The Mechanism Of Liquefaction Of Silkworm, Bombyx Mori Host Infected With Baculovirus

Infection of larvae by baculovirus resulted in liquefaction of insect host, which isconducive to the spread of the virus to the environment. This view was supported byinfection of silkworm (Bombyx mori) larvae with B. mori nucleopolyhedrovirus (BmNPV).Non-liquefaction of host, however, was detected in permissive B. mori larvae infected withAutographa california nucleopolyhedrovirus (AcMNPV). The activity of cathepsin(V-CATH) and chitinase (V-CHIA) are considered as the main factors for liquefaction ofthe insect host.To investigate the reason of non-liquefaction in the AcMNPV-infected silkworm larvae,in the first place, AcMNPV-CATH and BmNPV-CATH, AcMNPV-CHIA andBmNPV-CHIA were expressed using E.coli expression system, and the expressionproducts were purified using Ni-NTA affinity chromotography techniques. Western blotanalysis showed that, the two kinds of V-CATHs and V-CHIAs were expressed successfullyin E.coli BL21(DE3)plysS strain, but the expression products were detected mostly in theprecipitate and combined with Ni-NTA column poorly. When under denaturing condition,highly purified V-CATH were obtained, but V-CHIA were not obtained. Comparing theiractivity, it showed that the cathepsin activity of the unpurified BmNPV-CATH was about27%lower than that of AcMNPV-CATH, while the purified BmNPV-CATH was about2%lower than that of AcMNPV-CATH, and that it was no obvious difference between theunpurified BmNPV-CHIA and AcMNPV-CHIA in chitinase activity. These results suggestthat non-liquefaction in AcMNPV-infected silkworm larvae may be not caused by thedistinction of those two kinds of v-cath and v-chiA gene sequences.In the second place, BmNPV v-cath (Bmcath) and v-chiA gene (BmchiA) wereconstructed simultaneously or respectively into AcMNPV using Bac-to-Bac baculovirusexpression systems, in which v-cath and v-chiA genes were driven under the control of thep10promoter (Pp10) and polyhedrin promoter (Ppolh), respectively. Meanwhile, AcMNPVv-cath (Accath) and v-chiA gene (AcchiA) were constructed simultaneously or respectivelyinto AcMNPV with the same approach, and then six recombinant AcMNPVs weregenerated. Results showed that permissive infection and liquefaction of B. mori larvae hostby all of the recombinant AcMNPVs were observed, suggesting that the overexpression ofv-cath and v-chiA genes play an important role in the liquefaction of B. mori larvae host.Comparing the time of larvae liquefected and percentage of larvae liquefected to infected, we found that v-cath might have more obvious action than v-chiA in liquefaction. Theactivity of cathepsin and chitinase in the hemolymph of AcMNPV-and recombinantAcMNPVs-infected B. mori larvae indicated that the activity of cathepsin and chitinaseincreased by the cope number of v-cath and v-chiA genes, respectively. Our finding suggestthat non-liquefaction of B. mori larvae infected by AcMNPV is caused by the low activityof cathepsin.Subsequently, we investigated the degradation of the EGFP in recombinant AcMNPV-and BmNPV-infected cultured cells and silkworm larvae, respectively. Western blotanalysis of EGFP showed that the degradation occured in recombinant BmNPV-infectedsilkworm larvae strain54A as well as in hybrid species, indicating that degradation of EGFPin the hemolymph of baculovirus-infected silkworm larvae was not effected by thedistinction of silkworm strain but a common phenomenon. But it was not occured inrecombinant BmNPV-infected BmN cells, in recombinant AcMNPV-infected silkwormlarvae or in recombinant AcMNPV-infected Sf21cells. Degradation of EGFP-His in vitrowas preformed to analysis degradation degree of EGFP. The results showed that degradationappeared at position about2kDa from the C-terminal of EGFP. By examination of theproteinase activity, we found that proteinase we had detected was V-CATH, and it was themain reason for degradation of EGFP. The examination of V-CATH activity and thedegradation experiments in vitro showed that high activity of V-CATH in the recombinantBmNPV-infected B. mori larvae hemolymph was detected, which resulted in degradation ofEGFP. Whereas V-CATH might be inactive or lowly active in the recombinantAcMNPV-infected B. mori larvae haemolymph, which may be the reason fornon-degradation of EGFP even in the late stage of infection.The advantage of non-liquefaction of host and non-degradation of foreign gene productsin AcMNPV-infected silkworm larvae may be utilized to character the function of interestedgene by constructing recombinant AcMNPV as a gene delivery and transient expressionvector into silkworm in vivo.