Enterovirus D-68 Infection, Prophylaxis, and Vaccination in a Novel Permissive Animal Model, the Cotton Rat (Sigmodon hispidus)

Abstract

In recent years, there has been a significant increase in detection of Enterovirus D-68 (EV-D68) among patients with severe respiratory infections worldwide. EV-D68 is now recognized as a re-emerging pathogen; however, due to lack of a permissive animal model for EV-D68, a comprehensive understanding of the pathogenesis and immune response against EV-D68 has been hampered. Recently, it was shown that EV-D68 has a strong affinity for α2,6-linked sialic acids (SAs) and we have shown previously that α2,6-linked SAs are abundantly present in the respiratory tract of cotton rats (Sigmodon hispidus). Thus, we hypothesized that cotton rats could be a potential model for EV-D68 infection. Here, we evaluated the ability of two recently isolated EV-D68 strains (VANBT/1 and MO/14/49), along with the historical prototype Fermon strain (ATCC), to infect cotton rats. We found that cotton rats are permissive to EV-D68 infection without virus adaptation. The different strains of EV-D68 showed variable infection profiles and the ability to produce neutralizing antibody (NA) upon intranasal infection or intramuscular immunization. Infection with the VANBT/1 resulted in significant induction of pulmonary cytokine gene expression and lung pathology. Intramuscular immunization with live VANBT/1 or MO/14/49 induced strong homologous antibody responses, but a moderate heterologous NA response. We showed that passive prophylactic administration of serum with high content of NA against VANBT/1 resulted in an efficient antiviral therapy. VANBT/1-immunized animals showed complete protection from VANBT/1 challenge, but induced strong pulmonary Th1 and Th2 cytokine responses and enhanced lung pathology, indicating the generation of exacerbated immune response by immunization. In conclusion, our data illustrate that the cotton rat is a powerful animal model that provides an experimental platform to investigate pathogenesis, immune response, anti-viral therapies and vaccines against EV-D68 infection.

Nasal Infection of Enterovirus D68 Leading to Lower Respiratory Tract Pathogenesis in Ferrets (Mustela putorius furo)

“…Notably, despite having direct access to the nose upon infection, we observed less viral shedding from nasal discharges compared to fecal matter. Instead, the most impressive viral burdens were noted in the lung, which were similar to the experimental results for cotton rats [13]. While the EV-D68 infection was not measured directly by viral titration in our study, the results of qRT-PCR for viral genome and immunohistochemical analysis for viral antigens can support the EV-D68 virus replication in infected ferrets.…”

“…The neutralization antibody GMT exhibited a slight and slow increase of 1:16 levels at week 4 after inoculation ( Figure 6). With respect to the report by Patel et al, in the EV-D68 Cotton rat model, Fermon infection resulted in no detectable neutralization antibody (NA) response [13]. Findings in this study demonstrate that the EV-D68 Fermon strain infection may induce faint immunological response in the form of the neutralization antibody titer in ferrets.…”

“…Blood samples were collected before and after viral infection. Animal feces, nasal washes, and throat swabs were collected on days 1,3,5,7,9,11,13, and 15 after the challenge and frozen (−80 • C) until analysis. After infection, on days 3, 5, 7, 9, the ferrets were euthanized with an intramuscular injection of a ketamine (20 mg/kg), then the organs or tissues were harvested for histopathology and viral distribution analysis.…”

“…Viral load in feces (A) and nasal washes (B) and throat swabs (C) of infected ferrets were detected in the whole course of EV-D68 infection(1,3,5,7,9,11,13,15 days post-infection); (D) Viral RNA was picked up from blood samples and analyzed by Taqman real-time quantitative PCR assay, according to the protocols provided by the qPCR kit. Each blood samples were gathered during the whole EV-D68 infection course(1,3,5,7,9,11,13,15 days post-infection); (E)Viral load in lung of ferret at different day post-infection (3, 5, 7, 9 days post-infection); (F) Viral load in lymph nodes at different day post-infection(3, 5, 7, 9 days post-infection). The vitro-synthesized RNA was applied to quantify the viral copies of each RNA sample, and viral RNA's relative copies for each sample was calculated by the mathematical formula as follow: [(µg of RNA/µL)/(molecular weight)] × Avogadro's number = viral copy number/µL.…”

Contemporary Enterovirus D68 strains show enhanced replication and translation at 37°C

“…While some picornaviruses have been determined to have multiple open reading frames with an additional start codon in the 5' UTR, this is not the case for EV-D68 [61,62]. There has been substantial evolution in the EV-D68 5' UTR since its initial discovery in 1962, most notably in the form of heightened variability and large deletions in the spacer region flanking the AUG start codon [63,64]. Our studies identified a loss of temperature sensitivity in recent EV-D68 strains which was associated with increased translation of the viral genome.…”