Construction And Biological Characterization Of Recombinant Helicoverpa Armigera Nucleopolyhedrovirus(HearNPV) With Ar1b Gene

In this study, A per os infectivity recombined HearNPV, Ar1b-HearNPV, was constructedby repair the polyhedra gene (ph) to Ar1b-bacmid and HZ8-bacmid. The recombinantHearNPV was identified as an improved recombinant through bioassays. The virulence, dailycumulative weight gain and daily diet consumption from different instars larvae infected byAr1b-HearNPVwas investigated. And the differences of ALP activity and ALP expressionprofile under HearNPV infection to host larvae were studied also, following were the majorcollusions:The ph with its own promoter in the Ha.p-ph-HTb vector was repaired to Ar1b-bacmidand HZ8-bacmid facilitated by helper plasmid. The Ar1b-HearNPV and HZ8-HearNPV OBscan be found after transfection to HzAM1cell lines using, respectively. The viral growthcurve indicated that Ar1b-HearNPV could produce BVs as well as HZ8-HearNPV. The viralDNA replication curve showed that Ar1b-HearNPV DNA had less copies than HZ8-HearNPVhad in HzAM1cell lines.The lethal dose tests showed that the Ar1b-HearNPV owend a higher virulence thanHZ8-HearNPV. The lethal time tests indicated that the Ar1b-HearNPV can speed death oflarvae.The bioassay results of2nd,3rdand4thinstars larvae infected with high dose (120PIBs/larva) and low dose (30PIBs/larva) demonstrated that the high dose of Ar1b-HearNPVhad a similar final mortality to HZ8-HearNPV and wt-HearNPV infection. For larvae thatinfected with the low dose, Ar1b-HearNPV could kill the larvae much quicker thanHZ8-HearNPV and wt-HearNPV, although the HZ8-HearNPV and wt-HearNPV treatmentsobtained a higher final mortality.The daily cumulative weight gain of different instar larvae showed that Ar1b-HearNPVcan restrain the development of the infectec larvae. On the other hand, compared withwt-HearNPV and HZ8-HearNPV infections, Ar1b-HearNPV infected larvae had the lowestdaily diet consumption regardless the larvae instars, while CK owned the highest one. Totalaccumulation of diet consumption analysis illustrated that Ar1b-HearNPV had the lowest dietconsumption. This kind of inhibition on diet consumption would be deceased with the rise of the larvae's instars.The enzyme activity and gene expression level were determined at different stages afterviral infection with different infection titer. The results indicated that the ALP activity and itsgene expression level were inhibited significantly by HearNPV infection and this inhibitionstrength was co-related with viral infection titer and the time post infection. The viral invasionto host cell possibly could be facilitated by inhibition on ALP activity by virus. One of themechanisms of ALP activity inhibition by viral infection was down regulation of ALPexpression by virus.