key: cord-1002814-9lrwuk6z
authors: Bohmwald, Karen; Soto, Jorge A.; Andrade, Catalina; Fernández-Fierro, Ayleen; Espinoza, Janyra A.; Ríos, Mariana; Eugenin, Eliseo A.; González, Pablo A.; Opazo, María Cecilia; Riedel, Claudia A.; Kalergis, Alexis M.
title: Lung pathology due to hRSV infection impairs blood-brain barrier permeability enabling astrocyte infection and a long-lasting inflammation in the CNS
date: 2020-09-24
journal: Brain Behav Immun
DOI: 10.1016/j.bbi.2020.09.021
sha: 3c6bc6d24dcb9b075a4c230c1b8754769ae9bad9
doc_id: 1002814
cord_uid: 9lrwuk6z

The human respiratory syncytial virus (hRSV) is the most common infectious agent that affects children before two years of age. hRSV outbreaks cause a significant increase in hospitalizations during the winter season associated with bronchiolitis and pneumonia. Recently, neurologic alterations have been associated with hRSV infection in children, which include seizures, central apnea, and encephalopathy. Also, hRSV RNA has been detected in cerebrospinal fluids (CSF) from patients with neurological symptoms after hRSV infection. Additionally, previous studies have shown that hRSV can be detected in the lungs and brains of mice exposed to the virus, yet the potential effects of hRSV infection within the central nervous system (CNS) remain unknown. Here, using a murine model for hRSV infection, we show a significant behavior alteration in these animals, up to two months after the virus exposure, as shown in marble-burying tests. hRSV infection also produced the expression of cytokines within the brain, such as IL-4, IL-10, and CCL2. We found that hRSV infection alters the permeability of the blood-brain barrier (BBB) in mice, allowing the trespassing of macromolecules and leading to increased infiltration of immune cells into the CNS together with an increased expression of pro-inflammatory cytokines in the brain. Finally, we show that hRSV infects murine astrocytes both, in vitro and in vivo. We identified the presence of hRSV in the brain cortex and colocalizes with vWF, MAP-2, Iba-1, and GFAP, which are considered markers for endothelial cells, neurons, microglia, and astrocyte, respectively. hRSV-infected murine astrocytes displayed increased production of nitric oxide (NO) and TNF-α. Our results suggest that hRSV infection alters the BBB permeability to macromolecules and immune cells and induces CNS inflammation which can contribute to the behavioral alterations observed on infected mice. A better understanding of the neuropathy caused by hRSV could help to reduce the potential detrimental effects on the CNS in hRSV-infected patients.

Disease progression of mice infected with hRSV or UV-hRSV and mock-treated mice 307 were monitored by measuring the bodyweight of infected mice. As expected, hRSV-infected 308 mice displayed significant weight loss at day 2, 3 and 4 p.i. as compared to mock-treated and 309 UV-hRSV challenged animals ( Figure 1A ). Airway inflammation was also measured by 

It has been previously shown that cytokines, such as IL-6, TNF-α, IL-1β, IL-4, IL-10, 340 and CD200 can affect the behavior, learning and memory of animals (46-51). Moreover, the 341 high expression of GFAP by astrocytes has been related to astrocyte activation, inflammation 342 and neurodegenerative diseases (52-54). We think that it is possible that the content of these 343 cytokines and GFAP could be altered in the brain of mice infected with hRSV. Therefore, 344 the mRNA levels of IL-6, TNF-α, IL-1β, IL-4, IL-10, CD200 and GFAP were analyzed in 345 the brains from hRSV-infected mice 60 days p.i. Similar levels of IL-6, TNF-α, CX3CL1,

IL-1β and CD200 mRNAs were observed in the brains of hRSV-infected mice as compared 347 to mock-and UV-hRSV-treated mice ( Figure 3A ). Interestingly, 60 days p.i. a significant 348 increase was observed for IL-4, IL-10, CCL2 and GFAP mRNA levels in hRSV-infected 349 mice. The expression of TNF-α mRNA and protein content were alike among all 350 experimental groups ( Figure 3A and 3B, respectively). A significant increase in the protein 351 content of IL-4 ( Figure 3C ) and IL-10 ( Figure 3D ) and GFAP ( Figure 3E ) was observed in 352 hRSV infected mice as compared to UV-hRSV-and mock-treated mice. The protein levels 353 of these molecules were consistent with their mRNA levels.

354

Increased blood-brain barrier permeability and immune cell infiltration 355 into the brain of hRSV-infected mice

It has been observed an increase in the BBB permeability in mice infected with 357 influenza virus (55). Given that, we previously were able to identify hRSV in the brain cortex 358 (14) at 3 days p.i., we evaluated whether the hRSV infection promotes an alteration of the 359 BBB permeability and inflammatory cell infiltration. For that BALB/c mice were either 360 intranasally (i.n.) infected with 1x10 6 PFUs of hRSV or inoculated with non-infectious 361 supernatant (i.e., mock) or UV-inactivated hRSV. After 3 or 7 days of infection BBB 362 permeability was measured by Evans blue extravasation into the brain (see materials and 363 methods). Evans blue is a cationic dye that binds to albumin in the serum and forms a 364 complex that under normal conditions is not able to trespass an intact BBB (56). Thus, 365 extravasation of the Evans blue-albumin complex into the brain is indicative of increased 366 BBB permeability to macromolecules (57). Mock-or UV-hRSV treated mice showed no 367 significant extravasation of Evans blue into the brain ( Figure 4A and B) . However, hRSV-368 infected mice showed a significantly increased of Evans blue extravasation into the brain at 369 3 days p.i. (Figures 4A and 4C ). Even though, after seven days p.i. the amount of Evans Blue 370 detected at the CNS was reduced ( Figures 4B and 4D) , it is significantly high, suggesting 371 that the permeability of the BBB to macromolecules has increased in hRSV-infected mice. Moreover, it is possible that not only the BBB permeability has increased to 373 macromolecules but also the infiltration of inflammatory cells into the CNS. We assessed the 374 presence of inflammatory cells into the brain parenchyma of hRSV-infected mice. Immune 375 cell infiltration was evaluated by flow cytometry at days 3 and 7 p.i. with hRSV, UV-hRSV 376 or mock treatments ( Figure 5) . A significant increase in neutrophils ( Figure 5A and 5D 

It has been previously shown that hRSV can reach the brain at 3 days p.i. (14) and 451 enter the CNS utilizing a "Trojan horse" mechanism (66) However, whether hRSV can 452 infect and replicate in cells of the CNS remains unknown. Therefore, we evaluated whether 453 hRSV could infect astrocytes, neurons, microglia and endothelial cells at day 3 p.i. with 454 1x10 6 PFUs. Figure 7 shows representative pictures of immunofluorescence colocalization 455 analyses in brain cortex for hRSV and various markers for CNS cells. F-hRSV colocalized 456 with 1% of endothelial cells ( Figure 7B ), 1% of neurons ( Figure 7C ) and 1% of microglia 457 ( Figure 7D ). Whereas, F-hRSV colocalized with 5% of astrocytes ( Figure 7E ). Moreover, we 458 also detected approximately 4% of F-hRSV + free, which means that the viral protein was not 465 According to this notion and because we found that the percentage of hRSV-infected 466 astrocytes was higher as compared to the other cell types (Figure 7) , 

To determine whether astrocytes can be activated during hRSV infection, intracellular 480 levels of nitric oxide (NO) were quantified as a marker for astrocyte activation (50).

481 Importantly, the production of NO increased in hRSV-infected astrocytes 24, 48 and 72 h 482 p.i., as compared to UV-hRSV-treated cells ( Figure 8D ). These data further support the 483 notion that hRSV activates astrocytes and that this virus is likely that could induce an 484 inflammatory response in the CNS. Therefore, we also measured the secretion of cytokines 485 associated with inflammation, such as IL-4, IL-10, TNF-α and IL-6 in the supernatants of 496 DISCUSSION

Neurological complications in patients with severe bronchiolitis due to infection with 498 hRSV have been increasingly reported in the past years (4, 7, 8, 10-12) . Importantly, viral 499 mRNA has been detected in CSF samples of patients with neurological alterations, 500 suggesting that hRSV can reach the CNS (5, 6, 11, 70) . Furthermore, an increased cytokine 501 level detected in the CSF of hRSV-infected patients suggests that CNS infection could be 502 accompanied by encephalitis (7, 8, 10, 20, 70, 71 ). Consistently these important observations 503 in hRSV-infected patients, in this study we reported that neurological symptoms could also 504 take place in animal models. These data further support the notion that hRSV can access to 505 the CNS, in particular by infecting astrocytes. However, clinical signs associated with the 506 encephalopathy described in humans by hRSV, such as seizures (11, 12) were not observed 507 in the murine model. Importantly, our findings are in agreement with previous data 508 suggesting that hRSV may enter the CNS by a "Trojan horse" mechanism, by infecting Figure 6 ). These results 565 somewhat suggest that the increase in the expression of these pro-inflammatory cytokines 566 may be due to the activation of microglia and astrocytes, which could be enough to cause a 567 behavioral alteration in mice (55).

As mentioned above, both CD200 and CX3CL1 are immunomodulatory molecules 569 that are mainly expressed by neurons allowing their interaction with microglia, which express 570 the receptor for these molecules in the resting state (84-86). Also, it is known that CD200-571 deficient mice display cognitive impairment, suggesting that this molecule plays a significant 572 role in cognition processes (86). Similar to what was observed in the brains of influenza-573 infected mice, we found that CD200 expression was decreased starting day 1 p.i. in hRSV-574 infected mice, while in the study with influenza, this expression was only evaluated on day 575 7 p.i. (21). Furthermore, we found a decrease in CX3CL1 mRNA expression in hRSV-576 infected mice at 3 days p.i. suggesting that the alteration of the normal expression of CD200 577 and CX3CL1, mainly on day 3 p.i., could be due to the activation of microglia with 578 subsequent astrocytes activation and an inflammatory immune response.

Previously, it has been shown that CCL2 attracts inflammatory immune cells to sites 580 of injury, mainly monocytes (59). In the brain, CCL2 is constitutively expressed by neurons 581 yet, its levels increase in astrocytes and microglia under pathologic conditions (87). In line 582 with the findings of inflammatory monocytes in the brain of hRSV-infected mice, we found 583 that the CCL2 expression was elevated in this tissue. 

37 °C for 30 min. The cortex was centrifuged 300xg for 5 min, and the 192 supernatant was discarded. The pellet was dissociated in a single cell suspension in 10 ml of 193 DMEM supplemented with 20% Horse Serum (HS) by pipetting up and down 20 to 30 times

To perform an astrocyte primary culture, brain cells derived 201 from 1-3 postnatal day or 4-6 weeks old mice were used. Importantly, in both cases, only 202 astrocytes that are immature grow in culture and once they get confluent and make cell 203 contact, they begin to show a mature phenotype (40). In vitro, hRSV infection was performed 204 by the exposure of mouse astrocytes to hRSV (MOI: 5) for 2h at 37°C in DMEM 1% FBS 205 medium. After incubation, cells were washed 3 times with PBS and

Quantitative Real-Time qPCR

Total RNA was isolated from lungs and brains using TRIZOL reagent according to

Standard curves for qPCR were generated using 217 pTOPO-N-hRSV or pTOPO-β-actin as templates

Brain coronal slides of 30 for 20 min at -20°C. Tissue sections were transferred to 100% ethanol for 30 min and 228 dried for another 30 min at RT. Next, tissue sections were hydrated by incubating in 95% 229 ethanol for 30 min, followed by 75% ethanol for 5 min, then to 0.4% Triton X-100 in PBS 230 for 5 min and finally rinsed twice in PBS for 5 min. Sections were incubated in blocking 231 solution (5mM EDTA, 1% fish gelatin, 2% horse serum, and 1% mostly immunoglobulin 232 free BSA) for 1h at RT. After that, the sections were incubated with primary antibody 233

MAP-2 (10 μg/ml); vWF (10 μg/ml)

Fixed sections were washed four times with PBS for 5 min and 236 incubated with polyclonal goat α -mouse conjugated to Alexa Fluor 488 (10 μg/ml) and 237 polyclonal goat α -rabbit Alexa Fluor 555 (10 μg/ml) for 1h at RT, followed by 4 washes in 238 PBS for 5 min. Nuclei were stained with DAPI (1 μM) for 10 min

Images were processed using FV10-ASW 1.7 software (Olympus) and the

Flow cytometry was used to measure neutrophils infiltration in Bronchoalveolar 246 lavage fluid (BALF) as a means to quantify pulmonary disease in challenged animals. BALF 247 was obtained using 1X PBS, and lungs were collected after perfusion with 20 ml of PBS 1X

Lung samples were homogenized and filtered using 40-mm cell strainers. BALF and lung 249 cellular suspensions were centrifuged at 300xg for 5 min

BD Pharmingen) for 251 45 minutes at 4ºC. Data acquisition was performed on a FACS Canto-II flow cytometer (BD each BALF samples. To evaluate the infiltrating cells into the CNS, 255 brains from perfused animals were collected, and mononuclear cell suspensions were isolated 256 from brain tissue as follows. Brain tissue was digested using type IV collagenase (1 mg/ml) 257 and DNase I (50 µg/ml) for 30 min at 37°C. Brain samples were homogenized and filtered 258 using a 40 µm pore cell strainer. The homogenate was centrifuged at 200xg for 10 min at 259 4°C. The pellet was resuspended in 5 ml of 70% isotonic Percoll and loaded in a tube 395 brains of hRSV

A lack of a significant increase in the serum levels for IL-6 after hRSV 399 infection suggested that the increase of this cytokine was localized in the CNS and not a 400 systemic phenomenon due to viral infection (Supplementary Figure 3A). A significant 401 decrease of TNF-α mRNA was detected at day 9 p.i. for hRSV-infected as compared to UV-402 hRSV-and mock-treated mice (Figure 6B). In contrast, a significant increase of TNF-α 403 protein levels were detected at day 1 p.i. in hRSV-infected mice, as compared to mock-treated 404 animals and at day 3 p.i. in hRSV-infected mice, as compared to UV-hRSV-treated and 405 mock-treated animals (Figure 6G). When serum TNF-α was measured, no significant hRSV-infected mice at days 1 and 3 p.i. as compared to UV-hRSV-and mock-treated mice 409 (Figure 6C). Consistently, a significant increase in IL-4 protein was detected at days 1, 7 and 410 9 p.i. as compared to mock-treated mice. (Figure 6H)

In 415 this context, we analyze the levels of this molecule in the brains of hRSV-infected mice. An 416 increase in CCL2 mRNA and protein levels was observed at day 3 p.i. (Figure 6D, 6I)

During inflammatory conditions, the downregulation of CD200 leads to 426 increased activation of microglia (28). Moreover, it has been observed that the 427 downregulation of CD200 leads to cognitive impairment (27). Thus, the levels of CD200 428 mRNA and protein were measured in the brains of hRSV-infected mice. As shown in Figure 429 6E and, a significant decrease in CD200 expression was observed at days 1, 3, 7 and 9 p.i. in 430 hRSV-infected mice, as compared to UV-hRSV-and mock-treated mice

In the CNS, 435 the membrane CX3CL1is expressed by neurons and its receptor (CX3CR1), which is 436 expressed by microglial cells (61-63). Importantly, the CX3CL1-CX3CR1 axis keeps 437 microglia in a resting state and the loss of CX3CL1 expression promotes the activation of 438 microglial cells (61, 64). Interestingly, a significant decrease in CX3CL1 mRNA expression 439 was found at day 3 p.i. in the brains of hRSV-infected mice

TNF-α also increased in the supernatants of hRSV-489 infected astrocytes, but 12 h p.i., as compared to control cells (Figure 8G). Further, IL-6 490 secretion significantly increased in astrocytes infected with hRSV between, 12 h to 72 h, p.i. 491 as compared to control cells (Figure 8H). These results suggest that hRSV infection induces 492 an inflammatory response in astrocytes that is mediated by the secretion of molecules with 493 immunomodulatory and inflammatory properties

mRNA levels for IFN-α and IFN-β in the brains of hRSV-infected mice, which is consistent 590 with the notion that hRSV can inhibit the production of type-I IFNs to promote viral infection 591 (65)

In this work, we found that astrocytes which is one of the most 598 abundant cell type in the CNS (67, 89) can be infected by hRSV. These observations have 599 been reported for different viruses, such as the Venezuelan Equine Encephalitis virus (VEE) 600 (90), the Sindbis virus (SV) (91), and the Tick-borne Encephalitis virus (TBEV) (92)

Such a response is likely to promote inflammation in the CNS and may also cause 605 the activation of microglia. These cells in turn will secrete pro-inflammatory cytokines 606 affecting CNS function (95)

This is an important question to be addressed by future studies, as it goes beyond the scope 609 of this manuscript. Additionally, we found that not only astrocytes colocalized with hRSV 610 antigens (about 5%), but also did 1% of endothelial cells, 1% of microglia cells and 1% of 611 neurons (Figure 7). Regarding to that, it was previously shown that hRSV can infect 5 % of 612 lung neuronal cells and cortical neurons in culture

The infection of microglia also 615 could contribute to the increased amounts of cytokines found in the brains of hRSV-infected 616 mice. The detection of "free" virus probably is associated to the immune cells that infiltrate 617 the CNS of hRSV-infected mice. Nevertheless, additional studies would be needed to better 618 understanding the effects of hRSV-infection on other CNS cell types. 619 Based on the important role that astrocytes play in several CNS functions, the 620 response of astrocytes to hRSV infection was assayed by astrocyte infection in vitro and their 621 responses upon viral challenge. To approach this goal, primary glial cultures were used, 622 which were separated into microglia and astrocytes. Importantly, similar to the observations 623 in vivo

Additionally, we 626 observed that hRSV-infected astrocytes had a pro-inflammatory profile response 627 characterized by increased production of IL-4, TNF-α and IL-6. These data suggest that 628 hRSV-infection induces murine astrocyte activation and inflammatory responses by 629 secreting pro-inflammatory cytokines, likely involving these cells in neuroinflammation to 630 hRSV infection observed in the animal model. However, our results suggest that hRSV 631 replication may be somewhat impaired in astrocytes as viral RNA was found to decrease over 632 time. Taken together, our work provides new insights onto the effects of hRSV over the CNS 633 and calls for attention

This graph collects for 7 days the weight of 638 mice that were intranasally infected with 1x10 6 PFU of hRSV or UV-hRSV, or mock-639 infected. The results combine data from three independent experiments performed (N=3 mice 640 per experimental group per each experiment). Comparisons between groups were made using 641 one-way ANOVA test with Tukey´s post-test

was harvested 7 days pi. (b) Contour plots show the gating strategy to select the

Representative BALF contour plots of mock-treated and 644 hRSV-infected mice

One-way ANOVA test with Tukey´s post-test, * P<0.05. Graphs 646 show the quantification of N-hRSV mRNA in the lungs (c) and in the brain (d) of mice after 647 mock (white bar) or UV-hRSV (gray bar) treatment or hRSV infection

Light blue 655 circles indicate hidden marbles. (a) Representative photographs of the MB test performed 656 after 60 days. Two independent experiments were performed (N=2-3 mice per experimental 657 group per each experiment) and (b) 90 days (N=2 mice per group per experiment) p.i. with 658 hRSV. (c) The graph shows the quantification of the number of hidden marbles at 60 days 659 and (d) at 90 days p.i. Mann-Whitney U test

Expression of GFAP and molecules that contribute to inflammation 662 were increased in the brains of mice at 60 days post-infection with hRSV. mRNA and after performing the MB test (a) The graph shows mRNA relative expression for IL-6, 666 IL-1β, CD200, GFAP, TNF-α, IL-4, IL-10, CCL2 and CX3CL1 using β-actin as a 667 housekeeping control gene. Graphs show the quantification of TNF-α (b)

Figure 4. hRSV infection induces BBB permeability in vivo

Representative images of the brain of mice for each treatment and the quantification of Evans 676 blue dye in the brain of infected mice at 3 days p.i. (b) Representative images of the brain of 677 mice for each treatment and the quantification of Evans blue dye in the brains of infected 678 mice at 7 days p.i. Three independent experiments were performed (N=9 mice per group for 679 each experiment

Tukey´s post-test. ** P< 0.005, *P < 0.05 indicate that means show significant statistical 681 differences. Bars indicate mean ± SEM

Immune 683 cell infiltration in the brain of mice that were intranasally infected with 1x10 6 PFU of hRSV 684 or UV-hRSV and mock mice were analyzed by flow cytometry. (a-c) Representative contour 685 plot for the analyzed immune cells (1: Neutrophils, 2: Resident macrophages/microglial cells, 686 3: Inflammatory monocytes, 4: B cells, 5: CD4 + T cells, and 6: CD8 + T cells). The graph 687 shows the number of (d) neutrophils, (e) resident macrophages/microglial cells, (f) 688 inflammatory monocytes (j) B cells

Tukey´s post-test. *P < 0.05 indicates that means are significantly different. Bars indicate 693 mean ± SEM

Increased expression of inflammatory cytokines in the CNS of hRSV-695 infected mice. mRNA and protein levels of cytokines (analyzed by RT-qPCR and ELISA) hRSV or UV-hRSV and mock-697 treated mice. The following graphs show mRNA levels for: (a) IL-6

698 (d) CCL2; and (e) CD200. The following graphs show the content of protein levels for

IL-6; (g) TNF-α; (h) IL-4; (i) CCL2; and (j)

Figure 7. hRSV infects different CNS cell types in vivo

Representative immunofluorescence images of F-hRSV with (b) vWF, (c) Iba-1 (d) MAP-2 707 and (e) GFAP proteins were analyzed hRSV-infected mice at 20X magnification. The hRSV

F-protein is visualized in green, the cell markers in red and cell nuclei in blue. (f) The co-709 localizations of F-hRSV and the proteins with each cell type are visualized in yellow and 710 were quantified to determine the percentage of F-hRSV + cells in this

Figure 8. hRSV infects and activates astrocytes in vitro. Primary cultures of mouse 716 astrocytes were infected with hRSV at MOI 5 and cells analyzed to determine the percentage 717 of F-hRSV + cells (a), the number of N-hRSV copies analyzed by RT-qPCR (b)

cells (c) and the quantification of NO −2 in the supernatants of mice 719 astrocytes primary cultures (d)

0001*** P<0.0005, ** P< 0.005, * P<0.05 indicates that means are significantly

Supplementary Figure 1. Gating strategy to detect immune cell infiltration in 726 the brains of hRSV-infected mice. The contour plot shows the gating strategy to select the 727 cell population and then select singlet cells. Later, we selected the CD45 + cells and over this 728 selection we identified the CD19 + B220 + cells (B cells

Over the CD45 + cells, we selected the CD11b + and then Ly6C + cells

Supplementary Figure 2. hRSV-infected mice show an increased exploratory 731 behavior evaluated in Open Field Tests. Mice were intranasally infected with 1x10 6 PFU measured: (a) shows the total distance traveled and (b) the number of feces 735 averaged by experimental group. (c) Shows the percentage of time spent in traveling the 736 peripheral area and (d) the central area

Time course of mRNA expression and 740 immunomodulatory molecules in the CNS of hRSV-infected mice. A time-course analysis of cytokine levels in serum determined by ELISA: (a) IL-6; (b) TNF-α; (c) IL-4; and 744 (d) CCL2. The graphs show the time course of mRNA quantification in the brain for

AUC analyses for cytokines expression in the CNS of 750 hRSV-infected mice. The following graphs show the AUC analysis for: (a) IL-6 mRNA

TNF-α mRNA; (c) IL-4 mRNA; (d) CCL2 mRNA; and (e) CD200 mRNA; (f) IL-6 protein

752 (g) TNF-α protein; (h) IL-4 protein; (i) CCL2 protein; and (j) CD200 protein

Gating strategy to detect hRSV-infected astrocytes and 754 GFAP expression. Contour plot and histograms show the gating strategy to select the F

+ cell population after selecting singlet cells. The light grey line represents 756 mock-treated cells, the grey line represents UV-hRSV-treated cells and the dark grey line 757 hRSV

Supplementary Figure 6. AUC analyses for hRSV infected and activates 759 astrocytes in vitro. The following graphs show the AUC analysis for: the percentage of

MFI of GFAP (F-hRSV + GFAP + ) cells 761 (c) and the quantification of NO −2 in the supernatants of mice astrocytes primary cultures 762 (d)

CONICYT/FONDECYT 768 POSTDOCTORADO No. 3185070 for KB and No. 3190590 for JS. FONDECYT grants 769 number: 1150862. KB and AK designed the research

771 CR reviewed the manuscript and AK reviewed and approved the version to be published

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Regulation of proinflammatory cytokine 992 expression in primary mouse astrocytes by coronavirus infection

Role of pro-inflammatory cytokines 995 released from microglia in neurodegenerative diseases

Respiratory syncytial virus 998 (RSV) infects neuronal cells and processes that innervate the lung by a process 999 involving RSV G protein