Eastwood et al. Parasites Vectors (2020) 13:569 https://doi.org/10.1186/s13071-020-04440-4 Parasites & Vectors RESEARCH Open Access Local persistence of novel regional variants of La Crosse virus in the Northeast USA Gillian Eastwood1,2*, John J. Shepard1, Michael J. Misencik1, Theodore G. Andreadis1 and Philip M. Armstrong1 Abstract Background: La Crosse virus (LACV) (genus Orthobunyavirus, family Peribunyaviridae) is a mosquito-borne virus that causes pediatric encephalitis and accounts for 50–150 human cases annually in the USA. Human cases occur primar- ily in the Midwest and Appalachian regions whereas documented human cases occur very rarely in the northeastern USA. Methods: Following detection of a LACV isolate from a feld-collected mosquito in Connecticut during 2005, we evaluated the prevalence of LACV infection in local mosquito populations and genetically characterized virus isolates to determine whether the virus is maintained focally in this region. Results: During 2018, we detected LACV in multiple species of mosquitoes, including those not previously associ- ated with the virus. We also evaluated the phylogenetic relationship of LACV strains isolated from 2005–2018 in Con- necticut and found that they formed a genetically homogeneous clade that was most similar to strains from New York State. Conclusion: Our analysis argues for local isolation and long-term persistence of a genetically distinct lineage of LACV within this region. We highlight the need to determine more about the phenotypic behavior of these isolates, and whether this virus lineage poses a threat to public health. Keywords: Arbovirus, Vector, Mosquito species, La Crosse virus, Pathogen persistence, Genetic distinction, Public health risk Background socioeconomic burden associated [1–4]. In contrast, only La Crosse virus (LACV) (genus Orthobunyavirus, family one locally-acquired case of LACV has been reported in Peribunyaviridae) is a mosquito-borne virus, associated the northeastern USA during the last 20 years, occurring with clinical cases of pediatric encephalitis concentrated in upstate New York in 2010 and was non-neuroinvasive in the Midwest and Appalachian regions where it is [5]. In addition, two human cases were reported in Rhode also detected in mosquitoes during surveillance activi- Island, during 2018 and 2019, but both were imported as ties. Symptoms of La Crosse encephalitis include head- these individuals had a travel history outside that state ache, fever, vomiting, and disorientation which may lead (Rhode Island DPH, personal communication). LACV to seizure, coma, and death in severe cases [1]. Nota- was frst detected in the New England region in a pool ble clusters of illness have occurred in Ohio, Wiscon- of mosquitoes collected in Connecticut in 2005 during sin, Tennessee, and North Carolina with a substantial statewide surveillance for arboviral pathogens [6]. Phy- logenetic analysis indicated that the Connecticut isolate *Correspondence: [email protected] represented a genetically distinct lineage that diverged 1 Center for Vector Biology & Zoonotic Diseases, Department earliest from viruses circulating in other geographic of Environmental Sciences, The Connecticut Agricultural Experiment Station, New Haven, Connecticut, USA regions of the USA [6, 7]. Full list of author information is available at the end of the article © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Eastwood et al. Parasites Vectors (2020) 13:569 Page 2 of 8 Despite the disease being associated within the Mid- (iii) Tirdly, focused sampling was performed during west and Appalachian regions of the USA, LACV may 2017 and 2018 at the location where LACV was represent an unrecognized public health risk to residents frst detected in the region, (Fairfeld, CT; 41.19467, of New England and New York. Tere is a need to under- -73.32730), as well as two nearby sites: Easton, CT stand the transmission cycles, prevalence, and strains of (41.28032, -73.30308) and Redding, CT (41.30972, LACV in this region. Aedes triseriatus serves as the main -73.32178). Here, CDC light traps, BG-Sentinel vector of LACV which is broadly distributed throughout traps, MMX traps, and gravid traps, as well as GAT the eastern half of the USA [8]. Nevertheless, this species is traps (Biogents) were deployed overnight at sites likely under-sampled during statewide surveillance eforts for two consecutive nights each week, August- because it does not readily enter standard CDC light traps October 2017 and May-October 2018 targeting or gravid traps which are routinely used [3, 9]. Additional adult mosquitoes. In addition, as a pilot study of sampling and testing of Ae. triseriatus and other locally vertical transmission at those sites, six oviposition abundant mosquito species are needed to accurately esti- cups (black plastic casino cups, lined with seed mate the entomological risk of LACV in enzootic sites. germination paper), were placed in the feld and Here we report the results of mosquito monitor- checked weekly to collect container-breeding mos- ing activities detecting LACV in Connecticut. In 2018, quito eggs. we conducted two surveys that intensifed the extent of mosquito sampling and testing in central and southwest- Mosquitoes captured within each study were sorted ern Connecticut and highlighted increased detection of from other insect fauna, identifed morphologically to LACV activity [10]. In addition, routine statewide sur- species level using a key [11], with a cold-chain main- veillance in Connecticut, ongoing since 1997, detected tained throughout. four additional isolates of LACV since 2005. We pre- sent evidence for ongoing circulation of LACV in this Virus detection region, and report on the entomological and viral phylo- Adult mosquitoes were identified and screened for genetic data that supports local persistence of LACV in arbovirus as follows. Pools of up to 50 mosquitoes Connecticut. (grouped by species, location and capture date) were homogenized and inoculated on a Vero (African Green Methods monkey kidney) cell line, for evidence of arboviral Entomological data infection, as described Eastwood et al. [10]. Briefly, Mosquito collections mosquitoes were homogenized in a vial with 1 ml Isolates of LACV analyzed in the present investigation PBS-G (phosphate-buffered saline containing 0.5% were obtained from feld-collected mosquitoes that were gelatin, 30% rabbit serum and 1× antibiotic/antimy- procured from the following: cotic) and a copper BB pellet, using a mixer-mill set for (i) Connecticut state has conducted annual monitor- 4 min at 25 cycles/second. Samples were then centri- ing of mosquitoes at 36 locations since 1997 and fuged for 5 min at 7000× rpm at 4 °C. The supernatant was increased to a total of 91 locations (8 counties) (100 μl) was inoculated onto a confluent monolayer of since 2001. Field surveillance was conducted using Vero cells in 25-cm2 culture flasks, allowed to absorb CDC light traps and gravid traps, and at some sites, for 5 min on a plate rocker, then provided with 4 ml of BG-Sentinel traps (Biogents, Regensburg, Ger- minimum essential media supplemented with 10% fetal many), followed by mosquito identifcation and bovine serum, 1× antibiotic/antimycotic. Flasks were viral testing across the eight counties of Connecti- incubated at 37 °C with 5% CO 2 and examined daily cut. Each site was sampled at least once every 10 for cytopathic effect (CPE) for up to 7 days. Infected days from June-October (Fig. 1). We consider here, cell cultures showing CPE were harvested and stored data from the 2005–2018 surveillance seasons. at -80 °C. To identify viruses, RNA was extracted from (ii) A trapping-lure comparison study (reported by isolates using a QIAampViral RNA mini kit (Qiagen, Eastwood et al. [10]) was conducted in Stamford, Germantown, MD, USA), eluted in a final volume of CT and Hamden, CT, capturing 33,649 and 14,085 70 μl. A reverse transcription polymerase chain reac- individuals, respectively, using BG-Sentinel traps tion (RT-PCR) was performed in a 25 μl reaction using baited with CO2 and diferent chemical lures. A a Titan one-tube RT-PCR system (Roche Diagnostics, total of 47,734 mosquitoes (32 species of 8 genera) Indianapolis, IN, USA) with generic orthobunyavi- were trapped and identifed over 27 days of sam- rus primers [10, 12]. Amplification products of the pling in July and August 2018, and pooled for virus appropriate size, were purified as per Eastwood et al. detection. [10], then commercially sequenced (Science Hill DNA Eastwood et al. Parasites Vectors (2020) 13:569 Page 3 of 8 Analysis Facility, Yale University, New Haven, CT, and two isolates in the towns of Weston and Fairfeld USA). during 2018, all within Fairfeld county (Fig. 1, Table 1); Mosquito eggs collected using the oviposition cups, (ii) of 14,085 mosquitoes captured at the Hamden loca- as part of the third collection study, were reared to tion during the Lure evaluation study in July and August adults in the laboratory of the CAES at 25 °C with a 2018, 4 novel isolates of LACV were made from three 16:8 h light:dark photoperiod.