Officials with the NYSDOH and Suffolk County Department of Health Services contacted a Long Island, New York, resident for a follow-up investigation after receiving notification that a tick removed from the resident and submitted for comprehensive pathogen testing was positive for HRTV RNA. The resident, a man in his 60s, removed the tick on August 8, 2018, and recalled having a low-grade fever (maximum temperature 100.5°F) and fatigue for 5 days beginning on August 15, 2018. He noted no other symptoms.

Serum was provided at multiple time points for serologic analysis. We tested serum samples by using a standard 90% plaque reduction neutralization test (PRNT₉₀) for HRTV (strain M12-66) (8), provided by the Centers for Disease Control and Prevention. We tested samples at Wadsworth Center, NYSDOH, and results were confirmed by the Centers for Disease Control and Prevention. Neutralizing antibody titers were 1:20, 1:160, and 1:160 for samples collected at 8, 50, and 96 days after symptom onset (15, 57, and 103 days after removal of the tick), respectively, indicative of a recent infection with HRTV.

We initiated standardized drag and flag sampling of host-seeking A. americanum ticks on public lands for arbovirus surveillance during 2016, before HRTV detection. We found that 132 pools (containing 475 nymphs and 437 adults) from 4 Suffolk County locations were negative for HRTV by real-time reverse transcription PCR using established protocols (8). During 2018, tick surveillance at 5 locations yielded 102 pools (969 adults); all were negative for HRTV.

Increased efforts during the public health investigation conducted on August 23 and 24, 2018, yielded an additional 113 A. americanum ticks (92 larvae and 21 nymphs) from a location where tick exposure potentially occurred. All ticks collected during the investigation were negative for HRTV. No ticks were found during sampling of the property surrounding the residence of the case-patient.

Figure 1

Figure 1. Tick collection sites in study of heartland virus transmission, Suffolk County, New York, USA. Numbers within townships indicate sample size of deer tested for neutralizing antibody.

During 2019 and 2020, tick surveillance in the towns of Brookhaven and Riverhead yielded 1,123 pools of A. americanum ticks (2,788 adults and 6,728 nymphs) (Figure 1). We found that 3 pools of unengorged nymphs collected from the Brookhaven site on June 14 (n = 1) and June 24 (n = 2), 2019, and 2 pools of unengorged nymphs collected from the same location on July 25 and August 5, 2020, were positive for HRTV RNA. We isolated virus from 2 tick pools after incubation on Vero cells. We found that testing of >1,100 Ixodes scapularis ticks (199 pools) collected during the surveillance campaign in Suffolk County, during 2018–2020, were negative for HRTV.

Figure 2

Figure 2. Phylogenetic relationship among Heartland virus isolates, Suffolk County, Long Island, New York, USA. Separate alignments of large segments (A), medium segments (B), small segments (C), and partial nonstructural sequences (D)...

We extracted RNA from isolates by using established protocols (13). We developed primer pairs to amplify the small, medium, and large RNA segments by using a One-Step Superscript III Reverse Transcription PCR with Platinum Taq (Life Technologies, https://www.thermofisher.com) (Table 1). We performed 3 separate reactions using 5 μL of RNA, 1 μL of polymerase, and 0.2 μmol/L final concentration of primer pairs in a total reaction volume of 50 μL. We amplified products with the following thermocycler conditions: 55°C for 30 min; 94°C for 2 min; 40 cycles at 94°C for 30 s, 57°C for 45 s, and 68°C for 4 min; and a final extension at 68°C for 10 min. Amplicons were visualized by electrophoresis on a 1% agarose gel. Products were pooled and purified for next-generation sequencing at the Wadsworth Center, NYSDOH, Applied Genomics Core. We prepared libraries by using the Nextera XT Kit (Illumina, https://www.illumina.com) and performed sequencing using the MiSeq Illumina platform; we analyzed sequences by using Geneious Prime Software (https://www.geneious.com) (Table 2; Figure 2).

We conducted serologic testing of hunter-harvested white-tailed deer blood submitted for arbovirus serosurveys by using PRNT₉₀, as described (14). We screened 686 serum samples at a dilution of 1:20 for neutralizing antibodies to HRTV (Figure 1) and serially diluted positive serum samples for endpoint titers. Overall, 9.8% of the deer were seropositive and had titers ranging from 1:20 to >1:640; 76% of the seropositive deer had titers >1:20. We tested 1,641 A. americanum ticks collected from 145 sampled deer for HRTV RNA but did not detect any virus.

Evidence of widespread HRTV transmission was demonstrated throughout Suffolk County, New York. Consistent with previous studies, A. americanum ticks were implicated in local transmission of HRTV. All positive pools were nymphal stage ticks, including the tick originally submitted for testing at the University of Massachusetts. Tick minimal infection rates ranged from 0% to 1.1%. It is unclear whether flat nymphs had acquired the virus as larvae feeding on viremic hosts, through cofeeding transmission, or transovarially because each of these modes has been demonstrated in the laboratory (7).

The lack of HRTV detection in adult ticks is notable if one considers that collections occurred at the same site across 3 seasons. Higher numbers of positive nymph pools were observed in Missouri, where 53/60 HRTV-positive tick pools collected at sites near the first described human cases were nymphs (6). Complete genome sequence analysis of the HRTV strains isolated during this study showed >98% amino acid and >93% nucleotide identities to the original strains isolated from patients in Missouri during 2009 (1) and a strain isolated in Tennessee during 2013 (2).

White-tailed deer are a sensitive sentinel model for many arboviruses, given their abundance, limited home range, and the frequency on which they are fed upon by hematophagous arthropods (10,11,14). Approximately 10% of the deer sampled during this study were seropositive against HRTV. Our serologic testing strategy differed from those of previous studies by using a more stringent PRNT₉₀. Suffolk County deer seropositive rates were similar to those reported in Vermont (10%), Maine (11%), and Florida (4%) deer (12). The rates are lower than those reported for deer tested in midwestern and southeastern states, areas with burgeoning populations of A. americanum ticks (10,11). To date, no competent vertebrate host, including deer, has been implicated in HRTV amplification (15).

Results from this study emphasize the need to include HRTV in surveillance programs wherever A. americanum ticks are distributed. Furthermore, clinicians should be aware of this pathogen and the potential for overlapping symptomologies (fever, fatigue, and loss of appetite) with other tickborne infections. Providers should request HRTV testing for patients who have clinical symptoms, including leukopenia and thrombocytopenia, and a history of tick exposure or travel to regions where A. americanum ticks are reported.