Identification of Newcastle disease virus P-gene editing using next-generation sequencing

Abstract

Avian paramyxoviruses 1 has the ability to edit its P gene to generate three amino-coterminal proteins (P, V and W), but its kinetic change is unclear. In this study, next-generation sequencing (NGS) was used to analyze the P-gene editing of Newcastle disease virus (NDV). Transcriptome analysis of chicken embryonic tissues and bursa of fabricius showed the P-gene editing frequencies were 45.46-52.70%. To investigate the rules of P-gene editing along time, the ratio of PVW was determined by PCR based deep sequencing at multiple time points in cells infected with velogenic and lentogenic strain respectively. The results confirmed similar editing frequencies with transcriptome data and the PVW ratios were stable along time among different NDVs, but had a greater V-gene transcript on velogenic strain infection (P<0.001), which were different from previous reports. Also, it was shown that the number of inserted G residues in P-derived transcripts was not limited to +9G, and +10G transcripts were identified. These results confirmed the NDV P-gene editing frequencies and provided a novel point of view on NDV P-gene editing with NDV virulence.

Keywords: P-gene editing frequencies; V-gene transcript; next-generation sequencing; transcriptome analysis.

Fig. 1.

Transcriptional analysis of viral genes from transcriptome sequencing. A. Transcriptional analysis of viral genes from chicken embryonic tissues at 36 hr post F48E9 or La Sota infection. B. The percentages of P, V and W transcripts in Newcastle disease virus infected embryonic tissues. C. Transcriptional analysis of viral genes from chicken bursa fabricius at 72 hr and 96 hr post F48E9 infection. D. The percentages of P, V and W transcripts in F48E9 infected chicken bursa fabricius.

Fig. 2.

The P-gene editing frequencies of different time points post NDV infection. A. Flow diagram of high-throughput sequence. B. The number of G insertion during Newcastle disease virus infection. Origin 8.0 was used to create 3D picture, red line represents F48E9 infection, and blue represent La Sota infection. C. The relative percentages P-gene editing frequencies of F48E9 and LaSota. D. The dynamic changes of P, V and W of F48E9 and LaSota. ***P<0.001 between groups.