Rabies virus glycoprotein is an important determinant for the induction of innate immune responses and the pathogenic mechanisms

Abstract

Our previous studies have suggested that street and fixed rabies viruses (RABVs) induce diseases in the mouse model via different mechanisms. In the present study, attempts were made to determine if it is the glycoprotein (G) that is responsible for the observed differences in the pathogenic mechanisms. To this end, an infectious clone from fixed virus B2c was established and used as a backbone for exchange of the G from street viruses. The rate of viral replication, expression of viral proteins, and the induction of innate immune responses were compared in cells or in mice infected with each of the viruses. Furthermore, the infiltration of inflammatory cells into the CNS and the enhancement of blood-brain barrier (BBB) permeability were also compared. It was found that fixed viruses induced stronger innate immune responses (expression of chemokines, infiltration of inflammatory cells, and enhancement of BBB permeability) than street RABV or recombinant viruses expressing the G from street RABVs. Fixed viruses induce disease via an immune-mediated pathogenic mechanism while street viruses or recombinant viruses expressing the G from street RABVs induce diseases via a mechanism other than immune-mediated pathogenesis. Therefore, RABV G is an important determinant for the induction of innate immune responses and consequently the pathogenic mechanisms.

Fig. 1. Schematic diagram for the construction and characterization of rRABVs

Construction of the infectious B2c clone is accomplished by amplification of the indicated fragments from RNA extracted from B2c-infected mouse brain and ligation of these fragment into the pcDNA vector (A). Restriction enzyme Nhe I was used as a genetic marker. The G gene from the street DRV or SHBRV was inserted between the Pac I and Nhe I sites of the B2c full infectious clone (B). BSR or NA cells were infected with each of the viruses and virus titers determined at indicated time points (C). FFU, fluorescent focus units.

Fig. 2. Viral gene expression and induction of innate immune gene expression in cell cultures after infected with rRABVs

Mouse NA or BSR cells were infected with each of the viruses at MOI of 1.Total RNA was prepared from virus-infected cultures and used for qRT-PCR. Each reaction was carried out in duplicate and repeated for three times. RABV mRNA (G and N) (A, C) and the expression of chemokine mRNA (MIP-1! , RANTES, IP-10) (B, D) were analyzed by qRT-PCR. Data are given as mean values ± standard errors. Asterisks indicate significant differences between the indicated experimental groups as analyzed by One-Way ANOVA: (* p<0.05, ** p<0.01 and *** p<0.001)

Fig. 3. Detection of viral antigens in mice at day 6 p.i. with rRABV

RABV N and G antigens were detected by immunohistochemistry in mouse brain section (A, magnification ×20). RABV viral mRNAs were analyzed by qRT-PCR (B) and RVBV proteins (N and G) detected by Western blotting (D). The ratio between G and N was calculated with RNA copy number (C) or determined after measurement of the band density (D). As a loading control, β-tubulin was measured in the same sample preparation using anti-β-tubulin (anti-T) antibody in the Western blot assay. Significance of differences between the experimental groups was assessed by One-Way ANOVA (*, p<0.05; **, p<0.01 and *** p<0.001).

Fig. 4. Expression of chemokines and inflammatory responses in the CNS after infection with rRABV

Groups of 3 female ICR mice at the age of 4 to 6 weeks were infected with each of the viruses at 10ICLD₅₀ and transcardially perfused with 10% formalin at day 6 p.i. Brains were used for preparation of RNA and chemokine expression was assessed by qTR-PCR (A). Paraffin sections were subjected to immunohistochemistry for detecting CD3-positive cells (B). CD3-postive cells were quantified (C). Significance of differences between experimental groups was assessed by One-Way ANOVA. (*, p<0.05; **, p<0.01).

Fig. 5. Slow cytometry analysis of inflammatory cells in the CNS

Groups of 3 female BALB/c mice at the age of 6 to 8 weeks were infected with each of the viruses at 10 ICLD₅₀ and brains were collected after extensive perfusion at days 3, 6 and 9 p.i. Activated macrophages, neutrophils, CD19 B cells and CD3 positive cells were analyzed by flow cytometery. Four regions were identified: CD11b/CD45 (region R1, activated macrophages), Ly6G/CD45 (region R2, neutrophils), CD19/CD45 (region R3, CD19 B cells) and CD3/CD45 (region R4, CD3+ T cells). Total number of monocytes (per mouse brain) in regions R1, R2, R3 and R4 were shown in mice on 3, 6, or 9 dpi with B2c (A). Detailed analysis was performed for mice infected with each of the viruses and for each of the types of inflammatory cells (B). The results show the means±SE. The differences between the indicated experimental groups were analyzed by On-Way ANOVA (*, p<0.05; **, p<0.01.).

Fig.6. BBB permeability changes in the CNS tissues of mice infected with each of viruses

ICR mice (4-6 weeks of age) were infected with each of the viruses at 10 ICLD₅₀, BBB permeability in the cortex and the cerebellum was assessed by measuring NaF leakage from the circulation into CNS tissues at day 6 post infection. Data are given as mean values±SE. Asterisks indicate significant differences between the indicated experimental groups: *, p<0.05; **, p<0.01.

Fig. 7. Histopathological changes in mice after infection with rRABVs

Groups of 3 female ICR mice at the age of 4 to 6 weeks were infected with each of the viruses at 10ICLD₅₀ and were transcardially perfused with 10% formalin at 6 dpi. Pathological changes were observed in paraffin sections after H&E staining (A, magnification x20). Neurodegeneration/cell death and infiltration score were determined from 8 regions of three slides for each group (B). Significance of differences between different groups was assessed by One-Way ANOVA. *, p<0.05; **, p<0.01.