The S2 Subunit of QX-type Infectious Bronchitis Coronavirus Spike Protein Is an Essential Determinant of Neurotropism

Abstract

Some coronaviruses (CoVs) have an extra furin cleavage site (RRKR/S, furin-S2' site) upstream of the fusion peptide in the spike protein, which plays roles in virion adsorption and fusion. Mutation of the S2' site of QX genotype (QX-type) infectious bronchitis virus (IBV) spike protein (S) in a recombinant virus background results in higher pathogenicity, pronounced neural symptoms and neurotropism when compared with conditions in wild-type IBV (WT-IBV) infected chickens. In this study, we present evidence suggesting that recombinant IBV with a mutant S2' site (furin-S2' site) leads to higher mortality. Infection with mutant IBV induces severe encephalitis and breaks the blood-brain barrier. The results of a neutralization test and immunoprotection experiment show that an original serum and vaccine can still provide effective protection in vivo and in vitro. This is the first demonstration of IBV-induced neural symptoms in chickens with encephalitis and the furin-S2' site as a determinant of neurotropism.

Keywords: QX type; coronavirus; encephalitis; furin-S2′ site; infectious bronchitis virus; neurotropism.

Figure 1

Introduction of a furin cleavage site upstream of the fusion peptide. (A) Schematic representation of the IBV spike protein. The original cleavage site at the S1/S2 boundary and a furin cleavage site upstream of the FP are indicated by an arrow. (B) Growth curves of rYN and rYN-S2/RRKR in CEK cells. Virus (10^3.0EID₅₀) was inoculated in the CEK cells for 60 h. Viral loads were determined by measuring expression of the N gene in cell lysates using absolute RT-qPCR (n = 3 × 3). Two-way ANOVA was used for analysis of differences in GraphPad Prism 7.0 and the significance was considered as follows: highly significant at p < 0.01 (**), very highly significant at p < 0.001 (***) and extremely significant at p < 0.0001 (****).

Figure 2

Furin-S2′ site can cause high mortality in SPF chicken without obvious gross kidney injury. (A) Survival rate of 1-day-old SPF chickens inoculated with rYN or rYN-S2/RRKR. rYN-S2/RRKR was highly pathogenic to 1-day-old SPF chickens, with a mortality rate of 90% (9/10), while the mortality rate of rYN was 10% during the observation period (left). Results of necropsy at 9 dpi on 1-day-old chickens inoculated with rYN or rYN-S2/RRKR. Mucus and punctate hemorrhage in the larynx and trachea (black triangles), substantial urate deposits in the ureter and kidney swelling were observed (blank arrow), while only mucus and punctate hemorrhage in the larynx were observed in the rYN-S2/RRKR group (right). (B) Survival rate of 3-week-old SPF chickens inoculated with rYN or rYN-S2/RRKR. Diseased chickens in the rYN-S2/RRKR group appeared later than in 1-day-old chickens and the mortality rate was 20% (2/10). The mortality of rYN was still 10%, but disease onset was delayed (left). At necropsy of diseased chickens at 13 dpi, mucus and punctate hemorrhage in the larynx (black triangles) and mottled swelling kidney (blank arrow) were observed in the rYN-inoculated group. There were no obvious gross lesions in the rYN-S2/RRKR group and negative control group (right).

Figure 3

Microscopic lesions were observed in tissues of 1-day-old SPF chicks at 9 dpi. (A) Clear lesions could be observed in trachea and kidney in both virus-inoculated groups. The black triangles indicate clear mucosal thickening and infiltration of a large number of inflammatory cells in the trachea. The black arrows indicate cellular cast in the kidney. The blank arrow indicates inflammatory cell infiltration in kidney interstitium. (B) Results of H&E-stained brain sections from the rYN-S2/RRKR group. The panel indicates substantial microglial hyperplasia, forming microglial nodules in the brain (top). The panel indicates the formation of perivascular inflammatory infiltrates (bottom). No obvious lesions were observed in the brain of rYN and control groups (left). (C) Scores of 1-day-old chicken group. Sections stained with H&E were evaluated and the scores from 0 to 10, reflecting the severity of the lesions, were recorded. The indications for the scores were as follows: 0 = no microscopic lesions, 1–3 = mild lesions, 4–6 = moderate lesions and 7–10 = severe and extensive lesions. Both rYN and rYN-S2/RRKR caused serious injury in the respiratory and urinary systems of 1-day-old chicks, while rYN-S2/RRKR caused serious injury in the CNS (n = 4).

Figure 4

Microscopic lesions were observed in tissues of 3-week-old SPF chicks. (A) Clear lesions could be observed in the trachea and kidney in the rYN group. The black triangles indicate hemorrhage in mucosa and serosa in trachea. The blank triangle indicates hemorrhage in kidney interstitium. The black arrow indicates the cellular cast in kidney and the blank arrows indicate inflammatory cell infiltration in kidney interstitium. (B, Results of H&E-stained brain sections from the rYN-S2/RRKR group. The panel indicates substantial microglial hyperplasia in the brain (top). The panel indicates the formation of perivascular inflammatory infiltrates at a lower level than that in 1-day-old chickens (bottom). (C) Scores of the 3-week-old chicken group. Sections stained with H&E were evaluated and the scores from 0 to 10, reflecting the severity of the lesions, were recorded. The indications for the scores were as follows: 0 = no microscopic lesions, 1–3 = mild lesions, 4–6 = moderate lesions and 7–10 = severe and extensive lesions. rYN-S2/RRKR caused mild injury in the respiratory and urinary systems of 3-week-old chicks compared with rYN, but still caused severe injury in the CNS (n = 4).

Figure 5

Results of IHC. (A) Results of IHC for IBV antigen in kidney and brain sections. The presence of rYN or rYN-S2/RRKR antigen was detected in the renal tubular epithelial cells, while substantial IBV antigen was detected in neurons of the brain from the rYN-S2/RRKR-inoculated group. (B) The detection of IBV was evaluated as the number of positive cells per section from each microscopic field through a ranked score of 0–4. The indications for the scores were as follows: 0 = no positive cells, 1 = 1–10 positive cells, 2 = 11–30 positive cells, 3 = 31–50 positive cells and 4 = > 50 positive cells. There were more rYN-positive cells in the kidney than for rYN-S2/RRKR, while there were no positive cells in brains of both rYN and negative control groups. There were obvious rYN-S2/RRKR-positive cells in brains (n = 3, right).

Figure 6

Structure of the S protein was predicted. We used homologous modeling to predict the structure of original and mutant S proteins according to the Cryo-EM structure of IBV S protein.