Origin of 3 Rabid Terrestrial Animals in Raccoon Rabies Virus–Free Zone, Long Island, New York, USA, 2016–2017

During 2016–2017, three rabid terrestrial animals were discovered in the raccoon rabies virus–free zone of Long Island, New York, USA. Whole-genome sequencing and phylogenetic analyses revealed the likely origins of the viruses, enabling the rabies outbreak response (often costly and time-consuming) to be done less expensively and more efficiently.

Here, we describe our efforts to identify the origins of these rabid animals and our contingency plans to eliminate further cases and restore the rabies virusfree zone status.

The Study
In March 2016, a rabid raccoon with neurologic signs was trapped in Hicksville in Nassau County. Realtime reverse transcription PCR (6) and Sanger sequencing demonstrated that the animal was infected with a raccoon rabies virus variant. A phylogenetic analysis of the full nucleoprotein and partial glycoprotein gene sequences did not provide us the resolution required for us to confidently identify where the virus had originated. At the time of this original analysis, whole-genome sequencing (WGS) was not available in our laboratory. We initiated enhanced rabies surveillance for ≈12 months in Babylon and Huntington of Nassau County to determine if rabies virus was circulating locally by increasing the number of wild animals submitted for testing via trapping, roadkill collection, and animal control activities, but no animals were positive for rabies.
In December 2016, an employee of the New York State Department of Environmental Conservation (Albany, New York, USA) found a river otter on Sound Beach on the North Shore of Suffolk County; the otter was submitted to the Rabies Laboratory of the New York State Department of Health in February 2017. After the otter was determined to be rabies virus positive, our goal was to ascertain by WGS and sequence analysis where the otter was exposed, which would help us determine which supplementary contingency actions were required.
In November 2017, a cat with neurologic signs that bit a veterinary staff member in Nassau County was submitted for testing. After determining this cat was positive for rabies virus, we included this case in the study.
When the raccoon was originally discovered on Long Island, we had contacted multiple state and federal agencies to discuss contingency plans for containing a possible rabies outbreak. We agreed to limit the contingency actions at that juncture to 12 months' enhanced surveillance, public health alerts to educate citizens, and reminders about free rabies virus vaccine clinics for domestic animals. Only in the event that other rabid animals were discovered during this time would wildlife trap, vaccinate, and release programs be implemented and oral rabies virus vaccine baits be distributed (4,5,7). These vaccination programs can be expensive, so they are used conservatively as last resort efforts. The cost of the raccoon rabies virus elimination program that occurred on Long Island during 2006-2010 was ≈$2.6 million (4).
The contingency plan to respond to the rabid river otter was initially to deploy oral rabies vaccine around a 20-km radius of Sound Beach Long Island, covering nearly all of central Suffolk County ( Figure  1). Costs would have exceeded $200,000 as 120,000 baits would need to have been spread by foot, vehicle, and aircraft over a 636.6-km 2 area during spring 2017.

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Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 6, June 2020 Because Wadsworth Center Sequencing Core (Slingerlands, New York, USA) charged ≈$250/sample for WGS, we were able to save time and money by sam-pling animals and ruling out some areas as having local transmission and deescalating the initial plan. Applying WGS to priority samples has previously *Bold indicates rabies virus isolates that were found in the rabies virus-free zone. ID, identification. †Previously deposited in GenBank (8).
been shown to be a viable strategy for public health laboratories to use in epidemiologic applications (8).
Using phylogenetic analysis (Table; Figure 2), we found that the raccoon in Hicksville was infected with a rabies virus variant consistent with those found in southwestern Connecticut. Considering the absence of local rabid wildlife with a similar rabies virus variant, we hypothesized that this raccoon was either accidentally or purposefully translocated into Long Island by human activity. Such translocation events were previously demonstrated with the linkage of an outbreak in Canada to a clade of raccoon rabies virus from upstate and central New York (9) and the spread of raccoon rabies virus up the East Coast of the United States (2,8). Furthermore, although the range of the average raccoon is highly dependent on many factors, raccoon ranges have been established to rarely exceed 4 km (2.5 miles) (10,11), well short of the 56-km (35-mile) distance from the Connecticut panhandle to Hicksville, New York.
Our phylogenetic analysis could not be used to conclusively determine where the rabies virus variant found in the river otter circulates, although the clade from Connecticut demonstrated the highest sequence similarity. Otters are found on the South Shores of Connecticut, Massachusetts, and Rhode Island and the North Shore of Long Island (12). They can swim >32 km (20 miles) a day and occupy territories of considerable size (13). When found, the otter was extremely decomposed to the extent of being borderline untestable. We deduced that the otter was likely from coastal or even inland Connecticut on the basis of phylogenetic data, tissue quality, and species behavior and habitat, but we do not know for certain. Additional sampling from Connecticut could potentially result in the virus from the otter being nested definitively within clade 1 subclade 2.
The rabid otter and raccoon most likely represent 2 separate incursion events, evidenced by the phylogenetic analysis. Because enhanced surveillance did not lead to the discovery of additional rabid animals during January 2017-December 2019 in the entirety of Suffolk and Nassau Counties, we can confidently stipulate that these were isolated events. Although we cannot state with conviction that no other animals became infected, these incursions appear to be short lived and self-limiting. The rabid cat from November 2017 was briefly considered a continuation of this tentative Long Island rabies outbreak, but after several conversations with public health authorities and the pet owner, this cat was determined to have been unvaccinated and adopted ≈80 km (50 miles) away in Westchester County. Phylogenetic analysis confirmed the virus variant was consistent with those found in downstate New York (clade 1). Although international pet adoption often garners headlines, domestic animal adoption (14) and incidental rabies translocation remains a constant threat that can jeopardize the results of multimillion-dollar rabies elimination programs.

Conclusions
Using WGS of an assortment of viruses, we revealed the likely origins of 3 raccoon rabies virus variants from 3 animals that had unexpectedly broken into a rabies-free zone. Through intensified enhanced rabies surveillance and WGS, an expensive contingency plan to distribute baits on Long Island ultimately became unnecessary and was not implemented. This study demonstrates the utility of WGS and phylogenetics as part of a multifaceted rabies investigation with tangible real-world consequences.