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A three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines.

ABSTRACT: Tick-borne flaviviruses (TBFV) are sustained in nature through cycling between mammalian and tick hosts. In this study, we used African green monkey kidney cells (Vero) and Ixodes scapularis tick cells (ISE6) to compare virus-induced changes in mammalian and arthropod cells. Using confocal microscopy, transmission electron microscopy (TEM), and electron tomography (ET), we examined viral protein distribution and the ultrastructural changes that occur during TBFV infection. Within host cells, flaviviruses cause complex rearrangement of cellular membranes for the purpose of virus replication. Virus infection was accompanied by a marked expansion in endoplasmic reticulum (ER) staining and markers for TBFV replication were localized mainly to the ER in both cell lines. TEM of Vero cells showed membrane-bound vesicles enclosed in a network of dilated, anastomosing ER cisternae. Virions were seen within the ER and were sometimes in paracrystalline arrays. Tubular structures or elongated vesicles were occasionally noted. In acutely and persistently infected ISE6 cells, membrane proliferation and vesicles were also noted; however, the extent of membrane expansion and the abundance of vesicles were lower and no viral particles were observed. Tubular profiles were far more prevalent in persistently infected ISE6 cells than in acutely infected cells. By ET, tubular profiles, in persistently infected tick cells, had a cross-sectional diameter of 60-100 nm, reached up to 800 nm in length, were closed at the ends, and were often arranged in fascicle-like bundles, shrouded with ER membrane. Our experiments provide analysis of viral protein localization within the context of both mammalian and arthropod cell lines as well as both acute and persistent arthropod cell infection. Additionally, we show for the first time 3D flavivirus infection in a vector cell line and the first ET of persistent flavivirus infection.

SUBMITTER: Offerdahl DK

PROVIDER: S-EPMC3480448 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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A three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines.

Offerdahl Danielle K DK Dorward David W DW Hansen Bryan T BT Bloom Marshall E ME

PloS one 20121024 10

Tick-borne flaviviruses (TBFV) are sustained in nature through cycling between mammalian and tick hosts. In this study, we used African green monkey kidney cells (Vero) and Ixodes scapularis tick cells (ISE6) to compare virus-induced changes in mammalian and arthropod cells. Using confocal microscopy, transmission electron microscopy (TEM), and electron tomography (ET), we examined viral protein distribution and the ultrastructural changes that occur during TBFV infection. Within host cells, fla ...[more]

PMID: 23112871

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Project description:Innate immune signaling in the central nervous system (CNS) exhibits many remarkable specializations that vary across cell types and CNS regions. In the setting of neuroinvasive flavivirus infection, neurons employ the immunologic kinase receptor-interacting kinase 3 (RIPK3) to promote an antiviral transcriptional program, independently of the traditional function of this enzyme in promoting necroptotic cell death. However, while recent work has established roles for neuronal RIPK3 signaling in controlling mosquito-borne flavivirus infections, including West Nile virus and Zika virus, functions for RIPK3 signaling in the CNS during tick-borne flavivirus infection have not yet been explored. Here, we use a model of Langat virus (LGTV) encephalitis to show that RIPK3 signaling is specifically required in neurons of the cerebellum to control LGTV replication and restrict disease pathogenesis. This effect did not require the necroptotic executioner molecule mixed lineage kinase domain like protein (MLKL), a finding similar to previous observations in models of mosquito-borne flavivirus infection. However, control of LGTV infection required a unique, region-specific dependence on RIPK3 to promote expression of key antiviral interferon-stimulated genes (ISG) in the cerebellum. This RIPK3- mediated potentiation of ISG expression was associated with robust cell-intrinsic restriction of LGTV replication in cerebellar granule cell neurons. These findings further illuminate the complex roles of RIPK3 signaling in the coordination of neuroimmune responses to viral infection, as well as provide new insight into the mechanisms of region-specific innate immune signaling in the CNS.ImportanceInteractions between the nervous and immune systems are very carefully orchestrated in order to protect the brain and spinal cord from immune-mediated damage, while still maintaining protective defenses against infection. These specialized neuro-immune interactions have been shown to vary significantly across regions of the brain, with innate antiviral signaling being particularly strong in the cerebellum, although the reasons for this are poorly understood. Here, we show a specialized adaptation of programmed cell death signaling that uniquely protects the cerebellum from tick-borne flavivirus infection. These findings provide important new insight into the molecular mechanisms that promote the uniquely robust antiviral immunity of the cerebellum. They also provide new clues into the pathogenesis of tick-borne encephalitis, a zoonosis of significant global concern.

Project description:BackgroundIxodid ticks are important vectors of a wide variety of viral, bacterial and protozoan pathogens of medical and veterinary importance. Although several studies have elucidated tick responses to bacteria, little is known about the tick response to viruses. To gain insight into the response of tick cells to flavivirus infection, the transcriptomes and proteomes of two Ixodes spp cell lines infected with the flavivirus tick-borne encephalitis virus (TBEV) were analysed.MethodsRNA and proteins were isolated from the Ixodes scapularis-derived cell line IDE8 and the Ixodes ricinus-derived cell line IRE/CTVM19, mock-infected or infected with TBEV, on day 2 post-infection (p.i.) when virus production was increasing, and on day 6 p.i. when virus production was decreasing. RNA-Seq and mass spectrometric technologies were used to identify changes in abundance of, respectively, transcripts and proteins. Functional analyses were conducted on selected transcripts using RNA interference (RNAi) for gene knockdown in tick cells infected with the closely-related but less pathogenic flavivirus Langat virus (LGTV).ResultsDifferential expression analysis using DESeq resulted in totals of 43 and 83 statistically significantly differentially-expressed transcripts in IDE8 and IRE/CTVM19 cells, respectively. Mass spectrometry detected 76 and 129 statistically significantly differentially-represented proteins in IDE8 and IRE/CTVM19 cells, respectively. Differentially-expressed transcripts and differentially-represented proteins included some that may be involved in innate immune and cell stress responses. Knockdown of the heat-shock proteins HSP90, HSP70 and gp96, the complement-associated protein Factor H and the protease trypsin resulted in increased LGTV replication and production in at least one tick cell line, indicating a possible antiviral role for these proteins. Knockdown of RNAi-associated proteins Argonaute and Dicer, which were included as positive controls, also resulted in increased LGTV replication and production in both cell lines, confirming their role in the antiviral RNAi pathway.ConclusionsThis systems biology approach identified several molecules that may be involved in the tick cell innate immune response against flaviviruses and highlighted that ticks, in common with other invertebrate species, have other antiviral responses in addition to RNAi.

Dataset Information

A three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines.

Publications

A three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines.

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