Powassan Virus Strains Across North America Show Striking Laboratory Differences

Powassan virus, a tick-borne orthoflavivirus that has produced a steadily increasing number of human cases in North America over the past two decades, may be far more biologically varied than its modest case counts suggest. A new study led by researchers at Colorado State University, working with collaborators at Emory University and Tufts University, has systematically compared 17 virus isolates collected from 14 locations across the United States and Canada. The results, published in Virology Journal, reveal that laboratory-grown Powassan viruses differ substantially in plaque size and replication capacity, with patterns that track closely with the virus’s known genetic lineages.

Powassan virus exists in two distinct lineages. Lineage I corresponds to the classic Powassan virus, historically associated with Ixodes cookei ticks and small mammals in the northeastern and midwestern parts of the continent, while lineage II corresponds to deer tick virus, carried primarily by Ixodes scapularis, the same blacklegged tick that transmits Lyme disease bacteria. Despite high prevalence of infection in ticks and evidence of a broad geographic distribution, fewer than 50 human cases are detected annually, a figure that has long implied a substantial burden of undetected asymptomatic infections. What has been missing, the authors note, is any systematic experimental link between the virus’s genetic diversity and its disease potential.

To address that gap, the team assembled a geographically diverse panel of isolates and subjected each to a battery of in vitro assays. They measured plaque morphology, a classic virological readout in which the visible zones of cell death caused by viral spread reflect how efficiently a virus moves from cell to cell. They then quantified replication kinetics across multiple mammalian cell lines, including baby hamster kidney cells and deer mouse kidney cells, using standardized culture conditions and defined multiplicities of infection. These assays allowed the researchers to ask whether viruses drawn from different lineages and different places behave in measurably different ways when placed in identical laboratory environments.

The answer was emphatically yes. Plaque size varied significantly among the isolates, and the variation was not random. Viruses belonging to lineage II that were isolated in the Northeast generated significantly smaller plaques than both lineage I viruses and lineage II viruses from the Midwest. In flavivirology, plaque size can correlate with growth rate, cell-to-cell spread mechanisms, and sometimes virulence, although the relationship is context-dependent and should be interpreted cautiously. The fact that northeastern deer tick viruses form characteristically smaller plaques suggests that this geographically defined subset of the Powassan virus population carries functional differences that are detectable even under the uniform conditions of cell culture.

Replication assays reinforced the picture of a phenotypically diverse virus population. Across all cell lines tested, the isolates showed broad differences in how efficiently they multiplied, and lineage I viruses often reached significantly higher titers than their lineage II counterparts. This is a notable finding because it means that two viruses circulating in the same general region, infecting the same types of ticks and potentially the same host species, can differ markedly in their fundamental growth properties. If such differences carry over into infection of vertebrate hosts or tick vectors, they could influence transmission efficiency, tissue tropism, or the likelihood that an infection produces symptomatic disease.

To explore whether these viruses can adapt to a rodent host relevant to their natural ecology, the researchers serially passaged all of the isolates on deer mouse kidney cells. Deer mice, Peromyscus maniculatus, are plausible amplifying hosts in the sylvatic cycle of Powassan virus, so changes that emerge during adaptation to their cells may hint at host-specific selective pressures. After serial passage, sequencing revealed that two residues in the viral envelope protein, at positions 164 and 308, had changed in two of the viruses. The envelope protein is the major surface glycoprotein of flaviviruses, mediating receptor binding and membrane fusion, so substitutions in this protein can alter host range, entry efficiency, and antibody sensitivity. The authors are careful to state that the functional role of these particular mutations remains unknown, and further work will be needed to determine whether they represent genuine host adaptation or incidental changes arising during laboratory culture.

The study’s design has important strengths. By using 17 isolates from 14 locations rather than a handful of laboratory-adapted reference strains, the team captured a snapshot of natural variation that is often lost when virologists work with a single prototype virus. Serial passage experiments and standardized assays performed in parallel reduce the risk that observed differences are artifacts of inconsistent handling. The inclusion of a rodent cell line ties the work to the ecological context in which the virus actually circulates, between ticks and small mammals, with humans serving as incidental, dead-end hosts.

At the same time, the authors are appropriately measured about what the findings do and do not demonstrate. Differences in plaque size and replication titer are in vitro phenotypes, measured in cell culture conditions that simplify the enormously complex environment of a living host. The degree to which these laboratory traits reflect differences in transmission dynamics or human pathogenesis remains to be determined. Establishing that connection will require infection studies in animal models, comparisons with clinical outcomes in human cases, and possibly reverse genetics experiments in which the envelope substitutions at positions 164 and 308 are engineered into infectious clones to test their effects directly.

The public health stakes of this line of research are considerable. Powassan virus can cause severe encephalitis, and there is no approved antiviral treatment or widely deployed human vaccine. Roughly 10 percent of recognized severe cases are fatal, and many survivors suffer lasting neurological damage. As blacklegged ticks expand their range and infection prevalence in ticks rises in endemic areas, understanding whether certain viral lineages or geographic subpopulations pose greater risks becomes a pressing question. A virus population that is genetically and phenotypically diverse, as this study suggests, is also a population with more raw material for evolutionary change, including changes that could affect transmissibility or virulence.

The work also offers a foundation for surveillance. If in vitro phenotypes can eventually be mapped onto specific genetic signatures, public health laboratories could potentially gauge the biological characteristics of newly detected viruses from sequence data alone, without the need to culture each isolate. In the nearer term, the Colorado State team’s findings serve as a corrective to any assumption that Powassan virus is a monolithic threat. The virus circulating in North American ticks is a collection of distinct biological entities, and the ones infecting a hiker in Maine may behave quite differently, at least in the laboratory, from those in Wisconsin or Ontario. Sorting out which of those laboratory differences matter for human disease is now the field’s central task.

Subject of Research: Phenotypic diversity of North American Powassan virus isolates in cell culture

Article Title: North American Powassan virus encompasses diverse in vitro phenotypes

Article References: North American Powassan virus encompasses diverse in vitro phenotypes. (n.d.). https://doi.org/10.1186/s12985-026-03330-y

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03330-y

Keywords: Powassan virus, tick-borne virus, orthoflavivirus, deer tick virus, plaque size, viral replication, envelope protein, lineage I, lineage II, virus evolution, tick-borne encephalitis, in vitro phenotypes