RU2400536C1 - STRAIN OF BIRD FLU VIRUS OF A/common gull/Chany/2006 H5N1 SUBTYPE FOR PREPARATION OF ANTIGEN-CONTAINING DIAGNOSTIC OR VACCINE MEDICATION - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to strain of bird flue virus of A/common gull/ Chany/2006 H5N1 subtype for preparation of antigen-containing diagnostic or vaccine medication, deposited in collection of microorganism cultures of Federal state research institution "State Scientific Centre of Virology and Biotechnology "Vector" of Rospotrebnadzor under registration number V-368.

EFFECT: strain possesses higher productivity in comparison with known strains of H5N1.

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Description

The invention relates to medical virology and microbiology, in particular for the preparation of an antigen-containing substrate for serodiagnosis of the highly pathogenic H5N1 flu subtype in rtga, in particular the presence of antibodies in the blood serum of wild animals to assess the epizootic situation, as well as for the preparation of vaccine preparations.

Since 1959, when the highly pathogenic H5N1 subtype virus was first detected in chickens, a large number of viruses of this subtype were isolated from wild, poultry and mammals, including humans. Since the mid-1990s, the new HPAI H5N1 variant has become endemic in Southeast Asia. First isolated in 1996 from poultry in Hong Kong, it caused mortality in chickens over 90% and caused deaths in humans. As of September 10, 2008, a total of 387 cases of human infection with bird flu (H5N1) were recorded, in 245 cases the disease was fatal [http://www.who.int/csr/disease/avian_influenza/en/index.html] . Since 2005, several large outbreaks among wild birds have been recorded throughout Eurasia and North Africa. The largest epizootic among wild birds in the summer of 2006 was recorded on Lake Ubsu-Nur (Republic of Tuva) [Lvov D.K., Shchelkanov M.Yu., Deryabin P.G. et al. Isolation of highly pathogenic (HPAI) strains of influenza A / H5N1 virus from wild birds in the epizootic focus on Lake Ubsu-Nur (June 2006) and their deposition to the State Virus Collection of the Russian Federation (July 3, 2006). Zhurn. Questions of virology. 2006, 6: 14-18]. At this time, the H5N1 virus was isolated by the authors of the present invention from a clinically healthy specimen with the blue gull {Larus canus). The isolated strain is named A / common gull / Chany / 2006.

A known strain A / duck / Novosibirsk / 56/05 is the H5N1 subtype of avian influenza virus (analogue) isolated in Western Siberia, which is used to model viral infection in scientific experiments when studying the effectiveness of antiviral drugs in cell cultures (Patent RU 2309983, IPC C12N 7/00, published May 27, 2007).

However, this strain has the following disadvantages: the strain has reduced productivity when cultured on MDSK cells — the maximum virus concentration is 6.5 lg TCID ₅₀ / ml. The strain is intended for the production of preventive and diagnostic preparations and, based on the presented data on pathogenicity for cell cultures and the lack of data on pathogenicity for animals, including mice can be used to evaluate the antiviral activity of various compounds only on cell culture.

The closest strain (prototype) is strain A / LHC / RUSSIA / 05 (subtype H5N1) of avian influenza virus (Patent RU 2300564, IPC C12N 7/00, publ. 10.06.2007). The specified strain can be used to obtain antigen and the creation of vaccines and diagnostic drugs. Data on the pathogenic properties of the strain for laboratory animals, including no mice. However, it causes the death of RKE on the first day after infection, and the antigen titer in the RGA after the passage on the RKE is lower compared to the proposed strain.

The technical result of the invention is the production of such a producer strain of avian influenza virus, which would have a higher productivity compared to known strains of H5N1.

The specified technical result is achieved by obtaining a strain of avian influenza virus A / common gull / Chany / 2006 H5N1 subtype for the preparation of antigen-containing diagnostic or vaccine preparation.

Characterization of the claimed strain. Strain A / common gull / Chany / 2006 is a typical representative of avian influenza viruses of the genus Influenzavirus A of the family Orthomyxoviridae. The inventive strain was isolated from a cloacal wash from a clinically healthy individual with a blue-headed gull (Larus canus) on Lake Chany (Zdvinsky district of the Novosibirsk region) as a result of virological studies in July 2006.

The strain was deposited in the collection of cultures of microorganisms of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "(FGUN SSC WB" Vector ") Rospotrebnadzor under registration number No. V-368.

Cultural properties. Strain A / common gull / Chany / 2006 when cultured on a monolayer of MDSK cells causes a cytopathic effect (CPP) on days 2-4 (cultivation temperature 37 ° C, in an atmosphere of 5% CO ₂ ), with a titer of 7.2 ± 0 , 1 lg TCID ₅₀ / ml. When infected in a cell suspension, the titer was 7.8 ± 0.2 lg TCID ₅₀ / ml.

Strain A / common gull / Chany / 2006, when cultured in a cell suspension of a cat kidney PK-91, the titer is 8.4 ± 0.15 log TCID ₅₀ / ml.

To cultivate the strain on MDSC and PK-91 cell cultures, DMEM nutrient medium is used, with the addition of 2.5% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin.

Pathogenicity for chickens.

To confirm the pathogenic potential of the virus, we performed a chick test. In a chicken experiment, the pathogenicity index (IVPI) is 1.7. The previously studied highly pathogenic H5N1-subtype influenza viruses isolated in Western Siberia in 2005-2006 have a pathogenicity index> 2.5 [Onishchenko GG, Shestopalov AM, Ternova V.A. et al. Detection of highly pathogenic H5N1 viruses in Western Siberia influenza genetically related to viruses circulating in Southeast Asia in 2003-2005. Zhurn. Reports of the Academy of Sciences. 2006, 2 (406): 1-3.].

Pathogenicity for mammals. Strain A / common gull / Chany / 2006 refers to variants of the influenza virus pathogenic for mice, ferrets: it causes the death of outbred white mice and BALBc mice, as well as a serious disease in outbred ferrets during intranasal infection. At the same time, ferrets are capable of releasing the virus for a long time into the external environment when coughing and sneezing in titers corresponding to the infectious dose.

For long-term storage, the strain must be lyophilized using a solution of gelatin and sucrose (SAG) as a protective medium. Lyophilization with the addition of SAGE in a ratio of 1: 4 allows you to maintain stable infectious activity of the virus for at least 2 years when stored at -70 ° C.

Virus identification. Isolation of the virus in developing chicken embryos and its serological identification was carried out according to the previously described methods [The National Training Course on Animal Influenza Diagnosis and Surveillance. - Harbin, 2001. - 79 p.]. The inventive strain is produced on the RCE in titers of 9.8 ± 0.2 lg EID ₅₀ / ml The results of the comparative antigenic analysis are presented in table 1.

Table 1. Antigenic analysis of strain A / common gull / Chany / 2006 Serum Ck / Hidag / 95 Gs / hk / 99 HK / 156/97 HK / 213/03 VN / 1203/04 Prachirnbrr / 04 Tk / Suzdalka / 05 Reference antiserum (H5) CDC (USA) A / common gull / Chany / 2006 1280 1280 1280 640 640 320 160 2560 Note. Table 1 shows the inverse values of the dilution of serums that inhibit hemagglutination of 4 GAE virus.

The virus antigen showed a high affinity for polyclonal sera obtained on highly pathogenic H5N1 viruses circulating in countries of Southeast Asia. This allows the use of antigen to study sera for antibodies to a whole spectrum of highly pathogenic subtype H5N1 influenza viruses.

The sequence of the complete strain genome was determined. The nucleotide sequences of the genes are donated to Genbank under the numbers: EU 871936, EU 871937, EU 871938, EU 871939, EU 871940, EU 871941, EU871942, EU 871943.

Analysis of the full genome sequence of the isolated strain revealed its belonging to the Qinghai-Siberian genotype, circulating in the world since spring 2005 [Normil D. Avian influenza. Potentially more lethal variant hits migratory birds in China. Science. 2005, 309 (5732): 231.]. The nucleotide sequence of the site of proteolytic cutting of hemaggutinin determines the amino acid sequence PQGERRRKKRGLF characteristic of highly pathogenic type A influenza viruses [Ellis T.M., Bousfield R. B., Bissett L.A. et al. Investigation of outbreaks of highly pathogenic H5N1 avian influenza in waterfowl and wild birds in Hong Kong in late 2002. Avian Pathol. 2004 Oct; 33 (5): 492-505].

Phylogenetic analysis was performed according to the Maximum Parsinomy method, with bootstrap analysis (500 repetitions) based on the nucleotide sequence of the hemagglutinin gene. A fragment of a phylogenetic tree containing phylogenetically related viruses is presented. The strain we are studying is phylogenetically close to the highly pathogenic H5N1 viruses isolated during an outbreak among wild birds on Lake Ubsu-Nur (Republic of Tuva) [Lvov DK, Shchelkanov M.Yu., Deryabin PG et al. Isolation of highly pathogenic (HPAI) strains of influenza A / H5N1 virus from wild birds in the epizootic focus on Lake Ubsu-Nur (June 2006) and their deposition to the State Virus Collection of the Russian Federation (July 3, 2006). Zhurn. Questions of virology. 2006, 6: 14-18.], In the summer of 2006, which are somewhat different from all other H5N1 patented in 2005 (A / duck / Novosibirsk / 56/05, A / Chicken / Kurgan / 05/2005, A / LHC / RUSSIA / 05). The drawing shows a fragment of the phylogenetic tree of genes for hemagglutinin H5N1 subtype.

Strain A / common gull / Chany / 2006 has unique biological characteristics that can be used to prepare an antigen-containing substrate for serodiagnosis of the highly pathogenic H5N1 subtype in RTGA, in particular, the study of the presence of antibodies in the blood serum of wild birds to assess an epizootic situation (see example 2) . Moreover, he has a number of advantages. (see example 1).

Due to the molecular biological characteristics and effective cultivation of strain A / common gull / Chany / 2006, it can be used as a control when testing subtype influenza virus R5 RNA isolation systems and PCR diagnostic systems.

It is very important that during long-term storage of A / common gull / Chany / 2006 in the form of antigen inactivated with β-propiolactone, at a temperature of +4 - + 7C, it maintains without reduction the hemagglutinating titer within 1 / 256-1 / 1024 with various types of red blood cells mammals and birds (goose, chicken, rhesus monkey, guinea pig, horse, ram, ferret, man) for 14 months, (ongoing observation period).

Example 1. The study of the antigenic properties of strain A / common gull / Chany / 2006 influenza virus to create a panel of antigens.

The proposed strain has an advantage in rtga, for example, compared with the commercial reference antigen H5 CDC (USA).

Table 2. Results of rtga of the investigated antigens with commercial reference sera and sera obtained for H5N1 strains isolated in Russia. Antigen Serum Reference Serum H5 CDC Reference Serum H5N1 (St. Jude) Monoclone H5 CP62 & 364 / I H5N3 ¹ Krasnodar ² Tuva ³ Reference antigen H5 CDC 5120 5120 16384 160 80 <80 A / common gull / Chany / 2006 10240 10240 2048 160 640 640 - 1 - antiserum to influenza virus A / Anas platyrhynchs / Chany / 9/03 (H5N3) - 2 - antiserum to influenza virus A / Chicken / Krasnodar / 06 (H5N1) - 3 - antiserum to influenza virus A / duck / Tuva / 01/06 (H5N1)

From the data presented in table 2, it can be seen that in high-volume sera (reference serum from CDC and St. Jude) antibodies to H5 are detected in RTGA with all antigens used. However, titers with antigen of the proposed strain are higher. In serum of ferrets, who had the flu caused by highly pathogenic A / H5N1 strains isolated in Russia, in the RTGA with the reference antigen of the anti-H5 antibody, hemagglutinin is not detected (anti-A / duck / Tuva / 01/06) or detected in a limit titer of 80 (anti-A / Chicken / Krasnodar / 06).

In the low-volume serum obtained after immunization with the non-pathogenic strain A / Anas platyrhynchs / Chany / 9/03 (H5N3), anti-H5 hemagglutinin antibodies were determined equally using the antigens used.

Example 2. Retrospective serodiagnosis of sera of wild migratory birds.

A screening study of blood sera from wild birds using various antigens was carried out (table 3):

1. The antigen of the proposed strain A / common gull / Chany / 2006.

2. Antigen of strain A / Goose / Krasnoozerskoye / 627/05 (2008 patent No. 118903 from 05/13/2008).

3. Antigen for the diagnosis of avian influenza H5N3 (Pokrovsky biological products plant OJSC).

43 serum samples were tested.

Table 3. Values of reverse titers of positive blood serum of wild birds for the presence of antibodies to the H5 influenza virus subtype in RTGA. Serum Antigen for the diagnosis of avian influenza H5N3 (Pokrovsky biological products factory OJSC) Antigen A / common gull / Chany / 2006 Antigen A / Goose / Krasnoozerskoye / 627/05 one 512 ≥2048 128 2 64 1024 <16 3 64 512 <16 four 128 128 <16 5 64 256 <16 6 128 256 <16 7 128 512 64 8 128 256 32 9 128 256 <16 10 64 512 16 eleven 64 128 32 12 128 1024 16 13 128 512 <16 fourteen 64 256 <16 fifteen 64 512 <16

When using the antigen for the diagnosis of avian influenza H5N3 (OJSC Pokrovsky Biological Product Plant) 89% of the sera were positive, the antigen of the proposed strain A / common gull / Chany / 2006 - 100%, the antigen of the strain A / Goose / Krasnoozerskoye / 627/05 (application for the invention No. 2008118903 of May 13, 2008) - 33%.

Polyclonal sera from wild birds show a greater affinity for the antigen of the proposed strain, which allows more accurately detect the presence of antibodies. In accordance with WHO recommendations, serums with a titer in rtga≥1 / 40 are considered positive [Ellis T.M., Bousfield R.B., Bissett L.A. et al. Investigation of outbreaks of highly pathogenic H5N1 avian influenza in waterfowl and wild birds in Hong Kong in late 2002. Avian Pathol. 2004 Oct; 33 (5): 492-505.]. Thus, antigen A / common gull / Chany / 2006 reveals 100%.

Due to the reduced pathogenicity for chickens compared to other strains (see strain description), the characteristic of strain A / common gull / Chany / 2006 is its high reproductive ability in developing chicken embryos. The virus titer in the experiment reached 9.8 ± 0.2 lg EID ₅₀ / ml, the antigen titer in RGA 1: 1024 in 50 μl (20240 GAU / ml).

The unique biological properties of the proposed strain provide a technological advantage compared to others, since they allow to produce high-quality antigen in large quantities. The proposed strain A / common gull / Chany / 2006 can be used in conjunction with other known for a more detailed study of the antigenic characteristics of newly isolated strains of H5N1 and serodiagnosis of animal blood sera.

Example 3. The study of the possibility of using the inventive strain A / common gull / Chany / 2006 as a vaccine preparation. The strain has good immunogenic properties for both mammals and birds.

In experiments on intranasal infection of outbred ferrets with a dose of 10 ⁶ EID / ml, serum was taken after 21 days after infection. The presence of antihemagglutinating antibodies in titers 1 / 1280-1 / 2560 was shown by RTGA with the antigen of strain A / common gull / Chany / 2006.

In case of intranasal infection of mice with various doses, the serum was taken from the surviving mice on day 21 after infection. The presence of anti-hemagglutinating antibodies in titers 1 / 160-1 / 2560 was shown by RTGA with the antigen of strain A / common gull / Chany / 2006.

In accordance with WHO recommendations, serum titers in rtga≥1 / 40 are considered positive (WHO, 2002). Thus, the inventive strain has good immunogenic properties against mammals. Therefore, there is the possibility of further development of a vaccine preparation for mammals using an inactivated antigen based on the proposed strain, as one of the components of the vaccine against the H5N1 influenza virus.

Upon oral / intranasal infection of domestic ducks (Anas platyrhynchos domesticus) with a dose of 10 ⁶ EID / ml, blood serum was collected at 23 days after infection. The presence of antihemagglutinating antibodies in titers of 1 / 800-1 / 1600 was shown by RTGA with the antigen of strain A / common gull / Chany / 2006. In accordance with WHO recommendations, serum titers in rtga≥1 / 40 are considered positive (WHO, 2002). Thus, they have good immunogenic properties against birds. Therefore, there is the possibility of further development of a vaccine preparation for birds using an inactivated antigen based on the proposed strain, as one of the components of the vaccine against the H5N1 influenza virus.

As indicated above, the characteristic of strain A / common gull / Chany / 2006 is its high reproductive ability in developing chicken embryos (antigen titer in RSA 1: 1024 in 50 μl (20240 GAU / ml), which provides a technological advantage over this strain compared to others since it allows to produce high-quality antigen in large quantities.In combination with data on the reduced pathogenicity of the proposed strain A / common gull / Chany / P / 2006 for developing chicken embryos and the possibility of superproduction of the HA antigen, the prospect of a long-range our studies on the development of a vaccine preparation using antigen based on the proposed strain as one of the components of the vaccine against the H5N1 influenza virus.

Thus, the biological properties of this strain, determined by the unique primary structure, determine its high immunogenic activity. This allows us to consider the proposed strain as potential when used in the creation of a vaccine preparation.

Claims (1)

The strain of avian influenza virus A / common gull / Chany / 2006 H5N1 subtype for the preparation of antigen-containing diagnostic or vaccine preparation, deposited in the Collection of microorganism cultures of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "Rospotrebnadzor under registration number No. V-368.

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