Long-term surveillance defines spatial and temporal patterns implicating Culex tarsalis as the primary vector of West Nile virus.

West Nile virus (WNV) has become the most epidemiologically important mosquito-borne disease in the United States, causing ~50,000 cases since its introduction in 1999. Transmitted primarily by Culex species, WNV transmission requires the complex interplay between bird reservoirs and mosquito vectors, with human cases the result of epizootic spillover. To better understand the intrinsic factors that drive these interactions, we have compiled infection data from sentinel chickens, mosquito vectors, and human cases in Iowa over a 15 year period (2002-2016) to better understand the spatial and temporal components that drive WNV transmission. Supplementing these findings with mosquito abundance, distribution, and host preferences data, we provide strong support that Culex tarsalis is the most important vector of human WNV infections in the region. Together, our analysis provides new insights into WNV infection patterns in multiple hosts and highlights the importance of long-term surveillance to understand the dynamics of mosquito-borne-disease transmission.

A taxonomic checklist of the mosquitoes of Iowa.

The last published report of the mosquito species composition present in the state of Iowa was published in 1969 and included 43 species in 8 genera. Since that time, reassessment of specimens in the Iowa State Insect Collection and annual mosquito surveillance efforts have yielded 12 new species records, bringing the total to 55 species in 8 genera. In addition to providing an updated taxonomic checklist for the state of Iowa, abundance information is provided for each species using specimen counts from New Jersey light trapping events that span 45 years.

Susceptibility of fox squirrels (Sciurus niger) to West Nile virus by oral exposure.

Fox squirrels (Sciurus niger) (five of eight) were infected with West Nile virus (WNV) when challenged by the oral route with 10(2.3) or 10(3.4) plaque forming units (PFU). The mean maximum serum WNV titer of infected fox squirrels was 10(5.1) PFU/mL and ranged from 10(4.6) to 10(5.6) PFU/mL. These levels of viremia are infectious for several mosquito vectors of WNV. This virus was also isolated from swabs of the oral and rectal cavities, and urine swabs between day 5 and 9 postexposure (p.e.) in amounts as high as 10(2.0), 10(2.8), and 10(2) PFU, respectively. WNV RNA was detected in salivary gland and/or kidney tissue of three squirrels between day 65 and 72 p.e. in the presence of WNV neutralizing antibody, suggesting that long-term persistent infection occurs in fox squirrels. These observations justify further studies to determine if nonarthropod transmission and long-term persistent infection occur naturally in fox squirrels and contribute to trans-seasonal maintenance of WNV.

Antibodies to West Nile virus in raccoons and other wild peridomestic mammals in Iowa.

Surveillance for evidence of West Nile virus (WNV) infection in small- and medium-sized wild mammals was conducted in Iowa, USA, from May 2005 to June 2007. Sera were collected from 325 mammals belonging to nine species and tested for antibodies to WNV and other flaviviruses by epitope-blocking enzyme-linked immunosorbent assay (ELISA). All sera that had antibodies to flaviviruses by blocking ELISA were further examined by plaque reduction neutralization test (PRNT). Thirteen mammals were seropositive for WNV by PRNT, including 10 raccoons (Procyon lotor). The seroprevalence for WNV in raccoons was 34%. Although a moderately high seroprevalence for WNV has been detected in raccoons in other surveillance studies in the United States, this has not been reported previously in Iowa or most bordering states. Together, these data indicate that raccoons are exposed to WNV at high rates throughout the United States. Two Virginia opossums (Didelphis virginiana) and one fox squirrel (Sciurus niger) were also seropositive for WNV. Nineteen mammals had antibodies to an undetermined flavivirus(es). In summary, we provide serologic evidence that raccoons in Iowa are commonly exposed to WNV.

Fox squirrels (Sciurus niger) develop West Nile virus viremias sufficient for infecting select mosquito species.

The West Nile virus (WNV) viremia and shedding profiles of 11 adult fox squirrels (Sciurus niger) infected by needle inoculation or mosquito bite were characterized. Daily mean titers (95% confidence intervals) for all squirrels on days 1 through 6 postexposure (p.e.) were: 10(1.7 (1.32.1)), 10(4.4 (4.04.8)), 10(5.3 (5.05.6)), 10(4.4 (3.94.9)), 10(2.7 (2.03.4)), and 10(1.1 (0.52.1)) plaque-forming units (PFU)/mL. The highest WNV serum titers of individual squirrels infected by needle inoculation or mosquito bite ranged from 10(4.5) to 10(6.1) and from 10(5.1) to 10(5.3) PFU/mL, respectively. Nine (82%) squirrels, including all 4 squirrels infected by mosquito bite, had WNV serum titers > or =10(5.1) PFU/mL that persisted on average for 1.6 +/- 0.3 days. Infection and dissemination rates of Culex pipiens (L.) that fed on squirrels with serum titers of 10(4.4 +/- 0.1) PFU/mL were 56% and 13%, respectively. Both of these rates increased to over 80% when fed on squirrels with a mean WNV titer of 10(5.5 +/- 0.1) PFU/mL. Infection and dissemination also occurred in Aedes triseriatus (Say) but at a much lower rate. WNV was isolated from the oral and rectal cavities of all squirrels and from urine that was opportunistically collected from 5 squirrels. The largest quantity of WNV recovered from swabs of the oral cavity and urine was 10(3.1) PFU. The longest periods after exposure that WNV was isolated from the oral cavity and urine from a squirrel were 22 and 17 days p.e., respectively. WNV RNA was also detected in kidney tissue in 1 squirrel 29 days p.e., suggesting that fox squirrels can be persistently infected. Collectively these observations provide further evidence that squirrels can contribute to the natural history and epidemiology of WNV, especially in peridomestic environments.

West Nile virus viremia in eastern chipmunks (Tamias striatus) sufficient for infecting different mosquitoes.

In eastern chipmunks (Tamias striatus) inoculated intramuscularly with 101.5 to 105.7 PFU of West Nile virus (WNV), serum titers developed sufficient to infect Aedes triseriatus (Say), Ae. vexans (Meigen), and Culex pipiens (L.). Mean titers (95% confidence interval) of 8 chipmunks were 103.9(3.3-4.5), 106.7(6.4-7.0), and 105.8(4.1-7.5) PFU/mL on days 1-3 postinoculation (p.i.) and 105.8 PFU/mL in 1 chipmunk on day 4 p.i. Mean estimated days that WNV titers were >104.8 and >105.6 were 1.7 (1.1-2.3) and 1.4 (1.0-1.6). The longest period of viremia >104.8 PFU/mL was 3-4 days. WNV antigen was detected in the small intestine of 2 chipmunks and the kidneys of 4 chipmunks by immunohistochemistry. WNV also was detected in urine, saliva, and feces of some chipmunks. These data suggest chipmunks might play a role in enzootic WNV cycles and be an amplifying host for mosquitoes that could infect humans.

A comparision of West Nile Virus transmission by Ochlerotatus trivittatus (COQ.), Culex pipiens (L.), and Aedes albopictus (Skuse).

Transmission of West Nile virus (WNV) by Ochlerotatus trivittatus, Culex pipiens, and Aedes albopictus were compared 14 days after taking blood meals from viremic chickens with titers ranging from 10(2.5) to 10(9.5) cell infective dose (50)s (CID50s)/mL serum. Transmission occurred in one of four (25%) Oc. trivittatus and one of 25 (4%) Cx. pipiens that fed on chickens with titers of 10(5.5) CID50s/mL. No transmission occurred among two of 16 (13%) Oc. trivittatus or one of 25 (4%) Cx. pipiens that became infected after blood meals with titers of 10(5.0) and 10(4.5) CID50s/mL, the next lowest blood meal titers evaluated. Seventeen of 28 (61%) Ae. albopictus transmitted WNV after blood meals with titers of 10(7.0) CID50s/mL, but no infection or transmission was observed among 21 Ae. albopictus that fed on chickens with titers of 10(5.0) CID50s/mL, the next lowest titer evaluated. Transmission by all three species increased dramatically after blood meals with WNV titers of > or = 10(5.5) CID50s/mL. No significant differences occurred in dissemination and transmission rates of the three species after taking blood meals with titers of > 10(7.0) CID50s/mL. The cumulative mean +/- SE transmission rates of Oc. trivittatus, Cx. pipiens, and Ae. albopictus after blood meals with titers of > or = 10(7.0) CID50s/mL were 45.5 +/- 4.1%, 46.8 +/- 4.5%, and 72.4 +/- 5.5%. The cumulative mean dissemination rates of the three species were 78.3 +/- 6.7%, 74.8 +/- 2.6%, and 88.6 +/- 2.1%. The rates of transmission by the three species that developed disseminated infections after blood meals with titers of > or = 10(7.0) CID50s/mL were 58.8 +/- 4.4%, 62.6 +/- 5.8%, and 81.6 +/- 5.4%, respectively. In a previous study, we found that susceptibility of the three species to WNV was essentially the same when fed on chickens with WNV titers of > 10(7.0) CID50s/mL, but Oc. trivittatus and Cx. pipiens were more susceptible than Ae. albopictus to WNV at lower virus titers. The current study strongly suggests that Ae. albopictus is a more efficient vector than Oc. trivittatus and Cx. pipiens when fed blood meals with titers of > 10(7.0) CID50s/mL. However, Oc. trivittatus and Cx. pipiens might be more efficient as vectors when infected by blood meals with titers of < 10(7.0) CID50s/mL.

Eastern cottontail rabbits (Sylvilagus floridanus) develop West Nile virus viremias sufficient for infecting select mosquito species.

The potential of the eastern cottontail rabbit (CTR; Sylvilagus floridanus) to contribute to an enzootic West Nile virus (WNV) cycle was demonstrated by characterizing the WNV viremia profile of 15 CTRs and demonstrating that mosquitoes could become infected by feeding on these CTRs. Eight CTRs were infected with a titer of 10(5.0) cell-infectious dose 50% endpoints (CID50s) of WNV (NY99-Crow) by needle and seven CTRs by bite of one or more WNV-infected mosquitoes. There were no marked differences between the WNV viremia profiles of CTRs infected by either method. West Nile virus was detected in serums of all CTRs by 24 h p.i. The daily mean titers of all 15 CTRs on days 1-4 p.i. were 10(4.1+/-0.4), 10(4.7+/-0.3), 10(4.1+/-0.6), and 10(3.7+/-0.6) respectively, declining to 10(1.2+/-0.1) CID50s/ml of serum by day 6 p.i. No virus was detected in the blood of any CTR on day 7 p.i. The average duration of WNV titers of >or=10(4.3) and <10(5.0) CID50s/mL for all CTRs was 2.2 +/- 0.6 and 1.0 +/- 0.1 days, respectively. The minimum estimated infection rates (MEIRs) of Culex pipiens (L.) and Culex salinarius (Coq.) that fed on CTRs with titers of >or=10(4.3) and >10(5.0) were 11.5 +/- 5.5 and 21 +/- 6.0%, respectively. These rates increased to 20.5 +/- 6.4% and 25.0 +/- 3.0% when CTR serum titers were >10(5.0) CID50s/mL. Neither Aedes aegypti (L.) nor Aedes albopictus (Skuse) were infected by feeding on CTRs with titers of <10(5.0) CID50s/mL. The MEIRs of these two species were 11.5 +/- 3.5% and 1.5 +/- 0.5% after feeding on CTRs with titers of >10(5.0) CID50s/ml. None of the CTRs infected by mosquito bite or by needle showed any symptoms of WNV disease.

Susceptibility of Ochlerotatus trivittatus (Coq.), Aedes albopictus (Skuse), and Culex pipiens (L.) to West Nile virus infection.

The susceptibility of Ochlerotatus trivittatus (Coq.) to West Nile virus (WNV) was assessed by comparing it to the susceptibility of Aedes albopictus (Skuse), a likely bridge vector, and Culex pipiens (L.), a primary WNV amplifying species. The three species were infected with WNV (NY crow-1999) by feeding on 2-3-day-old chickens with serum virus titers ranging from 10(2.5) to 10(9.5) cell culture infective dose (CID) 50s/mL. The lowest infective titer for Oc. trivittatus and Cx. pipiens was 10(4.5) CID50s/mL. Thirteen percent (4/32) and 2% (1/45) of each species became infected postprandially. Infection rates of the two species increased to 43% (6/14) and 15% (6/40) after blood meals with a titer of 10(5.5) CID50s/mL. In contrast no infection was observed in nine Ae. albopictus that fed among three chickens with titers of 10(4.5) CID50s/mL nor in 41 Ae. albopictus that fed among three chickens with titers of 10(5.0) CID50s/mL. The infective dose 50s for Oc. trivittatus, Cx. pipiens and Ae. albopictus were 10(6.0), 10(6.2), and 10(6.6) CID50s/mL, respectively. Collectively these observations suggest that Oc. trivittatus and Cx. pipiens are more susceptible than Ae. albopictus to WNV when they feed on hosts with WNV titers of <10(7.5) CID50s/mL, but nearly as susceptible with blood meal titers of > or =10(7.5) CID50s/mL. Unpublished studies in our laboratory showed that cottontail rabbits fed on by WNV-infected Oc. trivittatus developed viremias as high as 10(5.5) CID50s/mL serum which exceeds 10 (4.2 (3.4-4.6)) CID50s/mL, the predicted ID10+/-95% CI of Oc. trivittatus. Consequently this mosquito, which also feeds on humans and birds has the potential to serve as a bridge vector and as a maintenance vector among mammals.