RU2309983C2 - Method for initial isolation of influenza virus a strain, strain virus a/duck/novosibirsk/56/05 h5n1 for preparation for diagnosis, prophylaxis and therapeutical agents, and for evaluation of antiviral activity of various compounds - Google Patents

Abstract

FIELD: medical virology.

SUBSTANCE: initial isolation of influenza virus A/H5N1 strain is carried out by contamination of cell culture of porcine embryo kidney. Contaminated culture is incubated in supporting medium 199 with penicillin and streptomycin addition in presence of medium TPCK treated tripsin. Disclosed is strain A/duck/Novosibirsk/56/05 H5N1 isolated by abovementioned method. Strain is capable of inducing virus neutralizing protective antibodies and is useful in preparation of test-systems and other diagnosis preparations.

EFFECT: strain of high antigen activity.

4 cl, 1 tbl, 4 ex

Description

The invention relates to medical virology, in particular to the problem of diagnosis, treatment and prevention of influenza A, and is intended to develop means and methods of biological protection.

Relatively few sensitive cell cultures are known for the primary isolation of influenza A viruses. Traditionally, chicken embryos are used to isolate influenza viruses from infected biological material. Currently, mammalian tissue cultures (MDCK) have been proposed for primary isolation of influenza virus. According to the Davis H.W. method e.a. (Bull. Wid. Htlh. Org., 1978, v.14, p.1445-1448) primary isolation of human influenza viruses is carried out in a spaniel dog kidney cell culture (MDCK) supplemented with TPCK-treated tripsin, XIII fraction, Sigma, at a final concentration of 2 μg / ml. In the literature, there is no evidence of isolation of influenza A virus using SPEV cell cultures. There is no data in the literature on the primary isolation of influenza A virus using SPEV cell cultures, and there are still no WHO recommendations for cultivating influenza A virus in SPEV cell cultures for the production of diagnostic and prophylactic drugs.

G.A.Danlybaeva et al. (Questions of Virology 1991, 1, p.10-13), studying the sensitivity of cells of tissue cultures of humans and animals, found that the influenza virus strain A / dove / Astrakhan / 31/76 (H5N3) actively reproduced not only in MDCK cells, but also in the transplanted culture of pig embryonic kidney cells (SPEV). The classic system for the production of diagnostic and prophylactic drugs is developing chicken embryos. A known method of culturing influenza virus in Vero cells (SU 614873, 10/15/1997, 849716).

Currently, for production purposes, on the recommendation of the World Health Organization (WHO), influenza A virus strains cultured in chicken embryos or in kidney cell cultures of dogs of MDSK are used.

However, the production of antigens for the diagnosis of avian influenza A or serum to it requires complex additional purification of the virus-containing allantoic fluid, and in MDSC cell cultures, the virus production is low, requires an additional concentration of the virus-containing material.

The inventive primary isolation by infection of the SPEV culture with biological material infected or suspicious of infection with the influenza A virus, which allowed to isolate the infectious influenza A virus, namely: the influenza virus strain A / duck / Novosibirsk / 56/05 H5N1, characterized by high ability to be produced in pig embryonic kidney cell cultures (SPEV). The resulting strain of avian influenza A virus H5N1 is characterized by high antigenic activity, the ability to induce neutralizing, protective antibodies and can be used to prepare test systems and other diagnostic drugs, to evaluate the antiviral activity of various compounds, to obtain a vaccine, highly immunogenic immunoglobulins against H5N1 influenza A virus suitable for the creation of diagnostic, therapeutic and prophylactic drugs.

Obtaining the original strain of influenza virus A / duck / Novosibirsk / 56/05 H5N1.

The strain of the influenza virus A / duck / Novosibirsk / 56/05 H5N1 was isolated during epizootics among poultry (ducks and chickens) in the Novosibirsk region (Zdvinsky district). From a pool of internal organs of a sick domestic duck positive for H5 RNA of influenza A virus, according to the polymerase chain reaction (RT PCR).

Material from a sick duck was used for primary isolation of influenza A virus by infection of cell cultures of SPEV.

To isolate the virus, a freshly prepared suspension of cell cultures of SPEV obtained from the collection of cell cultures of the Research Institute of Virology named after D.I. Ivanovo RAMS. The suspension contained 5 × 10 ⁶ cells in 1 ml of culture medium 199 with the addition of antibiotics and enzymes, trypsin. The prepared suspension of SPEV cells was poured into 24 well panels of 200 μl per well and 25 μl of each of the prepared samples of swabs or organs from birds was introduced directly into the cells. For each sample, 2 wells with a cell suspension were used. For infection of SPEV cell cultures, undiluted and diluted 10-fold samples from a pool of organs of a sick domestic duck were used. A portion of the cell suspension wells were left as controls. The panels were left at room temperature for 20 minutes, periodically shaking slightly.

At the end of this time, 0.4 ml of 199 medium was added to the wells with a suspension of panel cells and the panels were incubated at 37 ° C in a CO ₂ atmosphere. 24 hours after infection, a cytopathogenic effect of the agent was recorded.

Infectious titers of influenza A virus (H5N1) were recorded using the Reed and Mench method.

Polymerase chain reaction (RT-PCR) was used to determine the genome of the influenza A virus (H5) in samples taken from infected cultures of SPEV cells at the stage of maximum manifestation of the cytopathogenic activity of the virus.

Further determination of the type of hemagglutinin and neuraminidase was carried out using the microarray method.

Thus, the use of SPEV cell cultures for primary isolation of the virus made it possible to isolate the infectious influenza virus A / duck / Novosibirsk / 56/05 H5N1

Identification of strain A / duck / Novosibirsk / 56/05 H5N1.

The strain was identified by hemagglutination inhibition reaction, RT-PCR using primers for the influenza A virus genome, and sequencing of amplified sections of the influenza virus genome that reliably showed the presence of the gene encoding the influenza hemagglutinin H5. Using the microarray method, it was found that the isolated variant belongs to the H5N1 avian influenza virus. By the method of analysis of nucleic acid sequences, it was also shown that the gene encoding hemagglutinin 5 contains a cutting site characteristic of pathogenic strains of influenza A virus H5N1.

Strain A / duck / Novosibirsk / 56/05 H5N1 was deposited with the State Virus Collection, registration number 2371.

Physico-chemical signs.

The inactivation index of sodium deoxycholate is 5.2. The thermal inactivation index is 5.5 (when heated for 20 minutes at 50 ° C). When filtering a virus-containing suspension through millipore filters with a size of 100 nm, a decrease in the infectious titer was not observed. This indicates that the strain of the virus has sizes up to 100 nm and contains a lipoprotein membrane.

Based on the analysis of the primary structure of RNA, antigenic traits, the isolated strain of the virus is assigned to the Orthomyxoviridae family.

Cultural properties.

The interaction of the avian influenza A virus strain with cell cultures of various origins (SPEV, Vero, MDSK) occurs in the form of an acute infection, accompanied by cell destruction, usually occurring 24-48 hours after infection, with the virus accumulating in the culture medium in titers of up to 10 , 0 lg TCD50 / ML. In the culture fluid infected with the A / duck / Novosibirsk / 56/05 H5N1 strain, cell cultures accumulate HCV RNA, detected using primers to the region of genes encoding hemagglutinin H5, as well as hemagglutinins in titers up to 1: 256. During roller cultivation of chicken embryo fibroblasts infected with the HCV strain in the culture fluid, hemagglutinins accumulate agglutinating goose erythrocytes in titer up to 1: 128 on the 4-5th day after infection.

Antigenic properties.

The antigenic properties of strain A / duck / Novosibirsk / 56/05 H5N1 were studied in the RSA and RTGA, in the reaction of biological neutralization, by enzyme immunoassay. The reactions used polyclonal sera obtained for the H5N1 influenza A virus. The biological neutralization reaction was performed with 1:10 dilutions of the serum and ten-fold dilutions of the virus-containing material. The neutralization reaction was taken into account by the neutralization index.

Immunogenic properties.

The high antigenic properties of strain A / duck / Novosibirsk / 56/05 H5N1 allow it to be used to obtain highly immunogenic sera) or immune ascitic fluids. It was shown that a single infection of mice with inactivated ultraviolet virus-containing material allows the detection of antibodies to influenza A virus in blood serum that are actively reacting in RTGA and ELISA on days 14-16 (antibody titers 1:40, 1:80, respectively).

The diagnostic drug, the antigen from strain A / duck / Novosibirsk / 56/05 H5N1, is obtained by roller cultivation of the virus in SPEV cell cultures followed by clarification of the culture fluid, and, if necessary, precipitation by ultracetrifugation of the virus into a plaque at the peak of its hemagglutinating activity (24 hours after infection). In this case, the hemagglutinating activity of the virus from the plaque reaches a titer of 1: 5120.

A prophylactic preparation (culture inactivated vaccine) is prepared from the culture fluid collected from the SPEV cell cultures infected with the influenza A virus strain 2-3 days after infection by roller growth of the virus crop. The collection of culture fluid is inactivated with formaldehyde at a concentration of 1: 2000 for 60 days at + 4 ° C or with ultraviolet rays in the apparatus for 1.5 minutes, checking residual infectivity and maintaining antigenic activity.

Antiviral screening is based on the use of SPEV cell cultures infected with strain A / duck / Novosibirsk / 56/05 H5N1, which are treated with preparations before, after or at the time of infection with influenza A strain. The results are taken into account by the ability to suppress various compounds under different conditions of use Influenza A virus replication in SPEV cell cultures.

Example 1

The method of isolation of avian influenza virus A. For this purpose, a suspension of an inoculated pig cell line of a kidney of a pig embryo (SPEV) prepared on medium 199 with the addition of 100 PIECES of penicillin and streptomycin is used. To isolate the influenza virus, a suspension of cells is infected in a volume of 1-2 ml with field samples material in a volume of up to 0.2-0.4 ml in 25 cm ³ bottles followed by a 30-minute incubation of the virus-cell mixture at room temperature for optimal adsorption of the virus. At the end of the period of adsorption of the virus to the cells, the volume of the virus-containing suspension in the vial was adjusted with medium 199 with the addition of antibiotics and trypsin (TPCK-treated tripsin, XIII fraction, Sigma to 10 ml), incubated in a thermostat with a supply of CO ₂ at 37 ° C until cytodestruction manifested. The use of a series of ten-fold dilutions of samples of field material for infection of cell cultures for isolation of influenza A virus allows to increase the efficiency of isolation of influenza A virus.

Example 2

Obtaining a virus-containing culture fluid necessary for the production of diagnostic products based on the proposed highly productive strain of the virus A / duck / Novosibirsk / 56/05 H5N1. As a material for research, in the absence of an analogue, we offer strain A / duck / Novosibirsk / 56/05 H5N1, which infected a monolayer of cell cultures of SPEV grown by the roller method. For infection, use 10 TCD ₅₀ / per cell and after an hour of contact, the cells are washed three times from the non-adsorbed virus, after which they add 199 growth medium with 1% calf embryo serum and incubate on a roller machine at 37 ° C for 24-48 hours, when cultures, distinct phenomena of the cyto-destructive effect of the virus will be noted. The data obtained characterizing the discharge of culture fluid suitable for the production of influenza antigens are shown in the table.

Thus, a highly productive HCV strain can be used to produce diagnostic products.

Example 3

Obtaining a virus-containing culture fluid necessary for the production of prophylactic drugs (culture inactivated vaccine) based on the proposed highly productive strain of the virus A / duck / Novosibirsk / 56/05 H5N1. As a material for studies, in the absence of an analogue, we offer a strain A / duck / Novosibirsk / 56/05 H5N1 that is highly productive for cell cultures of SPEV and Vero-E6 (transplantable cultures of green monkey kidney cells), which infected a monolayer of cell culture Vero-E6 grown by roller method . For infection, 10.0 TCD ₅₀ / per cell is used and after an hour-long contact the cells are washed three times from the non-adsorbed virus, after which 199 growth medium is introduced with 0.1% human calf albumin and incubated on a roller machine at 37 ° C for 24-48 hours when in the cultures distinct phenomena of the cyto-destructive action of the virus will be noted. The data obtained characterizing the discharge of culture fluid suitable for obtaining a culture inactivated vaccine against H5N1 influenza are shown in the table.

Thus, a highly productive HCV strain can be used to produce prophylactic drugs.

Example 4

Obtaining an experimental model: avian influenza A virus (H5N1) and SPEV cell cultures sensitive to its replication suitable for screening antiviral drugs.

SPEV cell cultures are treated with non-toxic doses of drugs before, after, or at the time of infection with influenza A strain. Results are taken into account by the ability of certain compounds to suppress the replication of influenza A virus in SPEV cell cultures under different conditions. As control are: influenza A virus infected (strain A / duck / Novosibirsk / 56/05 H5N1) cell cultures without antiviral drugs, uninfected cultures of SPEV cells treated with the drugs at the same time and in the same doses.

Thus, the proposed method of primary isolation allows the isolation of influenza A viruses. The strain of influenza virus A / duck / Novosibirsk / 56/05 H5N1, (Bills No. 2371), the primary isolation of which was carried out using this method by infection of a kidney cell culture of a pig embryo (SPEV) ), is characterized by a high ability to reproduce in the SPEV culture, the ability to induce neutralizing, protective antibodies. The high antigenic activity of this strain allows it to be used to obtain highly immunogenic serums. The strain of the influenza virus A / duck / Novosibirsk / 56/05 H5N1, (Bills No. 2371) is suitable for the preparation of diagnostic, prophylactic drugs and for evaluating the antiviral activity of various compounds.

Table
Infectious and antigenic A / duck / Novosibirsk / 56/05 H5N1 by roller culture of infected cell cultures Cell culture Culture fluid sampling time Virus concentrate 6 o'clock 12 hours 24 hours 40 hours 24 hours GA JRC GA JRC GA JRC GA CPD ¹⁾ GA SPEV 1: 8 0 1:32 + 1: 128 +++ 1: 256 ++++ 1: 5120 Lg TCD ₅₀ 4.0 6.2 10.0 10.5 11.0 1) CPD - cytopathogenic effect of influenza virus: + - death of 10-15% of cells; ++ - damage to 50% of cells; +++ - death of 75-80% of infected cells, ++++ - death of 100% of cells.

Claims (4)

1. The method of primary isolation of strains of influenza A / H5N1 virus, including infection of cell cultures with biological material that is infected or suspicious of infection with the virus, characterized in that the primary isolation of the virus occurs by infection of the cell culture of the kidney of a pig embryo (SPEV). 2. The method of isolation of the influenza A / H5N1 virus according to claim 1, characterized in that for the incubation of the infected culture using support medium 199 with the addition of 100 PIECES of penicillin and streptomycin and in the presence of TRPC-treated tripsin support medium, fraction XIII, Sigma USA, at a final concentration of 2 μg / ml. 3. The method according to PP.1 and 2, characterized in that for infection using a series of ten-fold dilutions of samples of field material. 4. The strain of influenza virus A / duck / Novosibirsk / 56/05 H5N1, (Bills No. 2371), isolated by the method according to any one of claims 1 to 3, characterized by high ability to be produced in a culture of pig embryonic kidney cells (SPEV), suitable for diagnostic, prophylactic and therapeutic drugs and to evaluate the antiviral activity of various compounds.