Banked blood samples collected from white-tailed deer, raccoon, and (occasionally) moose (Alces alces) and coyote (Canis latrans) during 2009–2014 were analyzed by plaque-reduction neutralization test for HRTV neutralizing antibodies by using African green monkey kidney (Vero) cell culture. Only samples from healthy live-trapped animals or deceased animals from anthropogenic causes (i.e., hunting, culls, and automobile strikes) were tested. Inclusion of 19 states was opportunistic based on sample availability. We used the plaque-reduction neutralization test to evaluate HRTV seropositivity for white-tailed deer (n = 396), raccoon (n = 949), coyote (n = 61), and moose (n = 22) (Table 1). Samples consisted of whole blood dried onto Nobuto strips (Advantec MFS, Inc., Dublin, CA, USA), bloody body cavity fluids, or hemolyzed whole blood. Nobuto strip samples were eluted to 1:10 serum dilution in phosphate buffer solution in accordance with the manufacturer’s instructions. All samples were heat-inactivated at 56°C for 45 min.

We screened the inactivated samples at 1:20 dilution by mixing serum diluted 1:10 with equal volume of titrated HRTV to approximate a challenge dose of 50 PFUs. Treated Vero cells were incubated for 1 h at 37°C, 5% CO₂, before applying a nutrient-rich 0.5% agarose overlay. A second overlay containing Neutral Red was applied after 5–7 d of incubation. Viral plaques were counted 6–12 d after inoculation. A neutralization threshold of 70% relative to HRTV-only controls was used to determine positive samples. All screen-positive samples were repeat-tested to confirm results. Samples were considered seropositive if they were confirmed as positive at a dilution of >1:40. Comparative neutralization tests with related viruses were not performed, because we had previously found that murine antiserum developed against the other known phleboviruses in the United States—Sunday Canyon virus (7), Rio Grande virus (8), and Lone Star virus (9)—had no appreciable neutralizing activity against HRTV (Table 2). Human antiserum developed against HRTV exhibited weak 1-way neutralization of Lone Star virus and Sunday Canyon virus (Table 2).

Figure 1

Figure 1. State-level distribution of Heartland virus case reports in humans and seropositive wildlife, central and eastern United States, 2009–2014. Red indicates states with seropositive animals; gray indicates states with no seropositive animals....

Figure 2

Figure 2. Geographic groupings of confirmed seropositive animals for Heartland virus neutralizing antibodies, central and eastern United States, 2009–2014. Twenty groups were identified in 13 states. The geographic locations of the groups were...

Of 1,428 animals, 103 were seropositive: 55 deer, 33 raccoon, 11 coyotes, and 4 moose. Thirteen states had seropositive animals: Florida, Georgia, Illinois, Indiana, Kansas, Kentucky, Maine, Missouri, New Hampshire, North Carolina, Tennessee, Texas, and Vermont (Table 1; Figure 1). Within the 13 states, 20 geographic clusters of seropositive animals were mapped by plotting positive animals by the county where they were collected (Figure 2).

We provide evidence of widespread HRTV transmission activity across the central and eastern United States. Of 13 affected states, only Missouri and Tennessee had previous evidence of HRTV activity. A more stringent neutralization threshold of 80% would reclassify 14 positive samples to “equivocal,” but the number of positive states would remain unchanged. These findings should encourage clinicians and public health officials to consider HRTV as a potential source of illness throughout the eastern United States.

Surprisingly, seropositive white-tailed deer were detected in northern New England, where established populations of lone star ticks are unknown (6). Possible explanations include unreported lone star tick populations, immigration of seropositive deer, alternative tick vectors for HRTV, or presence of a serologically cross-reactive virus. Movement of deer across state boundaries is an unlikely explanation. Extensive lone star tick populations are not reported in neighboring states (6), and migration of deer from lone star tick–infested regions is unlikely (10). Savage et al. did not detect HRTV RNA in Dermacentor variabilis, the American dog tick (4), but additional tick species inhabit northern New England. Several tick species are reported to transmit severe fever with thrombocytopenia syndrome virus, a closely related phlebovirus found in eastern Asia (11). Further investigation of tick populations and their vector competence for HRTV is warranted, and production of HRTV neutralizing antibodies in response to a serologically similar virus should be investigated. Two new phleboviruses recently detected in Ixodes ticks in the northeastern United States are genetically unrelated to HRTV but raise the possibility that additional undiscovered phleboviruses exist (12). Severe fever with thrombocytopenia syndrome virus–reactive antibodies in wildlife were reported in Minnesota, also peripheral to the lone star tick geographic range, indicating the likely presence of HRTV or a similar virus there (13).

The finding of seropositive moose and coyotes indicates that these mammals are exposed to HRTV in certain situations and might be useful targets for serosurveillance, in addition to deer and raccoon. The full vertebrate host range and the reservoir competence of these mammals for HRTV remains unknown.

The chronology of dispersal of HRTV is unclear. Suggesting that HRTV emerged in northwestern Missouri and spread to neighboring states to the east and south is overly simplistic. Because animals were sampled at different points of time and space during this study, our data lack robustness to enable comparison of populations over time or between geographic locations. Thus, we are unable to evaluate the dynamics of HRTV spread. Furthermore, the proportions of HRTV-seropositive animal populations lack quantitative value because of our retrospective convenience sampling. Our results simply indicate that HRTV or a very similar virus has circulated in the sampled regions in the recent past and that this activity began as early as 2009. Adult seropositive white-tailed deer were detected in Maine and Florida in 2009, and based on the estimated ages of affected deer (data not shown), the infections could have occurred as early as 2003. A much larger retrospective serosurvey is necessary to elucidate HRTV’s history of emergence.