Natural attenuated strain of infectious laryngotracheitis virus with good safety and good immunogenicity and use

Abstract

A natural attenuated strain of infectious laryngotracheitis virus with good safety and good immunogenicity. The natural attenuated strain is named infectious laryngotracheitis virus ILTV FJ19, and has been deposited in the China Center for Type Culture Collection on November 29, 2023, with the deposit number of CCTCC NO: V2023109, and the deposit address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Experiments show that the avian infectious laryngotracheitis virus ILTV FJ19 strain has stable passage ability, can adapt to inoculation via the allantoic cavity route, has very good safety and immunogenicity when inoculated in 14-day-old SPF chickens, can be used for eye-drop inoculation or oral inoculation, and can be used as a candidate strain of a live attenuated vaccine against ILTV.

Description

A natural attenuated strain of infectious laryngotracheitis virus with good safety and immunogenicity and its application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 29, 2023, with application number CN202311854854.1 and invention name “A natural attenuated strain of infectious laryngotracheitis virus with good safety and immunogenicity and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] 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, and belongs to the field of microbial technology. The strain was isolated and obtained during routine pathogen monitoring of a domestic chicken flock. After isolation, identification, purification and cloning, it was named ILTV FJ19 strain. Its safety was determined using 14-day-old SPF chickens. 21 days after immunization of 14-day-old SPF chickens, the immunogenicity was determined by challenging them with the WF03 strain (patented strain, patent number: ZL202110385416.X). The test results showed that the infectious laryngotracheitis virus ILTV FJ19 strain in this application has good safety and immunogenicity, and can be used as a candidate strain for ILTV attenuated live vaccine. Background Art

[0003] Infectious laryngotracheitis virus (ILTV) belongs to the genus Infectious laryngotracheitis virus of the family Herpesviridae and the subfamily Alphaherpesvirinae. Infection mainly causes death and / or decreased egg production in chickens. Severe infection often manifests as difficulty breathing, wheezing, and coughing up bloody fluid, and is highly lethal. Mild infection mainly manifests in various forms such as mucotracheitis, rhinitis, conjunctivitis, reluctance to move, and low mortality. Both severe and mild infections bring serious losses and threats to production.

[0004] ILTV is a double-stranded DNA virus with a genome size of approximately 155 kilobases. Like other herpes viruses, its genome consists of a length-specific region, a short unique region, and inverted repeats at both ends. These regions encode numerous glycoproteins and proteins involved in virulence and viral replication. The virulence of wild-type ILTV strains varies greatly. Some strains cause high morbidity and mortality in chickens, making them highly pathogenic, while strains with mild or no symptoms and no mortality are considered less virulent.

[0005] Attenuated live vaccines are the main type of vaccine for preventing animal infectious diseases. However, most of the current poultry vaccine strains are weakened through avian embryo or cell passage. The attenuated strains obtained in this way for vaccine preparation have uncertainties such as the reversion of virulence. Natural attenuated strains have the advantages of low toxicity, few side effects, and good immunogenicity.

[0006] Summary of the Invention

[0007] The purpose of this application is to provide a natural attenuated strain of infectious laryngotracheitis virus with good safety and immunogenicity.

[0008] The technical solution adopted in this application is as follows: This application provides a natural attenuated strain of infectious laryngotracheitis virus with good safety and immunogenicity. The natural attenuated strain of the virus is named infectious laryngotracheitis virus ILTV FJ19 and classified as infectious laryngotracheitis virus (infectious laryngotracheitis virus). It was deposited in the China Center for Type Culture Collection on November 29, 2023, with the deposit number CCTCCNO: V2023109, and the deposit address is Wuhan University, Wuhan, China.

[0009] The present application provides the use of the natural attenuated strain of infectious laryngotracheitis virus described in the above technical solution in the preparation of a vaccine; the vaccine is a live attenuated vaccine; the live attenuated vaccine is used to prevent infectious laryngotracheitis; the infectious laryngotracheitis is infectious laryngotracheitis in chickens.

[0010] The present application provides an infectious laryngotracheitis vaccine, which contains the live virus of the above-mentioned natural attenuated strain of infectious laryngotracheitis virus; the immunization method of the infectious laryngotracheitis vaccine includes oral or eye drop immunization; the infectious laryngotracheitis vaccine is used to immunize chickens, and the immune protection rate can reach 100% when challenged with the virulent ILTV WF03 strain (patented strain, patent number: ZL202110385416.X) 21 days after immunization.

[0011] The present application provides a method for preparing the infectious laryngotracheitis vaccine described in the above technical solution, which uses the natural attenuated strain of the infectious laryngotracheitis virus as the vaccine production strain, inoculates SPF chicken embryos through the allantoic cavity route, harvests the infected embryos and / or the allantoic membranes, grinds them, and prepares a virus liquid to obtain the infectious laryngotracheitis vaccine.

[0012] The significant advantages of this application are: by isolating and identifying the pathogen from routinely tested chicken flocks, the application purified and cloned a genetically stable ILTV virus strain, designated FJ19, that is adaptable to inoculation via the allantoic cavity of the embryo. ILTV FJ19 strain P1 culture was found to be highly safe when inoculated into 14-day-old SPF chickens and provided excellent immune protection after challenge with the WF03 strain. Inoculation of 14-day-old SPF chickens with the adapted virus culture via the allantoic cavity with the ILTV FJ19 strain also demonstrated excellent safety and excellent immune protection after challenge with the WF03 strain. Therefore, the ILTV FJ19 strain isolated and cloned in this application exhibits stable passage capacity, is adaptable to inoculation via the allantoic cavity, and exhibits excellent safety and immunogenicity when inoculated into 14-day-old SPF chickens. It can be administered via eye drops or oral administration, and this virus strain can be used as a candidate strain for an attenuated live ILTV vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments.

[0014] Figure 1: Pathological changes in the allantoic membrane of 10-day-old SPF chicken embryos 7 days after suspected ILTV virus was inoculated through the allantoic membrane route, with grayish-white spots. DETAILED DESCRIPTION

[0015] In order to make the contents described in this application easier to understand, the technical solutions described in this application are further explained below in conjunction with specific implementation methods, but this application is not limited to this.

[0016] Example 1: Isolation, identification and purification of virus strains

[0017] During routine pathogen monitoring of chickens, suspected ILTV-positive tissue samples were collected, mixed with PBS (0.01M, pH 7.2-7.4) at a ratio of 1:5 m / v, ground, and then frozen and thawed three times. The samples were centrifuged at 12,000 rpm for 10 minutes and filtered through a 0.45 μm filter. The filtrate was inoculated into 10-day-old SPF chicken embryos via the allantoic membrane route. After incubation for 7 days, the allantoic membranes were aseptically harvested, mixed with PBS (0.01M, pH 7.2-7.4) at a ratio of 1:5 m / v, ground, and frozen and thawed three times. The supernatant was centrifuged and inoculated into 10-day-old SPF chicken embryos via the allantoic membrane route. After two more passages, the embryos were observed for pathological changes. After three blind passages, grayish-white spots appeared on the allantoic membranes of the embryos (Figure 1), indicating suspected ILTV infection. The samples were identified as ILTV virus according to the agricultural industry standard NY / T 556-2020, Diagnostic Techniques for Infectious Laryngotracheitis in Avian Chickens.

[0018] The identified ILTV virus strain was purified and cloned by inoculating 10-day-old chicken embryos with the limiting dilution method through the allantoic membrane route. The virus solution was diluted 10 times and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Six 10-day-old SPF chicken embryos were inoculated at each dilution. After 7 days of incubation, the chorioallantoic membranes were examined and considered positive if grayish-white spots appeared. The positive chorioallantoic membranes from the highest dilution were ground and prepared into a viral fluid. Purification and cloning were repeated four times using the same method. The two purified and cloned ILTV viruses were designated FJ19 and FJ22, respectively. They were expanded in 10-day-old SPF chicken embryos, and the chorioallantoic membranes were harvested to prepare viral fluids, designated as passage P1.

[0019] The virus solution was taken out from -70℃, thawed and equilibrated to room temperature, and 0.5mL was added to the corresponding 4.5mL PBS (0.01M, pH 7.2-7.4), and diluted 10-fold to 10 -6 Each dilution of virus solution was inoculated into 6 10-day-old SPF chicken embryos, and another 6 chicken embryos of the same type were used as negative controls. The cells were placed in a 37°C incubator and incubated for 7 days. The appearance of grayish-white spots on the chorioallantoic membrane was recorded as positive. The ILTV virus content was calculated according to the Reed-Muench method based on the number of positive results. 5.70 EID 50 / ml.

[0020] Example 2: Culture characteristics of ILTV FJ19 and ILTV FJ22 strains

[0021] ILTV FJ19 and ILTV FJ22 strains are cultured in 10-day-old SPF chicken embryos. The P1 generation is harvested via the allantoic membrane. The virus fluid prepared from the harvested allantoic membrane has the highest viral content. Seven days after inoculation, large grayish-white lesions or numerous grayish-white spots appear on the allantoic membrane. Testing of the harvested virus fluid for bacteria, mycoplasma, and exogenous virus contamination, as specified in the 2020 edition of the Chinese Veterinary Pharmacopoeia, revealed that the ILTV FJ19 and ILTV FJ22 strains were free of bacteria, mycoplasma, and exogenous virus contamination, demonstrating pure ILTV virus.

[0022] Example 3: Immunogenicity determination of ILTV FJ19 and ILTV FJ22 strains

[0023] Sixty 14-day-old SPF chickens were divided into 6 groups, namely G1, G2, G3, G4, G5 and G6. Groups G1 and G2 were not vaccinated, groups G3 and G4 were inoculated with ILTV FJ19 clone virus (P1 generation) by eye drop and oral administration, and groups G5 and G6 were inoculated with ILTV FJ22 clone virus (P1 generation) by eye drop and oral administration. The vaccination dose was 10 3.0 EID 50 After 21 days, the G2, G3, G4, G5 and G6 groups were challenged with the virulent strain ILTVWF03 in the trachea. The challenge dose was 0.2 mL / bird and the virus content was 10 4.0 EID 50 Group G1 was not challenged with the virus and served as the negative control group. Clinical symptoms were observed continuously for 14 days after the challenge.

[0024] The results showed that within 21 days after vaccination, the immunized chickens did not show visible side effects such as tearing, eye inflammation and respiratory tract reaction. During the 14-day observation period after the challenge, no visible specific clinical symptoms were observed in the chickens of G1 group. The morbidity of the chickens in G2 group was 100% (10 / 10) and the mortality rate was 40% (4 / 10), with specific clinical symptoms such as hemoptysis and open mouth breathing. The morbidity of the chickens in G3 group (ILTV FJ19 eye drop vaccination group) and G4 group (ILTV FJ19 oral vaccination group) was 0% (0 / 10) and the mortality rate was 0% (0 / 10), with no specific clinical symptoms. The morbidity of the chickens in G5 group (ILTV FJ22 eye drop vaccination group) was 60% (6 / 10) and the mortality rate was 30% (3 / 10), with specific clinical symptoms such as hemoptysis and open mouth breathing. The Chickens in the oral FJ22 vaccination group experienced morbidity of 70% (7 / 10) and mortality of 30% (3 / 10), manifesting with specific clinical symptoms such as hemoptysis and open mouth breathing. The results indicate that the ILTV FJ19 clone possesses strong immunogenicity and is suitable for use as a vaccine strain. However, ILTV FJ22 provided poor protection against challenge in chickens, making it unsuitable as a vaccine candidate.

[0025] Example 4: Safety determination of ILTV FJ19 strain

[0026] Thirty 14-day-old SPF chickens were divided into three groups: G1, G2, and G3. Group G1 was not vaccinated; Group G2 was inoculated with ILTV FJ19 clone virus (P1 generation) by eye drop at a dose of 10 4.0 EID 50 G3 was inoculated with ILTV FJ19 clone virus (P1 generation) by eye drop, with a dose of 10 5.0 EID 50, and observed continuously for 14 days after vaccination.

[0027] The results showed that within 14 days after vaccination, the chickens in group G1 did not show any visible clinical symptoms, the chickens in group G2 did not show any visible side effects such as tearing, eye inflammation and respiratory tract reactions, and 20% of the chickens in group G3 showed visible transient side effects such as tearing and eye inflammation 4 to 6 days after vaccination.

[0028] Example 5: Safety and immunogenicity assessment of ILTV FJ19 strain after adaptation via allantoic cavity inoculation

[0029] The P1 generation virus of ILTV FJ19 strain was continuously inoculated into 10-day-old SPF chicken embryos through the allantoic cavity. After 5-7 days of incubation, the allantoic membrane and embryoid bodies or the mixture of allantoic membrane and embryoid bodies were harvested, ground, added with appropriate amount of PBS (0.01M, pH 7.2-7.4), and then packaged and stored. The EID was determined. 50 The virus solutions of different generations were prepared by inoculation into the allantoic cavity, and virus solutions of generations P2, P3, P4 and up to P7 were obtained.

[0030] Fifty 14-day-old SPF chickens were divided into five groups, namely G1, G2, G3, G4 and G5. Group G1 was not vaccinated. Groups G2 and G3 were inoculated with the allantoic membrane virus and embryonic body virus of the P5 generation ILTV FJ19 clone harvested after adaptation via the allantoic cavity by eye drop. Groups G4 and G5 were inoculated with the allantoic membrane virus and embryonic body virus of the P7 generation FJ19 clone harvested after adaptation via the allantoic cavity by eye drop. The inoculation dose was 10 4.0 EID 50 , and observed continuously for 14 days after immunization.

[0031] The results showed that within 14 days after vaccination, the chickens in group G1 did not show any visible clinical symptoms, and the chickens in groups G2, G3, G4 and G5 did not show any visible side effects such as tearing, eye inflammation and respiratory reactions.

[0032] Sixty 14-day-old SPF chickens were divided into 6 groups, namely G1, G2, G3, G4, G5 and G6. Groups G1 and G2 were not vaccinated. Groups G3 and G4 were inoculated with the allantoic membrane toxin and embryonic body toxin of the P5 generation FJ19 clone harvested after adaptation via the allantoic cavity by eye drop. Groups G5 and G6 were inoculated with the allantoic membrane toxin and embryonic body toxin of the P7 generation FJ19 clone harvested after adaptation via the allantoic cavity by eye drop. The inoculation dose was 10 3.0 EID 5021 days after inoculation, the G2, G3, G4, G5 and G6 groups were challenged with the virulent strain ILTVWF03 in the trachea, with a challenge dose of 0.2 mL / bird and a virus content of 10 4.0 EID 50 The 10 birds in group G1 were not challenged with the poison and served as the negative control group. The clinical symptoms were observed for 14 consecutive days after the challenge.

[0033] The results showed that during the 14-day observation period after infection, no visible specific clinical symptoms were observed in the chickens of group G1; the morbidity rate of chickens in group G2 was 100% (10 / 10), and the mortality rate was 40% (4 / 10), with specific clinical symptoms such as hemoptysis and open mouth breathing; the morbidity rate of chickens in group G3 (P5 generation allantoin toxin) and group G4 (P5 generation embryonic toxin) was 0% (0 / 10), and the mortality rate was 0% (0 / 10), with no specific clinical symptoms; the morbidity rate of chickens in group G5 (P7 generation allantoin toxin) and group G6 (P7 generation embryonic toxin) was 0% (0 / 10), and the mortality rate was 0% (0 / 10), with no specific clinical symptoms.

[0034] Example 6: Sequencing of the major immunogenicity and virulence genes of ILTV FJ19 strain

[0035] Using the primers listed in Table 1, ILTV FJ19 strain P1 virus fluid was used as the genomic template to PCR amplify, clone, and sequence the ILTV virulence genes gE / gI, TK, ICP4, gC, and gG; the immunogenic genes gB, UL32, gD, and gC; and the viral replication-related genes gB, gD, gE, gI, gH, gL, gK, gM, and gN. The amplification system was as follows:

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

[0037] The recovered PCR products were ligated into a blunt-ended vector (pClone007 Blunt Simple Vector Kit, Qingke Biotechnology), transformed into competent DH5α cells, and plated on ampicillin-containing plates. Five clones (bacterial suspension) were selected for each gene and sent to Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing. Unnecessary upstream and downstream sequences of the target gene were deleted from the sequenced sequences before analysis and alignment. The sequences of the ILTV FJ19 virus gK, gG, gE, UL32, ICP4, gC, gL, gN, TK, gM, gD, gB, gH, and gI genes are shown in SEQ ID NOs. 1 to 14, respectively. The ILTV FJ19 virus gene sequence shows varying degrees of differences 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), and Nobilis Laringovac(R) (NCBI accession number: KP677881).

[0038] SEQ ID NO.1: gK (1020 bp)

[0039] SEQ ID NO. 2: gG (879 bp)

[0040] SEQ ID NO.3: gE (1500 bp)

[0041] SEQ ID NO.4: UL32 (1749 bp)

[0042] SEQ ID NO.5: ICP4 (4450 bp)

[0043] SEQ ID NO.6: gC (1245 bp)

[0044] SEQ ID NO.7: gL (708 bp)

[0045] SEQ ID NO.8: gN (354 bp)

[0046] SEQ ID NO.9: TK (1090 bp)

[0047] SEQ ID NO.10: gM (1182 bp)

[0048] SEQ ID NO.11: gD (1305 bp)

[0049] SEQ ID NO.12: gB (2620 bp)

[0050] SEQ ID NO.13: gH (2415 bp)

[0051] SEQ ID NO.14: gI (1089 bp)

[0052] Example 7: Determination of the duration of immunity of ILTV FJ19 strain

[0053] Seventy 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. The inoculation dose was 10 3.0 EID 50 At 4, 5 and 6 months after vaccination, 10 chickens from each group were challenged with the virulent strain ILTVWF03 in the trachea. The challenge dose was 0.2 mL / bird and the virus content was 10 4.0 EID 50 The clinical symptoms were observed continuously for 14 days after infection.

[0054] The results showed that during the 14-day observation period after challenge at 4, 5 and 6 months after vaccination, no visible specific clinical symptoms were observed in the non-challenged chickens. The incidence rates of the non-immune chickens during the observation period after challenge were 100% (10 / 10), 100% (10 / 10) and 90% (9 / 10), respectively, which showed specific clinical symptoms such as hemoptysis 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.

[0055] Although the above embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, 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 application.

Claims

1. A natural attenuated strain of infectious laryngotracheitis virus ILTVFJ19 with good safety and immunogenicity, with the preservation number of CCTCC NO: V2023109.

2. The application of the natural attenuated strain of infectious laryngotracheitis virus according to claim 1 in the preparation of a live vaccine.

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

4. The application according to claim 3, characterized in that The attenuated live vaccine is used for preventing infectious laryngotracheitis.

5. The application according to claim 4, characterized in that, The infectious laryngotracheitis is avian infectious laryngotracheitis.

6. An infectious laryngotracheitis vaccine, characterized in that, A live virus containing the natural attenuated strain of infectious laryngotracheitis virus according to claim 1.

7. The infectious laryngotracheitis vaccine according to claim 6, wherein The immunization methods of the infectious laryngotracheitis vaccine include oral immunization and eye drop immunization.

8. The infectious laryngotracheitis vaccine according to claim 6, wherein, The live virus is obtained by inoculating the natural attenuated strain of infectious laryngotracheitis virus into SPF chicken embryos via the allantoic cavity route for 5 - 7 days, and then harvesting the allantoic membrane and / or embryo body.

9. The infectious laryngotracheitis vaccine according to claim 7, characterized in that, When the infectious laryngotracheitis vaccine is used to immunize chickens, the immune protection rate against the virulent strain ILTVWF03 of infectious laryngotracheitis virus can reach 100%.

10. The preparation method of the infectious laryngotracheitis vaccine according to claim 6, characterized in that, Using the natural attenuated strain of infectious laryngotracheitis virus as the vaccine production strain, inoculating SPF chicken embryos via the allantoic cavity route, harvesting and grinding the infected embryo body and / or allantoic membrane after 5 - 7 days to prepare a virus solution, thus obtaining the infectious laryngotracheitis vaccine.

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