Incrimination of shrews as a reservoir for Powassan virus

Samples of host-seeking nymphal black-legged ticks were collected during 2018–2020 from Massachusetts and Rhode Island sites where DTV is enzootic (Fig. 1). Individual DTV-infected ticks were identified by RT-PCR; all ticks (including virus-negative ticks) were also analyzed for B. burgdorferi infection by PCR. Host bloodmeal remnant identification using assays targeting family and order specific retrotransposons was performed as described¹¹, with the addition of newly described primers (see Table 1). Assays targeted likely mammalian reservoir hosts within our study sites.

We identified 20 nymphal ticks that contained DTV RNA from 13 different sites (prevalence, 0.4–7%, Table 2) and confirmed viral identity by sequencing a 248 bp section of the NS5 gene and 286 bp section of the envelope gene. Cognate viral sequences from these ticks were assigned to the DTV lineage (Fig. 2). Sequences from ticks collected from field sites in close geographic proximity often clustered together. Borrelia burgdorferi prevalence was more variable, ranging from 0 to 21%., and B. burgdorferi infection was not associated with DTV infection (p = 0.5, Fig. 3), as sites with high numbers of ticks infected with spirochetes were not the same as those that had high numbers of DTV-infected ticks (Table 2).

The source of the infectious larval bloodmeal was identified from 16 of the 20 DTV-infected ticks (80%), 13 of which were identified as shrews (65%) (Table 3). The other DTV-infected ticks had fed on diverse hosts such as bird, squirrel, and cat. One tick showed evidence of having fed on multiple hosts (shrew and deer). None of the ticks had fed on a mouse. We conclude that in our sites, during the years that we sampled, larval ticks feeding on shrews were more likely to be infected by DTV than by feeding on any other animal. Using the 0.1% estimated rate of transmission of adult female ticks to larval progeny for the related tick-borne encephalitis virus¹², we calculated that up to four ticks (95% binomial confidence interval of 0 to 0.4% of ticks) from our study could derive from inheritance. Thus, we cannot exclude this as the source of the single infected ticks derived from a bird, squirrel, and cat. However, more than four infected ticks were derived from shrews, suggesting that inheritance alone cannot explain the apparent association.

During the years that we sampled our study sites, mice did not contribute as many larval bloodmeals as might be expected^13,14. The proportion of nymphal ticks that fed on mice ranged from 2 to 20% (median 10.5%) (Table 4). Our previous publication identified sites where the majority of ticks had fed on mice (Nantucket 2018, 100%, and Robin’s Island 2018 and 2019, 91% and 53%, respectively¹¹), but DTV was not identified from these collections. Squirrels, or other Sciuridae, contributed ticks at only two sites (median host contribution, 1%). In contrast, shrews were common hosts at our study sites, with a median host contribution of 40.5% (range, 0–68%). The proportion of nymphal ticks that fed on shrews as larvae at a site was associated with the prevalence of DTV infection in ticks at that site (R² = 0.44 p = 0.01, Fig. 3b), but not the prevalence of B. burgdorferi (R² = 0.04, p = 0.5, Fig. 3a). DTV-infected nymphs were highly likely to had fed on a shrew (OR = 139, 95% confidence interval 42–456, but not a mouse, squirrel (or other Sciuridae) or other host (Fig. 4a). By contrast, B. burgdorferi-infected ticks were likely to have fed on mice, but not shrews (OR = 1.1, 95% confidence interval 0.6–1.9) (Fig. 4b). This excludes the hypothesis that shrews were found to have served as virus sources simply because these hosts were the dominant host in these sites.

Three B. brevicauda shrews were trapped from two of our study sites in September of 2020. DTV was detected in the brain of one shrew. Attempts to isolate virus by suckling mouse inoculation failed. Sequencing of two gene targets demonstrates greatest similarity to virus found in a tick from the same site (Fig. 2) and not to standard laboratory strains.

DTV, like other tick-borne encephalitis viruses, may be perpetuated by three mechanisms¹⁵. Virus may be inherited by the tick, transovarial transmission¹⁶. We found that a greater number of ticks were associated with a specific host from all study sites than expected by vertical transmission, indicating that these ticks were not likely to have inherited the infection. There may be co-feeding or nonsystemic transmission in which an infected tick may serve as the direct source of infection for uninfected ticks attached to the skin around it, with no requirement for hematogenous viral dissemination¹⁷. Finally, horizontal transmission, in which a larval tick acquires infection from a viremic vertebrate host, requires a reservoir host that is susceptible to infection and allows for sufficient viremia to infect ticks as well as being sufficiently infested by the tick vector¹⁶. We focused solely on host-seeking nymphal ticks because they would only have one bloodmeal source, that of the larvae. Although adult ticks are also infected by DTV, they would have had two opportunities to become infected (a bloodmeal during the larval as well as the nymphal stage) and it would not be possible to determine whether the bloodmeal host that was identified from an adult was the source of the virus. Accordingly, we did not analyze adult ticks. Our analysis thus incriminates horizontal transmission between shrews and larval ticks, but we cannot exclude co-feeding transmission.

Shrews (likely Blarina brevicauda, the most common shrew in our study sites; our retrotransposon assay, however, may also detect Sorex spp.) were the larval bloodmeal host for the majority (65%) of DTV-infected ticks. The infected ticks were collected from eight different sites over the course of three field seasons, indicating that the finding is not spatiotemporally specific. Although our sample size is small, the positive association between the proportion of shrew-fed ticks and the prevalence of DTV infection in ticks also supports a general finding; no association was found between DTV-infected ticks and either mouse-fed or Sciuridae-fed ticks. Finally, we detected virus in the brain of a shrew and find that it is genetically similar to virus within ticks from that site. Shrews are thus the main candidate for the vertebrate DTV reservoir but we cannot now rank the contribution of horizontal transmission relative to other modes of perpetuation. Shrews may be more likely to sustain an infectious viremia, or be more likely to simultaneously serve as host to nymphs and larvae (co-feeding), than the other mammals present in our study sites. Virus has been detected from xenodiagnostic ticks removed from skunks, raccoons and opossum in New York¹⁸. As with other tick-transmitted infections, contributions to the DTV enzootic cycle are likely to be dependent on local conditions and other hosts than shrews may contribute to maintenance. However, the association of shrews with DTV-infected ticks across multiple transmission seasons and across diverse sites, suggests that additional studies of shrews would be useful. Further investigations, including laboratory transmission studies are necessary to quantify the reservoir capacity of these hosts.

Shrews have not previously been suggested as reservoir hosts for DTV or POWV, but they appear to be competent reservoirs for the related TBE virus in Eurasia^19,20,21. When DTV was identified, white-footed mice were considered the likely reservoir given that these rodents maintained the tick-transmitted agents of Lyme disease, babesiosis, and human granulocytic anaplasmosis^10,22. Shrews were considered to be poorly infested by ticks and thus were considered to have lower reservoir capacity for B. burgdorferi and B. microti²³; this suggestion has been reconsidered^24,25. Mammal surveys in DTV endemic sites have failed to detect virus or specific antibody in shrews¹⁸. Our use of host bloodmeal remnant analysis on infected ticks directly identified the source of the infecting animal reservoir without needing to extrapolate from indirect evidence such as comparative host density, tick infestation indices, and prevalence of pathogen exposure, and could be used to better understand the mode of perpetuation of other high consequence tick-borne pathogens such as the rickettsial agent of Rocky Mountain spotted fever, or those causing American tick-borne hemorrhagic fevers (Bourbon or Heartland virus).

Source: Ecology - nature.com