RU2348692C1 - Attenuated strain of nairobi sheep disease virus - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention concerns veterinary medicine, particularly attenuated strains of Nairobi sheep disease virus (NSDV). Attenuated cultural strain of Nairobi sheep disease virus is deposited in microbe strain collection of State Research Institution National Research Institute for Veterinary Virology and Microbiology of Russia by inventory No 2589. Invention can be applied in development and production of vaccines for Nairobi sheep disease in research institutions and biological industry enterprises.

EFFECT: obtaining attenuated strain of Nairobi sheep disease virus.

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Description

The invention relates to the field of veterinary medicine, in particular to attenuated strains of the Nairobi disease virus (VBI), and can be used to develop and manufacture vaccines against Nairobi disease in research institutions and biological enterprises.

According to the accepted classification, the Nairobi disease virus belongs to the Bunyaviridae family, the genus Nairovirus, a species of Nairobi sheep disease virus (Regenmortel MHV, Fauquet CM., Bishop DHL et al. / Virus Taxonomy The Classification and Nomenclature of Viruses. The Seven Report of the International Committee on Taxonomy of Viruses // Academic Press, San Diego, 613 p.).

Currently, in the disadvantaged areas of the African continent, for the prevention of Nairobi disease (ND), virus vaccines are used, made on the basis of attenuated strains of the Nairobi disease virus, obtained from the brain of newborn white mice.

R. Montgomery tried to immunize the sheep, introducing them different volumes of viral material after the introduction of serum from sick animals at different time intervals, but none of these attempts gave satisfactory results. He also tried to get a heat-inactivated vaccine, but the attempts were unsuccessful. The same author suggested attenuating the virus on goats. (Montgomery, R.E. On a tick-borne gastroenteritis of sheep and goats occurring in British East Africa. // J. Coll. Path., No. 30 1917, pp. 28-57.)

Weinbren M.P. (1956) showed the possibility of active immunization of sheep and goats attenuated in mice with a brain strain of the BN virus. For vaccination, we used the Entebbe strain (brain passage software) (Weinbren MP The developvent of a strain Nairobi sheep disease viruses non pathogenic for sheep a possible vaccine // Ann. Rep. EA Viruses Res. Inst, 1958, No. 8, p. 12) . The same strain (140-150 brain passages) is the most pathogenic and safe for animals. (Weinbren M.P., Gourlay R.N., Lumsden W.H., Weinbrein B.M. An epizootic of Nairobi sheep disease in Uganda // J. Comp PathoL, 1958, No. 68, pp. 174-187.)

Ansell (1957) reported that the Mugug-Tislon strain (22 brain passages) was avirulent and protected the sheep during challenge. (Ansell R.H. Attenuation of Nairobi sheep disease virus in the mouse brain // Veter. Rec. No. 69,1957, p. 410-412.)

Attempts to prepare an inactivated formol vaccine (Walker, 1932) based on virulent strains and a vaccine from virulent blood treated with crystal-violet according to the method of Danbney (1944) were unsuccessful. (Terpstra, C. Nairobi sheep disease-studies on virus properties, epizootiology and vaccination in Uganda // Diss., Univ. Utrecht. 1969.)

There are reports of cultivation of BN virus strains in cell cultures. The BN culture virus obtained in BNK-21 cell culture was precipitated with methanol, mixed with an adjuvant and used to immunize animals. Twice immunization protected the sheep during infection control. (Davies F.G., Otieno S. & Jessett D.M. The antibody response in sheep vaccinated with experimental Nairobi sheep disease vaccines // Trop. Anim. Health Prod., 1977, No. 9, pp. 181-183).

In 1980, at the VNIIVViM Safonov G.A. and Chistov Yu.V. a vaccine strain MM of BN virus was obtained by serial passage (134 passages) of a virulent virus in white mice, the activity of which was 6.5-7.0 lg MicLD ₅₀ / ml. Strain MM at a dose of 3.25 lg MicLD ₅₀ / ml protected the sheep by 70-80% from the control of infection with a virulent virus for up to six months and was recommended for the development of a live virus vaccine.

Live virus vaccines derived from cultured viral strains have several advantages over the use of virus vaccines obtained from the brain of newborn mice: firstly, it is possible to guarantee the absence of a foreign virus in the vaccine, and secondly, the concentration of the virus in such vaccines increases series to series, which leads to a reduction in dosage and a significant reduction in the amount of nonspecific antigenic material introduced; thirdly, methods have been developed for producing large-volume vaccine series, which is more economical nd, besides the risk of contamination in this case smaller than for the preparation of virus-vaccine of mice brain. The vaccine obtained in this way is cheaper due to the large scale of production, since one test for immunogenicity and lack of toxicity is sufficient to test a significant amount.

The aim of the invention is to obtain a new culture attenuated strain of BN virus, suitable for the development and manufacture of vaccines against this disease.

This goal is achieved by cloning and adaptation of the attenuated brain strain of MM BN virus to a primary trypsinized culture of lamb kidney cells (PJ) and transplanted saiga kidney cell line culture (PS).

The resulting attenuated culture strain of BN virus is designated as the strain "MM / K-05" and deposited in the collection of microorganisms GNU VNIIVViM under No. 2589 of 06.16.06 g.

The new culture strain has the following properties.

Morphological properties: during electron microscopic examination, virus particles are found to be rounded or slightly oval in viral containing material, sometimes pleomorphic in ultra-thin sections, with a diameter of 80-120 nm. Virions consist of a nucleocapsid of spiral symmetry and a lipoprotein membrane 10-12 nm thick, on the surface of which there are protrusions 5-10 nm long.

Cultural properties: strain “MM / K-05” of BN virus propagates in transplantable cultures of saiga kidney cells (PS) and sheep kidneys (PO-VNIIVViM) and in the primary trypsinized cell culture of lamb kidney cells (PN) with cytoplasmic changes characteristic of BN virus followed by destruction of the monolayer.

Growth method: The strain "MM / K-05" is cultivated in a monolayer of transplantable PS cell culture in a stationary and roller method. Under optimal conditions, the maximum accumulation of the virus in transplanted cell cultures is 6.5-7.0 lg TCD ₅₀ cm ^{3 is} achieved after 2-3 days of cultivation. The virus titer in the primary trypsinized cell culture of the cell is 4.5-5.0 lg TCD ₅₀ / cm ³ . As a supporting medium using the medium Needle MEM with 2% serum of cattle.

Infectious activity: Infectious activity is determined by titration in PS and PO-VNIIVViM cell cultures, as well as by intracerebral infection of sucking mice.

Antigenic properties: when administered subcutaneously to sheep and goats, the MM / K-05 strain causes the formation of virus-neutralizing, precipitating and complement-binding antibodies.

The specificity of the virus: the specificity of the virus is determined by direct immunofluorescence using diagnostic FITC-globulin.

Pathogenicity for humans: the virus is not pathogenic for humans.

Virulence for susceptible animals: strain "MM / K-05" is harmless to goats and sheep with subcutaneous and intramuscular injection.

Harmlessness for laboratory animals; for laboratory animals (rabbits, guinea pigs) it is not pathogenic, in white mice it causes death upon intracerebral infection.

Contagiousness: the strain "MM / K-05" is not transmitted from vaccinated non-immune sheep and goats when they are together.

Reactogenicity: the strain is weakly reactive for sheep and goats with subcutaneous administration at a dose of 3.0-5.0 lg TCD ₅₀ / cm ³ . In some vaccinated animals, an increase in body temperature to 40.1-40.5 ° C was noted for 2-3 days.

Immunogenic properties: strain "MM / K-05" with subcutaneous administration at a dose of 3.0 lg TCD ₅₀ / cm ³ . In vaccinated sheep and goats creates immunity for 21 days after vaccination.

Contamination by bacteria, fungi, mycoplasmas and extraneous viruses: the lyophilized strain “MM / K-05” of passage 9 in PS cell culture from 12/16/05 was not contaminated by bacteria, fungi, mycoplasmas and extraneous viruses.

Stability of genetic and immunobiological traits: The main biological properties of the strain are stably maintained for 15 passages (observation period) of cultivation in transplanted cell culture PS and PO.

Method and shelf life, frequency of passages: strain "MM / K-05" is stored in a lyophilized state at a temperature of minus 40 ° С and is "refreshed" by passage in PS cell culture once every five years.

Example 1. The cultivation of the strain "MM / K-05" VBN in a monolayer of transplantable cell cultures PS and PO-VNIIVViM grown in mattresses.

As a result of the studies, the optimal parameters of the strain cultivation were worked out, which were:

- the multiplicity of infection is 0.01-0.05 TCD ₃₀ / cell ',

- cattle serum content in the supporting medium Needle MEM-2%;

- pH of the medium is 7.2-7.4;

- the duration of cultivation 3-4 days;

- temperature condition 37.0 ± 0.5 ° C;

- filling volume of 1.5 liters of mattresses 0.2-0.25 liters.

These cultivation modes made it possible to obtain a virus-containing material with an activity of 6.5-7.0 lg TCD ₅₀ / cm ³ · The results of the production of viral raw materials of 3 experimental series based on the strain MM / K-05 of the BN virus are presented in table 1.

Table 1 Characterization of virus-containing culture raw materials (strain "MM / K-05") obtained in a monolayer of cell cultures PO and PS. Batch number Cell culture Number of mattresses Infecting Dose, TCD ₅₀ / cl Lasts. cult., day Series Volume The activity of the series, lg TCD ₅₀ / cm ³ one PS 5 0.05 2 1,0 7.0 2 PS 5 0.01 3 1,0 6.75 3 BY 5 0.05 3 1,0 7.0

As can be seen from the table, the activity of 3 series of viral raw materials obtained by the stationary method in PS and PO-VNIIVViM cell cultures was 6.75-7.0 lg TCD ₃₀ / cm ³ .

Example 2. The persistence of the lyophilized strain "MM / K-05" VBN at various temperature conditions.

Virus-containing material of the culture strain MM / K-05 VBN 9 of passage in PS cell culture was lyophilized with a peptone-lactose stabilizer.

The activity of the test sample after drying during intracerebral infection of sucking mice was 6.2 lg MicLD ₅₀ / cm ³ in the PS cell culture 6.5 lg TCD ₅₀ / cm ³ .

The results of studies of the biological activity of the strain "MM / K-05" stored at different temperatures are presented in table 2.

table 2 The study of the persistence of the culture strain "MM / K-05" virus of the Nairobi disease. Temperature mode (° С) Virus titer at x injured (lg MicLD ₅₀ / cm ³ ) Ref. 1 month 3 months 6 months 9 months 12 months Minus 40.0 ± 0.5 6.2 6.2 6.2 6.2 6.2 6.2 2.0 ± 2.0 6.2 6.2 5.9 5,6 5.3 4.9 18.0 ± 2.0 6.2 5.5 5,0 4.2 3.6 3.0 37.0 ± 0.5 6.2 - n / a n / a n / a n / a Note: n / a - not investigated
- - no virus detected

As can be seen from table 2, the lyophilized strain "MM / K-05" VBN retained its original activity for 12 months of storage at a temperature of minus 40 ± 0.5 ° C. At a storage temperature of 2.0 ± 2.0 ° C, the infectious activity of the virus decreased by 1.3 lg MicLD ₅₀ / cm ³ . At a temperature of (37.0 ± 0.5) ° C, the virus was not detected after 1 month.

The results obtained confirm the published data on the instability of VBI in the external environment when stored above 0 ° C. The optimal storage conditions of the lyophilized strain "MM / K-05" of the BN virus is a temperature of minus 40 ° C.

Example 3. Check the reversibility of the strain "MM / K-05" virus BN.

To this end, spend 5 passages of the strain "MM / K-05" on susceptible animals. During the first passage, the culture virus was introduced into a sheep of 8 months of age at a dose of 5.0 lg TCD ₅₀ / cm ³ (100 LDPE) subcutaneously in the axillary region. On the 7th day, a blood sample was taken and introduced to the next animal in a similar manner in a volume of 1.0 cm (second passage). Similarly spent the third, fourth and fifth passage.

Experimental animals did not have a clinical response to vaccination; they did not develop Nairobi disease within 21 days of observation after administration of the material. This indicates that the strain "MM / K-05" virus BN is not reversible.

Example 4. Verification of the immunobiological properties of the strain.

In the study of immunobiological properties for 1 vaccination dose was taken 3.0 lg TCD ₅₀ / cm ³ .

The safety of the strain was studied on four sheep and two goats 6-12 months of age. Strain "MM / K-05" VBN was injected subcutaneously into the hairless area of the axillary region. Two sheep and two goats were vaccinated with a virus at a dose of 5.0 lg TCD ₅₀ / cm ³ (100 LDPE), the remaining animals (two sheep) at a dose of 4.0 lg TCD ₅₀ / cm ³ (100 LDPE).

For 21 days, the immunized animals remained clinically healthy. In three vaccinated animals at a dose of 5.0 lg TCD ₅₀ / cm for 4-9 days, an increase in body temperature to 40.1-40.5 ° C was observed for 2-4 days (Table 3).

The immunogenicity of the strain was determined by the results of the control infection of vaccinated animals with a virulent strain of BN virus at a dose of 1000 LD ₅₀ .

For this purpose, two sheep and one goat of 7–9 months of age were inoculated with BN virus at a dose of 3.0 lg TCD _{50 /} cm ³ (1 LDPE). In one sheep, after vaccination, an increase in body temperature to 40.4 ° C was noted.

21 days after vaccination, all vaccinated animals and one control were infected with a virulent strain. Together with these animals, sheep and goats were used, used in the experiment to determine the safety. Before infection in animals, the level of BH antibodies was determined in sera, the accumulation of which was 1: 8-1: 32.

The results of the experiment are presented in table.3.

The control sheep died on the 17th day after infection with clinical signs of BN (body temperature 42 ° C, diarrhea, depression), while the vaccinated animals remained clinically healthy throughout the observation period.

Table 3 Immunobiological properties of the culture strain "MM / K-05" virus of the Nairobi disease, adapted to the culture of PS cells. Grafted Dose (TCD ₅₀ ) Kind of animal Vaccine response Control infection results Duration tamper. reactions, (days) Maksim. temperature Lasts. tamper. reactions, (days) Maksim. temperature 10 ^5.0 Number 1 sheep 3 40.5 - 40,0 Number 2 sheep four 40.3 - 39.9 No. 3 goat 2 40,2 - 39.7 No. 4 goat 3 40.1 - 39.8 10 ^4.0 Number 5 sheep 2 40,2 one 40.1 Number 6 sheep 2 40.1 - 40,0 10 ^3.0 Number 7 sheep 2 40,4 - 40,0 Number 8 sheep 2 39.9 one 40,4 No. 9 goat 2 39.8 one 40.3 The control Sheep - 9 42.0

The data obtained show that the MM / K-05 strain adapted to the transplanted PS cell culture creates after 21 days in animals vaccinated with a dose of 3.0 lg TCD ₅₀ / cm ³ immunity against the virulent virus of Nairobi disease. The strain is weakly reactive and harmless to sheep vaccinated at doses of 4.0 and 5.0 lg TCD ₅₀ / cm ³ , it is not reversible and induces the formation of BH antibodies.

Claims (1)

An attenuated culture strain of the Nairobi sheep disease virus deposited in the collection of microorganism strains of GNU VNIIVViM, inv. No. 2589 dated 06/16/06, suitable for the development and manufacture of vaccine preparations.