Volume 22, Issue 1, Pages 48-60.e5 (July 2017) Environmental Stress Causes Lethal Neuro-Trauma during Asymptomatic Viral Infections  Jonathan Chow, Zsuzsa Márka, Imre Bartos, Szabolcs Márka, Jonathan C. Kagan  Cell Host & Microbe  Volume 22, Issue 1, Pages 48-60.e5 (July 2017) DOI: 10.1016/j.chom.2017.06.010 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2017 22, 48-60.e5DOI: (10.1016/j.chom.2017.06.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 VSV-Infected Animals Have Immediate, Life-Threatening Sensitivity to CO2 Anesthesia Depending on Multiple Parameters (A) Drosophila melanogaster were infected using the standard method of pricking the thorax with a pin dipped in VSV. Flies were immediately collected following infection and homogenates were titrated on Vero cells to measure plaque forming units (PFU) (n = 3). (B and C) Adult flies were pricked with a sterile pin or infected with VSV. Survival was monitored during the course of infection (B), and acute recovery to 30 s of CO2 anesthesia was monitored 7 dpi (C). One experiment representative of three is shown. (D and E) Anopheles gambiae mosquitos were injected with buffer or VSV (107 PFU) in the thorax. After 3 days, animals were anesthetized for 30 s with CO2. Acute recovery after anesthesia (D) and survival (E) during the course of infection were monitored. One of three representative experiments is shown. (F) VSV-infected WT flies were incubated at 29°C or 21°C. At 7 dpi, flies were collected to measure PFU. (G) VSV titers were compared between 21°C infections of WT and Dcr2KO flies collected 7 dpi (n = 3). (H and I) WT and Dcr2KO flies were infected with VSV or received mock treatment. Acute recovery from CO2 anesthesia was monitored 7 dpi (H), and survival was tracked during the course of infection (I). One experiment representative of three is shown. (J) VSV titers were compared between 29°C infections of WT and Dcr2KO flies collected 3 dpi (n = 3). (K and L) VSV infected and mock treated WT and Dcr2KO flies were incubated at 29°C. At 3 dpi, flies were subjected to CO2 anesthesia and monitored for acute recovery (K). Flies were monitored for survival during infection (L). One experiment representative of three is shown. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Error bars represent SE. Cell Host & Microbe 2017 22, 48-60.e5DOI: (10.1016/j.chom.2017.06.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Climbing Behavior and Gait of Infected Animals Are Unaffected by VSV Infection (A) Dcr2KO flies were infected with VSV and kept at 29°C. Flies were anesthetized at the indicated days with CO2 for 30 s or left untreated. Survival was monitored. (B) WT and Dcr2KO flies were pricked on the thorax and then incubated at 29°C. On days 2 and 3, flies were assessed for their ability to climb up the vial after being startled. No statistical significance was observed between the two time points assessed (two-way ANOVA). A statistically significant difference in climbing activity was observed across the two genotypes (∗∗p < 0.01, two-way ANOVA, n = 3). (C) Dcr2KO flies were infected with VSV or mock treated and incubated at 29°C. Flies were assessed for climbing activity after being startled. No statistically significant differences were observed between infected and mock-treated flies nor between days 2 and 3 post-infection (two-way ANOVA, n = 3). (D–I) Dcr2KO flies were all pricked on the same side of the thorax and were either infected with VSV or mock treated. Flies were incubated at 29°C and tracked longitudinally 2 and 3 dpi. Video recordings of fly gait behavior were analyzed using FlyWalker software. No statistical differences were observed between mock (n = 8) and VSV (n = 11) infected groups at each time point for average step distance (D), tripod index (E), tetrapod index (F), wave gait index (G), noncanonical index (H), and speed (I) (t test). See also Movie S1. Error bars represent SE. Cell Host & Microbe 2017 22, 48-60.e5DOI: (10.1016/j.chom.2017.06.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Anesthesia Sensitivity Is Specific to Hypercapnia (A and B) WT and Dcr2KO flies were infected with VSV and kept at 21°C. At 8 dpi, flies were anesthetized with CO2 or N2. Acute recovery (A) and survival (B) are shown. One experiment representative of three is shown. ∗∗∗∗p < 0.0001; ns, not significant. Cell Host & Microbe 2017 22, 48-60.e5DOI: (10.1016/j.chom.2017.06.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 VSV Infection of the Nervous System Causes CO2 Sensitivity (A) Nanoject II was used to inject 9.2 nL volumes of VSV into the thorax of Dcr2KO flies at specified PFU. Infections were incubated at 29°C and anesthetized with 30 s of CO2 at 1, 2, 3, and 4 dpi. Flies were observed for 5 min to determine which had delayed CO2 anesthesia recovery before being collected to measure virus titers. (B) Ago2KO flies were infected with VSV-GFP via pricking of the thorax. Groups of infected flies were anesthetized with CO2 on the indicated days post-infection and survival was recorded. One of three experiments is shown. (C) Ago2KO flies were infected with VSV-GFP via pricking of the thorax and incubated at 29°C. At indicated times post-infection, flies were segmented as heads, thoraxes, and abdomens, homogenized, and blotted for GFP protein expression. Each lane represents a single fly. One of three representative experiments is shown. (D) WT and RNAi knockout flies were infected with VSV-GFP and incubated at 21°C for 11 days. Brains were dissected and stained for GFP expression. One of three representative experiments is shown. (E) Dcr2KO flies expressing GFP under the control of a glial, neuronal, or tracheal specific Gal4 were infected with VSV-Luc. After 11 days at 21°C, brains were dissected and stained for GFP and luciferase expression. DRAQ5 staining was used to detect nuclear DNA. Confocal images were taken with a 40× oil objective lens. Insets are 4× magnifications of selected areas. (F and G) Dcr2KO flies were bred to express functional Dcr2 in neurons or glia. Flies were infected with VSV and kept at 21°C. Flies were anesthetized with CO2 at 8 dpi, and recovery kinetics were measured (F). Animal survival was tracked throughout the infection (G). One experiment representative of three is shown. (H) VSV titers were measured 8 dpi from animals incubated at 21°C (n = 3). (I and J) Ago2 expression was knocked down ubiquitously or in specific cell types. VSV infected flies were incubated at 21°C. At 12 dpi, flies were assessed for acute recovery from CO2 anesthesia (I) and survival (J) throughout the experiment. One experiment representative of three is shown. (K) Infected animals kept at 21°C for 12 days were homogenized to measure VSV titers (n = 3). ∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant. Error bars represent SE. Cell Host & Microbe 2017 22, 48-60.e5DOI: (10.1016/j.chom.2017.06.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Sindbis Virus Infection Does Not Cause CO2 Sensitivity (A) Flies were pricked in the thorax with a pin swabbed in a concentrated stock of reporter virus, SINV-GFP (1011 PFU/mL), and infected flies were incubated at 29°C. At 3 dpi, brains were dissected and imaged for viral GFP expression. Representative dissections from one of three experiments are shown. (B) Dcr2KO flies were infected with SINV-GFP for 3 days at 29°C. Explanted brains were fixed and stained with antibodies against GFP and HRP to detect virus and neurons, respectively. DRAQ5 staining was used to detect nuclear DNA. Insets are 4× magnifications of selected areas. (C) Flies were injected in the thorax with 106 PFU of SINV. After 3 days at 29°C, viral titers were measured from infected flies (n = 3). (D and E) Flies were injected with buffer or 106 PFU of SINV and incubated at 29°C. At 3 dpi, flies were subjected to CO2 anesthesia and acute recovery (D) and survival (E) were assessed. One of three representative experiments is shown. ∗∗p < 0.01; ns, not significant. Error bars represent SE. Cell Host & Microbe 2017 22, 48-60.e5DOI: (10.1016/j.chom.2017.06.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 VSV G Expression in Glia Is Sufficient to Cause CO2 Sensitivity Flies in this figure were reared at 18°C. Flies were collected after eclosion by anesthetizing with N2 and sorting them on an ice-cold glass dish. (A) VSV transgene expression was driven by a heat shock-Gal4 (HS). After 8 days at 29°C, RNA was purified from flies and gene-specific mRNA was measured by qPCR (n = 3). (B) Dcr2KO were infected with VSV and incubated for 3 days at 29°C. VSV G and M transgene expression was driven by HS by incubating flies at 29°C for 8 days. Protein lysates of samples were western blotted for VSV G and M expression. Beta-actin was used as a loading control. (C and D) Flies were incubated at 29°C for 8 days to drive expression of VSV genes. Flies were subjected to CO2 anesthesia to assess acute recovery (C) and then were returned to 29°C to continue monitoring survival (D). One experiment representative of three is shown. (E) Glia and fat body-specific Gal4 transgenes drove VSV G expression. Flies were compared to ones containing only a Gal4 driver or VSV G transgene as controls. One of three representative experiments is shown for each blot. (F and G) A temperature-sensitive transcriptional inhibitory protein (TS) repressed VSV G expression in glial and fat body cells until flies were incubated at 29°C. After 5 days at 29°C, flies were subjected to CO2 anesthesia. Acute recovery to anesthesia (F) was assessed and survival (G) during the course of heat shock was monitored. One experiment representative of three is shown. ∗p < 0.05; ∗∗∗∗p < 0.0001; ns, not significant. Error bars represent SE. Cell Host & Microbe 2017 22, 48-60.e5DOI: (10.1016/j.chom.2017.06.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Fusogenic Activity of VSV G Causes Immediate Neuro-Trauma in the Nervous System Flies in this figure were reared at 18°C. Flies were collected after eclosion by anesthetizing with N2 and sorting them on an ice-cold glass dish. (A) Expression of WT and fusogenic mutants of VSV G were induced via 29°C incubation for 8 days. Flies were collected and VSV G expression was detected by western blotting. One of three representative experiments is shown. (B and C) Flies were heat shocked at 29°C for 8 days to induce expression of VSV GWT, GG124E, or GD137L before being assessed for acute recovery to CO2 anesthesia (B). Flies were then returned to the 29°C incubator to continue measuring viability (C). One experiment representative of three is shown. (D and E) Flies were incubated for 5 days at 29°C to induce VSV G expression. Flies were subjected to 30 s of CO2 or 60 s of N2 to assess recovery rate (D). Flies were then returned to 29°C incubation to continue tracking survival (E). One experiment representative of three is shown. (F) Apoliner and VSV G variants were expressed in glial cells by incubating flies at 29°C for 7 days. Flies were anesthetized with CO2 for 30 s and collected immediately, 3 hr later, or 6 hr later. Western blotting of fly protein lysates was used to assess Apoliner cleavage by GFP blotting. One experiment representative of three is shown. (G) VSV G and GFP expression was induced in glial cells by placing flies at 29°C for 7 days. Flies were anesthetized with CO2, and dissected brains were stained with GFP and HRP antibodies to stain for glia and neurons, respectively. DRAQ5 staining was used to stain for DNA. Confocal imaging was done using a 40× oil objective lens. Insets are 4× magnifications of selected areas. See also Figure S1 for additional staining control. ∗∗∗∗p < 0.0001; ns, not significant. Cell Host & Microbe 2017 22, 48-60.e5DOI: (10.1016/j.chom.2017.06.010) Copyright © 2017 Elsevier Inc. Terms and Conditions