Molecular determinants of vector specificity in Highlands J virus
Date
2014
Authors
Borland, Erin M., author
Quackenbush, Sandra, advisor
Powers, Ann, advisor
Chen, Chaoping, committee member
Journal Title
Journal ISSN
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Abstract
Highlands J Virus (HJV) is an understudied alphavirus that is closely related to western equine encephalitis virus (WEEV) and eastern equine encephalitis virus (EEEV). HJV is not known to cause disease in humans or equids, but it is a known avian pathogen with potential to significantly disrupt commercial production flocks. These studies compared the sequences of multiple strains of HJV in order to better characterize and compare the range of available strains. Strain B230, the prototype strain, was used to compare all other strains tested. Strain 64A-1519 was most similar to B230 with 22 nucleotide substitutions, only 5 of which resulted in a change in amino acid residues. Strain WX3-2AP was the most divergent with 167 nucleotide changes resulting in 8 amino acid residue changes. Four distinct lineages were identified through phylogenetic analysis. Lineage 1 consisted of strains B230 and 64A-15191. Lineage 2 consisted of strains AB-80-9, RU-M-80-259, and 73V-2540. Lineage 3 consisted of strains W17791 and two GenBank strains (744-01 and 585-01). Strain WX3-2AP was the sole member of Lineage 4. Vector capacity studies for HJV in live Culex tarsalis mosquitoes have not been published prior to these experiments. Cx. tarsalis mosquitoes were orally infected with one of four strains of HJV: B230, 64A-1519, WX3-2AP, or AB-80-9. The heads and bodies of mosquitoes were separated and processed independently to assess viral presence by cytopathic effects (CPE). The experiments were run in duplicate and at different times to ensure accuracy of results. Two infection patterns emerged: Lineage 1 strains had low infection and dissemination rates at all three time points, while Lineage 2 and 4 strains had high infection and dissemination rates which were more similar to those previously published for WEEV Imperial strain in Cx. tarsalis. Virus titrations were performed on mosquito heads and bodies, and Lineage 4 strain WX3-2AP was found to have the highest average titers. Mosquito bodies had the highest average titer on day 8 post infection and average titers for bodies ranged from 6.60 to 7.26 log10 pfu equivalents/body. Heads had no discernable pattern among titers or strains, but titers ranged between 6.01 and 6.80 log10 pfu equivalents/head. Saliva was collected from mosquitoes infected with Lineage 2 strain AB-80-9 to assess the potential presence of a salivary gland barrier resulting in lack of transmission. Twenty-two of 49 mosquitoes tested transmitted detectible levels of virus, meaning 44.9% of orally infected mosquitoes were able to actively transmit the virus. While the titer of the saliva on a per mL basis was impossible to compute since the volume of saliva could not be quantified, the titers of the samples collected ranged between 1.68 and 5.81 log10 pfu equivalents/saliva sample. By combining the data obtained by sequencing the strains with the data from the mosquito infections, a single amino acid difference between the attenuated Lineage 1 strains and the more virulent Lineage 2 and 4 strains was identified as being potentially responsible for the differences in infectivity. The mutation was located at genome nucleotide position 8605, E2 glycoprotein amino acid 69. This change caused the non-polar glycine in the attenuated Lineage 1 strains to be replaced with an acidic glutamic acid in the more virulent Lineage 2 and 4 strains. Additional studies are needed to assess the in vivo effects of this amino acid change in Cx. tarsalis mosquitoes.
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Subject
Highlands J virus
Culex tarsalis
E2 glycoprotein