Transcriptome data reveal beneficial effects of Rickettsia (Rickettsiales: Rickettsiaceae) on Bemisia tabaci(Hemiptera: Aleyrodidae) through nutritional factors and defense mechanisms.

Whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is a destructive insect pest of many crops. Rickettsia infection in different cryptic species of B. tabaci has been observed worldwide. Understanding the interactions between these 2 organisms is critical to developing Rickettsia-based strategies to control B. tabaci and thereby reduce the transmission of related vector-borne viruses. In this study, we investigated the effects of Rickettsia infection on the biological characteristics of the Middle East Asia Minor 1 (MEAM1) strain of B. tabaci through biological analysis of infected and uninfected individuals. The results of this study suggest that Rickettsia may confer fitness benefits. These benefits include increased fertility, improved survival rates, accelerated development, and resulted in female bias. We also investigated the transcriptomics impact of Rickettsia infection on B. tabaci by performing a comparative RNA-seq analysis of nymphs and adult females, both with and without the infection. Our analysis revealed 218 significant differentially expressed genes (DEGs) in infected nymphs compared to uninfected ones and 748 significant DEGs in infected female adults compared to their uninfected whiteflies. Pathway analysis further revealed that Rickettsia can affect many important metabolic pathways in whiteflies. The results suggest that Rickettsia plays an essential role in energy metabolism, and nutrient synthesis in the B. tabaci MEAM1, and depends on metabolites obtained from the host to ensure its survival. Overall, our findings suggest that Rickettsia has beneficial effects on B. tabaci and offered insights into the potential molecular mechanisms governing the interactions between Rickettsia and B. tabaci MEAM1.

The piRNA response to BmNPV infection in the silkworm fat body and midgut.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a DNA virus that causes huge losses to the silkworm industry but the piRNA responses during BmNPV infection in the silkworm remain uninvestigated. Here, silkworm piRNA profiles of uninfected and BmNPV-infected fat body and midgut were determined by high-through sequencing in the early stages of BmNPV infection. A total of 2675 and 3396 genome-derived piRNAs were identified from fat body and midgut, respectively. These genome-derived piRNAs mainly originated from unannotated instead of transposon regions in the silkworm genome. In total, 572 piRNAs were associated with 280 putative target genes in fat body and 805 piRNAs with 380 target genes in midgut. Compared to uninfected tissues, 322 and 129 piRNAs were significantly upregulated in BmNPV-infected fat body and midgut, respectively. In addition, 276 and 117 piRNAs were significantly downregulated. Moreover, differentially expressed (DE) piRNAs during BmNPV infection differed significantly between fat body and midgut. Putative DE piRNA-targeted genes were associated with "response to stimulus" and "environmental information processing" in fat body after infection with BmNPV, which may indicate an active piRNA response to BmNPV infection in fat body. This study may lay the foundation for future research of the potential roles of the piRNA pathway and specific piRNAs in BmNPV pathogenesis.

[Expression and assembly of rotavirus-like particles in insect cells mediated by recombinant Bombyx mori MultiBac].

OBJECTIVE: To construct recombinant baculoviruses co-expressing three structural genes vp2, vp6 and vp7 of rotavirus, and assemble rotavirus-like particles (VLPs) in BmN cells. METHODS: Human group A rotavirus was cultivated in MA104 cells, and the RNA was extracted and the three genes were obtained by RT-PCR. The PCR products were inserted into the transfer vectors pFBDM and pUCDM, respectively. A enhanced green fluorescent protein gene (egfp) driven by IE1 promoter was introduced into pFBDM to investigate the efficiency of infection. The expression baculoviruse was constructed by Tn7 and Cre-LoxP recombinant and transfected into BmN cells. The gene expression was determined by detecting 6-His tag fused into VP7 C-terminus, and the assembled VLPs were observed by transmission electron micrography. RESULTS: Three genes of rotavirus were cloned and BmMultiBac was constructed. The genes were expressed and the rotavirus-like particles assembled in BmN cells successfully as verified by ELISA and electron microscope. CONCLUSION: We have successfully constructed the recombinant baculovirus co-expressing the 3 structural genes of rotavirus, which provide the basis for producing protein complex containing multiple subunits and investigation of the structure of the macromolecules.

A novel economic method for high throughput production of recombinant baculovirus by infecting insect cells with Bacmid-containing diminopimelate-auxotrophic Escherichia coli.

Developing cost-effective methods for high throughput production of recombinant baculoviruses in insect cells is very challenging, because the baculovirus DNA preparation and the following transfection procedure are labour-intensive and time consuming. We developed a new method of introducing recombinant Bacmid DNA from bacteria into insect cells simply using invasive diaminopimelate (DAP) auxotrophic Escherichia coli to infectSpodoptera frugiperda 9 cells. The E. coli cells with recombinant Bacmids enter insect cells with the help of the invasion factor from Yersinia pseudotubercolusis. Without DAP in medium, the cell wall of DAP auxotrophic E. coli cannot be synthesized so that the bacterial cell will disrupt and release recombinant Bacmid. The released Bacmids will generate infective recombinant baculovirus particles in insect cells. We combined this E. coli invasion method with the zero background transposition system to generate recombinant baculovirus in a rapid and simple way. Without preparation and purification of recombinant Bacmids from E. coli and the labour-intensive and complex transfection procedure, this transfection reagent free method enables a convenient and economic high throughput production of recombinant baculoviruses.

[Biological characteristics of a chimeric rabies virus expressing canine parvovirus VP2 protein].

To obtain a bivalence vaccine against canine rabies virus and canine parvovirus, a chimeric rabies virus expressing canine parvovirus VP2 protein was generated by the technique of reverse genetics. It was shown that the chimeric virus designated as HEP-Flury (VP2) grew well on BHK-21 cells and the VP2 gene could still be stably expressed after ten passages on BHK-21 cells. Experiments on the mice immunized with the chimeric virus HEP-Flury (VP2) demonstrated that specific antibodies against rabies virus and canine parvovirus were induced in immunized mice after vaccination with the live chimeric virus.

A high efficient method of constructing recombinant Bombyx mori (silkworm) multiple nucleopolyhedrovirus based on zero-background Tn7-mediated transposition in Escherichia coli.

A high efficient way for generation of recombinant Bombyx mori (silkworm) multiple nucleopolyhedrovirus by Tn7-mediated transposition in Escherichia coli was performed. The new system consists of a conditional replication donor vector pRCDM and an attTn7 site blocked E. coli containing BmNPV-Bacmid. The donor vector contains a replication origin derived from R6Kgamma, which propagated only in host cells with pir gene expression decreased in the transposition background greatly. Compared with original vector derived from pUC, the transposition efficiency increased from 5.7 to 66% ( approximately 10 fold) when using conditional replication vector pRCDM transposition into original BmDH10Bac. A further effort to decrease the transposition background was made by blocking the attTn7 site in host E. coli genome. The resulting attTn7 occupied BmDH10BacDeltaTn7 resulted in a significant increase from 5.7 to 23% ( approximately 4 fold) in the efficacy of generate recombinant BmNPV Bacmid by transposition. Furthermore, the transposition of BmDH10BacDeltaTn7 with pRCDM resulted typically in 100% white colonies, and it indicated that a zero transposition background was accomplished. This high efficient and zero background transposition system provides a new simple and rapid method for construction of recombinant BmNPV used to express target genes or produce gene-delivery virus particles in silkworm.

[Rescue of chimeric rabies virus expressing green fluorescent protein].

Green fluorescent protein (GFP) gene was inserted into the pseudogene (psi) region of genome of rabies virus rHep-Flury strain, and a recombinant rabies virus carrying GFP, designated as HEP-GFP, was rescued by reverse genetics system. It was demonstrated that green fluorescent protein could be expressed in the chimeric virus after 5 passages in BHK-21 cell line. The research indicated that the pseudogene (psi) region in the genome of rHEP-Flury strain, as an independent functional unit in the process of virus assembly, could independently carry and express exogenous genes.

Entry of Bombyx mori cypovirus 1 into midgut cells in vivo.

In vivo entry of Bombyx mori cypovirus 1 into the silkworm midgut was studied by electron microscopy of ultrathin sections of midguts from silkworm larvae that had been administered virus-contaminated leaves. In 3 h, virions were observed outside and inside midgut cells, including columnar cells, goblet cells and muscle cells. Virions were seen adhering to the plasma membrane of microvilli, embedding in the membrane and settling themselves intact inside the microvilli of the columnar cells. These results suggested that intact virions entered columnar cells by means of direct penetration through the cell membrane. In 12, 24 and 48 h, virogenic stromata and progeny virions were observed in columnar cells, but not in other midgut cells.