Abstract
By utilizing cDNA microarrays including about 20,000 cDNAs, Gene expression levels of o’nyong nyong virus (ONNV)-infected female mosquitoes were compared to that of the uninfected control females harvested at 14 days post-infection. In response to ONNV infection, expression levels of 18 genes were significantly modulated, being at least two-fold up- or down-regulated. Quantitative real-time PCR analysis (qRT-PCR) further substantiated the differential expression of six genes of these genes in response to ONNV infection. These genes have similarity to a putative Heat shock protein cognate 70 (HSC70), DAN4, Agglutinin attachment subunit (Agglutinin), Elongation Factor 1 alpha (EF-1á) and Ribosomal protein L35 (RpL35). One gene, with sequence similarity to mitochondrial ribosomal protein L7 (MRpL7), was down regulated in infected mosquitoes. In addition, the phylogenetic analysis of HSC70 shows that it was in the same lineage to Heat shock protein cognate 70B (HSC70B) rather than other HSC70s or HSP70s of Drosophila. Thus we named it An. gambiae HSC70B.
Secondly, for a functional analysis, female An. gambiae were co-injected with ONNV and the respective double-stranded RNA (dsRNA) based on a cDNAs of the hsc70b and two other candidate genes, Agglutinin attachment subunit (Agglutinin), EF-1á from above cDNA microarray study (Sim et al., 2005) Among these post-transcriptional silencing of endogenous transcripts, only, dsRNAs of HSC70B (dsHSC70B) promoted ONNV replication in adult An. gambiae compared in the control mosquitoes that were co-injected with ONNV and dsRNA of â-galactosidase (dsâ-gal). ONNV titers from mosquitoes coinjected with dsHSC70B were 9-fold higher at 6 days post injection than in the control mosquitoes. To validate the results, by using recombinant ONNV that expresses enhanced GFP (ONNV-eGFP) as a marker (Keene et al., 2004), the coinjection with dsHSC70B shows approximately 2 or 3-fold higher GFP expression rates than in the controls at the three tissues (Head, Thorax, and Abdomen). Hence, these observations provide direct evidence that HSC70B impedes ONNV replication in An. gambiae.
In addition, to clarify the tissue tropism of An. gambiae in ONNV infection, ONNV infectious system (ONNV-eGFP) which is capable of express green fluorescent protein (Brault 2004), has been used to characterize tissue tropism and dissemination of ONNV in An. gambiae by intrathoracic injection. An. gambiae clearly showed the accumulation of GFP in the anterior midguts, foreguts, hindguts, cardia, ovaries, salivary glands and heads at 6 days post infection. However, interestingly, The GFP expression in intrathoracic injection of ONNV-eGFP did not show any infection foci in the posterior midgut at 6 and 12 days post-infection, implying that dissemination patterns of ONNV within An. gambiae posterior midguts are different from other Culicine infection with alphaviruses.
|
indicates that a file or directory is accessible from the campus network only.