Strain-dependent effects of clinical echovirus 30 outbreak isolates at the blood-CSF barrier

Abstract

BackgroundEchovirus (E) 30 (E-30) meningitis is characterized by neuroinflammation involving immune cell pleocytosis at the protective barriers of the central nervous system (CNS). In this context, infection of the blood-cerebrospinal fluid barrier (BCSFB), which has been demonstrated to be involved in enteroviral CNS pathogenesis, may affect the tight junction (TJ) and adherens junction (AJ) function and morphology.MethodsWe used an in vitro human choroid plexus epithelial (HIBCPP) cell model to investigate the effect of three clinical outbreak strains (13-311, 13-759, and 14-397) isolated in Germany in 2013, and compared them to E-30 Bastianni. Conducting transepithelial electrical resistance (TEER), paracellular dextran flux measurement, quantitative real-time polymerase chain reaction (qPCR), western blot, and immunofluorescence analysis, we investigated TJ and AJ function and morphology as well as strain-specific E-30 infection patterns. Additionally, transmission electron and focused ion beam microscopy electron microscopy (FIB-SEM) was used to evaluate the mode of leukocyte transmigration. Genome sequencing and phylogenetic analyses were performed to discriminate potential genetic differences among the outbreak strains.ResultsWe observed a significant strain-dependent decrease in TEER with strains E-30 Bastianni and 13-311, whereas paracellular dextran flux was only affected by E-30 Bastianni. Despite strong similarities among the outbreak strains in replication characteristics and particle distribution, strain 13-311 was the only outbreak isolate revealing comparable disruptive effects on TJ (Zonula Occludens (ZO) 1 and occludin) and AJ (E-cadherin) morphology to E-30 Bastianni. Notwithstanding significant junctional alterations upon E-30 infection, we observed both para- and transcellular leukocyte migration across HIBCPP cells. Complete genome sequencing revealed differences between the strains analyzed, but no explicit correlation with the observed strain-dependent effects on HIBCPP cells was possible.ConclusionThe findings revealed distinct E-30 strain-specific effects on barrier integrity and junctional morphology. Despite E-30-induced barrier alterations leukocyte trafficking did not exclusively occur via the paracellular route.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1061-4) contains supplementary material, which is available to authorized users.

Distinct migratory pattern of naive and effector T cells through the blood–CSF barrier following Echovirus 30 infection

“…To the upper compartment of the filter, 400 μl of MAM containing E-30 at a multiplicity of infection (MOI) of 0.7 was added and kept throughout the experiment (28 h). As previously shown the stimulation of HIBCPP with a MOI of 0.7 led to an effective infection and replication ([21], Additional file 3). For uninfected controls, only MAM was added to the upper compartment.…”

“…A potential limitation of our study is that it was solely performed in in vitro experiments. However, we could proof in several previous publications of our own group that our HIBCPP model can elegantly be used to study the pathogenesis of enteroviral meningitis [20, 21, 31]. Although some mouse studies on the neurovirulence of enteroviruses such as Coxsackie virus B [62] or Enterovirus 71 [63, 64] exist, no validated in vivo model for Echovirus 30, the most frequently isolated pathogen in enteroviral meningitis worldwide, exists so far.…”

“…In a recent review culture models to study leukocyte trafficking though the BCSFB were extensively described [19]. In an in vitro model of the BCSFB based on human immortalized brain choroid plexus papilloma (HIBCPP) cells, it has been shown that HIBCPP cells can be infected with human enterovirus, such as E-30 [20, 21]. The infection can cause a barrier alteration accompanied by a drop of the transepithelial electrical resistance (TEER), thus possibly promoting invasion of pathogens and leukocytes through HIBCPP cell layers.…”

Genetic characterization of a novel recombinant echovirus 30 strain causing a regional epidemic of aseptic meningitis in Hokkaido, Japan, 2017

“…The nucleotide and amino acid sequences of these strains were highly conserved (99.6-9% and 99.8-9% identity), suggesting that a single lineage of E30 had spread. The sequence of the 5' UTR and the P1 genomic region of the strain Hokkaido21208 showed the highest identity to those of the strain 13-759, which was isolated in Germany in 2013 [11] (Table 1). While a high degree of nucleotide sequence similarity was also found in the 2A and the 2B genes, the sequence identity values from the 2C gene to the 3' UTR were considerably lower (ranging from 71.2% to 84.0%).…”

“…Under these circumstances, it is important to explore whether the strains are pathogenic and able to spread beyond a regional scale in the near future. However, the strain-specific features that might be involved in disease are still not well understood [11,20], and more-detailed study is needed to evaluate the characteristics of each lineage and to understand the potential endemic risk and the dynamics of virus transmission.…”