Infectious laryngotracheitis virus natural attenuated strain with good safety and immunogenicity and application thereof

Abstract

The application discloses a natural weak strain of infectious laryngotracheitis virus with good safety and immunogenicity. The strain is named as infectious laryngotracheitis virus ILTV FJ19, and has been preserved in China Center for Type Culture Collection on November 29, 2013, with a preservation number of CCTCC NO: V2023109. Experiments show that the chicken infectious laryngotracheitis virus ILTV FJ19 strain of the application has stable passage ability, can be adapted to allantoic cavity inoculation, has good safety and immunogenicity when inoculating 14-day-old SPF chickens, and can be immunized by eye dropping or oral administration. The virus can be used as a weak live vaccine candidate strain of ILTV.

Description

TECHNICAL FIELD

[0001] The present application relates to the isolation and identification of a natural attenuated strain of infectious laryngotracheitis virus, and the determination of its immunogenicity and safety as a live vaccine, belonging to the field of microbial technology. The strain was isolated during routine pathogen monitoring of a domestic chicken flock, and was named ILTV FJ19 strain after isolation, identification, purification and cloning. Its safety was determined using 14-day-old SPF chickens, and its immunogenicity was determined by challenging with WF03 strain (patented strain, patent number: ZL202110385416.X) 21 days after immunizing 14-day-old SPF chickens. The determination results show that the infectious laryngotracheitis virus ILTV FJ19 strain in the present application has good safety and immunogenicity, and can be used as a candidate strain for ILTV attenuated live vaccine. BACKGROUND

[0002] Infectious laryngotracheitis virus (ILTV) belongs to the Herpesviridae family, the alphaherpesvirinae subfamily, and the laryngotracheitis virus genus, and mainly causes death and / or egg production decline in chickens. Severe infection often manifests as respiratory distress, panting, and coughing with blood-like fluid, and has a high mortality rate. Mild infection mainly manifests as mucous tracheitis, rhinitis, conjunctivitis, unwillingness to move, and low mortality, etc. Both severe infection and mild infection pose a serious threat and loss to production.

[0003] The genome of ILTV is about 155Kbp in size, and is a double-stranded DNA virus. Like other herpesviruses, the genome includes a long unique region, a short unique region, and inverted repeat sequences at both ends, and encodes a large number of glycoproteins and proteins related to virulence and virus replication. ILTV wild strains have large differences in virulence, with some strains having a high morbidity and mortality rate after infecting chickens, belonging to strong pathogenicity strains, and strains with mild or no symptoms after infection, and no mortality rate, belonging to attenuated strains.

[0004] Attenuated live vaccines are the main types of vaccines for preventing animal infectious diseases. However, most of the current poultry vaccine strains are attenuated by passage in avian embryos or cells, and there is uncertainty in the virulence reversion of the attenuated strains used for preparing vaccines obtained in this way. Natural attenuated strains have the advantages of low toxicity, small side effects, and good immunogenicity, etc. SUMMARY

[0005] The purpose of the present application is to provide a natural attenuated strain of infectious laryngotracheitis virus with good safety and immunogenicity.

[0006] The technical solution adopted by the present application is as follows:

[0007] The application discloses a natural attenuated strain of infectious laryngotracheitis virus (ILTV) with good safety and immunogenicity, and the strain is named as ILTV FJ19 and classified as infectious laryngotracheitis virus (ILTV), and has been preserved in the China Center for Type Culture Collection on November 29, 2023, with a preservation number of CCTCC NO: V2023109 and a preservation address of Wuhan, China.

[0008] The application of the natural attenuated strain of infectious laryngotracheitis virus in preparation of a vaccine, wherein the vaccine is a live attenuated vaccine, the live attenuated vaccine is used for preventing infectious laryngotracheitis, and the infectious laryngotracheitis is chicken infectious laryngotracheitis.

[0009] A vaccine for infectious laryngotracheitis, which contains live viruses of the natural attenuated strain of infectious laryngotracheitis virus, wherein the immunization mode of the vaccine includes oral or eye drop immunization, the vaccine is used for immunizing chickens, and the immunoprotection rate of the vaccine after 21 days of immunization and challenge of the ILTV virulent WF03 strain (a patent strain with a patent number of ZL202110385416.X) can reach 100%.

[0010] A preparation method of the vaccine for infectious laryngotracheitis, which uses the natural attenuated strain of infectious laryngotracheitis virus as a vaccine production strain, inoculates SPF chicken embryos through the allantoic cavity route, grinds the infected embryos and / or allantoic membranes to obtain virus liquid, and then the infectious laryngotracheitis vaccine is obtained.

[0011] The application has the following advantages:

[0012] The application separates and identifies pathogens of a conventional submitted chicken population, purifies and clones an ILTV virus with stable heredity and adaptability to inoculation through the allantoic cavity route of a chicken embryo, and names the ILTV virus as FJ19 strain. Inoculation of the P1 generation culture of the ILTV FJ19 strain into 14-day-old SPF chickens has good safety, and the chickens have good immunoprotection after challenge of the WF03 strain. Inoculation of the virus culture of the allantoic cavity route-adapted ILTV FJ19 strain into 14-day-old SPF chickens has good safety, and the chickens have good immunoprotection after challenge of the WF03 strain. Therefore, the ILTV FJ19 strain separated and cloned by the application has stable passage ability, adaptability to inoculation through the allantoic cavity route, good safety and immunogenicity in inoculation of 14-day-old SPF chickens, and can be used for eye drop or oral immunization, and the virus can be used as a live attenuated vaccine candidate strain of ILTV. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1: The allantoic membrane of the 10-day-old SPF chicken embryo inoculated with the suspected ILTV virus showed gray-white spots after 7 days. DETAILED DESCRIPTION

[0014] In order to make the content of the present application more convenient to understand, the technical solutions of the present application will be further described below in combination with specific embodiments, but the present application is not limited thereto.

[0015] Example 1: Isolation, identification and purification cloning of strains

[0016] During the routine pathogen monitoring of the chicken population, the suspected ILTV infection positive sample tissue was added to PBS (0.01M, pH 7.2-7.4) at a ratio of 1:5 m / v, ground and freeze-thawed for 3 times, centrifuged at 12000 r / min for 10 min, and then filtered with a 0.45 μm filter. The filtered liquid was inoculated into 10-day-old SPF chicken embryos by the allantoic membrane route, and the embryos were further incubated for 7 days. The allantoic membrane was aseptically harvested, added to PBS (0.01M, pH 7.2-7.4) at a ratio of 1:5 m / v, ground and freeze-thawed for 3 times, and centrifuged to take the supernatant for inoculation into 10-day-old SPF chicken embryos by the allantoic membrane route. After 2 more passages, the chicken embryo lesions were observed. It was found that the allantoic membrane of the chicken embryo showed gray-white spots (, suspected ILTV virus infection, and was identified as ILTV virus according to the agricultural industry standard NY / T 556-2020 diagnosis technology for chicken infectious laryngotracheitis. Figure 1

[0017] The identified ILTV virus strain was inoculated into 10-day-old chicken embryos by the allantoic membrane route for purification and cloning by the limited dilution method. The virus liquid was 10-fold serially diluted, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 dilutions were inoculated into 10-day-old SPF chicken embryos, 6 embryos for each dilution. After 7 days of further incubation, the allantoic membrane was checked, and the presence of gray-white spots on the allantoic membrane was determined as positive. The positive allantoic membrane with the highest dilution was ground to prepare virus liquid, and the same method was used for further purification and cloning for 4 times. The two ILTV virus strains purified and cloned were named FJ19 and FJ22, respectively, and the allantoic membrane was harvested for virus liquid preparation in 10-day-old SPF chicken embryos, which was recorded as P1 generation.

[0018] The virus liquid stored at -70°C was taken out, thawed and equilibrated to room temperature, and then 0.5 mL was added to the corresponding 4.5 mL PBS (0.01M, pH 7.2-7.4). It was diluted by 10 times in turn, and 10 -6 ​, each dilution of virus liquid was inoculated into 6 10-day-old SPF chicken embryos, and another 6 embryos were taken as negative controls and placed in a 37°C incubator for continuous incubation for 7 days. The appearance of gray-white spots on the allantoic membrane was recorded as positive. According to the number of positives, the ILTV virus content was calculated as 10 5.70 EID 50 / ml according to the Reed-Muench method.

[0019] Example 2: Culture characteristics of ILTV FJ19 strain and FJ22 strain

[0020] The culture of ILTV FJ19 strain and FJ22 strain requires 10-day-old SPF chicken embryos, and the P1 generation harvested by allantoic membrane inoculation has the highest virus content in the virus liquid prepared from the allantoic membrane. After inoculating the chicken embryos for 7 days, large gray-white lesions appear on the allantoic membrane or a large number of gray-white spots appear on the allantoic membrane. The harvested virus liquid was detected according to the bacterial, mycoplasma and exogenous virus detection method in the Chinese Veterinary Pharmacopoeia (2020 edition), and the results showed that the ILTV FJ19 strain and FJ22 strain virus liquid was not contaminated by bacteria, mycoplasma and exogenous virus, and was pure ILTV virus.

[0021] Example 3: Immunogenicity determination of ILTV FJ19 strain and FJ22 strain

[0022] 60 14-day-old SPF chickens were divided into 6 groups, G1, G2, G3, G4, G5 and G6, of which G1 and G2 were not inoculated, G3 and G4 were inoculated with ILTV FJ19 clone virus (P1 generation) by eye and oral inoculation, G5 and G6 were inoculated with ILTV FJ22 clone virus (P1 generation) by eye and oral inoculation, and the inoculation dose was 10 3.0 EID 50 , and continuous observation was carried out for 21 days after inoculation. After 21 days, G2, G3, G4, G5 and G6 were challenged with virulent strain ILTV WF03, the challenge dose was 0.2 mL / bird, and the virus content was 10 4.0 EID 50 , and G1 was not challenged as a negative control group, and clinical symptoms were observed continuously for 14 days after challenge.

[0023] The results show that within 21 days after inoculation, the chickens in the inoculation group did not show visible side effects such as tearing, ophthalmia and respiratory tract reaction, within the 14-day observation period after challenge, no specific clinical symptoms were observed in the chickens in the G1 group; the morbidity of the chickens in the G2 group was 100% (10 / 10), the mortality was 40% (4 / 10), and the chickens showed specific clinical symptoms such as coughing blood and open-mouth breathing; the morbidity of the chickens in the G3 group (ILTV FJ19 eye drop inoculation group) and the G4 group (ILTV FJ19 oral inoculation group) was both 0% (0 / 10), the mortality was both 0% (0 / 10), and no specific clinical symptoms were observed; the morbidity of the chickens in the G5 group (ILTV FJ22 eye drop inoculation group) was 60% (6 / 10), the mortality was 30% (3 / 10), and the chickens showed specific clinical symptoms such as coughing blood and open-mouth breathing; the morbidity of the chickens in the G6 group (ILTV FJ22 oral inoculation group) was 70% (7 / 10), the mortality was 30% (3 / 10), and the chickens showed specific clinical symptoms such as coughing blood and open-mouth breathing. The results show that the ILTV FJ19 clone has good virus immunogenicity and can be used as a vaccine immunization strain, while the ILTV FJ22 immunized chickens have poor protection effect after challenge, and is not suitable for use as a vaccine candidate strain.

[0024] Example 4: Safety determination of ILTV FJ19 strain

[0025] Thirty 14-day-old SPF chickens were divided into three groups, namely G1, G2 and G3 groups, wherein the G1 group was not inoculated; the G2 group was inoculated with the ILTV FJ19 clone virus (P1 generation) by eye drop, the inoculation dose was 10 4.0 EID 50 The G3 group was inoculated with the ILTV FJ19 clone virus (P1 generation) by eye drop, the inoculation dose was 10 5.0 EID 50 The chickens were continuously observed for 14 days after inoculation.

[0026] The results show that within 14 days after inoculation, the chickens in the G1 group did not show any visible clinical symptoms, the chickens in the G2 group did not show visible side effects such as tearing, ophthalmia and respiratory tract reaction, and 20% of the chickens in the G3 group showed visible side effects such as tearing and ophthalmia 4-6 days after inoculation.

[0027] Example 5: Safety and immunogenicity determination of the virus adapted by the allantoic cavity route of ILTV FJ19 strain The P1 generation virus of the ILTV FJ19 strain was continuously inoculated into 10-day-old SPF chicken embryos through the allantoic cavity route, and after 5-7 days of incubation, the allantoic membrane and embryo or the mixture of the allantoic membrane and embryo were harvested, ground, added with appropriate amount of PBS (0.01M, pH 7.2-7.4), and then divided and stored, and the EID 50, allantoic cavity inoculation passage to prepare different generations of virus liquid, P2, P3, P4 generation until P7 generation of virus liquid.

[0028] 50 14-day-old SPF chickens were divided into 5 groups, G1, G2, G3, G4 and G5, of which G1 was not inoculated, G2 and G3 were inoculated with P5 generation allantoic membrane virus and embryo virus of FJ19 clone harvested after allantoic cavity inoculation adaptation by eye-pointing, G4 and G5 were inoculated with P7 generation allantoic membrane virus and embryo virus of FJ19 clone harvested after allantoic cavity inoculation adaptation by eye-pointing, and the inoculation dose was 10 4.0 EID 50 , and the clinical symptoms were observed for 14 days after inoculation.

[0029] The results showed that within 14 days after inoculation, no visible clinical symptoms occurred in G1 group, and no visible side effects such as tearing, ophthalmia and respiratory tract reaction occurred in G2, G3, G4 and G5 groups.

[0030] 60 14-day-old SPF chickens were divided into 6 groups, G1, G2, G3, G4, G5 and G6, of which G1 and G2 were not inoculated, G3 and G4 were inoculated with P5 generation allantoic membrane virus and embryo virus of FJ19 clone harvested after allantoic cavity inoculation adaptation by eye-pointing, G5 and G6 were inoculated with P7 generation allantoic membrane virus and embryo virus of FJ19 clone harvested after allantoic cavity inoculation adaptation by eye-pointing, and the inoculation dose was 10 3.0 EID 50 , and the clinical symptoms were observed for 14 days after inoculation. 4.0 EID 50 , G1 group of 10 chickens was not attacked as a negative control group, and the clinical symptoms were observed for 14 days after inoculation.

[0031] The results showed that within 14 days after inoculation, no visible specific clinical symptoms were observed in G1 group; the morbidity of G2 group was 100% (10 / 10), the mortality was 40% (4 / 10), and the specific clinical symptoms such as coughing blood and open-mouth breathing were observed; the morbidity of G3 group (P5 generation allantoic membrane virus) and G4 group (P5 generation embryo virus) was 0% (0 / 10), the mortality was 0% (0 / 10), and no specific clinical symptoms were observed; the morbidity of G5 group (P7 generation allantoic membrane virus) and G6 group (P7 generation embryo virus) was 0% (0 / 10), the mortality was 0% (0 / 10), and no specific clinical symptoms were observed.

[0032] Example 6: Sequencing of major immunogenicity and virulence genes of ILTV FJ19 strain

[0033] The primers in Table 1 were used to amplify, clone and sequence the virulence genes gE / gI, TK, ICP4, gC, gG, the immunogenicity genes gB, UL32, gD, gC, and the virus replication related genes gB, gD, gE, gI, gH, gL, gK, gM, gN of the P1 generation virus liquid of ILTV FJ19 strain, respectively.

[0034] The amplification system is as follows:

[0035]

[0036] Table 1: Primer names and sequences used for PCR amplification

[0037]

[0038]

[0039] The recovered PCR products were ligated into a blunt-ended vector (pClone007 Blunt Simple Vector Kit, Genesky Biologies), and after transformation of competent cells DH5a, the plates containing ampicillin were coated, and 5 clones (bacterial liquid) of each gene were selected and sent to Sinobiology (Shanghai) Co., Ltd. for sequencing. After deleting the excess sequences of the target gene sequences in the sequencing sequences, analysis and comparison were performed. The sequence information of the ILTV FJ19 virus gK, gG, gE, UL32, ICP4, gC, gL, gN, TK, gM, gD, gB, gH, gI genes is shown in SEQ ID NO. 1-14, respectively. Compared with the domestic and foreign attenuated strains A20 (NCBI accession number: JN596963), K317 (NCBI accession number: JX458824), LT Blen (NCBI accession number: JQ083493), Laryngo Vac (NCBI accession number: JQ083494), Poulvac ILT(R) (NCBI accession number: KP677882), Rus / Ck / Tatarstan / 2009 / 1643 (NCBI accession number: MF405079), Nobilis Laringovac(R) (NCBI accession number: KP677881), the ILTV FJ19 virus gene sequence has different degrees of difference.

[0040] Example 7: Determination of the immune duration of ILTV FJ19 strain

[0041] 70 14-day-old SPF chickens were divided into two groups, one group of 30 chickens was inoculated with FJ19 clone virus (P1 generation) by eye drop, and the inoculation dose was 10 3.0 EID 50 The other group of 40 chickens was not immunized. Four, five and six months after immunization, 10 chickens from each group were challenged with the virulent ILTV WF03 strain by tracheal challenge, the challenge dose was 0.2 mL / chicken, and the virus content was 10 4.0 EID 50 Fourteen days after challenge, the clinical symptoms were continuously observed.

[0042] The results show that during the 14-day observation period after challenge at four, five and six months after immunization, no specific clinical symptoms were observed in the unchallenged chickens, and the incidence rates of the unimmunized chickens during the observation period after challenge were 100% (10 / 10), 100% (10 / 10) and 90% (9 / 10), respectively, and the chickens showed specific clinical symptoms such as coughing blood and open-mouth breathing, while the incidence rates of the immunized chickens during the observation period after challenge were 0% (0 / 10), 0% (0 / 10) and 10% (1 / 10), respectively.

[0043] The above only describes the preferred embodiments of the present application, and any equivalent changes and modifications made within the scope of the patent application of the present application shall be included in the scope of the present application.

Claims

1. A naturally attenuated strain of infectious laryngotracheitis virus, characterised in that: The strain is named Infectious Laryngotracheitis Virus (ILTV) Infectious laryngotracheitis virus ) ILTV FJ19, which was preserved in China Center for Type Culture Collection on November 29, 2023, and the preservation number is CCTCC NO: V2023109.

2. Use of the natural attenuated strain of infectious laryngotracheitis virus according to claim 1 in the preparation of a live vaccine for preventing infectious laryngotracheitis.

3. Use according to claim 2, characterized in that: The live vaccine is a natural attenuated live vaccine.

4. Use according to claim 2, characterized in that: The infectious laryngotracheitis is chicken infectious laryngotracheitis.

5. A vaccine against infectious laryngotracheitis, characterized in that: The live virus containing the natural attenuated strain of infectious laryngotracheitis virus according to claim 1.

6. The vaccine for infectious laryngotracheitis according to claim 5, characterized in that: The immunization mode of the infectious laryngotracheitis vaccine includes oral immunization or eye drop immunization.

7. The vaccine for infectious laryngotracheitis according to claim 5, characterized in that: The live virus is obtained by inoculating the natural attenuated strain of infectious laryngotracheitis virus into SPF chicken embryos through the allantoic cavity route, harvesting the allantoic membrane and / or embryo body after 5-7 days, and grinding the harvested allantoic membrane and / or embryo body.

8. A method of preparing a vaccine for infectious laryngotracheitis, characterized by: The natural attenuated strain of infectious laryngotracheitis virus according to claim 1 is used as a vaccine production strain, inoculated into SPF chicken embryos through the allantoic cavity route, the infected embryo body and / or allantoic membrane are harvested after 5-7 days, ground, and the virus liquid is prepared, thereby obtaining an infectious laryngotracheitis vaccine.

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