RU2096454C1 - Strain pestivirus for preparing lapinized virus-vaccine for control classic plague in pigs - Google Patents

Abstract

FIELD: biotechnology, veterinary virology. SUBSTANCE: invention relates to new strain Pestivirus "GM-94" "ВГНКИ" that is deposited in collection of viruses and microorganisms "ВГНКИ" at number "GM-94" - DEP. Safety for satisfactory vaccinated pigs is 100% and that for unsatisfactory ones - 97.7%. Mean titer of antibodies to serum blood in piglets and swines is (1:16)-(1: 64). Vaccine prepared from the strain Pestivirus "GM-94" "ВГНКИ" is harmless for piglets and swines and can be used for classic plague prophylaxis. Vaccine is administrated intramuscularly at effective single dose. New strain Pestivirus shows high immunogenicity and low reactivity. EFFECT: new strain indicated above, high effectiveness of vaccine.

Description

 The invention relates to the field of biotechnology and veterinary virology, in particular to a strain of classical swine fever virus used for the manufacture of a lapinized virus vaccine against this disease.

 For specific prophylaxis of classical swine fever (CSF), culture and lapinized strains of the CSF virus are mainly used to obtain vaccines.

Known lapinized strain "Hudson" Flavivirus suis "Hudson" virus CoES, which was used in the Soviet Union in the 60s for the manufacture of lapinized virus vaccines against CoES [1,2]
The disadvantage of this strain is that it has increased reactogenicity. After vaccination of piglets, the temperature reaction is observed in 70-80% of animals within 2-8 days and reaches 40.6-42 ^o C. In 20-30% of vaccinated animals, a depressed state, a decrease in feed intake are noted. The average weight gain of animals per month (compared with the control) is reduced by 4.2 kg.

 The aim of the invention is to obtain a highly immunogenic lapinized strain of CSF virus without increased reactogenicity.

 A new strain of CoES virus was obtained from the Hudson strain of CoES virus by sequential passages through the rabbit body and by moving sequential passages on the PPS cell culture, as well as rabbits using the “limit dilution” method with identification in the neutralization reaction with specific serum against reference virus strains CSF, selection of variants according to the criteria of harmlessness for piglets 1.5-3 months of age, immunogenicity by the method of control infection of vaccinated pigs with a virulent strain of CSF virus, and intensity of its reproduction in a sensitive system of rabbit body.

 The virus strain is deposited in the collection of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines and has a registration number "GM-94" DEP.

 The strain Pestivirus "GM-94" VGNKI is characterized by the following features and properties.

 Morphological signs.

 Electron microscopic examination of the strain "GM-94" VGNKI virus CoES presented typical for flaviviruses separate round virions, with a diameter of 40-44 nm. In ultrathin sections, the nucleocapsid value was determined to be 34-38 nm, and the thickness of the lipoprotein membrane was 3 nm. The sedimentation coefficient is 104 ± 18 according to Swedberg.

 Biological properties.

The strain of CSF virus "GM-94" VGNKI multiplies well in the rabbit. In the blood and spleen of a rabbit infected intravenously, the virus accumulates after 48-72 hours in a titer of 3.5-4.5 lg ICD ₅₀ / cm ³ (ICD infectious rabbit dose).

The strain of CSF virus "GM-94" VGNKI relatively weakly propagated in cell culture PPS and in transplantable cell culture PK-15. Infectious activity of the virus after 96 hours, when these cell cultures are infected at the rate of 0.015-0.03 ICD ₅₀ per cell, when incubated at 37 ^o C, reaches a value of 2.5-3.5 lg ICD ₅₀ / cm.

 In primary and transplantable cell cultures, the strain of cytopathic effect does not cause.

 Antigenic properties.

 With parenteral administration of the strain "GM-94" VGNKI CoES virus into the body of a pig and rabbit causes the formation of virus-neutralizing antibodies in a titer of 1: 16-1: 64.

 Reactogenic properties.

After injection of the strain "GM-94" VGNKI of the virus of CoES to piglets at a dose of 100-1000 ICD ₅₀ in 20-25% of vaccinated animals notes an increase in body temperature to 40.1-41 ^o C, which is maintained for 1-4 days without suppression of the total condition. The average weight gain of animals (per month) after injection of the strain decreases (in comparison with the control) by only 0.6-1.2 kg.

 Immunogenic properties.

 Strain "GM-94" VGNKI of the virus of CoES during parenteral administration to pigs stimulates the formation of intense immunity. In experiments on pigs, it was found that after intramuscular administration of a suspension of a strain of the virus on days 10-14, they, when tested in the neutralization reaction (PH), develop antibodies in a titer of 1:16 with a subsequent increase by 21 days to 1: 32-1: 64.

 Stability.

 Acquired properties (lack of reversion and weakened reactogenicity) strain "GM-94" VGNKI CoES virus stores for 8 consecutive passages in pigs.

 Persistence.

In the lyophilized state at 2-8 ^o C, the strain "GM-94" VGNKI of the virus of CoES is stored for a year without a significant decrease in infectious activity for rabbits. In the native state (rabbit blood) is stored at a temperature of 30 ^o C for 6 months.

 Additional information about the strain.

 Free of viral, bacterial and mycoplasma contaminants.

 Strain "GM-94 VGNKI used for the manufacture of lapinized virus vaccines against classical swine fever.

Example 1. Rabbits with a live weight of 2.5-3.0 kg are injected intravenously with a suspension of the strain "GM-94" VGNKI CoES virus in a dose of 100 ICD ₅₀ in a volume of 1.0-1.5 cm ³ . In animals, body temperature is measured daily. Rabbits reacting after 48-96 hours with an increase in temperature to 40.1-41.8 ^o C, bleed with extraction of the spleen and mesenteric lymph nodes. The resulting blood and organs (spleen and lymph nodes) of rabbits are homogenized in compliance with antiseptic rules. Antibiotics are added to the homogenate in an effective amount, after which it is poured into sterile vials (ampoules) of 2-4 cm ³ and subjected to freeze-drying, followed by capping.

The activity of the virus in the finished vaccine, when tested on rabbits, is 3.0-4.0 lg ICD ₅₀ / cm ³ .

The finished biological product is a dark red tablet that retains its immunobiological properties for 12 months at a temperature not exceeding 8 ^o C.

Example 2. Virus vaccine against CSF from strain "GM" VGNKI obtained in example 1, is checked for immunogenicity in piglets. For this purpose, from a mixture of the contents of three vials, a dilution of 1: 10, 1: 100, 1: 1000 and 1: 10000 is prepared in physiological saline. Each dilution of a 2.0 cm ³ virus vaccine inoculates two pigs intramuscularly, from the inside of the thigh or in the neck. As a control, leave two piglets to which the vaccine is not administered. After vaccination, piglets are monitored for 14 days, daily measuring body temperature. In vaccinated animals (30%), an increase in body temperature to 40.1-41.0 ^o C without signs of inhibition and a decrease in food intake is noted. A lack of incidence among vaccinated animals was noted.

After 14 days, the piglets that were immunized with a virus vaccine at a dilution of 1: 100, 1: 1000, 1: 10000 and two control (unvaccinated), are infected intramuscularly with a virulent strain of CoES virus "Chi-Myn" at a dose of 10 ⁵ LD ₅₀ .

Infected pigs are monitored for 14 days, measuring body temperature daily. The death of 2 experimental, vaccinated (breeding 1: 10000) and 2 control unvaccinated animals is established. The immunogenic activity of a vaccine prepared calculated by Kerber Ashmarina a modification which is in this case 3,5 lg IPD _50/2 cm ^3.

Example 3. In one of the CSF-disadvantaged pig complexes, piglets of 30 and 90 days of age are immunized with a dose of 2 cm ^{3 of a} lapinized virus vaccine against CSF from the strain "GM-94" VGNKI obtained in Example 1. A total of 81600 piglets are vaccinated. After vaccination with this vaccine (after 12-15 days), the death of piglets from the plague sharply decreased (to 0.4%), and after 30-40 days it stopped. Subsequently, there were no cases of plague among these animals. In total, 1870 goals fell from the CoES during the observation period. (2.3%).

 In two sectors of the same complex (growing group), 620 piglets are immunized with a 10-fold dose of culture virus vaccine against CSF LK-VNIIVViM. After 60-70 days, animals with plague are isolated. In total, 204 pigs (32%) fell from the plague and were forcedly killed in these sectors, which indicates the low efficiency of this vaccine in a pig-breeding complex unsuccessful in CSF. Due to the high percentage of the incidence of CoES piglets, the remaining number of animals is killed in a slaughterhouse.

 Example 4. Virus vaccine against CSF, made from strain "GM-94" VGNKI obtained according to example 1, is used in CSF-safe sectors of the dysfunctional complex. Piglets are vaccinated at the same time and in the same dose as in Example 3. The animals are monitored with selective thermometry. In some piglets (20-25%), a short-term (within 2-4 days) increase in body temperature without visible signs of inhibition or decrease in food intake is noted. Set the complete safety of the livestock after vaccination. On the 14th day, blood is sampled selectively from animals, serums are obtained, which are tested in the neutralization reaction. Antibody titers in the vaccinated population are 1: 16-1: 64, which indicates a high immunogenicity of the vaccine.

 Studies and tests show that the new Pestivirus strain "GM-94" VGNKI is highly immunogenic, harmless to piglets and pigs, which allows us to recommend it for the manufacture of an effective biological product against classical swine fever.

 The use of a vaccine obtained from this strain in veterinary practice will expand the arsenal of highly effective biological products and reduce the cost of measures to combat classical swine fever.

Claims (1)

 The strain Pestivirus "GM-94" VGNKI DEPT used for the manufacture of lapinized virus vaccines against classical swine fever.