# Evidence for Powassan virus deletions and defective RNA in field-collected ticks

## TL;DR

This study finds that Powassan virus produces defective RNA in ticks, which could impact its transmission and disease severity.

## Contribution

The study identifies and characterizes defective RNAs and structural variants in Powassan virus from field-collected ticks.

## Key findings

- Deletions in the methyltransferase domain of ns5 and ns2a-ns3 coding sequences are common in Powassan virus RNA from ticks.
- Defective RNAs are associated with microhomology and are enriched after one passage in mammalian cells.
- Certain deletion patterns suggest potential roles in viral persistence and immune evasion.

## Abstract

Powassan virus (POWV) is a tick-borne flavivirus endemic to the United States, Canada, and parts of Russia. POWV remains an under-studied pathogen, despite the potential for serious and life-threatening neurologic complications following infection. While prior studies have characterized viral diversity due to single nucleotide polymorphisms, little is known about POWV recombination, defective RNAs (D-RNAs), and functional structural variants (SVs). Understanding POWV recombination in its natural vector can provide important insights into its replication and evolution. We analyzed POWV sequence data from 53 ticks collected from the Northeastern United States to characterize and quantify recombination patterns in naturally infected ticks. We then compared these results to single-passage isolates. Deletions were common in POWV RNA from ticks, and several areas of the genome were enriched for recombination junctions. Deletions were often associated with areas of microhomology. While most deletions were sample-specific, two major deletion archetypes were observed across multiple tick samples. The first consisted of small 19–50 base deletions in the methyltransferase domain of the ns5 RNA-dependent RNA-polymerase coding sequence, resulting in a mixture of putative SVs and D-RNAs. The second consisted of approximately 1,600 base deletions spanning the ns2a-ns3 coding sequences, resulting in putative D-RNAs with abrogated viral protease function. Deletions in ns2a-ns3 were significantly enriched after one passage in baby hamster kidney cells, despite a decrease in overall deletions. These results demonstrate that POWV RNA recombines frequently, with certain variants more common than others. These findings may carry implications for virus immune evasion and persistence in ticks.

Powassan virus is a tick-borne flavivirus that can cause serious, life-threatening neurological disease. Understanding how Powassan virus replicates and evolves within its tick vector may elucidate factors important for persistence, transmission, and human disease. Defective RNAs (D-RNAs) are replication-incompetent viral genomes generated through internal deletions. D-RNAs are associated with disease severity and persistent infection in other viruses but have not been described for Powassan virus. Here, we show that Powassan virus produces abundant putative D-RNAs in field-collected ticks and that patterns of D-RNA expression change after one passage in mammalian cells. Although the function of these D-RNAs remains unknown, this work demonstrates that they occur under natural conditions and establishes a critical framework for investigating the role of D-RNAs in Powassan virus replication and transmission.