In 2005, mosquito eggs were collected weekly in Wise County, Virginia, by using ovitraps. The resultant larvae were reared to adults in a Biosafety Level 2 insectary at 24°C, 75% relative humidity, and a photoperiod of 16 hours of light and 8 hours of dark. In 2008 and 2009, adult mosquitoes were collected weekly from infusion-baited gravid traps in Montgomery and Craig Counties, Virginia. Mosquitoes were identified to the species level according to morphology and grouped in pools of <50 individuals according to species, collection location, and collection date. Adults were stored at −80°C until testing. Reverse transcription PCR (RT-PCR) was used for LACV detection in mosquitoes collected in 2005 and 2008. In 2009, mosquito pools were homogenized by using previously described methods for LACV isolation (6). Homogenate supernatant (150 μL) was inoculated onto Vero cells, incubated at 37°C, and monitored daily for cytopathic effect. Isolates that showed marked cytopathic effect were harvested and submitted to the Centers for Disease Control and Prevention (CDC) in Fort Collins, Colorado, USA, and the Wadsworth Center in Slingerlands, New York, USA, for quantitative RT-PCR (qRT-PCR).

In 2013, mosquito surveillance was conducted as part of the West Virginia Department of Health and Human Resources Mosquito Surveillance Program. Gravid traps, carbon dioxide–emitting light traps, and BG Sentinel (Biogents AG, Regensburg, Germany) traps baited with octenol lures were used to collect adult mosquitoes weekly from counties with high (Nicholas, Fayette, Raleigh) and low (Kanawha, Jackson, Wood) incidence of LACV among humans as defined (10) and on a somewhat regular basis in additional counties. Specimens were pooled by species, county, and collection date for qRT-PCR at the West Virginia Office of Laboratory Services, by use of previously described methods (11).

LACV RNA was detected in a pool of Ae. japonicus mosquitoes collected as eggs in August 2005 from Wise County, Virginia. LACV was also detected in a pool of Ae. japonicus mosquitoes from Montgomery County, Virginia, in July 2008. LACV RNA was detected in 5 separate pools of Ae. japonicus mosquitoes collected in West Virginia in 2013, representing 3 counties over a 4-month period. Of 3,529 Ae. japonicus mosquitoes collected from Montgomery County in 2009, we isolated LACV from 1 pool (n = 3). In that same year, of 796 Ae. japonicus mosquitoes from Craig County tested, LACV was isolated from 1 pool (n = 50) (Table). These isolations were verified by qRT-PCR at CDC (Montgomery County isolate) and the Wadsworth Center (Craig County isolate).

Figure 2

Figure 2. Phylogeny of La Crosse virus (LACV) based on the medium (M) segment of the viral polyprotein gene. State of isolate origin, isolation year, mosquito, or vertebrate isolate source and the National...

Nucleotide sequencing and a BLAST (http://blast.ncbi.nlm.nih.gov//Blast.cgi) query were performed on the amplified cDNA from both isolates. The LACV medium segment was used to infer phylogeny (Figure 2). Coding sequences were aligned in Mesquite version 2.75 with Opalescent version 2.10 (12,13). Phylogenetic trees for the polyprotein genes were estimated by using a maximum likelihood–based method and assuming a general time-reversible (GTR) model with gamma-distributed rate heterogeneity of nucleotide substitution GTR + Γ in RAxML version 8.0.0 (14). Support values for each clade were generated in RAxML by using 1,000 rapid bootstrap replicates. The Virginia 2009 isolates were within the previously described lineage I, which contains the reference LACV strain isolated from the brain tissue of a child in Wisconsin (National Center for Biotechnology Information accession no. U18979) (15). The 2 isolates from mosquitoes collected in Virginia in 2009 differed by only 1 bp.

The isolation of LACV from field-collected Ae. japonicus mosquitoes, and particularly from mosquitoes collected as eggs, is highly significant because of the pervasiveness of this species in the United States. The large number of LACV detections in this invasive species highlights the need for LACV mosquito surveillance and control efforts to include Ae. japonicus in addition to Ae. triseriatus mosquito populations. Most (3/5) detections of LACV in West Virginia were in mosquitoes from Fayette County, where incidence of LACV among humans is high, suggesting that Ae. japonicus mosquitoes may play a major role in transmission of LACV to humans. Our detection of LACV in Ae. japonicus mosquitoes from field-collected eggs in 2005 and the ability of LACV to be transmitted transovarially in Ae. triseriatus mosquitoes suggests that future research should examine the possibility of vertical transmission of LACV in invasive Ae. japonicus mosquitoes. In states east of the Mississippi River, where Ae. japonicus mosquitoes (9) and LACV (2) co-exist, this mosquito may play a major role in the maintenance, transmission, and range expansion of LACV.