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 cell death. 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 (C2+) 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 h 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 C2+, BHK-21, and SK-RST cells, at 72 hpi in PT-K75, and at 120 hpi in ST cells. CPE was visible in infected C2+ 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 C2+ 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.
Highlights
Arthropod-borne viruses are common causes of emerging infectious diseases that pose significant public health threats [1,2]
The objective of this study was to identify existing, established porcine cell lines for use as tools to study the pathogenesis of Japanese encephalitis virus (JEV) in pigs in vitro, with the hypothesis that they would be susceptible to the attenuated JEV strain, SA14-14-2
The selected commercial cell lines had no documented pre-existing infections, according to the vendor. These cell lines included a cell line with fibroblastic morphology derived from newborn piglet nasal turbinate mucosa (PT-K75), cells with epithelial morphology derived from the kidney cortices of 1-day-old pigs (SK-RST), and ST cells, as discussed above
Summary
Arthropod-borne viruses (arboviruses) are common causes of emerging infectious diseases that pose significant public health threats [1,2] Notable among these viruses is Japanese encephalitis virus (JEV), a zoonotic mosquito-borne flavivirus that is a leading cause of severe neurologic infections in humans in endemic regions [3,4,5]. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, including aquatic birds and pigs, giving the potential for extensive dissemination and maintenance upon introduction into new areas [10,22]. With numerous vectors identified as being competent for JEV, there is a potential for the introduction and spread of JEV to wild and domestic pig populations in new areas, and a risk of economic impacts on pig farming and production [31,32,33,34]
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