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Home , Ebola, Ebolavirus, Filoviridae, Reston virus

Reston causes severe respiratory in young domestic

Elaine Haddocka, Greg Saturdayb, Friederike Feldmannb, Patrick W. Hanleyb, Atsushi Okumuraa,c, Jamie Lovagliob, Dan Longb, Tina Thomasa, Dana P. Scottb, Mikayla Pulliamd, Jürgen A. Richte,f, Emmie de Wita, and Heinz Feldmanna,1

aLaboratory of , Division of Intramural Research, National Institute of Allergy and Infectious , NIH, Hamilton, MT 59840; bRocky Mountain Veterinary Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, NIH, Hamilton, MT 59840; cCenter for and Immunity, Mailman School of , Columbia University, New York, NY 10032; dResearch Technologies Branch, Rocky Mountain Laboratories, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH, Hamilton, MT 59840; eDepartment of Diagnostic Medicine & Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66502; and fCenter of Excellence for Emerging and Zoonotic Animal Diseases, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66502

Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved November 23, 2020 (received for review July 24, 2020) (RESTV), an , causes clinical disease in for necropsy at 3 d postinfection (dpi), at 6 dpi, or for survival macaques but has yet only been associated with rare asymptom- analyses. For the 7-wk-old animals, we had no survival group, atic in . Its 2008 emergence in pigs in the due to weight restrictions for pigs in high containment. Animals raised concerns about food safety, pathogenicity, and were infected by a combination of the oropharyngeal and nasal 5 zoonotic potential, questions that are still unanswered. Until route with 1 × 10 TCID50 (50% tissue culture infectious dose) today, the virulence of RESTV for pigs has remained elusive, with of RESTV, strain Reston 08-A, the strain isolated from a Phil- unclear pathogenicity in naturally infected animals and only one ippine in 2008 (4). Daily scoring of animals revealed signs of experimental study demonstrating susceptibility and evidence for disease after RESTV infection as early as 3 dpi (Fig. 1A), be- shedding but no disease. Here we show that combined oropha- ginning with and somnolence, followed by hunched ryngeal and nasal infection of young (3- to 7-wk-old) Yorkshire posture and piloerection. By 6 dpi, all infected animals showed cross pigs with RESTV resulted in severe respiratory disease, with respiratory distress including tachypnea or dyspnea with ab-

most animals reaching humane endpoint within a week. RESTV- dominal pumping and central cyanosis, particularly evident on MEDICAL SCIENCES infected pigs developed severe cyanosis, tachypnea, and acute in- the snout. Serous nasal discharge and productive cough were terstitial pneumonia, with RESTV shedding from oronasal mucosal noted in some animals. Pigs near endpoint noticeably changed membranes. Our studies indicate that RESTV should be considered positions to improve ventilation and were often lethargic and a livestock with zoonotic potential. difficult to arouse. Respiratory distress peaked at 6 dpi to 7 dpi, and two of four and three of four animals in the 3- and 5-wk-old Reston virus | RESTV | pig | infection | severe respiratory disease survivor groups were euthanized for significant breathing dif- ficulties according to humane endpoint criteria (Fig. 1B). Pig- eston virus (RESTV) was discovered in 1989/1990 in ma- lets surviving the acute stage quickly recovered to normal Rcaques imported into the from the Philippines activity levels. for research purposes (1). Since then, there have been several Radiographic changes in RESTV-infected animals were con- episodes of disease caused by RESTV in macaques and rare sistent with the observed clinical signs; mock-infected animals asymptomatic infections in humans (2, 3). Unexpectedly, in 2008, RESTV emerged in pigs in the Philippines, and, shortly there- Significance after, RESTV sequences were detected in Chinese swine, raising zoonotic and food safety concerns (4, 5). RESTV constitutes a separate species in the Ebolavirus, family , and The emergence of Reston virus (RESTV) in domestic pigs in the is generally thought of as the apathogenic filovirus (6). Philippines and, subsequently, the detection of RESTV se- Aside from humans (2, 3), RESTV has been shown to naturally quences in pigs in China are serious human and animal health and experimentally infect macaques, swine, ferrets, , and concerns. Food safety is an immediate fear, and pathogenicity several species (4, 5, 7–13). Upon experimental infection, and potential for zoonotic are important con- macaques and ferrets, as well as immunocompromised , cerns. To find answers for those problems, we performed a such as STAT-1 knockout mice, develop severe disease with le- pathogenicity study in young domestic pigs. We could dem- thal outcome, whereas immunocompetent rodents generally do onstrate that young pigs develop severe respiratory disease not (9–12). Whether RESTV itself causes disease in naturally upon RESTV infection and shed virus from mucosal membranes infected domestic pigs remains unknown, since the RESTV- of the respiratory tract. We conclude that RESTV should be infected pigs from the Philippines were coinfected with the vir- considered a livestock pathogen with zoonotic transmission ulent arterivirus porcine reproductive and respiratory syndrome impacting on animal and perhaps even human health. virus (PRRSV; now Betaarterivirus suid 1). In an initial experi- Author contributions: J.A.R., E.d.W., and H.F. designed research; E.H., G.S., F.F., P.W.H., mental study, domestic pigs infected with RESTV only exhibited A.O., J.L., D.L., T.T., D.P.S., M.P., and H.F. performed research; F.F., P.W.H., J.L., D.L., T.T., subclinical infections with evidence for virus shedding (7). We D.P.S., and M.P. contributed new reagents/analytic tools; E.H., G.S., F.F., P.W.H., A.O., J.L., studied RESTV infection in young (3- to 7-wk-old) Yorkshire D.L., T.T., D.P.S., M.P., J.A.R., E.d.W., and H.F. analyzed data; and E.H., G.S., F.F., P.W.H., cross pigs, a swine breed used frequently in commercial pig A.O., J.L., D.P.S., J.A.R., E.d.W., and H.F. wrote the paper. production systems around the world. The main objective was to The authors declare no competing interest. determine an age-dependent susceptibility to infection. This article is a PNAS Direct Submission. Published under the PNAS license. Results 1To whom correspondence may be addressed. Email: [email protected]. RESTV Infection of Young Pigs Causes Severe Respiratory Disease. This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Pigs aged 3, 5, or 7 wk were divided into predetermined group- doi:10.1073/pnas.2015657118/-/DCSupplemental. ings of four animals (one 7-wk-old group had only three animals) Published December 28, 2020.

PNAS 2021 Vol. 118 No. 2 e2015657118 https://doi.org/10.1073/pnas.2015657118 | 1of7 Downloaded by guest on September 24, 2021 5 Fig. 1. Clinical parameters for RESTV-infected pigs. Pigs were infected by the oropharyngeal and nasal route with 1 × 10 TCID50 of RESTV. Predetermined groups of mainly four animals were scheduled for necropsy at 3 dpi, at 6 dpi, or for survival analyses (7-wk-old animals had no survival group). Graphs represent a single biological experiment. (A) Clinical score, with mandated at a score of 30. Data are shown as individual points, with lines representing the mean response by animal age. Statistical analysis by one-way analysis of variants and a Dunnett’s multiple comparison test defines sig- nificance between RESTV-infected and naïve controls. Significant statistical differences were found between infected age groups and infected control animals but not between infected age groups. Analysis was performed only through 7 dpi, due to reduced group sizes after this point. Individual P values are as follows (95% CI): 7-wk 3 dpi = 0.0199, 7-wk 4 dpi to 6 dpi ≤ 0.0001, 5-wk 5 dpi = 0.0003, 5 wk 6 dpi = 0.0217, 5 wk 7 dpi = 0.0431, 3 wk 5–6dpi = 0.000. An unpaired t test with two-tailed P value defines significance in (B) survival curve. This graph only includes the survival group animals. All infected animals showed clear clinical signs of respiratory distress. (C) Radiography. Ventrodorsal thoracic X-rays, flipped horizontally for gross comparison. A marker (R) indicates right side of animal. (Left) Control animal 3 dpi: no pathologic changes. (Middle) RESTV-infected animal 3 dpi: Areas of mild increased pulmonary opacity are highlighted in the circles. (Right) RESTV-infected animal 6 dpi: Areas of moderate to severe pulmonary opacity with evidence of air bronchograms are highlighted in the circles. There were no significant age-dependent differences; data from 3-wk-old pigs are shown as representative for all others.

did not show any changes (Fig. 1C). In all pigs, mild interstitial dpi, lungs exhibited multifocal to focally extensive consolidation pulmonary infiltrations with variable lobar distribution were with dark red discoloration consistent with mild to moderate observed by 3 dpi, and, by 6 dpi, pulmonary infiltrations were interstitial pneumonia. By 6 dpi, lungs generally failed to col- evident in all lobes. Changes ranged from moderate interstitial lapse and showed diffuse consolidation and dark red discolor- infiltrates with cardiac border effacement to a severe mixed in- ation consistent with marked interstitial pneumonia. By 3 dpi, terstitial alveolar pattern with air bronchograms and consolida- postmortem lung−body weight ratios of infected animals were tion. Animals that survived acute infection tended to have lower not statistically higher than those of controls (Fig. 2B). However, radiographic scores than those which succumbed to infection. animals euthanized during acute respiratory disease demon- strated a significant increase in lung weights. Survivors, eutha- Young Pigs Show Evidence for Systemic RESTV Infection and Shedding. nized without overt clinical signs, still had lung−body weight Hematology and serum chemistry were not statistically different SI Appendix ratios significantly higher than controls. Virus titration deter- between mock- and RESTV-infected animals ( ,Fig. mined high viral loads in the lungs and draining lymph nodes of S1). Several animals had measurable RESTV-specific IgM re- RESTV-infected piglets, with lower titers in the and spleen sponses by 6 dpi, although only the surviving animals developed (SI Appendix, Fig. S2). Viral titers in the lungs ranged from 104 to RESTV-specific IgG responses (SI Appendix, Table S1). RESTV 109 TCID per g of tissue, with no notable difference between RNA was detectable in the of some animals as early as 3 dpi 50 age groups (Fig. 2C). While the two 3-wk-old survivors had and, in remaining animals, at 6 dpi or 7 dpi (SI Appendix,Table S1), although infectious RESTV was found at a 1:100 dilution of cleared infectious virus from the lungs by 16 dpi, the single blood only in some piglets (3-wk-old: 7/8, 5-wk-old: 5/8). Similarly, 5-wk-old survivor still had quantifiable virus at 13 dpi. RESTV RNA in oral, nasal, and rectal swabs was discernible at By 3 dpi, histopathologic evaluation showed mild to moderate low levels from some or all animals at various time points, but pneumonia characterized by thickening of alveolar septae by only oral swabs yielded infectious RESTV (3-wk-old: 5/12 at , fibrin, and mild to moderate numbers of and 3 dpi and 3/8 at 5 dpi; 5-wk-old: 1/12 at 3 dpi and 1/8 at 5 dpi) . Alveoli contained moderate to abundant numbers (SI Appendix, Table S1). of alveolar macrophages, fewer neutrophils, and rare necrotic debris (Fig. 3). At 6 dpi, lungs were more diffusely affected RESTV-Infected Young Pigs Develop Marked Interstitial Pneumonia. and showed moderate to severe interstitial pneumonia with Mock-infected animals did not show any gross pathologic more pronounced characteristics. Inflammation often surrounded changes at necropsy. Gross pathology lesions in RESTV-infected bronchioles and pulmonary vasculature. Type II pneumocyte pigs were confined to the lungs (firm and edematous) (Fig. 2A) hyperplasia was prominent and associated with numerous eo- and mediastinal lymph nodes (enlarged and edematous). By 3 sinophilic intracytoplasmic inclusions located within alveolar

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Fig. 2. Gross pathology and tissue viral loads of RESTV-infected pigs. Animal groups, infection, and examinations are the same as described in the legend of Fig. 1. Graphs represent a single biological experiment. (A) Dorsal aspect of lungs. (Left) Control animal 3 dpi: no pathologic changes. (Middle) RESTV-infected animal 3 dpi: focally extensive consolidation and dark red discoloration of the caudal lung lobes and failure to collapse. (Right) RESTV-infected animal 6 dpi: diffuse consolidation with dark red discoloration and diffuse failure to collapse. There were no significant age-dependent differences; data from 3-wk-old pigs are shown as representative for all others. (B) Lung to body weight calculations at terminal time points (3-wk survivors were euthanized at 16 dpi, 5-wk survivor at 13 dpi). Data are shown as individual points, with bars representing the mean response per group; error bars represent the SD of the individual points; horizontal dotted line at 0.11 represents the average lung weight to body weight ratio of control, mock-infected animals. An unpaired t test with two- tailed P value defines significance (denoted with asterisk) between infected and mock-infected groups. Individual P values are as follows (95% CI): all 3-wk-aged animals euthanized at 6 dpi to 7 dpi = 0.0197, all 5-wk-aged animals euthanized at 6 dpi to 7 dpi = 0.0001, all 7-wk-aged animals euthanized at 6 dpi = 0.0006, all study survivors = 0.0027. (C) The following tissue samples were titered for RESTV: lungs (right upper lung lobe [RUL], right middle lung lobe [RML], right lower lung lobe [RLL], left upper lung lobe [LUL], left middle lung lobe [LML], left lower lung lobe [LLL]), as well as the mediastinal (MLN), liver, and spleen of each animal in the study. Results for each animal are displayed individually, and data represent a single technical measurement. Mean and SD are shown for each animal group for each tissue.

Haddock et al. PNAS | 3of7 Reston virus causes severe respiratory disease in young domestic pigs https://doi.org/10.1073/pnas.2015657118 Downloaded by guest on September 24, 2021 Fig. 3. Histopathology of lungs from RESTV-infected pigs. Animal groups, infection, and examinations are the same as described in the legend of Fig. 1. Control (mock-infected animal 3 dpi): hematoxylin and eosin (H&E) 100×—no pathologic changes; immunohistochemistry (IHC) 100×—no immunoreactivity. Day 3 (RESTV-infected animal 3 dpi): H&E 100×, Inset 400×—mild acute interstitial pneumonia with increased alveolar macrophages (arrow); IHC 100×, Inset 400×—strong immunoreactivity in alveolar macrophages. Day 6 (RESTV-infected animal 6 dpi): H&E 100×, Inset 400×—marked in- terstitial pneumonia with exudate (arrow), type II pneumocyte hyperplasia (arrow head), and edema (asterisk); IHC 100×, Inset 400×—strong immunore- activity in alveolar macrophages.

macrophages (Fig. 3). Also noted were occasional alveolar subclinical infection with virus shedding (7). Despite the obvious multinucleated cells as previously reported in pigs experimen- differences in clinical outcome, infection parameters such as tally infected with the related virus (EBOV) (Fig. 3) (14). tropism, pathology, and pathophysiology were, in general, The three surviving animals necropsied on 13 dpi or 16 dpi similar in both studies but much more pronounced in the animals showed pulmonary pathology ranging from mild to severe in- described here. The difference in infection outcome was not terstitial pneumonia within various lung lobes. By 6 dpi, medi- related to age of the pigs but could be related to the pig breed or astinal lymph nodes contained moderate numbers of draining the status of comorbidities such as infections with unrelated re- neutrophils and necrotic debris. RESTV was de- spiratory or nonrespiratory as discussed previously tected by immunohistochemistry predominately in alveolar (7). We tested our pigs for the presence of PRRSV, porcine macrophages on 3 dpi and 6 dpi (Fig. 3). Alveolar macrophages (PCV) types 2 and 3, and other common swine re- were the only leukocytes in the lungs to stain with RESTV- spiratory pathogens (see Methods) and did not find any evidence specific (Fig. 3), similar to previous reports from for , with the exception of a single animal that was RESTV- and EBOV-infected pigs (7, 14). Rare immunoreac- PCR positive for PCV-2 (SI Appendix, Table S1). Future studies tivity was also identified in type II pneumocytes, bronchiolar need to more deeply investigate the impact of and epithelial cells, and endothelial cells (Fig. 3). Virus particles pig genetics on susceptibility to RESTV infections. were detectable in the lung tissue by transmission electron mi- Both studies used the oropharyngeal/oronasal route of infec- croscopy (Fig. 4). Type II pneumocyte hyperplasia was associ- tion likely mimicking best natural exposure of pigs to RESTV- ated with 1- to 3-μm intracytoplasmic inclusions in alveolar contaminated excretions or secretions from the postulated fruit macrophages that were identified by transmission electron mi- reservoir (3, 13). However, the initial study (7) used a 10-fold 6 croscopy as membrane-bound enlarged (Fig. 4). higher inoculation dose (10 TCID50), still not producing disease in pigs. Virus load in secretions and excretions from EBOV- Discussion infected bats are unknown, but, for virus, they are es- Here we report that RESTV infection of piglets aged 3 wk to 7 timated to be <104 plaque-forming units per mL (15, 16), which wk resulted in severe respiratory disease, which is in contrast to is closer to what we used in this study. Notably, studies with the only previous experimental RESTV pig study that reported EBOV in mice have resulted in disease attenuation when higher

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Fig. 4. Transmission electron microscopy of lungs from RESTV-infected pig. Animal groups, infection, and examinations are the same as described in the legend of Fig. 1. (A) Alveolus with type I pneumocyte and a degenerate alveolar containing membrane-bound lysosomes (asterisk) and virus particles (arrowhead). (Scale bar = 1 μm.) (B) Endothelial cells surrounding a red blood (asterisk), with virus particles in the vascular and subendothelial space (arrowhead). (Scale bar = 600 nm.) (C) Alveolar macrophage with cytoplasmic complex (asterisk) and adjacent virion (arrowhead). (Scale bar = 1 μm.) (D) REST particle cross-sections. (Scale bar = 100 nm.) (E) RESTV particle longitudinal section with polar vesicle. (Scale bar = 400 nm.)

infectious doses were used. The authors (17) speculated that attenuation. This was shown for Sin nombre ,wherea high viral loads may mediate stronger innate immune responses, single tissue culture passage did attenuate the virus for rhesus ma- leading to disease attenuation and survival. Further studies are caques (18). Both RESTV seed stocks were prepared by a single needed to confirm this phenomenon. passage, and future sequence comparison, once information becomes The virus strain used for infections in both pig studies was derived available from the previous study (7), should clarify any issues. from the same original source in the Philippines, but in-house tissue The role of pigs as an interim or amplifying for ebolavi- culture passaging could have led to genetic changes resulting in ruses has been discussed and remains a matter of concern (19).

Haddock et al. PNAS | 5of7 Reston virus causes severe respiratory disease in young domestic pigs https://doi.org/10.1073/pnas.2015657118 Downloaded by guest on September 24, 2021 × 5 Pigs are experimentally susceptible to mucosal exposure of dorsal recumbency with either 1 10 TCID50 RESTV 08 or DMEM EBOV resulting in acute respiratory disease (20, 21). EBOV (mock-infected) by nasal (1 mL per nare) and oropharyngeal (5 mL) inocu- shedding from the oronasal mucosa resulted in transmission to lation. The challenge dose was confirmed by back-titration of the inoculum naïve pigs and macaques, indicating the potential for transmis- on Vero cells. Clinical examinations including blood collection, radiographs (ventrodorsal, right and left laterals), and mucosal swabs were conducted on sion (20, 21). Recently, pigs in have been tested predetermined days (0, 1, 3, 5, 7, and subsequent) and at terminal end points positive for (22). All of this may favor a defined and approved by the IACUC based on a previous publication (28). role of pigs in the ecology of filoviruses, but further investiga- Radiographs were scored using a published scoring matrix adapted to pigs. tions need to confirm this hypothesis. Animals were euthanized either at predetermined time points (day 3 RESTV is considered a human apathogenic filovirus but is still and day 6) or at study endpoint, which was day 13 and day 16 for the classified as a 4 pathogen in many countries and a 5-wk-old and 3-wk-old groups, respectively. Full necropsies were performed select agent in the United States (23). In a few human cases, for gross pathology evaluation, and tissue was harvested for histopathology evidence of RESTV infection has been documented by sero- and virology. Lung tissues from animals tested negative by PCR for PRRSV, A virus, Mycoplasma spp. and bacterial ribosomal RNA. Serum conversion and detectable viremia (2, 3). This is in strong con- samples from all animals were PCR negative for PCV-3. A single animal in the trast to the other highly pathogenic ebolaviruses (6). The 5-wk-old group was PCR positive for PCV-2 (SI Appendix, Table S1). emergence of RESTV in pigs is a wake-up call, as transmission into humans through direct pig contact or the food chain is a Hematology and Serum Biochemistry Analyses. Hematology was completed on possibility. As RESTV can infect humans (3), replication could a Procyte DX (IDEXX Laboratories), and the following parameters were result in changes in virulence, of which would be most evaluated: red blood cells, hemoglobin, hematocrit, mean corpuscular vol- worrisome for public health. Adaptation as a result of multiple ume, mean corpuscular hemoglobin, mean corpuscular hemoglobin con- transmission chains in humans has been controversially discussed centration, red cell distribution weight, , mean volume, during the West African Ebola outbreak (24–26). white blood cells, count (abs and percent), count (abs and percent), count (abs and percent), eosinophil count (abs and Finally, current data support the notion that RESTV should percent), and basophil count (abs and percent). Serum chemistries were be considered a livestock pathogen with unknown zoonotic po- completed on a Vetscan VS2 Chemistry Analyzer (Abaxis), and the following tential. There does not seem to be an immediate threat of parameters were evaluated: glucose, blood urea nitrogen, creatinine, cal- RESTV introduction into other countries, including the United cium, albumin, total , alanine aminotransferase, aspartate amino- States, but the RESTV situation in pigs in the Philippines and transferase, alkaline phosphatase, total bilirubin, globulin, sodium, potassium, other Southeast Asian countries should be closely monitored. chloride, and total carbon dioxide. Regulations for import/export of life pigs and pork products should likely consider RESTV as a potential transboundary Antibody Detection. RESTV-specific IgM and IgG were measured by enzyme- animal disease. linked immunosorbent assay. Recombinant RESTV lacking the transmembrane region (rRESTV GPdTM) (IBT Bioservices) was diluted in PBS μ Methods and then adsorbed to Nunc Maxisorp plates at 0.05 g/mL. Plates were washed with 5% skim milk in PBS + 0.05% Tween-20 and blocked with the Biosafety and Animal Ethics. All infectious work with RESTV and sample in- same. Serial fourfold dilutions of serum in blocking buffer (5% skim milk in activation was performed in the maximum containment laboratory in ac- PBS + 0.05% Tween-20) beginning with 1:100 were then applied to plates. cordance with standard operating procedures approved by the Rocky Plates were washed with PBS + 0.05% Tween-20, and bound antibody was Mountain Laboratories (RML) Institutional Biosafety Committee (IBC), Divi- detected with either peroxidase-labeled rabbit anti-pig IgG (1:1,000 dilution) sion of Intramural Research, National Institute of Allergy and Infectious (Invitrogen) or goat anti-pig IgM (1:5,000) (Bio-Rad Laboratories). Absor- Diseases, NIH. All animal work was performed in strict accordance with the bance at 405 nm was measured. recommendations described in Guide for the Care and Use of Laboratory Animals (27) of the NIH, the Office of Animal Welfare, and the United States Nucleic Acid Detection. Total RNA was extracted from blood, swabs, and urine Department of Agriculture in an Association for Assessment and Accredi- samples using QIAamp Viral RNA extraction kits or from tissue samples using tation of Laboratory Animal Care International (AAALAC) accredited facility. RNeasy extraction kits (Qiagen) [IBC-approved and published protocol (29)] Animals were group housed in cages that enabled social interaction, under and tested for the presence of RESTV RNA by real-time one-step qRT-PCR run controlled conditions of humidity, temperature, and light (12-h light/12-h on a Rotor- RG-3000 instrument using a QuantiFast Probe RT-PCR +ROX dark cycles). Food and water were available ad libitum. Animals were Vial Kit (Qiagen) with primers specific to RESTV (forward 5′-CCCTTTGGCCCG- monitored at least twice daily and fed commercial pig chow by trained AACAG and reverse 5′-GGGCGGCCTTAAATGCAT; 0.25 μM final concentra- personnel. Environmental enrichment consisted of manipulanda and audio tion; IDT) and a dual-labeled fluorescent probe (6FAM-CAAAGTGCG- enrichment. Humane endpoints specified and approved by the Institutional TAAT+GA+G+G-BBQ; 0.125μM final concentration; TIB Molbiol). RT-PCR was Animal Care and Use Committee (IACUC) were applied to determine when carried out in three stages: reverse (50 °C for 10 min), Taq ac- animals should be euthanized. tivation (95 °C for 5 min), and amplification (40 cycles of 95 °C for 10 s and 60 °C for 45 s) of each amplification cycle. Samples were quantified against a RESTV Virus Stock. RESTV, strain 08-A, was isolated from a Philippine pig in standard curve of RESTV 2008 RNA extracted from viral stock with pre-

2008 (4) and kindly provided by the Viral Special Pathogens Branch of the determined titer and calculated as infectivity equivalents (TCID50/mL or Centers for Disease Control and Prevention. The virus was propagated in TCID50/gram). Vero cells (passage 3) with 2% fetal bovine serum (FBS), L-glutamine (40 μM), and penicillin/streptomycin (500 U/mL and 500 μg/mL), then harvested, spun Virus . Isolation was conducted on Vero cells plated for 80% con- for clarification, aliquoted, and frozen in liquid nitrogen with 10% FBS. Viral fluency in 48-well plates. Medium was removed from cells and replaced with stocks were diluted to challenge dose in Dulbecco’s modified Eagle’s me- 1:100 and 1:1,000 dilutions of blood or swab medium in 0.1 mL DMEM, with dium (DMEM; Sigma-Aldrich). The stock was analyzed by next-generation an infection time of 45 min. Following inoculation, DMEM with 2.5% FBS, sequencing (NGS), resulting in no mutation to the original GenBank entry penicillin/streptomycin, and L-glutamine was added for a total volume of (MT796851); contaminations were not detected. 0.5 mL per well. Tissues were weighed in 1 mL of DMEM with a 5-mm stainless steel bead, homogenized at 30 Hz for 10 min, spun at 8,000 rpm for Animal Studies. Commercially available Yorkshire cross piglets (male and 10 min, diluted 1:102 to 1:108, and used to infect Vero cells as above. Cell female) were weaned and shipped at ∼2 wk of age. Pigs were group housed cultures were read for cytopathic effect at day 14 for evidence of virus. in caging until the challenge ages of 3, 5, or 7 wk. For 3- and 5-wk-old pigs, animals were grouped as follows: two controls, four early pathology (3 dpi), Histopathology. Histopathology was performed on pig tissues. Following four late pathology (6 dpi), and four survival. For 7-wk-old pigs, animals fixation and inactivation of tissues (<1cm3) for a minimum of 7 d in 10% were grouped as follows: three early pathology (3 dpi) (one animal had to neutral-buffered formalin and one formalin exchange [IBC-approved and be euthanized for unrelated medical conditions before study start) and four published protocol (29)], tissues were removed from BSL4 and processed late pathology (6 dpi); there was no survival group, due to animal weight using a VIP-6 Tissue Tek (Sakura Finetek) tissue processor and embedded in restrictions in maximum containment at RML. Animals were challenged in Ultraffin paraffin polymer (Cancer Diagnostics). Samples were sectioned at

6of7 | PNAS Haddock et al. https://doi.org/10.1073/pnas.2015657118 Reston virus causes severe respiratory disease in young domestic pigs Downloaded by guest on September 24, 2021 5 μm, and resulting slides were stained with hematoxylin and eosin or used Statistical Analysis. Data were analyzed with one-way analysis of variance for immunohistochemistry. Specific immunoreactivity was detected using and a Dunnett’s multiple comparison test comparing differences between polyclonal rabbit serum against RESTV nucleoprotein diluted 1:250 (kindly RESTV-infected age groups and mock-infected controls. Data were not an- provided by Ayato Takada, Hokkaido University, Hokkaido, Japan), followed alyzed past 7 dpi, as overall group numbers were greatly reduced. Lung to by a Biogenex biotinylated anti-rabbit antibody (SS Rabbit Link Biogenex) weight ratios were analyzed with an unpaired t test with two-tailed P value and the 3,3’-Diaminobenzidine (DAB) chromogen (Discovery DABMap de- to compare values between RESTV-infected and mock-infected groups. This tection kit; Roche Tissue Diagnostics). study was not specifically designed for statistical evaluation, as group numbers were small, and any animal loss therefore negatively impacts Transmission Electron Microscopy. Small segments of lung tissue were fixed for statistical testing. 7 d in Karnovsky’s fixative [IBC-approved and published protocol (29)]. Specimens were fixed overnight at 4 °C with 2.5% glutaraldehyde/4% Data Availability. All study data are included in the article and SI Appendix. paraformaldehyde in 0.1 M Sorensen’s buffer, pH 7.4. Samples were post- fixed for 1 h with 1.0% osmium tetroxide/0.8% potassium ferricyanide, 1 h ACKNOWLEDGMENTS. We thank the Rocky Mountain Veterinary Branch, Division on Intramural Research (DIR), National Institute of Allergy and with 1% tannic acid in dH2O, and 2% osmium tetroxide for an additional hour. The tissues were further stained overnight with 1% uranyl acetate at Infectious Diseases (NIAID), NIH for help with animal husbandry and veter- inary clinical and pathology support. We thank Elizabeth R. Fischer (Research 4 °C, dehydrated with a graded ethanol series, and embedded in epon/ Technologies Branch, DIR, NIAID, NIH) for assistance with processing for araldite resin. Thin sections were cut with a Leica UC6 ultramicrotome transmission electron microscopy, and Anita Mora (Visual and Medical Arts, (Leica), and viewed at 120 kV on an FEI BT Tecnai transmission electron DIR, NIAID, NIH) for aid in figure development. The study was financially microscope (Thermofisher/FEI). supported by the Intramural Research Program of NIAID, NIH.

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