The strain vl-94" parvovirus pigs for the manufacture of vaccines


The invention relates to biotechnology, in particular for veterinary Virology. A new vaccine strain VL-94" swine parvovirus (PVA) has a high immunogenic activity. The strains propagated in human cell cultures kidney pig (CPD) and accumulates in the title 8,0 Ig TCD50/cm3after 18-24 h of cultivation. The resulting strain has a high biological, antigenic and immunogenic activity and can be used for production of effective vaccines. 1 Il., 6 table.

The invention relates to the field of veterinary Virology and biotechnology and can be used in the development and manufacture of vaccines for prevention of parvovirus infection of pigs (PVIS).

PVIS - contagious viral disease, which is manifested clinically only in pregnant sows and characterized by pogolotti, small litters, the birth of mummified fetuses, dead and weak piglets and early (in the first half of pregnancy) abortion.

The relationship of parvovirus pigs (PVA) with impaired reproductive function sows in vivo was first established in 1967 in the UK, tnlo, without visible symptoms. There is only disorder of the reproductive function. The virus crosses the placenta and infects the fruit in the first half of gestation, causing their mummification and death. Clinically the disease manifests with pogolotti, early in the first half of gestation, abortion and prezhdevremennye childbirth. In the event of illness in previously prosperous farms in the birth of live piglets per sow per year is reduced by 50-60% in fixed-disadvantaged households - 10-20%.

For specific prevention PVIS are used both inactivated and live vaccines. Currently the main means of combating PVIS are inactivated vaccines, which are subjected to immunization of young sows not later than the 30th day before the first insemination. Vaccination creates sows expressed immunity and prevents transmission of the virus to the embryos and fruits (1).

It is known that all known PVS isolates were antigenically similar, if not identical (2).

In the manufacture of inactivated vaccine PVIS the only requirement to the original strain PVA is a good accumulation of the virus in a sensitive biological system used for its cultivation.

<'s highly productive industrial strains PVA for the manufacture of vaccines remains relevant.

A known strain NADL-2 (collection of ATSS VR-742) PVA used for the manufacture of inactivated vaccines (3).

The closest to the proposed invention, the essential features is the strain-82 PVA used for the manufacture of inactivated vaccines (4).

A common disadvantage of the known strains is their low productivity in known biological systems of cultivation.

In the task of creating the present invention was to enlarge the Arsenal of strains of PVA having a high biological, antigenic and immunogenic activity and suitable for the manufacture of effective vaccines, creating intense and long-lasting immunity in vaccinated animals.

The technical result of the proposed use of the invention consists in expanding Arsenal of new high-yield vaccine strains PVA having a high biological, antigenic and immunogenic activity and is suitable for production of effective vaccines.

This technical result is achieved by obtaining strain VL-94" PVS. The strain VL-94 is a new, previously unknown. The original virus to obtain strain VL-94" highlighted in the research Institute of animal protection is omatek, brought from the farm "Vladimir, Vladimir region. The production strain VL-94" PVA obtained by serial passages on transplantable cell cultures kidney pig "Kazan" (CPD).

The resulting strain deposited at the Russian state collection of strains of microorganisms used in veterinary medicine and animal husbandry, all-Russian state research Institute for control, standardisation and certification of veterinary preparations Ministry of agriculture of the Russian Federation (VGNKI) July 1, 1996 under registration code "VL-94-DEPT".

The strain VL-94" PVA has a high biological, antigenic and immunogenic activity in the native form and after inactivation and has high productivity in sensitive biological systems of cultivation. Experimentally confirmed the possibility of its use for the preparation of inactivated emulsion vaccine PVIS, creating intense and long-lasting immunity in vaccinated animals.

The invention is illustrated in the drawing, which shows to something called a microarray findings hemagglutinin activity (HA activity) strain "VL-94" in the reaction of haemagglutination (DSA). the m VL-94" PVA belongs to the family Parvoviridae, the genus Parvovirus. Mature virion has a cubic symmetry, includes 32 capsomere, 2 or 3 capsid protein, the diameter of the virion is about 20 nm, does not contain membranes and lipids, mol. m is 5.3106D. Viral genome presents 1-stranded DNA mol. m a 1.4 KD, i.e. 26.5% of the mass of the complete virion, the proportion of guanine + cytosine is 4153%. Buoyant density in CsCl full of infectious virus, incomplete ("empty") of the virion and extracted mirinoi DNA at 1.381,395; 1,301,315 and 1,724 g/cm3respectively. The sedimentation coefficient 110122S.

Antigenic properties of the Virus contains three polypeptide a, b and C (mol. weight 6090 KD. All sravnivayete isolates PVA pigs were antigenically similar, if not identical. Their identity is established in PH, rtha. The virus has expressed antigenic activity and induces the synthesis of neutralizing, complimentative and precipitating antibodies, as well as antihemagglutinin. The study of the antigenic properties of isolated individual virionyx polypeptides of the pathogen showed that anticavity to karamja structural proteins of the virus. Each individual protein interacts with neutralizing antibodies obtained on the full introduction of the virus. Virousspecificakih antigenic determinants are, presumably, all virionyx proteins PVA. The biological half-life of antibodies in piglets depends on their mass, the average is 18 to 20 days.

Biotechnological characteristics of the Strain VL-94" PVS well reproduced in the transplantable cell culture CPD and accumulates in the title of 8.0 lg TCD50/cm3. Transplantable cell culture spew insensitive to the virus. A feature of the cultivation and accumulation of the virus is its dependence on cell division. Curve propagation of the virus depends on the number of mitoses. He is well reproduced in the dividing cells of the kidney of the fetal pig when infection within 48 hours after planting culture. The highest concentration of the virus is achieved in 2-3 days of cultivation. JRS is characterized by vacuolation and rounding of cells, diffuse granulation. When studying the breeding cycle method immunoflourescence it was found that the antigen is found in the nucleus of the cells after 6 hours after infection and stored in maximum quantities for 18-24 hours. The strain VL-94" PVA prednasky stable and retains immunobiological properties for 10 passages (observation period).

Chemo-geotectonically characteristic of PVS Genome of strain VL-94" presents 1-spiral linear DNA molecule mol. m 1,4 KD. 1-spiral DNA consists of about 5 thousand nucleotides and encodes 4 protein - 3 structural and 1 non-structural. On both ends of the DNA are not identical, semicomplete sequence of nucleotides (palindromes), forming 2-helical hairpins that are resistant to nuclease S1; 3'-terminal hairpin consists of 115116 nucleotides, the 5'-end - from 200242 nucleotides. Hairpin structures play an important role in DNA replication. The genes encoding the structural proteins are located in the 5'-terminal half, and the gene encoding non-structural protein 3'-terminal half of the DNA. In the virions of strain VL-94 contains a minus-DNA. The virus strain VL-94 is an independent (non-defective).

Pathogenic properties of the Strain VL-94" PVA pathogenic for cattle and apathogenic for hamsters and mice. All infected piglets symptoms and pathological changes are usually absent, however, are detected specific antibodies. The virus isolated from the seminal vesicles on day 7 to day 9 after infection. With 12 days in the serum of animals reveal antihemagglutinin, which indicates the contagiousness of the infection. After intracerebral, oral and intranasal infection of piglets specific reproducible clinical symptoms were observed. However, these animals were able to isolate the virus from stool, blood and most tissues. To 7 days were allocated specific antibodies. When infection of pigs with the virus of strain VL-94" at different stages of pregnancy were observed transplacental infection of fetuses.

Hemagglutinins properties of the Strain VL-94" PVA agglutinate erythrocytes of Guinea-pig, chicken, cat, rat, mouse, monkey, human and not agglutinate erythrocytes of sheep, horses, pigs, cattle, dog, rabbit, hamster and ducks. To determine the HA activity using Guinea pig erythrocytes, the reaction is carried out at 4oAnd as diluent used saline solution, pH of 7.2. HA activity in DSA was 1:2048 GUY. The virus is not released from erythrocytes at 37-40oC for one hour, but in alkaline buffer (pH 9,0) he fully aluinum with them for 30 minutes at room temperature. 62 Physical properties Molecular weight of virions is 5.56,2 MD, buoyant density in CsCl gradient 1 is ity to external factors Strain "VL-94" PVA resistant to various physical and chemical factors. Retains infectivity at pH9 and 37oC for 1.5 h, but completely inactivated at pH 2 under the same conditions. The virus is stable at 56oC for 2 days at 70oC - 2 h and at 80oWith inactivated for 5 minutes without losing infectivity in culture fluid with 20% fetal cattle serum at 3537oWith over 15 weeks. Sustainable handling chloroform, ether and freon 1050% concentration for 1030 min at room temperature. The virus is resistant to the action of RNA - and DNA-ases and proteases (papain, chemotrypsin, trypsin, pepsin), resistant to the action of ultrasound, is inactivated by UV rays, formalin, ethylenimines and its derivatives;-propiolactone, glutaraldehyde. The virus remains active for more than 4 months, in the pens of up to 46 months.

Additional characteristics and properties Free from contamination by bacteria, fungal microflora, mycoplasmas and viruses.

On the basis of the obtained data it can be argued that the strain VL-94" antigenic and immunological spectra is original, in taxonomic relation epilectic emerging foci of disease, and this requires high-level and harmless vaccine.

According to the applicant, the proposed strain has established Patent law of the Russian Federation signs of the patentability of "novelty" and "inventive step".

The essence of the invention is illustrated by examples of its execution, which do not limit its scope.

Example 1.

In 1994, when virological examination of the internal organs of an aborted (up to 15 cm long) fruit sows brought from the farm "Vladimir, Vladimir region, transplantable cell culture CPD was isolated PVS with hemagglutinins a titer of 1:16. In the following serial passages on ACC title PVA was 1:64 (2 arcade), 1:128 (3rd passage), 1:256 (4th passage), 1:1024 (5th passage), 1:20481:4096 (6th passage).

For the cultivation of the isolate were tested cell culture spew, VIRO, MARC-145 and KSS-21, but some of them were not noted JRS and HA-virus activity (KSS-21, MARS-145), on the other manifested minor JRS, however, HA-activity remained at the level of 1:16 (SPM, VIRO). Optimal protein isolate results were obtained for 3-6 passages in 2-3 days transplantable cell cultures kidney pig "Kazan" (CPD), grown at the temperature of cultivation within 36-37oC and a pH of 7.2 to 7.4, and contact within 50-60 min vaccinated suspension with monolayer cells CPD during infection. To release the PVS cells CPD destroyed 3x freezing at -20oC and thawing at 37oC. the resulting virus was subjected to a comprehensive inspection in accordance with the guidelines of the OIE standard diagnostic methods and vaccines (1996).

The results of the tests showed that the isolate PVS meets the requirements of industrial strains of PVA. The infectious virus activity in cell culture of 7.5 CPD lg TCD50/cm3and with HA activity in RSA 1: 2048 laid deposited as motroway of the seed under the author name "VL-94".

Strain stored at -40oWith or -70oIn the native form in vials of 10 cm3under hermetically sealed tubes and metal caps. The strain in the native form refresh every 12 months in culture cells CPD. The titer of virus in the bottle for 12 months of storage has dropped to less than 0.5 lg TCD50/cm3. This consistently is active in the DSA and immunogenic activity.

Example 2.

For the manufacture of emulsion inactivated vaccines use strain "VL-94" PVS,">1024 the GUY and infectious activity of not less than 7.5 lg TCD50/cm3. To obtain vaccines use grown in mattresses cell culture CPD with a good monolayer 2-3 days age. To obtain 20 liters of vaccinated suspension PVA take from 110 to 120 mattresses cell culture capacity of 1.5 DM3. After draining the growth environment in mattresses contribute 5-7 cm3PVA matrix series. After 40-60 min of incubation at (372)oC in mattresses make 150-200 cm3supporting medium without serum of cattle. Your mattress is placed in a thermostat for cultivation in (372)oC. the Period of cultivation from 48 to 72 hours To control uninfected leave 3 of the mattress, in which the growth medium is changed to 200 cm3supporting medium without serum. Starting from 24 h incubation, infected and control mattresses daily mikroskopiruyut. Mattresses that are experiencing JRS defeat to 60% of the cell monolayer (usually 48-72 hours), taken 3 times frozen at a temperature not higher than -20oC and thawed at 37oC. Then vaccinated material cleared from cellular debris. To do this, the contents of mattresses merge into one container and algherovia at 1000 rpm for 10 minutes Purified from detritus suspension should be transparent liquid pinkish-red color. The purified suspension contribute gentamicin (100 U) and stirred for 20 minutes Then out of the container take a sample for laboratory and production control for sterility, infection and HA activity of the virus. Production series must have the HA activity of not less than 11024 and infectious activity of not less than 7.5 lg TCD50/cm3. Purified vaccinated suspension is subjected to inactivation. Inactivation of the virus are using a 1% aqueous solution aminoethylethanolamine (AAAI) made up to a final concentration of 0.050,08%. For this purpose, the suspension is heated up to 26-28oTo contribute with constant stirring solution AAAI and set the pH value of 7.6 to 7.8 by adding a 5% solution of succinic acid. The inactivation is carried out in the unit (372)oC for 20-22 hours with periodic mixing. After inactivation antigenaemia suspension is cooled to 46oTo set the value of its pH in the range of 7.6-7.8 and take samples of the antigen to test for sterility, the avirulence and HA activity, use terasawa material and oil adjuvant, containing mineral oil with an emulsifier.

As oil adjuvant use drugs firm "seppic" (France): Montane IZA 70 and Montanide ISA 206. Oil adjuvant and vaccine antigen is mixed on the homogenizer according to the following modes:
1) when using Montanide IZA 70 - homogenization for 4-5 minutes at a speed of screw rotation 3000 rpm and a temperature of 10oWith, you get a homogeneous white emulsion of the type water-in-oil";
2) when using Montanide ISA 206 - homogenization for 3-4 min at a rotation speed of the screw 600 rpm and a temperature of 30oC. the Type of the obtained emulsion, water-oil-water. In the manufacture of vaccines, the antigen is combined with an oil Advanta in the following ratio: the adjuvant Montanide IZA 70 at least 1:2, and with the adjuvant Montanide ISA 206 at least 1:1, respectively.

The finished emulsion vaccine gather in one sterile container, Packed in glass bottles and is controlled in accordance with the technical specifications.

To assess the antigenic activity of the drug use quantitative method of control by which to determine the number of parvovirus antigen in pravilnoy dose of vaccine (1 cm3). The methods of the ri 2000 rpm Adosados examined in DSA. The vaccine is immunogenic, if HA-titer isolated from vaccine drug parvovirus antigen is not lower than 1: 256 in pravilnoy dose of 1 cm3. In most series, the amount of antigen equals 512 GUY, 1024 GUY and 2048 the GUY.

The vaccine is an emulsion of white or white with a pink tint, slightly viscous consistency. When storing vaccines slight peeling of mineral oil on the surface of the emulsion. With shaking, the vaccine becomes homogeneous structure.

Received the vaccine has an optimal component composition, wt. %:
Antihistamime material from strain "VL-94" PVA infectious activity of not less than 7.5 lg TCD50/cm3- 40,0-60,0
AAAI - 0,050,08
Oil adjuvant - Up to 100.0
The vaccine is used for prophylactic immunization of pigs against PVIS. The vaccine promotes the formation of active immunity against PVIS through 21 days after 2 times of using the drug a duration of at least 6 months.

Sows replacement gilts and boars-producers vaccinated twice with an interval of 20-30 days for three weeks before insemination. Piglets vaccinated at 2-2,5 months old 2-fold animals. The vaccine is suitable for use within 12 months after production if stored in a dark room at a temperature of (1-8)oC.

Example 3.

Conducted research on testing the sterility of the production strain "VL-94" PVA (4th passage). To do this, the 3 bottles of native virus was prepared by the average sample was made on 1 cm3three tubes containing thioglycolate environment. One sowed the test tube was kept in an incubator for 14 days at 370,5)oC, the other at room temperature of 21oWith up to 25oWith during this same period. The third test tube was kept for 7 days at 370,5)oC and then made her subcultures 0.5 cm3in each tube with the following environments: mastopathy broth (BCH) and agar (MPA), mastopathy liver broth under vaseline oil (MPB), agar and broth Saburo. Environment BCH, MPA, MPB crops and kept for 7 days at 370,5)oC, and agar and broth Saburo is at a temperature of 21oWith up to 25oC. the results of the studies are given in table. 1.

Tests showed that the strain VL-94" (4 passages) PVA is not contaminated by bacterial and fungal microflora. On the Sabbath.

Studies to test the biological activity of the production strain VL-94" PVA (4th passage) titration in transplantable cell cultures kidney pig CPD. To do this, the contents of 3 bottles of strain VL-94" (4 passages) were mixed and prepared dilution from 10-3up to 10-10on the environment of the SRP.

From bottles of 0.05 cm3poured growth environment, then in 4 test tubes with monolayer cell culture of CPD was made 2 cm3appropriate dilutions of the virus. The contact was 40-60 minutes After which the vials were made of the SRP.

Infected and control cell culture was kept in a thermostat at 372oC for 144 h without changing the medium. Titration of virus in cell culture CPD was determined by the characteristic of the JRS and the destruction of the monolayer of cells. Microscope monolayer in vials was carried out daily. In the control vials (4 pieces ) did not report any destructive changes of the cells. Final analysis was carried out for 6 days. Simultaneously viewed and control vials. Titration results were considered significant when saving monolayer in control. The results are shown in table. 2. Thus, tests have shown that the emer 5.

Conducted studies to determine the HA activity of the production strain VL-94" PVS. HA activity of the strain was determined in the DSA.

The virus for the study was taken from the averaged samples, made from native virus 3 penicillin vials. The virus was titrated in triplicate in 96-well round-bottom microarrays. The wells were made in 50 ál saline pH of 7.27.4, was prepared by 2-fold dilution of the virus, ranging from 1:2 to 1:4096. Then to each well was made in 50 µl of 0.6% suspension of Guinea pig erythrocytes. To something called a microarray was shaken and left at 1-1 .5 h at 42oC. HA-titer strain PVA considered the highest dilution that caused a complete agglutination of erythrocytes. The results of the DSA reliable, because microarrays were present controls:
control specific parvovirus antigen with known titer;
control of Guinea pig erythrocytes (50 μl of saline was introduced into 50 μl of 0.6% suspension of erythrocytes).

The results of the tests shown in the drawing microarrays.

Thus, tests have shown that hemagglutinine titer of strain VL-94" 5 passage in three replications was 1:2048.

Example 6.

The Prov is uma methods: molecular and serological. Using PCR strain VL-94" found only genome PVA.

Determination of the specificity of serological strain method was staged by rtha with specific swine serum obtained at 28 days after injection of strain VL-94" 5 passage. The results of rtha showed that specific porcine serum inhibited hemagglutinins properties (8 AI) virus titer 1:1024. Delay of haemagglutination indicates the type of virus and taken (known) specific serum. Thus, the results of PCR and rtha demonstrate the specificity of the strain VL-94" PVS.

Example 7.

Studies to test immunogenic production strain "VL-94" PVS. To do this, the 3 bottles of strain VL-94" (5th passage) was the average sample was prepared by dilution of 10-3up to 10-10on the environment of the SRP. To assess the antigenic activity of the strain used 5 pigs weighing 25-30 kg, two pigs for the control and three introduced the virus infectious activity of 8.0 lg TCD50/cm3at a dose of 1 cm3each (intramuscularly in the neck behind the ear). For infected and control animals were daily clinical observation, conducted thermometry. Within 12 redelf norms. After 14, 21 and 28 days after infection, all animals were taken blood samples. Serum was investigated in rtga. The results of the study of blood sera are presented in table. 3. Thus, specific to the strain VL-94" antibodies have been identified as infected and control (contact) of the pigs.

Example 8.

Effectiveness of inactivated emulsion vaccine PVIS of strain VL-94", manufactured as described in example 2, was tested in speciose JSC "Quiet flows the don", Ostrogozhsk district of the Voronezh region in repair pigs in the amount of 22 goals, grafted mentioned vaccine twice with an interval of 20 days at a dose of 1 cm3. The results of the study in rtga blood sera from repair sows immunized with this vaccine, given in table. 4. The replacement gilts farrowing was installed in physiological terms 114116 day of gestation. Received on 9-10 piglets per sow. Piglets had a body weight of 0.91.1 kg, well developed and with a good sucking reflex.

Among farrowing sows were observed: the number of litters with stillborn piglets - no, number of litters with partially stillborn piglets - no, collisional vaccine PVIS of strain VL-94", manufactured as described in example 2, was tested in C/C "Kulikovo" Somethinggo district, Penza region 12 sows vaccinated mentioned vaccine twice with an interval of 20 days at a dose of 1 cm3.

The results of the research in rtga blood sera from sows immunized with this vaccine, given in table. 5.

Complications after vaccination were observed. Vaccine efficacy was 97%.

Example 10.

Effectiveness of inactivated emulsion vaccine PVIS of strain VL-94", manufactured as described in example 2, was tested in JSC "Wisniewski", Voronezh region 10 sows vaccinated mentioned vaccine twice with an interval of 20 days at a dose of 1 cm3.

The results of the research in rtga blood sera from sows immunized with this vaccine, given in table. 6.

Complications after vaccination were observed. Vaccine efficacy was 98%.

Thus, the above information shows the implementation of the use of the present invention, the following cumulative conditions:
a strain of "VL-94" PVA embodying the invention, intended for use in agriculture, namely in veterinary WEIMA the claim, confirmed the possibility of its implementation using the steps described in the application or known before the priority date tools and techniques;
a strain of "VL-94" PVA obtained in accordance with the invention, expands the Arsenal of new high-yielding strains of homologous virus with high biological, antigenic and immunogenic activity and is suitable for production of effective vaccines. Therefore, the present invention meets the condition of patentability "industrial applicability".

Sources of information
1. Syurin Century. N. and other Viral diseases of animals. M, UNITEMP, 1998, 573-584.

2. G. M. Ruckenbauer et al. Can. J. List. Med., 1978, 42, 278.

3. Application France 2781159 And 61 To 39/23, 21.03.2000,

4. Pat. Russia 1538305 And 61 To 39/295, 15.12.94, (prototype).


The strain of Parvovirus suum, collection VGNKI "VL-94-DEPT", for the manufacture of vaccines.


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FIELD: veterinary virology and biotechnology.

SUBSTANCE: claimed vaccine contains antigenic material from PRRS virus strain, reproduced in passaged cell culture Marc-145 and inactivated with aminoethylethyleneimine (AEEI), antigenic material from PPVI virus strain reproduced in passaged YPK cell culture and inactivated with AEEI, and oil adjuvant in effective ratio. Vaccine represents white or white-rose emulsion. Vaccine is intramuscularly (behind ear) administered to animal in dose of 2 cm3 independently of age thereof. Immunity in vaccinated animals appears over 21-30 days after vaccination and remains for 6 months or more. Vaccine of present invention induces high levels of antibodies against PRRS and PPVI and provides durable passive immunity in young stocks up to 20-25-days age.

EFFECT: innoxious vaccine useful in animal protection of neonatal age from field viral PRRS and PPVI agents.

14 cl, 8 tbl, 11 ex