Langat Virus Infection Affects Hippocampal Neuron Morphology and Function in Mice Without Disease Signs Angela D
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Cornelius et al. Journal of Neuroinflammation (2020) 17:278 https://doi.org/10.1186/s12974-020-01951-w RESEARCH Open Access Langat virus infection affects hippocampal neuron morphology and function in mice without disease signs Angela D. A. Cornelius1,2†, Shirin Hosseini3,4†, Sarah Schreier5†, David Fritzsch5†, Loreen Weichert1,5, Kristin Michaelsen-Preusse3, Markus Fendt6,7 and Andrea Kröger1,5,7,8* Abstract Background: Tick-borne encephalitis virus (TBEV) is an important human pathogen that can cause the serious illness tick-borne encephalitis (TBE). Patients with clinical symptoms can suffer from severe meningoencephalitis with sequelae that include cognitive disorders and paralysis. While less than 30% of patients with clinical symptoms develop meningoencephalitis, the number of seropositive individuals in some regions indicates a much higher prevalence of TBEV infections, either with no or subclinical symptoms. The functional relevance of these subclinical TBEV infections and their influence on brain functions, such as learning and memory, has not been investigated so far. Methods: To compare the effect of low and high viral replication in the brain, wildtype and Irf-7−/− mice were infected with Langat virus (LGTV), which belongs to the TBEV-serogroup. The viral burden was analyzed in the olfactory bulb and the hippocampus. Open field, elevated plus maze, and Morris water maze experiments were performed to determine the impact on anxiety-like behavior, learning, and memory formation. Spine density of hippocampal neurons and activation of microglia and astrocytes were analyzed. Results: In contrast to susceptible Irf-7−/− mice, wildtype mice showed no disease signs upon LGTV infection. Detection of viral RNA in the olfactory bulb revealed CNS infections in wildtype and Irf-7−/− mice. Very low levels of viral replication were detectable in the hippocampus of wildtype mice. Although wildtype mice develop no disease signs, they showed reduced anxiety-like behavior and impaired memory formation, whereas Irf-7−/− mice were not affected. This impairment was associated with a significant decrease in spine density of neurons in the hippocampal CA1 region of wildtype mice. Microglia activation and astrogliosis were detected in the hippocampus. Conclusion: In this study, we demonstrate that subclinical infections by viruses from the TBEV-serogroup affected anxiety-like behavior. Virus replication in the olfactory bulb induced far-reaching effects on hippocampal neuron morphology and impaired hippocampus-dependent learning and memory formation. Keywords: Tick-borne encephalitis virus, Langat virus, Type I interferon, Inapparent infection, Hippocampus, Learning and memory * Correspondence: [email protected] †Angela D. A. Cornelius, Shirin Hosseini, Sarah Schreier and David Fritzsch contributed equally to this work. 1Innate Immunity and Infection, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany 5Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University, Leipziger Strasse 44, D-39120 Magdeburg, Germany Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cornelius et al. Journal of Neuroinflammation (2020) 17:278 Page 2 of 17 Background formation, receives input from the entorhinal cortex, Tick-borne encephalitis virus (TBEV) is a member of the which relays through the dentate gyrus (DG) and the genus Flavivirus in the family of Flaviviridae. Flavi- Cornu Ammonis regions CA1 and CA3. Inflammatory cy- viruses comprise many human pathogens, including tokines can negatively affect long-term potentiation (LTP) Dengue virus (DENV), Japanese encephalitis virus (JEV), [14, 15], neuronal survival, synaptic plasticity, and memory West Nile virus (WNV), Yellow fever virus (YFV), and formation [16–19]. It was shown that acute viral or bac- Zika virus (ZIKV) [1]. terial infections of the CNS or the periphery could lead to TBEV is transmitted to humans primarily by infected changes in the neuronal complexity and functional deficits ticks but can also be transmitted by dairy products from in hippocampal-dependent learning and memory [20, 21]. unpasteurized milk produced from infected livestock [2, Whereas severe TBEV infection has long-term in- 3]. Infection with TBEV causes tick-borne encephalitis fluence on behavioral function [6], the question (TBE) which affects the central nervous system (CNS). arises, whether also inapparent infections of the CNS TBE is a typically biphasic disease. In the first phase of alter neural circuits and lead to learning and behav- viremia, the dominant symptoms are fever, fatigue, general ioral deficits. The aim of this study was to malaise, headache, and pain followed by a second phase characterize the impact of low-pathogenic flavivirus with a clinical spectrum that ranges from mild meningitis infection, caused by LGTV in wildtype mice, on be- to severe encephalitis with or without myelitis and spinal havior and spatial memory. We compared findings −/− paralysis. More than 30% of patients with clinical symp- with Irf-7 mice, which showed high viral replica- toms caused by a TBEV infection develop prolonged se- tion in the brain and weight loss but recovered from quelae that include neuropsychiatric symptoms, severe an infection. We found that inapparent infections headaches, and a general decrease in quality of life [4, 5]. lead to alterations in hippocampal structure and The total number of cases worldwide has been estimated function and are associated with impairments in to be up to 13,000 per year, and as such, the infection con- spatial learning in infected mice. Our findings indi- stitutes the most important tick-borne viral disease. Sero- cate that non-recognized infections with neurotropic conversion without prominent morbidity is common. In viruses can cause neurological changes. two-thirds of infected humans, the infection is inapparent and without any disease signs [5–7]. Thus, possible cogni- tive impairments and impact on the behavior of patients Material and methods with unrecognized infections are unknown. Mice To prevent TBEV infection, the Langat virus (LGTV), Allanimalexperimentswereperformedincompli- a member of the TBEV serogroup, was used as a vaccine ance with the relevant animal welfare law (EU-Dir- against TBEV. In humans, LGTV is a low pathogenic fla- ective 2010/63/EU). The mice were housed and vivirus, and it shares 82–88% amino acid homology with handled in accordance with good animal practice as TBEV. Vaccination with LGTV resulted in a neuro- defined by FELASA. All animal experiments were ap- logical disease, with an incidence of approximately 1:18, provedbytheLowerSaxonyStateOfficeofCon- 500 and caused permanent neurologic sequelae among sumer Protection and Food Safety under permit the vaccinated. In adult mice, LGTV generally has a low number AZ 33.9-42502-04-15/1950 or by the Land- pathogenicity when inoculated subcutaneously or intra- esverwaltungsamt Sachsen-Anhalt AZ 42502-2-1344, cerebrally but is still associated with low-level viral repli- University Magdeburg. Wildtype C57BL/6 J (WT) −/− cation in the brain [8]. and Irf-7 mice were bred under SPF conditions at The innate immune system is essential for the control the Helmholtz Centre for Infection Research in of TBEV and LGTV infection. The type I interferon Braunschweig or at the Otto-von-Guericke University (IFN) system plays a pivotal role in the antiviral response Magdeburg, Germany. To exclude gender effects, all against TBEV infections. The loss of type I IFN re- experiments were performed with male and female sponses leads to higher viral replication and death of in- mice. Six- to 10-week-old mice were infected intra- fected mice [8]. Interferon regulatory factor-7 (IRF-7) is peritoneally (i.p.) with 104 focus-forming units (FFU) −/− a master regulator of type I IFN production, and Irf-7 LGTV in 100 μl PBS or mock injected with 100 μl mice are highly susceptible to various viruses [9]. PBS. Mice that lost 20% or more of their body Activation of the innate immune system induces pro- weight or showed pain were sacrificed. For analysis, duction of cytokines including interleukin-1β (IL-1β), IL- all mice were killed by transcardial perfusion with 6, and tumor necrosis factor-α (TNF-α) and IFNs in the 30 ml PBS during anesthesia (100 μl/10 g body weight brain where they can have deleterious effects on cognitive ketamine (150 mg)/xylazine (9 mg)). For histological and emotional behavior