A Muscovy duck chapama virus strain, applications and vaccines

Abstract

The present invention belongs to the field of biotechnology, and discloses a Muscovy duck Chaphama virus strain MuChPv-GD2022, characterized in that the classification name is Muscovy duck Chaphama virus (Chaphamaparvovirus MuChPv-GD2022), the deposit number is: CCTCC NO: V202311; the deposit date is: March 1, 2023, and the deposit unit is: China Center for Type Culture Collection. The homology between the strain and the existing duck-origin Chaphama virus in the GenBank database is 76.77%-78.36%, indicating that this is a new strain, which provides a basis for vaccine preparation for the subsequent prevention and control of duck-related Chaphama virus infection; at the same time, the present invention also provides a preparation method, application and vaccine of the strain.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a Muscovy duck chapama virus strain, application and vaccine. Background Art

[0002] In recent years, with the transformation and upgrading of the waterfowl farming model, the breeding density has increased significantly, but the level of feeding and management is relatively backward. The isolation measures are not strict and the epidemic prevention is not implemented effectively, which leads to the prevalence and increasing complexity of waterfowl diseases. Various pathogens are cross-infected in waterfowl groups, and secondary infections are also increasing. Waterfowl are the common natural carriers and storage hosts of many viruses such as avian influenza. Various pathogens are transmitted among waterfowl, resulting in the continuous emergence of new diseases or new symptoms of old diseases, and even large-scale outbreaks of diseases, which seriously endanger the healthy development of the waterfowl industry.

[0003] Muscovy duck chapama virus is a member of the Parvoviridae family, Hamaparvovirinae subfamily, and Chaphamaparvovirus genus. Currently, there is almost no research on Muscovy duck chapama virus. Summary of the invention

[0004] The purpose of the present invention is to provide a Muscovy duck Chapama virus strain, which has a homology of 76.77%-78.36% with the existing duck-origin Chapama virus in the GenBank database, indicating that this is a completely new strain, which provides a basis for vaccine preparation for the subsequent prevention and control of duck-origin related Chapama virus infection.

[0005] At the same time, the invention also provides the application of the strain and a vaccine.

[0006] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a Muscovy duck chapama virus strain MuChPv-GD2022, classified as Muscovy duck chapama virus (Chaphamaparvovirus MuChPv-GD2022), with a preservation number of: CCTCC NO: V202311; preservation date: March 1, 2023, and preservation unit: China Center for Type Culture Collection.

[0007] The strain is isolated from the liver tissue of a sick Muscovy duck. The full genome sequence of the Muscovy duck Chapama virus strain MuChPv-GD2022 is shown in SEQ ID No.: 1.

[0008] After studying its genomic characteristics, the genomic analysis of the evolutionary tree showed that the Muscovy duck chapama virus was clustered into a separate branch. The acquisition of this strain enriched the types of pathogens that cause diseases in Muscovy ducks.

[0009] At the same time, the present invention also discloses the use of the Muscovy duck chapama virus strain MuChPv-GD2022 as described above for preparing a vaccine.

[0010] Finally, the present invention also discloses a vaccine, comprising inactivated or attenuated Muscovy duck chapama virus, wherein the Muscovy duck chapama virus is the Muscovy duck chapama virus strain MuChPv-GD2022 as described above.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The present invention discloses a strain of Muscovy duck chaphama virus (named as MuChPV-GD2022), which is isolated from a liver sample of Muscovy duck for the first time, and its genome characteristics and genome analysis evolution tree are studied for the first time. The results show that the sequence similarity between MuChPV-GD2022 and Pacific black duck chaphamaparvovirus 1 (GenBank accession number MT247730) is the highest, which is 76.8%. The whole genome length of MuChPV-GD2022 is 4407bp, and the G+C content is 39.1%. Two different ORFs encode non-structural protein NS1 (nt 640-2703) and structural protein VP1 (nt2696-4282). The NS1 of MuChPV-GD2022 is 2064bp in length, encodes a non-structural protein of 687 amino acids, and has an amino acid homology of less than 71.5% with NS1 of other members of the Parvoviridae family. The VP1 of MuChPV-GD2022 is 1587 bp in length and encodes a capsid protein of 528 amino acids, with an amino acid homology of less than 69.4% with other members of the Parvoviridae family.

[0013] The Muscovy duck Chapama virus is clustered into a separate branch, and the acquisition of this strain has enriched the types of pathogens that cause Muscovy duck diseases.

[0014] After the isolated Muscovy duck chapama virus strain MuChPV-GD2022 is prepared into an inactivated vaccine, the viral load in Muscovy duck tissues can be significantly reduced, and the vaccine has a good immune protection effect, and is of great value in the application research of inactivated vaccines and attenuated vaccines of Muscovy duck chapama virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a picture of normal liver tissue of a Muscovy duck.

[0016] Figure 2 The figure is a picture of the lesion of liver tissue of a Muscovy duck infected with the Muscovy duck chapama virus of the present invention.

[0017] Figure 3 It is a homology comparison diagram between the virus of the present invention and other related viruses.

[0018] Figure 4Phylogenetic tree analysis of amino acid sequence of parvovirus NS1 protein; Figure 4 The black upper triangle is the location of the NS1 protein of the virus of the present invention.

[0019] Figure 5 This is an electron microscopic observation of the morphology of virus particles after negative staining of the supernatant of the diseased sample.

[0020] Figure 6 The liver lesions of Muscovy ducks immunized with the inactivated vaccine prepared from the Muscovy duck chapama virus MuChPV-GD2022 isolate of the present invention and after challenge, wherein A is the negative control group, B is the challenge group after immunization, and C is the non-immune challenge group.

[0021] Figure 7 This is a graph showing the results of viral load detection in the liver of Muscovy ducks after immunization and challenge with the inactivated vaccine prepared from the Muscovy duck chapama virus MuChPV-GD2022 isolate of the present invention. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1 Virus isolation and identification

[0024] Since February 2022, some Muscovy duck farms in Guangdong have experienced outbreaks with hepatitis as the main pathological feature (refer to the Figure 1 and Figure 2 , Figure 1 Normal liver tissue. Figure 2 The diseased ducks had no obvious respiratory symptoms and their feces were green. At autopsy, hepatitis, swelling and bleeding or yellowing were mainly observed in the liver. The liver of the diseased duck was ground, and the supernatant was taken after repeated freezing and thawing for 3 times. After centrifugation at 10,000 rpm, the supernatant was taken, negatively stained, and observed by transmission electron microscopy. Virus particles with a diameter of about 25 nm were seen ( Figure 5 )

[0025] A new virus was found through macrovirome sequencing and sequencing result analysis. Blast analysis of the newly discovered viral nucleic acid showed that it was a new parvovirus - Muscovy duck chapama virus. The full genome sequence of the virus is the sequence shown in SEQ ID No.: 1.

[0026] The complete viral genome was obtained through metaviromics and genome sequencing. A BLAST search of the genome sequence of MuChPV-GD2022 strain was performed in the GenBank database, and the results showed that the sequence similarity between MuChPV-GD2022 and Pacific black duck chaphamaparvovirus 1 (GenBank accession number MT247730) was the highest, at 76.8% ( Figure 2 ). The whole genome of MuChPV-GD2022 is 4407bp long and has a G+C content of 39.1%. Two different ORFs encode the nonstructural protein NS1 (nt 640–2703) and the structural protein VP1 (nt2696–4282). The NS1 of MuChPV-GD2022 is 2064bp long and encodes a nonstructural protein of 687 amino acids, with an amino acid homology of less than 71.5% with the NS1 of other members of the Parvoviridae family. The VP1 of MuChPV-GD2022 is 1587bp long and encodes a capsid protein of 528 amino acids, with an amino acid homology of less than 69.4% with other members of the Parvoviridae family. The homology comparison of the virus of the present invention with other related viruses is referenced Figure 3 ;

[0027] The Neighbor-joining (NJ) method was used to construct the phylogenetic tree. Figure 4 It can be seen that on the phylogenetic tree, the MuChPV-GD2022 strain is clustered with the duck-derived Chaphamapavovirus. The MuChPV-GD2022 strain has a 47.1%-76.8% genome sequence similarity with other avian parvoviruses and a 61.8-76.8% genome sequence similarity with other duck-derived parvoviruses. The amino acid sequence of its NS1 protein has a 29.3–71.5% similarity with all known parvoviruses. The newly discovered Muscovy duck Chaphamapavovirus is far from the known duck Chaphamapavovirus and duck parvovirus in evolution, forming an independent branch.

[0028] Example 2 Verification of viral sequences

[0029] The genome sequence of the virus was obtained by macrovirome sequencing. To verify the full genome sequence of this new Muscovy duck parvovirus MuChPV-GD2022, a 4407bp sequence was amplified using four pairs of primers, and the sequences of the four fragments and two UTRs were assembled together. Finally, a 4407bp viral genome sequence of the MuChPV-GD2022 strain was obtained.

[0030] The primers used can be seen in Table 1

[0031] Table 1

[0032]

[0033]

[0034] The above amplification procedure is as follows:

[0035] The 50 μL reaction system is as follows: 2×One Step Mix 25 μL, One Step Enzyme Mix 2.5 μL, 10 μmol / L upstream and downstream primers 2 μL each, positive nucleic acid 5 μL, DEPC H 2 O 13.5 μL. PCR amplification conditions were: 94°C for 3 min; 94°C for 30 s, 52°C for 30 s, 72°C for 1 min / Kb, 35 cycles; 72°C for 5 min.

[0036] Example 3 Preparation of inactivated vaccine for Muscovy duck Chapama virus

[0037] MuChPV-GD2022 was cultured in duck embryo kidney primary cells to obtain virus liquid, and the harvested virus liquid was introduced into the inactivation tank, and 10% formaldehyde solution was slowly added, stirring while adding, and finally the final concentration of formaldehyde in the virus liquid was 0.2% (V / V). Then when the temperature in the tank rises to 37°C, inactivate for 20 hours, stirring once every 4 to 6 hours during the period. The inactivated virus liquid was inoculated into duck embryo kidney primary cells for inactivation test, and blindly propagated for two generations to observe the cell pathological changes. Take 2 portions of the oil phase and put them into the emulsification tank, stir slowly, and slowly add 1 portion of the water phase at the same time, and then stir at 4000r / min for 30 to 40 minutes. After emulsification, take 10mL of the vaccine and add it to a 15mL centrifuge tube, centrifuge at 3000r / min for 15 minutes, and the water phase precipitated at the bottom of the tube should not exceed 0.5mL. Subsequently, quantitatively divide and store at 2 to 8°C.

[0038] Thirty healthy 1-day-old Muscovy ducks were selected and randomly divided into 3 groups, namely, negative control group, immunized challenge group and non-immunized challenge group. The first immunization was carried out at 7 days of age. The ducks in the immunized challenge group were subcutaneously inoculated with 0.2 mL of inactivated vaccine in the neck, and the ducks in the non-immunized challenge group were inoculated with 0.2 mL of PBS. The negative control group was not treated. The second immunization was carried out at 21 days of age, and the vaccination method and dose were the same as described above. Clinical abnormalities were observed daily to evaluate the safety of the vaccine. The ducks in the immunized challenge group and the non-immunized challenge group were challenged with Chapama virus at 35 days of age, and the negative control group was not treated.

[0039] The results of animal experiments showed that the MuChPV-GD2022 inactivated vaccine has good safety, can significantly reduce the viral load in the liver of Muscovy ducks, alleviate the degree of tissue lesions, has good immune protection effects, and is an ideal vaccine candidate strain.

[0040] Through the above experiments, we can find that:

[0041] 1. The Muscovy duck Chapama virus of the present invention is clustered into a separate branch, and the acquisition of this strain enriches the types of pathogens that cause diseases in Muscovy ducks.

[0042] 2. The discovery of the strain of the present invention, and the inactivated or attenuated vaccine prepared by using the strain, are of great significance to the rapid development of the Muscovy duck breeding industry.

[0043] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A Muscovy duck chapama virus strain, characterized in that: The Muscovy duck chapama virus strain is the Muscovy duck chapama virus MuChPv-GD2022 ( Chaphamaparvovirus MuChPv-GD2022), the deposit number is: CCTCC NO: V202311; the deposit date is March 1, 2023, and the depository unit is: China Center for Type Culture Collection.

2. The Muscovy duck chapama virus strain according to claim 1, characterized in that The complete genome sequence of the Muscovy duck chapama virus MuChPv-GD2022 is shown in SEQ ID No.

1.

3. Use of the Muscovy duck chapama virus MuChPv-GD2022 as claimed in claim 1 to prepare a vaccine.

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