共表达膜结合型与可溶性H9N2亚型禽流感病毒HA蛋白的重组基因Ⅶ型新城疫病毒的构建及免疫效果评价

doi:10.11843/j.issn.0366-6964.2024.05.030

Abstract

Abstract: The purpose of this study was to construct a recombinant genotype Ⅶ Newcastle disease virus (NDV) that can efficiently and stably express H9N2 subtype avian influenza virus (AIV) HA protein. It is expected to develop a bivalent vaccine against NDV and H9N2 AIV to provide a new defense idea for the control of H9N2 AIV and NDV, and has important reference significance for the construction and optimization of NDV vaccine vector. A reverse genetic technique was used to insert the full-length membrane-bound HA and another soluble HA protein with truncated transmembrane domain gene into the non-coding region between the P and M genes of NDV using the genotype Ⅶ NDV-weakening strain rDHN3-mF as the backbone. The recombinant virus rDHN3mF-HA and rDHN3mF-2HA of gene type VII NDV attenuated virus which can express HA protein stably and efficiently was obtained. The stability and biological characteristics of recombinant virus were detected by chicken embryo inoculation, Western blot, HA, MDT and other experiments. Recombinant virus was immunized with 7 d SPF chickens and serum HI assay were measured. And the group was subjected to challenge protection experiments 28 d after immunization. The recombinant viruses were passed through fifteen generations in chicken embryos without mutations.Western blot results demonstrated that recombinant viruses can express specific HA proteins stably and efficiently; The growth curves illustrates the recombinant viruses have similar replication and low virulence characteristics to parental strains; Groups of SPF chickens immunized with recombinant virus produce specific HI antibodies against NDV or H9N2 AIV.The challenge protection test showed that the recombinant viruses can produce 100% protection against chickens after challenge, and the results of throat and swab detection indicate that recombinant virus can significantly reduce the amount of NDV and H9N2 AIV shedding. The results of tracheal and lung qPCR showed that rDHN3mF-2HA significantly reduced the shedding of H9N2 AIV in organs than rDHN3mF-HA 3 dpc. The results indicated that the recombinant NDV of genotype Ⅶ coexpressing membrane-bound and soluble H9N2 subtype avian influenza HA protein was successfully constructed, which laid a foundation for the development and application of a new type of recombinant genetic engineering live vector vaccine to prevent H9N2 AIV and NDV.

Key words: H9N2 subtype avian influenza, HA protein, Newcastle disease virus, immunogenicity, vector vaccines, immunoprotective efficacy

CLC Number:

S858.315.3

LÜ Yadi, YANG Jie, XIE Wenting, XU Ting, CHEN Ruiai. Construction and Evaluation of the Immune Effect of Recombinant Genotype Ⅶ NDV Strain Co-expressing Membrane-bound and Water-soluble HA Protein of Avian Influenza Virus H9N2[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2123-2134.

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Construction and Evaluation of the Immune Effect of Recombinant Genotype Ⅶ NDV Strain Co-expressing Membrane-bound and Water-soluble HA Protein of Avian Influenza Virus H9N2

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