A pigeon Newcastle disease virus strain, hemagglutination inhibition test antigen, and preparation method and application thereof

Abstract

The present invention belongs to the field of veterinary biotechnology detection technology, and specifically relates to a virus strain of pigeon Newcastle disease virus, a hemagglutination inhibition test antigen, and a preparation method and application thereof. The present invention provides an LD strain of pigeon Newcastle disease virus, and the deposit number of the LD strain is CCTCC NO: V202468. The pigeon Newcastle disease virus LD strain of the present invention is a weak strain with the characteristics of low pathogenicity, good immunogenicity and good safety, which is more in line with the domestic market and production needs. The present invention provides a method for preparing a pigeon Newcastle disease virus hemagglutination inhibition test antigen. The present invention adopts the pigeon Newcastle disease virus LD strain to prepare the pigeon Newcastle disease virus hemagglutination inhibition test antigen. The obtained antigen has the characteristics of high specificity, high stability and high sensitivity, and can be used for rapid detection of clinical serum samples within 1 hour, thereby improving the detection and prevention and control level of pigeon Newcastle disease.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary biotechnology detection, and particularly relates to a virus strain of pigeon Newcastle disease virus, a hemagglutination inhibition test antigen, and a preparation method and application thereof. Background Art

[0002] Pigeon Newcastle disease (ND) is a highly contagious and lethal disease caused by the pigeon-derived ND virus (PNDV). NDV is also known as pigeon paramyxovirus type I (PPMV-I). ND is characterized by enteritis, severe diarrhea, and neurological symptoms, similar to the neurological form of NDV in chickens. It is a major threat to the pigeon industry. NDV carries high morbidity and mortality rates. Currently, there is no approved vaccine for NDV in China. NDV vaccines are often used for pigeon immunization. Currently, the majority of NDV viruses belong to genotype VI, which is genetically distant from the traditional NDV vaccine strains, LaSota and B1. This makes them ineffective against prevalent strains, leading to frequent NDV outbreaks in pigeon farms. Therefore, to effectively prevent and control NDV, the development of a new genotype VI vaccine to prevent NDV infection in pigeons is urgent.

[0003] The hemagglutination inhibition (HI) test is a rapid, sensitive, specific, and widely used serological test method. Newcastle disease (ND) HI antigen is required for ND vaccine development, immune efficacy evaluation, flock serum level monitoring, and serological surveys. However, standard materials such as ND HI antigen are not available on the market. Standard diagnostic antigens for ND are commonly used, but the genetic distance between the two is large, resulting in frequent false negatives in production practice. Therefore, the accuracy of ND HI antigen in ND testing is significantly reduced, hindering the development of ND vaccines and the diagnosis of the disease. Therefore, there is an urgent need for ND HI antigens and the establishment of HI detection methods. Summary of the Invention

[0004] The purpose of the present invention is to overcome the defects of the prior art and to ensure a higher detection rate of pigeon Newcastle disease virus (PPMV-I) and the accuracy of the test results. The present invention provides an LD strain of pigeon Newcastle disease virus, a hemagglutination inhibition test antigen, and a preparation method and application thereof. The LD strain of pigeon Newcastle disease virus can be used to prepare a high-titer pigeon Newcastle disease hemagglutination inhibition test antigen. The pigeon Newcastle disease hemagglutination inhibition test antigen has high specificity and high titer, thereby improving the accuracy of the test results.

[0005] The invention provides a pigeon Newcastle disease virus LD strain, and the deposit number of the pigeon Newcastle disease virus LD strain is CCTCC NO: V202468.

[0006] The present invention provides a method for preparing a pigeon Newcastle disease virus hemagglutination inhibition test antigen, comprising:

[0007] The LD strain described in the above technical solution was inoculated into chicken embryos to obtain the pigeon Newcastle disease virus hemagglutination inhibition test antigen.

[0008] Preferably, including:

[0009] Inoculating the LD strain described in the above technical solution into chicken embryos for incubation to obtain chicken embryos incubated for 60 to 96 hours;

[0010] Collect chicken embryo allantoic fluid with a hemagglutination test titer greater than 7;

[0011] The chicken embryo allantoic fluid is inactivated to obtain the pigeon Newcastle disease virus hemagglutination inhibition test antigen.

[0012] Preferably, the inactivation reagent includes formaldehyde, and the working concentration of the formaldehyde is 0.15% to 0.20%; the inactivation temperature is 37° C., and the inactivation time is 16 to 24 hours.

[0013] Preferably, the virus content in the chicken embryo is 10 5.0 EID 50 / 0.1mL~10 6.0 EID 50 / 0.1mL.

[0014] Preferably, cooling is further included before the inactivation, the cooling temperature is 2 to 8° C., and the cooling time is 12 to 24 hours.

[0015] The present invention provides a pigeon Newcastle disease virus hemagglutination inhibition test antigen obtained by the method described in the above technical solution.

[0016] The present invention provides the use of the pigeon Newcastle disease virus hemagglutination inhibition test antigen in the above technical solution in the preparation of a hemagglutination inhibition test detection reagent and / or a kit for PPMV-Ⅰ.

[0017] The present invention provides a hemagglutination inhibition test reagent or a hemagglutination inhibition test kit, comprising the PPMV-I hemagglutination inhibition test antigen described in the above technical solution.

[0018] The present invention provides the use of the pigeon Newcastle disease virus hemagglutination inhibition test antigen described in the above technical solution or the hemagglutination inhibition test detection reagent or hemagglutination inhibition test detection kit described in the above technical solution in one or more of the following 1) to 3);

[0019] 1) Screening of Newcastle disease-negative pigeons in the pigeon flock;

[0020] 2) Detection of PPMV-Ⅰ serum antibody titer;

[0021] 3) Evaluate the immune effect of pigeon Newcastle disease vaccine.

[0022] The present invention has the beneficial effects of providing a pigeon Newcastle disease virus LD strain, the deposit number of which is CCTCC NO: V202468. The present invention uses the pigeon Newcastle disease virus LD strain to prepare a pigeon Newcastle disease virus hemagglutination inhibition test antigen. The pigeon Newcastle disease virus LD strain of the present invention is a weak strain with low pathogenicity, good immunogenicity, and good safety. The pigeon Newcastle disease virus LD strain has a high sequence homology with domestically prevalent strains, and is more suitable for domestic market and production needs.

[0023] The present invention provides a method for preparing a pigeon Newcastle disease virus hemagglutination inhibition test antigen, comprising: inoculating the LD strain described in the above technical solution into chicken embryos to obtain the pigeon Newcastle disease virus hemagglutination inhibition test antigen. The present invention uses the pigeon Newcastle disease virus LD strain to prepare the pigeon Newcastle disease virus hemagglutination inhibition test antigen, and collects the allantoic fluid of dead chicken embryos and the allantoic fluid of live chicken embryos incubated for 60 to 96 hours, which can increase the titer of the pigeon Newcastle disease virus hemagglutination inhibition test antigen, improve the sensitivity of detection, and can quickly detect clinical serum samples within 1 hour. The pigeon Newcastle disease virus hemagglutination inhibition test antigen (i.e., PPMV-ⅠHI antigen) prepared by the present invention improves the reliability and operability of detecting PPMV-Ⅰ using the HI test method, and can be widely used for the screening of pigeons with negative Newcastle disease, the determination of HI antibodies after vaccine immunization, and on-site diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0025] Figure 1 This is the result of constructing the phylogenetic tree of the PPMV-ⅠLD strain in Example 1 and other currently prevalent strains in China;

[0026] Figure 2 This is a diagram showing the homology analysis results of the PPMV-ⅠLD strain in Example 1 (analysis based on the F gene);

[0027] Figure 3 The results of autopsy on pigeons after challenge with the PPMV-ⅠLD strain in Example 10 are as follows;

[0028] Figure 4 These are the autopsy results of pigeons after challenge with PPMV-ⅠPi / NT / 2011 strain in Example 10.

[0029] Biological deposit certificate

[0030] The pigeon Newcastle disease virus LD strain (PPMV-Ⅰ, LD) was deposited in the China Center for Type Culture Collection on June 28, 2024, with the deposit number CCTCC NO: V202468. The depository address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0031] The invention provides a pigeon Newcastle disease virus LD strain, and the deposit number of the pigeon Newcastle disease virus LD strain is CCTCC NO: V202468.

[0032] In the present invention, the LD strain of the pigeon Newcastle disease virus is genotype VI, which is a weak strain with the advantages of low pathogenicity, good immunogenicity and good safety. The LD strain of the pigeon Newcastle disease virus has a high sequence homology with the domestic prevalent strains and is more in line with the domestic market and production needs.

[0033] The present invention provides a method for preparing a pigeon Newcastle disease virus hemagglutination inhibition test antigen, comprising:

[0034] The LD strain described in the above technical solution is inoculated into chicken embryos for incubation to obtain pigeon Newcastle disease virus hemagglutination inhibition test antigen; more preferably, the method comprises: inoculating the LD strain described in the above technical solution into chicken embryos for incubation to obtain chicken embryos incubated for 60 to 96 hours;

[0035] Collect chicken embryo allantoic fluid with a hemagglutination test titer greater than 7;

[0036] The chicken embryo allantoic fluid is inactivated to obtain the pigeon Newcastle disease virus hemagglutination inhibition test antigen.

[0037] The present invention preferably inoculates the LD strain described in the above technical solution into chicken embryos for incubation to obtain chicken embryos incubated for 60 to 96 hours; the chicken embryos of the present invention preferably include SPF chicken embryos; the chicken embryos are preferably 9 to 12 days old; the virus content in the SPF chicken embryos of the present invention is 10 5.0 EID 50 / 0.1mL~10 6.0 EID 50 / 0.1mL, more preferably 10 5.5 EID 50 / 0.1mL.

[0038] The incubation temperature of the present invention is preferably room temperature, and dead chicken embryos within 60 hours of incubation are preferably discarded; the present invention uses chicken embryos incubated for 60 to 96 hours; the chicken embryos incubated for 60 to 96 hours preferably include live chicken embryos and dead chicken embryos incubated for 60 to 96 hours; after obtaining the live chicken embryos and dead chicken embryos incubated for 60 to 96 hours, the present invention preferably tests the hemagglutination test titer of the allantoic fluid of each live chicken embryo and dead chicken embryo, and selects and collects chicken embryo allantoic fluid with a hemagglutination test titer greater than 7. The present invention collects chicken embryo allantoic fluid with a high hemagglutination test titer, which can increase the hemagglutination test titer of the pigeon Newcastle disease virus hemagglutination inhibition test antigen, and can ensure the accuracy of the test results.

[0039] A chicken embryo allantoic fluid with a hemagglutination test titer greater than 7 is obtained. The present invention preferably cools the chicken embryo allantoic fluid and then inactivates it. The resulting inactivated chicken embryo allantoic fluid is the antigen for the pigeon Newcastle disease virus hemagglutination inhibition test. The chicken embryo cooling temperature of the present invention is preferably 2-8°C, more preferably 4°C; the cooling time is preferably 12-24 hours, more preferably 12-16 hours, and more preferably 12 hours. Cooling can freeze the chicken embryo to death, causing the blood to coagulate, preventing the red blood cells from flowing out and mixing with the allantoic fluid during harvest. When mixed with the allantoic fluid, the red blood cells will agglutinate with the virus in the allantoic fluid, causing a decrease in viral titer. The reagent used in the inactivation of the present invention preferably includes formaldehyde, and the working concentration of the formaldehyde is preferably 0.15%-0.20%, more preferably 0.18%-0.20%; the inactivation temperature is preferably 37°C; and the inactivation time is preferably 16-24 hours, more preferably 16-20 hours. When using formaldehyde for inactivation, if the formaldehyde concentration is too low, the virus will be inactivated incompletely, posing a risk of spreading the virus; if the formaldehyde concentration is too high, it will easily lead to high formaldehyde residual levels and increase costs. During formaldehyde inactivation, the inactivation time must also be controlled. If the inactivation time is too long, the virus is easily over-processed, while if the inactivation time is too short, the inactivation will be incomplete. The selection of formaldehyde concentration and inactivation time of the present invention can ensure that pigeon Newcastle disease virus is completely inactivated with low formaldehyde residual levels.

[0040] The present invention provides a method for preparing a pigeon Newcastle disease virus hemagglutination inhibition test antigen (i.e., PPMV-I hemagglutination inhibition test antigen, abbreviated as HI antigen). The method collects chicken embryo allantoic fluid with a hemagglutination test titer greater than 7. The method uses the LD strain of the pigeon Newcastle disease virus, sets an incubation time and incubation temperature, and sets cooling and inactivation parameters, all of which are aimed at improving the titer of the prepared PPMV-I hemagglutination inhibition test antigen.

[0041] The present invention provides a pigeon Newcastle disease virus hemagglutination inhibition test antigen prepared by the method described in the above technical solution. The pigeon Newcastle disease virus hemagglutination inhibition test antigen is recorded as PPMV-ⅠLD strain HI antigen. The PPMV-ⅠLD strain HI antigen described in the present invention can be used to detect PPMV-Ⅰ (i.e., pigeon Newcastle disease virus) without cross-reaction to other viruses. It is convenient and efficient, highly operable, and has high specificity and sensitivity. The HI antigen prepared by the present invention is stored at -20°C and maintained for at least 12 months, indicating that the antigen has good stability; the HI antigen shows a negative reaction to SPF chicken serum, avian influenza virus (H5, H7, H9 subtypes) positive serum, EDS positive serum, and IBV positive serum, indicating that the HI antigen has good specificity.

[0042] The present invention provides the use of the pigeon Newcastle disease virus hemagglutination inhibition test antigen described in the above technical solution in the preparation of a hemagglutination inhibition test reagent and / or kit for PPMV-I. In the present invention, the hemagglutination unit of the pigeon Newcastle disease virus hemagglutination inhibition test antigen used in the detection of PPMV-I serum antibody titer is preferably 4 HAU. The use of the hemagglutination inhibition test detection reagent and / or kit of the present invention preferably includes the following steps: taking the serum to be tested and placing it in a test plate for dilution to obtain diluted serum to be tested; mixing the diluted serum to be tested with HI antigen and conducting an antigen-antibody binding reaction to obtain a reacted sample; mixing the reacted sample with a chicken red blood cell suspension and observing the agglutination of the chicken red blood cells. The present invention preferably performs the antigen-antibody binding reaction after mixing the diluted serum to be tested with HI antigen. The temperature of the antigen-antibody binding reaction is preferably room temperature, and the time of the antigen-antibody binding reaction is preferably 35 minutes. The present invention preferably performs the antigen-antibody binding reaction after mixing the sample with a chicken red blood cell suspension and observing the agglutination of the chicken red blood cells. The concentration of the chicken red blood cell suspension is preferably 0.8%.

[0043] The present invention provides a hemagglutination inhibition test reagent or a hemagglutination inhibition test kit, comprising the PPMV-I hemagglutination inhibition test antigen described in the above technical solution.

[0044] The present invention has no particular limitation on the structure of the hemagglutination inhibition test kit, and any conventional structure may be used.

[0045] The present invention provides the use of the pigeon Newcastle disease virus hemagglutination inhibition test antigen described in the above technical solution or the hemagglutination inhibition test detection reagent or hemagglutination inhibition test detection kit described in the above technical solution in screening Newcastle disease negative pigeons in a pigeon flock.

[0046] The present invention provides the use of the Newcastle disease virus hemagglutination inhibition test antigen described in the above technical solution, or the hemagglutination inhibition test detection reagent or hemagglutination inhibition test kit described in the above technical solution, for detecting PPMV-I serum antibody titers. In the present invention, the hemagglutination unit of the Newcastle disease virus hemagglutination inhibition test antigen used in detecting PPMV-I serum antibody titers is preferably 4 HAU. The use of the present invention for detecting PPMV-I serum antibody titers preferably includes the following steps: taking the serum to be tested and placing it in a test plate for dilution to obtain diluted serum to be tested; mixing the diluted serum to be tested with HI antigen and performing an antigen-antibody binding reaction to obtain a reacted sample; and mixing the reacted sample with a chicken red blood cell suspension and observing the agglutination of the chicken red blood cells. The present invention preferably performs the antigen-antibody binding reaction after mixing the diluted serum to be tested with HI antigen, the reaction temperature is preferably room temperature, and the reaction time is preferably 35 minutes. The present invention preferably performs the agglutination of the chicken red blood cells after mixing the reacted sample with a chicken red blood cell suspension, and the concentration of the chicken red blood cell suspension is preferably 0.8%.

[0047] The present invention provides the use of the hemagglutination inhibition test antigen of pigeon Newcastle disease virus described in the above technical solution or the hemagglutination inhibition test detection reagent or hemagglutination inhibition test detection kit described in the above technical solution in evaluating the immune effect of pigeon Newcastle disease vaccine. When the HI antibody titer of the immunized pigeon is not less than 1:32, it means that the immunization is successful and can resist the attack of the strong toxic pigeon Newcastle virus. The higher the HI antibody titer, the better the immune effect. When the hemagglutination inhibition test detection reagent and / or kit of the present invention is used to evaluate the immune effect of the pigeon Newcastle disease vaccine, if the immunized pigeon group can react positively with the prepared HI antigen, it means that the pigeon Newcastle disease vaccine can produce antibodies after immunization.

[0048] The HI titer of the positive serum of the La Sota strain of Newcastle disease virus of chicken detected by the HI antigen of the present invention is referred to as titer 1, and the HI titer of the positive serum of the PPMV-ⅠLD strain of pigeon Newcastle disease virus of pigeon detected by the HI antigen is referred to as titer 2. Titer 1 is 1 to 2 titers lower than titer 2, thereby distinguishing between chicken Newcastle disease virus and pigeon Newcastle disease virus.

[0049] The present invention prepares a pigeon Newcastle disease hemagglutination inhibition test antigen and establishes an HI detection method. The prepared PPMV-Ⅰ HI antigen and the established HI method are mainly used for diagnosing PPMV-Ⅰ and monitoring antibody levels, have the characteristics of high specificity, high sensitivity and low cost, can quickly detect serum samples within 1 hour, overcomes the disadvantage of traditional detection methods that are time-consuming, is easy to promote at the grassroots level, and provides a reliable material basis and technical means for early rapid diagnosis of PPMV-Ⅰ, molecular epidemiological research and pigeon Newcastle disease vaccine development.

[0050] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] The biological materials used in the following examples are as follows: avian influenza virus (H5, H7, H9) positive serum was purchased from Harbin Weike Biotechnology Co., Ltd.; Newcastle disease virus La Sota strain positive serum, egg drop syndrome positive serum, chicken infectious bronchitis positive serum, pigeon Newcastle disease positive serum and negative serum, etc., were all provided by the China Veterinary Drug Administration.

[0052] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0053] Example 1 Preparation of PPMV-ⅠLD strain virus solution

[0054] Propagation of PPMV-ⅠLD strain: The allantoic fluid of PPMV-ⅠLD strain stored at -80℃ was cultured through the allantoic cavity according to the 10 5.5 EID 50 / 0.1mL is inoculated into 9-11 day old SPF chicken embryos with the PPMV-ⅠLD strain, with a total inoculation of 0.2mL per chicken embryo. The chicken embryos are then placed in a 37°C incubator for incubation and observed every 12 hours. Chicken embryos that die within 60 hours of incubation are discarded; dead embryos (recorded as dead embryos) are removed at any time during the 60-96 hours of incubation time, and all living embryos within 96 hours of incubation time (recorded as living embryos) are removed, and the dead and living embryos are cooled at 4°C for 12 hours. The allantoic fluid of each chicken embryo is collected under sterile conditions, the HA titer of each chicken embryo allantoic fluid is determined, and the chicken embryo allantoic fluid with an HA titer greater than 7 is collected; the chicken embryo allantoic fluid can be used immediately or stored in a -80°C refrigerator for later use.

[0055] Example 2

[0056] Preparation of LD strain hemagglutination inhibition antigen (i.e., PPMV-Ⅰ LD strain hemagglutination inhibition test antigen): The chicken embryo allantoic fluid with an HA titer greater than 7 prepared in Example 1 was inactivated to obtain the pigeon Newcastle disease virus hemagglutination inhibition test antigen.

[0057] Specifically, 10% formaldehyde was added to the allantoic fluid of PPMV-ⅠLD chicken embryos, resulting in a formaldehyde concentration of 0.2%. The mixture was thoroughly mixed and incubated at 37°C with constant shaking. Samples were taken at 12, 16, 20, and 24 hours of shaking and inoculated into SPF chicken embryos to test for inactivation efficacy and HA titer. The results showed that the best inactivation efficacy was achieved within 16 to 24 hours. The results of the inactivation efficacy and HA titer determinations are shown in Table 1.

[0058] Table 1 Inactivation effect of HI antigen and HA titer of PPMV-ⅠLD strain at different inactivation times

[0059] Inactivation time 12h 16h 20h 24h Inactivation effect +---- ----- ----- ----- HA titer <![CDATA[1:2 10 ]]> <![CDATA[1:2 10 ]]> <![CDATA[1:2 10 ]]> <![CDATA[1:2 10 ]]>

[0060] Note: 5 chicken embryos are used for inactivation test. The number of dead chicken embryos is indicated by “+”, and the number of live chicken embryos is indicated by “-”.

[0061] Example 3 Optimization of the Optimal Reaction Temperature and Time for PPMV-ⅠLD Strain HI Antigen HA Test Preparation of Pigeon Newcastle Disease Virus Hemagglutination Inhibition Test Antigen:

[0062] The pigeon Newcastle disease virus LD strain was inoculated into SPF chicken embryos for incubation. The initial virus content of SPF chicken embryos was 10 5.5 EID50 / 0.1mL; all SPF chicken embryos incubated for 60 to 96 hours were retained, the incubated SPF chicken embryos were cooled, and the chicken embryo allantoic fluid was collected;

[0063] The chicken embryo allantoic fluid with an HA titer greater than 7 is mixed and inactivated. The inactivation step is: add 10% formaldehyde to the PPMV-ⅠLD strain chicken embryo allantoic fluid until the final formaldehyde concentration in the chicken embryo allantoic fluid is 0.2%, mix thoroughly, and place at 37°C for constant temperature oscillation for 16 hours. The inactivated chicken embryo allantoic fluid obtained after inactivation is the pigeon Newcastle disease virus hemagglutination inhibition test antigen (i.e., PPMV-ⅠLD strain HI antigen, referred to as HI antigen) prepared in this experiment.

[0064] The optimal reaction temperature and time for the HI antigen HA test of PPMV-ⅠLD strain were optimized as follows: 8 treatment groups were divided into the following groups:

[0065] Process 1

[0066] ①In a 96-well microtiter plate, add 25 μL of PBS buffer to each well 1 to 12.

[0067] ② Add 25 μL of the prepared HI antigen to the first well on the left, mix well, pipette 25 μL to the second well, dilute serially to the 12th well, mix well, discard 25 μL from the 12th well, and retain 25 μL.

[0068] ③ Add 25 μL of 1% chicken red blood cell suspension to each well from right to left, and set up a red blood cell control well.

[0069] ④ The blood coagulation plate was shaken and mixed on a micro-oscillator, and then placed at 4°C for 30 minutes.

[0070] ⑤ Result determination: Tilt the microplate 45 degrees to observe the agglutination of chicken red blood cells. The highest dilution factor of HI antigen that causes complete agglutination of chicken red blood cells is the HA titer of the HI antigen.

[0071] Treatment 2: Same as treatment 1, except that the cells were placed at 4°C for 45 min in step ④.

[0072] Treatment 3: Same as treatment 1, except that in step ④, the mixture was allowed to stand at room temperature for 15 minutes.

[0073] Treatment 4: Same as treatment 1, except that in step ④, the mixture was allowed to stand at room temperature for 25 minutes.

[0074] Treatment 5: Same as treatment 1, except that in step ④, the mixture was allowed to stand at room temperature for 35 minutes.

[0075] Treatment 6: Same as treatment 1, except that the cells were incubated at 37°C for 15 min in step ④.

[0076] Treatment 7: Same as treatment 1, except that the cells were incubated at 37°C for 20 min in step ④.

[0077] Treatment 8: Same as treatment 1, except that the cells were incubated at 37°C for 35 min in step ④.

[0078] The results of treatments 1 to 8 were observed, and three parallel experiments were set up for each treatment.

[0079] The results showed that the HA titer of HI antigen reached a maximum of 10 at room temperature for 25 min in treatment 4. 11 The titer of HI antigen under other treatment conditions was 10 10 Therefore, the optimal reaction temperature for the HA test was determined to be room temperature, and the reaction time was 25 min.

[0080] Example 4 PPMV-ⅠLD strain HI antigen HA test and optimization of the optimal red blood cell concentration for the HI test

[0081] The PPMV-ⅠLD strain HI antigen is the PPMV-ⅠLD strain HI antigen prepared in Example 3.

[0082] The specific methods for the LD strain HI antigen HA test and the optimal red blood cell concentration for the HI test were optimized as follows:

[0083] ① In a 96-well microtiter plate, add 25 μL of PBS to each well from wells 1 to 12; wells 1 to 12 are considered one row, and a total of 6 rows are added;

[0084] ② In each row, add 25 μL of the prepared HI antigen to the first well on the left side, mix well, pipette 25 μL to the second well, dilute in multiple proportions to the 12th well, and discard 25 μL.

[0085] ③ Add 25 μL of chicken red blood cell suspension to each well in each row from right to left. Among them, add a 0.5% chicken red blood cell suspension to rows 1 and 2, a 0.8% chicken red blood cell suspension to rows 3 and 4, and a 1% chicken red blood cell suspension to rows 5 and 6. Set the 7th row as the red blood cell control well; set up two more 96-well microplates according to steps ① to ③ to repeat the experiment.

[0086] ④ Three 96-well microplates were shaken and mixed on a micro-oscillator, and the three 96-well microplates were placed at room temperature for 25 min to observe the aggregation of chicken red blood cells. According to the results, the aggregation results of chicken red blood cells were more clearly and intuitively observed at a concentration of 0.8%. Therefore, the chicken red blood cell concentration in the hemagglutination test (HA) and the hemagglutination inhibition test (HI) was 0.8%.

[0087] Example 5 Determination of the stability of HI antigen of PPMV-ⅠLD strain

[0088] The PPMV-I LD strain HI antigen was prepared in Example 3 and stored at -20°C. HA titer was determined using the hemagglutination plate storage time and temperature determined in Example 3 (i.e., room temperature, reaction time of 25 minutes) and the 0.8% chicken red blood cell concentration determined in Example 4. The specific HA titer determination method is as follows:

[0089] ①In a 96-well microtiter plate, add 25 μL of PBS to each well from well 1 to well 12.

[0090] ② Add 25 μL of the prepared HI antigen to the first well on the left, mix well, pipette 25 μL to the second well, dilute in multiple proportions to the 12th well, and discard 25 μL.

[0091] ③ Add 25 μL of 0.8% chicken red blood cell suspension to each well from right to left, and set up a red blood cell control well without HI antigen.

[0092] ④ Oscillate and mix on a micro-oscillator, place the blood coagulation plates at room temperature for 25 minutes, and observe the results.

[0093] ⑤ Result determination: Tilt the microplate 45 degrees to observe the agglutination of chicken red blood cells. The highest dilution factor of HI antigen that causes complete agglutination of chicken red blood cells is the HA titer of the HI antigen.

[0094] Three parallel experiments were conducted, with the HA titer of the HI antigen measured every two months. The HA titer test results of the HI antigen stored at -20°C are shown in Table 2. It can be seen that the HA titer of the HI antigen was stable for 12 consecutive months and did not decrease. The shelf life of the antigen is at least 12 months. This shows that the HI antigen obtained by the present invention has good stability.

[0095] Table 2 HI antigen stability period record

[0096] Save time 2 months 4 months 6 months 8 months 10 months 12 months HA titer <![CDATA[1:2 11 ]]> <![CDATA[1:2 11 ]]> <![CDATA[1:2 11 ]]> <![CDATA[1:2 11 ]]> <![CDATA[1:2 11 ]]> <![CDATA[1:2 11 ]]>

[0097] Example 6 Optimization of the optimal binding temperature and time for antigen and antibody in PPMV-ⅠLD strain HI test

[0098] The HI antigen of the PPMV-I LD strain was prepared in the same manner as in Example 3. The HA titer of the HI antigen was determined using the hemagglutination plate incubation time and temperature determined in Example 3 and the chicken red blood cell concentration conditions determined in Example 4. PBS was used to prepare 4 HA units of HI antigen. For example, if the HA antigen agglutination titer was 1:1024, then 4 HA units would be 1:256. The antigen was diluted 256-fold with PBS to produce a 4 HA unit HI working antigen solution.

[0099] Validation of 4HAU hemagglutinin, the highest dilution of antigen represents 1 hemagglutination unit (HAU):

[0100] To check for the correct hemagglutination titer of 4 HAU, add 1 mL of the prepared HI antigen solution containing 4 HA units to 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL of PBS, respectively, for final dilutions of 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7. Then, take 25 μL of each dilution and add 25 μL of a 0.8% chicken red blood cell suspension, followed by 25 μL of PBS and mix thoroughly. The resulting hemagglutination plate should then be allowed to stand at room temperature for 25 minutes. If the prepared hemagglutinin dilution is 4 HAU, a 1:4 dilution will produce a complete agglutination endpoint. If the 4 HAU is higher than 4 HA units, the endpoint may be 1:5 or 1:6; if lower, the endpoint may be 1:2 or 1:3. The hemagglutinin dilution should be adjusted appropriately based on the test results to ensure that the HI antigen working solution is indeed 4 HAU.

[0101] PPMV-Ⅰ-HI test operation:

[0102] ① Take a microplate and add 25 μL PBS to wells 1 to 11, and add 50 μL PBS to well 12.

[0103] ② Pipette 25 μL of the serum to be tested, add it to the first well, mix thoroughly, then pipette 25 μL to the second well, dilute it 2-fold to the tenth well in sequence, and draw 25 μL from the tenth well and discard it. At the same time, set up a pigeon Newcastle disease virus positive serum control and a pigeon negative serum control on each plate, and also make a 2-fold serial dilution with reference to the serum to be tested. The LD strain of the present invention belongs to genotype VI, and other isolated pigeon Newcastle disease viruses are also type VI. Pigeon Newcastle disease virus positive serum prepared by any method can be used. The serum HI titer of the pigeon negative serum control is not higher than 1:4.

[0104] ③ Add 25 μL of HI antigen containing 4 HA units to wells 1 to 11 and shake on a microplate for 30 seconds. Then, incubate the HI antigen-antibody hemagglutination plate under different conditions: 4°C for 30 minutes; 4°C for 45 minutes; room temperature for 30 minutes; room temperature for 45 minutes; 37°C for 30 minutes; and 37°C for 45 minutes.

[0105] ④ Add 25 μL of 0.8% chicken red blood cell suspension to each well from right to left, mix gently, and let stand at room temperature for 25 minutes. The control red blood cells will appear as distinct buttons.

[0106] 5. Determination of results: Tilt the microplate at 45 degrees and read within 3 minutes. The test is considered valid when the negative control serum HI titer is no higher than 1:4 and the positive control serum titer is within 1 titer of the known result. The highest dilution of the tested serum that inhibits 100% agglutination is the agglutination inhibition titer, which is the HI antibody titer.

[0107] The results showed that the titer of HI antibody for the same positive serum was 1:2 at different reaction temperatures and times. 10 , the effect is not significant. The HI titer of serum samples obtained under other conditions when the antigen and antibody are reacted for 30 minutes at room temperature is consistent. Reacting for 30 minutes at room temperature can save time and is easier to operate. Therefore, reacting for 30 minutes at room temperature is selected as the optimal antigen-antibody reaction condition.

[0108] Example 7 Detection of HI Antigen Specificity of PPMV-ⅠLD Strain

[0109] (1) Utilizing the HI antigen prepared in Example 3;

[0110] (2) PPMV-Ⅰ-HI test operation steps:

[0111] ① Take a microplate and add 25 μL PBS to wells 1 to 11, and add 50 μL PBS to well 12.

[0112] ② Pipette 25 μL of the serum to be tested into well 1. After thorough mixing, pipette 25 μL into well 2. Dilute the serum 2-fold to well 10. Pipette 25 μL from well 10 and discard. Simultaneously, set up a pigeon Newcastle disease virus-positive serum control and a pigeon negative serum control on each plate and perform the same 2-fold serial dilutions.

[0113] ③ Add 25 μL of HI antigen containing 4 HA units to wells 1 to 11 respectively, shake on a micro-oscillator for 30 seconds, and let it stand at 37°C for 30 minutes.

[0114] ④ Add 25 μL of 0.8% chicken red blood cell suspension to each well from right to left, mix gently, and let stand at room temperature for 25 minutes. The control red blood cells will appear prominently button-shaped.

[0115] 5. Result determination: Tilt the microplate 45 degrees and complete the reading within 3 minutes. The test is considered valid when the negative control serum HI titer is no higher than 1:4 and the positive control serum does not differ from the known result by more than 1 titer. The highest dilution of the test serum that results in 100% inhibition of agglutination is the agglutination inhibition titer, which is also known as the HI antibody titer (HI antibody titer is referred to as HI titer).

[0116] The steps of step (2) are used to detect common hemagglutinating poultry pathogens: specifically, positive sera of H5, H7 and H9 subtype avian influenza viruses, positive sera of NDV La Sota strain, positive sera of chicken infectious bronchitis virus IBV (type M41) and egg drop syndrome (EDS) virus.

[0117] The results are shown in Table 3. Except for the positive serum of PPMV-ⅠLD strain and NDV La Sota strain, the HI antigen showed negative reaction with other viruses, indicating that the prepared HI antigen had no cross reaction with other hemagglutinating viruses (H5, H7 and H9 subtype avian influenza viruses, IBV, EDS), indicating that the method has good specificity.

[0118] Table 3 HI antigen and other poultry disease pathogens HI reaction results

[0119]

[0120]

[0121] Note: The symbols in Table 3 represent pigeon Newcastle disease virus LD strain, avian influenza virus AIV-(H5, H7 and H9 positive serum), Newcastle disease virus NDV-LaSota strain positive serum, chicken egg drop syndrome virus positive serum EDS-76, negative serum, chicken infectious bronchitis virus positive serum IBVM41 strain.

[0122] Example 8 Detection of HI Antibodies in Serum Samples after Inactivated Vaccine Immunization

[0123] Two groups of pigeons, one group of 10 pigeons as the immunized group and one group of 5 pigeons as the blank control group, i.e., the non-immunized group. After the immunized group was immunized with an inactivated Newcastle disease vaccine, sera from the blank and immunized pigeons were collected for HI antigen specificity detection. The HI antigen specificity detection was performed according to the PPMV-Ⅰ-HI test procedure of step (2) of Example 7. The results are shown in Table 4. It can be seen that the immunized pigeon group can react positively with the HI antigen obtained in Example 3, while the HI antigen obtained in Example 3 reacts negatively with the non-immunized pigeon group. This indicates that the detection method established by the present invention can be used to evaluate the immune effect of the vaccine, and the immune efficacy of the vaccine can be evaluated based on the level of HI antibody titer. This operation method is simple, accurate, and easy to determine.

[0124] When the HI antibody titer of the immunized pigeons is not less than 1:32, it means that the immunization is successful and the pigeons can resist the attack of the strong Newcastle virus. The higher the HI antibody titer, the better the immunization effect.

[0125] Table 4HI antigen detection of pigeon serum after inactivated vaccine immunization

[0126]

[0127] Example 9 Detection of 23 clinical serum samples using LD strain HI antigen

[0128] Twenty-three clinical serum samples from pigeons were collected from three pigeon farms in Guangdong Province and tested for HI antibody titers using the antigen prepared in Example 3. HI antigen specificity testing was performed according to the PPMV-I-HI test procedure in step (2) of Example 7. The results are shown in Table 5. Four sera tested positive for Newcastle disease antibodies. These four sera were collected from the same pigeon farm, indicating that the farm had been or was currently infected with Newcastle disease virus. No Newcastle disease antibodies were detected in the remaining 19 sera.

[0129] Reference national standard GB / T 16550-2020 "Newcastle Disease Diagnostic Technology" states: A serum HI titer greater than 1:16 can be considered antibody positive, and a serum HI titer less than 1:8 can be considered antibody negative. However, in vaccine testing, it is stricter than the national standard. A serum HI titer less than 1:4 is considered antibody negative, and a serum HI titer greater than 1:16 is considered antibody positive. However, a serum HI titer greater than 1:32 indicates a good immune effect.

[0130] Table 5 PPMV-ⅠHI antigen detection clinical pigeon serum samples

[0131] Serum number Serum HI antibody titer 1 1:256 2 ＜1:2 3 ＜1:2 4 ＜1:2 5 ＜1:2 6 ＜1:2 7 ＜1:2 8 ＜1:2 9 ＜1:2 10 ＜1:2 11 ＜1:2 12 ＜1:2 13 1:128 14 ＜1:2 15 ＜1:2 16 ＜1:2 17 ＜1:2 18 ＜1:2 19 ＜1:2 20 ＜1:2 21 ＜1:2 22 1:128 23 1:32

[0132] Example 10 Optimization of PPMV-I LD strain for preparation of HI antigen

[0133] 1. Typing of strains used to prepare HI antigens

[0134] Based on the F gene segment sequence, the PPMV-ⅠLD strain was combined with 16 representative Newcastle disease virus strains downloaded from GenBank to construct a phylogenetic tree. Figure 1 The results of homology analysis are shown in Figure 2 The 16 representative Newcastle disease virus strains were divided into seven different genotypes, with the LD strain belonging to genotype VI. It had the highest homology with the CH-A796 strain (GenBank accession number AY028995.1) and the jiangxi072009 strain (GenBank accession number KJ450977.1), at 91.1% and 90.3%, respectively.

[0135] The nucleotide sequence of the F gene is shown in SEQ ID NO.1:

[0136]

[0137] 2. Safety and immunogenicity studies of PPMV-ⅠLD strain

[0138] Thirty non-immune pigeons aged 32 days were divided into three groups, with 10 pigeons in each group. The first group was injected intramuscularly with 0.2 ml of PPMV-ⅠLD strain (containing 10 6.0 EID 50 ), the second group was injected intramuscularly with 0.2 ml of Newcastle disease virus Pi / NT / 2011 strain (a gift from a certain enterprise, containing 10 6.0 EID 50 The third group received an intramuscular injection of 0.2 ml of sterile saline. Each chicken was marked with a footmark. The chickens were observed for 14 days, with daily records of morbidity and mortality. After the observation period, antibody levels were measured, and the pathogenicity of the LD strain was comprehensively evaluated based on clinical symptoms and postmortem changes.

[0139] The PPMV-ⅠLD strain challenge group showed no obvious clinical symptoms after infection. All chickens were alive and well during the observation period. Five pigeons were selected for autopsy, and no abnormalities were found. Figure 3 , Figure 3 The numbers in the figure represent the pigeon's foot number. In the Pi / NT / 2011 strain challenge group, 100% of the pigeons showed clinical symptoms (ruffled feathers, lethargy, loss of appetite, and yellow-green loose stools) after infection. All 5 pigeons died. The autopsy results of the 5 dead pigeons are shown in Figure 4 , Figure 4 The numbers in the figure represent the pigeon's foot numbers. Gastric and intestinal mucosal bleeding occurred. Antibody testing showed that the LD strain had higher antibody levels than the Pi / NT / 2011 strain after 14 days. Overall, the LD strain is a weak strain with low pathogenicity and produces high antibody levels. High antibody levels indicate good immunogenicity and safety. Therefore, the present invention selected the PPMV-I LD strain for the preparation of hemagglutination inhibition test antigens.

[0140] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A pigeon Newcastle disease virus LD strain, the deposit number of which is CCTCC NO: V202468.

2. A method for preparing pigeon Newcastle disease virus hemagglutination inhibition test antigen, characterized in that: include: inoculating the LD strain according to claim 1 into chicken embryos for incubation to obtain chicken embryos incubated for 60 to 96 hours; The chicken embryo allantoic fluid with a hemagglutination test titer greater than 7 is collected; the chicken embryo allantoic fluid is inactivated to obtain a pigeon Newcastle disease virus hemagglutination inhibition test antigen.

3. The method according to claim 2, characterized in that The reagent used for the inactivation includes formaldehyde, and the working concentration of the formaldehyde is 0.15% to 0.20%; the inactivation temperature is 37° C., and the inactivation time is 16 to 24 hours.

4. The method according to claim 2, characterized in that The virus content in the chicken embryo is 10 5.0 EID 50 / 0.1mL~10 6.0 EID 50 / 0.1mL.

5. The method according to claim 2, characterized in that The inactivation step also includes cooling, wherein the cooling temperature is 2-8° C. and the cooling time is 12-24 hours.

6. Use of the pigeon Newcastle disease virus LD strain according to claim 1 in preparing a pigeon Newcastle disease virus hemagglutination inhibition test antigen.

7. Use of the pigeon Newcastle disease virus LD strain according to claim 1 in the preparation of a hemagglutination inhibition test reagent and / or kit for PPMV-Ⅰ.

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