WO2006025218A1 - Method of examining infection with avian influenza - Google Patents

Abstract

A method of examining infection with avian influenza by contacting the serum obtained from a test bird with a viral antigen to induce an immune reaction characterized in that an avian influenza non-structural protein NS1, which is less reactive with the serum of a vaccine-inoculated bird, or a polypeptide comprising a part of its amino acid sequence is employed as a viral antigen to distinguish naturally infected birds alone. According to this method, naturally infected birds can be distinguished from vaccine-inoculated birds.

Description

 Technical details Testing methods for avian influenza infection Technical field

 The present invention relates to an inspection method for avian influenza infection, and particularly to an inspection method for distinguishing poultry naturally infected with avian influenza from vaccinated poultry. Background art

 In recent years, avian influenza infection has been prevalent in various parts of the world, and countermeasures against it have been demanded. One of the measures to control avian influenza is vaccination of birds (poultry). However, when vaccination is performed, the increase in antibody due to the vaccine occurs, so it is not possible to identify naturally infected birds that could be identified in the past. The problem arises. Therefore, the administration of vaccinations for birds should be carried out after the introduction of the DI VA (Differntiatmg infected, from vaccinated animals) system under government control. (Technical Consultation on the Control of Avian Influenza (February 2004)).

The DI VA system currently used is a method that uses a hetero-vaccine with a different NA subtype from the virus that is prevalent. In general, influenza viruses have protrusions consisting of HA (hemagglutinin) and NA (neuraminidase) on the outer shell of the virus particle, and this combination results in HIN 1 (HA subtype = 1, NA subtype). = 1), H3N2 (HA subtype = 3, NA subtype = 2) and other antigen types. If an influenza virus belonging to an serotype (eg H 1 N 1) is prevalent, a vaccine with the same serotype Once inoculated, the antibody titer against the antigen (H l, N 1) is equally elevated in both vaccinated and naturally infected birds, making it difficult to distinguish between them (this is why DIVA is required).

 In the current DIVA system, discrimination between the two is realized by using hetero vaccines with different NA subtypes (for example, NA subtype = 2). However, in the above method, when the epidemic of the same NA subtype virus as the hetero vaccine starts, it becomes impossible to distinguish between vaccinated birds and naturally infected birds, and it is difficult to completely distinguish naturally infected birds. Also, with this method, it is necessary to keep the non-inoculated group as a decoy bird in the vaccinated group and check for the presence of natural infection. Furthermore, in Southeast Asian countries, etc., homo-vaccination with the same antigen type as the endemic species is carried out in an uncontrolled state without receiving administrative control.

Even if the DIVA system is introduced afterwards, it is difficult to identify naturally infected birds. On the other hand, the use of nonstructural proteins of viruses as markers for virus infection diagnosis has been proposed for several diseases [specifically, type C hepatitis virus (Inoue et al., J Gen. Virol. 73, 2151-2154 (1992)), hand-foot-and-mouth disease (Neitzert et al., Virology 184, 799-804 (1991)), Mainfluenza (Birch et al., J. Virol. Methods 65, 255-263 (1997), Ozaki et al., Vet. Microb. 82, 111-119 (2001))]. However, there are no examples of avian influenza nonstructural proteins proposed for the diagnosis of viral infection. In addition, in order to use a non-structural protein of avian influenza virus as a marker for D IVA, the antibody titer must increase in natural infections, but the antibody titer must not increase in vaccination with vaccination. These studies have not been conducted on avian influenza. Disclosure of the invention

Therefore, the present invention is intended to distinguish and distinguish naturally infected birds from vaccinated birds. The objective is to provide an avian influenza virus inspection method.

 As a result of intensive studies on the above problems, the present inventors have examined the possibility of both avian influenza virus nucleoprotein (N P) and nonstructural protein NS 1 as virus infection identification markers. As a result, NP, which is a structural protein, reacts indifferently between vaccinated birds and naturally infected birds, but non-structural protein NS 1 does not react with vaccinated birds, but only with naturally infected birds. I found out. That is, the present inventors have found that the nonstructural protein NS 1 can be used as an antigen for identification of the D I V A system, and completed the present invention.

 That is, the present invention provides the following inspection method for avian influenza virus infection.

 1. In a method for testing avian influenza virus infection by immunological reaction by contacting serum obtained from a test bird with a viral antigen, a non-structural protein NS 1 or avian influenza virus having low reactivity to sera inoculated with acupuncture A method for examining avian influenza virus infection, characterized in that only a naturally infected bird is identified using a polypeptide consisting of a part of the amino acid sequence as a virus antigen.

 2. In a method for testing avian influenza virus infection by immunological reaction by contacting serum obtained from a test bird with a viral antigen,

(a) screening of naturally infected bird specimens and bird specimens inoculated with acupuncture using the structural protein of avian influenza virus as a viral antigen; and (b) a bird with low reactivity to vaccinated serum from specimens positive in the screening process. A method for examining avian influenza virus infection, comprising the step of identifying only a naturally-infected bird specimen by contacting with a non-structural protein of influenza virus NS1 or a polypeptide comprising a part of its amino acid sequence. 3. The NS 1 protein or a polypeptide comprising a part of the amino acid sequence thereof is a recombinant protein or polypeptide obtained by expressing the NS 1 gene in a host other than influenza virus. The inspection method for the avian fluenza virus infection as described.

4. The method for examining avian influenza virus infection according to 3 above, wherein the host is an insect cell.

 5. The method for examining avian influenza virus infection according to 3 or 4 above, wherein the NS1 protein is a modified NS1 protein.

 6. The method for examining avian influenza virus infection according to 5 above, wherein the modified NS1 protein is a modified protein having a histidine tag at the C-terminus.

 7. The method for examining avian influenza virus infection according to any one of the above items, wherein the immunological reaction is performed by Western plotting, ELISA, immunoprecipitation, immunoagglutination, or a combination thereof.

 8. The inspection method for avian influenza virus infection according to any one of the above items, wherein the bird is a poultry.

 9. The method for inspecting avian influenza virus infection as described in 8 above, wherein the poultry are chickens.

The invention's effect

 According to the method of the present invention, infection with avian influenza virus can be determined by a simple test. In addition, according to the method of the present invention, it is possible to discriminate between naturally infected birds and pox-inoculated birds even if an inactivated homo vaccine is used, so that it can be used not only in developed countries but also in Southeast Asia. BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a non-structural protein NS 1 of avian influenza virus as an antigen. A bird flu virus infection test method for DI VA system is provided.

 A-type influenza virus, including avian influenza virus, contains nucleoprotein (NP), RNA, and RNA in a 100-250 nm diameter particle surrounded by a lipid bilayer envelope derived from the host cell membrane. Contains viral RNA polymerase (PB2, PB1, PA). RNA is an 8-segment type, PB2, PB 1, PA (viral RNA polymerase), HA (hemadaltinin spike), NP (nucleoprotein), NA (neuraminidase spike), M (coat and ion channel), NS The code area is divided. NS encodes NS 1, a nonstructural protein, and NS 2, which is now thought to be a structural protein.

 In the present invention, a non-structural protein NS 1 or a polypeptide comprising a part of its amino acid sequence (hereinafter sometimes abbreviated as NS 1 polypeptide) is used as a viral antigen. NS1 has the theory that it has the function of promoting translation of Ml protein (protein lining the outer coat) and regulating the nuclear export of mRNA for equine influenza. There is also a report that it is not necessary for the expression of lysozyme (Huang et al., J. Virol. 64, 5669-5673 (1990)), and the details of its function are unknown. In any case, as can be seen in the experimental examples described below, NS 1 protein is expressed in a live virus infection state, but its content in the vaccine is very small.

 In the present invention, NS 1 protein may be obtained from avian influenza virus in prevalence or may be obtained from other antigenic avian influenza virus. NS 1 protein is preferably obtained by expressing NS 1 gene in a host other than influenza virus.

Isolation of NS1 gene from avian influenza virus can be performed by conventional methods. Typically, cD from total RNA of avian influenza virus NA is synthesized by reverse transcription, and a polynucleotide containing the NS1 coding region is obtained using a primer specific for the eighth segment encoding NS1. A primer specific to the eighth segment is described, for example, in Fouchier et al., Arch. Virol. 146: 2275-89 (2001).

 In addition to insect cells, yeast, bacteria such as Escherichia coli and Bacillus, and mammalian cell systems, the expression system can be any post-translational modification or three-dimensional structure of the expressed protein. Insect cells are preferably used because the obtained antigen can be obtained.

 NS 1 gene expression in insect cells can be achieved by inserting NS 1 gene into a baculovirus transfer vector, cotransforming it into insect cells along with baculovirus DNA, and recombinant baculovirus containing NS 1 gene. Bac-to-Bac system (Luckow, et al., J. Virol., 67, 4566 (1993)) or the like may be used. By infecting the insect cells with the obtained recombinant baculovirus, recombinant NS 1 protein can be obtained in the insect cells.

NS 1 protein may have the same structure as natural NS 1 protein, or may be modified to be suitable for purification or immunological reaction. For example, by adding a polyhistidine tag (usually H ₆ ) to the C-terminus of NS 1 protein, purification can be easily performed without affecting antigenicity. Other fusion proteins (for example, those with a FL AG tag (DYKDDDDK) ヽ My c tag (EQKLISEEDL) etc. added) may be used as long as the antigenicity is not affected.

The immunological method for discriminating avian influenza virus using the NS1 protein thus obtained is any antigen antibody reaction using NS1 protein as an antigen. In other words, NS 1 protein is fixed to a membrane, column, substrate, particle, molecule, etc. or placed in a specific area (eg within a well) and infected Avian serum suspected of being contacted with this is identified by reacting NS 1 protein antibody with NS 1 protein in the bird serum. Identification may be any of optical, chemical, and physical methods.

 Preferred antigen-antibody reactions include Western Proto method, ELI SA method, immunoprecipitation method, immunoagglutination method and the like.

 A competitive EL I SA method using a monoclonal antibody against NS 1 protein may be used. This is a method of reacting NS 1 antigen affixed to the plate with test serum, and then adding NS 1-specific monoclonal antibody. If the test serum contains antibodies against NS 1 protein Thus, NS 1 antigen is covered thereby, and NS 1-specific monoclonal antibody added later cannot stick to NS 1 protein. Using this competitive ELI SA method, any animal (duck, goose, dachyo, chicken, duck, etc.) can be detected with anti-mouse antibodies that recognize monoclonal antibodies. .

 The Western plot method can be performed by a conventional method. That is, the whole protein is taken out from the host cell (for example, insect cell) in which the NS1 gene is expressed as described above, and this is developed on an acrylamide gel and transferred onto an appropriate transcription membrane. After blocking, contact the serum and add a coloring reagent to check if NS 1 band is detected. NS 1 protein (27-28 kDa) can be identified by comparison with a control (total protein sample obtained from a host cell into which NS 1 gene has not been introduced). The control sample is obtained, for example, by infecting insect cells with a baculovirus that does not have the NS1 gene (such as wild-type AcNP V).

The Western blot method may be performed by reaction with an NS 1 protein standard sample. NS 1 protein standard sample is preferably NS 1 protein (NS IHi s) with His-tag that is easy to purify! ^ Those purified by infinity chromatography are used.

 For the purposes of the present invention, SDS-PAGE can be used for Western blotting. The acrylamide concentration in the acrylamide gel is about 10 to 15%. Nitrocellulose membrane can be used as the transfer membrane, but nylon membrane or PVDF membrane may also be used. After membrane transfer, react with a commercially available enzyme label-anti-chicken Ig G (if the specimen is derived from chicken), and then add a chromogenic substrate to examine the presence or absence of coloration of the NS1 band.

 Changes and modifications to the ELISA method and other antigen-antibody reactions can be easily made by those skilled in the art.

 In the test method of the present invention, a test using a structural protein, particularly a nucleoprotein (N P) as an antigen may be preceded as a screening step. In other words, a test using NP as an antigen distinguishes infected or vaccinated specimens from non-infected and non-vaccinated specimens, and positive specimens (infected / vaccinated specimens) are unstructured proteins NS 1 or NS 1 A test using a polypeptide as an antigen is performed to discriminate between naturally infected samples and vaccinated samples.

 The method of the invention can be applied to birds, preferably poultry, especially chickens. Brief Description of Drawings

 Figure 1 is a photograph showing the results of Kumashi brilliant blue staining (Figure 1 (A)) and Western mouth (Figure 1 (B)) showing the expression of the avian influenza virus structural protein NS 1 used in the present invention. is there.

 Fig. 2 is a photograph showing the results of Coomassie primiant blue staining (Fig. 2 (A)) and Western blotting (Fig. 2 (B)) showing the expression of avian influenza virus nucleoprotein NP that can be used in the present invention. is there.

FIG. 3 is a photograph showing the results of a Western plot in which avian influenza virus-infected chickens and vaccinated chickens were identified by the method of the present invention. Example

 Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. In Example 1 and Reference Example 1 below, the avian influenza virus is an isolate # 478 (H1N1) (A / Duck / Aomori / 478/03: H IN 1) was used. In addition, as the naturally infected serum of avian influenza virus in Example 2, serum collected 6 weeks after infection with AIV (stock name unknown H9N2) at the Institute of Animal Health was used. The inactivated virus of Example 2 is a form of A / Duck / HokkaidoZ 9/99: H 9 N2 distributed from Hokkaido University, treated with formalin. A highly immunized chicken is treated with Freund's inactivated virus. Serum obtained by immunizing chickens three times with the complete adjuvant. Example 1: Production of N S 1 protein

 (1) Cloning and modification of NS 1 gene

 A test bird influenza virus (A IV) solution was inoculated into the allantoic cavity of 10-day-old hen's eggs using a syringe and cultured at 37 ° C for 2 days. The eggshell was disinfected and then opened to collect chorioallantoic fluid and centrifuged to precipitate cell debris. Total RNA was extracted from the resulting supernatant using ISOGEN-LS (Nippon Pongene Co., Ltd.) according to the protocol described in the manual. A IV 8 segments (Segment 3) Using a primer (Uni 12: (age aaaagcagg (SEQ ID NO: 1)) common to all sequences of RNA, cDNA was reverse transcriptase (Perkin Elma Japan). Reverse transcription (RT) was performed at room temperature (25 ° C) for 15 minutes, followed by 42 ° C for 60 minutes, and the enzyme was inactivated at 99 ° C.

Next, a primer specific for the eighth segment encoding NS 1 (Fouchier et al., Arch. Virol. 146, 2275-89 (2001)):

5, — t a t t cgt c t cagggagcaaaagcagggt g— 3 ′ (SEQ ID NO: 2);

 5, — atat cgt ct cgtat tagt agaaacaagggtg t t t t 1 3, (SEQ ID NO: 3)

Using the above cDNA as a template, PCR was performed to amplify the 8th segment cDNA. PCR was started at 94 ° C for 4 minutes, followed by 30 cycles of 94 ° C for 20 seconds, 58 ° C for 30 seconds, 72 ° C for 2 minutes, and the final step was 72 ° C for 5 minutes.

 The PCR amplified fragment was cloned into the cloning vector pCR2.1 using the TA cloning kit. After sequencing (SEQ ID NO: 4), a new primer (SEQ ID NO: 5: cggat c CATAATGGATTCCAACACTG ^ SEQ ID NO: 6: gagaa ca att agt cagaagttCATCATCATCATCATCATt gaggat ccg) is designed and cloned into the plasmid. Only the NS 1 region was amplified from the resulting 8th segment. PCR conditions started at 94 ° C for 4 minutes, followed by 30 cycles of 94 ° C for 20 seconds, 58 ° C for 30 seconds, 72 ° C for 2 minutes, and the final step was 72 ° C for 5 minutes. One of the above primers (SEQ ID NO: 6) was modified so that the NS 1 gene was modified at the 3 and ends, and a His-Tag consisting of 6 histidines was added to the NS 1 carboxy terminus. Imaichi was designed.

 (2) Production of baculovirus expressing NS 1

After PCR, the amplified gene was cloned and transferred to the baculovirus transfer vector pAc YM 1 (NERC Institute of Virology, Oxford, UK). The obtained plasmid was combined with baculovirus DNA (AcRP 23-1 acZ) and insect cells Sf Transfected 21 (Virology Institute). The experimental procedure was based on the protocol of the A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures: Summers & Smith, Texas A & M. Recombinant baculovirus AcAI NS lHis was obtained by homologous recombination.

 (3) NS 1 protein expression

 A transfer vector having the NS1 gene was transfected into insect cells Sf21 using lipofectin (Gibco BRL) together with DNA of baculovirus Ac R P 23-1 a c Z. Four days later, the culture supernatant was collected, and the virus was cloned by the black method. The parent strain AcRP 23_lacZ forms a blue black in the presence of X-gal, but forms a white plaque in the recombinant baculovirus. Plaque cloning was repeated twice to obtain the recombinant virus Ac A I NS lHis.

 To confirm NS 1 expression, infect Sf 21 cells with AcAINS lHis and, after SDS-PAGE, stain with Kumashi Brilliant Blue or transfer to nitrocellulose membrane and use anti-polyhistidine antibody (SIGMA) A histidine-tagged protein was detected. As a result, as shown in Fig. 1 (A), protein expression was specifically observed at a position of about 27 kDa, and this protein was detected with an anti-polyhistidine antibody as shown in Fig. 1 (B). Reference Example 1: Production of NP protein

Recombinant baculovirus for NP protein expression was prepared in the same manner as NS1. In order to clone the 5th segment encoding the NP protein gene, the 錶 type was # 478 cDNA and the primers were referred to Fouchier et al., Arch. Virol. 146, 2275-89 (2001). : TATTCGTCTCAGGGa gcaaaagcaggGTA (SEQ ID NO: 7) and ATAT C GT CTC GT ATTagt gaaacaaggGTATTTTT (SEQ ID NO: 8) was used. After sequencing, gagga t ccat catggcgt cccaaggcac (SEQ ID NO: 9) and gag gat cctt aat tgt catact cct ctgc (layout!) Number 10 were used to amplify only the NP protein gene.

The obtained NP gene was put into a baculovirus like NS 1 to obtain a recombinant baculovirus Ac AI NP. Ac AI NP was infected into insect cells to obtain NP protein. Example 2: Identification of avian influenza virus infection using NS1 protein The recombinant virus Ac AINS lHis obtained in Example 1 was inoculated into Sf21 cells, and the cells were collected after 3-4 days of culture. Cells (about 10 ⁶ cells) were washed with phosphate buffered saline (PBS), resuspended in 80-1 of Laemmli electrophoresis buffer, and heated in boiling water at 100 ° C for 5 minutes.

 The obtained cell lysate was subjected to 12% 803-acrylamide gel slab electrophoresis (100 for 1 hour) to develop all proteins. After development, the protein was transferred to a nitrocellulose membrane. Broking solution (Skim milk one

10 g, Tween20 -0.2 ml, PBS—200 ml) was used to block unadsorbed protein. As a control, Sf 21 cells were infected with the parent strain Ac RP 23-lacZ, and the same operation was performed.

Avian influenza-infected chicken sera and inactivated virus hyperimmune chicken sera are diluted 25-fold, 50-fold, and 250-fold with the above blocking solution, then applied to the above-mentioned protein-transferred cellulose membrane and 1 at room temperature (about 20 ° C). Reacted for hours. Next, after washing the membrane with a blocking solution, the secondary antibody (enzyme label—anti-chicken IgG (Cappel)) was diluted with a blocking solution and reacted, and after washing the membrane with a blocking solution, the chromogenic substrate ( Bm blue POD substrate (Boehringer Mannheim)) To see if the NS IHi s band is recognized.

Avian influenza-infected chicken sera developed NS IHis band (approximately 27 kDa) up to 50-fold dilution, whereas inactivated virus hyperimmune chicken sera developed NS 1 His band at any dilution. Was not recognized. In addition, AcRP23-lacZ-infected cells were not colored at all and were confirmed to have no antigenicity. Example 3: Identification of avian influenza virus infection using NS 1 protein The experiment was conducted in the same manner as in Example except that the dilution ratio was 50 to 1250 times. The results are shown in Figure 3. In the avian influenza (AIV) -infected serum, a clear NS IHi s band color can be seen at a dilution factor of 50 (enlarged part in the figure). Comparative Example 2: Identification of avian influenza virus infection using NP protein Sf21 cells were inoculated with the recombinant virus AcAINP obtained in Reference Example 1, and the cells were collected after 3-4 days of culture. Cells (approx. 10 ⁶ cells) are resuspended in Laemmli's electrokinetic buffer 80 ^ 1 and, similar to NS IHi s protein, after SDS-PAGE, stained with Kumashi-brilliant bluish or transferred to nitrocellulose membrane Thereafter, expression was confirmed by Western blotting.

As a result, NP band (56 kDa) coloration was observed in avian influenza-infected chicken sera and inactivated virus highly immunized chicken serum at all dilutions. Control (non-infected 'non-immune specimen) No color development was seen in serum. Thus, immunological reactions with NP antigens cannot distinguish between naturally infected and virus-inoculated chickens, but they are highly sensitive to non-infected and non-immunized chickens. Therefore, it is useful for screening naturally infected chicken samples and virus-inoculated chicken samples before identification by NS1. It is also effective for ELI SA reactions. Industrial applicability

 According to the present invention, a bird flu virus naturally infected bird can be distinguished from a vaccinated bird by a simple method. For this reason, it is highly useful as an inspection method for implementing a wide range of DIVA systems where international compliance is an urgent issue.

Claims

The scope of the claims 1. In a method for testing avian influenza virus infection by immunological reaction by contacting serum from a test bird with a viral antigen, a non-structural protein NS1 or avian influenza virus that is less reactive against vaccinated serum A method for examining avian influenza virus infection, wherein a polypeptide comprising a part of the amino acid sequence is used as a virus antigen to identify only naturally infected birds. 2. In a method of testing avian influenza virus infection by immunological reaction by contacting serum obtained from a test bird with a viral antigen,  (a) screening a naturally infected bird specimen and a sputum inoculated bird specimen using the structural protein of avian influenza virus as a viral antigen; (b) Naturally infected bird specimens by contacting the specimen serum positive in the screening process with a non-structural avian influenza virus non-structural protein NS 1 or a part of its amino acid sequence, which has low reactivity to the vaccinated serum. A method for examining an avian influenza virus infection, comprising a step of identifying only a virus. 3. The polypeptide consisting of NS 1 protein or a part of its amino acid sequence is a recombinant protein or polypeptide obtained by expressing NS 1 gene in a host other than influenza virus. 2. The inspection method for avian influenza virus infection according to 2. 4. The method for examining an avian influenza virus according to claim 3, wherein the host is an insect cell. 5. The method for examining avian influenza virus infection according to claim 3 or 4, wherein the NS 1 protein is a modified NS 1 protein. 6. The method for examining avian influenza virus infection according to claim 5, wherein the modified NS 1 protein is a modified protein having a histidine tag at the C-terminus. 7. The method for examining avian influenza virus infection according to any one of the preceding claims, wherein the immunological reaction is performed by Western plotting, ELI SA, immunoprecipitation, immunoagglutination, or a combination thereof. 8. The method for examining avian influenza virus infection according to any of the preceding claims, wherein the bird is a poultry. 9. The inspection method for avian influenza virus infection according to claim 8, wherein the poultry are chickens.