Variation in infectivity and genetic diversity in the structural proteins were compared among eight strains of Western equine encephalitis virus (WEEV) to investigate WEEV virulence at the molecular level. A lethal intranasal infectivity model of WEEV was developed in adult BALB/c mice. All eight strains examined were 100 % lethal to adult mice in this model, but they varied considerably in the time to death. Based on the time to death, the eight strains could be classified into two pathotypes: a high-virulence pathotype, consisting of strains California, Fleming and McMillan, and a low-virulence pathotype, comprising strains CBA87, Mn548, B11, Mn520 and 71V-1658. To analyse genetic diversity in the structural protein genes, 26S RNAs from these eight strains were cloned and sequenced and found to have > 96 % nucleotide and amino acid identity. A cluster diagram divided the eight WEEV strains into two genotypes that matched the pathotype grouping exactly, suggesting that variation in infectivity can be attributed to genetic diversity in the structural proteins among these eight strains. Furthermore, potential amino acid differences in some positions between the two groups were identified, suggesting that these amino acid variations contributed to the observed differences in virulence.

Highlands J virus (HJV) is an alphavirus closely related to western equine encephalitis virus (WEEV) and eastern equine encephalitis virus (EEEV). HJV is an avian pathogen with the potential for disruption of poultry operations, but is not known to cause human or equine disease. HJV has only been identified in the eastern United States and is thought to have a transmission cycle similar to that of EEEV involving Culiseta melanura mosquitoes and birds. However, HJV is more genetically similar to WEEV and it remains unclear if it may be transmitted by Culex species mosquitoes like WEEV. Seven strains of HJV were characterized to assess this potential. Phylogenetic analysis of whole genome sequences revealed four distinct HJV lineages (lineages 1-4), and vector competence studies in Cx. tarsalis with four of the HJV strains from different lineages yielded two distinct infection patterns. Lineage 1 strains had low infection rates, while lineages 2 and 4 had significantly higher infection rates similar to those previously published for WEEV. The average mosquito body viral titer was highest at 8 dpi (6.60-7.26 log(10) pfu equivalents/body), and head titers at all time points ranged between 6.01 and 6.80 log(10) pfu equivalents/head. Nearly 45% of mosquitoes infected with strain AB-80-9 were able to transmit virus in saliva with an average titer of 5.02 log(10) pfu equivalents/saliva. A single amino acid difference between high and low infectivity phenotypes was identified at genome position 8605, in the E2 gene. A nonpolar glycine was present in the low infectivity lineage 1 strains, while an acidic glutamic acid was present in the higher infectivity lineage 2 and 4 strains. This study demonstrates HJV transmission by Cx. tarsalis mosquitoes and clearly identifies the potential for transmission in the western United States. Two infection phenotypes were exhibited, indicating the need for further studies to understand Culex species transmission patterns.