Imaging the Transport Dynamics of Single Alphaherpesvirus Particles in Intact Peripheral Nervous System Explants from Infected Mice

ABSTRACT Alphaherpesvirus particles travel long distances in the axons of neurons using host microtubule molecular motors. The transport dynamics of individual virions in neurons have been assessed in cultured neurons, but imaging studies of single particles in tissue from infected mice have not bee...

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Autores principales:	Andrea E. Granstedt, Bingni W. Brunton, Lynn W. Enquist
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2013
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/31f615d20c5e46acafe63fc0f1afac87
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spelling	oai:doaj.org-article:31f615d20c5e46acafe63fc0f1afac872021-11-15T15:40:05ZImaging the Transport Dynamics of Single Alphaherpesvirus Particles in Intact Peripheral Nervous System Explants from Infected Mice10.1128/mBio.00358-132150-7511https://doaj.org/article/31f615d20c5e46acafe63fc0f1afac872013-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00358-13https://doaj.org/toc/2150-7511ABSTRACT Alphaherpesvirus particles travel long distances in the axons of neurons using host microtubule molecular motors. The transport dynamics of individual virions in neurons have been assessed in cultured neurons, but imaging studies of single particles in tissue from infected mice have not been reported. We developed a protocol to image explanted, infected peripheral nervous system (PNS) ganglia and associated innervated tissue from mice infected with pseudorabies virus (PRV). This ex vivo preparation allowed us to visualize and track individual virions over time as they moved from the salivary gland into submandibular ganglion neurons of the PNS. We imaged and tracked hundreds of virions from multiple mice at different time points. We quantitated the transport velocity, particle stalling, duty cycle, and directionality at various times after infection. Using a PRV recombinant that expressed monomeric red fluorescent protein (mRFP)-VP26 (red capsid) and green fluorescent protein (GFP)-Us9 (green membrane protein), we corroborated that anterograde transport in axons occurs after capsids are enveloped. We addressed the question of whether replication occurs initially in the salivary gland at the site of inoculation or subsequently in the neurons of peripheral innervating ganglia. Our data indicate that significant amplification of infection occurs in the peripheral ganglia after transport from the site of infection and that these newly made particles are transported back to the salivary gland. It is likely that this reseeding of the infected gland contributes to massive invasion of the innervating PNS ganglia. We suggest that this “round-trip” infection process contributes to the characteristic peripheral neuropathy of PRV infection. IMPORTANCE Much of our understanding of molecular mechanisms of alphaherpesvirus infection and spread in neurons comes from studying cultured primary neurons. These techniques enabled significant advances in our understanding of the viral and neuronal components needed for efficient replication and directional spread between cells. However, in vitro systems cannot recapitulate the environment of innervated tissue in vivo with associated defensive properties, such as innate immunity. Therefore, in this report, we describe a system to image the progression of infection by single virus particles in tissue harvested from infected animals. We explanted intact innervated tissue from infected mice and imaged fluorescent virus particles in infected axons of the specific ganglionic neurons. Our measurements of virion transport dynamics are consistent with published in vitro results. Importantly, this system enabled us to address a fundamental biological question about the amplification of a herpesvirus infection in a peripheral nervous system circuit.Andrea E. GranstedtBingni W. BruntonLynn W. EnquistAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 4, Iss 3 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Andrea E. Granstedt Bingni W. Brunton Lynn W. Enquist Imaging the Transport Dynamics of Single Alphaherpesvirus Particles in Intact Peripheral Nervous System Explants from Infected Mice
description	ABSTRACT Alphaherpesvirus particles travel long distances in the axons of neurons using host microtubule molecular motors. The transport dynamics of individual virions in neurons have been assessed in cultured neurons, but imaging studies of single particles in tissue from infected mice have not been reported. We developed a protocol to image explanted, infected peripheral nervous system (PNS) ganglia and associated innervated tissue from mice infected with pseudorabies virus (PRV). This ex vivo preparation allowed us to visualize and track individual virions over time as they moved from the salivary gland into submandibular ganglion neurons of the PNS. We imaged and tracked hundreds of virions from multiple mice at different time points. We quantitated the transport velocity, particle stalling, duty cycle, and directionality at various times after infection. Using a PRV recombinant that expressed monomeric red fluorescent protein (mRFP)-VP26 (red capsid) and green fluorescent protein (GFP)-Us9 (green membrane protein), we corroborated that anterograde transport in axons occurs after capsids are enveloped. We addressed the question of whether replication occurs initially in the salivary gland at the site of inoculation or subsequently in the neurons of peripheral innervating ganglia. Our data indicate that significant amplification of infection occurs in the peripheral ganglia after transport from the site of infection and that these newly made particles are transported back to the salivary gland. It is likely that this reseeding of the infected gland contributes to massive invasion of the innervating PNS ganglia. We suggest that this “round-trip” infection process contributes to the characteristic peripheral neuropathy of PRV infection. IMPORTANCE Much of our understanding of molecular mechanisms of alphaherpesvirus infection and spread in neurons comes from studying cultured primary neurons. These techniques enabled significant advances in our understanding of the viral and neuronal components needed for efficient replication and directional spread between cells. However, in vitro systems cannot recapitulate the environment of innervated tissue in vivo with associated defensive properties, such as innate immunity. Therefore, in this report, we describe a system to image the progression of infection by single virus particles in tissue harvested from infected animals. We explanted intact innervated tissue from infected mice and imaged fluorescent virus particles in infected axons of the specific ganglionic neurons. Our measurements of virion transport dynamics are consistent with published in vitro results. Importantly, this system enabled us to address a fundamental biological question about the amplification of a herpesvirus infection in a peripheral nervous system circuit.
format	article
author	Andrea E. Granstedt Bingni W. Brunton Lynn W. Enquist
author_facet	Andrea E. Granstedt Bingni W. Brunton Lynn W. Enquist
author_sort	Andrea E. Granstedt
title	Imaging the Transport Dynamics of Single Alphaherpesvirus Particles in Intact Peripheral Nervous System Explants from Infected Mice
title_short	Imaging the Transport Dynamics of Single Alphaherpesvirus Particles in Intact Peripheral Nervous System Explants from Infected Mice
title_full	Imaging the Transport Dynamics of Single Alphaherpesvirus Particles in Intact Peripheral Nervous System Explants from Infected Mice
title_fullStr	Imaging the Transport Dynamics of Single Alphaherpesvirus Particles in Intact Peripheral Nervous System Explants from Infected Mice
title_full_unstemmed	Imaging the Transport Dynamics of Single Alphaherpesvirus Particles in Intact Peripheral Nervous System Explants from Infected Mice
title_sort	imaging the transport dynamics of single alphaherpesvirus particles in intact peripheral nervous system explants from infected mice
publisher	American Society for Microbiology
publishDate	2013
url	https://doaj.org/article/31f615d20c5e46acafe63fc0f1afac87
work_keys_str_mv	AT andreaegranstedt imagingthetransportdynamicsofsinglealphaherpesvirusparticlesinintactperipheralnervoussystemexplantsfrominfectedmice AT bingniwbrunton imagingthetransportdynamicsofsinglealphaherpesvirusparticlesinintactperipheralnervoussystemexplantsfrominfectedmice AT lynnwenquist imagingthetransportdynamicsofsinglealphaherpesvirusparticlesinintactperipheralnervoussystemexplantsfrominfectedmice
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Imaging the Transport Dynamics of Single Alphaherpesvirus Particles in Intact Peripheral Nervous System Explants from Infected Mice

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