Direction and relative scale of sRNA counts for a given target are marked by red bar indicators near the corresponding target genes. Bar 1 check details indicates ASK inhibitor un-infected controls; Bar 2 indicates DENV2-infected pools. The legend to GeneGo Metacore pathway maps is given in Additional File 4. Small non-coding RNAs (ncRNAs), such as tRNAs and small nucleolar RNAs (snoRNAs),

are cleaved by Dicer-dependent mechanisms [28, 32]. Changes to tRNA and other ncRNA levels could be one mechanism used by hosts in anti-viral defense to slow viral replication. This is supported by the observation that codon usage bias differs among mosquitoes and flaviviruses [45]. Distinct subsets of tRNA and U spliceosomal ncRNAs are affected during DENV infection (Additional File 2). Further study is needed to determine the mechanisms by which ncRNA

pattern changes would affect DENV replication. Conclusions Together, these data indicate that profound changes occur in mosquito metabolic pathways early in the DENV2-infection process. Mosquitoes use SRRPs in multiple lines of defense against arboviruses but remain unable to prevent persistent infections. The important features of the DENV2-infection process described here provide a context selleck for future studies to define cell autonomous host responses to arbovirus infection in vector mosquitoes. Methods Mosquito Infections/Virus stocks Colonized Ae. aegypti, Puerto Rico Rexville D or HWE strains, were reared under PIK-5 standard conditions at 28°C, 80% relative humidity, with a photoperiod of 14:10 (L:D). HWE is a white eye genetic variant of the RexD strain. Adults were provided with a sugar source and water and held in the same conditions during the virus infection

extrinsic incubation period. High passage Dengue serotype 2 Jamaica 1409 (DENV) cultures were prepared by infecting C6/36 Ae. albopictus cell culture at an MOI of 0.01 and incubating for 12 days at 28°C at 5% CO2 in Minimal Eagles medium. RexD mosquitoes at 4-7 days of age were fed a blood meal containing a 1:1 dilution of DENV in C6/36 cell culture medium and defibrinated sheep blood. Samples harvested at days indicated. Un-infected controls were fed blood diluted 1:1 with C6/36 cell culture medium. Three biological replicates were performed for deep sequencing libraries. DENV2-blood meal titers ranged from 6.7 to 7.8 log plaque forming units (pfu) per ml. Whole mosquito pools were stored in Trizol reagent (Invitrogen) at -80°C. Ten mosquitoes were titered individually using standard methods [3]. Libraries and Sequencing Total RNA was extracted from each RexD pool using Trizol (Invitrogen). Small RNAs were isolated from the total RNA using the FLASHPAGE system (Applied Biosystems) and the manufacturer’s recommendations. Individual sequencing libraries were prepared using the Applied Biosystem’s Small RNA Expression kit. Use of bar-coded primers allowed library pools to be sequenced simultaneously on two slides.

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