RU2631925C2 - Strain "shevchenko-2014" of rabbit hemorrhagic disease virus for preparing vaccines, diagnostic and therapeutic preparations - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: invention relates to veterinary virology and biotechnology and concerns the strain of rabbit hemorrhagic disease virus, Caliciviridae family, genus Lagovirus. Presented strain is deposited under number 55 in autonomous noncommercial organization "Scientific Research Institute of diagnosis and prevention of human and animals diseases". Registration name of strain "Shevchenko-2014".

EFFECT: invention can be used for preparing vaccines, diagnostic and therapeutic preparations in viral hemorrhagic disease in rabbits.

1 cl, 5 tbl

Description

The invention relates to the field of veterinary virology, biotechnology and can be used for the manufacture of vaccine, diagnostic and therapeutic drugs.

Rabbit viral hemorrhagic disease - HBVC (rabbit hemorrhagic pneumonia, necrotic hepatitis) is a highly contagious, acute leaking infectious disease characterized by hemorrhagic syndrome in all organs, especially in the lungs and liver, caused by the virus of the family Caliciviridae, a genus of Lagovirus, causing significant economic damage to rabbits. Rabbits of different age groups are affected by HBV, but more often older than 1.5 months of age. Other types of animals and humans do not get sick. With HBV, the incidence reaches 70-80%, and mortality - 90-100%. The disease is widespread in the world among European rabbits (Oryctolagus cuniculus), and hares can also get sick.

The economic damage from HBVC is significant, which is due to the mass incidence and death of rabbits of all age groups.

The most reliable protection of rabbits against HBV is prophylactic vaccination, which takes the main place when performing anti-epizootic measures. For specific prophylaxis of rabbit viral hemorrhagic disease, inactivated mono- and associated vaccines, previously developed by us, are used [1, 2]. Recombinant vaccines against HBV have not yet been found commercial use.

Studies of foreign and domestic scientists over the past decade have established that all isolated isolates of the HBVC virus during the epizootic period belong to the same serotype. When sequencing the selected isolates of HBVV virus, it was found that in the conservative regions of the genome they have differences in amino acid composition in the range from 2 to 7%. Foreign researchers have found that the temperature-dependent isolates of HBV virus isolated in recent years in Germany and Italy differ significantly in hemagglutinating properties at a lower temperature of + 4 ° C and in amino acid composition from previously known HBV viruses in region E of the genome, where representatives of the family Caliciviridae have main antigenic determinants [3, 4].

Known strains "Voronezh-87", "Belgorod-03" and "Manikhino-09", officially deposited in the collections of the Federal State Institution "VGNKI" and GNU VNIIVVIM for the manufacture of inactivated vaccines against VGBK [5, 6, 7].

However, rabbits vaccinated with a vaccine made from previously isolated virus isolates do not have specific protection against isolated HBV virus isolates in recent years. Therefore, it is necessary to conduct epizootological, virological, genetic monitoring and comparison of the hemagglutinating and immunological properties of newly isolated HBV virus isolates in the foci of infection, to have the rabbit hemorrhagic disease virus strains in museum collections, which will make it possible to produce specific diagnostic, therapeutic drugs and highly effective vaccines for protecting rabbits from them against VGBK.

The aim of the present invention was to obtain a new production strain of the virus of hemorrhagic disease of rabbits, retaining its immunological properties after inactivation and suitable for the manufacture of highly immunogenic vaccines, diagnostic and therapeutic drugs.

The technical result from the use of the present invention is to expand the arsenal of strains of the virus of hemorrhagic disease of rabbits, differing in genome and immunological properties, with high biological, antigenic and immunogenic activity, retaining their immunobiological properties after inactivation, suitable for the manufacture of highly effective vaccines, diagnostic and therapeutic drugs.

This technical result was achieved by obtaining the strain "Shevchenko-2014" virus of hemorrhagic disease of rabbits. The strain "Shevchenko 2014" is new and previously unknown.

The resulting strain was deposited at number 55 in the Autonomous non-profit organization "Research Institute for Diagnosis and Prevention of Human and Animal Diseases" in Moscow and is stored in the Armavir Biofactory Federal State Unitary Enterprise. The strain Shevchenko 2014 differs from the previously deposited strains of VGBK Voronezhsky-87, Belgorodsky-03, and Manikhino-09 by 5-7% in the nucleotide sequences of the conserved region of the VP-60 gene. The strains indicated above, based on the results of phylogenetic analysis, belong to different genogroups.

Strain "Shevchenko-2014" provides a serological diagnosis of viral hemorrhagic disease of rabbits and the production of highly effective inactivated vaccine against HBV, which creates reliable protection for wild and domestic European rabbits against HBV.

The strain "Shevchenko-2014" virus of hemorrhagic disease of rabbits is characterized by the following signs and properties.

The taxonomic characteristic is that the Shevchenko-2014 strain has the shape and dimensions typical of the Caliciviridae family. In serological reactions (RSHA, RSK, ELISA, RDP) antigens are detected that are common to all strains of the rabbit hemorrhagic disease virus. The main marker characteristics of the strain "Shevchenko-2014" virus of hemorrhagic disease of rabbits are shown in table 1.

The essence of the invention is illustrated by examples of its implementation.

Example 1

To obtain the virus of hemorrhagic disease of rabbits, the Shevchenko-2014 strain uses three ampoules with the virus, at the level of the first passage, on rabbits who died from the introduction of a 10% suspension of the liver from rabbits that fell on a farm in the Krasnodar Territory in January 2014 (infectious activity below 10 ^4.0 LD ₅₀ / cm ³ ). A sterile solvent (distilled water or physiological saline, pH 7.0-7.4) is added to each ampoule to the volume indicated on the label. Then the contents of the ampoules are combined and the virus is diluted in physiological saline in such a way that 100 LD ₅₀ / cm ^{3 is} contained in the final dilution. The indicated dilution of the virus is administered intramuscularly to 5 rabbits in a volume of 1.0 cm ³ . Infected rabbits are kept in equipped infectious vivarium in individual cages.

In rabbits infected with HBV virus, characteristic clinical signs of the disease are noted after 14-24 hours: depression, refusal to feed, fever up to 40.7 ° C, nosebleeds and death within 24-96 hours. The corpses of animals in the opening room of the infectious vivarium suspended on hangers, removed the skins, treated the abdominal cavity with a solution of potassium permanganate (1: 10000), cut the abdominal muscles along the white line, remove the liver, and separate the gall bladder. The liver is used only from corpses, the autopsy of which revealed specific pathological and morphological changes characteristic of HBVC: hemorrhage in the trachea, under the capsule of the kidneys, spleen, hemorrhagic pneumonia, liver of loose texture, is easily torn and has a yellow-brown color, sometimes with a reddish tint. Liver necrosis and enlarged spleen are noted. Heart attacks are observed in almost all organs, the kidneys are dark brown in color.

The liver is placed in sterile vials, sealed tightly, disinfected externally with a 5% solution of chloramine, placed in a thermos with ice and delivered to a sterile box.

Each liver sample is prepared separately using separate sterile dishes. A liver sample is ground with scissors, sterile FSB is added (ratio 1:10 - weight / volume), homogenized in a blender with cooling for 10 minutes. Selected into a separate sterile tube type "Eppendorf" for identification in PCR. Then the liver suspension is centrifuged for 1 h at 6000 g at 4 ° C. The supernatant is ultracentrifuged at 80,000 g for 2 hours at 4 ° C through a 20% sucrose solution. The precipitate was resuspended in FSB 1/100 of the initial volume.

To obtain the results of the molecular genetic characteristics of the Shevchenko 2014 strain of the rabbit hemorrhagic disease virus, nucleotide sequencing of the conserved region of the VP-60 gene, 510 nucleotides long, was performed.

RNA from the sample was isolated using a sorbent (Silica). The resulting RNA was used for reverse transcription amplification (RT-PCR). Reverse transcription was performed together in one tube. The reaction mixture contained: 16 mM TrisHCl, 82 mM KCl, 2.4 mM MgCl2, Triton × 100 1%, primers 10 pmol each, 0.25 mMdNTPs, 5 units. taq polymerase, 25 units M-MLV Reverse Transcriptase.

Reverse transcription was performed for 50 minutes. at 50 ° C. PCR included 35 cycles (94 ° C × 20 s, 50 ° C × 20 s, 72 ° C × 30 s). The primers used amplify a portion of the gene encoding the VP60 HBVA structural protein with a length of 510 base pairs (positions 5182-5692 in the virus genome). The amplified gene fragment encodes the N-terminal region of the protein. Primers have the following composition:

P1 (sense) 5'gagctcgagcgacaacaggc3 ' P2 (antisense) 5'caaacacctgacccggcaac3 '

Electrophoresis obtained during PCR amplification showed the presence of a fragment of a specific size. The PCR fragment, after purification from agarose gel using a commercial Silica Bead DNA Gel Extraction kit (Fermentas, Latvia), was used for sequencing. A 4-channel capillary sequencer Applied Biosystems 3130 Genetic Analyzer (USA) was used. The resulting sequence was further analyzed using the BLAST program (http://blast.bemd.ncbi.nlm.nih.gov). The results allow us to unequivocally talk about the correspondence of the studied fragment of the nucleotide sequence of the VP60 gene of HBBC. The closest were the corresponding regions of the genomes of the following HBVC strains: HYD isolate (JF412629), NJ-2009 isolate (NM623309), Erfurt strain (EF558581)

The sequence of the gene encoding the structural protein VP60 HBBC

Virus-containing material is examined in accordance with the recommendations of the OIE for standard diagnostic methods and vaccines (2004) for the absence of contamination with mycoplasmas, bacteria and fungi by plating on sensitive nutrient media. The absence of contamination with extraneous viruses and the identification of the rabbit hemorrhagic disease virus are determined by negative contrast electron microscopy. Infectious activity in rabbits, hemagglutinating activity in RGA, antigenic activity in TF ELISA, specificity in TF ELISA and RHA and at the level of passage 2 on rabbits are determined, the virus was packaged, lyophilized and sealed in ampoules under vacuum and stored at a temperature not exceeding minus 40 ° C as a Master seed (Ms) with a titer of infectious activity of at least 5.0 lg LD ₅₀ / cm ³ . The resulting strain of the virus of hemorrhagic disease of rabbits is assigned the copyright name "Shevchenko-2014".

Example 2

In order to determine the biological, antigenic and immunogenic activity of the virus of hemorrhagic disease of rabbits of the Shevchenko 2014 strain, hyperimmunization of clinically healthy rabbits from farms that are safe for infectious and invasive diseases is carried out, with a live weight of at least 2.0 kg, according to the 2nd scheme for obtaining specific serum for the treatment, diagnosis and prevention of HBVC (patent No. 2077340 from 08/06/93, authors: Shevchenko A.A. et al.). For hyperimmunization of rabbits, a tissue inactivated vaccine made from the Shevchenko 2014 strain of rabbit hemorrhagic disease virus is used at a dose of 0.5 ml per head by intramuscular injection twice with an interval of 14 days. 14 days after the second immunization, blood serum is obtained from hyperimmunized rabbits. Specific serum is tested for activity and specificity in the hemagglutination inhibition reaction. The research results are shown in table 2.

The data in Table 2 show that already after the first vaccination of rabbits with the Shevchenko-2014 strain of HBV virus in rabbits, specific hemagglutination-inhibiting antibodies are formed in the titer from 5 to 8 log ₂ , in the future the antibody level increases to 10-12 log ₂ . This confirms the high biological, antigenic and immunogenic activity of the Shevchenko-2014 virus strain.

Example 3

To study the therapeutic effect of a specific immune serum against HBV, adult rabbits were taken free of antibodies to the HBV virus, they were infected with a virulent virus at a dose of 1000 LD ₅₀ / cm ³ and after 24-30 hours, during the development of primary clinical signs (depression, disheveled, denial from feed, drooping ears), a specific immune serum of 0.5 cm ^{3 was} administered subcutaneously once, the control rabbits were injected with normal rabbit serum. The results are presented in table 3.

From the data in Table 3 it can be seen that a single administration of native immune serum to rabbits with activity in RHCA from 6 to 8 log ₂ within 24-30 hours after infection with virulent virus of HBV allows treating all rabbits with complete death of animals in the control.

To obtain the virus used in the manufacture of tissue inactivated aluminum hydroxide vaccine against HBVC from the Shevchenko-2014 strain, the obtained and characterized master seed seedling of rabbit hemorrhagic disease virus from the above strain is used. First, the virus is diluted in physiological saline so that the final dilution contains 100 LD ₅₀ / cm ³ . The indicated dilution of the virus is administered intramuscularly to rabbits in a volume of 1.0 cm ³ . Previously, the blood serum of rabbits was examined for the presence of antibodies to the Shevchenko-2014 HBVC virus in RHCA at + (4 ± 2) ° C and at + (20 ± 2) ° C with human blood erythrocytes of group 0 (I). In the presence of specific antibodies to the specified virus in a titer of 1: 4 and higher, rabbits are not taken into the experiment. Infected rabbits are kept in equipped infectious vivarium in individual metal cells.

From a liver obtained from dead rabbits, a 10% viral suspension is prepared in buffered saline (pH 7.0-7.4). For clarification, chloroform (final concentration of 2%) is added to the prepared liver suspension and placed in a refrigerator at 4 ° C for 18 hours. Then the liver suspension is centrifuged for 1 h at 6000 g at 4 ° C. The viral suspension used to make the tissue inactivated vaccine should have an activity in the RGA at + (4 ± 2) ° C of at least 1: 1280 GAE.

Inactivation of the virus is carried out using a 4% formalin solution. Based on the concentration of formaldehyde, a 4% formalin solution (i.e., a 1.1-1.4% formaldehyde solution) is prepared in sterile distilled water. A formalin solution is prepared in glass bottles on the day the vaccine is prepared and introduced into the viral suspension to a final concentration of at least 0.1% formaldehyde.

Inactivate the virus at a temperature of 37 ± 1 ° C for 3 days with periodic stirring (1 time per hour for 5 minutes).

The start of inactivation is considered the time after bringing the temperature in the tank to 37 ± 1 ° C and adding formalin. A sample was taken from inactivated material to verify sterility, activity, completeness of inactivation and safety. To check the completeness of inactivation and harmlessness, two rabbits weighing at least 2.0 kg, which do not have specific antibodies to the HBV virus, are injected with 3.0 cm ³ intramuscularly inactivated material. Animals remained healthy for 10 days of observation.

After the inactivation of the virus-containing suspension is completed, an alumina hydrate gel (GOA) with 3% solids is added to the container with the virus suspension after grinding based on 1000 cm ^{3 of a} suspension of 150 cm ³ GOA (20%) at pH 7.2-7.4 and a temperature of 4-8 ° C (RF patent No. 2039570 from 07.20.95, authors: A. Shevchenko and others). Sorption is carried out for 1-2 hours with periodic stirring. The vaccine is stored at 10 ± 2 ° C until the results of the control of the drug. Then the vaccine is stirred at a temperature of 10 ± 2 ° C for 1 hour and Packed in sterile vials with a capacity of 20 or 50 cm ³ .

The antigen content at + (4 ± 2) ° C in the vaccine dose of at least 640 GAE is its optimal amount in the preparation, ensuring the achievement of a technical result.

The resulting vaccine is checked for appearance, sterility, pH, completeness of virus inactivation, immunogenicity and harmlessness.

The vaccine is a clear, gray-brown liquid with a loose precipitate formed during storage, which, when shaken, easily breaks into a homogeneous suspension.

Sterility of the vaccine is determined in accordance with GOST 28085-89. The safety of the drug is determined by its administration to four rabbits intramuscularly at 3 cm ³ . The vaccine is considered harmless if it does not cause death or disease of the rabbits, as well as changes in the necrotic nature at the injection site within 14 days of observation.

The completeness of the inactivation of the virus in the vaccine is checked on 4 rabbits, which are injected intramuscularly in the region of the middle third of the thigh 3.0 cm ³ . Rabbits are monitored for 10 days. A vaccine is considered not containing an infectious virus if it does not cause the death of rabbits from viral hemorrhagic disease within 10 days after administration of the drug.

In the used vaccine vials, the pH is determined, the average value of the indicators should be in the range of 7.2-7.4. The hemagglutinating activity of the antigen of each vaccine series is determined in the hemagglutination reaction (RGA) at + (4 ± 2) ° C, with human erythrocytes of group 0 (I) and expressed in hemagglutinating units. The hemagglutinating titer in the vaccine should not be lower than 8.0 log ₂ , which corresponds to a dilution of 1: 256 GAE.

The data in table 4 indicate that the tissue inactivated aluminum hydroxide vaccine against HBV from strain "Shevchenko 2014" has a high hemagglutinating activity.

When studying the immunogenic activity of tissue inactivated aluminum hydroxide vaccine against HBV from strain "Shevchenko-2014" it was found that in rabbits older than 1.5-2 months. age, vaccinated once intramuscularly at a dose of 0.5 cm ³ , already on day 5 after administration of the drug, an immune response is formed, i.e. specific antibodies are formed to protect vaccinated rabbits against HBV. It has been experimentally shown that one vaccination dose in a volume of 0.5 cm ³ contains at least 640 GAE. The results of studies of immunogenic activity are presented in table 5.

The data in table 5 confirm the high immunogenic activity and effectiveness of the inactivated vaccine against HBV from strain "Shevchenko-2014".

Thus, the Shevchenko 2014 strain of the rabbit hemorrhagic disease virus obtained in accordance with the invention has high infectious, antigenic and immunogenic activity and is suitable for the manufacture of vaccines, diagnostic and therapeutic drugs against HBV.

Information sources

1. Shevchenko A.A. Specific prevention of infectious diseases of rabbits: viral hemorrhagic disease, myxomatosis and pasteurellosis: dis. ... dr. wind. Sciences: 16.00.03 / Shevchenko Aleksandr Alekseevich. - Pokrov, 1995 .-- 233 p.

2. Shevchenko A.A. New associated vaccine / A.A. Shevchenko // Rabbit breeding and fur farming. - 1994. - No. 2.

3. Molecular epidemiology of Rabbit haemorrhagic disease virus. Moss S.R., Turner S.L., Trout R.C., White P.J., Hudson P.J., Desai A., Armesto M., Forrester N.L., Gould E.A .; J. Gen. Virol. - 2002. - Vol. 83. - P. 2461-2467.

4. Manual of standards Diagnostic Tests and Vaccines 2004; PART 2; SECTION 2.8 .; CHAPTER 2.8.3. RABBIT HAEMORRHAGIC DISEASE.

5. Burmakina G.S. Development of molecular genetic identification tools for the rabbit hemorrhagic disease virus: Diss. ... cand. biol. Sciences: 03.01.06 / Burmakina Galina Sergeevna. - Pokrov, 2012 .-- 150 s.

6. RF patent No. 2077340 “A method for producing specific serum for the treatment, diagnosis and prevention of rabbit viral hemorrhagic disease and a kit for diagnosing the disease”, authors: A. Shevchenko and other priority of 08/06/1993, application No. 93039551 registered in the state register of inventions 04/20/1997

7. RF patent No. 2039570 "A method of manufacturing an inactivated vaccine against viral hemorrhagic disease", authors: A. Shevchenko et al., priority dated 05/26/1992, application No. 5043431 was registered in the state register of inventions on July 20, 1995.

Claims (1)

The strain of the virus of hemorrhagic disease of rabbits, family Caliciviridae, genus Lagovirus, deposited at number 55 in the Autonomous non-profit organization "Research Institute for Diagnosis and Prevention of Diseases of Man and Animals", the registration name "Shevchenko-2014" for the manufacture of vaccines, diagnostic and therapeutic drugs for rabbit viral hemorrhagic disease.