In vitro infection dynamics of Japanese encephalitis virus in established porcine cell lines

Authors: ADETUNJI, SHAKIRAT - Kansas State University; Smolensky, Dmitriy; Mitzel, Dana; Chitko-Mckown, Carol; CERNICCHIARO, NATALIA - Kansas State University; Noronha, Leela; Owens, Jeana

Submitted to: Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/27/2021
Publication Date: 11/12/2021
Citation: Adetunji, S.A., Smolensky, D., Mitzel, D.N., Chitko-Mckown, C.G., Cernicchiaro, N., Noronha, L.E., Owens, J.L. 2021. In vitro infection dynamics of Japanese encephalitis virus in established porcine cell lines. Pathogens. 10(11). Article 1468. https://doi.org/10.3390/pathogens10111468.
DOI: https://doi.org/10.3390/pathogens10111468

Interpretive Summary: Japanese encephalitis virus (JEV) causes severe neurological disease in humans, mostly in Southeast Asia and the Western Pacific region. It is transmitted from infected domestic and wild animals through the bites of mosquitoes. Pigs play a central role in the JEV transmission cycle because they can amplify the virus to high levels following infection. The host and virus factors associated with JEV infection in pigs are poorly understood. Advances in JEV treatment and prevention in pigs have been held back by a lack of thorough understanding of how the virus is able to reproduce in, and interact with, the porcine host. In vitro cell model systems can serve as useful tools to investigate features of these interactions. Here, an in vitro system was established to evaluate the susceptibility of several porcine cell lines to an attenuated strain of JEV. Findings from this study demonstrate that these porcine cell lines have the potential to be useful research tools for in vitro investigation of JEV infections in a natural host species and can serve as resources for researchers with limited access to animal models or fresh porcine samples.

Technical Abstract: Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne pathogen that regularly causes severe neurological disease in humans in Southeast Asia and the Western Pacific region. Pigs are one of the main amplifying hosts of JEV and play a central role in the virus’ transmission cycle. The objective of this study was to identify in vitro cell systems to investigate early effects of JEV infection including viral replication and host response. Here, we demonstrate the susceptibility of several porcine cell lines to the attenuated genotype III JEV strain SA14-14-2. Monolayers of porcine nasal turbinate (PT-K75), kidney (SK-RST), testis (ST), and monocyte-derived macrophage (C-DELTA-2+) cells were infected with SA14-14-2 for up to five days at a multiplicity of infection (MOI) of 0.1. The hamster kidney cell line BHK-21, previously shown to be susceptible to SA14-14-2, was used as a positive control. Culture supernatants and cells were collected between 0 and 120 hours post infection (hpi), and monolayers were observed for cytopathic effect (CPE) using brightfield microscopy. The number of infectious virus particles was quantified by plaque assay and cell viability was determined using trypan blue staining. An indirect immunofluorescence assay was used to detect the presence of JEV NS1 antigens in cells infected at 1 MOI. All four porcine cell lines demonstrated susceptibility to SA14-14-2 and produced infectious virus by 12 hpi. Virus titers peaked at 48 hpi in C-DELTA-2+, BHK-21, and SK-RST cells, at 72 hpi in PT-K75, and at 120 hpi in ST cells. CPE was visible in infected C-DELTA-2+ and BHK-21 cells, but not the other three cell lines. The proportion of viable cells, as measured by trypan blue exclusion, declined after 24 hpi in BHK-21 and 48 hpi in C-DELTA-2+ cells, but did not substantially decline in SK-RST, PT-K75 or ST cells. At 48 hpi, JEV NS1 was detected in all infected cell lines by fluorescence microscopy. These findings demonstrate several porcine cell lines which have the potential to serve as useful research tools for investigating JEV infection dynamics and host cell mechanisms in a natural amplifying host species, such as pigs, in vitro.