Degree Type

Dissertation

Date of Award

2014

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Bradley J. Blitvich

Abstract

Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts; thus, they are dual-host viruses. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) are single-host viruses because they have host ranges restricted to vertebrates and insects, respectively. Numerous insect-specific flaviviruses (ISFs) including CxFV have been discovered in the last decade and most are widely spread in nature. However, little is known about the mechanism(s) by which ISFs are maintained in nature. In a previous study, CxFV was detected in both female and male mosquitoes collected in the field suggesting that this virus is maintained in nature by vertical transmission. The experiments outlined in chapter 2 were designed to test the hypothesis that efficient transovarial transmission (TOT) of CxFV occurs in the mosquito host. CxFV RNA was detected in 526 of 540 Culex pipiens progeny derived from CxFV-infected females and thus, the filial infection rate was 97.4%. Because all positive females produced infected offspring, the TOT prevalence was 100%. These data indicated that extremely efficient TOT of CxFV occurs in mosquitoes in nature. Tissue tropisms of CxFV were also defined. CxFV RNA was detected in all tissues tested: salivary glands, ovaries, testes, head, fat bodies and midguts. Time course experiments demonstrated that CxFV disseminates to the ovaries as early as 4 days post-inoculation. In chapter 3, the host range and genetic diversity of CxFV was investigated. Previously, a high prevalence of CxFV was reported in Cx. quinquefasciatus in the Yucatan Peninsula of Mexico. To determine whether other Culex spp. mosquitoes in this region are susceptible to natural CxFV infection, five other Culex spp. mosquitoes were tested for evidence of CxFV infection. Two pools of Cx. interrogator were positive. The envelope protein genes of these isolates and 16 isolates from Cx. quinquefasciatus were sequenced and shown to have >99.2% nucleotide identity. These data suggest that there is limited genetic diversity among CxFV isolates in Yucatan Peninsula of Mexico. In chapter 4, studies were performed to increase our knowledge of the genetic elements that condition the differential host ranges of flaviviruses. Although flaviviruses possess a similar genomic organization, they differ in terms of their host specificity; some flaviviruses infect both vertebrates and arthropods whereas others have a vertebrate-specific or arthropod-specific phenotype. The genetic elements that condition these differential host ranges and transmission cycles have not been identified. Therefore, chimeric viruses were constructed by replacing the capsid (C), premembrane (prM) and envelope (E) genes or the prM-E genes of MODV with the corresponding regions of WNV and CxFV. Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV in a MODV backbone. The virus could infect vertebrate but not mosquito cells, indicating that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype. The three other chimeras did not produce detectable virus. Comparative studies between flaviviruses that possess differential host range profiles will help us understand why some flaviviruses can infect only vertebrate or only invertebrate organisms while other flaviviruses can infect both insect and vertebrate hosts and cause devastating disease in humans and animals.

Copyright Owner

Rungrat Saiyasombat

Language

en

File Format

application/pdf

File Size

131 pages

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