The hPPMV-1/Netherlands/579/2003 isolate was obtained from an adult female patient from the Netherlands with multiple myeloma, who died from respiratory failure after receiving allogenic bone marrow transplantation and immunosuppressive treatment  (unpublished observations). The virus isolate was passaged twice on human rhabdomyosarcoma cells and once on Vero cells. Prior to inoculation, the virus isolate was cultured in 10-day-old specific-pathogen-free embryonated chicken eggs using standard techniques, and harvested at 2 dpi. The virus stock was titrated by end-point dilution assay in Vero clone 118 cells. To read out infection, cells were stained with chicken polyclonal anti-APMV-1 antibody and rabbit FITC-labeled anti-chicken IgG antibody (1:2000 and 1:1000 dilution respectively; both from Abcam, Cambridge, UK) 72 h after inoculation. Viral titers were calculated using the method of Reed and Muench . The infectious virus titer of this stock was 5 × 108 median tissue culture infective dose (TCID50) per mL.
Three male cynomolgus macaques, 3.5 years old, were colony bred and had been maintained in group housing, where they were screened annually—and tested negative—for the following infections: simian virus 40, polyomavirus, Mycobacterium tuberculosis, measles virus, mumps virus, simian immunodeficiency virus, simian retrovirus type D, and simian T cell leukemia virus. Prior to infection, they were examined clinically and determined as healthy by a registered veterinarian. One month before the start of the experiment, a Data Storage Tag centi-Temperature probe (Star-Oddi, Brussels, Belgium) was implanted intraperitoneally, set to register temperature every 10 min. One week before inoculation, the macaques were placed together in a negatively pressurized, HEPA-filtered isolator cage. They were provided with commercial food pellets and water ad libitum. The macaques were inoculated with 1.0 × 108 TCID50 of hPPMV-1/Netherlands/579/2003, which was suspended in 4.5 mL of phosphate-buffered saline (PBS). Approximately 3 mL was applied intratracheally by use of a catheter, 0.5 mL in each nare, and 0.25 mL on each of the conjunctivae. The macaques were observed daily for the occurrence of malaise, coughing, exudate from the eyes or nose, forced respiration, and any other signs of illness. The macaques were euthanized at 3 dpi by exsanguination under ketamine and medetomidine hydrochloride anesthesia. Just before infection and daily until euthanasia, the macaques were anesthetized with ketamine and medetomidine hydrochloride, and pharyngeal, nasal, ocular, and rectal swabs were collected in 1 mL transport medium  and stored at −70 °C until quantitative RT-PCR (qRT-PCR) and/or virus isolation. In addition, 4 mL blood was taken from an inguinal vein at each of these time points and collected in Vacuette Z Serum Sep Clot Activator tubes (Greiner Bio One, Alphen aan den Rijn, The Netherlands).
Clotted blood samples were centrifuged and 100 µL separated serum was assayed with Piccolo BioChemistry Panel Plus Reagent Discs (Abaxis, Darmstadt, Germany), which were processed using a Piccolo Xpress chemistry analyzer (Abaxis) following the manufacturer’s instructions. Measurements were obtained for glucose, blood urea nitrogen, creatinine, calcium, albumin, total protein, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase, amylase and C-reactive protein. Reference values (if indicated) were obtained from the supplemental data of a publication by Xie et al. .
Autopsy and tissue sampling
Autopsies were carried out according to a standard protocol. Organs were examined for lesions and sampled for laboratory analyses. For histopathology and immunohistochemistry, samples of adrenal gland, brain stem, cerebellum, cerebrum, conjunctiva, ethmoid bone, eye, eyelid, heart (left and right ventricle), kidney, large intestine, lung (left and right; upper, middle and lower lobes), liver, mesenteric lymph nodes, nasal conchae, pancreas, small intestine, spleen, tonsil and tracheo-bronchial lymph nodes were collected in 10% neutral-buffered formalin (lungs after inflation with formalin) and allowed to fix for 1 week. For qRT-PCR, broncho-alveolar lavage (BAL) was performed by direct infusion of PBS into the right main bronchus. Recovered BAL fluid was centrifuged and the cellular pellet resuspended in TRIzol and stored at −80 °C until further analysis. In addition, samples of adrenal gland, brain, cerebrospinal fluid, conjunctivae, heart, kidney, mesenteric lymph nodes, lung (right upper and lower lobes), large intestine, liver, nasal conchae, nasal septum, pancreas, primary bronchus, small intestine, spleen, trachea, and tracheo-bronchial lymph nodes were collected in RNAlater (Life Technologies, Bleiswijk, The Netherlands) and stored at −80 °C until further analysis. For virus isolation, samples from the same tissues as for qRT-PCR were stored without additives at −80 °C until further analysis.
Samples for histopathological analysis were embedded in paraffin, sectioned at 3 µm, and stained with hematoxylin and eosin (H&E) for examination by light microscopy. Tissue sections of a clinically healthy juvenile cynomolgus macaque that had not been infected with PPMV-1 were used as a negative control.
Formalin-fixed, paraffin-embedded, 3-µm-thick sections of the same tissues examined histopathologically were stained using an immunoperoxidase method. Tissue sections were mounted on coated slides (Klinipath, Duiven, The Netherlands), deparaffinized and rehydrated. Endogenous peroxidase was blocked by incubating sections in 3% H2O2 in PBS for 10 min at room temperature (RT). Antigen was retrieved by Tris–EDTA buffer (pH 9) for 15 min. Sections were subsequently washed with PBS containing 0.05% Tween 20 (Fluka, Chemie AG, Buchs, Switzerland) and incubated in PBS with 0.1% BSA (Aurion, Wageningen, The Netherlands) for 10 min at RT. After this, sections were incubated in PBS with 0.1% BSA with a monoclonal mouse antibody IgG2a to APMV-1 (dilution 1:100, MAb 6H12, specific to ribonucleoprotein; La Sota strain, Hytest Ltd, Turku, Finland) or with a negative control isotype mouse monoclonal antibody (dilution 1:100, MAb 003, R&D System, Minneapolis, USA) for 1 h at RT. After washing, sections were incubated with goat anti-mouse antibody (dilution 1:400, Southern Biotech, Birmingham, AL, USA) labeled with horseradish peroxidase (HRP) for 1 h at RT. HRP activity was revealed by incubating the sections in 3-amino-9-ethylcarbazole (Sigma Chemical Co., St. Louis, USA) in N,N-dimethylformamide (Sigma Chemical Co.) solution for 10 min at RT, resulting in a red precipitate. Sections were counterstained with hematoxylin. Brain tissue sections from a cormorant (Phalacrocorax auritus) known to be infected with APMV-1 were used as a positive control. Tissue sections of a clinically healthy juvenile cynomolgus macaque that had not been infected with PPMV-1 were used as a negative control.
RNA isolation and qRT-PCR assay
BAL samples stored in TRIzol were processed according to the manufacturer’s instructions to isolate RNA. Tissue samples stored in RNAlater were weighed, thawed, transferred to tubes containing a quarter-inch-diameter ceramic sphere in virus transport medium, and homogenized using a FastPrep 24 tissue homogenizer (MP Biomedicals, Eindhoven, The Netherlands). The homogenates were centrifuged, and the cleared supernatants were used for RNA isolation. 200 µL of the cleared supernatant of tissue samples, as well as 200 µL of other samples (transport medium of swabs, plasma) were combined with 300 µL lysis buffer of the Total Nucleic Acid Isolation kit (Roche, Woerden, The Netherlands) and RNA was isolated in a volume of 50 µL using a MagNA Pure LC machine (Roche) following the manufacturer’s instructions.
NDV-specific qRT-PCR was performed on 5 µL (TRIzol samples) or 19.5 µL (MagNA Pure samples) RNA in an ABI PRISM 7000 Sequence Detection System using TaqMan Fast Virus 1-Step Master Mix (both from Life Technologies) in a total volume of 30 µL, using primers as described by Wise et al. . The RT step was 5 min at 50 °C, followed by 95 °C for 20 s. Cycling consisted of 45 cycles of 3 s denaturation at 95 °C, 5 s annealing at 54 °C and 31 s extension at 60 °C.
200 µL transport medium from collected swabs or 200 µL supernatant from homogenized tissue samples was injected in duplicate into 10-day-old specific-pathogen-free embryonated chicken eggs. After 2 days, allantoic fluid was harvested and tested for presence of virus by hemagglutination assay.