| Virulent strains of Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) can cause serious respiratory diseases in poultry worldwide. These viruses are highly contagious and possess serious threats to food safety, public health, and biosecurity. The aMPV disease is usually accompanied by secondary bacterial infection that can increase high incidence and mortality, resulting in the significant economic losses to the poultry industry. Vaccination combined with strict biosecurity practices has been the recommendation for controlling both NDV and aMPV diseases in the field. Naturally occurring lentogenic NDV strains, such as B1, VG/GA, and LaSota strains, are routinely used as live vaccines throughout the world to prevent Newcastle disease. However, there are no low-virulence strains of aMPV that can be directly used as live vaccines. Live, attenuated aMPV vaccines appear to be effective against aMPV disease, but the stability and safety of these live attenuated vaccines remain a concern because of the potential of virulence revertance. Thus, there is a pressing need to develop a save, effective and economic bivalent vaccine that can be readily administered in the field to control these viral diseases. Therefore, this research proposed to develop NDV LaSota vaccine strain-based recombinant viruses that express aMPV fusion (F) protein and/or glycoprotein (G) as a bivalent vaccine against these viral diseases.The NDV LaSota vaccine strain was chosen as a vector to generate recombinant viruses in this study. Firstly, a reporter gene, green fluorescence protein (GFP), was inserted into the intergenic region between F and HN genes of NDV genome, and successfully rescued a recombinant virus expressing GFP by using a reverse genetics approach. Secondly, the recombinant LaSota virus expressing the aMPV-C G protein was generated. Subsequently, the recombinant virus was characterized in vitro and in vivo. The results showed that the recombinant virus, rLS/aMPV-C G, was slightly attenuated in vivo, yet maintained similar growth dynamics, cytopathic effects, and virus titers in vitro when compared to the parental LaSota virus. Vaccination of turkeys with rLS/aMPV-C G induced complete protection against velogenic NDV challenge and partial protection against pathogenic aMPV-C challenge. The results, suggest that expression of the aMPV-C G protein alone is not sufficient to provide full protection against an aMPV-C infection. Expression of additional aMPV-C immunogenic protein(s) may be needed to induce stronger protective immunity against the aMPV-C disease. Thirdly, to prove whether the LaSota vaccine can express two foreign genes, another reporter gene, the red fluorescence protein (RFP), was added into the rLS/GFP vector. The RFP and an internal ribosome entry site sequence (IRES) were inserted into the F gene end as a second open reading frame (ORF). A recombinant NDV LaSota virus that expressed both RFP and GFP was rescued. Based on this rLS/I-RFP-GFP construct. Finally, the NDV recombinant virus expressing the aMPV-C F and G protein was successfully generated by replacing the RFP and GFP with the F and G genes, respectively. This recombinant virus vectoring the aMPV-C F and G displayed similar growth dynamics, virus titer and lentogenic pathotype as the parental LaSota strain. SPF turkeys vaccinated with rLS/aMPV-C F G were completely protected against the challenge with a virulent NDV strain, NDV/CA02. About 60-80% of rLS/aMPV-C G vaccinated birds developed an aMPV-C specific antibody response following the first vaccination and all birds seroconverted after the booster. After challenge with the pathogenic aMPV-CO virus, vaccinated turkeys displayed milder clinical signs and significantly less virus shedding in tracheal tissues than the control birds that were vaccinated with the parental LaSota strain.In summary, the results demonstrated that vaccination of SPF turkeys with the NDV LaSota vaccine strain-based recombinant virus expressing the aMPV-C F and G proteins provided complete protection against the virulent NDV/CA02 strain challenge and significant protection against the pathogenic aMPV-C challenge. Thus, this recombinant virus could be used as a bivalent vaccine against NDV and aMPV-C diseases. In addition, the recombinant NDV LaSota vaccine vector can be used to develop multivalent vaccines against other avian viral diseases and may also have potential applications in gene therapy or anti- cancer research.?...
|
PDF Full Text Request