and Nucleotide is according to the data record of VR-2332.Fig. 1,2 and 3 provides the information of representing all PRRS strains, thereby makes the invention imagery, can also differentiate preferred amino acids and preferred site in all PRRS viruses of carrying out different numberings.Differentiate that these sites can carry out by the following method: differentiate target P RRS virus strain and list with reference to the identical amino acid of feature conservative in the virus strain, on then definite target viral respectively corresponding to the position in site among Fig. 1,2 and/or 3.
ORF1a, the ORF1b of VR-2332 shown in Fig. 1,2 and/or 3 and/or three above-mentioned sites in the ORF2 encoded protein have been identified.
Another aspect of the present invention relates to attenuation PRRS virus alive, this virus comprises basically as VR-2332 but not the ORF1a among the RespPRRS, ORF1b and ORF2, at least one amino acid that it is characterized in that the 321-341 position of ORF1a is not equal to the amino acid of VR-2332 on described corresponding position of describing among Fig. 1, and/or at least one amino acid of the 936-956 position of ORF1b is not equal to the amino acid of VR-2332 on described corresponding position of describing among Fig. 2, and/or at least one amino acid of the 1-20 position of ORF2 is not equal to the amino acid of VR-2332 on described corresponding position of describing among Fig. 3.Therefore, on the position of the 1-20 position (respectively referring to Fig. 4,5 or 6) of the 936-956 position of the 321-341 position of ORF1a, ORF1b and/or ORF2, one or more amino acid whose nucleotide triplets of encoding morph, it causes the sequence generation nucleotide level in described site or also has in addition and one or more variation of preferred amino acid level in addition, to such an extent as to all Nucleotide in described site or amino acid position can be undergone mutation.The viruses of sudden change have taken place in one or two or all three that the present invention includes described site." sudden change " refers to that an amino acid replaces to another amino acid or replaces coding nucleotide (being replaced by T as C) by another Nucleotide, promptly so-called substituting, and preferably it is changed amino acids coding, or any other sudden change as " disappearance " or " insertion ".Sudden change is always carried out in coding nucleotide sequence.Described sudden change can be undertaken by using this area standard method, as (Adv.Exp.Med.Biol such as Meulenberg, 1998,440:199-206) describe to a site-directed mutagenesis that infects copy (referring to as Sa nurse Brooker etc., 1989, molecular cloning: test guide, second edition, press of cold spring harbor laboratory, cold spring harbor laboratory, New York)." basically " refer at least 75% sequence, preferred 85%, more preferably all sequences except " site " of the present invention is equal to VR-2332, but also can the Nucleotide of other coded amino acid outside the site of the present invention be suddenlyd change.This class is according to virus of the present invention standard still according to the invention, promptly unlikely reverts back to wild-type and than a little less than the VR-2332 virulence.
Refer to PRRS virus as (with reference to the same) definition such as E.J.Snijder, the virus that can infect pig and can in pig, duplicate according to the PRRS virus of work of the present invention.The RespPRRS virus of conventional attenuation will be abandoned especially, and it does not belong to the present invention and not in protection scope of the present invention.RespPRRS virus can obtain by commercial sources, as buying (Ingelvac from Boehringer Ingelheim Vetmedica company
@PRS MLV), its most of sequence also is the public available (Genebank AJ223082).
The site of particularly important and amino acid are in no way limited to specifically described position in the VR-2332 strain for the present invention, but are used for pointing out preferred amino acid on this site or other corresponding site of PRRS strain in a kind of mode exemplary.For different PRRS virus, the Position Number mode of preferred amino acid can be different, but are easy to differentiate them by the dependency of these preferred amino acid and described proteic other conserved amino acid as the expert of the biology field of Arteriviridae Viraceae virus.
It is the clinical symptom of virus interested and VR-2332 that term " a little less than PRRS virus VR-2332 virulence " can be understood as.Determine whether a kind of PRRS virus is set forth by embodiment 1 than the preferred process a little less than the PRRS virus VR-2332 virulence.Be not to suddenly change in all possible preferred amino acid in virulence specificity site all to cause virulence to reduce.The method of embodiment 1 provides accurately and whether has directly determined to contain proteic PRRS live virus of the present invention than the experimental strategy a little less than the VR-2332 of the street strain virulence.
Virulence specificity site can be differentiated to the amino acid whose answer of VR-2332 by at least one amino acid of attenuated virus.This specific amino acid is the bigger secondary peptide structure such as the part of α spiral, βZhe Die or hair clip β motif or other structure.Therefore very likely be that adjacent amino acid also participates in regulating this proteic toxicity, the expert in protein chemistry field therefore expectation identifies other amino acid relevant with virulence at 10 of the initial amino acid sites left and right sides of identifying probably in amino acid.10 to 20 amino acid are typical ranges of peptide motif in the albumen.Therefore, the preferred virus of the present invention contains just like the illustrative albumen of Fig. 1 or its corresponding protein in other virus strain, and the virulence specificity site in these albumen is included in 10 amino acid of amino acid sites upstream of initial evaluation and 10 amino acid in downstream.Preferred aforesaid virus, virulence specificity site wherein are included in 5 amino acid of amino acid sites upstream of initial evaluation and 5 amino acid in downstream.Most preferred aforesaid virus, virulence specificity site wherein are included in 3 amino acid of amino acid sites upstream of initial evaluation and 3 amino acid in downstream.
According to instruction of the present invention,, the amino acid on the virulence specificity site might produce the PRRS strain of attenuation from street strain by being suddenlyd change.Yet the safety problem due to the high frequency of RNA viruses suddenlys change still exists.This problem can be alleviated greatly by the specific amino acid on the virulence specificity site of disappearance viral protein.Therefore the present invention relates to above-mentioned PRRS virus, it is characterized in that at least one amino acid is lacked on the virulence specificity site of this viral protein.Term " disappearance " is appreciated that into the corresponding aminoacid deletion on one or more site shown in the figure.Therefore, according to the present invention, " disappearance " refers to remove one or several Nucleotide or amino acid.
In a preferred scheme, the present invention relates to the attenuation PRRS virus of living, at least one amino acid that it is characterized in that the 321-341 position of ORF1a is lacked and/or at least one amino acid of the 936-956 position of ORF1b is lacked and/or at least one amino acid of the 1-20 position of ORF2 is lacked.Therefore, no matter in the 321-341 position of ORF1a, the 936-956 position of ORF1b or the 1-20 position of ORF2, or all three site disappearances of one or more amino acid whose nucleotide triplet of encoding have caused the one or more disappearances in described site.The virus that only lacks a Nucleotide or one of an amino acid whose virus and described site or all lack all comprises in the present invention (respectively referring to Fig. 4,5 or 6).
In order to identify the virulence specificity site on the specific PRRS viral protein, confirmed that in a preferred embodiment at least one amino acid is to be under the high pressure of sudden change and relevant with the virulence characteristic of the revertant of the VR-2332 of street strain.This indivedual amino acid sites is the most preferred scheme of the present invention.
Therefore, in most preferred scheme, the present invention relates to a kind of attenuated virus according to work of the present invention, this virus be not RespPRRS and virulence than ACTT VR-2332 a little less than, it is characterized in that amino acid and/or ORF2 the 10th amino acids of the 946th of the amino acid of the 331st of ORF1a and/or ORF1b is not equal to the amino acid of ACTT VR-2332 strain on corresponding site.Therefore, undergo mutation in all three sites of the 946th of the 321st of ORF1a, ORF1b or the 10th of the ORF2 or the amino acid whose nucleotide triplet of encoding or at this amino acid and cause one to three kind of sudden change (respectively referring to Fig. 4,5 or 6) in described site.
Because above-mentioned reason, only be easy to the type that amino acid that regressive amino acid just can avoid changing safer and better reverts back to virulence by disappearance.
Therefore, the present invention most preferably relates to attenuation PRRS live virus of the present invention in the scheme at this, the amino acid that it is characterized in that the 10th of the amino acid of the 946th of the amino acid of the 331st of ORF1a and/or ORF1b and/or ORF2 is lacked, that is, do not exist when comparing with the site of VR-2332.
Another most preferred scheme is the attenuation PRRS virus according to work of the present invention, and the amino acid that it is characterized in that the 321st of ORF1a is its coding triplet disappearance.Another most preferred scheme is the attenuation PRRS virus according to work of the present invention, and the amino acid that it is characterized in that the 946th of ORF1b is its coding triplet disappearance.Another most preferred scheme is the attenuation PRRS virus according to work of the present invention, and the amino acid that it is characterized in that the 10th of ORF2 is its coding triplet disappearance (respectively referring to Fig. 4,5 or 6).Described most preferred virus is equal to VR-2332 on all other sites.
Instruction of the present invention makes the expert to recombinate to produce the infections clone of PRRS virus, this clone than VR-2332 virulence a little less than and can be used for preparing a kind of pharmaceutical composition of work.All required all informations of reorganization infections clone that produce positive chain RNA virus can obtain from document easily, and are especially all the more so for PRRSV.For example, the European patent application EP 0839912 of Meulenberg etc., as a reference, it provided the clearly instruction of preparation reorganization PRRS virus alive during this invention was incorporated herein in full.Therefore, on the other hand, the present invention relates to the nucleotide sequence of coding basis virus of the present invention.Because the degeneracy of genetic code, the polynucleotide varient will cause being equal to amino acid whose translation.The present invention comprises the varient of those degeneracys equally.
As mentioning in the lobby page, very important to the health control of pig, can distinguish the weak virulence living vaccine strain in the pharmaceutical composition and the infection of street strain's virus like this.This usually is difficult, especially the clinical symptom that wild-type infects be not the sort of specific or with other infect form superinfection or the time of observation and assessment too short in.The reorganization of target viral produces and makes that importing is modified in the genetic code that can set up serology mark or virulence mark.The serology mark refers to that antigenicity can detected molecule such as peptide, albumen, glycoprotein etc., these can from infected cells or body fluid as but be not limited to pharynx, nose liquid or the urine and isolate.The virulence mark can be understood as can by the recombination analysis method as but be not limited to mark in the genetic code that PCR and conventional order-checking differentiate.Therefore, in a preferred version, the present invention relates to according to nucleotide sequence of the present invention, wherein this nucleotide sequence is modified to a kind of virulence mark of coding and/or serology mark.Especially, sudden change that imports in order to make virulence attenuation of or disappearance can be used for as virulence and serology mark.In on disclosed virulence specificity site, monitor these sudden changes, the appearance of the possible virulence revertant that might predict in early days.
The nucleotide sequence of the described mark of coding is arranged in the arbitrary open reading frame of coding structure venereal disease toxalbumin in the nucleotide sequence more preferably of the present invention.
Another aspect of the present invention relates to the method for generation according to infectivity attenuation PRRS live virus of the present invention, and described method comprises a kind of RNA copy of at least one full length DNA copy or in-vitro transcription or recombinant nucleic acid of any derivative of comprising as described above of generation.
Another preferred scheme of the present invention relates to according to method of the present invention, wherein introduce specific mutant with molecular biology method, it is characterized in that corresponding to ORF1a amino acid sites 321-341 position, undergo mutation corresponding to the amino acid sites 936-956 position of ORF1b and/or corresponding to the nucleic acid of the amino acid sites 1-20 position of ORF2, this sudden change is that at least one Nucleotide in described site takes place to replace or disappearance.
Another important aspect of the present invention is the pharmaceutical composition that contains the PRRS virus of with good grounds invention and pharmaceutically can accept carrier.
" pharmaceutical composition " formed one or more changes and accepted the biological of its administration or grow in wherein or the physiological function of the biology on its surface such as immunologic function but be not limited to microbiotic or the composition of antiparasitic by following basically, with other component that adds in order to obtain some other target, they be not limited to processing characteristics, sterility, stability, through in enteron aisle or parenteral route such as oral, the nose, the feasibility of intravenously, intramuscular, subcutaneous, intracutaneous or other suitable administration, and tolerance and sustained release characteristic after the administration.
Pharmaceutically acceptable carrier can contain acceptable compound on the physiology, and this compound is used for as stable or increase and absorb or become a part according to the slow delivery formulations of PRRS virus of the present invention.Acceptable compound comprises on this physiology, carbohydrate for example, as glucose, sucrose or dextran, antioxidant such as xitix or gsh, sequestrant, low molecular weight protein (LMWP) or other stablizer or vehicle are (referring to as Remington ' s Pharmaceutical Sciences, 1990,18
ThEd.Mack Publ., Easton).Skilled in the art will recognize that how to select a kind of pharmaceutically acceptable carrier, comprise as according to as described in the route of administration of composition select the acceptable compound of physiology.
Another aspect of the present invention relates to the application of virus of the present invention.By the attenuation PRRS virus of work of the present invention is provided, might be used to prevent and treat the vaccine that PRRS infects with they preparations now.Their specific molecular basises aspect attenuation make them be better than conventional at present attenuated virus.Especially preferably use according to the virus that disappearance is arranged in virulence specificity site of the present invention, because disappearance is difficult for replying.
The following example is used for further explaining the present invention, but should not be construed as restriction protection scope of the present invention disclosed herein.
Reference 1.Snijder E.J., Meulenberg J.J.M., 1998.The molecular biology of arteriviruses, Journalof General Virology May 79 (5): 961-971.2.Meulenberg J.J.M., Hulst, M.M.et al., 1993b.Lelystad virus, the causative agent ofporcine epidemic abortion and respiratory syndrome, Virology 192,62-72.3.Conzelmann K.K, Visser N., Van Woensel P., Thiel H.J., 1993.Molecularcharacterization of porcine reproductive and respiratory syndrome virus, a member of thearterivirus group, Virology 103,329-339.4.Murtaugh M.P., Elam M.R., Kakach L.T., 1995.Comparison of the structural proteincoding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus, Archives ofVirology 140,1451-1460.5.Kapur V., Elam M.R., Pawtovich T.M.Murtaugh M.P., 1996.Genetic variation inporcine reproductive and respiratory sydrome virus isolates in the midwestem United States, Journal of General Virology 77,1271-1276.6.Rossow K.D., 1998.Porcine reproductive and respiratory syndrome, Vet Pathol 35:1-20.
The establishment of embodiment 1 attenuation
This embodiment clearly indicates the virulence characteristic that how to compare two kinds of different PRRS virus strain.
At least 10 every group gilt that do not infect PRRS virus in each experiment.
The animal via check does not contain PRRS virus-specific serum antibody and PRRSV is negative.
All animals in the experiment have identical source and kind.The animal grouping is at random.
In the time of conceived 90 days, in each nostril, use 1ml 10
5TDCID
50The PRRSV of (third generation), at least three groups during every cover detects.
One VR-2332 attack group, the group that the possible attenuated virus of usefulness is attacked, a strict control group.
If strict control group keeps the PRRS feminine gender in the experiment whole process, and the gilt of the life and health of being born in the VR-2332 attack group lacks 25% at least than strict control group, and it is effective then testing.
Attenuation, virulence is low in other words, is defined as the parameter noticeable change statistically of one or more definite reproductive performances.
The optimization experiment group has at least one following parameters significantly to reduce:
-stillborn foetus frequency
-in conceived 112 days or before abortion ratio
The quantity of-mummification (mumified) gilt
The quantity of-survival and weak gilt
Mortality ratio before the-wean
Or go back the optimization experiment group and have at least one following parameters significantly to increase:
The quantity of the wean gilt of-every sow
-every sow bears the quantity of healthy gilt
Compare with the VR2332 infected group
Claims (11)
1. the attenuation PRRS virus of a work, this virus comprises basically as the ORF1a among the VR-2332, ORF1b and ORF2 but is not RespPRRS, at least one amino acid that it is characterized in that the 321-341 position of ORF1a is not equal to the amino acid of VR-2332 on described corresponding position, and/or at least one amino acid of the 936-956 position of ORF1b is not equal to the amino acid of VR-2332 on described corresponding position, and/or at least one amino acid of the 1-20 position of ORF2 is not equal to the amino acid of VR-2332 on described corresponding position.
2. the attenuation PRRS virus of a work as claimed in claim 1, at least one amino acid that it is characterized in that the 321-341 position of ORF1a are lacked and/or at least one amino acid of the 936-956 position of ORF1b is lacked and/or at least one amino acid of 1-20 position is lacked.
3. the attenuation PRRS virus of a work as claimed in claim 1 or 2 is characterized in that the amino acid of the 10th of the amino acid of the 946th of the amino acid of the 331st of ORF1a and/or ORF1b and/or ORF2 is not equal to the amino acid of ACTT VR-2332 virus strain on corresponding site.
4. attenuation PRRS virus as each described work of claim 1 to 3 is characterized in that the amino acid of the 10th of the amino acid of the 946th of the amino acid of the 331st of ORF1a and/or ORF1b and/or ORF2 is lacked.
5. a coding is as the nucleotide sequence of claim 1 to 4 virus as described in each.
6. nucleotide sequence as claimed in claim 5, wherein this nucleotide sequence is modified to a kind of virulence mark of coding and/or serology mark.
7. nucleotide sequence as claimed in claim 6, the nucleotide sequence of the described mark of wherein encoding is arranged in the open reading frame of any coding structure venereal disease toxalbumin.
8. method that produces infectious attenuation PRRS live virus, described method comprises a kind of RNA copy of at least one full length DNA copy or in-vitro transcription or recombinant nucleic acid sequence of any derivative of comprising of generation, it is characterized in that described nucleotide sequence is as each described nucleotide sequence of claim 5-7.
9. method as claimed in claim 8, wherein insert specific mutant by molecular biological method, it is characterized in that undergoing mutation, make that at least one Nucleotide on the described site is substituted or lacks corresponding to the nucleic acid of the amino acid sites of the amino acid sites of the 936-956 position of the amino acid sites of the 321-341 position of ORF1a and/or ORF1b and/or 1-20 position.
10. pharmaceutical composition that contains just like each described PRRS virus of claim 1-4 and pharmaceutically acceptable carrier.
11. be used for preventing and treating the application of the vaccine of PRRS infection in preparation as each described PRRS virus of claim 1-4.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2000103372 DE10003372A1 (en) | 2000-01-26 | 2000-01-26 | Novel live porcine reproductive and respiratory syndrome virus attenuated by mutations in specific site of viral protein coded by specified open reading frames, useful for prophylaxis/treatment of viral infections |
| DE10003371.7 | 2000-01-26 | ||
| DE10003373.3 | 2000-01-26 | ||
| DE10003372.5 | 2000-01-26 | ||
| DE2000103371 DE10003371A1 (en) | 2000-01-26 | 2000-01-26 | Novel live porcine reproductive and respiratory syndrome virus attenuated by mutations in specific site of viral protein coded by specified open reading frames, useful for prophylaxis/treatment of viral infections |
| DE2000103373 DE10003373A1 (en) | 2000-01-26 | 2000-01-26 | Novel live porcine reproductive and respiratory syndrome virus attenuated by mutations in specific site of viral protein coded by specified open reading frames, useful for prophylaxis/treatment of viral infections |
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|---|---|
| CN1406277A true CN1406277A (en) | 2003-03-26 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN01804245A Pending CN1406277A (en) | 2000-01-26 | 2001-01-26 | Recombinant attenuation of PRRSV |
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| EP (1) | EP1254213A2 (en) |
| JP (1) | JP2003520601A (en) |
| KR (1) | KR20030032910A (en) |
| CN (1) | CN1406277A (en) |
| AR (1) | AR027291A1 (en) |
| AU (1) | AU2001228502A1 (en) |
| BR (1) | BR0107827A (en) |
| CA (1) | CA2396454A1 (en) |
| CZ (1) | CZ20022554A3 (en) |
| EA (1) | EA200200743A1 (en) |
| HU (1) | HUP0204176A3 (en) |
| MX (1) | MXPA02007198A (en) |
| PL (1) | PL356274A1 (en) |
| SK (1) | SK10932002A3 (en) |
| WO (1) | WO2001055353A2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008110056A1 (en) * | 2007-03-14 | 2008-09-18 | China Animal Disease Control Center | A vaccine for porcine reproductive and respiratory syndrome, a preparion method and use thereof |
| CN101307305B (en) * | 2008-04-30 | 2010-06-16 | 中国动物疫病预防控制中心 | Low virulent strain of porcine reproductive and respiratory syndrome virus, immunogenicity immunogenicity material and vaccine |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SK11282003A3 (en) * | 2001-03-09 | 2004-02-03 | Boehringer Ingelheim Vetmedica Gmbh | Life attenuated European PRRS virus, nucleotide sequence coding said virus, method of generating such virus, cell line, and a pharmaceutical composition comprising said PRRS viruses and the use thereof |
| US6841364B2 (en) | 2002-01-22 | 2005-01-11 | Protatek International, Inc. | Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and expression vectors thereof |
| ATE539162T1 (en) | 2003-03-27 | 2012-01-15 | Ottawa Hospital Res Inst | MUTATED VIRUSES OF VESICULAR STOMATITIS AND THEIR USE |
| RU2381035C2 (en) * | 2005-02-25 | 2010-02-10 | Пфайзер Продактс Инк. | Composition for pigs protection against prrs-virus infection (versions) and vaccine containing specified composition (versions) |
| US7666585B2 (en) | 2007-06-15 | 2010-02-23 | Protatek International, Inc. | Construction of chimera PRRSV, compositions and vaccine preparations |
| UA108902C2 (en) * | 2010-11-10 | 2015-06-25 | NORTH AMERICAN REPRODUCTIVE AND RESPIRATORY PIG SYNDROME (PRRS) VIRUS | |
| US10072046B2 (en) * | 2014-03-21 | 2018-09-11 | Nutech Ventures | Non-naturally occurring porcine reproductive and respiratory syndrome virus (PRRSV) and methods of using |
Family Cites Families (3)
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|---|---|---|---|---|
| US6251397B1 (en) * | 1992-10-30 | 2001-06-26 | Iowa State University Research Foundation, Inc. | Proteins encoded by polynucleic acids isolated from a porcine reproductive and respiratory syndrome virus and immunogenic compositions containing the same |
| WO1996004010A1 (en) * | 1994-08-05 | 1996-02-15 | Regents Of The University Of Minnesota | Vr-2332 viral nucleotide sequence and methods of use |
| EP0839912A1 (en) * | 1996-10-30 | 1998-05-06 | Instituut Voor Dierhouderij En Diergezondheid (Id-Dlo) | Infectious clones of RNA viruses and vaccines and diagnostic assays derived thereof |
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2001
- 2001-01-26 HU HU0204176A patent/HUP0204176A3/en unknown
- 2001-01-26 SK SK1093-2002A patent/SK10932002A3/en unknown
- 2001-01-26 WO PCT/EP2001/000865 patent/WO2001055353A2/en not_active Application Discontinuation
- 2001-01-26 MX MXPA02007198A patent/MXPA02007198A/en unknown
- 2001-01-26 AR ARP010100333A patent/AR027291A1/en not_active Suspension/Interruption
- 2001-01-26 CA CA002396454A patent/CA2396454A1/en not_active Abandoned
- 2001-01-26 JP JP2001554384A patent/JP2003520601A/en active Pending
- 2001-01-26 EP EP01946891A patent/EP1254213A2/en not_active Withdrawn
- 2001-01-26 CZ CZ20022554A patent/CZ20022554A3/en unknown
- 2001-01-26 PL PL01356274A patent/PL356274A1/en not_active Application Discontinuation
- 2001-01-26 AU AU2001228502A patent/AU2001228502A1/en not_active Abandoned
- 2001-01-26 EA EA200200743A patent/EA200200743A1/en unknown
- 2001-01-26 CN CN01804245A patent/CN1406277A/en active Pending
- 2001-01-26 BR BR0107827-5A patent/BR0107827A/en active Pending
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008110056A1 (en) * | 2007-03-14 | 2008-09-18 | China Animal Disease Control Center | A vaccine for porcine reproductive and respiratory syndrome, a preparion method and use thereof |
| CN100439494C (en) * | 2007-03-14 | 2008-12-03 | 中国动物疫病预防控制中心 | Swine reproduction and respiration syndrome vaccine and its prepn process and application |
| CN101307305B (en) * | 2008-04-30 | 2010-06-16 | 中国动物疫病预防控制中心 | Low virulent strain of porcine reproductive and respiratory syndrome virus, immunogenicity immunogenicity material and vaccine |
Also Published As
| Publication number | Publication date |
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| MXPA02007198A (en) | 2002-12-09 |
| HUP0204176A3 (en) | 2003-12-29 |
| PL356274A1 (en) | 2004-06-28 |
| KR20030032910A (en) | 2003-04-26 |
| EP1254213A2 (en) | 2002-11-06 |
| CA2396454A1 (en) | 2001-08-02 |
| SK10932002A3 (en) | 2002-11-06 |
| WO2001055353A3 (en) | 2001-12-27 |
| AR027291A1 (en) | 2003-03-19 |
| CZ20022554A3 (en) | 2002-10-16 |
| BR0107827A (en) | 2002-11-05 |
| HUP0204176A2 (en) | 2003-04-28 |
| JP2003520601A (en) | 2003-07-08 |
| EA200200743A1 (en) | 2002-12-26 |
| WO2001055353A2 (en) | 2001-08-02 |
| AU2001228502A1 (en) | 2001-08-07 |
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