Reston virus causes severe respiratory disease in young domestic pigs

Significance

The emergence of Reston virus (RESTV) in domestic pigs in the Philippines and, subsequently, the detection of RESTV sequences in pigs in China are serious human and animal health concerns. Food safety is an immediate fear, and pathogenicity and potential for zoonotic transmission are important concerns. To find answers for those problems, we performed a pathogenicity study in young domestic pigs. We could demonstrate that young pigs develop severe respiratory disease upon RESTV infection and shed virus from mucosal membranes of the respiratory tract. We conclude that RESTV should be considered a livestock pathogen with zoonotic transmission impacting on animal and perhaps even human health.

Abstract

Reston virus (RESTV), an ebolavirus, causes clinical disease in macaques but has yet only been associated with rare asymptomatic infections in humans. Its 2008 emergence in pigs in the Philippines raised concerns about food safety, pathogenicity, and zoonotic potential, questions that are still unanswered. Until today, the virulence of RESTV for pigs has remained elusive, with unclear pathogenicity in naturally infected animals and only one experimental study demonstrating susceptibility and evidence for shedding but no disease. Here we show that combined oropharyngeal and nasal infection of young (3- to 7-wk-old) Yorkshire cross pigs with RESTV resulted in severe respiratory disease, with most animals reaching humane endpoint within a week. RESTV-infected pigs developed severe cyanosis, tachypnea, and acute interstitial pneumonia, with RESTV shedding from oronasal mucosal membranes. Our studies indicate that RESTV should be considered a livestock pathogen with zoonotic potential.

Reston virus (RESTV) was discovered in 1989/1990 in macaques imported into the United States from the Philippines for research purposes (1). Since then, there have been several episodes of disease caused by RESTV in macaques and rare asymptomatic infections in humans (2, 3). Unexpectedly, in 2008, RESTV emerged in pigs in the Philippines, and, shortly thereafter, RESTV sequences were detected in Chinese swine, raising zoonotic and food safety concerns (4, 5). RESTV constitutes a separate species in the genus Ebolavirus, family Filoviridae, and is generally thought of as the human apathogenic filovirus (6). Aside from humans (2, 3), RESTV has been shown to naturally and experimentally infect macaques, swine, ferrets, bats, and several rodent species (4, 5, 7–13). Upon experimental infection, macaques and ferrets, as well as immunocompromised rodents, such as STAT-1 knockout mice, develop severe disease with lethal outcome, whereas immunocompetent rodents generally do not (9–12). Whether RESTV itself causes disease in naturally infected domestic pigs remains unknown, since the RESTV-infected pigs from the Philippines were coinfected with the virulent arterivirus porcine reproductive and respiratory syndrome virus (PRRSV; now Betaarterivirus suid 1). In an initial experimental study, domestic pigs infected with RESTV only exhibited subclinical infections with evidence for virus shedding (7). We studied RESTV infection in young (3- to 7-wk-old) Yorkshire cross pigs, a swine breed used frequently in commercial pig production systems around the world. The main objective was to determine an age-dependent susceptibility to infection.

Results

RESTV Infection of Young Pigs Causes Severe Respiratory Disease.

Pigs aged 3, 5, or 7 wk were divided into predetermined groupings of four animals (one 7-wk-old group had only three animals) for necropsy at 3 d postinfection (dpi), at 6 dpi, or for survival analyses. For the 7-wk-old animals, we had no survival group, due to weight restrictions for pigs in high containment. Animals were infected by a combination of the oropharyngeal and nasal route with 1 × 10⁵ TCID₅₀ (50% tissue culture infectious dose) of RESTV, strain Reston 08-A, the strain isolated from a Philippine pig in 2008 (4). Daily scoring of animals revealed signs of disease after RESTV infection as early as 3 dpi (Fig. 1A), beginning with anorexia and somnolence, followed by hunched posture and piloerection. By 6 dpi, all infected animals showed respiratory distress including tachypnea or dyspnea with abdominal pumping and central cyanosis, particularly evident on the snout. Serous nasal discharge and productive cough were noted in some animals. Pigs near endpoint noticeably changed positions to improve ventilation and were often lethargic and 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 survivor groups were euthanized for significant breathing difficulties according to humane endpoint criteria (Fig. 1B). Piglets surviving the acute stage quickly recovered to normal activity levels.

Fig. 1.

Clinical parameters for RESTV-infected pigs. Pigs were infected by the oropharyngeal and nasal route with 1 × 10⁵ 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 euthanasia 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 significance 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.

Radiographic changes in RESTV-infected animals were consistent with the observed clinical signs; mock-infected animals did not show any changes (Fig. 1C). In all pigs, mild interstitial pulmonary infiltrations with variable lobar distribution were observed by 3 dpi, and, by 6 dpi, pulmonary infiltrations were evident in all lobes. Changes ranged from moderate interstitial infiltrates with cardiac border effacement to a severe mixed interstitial alveolar pattern with air bronchograms and consolidation. Animals that survived acute infection tended to have lower radiographic scores than those which succumbed to infection.

Young Pigs Show Evidence for Systemic RESTV Infection and Shedding.

Hematology and serum chemistry were not statistically different between mock- and RESTV-infected animals (SI Appendix, Fig. S1). Several animals had measurable RESTV-specific IgM responses by 6 dpi, although only the surviving animals developed RESTV-specific IgG responses (SI Appendix, Table S1). RESTV RNA was detectable in the blood of some animals as early as 3 dpi 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 blood only in some piglets (3-wk-old: 7/8, 5-wk-old: 5/8). Similarly, RESTV RNA in oral, nasal, and rectal swabs was discernible at low levels from some or all animals at various time points, but only oral swabs yielded infectious RESTV (3-wk-old: 5/12 at 3 dpi and 3/8 at 5 dpi; 5-wk-old: 1/12 at 3 dpi and 1/8 at 5 dpi) (SI Appendix, Table S1).

RESTV-Infected Young Pigs Develop Marked Interstitial Pneumonia.

Mock-infected animals did not show any gross pathologic changes at necropsy. Gross pathology lesions in RESTV-infected pigs were confined to the lungs (firm and edematous) (Fig. 2A) and mediastinal lymph nodes (enlarged and edematous). By 3 dpi, lungs exhibited multifocal to focally extensive consolidation with dark red discoloration consistent with mild to moderate interstitial pneumonia. By 6 dpi, lungs generally failed to collapse and showed diffuse consolidation and dark red discoloration consistent with marked interstitial pneumonia. By 3 dpi, postmortem lung−body weight ratios of infected animals were not statistically higher than those of controls (Fig. 2B). However, animals euthanized during acute respiratory disease demonstrated a significant increase in lung weights. Survivors, euthanized without overt clinical signs, still had lung−body weight ratios significantly higher than controls. Virus titration determined high viral loads in the lungs and draining lymph nodes of RESTV-infected piglets, with lower titers in the liver and spleen (SI Appendix, Fig. S2). Viral titers in the lungs ranged from 10⁴ to 10⁹ TCID₅₀ per g of tissue, with no notable difference between age groups (Fig. 2C). While the two 3-wk-old survivors had cleared infectious virus from the lungs by 16 dpi, the single 5-wk-old survivor still had quantifiable virus at 13 dpi.

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 lymph node (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.

By 3 dpi, histopathologic evaluation showed mild to moderate pneumonia characterized by thickening of alveolar septae by edema, fibrin, and mild to moderate numbers of macrophages and neutrophils. Alveoli contained moderate to abundant numbers of alveolar macrophages, fewer neutrophils, and rare necrotic debris (Fig. 3). At 6 dpi, lungs were more diffusely affected and showed moderate to severe interstitial pneumonia with more pronounced characteristics. Inflammation often surrounded bronchioles and pulmonary vasculature. Type II pneumocyte hyperplasia was prominent and associated with numerous eosinophilic intracytoplasmic inclusions located within alveolar macrophages (Fig. 3). Also noted were occasional alveolar multinucleated cells as previously reported in pigs experimentally infected with the related Ebola virus (EBOV) (Fig. 3) (14). The three surviving animals necropsied on 13 dpi or 16 dpi showed pulmonary pathology ranging from mild to severe interstitial pneumonia within various lung lobes. By 6 dpi, mediastinal lymph nodes contained moderate numbers of draining neutrophils and necrotic debris. RESTV nucleoprotein was detected by immunohistochemistry predominately in alveolar macrophages on 3 dpi and 6 dpi (Fig. 3). Alveolar macrophages were the only leukocytes in the lungs to stain with RESTV-specific antibodies (Fig. 3), similar to previous reports from RESTV- and EBOV-infected pigs (7, 14). Rare immunoreactivity was also identified in type II pneumocytes, bronchiolar epithelial cells, and endothelial cells (Fig. 3). Virus particles were detectable in the lung tissue by transmission electron microscopy (Fig. 4). Type II pneumocyte hyperplasia was associated with 1- to 3-μm intracytoplasmic inclusions in alveolar macrophages that were identified by transmission electron microscopy as membrane-bound enlarged lysosomes (Fig. 4).

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 interstitial pneumonia with exudate (arrow), type II pneumocyte hyperplasia (arrow head), and edema (asterisk); IHC 100×, Inset 400×—strong immunoreactivity in alveolar macrophages.

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 macrophage containing membrane-bound lysosomes (asterisk) and virus particles (arrowhead). (Scale bar = 1 μm.) (B) Endothelial cells surrounding a red blood cell (asterisk), with virus particles in the vascular and subendothelial space (arrowhead). (Scale bar = 600 nm.) (C) Alveolar macrophage with cytoplasmic viral replication 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.)

Discussion

Here we report that RESTV infection of piglets aged 3 wk to 7 wk resulted in severe respiratory disease, which is in contrast to the only previous experimental RESTV pig study that reported subclinical infection with virus shedding (7). Despite the obvious differences in clinical outcome, infection parameters such as organ tropism, pathology, and pathophysiology were, in general, similar in both studies but much more pronounced in the animals described here. The difference in infection outcome was not related to age of the pigs but could be related to the pig breed or the status of comorbidities such as infections with unrelated respiratory or nonrespiratory pathogens as discussed previously (7). We tested our pigs for the presence of PRRSV, porcine circovirus (PCV) types 2 and 3, and other common swine respiratory pathogens (see Methods) and did not find any evidence for coinfection, with the exception of a single animal that was PCR positive for PCV-2 (SI Appendix, Table S1). Future studies need to more deeply investigate the impact of coinfections and pig genetics on susceptibility to RESTV infections.

Both studies used the oropharyngeal/oronasal route of infection likely mimicking best natural exposure of pigs to RESTV-contaminated excretions or secretions from the postulated fruit bat reservoir (3, 13). However, the initial study (7) used a 10-fold higher inoculation dose (10⁶ TCID₅₀), still not producing disease in pigs. Virus load in secretions and excretions from EBOV-infected bats are unknown, but, for Marburg virus, they are estimated to be <10⁴ plaque-forming units per mL (15, 16), which is closer to what we used in this study. Notably, studies with EBOV in mice have resulted in disease attenuation when higher infectious doses were used. The authors (17) speculated that high viral loads may mediate stronger innate immune responses, leading to disease attenuation and survival. Further studies are needed to confirm this phenomenon.

The virus strain used for infections in both pig studies was derived from the same original source in the Philippines, but in-house tissue culture passaging could have led to genetic changes resulting in attenuation. This was shown for Sin nombre orthohantavirus, where a single tissue culture passage did attenuate the virus for rhesus macaques (18). Both RESTV seed stocks were prepared by a single passage, and future sequence comparison, once information becomes available from the previous study (7), should clarify any issues.

The role of pigs as an interim or amplifying host for ebolaviruses has been discussed and remains a matter of concern (19). Pigs are experimentally susceptible to mucosal exposure of EBOV resulting in acute respiratory disease (20, 21). EBOV shedding from the oronasal mucosa resulted in transmission to naïve pigs and macaques, indicating the potential for transmission (20, 21). Recently, pigs in Sierra Leone have been tested antibody positive for ebolaviruses (22). All of this may favor a role of pigs in the ecology of filoviruses, but further investigations need to confirm this hypothesis.

RESTV is considered a human apathogenic filovirus but is still classified as a biosafety level 4 pathogen in many countries and a select agent in the United States (23). In a few human cases, evidence of RESTV infection has been documented by seroconversion and detectable viremia (2, 3). This is in strong contrast to the other highly pathogenic ebolaviruses (6). The emergence of RESTV in pigs is a wake-up call, as transmission into humans through direct pig contact or the food chain is a possibility. As RESTV can infect humans (3), replication could result in changes in virulence, of which adaptation would be most worrisome for public health. Adaptation as a result of multiple transmission chains in humans has been controversially discussed during the West African Ebola outbreak (24–26).

Finally, current data support the notion that RESTV should be considered a livestock pathogen with unknown zoonotic potential. There does not seem to be an immediate threat of RESTV introduction into other countries, including the United States, but the RESTV situation in pigs in the Philippines and other Southeast Asian countries should be closely monitored. Regulations for import/export of life pigs and pork products should likely consider RESTV as a potential transboundary animal disease.

Methods

Biosafety and Animal Ethics.

All infectious work with RESTV and sample inactivation was performed in the maximum containment laboratory in accordance with standard operating procedures approved by the Rocky Mountain Laboratories (RML) Institutional Biosafety Committee (IBC), Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH. All animal work was performed in strict accordance with the 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 Department of Agriculture in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited facility. Animals were group housed in cages that enabled social interaction, under controlled conditions of humidity, temperature, and light (12-h light/12-h dark cycles). Food and water were available ad libitum. Animals were monitored at least twice daily and fed commercial pig chow by trained personnel. Environmental enrichment consisted of manipulanda and audio enrichment. Humane endpoints specified and approved by the Institutional Animal Care and Use Committee (IACUC) were applied to determine when animals should be euthanized.

RESTV Virus Stock.

RESTV, strain 08-A, was isolated from a Philippine pig in 2008 (4) and kindly provided by the Viral Special Pathogens Branch of the Centers for Disease Control and Prevention. The virus was propagated in 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 for clarification, aliquoted, and frozen in liquid nitrogen with 10% FBS. Viral stocks were diluted to challenge dose in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich). The stock was analyzed by next-generation sequencing (NGS), resulting in no mutation to the original GenBank entry (MT796851); contaminations were not detected.

Animal Studies.

Commercially available Yorkshire cross piglets (male and female) were weaned and shipped at ∼2 wk of age. Pigs were group housed 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), four late pathology (6 dpi), and four survival. For 7-wk-old pigs, animals were grouped as follows: three early pathology (3 dpi) (one animal had to be euthanized for unrelated medical conditions before study start) and four late pathology (6 dpi); there was no survival group, due to animal weight restrictions in maximum containment at RML. Animals were challenged in dorsal recumbency with either 1 × 10⁵ TCID₅₀ RESTV 08 or DMEM (mock-infected) by nasal (1 mL per nare) and oropharyngeal (5 mL) inoculation. The challenge dose was confirmed by back-titration of the inoculum on Vero cells. Clinical examinations including blood collection, radiographs (ventrodorsal, right and left laterals), and mucosal swabs were conducted on predetermined days (0, 1, 3, 5, 7, and subsequent) and at terminal end points defined and approved by the IACUC based on a previous publication (28). Radiographs were scored using a published scoring matrix adapted to pigs. Animals were euthanized either at predetermined time points (day 3 and day 6) or at study endpoint, which was day 13 and day 16 for the 5-wk-old and 3-wk-old groups, respectively. Full necropsies were performed for gross pathology evaluation, and tissue was harvested for histopathology and virology. Lung tissues from animals tested negative by PCR for PRRSV, influenza A virus, Mycoplasma spp. and bacterial ribosomal RNA. Serum samples from all animals were PCR negative for PCV-3. A single animal in the 5-wk-old group was PCR positive for PCV-2 (SI Appendix, Table S1).

Hematology and Serum Biochemistry Analyses.

Hematology was completed on a Procyte DX (IDEXX Laboratories), and the following parameters were evaluated: red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution weight, platelets, mean platelet volume, white blood cells, neutrophil count (abs and percent), lymphocyte count (abs and percent), monocyte count (abs and percent), eosinophil count (abs and percent), and basophil count (abs and percent). Serum chemistries were completed on a Vetscan VS2 Chemistry Analyzer (Abaxis), and the following parameters were evaluated: glucose, blood urea nitrogen, creatinine, calcium, albumin, total protein, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, globulin, sodium, potassium, chloride, and total carbon dioxide.

Antibody Detection.

RESTV-specific IgM and IgG were measured by enzyme-linked immunosorbent assay. Recombinant RESTV glycoprotein lacking the transmembrane region (rRESTV GPdTM) (IBT Bioservices) was diluted in PBS and then adsorbed to Nunc Maxisorp plates at 0.05 μg/mL. Plates were washed with 5% skim milk in PBS + 0.05% Tween-20 and blocked with the same. Serial fourfold dilutions of serum in blocking buffer (5% skim milk in PBS + 0.05% Tween-20) beginning with 1:100 were then applied to plates. Plates were washed with PBS + 0.05% Tween-20, and bound antibody was detected with either peroxidase-labeled rabbit anti-pig IgG (1:1,000 dilution) (Invitrogen) or goat anti-pig IgM (1:5,000) (Bio-Rad Laboratories). Absorbance at 405 nm was measured.

Nucleic Acid Detection.

Total RNA was extracted from blood, swabs, and urine samples using QIAamp Viral RNA extraction kits or from tissue samples using RNeasy extraction kits (Qiagen) [IBC-approved and published protocol (29)] and tested for the presence of RESTV RNA by real-time one-step qRT-PCR run on a Rotor-Gene RG-3000 instrument using a QuantiFast Probe RT-PCR +ROX Vial Kit (Qiagen) with primers specific to RESTV (forward 5′-CCC​TTT​GGC​CCG​AAC​AG and reverse 5′-GGG​CGG​CCT​TAA​ATG​CAT; 0.25 µM final concentration; IDT) and a dual-labeled fluorescent probe (6FAM-CAAAGTGCGTAAT+GA+G+G-BBQ; 0.125μM final concentration; TIB Molbiol). RT-PCR was carried out in three stages: reverse transcription (50 °C for 10 min), Taq activation (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 standard curve of RESTV 2008 RNA extracted from viral stock with predetermined titer and calculated as infectivity equivalents (TCID₅₀/mL or TCID₅₀/gram).

Virus Isolation.

Isolation was conducted on Vero cells plated for 80% confluency in 48-well plates. Medium was removed from cells and replaced with 1:100 and 1:1,000 dilutions of blood or swab medium in 0.1 mL DMEM, with an infection time of 45 min. Following inoculation, DMEM with 2.5% FBS, penicillin/streptomycin, and l-glutamine was added for a total volume of 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 10 min, diluted 1:10² to 1:10⁸, and used to infect Vero cells as above. Cell cultures were read for cytopathic effect at day 14 for evidence of virus.

Histopathology.

Histopathology was performed on pig tissues. Following fixation and inactivation of tissues (<1 cm³) for a minimum of 7 d in 10% neutral-buffered formalin and one formalin exchange [IBC-approved and published protocol (29)], tissues were removed from BSL4 and processed using a VIP-6 Tissue Tek (Sakura Finetek) tissue processor and embedded in Ultraffin paraffin polymer (Cancer Diagnostics). Samples were sectioned at 5 μm, and resulting slides were stained with hematoxylin and eosin or used for immunohistochemistry. Specific immunoreactivity was detected using polyclonal rabbit serum against RESTV nucleoprotein diluted 1:250 (kindly provided by Ayato Takada, Hokkaido University, Hokkaido, Japan), followed by a Biogenex biotinylated anti-rabbit antibody (SS Rabbit Link Biogenex) and the 3,3'-Diaminobenzidine (DAB) chromogen (Discovery DABMap detection kit; Roche Tissue Diagnostics).

Transmission Electron Microscopy.

Small segments of lung tissue were fixed for 7 d in Karnovsky’s fixative [IBC-approved and published protocol (29)]. Specimens were fixed overnight at 4 °C with 2.5% glutaraldehyde/4% paraformaldehyde in 0.1 M Sorensen’s buffer, pH 7.4. Samples were postfixed for 1 h with 1.0% osmium tetroxide/0.8% potassium ferricyanide, 1 h 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 4 °C, dehydrated with a graded ethanol series, and embedded in epon/araldite resin. Thin sections were cut with a Leica UC6 ultramicrotome (Leica), and viewed at 120 kV on an FEI BT Tecnai transmission electron microscope (Thermofisher/FEI).

Statistical Analysis.

Data were analyzed with one-way analysis of variance and a Dunnett’s multiple comparison test comparing differences between RESTV-infected age groups and mock-infected controls. Data were not analyzed past 7 dpi, as overall group numbers were greatly reduced. Lung to weight ratios were analyzed with an unpaired t test with two-tailed P value to compare values between RESTV-infected and mock-infected groups. This study was not specifically designed for statistical evaluation, as group numbers were small, and any animal loss therefore negatively impacts statistical testing.

Data Availability.

All study data are included in the article and SI Appendix.