Cooperative effect of the attenuation determinants derived from poliovirus sabin 1 strain is essential for attenuation of enterovirus 71 in the NOD/SCID mouse infection model

Abstract

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and is also associated with serious neurological disorders. An attenuated EV71 strain [EV71(S1-3')] has been established in the cynomolgus monkey infection model; this strain contains the attenuation determinants derived from the type 1 poliovirus vaccine strain, Sabin 1 [PV1(Sabin)], in the 5' nontranslated region (NTR), 3D polymerase, and 3' NTR. In this study, we analyzed the effect of the attenuation determinants of PV1(Sabin) on EV71 infection in a NOD/SCID mouse infection model. We isolated a mouse-adapted EV71 strain [EV71(NOD/SCID)] that causes paralysis of the hind limbs in 3- to 4-week-old NOD/SCID mice by adaptation of the virulent EV71(Nagoya) strain in the brains of NOD/SCID mice. A single mutation at nucleotide 2876 that caused an amino acid change in capsid protein VP1 (change of the glycine at position 145 to glutamic acid) was essential for the mouse-adapted phenotype in NOD/SCID mice. Next, we introduced attenuation determinants derived from PV1(Sabin) along with the mouse adaptation mutation into the EV71(Nagoya) genome. In 4-week-old mice, the determinants in the 3D polymerase and 3' NTR, which are the major temperature-sensitive determinants, had a strong effect on attenuation. In contrast, the effect of individual determinants was weak in 3-week-old NOD/SCID mice, and all the determinants were required for substantial attenuation. These results suggest that a cooperative effect of the attenuation determinants of PV1(Sabin) is essential for attenuated neurovirulence of EV71.

FIG. 1.

Adaptation of EV71 in NOD/SCID mice. Three-week-old NOD/SCID mice were inoculated with 10⁶ CCID₅₀ of EV71(Nagoya) via the intracerebral route. The virus was isolated from the brains of inoculated mice in RD cells. After three rounds of passage, EV71(NOD/SCID) was isolated after limiting dilution in RD cells.

FIG. 2.

Identification of the mouse adaptation determinant of EV71(NOD/SCID). (A) Nucleotide and amino acid changes from the parental EV71(Nagoya) to EV71(NOD/SCID). (B) Mouse-adapted phenotypes of EV71 mutants with mouse adaptation mutations. Sequences derived from the parental EV71(Nagoya) genome are represented by the closed regions, and the synonymous and nonsynonymous mutations derived from EV71(NOD/SCID) are represented by the open regions with white and black stars, respectively, above them. + and −, EV71 mutants that did and did not cause paralysis in inoculated mice, respectively.

FIG. 3.

Effect of mouse adaptation mutation at nt 2876 on EV71 infection in RD cells. (A) Purification of EV71 pseudovirions. The number of copies of viral RNA in purified TE-EV71-Fluc mc and TE-EV71(2876A)-Fluc mc pseudovirions was determined by real-time PCR. The amount of nonencapsidated viral RNA in the sample was determined from samples prepared from 293 cells that were not pretransfected with the DNA of pKS435-EGFP-EV71(Nagoya) capsid vector for capsid proteins expression before RNA transfection with RNA transcript of EV71-Fluc mc. (B) Electron microscopy observation of purified EV71 pseudovirions. Bar, 100 nm. (C) Replication kinetics of EV71 replicon in RD cells infected with pseudovirions. RD cells (1.4 × 10⁴ cells) were infected with 6.9 × 10⁴ to 6.9 × 10⁶ pseudovirions. The cells were washed at 2 h p.i., and then the luciferase activity in the cells was measured at the indicated times. Total luciferase activity with standard deviations is shown. (D) Binding of mouse-adapted EV71 mutant to RD cells. EV71(Nagoya) or EV71(Nagoya-2876A) virions were incubated with RD cells at room temperature for 30 min in the presence of 2 mM guanidine hydrochloride. The amount of virions bound to RD cells was determined by real-time PCR. The binding activity of EV71(Nagoya) was taken as 100%.

FIG. 4.

Infection of NOD/SCID mice with EV71(Nagoya-2876A). (A) Time course of virus dissemination in NOD/SCID mice after intracerebral inoculation. NOD/SCID mice were inoculated with 10⁶ CCID₅₀ of EV71(Nagoya-2876A) via the intracerebral route, and then the tissues were collected at the time indicated. To determine the primary infection sites after the establishment of viremia, NOD/SCID mice were inoculated with 10^6.5 CCID₅₀ of EV71(Nagoya-2876A) via the intravenous route, and then the tissues were collected at 24 h p.i. The number of copies of viral RNA in each tissue was determined by real-time PCR. The number of copies of viral RNA in 0.1 g of each tissue or in 5 μl of serum are shown. (B) Detection of viral antigen in NOD/SCID mice. The viral antigen was detected in the skeletal muscles and hearts of infected mice. The cells with viral antigen are shown with an asterisk.

FIG. 5.

Characterization of the effect of the attenuation determinants of PV1(Sabin) on EV71 infection in NOD/SCID mice. (A) Mouse-adapted phenotype of EV71(Nagoya-2876A) with the attenuation determinants of PV1(Sabin). The sequences derived from the parental EV71(Nagoya) genome are represented by the closed regions, and the introduced mutations are represented by the open regions with stars above them. White stars represent the attenuation determinants of PV1(Sabin) and black stars represent a mouse adaptation mutation derived from EV71(NOD/SCID) at nt 2876. + and −, EV71 mutants that did and did not cause paralysis, respectively, in the inoculated mice (3 and 4 weeks old). (B and C) Tissue specificity of EV71 mutants in 3- and 4-week-old NOD/SCID mice. NOD/SCID mice were inoculated with 10⁶ CCID₅₀ of each mutant via the intracerebral route, and the tissues were collected when the mice showed paralysis or at 4 weeks p.i. The number of copies of viral RNA in each tissue was determined by real-time PCR. The number of copies of viral RNA in 0.1 g of each tissue or in 5 μl of serum is shown.