CN104388399A - PRRSV attenuated strain which is capable of inducing pig body to relatively early generate interferon and neutralizing antibody and possesses wide-spectrum immunogenicity - Google Patents
PRRSV attenuated strain which is capable of inducing pig body to relatively early generate interferon and neutralizing antibody and possesses wide-spectrum immunogenicity Download PDFInfo
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- CN104388399A CN104388399A CN201410407284.6A CN201410407284A CN104388399A CN 104388399 A CN104388399 A CN 104388399A CN 201410407284 A CN201410407284 A CN 201410407284A CN 104388399 A CN104388399 A CN 104388399A
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Abstract
The invention discloses a PRRSV attenuated strain which is capable of inducing pig body to relatively early generate interferon and a neutralizing antibody and possesses wide-spectrum immunogenicity. The porcine reproductive and respiratory syndrome virus (PRRSV) attenuated strain obtained through separation, passage and attenuation is named as HP-PRRSV-LZ-F65, and is prepared by subculturing PRRSV isolated virus in primary porcine alveolar macrophage for 5 generations and in MARC-145 cell for 60 generations and then performing attenuation, and the homologous rates of HP-PRRSV-LZ-F65 with PRRSV VR-2332 and VR-2385 are respectively 97.50% and 98.2%. Experiments prove that PRRSV-LZ-F65 is capable of relatively early inducing a pig body to generate an anti-PRRSV neutralizing antibody with the level substantially higher than that of other same-kind vaccines, and also is capable of preventing challenge infection of homologous type and heterogenous type PRRSV, and possesses stable gene after being subcultured in vitro for 50 generations. The attenuated strain HP-PRRSV-LZ-F65 and passage strains thereof possess wide application prospect to prevent porcine reproductive and respiratory syndrome as safe efficient wide-spectrum vaccine strain candidates.
Description
Technical field
The present invention relates to the strain of a kind of porcine reproductive and respiratory syndrome virus attenuation and vaccine thereof, particularly a kind ofly pig body can be induced comparatively early to produce Interferon, rabbit and neutralizing antibody and there is the porcine reproductive and respiratory syndrome virus attenuation strain of broad-spectrum immunogenicity and to be gone down to posterity the vaccine that strain prepares by this attenuated strain or its.The invention belongs to technical field of pharmaceutical biotechnology.
Background technology
Porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV), belong to the member of the Arteriviridae Arterivirus of shell type virales, it is the cause of disease causing porcine reproductive and respiratory syndrome, pregnant sow late abortion, premature labor and product stillborn foetus can be caused, piglet and growing and fattening pigs respiratory tract disease.
The genome of porcine reproductive and respiratory syndrome virus is strand forward RNA, and its genome length is about 15kb, has 5 ' end cap and 3 ' end Poly (A) structure, has had been found that 9 open reading frame (ORF1-9).ORFs-1 coding is responsible for the enzyme that virus replication is transcribed, ORFs2-4 is encode viral particle associated protein GP2, GP2b, GP3 and GP4 respectively, ORFs5-7 encodes major structural protein respectively: glycosylation envelope protein (E), membranin (M) and nucleocapsid protein (N), envelope protein (E) is also known as GP5 albumen.Current PRRSV has 2 main genotypes, Europe class (1 type) PRRSV and american type (2 type) PRRSV, genetic homology 55-70%, gene 1 type PRRSV virus has 3 genetic pedigrees at least, and gene 2 type PRRSV virus has 9 different genetic pedigrees at least.
The reproductive disease that porcine reproductive and respiratory syndrome virus can cause sow serious, as the respiratory tract disease of stillborn foetus, lopsided piglet and superinfection, causes the loss of about 500,000,000 dollars every year, causes the loss of about 20,000,000,000 Renminbi in China every year in the U.S..Existing attenuated live vaccine and inactivated vaccine are used for the prevention and corntrol of pig porcine reproductive and respiratory syndrome at present.
Molecular epidemiology investigation finds the highly pathogenic breeding of pig of the multiple genetic pedigree of domestic popular 2 and 1 genotype at present and breathes comprehensive virus.The appearance of genotype, antigenic various, continuous variation new variant is popular, and has vaccine strain and street strain's reprovision, vaccine immunity pig body to be separated to variant and the low phenomenon of attenuated live vaccine immunoprotection.
The effect that porcine reproductive and respiratory syndrome viral escape immunity of organism and Viral diversity result in attenuated live vaccine and the inactivated vaccine gone on the market at present is both at home and abroad not satisfactory, not good to heterologous gene type virus strain infection immune effect especially.The experimental porcine reproductive and respiratory syndrome virus vaccines adopting reverse molecular genetics to develop, attenuated live vaccine, recombinant vectors express PRRSV protein vaccine, DNA vaccination, plant production subunit vaccine all attempt on different tests animal, but become the significant challenge of these tentative Vaccine Developments very less due to the diversity of the gene of virus own and antigen and understanding that is pathogenic to it and immunity.Along with the research of anti-PRRSV immunity and the reverse molecular genetics of PRRSV is goed deep into, appropriate design researches and develops the Main way that the porcine reproductive and respiratory syndrome virus vaccines with broad-spectrum immunogenicity becomes vaccine research.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of attenuated strain of porcine reproductive and respiratory syndrome virus; this strain more early can activate pig body Interferon, rabbit immune defense system; pig body is made comparatively early to produce I interferoid; comparatively early produce neutralizing antibody and cellular immunization simultaneously; excite the protective immunity of the porcine reproductive and respiratory syndrome virus to homology type and Allotype simultaneously; using this virus as antigen injection body; compare with existing attenuated live vaccine, there is the advantages such as safer, more stable, immunoprotection level is high, immunity spectrum is wide, lasting immunity.
In order to achieve the above object, present invention employs following technique means:
The separation of porcine reproductive and respiratory syndrome virus virulent strain (HP-PRRSV-LZ): the sick pig breaking out high fever syndrome of pigs pig farm from Baoding, Hebei province of China, gathered pathological material of disease is ground, centrifugal, filter, to get filtrate frozen stand-by.After passing for 4 generations with primary porcine alveolar macrophage amplification strain, carry out RT-PCR amplification with N, GP5 primer of PRRSV; Identified by indirect immunofluorescence method with N, GP5 protein-specific monoclonal antibody of PRRSVCH-1a strain, test proves that this isolated strain is PRRS virus, called after HP-PRRSV – LZ-F4 strain; Find that this strain virus has stronger lethality to piglet by the mensuration of the virulence to HP-PRRSV – LZ-F4 strain, pcr amplification result is positive.HP-PRRSV – LZ-F4 strain and PRRSV N, GP5 protein specific monoclonal antibodies positive reacts, and and Pestivirus suis, pig circular ring virus, pig parvoviral, pig encephalitis b virus, pig Hepatitis E virus, the specific monoclonal antibody no cross reaction of gastroenteritis virus of swine and Porcine epidemic diarrhea virus, ORF7 and the ORF5 gene of RT-PCR technology to the HP-PRRSV – LZ-F4 strain be separated is adopted to increase, amplify 2 goal gene fragments respectively, the sequence being PRRSVNsp2 and GP5 to the HP-PRRSV – LZ-F4 strain be separated on this basis is carried out analytical proof HP-PRRSV – LZ-F4 strain and is belonged to american type (gene 2 type), with isolation identification VR-2332, there is higher homology.
PRRSV attenuation strain HP-PRRSV – LZ-F4 goes down to posterity continuous passage on Marc-145 cell again once by the present invention on primary porcine alveolar macrophage, through 3 Plaque Clones, to the cultural characters of generation virus different in succeeding generations, pure property and biological characteristics detect, result shows that each generation virus is without exogenous virus, bacterium, the pollutions such as mould, and can be neutralized by high-pathogenicity porcine reproductive and the specific positive serum of breathing syndrome virus, the sequencing results according to above result and in conjunction with different generation virus compares, the present invention selects porcine reproductive and respiratory syndrome virus strain the 65th generation cell toxicant, called after HP-PRRSV – LZ-F65, Classification And Nomenclature is high-pathogenicity porcine reproductive and breathing syndrome virus, criticize as high-pathogenicity porcine reproductive and respiration syndrome attenuated live vaccine primordial seed, continuous passage 50 generation, every 5-10 generation order-checking, result shows that its genome is stable within 50 generations.This attenuated strain called after HP-PRRSV-LZ-F65, Classification And Nomenclature is highly pathogenic PRRSV, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the preservation time is: on July 8th, 2014, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, preserving number CGMCC No.9454.This attenuated strain be by HP-PRRSV-LZ be separated poison passed for 5 generations at primary porcine alveolar macrophage, MARC-145 cell upload 60 generation attenuation.
Homology analysis result shows: the homology of HP-PRRSV-LZ-F65 and VR-2332 on nucleotide level is 97.50%, be 98.2% with the porcine reproductive and respiratory syndrome virus homology of VR-2385 on nucleotide level, the ORF5 gene coding amino acid homology of HP-PRRSV-LZ-F65 ORF5 gene coding amino acid and VR-2332 is 86.9%, be 84.6% with the amino acid identity of V2385 ORF5 genes encoding, the nucleotide acid sequence total length of the Nsp2 gene of HP-PRRSV-LZ-F65 strain is 2490bP, consecutive miss 600-728 amino acids compared with the nucleotide sequence of HP-PRRSV-LZ-F2 strain Nsp2 gene, lack 128 amino acid altogether.
Security, Study On Immunogenicity result show: the HP-PRRSV – LZ-F65 that the present invention obtains go down to posterity in vitro still can to keep after at least 1-45 generation attenuation no pathogenicity stable, without atavism.Show its security used at nature, even if continuous passage in pig body, at least 6 generations, are without phenotypic variation.Attenuated strain of the present invention and strain of going down to posterity is all safe to pig of each age such as pregnant sow, piglet, the pig body at various age can be induced without side reaction comparatively early to produce Interferon, rabbit, comparatively early produce neutralizing antibody and cell immune response.
The morphological observation of attenuation strain HP-PRRSV – LZ-F65 of the present invention: HP-PRRSV – LZ reaches 65 generations its virus titers and reaches the highest and keep stable, electron microscopic observation finds that low generation virus changes to some extent with the viral particle morphology of high generation, plaque form compares and finds that the plaque of low generation virus is not of uniform size, and the plaque size that high generation virus is formed is homogeneous, and it is less than low generation, negative staining electron microscope is observed and is found that HP-PRRSV – LZ-F65 virus particle is based on circle, and diameter is 40-100nm.
Further, the invention allows for described porcine reproductive and respiratory syndrome virus attenuation strain or its application of strain in preparation prevention porcine reproductive and respiratory syndrome medicine of going down to posterity.
By attenuated strain HP-PRRSV – LZ-F65 of the present invention its go down to posterity strain and pharmaceutically acceptable adjuvant compatible, a kind of vaccine composition preventing porcine reproductive and respiratory syndrome can be obtained.
Preferably, contained porcine reproductive and respiratory syndrome virus attenuation strain or the significant quantity of its strain of going down to posterity are 2.0-5.0lgTCID
50/ agent.
Concrete vaccine composition of the present invention can prepare with reference to following methods:
Set up Marc-145 cell work cell bank and identify by relevant code, by the virus of 0.01-0.01 infection multiplicity inoculation HP-PRRSV – LZ-F65 working seed lots, change cell culture fluid, add the viral maintenance medium containing Small Volume Serum, when after 2-3 days there is pathology in 80% cell, after freeze thawing 3 times, centrifuging and taking supernatant liquor-20 DEG C is for subsequent use, and tests to virus titer, sterility test.Virus stock solution used after qualified adds suitable lyophilized vaccine freeze-drying by 1:3 and prepares attenuated vaccine preparation, adds suitable inactivator can be made into inactivated vaccine by suitable proportion.
The using method of vaccine composition of the present invention:
(1) route of inoculation: musculi colli is injected
(2) dosage of inoculation: sucking piglets 0.5ml, weanling pig and feeder pig 1ml.
Compared to prior art, the invention has the advantages that:
1, attenuated strain provided by the invention or its strain of going down to posterity overcome current commercial vaccine immune effect low, reduce or suppress pig body Interferon, rabbit to generate, the problem low to homogenic type heterologus virus level of protection, the efficient porcine reproductive and respiratory syndrome attenuated vaccine strain provided, the immunosuppression of existing attenuated live vaccine and tardy immune response can be improved, body can be induced to produce high-caliber immune response in early days after injecting body, the level of neutralizing antibody is significantly higher than the neutralizing antibody of other porcine reproductive and respiratory syndrome virus inductions;
2, pig body immunity HP-PRRSV – LZ-F65 or its strain of going down to posterity can induce pig body to produce I interferoid, early stage generation neutralizing antibody and cellular immunization; immunoprotection is lasting; significantly reduce porcine reproductive and respiratory syndrome virus infection; comprise homology high-pathogenicity porcine reproductive and breathing syndrome virus and allos high-pathogenicity porcine reproductive and breathing syndrome virus, and without pulmonary lesion.Immunoprotection can reach the 6-8 month, and 50 generations gene maintenance of going down to posterity in vitro is stable.
3, in succeeding generations, on primary pig pulmonary macrophage, I interferoid generates enhancing, cytopathic effect reduces, in pig body, I interferoid generates and strengthens, Marc-145 cell produces Interferon, rabbit and complete HP-PRRSV – LZ-F65 virus can be produced, virus titer is high, is so not only beneficial to this original attenuation being separated poison and tames and improve the titre of intact virus.
Embodiment
The present invention is further described below in conjunction with specific embodiment; advantage and disadvantage of the present invention will be more clear along with description; but these embodiments are only exemplary; any restriction is not formed to scope of the present invention; what those skilled in the art should understand that is; can modify to the details of technical solution of the present invention and form lower without departing from the spirit and scope of the present invention or replace, but these amendments and replacement all fall within the scope of protection of the present invention.
The separation of embodiment 1 porcine reproductive and respiratory syndrome virus, to go down to posterity and pathogenic and immunogenic relevant qualification
1, materials and methods
1.1 material
3 week age of piglet, specific pathogen free is without PRRSV antibody, commercial detection PRRSV antibody ELISA test kit (IDEXX Laboratories, Inc., Westbrook, Mass.), test strain ATCC VR2385, ATCC VR2332 purchased from American ATCC, HP-PRRSV-HuN4-F112 (lot JA-621C-416) buys from market, and 2ml is 1 part.
1.2 method
1.2.1 virus purification, to go down to posterity
Typical case has been broken out and serious porcine reproductive and respiratory syndrome in a certain pig farm, Baoding, and 300, pig farm sow has 80 to produce at 5 inner peripheral flows, and 50% weans in advance, and the growing and fattening pigs of 15% are dead, the mortality ratio 78% of pig.Sick pig is taken a blood sample, the frit of serum 0.45um, filtrate be positioned over-60 DEG C for subsequent use.75cm
2flask culture PAMs cell, cell culture fluid is DMEM, 10% foetal calf serum, 37 DEG C of cultivations, inoculate 1ml serum after cell monolayer, 32 DEG C of cultivations in the DMEM nutrient solution of 2% foetal calf serum, observation of cell pathology effect every day (cytopathic effect, CPE).After 3 days, culturing cell 70% comes off, and collects virus liquid, and inoculation 1ml covers with the 75cm of individual layer to another ready PAMs cell
2square vase, it is for subsequent use that all the other virus liquids are stored in-80 DEG C of refrigerators.Repeat to go down to posterity 4 times, virus titer reaches 5.5lg TCID
50/ ml.MARC-145 cell is at 75cm
2square vase in cover with individual layer, cell culture fluid is the DMEM nutrient solution containing 8-10% foetal calf serum, virus inoculation liquid 1 milliliter, and cultivate 4 days for 37 DEG C, blind passage 2-3 time, serum isolated viral has pathology on Marc-145 cell.The virus be separated reaches many generations at MARC-145 cell continuously, and produce continual and steady cytopathic effect, virus titer reaches 8.20lgTCID
50/ ml.
Often all need to confirm virus by titre and Immunofluorescent Antibody detection method for the virus gone down to posterity.Immuno-fluorescent antibody technique concrete operations are: the serum with porcine reproductive and respiratory syndrome clinical symptom, through saturated ammonium sulphate method, G or A albumen affinity chromatography column purification as discriminating polyclonal antibody.96 orifice plate inoculation MARC-145 cells, nutrient solution is the MEM containing 5-10% foetal calf serum, at 37 DEG C, 5%CO
2incubator overnight incubation, adds different dilution virus liquid and cultivates 48 hours, cell ethanol is fixed, dries in air.The purified polyclonal antibodies of pig is as first antibody, and the lsothiocyanates of mouse-anti pig is second antibody in conjunction with IgG antibody.
1.2.2 go down to posterity poison pathogenic and immunogenic relevant qualification
After selecting the poisons plaque purification that goes down to posterity in the 4th generation (HP-PRRSV-LZ-F4), the 20th generation (HP-PRRSV-LZ-F20) and the 65th generation (HP-PRRSV-LZ-F65), the MARC-145 cellular replication of individual layer is covered with in inoculation, and after results, adjustment virus titer is 10
5.5tCID
50/ 2ml.Gilt (before 6 months Pigs Inoculated reproductive and respiratory syndrome vaccine (HuN4-F112 strain)) is divided into 7 groups at random, often organize 19-20 head, isolated rearing, the virus liquid 2 milliliters that negative control group inoculation does not contain viral cell pyrolysis liquid 2 milliliters, experimental group inoculates HP-PRRSV-LZ-F4, HP-PRRSV-LZ-F20 and HP-PRRSV-LZ-F65 respectively, positive controls inoculates VR2332 and VR2385 virus liquid 2 milliliters respectively, and vaccine group inoculates HP-PRRSV-HuN4-F112 virus liquid 2 milliliters respectively.
Clinical observation comprises Respiratory symptoms (0-6 level), rectal temperature record (0-28 days), within 6,14,21,28 days, gets blood and carry out virus and Virus monitory after inoculation.Within 10 days, often organize in inoculation and select 10 dissections at random, remaining in inoculation dissection in 28 days, and within 10 days and 28 days, collect bronchoalveolar lavage fluid after inoculation.Commercial PRRSVELISA (IDEXX Laboratories, Inc.) detects the PRRSV antibody response of pig before and after inoculation, and the serum sample positive and negative ratio are 0.4, then can think that antibody response is positive.
Pathological observation is carried out to all pigs and organ.Observe lung, heart, spleen, lymphoglandula etc., organ fixedly uses microscopic examination by 10% neutral formalin after collecting.Lung, lymphoglandula wax embed, and monoclonal antibody detectable antigens, monoclonal antibody SDOW-171/5000 dilutes, and monoclonal antibody SR-301/1500 dilution mixture becomes antibody.Bronchial perfusate reverse transcription-pcr detects PRRSV virus, if detect negative, then detects porcine blood serum with PCR, if blood-serum P RRSV is negative, then detects the tonsilla of this pig with PCR.Every pig sterile collection bronchial perfusate 50ml, with Earle balanced salt solution (Sigma Chemical Co., St.Louis, Mo.) lavation obtains, be stored in-70 DEG C in order to RT-PCR., concrete grammar is: irrigating solution freeze thawing 3 times, 4 DEG C, 2, centrifugal 10 minutes of 000 × g, get 140 microlitre supernatant QIAamp viral RNA minikit (Qiagen Inc., Valencia, Calif.) by specification extraction RNA One Step RT-PCR kit (Qiagen Inc.) increases, 8 microlitre RNA templates are added in system, add 42 microlitre RT-PCR mixed solutions, add ORF7 primer.Virus sequence is analyzed, and often organizes 2 amplified productions, and one is inoculate latter 10 days, and another is the inoculation ORF5 sequence amplification product of 28 days, primer P5F:13696 to 13714; Primer P5R:14459 to 14437; PCR primer length 764bp.Product delivers to company's order-checking, contrasts with DNAstar (DNASTAR Inc., Madison, Wis.) computer program analysis.
Statistical study: rectal temperature data multivariate analysis of variance, how often pulmonary lesion per-cent, group sample size are applicable to Kruskal-Wallis analysis of variance (ANOVA), if P < 0.05, then significant difference.
2, result
2.1 virus purification, to go down to posterity
By constantly going down to posterity on variant cell, HP-PRRSV-LZ adapts to copy gradually on cell, and cytopathy progressively expands, but after reaching certain generation, cytopathy reduces and stable, the sudden change of virus also accumulation and cause virus virulence, pathogenicly to weaken in continuous passage copies.Observing this separation poison in succeeding generations is 8-10 hour at the replicative cycle of MARC-145 cell, and infection multiplicity is 0.0001-0.01,3 days 1 generations, and 200 generations of going down to posterity need about 400 day time.
2.2 antibody response
Selected pig detected through antibody assay kit 4 week age in experiment, and result is indicated as feminine gender.The all pig of VR2332 and VR2385 group was at 7-14 days antibody male rotaries, the PRRSV antibody response of HP-PRRSV-LZ-F4, HP-PRRSV-LZ-F20, HP-PRRSV-LZ-F65 group pig is different from VR2332, inoculate latter 6 days pig PRRSV antibody male rotaries, inoculate 14 days antibody titerss to peak, 4/9 pig detects PRRSV antibody in latter 7 days in immunity, 7/9 pig is 14 days antibody male rotaries after inoculation, and 8/9 pig is 21 days antibody male rotaries after inoculation.Latter 6 days of HP-PRRSV-HuN4-F112 inoculation, the antibody response of pig is 0,14 days 2/9 antibody male rotaries, and after 21 days, 4/9 pig antibody response sun turns, the 5/9 pig 28 days antibody response positive, and result is as shown in table 1:
Table 1 is separated the PRRSV antibody response (S/P=0.4 is sun turn) of passaged virus inoculation pig in 4 week age
| Group | 0DPI | 6DPI | 14DPI | 21DPI | 28DPI |
| Negative control | 0 | 0 | 0 | 0 | 0 |
| HP-PRRSV-HuN4-F112 | 0 | 0 | 0 | 0.35 | 0.38 |
| HP-PRRSV-LZ-F65 | 0 | 0.4 | 2.0 | 1.8 | 1.8 |
| HP-PRRSV-LZ-F20 | 0 | 0.5 | 2.0 | 1.7 | 1.5 |
| HP-PRRSV-LZ-F4 | 0 | 0.5 | 2.5 | 2.0 | 1.2 |
| VR2332 | 0 | 0 | 1.2 | 2.1 | 1.9 |
| VR2385 | 0 | 0 | 1.6 | 2.2 | 2.0 |
2.2 Viral diagnosis
Control group pig is under study for action without PRRSV infection, the pig of VR2332 and VR2385 group was infection 10 and 28 days, and bronchoalveolar lavage fluid finds that PRRSV nucleic acid is positive, HP-PRRSV-LZ-F56 group after inoculation 10 days, RT-PCR detected result 6/10 is positive, and it 28 days 8/10 is positive for infecting; HP-PRRSV-LZ-F20 10 days after infection, 8/10 is positive, infects after 28 days, and the pig RT-PCR of 9/10 is positive; HP-PRRSV-LZ-F4 group infection after 10 days 9/10 pig RT-PCR result positive, after 28 days, the RT-PCR result of pig of 10/10 is positive.In HP-PRRSV-HuN4-F112 group, infect after 10 days, the pig RT-PCR result of 6/10 is positive, and after 28 days, the pig detected result of 5/9 is positive.
2.3 are separated malicious, to go down to posterity poison sequential analysis
The porcine reproductive and respiratory syndrome virus be separated, passage cell adapt to malicious ORF5 gene sequencing result and show, HP-PRRSV-LZ-F56, HP-PRRSV-LZ-F20, HP-PRRSV-LZ-F4 belong to same gene evolution race, HP-PRRSV-LZ-F56 ORF5 gene and HP-PRRSV-HuN4-F112 amino acid identity are 96.5%, HP-PRRSV-LZ-F4ORF5 gene and HP-PRRSV-HuN4-F112 amino acid identity are the homology of 99.5%, HP-PRRSV-LZ-F4 virus O RF5 gene coding amino acid and HP-PRRSV-HuN4-F112 is 96%.The sequence of its ORF5 of virus that HP-PRRSV-LZ-F56, HP-PRRSV-LZ-F20, HP-PRRSV-LZ-F4 are once separated afterwards at pig interior generation and have 99.5-100% homology before going down to posterity.
2.4 go down to posterity poison pathogenic and immunogenic relevant qualification
The pig of negative control group, HP-PRRSV-LZ-F65 and vaccine group is at any time all without the symptom of blue otopathy, and rectal temperature and respiratory symptom classification are in normal range.The pig of inoculation VR2332 and VR2385 virus group starts to occur that expiratory dyspnea is short of breath and lethargic sleep symptom after inoculation for 5-7 days, and after 10 days, illness increases the weight of, and within 14-21 days, disappears.The pig of inoculation HP-PRRSV-LZ-F4 virus liquid has respiratory symptom to occur (dyspnea and tachypnea) for 21 days in inoculation, and fever and lethargic sleep, 21 and 28 days after inoculation, symptom slightly developed into moderate.The symptom of the pig of these groups of the statistical study of clinical symptom ranked data is without significant difference.Point progression of negative control group and HP-PRRSV-LZ-F65 and vaccine group is lower than HPRRSV-LZ-F4 and HP-PRRSV-LZ-F20 group, and the clinical scale difference between group reduces with days post inoculation.The statistics of the rectal temperature duplicate detection result of each group of pig shows, the rectal temperature of each group of pig has significant difference sometime, P < 0.05, often organize rectal temperature mean value to change with days post inoculation, the change often organized is different, trendless can be followed, but 14,16,18 days temperature have decline after inoculation.
Lung's mass lesions is in table 2, and the mean value (standard deviation) of pulmonary lesion valuation, the pulmonary lesion type that each group PRRSV produces is the same, but degree varies, and damage characteristic shows as at pneumonia position ubiquity lung failure, mottled brown.The most common site that this damage occurs is cranium belly, but finds that it is multifocal appearance in whole lung.10 days after inoculation, VR2385 and VR2332 group pig serious lung injury degree was significantly higher than other groups, P < 0.0001.Inoculate latter 28 days, HP-PRRSV-LZ-F2 group lung damage far above other groups, P < 0.002.
The table 2 PRRSV lung damage that 10 days and 28 days produce after inoculation compares
Lung's microdamage classification is divided into 0-6 level by intermittence pneumonia: 0, without microdamage; 1, slight multiple spot damage; 2, slight spread is damaged; 3, moderate multiple spot damages; 4, moderate multiple spot diffuse injury; 5, serious multiple spot damage; 6, serious multiple spot diffuse injury or serious disperse intermittence pneumonia.Lung's microscopic lesion that each group of PRRSV causes is the same with type of impairment, but severity, generation, cure time are different.Pulmonary lesion is characterized as 2 type pneumonocyte size degradations, hypertrophy and hyperplasia, and monocyte is aseptic invades profit, and alveolar exudate and Necrotic Debris quantity increase.VR2332 and VR2385 group pig has slight and moderate lesion after inoculation for 28 days, and after inoculation in 10 days, injury of lung has moderate, major injury.The pulmonary lesion of HP-PRRSV-LZ-F4, HP-PRRSV-LZ-F20 group almost not to have after 10 days or very little in inoculation, 28 days after inoculation, has slight and moderate lesion.HP-PRRSV-LZ-F65, HP-PRRSV-HuN4-F112 and negative control group inoculation after 10 days and 2 days microdamage not obvious.The results are shown in Table 2.The inoculation of VR2332 and VR2385 group after 10 days lung microdamage be seriously significantly higher than other groups P < 0.0001, inoculate VR2332 VR2385, HP-PRRSV-LZ-F4 after 28 days, the injury of lung of HP-PRRSV-LZ-F20 group far above negative control group, HP-PRRSV-HuN4-F112 vaccine group, HP-PRRSV-LZ-F65 group, P < 0.0001.Without myocarditic micro-symptom in the pig of negative control group, HP-PRRSV-HuN4-F112, HP-PRRSV-LZ-F65 group, and VR2332, VR2385, HP-PRRSV-LZ-F4, in HP-PRRSV-LZ-F20 group pig, 8/20,8/20,8/19,7/20 is had to have slight lympho-plasmacytic myocarditis respectively.
2.3 antibody response
Selected pig detected through antibody assay kit 4 week age in experiment, and result is indicated as feminine gender.The all pig of VR2332 and VR2385 group was at 7-14 days antibody male rotaries, the PRRSV antibody response of HP-PRRSV-LZ-F4, HP-PRRSV-LZ-F20, HP-PRRSV-LZ-F65 group pig is different from VR2332, inoculate latter 6 days pig PRRSV antibody male rotaries, inoculate 14 days antibody titerss to peak, 4/9 pig detects PRRSV antibody in latter 7 days in immunity, 7/9 pig is 14 days antibody male rotaries after inoculation, and 8/9 pig is 21 days antibody male rotaries after inoculation.Latter 6 days of HP-PRRSV-HuN4-F112 inoculation, the antibody response of pig is 0,14 days 2/9 antibody male rotaries, and after 21 days, 4/9 pig antibody response sun turns, the 5/9 pig 28 days antibody response positive, and result is as shown in table 3:
Table 3 is separated the PRRSV antibody response (S/P=0.4 is sun turn) of passaged virus inoculation pig in 4 week age
| Group | 0DPI | 6DPI | 14DPI | 21DPI | 28DPI |
| Negative control | 0 | 0 | 0 | 0 | 0 |
| HP-PRRSV-HuN4-F112 | 0 | 0 | 0 | 0.35 | 0.38 |
[0062]
| HP-PRRSV-LZ-F65 | 0 | 0.4 | 2.0 | 1.8 | 1.8 |
| HP-PRRSV-LZ-F20 | 0 | 0.5 | 2.0 | 1.7 | 1.5 |
| HP-PRRSV-LZ-F4 | 0 | 0.5 | 2.5 | 2.0 | 1.2 |
| VR2332 | 0 | 0 | 1.2 | 2.1 | 1.9 |
| VR2385 | 0 | 0 | 1.6 | 2.2 | 2.0 |
Embodiment 2 primordial seed criticizes the stability of PRRSV-LZ-F65 in pig body (pathogenic and attenuation) research
1, method:
PRRSV-LZ-F65 Pigs Inoculated body, pig is divided into 6 groups, the 1st group 5 pig intranasal vaccination PRRSV-LZ-F65, and control group 3 pig intranasal vaccination sterile liquids, every day observes clinical symptom: outward appearance, breathing, ight soil etc.Weigh after 6 days, bloodletting, to slaughter.Collect the lung lavage thing of each pig body, through freeze thawing-thawing; Collect porcine blood serum, prepare every pig lung lavage thing 2ml, serum 2ml mixture, be used for the pig body of inoculation second group and the second control group, continuous passage was inoculated into for the 6th generation, often for repeated observation, collect pulmonary lavage thing and serum.Often group is raised separately but condition is the same.
After pig virus inoculation, 1-6 days sample of blood, drawn after inoculation, periodic detection body temperature, during research, every day detects the healthy state of every pig, and record description is as follows:
1. appearance normally=0; Dejected depression=1; Excited=2; Lethargic sleep/death=30.
2. breathe normal=0; Sneeze=1; Cough=1; Breathe fast/very brief=2; Breathe with difficulty=3.
3. ight soil normally=0; Dry=1; Loose=2; Fluid=3.
4. eyes normally=0; Moist=1; Unglazed=2; Depression=3.
5. nostril normally=0; Watery secretion=1; Red/red and swollen=2; Crust ulcer=3.
6. mouth normally=0; Slobber=2; Ulcer=3.
7. appetite normally=0; Decline=1; Apocleisis=3.
Pig is weighed before immunity and postmortem, by often organize mean body weight compare acquisition often organize body weight increase situation, experimental group, control group serum ELISA and neutralizing antibody method detect, in serum, the separation of PRRSV virus adopts MA-104 cell, overall white blood cell count(WBC) COULTER COUNTER (Beckman company), postmortem often organizes the lung of pig body and record, pig lung is divided into 7 parts, detect the per-cent that each several part damages, redness occupies whole lung, the pathological parameter of lung is as follows: lobus sinister lung relates to redness, percent injury; Left cardiopulmonary portion relates to pathology damage per-cent; Lung left spaced design pathology per-cent; The per-cent of lung's right part design pathology; Right cardiopulmonary portion relates to pathology damage per-cent; Lung right septum design pathology per-cent; Amount to.
2, result
Poison is passed by cell and pig body, the body temperature of each group of pig body, weight does not change, lung loses without pathology, each group of pig body is negative antibody by ELISA and neutralization test inspection at duration of test at duration of test, infect in the porcine blood serum of viral HP-PRRSV-LZ-F65 and passback and pulmonary lavage liquid at the 6th day 60-100% and be separated to virus, and control group virus is negative, in the serum of initial continuous 3 pigs of going down to posterity and lung-douching fluid, the pig of 20-40% has virus, go down to posterity for follow-up 3 times, 50-80% is separated to PRRSV virus, based on these data, can find out that HP-PRRSV-LZ-65 goes down to posterity 6 times at pig body, its virulence does not reverse, its security aspect virulence attenuation of is described, without atavism.
The virus of embodiment 3 porcine reproductive and respiratory syndrome virus attenuation strain HP-PRRSV-LZ-F65 seed lot and go down to posterity malicious immunogenicity and safety experiment
1, material
The pig in 1.1 laboratory animal: 100-120 4 week age, PRRSV antibody, mycoplasma pneumonia negative antibody, pig annulus 2 C-type virus C PCR detects feminine gender.Be divided into the isolated rearing of A-F group.
Experimental vaccine: porcine reproductive and respiratory syndrome vaccine HP-PRRSV-LZ-F95, HP-PRRSV-LZ-F80, (HP-PRRSV-LZ-F65 is the original seed lot of vaccine to HP-PRRSV-LZ-110,80 on behalf of main seed lot, 110 on behalf of working seed lots, MARC-145 passage is adopted to cultivate, concrete grammar is as embodiment 1), domestic porcine reproductive and respiratory syndrome vaccine HP-PRRSV-HuN4-F112, external import vaccine Ingelvac PRRS MLV (BoehringerIngelheim, USA) is purchased from market.Porcine reproductive and respiratory syndrome culturing cell MARC-145 purchased from American ATCC.
2, method
Reject underproof piglet for each group, often group respectively intramuscular immunisation experimental vaccine and domestic, import commercial vaccine, the immune MARC-145 cell culture supernatant of F group, as placebo, after immune group immunoprophylaxis 3 days, introduces 9 qualified pigs unavoidably in contrast.Often group immunity and pig daily inspection clinical symptom rather, rectal temperature and situation of ingesting, rectal temperature and situation of ingesting start to observe in immunity for first 3 days.Immune swine blood sample gathers for 0,3,6,9,12,15 and 18 day after exempting from, and the blood sample often organizing non-immune swine gathers for 3,6,9,12,15,18 and 24 days after exempting from, serum be stored in-80 DEG C for subsequent use.
Isolated viral and detection antibody, non-immune swine then detects PRRSV virus with RT-PCR, and method is as embodiment 1.Shift out from group as early as possible after ill toxemic non-immune swine finds.Also collect nasal cavity, oral cavity, anal swab when the pig of immune group is taken a blood sample simultaneously, immerse 2mL DMEM immediately and be stored in-80 DEG C of refrigerators in order to isolated viral.Weigh when pig gets blood at every turn, estimate 0-6 days, 7-15 days, every day weight gain (DWG) after 16-21 immunization.7,14,21 days that test, often organize pig and the excessive Veronal sodium (Dolethal of control group 5 pigs of immunity, Vetoquinol, France) intravenous injection anesthesia, under getting lung, tonsilla, thymus gland, ileum, jaw, inguinal region is shallow, breastbone, and mediastinum and mesenteric lymph nodes are stored in-80 DEG C in order to isolated viral.Lung is used as pathological tissue research.Dissection pig blood, nasal cavity, buccal swab also store-80 DEG C in order to isolated viral.At 24 days of test, the clinical sample of all contact pigs carried out virus purification and titration, inoculation individual layer PAM and MARC-145 cell, every part is repeated 4 times, and 37 DEG C adsorb 90 minutes, wash 2 times with cell culture fluid, cultivate with containing 10% foetal calf serum DMEM, 37 DEG C, 5%CO
2incubator cultivates 6 days, estimates cytopathic effect during 4-6 days, and positive control is porcine reproductive and respiratory syndrome virus TJM-F92, and every hole is respectively according to 10
3tCID
50, 10
2tCID
50and 10TCID
50inoculation, result 10TCID
50pAM sensitivity is infected.Virus-free DMEM or serum as negative control, PRRSV virus titer Reed and Muench method.The PRRSV antibody test of blood serum special is as embodiment 1, and respiratory organs damage pathology detection method is by embodiment 1, and immunohistochemical observation is by embodiment 1, and statistical test adopts embodiment 1 method.
3, result
3.1 clinical symptom and growth property
Observe and find, healthy after the pig immunity of group A-F, observe, also without porcine reproductive and respiratory syndrome clinical symptom without local and common side reaction.In whole experimentation, the body temperature of each group pig is in normal range, increases without significance, and the pig of contact is healthy at duration of test, without clinical symptom and fever phenomenon.The weightening finish change of each group increases in time, and each group weightening finish is at any time without significant difference.
3.2 lung's mass lesions
A-F group pig obviously damages without any lung and observes, the each group of pulmonary lesion rank value no significant difference observed, as table 4, each group of pulmonary lesion reduces in time, at 3 time period no significant differences, show HP-PRRSV-LZ-F65 by passage to 80-110 for rear (HP-PRRSV-LZ-F95, HP-PRRSV-LZ-F80, HP-PRRSV-LZ-110), pathogenic without replying.
Lung's mass lesions rank value (average ± standard deviation) dissected by table 4 respectively group pig
3.3 microdamage
Control group Zhu Ti lung microscopic inspection without any damage, in experimental group pig some to have intermittence pneumonia disease to get a haircut raw, thicken with slight or moderate alveolus wall, between alveolar full scavenger cell, the disperse of many stoves position for feature.Intermittence pneumonia increases the weight of in time, and as table 5, the number of A-E Zu Zhu lung microdamage and grade are without significant difference during experimental observation, and impairment scale is all slight 1 grade.
Lung's microdamage rank value (average ± standard deviation) dissected by table 5 respectively group pig
The viremia of 3.4 immune group pigs
Control group sample is all viral without PRRS by which kind of cell detection, after the immunity of A-F group, the virus purification of different serum the results are shown in Table 6, all vaccine strain virus can induce pig body to produce viremia, although often group produces the pig number of individuals difference of viremia, but adopt MARC-145 cellular segregation virus, most pigs detects result for positive at immunity latter 3 days isolated virals, each group of the time that viremia occurs there is no significant difference, P > 0.05, the immunity A papova mass formed by blood stasis positive rate day after tomorrow is 93.8%, B group positive rate is 86.7%, C group positive rate 73.3%, D group positive rate is 100%, E group positive rate is 80%.Use PAM isolated viral, most sample virus detects result for negative, and A-F group pig detects positive rate and is respectively and all becomes virus-positive afterwards in 4.2%, 5.9%, 5.0%, 4.9%, 6.1%, 12 days.The pig virus titer of all groups peaks in off-test the last week, and virus titer, the viral time of occurrence of each group pig there is no significant difference, P > 0.05.
Table 6 MARC-145 is separated each group of porcine blood serum virus results with PAM cell cultures
The distribution in the tissue of 3.5 viruses
Different tissues organ sample is through MRAC-145 cell cultures, all tissues obtained from control group fail to be separated to virus, in immune group, the histoorgan of different time all can be separated to virus, the frequency that lung, tonsilla, thymus gland, ileum, lymphoglandula, spleen are separated to virus is respectively 44.4%, 81.7%, 37.8%, 33.9%, 36.8%, 21.7%, illustrates that virus is main at tonsilla.Each group of porcine tissues isolated viral positive rate without significant difference, P > 0.05.。Respectively organize the PRRSV of pig body organ by PAM cellular segregation, each group is separated to virus in the tonsilla of 21 days after exempting from, and virus-positive rate is very low.
3.6 ImmunohistochemistryResults Results
Control group pig lung sections does not have PRRSV Detection of antigen to arrive, each group of pig lung sections detects that the probability of PRRSV antigen is extremely low, and A-F group 7 and 14 days all organ-tissues section Detection of antigen after exempting from are negative, but after exempting from 21 days, A group has 3/5, B group 2/5, C group 2/5, D group 1/5, E group 2/5, F group 3/5 pattern detection, to PRRSV antigen, is classified as 1, mainly in pulmonary alveolar macrophage tenuigenin.
3.7 virus discharges
Immunity each group pig is negative at duration of test isolated viral from the body fluid negative control group, 6-15 days after immunity, identical from the frequency of nasopharynx isolated viral, total separation positive rate is 8.33%, exempt from latter 3 days to exempting from latter 18 days, the positive rate being separated to virus from nasal cavity is 4.67%, immune latter 6 days until off-test, it is 5.67% that ight soil is separated to PRRSV virus-positive rate, the positive rate of A group excretion virus is 8.0%, it is 4.89% that B papova is separated positive rate, it is 5.33% that C papova is separated positive rate, D group is 6.67%, E group is 6.63%, each group of isolated viral compares, P > 0.05, without significant difference.
The infectivity of 3.8 vaccine strains
All vaccine strains all can pass to non-immune pig from the pig of immunity, latter 9 days of immunity, just has viremia to find, within 12 days, respectively organizes non-immune swine majority and occur viremia in non-immune pig, 18 days there is viremia in whole non-immune pig, and still have viremia when off-test.Each group occurs that the per-cent of viremia compares, P > 0.05, there was no significant difference.Respectively organize the serum of pig rather by PAM separation and Culture, after immunity, within 18-21 days, be separated to virus, A-E group titre is respectively 2.6,2.5,3.2,1.8,1.5TCID
50/ mL, the toxemic incidence there was no significant difference of each group swine disease.
3.9 antibody male rotary
A-C group is at immunity latter 7 days antibody male rotaries, wherein animal starts sun turn at immunity latter 12 days antibody rather, A group have 1, B group 2, C group 2 pig antibody male rotaries, 15 days, each group 5 pig antibody rather all sun turn, D-E group 15 days antibody starts sun and turns, D group 2, E group 2, within 24 days, 5 whole sun turn.
3.10 virus discharge
9-24 days, A-C group detection 102 samples after immunity, wherein 12 detect vaccine virus, account for 11.77%, and draining viral probability height is nasal cavity, oral cavity, ight soil successively.D group detection 45 samples, isolated viral 8, accounts for 17.8%, and in ight soil, isolated viral frequency is the highest, and be about 11.1%, E group detection, 115 samples, isolated viral 11.3% in ight soil, nasal cavity isolated viral is 6.9%, and oral cavity is 4.3%.Each papova excretion is compared without significant difference.
In conjunction with the embodiments 2 and 3, can HP-PRRSV-LZ-F65 be found out, at least 65-110 generation keep attenuation no pathogenicity stable, without atavism.Show its security used at nature, even if consecutive infection in pig body, at least 6 generations, are without phenotypic variation.Also show that it is reasonable and safe for being no more than 45 generations from primordial seed to work seed restriction passage number at preparation engineering.
Embodiment 4 porcine reproductive and respiratory syndrome is separated malicious HP-PRRSV-LZ-F65 and pig body can be induced on PAMs cell comparatively early to produce Interferon, rabbit and neutralizing antibody
1, materials and methods:
The preparation of 1.1 porcine alveolar macrophages: cell culture fluid composition, RPMI-1640 substratum (Gibco Laboratories, Grand Island, NY, USA), 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, Laboratories, Logan, UT) the two property mycin of 10,000 units per ml penicillin, 10,000 mcg/ml Streptomycin sulphate, 25 microgram milliliters.20 age in days pigs are got pig alveolar cell and grow up to individual layer, cell dissociation buffer is 0.25% trypsinase and EDTA mixed solution (0.2 mg/ml), centrifugal removing trypsinase, EDTA after primary monolayer cell digestion, add cell culture fluid and make cell concn be 2X10
5cells/ml, adds 100 microlitre to 96 orifice plates, 37 DEG C, cultivate in 5% carbonic acid gas incubator.
1.2 attack poison: the preparation of encephalitis b virus (SA-14-14-2Vero cell adapted strain is provided for studying by Chinese medicine and biological products assay institute research institute), use 25cm
2cell bottle Cultivation of Vero, after cell monolayer, inoculate encephalitis b virus by MOI0.01, cultivate after 4 days, collect virus liquid, routinely mensuration virus titer TCID
50,-80 DEG C frozen for subsequent use.
1.3 experimental vaccine
VR-2385、HP-PRRSV-LZ-F2、HP-PRRSV-LZ-F65、HP-PRRSV-TJM-F92
1.4 Interferon, rabbit methods for measuring
Porcine reproductive and respiratory syndrome virus HP-PRRSV-LZ-F65, VR-2385, HP-PRRSV-LZ-F2, HP-PRRSV-TJM-F92 is at MARC-145 cell, the virus liquid cell culture fluid doubling dilution gone down to posterity in PAMs primary cell, 10 times of dilutions again, draw 100 microlitres respectively to add in 96 orifice plate monolayer cells (discard enchylema and repeatedly pat and remove residual liquid several times), each extent of dilution repeats 1-2 hole, 6-12 hole is had to add 100 microlitres of cells nutrient solutions as cell or virus control in every plate, after incubated overnight, remove liquid, every hole adds 100 microlitres containing 400TCID
50encephalitis b virus liquid, comprise virus control wells, cell control well adds the same cell culture fluid of 100 microlitres, hatches after 24 hours, when virus control wells observes the cytopathic effect of 50-90%, cell plate stop hatching, discard liquid, wash 2 times with 0.85% physiological saline, add dyeing stationary liquid and fix 20 minutes, stationary liquid distilled water is prepared, containing 0.5% Viola crystallina, 5% formalin (v/v), 50% ethanol (v/v), 0.85% sodium-chlor.Discard stationary liquid, every hole dyestuff 200 microlitre 2-methyl ethanol wash-outs after dry, stir wash-out 20 minutes, get each extent of dilution 2 hole elutriant and put into the absorbance value that 96 orifice plates measure 550 nanometers on spectrophotometer, the dilution logarithmic value of Interferon, rabbit and absorbance value mapping, reduce extent of dilution=0.5 × (the cell controls absorption value+virus control absorption value) of 50% cytopathic Interferon, rabbit.Recombinant Swine Interferon, rabbit IFN-α (purchased from Chemicon, Temecula, California, USA) is as standard, and interferon activity 1 unit is the dilution inverse of generation 50% cytopathic effect inhibition.Vero cell, Latex agglutination test are used for the detection of interferon activity, are separated the supernatant liquor that poison is cultivated on MARC-145 cell, porcine alveolar macrophage, detect by the measuring method of Interferon, rabbit,
2, result
2.1 HP-PRRSV-LZ-20 on MARC-145 cell on the Interferon, rabbit of induced high levels
By monkey ELISA kit, HP-PRRSV-LZ-F65, VR-2385, HP-PRRSV-LZ-F2, HP-PRRSV-TJM-F92 are measured respectively at the supernatant liquor of MARC-145 cell cultures, the amount average out to 38.5pg/ml of result display HP-PRRSV-LZ-F65 inducing interferon, far away higher than the amount of VR-2385, HP-PRRSV-LZ-F2, HP-PRRSV-TJM-F92 induction.These results show: HP-PRRSV-LZ-F65 can induce MARC-145 Cells Interferon Production, and VR-2385, HP-PRRSV-LZ-F2, HP-PRRSV-TJM-F92 generation to MARC-145 Cell Interferon has certain restraining effect.
The Interferon, rabbit of 2.2 HP-PRRSV-LZ-20 induced high levels on primary pulmonary alveolar macrophage
HP-PRRSV-LZ-F65, VR-2385, HP-PRRSV-LZ-F2, HP-PRRSV-TJM-F92 inoculates PAMs cell cultures respectively by MOI=0.01, VR-2385 HP-PRRSV-TJM-F92s in contrast, carry out interferon activity and detection by quantitative, result shows the interferon activity containing 10u/ml in HP-PRRSV-LZ-F65 supernatant liquor, the pulmonary alveolar macrophage repeating 3 young pigs cultivates the Interferon, rabbit of HP-PRRSV-LZ-F20 supernatant containing 9U ± 2/ml, various viral supernatants is detected with pig interferon test kit, HP-PRRSV-LZ-F20 induces porcine alveolar macrophage to produce average 30.5pg ± 1.3 of Interferon, rabbit.HP-PRRSV-TJM-F92 is at the Interferon, rabbit of PAMs primary cell culture supernatant containing 1.5U ± 0.8/ml, content 8 ± pg/ml, the supernatant liquor that VR-2385, HP-PRRSV-LZ-F2 cultivate on scavenger cell containing Interferon, rabbit, can not suppress the pathology of pig encephalitis b virus on Vero cell.
Result shows HP-PRRSV-LZ-F65 and the poison that goes down to posterity is compared on pig body induction of high-caliber Interferon, rabbit with reference to vaccine strain.
The growth of embodiment 5 HP-PRRSV-LZ-F65 on vaccine stroma cell
1, HP-PRRSV-LZ-F65 virus liquid by infection multiplicity (MOI) 0.01,0.1,1.0TCID
50inoculating cell, collecting cell supernatant liquor, amounted to 5 days and measured virus titer every day, and the cell of inoculation 0.01MOI is after 3 days, and virus titer can reach 6.6-7.0log TCID
50/ ml is 3.0logTCID by the titre of the product poison of MOI=1 inoculating cell
50/ ml, the 5th day titre 2.0logTCID
50the titre that/ml produces poison on the 3rd day by the cell that 0.1MOI inoculates is 6.67log TCID
50, the 1st day is 3.0logTCID
50/ ml, the 5th day is 6.0logTCID
50/ ml, the virus of therefore pressing 0.01MOI inoculation 3-5 days titres apparently higher than by 0.1, the cell of 1MOI inoculation produces malicious.
2, Virus plaque clone purification method: by viral dilution to 10 to be purified
-6, get 10
-2, 10
-3, 10
-4, 10
-5, 10
-6extent of dilution inoculates the MARC-145 cell that 6 well culture plates have covered with individual layer, each extent of dilution inoculates 1 hole, establish 1 cell control well simultaneously, 100 microlitres are inoculated in every hole, and 37 DEG C adsorb 1 hour, remove adsorption liquid, 2 times are cleaned with serum-free MEM, heat the low melting point agar of water-bath to 37 DEG C, 3 milliliters, every hole, is positioned over 37 DEG C of 5%CO after room temperature cooling
2incubator cultivates 4-5 days, observes plaque.Select most high dilution virus inoculation hole picking independence plaque, become the MARC-145 cell of F+1 pickup kind 6 well culture plate well-grown individual layer, continue to go down to posterity, pass after 1 generation through 6 orifice plates and be F+2 generation.In every 10 generations of biography continuously, return 1 time, carry out a plaque clone purification.
Respectively respectively Plaque assays is carried out to HP-PRRSV-LZ-65, HP-PRRSV-LZ-F20, HP-PRRSV-TJM-F92 according to the method described above, 4 days cell plate neutral red stainings after virus inoculation, the plaque that HP-PRRSV-LZ-F20, HP-PRRSV-LZ-F65 produce is less, diameter is about 2-3 millimeter, it is larger that HP-PRRSV-TJM-F92 produces plaque, diameter is 5 millimeters, and result shows that HP-PRRSV-LZ-F20 copies on MARC-145 cell and is different from HP-PRRSV-TJM-F92.3 kinds of viruses are pressed 0.05MOI and are inoculated MARC-145 cell and primary PAM cell, and HP-PRRSV-LZ causes primary PAM necrocytosis, and HP-PRRSV-TJM-F92 is induction of visible cell pathology, and HP-PRRSV-LZ-F65 induced pathologies is smaller not obvious.
3, virus titer measures
It is 3.8lg TCID respectively that HP-PRRSV-LZ-F20, HP-PRRSV-LZ-F95, HP-PRRSV-TJM-F92 infection PAMs cell nutrient solution of 24 hours measures titre on MARC-145 cell
50/ ml, 5.5lgTCID
50/ ml and 3.2lgTCID
50/ ml.Result illustrates that the output of HP-PRRSV-LZ-F95 on pig alveolar cell is higher than the malicious HP-PRRSV-LZ of original separation, higher than with reference to malicious HP-PRRSV-TJM-F92, describe the adaptability of HP-PRRSV-LZ-F95 on cell and Seedling height, attenuation process is successful, is suitable for producing vaccine.
Embodiment 6 HP-PRRSV-LZ-65 induces the antibody of anti-homotype or special-shaped virus to produce test at pig body
(1) the antibody production of HP-PRRSV-LZ-F65, VR-2385, HP-PRRSV-TJM-F923 kind virus induction homology virus:
1, method
The 3-8 kilogram of negative piggy of PRRSV is divided into 8 groups, (I.N) immune HP-PRRSV-LZ-F65, HP-PRRSV-TJM-F92, VR-2385 virus liquid 1 milliliter respectively in 1-3 group nose, titre 10
5tCID
50/ milliliter, immune HP-PRRSV-LZ-F65, HP-PRRSV-TJM-F92, VR-2385 virus liquid of 5-7 group muscle (I.M) 1 milliliter, titre 10
5tCID
50/ milliliter, 4 groups, 8 groups as a control group, and each group virus inoculation is after 3 days, and coming off and infecting for assessment porcine reproductive and respiratory syndrome virus, often organizes increase by 2 and do not contact the pig of virus for contrast, PBS, pH=7.2 injection liquid as a control group.Pig is observed every day, inoculates first 3 days and infects and weigh for 14,56 days.Blood sample before inoculation, postvaccinally to take weekly, the serum of every pig be stored in separately-80 DEG C detect in order to serum virus antibody, virus purification.Infect virus latter 14 days, 56 days anesthesia seen at post-mortem pulmonary lesions and classification with Sodital pig, collect pulmonary lavage liquid, be separated pulmonary alveolar macrophage and add dimethyl sulfoxide (DMSO) (DMSO).Also observe lung pathologies change after postmortem, intermittence pneumonia presses 0-6 marking, and 0 represents do not have symptom, and 6 represent the intermittent pneumonia of serious diffusion, and result is as shown in table 7.
Table 7 test design and marking result
| 1 group | 2 groups | 3 groups | 4 groups | 5 groups | 6 groups | 7 groups | 8 groups | |
| Approach | I.N | I.N | I.N | I.N | I.M | I.M | I.M | I.M |
| PRRSV | LZ-F65 | TJM-F92 | VR-2385 | PBS | LZ-F65 | TJM-F92 | VR-2385 | PBS |
| Head number | 6 | 6 | 6 | 6 | 3 | 3 | 3 | 3 |
| Control group head number | 2 | 2 | 2 | 0 | 2 | 2 | 2 | 0 |
| Infect postmortem in 14 days | 3 | 3 | 3 | 3 | 0 | 0 | 0 | 0 |
| Infect postmortem in 56 days | 5 | 5 | 5 | 3 | 2 | 2 | 2 | 2 |
2, result
In each group, pig average weight gain every day of inoculation HP-PRRSV-LZ-F65 is greater than the pig of inoculation HP-PRRSV-TJM-F92, equal with the pig of inoculation PBS, but without significant difference.Result illustrates that the growth effect of HP-PRRSV-LZ-F65 to pig is little.
MARC-145 cell to measure in serum and the antibody titers of HP-PRRSV-LZ-F65, no matter in nose or intramuscular infection, the pig that HP-PRRSV-LZ-F65 infects, all induction of the generation of the neutralizing antibody of high titre.At intranasal infection after 49 days, the neutralizing antibody average titer that HP-PRRSV-LZ-F65 induces is the neutralizing antibody average titer that the neutralizing antibody average titer 10, VR-2385 of 90, HP-PRRSV-TJM-F92 induction is induced is 18.The intranasal infection neutralizing antibody average titer that after 56 days, HP-PRRSV-LZ-F65 induces is the neutralizing antibody average titer that the neutralizing antibody average titer 8, VR-2385 of 90.0, HP-PRRSV-TJM-F92 induction is induced is 32.Contact control group pig 49 days after infection, the serum neutralizing antibody average titer of HP-PRRSV-LZ-F65 induction is 45, and infecting latter 56 days neutralizing antibody average titers is 48; Contact control group pig TJM49 days after infection, 56 days neutralizing antibody average titers are 6, and the porcine blood serum of the touch controls that VR-2385 infects all did not detect antibody at 49 days, 56 days.
Intramuscular infection 49 days, the neutralizing antibody average titer that HP-PRRSV-LZ-F65 induces is the neutralizing antibody average titer that the neutralizing antibody average titer 20.0, VR-2385 of 90.0, HP-PRRSV-TJM-F92 induction is induced is 10.0.The neutralizing antibody average titer infecting the induction of HP-PRRSV-LZ-F65 after 56 days is the equal titre of neutralizing antibody that the neutralizing antibody average titer 5, VR-2385 of 68.0, HP-PRRSV-TJM-F92 induction is induced is 20.0.Contact control group pig 49 days after infection, the serum neutralizing antibody average titer of HP-PRRSV-LZ-F65 induction is 78, and infecting latter 56 days neutralizing antibody average titers is 70.0; Contact control group pig TJM-F9249 days after infection, 56 days NAT average out to 4, and the porcine blood serum of the touch controls that VR-2385 infects did not detect antibody at 49 days, 56 days.
These results show, HP-PRRSV-LZ-F65 infection induced pig body comparatively early produces the neutralizing antibody of anti-HP-PRRSV-LZ-F65, and titre is higher, find with One-way ANOVA, HP-PRRSV-LZ-F65 is no matter in nose or intramuscular infection pig body, and the antibody of generation is significantly higher than the antibody (P<0.01) that HP-PRRSV-TJM-F92 infects (P<0.05), VR-2385 infected group.HP-PRRSV-LZ-F65 just has the antibody tormation that can detect in 28 days after infection, within latter 56 days, reaches maximum to infection.The antibody titers of HP-PRRSV-LZ-F65 intranasal infection pig higher than HP-PRRSV-TJM-F92 intranasal infection pig 2-6 doubly, only after infection the NAT of 28 days there were significant differences.2 pigs that HP-PRRSV-LZ-F65 infects created at 28 days and just can neutralizing antibody be detected, and in infection, within 56 days, all neutralizing antibody is positive afterwards.The pig of HP-PRRSV-TJM-F92 intranasal infection had the neutralizing antibody that can detect at 35 days, and the pig of VR-2385 intranasal infection had a pig to create and can neutralizing antibody be detected at 56 days.
In the pig of intramuscular infection, HP-PRRSV-LZ-F65 creates higher neutralizing antibody, the antibody titers produced afterwards for 49 days is significantly higher than the NAT of the generation of HP-PRRSV-TJM-F92 infected pigs, infect 56 days, the antibody of HP-PRRSV-LZ-F65 infected pigs is 128.0,16.0,64.0 respectively, and HP-PRRSV-TJM-F92 infected pigs only had the antibody of 1 pig to reach 32, VR-2385 infected group at 56 days also has 1 to measure antibody at 56 days.HP-PRRSV-LZ-F65, HP-PRRSV-TJM-F92 intranasal infection is induced the neutralizing antibody of pig higher than intramuscular infection and is had significant difference (P < 0.05), and nose is interior or intramuscular infection VR-2385 is very micro-on the impact of induction neutralizing antibody.HP-PRRSV-LZ-F65 Pigs Inoculated detected neutralizing antibody at 35 days, lasted till 56 days, and the group Pigs Inoculated of HP-PRRSV-TJM-F92 only has 1 neutralizing antibody to be detected in infection after 56 days.Result is as shown in table 8.
Table 8 respectively group induction produces the situation inverse of antibody greatest dilution of 50% virus (in and) of PRRSV neutralizing antibody
Above-mentioned is the antibody production of HP-PRRSV-LZ-F65, VR-2385, HP-PRRSV-TJM-F923 kind virus induction homology virus.
(2) the antibody production of HP-PRRSV-LZ-F65, VR-2385, HP-PRRSV-TJM-F923 kind virus induction heterologus virus
To HP-PRRSV-LZ-F65 intranasal infection group after 49,56 days in serum and the ability of allos PRRSV strain VR-2385 detect, result shows: the pig of 3/4 creates anti-VR-2385 neutralizing antibody for 56 days after infection in latter 49 days of immunity, all pig.The pig of VR-2385 intranasal infection group has the antibody that can detect in 49,56 days after infection.The anti-VR-2385 NAT that in VR-2385, HP-PRRSV-LZ-F65, HP-PRRSV-TJM-F92 nose, dye produces for latter 49 days is respectively 80,24,0, and in 56 days serum, the antibody average titer of anti-VR-2385 is 64,32,0 respectively after infection.HP-PRRSV-LZ-F65, HP-PRRSV-TJM-F92 infects the allos NAT produced significant difference (p < 0.01, equally in contact control group, VR-2385, HP-PRRSV-LZ-F65, the anti-VR-2385 NAT that after HP-PRRSV-TJM-F92 intranasal infection, 49 days produce is respectively 48, 16, 0, in 56 days serum, the antibody average titer of anti-VR-2385 is 42 respectively after infection, 16, 0, HP-PRRSV-LZ-F65, the allos NAT that HP-PRRSV-TJM-F92 intranasal infection produces has significant difference.The anti-VR-2385 NAT that after VR-2385, HP-PRRSV-LZ-F65, HP-PRRSV-TJM-F92 intramuscular infection, 49 days produce is respectively 80,22,0, after infection in 56 days serum the antibody average titer of anti-VR-2385 be 62 respectively, 28,0, HP-PRRSV-LZ-F65, HP-PRRSV-TJM-F92.
The allos NAT that intramuscular infection produces has significant difference.The anti-VR-2385 NAT that in the contact pig of intramuscular infection, after VR-2385, HP-PRRSV-LZ-F65, HP-PRRSV-TJM-F92 intramuscular infection, 49 days produce is respectively 76,8,0, after infection in 56 days serum the antibody average titer of anti-VR-2385 be 72 respectively, 8, the allos NAT that produces of 0HP-PRRSV-LZ-F65, HP-PRRSV-TJM-F92 intramuscular infection has significant difference (p < 0.01).
These results show compared with vaccine Reference Strains HP-PRRSV-TJM-F92, and HP-PRRSV-LZ-F65 virus strain can induce body to produce the neutralizing antibody of the anti-allos PRRSV of high titre.
The pig of infection virus is anaesthetized after 14 days and gets lung tissue's microscopy, has change in various degree, and slightly, average lesion mark is 24,18 respectively, there was no significant difference for HP-PRRSV-LZ-F65 group, the macroscopic view change of HP-PRRSV-TJM-F92 lung.The average lesion mark of VR-2385 infected pigs is 54.But this change disappeared after 56 days.The average rank value of HP-PRRSV-LZ-F65 infected pigs interstitial pneumonia is 2.8, HP-PRRSV-TJM-F92 interstitial pneumonia classification mean value is 1.8, be 1.2 with PBS blank group, there was no significant difference, but with VR-2385 group has significant difference, VR-2385 infects interstitial pneumonia pig mean value 5.2.Infect after 56 days, each group occurs without can be observed interstitial pneumonia, illustrates that the security of HP-PRRSV-LZ-F65 is the same with HP-PRRSV-TJM-F92, and has the ability comparatively early producing neutralizing antibody and Interferon, rabbit generation, but pathogenic the same with vaccine Reference Strains.
Embodiment 7 HP-PRRSV-LZ-F65 is on the impact of sow reproduction ability
1, materials and methods
External attenuation makes the growth of HP-PRRSV-LZ-F65 strain and pathogenic change, observe it by immune sow and can assess the pathogenic of isolated viral or immunity to the impact of the fecundity of pig, make attenuated strain of the present invention reach its immune effect, this requires animal model sensitivity and it can be checked to cause a disease or virulence.
The present embodiment chooses 28 sows, often organizes 7, is divided into A, B, C, D group.Passback passaged virus HP-PRRSV-LZ-F65-B6 in 6 bodies of A group intranasal vaccination HP-PRRSV-LZ-F65, passback passaged virus HP-PRRSV-LZ-F65-B6 in 6 bodies of B group intramuscular inoculation HP-PRRSV-LZ-F65, C group intranasal vaccination water for injection shines group as to normal, the passage strain HP-PRRSV-LZ-F65-P4 of D group intranasal vaccination HP-PRRSV-LZ-F65, inoculation in conceived 90 days.Viewing duration, carries out the sow immunity temperature check of latter 7 days, childbirth is front to blood examination weekly during farrowing.Collect piglet in the blood sample of birth, 7 ages in days, 14 ages in days and body weight information, to dividing observe healthy state an every day 14 day puerperium during studying.
The commercial PRRSV ELISA kit of serum after sow exposes, MA-104 cell cultures detect, and within the postvaccinal 0-7 of sow days, observation body temperature every day obtains the mean body temperature often organized; Before and after immunity, total white blood cells detection, clinical observation classification are as embodiment 2, and the sow clinical observation time is that immunity arrives childbirth in first 1 day; Rectal temperature monitoring time is the 0-7 days of immunity, and postmortem detects lung and relates to per-cent.Detect serum by PRRSV ELISA kit weekly after same piglet birth, the piglet clinical observation time is from birth to 14 week age.
2, result
Sow is before virus inoculation, serum is without pig breeding and respiratory complication antiviral antibody, also without porcine reproductive and respiratory syndrome virus infection, at inoculation HP-PRRSV-LZ-F65 and different passaged virus after 14 days, the sow antibody male rotary of A, B, D group, B group has 3 pigs, 14 negative antibodies after exempting from, but give a birth time antibody male rotary.
The serum ELISA result display of A group, the most piglet of B group, piglet is born 0 day negative antibody, feeds antibody positive after 7 days through sow.C group piglet antibody used is negative, because stillbirth only has the several Virus monitory of D group, and half is positive.The piglet that D group sow produces is in birth all death afterwards in 7 days, and it is negative that A, B two groups of sow serum are separated porcine reproductive and respiratory syndrome virus.A, B two groups of piglet serum are separated porcine reproductive and respiratory syndrome virus-positive, and in A group, the porkling virus purification of 1 sow is negative all the time, and B group only has 2 porkling virus purification positive; C group porkling does not have virus purification to arrive, and 71% porkling virus purification of D group is positive.
After virus inoculation sow, temperature variation is not remarkable, and A, B group sow and C group sow temperature are no more than 101.7 Fahrenheit degrees, and 4 pigs of D group have 1 day temperature more than 102 Fahrenheit degrees, and wherein a temperature is more than 103.4 Fahrenheit degrees.
The weight gain of piglets of A group, B group, higher than C group weight gain of piglets, is observed 14 days, average weight gain 7.9 pounds, A group average weight gain 7.7 pounds, B group, C group average weight gain 6.9 pounds, and how many this and farrowing of sow brood have nothing to do.The a brood of bearing average out to 9 of A group sow, B group is 7, and C group is 10, D group institute produce porkling after birth 3 days with regard to death.
The leukocyte count of A, B, C tri-groups of sows is relatively stable, and the white corpuscle mean number before immunity is equal to or higher than 3 sows of 92%, D group of viewing duration at viewing duration, and leukocyte count is lower than range of normal value (7-20 × 10
6/ ml).
After A, B group sow virus inoculation, clinical symptom is not obvious, has several sows to have obvious clinical symptom in for some time in C group, and cardinal symptom is appetite stimulator, respiratory symptom, dispirited.6 pigs of D group show obvious clinical symptom, are mainly apocleisis in various degree, apositia.
Piglet clinical symptom is mainly death and appetite stimulator, if calculated by every nest Mean Death, A, B, C3 group is without significant difference, A group every nest Mean Death number (average deaths per litter DPL) 1.3DPL, B group 2.4DPL, the survival of C group 2.0DPL, D group porkling is no more than 3 days postpartum.
Because virus inoculation does not affect the farrowing amount of sow, the assessment of breeding of sow used adopts percentage to be suitable for, A group piglet average viability is 85% (SD ± 9.6), C group piglet average viability is 83.4% (SD ± 7.9), and B group piglet average viability is 89% (SD ± 11.6).A group mean over frequency of embryonic death 8.8 (SD ± 9.66), B group mean over frequency of embryonic death is 6.6 (SD ± 9.7), and C group mean over frequency of embryonic death is 14 (SD ± 11.39).A group average mummy tire rate 6.1 (SD ± 6.01), B group average mummy tire rate 3.9 (SD ± 4.45), C group average mummy tire rate 2.6 (SD ± 4.01), the young survival rate 8.7 (SD ± 8.92) of D group, D group mean over frequency of embryonic death 10.7 (SD ± 11.39), D group average mummy tire rate 81.9 (SD ± 17.18).
The present embodiment explanation, the attenuated strain HP-PRRSV-LZ-F65-B6 in HP-PRRSV-LZ-F65 and passback pig body 6 generation, attenuation is stablized, inoculation pig and piglet in reproductive behavior, oligoleukocythemia, rectal temperature, clinical symptom without marked difference, safety and low toxicity, the little reacting phase almost caused with water for injection of toxicity is same, the not reversion on sow of HP-PRRSV-LZ-F65 virulence.
Embodiment 8 high dosage HP-PRRSV-LZ-F65 is to the proof test of sow
1, materials and methods
Current porcine reproductive and respiratory syndrome attenuated live vaccine using dosage is 1-2 × 10
5lgTCID50, the present embodiment adopts HP-PRRSV-LZ-F6510 times of concentrated solution injection sow, observe the impact on sow reproductive behavior, sow is divided into A, B, D tetra-groups, at the virus liquid inoculating various dose for 90 days respectively of pregnancy, the intranasal vaccination of A group, HP-PRRSV-LZ-F65, C group intranasal vaccination water for injection as Normal group, the intranasally inoculated attenuated strain of D group 10 times of concentrated solutions.
Viewing duration, carries out the sow immunity temperature check of latter 7 days, childbirth is front to blood examination weekly during farrowing.Collect piglet in the blood sample of birth, 7 ages in days, postmortem and body weight information, to dividing observe healthy state an every day 14 day puerperium during studying.
The commercial PRRSV ELISA kit of serum after sow exposes, MA-104 cell cultures detect, and within the postvaccinal 0-7 of sow days, observation body temperature every day obtains the mean body temperature often organized; Before and after immunity, total white blood cells detection, clinical observation classification are as embodiment 2, and the sow clinical observation time is that immunity arrives childbirth in first 1 day; , rectal temperature monitoring time is the 0-7 days of immunity, and postmortem detects lung and relates to per-cent.Detect serum by PRRSV ELISA kit weekly after same piglet birth, the piglet clinical observation time is from birth to 14 week age.
2, result
The result display farrowing sow of this embodiment injects HP-PRRSV-LZ-F times of concentrated solution HP-PRRSV-LZ-F65 virus stock solution used, water for injection in security and to procreative effect without significant difference, therefore, HP-PRRSV-LZ-F65 is pathogenic to be weakened, use safety, 10 times of concentrated solutions use the reproductive behavior also not affecting farrowing sow, these results illustrate the highly attenuated and use safety of HP-PRRSV-LZ-F65 strain, and are highly resistant to highly pathogenic HP-PRRSV and infect the reproductive syndrome caused.
The immunogenicity of embodiment 9 HP-PRRSV-LZ-F65 in growing and fattening pigs
1, materials and methods
Pig is divided into 3 groups, organize the virus liquid of 1 intramuscular inoculation HP-PRRSV-LZ-F65,1 dose of 2.0lg10TCID50, organize 2 intramuscular inoculation waters for injection, group 3 is normal control, the pig of group 1 and group 2 attacks poison in latter 28 days in immunity, attacks poison from HP-PRRSV-LZ 2 generation virus (HP-PRRSV-LZ-F2).
First 7 days after immunity and the body temperature detecting pig every day after attacking poison, pig detects body weight weekly after attacking poison, takes a blood sample weekly 1 time before attacking poison, within after attacking poison every 2 days, takes a blood sample, the lung of postmortem relates to percentage calculation and is undertaken by embodiment 2, and during research, every day observes the healthy state of every pig.Pig immunity and get serum after attacking poison and carry out PRRSV ELISA and detect pig antibody, attack the virus of the serum MA-104 cellular segregation after poison, the ELISA detected result of each group and virus purification result adopt chi square test to analyze, and white blood cell count(WBC) method is as embodiment 2.
2, result and discussion
Every Testing index of immune group 1 is all better than water for injection immune group.Immune group attacked poison after 4 days, and viremia significantly declines, 10 days after attacking poison, 11 days, has the number of viremia pig to reduce further.Weightening finish and the leukocyte count of organizing 2 pigs are lower, and lung relates to per-cent, clinical scale, rectal temperature are higher, 10 days after attacking poison, 11 days, has the number of viremia pig to reduce further.
Immunity before and after, attack poison before and after all pigs serum ELISA detected result display, all pigs are PRRSV negative antibody (S/P ratio <0.4) when immunity, immunity latter 7 days is still negative, immunity is after 7 days, 21 antibody male rotaries in 22 immune pigs, 2 pigs negative antibody before attacking poison of group 1, is attacking poison latter 8 days antibody male rotaries.Organize 2 all pigs in on-test to negative antibody before attacking poison, but attacking poison latter 8 days, 17 antibody male rotaries in 22 pigs, group 3 all pigs are at whole duration of test negative antibody.
Pig has all carried out serum-virus and has been separated before exempting from after exempting from, and in 22 immune pigs, 17 are separated to virus in 2 days after exempting from, and 18 are separated to virus in latter 7 days in immunity, have 1 pig immunity to be separated all the time less than virus to when attacking poison afterwards.These results are consistent with Post-immunisation serum state outcome.
When attacking poison, the pig of 55% immunity there occurs viremia, attack poison latter 2 days, the pig of viremia rises to 82%, declines gradually later, attack poison within latter 11 days, drop to 9%.Organize 2 all pigs, do not have viremia when attacking poison, attacking poison latter 2 days, there is viremia in the pig of 82%, attacks poison latter 4 days, the pig generation viremia of 91%.After attacking poison 6 days, 10 days of pig of group 2, have 82% swine disease poison to be separated to virus, and attacking poison latter 11 days, 71% swine disease poison is separated to virus.The pig of group 3 is at experimental session, and virus purification is negative all the time.
All temperature of pig body of temperature check result display group 2 rise, and have the pig of half that more than 2 days high heat occur, exceed the medial temperature before attacking poison 1 Fahrenheit degree.Immune group 22 pigs have the high heat of 4 pigs, and body temperature exceedes attacks front medial temperature 1 Fahrenheit degree of poison.The mean time of 1 group of temperature of pig body rising is 2.2 days, and 2 groups average 4 days, and 3 groups of body temperature are normal, body temperature does not occur and continues to raise phenomenon in 2-3 days.
The weightening finish of experimental session pig observes 11 days, organize 3 pig average weight gain 1.06 lbs/day, organize 2 pig average weight gain 0.94 lb/day, organize 1 pig average weight gain 0.53 lb/day, so there is no immunity but the pig weightening finish infecting PRRSV virus only has 57% of the weightening finish of immune attack pig, is 50% of Normal group.
After attacking poison, the oligoleukocythemia situation of pig detects, after attacking poison 2 days of pig of group 3, the leukocyte count of every pig and attack the mean value before poison and compare, minimizing 5%.Group 2 porcine leukocyte count drop to 41%, to attack poison after 11 talentes return to attack poison before level.Immune group pig attacking poison latter 3 days, leukocyte count decline 12%, but attack poison within latter 4 days, return to the leukocyte count level before exempting from.
Attacking malicious first 4 days to attacking poison latter 11 days, every day carries out clinical observation, and all pigs are without visible clinical symptom before attacking poison, and the pig of group 3 is attacking the rear viewing duration of poison also without clinical symptom, 5 pigs of group 2 show as and attack malicious clinical symptom, and after attacking poison, 6 light get brighter aobvious but not serious.Only have in the immune swine of 1 group 1 attack poison within latter 11 days, have obvious clinical symptom.
In research latter stage, seen at post-mortem pig injury of lung situation, 3 groups of pig lungs are normal, average mark level score value be the lung classification score value of 0.02,2 groups of pigs between 33 and 98, average 78.33, the classification score value of 1 group of pig between 30 and 90, average 53.20.
These data declarations, HP-PRRSV-LZ-F65 as the effect of vaccine activity composition, minimum dose 2.0logTCID
50, can significantly reduce pig lung damage, reduce infect cause oligoleukocythemia symptom, reduce pig heating.In addition, immune HP-PRRSV-LZ-F65 vaccine, does not affect the growth of pig, is significantly higher than and infects non-immune pig.
The using dosage of embodiment 10 HP-PRRSV-LZ-F65 and effect
1, materials and methods
70 pigs, are divided into 4 groups, each 20 of 1-3 group, 4 groups 10.HP-PRRSV-LZ-F65 virus liquid, attacking poison is HP-PRRSV-LZ-F2.1 group of intramuscular injection HP-PRRSV-LZ-F65 virus liquid, 2.5lgTCID
50/ agent.2 groups of intranasal vaccination HP-PRRSV-LZ-F65 virus liquids, 5.0lgTCID
50/ agent, 3 groups of intramuscular injection diluents, 4 groups as strict contrast.The pig of 1 group, 2 groups, 3 groups immunity within latter 28 days, attack poison attack poison after the body temperature detecting pig every day, pig immunity, attack poison, attack poison after detect body weight weekly, take a blood sample weekly before attacking poison 1 time, within after attacking poison every 2 days, take a blood sample, the lung of postmortem relates to percentage calculation and is undertaken by embodiment 2, and during research, every day observes the healthy state of every pig.Pig immunity and get serum after attacking poison and carry out PRRSV ELISA and detect pig antibody, attack the virus of the serum MA-104 cellular segregation after poison, white blood cell count(WBC) method is as embodiment 2.
2, result
Serum ELISA detected result shows, and all pigs are PRRSV negative antibody (S/P ratio <0.4) when immunity, latter 14 days of immunity, the pig antibody male rotary of 1 group 70%, the 95% pig antibody male rotary of 2 groups.1 group is only had 1 pig negative antibody before attacking poison, and group 2 all pigs to negative antibody before attacking poison, but are attacking malicious latter 8 days in on-test, attacks antibody male rotary on malicious same day.17 antibody male rotaries in 22 pigs.3 groups and 4 groups of all pigs attack negative antibody before poison, the 9th day after attacking poison whole, and 3 groups of all pigs detect antibody positive through ELISA, 4 groups in experimental stage, antibody is still negative.
The separating resulting of virus is relevant to Serological, only has 1 pig virus purification negative, corresponding with its Post-immunisation serum negative antibody, these results illustrate the immunizing dose relation of the rear viremia of immunity and antibody male rotary, 2 groups of pigs after exempting from 14 days, 100% sun turns, and 1 group of 70% sun turns; Immunity latter 14 days, 21 days, high dose group, had the pig of 85% and 90% to occur viremia, in low dose group, exempted from have the pig of 55% to occur viremia in latter 14 days, exempted from have the pig of 85% to occur viremia in latter 21 days.
After attacking poison, the pig of 3 group 89% has high thermal phenomenon to occur, and body temperature exceedes attacks front medial temperature 1 Fahrenheit degree of poison, continues more than 2 days.In 1 group of pig, there is high heat in the pig of 75%, body temperature exceedes medial temperature 1-2 Fahrenheit degree, continues more than 2 days, and 2 groups are only had the high heat of 45% pig, Normal group pig to have the pig of 30% that the high heat of more than 2 days occurs.Exempt from first 3 days to attacking poison latter 28 days, high dosage and low dosage immunity produce harm to the growth of pig.1 group and 2 groups of average daily gains are respectively 0.77 lb/day and 0.76 lb/day, and 3 and 4 groups of average daily gains are 0.77 and 0.78 respectively.After attacking poison, the pig Average weight increasing a day of 1 group exceeds 3 groups of pigs 0.05 lb/day, and 2 groups of pig Average weight increasing a days exceed 0.01 lb/day, and Normal group day weight gain exceeds immune group 0.4-0.5 lb/day.After attack 1 day or many days of the pig of 3 group 84%, leukocyte count declines more than 25%, has 30% pig in Normal group, also has similar leukocyte count to decline.After attacking poison, high dose group pig 15% (3/20), low dose group 55% pig occur that leukocyte count declines, and decline 25%, continue 1 day or several days.
Clinical observation shows all pigs before attacking poison, is in good health state, and attack the state of health level of the rear 1-3 group pig of poison without considerable change, lethargic sleep, respiratory symptom, loss of appetite are as mild.
Postmortem lung observes porcine reproductive and respiratory syndrome sample injury scale and calculates lung and relate to per-cent, and it be zero, 3 groups is 70.08 that Normal group Mean pulmonary relates to per-cent, and 1 group is 48.83,2 group 17.76.
From the result of this embodiment, high dosage HP-PRRSV-LZ-F65, titre 5.0lgTCID
50/ agent, as vaccine, can reduce the Respiratory Apparatus symptom that HP-PRRSV-LZ causes, and obviously can alleviate pulmonary lesion.Therefore vaccine effective dose is 2.0-5.0lgTCID
50/ agent.
The gene sequencing of embodiment 11 HP-PRRSV-LZ-F65 and molecule marker
The present embodiment, by gene sequencing, the molecular mechanism of external attenuation is described and genome goes down to posterity in vitro and whether stablize in pig body, for the restriction safety range that HP-PRRSV-LZ-F65 practical application provides seed culture of viruses to go down to posterity, also thoroughly do not reverse for HP-PRRSV-LZ-F65 attenuation, HP-PRRSV-LZ-F65 keeps stable attenuation, immunity, security, broad spectrum (to allos PRRSV virus), distinguish the early stage generation Interferon, rabbit of other vaccine strains, the directed attenuation of early stage generation specificity neutralizing antibody and manual method provides genetics or molecular biological partly cause and mechanism.
The present embodiment illustrates the difference of HP-PRRSV-LZ-F65 and open country poison, the molecular basis that this characteristic of attenuated strain provides porcine reproductive and respiratory syndrome prevention, diagnoses, and also can distinguish the gene of existing vaccine strain and variation, restructuring.HP-PRRSV-LZ-F65, in the change of GP5 gene, NSP1 gene, can be used as the immunity of pig body or infects wild malicious molecular diagnosis.Provide porcine reproductive and respiratory syndrome to infect and HP-PRRSV-LZ-F65 immunity district method for distinguishing.
1, method
Cell culture processes: HP-PRRSV-LZ-F65 is obtained by MARCK-145 cell, pig body passback attenuation, plaque clone.Cell cultures 75cm
2t cell bottle, substratum DMEM50 milliliter, containing the gentamicin of 5-10% foetal calf serum, 50 mcg/ml, the virus that cell inoculation is separated, the incubator of 5% carbonic acid gas cultivate 5-7 days as 1 generation viral passages.By 1:4 cell dispersion when going down to posterity, with pancreatin-Versene as Digestive system.Virus liquid individual layer inoculates 1 milliliter, containing 10
5-10
6tCID
50virus liquid, adsorbs 1 hour, cultivates and receives poison when cytopathy is obvious, centrifugal removing cell debris in 5 days, frozen for subsequent use in-80 DEG C.
RNA extracts and RT-PCR method: virus liquid 280 microlitre adds 1120 microliter of buffer liquid AVL (QIAamp viral RNA separating kits, Qiagen), room temperature vortex mixed 10 minutes, add 1120 il of ethanol, repeatedly put upside down several times, within 1 minute, the centrifugal viral RNA that makes of 6000g is adsorbed onto on rotating separation pillar, with buffer A W washing in test kit, and 60 microlitre diethyl coke sour water wash-outs.Superscript II RNase H.sup.-reverse transcriptase (RT) reversed transcriptive enzyme (Life Technologies, Inc.), PRRSV special primer or random primer, purified virus RNA67 DEG C heating 7 minutes, the reaction system of 40 microlitres comprises 5 millis and to rub MgCl
2, 1 × standard buffer solution II (Perkin Elmer Corp.), 1 milli rubs dNTP, 1 units of RNAase inhibitor, 2 unit reversed transcriptive enzymes, 1 microlitre RNA, within 5 minutes, hatches for 42 DEG C 15 minutes, 99 DEG C 5 minutes, 5 DEG C.Polymerase joins 25 microlitre reaction mixtures: 10 microlitre cDNA products, 2 millis rub MgCl
2, 1 × standard buffer solution II (Perkin Elmer Corp.), 0.2 milli rub dNTP, 0.375 unit Taq enzyme, 0.3 micro-rub 5 ' end primer, 0.3 micro-rub 3 ' end primer.Reaction conditions: 93 DEG C of sex change in 4 minutes once, enter 35 circulating reactions, each circulation 94 DEG C of sex change 30 seconds, 59 DEG C annealing 30 seconds, 73 DEG C extend 30 seconds, and 35 circulation after products, 72 DEG C of extensions 10 minutes, are placed in 4 DEG C.PCR primer purifying can carry out with commercial kits by specification.
The RNA of the PCR reaction of virogene group end is separated, cDNA synthesizes, purifying takes aforesaid method or easier test kit, react further at pcr amplification terminal fragment and add end, 20 microlitre reaction systems: 10 microlitre cDNA, 1 × damping fluid 2 (New England Biolabs), 2.5 micro-CoCl2 that rub, 0.5 milli rubs dATP, 2 units terminal transferase (New England Biolabs).37 DEG C are heated 15 minutes, and 65 DEG C of 5 minutes termination reactions, are diluted with water to 200 microlitres.
The PCR reaction system of long segment, volume 50 microlitre, comprise the cDNA of 10 microlitre band poly-A tails, dNTP that 1 × damping fluid, 0.35 milli rub, 0.625 milli rub MgCl2,0.04 micro-Qt primer (Frohman that rubs, 1994), 0.3 micro-Qo primer (Frohman that rubs, 1994), 0.3 micro-5'-CGCCCTAATTGAATAGGTGAC-3' that rubs, 0.75 microlitre enzyme.Mixture 93 DEG C of sex change 2 minutes, then carry out the circulation of 25 times, each circulation comprises 93 DEG C of sex change 10 seconds, 63 DEG C of annealing 30 seconds, 68 DEG C 12 minutes.Circulate rear 68 DEG C 7 minutes, product is placed in 4 DEG C.Next round amplified reaction, 50 microlitres reactions comprise: the dNTP that 5 microlitre previous step PCR primer, 100 times of diluents, 1 × damping fluid, 0.35 milli rub, 0.625 milli rub MgCl2,0.04 micro-Qi primer (Frohman that rubs, 1994), 0.3 micro-primer 5'-CCTTCGGCAGGCGGGGAGTAGTGTTTGAGGTGCTCAGC-3' that rubs, 0.75 microliters polymerase.Mixture 93 DEG C of sex change 2 minutes, then carry out the circulation of 25 times, each circulation comprises 93 DEG C of sex change 10 seconds, 63 DEG C of annealing 30 seconds, 68 DEG C 4 minutes.Circulate rear 68 DEG C 7 minutes, product is placed in 4 DEG C.
Amplified production is shown as the band of 1000-1500bp in 1% agarose electrophoresis, uses gel purification kit purified colonies and plasmid pGEM-T (Promega), and being separated checks order clones and increases, check order, splice.About there are 100-105 PCR reaction and 450 DNA sequencing reactions to carry out splicing, analyzing, compare with the standard virus shown in table 9.
Table 9 complete genome sequence compares the standard virus involved by PRRSV strain
2, result
Through sequence homology compare of analysis, found that HP-PRRSV-LZ-F65 and VR-2332 is 97.50% in nucleotide level homology, 98.2% is had at nucleotide level with porcine reproductive and respiratory syndrome virus VR-2385, the ORF5 gene coding amino acid homology of HP-PRRSV-LZ-F65ORF5 gene coding amino acid and VR-2332 is 86.9%, with V2385ORF5 gene coding amino acid homology 84.6%, the nucleotide acid sequence total length of the NsP2 gene of HP-PRRSV-LZ-F65 strain is 2490bP, consecutive miss 600-728 amino acids compared with the nucleotide sequence of PRRSV-LZ-F2 strain NsP2 gene, lack 128 amino acid altogether.
Find that the street strain that most PRRSV is separated only contains a NspI restriction enzyme site, the RT-PCR product of 1Kb has cut 2 fragments through enzyme, and HP-PRRSV-LZ-F65 attenuated strain has 2 NspI restriction enzyme sites, and enzyme shows 3 fragments after cutting in electrophoresis.Some open countries poison strain isolated is not containing NspI restriction enzyme site.
Reverse transcription PCR gene amplification, cDNA sequencing result show, its sequence in Nucleotide with HP-PRRSV-LZ-F65 with original be separated that poison cell 2 generation goes down to posterity poison delete the homology that other nucleotide sequencings have 99.8% except NSP2 amino acid, 34 Nucleotide are had to there occurs change, cause 19 amino acid mutations, the position of change is as shown in table 10.Except above nsp deletes, the amino acid that HP-PRRSV-LZ is separated poison there occurs change, discloses the possible cause of attenuation, and HP-PRRSV-LZ-F65 is stable gene between 65-115 generation.Illustrate the moieties mechanism why with early evoking neutralizing antibody and Interferon, rabbit simultaneously.
Table 10 HP-PRRSV-LZ-F65 is separated poison with original, different generation goes down to posterity malicious amino acid whose change
Claims (5)
1. pig body can be induced comparatively early to produce Interferon, rabbit and neutralizing antibody and there is the strain of porcine reproductive and respiratory syndrome virus attenuation or its strain of going down to posterity of broad-spectrum immunogenicity for one kind, described attenuated strain called after HP-PRRSV-LZ-F65, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.9454.
2. pig body can be induced as claimed in claim 1 comparatively early to produce Interferon, rabbit and neutralizing antibody and there is the strain of porcine reproductive and respiratory syndrome virus attenuation or its strain of going down to posterity of broad-spectrum immunogenicity, it is characterized in that described strain of going down to posterity is the poison that goes down to posterity in 1-45 generation of HP-PRRSV-LZ-F65.
3. the porcine reproductive and respiratory syndrome virus attenuation strain described in claim 1 or 2 or its application of strain in preparation prevention porcine reproductive and respiratory syndrome medicine of going down to posterity.
4., for preventing a vaccine composition for porcine reproductive and respiratory syndrome, it is characterized in that going down to posterity strain and medically acceptable adjuvant containing the porcine reproductive and respiratory syndrome virus attenuation strain described in claim 1 or 2 or its.
5. vaccine composition as claimed in claim 4, is characterized in that the significant quantity of contained porcine reproductive and respiratory syndrome virus attenuation strain or its strain of going down to posterity is 2.0-5.0lgTCID
50/ agent.
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| CN109943536A (en) * | 2019-03-26 | 2019-06-28 | 昆明理工大学 | A kind of Hepatitis E virus, the preparation method of cultural method and its inactivated vaccine |
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| EP0676467A2 (en) * | 1994-04-11 | 1995-10-11 | Akzo Nobel N.V. | European vaccine strains of the porcine reproductive respiratory syndrome virus (PRRSV) |
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| CN109943536A (en) * | 2019-03-26 | 2019-06-28 | 昆明理工大学 | A kind of Hepatitis E virus, the preparation method of cultural method and its inactivated vaccine |
| CN109943536B (en) * | 2019-03-26 | 2021-09-14 | 昆明理工大学 | Method for culturing hepatitis E virus and method for preparing inactivated vaccine thereof |
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