Reverse Genetics And Thermostable Application Of Newcastle Disease Virus NDV4-C Strain

Newcastle disease (ND), caused by Newcastle disease virus (NDV), is an acute and highlycontagious disease which affects many domestic and wild avian species and causes severe economiclosses to the poultry industry in the world. Currently, vaccination remains a primary strategy for NDcontrol.NDV4vaccine strain is being widely used in the tropical and sub-tropical countries of SoutheastAsia and Africa due to its thermostability and long-lasting immunity. NDV4-C is an avirulent andthermostable strain which is screened by passaging in specific-pathogen-free (SPF) chicken eggs. Todate, the reverse genetics of NDV4strain is unavailable, which accounts for the limited research ofNDV4strain. Based on the systemic sequence analysis of NDV4-C, in this study, we established thereverse genetics system for the avirulent and thermostable NDV4-C strain. Successful recovery ofNDV4-C was achieved by using either T7RNA polymerase or cellular RNA polymerase II to drivetranscription of the full-length virus antigenome from cloned cDNA. The recovered viruses rNDV4-C(T7) and rNDV4-C (CMV) showed similar thermostability and biological properties as the parentalstrain NDV4-C. The potential of rNDV4-C (T7) to serve as a viral vector was assessed by generating arecombinant virus, rNDV4-eGFP, which expressed enhanced green fluorescent protein (eGFP). TherNDV4-eGFP could stably carry and express eGFP for at least fifteen passages. These results indicatedthat the rNDV4-C (T7) has the potential to serve as a live vaccine vector.It is reported that the large protein is associated with the temperature-sensitive in theparamyxovirus. Here, we engineered these chimeras rNDV4-La-L and rLaSota-V4-L by exchanging theL gene between the thermostable NDV4-C strain and the thermolabile LaSota strain to evaluate the roleof large protein in NDV4-C thermostability. Thermostability of chimeric viruses was examined bydetermining the infectious titre after incubation for different periods of time at55°C. The characteristicof chimeric viruses was also evaluated in embryonated SPF chickens and eggs. Chimera rNDV4-La-Lposses decreased thermostability and lower viral titre, and rather than vise versa, suggesting that the Lgene contributes to the thermostability of NDV4-C. Furthermore, the in vitro replication assay with aminigenome system expressing luciferase showed that the L activity of NDV4-C was higher than that ofLaSota, which contributes to the thermostability of NDV.Routine and/or intensive vaccinations have been shown to be effective in preventing disease andmortality but not in preventing viral shedding, transmission, and infection, NDV4vaccine inducedlower humoral immune response antibody level than the LaSota, Clone30, and Hitchner B1, but couldinduce good mucosal immune response. We engineered two chimeras, rNDV4-HNLL01andrNDV4-MuF/HNLL01, which combined the thermostability of the NDV4-C strain with the goodimmunogenicity of the velogenic sub-genotype VIId Go/CH/HLJ/LL01/08(LL01/08) strain.Thermostability of these chimeras was confirmed by determining the infectious titre after incubation for different periods of time at55°C. Both chimeras fulfilled the criteria for lentogenic virus.Two-week-old SPF chickens, which were inoculated by the oculonasal route with each of thesechimeras, the rNDV4-C strain, or the widely used NDV vaccine strain LaSota, respectively, produceddifferent levels of humoral IgG and mucosal IgA antibodies. The IgG antibody level induced by LaSotawas higher than that induced by the other groups, while these chimeras induced higher levels of IgAantibody than that of rNDV4-C in sera and dodecadactylon. The animal experiments demonstrated thatall vaccinated groups were fully protected from morbidity and mortality. Importantly, bothrNDV4-MuF/HNLL01-and LaSota-vaccinated chickens showed a significant reduction in viralshedding in the cloaca compared to chickens of the other vaccinated groups. In summary, our datasuggest that the lentogenic and thermostable rNDV4-MuF/HNLL01strain, which induces enhancedmucosal immune response, could be a promising vaccine candidate for the control of current NDepidemics.