In Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines

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 objectiv...

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Autores principales:	Shakirat A. Adetunji, Dmitriy Smolensky, Dana N. Mitzel, Jeana L. Owens, Carol G. Chitko-McKown, Natalia Cernicchiaro, Leela E. Noronha
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	arboviruses cell culture Japanese encephalitis infection in vitro porcine Medicine R
Acceso en línea:	https://doaj.org/article/f20ce9954d1c44549a01dc382bc0191b
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spelling	oai:doaj.org-article:f20ce9954d1c44549a01dc382bc0191b2021-11-25T18:38:29ZIn Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines10.3390/pathogens101114682076-0817https://doaj.org/article/f20ce9954d1c44549a01dc382bc0191b2021-11-01T00:00:00Zhttps://www.mdpi.com/2076-0817/10/11/1468https://doaj.org/toc/2076-0817Japanese 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 (C<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mo>Δ</mo></semantics></math></inline-formula>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 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 C<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mo>Δ</mo></semantics></math></inline-formula>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<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mo>Δ</mo></semantics></math></inline-formula>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<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mo>Δ</mo></semantics></math></inline-formula>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.Shakirat A. AdetunjiDmitriy SmolenskyDana N. MitzelJeana L. OwensCarol G. Chitko-McKownNatalia CernicchiaroLeela E. NoronhaMDPI AGarticlearbovirusescell cultureJapanese encephalitisinfectionin vitroporcineMedicineRENPathogens, Vol 10, Iss 1468, p 1468 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	arboviruses cell culture Japanese encephalitis infection in vitro porcine Medicine R
spellingShingle	arboviruses cell culture Japanese encephalitis infection in vitro porcine Medicine R Shakirat A. Adetunji Dmitriy Smolensky Dana N. Mitzel Jeana L. Owens Carol G. Chitko-McKown Natalia Cernicchiaro Leela E. Noronha In Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines
description	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 (C<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mo>Δ</mo></semantics></math></inline-formula>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 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 C<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mo>Δ</mo></semantics></math></inline-formula>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<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mo>Δ</mo></semantics></math></inline-formula>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<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mo>Δ</mo></semantics></math></inline-formula>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.
format	article
author	Shakirat A. Adetunji Dmitriy Smolensky Dana N. Mitzel Jeana L. Owens Carol G. Chitko-McKown Natalia Cernicchiaro Leela E. Noronha
author_facet	Shakirat A. Adetunji Dmitriy Smolensky Dana N. Mitzel Jeana L. Owens Carol G. Chitko-McKown Natalia Cernicchiaro Leela E. Noronha
author_sort	Shakirat A. Adetunji
title	In Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines
title_short	In Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines
title_full	In Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines
title_fullStr	In Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines
title_full_unstemmed	In Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines
title_sort	in vitro infection dynamics of japanese encephalitis virus in established porcine cell lines
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/f20ce9954d1c44549a01dc382bc0191b
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In Vitro Infection Dynamics of Japanese Encephalitis Virus in Established Porcine Cell Lines

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