RU2176520C2 - Industrial strain enterovirus suis of swine encephalomyelitis pathogen (teshen's disease) useful for biological preparation preparing - Google Patents

Abstract

FIELD: veterinary virology. SUBSTANCE: invention relates to a novel strain of swine enteroviruses used for preparing vaccines and diagnostic preparations. Strain is obtained from swine pathological material, in transplanted cell cultures with signs of enteroviral encephalomyelitis in period of epizooty of Teshen's disease virus. Strain is accumulated in titers 8.0-9.0 lg TCD 50/ml, it shows contagiousness inducing Teshen's disease in pigs at intracerebral and contact infection and virus persistence in infected rodents. Strain shows the expressed antigenous and immunogenous activity and does not contain biological contaminants. The inactivated emulsified vaccine against Teshen's disease prepared from the strain "Navlya-96" shows 100% antiepizootic efficiency. Strain "Navlya-96" in complex with strain "Zakarpatsksky" is antibody diagnosticum used for subtype identification of Teshen's disease pathogen. EFFECT: pathogen strain indicated above, valuable properties. 3 tbl, 2 ex

Description

 The invention relates to the field of veterinary virology, in particular to production strains of pig enteroviruses, which can be used for the manufacture of diagnostic and vaccine preparations in enterprises of the biological industry.

 Based on the results of the neutralization reaction, pig enteroviruses isolated in different regions of the world are assigned to 11 serotypes (Derbishire J.D. Porcine Enterovirus Polioencephamielitis. In: Virus infections of porcine, Amsterdam etc., 1989, pp. 225-233). Swine enteroviruses of serotype 1 cause pig enterovirus encephalomyelitis in this animal species (Teshen disease).

 In the territory of the Russian Federation and some CIS countries, the Transcarpathian strain of Teshen disease virus is used as an industrial strain. In particular, the Transcarpathian strain is the production strain, the closest analogue of the present invention, in the manufacture of "Inactivated emulsified vaccines against enterovirus encephalomyelitis of pigs" according to TU 9384-020-00008064-96 and "Drug kit for identifying Teshen disease virus by neutralization reaction" according to TU 9388-022-00495549-89.

The Transcarpathian strain propagates in transplantable cultures of cells of porcine origin: transplantable cells of a pig embryo (SPEV), transplantable cells of a piglet testicle (PTP), a transplantable cell line of a kidney of a pig embryo (PPC), accumulating in titers 10 ⁷ - 10 ⁹ TCD _{50 / ml} , as well as in the body of the pig, causing an intracerebral infection of piglets of 2-4 months of age with a disease with damage to the brain and spinal cord.

 When studying the regional epizootology of Teshen disease in the Bryansk region, in 1995-1996. it was found that the inactivated vaccine prepared on the basis of the Transcarpathian strain, when vaccinated once according to the instructions for use, did not provide complete protection of the animals from the disease. Virological studies showed the presence of immunological mismatch of the Transcarpathian strain to the epizootic virus of the Teshen disease pathogen circulating in the Bryansk region. This necessitated the search for a new production strain for the improvement of antiepizootic drugs and methods of specific protection against Teshen disease.

 The aim of the invention is to obtain a new, immunologically different from the known, industrial strain of enterovirus pigs of the 1st serotype, homologous to the protective antigen of epizootic strains of Teshen disease virus circulating in the central regions of the Russian Federation.

The goal was achieved by isolating the epizootic (virulent) virus of Teshen disease in the focus of infection in 1996 among the pigs in the Navlinsky district of the Bryansk region, vaccinated against the Teshen disease with a vaccine from the Transcarpathian strain. For this, pathological material of the brain from a fallen gilt with signs of enterovirus encephalomyelitis inoculated a transplanted culture of SPEV in test tubes, kept it for 5 days at 37 ^° C, frozen, thawed and carried out another passage until the development of a cytopathic effect (CPD). An isolated cytopathic agent was identified in the neutralization reaction, according to the requirements of the Guidelines for the diagnosis of Teshen disease. The strain-specific features of the isolate were studied in the neutralization reaction according to the Guidelines for typing and subtyping of FMD virus. The isolated strain of the Teshen disease virus at the place and time of isolation was called the strain Navlya-96.

 According to the results of the neutralization reaction, it was established that this virus, like the Transcarpathian strain, belongs to the 1st serotype of pig enteroviruses, but differs from this strain in the neutralization index (Table 1).

The difference between the neutralization index of viruses of the strain "Transcarpathian" and strain "Navlya-96" specific serum to pc. "Transcarpathian" does not exceed 1.0 lg TCD _{50 / ml} and is in the range of 0.75-1.0 lg TCD _{50 / ml} :
in experiment N 1 7.25-6.25 = 1.0 lg TCD _{50 / ml} ;
in experiment N 2 5.5-4.75 = 0.75 lg TCD _{50 / ml} ;
in experiment N 3 5.5-4.5 = 1.0 lg TCD _{50 / ml} .

 From this it follows that the compared strains are antigenically related, while at the same time there are significant immunological differences between them.

 The data table. 2 also indicate an incomplete serological correspondence of strains "Transcarpathian" and "Navlya-96". So whey per pc. Navlya-96 neutralized equally both strains of Transcarpathian and Navlya-96 at a dilution of 1: 256. Whereas the serum for the Transcarpathian strain neutralized the homologous strain of the virus at a dilution of 1: 1024, and the heterologous strain of Navlya-96 at a dilution of 1: 512.

Strain "Navlya-96" was adapted to transplantable cell cultures PSGK (transplantable cells of the Siberian mountain ibex), PTP (transplantable cell culture of testicles of a piglet), PPK-66 (transplantable cell line of a kidney of a pig embryo). In cell cultures, the virus accumulates in high titers (8.0-9.0 lg TCD _{50 / ml} ), produces a highly active protective antigen that is effectively detected in immunological tests (in CSC, ELISA, RDP) and induces the formation of virus-neutralizing antibodies in high titers after a single administration to guinea pigs and rabbits. The strain Navlya-96 was deposited at the Institute’s Museum of Microorganisms and assigned registration number 1970.

 The strain "Navlya-96" is characterized by the following features.

TAXONOMIC CHARACTERISTIC
The virus is resistant to fat solvents (chloroform, freon), and with electron microscopy it has the shape and size typical of enteroviruses. In the reactions of neutralization, complement binding (CSC), diffusion precipitation (RDP) and enzyme-linked immunosorbent assay (ELISA) it detects the presence of antigens that are common to all pig serum enteroviruses of the 1st serotype.

MARKER CHARACTERISTICS
Adaptation properties and contagiousness: the virus is reproduced in the culture system of primary (PS) and transplanted pig cell lines (SPEV, PK-15, SK-6, PTP), kidneys of the Siberian mountain ibex (PSGC) at the optimum temperature (37.5 ± 0, 5 ^o C) to infectious titers of 8.0 -9.0 lg TCD _{50 / ml} . The strain is contagious to pigs.

 Pathogenic and neurovirulent properties: the virus does not show pathogenicity for rodents with intramuscular and subcutaneous methods of infection, but causes short-term viremia and persistence of the virus in them. It has neurovirulent properties for pigs, causing disease and death of some animals, which increase during passage through the body of rodents.

 Properties of the beneficial substance produced by the virus: causes the formation of virus-neutralizing protective, complement-binding, precipitating antibodies. An inactivated virus induces immunity in pigs against epizootic variants of the Teshen disease pathogen currently circulating in the Russian Federation.

 Agglutination properties: does not agglutinate red blood cells of mouse, guinea pig, pig, chicken, duck without additional processing.

Thermal resistance (T ₅₆ - sign): completely loses infectious properties as a result of heating at 56 ^o C for 2 hours, partially retaining antigenic activity.

 Inhibitor sensitivity: chloroform, ether, freon and other fat solvents do not inhibit the infectious activity of the virus. It is sensitive to antibodies against heterologous enteroviruses in pigs, to thermolabile B-inhibitors of native cattle and pig sera. The virus inactivates solutions of formaldehyde, copper sulfate, theotropin, crystal violet.

 Stability of genetic and immunobiological traits: the stability of genetic and immunobiological traits was maintained when it was passaged in a culture of cells sensitive to Teshen disease, for at least 21 passages (observation period).

 Contamination by bacteria, fungi, mycoplasmas and extraneous viruses: not contaminated by bacteria, fungi, mycoplasmas and extraneous viruses.

STANDARD GROWING CONDITIONS
Transplanted cultures of kidney cells of the Siberian mountain ibex (PSGC) or piglet testicle (LTP) grown in mattresses, test tubes, tablets or in a roller method in glass or plastic bottles are inoculated with Teshen disease virus pcs. "Navlya-96" and incubated at 37 ^o C until the development of the cytopathic effect (CPP), i.e. until the complete destruction of the monolayer of cells, manifested in the form of rounding and combining of the affected cells into clusters, followed by exfoliation into the culture fluid. With a multiplicity (M) of infection of 0.001 TCD 50 /, the CPD cell develops after 96 hours of incubation under stationary conditions and after approximately 72 hours with roller culture. With an increase in M to 0.01 TCD 50 / cell, the CPD occurs after 48, and at M 0.1 TCD 50 / cell in 24 hours.

The strain is stabilized with skim milk or other stabilizers and stored in a lyophilized state at a temperature not exceeding minus 40 ^o C. The virus is refreshed by passaging it in PTP or PSGK cell cultures once every five years.

 Isolated strain of Navlya-96 Enterovirus suis in the Bryansk Region, the pathogen of porcine enterovirus encephalomyelitis (Teshen disease), is currently used at the institute as a production facility for the development and manufacture of biological preparations.

Example 1. The use of strain for the manufacture of vaccines
An inactivated emulsified vaccine against Teshen disease is made from the virus of the Navlya-96 strain and controlled in accordance with the requirements of TU 9384-020-00008064-96 "Inactivated emulsified vaccine against Teshen disease." At the same time, the presence of virus-neutralizing antibodies in vaccinated rabbits and gilts is checked using 100-1000 TCD _{50 / ml} of Navlya-96 virus. 14-21 days after vaccination, antibody titers range from 1/16 to 1/64. The vaccine from the Navlya-96 strain in the enzootic foci of Teshen disease in the Bryansk region, as part of production tests commissioned by the Department of Veterinary Medicine of the Russian Federation, showed 100% antiepizootic efficacy. At the same time, vaccines from the Transcarpathian strain of various manufacturing firms in 15-40% of cases did not show a protective effect due to the incomplete immunological correspondence of the epizootic and vaccine strain (see table. 3)
Example 2. The use of strain for the development of subtype diagnosticum.

The Navlya-96 virus with an activity of 7.5 lg TCD _{50 / ml is} inoculated with guinea pigs (n = 5) and placed in a cage with a mesh floor over the feed zone of the pig machine. After 2-4 weeks from the onset of contact, pigs develop Teshen disease. Some of them die, and some (up to 25%) survive. In pigs in the terminal stage of the disease, the titer of serum virus-neutralizing antibodies is from 1:32 to 1: 256, and in pigs-convalescents - from 1:64 to 1: 1024. Pigs with the most active sera in the neutralization reaction are bled, the obtained blood serum is clarified by centrifugation or by microfiltration through membrane filters with a pore diameter of 22 μm, pack 0.5-1.0 ml in ampoules and lyophilize in standard mode in any type of freeze-drying apparatus. Antibody diagnosticum activity is monitored in a neutralization reaction, enzyme-linked immunosorbent assay, etc. A worker is considered a serum dilution of 4 times the titer limit. For example, if the maximum titer of serum in the neutralization reaction is 1: 1024, then its working dilution is 1: 256. Antibody diagnosticum is developed similarly for subtype identification of the Transcarpathian virus. Both antibody diagnosticums complete the kit for subtype identification of the causative agent of Teshen disease.

 The serum was deposited in the Museum of VNIIVViM strains (inv. N. 1971 dated 11/06/1997) and is used for the subtype identification of the Teshen disease pathogen during epizootological studies and to optimize vaccine prophylaxis.

Claims (1)

 Production strain of Enterovirus suis of the pathogen of pig encephalomyelitis (Teshen disease), deposited in the collection of microorganisms of the All-Russian Research Institute of Veterinary Virology and Microbiology from 1970 November 6, 1997, suitable for the manufacture of biological preparations.

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