Mosquito Collections. Isolates of LACV analyzed in the present investigation were obtained from field-collected mosquitoes that were procured from the following:
a. Connecticut state has conducted annual monitoring of mosquitoes at 36 locations since 1997 and was increased to a total of 91 locations (8 counties) since 2001. Field surveillance was conducted using CDC light traps and gravid traps, and at some sites, BG Sentinel traps (Biogents, Germany), followed by mosquito identification and viral testing across the eight counties of Connecticut. Each site was sampled at least once every 10 days from June-October (Figure 1). We consider here, data from the 2005-2018 surveillance seasons.
b. A trapping-lure comparison study (reported Eastwood et al, 2019) was conducted in Stamford, CT and Hamden, CT, capturing 33,649 and 14,085 individuals respectively using BG-Sentinel traps baited with C02 and different chemical lures. A total of 47,734 mosquitoes (32 species of 8 genera) were trapped and identified over 27 days of sampling in July and August 2018, and pooled for virus detection.
c. Thirdly, focused sampling was performed during 2017 and 2018 at the location where LACV was first detected in the region, (Fairfield, CT; 41.19467, -73.32730), as well as two nearby sites - Easton, CT (41.28032, -73.30308) and Redding, CT; 41.30972, -73.32178). Here, CDC light traps, BG sentinel traps, MMX traps, and gravid traps, as well as GAT traps (Biogents) were deployed overnight at sites for two consecutive nights each week, August-October 2017 and May-October 2018 targeting adult mosquitoes. In addition, as a pilot study of vertical transmission at those sites, six oviposition cups (black plastic casino cups, lined with seed germination paper), were placed in the field and checked weekly to collect container-breeding mosquito eggs.
Mosquitoes captured within each study were sorted from other insect fauna, identified morphologically to species level using a key , with a cold-chain maintained throughout.
Adult mosquitoes were identified and screened for arbovirus as follows. Pools of up to 50 mosquitoes (grouped by species, location, and capture date) are homogenized and inoculated on a Vero (African Green monkey kidney) cell line, for evidence of arboviral infection, as described Eastwood et al. 2019 . Briefly, mosquitoes were homogenized in a vial with 1 mL PBS-G (phosphate-buffered saline containing 0.5% gelatin, 30% rabbit serum, 1× antibiotic/antimycotic) and a copper BB pellet, using a mixer-mill set for 4 min 25 cycles/second. Samples were then centrifuged for 5 min at 7,000 rpm at 4°C. The supernatant (100 μl) was inoculated onto a confluent monolayer of Vero cells in 25-cm2 culture flasks, allowed to absorb for 5 min on a plate rocker, then provided with 4 mL of minimum essential media supplemented with 10% fetal bovine serum, 1× antibiotic/antimycotic. Flasks were incubated at 37°C with 5% CO2 and examined daily for cytopathic effect (CPE) for up to 7 d. Infected cell cultures showing CPE were harvested and stored at −80°C. To identify viruses, RNA was extracted from isolates using a QIAampViral RNA mini kit (Qiagen, Germantown, MD), eluted in a final volume of 70 μL. A reverse transcription polymerase chain reaction (RT-PCR) was performed in a 25 μL reaction using a Titan One-Tube RT-PCR system (Roche Diagnostics, Indianapolis, IN) with generic orthobunyavirus primers [10, 12]. Amplification products of the appropriate size, were purified as per Eastwood (2019), then commercially sequenced (Science Hill DNA Analysis Facility, Yale University, New Haven, CT).
Mosquito eggs collected using the oviposition cups, as part of the third collection study, were reared to adults in the laboratory of the CAES at 25°C with 16/8 h light/dark photoperiod. This F1 generation was screened for virus as above, to test for evidence of LACV having been vertically transmitted.
Minimum infection rates [MIR] of LACV were determined parsed by mosquito species/site/year using the CDC-provided Excel Add-in tool for calculating bias-corrected maximum likelihood estimate pooled infection rates  .
Nucleotide sequencing and genetic analyses
Viral RNA was isolated from virus cultures using the QIamp viral RNA Kit (Qiagen, Valencia, CA). RT-PCR was performed using the Titan One-Tube RT-PCR System (Roche Diagnostics, Indianapolis, IN) with primers targeting each of the three genomic segments of LACV. Primer pairs BUNS+new/BUNS-new and M14C/M4510r were used to amplify the entire S and M segments as previously described (Armstrong and Andreadis 2006). In addition, a portion of L segment was amplified using primers LACL2fwd (GTAGTGTACTCCTATCTACAAAAC) and LACL1077rev (GTTGATATACCCTTTATGCTTTG) . Amplification products were purified using the PCR purification kit (Qiagen, Valencia, CA) and sequenced at the DNA Analysis Facility on Science Hill (New Haven, CT) using an Applied Biosystems 3730xl 96-capillary genetic analyzer (Foster City, CA). Overlapping sequence chromatograms were aligned and edited using ChromasPro (Technelysium Ltd. Tewantin, Australia) and virus sequences were deposited in GenBank (S1 Table).
Edited nucleotide sequences were compared to those available on GenBank using the Blastn search algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments were generated by the ClustalW algorithm and phylogenetic relationships were evaluated by maximum-likelihood analysis in Mega 6.0. The optimal nucleotide substitution was selected by performing ML fits of 24 different models in Mega. Support for individual nodes was evaluated by performing 1000 bootstrap replicates.