Dengue virus serotype 2 (DENV-2) RNA replication profiles and tropisms were studied by using quantitative RT-PCR (q-RTPCR) in intrathoracically infected Aedes albopictus. The virus RNA replication profiles were diverse in mosquito organs. In fat body, brain, salivary gland, and malpighian tubes, it peaked at 8, 23, 23, and 27 days post-infection, respectively, and then, all declined. In midgut, it increased all the time and had no trend of decline. In ovary, it had no apparent increase. Subsequent Western blotting of DENV-2 E protein had similar results. Using ribosomal protein 7 (rpS7) as an internal control, we found that, in salivary gland, brain, fat body, and midgut, the average DENV-2 RNA levels (DENV-2 RNA/rpS7 mRNA) were 1,028, 464, 5.6, and 6.2, respectively; in malpighian tubes, it was 1, and in ovary, it was far less than 1. These results suggest that infection profiles and tropism of DENV-2 RNA in Ae. albopictus organs are significantly different.

Laccase (EC 1.10.3.2) is a member of multicopper oxidases that have been found in higher plants, fungus, bacterium, and insects. Two types of laccase genes have been detected in many species of insects: laccase1 and laccase2. It has been identified that laccase2 enzyme may play a key role in sclerotization and pigmentation of insect cuticle. But few attentions were given to the biological role of laccase2 in the synthesizing of similar structures, such as oothecae, eggshell, or silk cocoons. We cloned laccase2 gene from Aedes albopictus, one main mosquito vector of dengue virus in China. An upregulation of laccase2 gene was observed after a blood meal in female adult mosquitoes, suggesting that laccase2 gene may have an involvement in the development of ovary. RNA interference experiment was performed by using adult female mosquitoes. Female mosquitoes were injected with 20 ng of double-strain RNA into the thorax. Pigmentation of mosquito eggshell was blocked that these eggs never became dark. And the incomplete sclerotization of eggshell weakened the stability and flexibility of the eggs. These eggs without enough protection were deformed and died in water. These results demonstrate that laccase2 plays a critical role in the development of eggs of A. albopictus. Laccase2 may provide a novel target for mosquito control and management.

The interaction between dengue virus (DENV) and vector mosquitoes are still poorly understood at present. In this study, 2-D DIGE combined with MS was used to analyze the differential proteomes of Aedes albopictus salivary gland, midgut and C6/36 cells induced by DENV-2. Our results indicated that the virus infection regulated several functional classes of proteins. Among them, 26 were successfully analyzed by real-time RT-PCR. The mRNA levels of 15 were the highest in salivary gland, 2 in midgut and none in C6/36 cells, however, 18 were the least in fat body compared to other organs. Interestingly, the changes of differential proteins mRNA were the most obvious in fat body post-infection. Chaperone, cytoskeleton and energy metabolism enzyme were the most down- or up- regulated proteins after DENV-2 infection. The abundant expression of these proteins in salivary gland may relate to its high susceptibility.

The transmembrane microneme protein MIC6 and its partner MIC1, MIC4 comprise an adhesive complex that play important roles in host cell attachment by the obligate intracellular parasite Toxoplasma gondii. Successful penetration of host cells by T. gondii depends on coordinated interactions between MICs complex and the parasite's cytoskeleton. We have identified that the carboxy-terminal cytoplasmic domain (C domain) of MIC6 interacts with aldolase and the parasite cytoskeleton. Our finding uncovers new features regarding MIC6-aldolase interactions in host cell invasion by T. gondii.

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