Studies On Proteins Involved In The Silkworm Tissues Against Infection Of Densovirus China (Zhenjiang) Strain

Bombyx mori Densovirus (BmDNV) is one of the four main viruses infecting silkworm. The BmDNV infects silkworm mainly through oral administration, and replicates only in the columnar cells of midgut epithelium, which is different from other densoviruses. The symptom of diseased silkworm includes body transparency, diarrhea, vomiting and so on. Thus the densonucleosis is a kind of severe viral diseases to the silkworm in sericultural production. The susceptibility to BmDNV differs according to the silkworm strains. Some strains are absolutely non-susceptible to BmDNV, while few are easily to be infected. The molecular mechanism of the silkworm susceptibility to BmDNV at protein level is unknown presently. In this paper, with two near isogenic silkworm strains, JS and NIL, respectively susceptible and non-susceptible to BmDNV Zhenjiang Strain (BmDNV-ZJ) as materials, proteins in the silkworm tissues related to the infection of BmDNV-ZJ were studied by bioassay, DIG-labeled ELISA, two dimensional electrophoresis (2-DE) and matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF MS).The results will be helpful for the understanding of the molecular mechanism of silkworm susceptibility to BmDNV and the pathogenesis of the silkworm densonucleosis.The purified virus was incubated with extract from different tissues of susceptible silkworm strain JS and non-susceptible silkworm strain NIL, then the infectivity of the virus was tested by bio-assay and the combination activity of the virus with proteins from the extract measured by DIG-labeled ELISA. The results indicated that there were no protein factors affecting the invasion of the virus in digestive juice and blood. Some protein factors enhancing the invasion probably exist in the midgut of JS but not in NIL. It is speculated that protein factor as virus receptor may exist in the midgut of JS, but not in the NIL. Meanwhile,the combination activity was tissue-specific to the midgut of JS.Proteins in the midgut of JS and NIL were analyzed comparatively by two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results showed that there was less difference in the number, location and shape of the protein spots on the 2-DE image between the two strains. Protein peptide mass fingerprints (PMF) of all the 9 distinguished specific spots, six in JS and three in NIL , showed high intensity when analyzed by MALDI-TOF MS.Among the six specific spots in JS, four might be H+ transporting ATP synthase beta subunit isoforms 1 and 2, cathepsin D or 3-hydroxyacyl-CoA dehydrogenase, respectively. Of the three specific spots in NIL, two spots might be cathepsin.Comparative analysis of proteins in the hemolymph and peritrophic membrane (PM) between JS and NIL indicated that there was less difference in tissue proteins from hemalymph or PM between the two strains, the number, location and shape of the protain spots on the 2D image were very similar. The proteins from the blood of both the two silkworm strains were mainly neutral and slightly alkaline ones with very few extremely acid or extremely alkaline ones. The proteins from PM was mostly middle or low in molecular weight and neutral in isoelectric point value. Total five differentiated spots were distinguished on 2-D image of the hemolymph proteins. One spots distinguished in the susceptible strain might be casein kinase and another be an un-reported protein. Spots distinguished in the non-susceptible strain might be masquerade-like serine proteinase and mitochondrial elongation factor respectively. Moreover, the protein content in the hemolymph of non-susceptible strain was remarkably higher than that in the susceptible strain. According to the probable function of these differentiated spots, it is suggested that the susceptibility of silkworm strains against BmDNV-ZJ may be not related with the difference of their hemolymph proteins.Total four differentiated spots were detected in PM proteins. A specific spot distinguished in the PM of susceptible strain might be an un-reported protein. The protein content of each counterpart spots of the rest three is remarkably different between the susceptible and non-susceptible strains. It is not clear whether the four differentiated spots were related with the difference of susceptibility to BmDNV-ZJ among the silkworm strains.In the susceptible strains, serpin-2 (regulating protease activity and apoptosis) and thiol peroxiredoxin (anti-oxidation and eliminating free radicels) was detected in the midgut in the initial stage of infection by BmDNV-ZJ. They might be the products by the induction of the virus infection. The proteins specifically expressed in the hemolymph against the infection might be mitogen-activated protein kinase (regulating apoptosis) or peroxiredoxin-like protein (anti-oxidation and eliminating free radicels). According to the probable functions of these differentiated proteins, it is speculated that the silkworm of susceptible strain may prevent the BmDNV-ZJ infection from further development in the initial stage of infection through endogenous immune defense ways such as the apoptosis and the secretion of resistant protein factors in midgut (the infected position) and blood (the tissue of immune defense), like anti-oxidation proteins.In the advanced stage of infection by BmDNV-ZJ, the amount of proteins extracted from midgut and hemolymph of the silkworm of susceptible strain decreased rapidly. About 30% spots disappeared and the down-regulation of the rest spots was more than 50%. This implies that the protein synthesis is significantly reduced due to destruction of the digestion and absorption function of the midgut caused by the virus infection.