Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019;8(1):1076-1085.
doi: 10.1080/22221751.2019.1644142.

Intra-host emergence of an enterovirus A71 variant with enhanced PSGL1 usage and neurovirulence

Liang Sun  1 , Aloys Tijsma  1 , Carmen Mirabelli  1 , Jim Baggen  2 , Maryam Wahedi  2 , David Franco  1 , Armando De Palma  1 , Pieter Leyssen  1 , Erik Verbeken  3 , Frank J M van Kuppeveld  2 , Johan Neyts  1 , Hendrik Jan Thibaut  1   2
Affiliations

Intra-host emergence of an enterovirus A71 variant with enhanced PSGL1 usage and neurovirulence

Liang Sun et al. Emerg Microbes Infect. 2019.

Abstract

Enterovirus A71 (EV-A71) is one of the main causative agents of hand-foot-and-mouth disease and is occasionally associated with severe neurological complications. EV-A71 pathophysiology is poorly understood due to the lack of small animal models that robustly support viral replication in relevant organs/tissues. Here, we show that adult severe combined immune-deficient (SCID) mice can serve as an EV-A71 infection model to study neurotropic determinants and viral tropism. Mice inoculated intraperitoneally with an EV-A71 clinical isolate had an initial infection of the lung compartment, followed by neuroinvasion and infection of (motor)neurons, resulting in slowly progressing paralysis of the limbs. We identified a substitution (V135I) in the capsid protein VP2 as a key requirement for neurotropism. This substitution was also present in a mouse-adapted variant, obtained by passaging the clinical isolate in the brain of one-day-old mice, and induced exclusive neuropathology and rapid paralysis, confirming its role in neurotropism. Finally, we showed that this residue enhances the capacity of EV-A71 to use mouse PSGL1 for viral entry. Our data reveal that EV-A71 initially disseminates to the lung and identify viral and host determinants that define the neurotropic character of EV-A71, pointing to a hitherto understudied role of PSGL1 in EV-A71 tropism and neuropathology.

Keywords: CNS; Enterovirus A71; SCID; mouse model; pathogenesis; receptor; tropism.

PubMed Disclaimer

Conflict of interest statement

No potential conflict of interest was reported by the authors.

Figures

Figure 1.
Figure 1.
A mouse-adapted EV-A71 strain (#812MA) exhibits increased neurovirulence. (A) In vivo adaptation scheme of the clinical isolate EV-A71 #812. EV-A71 #812 was intracranially injected in C57Bl6/J suckling mice (1 d old). Two days post infection (p.i.), mice were sacrificed and the brain homogenate was used to infect new C57Bl6/J 1-day-old-mice. After five passages, the virus was passaged twice in RD cells to obtain the EV-A71 #812MA. (B, left) Survival of mice infected with EV-A71 #812 and EV-A71 #812MA. Six weeks old SCID mice (n = 5) were inoculated i.p. with EV-A71 #812, EV-A71 #812MA or PBS (mock) and were monitored daily for signs of disease. (B, right) Relative body weight at the moment of sacrifice. Horizontal bars represent median ± SEM. (C-E) EV-A71 RNA load in various organs. Viral genome copies were determined by means of RT-qPCR (C) in SCID mice infected with 2.4*105 PFU EV-A71 #812MA and sacrificed when paralysis was first observed, (D) 2.4*105 PFU EV-A71#812 and sacrificed when paralysis was first observed, (E) 2.4*105 PFU EV-A71 #812 and sacrificed at six days p.i. (no signs of paralysis). n = 5 mice for all studies. Horizontal bars represent median ± SEM. The dotted line indicates the detection limit of the assay.
Figure 2.
Figure 2.
VP2 substitution V135I in mouse-adapted EV-A71 (#812MA) is a genetic determinant of neurotropism (A) Side view of a protomeric structure of EV-A71 (3VBS) along the 2-fold symmetry axis (USCF Chimera). The four capsid proteins VP1-4 are depicted in blue, green, red and purple, respectively. A close up view of the EF loop and amino acid V135 of VP2 (in red) is also shown. (B) Scheme of EV-A71 #812 in vitro adaptation. EV-A71 #812 was used to infect neuroblastoma murine N2a cell line (MOI = 1). Three days p.i., supernatant (diluted 1:5 was used to infect a fresh monolayer of N2a cells). At passage 0, 3 and 8, viral RNA was extracted and VP2 was sequenced. (C) Replication kinetics of EV-A71 #812 and #812MA on N2a cells. At the indicated times p.i., N2a cells were harvested and viral levels were determined by means of end-point titration on RD cells. Error bars represent the mean ± SEM of 3 biological replicates. P values were calculated by two-way ANOVA in GraphPad, *P < .05; ns, P > .05.
Figure 3.
Figure 3.
EV-A71 infects (motor)neurons and type II pneumocytes. EV-A71 antigen expression in SCID mouse tissue. Mice were either mock infected or infected with EV-A71 #812MA or EV-A71 #812 and sacrificed when paralysis was first observed. Paraffin embedded, formalin fixed tissue sections were stained with an anti-EV-A71 antibody. Representative pictures of (A-C) cerebrum, (D-F) spinal cord and (G-I) lungs. (J-K) Infection of bone-marrow-derived macrophages. Cells were differentiated in M-CSF-containing medium for 6 days and infected with #812 and #812MA (MOI = 1). At selected times p.i., cells were harvested. Infection was quantified by RT-qPCR (J) and TCID50 determination on RD cells (K). (L) MBEC and RD cells were infected with EV-A71 #812 or EV-A71 #812MA for 1 h. Following fresh medium replacement, the cells were further incubated for the indicated times. After fixation, the infected or mock cells were stained by dsRNA antibody, followed by high-content imaging counting. Images shown are representative fluorescent micrographs. The data shown are mean ± SEM of one (J-K) or two (L) independent experiments each containing three (J-K) to six (L) biological replicates.
Figure 4.
Figure 4.
Substitution V135A enhances PSGL1 usage by mouse-adapted EV-A71. The relative levels of mRNA of (A) SCARB2 and (B) PSGL1 in ten different tissues was quantified by means of RT-qPCR. Data were normalized to β-actin (housekeeping gene). RT-qPCR data are expressed as relative levels. Horizontal bars represent the mean. (C) SCARB2 and PSGL1 in various organs tissues assessed by means of immunohistochemistry. SCARB2 protein expression in CNS, intestine, pancreas, liver and lung. (D-E) Infection of EV-A71 #812 and #812MA in L929 cells transfected with mouse SCARB2 and PSGL1 expression plasmids; Cells were infected with #812 and #812MA 24 h post transfection and frozen at 0 (D) or 24 (E) hours p.i. Following three freeze-thaw cycles, infectious virus titres (CCID50/ml) were determined by means of end-point titration on RD cells. (F) Mouse SCARB2 and PSGL1-transfected L929 cells or RD cells were infected with #812 and #812MA; L929 cells were fixed at 48 h p.i., RD cells at 24 h p.i., followed by dsRNA (green) and nuclei (blue) staining. Images shown are representative fluorescent micrographs. The data shown are mean ± SEM of two (F) or three (D-E) independent experiments each containing three (D-E) or four (F) biological replicates. P values were calculated by two-way ANOVA in GraphPad, **P < .01; ***P < .001; ns, P > .05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ooi MH, Wong SC, Lewthwaite P, et al. . Clinical features, diagnosis, and management of enterovirus 71. Lancet Neurol. 2010;9:1097–1105. DOI:10.1016/S1474-4422(10)70209-X. - DOI - PubMed
    1. Wong KT, Munisamy B, Ong KC, et al. . The distribution of inflammation and virus in human Enterovirus 71 encephalomyelitis suggests possible viral spread by neural pathways. J Neuropathol Exp Neurol. 2008;67:162–169. DOI:10.1097/nen.0b013e318163a990. - DOI - PubMed
    1. Puenpa J, Mauleekoonphairoj J, Linsuwanon P, et al. . Prevalence and characterization of enterovirus infections among pediatric patients with hand foot mouth disease, herpangina and influenza like illness in Thailand, 2012. PLoS One. 2014;9:e98888. DOI:10.1371/journal.pone.0098888. - DOI - PMC - PubMed
    1. Liu W, Wu S, Xiong Y, et al. . Co-circulation and genomic recombination of coxsackievirus A16 and enterovirus 71 during a large outbreak of hand, foot, and mouth disease in Central China. PLoS One. 2014;9:e96051. DOI:10.1371/journal.pone.0096051. - DOI - PMC - PubMed
    1. Thoa LPK, Chiang P-S, Khanh TH, et al. . Genetic and antigenic characterization of enterovirus 71 in Ho Chi Minh City, Vietnam, 2011. PLoS One. 2013;8:e69895. DOI:10.1371/journal.pone.0069895. - DOI - PMC - PubMed

MeSH terms

Grants and funding

L.S. received funding of the China Scholarship Council (CSC) [grant number 201403250056). Funding was provided by European Union 7th Framework Program (EUVIRNA Marie Curie Initial Training Network), grant agreement number 264286. J.N. received funding from BELSPO (Belgian Science Policy Office) [grant number IUAP-BELVIR]. F.J.M.v.K. received funding from the Netherlands Organization for Scientific Research (grant number NWO-VICI-91812628). Part of this research work was performed using the ‘Caps-It’ research infrastructure [Fonds Wetenschappelijk Onderzoek; project ZW13-02) that was financially supported by the Hercules Foundation (FWO) and Rega Foundation, KU Leuven.

LinkOut - more resources