Quantitative analysis of poliomyelitis-like paralysis in mice induced by a poliovirus replicon

Abstract

Poliovirus (PV) infection causes severe paralysis, typically of the legs, by destruction of the motor neurons in the spinal cord. In this study, the relationship between PV replication in the spinal cord, damage in the motor neurons and poliomyelitis-like paralysis was analysed in transgenic mice expressing the human PV receptor (TgPVR21). First, a PV replicon encoding firefly luciferase in place of the capsid genes (PV-Fluc mc) was trans-encapsidated in 293T cells and the trans-encapsidated PV-Fluc mc (TE-PV-Fluc mc) was then inoculated into the spinal cords of TgPVR21 mice. TE-PV-Fluc mc was recovered with a titre of 6?3610 7 infectious units ml "1 , which was comparable to those of PV1 strains. TgPVR21 mice inoculated with TE-PV-Fluc mc showed non-lethal paralysis of the hindlimbs, with severity ranging from a decline in grip strength to complete flaccid paralysis. The replication of TE-PV-Fluc mc in the spinal cord reached peak levels at 10 h post-inoculation (p.i.), followed by the appearance of paralysis at as early as 12 h p.i., reaching a plateau at 16 h p.i. Histological analysis showed a correlation between the lesion and the severity of the clinical symptoms in most mice. However, severe paralysis could also be observed with an apparently low lesion score, where as few as 5?3610 2 motor neurons (1?4 % of the susceptible cells in the lumbar cord) were infected by TE-PV-Fluc mc. These results indicate that PV replication in a small population of the motor neurons was critical for severe residual poliomyelitis-like paralysis in TgPVR21 mice. INTRODUCTIONPoliovirus (PV) is a small, non-enveloped virus with a single-stranded, positive-sense genomic RNA; it is known as the causative agent of poliomyelitis and belongs to the family Picornaviridae. The motor neurons are the major target of PV infection in the central nervous system (CNS) (Bodian, 1949). The tropism of PV to the motor neurons is attributable in part to the expression of the PV receptor (PVR) (Crotty et al., 2002;Ida-Hosonuma et al., 2002;Koike et al., 1994;Ren & Racaniello, 1992).Tissues susceptible to PV infection in the CNS are limited: the brainstem, the roof nuclei of the cerebellum, the precentral gyrus of the cerebrum and the spinal cord (the cervical and lumbar cords) (reviewed by Minor, 1997). Among these tissues in the CNS, the spinal cord seemed to have a high susceptibility to PV infection: PV can adapt to the spinal cord with an increased tropism (Nathanson & Bodian, 1961) and a PV mutant that has a tropism for the spinal cord, but not for the brain, has been isolated (Jia et al., 1999). The adaptation of PV was partly supported by an enhanced efficiency in the uncoating step with decreased thermostability in the virion (Couderc et al., 1996). The lumbar cord supported stable replication of a PV mutant with a severe defect in viral protein synthesis, which showed unstable replication in in vitro-cultured cells and also in the brain (Arita et al., 2004). Experimental infections of other enteroviruses, including coxsackievirus A21 (D...

Characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus 71 infectivity

“…RD cells were used for titration of pseudoviruses and for drug screening. PV and EV71 pseudoviruses, which encapsidated luciferase-encoding PV and EV71 replicons with capsid proteins derived from PV (Mahoney) and EV71 (Nagoya), respectively, were prepared as reported previously (Arita et al, 2006(Arita et al, , 2008, except that the EcoRI site in the 59NTR of PV replicon was removed by site-directed mutagenesis. A Sendai virus (SeV) mutant (SeV/luc), which encodes luciferase (Hasan et al, 1997), was a generous gift from Atsushi Kato, Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan.…”

“…In this study, we performed drug screening by utilizing a pharmacologically active compound library with partially characterized targets and pseudoviruses, which have PV and EV71 replicons encoding firefly luciferase as a replication marker encapsidated in the PV and EV71 capsid proteins, respectively (Arita et al, 2006, Porter et al, 1998. We have identified four compounds and characterized their inhibitory effects on PV and EV71 infection.…”

Cellular kinase inhibitors that suppress enterovirus replication have a conserved target in viral protein 3A similar to that of enviroxime

“…RD cells (a human rhabdomyosarcoma cell line) were cultured as monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal calf serum (FCS) and used for titration of pseudoviruses and screening of anti-PV and anti-EV71 compounds. PV and EV71 pseudoviruses (TE-PV-Fluc mc and TE-EV71-Fluc mc), which encapsidated luciferase-encoding PV and EV71 replicons with capsid proteins derived from PV (Mahoney) and EV71 (Nagoya), respectively, were prepared as reported previously (Arita et al, 2006(Arita et al, , 2008a. A PV pseudovirus mutant [TE-PV-Fluc mc (5318A)] that has adenine at nt 5318 was prepared with cDNA of a luciferase-encoding PV replicon by site-directed mutagenesis as described previously (Arita et al, 2006(Arita et al, , 2008a.…”

“…PV and EV71 pseudoviruses (TE-PV-Fluc mc and TE-EV71-Fluc mc), which encapsidated luciferase-encoding PV and EV71 replicons with capsid proteins derived from PV (Mahoney) and EV71 (Nagoya), respectively, were prepared as reported previously (Arita et al, 2006(Arita et al, , 2008a. A PV pseudovirus mutant [TE-PV-Fluc mc (5318A)] that has adenine at nt 5318 was prepared with cDNA of a luciferase-encoding PV replicon by site-directed mutagenesis as described previously (Arita et al, 2006(Arita et al, , 2008a. Kinase inhibitors included in the LOPAC 1280 drug library (Sigma-Aldrich), ZM336372 (Calbiochem), Akt inhibitor VIII (Sigma-Aldrich), STO-609 (Calbiochem), FK506 (Fermentek), SL327 (Sigma-Aldrich) and U0126 (Sigma-Aldrich) were used for screening of kinase inhibitors (see Supplementary Table S1, available in JGV Online).…”

“…Therefore, we used RD cells for the present screenings because this cell line is susceptible to both PV and EV71. Cells were inoculated with PV pseudoviruses that have a luciferase-encoding PV replicon genome encapsidated in a PV capsid, as described previously (Arita et al, 2006(Arita et al, , 2008b, and then the luciferase activity in the cells was measured at 7 h p.i. The mean luciferase activity in the mock-treated cells infected with PV pseudoviruses was 1.3610 6 relative light units (RLU), and that in GW5074-treated cells was 2.4610 4 RLU.…”