Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody

<div><p>The Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV.</p></div

CD3+ cells in the lungs following primary infection and reinfection.

<p>DAB images from primary infection (A) and reinfection (B). Immunofluorescence (IF) image of CD3 (green) and virus antigen (red) within the same perivascular region following reinfection (C). DAB images from day 3 post-infection at 10x, bar equivalent to 100μm. IF images at 40x, bar equivalent to 20μm.</p

Viral RNA titers and histopathology in the lungs following MERS-CoV infection in rabbits that received passive transfer (PT) of serum from infected rabbits.

<p>Viral RNA titers in the lungs upon infection with 10⁵ TCID₅₀ of MERS-CoV in rabbits either previously infected with a low dose of MERS-CoV (10³ TCID₅₀) four weeks prior or naïve rabbits following PT of post-infection sera at either a full dose or 1:10 dilution (A). Images show the H&E staining (left) and IHC with an antibody against the MERS-CoV N protein (right) following infection for the 10^3//10⁵ TCID₅₀ (reinfection) group (B,E), the group that received passive transfer of undiluted post-infection serum (C, F), and the group that received passive transfer of post-infection serum at 1:10 dilution (D,G). n = 3 rabbits per group. Images from day 3 post-infection at 10x, bar equivalent to 100μm.</p

Schematic of rabbit infection studies.

<p>Rabbits were inoculated intranasally with EMC/2012 strain of MERS-CoV (green arrows) and tissue samples were collected for viral titration and histopathology at necropsy (blue arrows). Three rabbits were necropsied at each time point. Numbers indicate days since virus administration for primary, (secondary), or [tertiary] infections.</p

Viral RNA titers and histopathology in lungs following infection with MERS-CoV when neutralizing antibodies are present.

<p>Viral RNA titers in the lungs following tertiary infection with EMC/2012 strain (A). Images show H&E staining (B) and IHC with an antibody against the MERS-CoV N protein (C) in the 10^5//10^5//10⁵ TCID₅₀ group. Images are representative of all rabbits following tertiary infection. All images at 10x, (bar equivalent to 100μm) with 40x inset (bar equivalent to 20μm). n = 3 rabbits per group. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test. Images from day 3 post-infection. p values **<0.01, ***<0.001.</p

Functional genomic analysis of transcriptional regulation of immune responses in the brain of patients with AstV-ND.

(A) Coordinated shifts in transcriptional regulation of immune processes in the brain of AstV-ND-1-NIH compared to a normal age-matched control. Plotted are negative log10 FDR-adjusted p-values (-log10 p-adj) for major Gene Ontology (GO) terms of interest determined by SET. Dashed line indicates the significance cut-off. Inferred changes in AstV-ND-1-NIH compared to normal control for each term are indicated in the gray box. (B) Multi-source functional enrichment for upregulated gene expression in AstV-ND. The plot shows significantly enriched terms of interest across multiple ontologies (x-axis) with their p-adj values (left y-axis) and the number of upregulated genes annotated to each term (right y-axis). Genomic ontology sources and their corresponding terms are indicated by the same color.</p

gProfiler Multiquery Upregulated Genes.

Astroviruses (AstVs) can cause of severe infection of the central nervous system (CNS) in immunocompromised individuals. Here, we identified a human AstV of the VA1 genotype, HAstV-NIH, as the cause of fatal encephalitis in an immunocompromised adult. We investigated the cells targeted by AstV, neurophysiological changes, and host responses by analyzing gene expression, protein expression, and cellular morphology in brain tissue from three cases of AstV neurologic disease (AstV-ND). We demonstrate that neurons are the principal cells targeted by AstV in the brain and that the cerebellum and brainstem have the highest burden of infection. Detection of VA1 AstV in interconnected brain structures such as thalamus, deep cerebellar nuclei, Purkinje cells, and pontine nuclei indicates that AstV may spread between connected neurons transsynaptically. We found transcriptional dysregulation of neural functions and disruption of both excitatory and inhibitory synaptic innervation of infected neurons. Importantly, transcriptional dysregulation of neural functions occurred in fatal cases, but not in a patient that survived AstV-ND. We show that the innate, but not adaptive immune response was transcriptionally driving host defense in the brain of immunocompromised patients with AstV-ND. Both transcriptome and molecular pathology studies showed that most of the cellular changes were associated with CNS-intrinsic cells involved in phagocytosis and injury repair (microglia, perivascular/parenchymal border macrophages, and astrocytes), but not CNS-extrinsic cells (T and B cells), suggesting an imbalance of innate and adaptive immune responses to AstV infection in the brain as a result of the underlying immunodeficiencies. These results show that VA1 AstV infection of the brain in immunocompromised humans is associated with imbalanced host defense responses, disruption of neuronal somatodendritic compartments and synapses and increased phagocytic cellular activity. Improved understanding of the response to viral infections of the human CNS may provide clues for how to manipulate these processes to improve outcomes.</div

Serum ELISA and neutralizing antibody titers in rabbits following primary and secondary infection.

<p>Serum ELISA and neutralizing antibody titers in rabbits following primary and secondary infection.</p

Analysis of disruption of neurophysiology in AstV-ND at the levels of transcriptional regulation, protein expression, and cell morphology/topology/function.

Analysis of disruption of neurophysiology in AstV-ND at the levels of transcriptional regulation, protein expression, and cell morphology/topology/function.</p

Viral RNA titers and histopathology in the lungs of rabbits following reinfection with MERS-CoV.

<p>Viral RNA titers in the lungs of rabbits following reinfection with EMC/2012 (A). Images show H&E (left) and IHC for the MERS-CoV N protein (right) following reinfection for the 10^3//10⁵ TCID₅₀ reinfection group (B,E) and 10^5//10⁵ TCID₅₀ reinfection group (C,F). The 10⁵ TCID₅₀^//media control group was included to demonstrate that the observed inflammation was not residual from the primary infection (D,G). n = 3 rabbits per group. All images at 10x, (bar equivalent to 100μm) with 40x inset (bar equivalent to 20μm). Images from day 3 post-infection.</p