Using a mycovirus to improve surveillance for the fungus causing white-nose syndrome in bats

White-nose syndrome (WNS) has devastated populations of cave-hibernating bats in the wake of its westward invasion across North America. Early detection of the fungus that causes WNS (Pseudogymnoascus destructans, or Pd) is critical, but efforts are complicated in the West because winter roost locations are largely unknown. While environmental sampling methods have been developed for Pd, they can yield inconclusive results in western states that are hard to interpret and unreproducible, complicating management decisions shared by multiple stakeholders.

We propose using an assay to detect a Pd mycovirus as an indicator of Pd presence to greatly increase the sensitivity of Pd detection in environmental samples. Use of the mycovirus to detect the presence of Pd in theory promises to increase sensitivity of detection 100-1000-fold compared to the current gold standard. We also plan to conduct rigorous testing to validate the use of the mycovirus assay to increase sensitivity while maintaining specificity for Pd detection.

This research would be critical for early Pd detection in vulnerable bat populations and ideally improve decision making among multiple stakeholder groups in the West.

Approach

For PdPV-pa to be useful as a proxy for Pd detection, the following biological conditions should be met:

   A. The assay is specific to PdPV-pa, i.e. it will not amplify related viruses (PdPV-pa is always absent when Pd is absent)

   B. PdPV-pa is specific to Pd, i.e. no other fungi are host to PdPV-pa (PdPV-pa is always absent when Pd is absent)

   C. All North American Pd isolates are infected with PdPV-pa (PdPV-pa is always present when Pd is present)

This study follows an iterative design to first, assess whether the conditions above are met; second, optimize conditions to maximize assay sensitivity to detect targets; and third, compare the performance of the PdPV-pa assay to the gold standard assay for Pd detection used in national surveillance.

Image: Magnified fungal stocks of Pseudogymnoascus destructans, the fungus that causes white-nose syndrome in bats.

To address assay specificity (condition A) and host species specificity (condition B), sympatric and closely related fungal species isolated from bat hibernacula in Pd-endemic states and in states where Pd had not yet been observed will be screened with a PdPV-pa assay. To assess whether all North American Pd isolates are infected with the mycovirus (condition C), known Pd-positive bat wing and environmental swab extracts from across North America will be screened with the assay. Viral and fungal load will be quantified in each sample to provide a relative comparison of the number of virus copies to copies of the fungal genomes.

If the PdPV-pa assay meets conditions A-C, protocols will be optimized to maximize assay sensitivity.

Finally, the performance of the optimized PdPV-pa assay will be compared to that of the gold standard assay for Pd DNA detection. The PdPV-pa assay will be applied to environmental samples, previously screened with the Pd assay, representing environments with varying pathogen loads. Relative comparisons will be made between the strength of the signal between the two assays and an occupancy framework will be used to estimate any difference in their probabilities of detection.

The result of this research optimally will be a novel tool to facilitate the early detection of the fungus that causes white-nose syndrome in ecologically important bat populations. This tool would be a critical addition to the existing toolbox for Pd monitoring in the West where overwintering sites are unknown, and aid in decision making by multiple stakeholders.

Key Findings

This study is in progress. Preliminary findings include:

• An assay to detect PdPV-pa was designed and preliminary tests show that it is efficient and sensitive, and detects 10x more targets than the Pd assay on fungal stocks.
• The assay detected PdPV-pa in known Pd-positive environmental samples collected at 4 bat hibernacula in Wisconsin (supports that PdPV-pa is present when Pd is present, and PdPV-pa is present in all North American Pd isolates).
• The assay did not detect PdPV-pa in environmental samples in which Pd was not detected (supports that PdPV-pa is absent when Pd is absent).
• We cultured 111 nontarget fungi to assess whether PdPV infected other fungal species in bat hibernacula. Of 56 isolates that have been tested so far, 0 have been positive for PdPV. This observation supports that, in samples tested to date, conditions A and B are met.
• Recent paper by Thapa et. al. 2021 found 100% prevalence of PdPV in Pd isolates collected across 17 US states and 1 Canadian province, supporting that condition C, that PdPV is always present when Pd is present may be met in North America.