Functional Analysis Of Helicoverpa Armigera Nucleopolyhedrovirus (HearNPV) ORF33

Helicoverpa armigera single nucleopolyhedroviru(sHearNPV)orf33 is a functional gene which function is not yet entirely clear. The gene is a 717bp open reading frame located between 27566bp-28282bp, that encodes a 238-residue protein with a predicted molecular mass 28.4kDa. HA33 protein was located in the BV capsule shell of HearNPV, which is the structural protein BV-e31. For further research the gene function in the viral life cycle, we study its effect on the virus titer, DNA replication and virulence through the deletion of 33rd gene. Our study provide a theoretical basis for the baculovirus genetic improvement, genetic engineering and the development of more effective insecticides and virus vectors.The orf33 deletion recombination Bacmid (HaBacHZ8^△33-egfp) checked by PCR and restriction enzyme digestion were transfected into insect cell HzAM1 by lipofection and observed by fluorescent microscope, wild-type Bacmid (HaBacHZ8^wt) as control. 3 -5 days after culture, cytopathic effect and the green fluorescent cells were observed. The result shows that HaBacHZ8^△33-egfp has replicated and expressed in the HzAM1 cell, at the same time it proved a successful orf33 deletion. Through the re-infection HzAM1 cells after collecting the supernatants, a large number of cells showed green fluorescence and cell number and fluorescence intensity increased with time goes on.The result indicates that it is still producing infectious virus particles even though orf33 was deleted, and that reportor gene EGFP of deletion virus is highly expressed. The Western Blot test demonstrated orf33 had been deleted in orf33 deletion recombination virus (HearNPV^△33-egfp). We infected HzAM1 cells with multiplyed HearNPV^△33-egfp ,wild type virus (HearNPV^wt) as the contro, and collected the virus at different times after infection.The virus titer was determined by TCID₅₀ methed to draw the virus gro^wth curve.The result shows that HearNPV^△33-egfp had lower capacity of producing virus particles than HearNPV^wt. To further assess the virus replication, we collected the HzAM1 cells at different time after infection and extracted the viral genomic DNA, qPCR technology was used to quantified the viral genome DNA.The result shows that the DNA replication level of HearNPV^△33-egfp is similar to HearNPV^wt in infected cells. Bioassay result shows that the LT₅₀ of HearNPV^△33-egfp is significantly longer than that of HearNPV^wt .In summary, orf33 is not a responsible gene for replication of viral genome DNA, but it plays an important role in virion production, and is an important functional gene for viral infections.