RU2100433C1 - Strain coronavirus canine used for control over vaccine immunogenic activity and serum preparing for treatment and diagnosis of coronaviral infection in dogs - Google Patents

Abstract

FIELD: biotechnology, veterinary virology. SUBSTANCE: new strain Coronavirus canine "Karat" shows stable infectious activity, high virulent property and provides objective control over vaccine immunogenic activity with respect to coronaviral infection in dogs. Strain is used also for preparing curative serum and diagnostic specific antiserum. Strain Coronavirus canine "Karat" is deposited in collection VGNKI of veterinary preparations at N 29. EFFECT: new strain indicated above, enhanced effectiveness of vaccine.

Description

 The invention relates to the field of veterinary virology, in particular, to a strain of dog coronavirus, used to control the immunogenic activity of vaccines and to obtain serums for the treatment and diagnosis of coronavirus infection of dogs.

 Known dog coronavirus strains "Toda" and "I-71" used to obtain sera for the diagnosis of coronavirus infection of dogs [1,2] Known strains are not sufficiently virulent, which does not allow them to be used as a control for evaluating the immunogenic properties of the coronavirus vaccine dog infections.

 The aim of the invention is to obtain a culture strain of dog coronavirus with stable infectious activity, high virulent properties, the ability to indicate the formation of specific antibodies in laboratory animals, which can be used as a control to evaluate the immunogenic activity of vaccines and to obtain therapeutic and diagnostic sera against dog coronavirus infection.

 The Coronavirus canine "Karat" strain was obtained from a coronavirus enteritis strain isolated in 1993 from a dead four-month-old German Shepherd puppy by alternating successive passages on a transplanted cat kidney cell line (CRFK) and a primary trypsinized puppy kidney cell culture (PSC).

 The strain is deposited in the collection of microorganisms of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines and has a registration number 29-DEP.

 The strain Coronavirus canine "Carat" N 29-DEPT is characterized by the following features and properties.

 Morphological properties. Virions have a spherical shape, their diameter ranges from 90 to 150 nm. The surface of the viral particles is covered with club-shaped protrusions forming the likeness of the solar corona, with a height of 15-20 nm.

Cultural properties. The strain multiplies well in cell cultures CRFK, PG, MDCK, causes a cytopathic effect. When cultured in a transplanted CRFK cell culture, the virus titer is 6.5 lq TCD 50 / cm ³ , and in a primary trypsinized culture of PG cells and in a transplanted culture of dog kidney cells MDCK 6.0 lq TCD 50 / cm ³ . The cytopathic effect of the Karat virus strain in the cultures used is manifested by the formation of symplasts, thinning of the cytoplasm of the cell processes, the appearance of clusters of rounded cells, gradually degenerated cells are separated from the glass and form windows.

 The antigenic properties of the Karat strain of dog coronavirus has antigenic affinity for the reference pathogenic strain of porcine transmissible gastroenteritis isolated by L. A. Melnikova et al. 1978. With intravenous and subcutaneous administration to rabbits, the virus of the Karat strain induces the formation of virus-neutralizing antibodies in titers of 1: 64-1: 128.

 Harmless to laboratory animals. The strain of coronavirus dogs "Carat" is harmless to white mice, guinea pigs and rabbits with intramuscular, intravenous and subcutaneous administration.

Virulence. With intraperitoneal infection of 2-4 month-old puppies of dogs with a culture seed of a strain at a dose of 6.5 lq TCD 50 / cm ^3, clinical signs of the disease appear in 75% of puppies on 3 days. In animals, depression, drowsiness, refusal to feed, diarrhea of varying severity and dehydration are noted. On 6-7 days, 20% of the animals die.

 Serological properties. The titer of virus-neutralizing antibodies of hyperimmune serum against coronavirus infection of dogs in a neutralization reaction of 1: 64-1: 128.

 The stability of the basic properties of the strain. The strain "Carat" maintains a stable infectious and antigenic activity for 15 passages.

 Additional information about the strain. Free from bacterial, fungal, mycoplasma and viral contaminants.

 The ability of the strain to spread in vivo. With experimental intraperitoneal and oral infection of puppies of dogs from 3 to 10 days, the virus is excreted with feces. Infection of animals in the environment occurs through contact with sick animals and their excreta.

Basic storage conditions. The strain "Carat" of dog coronavirus, lympholyzed with stabilizing additives, remains active at 20-22 ^° C for 40 days, at +4 ^° C for 280 days, at -40 ^° C for more than a year.

 The invention is illustrated by the following examples.

Example 1. Assess the antigenic activity of the multivalent vaccine "Multican-4" against plague, parvovirus and coronavirus enteritis and adenovirus infection of dogs. For this, puppies of dogs 3-4 months of age are vaccinated with the indicated vaccine in a dose of 1 cm ³ according to the instructions for use. After 21 days, blood samples are taken from all vaccinated puppies and blood serums are obtained. To determine the titer of virus-neutralizing antibodies to dog coronavirus, the obtained serum is examined in a neutralization reaction with 100 TCD 50 / cm ^{3 of} the Karat strain. Evaluation of the reaction is carried out after 7 days for the development of a cytopathic effect in an inoculated monolayer CRFK cell culture. The control is an infected and non-infected CRFK cell culture. The titer of neutralizing antibodies in blood serum is 1: 64-1: 256. Thus, the vaccine "Multican-4." It is immunogenic and suitable for use.

Example 2. Two groups of puppies of dogs of 3 months of age are formed, 4 heads each. Puppies of the experimental group are immunized twice with an interval of 14 days with the vaccine "Multican-4" in a dose of 1 cm ³ . Animals of the control group are not immunized. On the 15th day after the second immunization, all animals are infected intramuscularly with a virus-containing suspension of the Karat strain at a dose of 6.5 loq TCD 50 / cm ³ . In animals of the experimental group during the entire observation period, deviations from the physiological state are not noted. In 3 puppies of dogs in the control group, on the 3rd day after infection, the phenomena of refusal of feed, an inhibited state, signs of diarrhea of varying severity with subsequent development of signs of dehydration by the 7th day of the disease are noted. On the 7th day of the disease, the death of 1 puppy was noted. In fecal samples, the presence of the causative agent of the dog coronavirus disease is established.

 Thus, the vaccine "Multican-4" is immunogenic and suitable for use, and the Coronavirus canine strain "Carat" VGNKI N 29 reveals the correspondence of the severity of clinical signs of coronavirus infection and can serve to control the immunogenicity of vaccines against coronavirus infection of dogs.

Example 3. Get serum for the treatment of coronavirus infection of dogs. For this, goats are immunized 3 times with an interval of 21 days. For immunization of goats using native coronavirus of dogs from the strain "Carat" with a titer of 6.5 lq TCD 50 / cm ³ . In order to increase the specificity of the serum, the virus is used clarified and concentrated by ultracentrifugation at a speed of 35,000 rpm for 2 hours. The concentrated virus is administered to the goats initially subcutaneously with Freund's adjuvant 1: 1 in a volume of 1.5 cm ³ , subcutaneously at a dose of 5 cm ³ and a third time intravenously in the same dose.

 Serum is obtained by the conventional method. Set the titer of the obtained serum in the neutralization reaction, which is 1: 256-1: 512.

 The finished treatment serum is a clear yellow liquid.

The resulting serum is poured into sterile penicillin vials of 4 cm ³ and stored at a temperature of 4 ^o C for 12 months.

 Example 4. Testing the therapeutic properties of hyperimmune serum against coronavirus disease of dogs obtained in example 3.

Previously, 6 outbred puppies at the age of 3 months are injected with a culture suspension from the Karat dog coronavirus strain with an infectious activity titer of 6.5 lq TCD 50 / cm ³ in a volume of 3 cm ³ . 3 days after experimental infection, puppies show clinical signs of coronavirus disease. For treatment of 4 puppies, the serum is administered at a dose of 2 cm ³ for three consecutive days. Two other puppies (control) - saline in a dose of 2 cm ³ for 3 days. On the fifth day from the beginning of the experiment, active behavior, improvement in physical condition, and cessation of diarrhea are noted in puppies receiving serum. Control animals continue to hurt. On day 6, one puppy of the control group died, and the second fell ill and recovered on day 8. On day 14 of observation, all five puppies were clinically healthy.

Example 5. Prepare a luminescent specific serum for the diagnosis of coronavirus infection of dogs. First, hyperimmune serum is obtained against dog coronavirus enteritis in rabbits weighing 2.5-3.0 kg. For immunization of animals using a culture virus strain "Carat" with a titer of 6.5 lg TCD 50 / cm ³ . Before immunization, the virus is clarified by centrifugation at 3000 rpm for 20 minutes. The virus is administered intravenously at 2 cm ³ and then subcutaneously at 6 cm ³ with incomplete Freund's adjuvant in a ratio of 1: 1, after 3 and 6 weeks. 2 weeks after the last immunization, the animals are bled. Serum is obtained by the conventional method. The specific activity of the sera is determined in the neutralization reaction against 100 TCD 50 / cm ³ dogs coronavirus in a test tube culture of CRFK cells according to the standard method. As a control, normal non-immune rabbit serum is used.

 A hyperimmune serum is obtained having specific activity in the neutralization reaction with a titer of 1:64.

Hyperimmune rabbit serum to dog coronavirus is twice reprecipitated with a saturated solution of ammonium sulfate. Salting out of globulins from serum is carried out at a temperature of 0-2 ^o C. The precipitate of globulins obtained after reprecipitation, dissolved in an isotonic sodium chloride solution, is freed from ammonium sulfate by dialysis against a 0.15 M solution of sodium chloride with 0.01 M phosphate buffer, pH 7 ,4.

After dialysis, the rabbit serum globulin is conjugated to fluorescein isothiocinate. The conjugate was purified from free dye using Sephadex G-25 and DEAE cellulose ion exchange resin. The fluorescent specific antiserum obtained after purification is poured into sterile ampoules of 1 cm ³ and sealed, or into sterile penicillin vials of 3 cm ³ and freeze-dried. The finished fluorescent specific antiserum is a yellow opalescent liquid; dry form beige colored tablet.

The antiserum (liquid form) retains its activity for 12 months, provided that it is stored at a temperature of -20 ^o C. The dry form of the biological product is stored at a temperature of + 4 ^o C.

Example 6. For the study, fresh samples of feces are taken from 8 dogs experimentally infected with a culture suspension of the Karat strain with severe signs of diarrhea on the 3rd day from the onset of the disease. Fecal samples from 4 uninfected dogs were taken as a control. An equal volume of phosphate-buffered saline pH 7.2-7.4 is added to feces samples and mixed thoroughly. The feces suspension is centrifuged at a speed of 8 thousand rpm for 20 minutes A drop of sample is taken from the obtained supernatant, placed on a glass slide and a thin smear is made. The smears are dried and fixed in ethanol or acetone for 15-20 minutes. After fixation, the drug is washed in two shifts of phosphate-saline buffer pH 7.2-7.4 for 5 minutes in each shift. Dry strokes in the air. After drying, the fixed smears are coated with a fluorescent specific antiserum obtained according to Example 5 (in working dilution) and kept in a humid chamber at 37 ^° C for 20 minutes. After coloring, shake off the drops of the remaining serum and wash with saline and distilled water. After drying, the smears are enclosed in a 9: 1 neutral glycyrin mixture with a pH 8.0 phosphate buffer under a coverslip and examined under fluorescence microscopy for fluorescence. In seven smears, a bright yellow-green glow of rounded epithelial cells is noted. The diagnosis is positive, consistent with the data of epizootic, clinical and pathological and morphological studies. In 5 strokes, a yellow-green glow is not noted. The diagnosis is negative.

 At the same time, the specificity of the fluorescent specific antiserum is monitored by neutralizing the viral antigen from the Karat strain in a prepared smear of unlabeled specific antiserum at a dilution of 1: 10 for 30 min and staining with a fluorescent specific antiserum for 30 min and scanning under a luminescent microscope for fluorescence. Note the absence of a yellow-green glow of the test object, which indicates the specificity of immunofluorescence.

Example 7. For the study, from 3 dead puppies, after experimental infection, autopsy sites take sections of the small intestine. Make smears imprints of its affected fragments. Imprint smears are fixed in ethanol for 15 minutes or acetone for 15-20 minutes at 0-2 ^o C. Fixation and staining of the smears is carried out by analogy with example 6. In the smears note a bright yellow-green glow of the cytoplasm of individual cells. The control of the specificity of the fluorescent specific antiserum obtained for the strain "Carat" is carried out similarly as in example 6.

 In this case, a positive diagnosis of coronavirus enteritis in dogs is consistent with clinical and pathological morphological studies.

 The use of the Coronavirus canine "Carat" VGNKI N 29-DEP in production conditions will make it possible to judge with high reliability the quality of the manufactured and used biological products against coronavirus infection of dogs, and its use to obtain therapeutic serum and fluorescent specific antiserum makes it possible to timely treat and diagnose this disease .

Claims (1)

 The strain Coronavirus canine "Carat" VGNKI N 29-DEPT used to control the immunogenic activity of vaccines and to obtain sera for the treatment and diagnosis of coronavirus infection of dogs.