# The free-living ciliate Tetrahymena pyriformis inactivates engulfed influenza A(H1N1)pdm09 virus via two distinct types of endosomes

## TL;DR

This study shows that the ciliate Tetrahymena pyriformis can inactivate the influenza A(H1N1)pdm09 virus through two types of endosomes, suggesting protozoa play a key role in controlling viral spread in ecosystems.

## Contribution

The study reveals two distinct endosomal mechanisms in Tetrahymena pyriformis for inactivating influenza A virus, a novel insight into protozoan-virus interactions.

## Key findings

- Tetrahymena pyriformis inactivates influenza A(H1N1)pdm09 virus through endocytosis within 72 hours.
- Two types of endosomes are involved in virus inactivation at different time points.
- Protozoa may act as both predators and vectors of influenza A viruses in natural ecosystems.

## Abstract

Wild aquatic birds are a major reservoir of the influenza A virus in natural ecosystems, facilitating its entry into the aquatic microbial food web through their feces. Free-living protozoa and particularly bacterivorous ciliates are essential players of the microbial food web. This study investigates the interactions between the Influenza A(H1N1)pdm09 virus and the ciliated protozoan Tetrahymena pyriformis at the population and ultrastructural levels. Co-cultivation of Influenza A(H1N1)pdm09 and T. pyriformis resulted in a decline and eventual complete elimination of the viral population. The inactivation of the virus was not mediated by products excreted by T. pyriformis but required A(H1N1)pdm09 endocytosis. Viruses ingested by protozoa lost their virulence within 48 hours post infection (hpi) and, as determined by hemagglutination assays, were entirely inactivated within 72 hpi. When lysates infected with A(H1N1)pdm09 T. pyriformis were applied to MDCK cells 1.5 and 24 hpi the undamaged part of ingested virions caused a cytopathic effect. Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) of infected T. pyriformis cells revealed large food vacuoles, including multiple undamaged and partly processed virus particles, at 1.5 and 24 hpi. Furthermore, TEM identified coated and half-coated small one-virus endosomes that predominated at 48 hpi. These results demonstrated that A(H1N1)pdm09 inactivation by T. pyriformis includes two types of endosomes that dominated at different periods of interpopulation interactions. The process of A(H1N1)pdm09 inactivation in protozoan cells occurs rapidly, but not instantaneously, that suggesting a dual role of protozoa in the fate of influenza A viruses in natural ecosystems, both as predators and as potential vectors.