Ex vivo promoter analysis of antiviral heat shock cognate 70B gene in Anopheles gambiae

<p>Abstract</p> <p>Background</p> <p>The <it>Anopheles gambiae </it>heat shock cognate gene (<it>hsc70B</it>) encodes a constitutively expressed protein in the <it>hsp70 </it>family and it functions as a molecular chaperone for protein folding. However, the expression of <it>hsc70B </it>can be further induced by certain stimuli such as heat shock and infection. We previously demonstrated that the <it>An. gambiae hsc70B </it>is induced during o'nyong-nyong virus (ONNV) infection and subsequently suppresses ONNV replication in the mosquito. To further characterize the inducibility of <it>hsc70B </it>by ONNV infection in <it>An. gambiae</it>, we cloned a 2.6-kb region immediately 5' upstream of the starting codon of <it>hsc70B </it>into a luciferase reporter vector (pGL3-Basic), and studied its promoter activity in transfected Vero cells during infection with o'nyong-nyong, West Nile and La Crosse viruses.</p> <p>Results</p> <p>Serial deletion analysis of the <it>hsc70B </it>upstream sequence revealed that the putative promoter is likely located in a region 1615–2150 bp upstream of the <it>hsc70B </it>starting codon. Sequence analysis of this region revealed transcriptional regulatory elements for heat shock element-binding protein (HSE-bind), nuclear factor κB (NF-κB), dorsal (Dl) and fushi-tarazu (Ftz). Arbovirus infection, regardless of virus type, significantly increased the <it>hsc70B </it>promoter activity in transfected Vero cells.</p> <p>Conclusion</p> <p>Our results further validate the transcriptional activation of <it>hsc70B </it>during arbovirus infection and support the role of specific putative regulatory elements. Induction by three taxonomically distinct arboviruses suggests that the HSC70B protein may be expressed to cope with cellular stress imposed during infection.</p

Anopheles gambiae heat shock protein cognate 70B impedes o'nyong-nyong virus replication

Background Phylogenetic and functional analysis was conducted on an Anopheles gambiae gene, ENSANGG00000017398. Based on phylogenetic analysis, this gene belongs to the same lineage as Heat shock protein cognate 70-4 (Hsc70-4) in Drosophila. Accordingly, we propose to name this gene Heat shock protein cognate 70B (HSC70B). We previously reported that expression of HSC70B and other genes including elongation factor-1α (EF-1α) and the agglutinin attachment subunit (agglutinin) were up-regulated in o'nyong-nyong virus (ONNV)-infected female An. gambiae. Double-stranded RNA interferences have been applied to further investigate HSC70B, EF-1α and the agglutinin functions in ONNV replication in An. gambiae. Results Among these three RNAi silenced genes, only dsRNAs of HSC70B (dsHSC70B) promoted ONNV replication in adult An. gambiae compared to the control mosquitoes that were co-injected with ONNV and dsRNA of β-galactosidase (dsβ-gal). ONNV titers from mosquitoes co-injected with dsHSC70B were about 9-fold higher at 6 days post-injection (d.p.i.) as compared to the control mosquitoes. By using ONNV tagged with enhanced green fluorescent protein (ONNV-eGFP), co-injection of ONNV-eGFP with dsHSC70B also showed approximately 2 ~ 3-fold higher GFP expression rates than the controls in the head, thorax, and abdomen of the mosquito. Furthermore, co-injection of ONNV with dsHSC70B significantly reduced the lifespan of adult mosquitoes as compared with the control, co-injection of ONNV with dsβ-gal treated mosquitoes. Conclusion These results indicate that HSC70B plays important roles in homeostasis and suppression of ONNV replication in the vector, An. gambiae. Biological implications of these findings are that while mosquitoes allow ONNV to replicate in them, they also check viral titers so that ONNV infection will result in no harmful effect on mosquitoes. Therefore, mosquitoes can function as vectors of ONNV transmission to humans while ONNV infection in An. gambiae remains asymptomatic.We wish to thank Dr. K. E. Olson and B. D. Foy for his kind gift of infectious clone pONNic-Foy. This research would not have been possible without the assistance of Dr. Mabel Berois with helpful guide. This project was supported by grants R01-AI44273 from NIH/NIAID to F.H.C. D.L.V. was supported by NIH T32 A10753

Genomic Resources Notes Accepted 1 August 2014–30 September 2014

This article documents the public availability of (i) transcriptome sequence data, assembly and annotation, and single nucleotide polymorphisms ( SNP s) for the cone snail Conus miliaris ; (ii) a set of SNP markers for two biotypes from the Culex pipiens mosquito complex; (iii) transcriptome sequence data, assembly and annotation for the mountain fly Drosophila nigrosparsa ; (iv) transcriptome sequence data, assembly and annotation and SNP s for the Neotropical toads Rhinella marina and R. schneideri ; and (v) partial genomic sequence assembly and annotation for 35 spiny lizard species (Genus Sceloporus ).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110107/1/men12340-sup-0004-AppendixS4.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110107/2/men12340-sup-0003-AppendixS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110107/3/men12340-sup-0002-AppendixS2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110107/4/men12340-sup-0005-AppendixS5.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110107/5/men12340.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110107/6/men12340-sup-0001-AppendixS1.pd

Genome sequence of the tsetse fly (Glossina morsitans):Vector of African trypanosomiasis

Tsetse flies are the sole vectors of human African trypanosomiasis throughout sub-Saharan Africa. Both sexes of adult tsetse feed exclusively on blood and contribute to disease transmission. Notable differences between tsetse and other disease vectors include obligate microbial symbioses, viviparous reproduction, and lactation. Here, we describe the sequence and annotation of the 366-megabase Glossina morsitans morsitans genome. Analysis of the genome and the 12,308 predicted protein-encoding genes led to multiple discoveries, including chromosomal integrations of bacterial (Wolbachia) genome sequences, a family of lactation-specific proteins, reduced complement of host pathogen recognition proteins, and reduced olfaction/chemosensory associated genes. These genome data provide a foundation for research into trypanosomiasis prevention and yield important insights with broad implications for multiple aspects of tsetse biology.IS

Data from: "Identification and assessment of single nucleotide polymorphisms (SNPs) between Culex complex mosquitoes." in Genomic Resources Notes Accepted 1 August 2014-30 September 2014

Culex pipiens complex mosquitoes are important vectors for many human pathogens including West Nile encephalitis, Rift Valley fever and Lymphatic filariasis. In this study we characterized a set of SNP markers between two biotypes of the Culex pipiens complex, Culex pipiens form molestus and Culex pipiens form pipiens, for use in a high-resolution genetic mapping and population genetics. DNA pooled from 10 specimens of each biotype were sequenced and analyzed for variation in 28 genes. The total of 4714 bp across orthologs of C. pipiens form pipiens and C. pipiens form molestus revealed 44 SNPs in the 3279 bp coding regions, 48 SNPs in the 1435 bp non-coding regions, and 10 indels. The ratio of transitions to transversions approached 2:1, with transitions constituting the majority of synonymous coding substitutions. The informative SNP markers were successfully identified and assessed from both C. pipiens biotypes. We expect that novel SNPs characterized in this study would be useful for genetic studies to elucidate the genetic basis of diverged eco-physiological traits between the two biotypes of the C. pipiens complex

08.25.14 - Dryad submission

08.25.14 - Dryad submissio

Comparative Transcriptomics Reveals Key Gene Expression Differences between Diapausing and Non-Diapausing Adults of Culex pipiens.

Diapause is a critical eco-physiological adaptation for winter survival in the West Nile Virus vector, Culex pipiens, but little is known about the molecular mechanisms that distinguish diapause from non-diapause in this important mosquito species. We used Illumina RNA-seq to simultaneously identify and quantify relative transcript levels in diapausing and non-diapausing adult females. Among 65,623,095 read pairs, we identified 41 genes with significantly different transcript abundances between these two groups. Transcriptome divergences between these two phenotypes include genes related to juvenile hormone synthesis, anaerobic metabolism, innate immunity and cold tolerance

qRT-PCR primers and associated genes.

<p>qRT-PCR primers and associated genes.</p

Total genes upregulated and downregulated in diapausing females of <i>Cx</i>. <i>pipiens</i>.

<p>Total genes upregulated and downregulated in diapausing females of <i>Cx</i>. <i>pipiens</i>.</p