CN101090981A - Porcine reproductive and respiratory syndrome isolates and methods of use - Google Patents

Abstract

A method of predicting the virulence of a new or uncharacterized PRRS virus isolate is provided wherein the isolate is injected into swine and allowed to replicate for a period of from about 3-15 days. During this period, the rate of virus growth and/or the magnitude of viremia is determined, and this data is compared with a corresponding growth rate and/or viremia magnitude of a PRRS virus isolate of known virulence, as a measure of the virulence of the new or uncharacterized isolate. Additionally, a method of selecting an isolate for inclusion in an immunogenic composition based on the predicted virulence is also provided, together with compositions incorporating attenuated forms of viruses predicted to be virulent.

Description

Pig reproduction and respiration syndrome strain isolated and using method thereof

Related application

Present application for patent is the part of the application case that proposed on December 24th, 2004 (its application case number be 11/022, the 262) application case that continues, the interests of its provisional application case of advocating to be proposed on September 21st, 2004 (its application case number be 60/611,824).The teaching and the content of these previous application cases are incorporated into herein with way of reference.

Technical field

The invention relates to new wild-type PRRS virus isolated strain and corresponding modified form attenuation PRRS virus thereof, the purposes of this attenuated virus in vaccine and immune composition, and detect this virus isolated strain viremia level, the speed of growth, antibody response, or the method for its combination.More specifically, the invention provides a kind of pathogenic method new or the previous PRRS strain isolated that does not characterize, method of this strain isolated of attenuation, and the purposes of this virus isolated strain in vaccine and immune composition predicted.

Background technology

Pig reproduction and breath syndrome virus (PRRSV) are a kind of single strand RNA viruses of tool coating, are to belong to coating Viraceae (Arteriviridae) (Cavanaugh, 1997).This virus can cause a kind of pig disease of ubiquity, and this disease at first came across U.S.'s (Hill, nineteen ninety) and is called as mystery swine disease (mystery swine disease) in 1987.Its clinical symptom is to cause the respiratory tract disease of all ages pig, and causes the death of some younger pig, and the sow that is in the reproductive age is caused serious reproductive problems.

The kinetic characteristic of PRRSV changes it constantly in disease, the chance that fully provides for the appearance of new virus isolated strain (people such as Andreyev, 1997; People such as Murtaugh, 1998; Meng, 2000).Because PRRSV constantly develops, add ability to damaging property of a pig farmer difficult problem, make it become an important theme and obtain research (people such as Mengeling, 1998; People such as Pejsak, 1997), exploitation vaccine and seek the method that other reduces infectivity.The difference of the pathogenic standard of virus isolated strain, in the pulmonary lesion of pig and dead situation, proved (people such as Halbur, 1996), but the relevant trial of the difference on these biology and the immunology and specific gene variation is then failed mostly (people such as Albina, 1998; People such as Key, calendar year 2001; People such as Yuan, calendar year 2001; People such as Murtaugh, 2002; People such as Grebennikova, 2004).People such as Labarque (2003), and people such as Mengeling (2003 a) and in people's's (calendar year 2001) such as Nodelijik the research, the security and the efficient of having tested the PRRS vaccine.These studies have shown that under the experiment situation, the PRRS living vaccine of improvement can reduce the amount and the persistence of viremia, and alleviates pyreticosis and the pulmonary lesion that causes behind the virus attack.

People such as Opriessing (2002) prove, when virus isolated strain has when highly aminoacid sequence is similar in open reading frame 5 (open readingframe 5, abbreviation ORF5), can cause pig to have pneumonia (pneumonia) significantly in various degree to produce.Host's difference also can influence pig for PRRSV have differential responses (people such as Mengeling, 2003b).Research proves, pathogenic and PRRSV multiple-copy rate and the distribution (people such as Hayne in pig, 1997), that the cupric ion of scavenger cell is removed severity of anemia people such as (, 2002) Halbur of ability people such as (, 1998) Thanawongnuwech and host animal is relevant.Yet these methods also can't provide the prediction pathogenic method new or the previous PRRS virus isolated strain that does not characterize of imitating.

According to aforementioned, this area needs a kind of method of predicting that the PRRS virus isolated strain is pathogenic.This area further need give virus isolated strain or expose or after previously do not have the pig of PRRS, according to the growth velocity and/or the viremia level of PRRS virus in the pig body, predict the pathogenic method of virus isolated strain.

Summary of the invention

The present invention overcomes the problems referred to above, and the new wild-type PRRS virus isolated strain and the attenuation type of this virus isolated strain are provided, and corresponding vaccine, and this vaccine is the pharmaceutical composition that contains attenuation type PRRS virus.Particularly, wild-type virus strain isolated name of the present invention is called SDSU73,17198-6, MN 184, and mixes strain.The attenuation type of these virus isolated strains, and comprise the complete vaccine of attenuation type strain isolated, the immunizing power of antagonism PRRS in the pig body can both be provided, and can not produce tangible PRRSV clinical symptom.

Another feature of attenuation type PRRS virus isolated strain of the present invention is, their major parts kept wild-type or not the attenuated virus strain isolated produce the situation of viremia.Therefore, the viremia that the attenuation C-type virus C produces is not at least 50% of a viremia that attenuated virus produces of wild-type, preferably at least about 75%.By this way, the attenuation C-type virus C can be given pig PRRS immunizing power faster and completely.

The accompanying drawing summary

Fig. 1 is the figure of the pig test among the embodiment 1, and it is the average serum virus titer to the variation of time, and described virus titer is with log10 TCID ₅₀/ ml represents;

Fig. 2 is the figure of the pig test among the embodiment 1, and it is the average serum PRRSV virus concentration that utilizes real-time RT-PCR to measure;

Fig. 3 uses the average S/P ratio of commercial ELISA assay method mensuration with respect to the figure to the time;

Commercial ELISA chemical examination and log10 TCID in Fig. 4 illustrated embodiment 1 ₅₀The replicate measurement analysis of/ml data, wherein the cell mean under the usefulness ELISA S/P ratio curve is to log10 TCID ₅₀Group average area mapping under the/ml;

The replicate measurement analysis of commercial ELISA chemical examination and RT-PCR concentration data in Fig. 5 illustrated embodiment 1 is wherein mapped to the group average area under the RT-PCR concentration curve with the group average area under the ELISA S/P ratio curve;

Fig. 6 a is the figure that absorption value was mapped to the time among the embodiment 1;

Fig. 6 b is with the figure of absorption value to the time mapping, and the influence of PRRSV strain isolated to nsp-4 IgG reaction is described, wherein data are mean values of ten animals, but then do not list calculating in as animal dead;

Fig. 7 illustrates nsp-4 and the log10 TCID that utilizes among the embodiment 1 ₅₀The replicate measurement analysis of/ml data is to log10 TCID with the group average area under the nsp-4 curve wherein ₅₀Group average area mapping under the/ml curve;

Fig. 8 is clinical score and the log10 TCID that utilizes embodiment 1 ₅₀The figure that the replicate measurement of/ml data is analyzed, wherein the cell mean under the usefulness clinical score curve is to log10 TCID ₅₀Group average area mapping under the/ml curve; And

Fig. 9 is the figure of the IHC scoring of each strain isolated among the application.

Embodiment

Wild-type virus can carry out attenuation by many methods, for example makes virus repeat continuous subculture, gene insertion, gene conversion (gene switching), genetically deficient, base pair replacement and temperature sensitive mutation etc. in cell cultures.Use known in the art method, described attenuation C-type virus C can be used for various immune compositions, comprises vaccine.In some cases, these attenuation C-type virus Cs can utilize method well known in the art further to be killed or deactivation, mix then or be included in according to of the present invention to cause in the immune composition.Can be used for preventing that PRRS from infecting or reducing the example that causes immune composition that PRRS infects the severity of back clinical symptom and comprising and utilize Abst-1 or the prepared vaccine that forms of JA-142.

Find that also vaccine of the present invention can store for future use with freezing state, and the vaccine that can not detract desire feature.This solves long problem in this technology, because previous PRRS vaccine and be not suitable for refrigerated storage.

The present invention further provides the method for the prediction pathogenic grade of PRRS virus isolated strain new or that do not characterize.This method is usually directed to the new or previous PRRS virus isolated strain that does not characterize carried out in following several the parameters at least one assessment: viral growth speed, viremia level, antibody response or its combination.The result who utilizes assessment to obtain predicts pathogenic grade new or the previous virus isolated strain that does not characterize then.This method is usually directed to give or will be subjected to the pig of PRRS virus infection to be exposed to a certain amount of new or previous PRRS virus isolated strain that does not characterize, make virus replication reach about 15 days for some time, preferably from about 2 to 12 days, more preferably from about 3 to 10 days, more preferably from about 3 to 7 days.The mode that gives virus can realize with usual manner, comprise in oral, the nose, in the intramuscular, lymphoglandula, intracutaneous, intraperitoneal, subcutaneous or its combination, but best mode then is to see through interanasal administration.Nasal cavity dispensing institute using dosage preferably reaches about 5 milliliters, between 0.5 to 4 milliliter, between 1 to 3 milliliter, more preferably then is about 2 milliliters more preferably from about more preferably from about.Every dose virus concentration should reach about 5.0 Log10 TCID ₅₀/ ml is more preferably from about at 1.0 to 4.0 Log10 TCID ₅₀Between/the ml, more preferably from about at 2.5 to 3.5 Log10 TCID ₅₀Between/the ml, most preferably be about 3.0 Log10TCID ₅₀/ ml.The time point of selecting during this virus replication obtains biological sample from pig, measures growth velocity, viremia level, antibody response and/or its combination of the virus that gives.To analyze data and other viral growth speed, viremia level, antibody response or its combination known or confirmed virus isolated strain of collecting from these subsequently and compare, as the pathogenic standard of prediction virus isolated strain new or that do not characterize.

Utilize method of the present invention, measure it has been bestowed PRRS viral growth, viremia level, antibody response and/or its combination in a kind of pig in eight kinds of different PRRSV strain isolateds.In these virus isolated strains each all has known pathogenic level and clinical symptom.Also measure and given above-mentioned characteristic in the pig of combination of all eight kinds of strain isolateds it.

More particularly, with 100 2 to 3 all big pigs, be divided into 10 groups at random according to its body weight, 10 every group.All pigs all utilize HerdChek ^(ME) test PRRS infects PRRS ELISA 2XR for IDEXX LaboratoriesInc., Westbrook.Wherein give a kind of in eight kinds of virus isolated strains to eight groups, one group of combination that gives all eight kinds of virus strain, last group then gives Eagle ' s minimum essential medium (EMEM) and is used as control group.Every kind of virus inoculation taken a sample carry out again quantitatively, to determine virus titer.Preferably, designing the virus titer that gives makes its natural exposure level to virus similar.In the different steps that whole experiment is carried out, collect the biological sample of blood form.Separate quantitative RT-polymerase chain reaction (RT-PCR), HerdChek by virus ^PRRS ELISA 2XR, and each sample of PRRSV protein-specific elisa assay.

It is by serial dilution serum and with itself and EMEM, Geneticin (gentamicin) (Sigma Chemical Co. that virus is separated, St.Louis, MO) and the mould B of both sexes (Fungizone) (Invitrogen Corp., Grand Island NY) is blended in and carries out on CL2621 (a kind of MA 104 cell strains) cell.Be incubated diluent subsequently, check its cytopathic effect (cytopathiceffect) (CPE).Utilize the Reed-Muench method to calculate virus titer.

RT-PCR utilizes QIAamp Viral RNA Mini-Kit ^(available from Qiagen, Inc., Valencia CA) carries out, and PRRSV utilizes Tetracore company (Gaithersburg, single tube analysis MD) (single-tube assay) detects.In order to determine virus quantity, at first set up typical curve, by carrying out the concentration that unknown sample is tried to achieve in linear extrapolation to the cycle threshold that 3 ' UTR transcription product of concentration known is mapped.

Using the antibody test of ELISA S/P ratio is to utilize HerdChek ^PRRS ELISA 2XR produces according to manufacturer specification.PRRSV protein-specific ELISA be utilize BL21 (DE3)-RP cell (available from Stratagene, La Jolla, nucleocapsid protein (N) that recombinant virus strain isolated VR2332 CA) expresses and unstructuredness albumen 4 (nsp4) carry out.

Beginning and terminal point in research carry out measured body weight to all pigs.In addition, the every day in research process, the clinical symptom of the PRRS disease of every pig is assessed and marked by the animal doctor.All resulting data are analyzed by statistics, and compare based on group.

Attack with highly pathogenic virus isolated strain once more subsequently for nine groups in ten groups, further assess the relative wild-type allos of homology provide protection and vaccine provide protection (notion that live virus exposes).Through this attack, assessment pig and autopsy are analyzed to use viral separation and immunohistochemical method.

Therefore, the present invention also provides the immune composition that causes that comprises attenuation type PRRS virus isolated strain.In a preferred form, said composition also can comprise the carrier and/or the adjuvant of pharmaceutically compatible.One of them example of said composition comprises Abst-1 and/or utilizes the attenuation type of the pathogenic PRRS virus of tool that aforesaid method is predicted as.

The present invention also provides a kind of method of improvement, selects to be used for attenuation and to be incorporated into the PRRS virus isolated strain that causes immune composition.This method generally includes following steps: the PRRS virus isolated strain of a) obtaining pathogenic the unknown; B) give a certain amount of this PRRS virus isolated strain to the pig that does not infect PRRS; C) make this virus isolated strain in pig, duplicate about 3 to 15 days time; D) measure inner virus growth velocity and/or viremia level during this period; E) growth velocity and/or the viremia level of this growth velocity or viremia level and pathogenic known PRRS virus isolated strain are made comparisons; F), select virus isolated strain, in order to attenuation and be incorporated into and cause immune composition according to the growth velocity and/or the viremia level of comparing with pathogenic known virus isolated strain.Although adopt the virus isolated strain be predicted as pathogenic lower (comprising those no pathogenicities) sometimes, preferably select and be predicted as the highly pathogenic virus isolated strain of tool and cause immune composition in order to be incorporated into.This is because being predicted as the highly pathogenic virus isolated strain of tool has than high growth rates and/or viremia level, so causes immune response more vigorous and that provide protection is stronger usually.Utilize method of the present invention, simplified and selected in order to being incorporated into the process of the virus isolated strain that causes immune composition, and increased and obtain the possibility that effective vaccine resists pathotype PRRS virus isolated strain.

" growth velocity " used herein means and measures virus duplicating over time in pig, and embodiment 1 provides preferred embodiment." viremia level " used herein means the viral circulation composition of pig blood, and embodiment 1 also provides preferred embodiment.

The brief description of reference implementation scheme

Following examples have illustrated according to preferred virus strain isolated of the present invention and step.Should be understood that these embodiment only for exemplary, it should not be considered as the restriction to category of the present invention.

Embodiment

Embodiment 1

Materials and methods

100 2 to 3 week big health pig available from the swinery of buying that is not subjected to the PRRS virus infection, and under veterinarian's management, raise Iowa An Musi animal doctor's resource company (VeterinaryResouces Inc., Ames, Iowa).Drinking-water and the food of pig provide arbitrarily.All animals treatment personnel and the lab assistant that participate in this research are all ignorant for the processing mode of each treated animal.Utilize HerdCheh ^(IDEXX Laboratories Inc., Westbrook ME) detects pig PRRS ELISA 2XR, determines whether pig is subjected to PRRSV and infects.Pigs all in the present embodiment all are negative after tested.According to body weight pig is divided into 10 groups at random then, every group has 10 pigs.

The used PRRSV strain isolated of present embodiment always has eight kinds, and these virus isolated strains have been named as VR-2332, Ingelvac ^PRRS MLV, JA 142, Ingelvac ^PRRS ATP, SDSU 73, Abst-1, MN 184 and 17198-6.These eight kinds of virus isolated strains show various pathogenic grades in various degree, and present relevant clinical symptom across the medical history of whole PRRS disease.All virus isolated strains all are grown in (CL2621 is from NVSL, Ames, the privately owned cell strain of IA) on the ATCC clone CL2621 cell (a kind of MA-104 monkey kidney cell is).The original wild-type virus strain isolated of three strains VR-2332, JA-142 and SDSU 73 also have its corresponding attenuation type, are called Ingelvac respectively ^PRRS MLV (Boehringer Ingelheim Vetmedica Inc., St.Joseph, MO), Ingelvac ^PRRS ATP (Boehringer Ingelheim Vetmedica Inc., St.Joseph, MO) and Abst-1.These attenuation C-type virus C strain isolateds all show low maybe can not detect pathogenic, and it is deutero-by going down to posterity attenuation in vitro.PRRSV isolate A TCC VR-2332 be 1991 in the Minnesota State (Minnesota) separates, use be third generation passage culture.This viral attenuation type can commerce be buied, and trade name is Ingelvac ^PRRS MLV.PRRSV strain isolated JA142 (ATCC is numbered PTA-6504) is provided by the William Mengeling of Iowa An Musi country Animal diseases centers (NationalAnimal Disease Center), be 1997 in the Iowa, from serious " miscarriage storm (the abortion-storm) " case that causes breeding difficulty, separate, use the 5th generation the passage culture.The attenuation type of JA 142 can commerce be buied, and its trade name is Ingelvac ^PRRS ATP, and ATCC is numbered VR-2638.PRRSV SDSU 73 (ATCC is numbered PTA-6322) be 1996 in the Iowa, from serious breeding disease case, find, use first-generation passage culture.The attenuation type of SDSU 73 is called Abst-1 (ATCC is numbered PTA-6320), by 52 acquisitions of going down to posterity.PRRSV strain isolated 17198-6 (ATCC is numbered PTA-6321) is to obtain Oklahoma (Oklahoma) suffers from the pig of serious breeding disease from a group in 1997, use the 4th generation the passage culture.PRRSV MN184 (ATCC is numbered PTA-6319) obtained from the pig farm that meets with serious breeding disease and sow death in southern Minnesota in calendar year 2001, by (the University of Minnesota of Sao Paulo University of Minnesota, St.Paul) KurtRossow provides, and uses the first-generation passage culture of virus isolated strain.In addition, we have also prepared the mixing strain of the combination that comprises all virus isolated strains.

At the 0th day, each and PRRSV mixing strain in eight kinds of PRRSV strain isolateds are being contained 4% foetal calf serum (FBS) (JRH Bioscience, Lenexa, KS) Eagle ' s minimum essential medium (EMEM) (JRH Bioscience, Lenexa is diluted to about 3.0 log10 TCID in KS) ₅₀/ ml offers medicine in the intranasal then to pig, and dosage is 2 milliliters of potions (1 milliliter in every nostril).Give the nutrient solution of 2 milliliters of untreated control groups.The titration inoculum is determined to tire to utilize Reed-Muench method (Reed etc., 1938) on 96 orifice plates that contain 3 age in days CL2621 cells.Observed to being applied to tiring of pig, and the description of pathogenic level and separate information are listed in the table 1.

Pathogenic and the inoculation of table 1 strain isolated is tired

Group	Strain isolated	Separate the age	Pathogenic ***	Log tires 10?TCID 50/ml
					1	VR?2332	1991	Moderate	3.43
2	IngelvacPRRS?MLV *	USDA permission 1996	The VR2332 of attenuation	3.02
					3	JA?142	1997	High	3.13
4	IngelvacPRRS?ATP *	USDA permission 1999	The JA 142 of attenuation	4.14
					5	SDSU?73	1996	High	2.75
6	Abst-1 *	1999 of attenuation	The SDSU 73 of attenuation	4.18
					7	MN?184	2001	High	4.10
8	17198-6	1997	High	2.81
					9	Mixture **	N/A	High	3.71
10	Contrast	N/A	N/A	N/A

^*Represent attenuation type RRSV virus

^*The mixture that contains eight kinds of virus isolated strains

^{* *}Be published among the Symposium on Emerging Diseases (Rome, 2003) summary about pulmonary lesion

Compare strain isolated to determine its gene similarity by analytical percentage sequence identity then.Sequence identity entrusts University of Minnesota diagnostic test chamber (Diagnostic Laboratory) to carry out sequential analysis viral sample to determine.The result of ORF 5-6 is provided, and the consensus sequence with itself and PRRS virus compares then.The difference of indivedual base pairs is indicated, and compares the % sequence identity of each virus isolated strain then.As is known to the person skilled in the art, blast searches and also can carry out on different websites.For example, University of Minnesota provides a PRRSV database (ccgb.umn.edu/cgi-bin/common/web_blast.cgi), wherein lists the sequence of virus isolated strain in from 1989 to 2003.Another website that often is used is NCBI BLAST, and network address is ncbi.nlm.nih.gov/BLAST.

According to per-cent sequence identity and dendrogram that table 2 shows, the gene difference of pathogenic wild-type virus strain isolated is very obvious, and represents a plurality of groups PRRSV strain isolated.On the contrary, parental generation and vaccine PRRSV are to identical on gene.The paired comparison of table 2 (pairwise comparison) is that (Madison WI) produces for DNASTAR, Inc. with the Lasergene software suite in the sequential analysis instrument with dendrogram.

Table 2, the paired comparison of the ORF5 nucleotide sequence of pathotype that this institute uses and attenuation type PRRSV strain isolated.Similar percentages show is in the form upper right corner, and difference percentage is presented at the form lower left corner.Dendrogram shows the genetic correlation degree of virus isolated strain.On behalf of each hundred residue, band have a Nucleotide to change.VR2332 is the parental virus strain isolated of Ingelvac PRRS MLV, and JA 142 is parental virus strain isolateds of Ingelvac PRRS ATP, and SDSU 73 is parental virus strain isolateds of Abst-1.

Similar per-cent

	VR?2332	?Ingelvac ?PRRS ?MLV	JA-142	Ingelvac PRRS ATP	SDSU?73	Abst-1	MN?184	17198-6
									VR?2332 Ingelvac ? PRRS?MLV JA-142 Ingelvac ? PRRS?ATP SDSU?73	? ? 0.3 ? 9.7 ? 10.3 ? 10.9	?99.7 ? ? ? ?10.1 ? ?10.7 ? ?11.3	91.0 ? 90.7 ? ? ? 0.8 ? 7.8	90.5 ? 90.2 ? 99.2 ? ? ? 8.8	90.0 ? 89.7 ? 92.7 ? 91.9 ? ?	89.6 ? 89.2 ? 92.2 ? 91.4 ? 99.5	86.4 ? 86.4 ? 87.2 ? 86.4 ? 87.2	90.4 ? 90.0 ? 92.2 ? 91.4 ? 91.7

?Abst-1 ?MN?184 ?17198-6

11.5 15.5 10.5

11.9 15.5 10.9

8.4 14.4 8.8

9.4 15.5 9.7

0.5 14.4 9.0

? 15.1 9.6

86.7 ? 15.9

91.2 86.1

Difference percentage

At the 49th day, with the to the 1st to 9 group of dispensing in 2 milliliters of pathotype strain isolated PRRS MN 184 intranasals.MN 184 strain isolateds systems is diluted among the MEM+4%FCS, makes its concentration equal every milliliter of log3.0+/-0.5.Diluted challenge virus (challenge virus) titration in 96 orifice plates that include the three age in days CL2621 cells that are used to test.Evaluated 14 days of animal carries out autopsy then.

The assessment of viremia

The 0th, 1,3,7,15,21,28,35,42, with 49 days, utilize vacuum test tube (vacutainer) from every pig of each group, to gather blood sample.With centrifugal 20 minutes separation of serum from the agglutinative whole blood of 6000RPM.Then with the serum sample packing be used for by virus separate, TCID ₅₀Analysis, quantitative RT-polymerase chain reaction (RT-PCR), HerdChek ^PRRS ELISA 2XR and PRRSV protein-specific ELISA analyze.The serum sample of this research is handled after collection immediately, and is obtaining placement cooling on ice in back three hours.Sample is stored in 4 ℃ and is no more than 24 hours at most, is stored in-70 ℃ afterwards.The serum sample that detects with RT-PCR froze at-70 ℃ collecting the same day, and stored and be enough to test in 24 hours up to the sample number, sample was thawed again, extract, and test.

For TCID ₅₀Analyze, 100 microlitres are added to from the serum of every pig contain every milliliter of Geneticin of 900 microlitre EMEMm+2%FBS+50 micrograms (Sigma Chemical Co.St.Louis, MO)+2.5 every milliliter of amphotericin B of microgram (Invitrogen Corporation, Grand Island is in dilution tube NY).With dilution tube vortex concussion, get in 100 microlitres contain every milliliter of Geneticin of 900 microlitre EMEMm+2%FBS+50 micrograms+every milliliter of amphotericin B of 2.5 micrograms to another the dilution tube.Repeat this step up to reaching final extent of dilution 10 ^-64 kinds of repeat bodies of dilution at every turn are laid in 96 porose discs that include every hole 100 microlitre CL2621 (MA 104 cells), place 37 ℃ and 4%CO ₂Condition under cultivated eight days.Then check the cytopathic effect (CPE) in each hole, and utilize the Reed-Muench computing method to measure and tire.Separate for virus, 100 microlitre serum are added in each of the bipartite test hole that contains MA 104 cells.Then plate is placed 37 ℃, 4%CO ₂Under be incubated 1 hour.Connect down every milliliter of Geneticin of 500 microlitre EMEM+2%FBS+50 micrograms+every milliliter of amphotericin B of 2.5 micrograms is added each hole.Plate is placed the insulation eight days down of 37 ℃, 4%CO2, check CPE then.

In order from serum, to extract viral RNA, as described in the indication in the test kit, use QIAamp Viral RNA Mini-kit to be used for quantitative RT-PCR ^(available from Qiagen Inc., Valencia, CA).For PCR in real time, the real-time single tube RT-PCR test (it is used to detect U.S. PRRSV) that commerce can be buied is that (Gaithersburg MD) provides, and is used to detect PRRSVRNA by Tetracore company.By comparison GenBank PRRSV strain isolated and based on the conservative region of 3 ' UTR primer and probe region, design ditch mating type (minorgroove binding, MGB) 5 ' nuclease probe and primer from 3 ' non-translational region (UTR) PRRSV genome area.The 25 microlitre reaction volumes that the PRRSV RNA that use is extracted by Tetracore U.S.PRRSVMaster Mix (18.9 microlitre Master mix, 2 microlitre Enzyme mix 1, with 0.1 microlitre Enzymemix 2) and 4 microlitres is formed are transcribed PRRSV RNA in single pipe.Reaction tubes is inserted Smart Cycler II ^Block (Cepheid, Sunnyvale, CA) in, the Software tool of fluoroscopic examination is set at the automatic calculating that is used for baseline, and opens background deduction.The temperature cycle program is made up of following: 52 ℃ 1800 seconds, 95 ℃ 900 seconds, with 94 ℃ 30 seconds, 61 ℃ of 60 seconds and 72 ℃ carried out 45 circulations in 60 seconds.If cycle threshold (Ct) level is being less than or equal to 45 circulation acquisitions, the PCR reaction is considered to positive.For quantitatively, use the in-vitro transcription RNA product (1 * 10 of the serial dilution of known quantity ^-1To 1 * 10 ⁸Copy every microlitre) produce typical curve.The copy of unknown sample/ml concentration is measured by carrying out linear extrapolation to the Ct value of known 3 ' the UTR transcription product mapping of concentration.

Antibody test

ELISA S/P ratio is by carrying out HerdChek by the explanation according to the manufacturer ^(INDEXX Laboratories, Westbrook MA) produces PRRS ELISA2XR.Be used for HerdChek ^PRRSV protein-specific ELISA utilize the nucleocapsid protein (N) of recombinant virus strain isolated VR2332 and unstructuredness protein 4 (nsp4) to carry out, it is expressed as from plasmid pET 24b in BL21 (DE3)-RP cell (available from stratagene) and contains N-terminal myc label and the fusion rotein of carboxylic end 6x histidine-tagged (6x histidine tag).Denatured protein is at 0.1M Tris HCl, pH8.0, and 6M guanidine-HCl dialyses among the 2mM EDTA, and adjusts concentration to 3 milligram every milliliter.DTT is added to 300mM, with 0.45 micron membranes filtering solution.Be added to refolding damping fluid (100mM Tris HCl, pH8.0, the acid of 0.5ML-spermine through reductive protein, the 8mM Sleep-promoting factor B, 2mM EDTA, 10 μ M pepstatin A, 10 μ M leupeptin, 1mM PMSF) in, filtration (0.22 μ m) is also stirred and is spent the night.(Pellicon XLUltracel PLC 5kd Millpore) concentrates purified protein, uses 20mM Tris HCl (pH8.0) dialysis again by tangential flow filtration (tangential flow filtration).Protein is analyzed on Agilent 2100 Bioanalyzer with Protein LabChip.Purified protein soln is stored in-80 ℃.

Protein-specific ELISA is by being used in the 100ng recombinant protein in the pH9.6 carbonic acid buffer or being undertaken by microtiter plate with independent damping fluid bag.This plate seals (block) with 2.5% skimmed milk in the phosphate buffered saline (PBS) that contains 0.1%Tween20 (PBST).1: 2000 serum of 100 microlitre extent of dilution was added in the duplicate hole 2 hours, then with the PBST cleaning disc, the combination of antibody detects by the following method: with 1: 5000 the dilution horseradish peroxidase link coupled goat-(weight+light chain is (available from KPL for anti-pig IgG, Gaithersburg MD)) insulation is 1 hour, then washing, and with 100 microlitre tmb substrate (available from KPL) colour generations.Reaction utilizes 1M phosphoric acid to stop, and reads the plate value at wavelength 450nm.

Body weight

The 0th day (studying the 1st day) and the 49th day (research the last day) all pigs are weighed.Pig is at portable electronic weight-whippletree balance system Weigh-Tronix ^TM(Fairmont MN) goes up weighing to pattern 615XL for Wight-Tronix, Inc..Balance is proofreaied and correct with the acceptance test counterweight before and after each the use.

Clinical score

Under study for action every day, each pig is marked to respiratory symptom, behavior and cough by the veterinarian, and the scoring of each clinical symptom is 1 to 4 minute.Intact animal is given 3 fens, and the most serious clinical disease gives 9 fens, and dead animal obtains 12 fens.The sample of all dead pigs is sent to Iowa State University animal doctor's diagnostic test chamber and carries out pathological examination in the research.

The assessment of immuning tissue of lung

On autopsy same day, will be from lung's sample formalin fixed of every pig, to be used for meeting the damage of PRRSV by immunohistochemistry and microscopy.This check is undertaken by Iowa State University animal doctor's diagnostic test chamber.

Statistical study

All data are all imported SAS (version 8.02) and are carried out data management and analysis.All target variables (out come variable) that are fit to are produced statistical abstract, comprise mean value, standard deviation, standard error, median, reach frequency distribution.For body weight, RT-PCR, and Log10 TCID ₅₀The data of/ml by the whole difference between unidirectional ANOVA analyzing and processing group, and are carried out to detecting the difference between treatment group by minimum significant difference t assay method (Least Significant Difference t test).All Test Design of group difference are two-way detection (two-sided test).Difference is less than or equal at 0.05 o'clock in the p value and is considered to statistically evident.

Data can be carried out some changes and are beneficial to correlation analysis.Log10 TCID ₅₀The numerical value of/ml can be set to 1.0 less than 2.00 o'clock.When RT-PCR numerical value is negative, be set at 1.0, and all RT-PCR numerical value carry out normalization (normalize) by being converted to denary logarithm (log base 10) before analysis.The result of control group is not included in the correlation analysis.The result of every pig utilizes trapezoidal rule (Trapezoidal Rule) to be converted to approximate area under the curve.Calculate whole research cycle, from observing for the first time the 15th day and the area under curve of observation once to the end from the 15th day, but only show whole research cycle in the drawings.

The result

Virus is separated and Log10 TCID ₅₀/ ml's is quantitative

Before infecting being exposed to of the same day, there is not animal positive to the PRRSV test.See through back the 1st day of intranasal infection, have only the virus test of 13 animals positive in five groups.Yet after infection the 3rd day, except the animal that infects the 17198-6 virus isolated strain, other all animals that infect wild-type (field) virus isolated strain all were and are virus-positive, average Log10 TCID ₅₀/ ml value is to 3.9 (MN 184) from 2.1 (SDSU-73).On the contrary, the cell cultures of the pig of inoculation attenuation C-type virus C strain isolated presents feminine gender without exception.These results show at Fig. 1.For 4 strains in the 5 strain pathogenic virus strain isolateds, reached the viremia peak level at the 7th day, it is from 3.6 to 4.6 Log10 TCID ₅₀/ ml.The 17198-6 virus isolated strain reached the highest at the 15th day.Tiring in all pathogenic virus groups maintains and is close to or higher than 2 Log10 TCID ₅₀/ ml 21 days, but be lower than this level except JA 142 infected group have.

In the pig that has inoculated attenuation type PRRSV strain isolated, the viremia degree is than low in the pig of inoculation pathotype wild-type virus strain isolated.Low log value of maximum that the average maximum of the group of tiring of attenuation C-type virus C strain isolated is tired than minimum pathotype group.Except after inoculation the 3rd beyond the highest heavens, the Abst-1 strain isolated is never separated again.The from the 7th to 28 day, Ingelvac ^The viremia of PRRS MLV is at 0.5 to 1.0 Log10 TCID ₅₀Change between/the ml, and the from the 7th to 28 day, Ingelvac ^The viremia of PRRSATP is at 0.4 to 1.2 Log10 TCID ₅₀Change between/the ml.The virus of attenuation C-type virus C strain isolated is after the 28th day, can't from serum, reclaim, and by the 35th day, virus only can reclaim (result is also illustrated in Fig. 1) from two pathotype wild-type virus strain isolated groups (pigs that infect with MN 184 of strain isolated mixture-infection).At the 42nd and 49 day, viral isolating result showed that nearly all pig flaviviridae viral mass formed by blood stasis takes place.

Generally, it is faster than attenuation C-type virus C strain isolated reproduction speed in pig to observe pathogenic high more virus isolated strain, and it is higher to tire.Particularly, the pig that MN 184 virus isolated strains infect shows the virus replication (it began) of very fast increase before the 3rd day, reaches at the 7th day to surpass 4.5 Log10TCID ₅₀The peak value of/ml.After reaching peak value, MN 184 viremias constantly descend, but at the 28th and 35 day, compared with other virus isolated strain, still maintain significantly higher tiring (t check, the p value is less than or equal to 0.05).In all other pathotype groups (VR2332, JA 142, SDSU 73, with strain isolated mixture), all observe similar trend (see figure 1).Infect the pig of 17198-6, identical with the trend of MN 184 infected group, but and it is not really approaching.

Bestow attenuation C-type virus C strain isolated (Ingelvac ^PRRS MLV, Ingelvac ^PRRS ATP and Abst-1) the trend of group of pig different.After the 3rd day, they begin to demonstrate moderate viral equivalent and increase, and reach maximum value between the 7th to 15 day, and any pathotype exposure of its viral equivalence ratio group is hanged down a logarithmic value, and than low several orders of magnitude of MN 184 infected group.Then, viewed the tiring of these attenuation type exposure groups, the 35th day or preceding meeting drop to 0 (see figure 1).

During group mean value after exposing relatively for the second time, the virus titer of attenuation type group has very rapid increase, and the pathotype group only manifests less increase or even do not have increase fully.For instance, exposing back 7 days for the second time, tiring of Abst-1 is 3.76logs, and tiring of MN 184 is 0.It tires and reaches the highest other attenuation type group for the second time infecting back the 3rd day.After infecting for the second time any pathotype group the highest observed tiring is 1.32logs, described situation JA 142 exposes back 3 days and sees.

Quantitatively viral by real-time RT-PCR

The viremia level also can utilize real-time RT-PCR to measure because the growth on the CL2621 cell may be inequality for all virus isolated strains, and because for viremia RT-PCR comparable on cell growth more sensitive.As shown in Figure 2, pathotype exposure group manifested mean concns at the 1st day sharply to be increased, and the maximum value of all groups between the 7th to 15 day reaches above every milliliter of 8logs.Pathotype exposure group concentration then at step-down gradually of following several weeks, is lower than every milliliter of 4logs the 49th day concentration.

The concentration of attenuation C-type virus C strain isolated exposure group began at about the 1st day to show far beyond unconspicuous increase equally, and its average group is tired from no show or above every milliliter of 7logs (Fig. 2).Viewed concentration is organized in exposure to the attenuation type, and a few weeks longer maintains and manifests on a large scale the level of fluctuation up and down after exposure.This fluctuation mainly is owing to can detect high value once in a while in single pig.Three kinds of attenuation type strain isolated exposure groups all reach maximum value in the different number of days of research.Ingelvac ^PRRS MLV infected group reached every milliliter of peak concentration 4.31logs, Ingelvac at the 28th day ^PRRS ATP infected group reached every milliliter of maximum value 6.58logs at the 3rd day, and the Abst-1 infected group reached every milliliter of maximum value 6.85logs at the 35th day, and it is the highest the tiring (Fig. 2) that attenuation type strain isolated reaches.In addition, at the 3rd and 15 day, the mean concns of observing pathotype strain isolated group is apparently higher than attenuation type strain isolated group mean concns (the p value is less than 0.05), but at the 49th day, the mean concns of pathotype strain isolated group was starkly lower than the mean concns (the p value is less than 0.05) of attenuation type strain isolated group.

HerdChek ^PRRS?ELISA?2XR

As shown in Figure 3, by HerdChek ^The measured body fluid immune response to PRRS virus of PRRS ELISA 2XR S/P ratio shows that in the time of the 15th day, the positive findings of pathotype strain isolated exposure group on average improves above 0.4 section (cut off).On the contrary, attenuation type strain isolated expose cell mean negative and all three groups all keep and be lower than 0.4 after the 21st day.Ingelvac ^PRRS MLV and Ingelvac ^PRRS ATP group showed positive findings at the 28th day, but up to the 42nd day, the Abst-1 infection group and viral infection group did not show the average S/P ratio above 0.4.

In the immunoreactive comparison of body fluid of pathotype virus isolated strain or strain isolated mixture group that infects and the group of having inoculated attenuation C-type virus C strain isolated, know that very the kinetics of antibody response and magnitude and viremia level have dependency, particularly after the infection between the 14th to 35 day.This observes further by HerdChek ^Dependency between determined viremia level of the paired comparison of PRRS ELISA 2XR S/P ratio and virus titer or RT-PCR and humoral antibody reaction is supported.Fig. 4 and Fig. 5 display body fluidity antibody response are closely related with viral load during whole research, the relation conefficient γ of virus titer=0.858, the relation conefficient γ of RT-PCR=0.794.This dependency is highly significant (the p value is less than 0.0001 in every kind of situation), and in addition, attenuation C-type virus C strain isolated shows low antibody response and low viral load, and the pathotype virus isolated strain shows high reaction.

PRRSV protein-specific ELISA

In order to obtain further understanding, measure antibody titer at N (primary structure albumen) and nsp4 (important but accessory Nonstructural Protein enzyme) to the dependency between PRRSV inoculum and the immunoreactive difference of body fluid.The kinetics that Fig. 6 a shows the anti-NIgG antibody response of nucleocapsid protein reached maximum at the 28th day and tires to all groups much at one, immediately back 7 to 14 days after reduce rapidly, between the 42nd to 49 day, remain unchanged afterwards or slightly increase.

The level of response of each strain isolated and HerdChek ^Gained is similar as a result for PRRS ELISA 2XR, and consistent with the viremia level.At the 28th day, minimum peak valence value observed in the group of having inoculated attenuation C-type virus C strain isolated, and the highest valence value observes in the pig that has infected height pathotype MN 184 virus isolated strains.By the 49th day, except that MN 184 and strain isolated mixture group, in each group anti-N antibody tire all identically, showing may be different in nature to the humoral response of MN 184.In addition, by the 49th day, every group was had only 5 pigs survivals in these two groups, and when reflecting the 49th day, the standard error in 184 groups of MN increases.

Shown in Fig. 6 b, different with to N in fact to the IgG of nsp4 reaction.Do not detect anti-nsp4 antibody before the 21st day, general reaction is much lower, and is accepting Ingelvac ^Do not detect significant reaction in the group of PRRS MLV and Abst-1.In addition, anti-nsp4 level of response and viremia level do not have dependency.Reaction for VR 2332, JA 142, MN 184 and strain isolated mixture is all identical, arrives maximum at the 28th day, descends at the 35th day subsequently, rose once again at the 42nd day then, and in these four groups, the magnitude of viremia, time-histories all has different variations with the time length.Fig. 7 represents when checking replication analysis (repeated measures analysis), the data of nsp4 ELISA and Log10 TCID ₅₀/ ml data are compared, and can find that the reaction of viremia level and nsp4 humoral antibody there is no dependency.After exposing the 49th day second time to MN 184 virus isolated strains, reaction is also very low for the second time.

Body weight

When testing the 0th day, arbitrary group mean body weight and no significant difference (the p value is 0.099).In the time of the 49th day, the pig that has inoculated attenuation isolate A bst-1 virus has the highest mean body weight, except that control group, apparently higher than other all groups (table 3).In addition, in the time of the 49th day, except that the 17198-6 group, the mean body weight of all pathotype strain isolated exposure groups is starkly lower than control group (table 3).Attenuation type strain isolated exposure group Ingelvac ^PRRS MLV and Ingelvac ^The mean body weight of PRRS ATP and control group equates (table 3) on statistics.

Table 3 mean body weight

Virus isolated strain the 0th day the 49th day

VR2332 6.381 33.5b

Ingelvac ^PRRS?MLV 6.56 34.6 ^*

JA?142 6.42 32.7b

Ingelvac ^PRRS?ATP 6.24 35.0 ^*

SDSU-73 6.59 32.9b

Abst-1 6.69 39.4a

MN?184 6.73 23.7c

17198-6 6.36 34.5 ^*

Mixture ^*6.51 23.0c

Control organizes 6.48 38.4 ^*

¹Body weight is unit with the kilogram.Mean body weight in the time of the 0th day there is no significant difference.

^*When representing the 49th day, this body weight of several groups is identical on statistics.

^*The mixture representative contains the mixture of all eight strain isolateds.

A is significantly higher than all groups (the p value is less than or equal to 0.05) except that control group.

The b representative significantly is lower than control group.

The c representative significantly is lower than all groups.

Clinical score

Only having in pathotype exposure group four groups to observe average clinical score increases: JA 142, SDSU 73, MN 184, with the mixture group.In whole research, keep higher scoring, and remaining group (comprising pathotype and attenuation type exposure group) has the normal clinical scoring basically during studying always.Observedly in this research change unique main factor to average clinical score and be, one or many animals dead (table 4) in relevant treatment group.

Table 4 infects back pig mortality ratio

The dead fate of group virus isolated strain mortality ratio

1 VR 2,332 0/10 does not have

2 Ingelvac ^PRRS MLV 0/10 does not have

3 JA?142 1/10(10％)?17

4 Ingelvac ^PRRS ATP 0/10 does not have

5 SDSU-73 2/10(20％)?9，23

6 Abst-1 0/10 do not have

7 MN?184 5/10(50％) 14，14，17，23，41

8 17198-6 0/10 do not have

9 combined group ^*5/10 (50%) 12,16,17,21,21

10 control groups 2/10 ^*41,48

Attenuation type PRRSV 0/30 (0%)

Pathotype PRRSV 13/60 (22%)

All death all are because moderate that the PRRS virus and the second time, infectation of bacteria caused or serious apyetous interstitial pneumonia (non-suppurative interstitial pneumonia) in treatment group.

^*Cause death by bacterial pneumonia, irrelevant with PRRS virus.

^*Mixture is the mixture that contains all eight strain isolateds.

The seriousness of clinical disease and viral load height correlation (the p value of virus titer is less than 0.001).As shown in Figure 8, the highest clinical score of the group of MN 184 and mixture infection.Every group has 50 percent pig death, and virus titer shows that other group is high than all in fact for infection level.Difference according to the viral load that RT-PCR measured more not obvious (data not shown), and clinical symptom with by the dependency of the viral load that RT-PCR measured than with the dependency low (being respectively γ=0.556) of virus titer with respect to γ=0.803.The clinical score of the 10th group (control group) increases after two pigs die from bacterial pneumonia.Separate and the real-time RT-PCR analysis by immuning tissue's dyeing of lung tissue, negative virus, show that two pigs all are the PRRSV feminine gender, and HerdChek ^PRRS ELISA 2XR or protein-specific ELISA show does not have seroconversion (seroconversion) fully.Discovery afterwards shows that there are many bacterial pathogens in Yi Waisiwang pig under study for action, and therefore infectation of bacteria causes death (table 5) to possibility owing to being subjected to for the second time.

Table 5 exposes the dead reason in back

The dead fate of the pig numbering group cause of death

993 JA, 142 PRRS and swine streptococcus (Streptococcus suis) 17

Suppurative concealed bacillus of 948 negative control group (Arcanobacterium pyogenes) and sepsis 41

Property pasteurellosis bacillus (Pasteurella multocida)

Suppurative concealed bacillus of 983 negative control group and sepsis pasteurellosis bacillus 48

922 SDSU, 73 PRRS and bacterial pneumonia ^*9

918 SDSU, 73 PRRS and dust Xi Shi intestinal bacteria (Escherichia coli) 23

973 MN, 184 PRRS and pig radiation bacillus (Actinobacillus suis) 14

992 MN, 184 PRRS and pig radiation bacillus 14

980 MN, 184 PRRS and dust Xi Shi intestinal bacteria 17

971 MN, 184 PRRS and dust Xi Shi intestinal bacteria 23

958 MN, 184 PRRS and dust Xi Shi intestinal bacteria 41

976 mixture PRRS and pig radiation bacillus 12

970 mixture PRRS and swine streptococcus 16

972 mixture PRRS and swine streptococcus 17

995 mixture PRRS and streptococcus suis 21

969 mixture PRRS and suppurative concealed bacillus 21

^*Diagnosis report represents not have " bacterial pneumonia " of listed specificity factor.

Lung's immunohistology assessment

Though respectively average IHC scoring of group and observed to no significant difference between the PRRSV damage when marking according to the pathogenic comparison IHC of coming, has evident difference to exist.Average IHC scoring and PRRSV damage are shown in Fig. 9 and table 6.

Table 6 is according to pathogenic average IHC scoring

Variable (variable)	AVIR mean value	VIR mean value	ANOVA p value
				Immuning tissue's dyeing	2.36	1.49	Less than 0.0001 *

^*=p≤0.05 is o'clock remarkable.

Discuss

With in its body whether a purpose of present embodiment is to check the known different PRRSV strain isolateds of pathogenic level, to measure and duplicated relation, and it can be used for predicting the pathogenic of PRRSV strain isolated, and need not carry out in check attack experiment.In addition, interest be to measure virus isolated strain pathogenic, viremia level, and the humoral antibody reaction between relation.At last, interest is to utilize method of the present invention, and the exploitation antagonism is found the vaccine with pathogenic PRRSV strain isolated.Target is the vaccine that obtains other pathogenic virus strain isolated is provided protection in a way; Yet it may not be at large at all PRRSV strain isolateds that this kind intersects effectiveness, and needs further test again.In any case, the invention provides a kind of effective instrument significantly, identify the main material standed for of vaccine development.

For test PRRSV strain isolated under same condition, the dosage of employed vaccine (licensed vaccine) through permitting must be lower than the determined minimum immune dosage of USDA (USDA), and must not be typical commercial dosage.In addition, this interior dosing way of intranasal of studying employed MLV vaccine is not to meet USDA to indicate, and only is the exposure of nature of imitation.The typical commercial dosage of the PRRS vaccine alive (Ingelvac PRRS MLV and Ingelvac PRRS ATP) through improveing is more much higher than this test use.These are not represented employed actual product dosage and pattern in this field, and are easy to explain the serum response of being reported through improveing PRRS vaccine alive employed low dosage in experiment.When using the commercial dosage of vaccine, utilize IDEXX to measure to record serology at the 14th day and tire.Tiring in use is about in this test of 3logs, and this sero-reaction can postpone and be lower.This phenomenon expected originally, but carry out it throw to guarantee between group, to tire and consistence, and promote analysis and comparison between pathotype and the attenuation C-type virus C strain isolated.Though do not particularly point out in the present embodiment, but the influence that dosage caused is much more obvious probably for pathogenic wild-type virus strain isolated than it for attenuation type or pathogenic lower virus, described pathogenic wild-type virus can be grown in porcine blood serum fast, just can gather in the crops the virus quantity above every milliliter of 4logs after exposure in 3 to 7 days.The commercial intramuscular dosage of higher recommendation, back 14 days of inoculation (observe this institute with time of dosage 1/2nd) HerdChek that obtains ^PRRS ELISA 2XR S/P ratio is higher than 0.4 section (cutoff) (people such as Roof, 2003).Customary dosage in this research (every pig 2 * 10 ³TCID ₅₀), in the group of accepting MN 184 virus isolated strains, cause 50% lethality rate, and cause anti-nucleocapsid protein reaction in all groups.Higher dosage is not tested, because the too much death in the group of attacking with highly pathogenic virus isolated strain can hamper the purpose of this research.In addition, previous research is verified, the young pig of inoculation PRRSV strain isolated VR2332, is every animal 10 at dosage ^2.2, 10 ^3.2With 10 ^4.2TCID ₅₀The time, clinical symptom and viremia and indifference.

Log10 TCID ₅₀The result of/ml and real-time RT-PCR shows that the viremia level that exposes between the group of back at PRRSV has considerable change.This represents PRRSV growth velocity in the pig is a kind of phenotypic characteristic of virus, and it has nothing to do with the possible difference of pig for the tolerance of virus infection.In addition, growth makes the PRRSV attenuation in the CL2621 cell strain by adapting to, and not only reduces virus and grows in pig, and change the kinetics of virus replication, makes the later appearance of viremia peak value.People such as Chang (2002) also have similar observation, even they cultivate one limited period at passage at proof, the viral growth of moderate pathotype PRRSV strain isolated VR 2332 in pig also can slow down, and the time that reaches the viremia peak value obviously postpones.Yet, postpone the viremia time to peak, go down to posterity for cell in vitro and cultivate or attenuation there is no diagnostic significance, because also showing the viremia time to peak, height pathotype virus isolated strain 17198-6 postpones.

Generally speaking, when the same dosage of inoculation, the pathotype virus isolated strain is compared the higher in fact serum-virus mass formed by blood stasis level of performance with attenuation C-type virus C strain isolated.For instance, at the 15th day for giving Ingelvac ^The pig of PRRS ATP, observed in arbitrary attenuation C-type virus C strain isolated exposure group is 1.22logs to maximum virus titer, however any pathogenic group minimum tiring was 2.40logs among 73 groups of the SDSU at the 15th day.In pathotype PRRSV strain isolated, viremia appears at 3 to 7 days in maximum value, and detected virus levels (all greater than every milliliter of 3.5logs) have the height consistence, though wherein still there was virus titer in MN 184 in degree and obviously higher on the time length at the 28th and 35 day.This supports following notion: height pathotype PRRSV strain isolated is compared with attenuation type or low pathotype virus isolated strain, copy to higher tiring in vivo, but in wild-type PRRSV and can't set up the direct quantitative relationship between the speed of growth in pathogenic level and body people such as (, 1997) Haynes.Described group of protective effect that shows the wide model that the secondary to MN 184 strain isolateds exposes.For example, do not detect virus at MN 184 and mixing strain group, both all accept the MN184 virus isolated strain at first.Can see Abst-1 and after being exposed to MN894, give minimum protection.These are observed and further consolidate following notion: homology exposes than the allos exposure provides more provide protection.The homology pathotype exposes system than protective, and the allos pathotype exposes then low protectiveness, but still exposes high than allos attenuation type.

The real-time RT-PCR result the statistics on Log10 TCID ₅₀/ ml result is very similar, the relative level of infectious virus between all measurement groups of two kinds of methods being described and organizing.In the 7th day measured data, RT-PCR and Log10 TCID ₅₀Pearson's relation conefficient of/ml value (Pearson correlationcoeffcient) is 0.89, and average real-time RT-PCR and Log10 TCID ₅₀Pearson's relation conefficient of/ml value result is 0.88.The concentration value that real-time RT-PCR is measured is than Log10 TCID ₅₀The high several magnitudes of/ml value, reason comprises the difference of the frequency of virion that contains target amplification (amplicon) and the virion that infects the CL2621 cell fully, and can reduce infectivity in and the existing of type antibody people such as (, 2002) Dianzani.Yet, in and type antibody unlikely explain its difference because all can be observed described difference at all time points (comprising the preceding time that anti-PRRSV antibody response produces).

Every milliliter of value of the copy that real-time RT-PCR records, than in cell cultures by TCID ₅₀The measured infectious titer value of/ml is also high.This is because use in quantitative RT-PCR based on the typical curve of virogene body copy number is customary, its direct amplicon virus genome sequence but not known infectious viral particle.Bioanalysis is the existence of cell cultures measurement infectivity for example, yet it can't count all infectious particles that exist in the prepared product.(for example cell culture condition and internal antibody (this antibody can neutralize virus) are observed in other is studied the infectious factor of tiring of influence, cause underestimating in serum with TCID ₅₀The amount of the infectious virus that/ml is measured.Perhaps, some non-infectious or replication-defective virus may exist, and it can be by reflecting than high copy number.

General, the ELISA observations is supported following idea: the virus replication amount during body fluid immune response scale and the acute infection is relevant.Fig. 4 and trend shown in Figure 5 illustrate this dependency.Utilize the attenuation C-type virus C strain isolated of cell cultures to cause more slowly, lower body fluid immune response, the pathotype virus isolated strain then causes comparatively fast, stronger body fluid immune response.In addition, these observe also proof, have the relative S/P ratio of two factors (virus isolated strain kind and infective dose) influence in HerdChekPRRS ELISA 2XR at least.Though ELISA result shown in Figure 3 shows the reaction of the average group of tangible positive or negative, is important to note that the difference between individual animals.Some pig in attenuation C-type virus C group was presenting positive reaction always up to the 21st day, and the pig in some pathotype virus group was up to still negative reaction in the 21st day.

Analysis is to N and nsp4 protein specific antibody reaction and display, and is irrelevant to the variation of immune response on degree and the virus isolated strain of being inoculated of PRRSV.In the animal of inoculation height pathogenic virus strain isolated MN 184 and JA 142, for the proteic antibody response of N, shown the trend similar to other all virus isolated strains, but higher on degree.Show that as Fig. 1 and 2 inoculation MN 184 also has the highest virus titer with the pig of JA 142.This shows that the immunoreactive degree of body fluid may be relevant with viral load (measuring as virus titer) in acute infection.What is interesting is that (time course) is identical for the time course of the reaction of all groups, though in height pathogenic virus strain isolated 17198-6 and attenuation C-type virus C strain isolated, postpone the highest time of arrival of tiring.Otherwise the nsp4 antibody response is at all time points and all very low for all virus isolated strains (attenuation type and pathotype).The time flow of anti-nsp4 reaction is all identical in all groups, although identical with the observations of anti-N protein antibodies reaction, every group of time that arrives the highest viral load is difference to some extent.Fig. 7 shows that the anti-nsp4 reaction of all pigs is all very low.

These observations show that some PRRSV albumen causes the reaction that host immune system is stronger, and have nothing to do with the pathogenic of the virus isolated strain that is exposed.Yet, observe also and show that for the stronger albumen of immunogenicity, it causes immunoreactive scale, may be with the virus isolated strain that is exposed pathogenic, or virus isolated strain replication is relevant in vivo.Another possibility is, the difference of antibody response may be because the gene difference of virus isolated strain, thereby cause the difference of antigen reactivity, so the N of anti-other virus isolated strain and the antibody of nsp4 do not react with the recombinant protein (it is used for bag by elisa plate) that the VR2332 virus isolated strain is expressed, or react relatively poor.Yet many evidences show that observed difference to antibody horizontal reflects immune-related reaction.According to the ORF5 comparative measurement, MN 184 and VR2322 virus isolated strain gene difference maximum, but but show the highest anti-N antibody response.People (1996) such as previous Kapuer show, the relative different between the PRRSV strain isolated in an open reading frame exists in other open reading frame too.In addition, single albumen contains conservative and non-conservative region (for example people such as Kapur, 1996), and intensive becomes the immunoreactivity may be at conservative property antigen determining area people such as (, 2002) Ostrowski.Even so, be subjected to gene and antigenic difference influence according to ELISA the possibility of result, and these effects need be considered into to the proteic antibody response of purified PRRSV.Recombinant protein is carried out refolding, but do not observed difference without refolding or between the proteic elisa plate of refolding at bag.

It should be noted that 4 to 5 weeks after inoculation greatly, take place the antibody response of N and nsp4 is gone up bigger reduction relatively.People such as Foss (2002) had before also observed the antibody response peak antibody response reduction then in 1 to 2 similar week to GP5 (main film glucoprotein).In a word, these observations promptings possibly can't be represented the immunoreactive overall picture of pig to PRRS virus for the reaction of single viral protein, because by HerdChek ^The body fluid immune response that PRRS ELISA 2XR is measured does not manifest the similar of short duration peak value of antibody response.

Decreased growth and death are to make the interior growth velocity of the pathogenic and viral body key that is mutually related.Observedly in pathogenic virus exposure group in the pig body, duplicate and induce difference than the ability of serious disease than harmonic(-)mean weight most probable reflection PRRSV strain isolated.These are observed with previous report data consistent (Thacker, 2003): PRRS infection may cause poor appetite, and day weight gain (daily weightgain) reduces by 25 to 40 per-cents.The clinical score that is exposed to the pig of pathotype virus manifests quick increase soon after inoculation, but this moment, in fact the clinical score that the attenuation C-type virus C exposes treated animal there is no change.Being increased in of clinical symptom accept PRRS strain isolated MN 184, SDSU 73, with the pathotype exposure group of JA 142 in show as viewed mortality ratio and be respectively 50%, 20% and 10%.Otherwise attenuation type exposure group does not cause pig death.When relatively being exposed to the group of MN 184 and Abst-1, expose under the condition in identical virus sense, fast viral growth and virus pathogenic between concern the most obvious.Inoculation is tired practically identical, is respectively every milliliter of every milliliter of 4.10logs and 4.18logs, but shows that as Fig. 8 there is significant difference in the mode that two virus isolated strains influence pig.The Abst-1 virus isolated strain is non-activity almost, duplicates in vivo hardly, and does not cause clinical symptom to occur.On the contrary, MN 184 virus isolated strains are copied to height in vivo and tire, and cause serious clinical symptom, make 50% animal dead that exposes.Be noted that in addition, be exposed to the pig of the mixture of all virus isolated strains, show and the identical virusology of the pig that is exposed to MN 184, clinical and immunological response.This discovery shows that in polyinfection, duplicating the most fast, virus may cause the situation of the highest single virus isolated strain of net result and infection growth potential identical better than its kind virus isolated strain.

Pathotype and the attractive in vivo difference of attenuation type PRRSV strain isolated, make we understand grow in pathogenic and its body of virus and duplicate between dependency.When giving the pig equal dose, the Log10 TCID that pathogenic high more virus isolated strain shows ₅₀/ ml equivalent value and RT-PCR concentration value are compared with attenuation C-type virus C strain isolated and are the exponential form rising.The pathotype virus isolated strain brings out faster, stronger body fluid immune response.The pathotype virus isolated strain is compared with attenuation C-type virus C strain isolated, and influence obtains heavy and causes than high mortality and more serious clinical symptom negatively.

In summary, the embodiment of the application's case and experiment expression, attenuation type and pathotype PRRSV strain isolated cause significantly different clinical symptom, and the immune response of different scales.The reason of these differences is virus replicatioies in vivo, its be can be in serum sample detection by quantitative, and can set up with the pathogenic phenotypic characteristic of prediction PRRSV strain isolated.

Embodiment 2

Present embodiment provides several and is incorporated into the method that causes immune composition to the attenuation of PRRS virus isolated strain and with it.

Materials and methods

Prepare the vaccine production thing, wherein mixed the live virus of modified or attenuation, to be used to making pig obtain immunity by infecting PRRS virus.The PRRSV virus that is used for the vaccine production thing is to breed at MA-104 persistence cell strain, preferred ATCC No.CL2621.Cell line growth is in the culturing bottle that contains the MEM that adds 10% foetal calf serum.It is about 7.2 that the pH value of substratum is adjusted to, and cultivate at about 37 ℃.Afterwards by adding about 1 milliliter of freezing inoculum to liquid culture medium matrix virus inoculation cell.Virus is adsorbed onto on the cell, kept 24 hours.At this moment, growth medium is replaced by keeps substratum (by the MEM that adds 4% foetal calf serum, pH7.6 forms).Environmental optimization is 35 to 37 ℃.Make viral growth, up to the 50%MA-104 cell thin by virus damage.Sample is freezing, and preparation is passaged to the MA-104 cell of another flask.This process of continuing went down to posterity for 25 generations virus in clone.Afterwards at 31 ℃, but not 35 to 37 ℃,, allow virus continue 12 generations of breeding with above-mentioned constructed.In the 12nd generation, carried out packing, freezing, be called kind of a provirus strain (Master SeedVirus).

Below describe preferred methods in detail.

I. substratum:

A.Eagles minimum essential medium (MEM), available from JRH Biosciences, article No. is #200-2041.

B. foetal calf serum (FCS) is available from JRH Biosciences.

C. the growth medium of cell cultures-MEM+10% foetal calf serum

D. keep substratum-MEM 4% foetal calf serum

E. trypsinase-Versene IX

F.5% or saturated sodium bicarbonate (Sodium Bicarbonate).

II. tissue culture

(cercopithecus aethiops (African Green Monkey) nephrocyte maintains in the 20 passage number levels (about 58 to 78 generations of subculture number) clone of using: MA-104.

III. equipment

The 75cm tissue culture flasks

Be set in 35 to 37 ℃ incubator

Be set in 31 ℃ incubator

Whizzer

IV. be used to be grown in the method for 25 to 37 ℃ attenuation PRRS virus.

A. the mother liquor (stock) for preparing tissue culture:

With 5 to 7 days MA-10475cm storage bottle, according to following manner with 1 to 4 mode packing:

A. outwell all substratum (50 milliliters on each culturing bottle).

B. use 10 milliliters of trypsinase-Versene, 37 ℃ of insulations 5 to 10 minutes, with the emigrated cells thin layer.

C. cell is taken out centrifugal 5 to 10 minutes with 270xg from bottle.

D. outwell supernatant liquor, and cell is resuspended in 5 to 10 milliliters of growth mediums (MEM10%FCS).

E. all cells is added among 200 milliliters of MEM 10%FCS, is assigned to then in four 75cm culturing bottles, 50 milliliters every bottle with 1 to 4 packing.Afterwards culturing bottle is placed 35 to 37 ℃, up to experiment required (can under no carbon dioxide environment, carry out).

F. 3 to 4 days the culturing bottle that has formed cell thin can use immediately.

G. adjust the pH value to 7.2 of 50 milliliters of nutrient solutions in the culturing bottle, add 1 milliliter of virus then in substratum, culturing bottle is placed 35 to 37 ℃ (can carry out) under no carbon dioxide environment.

H.24 hour after, outwell substratum, add 50 milliliters of MEM 4%FCS (pH7.6) again in culturing bottle, place 35 to 37 ℃ again.

I. this time liquid changing is after 24 hours, and CPE should occur, and when 50 to 60% holes appear in the cell thin, it is freezing.

J. above-mentioned culturing bottle is thawed, get 1 milliliter of wherein liquid and it is moved on to new 75cm culturing bottle, as above-mentioned, to make next viral subculture.

Carry out this above step 25 times altogether, be grown in 35 to 37 ℃.

V. be used for the method that attenuation is grown in 31 ℃ PRRS virus.Use prepares the 1st subculture that is grown in 31 ℃ of viruses in the virus of 35 to 37 ℃ of subcultures 25 times.

A. prepare the mother liquor of tissue culture:

5 to 7 days MA-104 cell 75cm storage bottle, according to following manner with 1 to 4 mode packing:

A. outwell all substratum (50 milliliters on each culturing bottle).

B. use 10 milliliters of trypsinase-Versene 37 ℃ of insulations 5 to 10 minutes, with the emigrated cells thin layer.

C. cell is taken out centrifugal 5 to 10 minutes with 270xg from bottle.

D. outwell supernatant liquor, and cell is resuspended in 5 to 10 milliliters of growth mediums (MEM10%FCS).

E. all cells is added among 200 milliliters of MEM 10%FCS, is assigned to then in four 75cm culturing bottles, 50 milliliters every bottle, 1 to 4 packing.Afterwards culturing bottle is placed 35 to 37 ℃, up to experiment required (can under no carbon dioxide environment, carry out).

F. 3 to 4 days the culturing bottle that has formed cell thin can use immediately.

G. adjust 50 milliliters of medium pH values to 7.2 in the culturing bottle, add 1 milliliter of virus then in substratum, culturing bottle is placed 31 ℃ (can carry out) under no carbon dioxide environment.

H.24 hour after, outwell substratum, add 50 milliliters of MEM 4%FCS (pH7.6) again in culturing bottle, place 31 ℃.

I. this time liquid changing is after 24 hours, and CPE should occur, and when 50 to 60% holes appear in the cell thin, it is freezing.

J. above-mentioned culturing bottle is thawed, and take out 1 milliliter of this liquid to prepare next SIRSVR-2332 virus subculture.

Carry out above step 12 times altogether at 31 ℃.The 12nd subculture that is grown in 31 ℃ of viruses is called kind of a provirus strain (Master Seed Virus), is used for producing vaccine.

Utilize genetically deficient to make PRRS virus attenuation

Haveing the knack of this operator can utilize traditional genetically deficient mode to make the PRRSV attenuation.In general, this method comprises the genes involved disappearance of the pathogenic phenotype that makes coding virus.This method can be adapted people's (U.S.Pat.No.6,740,324) such as people (U.S.Pat.App.No.20020012670) such as Elbers freely or Schall reference, and its teaching and content system are incorporated into herein with way of reference.

Utilize the temperature sensitive type sudden change to make PRRS virus attenuation

Haveing the knack of this operator also can utilize traditional temperature sensitive type mutation method to make the PRRSV attenuation.In general, this method comprises and causes sudden change, its limiting virus activatory temperature range.For example, can adapt people's (" Identification of Mutations Contributing to theTemperature-Sensitive such as Skiadopoulos; Cold-Adapted; Cold-Passage 45 (cp45) Human Parainfluenza Virus 3 CandidateVaccine of and Attenuation Phenotypes of the Live-attenuation ", 73 No.2 Journal of Virology 1374, (February 1999)) method to be used for PRRSV.People's such as Skiadopoulos teaching and content system are incorporated into herein with way of reference.

It is to make PRRS virus attenuation that utilization sets up that infectivity grows

Haveing the knack of this operator also can utilize tradition to set up infectiously to grow the method that is and make the PRRSV attenuation.In general, this method comprises sets up the full length cDNA clone that contains required virogene type, and this clone also has pathogenic to target animal.For example, can adapt people's (" Generation of anInfections Clone of VR-2332 such as Nielson; the high ly Virulent of a North American-type Isolate ofProcine Reproductive and Respiratory Syndrome Virus ", 77 Journal of Virology3702 (Mar.2003)) method to be used for the attenuation type of PRRSV.People's such as Nielson teaching and content system are incorporated into herein with way of reference.

Utilize gene to insert and make PRRS virus attenuation

Haveing the knack of this operator also can utilize traditional gene insertion method to make the PRRSV attenuation.In general, this method comprises and a fragment gene is inserted in the virus the pathogenic phenotype of this gene inhibition virus.For example, can adapt people's (U.S.Pat.No.6,740,324) such as Schall method to be used for the attenuation type of these PRRS viruses.People's such as Schall teaching and content system are incorporated into herein with way of reference.

Utilize base pair replacement to make PRRS virus attenuation

Haveing the knack of this operator also can utilize conventional cipher to make the PRRSV attenuation with the base pair replacement method.In general, codon or base pair replacement comprise with different codons or base pair replacement codon and base and make the pathogenic protein of virogene coding limiting virus.For example, can adapt people's (" Codon Substitution Mutations at Two Positions in the LPolymerase Protein of Human Parainfluenza Virus Type 1 Yield Viruses with aSpectrum of Attenuation In Vivo and Increased Phenotypic Stability In vitro such as McAuliffe ", 78Journal of Virology 2029 (February 2004)) codon method of replacing is set up the gene substitution method that is used for attenuation PRRS virus.In addition, Hurrelbrink and McMinn (" Attenuationof Murray Valley Encephalitis Virus By Site-Directed Mutagenesis of the Hingeand Putative Receptor-Binding Regions of the Envelope Protein ", 75 Journal ofVirology 7692 (Auguest 2001)) and people (U.S.Pat.App.No.20020012670) such as Elbers the base pair replacement method is provided, it can be set up the virus isolated strain through attenuation PRRS by reorganization.People's such as people such as McAuliffe, Hurrelbrink and McMinn and Elbers teaching and content system are incorporated into herein with way of reference.

Utilize mosaic type PRRS construct to make PRRS virus attenuation

Have the knack of the method that this operator also can utilize traditional mosaic type construct and make PRRS virus attenuation.In general, the mosaic type constructive system has gene not of the same race to bring out the reaction of generation antigenicity in host cell.For example, can adapt people's (U.S.Pat.App.No.20040157307) such as Harris method, set up mosaic type construct with PRRS gene.

Attenuation type PRRS virus isolated strain mixed cause immune composition

After new virus isolated strain is carried out attenuation (preferably with one of aforesaid method), this attenuation type strain isolated is mixed the immunoreactive composition that antagonism PRRS pathogenic strain isolated can effectively be provided.Optimal way is that for the animal of accepting the significant quantity composition, said composition can provide antagonism PRRS the protective immunity of virus infection.Expose afterwards or be subjected in the postvaccinal animal that the pathotype virus isolated strain infects, the immunity of this kind protective can reduce the severity of the clinical symptom of PRRS virus infection.Preferably, described clinical symptom can by the dispensing significant quantity cause immune composition or vaccine is prevented.In some form, attenuation type PRRS strain isolated can be used to prepare the live-virus vaccine through improvement, and wherein in causing immune composition, the attenuation strain isolated uses with its existing state.In other form, the attenuation strain isolated can be killed or deactivation before causing immune composition being incorporated into.

Reference

Below the teaching of each reference and content system be incorporated into herein with way of reference.

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Claims (20)

1. the pathogenic method of PRRS virus isolated strain of pathogenic the unknown of prediction, its step comprises: a certain amount of this PRRS virus isolated strain is administered to the pig that is not subjected to the PRRS virus infection, make virus in described pig, duplicate about 3 to 15 days length, measure inner virus growth velocity and/or viremia level during this period, and the relatively growth velocity and/or the viremia level of this growth velocity and/or viremia level and pathogenic known PRRS virus isolated strain, with its pathogenic prediction index as the PRRS strain isolated of pathogenic the unknown.

2. according to the method for claim 1, described during greatly between 3 to 7 days.

3. according to the method for claim 1, be included in and measure the viremia level during described, and the step of the viremia level of this level and described known PRRS virus isolated strain relatively.

4. according to the method for claim 1, the amount of the virus that this gives is similar to animal and exposes the amount of being accepted naturally.

5. according to the method for claim 1, this PRRS virus is oral by being selected from, in the nose, in the intramuscular, lymphoglandula, the method for intracutaneous, intraperitoneal, the subcutaneous or group that its combination is formed and giving.

6. according to the method for claim 1, this growth velocity or viremia level are measured in the biological sample from described pig.

7. according to the method for claim 1, Log is passed through in the measurement of this viremia ₁₀TCID ₅₀/ ml, reverse transcriptase-polymerase chain reaction, PRRS specific ELISA, PRRS albumen-specific ELISA or its make up and carry out.

8. according to the method for claim 1, further be included in the step that gives to observe behind the described PRRS virus isolated strain clinical symptom that this pig PRRS infects.

9. method according to Claim 8, this clinical symptom comprises respiratory symptom, behavior, cough, or its combination.

10. according to the method for claim 1, further comprise when the growth velocity of this PRRS strain isolated or viremia level to have highly pathogenic virus isolated strain and predict that this PRRS virus isolated strain has highly pathogenic step when similar.

11., further comprise when the growth velocity of this PRRS strain isolated or viremia level are similar to the virus isolated strain with low pathogenicity and predict that this PRRS virus isolated strain has the step of low pathogenicity according to the method for claim 1.

12. according to the process of claim 1 wherein that the administered dose of this PRRS virus can reach about 5 milliliters of inoculums, its virus concentration can reach 5.0 Log ₁₀TCID ₅₀/ ml.

13. one kind causes immune composition, it comprises attenuation type PRRS virus isolated strain and pharmaceutically-compatible carrier, and this attenuation type PRRS virus isolated strain is selected from by Abst-1 and according to the method for claim 1 and is predicted to be the group that the attenuation form of the pathogenic PRRS virus of tool is formed.

14. according to the composition of claim 13, this attenuation C-type virus C strain isolated is Abst-1.

15. according to the composition of claim 13, described method according to claim 1 is predicted to be the pathogenic PRRS virus isolated strain of tool and is selected from the group of being formed by ATCC VR-2332, ATCC PTA-6504, ATCC PTA-6319, ATCC PTA-6321, with ATCC PTA-6322.

16. select the method that is used for attenuation and is incorporated into the PRRS virus isolated strain that causes immune composition for one kind, it may further comprise the steps:

A) the PRRS strain isolated of the pathogenic the unknown of acquisition;

B) pig that a certain amount of this PRRS virus isolated strain is given not to be subjected to the PRRS virus infection;

C) this strain isolated is duplicated from about 3 to 15 days length in this pig;

D) measuring viral growth speed and/or viremia level during this period;

E) growth velocity and/or the viremia level with this growth velocity and/or viremia level and pathogenic known PRRS virus isolated strain compares; And

F), select and be used for attenuation and be incorporated into the strain isolated that causes immune composition according to the growth velocity of strain isolated and/or the comparison of its viremia level and pathogenic separation known strain.

17. according to the method for claim 16, step (f) further comprises selects the strain isolated that has similar growth velocity and/or viremia level to highly pathogenic strain isolated.

18., further comprise the step that makes selected viral attenuation according to the method for claim 16.

19. according to the method for claim 18, this virus is selected from by the method for the group that repeats continuous passage, gene insertion, gene conversion, genetically deficient, base pair replacement in cell culture, form with temperature sensitive mutation comes attenuation.

20., further comprise this selected virus is incorporated into the step that causes immune composition according to the method for claim 18.

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