Admittedly, there are a majority of infectious poultry diseases not only Newcastle disease (ND), but also other disease such as infectious bursal disease (IBD)[19, 20], infectious laryngotracheitis(ILT) [21, 22], hypervirulent fowl adenovirus (FADV) infection and avian influenza (AI), which endanger the poultry industry. Vaccination is a powerful and necessary mean to the controlling of these diseases. Vaccines of Newcastle disease (ND), as a compulsory immunization to most chicken farms, have been used worldwide. Recombiant NDVs have been proven to be an excellent viral vector to delivery various heterogenous genes against a serial of poultry diseases including IBD, ILT, and AI[25–27]. Therefore, the development of ND as a vaccine vector may be a powerful mean to the poultry disease control. By now, several NDV strains have been rescued by reverse genetic techniques. This manipulation has facilitated the study of the molecular mechanisms of the viral pathogenicity and also provided a powerful platform for the research using NDV as vaccines vectors.
The reverse genetics system for NDV is difficult and complex compared to DNA viruses or positive-sense RNA viruses [28–30]. First of all, the naked genomic RNA of NDV is not infectious. It is only functional when being encapsidated with protein complex, ribonucleoprotein (RNP) formed by the viral NP P and L proteins. Secondly, the genome length and genome complexity have made that the cloning of the full-length NDV genome remains the most challenging and laborious of all the steps[29, 30]. Moreover, the NDV genome has to meet particular requirements for successful rescue such as the rule-of-six[31, 32] and generation of the precise 3’ and 5’ ends[30, 32]. To get the precise integral NDV genome, The traditional method is dividing the NDV genome into a set of short fragments from 6 to 11, and it takes months even years to complete for the multiple DNA fragments cloning[10, 13, 33–37]. In this study, the genome of NDV was divided into three fragments 7bp, 5kb and 3kb. Compared to the approaches before, the design in this study needs much shorter time for the full-length genome cloning. As the amplification of a cDNA fragment around 10 kb is available by using the commercial high fidelity reverse transcription PCR by now, it seems the strategy of dividing the whole NDV genome into two parts is possible, which need to be explored in the future.
A majority of recombinant NDVs expressing one foreign gene (FG) as vaccines or biological therapies express through either an independent transcription unit (ITU) or an internal ribosomal entry site (IRES) approach[27, 35, 38]. The efficacy of these two strategies has been investigated, showing that the ITU approach was more efficient than that through the IRES approach. The expression levels of FGs through ITU at different genomic locations were variable and the optimal insertion site in NDV genome for efficient expression has been identified as the non-coding region between the P and M genes[15, 16, 39]. Here, the RFP gene was inserted into the NDV genome at the site of the non-recoding region between P and M gene. Our data showed that the RFP could be stably expressed in a high level in this region, and the recombinant virus rZM10-RFP did not showed any significantly differences in MDT and ICPI, indicating that the noncoding region between the P and M genes is a suitable position for rZM10 to express an FG and provides the design strategy for the recombinant bivalent vaccines.
Pathogens especially the viruses could infect corresponding host cell types, results in replicating in specific host organs[40–42]. The ZM10 strain has an obvious preference for replication the intestine though it could infect both the respiratory and intestinal tract. This characteristic makes the ZM10 to be an ideal vector to express the protective antigen of intestinal pathogens, therefore, an important application of our NDV rescue platform would be to use the recombinant viruses as vaccine vector for the expression of protective antigens against avian or other animals’ diseases to develop multivalent vaccines.