The Replication Of HaSNPV In HzAm1 Cells

The real-time quantitative PCR method was used in this thesis to study the the difference of HaSNPV replication in HzAm1 cells infected at exponential phase and at stationary phase, as well as the transcription profiles of nine HaSNPV genes in infected HzAm1 cells. The effect of HaSNPV infection on the transcription of RpL13 in host cells was also studied. Chapter 1 was a literature review. It contained the general introductions of taxonomy, replication, molecular biology and application of baculovirus. The principle of real-time quantitative PCR was introduced. And the purpose of this thesis was introduced. In chapter 2, we described the use of real-time quantitative PCR to characterize viral DNA replication in HzAM1 cells. In general, HaSNPV started to replicate at 7 hours post infection (hpi) and budded virus (BV) was released firstly from infected HzAm1 cells at 18hpi. The amounts of entered BVs released BVs, progeny virions and viral protein product in the infected exponential phase cells were significantly higher than that of the stationary phase. But there were almost no difference on the HaSNPV replication rate between the two different phases of the cells. The doubling time of HaSNPV replication during 14-20hpi was 1.7h in infected exponential cells and 1.9h in stationary cells. Therefore, the major reason for higher amounts of viral DNA synthesized in exponential cells may due to the higher amounts of BV entered the cells rather than the difference of viral replication rate. Up to 144hpi, 25% of the progeny virions budded from the cells infected at exponential phase, but only 13% of the progeny virions budded from the cells infected at stationary phase. It was suggested that cells infected at the exponential phase were more propitious for BV entry and BV release than that at the stationary phase. The data of membrane fluidity determination of the cells at exponential and stationary phases suggested that the fluidity of cell membrane may played an important role in BV entry and BV release. In Chapter 3, we studied the transcription profiles of nine HaSNPV genes by using real-time quantitative PCR. The transcription profiles of nine selected HaSNPV genes including predicted early phase genes, early and late phase genes, late phase genes and very late phase genes was analyzed using reverse transcription and real-time quantitative PCR. The result indicated for most of these genes the transcriptional start point was coincident with the prediction of their promoter. But the predicted early phase gene pkip started to be transcribed at late phase, and the predicted early and late phase gene ha107 was only transcribed at late phase. At 72hpi, the transcription level of these genes was generally the highest. The transcription level of polyhedrin was obviously higher than other genes. The iap2 was the second highly transcribed gene indicating that it might be functional. The HaSNPV p10, unlike its homologue in AcMNPV, was not highly transcribed. Chapter 4 was about sequence analysis of RpL13 gene from HzAm1 cells and the effect of HaSNPV infection on the transcription of RpL13. The cDNA and genome DNA of ribosomal protein L13 (RpL13) gene were cloned from Helicoverpa zea cells (HzAM1). Sequence analysis indicated that the open reading frame of RpL13 was composed of 666bp without intron, encoding a protein of 25 kDa. The sequence was used to construct phylogentic trees with other known sequences of 15 animal RpL13s. The result suggested it has similar topology with the classical taxonomy. Quantitative RT-PCR assay revealed that the transcription level of RpL13 declined in the HzAm1 cells infected by HaSNPV. At 96hpi, the mRNA copies of RpL13 were about 8% to that of healthy cells.