| It has been demonstrated that optical brightener (or fluorescent brightener) can enhance viral activity, provide UV protection for nucleopolyhedrovirus (NPV) and increase host susceptibility. The mechanism enhancement of the optical brightener is not known. Shapiro et al. postulated that selected brightener including M2R inhibit or alter the chitinous peritrophic membrane (PM), creating gaps in the membrane or gut lining and perhaps allowing more virions to pass from the gut lumen into the hemocoel. We used a reporter gene recombinant of Autographa californica, MNPV(AcMNPV-hsp70/lacZ) to investigate the enhancing effects of FB-28 on pathogenesis in midgut, mortality and time to death in larvae of Spodoptera exiguaStudies were designed to determine the effect of the selected optical brighter FB-28 on the infectivity of the AcMNPV-hsp70/lacZ.the viral preparation were diluted in water (l%)and applied to the surface of artificial diet, the addition of the FB-28 to the virus in the bioassay greatly enhanced infectivity of the AcMNPV-hsp70/lacZ of FB-28 to the AcMNPV-hsp70/lacZ, reduced the LC50 values from 2.31 X 107 PIB per ml to 5.18 X 104 PIBs/ml and at the highest virus concentration 1.55 X 107PIBs/ ml, the LT50 values was reduced from 7.0d to 4.6d. suggesting that the larvae of Spodoptera exigua died more quickly when FB-28 were added to the virus .The larvae of Spodoptera exigua inoculated as newly molted fourth instars (40s) and at various hours after molting (316s), we sacrificed larvae at various time intervals after inoculation to process the larvae for lacZ expression in midgut by using tissue section techniques on early phase infection. Optical brighteners can't change the part of infection, but it improves viral enhancement on pathogenesis of midgut. We detect the differentiation of the control and the test to study synergism of optical brighteners to viral enhancement and its mechanisms. Fixed with gluteraldehyde, Stained with pH 7.4 phosphate buffered X-gal Dye Solution,observed by reverse microscope, the results are as follow: the percent of larvae expressing lacZ in midgut showed that FB-28 improved the sensitivity of larvae to virus. The percent of larvae expressing lacZ gradually increased for various time points after inoculation, but the percent of larvae expressing lacZ reduced after 20 hours post inoculation, we thought the infection spots transformed.The number of foci in midgut showed: foci became more with the delay of infection times. The foci were more in control than in test. Which stated the addition of optical brighteners fastened the infections of virus and enhanced the ability.The foci located the basal in midgut not in control but also in test, the results showed that the addition of optical brighteners didn't change the spots of infection but improved the viral enhancement, in addition, Infection of hemolymph cell showed that optical brightener increased sensitivity of larvae to virus, forming more foci.In addition, We designed a test: The midgut was removed from Spodoptera exigua larvae and was cut longitudinally into halves, the midgut epithelia were cleaned and digested with dispase II ,the pelleted cells in Grace medium with 7% FBS supplemented with 100units/ml penicillin,100ug/ml streptomycin were cooled and then suspended in R18-labelled virus. Aliquots of cells were mixed 0.15%mg/ml FB-28,and kept at 4C for 30min,fusion assays were conducted: fluorescence was measured immediately at regular time-points with fluorescence spectrophotometer with an excitation wave length of 560nm and emission wave length of 590nm. the percentages of membrane fusion was calculated. By monitoring fusion using the R18 assay, We found that the fluorescent brightener 28 influenced membrane fusion of virus and midgut epithelia cells.
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