A Focus of Deer Tick Virus Transmission in the Northcentral United States

Abstract

We screened salivary glands from adult deer ticks collected near Spooner and Hayward, Wisconsin, to determine whether deer tick virus, a recently described flavivirus, occurs with other tickborne agents in the upper Midwest. Intraacinar inclusions suggestive of replicating virus were detected in 4 (4.6%) of 87 ticks. The virus was isolated by suckling-mouse inoculation. failed to right themselves when placed on their backs. By mid-afternoon they were moribund and died shortly thereafter. The time between inoculation and death was similar to that seen in DTV and POW infections, which kill suckling mice in 5 to 7 days (5,9). To determine whether the putative viral agent was DTV, POW, or a related flavivirus, salivary glands that appeared infected were analyzed by RT-PCR. The clarified homogenate used for mouse inoculation was added to 350 µl of a lysis buffer, and RNA was extracted by RNEasy spin columns (Qiagen) as directed by the manufacturer. The resulting RNA-containing solution was reverse transcribed by using random hexamers at 42°C for 20 minutes. We added 5 µl of cDNA to an amplification mixture containing primers (TBE-1[5'-3' ACATGGCAGTACTGGGG) and TBE-2 (5'-3' CCCATCATGTTGTACAC]) designed to amplify an approximately 450-bp fragment of the ns5 gene of Central European tickborne encephalitis (TBE). Reaction conditions were as follows: initial denaturation at 94°C for 1 minute, followed by 35 cycles of 94°C for 45 seconds, 40°C for 45 seconds, and 72°C for 1 minute. A final extension step of 72°C for 6 minutes was also performed. Bands of the correct size were excised and sequenced by the dideoxychain termination method and an automated DNA sequencer (Applied Biosystems, Foster City, CA). Sequences were compared with those accessioned in GenBank. Sequence alignments were generated with the PILEUP program of the Wisconsin Genetics Computer Group and then analyzed by the distance method using MEGA (10). Third codon positions were omitted to minimize sequence convergence due to homoplasy and to better reflect nucleotide changes resulting in amino acid differences. Analyses including third codon positions resulted in identical topologies with higher bootstrap values and longer branch lengths. Evolutionary distances were computed by the Kimura 2-parameter method, including both transitions and transversions. Distance trees were constructed by the neighbor-joining method, and their robustness was estimated by performing 500 bootstrap replicates. Phylogenetic analysis of this fragment (GenBank accession af135459) indicated that the virus was most closely related, but not identical, to DTV from northwestern Connecticut (af135460) (Figure 1). Because the ns5 gene from our Spooner isolate was more closely related to DTV than to POW, this isolate is provisionally designated as deer-tick virusSpooner (DTV-SPO).572 572 572 572 572

Partial Genetic Characterization of West Nile Virus Strains, New York State, 2000

“…Although complete genome sequences may have provided additional information, short sequence fragments have often been used in population studies of arboviruses (10)(11)(12)(13). Additional criteria (maximum parsimony and maximum likelihood) may have provided corroboration for the close relationships observed; however, the sequences are so similar and the nodes on the neighbor-joining tree so poorly supported that additional analyses seemed unwarranted.…”

Prevalence and Genetic Characterization of Powassan Virus Strains Infecting Ixodes scapularis in Connecticut

“…25 The DTV lineage was first detected by dissecting salivary glands from field-collected ticks removed from carcasses of dead deer or that were fed for 4 to 5 days on a naive New Zealand rabbit in the laboratory, staining one salivary gland of each tick with Feulgen reaction, and examining the salivary glands for evidence of virus growth. 12,26 The remaining salivary gland of each Feulgen-positive tick was pooled with those of four other ticks, placed in Hanks Balanced Salt Solution with 15% fetal bovine serum, homogenized, and intracerebrally inoculated into outbred CD-1 suckling mice. Injected mice subsequently were examined for illness.…”

Seroprevalence of Powassan Virus in New England Deer, 1979–2010

“…1 A subtype of POWV, frequently termed deer tick virus (DTV), was first identified in 1995 2 and has subsequently been recognized as a human pathogen. 3 Because it is associated with the aggressively human-biting blacklegged or deer ticks (Ixodes scapularis), 4,5 this virus has been considered to pose a more significant threat to public health than the prototype virus, POWV, that is associated mainly with the relatively host-specific ticks Ixodes cookei and I. marxi. 6,7 Recent studies have suggested that the incidence of human POWV infection is increasing in the United States, 8 raising the possibility that POWV, like other members of the deer tick-associated guild of emerging zoonoses (Lyme disease, human babesiosis, and human granulocytic anaplasmosis), constitutes a mounting threat in regions where it is enzootic and where I. scapularis ticks are abundant.…”