Enterovirus D68 infection in cotton rats results in systemic inflammation with detectable viremia associated with extracellular vesicle and neurologic disease

Abstract

Enterovirus D68 (EV-D68) is a non-polio enterovirus that causes respiratory illness and is linked to acute flaccid myelitis (AFM) in infants and children. Recent demonstration of association of EV-D68 with extracellular vesicles (EVs) released from infected cells in vitro suggests a role for these vesicles in non-lytic dissemination of virus beyond the respiratory tract. We previously reported the permissiveness of cotton rat (Sigmodon hispidus) to infection with different EV-D68 strains of clades A and B, but did not investigate the virus association with EVs. We present a model of acute respiratory infection with a clinical isolate of EV-D68 of clade B3 in immunocompetent cotton rats featuring systemic dissemination of the virus. EV-D68 was detected in circulation and organs outside of the respiratory tract with the inflammatory response accompanying dissemination. Further analysis demonstrated that the virus was associated with extracellular vesicles purified from plasma. We also present a model of intraperitoneal infection with EV-D68 in young cotton rats featuring dissemination of the virus to spinal cord and brain with associated clinical signs of neurologic disease. EV-D68-associated with EVs produced in cotton rat cells and injected intraperitoneally into young cotton rats also resulted in detection of virus in the CNS. Our results provide the first in vivo experimental support for the notion that respiratory infection with EV-D68 generates virus associated with extracellular vesicles that disseminate outside the respiratory tract. These models of infection could be used to investigate the role of EVs-associated EV-D68 in the pathogenesis of EV-D68 infection and to assess therapeutic interventions.

Fig. 1

Enterovirus D68 MN/18/23263 replication in cotton rats. Sigmodon hispidus cotton rats (males and females, 4 weeks old) were infected I.N. with 10⁷ PFUs of EV-D68 isolate MN/18/23263. Animals were sacrificed at the indicated times post-infection (p.i.), or before infection (0 h p.i.). Viral titers in the lung (A) and in the nose (B) were expressed as total PFU per gram of tissue (n = 6). Limit of detection for lung and nose were 2.3 and 2 Log₁₀ PFU/gram, respectively. (C) Detection of viral mRNA in the lung of infected cotton rats. Controls (0 h p.i.) were lungs of mock-infected animals (PBS) sacrificed before infection.

Fig. 2

Enterovirus D68 MN/18/23263-associated lung pathology in cotton rats. (A) Pulmonary histology scores measured in lungs of animals infected with EV-D68 isolate US/MN/18/23263 and sacrificed at the indicated times p.i. Control animals were inoculated I.N. with the same dose of UV-inactivated virus (UV) and sacrificed at the indicated times, or mock-infected with PBS and sacrificed 7 days later (Control). Scores for peribronchiolitis, perivasculitis, interstitial pneumonia, and alveolitis were measured blindly (n ≥ 5 for each time point). Percentage pulmonary pathology is presented as a mean ± SE of each score for each group. (B) Pictures of lung tissue of animals inoculated with UV-inactivated virus (a) or with 10⁷ PFU of EV-D68 isolate US/MN/18/23263 (b-f) sacrificed on day 1 (b, c), day 4 (d, e), or day 7 (f) p.i. Animals infected with EV-D68 and sacrificed on day 1 showed the strongest lung pathology with increases in peribronchiolitis (b) and alveolitis (c). Alveolitis remained strong on day 4 p.i. (d), featuring infiltrates of macrophages and granulocytes distinguishable at higher magnification (e). Residual lung pathology could still be detected on day 7 p.i. (f). Magnification in a to d and f, 100x; e, 400x. (C) Immunohistochemistry of lung tissue of a cotton rat infected with 10⁷ PFU of EV-D68 isolate US/MN/18/23263 and sacrificed on day 2 p.i. Staining was performed using picornavirus specific antibody LS-C760118 (LSBio), recognizing viral protein VP2 (a and d) or mouse antibody control (b and e). Epithelium of small bronchus (a) and alveolar macrophages (d) showed large content of viral antigens. H&E staining showing equivalent area of tissue (c and f). Magnification, 1,000x.

Fig. 3

Kinetics of mRNA expression for the indicated cytokines and the chemokine IP-10 in the lungs of EV-D68 MN/18/23263-infected cotton rats. Animals were infected I.N. with 10⁷ PFU of EV-D68 and sacrificed at the indicated times p.i. (black symbols). Mock-infected animals (M, red symbols) and animals inoculated with the UV-inactivated preparation of virus and sacrificed 8 h p.i. (UV, green symbols) were included as controls. Each symbol corresponds to an individual animal. Cross lines represent mean +/- SEM. n = 5–6. ANOVA comparison was performed against mock-infected animals. *, ***, **** for p < 0.05, 0.001, and 0.0001, respectively.

Fig. 4

Detection of EV-D68 and kinetics of host gene expression in additional organs. mRNA expression for the indicated genes was evaluated in the spleen (A), brain (B), or liver (C) of EV-D68 US/MN/18/23263-infected cotton rats. Animals were inoculated I.N. with 10⁷ PFU of EV-D68 and sacrificed at the indicated times p.i. (black symbols). Animals inoculated with the UV-inactivated preparation of virus and sacrificed 8 h p.i. (UV, green symbols) were included as controls. Cross lines represent mean +/- SEM. n = 5–6. ANOVA comparison was performed against mock-infected animals. *, **, **** for p < 0.05, 0.01, and 0.0001, respectively.

Fig. 5

Detection of circulating EV-D68 and expression of inflammation markers. Whole blood (A–E), serum (F), and plasma (G–K) were obtained from US/MN/18/23263-infected cotton rats at the indicated times p.i. (A) Detection of EV-D68 in whole blood by qRT-PCR. Black symbols denote EV-D68-infected animals. Red and green symbols represent animals mock-infected with PBS (time 0 h) or inoculated with the UV-inactivated virus (UV, green symbol), respectively. (B) Detection of SS (+) and SS (-) EV-D68 RNA in whole blood on day 1 p.i. (C–E) mRNA levels of Mx-1, IP-10, and IFNγ, respectively. (F) Circulating HMGB1 in serum of EV-D68 US/MN/18/23263-infected cotton rats at the indicated times p.i. (G–K) EV-D68 detection in plasma samples was performed by plaque assay (G and J) and qRT-PCR (H, I, and K). (I) Detection of SS (+) and SS (-) EV-D68 RNA in plasma on day 1 p.i. Panels A-I display data for each animal individually, males and females, whereas panels J and K show group results segregated by sex. *, **, ***, **** for p < 0.05, 0.01, 0.001, and 0.0001, respectively.

Fig. 6

Plasma EVs from EV-D68-infected animals contain associated virus. Plasma samples obtained on days 1 and 2 p.i. from male and female cotton rats infected with US/MN/18/23263 were pooled by sex and day p.i. and subjected to size exclusion chromatography. (A) Fractions were used to investigate the presence of virus by plaque assay on HeLa OH cell monolayers. The results were graphically depicted for individual pools (5 females and 5 males per pool per day p.i.). (B) Transmission electron microscopy (TEM) images depicting vesicles and associated virus-like structures (a and b, arrowheads) in samples of fraction 3 of plasma of a female-infected cotton rat, or in samples of fraction 2 of a pool of females’ cotton rat plasma (c). Inset in c shows a TEM image of a purified EV-D68 preparation, under the same magnification. (d) TEM image of purified plasma fraction 3 from an uninfected animal showing EVs.

Fig. 7

Re-infection model. (A) Scheme of the experimental design. (B) Measurement of serum NA against EV-D68 US/MN/18/23263. Blue symbols represent animals infected on day 0 and re-infected on day 45 with EV-D68 (Group A). Red symbols represent control animals infected once on day 45 (Group B). Panels C-J show comparison of viral load and gene expression in animals infected with EV-D68 once and sacrificed one day later (red symbols) and in EV-D68-infected animals re-infected with EV-D68 on day 45 p.i. and sacrificed one day later (blue symbols). (C) Plasma viral titers. (D) Lung viral titers. (E) Nose viral titers. (F) Lung virus detection by qRT-PCR. (G–J) Lung mRNA expression for IFNβ, MX-1, IFNγ, and MCP-1, respectively. Line represents mean +/- SEM. n = 8, Student t-test. *, **, ***, **** for p < 0.05, 0.01, 0.001, and 0.0001, respectively.

Fig. 8

Dissemination of EV-D68 to CNS depends on the route of infection. (A and B) Detection of EV-D68 by qRT-PCR in the brain and in the spinal cord. Three-week-old weanling cotton rats were infected with 10⁷ PFU of EV-D68 US/MN/18/23263 intraperitoneally (EV-D68 I.P.) or intranasally (EV-D68 I.N.). Control animals were inoculated I.P. or I.N. with UV-inactivated EV-D68 US/MN/18/23263 (EV-D68 UV, I.P. or EV-D68 UV, I.N., respectively). All animals were sacrificed on day 6 post-inoculation. Brain (A) and spinal cord (B) tissues were isolated from all animals and used for quantification of mRNA of EV-D68 and Mx-1. n = 3–7, Mann-Whitney test, * p < 0.05. (C) Percentage body weight gain in animals infected I.P. with EV-D68 compared to control PBS-inoculated animals. *, p < 0.05. (D) Representative picture of an EV-D68-induced hind leg paralysis (arrow) detected in an I.P.-infected animal on day 4 p.i. Table shows the number of animals (and percentage given in parentheses) with neurologic signs of disease seen in two independent experiments. (E and F) Time course of detection of EV-D68 mRNA in the brain and spinal cord of animals infected with EV-D68 I.P. (black symbols). Numbers in parentheses represent animals with virus detected vs. total number of animals analyzed. Controls (red symbols) were animals inoculated with PBS I.P.

Fig. 9

Characterization of EV-D68-containing EVs purified from cotton rat CCRT cells. Fractionation of EVs produced in the cotton rat cell line CCRT. CCRT monolayers were infected at an m.o.i. of 10. Supernatants were subjected to 3 consecutive rounds of centrifugation following the previously described method. S1 and S3, and P1 and P3 represent the supernatant and the pellet after the first and the third centrifugation of cell culture media from infected cells, respectively. B3 is the result of sample P3 after its treatment with Triton X-100 detergent. (B) EM obtained from P3 fractions of EV-D68 US/MN/18/23263-infected CCRT supernatant. (C) Detection of SS (+) and SS (-) EV-D68 RNA in EVs purified from cotton rat CCRT cells or from HeLa cells. (D) Neutralization of the infectivity of EVs containing EV-D68 purified from cotton rat CCRT cells using cotton rat immune serum against US/MN/18/23263. (E, F) CNS infection after challenge of cotton rats with EVs-associated EV-D68 purified from CCRT cells. Two-week-old cotton rats kept with the dams were separated into indicated groups and infected with 5.6 × 10⁵ EV-D68-associated EVs (EVs-EV-D68), with the same dose of the free virus, or with a matching dose of the UV-inactivated virus. Brain (E) and spinal cord (F) tissues were isolated from all animals and used for quantification of mRNA of EV-D68 and Mx-1 by qRT-PCR. n = 5–7.