Location: Foreign Arthropod Borne Animal Disease Research
Project Number: 3022-32000-024-003-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2021
End Date: Jul 31, 2026
Objective:
Understanding the factors that are associated with arbovirus infection pathogenesis and maintenance is needed to develop effective control strategies. Environmental temperature is known to effect the ability of insect vector become infected an transmit arboviruses. Mosquito species such as Culex species are known to transmit two important exotic zoonotic arboviruses, Japanese encephalitis and Rift Valley fever viruses, that are of potential concern to the U.S. animal and public health. The objective is to determine how temperature influences vector competence of Culex quinquefasciatus and Culex tarsalis and potentially other relevant mosquito species for these arboviruses. In addition, samples will be collected to study genetic interactions between the mosquito and virus by performing transcriptomic sequencing and gene expression analyses of arbovirus infected and control mosquitoes.
Approach:
Culex quinquefasciatus and Culex tarsalis will be utilized for these studies and will be maintained under standard insectary condidtions. Adult female mosquitoes at five days of age (Cx. tarsalis) and 12 days of age (Cx. quinquefasciatus) will be maintained on 10% sucrose and fasted 24 h before virus infection. Mosquitos will be provided an artificial bloodmeal for each treatment group (virus infected and sham-infected). The artificial blood meal will contain at least 5.0 log TCID50/ml of Japanese encephalitis virus (JEV), Rift Valley fever virus or media (sham-infected control). Engorged females with visible blood in the abdomen will be collected from each treatment group, transferred into cardboard containers, and provided with 10% sucrose solution. Engorged mosquitoes will be maintained at one of three temperatures, 22°-23° C, 28°-31° C, and 35°-37°. Mosquitoes will be collected at 0, 7 and 14 d post feeding to examine replication and dissemination (n=4 pools of 5 mosquitoes/time point) and for transcriptomics (n=10 mosquitoes/timepoint). The 0 d timepoint will be collected immediately after feeding to demonstrate the uptake of virus. Samples at day 7 and 14 will demonstrate infection and dissemination of the virus. At the time of collection, mosquitoes will be held at -20°C for 20 min to freeze kill the insect. Some of the mosquitoes will be collected whole for transcriptomics analysis. The remaining mosquitoes will be dissected to collect the midgut, salivary glands and remaining body parts examined for virus replication and dissemination by virus isolation/titration and reverse transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR).
For transcriptomic analysis, RNA will be extracted from the pool of 10 mosquitoes collected from infected and control samples at 0, 7, 14 d post infection. The mRNA libraries will be constructed, sequenced and assembled. Annotation and differential gene expression (DGE) will be performed with use of available genomes for the two mosquito species: if not accessible for Cx. tarsalis, the de novo transcriptome itself will be used in place of a reference genome for read mapping and DE analysis. Validation of DGE will be performed on select genes of interest (to be determined) by qRT-PCR as previously described.
