Limited trafficking of a neurotropic virus through inefficient retrograde axonal transport and the type I interferon response

Abstract

Poliovirus is an enteric virus that rarely invades the human central nervous system (CNS). To identify barriers limiting poliovirus spread from the periphery to CNS, we monitored trafficking of 10 marked viruses. After oral inoculation of susceptible mice, poliovirus was present in peripheral neurons, including vagus and sciatic nerves. To model viral trafficking in peripheral neurons, we intramuscularly injected mice with poliovirus, which follows a muscle-sciatic nerve-spinal cord-brain route. Only 20% of the poliovirus population successfully moved from muscle to brain, and three barriers limiting viral trafficking were identified. First, using light-sensitive viruses, we found limited viral replication in peripheral neurons. Second, retrograde axonal transport of poliovirus in peripheral neurons was inefficient; however, the efficiency was increased upon muscle damage, which also increased the transport efficiency of a non-viral neural tracer, wheat germ agglutinin. Third, using susceptible interferon (IFN) alpha/beta receptor knockout mice, we demonstrated that the IFN response limited viral movement from the periphery to the brain. Surprisingly, the retrograde axonal transport barrier was equivalent in strength to the IFN barrier. Illustrating the importance of barriers created by the IFN response and inefficient axonal transport, IFN alpha/beta receptor knockout mice with muscle damage permitted 80% of the viral population to access the brain, and succumbed to disease three times faster than mice with intact barriers. These results suggest that multiple separate barriers limit poliovirus trafficking from peripheral neurons to the CNS, possibly explaining the rare incidence of paralytic poliomyelitis. This study identifies inefficient axonal transport as a substantial barrier to poliovirus trafficking in peripheral neurons, which may limit CNS access for other viruses.

Figure 1. Identification of the barrier site between the periphery and CNS and the effect of type I interferon.

(A) Detection of virus in peripheral nerves after oral infection. PVR-IFNAR^−/− mice were orally inoculated with 2×10⁷ PFU of poliovirus, tissues were harvested upon disease onset, and the percentage of mice with detectable virus was quantified by RT-PCR. Mean with SEM from 13–14 mice is shown; more details are shown in Figure S1. (B) A representative blot from the hybridization-based viral diversity assay. PVR mice were intramuscularly inoculated with 2×10⁷ PFU of the 10-marked viruses, tissue were collected upon disease onset (ranging from 2 to 6 days post-inoculation, see Figure 6), and viral pool members were detected with the viral diversity assay. Results from one representative mouse are shown. Viral population diversity in intramuscularly inoculated (C) PVR mice or (D) PVR-IFNAR^−/− mice. Mice were intramuscularly inoculated as in (B), and the number of pool members in each tissue for each mouse is shown. Horizontal lines represent mean diversity for each tissue. Asterisks denote statistically significant reductions in viral population diversity (Student's t test, p<0.01). Viral population diversity in PVR-IFNAR^−/− mice was significantly higher than in PVR mice across all tissues (p<0.0001, 2 way ANOVA).

Figure 2. Viral titers from intramuscularly inoculated PVR and PVR-IFNAR^−/− mice.

Mice were inoculated with 2×10⁷ PFU, tissues were harvested upon disease onset, and viral titers were determined by plaque assay. Titers from PVR mice are depicted as closed circles, and titers from PVR-IFNAR^−/− mice are depicted as open squares, with mean indicated by horizontal lines. The magnitude of the titer difference between PVR and PVR-IFNAR^−/− mice is indicated below each tissue, and asterisks denote statistically significant differences (p<0.0001, Student's t test).

Figure 3. Viral replication kinetics.

Mice were intramuscularly inoculated with 2×10⁷ PFU of light-sensitive/neutral red poliovirus in the dark, tissues were harvested at 2, 6, 30 and 72 hpi in the dark, and virus was extracted in the dark. Half of the virus sample for each tissue was exposed to light (inactivating non-replicated viruses, to quantify replicated viruses), and half was maintained in the dark (to quantify all viruses). Virus in light-exposed and non-exposed samples was quantified by plaque assay. Total titer represents titer from non-light-exposed sample and is expressed as PFU/tissue (grey lines). Therefore, the grey lines represent titer from inoculum virus plus replicated virus. The percent of replicated viruses was obtained by dividing the light-exposed titer by the non-light-exposed titer for each individual sample (black bars). Mean with SEM from 3–7 mice is shown.

Figure 4. Replication status for individual viral population members.

(A) Schematic of light-sensitive/neutral red poliovirus hybridization-based diversity assay for determining viral replication status. In the dark (using a red safety light; black star/grey boxes), the 10 marked viruses containing neutral red dye were intramuscularly injected into the left gastrocnemius muscle of PVR or PVR-IFNAR^−/− mice. At 72 hpi, tissues were harvested and processed in the dark. The viral sample for each individual tissue was split, and half of the sample was exposed to light to inactivate non-replicated virus (unfilled star/unfilled boxes), and the other half of the sample was kept in the dark. Both virus samples were amplified by a single cycle of replication in HeLa cells to expand surviving virus and decrease the prevalence of light-inactivated virus. Total RNA was extracted, and the tagged region of the viral genome was amplified by RT-PCR. DNA was then spotted on a membrane, and the viral diversity assay was performed. Signal for each virus from the light-exposed and non-light exposed samples was compared for each tissue; sciatic nerve from a representative mouse is shown as an example. Light-sensitive viruses were scored as ‘Non-replicated’ (in this case, viruses 3/4/5/6/7/8/9/10), and light-insensitive viruses were scored as ‘Replicated+Non-replicated’ (in this case, viruses 2 and 11) because they may contain a sub-population of non-replicated viruses, which would be masked by the signal from replicated viruses. Pooled results from each tissue for 10 PVR mice (B) and 5 PVR-IFNAR^−/− mice (C) are shown. Bars represent the mean diversity in each tissue, and the proportion of non-replicated virus is indicated (unfilled bar).

Figure 5. Quantification of retrograde axonal transport efficiency.

(A) Efficiency of poliovirus transport and the effects of type I interferon and muscle damage. PVR and PVR-IFNAR^−/− mice were intramuscularly inoculated with 2×10⁷ PFU of the ten marked viruses, and were treated with or without needle sticks twice per day to induce muscle damage. Tissues were harvested at disease onset, and viral diversity was quantified. PVR mice, solid black bars; PVR mice with needle sticks, white hatched bars; PVR-IFNAR^−/− mice, grey bars; PVR-IFNAR^−/− mice with needle sticks, grey hatched bars. Results are expressed as mean with SEM, representing 4-13 mice per group. Statistically significant differences between groups are described in the text. (B) Efficiency of wheat germ agglutinin (WGA) transport and the effects of muscle damage. PVR mice were intramuscularly injected with 5 µg of WGA and treated with or without needle sticks. Tissues were harvested at 6 hours post-injection, and were processed for “dot-blot” protein immunoblot analysis. A WGA immunoblot, representative of four experiments, is shown under densitometry quantification (arbitrary units). Signal was normalized to sciatic nerve tissue from PVR mice not injected with WGA (denoted ΔWGA). Total WGA signal in the sciatic nerve represented approximately 1% of the WGA signal in muscle (data not shown).

Figure 6. The effect of barriers on viral pathogenesis.

PVR or PVR-IFNAR^−/− mice were intramuscularly inoculated with 2×10⁷ PFU of the 10-marked viruses, and were treated with or without needle sticks twice per day. Mice were euthanized upon disease onset; therefore, results are depicted as percent of mice without symptoms. Untreated PVR mice, solid black line with circles; PVR mice with needle sticks, dashed black line with squares; PVR-IFNAR^−/− mice, solid grey line with inverted triangle; PVR-IFNAR^−/− mice given needle sticks, grey dashed line with triangles. Data represent 12–36 mice per condition. Statistically significant differences between groups are indicated by asterisks (Mantel-Cox test, p<0.0001).