Interspecies co-feeding transmission of Powassan virus between a native tick, Ixodes scapularis, and the invasive East Asian tick, Haemaphysalis longicornis

Abstract

Background: Powassan virus, a North American tick-borne flavivirus, can cause severe neuroinvasive disease in humans. While Ixodes scapularis are the primary vectors of Powassan virus lineage II (POWV II), also known as deer tick virus, recent laboratory vector competence studies showed that other genera of ticks can horizontally and vertically transmit POWV II. One such tick is the Haemaphysalis longicornis, an invasive species from East Asia that recently established populations in the eastern USA and already shares overlapping geographic range with native vector species such as I. scapularis. Reports of invasive H. longicornis feeding concurrently with native I. scapularis on multiple sampled hosts highlight the potential for interspecies co-feeding transmission of POWV II. Given the absence of a clearly defined vertebrate reservoir host for POWV II, it is possible that this virus is sustained in transmission foci via nonviremic transmission between ticks co-feeding on the same vertebrate host. The objective of this study was to evaluate whether uninfected H. longicornis co-feeding in close proximity to POWV II-infected I. scapularis can acquire POWV independent of host viremia.

Methods: Using an in vivo tick transmission model, I. scapularis females infected with POWV II ("donors") were co-fed on mice with uninfected H. longicornis larvae and nymphs ("recipients"). The donor and recipient ticks were infested on mice in various sequences, and mouse infection status was monitored by temporal screening of blood for POWV II RNA via quantitative reverse transcription polymerase chain reaction (q-RT-PCR).

Results: The prevalence of POWV II RNA was highest in recipient H. longicornis that fed on viremic mice. However, nonviremic mice were also able to support co-feeding transmission of POWV, as demonstrated by the detection of viral RNA in multiple H. longicornis dispersed across different mice. Detection of viral RNA at the skin site of tick feeding but not at distal skin sites indicates that a localized skin infection facilitates transmission of POWV between donor and recipient ticks co-feeding in close proximity.

Conclusions: This is the first report examining transmission of POWV between co-feeding ticks. Against the backdrop of multiple unknowns related to POWV ecology, findings from this study provide insight on possible mechanisms by which POWV could be maintained in nature.

Keywords: Haemaphysalis longicornis; Co-feeding transmission; Nonviremic transmission; Powassan virus.

Fig. 1

Tick infestation strategies used for the in vivo experiments. a “Staggered (donor first) tick infestation” timeline. Each mouse was infested with a single POWV II-injected donor Ixodes scapularis at 0 d.p.a. At 2 d.p.a., each mouse was then infested with recipient Haemaphysalis longicornis nymphs. Submandibular blood was collected from each mouse at −1, 2, and 5 d.p.a. Mice were euthanized at 14 d.p.a. or earlier if they met humane endpoints. b “Simultaneous donor and recipient tick infestation” timeline. Each mouse was infested with a single POWV II-injected donor I. scapularis and recipient H. longicornis nymphs at the same time (0 d.p.a.). Submandibular blood was collected every other day from each mouse. Mice were euthanized 2–3 days after completion of H. longicornis feeding for mice that did not reach humane endpoints. c “Staggered (recipient first) tick infestation” timeline. At −2 d.p.a., each mouse was infested with recipient H. longicornis larvae or nymphs, and 2 days later (0 d.p.a.), each mouse was then infested with a single POWV II-injected donor I. scapularis. Submandibular blood was collected every other day from each mouse. Mice were euthanized 2–3 days after completion of H. longicornis feeding for mice that did not reach humane endpoints. d “Staggered (recipient first) tick infestation with daily bleeds” and 2 or 4 d.p.a. necropsy timeline. At −2 d.p.a., each mouse was infested with recipient H. longicornis larvae or nymphs, and 2 days later (0 d.p.a.), each mouse was then infested with a single POWV II-injected donor I. scapularis. Submandibular blood was collected daily from each mouse until euthanasia at 2 or 4 d.p.a. d.p.a. days post-attachment, consistently indicates the attachment day of the donor I. scapularis female ticks, POWV II Powassan virus lineage II.

Fig. 2

Detection of POWV II RNA in the blood of mice that yielded recipient H. longicornis positive for POWV II RNA. a Mice 1–5 are from the “Staggered (recipient first) tick infestation.” Each of these mice were bled every other day. b Mice 6–9 are from the “Staggered (recipient first) tick infestation with daily bleeds.” These mice were bled daily and euthanized at 2 d.p.a. c Mice 10–14 are from the “Staggered (recipient first) tick infestation with daily bleeds.” These mice were bled daily and euthanized at 4 d.p.a. POWV II RNA was detected in mouse blood and recipient H. longicornis samples via q-RT-PCR. Viral RNA quantities are expressed as the number of NS5 gene fragment copies per ng of RNA after normalization to a standard curve. Each bar represents the viral RNA quantity in the blood for that mouse at a specific time point. “ND” means that POWV II RNA was Not Detected in the blood. “NB” means that No Blood was collected on that day. The “+” symbol means that fed recipient H. longicornis screened positive for POWV II RNA at that timepoint. d.p.a. days post-attachment, consistently indicates the attachment day of the donor I. scapularis female ticks, POWV II Powassan virus lineage II, RNA ribonucleic acid, q-RT-PCR quantitative reverse transcription polymerase chain reaction.

Fig. 3

Detection of POWV II RNA in skin biopsies proximal and distal to the tick co-feeding site. a Location of skin biopsies #1, #2, and #3. b Mice 6–14 are from the “Staggered (recipient first) tick infestation with daily bleeds”. These mice were bled daily and euthanized at 2 d.p.a. or 4 d.p.a. POWV II RNA was detected in mouse skin biopsy samples via q-RT-PCR. Viral RNA quantities are expressed as the number of NS5 gene fragment copies per ng of RNA after normalization to a standard curve. Each point represents the viral RNA quantity in a skin biopsy for a specific mouse, as indicated in the legend. d.p.a. days post-attachment, consistently indicates the attachment day of the donor I. scapularis female ticks, POWV II Powassan virus lineage II, RNA ribonucleic acid, q-RT-PCR quantitative reverse transcription polymerase chain reaction.