Expression of two foreign genes by a Newcastle disease virus vector from the optimal insertion sites through a combination of the ITU and IRES-dependent expression approaches

Authors: HE, LEI - Hunan University Of Science And Technology; ZHANG, ZHENYU - Harbin Veterinary Research Institute; Yu, Qingzhong

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/31/2020
Publication Date: 4/28/2020
Citation: He, L., Zhang, Z., Yu, Q. 2020. Expression of two foreign genes by a Newcastle disease virus vector from the optimal insertion sites through a combination of the ITU and IRES-dependent expression approaches. Frontiers in Microbiology. 11:769. https://doi.org/10.3389/fmicb.2020.00769.
DOI: https://doi.org/10.3389/fmicb.2020.00769

Interpretive Summary: Newcastle disease (ND), caused by virulent strains of Newcastle disease virus (NDV), is one of the most devastating infectious diseases of poultry. Vaccination with live attenuated NDV vaccines has been a common practice around the world to prevent poultry from ND for over sixty year. Since the reverse genetics technology was used to manipulate the NDV genome, many NDV vaccine strains have been developed as vectors to express foreign genes for vaccine and gene therapy purposes. A majority of these NDV vectors express only a single foreign gene or two foreign genes but from suboptimal insertion sites in NDV genome. Clinical trials with these vaccine candidates obtained various levels of protections against the targeted pathogen. In the present study, we generated NDV LaSota vaccine strain-based recombinant viruses to express two foreign genes from the identified optimal insertion sites. Evaluation of these recombinant viruses showed that they maintained their growth ability in chicken embryos and were slightly attenuated in chickens when compared with their parental virus. These viruses expressed two foreign gene products in cell cultures as efficient as the recombinant viruses vectoring only a single foreign gene. These results suggest that the NDV LaSota vector is a safe and promising vector to express two foreign genes from the identified optimal insertion site for use as a multivalent vaccine.

Technical Abstract: Many Newcastle disease virus (NDV) strains have been developed as vectors to express a foreign gene (FG) for vaccine and cancer therapy purposes. The non-coding region between the phosphoprotein (P) and matrix protein (M) genes and the non-coding region behind the NP gene open reading frame (ORF) in the NDV genome have been identified as the optimal insertion sites for efficient FG expression through the independent transcription unit (ITU) and the internal ribosomal entry site (IRES) dependent expression approaches, respectively. To date, however, the majority of these NDV vectors express only a single or two FGs from suboptimal insertion sites in the NDV genome, obtaining various levels of FG expression. To improve the FG expression, we generated NDV LaSota vaccine strain-based recombinant viruses expressing two FGs, GFP, and RFP, from the identified optimal insertion sites through a combination of the ITU and IRES-dependent approaches. Biological assessments of the recombinant viruses indicated that the recombinants expressing two FGs were slightly attenuated with approximately one order of magnitude lower in virus titers when compared to the viruses containing a single FG. The FG expression efficiencies from the two-FG viruses were also lower than those from the single-FG viruses. However, the expression of two FGs from the optimal insertion sites was significantly (p < 0.05) higher than those from the suboptimal insertion sites. The expressions of FGs as monocistronic ITU were approximately 4-fold more efficient than those expressed by the bicistronic IRES-dependent approach. These results suggest that the NDV LaSota vector could efficiently express two FGs from the identified optimal insertions sites. The ITU strategy could be used for “vectoring” FGs in circumstances where high expression of gene products (e.g., antigens) is warranted, whereas, the IRES-dependent tactic might be useful when lower amounts of IRES-directed FG products are needed.