Interspecies protein substitution to investigate the role of the lyssavirus glycoprotein

Abstract

European bat lyssaviruses type 1 (EBLV-1) and type 2 (EBLV-2) circulate within bat populations throughout Europe and are capable of causing disease indistinguishable from that caused by classical rabies virus (RABV). However, the determinants of viral fitness and pathogenicity are poorly understood. Full-length genome clones based on the highly attenuated, non-neuroinvasive, RABV vaccine strain (SAD-B19) were constructed with the glycoprotein (G) of either SAD-B19 (SN), of EBLV-1 (SN-1) or EBLV-2 (SN-2). In vitro characterization of SN-1 and SN-2 in comparison to wild-type EBLVs demonstrated that the substitution of G affected the final virus titre and antigenicity. In vivo, following peripheral infection with a high viral dose (10(4) f.f.u.), animals infected with SN-1 had reduced survivorship relative to infection with SN, resulting in survivorship similar to animals infected with EBLV-1. The histopathological changes and antigen distribution observed for SN-1 were more representative of those observed with SN than with EBLV-1. EBLV-2 was unable to achieve a titre equivalent to that of the other viruses. Therefore, a reduced-dose experiment (10(3) f.f.u.) was undertaken in vivo to compare EBLV-2 and SN-2, which resulted in 100 % survivorship for all recombinant viruses (SN, SN-1 and SN-2) while clinical disease developed in mice infected with the EBLVs. These data indicate that interspecies replacement of G has an effect on virus titre in vitro, probably as a result of suboptimal G-matrix protein interactions, and influences the survival outcome following a peripheral challenge with a high virus titre in mice.

Fig. 1.

Three-dimensional antigenic map showing the antigenic relationship between recombinant viruses and wild-type viruses. Viruses (spheres) and sera (open boxes) are positioned such that the distance from each serum to each virus is determined by the neutralization titre. Antigenic cartography is used to position both sera and viruses relative to each other, so orientation of the map within the axes is free. Bar, 1 AU (antigenic unit), equivalent to a twofold dilution in antibody titre. Green spheres, RABV strains [Flury low egg passage (LEP) vaccine strain is labelled]; yellow spheres, EBLV-1 strains (RV9 is labelled); red spheres, EBLV-2 srains (RV1787 is labelled); labelled white spheres, recombinant viruses.

Fig. 2.

Single-step (a) and multi-step (b) growth curves of recombinant viruses SN-1 and SN-2 with EBLV-1 and EBLV-2 and SN backbone. Cells were infected at an m.o.i. of 1 for the single-step or 0.01 for the multi-step growth curve. Aliquots of the cultured supernatants were collected at the indicated time points and virus titres were determined in triplicate on BHK cells. For SN (▴), SN-1 (▪) and EBLV-1 (□), both time courses were undertaken twice and the mean of the combined results are displayed. For SN-2 (•) and EBLV-2 (○), the time courses were undertaken once.

Fig. 3.

Pathogenicity of SN (▴), SN-1 (▪), SN-2 (•), EBLV-1 (□) and EBLV-2 (○) in mice. Groups of CD1 mice were injected via fp with 1×10³ f.f.u. of each virus. Mice were observed twice daily and euthanized when neurological signs were observed. The experiment was terminated after 28 days, but no clinical signs were observed past day 18. *Significant difference in survivorship as determined by Fisher’s exact test (CI = 90 %).

Fig. 4.

Pathogenicity of SN (▴), SN-1 (▪) and EBLV-1 (□) in mice. Groups of CD1 mice were injected via fp with 10⁴ f.f.u. of SN (n = 25), SN-1 (n = 30) and EBLV-1 (n = 15). Mice were observed twice daily and euthanized when neurological signs were observed. The experiment was terminated after 28 days, but no clinical signs were observed past day 17. *Significant difference in survivorship as determined by Fisher’s exact test (CI = 90 %).

Fig. 5.

Histopathology and immunohistochemical demonstration of lyssavirus antigen. (a–e) Detection of viral antigen in cortex of ic-inoculated mice. EBLV-1 (a) and EBLV-2 (b) display abundant intraneuronal labelling (brown), while SN (c), SN-1 (d) and SN-2 (e) infection resulted in fewer infected neurons but more severe inflammatory changes including numerous perivascular cuffs (arrows). (f–j) Demonstration of lyssavirus antigen in cortex of peripherally (fp) inoculated animals. EBLV-1-challenged animals displayed abundant positive neurons (f); a smaller number in EBLV-2-challenged mice (g) and neurons were negative for SN (h) and SN-1 (i). Viral antigen demonstration in the vestibular nuclei of SN-1 peripherally challenged mice (j). (k–o) Histopathological changes in the thoracic spinal cord (DRG). Mild poliomyelitis was observed following EBLV-1 inoculation (k), while more severe changes were observed after (k) EBLV-1, (l) SN and (m) SN-1 infection with marked gliosis and glial nodule formation (*). (n, o) Viral antigen detection in thoracic spinal cord of peripherally challenged mice. Abundant immunolabelled cells after EBLV-1 infection (n) contrast with the very limited immunolabelling observed with SN (o).