Free-living Amoebae as Reservoirs for the Transmission of Norovirus

Introduction: Human noroviruses (HuNoV) are the most common cause of foodborne disease outbreaks in the United States. The most common food commodities implicated in HuNoV outbreaks are leafy greens; however, the vehicle of transmission and point of contamination are often unknown. Here, we hypothesize that common free-living amoebae (FLA) ubiquitous in the environment may act as reservoirs of HuNoV and facilitate the transmission of these pathogens to fresh produce.

Purpose: To investigate FLA as reservoirs for HuNoV transmission the interaction between two Acanthamoeba species and two HuNoV surrogates were evaluated. The Acanthamoeba species include A. castellanii and A. polyphaga. In addition, two HuNoV surrogates – murine norovirus type 1 (MNV-1) and feline calicivirus (FCV) were evaluated and compared in this study.

Methods: Initially, 3 × 10⁶ amoebae were inoculated with 3 × 10⁶ PFU of each virus and incubated at 25°C for 1 hour. Amoebae were washed 3 times with 1×PBS to remove residual viruses followed by resuspension in 1 ml amoeba culture medium. Virus titers were then analyzed by plaque assay to determine the number of virus associated with amoebae. Also, amoeba growth curves at 25°C were established after virus inoculation.

Results: After amoeba-virus incubation, 490 PFU and 337 PFU of MNV-1 were associated with A. castellanii and A. polyphaga, respectively, via plaque assay while only few or none FCV were detected. Moreover, the growth curves of amoebae with and without virus inoculation were not statistically significantly different. To clarify the interaction between amoebae and HuNoV surrogates, further experiments including localization and longitudinal analyses of viruses in amoebae are underway.

Significance: The findings of this study indicate that MNV-1 associate with A. castellanii and A. polyphaga without affecting the normal growth of these amoebae. This is significant in understanding the HuNoV survival in the environment and the future control of HuNoV outbreaks.