Human IVIG treatment in a neurological disease model for Enterovirus A71 infection in 28-day-old AG129 mice.

Enterovirus A71 can cause serious neurological disease in young children. Animal models for EV-A71 are needed to evaluate potential antiviral therapies. Existing models have limitations, including lack of lethality or crucial disease signs. Here we report the development of an EV-A71 model in 28-day-old mice. Virus was serially passaged until it produced consistent lethality and rear-limb paralysis. Onset of disease occurred between days 6-9 post-infection, with mortality following weight loss and neurological signs on days 9-14. In addition, a single administration of human intravenous immunoglobulin at doses of 200, 400 and 800 mg/kg at 4h post-infection was evaluated in the model. Protection from weight loss, neurological signs, and mortality (between 50 and 89%) were observed at doses of 400 mg/kg or greater. Based on these results, IVIG was selected for use as a positive control in this acute model, and suggest that IVIG is a potential therapeutic for EV-A71 infections.

Evaluation of antiviral therapies in respiratory and neurological disease models of Enterovirus D68 infection in mice.

Enterovirus D68 (EV-D68) is unique among enteroviruses because of the ability to cause severe respiratory disease as well as neurological disease. We developed separate models of respiratory and neurological disease following EV-D68 infection in AG129 mice that respond to antiviral treatment with guanidine. In four-week-old mice infected intranasally, EV-D68 replicates to high titers in lung tissue increasing the proinflammatory cytokines MCP-1 and IL-6. The respiratory infection also produces an acute viremia. In 10-day-old mice infected intraperitoneally, EV-D68 causes a neurological disease with weight-loss, paralysis, and mortality. In our respiratory model, treatment with guanidine provides a two-log reduction in lung virus titers, reduces MCP-1 and IL-6, and prevents histological lesions in the lungs. Importantly, viremia is prevented by early treatment with guanidine. In our neurological model, guanidine treatment protects mice from weight-loss, paralysis, and mortality. These results demonstrate the utility of these models for evaluation of antiviral therapies for EV-D68 infection.

Development of a respiratory disease model for enterovirus D68 in 4-week-old mice for evaluation of antiviral therapies.

Enterovirus D68 (EV-D68) is a non-polio enterovirus that affects the respiratory system and can cause serious complications, especially in children and older people with weakened immune systems. As an emerging virus, there are no current antiviral therapies or vaccines available. Our goal was to develop a mouse model of human EV-D68 infection that mimicked the disease observed in humans and could be used for evaluation of experimental therapeutics. This is the first report of a respiratory disease model for EV-D68 infection in mice. We adapted the virus by 30 serial passages in AG129 mice, which are deficient in IFN- α/ß and -γ receptors. Despite a lack of weight loss or mortality in mice, lung function measured by plethysmography, showed an increase in enhanced pause (Penh) on days 6 and 7 post-infection. In addition, as virus adapted to mice, virus titer in the lungs increased 50-fold, and the pro-inflammatory cytokines MCP-1 and RANTES increased 15-fold and 2-fold in the lung, respectively. In addition, a time course of mouse-adapted EV-D68 infection was determined in lung, blood, liver, kidney, spleen, leg muscle, spinal cord and brain. Virus in the lung replicated rapidly after intranasal inoculation of adapted virus, 106 CCID50/mL by 4 h and 108.3 CCID50/mL by 24 h. Virus then spread to the blood and other tissues, including spinal cord and brain. This mouse model for EV-D68 infection includes enhanced pause (Penh) as an indicator of morbidity, and viremia, virus titers and proinflammatory cytokines in the lung, and lung histopathology as indicators of disease. Our mouse-adapted virus has a similar antiviral profile to the original isolate as well as another respiratory picornavirus, rhinovirus-14. This model will be valuable in evaluating experimental therapies in the future.

Evaluation of cell viability dyes in antiviral assays with RNA viruses that exhibit different cytopathogenic properties.

Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic properties. Dyes included alamarBlue® measured by absorbance (ALB-A) and fluorescence (ALB-F), neutral red (NR), Viral ToxGlo™ (VTG), and WST-1. Viruses were chikungunya, dengue type 2, and Junin, which generally cause 100, 80-90, and 50% maximal cytopathic effect (CPE), respectively, in Vero or Vero 76 cells Compounds evaluated were 6-azauridine, BCX-4430, 3-deazaguanine, EICAR, favipiravir, infergen, mycophenolic acid (MPA), ribavirin, and tiazofurin. The 50% virus-inhibitory (EC50) values for each inhibitor and virus combination did not vary significantly based on the dye used. However, dyes varied in distinguishing the vitality of virus-infected cultures when not all cells were killed by virus infection. For example, VTG uptake into dengue-infected cells was nearly 50% when visual examination showed only 10-20% cell survival. ALB-A measured infected cell viability differently than ALB-F as follows: 16% versus 32% (dengue-infected), respectively, and 51% versus 72% (Junin-infected), respectively. Cytotoxicity (CC50) assays with dyes in uninfected proliferating cells produced similar CC50 values for EICAR (1.5-8.9µM) and MPA (0.8-2.5µM). 6-Azauridine toxicity was 6.1-17.5µM with NR, VTG, and WST-1, compared to 48-92µM with ALB-A and ALB-F (P<0.001). Curiously, the CC50 values for 3-deazaguanine were 83-93µM with ALB-F versus 2.4-7.0µM with all other dyes including ALB-A (P<0.001). Overall, ALB minimized the toxicities detected with these two inhibitors. Because the choice of dyes affected CC50 values, this impacted on the resulting in vitro selectivity indexes (calculated as CC50/EC50 ratio).

Susceptibilities of enterovirus D68, enterovirus 71, and rhinovirus 87 strains to various antiviral compounds.

Compounds were evaluated for antiviral activity in rhabdomyosarcoma (RD) cells against a recent 2014 clinical isolate of enterovirus D68 (EV-D68), a 1962 strain of EV-68D, rhinovirus 87 (RV-87, serologically the same as EV-D68), and enterovirus 71 (EV-71). Test substances included known-active antipicornavirus agents (enviroxime, guanidine HCl, pirodavir, pleconaril, and rupintrivir), nucleobase/nucleoside analogs (3-deazaguanine and ribavirin), and three novel epidithiodiketopiperazines (KCN-2,2'-epi-19, KCN-19, and KCN-21). Of these, rupintrivir was the most potent, with 50% inhibition of viral cytopathic effect (EC50) and 90% inhibition (EC90) of virus yield at 0.0022-0.0053 µM against EV-D68. Enviroxime, pleconaril and the KCN compounds showed efficacy at 0.01-0.3 µM; 3-deazaguanine and pirodavir inhibited EV-D68 at 7-13 µM, and guanidine HCl and ribavirin were inhibitory at 80-135 µM. Pirodavir was active against EV-71 (EC50 of 0.78 µM) but not against RV-87 or EV-D68, and all other compounds were less effective against EV-71 than against RV-87 and EV-D68. The most promising compound inhibiting both virus infections at low concentrations was rupintrivir. Antiviral activity was confirmed for the ten compounds in virus yield reduction (VYR) assays in RD cells, and for enviroxime, guanidine HCl, and pirodavir by cytopathic effect (CPE) assays in A549, HeLa-Ohio-1, and RD cells. These studies may serve as a basis for further pre-clinical discovery of anti-enterovirus inhibitors. Furthermore, the antiviral profiles and growth characteristics observed herein support the assertion that EV-D68 should be classified together with RV-87.