La Crosse virus (LACV) is a member of the California serogroup viruses, which include Jamestown Canyon virus, California encephalitis virus, and snowshoe hare virus. LACV is most commonly associated with the Midwest and Appalachian regions of the USA. No locally acquired clinical cases have been documented in New England, and only one human case reported in New York State a decade ago. However, we show that through intensive targeted trapping, further evidence of LACV activity in mosquitoes in the Northeast is produced. Following the original detection in 2005, no isolates of LACV were obtained in Connecticut for 10 further years. Corroborating data from a focused-surveillance studies with routine monitoring data in Connecticut, and isolates from previous years (2005, 2015 and 2016) enabled evidence of localized persistence of novel regional variants of LACV. Twelve of the fourteen new isolates were detected in 2018, when species-directed sampling efforts were increased to assess the persistence of this virus. To be noted, 2018 was a year when higher than usual numbers of mosquitoes, as well as arbovirus detections, were observed in general under the standard routine sampling in Connecticut (unpublished data, (14)), and this increased catch size may also have contributed to our detection of LACV.
Aedes triseriatus has been implicated as the main vector involved in the maintenance and transmission of LACV (15). LACV isolates described here were detected between July and October from four different mosquito species but were most frequently detected in Ae. triseriatus, providing further evidence to support its role as the primary vector. Our detection of LACV in Ae. cinereus and Ae. trivittatus represents a novel finding in the USA. Aedes cinereus is frequently captured in Connecticut, and a source of several other arbovirus isolations as well, including Cache Valley, Eastern equine encephalitis, Jamestown Canyon, and West Nile viruses (11). Nevertheless, detection of LACV in a particular mosquito species does not necessarily incriminate it as a competent vector for the virus, and further studies are needed to elucidate its respective role in LACV transmission in the region. Outside of the northeastern US, LACV has been isolated from a number of mammalian-biting mosquito species in addition to Ae. triseriatus, including Ae. albopictus, Ae. canadensis, Ae. japonicus, Ae. vexans and Psorophora howardii (6, 16–20). Aedes albopictus and Ae. japonicus are invasive mosquito species emerging in Connecticut (21, 22); however, no LACV isolates were found from either of these species in this study. Our detection of LACV in a pool of Ae. canadensis reinforces findings in Ohio which showed this species to be a secondary vector of LACV, via both field isolations of the virus and vector competence studies (23); Aedes vexans is also thought to be an accessory vector for LACV in Virginia (24). There is also experimental evidence that Culex spp. may play a limited role in LACV transmission dynamics (25).
Sciurids (chipmunks and squirrels) serve as the main vertebrate hosts for horizontal transmission and amplification of LACV, and a high rate of transovarial transmission is also reported in Ae. triseriatus in other regions of the USA (26, 27). The contribution of both vertical and horizontal transmission requires further study to better understand LACV dynamics in the northeastern USA. Our study here included limited testing for evidence of vertical transmission in F1 mosquitoes reared from field-collected eggs, none of which tested positive. Finding no evidence of LACV in F1 mosquitoes could be surprising, given the high rate of vertical transmission reported elsewhere in the USA. For example, minimal infection rates of LACV in mosquitoes collected as eggs ranged from 0.4 to 7.5/1,000 in study sites in West Virginia (28). However, greatly increased numbers of field samples are required, as well as controlled experiments in the laboratory, to fully assess the role of vertical transmission. Reduced levels of vertical transmission of virus in mosquitoes could be one reason why LACV is infrequently detected in the northeastern USA. Miller et al (1977) report that LACV remained infective to vertebrate hosts after eight transovarial passages in Ae. triseriatus with infection rates of up to 71% in the offspring of an infected female (25). From this, they estimated that LACV can persist four years or longer in the vector population in the absence of horizontal amplification in vertebrate hosts. Transovarial transmission (TOT) is ecologically significant in the persistence of many vector-borne pathogens and used frequently by members of Bunyavirales when classical horizontal transmission is not possible (29).
MIRs can be seen to be high in 2015 and 2016, reflecting that the small quantity of Ae. triseriatus captured also yielded LACV. Our focused study in 2017-18 aimed to improve the catch rate of this mosquito species to examine LACV in the region. Routine mosquito surveillance in Connecticut and throughout much of the northeast, largely focuses on the use of CDC light and gravid traps. Few adult Ae. triseriatus were captured during routine mosquito surveillance using CDC light traps, gravid traps, or standard deployment of BG sentinel traps, and it was not until our focused-LACV study 2017-18 using a wide variety of different traps, that we saw numbers of this species increase. We suggest BG-Sentinel baited not only with BG-lure, but additionally with CO2, may enable increased collections of adult Ae. triseriatus, and thus enhanced chance of detecting LACV if it is present in an area (3); once commercially available, additional baits described Eastwood et al. (2020) are even more effective than BG-Lure (10). Minimal field infection rates reported elsewhere have ranged from 0.26 to 27 per 1,000 mosquitoes tested (15, 28, 30, 31).
Variants of LACV identified during this study represent a distinct third lineage. In other geographic regions of the USA, where lineages one and two occur, the virus is associated with clinical symptoms of human disease. Conversely and to our knowledge, locally acquired clinical cases of La Crosse encephalitis are exceedingly rare in the northeastern US. Whether this is due to under-diagnosis of clinical illness to LACV (lack of case recognition), differences in virus strain virulence, a low prevalence of infection in mosquito vectors that effectively limits human exposure to biting activity in regions where the virus circulates, or limited vector competence by these mosquito species, remains to be determined. Clearly, given the presence of this arbovirus in mosquitoes in several areas of CT, there appears to exist an entomological risk. Furthermore, this risk occurs where there is currently no reported human disease, yet a sizeable human population which could come into contact with LACV-infected mosquitoes.
Our findings clearly warrant that further investigation be taken to assess the public health risk that LACV (lineage III) may pose to this region of the USA. There is a requirement for 1) vector competency studies with local populations of Ae. triseriatus and other identified mosquito species; 2) infection and virulence studies in an animal model; 3) assessment of the role of enzootic vertebrate reservoir hosts for this lineage of LACV, and 4) vertical transmission studies in Ae. triseriatus