Abstract
Avian paramyxovirus serotype 3 (APMV-3) causes infection in a wide variety of avian species, but it does not cause apparent diseases in chickens. On the contrary, APMV-1, also known as Newcastle disease virus (NDV), can cause severe disease in chickens. Currently, natural low virulence strains of NDV are used as live-attenuated vaccines throughout the world. NDV is also being evaluated as a vaccine vector against poultry pathogens. However, due to routine vaccination programs, chickens often possess pre-existing antibodies against NDV, which may cause the chickens to be less sensitive to recombinant NDV vaccines expressing antigens of other avian pathogens. Therefore, it may be possible for an APMV-3 vector vaccine to circumvent this issue. In this study, we determined the optimal insertion site in the genome of APMV-3 for high level expression of a foreign gene. We generated recombinant APMV-3 viruses expressing the green fluorescent protein (GFP) by inserting the GFP gene at five different intergenic regions in the genome. The levels of GFP transcription and translation were evaluated. Interestingly, the levels of GFP transcription and translation did not follow the 3′-to-5′ attenuation mechanism of non-segmented, negative-sense RNA viruses. The insertion of GFP gene into the P-M gene junction resulted in higher level of expression of GFP than when the gene was inserted into the upstream N-P gene junction. Unlike NDV, insertion of GFP did not attenuate the growth efficiency of AMPV-3. Thus, APMV-3 could be a more useful vaccine vector for avian pathogens than NDV.
Highlights
Among the members of the order Mononegavirales, the family Paramyxoviridae includes pathogenic and non-pathogenic viruses whose natural hosts include avian and aquatic animals as well as humans (Falk et al, 2008; Nylund et al, 2008; Lamb and Parks, 2013)
Our results showed that the level of green fluorescent protein (GFP) expression did not follow the 3 -to-5 attenuation, since the insertion of GFP gene into the P-M gene junction resulted in an expression level higher than that when the gene was inserted into the upstream N-P gene junction
The GFP mRNA transcription levels detected in the P/M-GFP virus-infected cells were 2.0- and 8.8-fold greater than those of the N/P-GFP virus-infected cells, respectively (Figures 3C,D). These results indicate that the higher level of GFP expression observed in the P/M-GFP virus-infected cells compared to the N/P-GFP virus-infected cells is due to a higher transcription level of the GFP gene, suggesting that the P-M gene junction is the optimal insertion site for foreign gene expression in the Avian paramyxovirus serotype 3 (APMV-3) vector
Summary
Among the members of the order Mononegavirales, the family Paramyxoviridae includes pathogenic and non-pathogenic viruses whose natural hosts include avian and aquatic animals as well as humans (Falk et al, 2008; Nylund et al, 2008; Lamb and Parks, 2013) Members of this family are primarily characterized by possessing a linear, non-segmented negative-sense (NNS) RNA genome, which encodes 6–10 viral genes tandemly (Lamb and Parks, 2013). Replication of paramyxoviruses is generally limited to the respiratory tract, and do not spread to other organs These viruses are highly safe to use as viral vectors (Lamb and Parks, 2013)
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