JP4084403B2 - Testing method for avian influenza infection - Google Patents

Description

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

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 carried out, antibody increases due to the vaccine, so naturally infected birds that could previously be identified cannot be identified. The problem arises. For this reason, it is recommended by international organizations that bird vaccination should be carried out with the introduction of the DIVA (Differentiating Infected from Vaccinated Animals) system under government control. (FAO / OIE / WHO Expert Consultation on the Avian Influenza (February 2004)).
The DIVA system currently employed is a technique that uses a hetero-vaccine with a different NA subtype from the virus in prevalence. That is, in general, influenza viruses have protrusions made of HA (hemagglutinin) and NA (neuraminidase) on the outer surface of the virus particle, and H1N1 (HA subtype = 1, NA subtype = 1), H3N2 by this combination. It is classified into many antigen types such as (HA subtype = 3, NA subtype = 2). When influenza viruses belonging to a certain antigen type (eg, H1N1) are prevalent, when vaccinated with the same antigen type, the antibody titer against the antigen (H1, N1) is equally increased in both vaccinated and naturally infected birds Therefore, it becomes difficult to distinguish the two (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 an epidemic by the virus of the same NA subtype as the hetero vaccine starts, it becomes impossible to distinguish between the vaccinated bird and the naturally infected bird, and it is difficult to completely identify the naturally infected bird. In this method, it is necessary to keep the non-inoculated group as a decoy bird in the vaccinated group and to check for the presence of natural infection. Furthermore, in Southeast Asian countries and the like, homo-vaccination with the same antigen type as the endemic species is carried out in an unmanaged state without administrative control, and even if the above-mentioned DIVA system is introduced afterwards, it is natural. It is difficult to identify infected birds.
On the other hand, utilization of nonstructural proteins of viruses as markers for virus infection diagnosis has been proposed in several diseases [specifically, hepatitis C virus (Inoue et al., J. Gen. Virol 73, 2151-2154 (1992)), hand-foot-and-mouth disease (Neitzert et al., Virology 184, 799-804 (1991)), equine influenza (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 DIVA, the antibody titer must increase in natural infection, and the antibody titer must not increase in vaccination. Conventionally, these studies have not been made on avian influenza.

Therefore, an object of the present invention is to provide an avian influenza virus test method for distinguishing and distinguishing naturally infected birds from vaccinated birds.
As a result of intensive studies on the above problems, the present inventors examined the possibility of both avian influenza virus nucleoprotein (NP) and nonstructural protein NS1 as virus infection identification markers. As a result, NP, which is a structural protein, reacts without distinction between vaccinated and naturally infected birds, whereas NS1, which is a non-structural protein, does not react with vaccinated birds, but only with naturally infected birds. I found it. That is, the present inventors have found that the nonstructural protein NS1 can be used as an identification antigen for the DIVA system, and have 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, one of the amino acid sequences of the avian influenza virus nonstructural protein NS1 having low reactivity to vaccinated serum A method for examining an avian influenza virus infection, wherein only a naturally infected bird is identified using a polypeptide comprising a part as a virus antigen.
2. In a method for examining avian influenza virus infection by immunological reaction by contacting serum obtained from a test bird with a viral antigen,
(A) screening naturally infected bird specimens and vaccinated bird specimens using avian influenza virus structural proteins as viral antigens;
(B) Contact the specimen serum positive in the screening process with the non-structural avian influenza virus non-structural protein NS1 having a low reactivity to the vaccinated serum or a polypeptide comprising a part of its amino acid sequence to identify only naturally infected bird specimens A test method for avian influenza virus infection, comprising a step.
3. 3. The avian influenza virus infection according to 1 or 2 above, wherein the NS1 protein or a polypeptide comprising a part of its amino acid sequence is a recombinant protein or polypeptide obtained by expressing the NS1 gene in a host other than influenza virus. Inspection method.
4). 4. The method for examining avian influenza virus infection according to 3 above, wherein the host is an insect cell.
5. 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). 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 preceding items, wherein the immunological reaction is performed by Western blotting, ELISA, immunoprecipitation, immunoaggregation, or a combination thereof.
8). The inspection method for avian influenza virus infection according to any one of the preceding items, wherein the bird is a poultry.
9. 9. The inspection method for avian influenza virus infection according to 8 above, wherein the poultry are chickens.
Effects of the Invention According to the method of the present invention, infection with avian influenza virus can be determined by a simple test. Furthermore, according to the method of the present invention, it is possible to distinguish between naturally infected birds and vaccinated 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.

The present invention provides a method for examining an avian influenza virus infection for a DIVA system using the nonstructural protein NS1 of an avian influenza virus as an antigen.
Influenza A viruses, including avian influenza virus, are nucleoprotein (NP), RNA, and viral RNA polymerase in a particle having a diameter of 100 to 250 nm surrounded by an envelope (envelope) made of a lipid bilayer derived from the host cell membrane. (PB2, PB1, PA). RNA is an 8-segment type, PB2, PB1, PA (viral RNA polymerase), HA (hemagglutinin spike), NP (nucleoprotein), NA (neuraminidase spike), M (coat and ion channel), NS coding region It is divided into. NS encodes NS1 which is a nonstructural protein and NS2 which is now considered to be a structural protein.
In the present invention, a non-structural protein NS1 or a polypeptide consisting of a part of its amino acid sequence (hereinafter sometimes abbreviated as NS1 polypeptide) is used as a viral antigen. NS1 has the theory that it has the function of promoting translation of M1 protein (protein lining the outer coat) and regulating nuclear export of mRNA for equine influenza, but it is not necessary for replication or expression of viral ribonuclease. (Huang et al., J. Virol. 64, 5669-5673 (1990)), and the details of the function are unknown. In any case, as can be confirmed in the experimental examples described later, NS1 protein is expressed in a live virus infection state, but the content in the vaccine is very small.
In the present invention, NS1 protein may be obtained from a prevalent avian influenza virus or may be obtained from other antigenic avian influenza viruses. NS1 protein is preferably obtained by expressing NS1 gene in a host other than influenza virus.
Isolation of NS1 gene from avian influenza virus can be performed by a conventional method. Typically, cDNA is synthesized by reverse transcription from total RNA of avian influenza virus, and a polynucleotide containing the NS1 coding region is obtained using a primer specific for the eighth segment encoding NS1. Primers specific for the eighth segment are described in, for example, Fuchier et al. , Arch. Virol. 146, 2275-89 (2001).
The expression system may be insect cells, yeast, bacteria such as Escherichia coli and Bacillus, and mammalian cell systems. However, post-translational modifications and three-dimensional structures of expressed proteins are close to the original ones, and a large amount of stable antigens. Insect cells are preferably used.
NS1 gene expression in insect cells can be achieved by inserting NS1 gene into a baculovirus transfer vector and co-transfecting insect cells with baculovirus DNA to obtain a recombinant baculovirus having NS1 gene. A Bac system (Luckow, et al., J. Virol., 67, 4566 (1993)) or the like may be used. By infecting host insect cells with the obtained recombinant baculovirus, recombinant NS1 protein can be obtained in insect cells.
NS1 protein may have the same structure as natural NS1 protein, or may be modified to be suitable for purification or immunological reaction. As an example, by adding a polyhistidine tag (usually H ₆ ) to the C-terminus of NS1 protein, purification can be easily performed without affecting antigenicity. Other fusion proteins (for example, FLAG tag (DYKDDDDK), Myc tag (EQKLISEEDL) etc. added) may be used as long as 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. That is, NS1 protein is immobilized on a membrane, column, substrate, particle, molecule or the like, or placed in a specific region (for example, in a well), suspected to be infected with bird serum, The NS1 protein antibody and NS1 protein are reacted to be identified. The identification may be any of optical, chemical and physical methods.
Preferred antigen-antibody reactions include Western blotting, ELISA, immunoprecipitation, immunoaggregation, and the like.
A competitive ELISA method using a monoclonal antibody against NS1 protein may be used. This is a method of reacting the NS1 antigen affixed to the plate and the test serum, and then adding an NS1-specific monoclonal antibody. If the test serum contains an antibody against NS1 protein, NS1 antigen is covered thereby, and subsequently added NS1-specific monoclonal antibody cannot stick to NS1 protein. When this competitive ELISA method is used, it is possible to detect the serum to be examined from any animal (duck, goose, ostrich, chicken, duck, etc.) using an anti-mouse antibody that recognizes a monoclonal antibody.
Western blotting can be performed by a conventional method. That is, the entire 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 transfer membrane. After blocking, serum is contacted, and a coloring reagent is added to check whether NS1 band is detected. The NS1 protein (27-28 kDa) can be identified by comparison with a control (total protein sample obtained from a host cell that does not introduce the NS1 gene). 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 AcNPV).
Western blotting may be performed by reaction with an NS1 protein standard. As the NS1 protein standard sample, a NS-protein with NS-His (NS1His), which is easily purified, is preferably purified by Ni-NTA affinity chromatography or the like.
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%. A nitrocellulose membrane can be used as the transfer film, but a nylon membrane, a PVDF membrane, or the like may be used. After membrane transfer, a commercially available enzyme-labeled anti-chicken IgG (when the specimen is derived from chicken) is reacted, and the presence or absence of coloring of the NS1 band is examined by adding a chromogenic substrate.
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 (NP), as an antigen may be preceded as a screening step. That is, a test using NP as an antigen distinguishes an infected or vaccinated specimen from an uninfected non-vaccinated specimen, and a positive specimen (infected / vaccinated specimen) uses a nonstructural protein NS1 or NS1 polypeptide as an antigen To identify natural and vaccinated specimens.
The method of the invention can be applied to birds, preferably poultry, especially chickens.

FIG. 1 is a photograph showing the results of Coomassie brilliant blue staining (FIG. 1 (A)) and Western blot (FIG. 1 (B)) showing the expression of the avian influenza virus structural protein NS1 used in the present invention.
FIG. 2 is a photograph showing the results of Coomassie brilliant blue staining (FIG. 2 (A)) and Western blot (FIG. 2 (B)) showing the expression of avian influenza virus nucleoprotein NP that can be used in the present invention.
FIG. 3 is a photograph showing the results of Western blotting in which avian influenza virus-infected chickens and vaccinated chickens were identified by the method of the present invention.

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, as avian influenza virus, isolate # 478 (H1N1) (A / Duck / Aomori / 478/03: H1N1) at Kitasato University School of Veterinary and Animal Science Was used. Moreover, as the avian influenza virus natural infection serum of Example 2, serum collected 6 weeks after AIV (stock name unknown H9N2) infection at the Institute of Animal Health was used. The inactivated virus of Example 2 is a formaldehyde-treated A / Duck / Hokkaido / 9/99: H9N2 distributed from Hokkaido University, and a highly immunized chicken is a complete Freund's inactivated virus. Serum obtained by immunizing chickens three times with an adjuvant.
Example 1 Production of NS1 Protein (1) Cloning and Modification of NS1 Gene Test bird influenza virus (AIV) solution was inoculated into the allantoic cavity of 10-day-old embryonated chicken eggs using a syringe, and 2 at 37 ° C. Cultured for 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 Gene) according to the protocol described in the manual. CDNA was synthesized by reverse transcriptase (Perkin Elmer Japan) using a primer (Uni12: (agcaaaaagcagg (SEQ ID NO: 1)) common to all 3 ′ sequences of 8 segment (segment) RNAs of AIV. 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 NS1 (Foucher et al., Arch. Virol. 146, 2275-89 (2001)):
5'-tattcgtctcagggaggcaaaaggcagggt-3 '(SEQ ID NO: 2);
5'-atatcgtctgtgttagtagagaaaagagggtgtttt-3 '(SEQ ID NO: 3)
Using the above cDNA as a template, PCR was performed to amplify the eighth segment cDNA. The 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 fragment amplified by PCR was cloned into the cloning vector pCR2.1 using the TA cloning kit. After sequencing (SEQ ID NO: 4), a primer (SEQ ID NO: 5: cggatcCATAATGGATTCCAACACTG, SEQ ID NO: 6: gagaacaattgagtcagaagttCATCATCATCATCATCATtgagcccc) was designed from the Nth plasmid, and the first clone was designed from the Nth plasmid. I let you. 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) is a primer designed by modifying the 3 ′ end of the NS1 gene and adding His-Tag consisting of 6 histidines to the carboxy terminus of NS1. .
(2) Preparation of NS1 expressing baculovirus After PCR, the amplified gene was cloned and inserted into baculovirus transfer vector pAcYM1 (Virology Institute, Oxford, UK: NERC Institute of Virology, Oxford, UK). The resulting plasmid was transfected into insect cells Sf21 (Virology Laboratories) together with baculovirus DNA (AcRP23-lacZ). The experimental procedure was in accordance with the protocol of the baculovirus expression experiment manual (A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures: Summers & Smith, Texas A & M). Recombinant baculovirus AcAINS1His was obtained by homologous recombination.
(3) Expression of NS1 protein A transfer vector having the NS1 gene was transfected into insect cells Sf21 using lipofectin (Gibco BRL) together with DNA of baculovirus AcRP23-lacZ. Four days later, the culture supernatant was collected, and the virus was cloned by the plaque method. The parent strain AcRP23-lacZ forms a blue plaque in the presence of X-gal, but forms a white plaque in the recombinant baculovirus. Plaque cloning was repeated twice to obtain recombinant virus AcAINS1His.
In order to confirm the expression of NS1, AcAINS1His was infected to Sf21 cells, and after SDS-PAGE, stained with Coomassie brilliant blue, or transferred to a nitrocellulose membrane, and an anti-polyhistidine antibody (SIGMA) was used to detect the histidine-tagged protein. 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 A recombinant baculovirus for NP protein expression was produced in the same manner as NS1. In order to clone the fifth segment encoding the NP protein gene, the template was # 478 cDNA and the primer was used by Fuchier et al. , Arch. Virol. 146, 2275-89 (2001): TATTCGTCTCAGGGGagcaaaaagcaggGTA (SEQ ID NO: 7) and ATATCGTCTCGTATTagagaagaagGTATTTTTT (SEQ ID NO: 8) were used. After sequencing, gaggatcccatcatgcgtccccagaggcac (SEQ ID NO: 9) and gaggacccctaattgtcatactcctctgc (SEQ ID NO: 10) were used to amplify only the NP protein gene.
The obtained NP gene was put into a baculovirus like NS1 to obtain a recombinant baculovirus AcAINP. AcAINP was infected into insect cells to obtain NP protein.
Example 2: Identification of avian influenza virus infection using NS1 protein The recombinant virus AcAINS1His obtained in Example 1 was inoculated into Sf21 cells, and the cells were collected after 3-4 days in culture. Cells (about 10 ⁶ cells) were washed with phosphate buffered saline (PBS), resuspended in 80 μl of Laemmli electrophoresis buffer and heated in 100 ° C. boiling water for 5 minutes.
The obtained cell lysate was subjected to 12% SDS-acrylamide gel slab electrophoresis (100 V for 1 hour) to develop the total protein. After development, the protein was transferred to a nitrocellulose membrane. The protein non-adsorbed portion was blocked with a blocking solution (skimmed milk-10 g, Tween 20-0.2 ml, PBS-200 ml). As a control, Sf21 cells were infected with the parent strain AcRP23-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, applied to the protein-transferred nitrocellulose membrane, and allowed to react at room temperature (about 20 ° C.) for 1 hour. It was. Next, after washing the membrane with a blocking solution, a secondary antibody (enzyme-labeled anti-chicken IgG (Cappel)) is appropriately diluted and reacted with the blocking solution. Further, after washing the membrane with the blocking solution, the chromogenic substrate (Bm blue POD) is obtained. Substrate (Boehringer Mannheim)) was added to see if the NS1His band was recognized.
Avian influenza-infected chicken sera developed NS1His band (approximately 27 kDa) until 50-fold dilution, whereas inactivated virus hyperimmune chicken sera did not develop NS1His band at any dilution ratio . In addition, AcRP23-lacZ-infected cells were not colored at all and were confirmed to have no antigenicity.
Example 3: Discrimination of avian influenza virus infection using NS1 protein The experiment was conducted in the same manner as in Example except that the dilution factor was 50 to 1250 times. The results are shown in FIG. In the avian influenza (AIV) -infected serum, a clear NS1His band color is observed at a dilution factor of 50 (enlarged portion in the figure).
Comparative Example 2: Identification of avian influenza virus infection using NP protein The recombinant virus AcAINP obtained in Reference Example 1 was inoculated into Sf21 cells, and the cells were collected after 3-4 days in culture. Cells (approx. 10 ⁶ cells) are resuspended in 80 μl of Laemmli's electrophoresis buffer and, like NS1His protein, after SDS-PAGE, stained with Coomassie Brilliant Blue, or transferred to a nitrocellulose membrane and expressed by Western blotting. confirmed.
As a result, NP band (56 kDa) color was observed in the avian influenza-infected chicken serum and the inactivated virus hyperimmune chicken serum at any dilution rate. No color was observed in the control (non-infected / non-immunized specimen) serum. As described above, the immunological reaction by the NP antigen cannot distinguish between naturally infected chickens and virus-inoculated chickens, but has high sensitivity for discrimination from non-infected chickens and non-immunized chickens. Therefore, it is useful as a step of screening naturally infected chicken specimens and virus-inoculated chicken specimens before identification by NS1. It is also effective in an ELISA reaction.

  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 has high utility as an inspection method for carrying out a wide range of DIVA systems in which international compliance is an urgent issue.

Claims (6)

In a method for examining avian influenza virus infection by immunological reaction by contacting serum obtained from a test bird with a viral antigen,
(A) The structural protein NP of avian influenza virus is a recombinant protein obtained by expressing the NP gene in Sf21 cells, and screening for naturally infected bird specimens and vaccinated bird specimens using the protein as a viral antigen And a process of
(B) A recombinant protein or polypeptide obtained by expressing a NS1 gene in Sf21 cells, which is a non-structural protein NS1 having a low reactivity with vaccinated serum or a part of its amino acid sequence. A bird comprising the step of contacting a sample serum positive in the screening step with a polypeptide comprising a part of the NS1 protein or its amino acid sequence to identify only a naturally infected bird sample. Inspection method of influenza virus infection.   The polypeptide consisting of NS1 protein or a part of its amino acid sequence is a recombinant protein or polypeptide obtained by transfecting a transfer vector having NS1 gene together with baculovirus DNA into Sf21. Inspection method for avian influenza virus infection.   The method for examining avian influenza virus infection according to claim 2, wherein the transfer vector is pAcYM1 and the baculovirus is AcRP23-lacZ.   The method for examining avian influenza virus infection according to any one of claims 1 to 3, wherein the immunological reaction is performed by Western blotting, ELISA, immunoprecipitation, immunoaggregation, or a combination thereof.   A bird is poultry, The inspection method of the avian influenza virus infection of any one of Claims 1-4.   The inspection method for avian influenza virus infection according to claim 5, wherein the poultry are chickens.

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