Plasmid design and construction
RNA was isolated from NiVB virus stocks (NiVB #200401066 was obtained from a patient from the 2004 outbreak in Bangladesh (kindly provided by Dr. Thomas Ksiazek) using approved protocols at biosafety level 4 (BSL-4) in the Galveston National Laboratory (GNL) at the University of Texas Medical Branch (UTMB). The RNA was used to make cDNA with gene specific primers and the genes were amplified using gene specific primers (NiVB F or G) containing MluI and NheI restriction sites at the 5′ and 3′ ends respectively. The PCR amplified products and pΔG-VSV-2.6 plasmid were restriction digested with MluI and NheI and gel purified. Purified products and vector were ligated and positive colonies were screened and sequenced for positive constructs.
rVSV vaccine vectors and challenge virus
The rVSV NiVB vaccines (rVSV-ΔG-NiVB/F-GFP and rVSV-ΔG-NiVB/G-GFP) and rVSV-ΔG-GFP were recovered using methods previously described . The rVSVΔG viruses were propagated on BHK-21 cells transfected with 2 μg of pCAGGS-GInd expressing the VSV glycoprotein (GInd) and titered as previously described . Viruses complemented with VSV GInd are denoted as GInd* rVSV-ΔG-GFP, G*rVSV-ΔG-NiVB/F-GFP, and G* rVSV-ΔG-NiVB/G-GFP. To make the vaccine in Group 4 (Figure 1A), Vero cells were co-infected with G*rVSV-ΔG-NiVB/F-GFP and G* rVSV-ΔG-NiVB/G-GFP at MOI 5 for each virus. Supernatants were collected 24 h.p.i. and titered on BHK-21 cells complemented with VSV GInd.
NiVM #1999011924 was obtained from a patient from the 1999 outbreak in Malaysia (kindly provided by Dr. Thomas Ksiazek). NiVM was chosen for challenge in ferrets based on our lethality data with this particular stock at the time of the study. The virus was propagated on Vero E6 cells in Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal calf serum. The NiVM challenge virus stock was assessed for the presence of endotoxin using The Endosafe®-Portable Test System (PTS) (Charles River, Wilmington, MA). Virus preparations were diluted 1:10 in Limulus Amebocyte Lysate (LAL) Reagent Water (LRW) per manufacturer’s directions and endotoxin levels were tested in LAL Endosafe®-PTS cartridges as directed by the manufacturer. Each preparation was found to be below detectable limits while positive controls showed that the tests were valid.
Conducting animal studies in BSL-4 severely restricts the number of animal subjects, the volume of biological samples that can be obtained and the ability to repeat assays independently and thus limit statistical analysis. Consequently, data are presented as the mean calculated from replicate samples, not replicate assays, and error bars represent the standard deviation across replicates.
Animal studies were performed in BSL-4 biocontainment at the GNL at the UTMB at Galveston and were approved by the UTMB Institutional Animal Care and Use Committee (IACUC). Animal research was conducted in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to the principles stated in the eighth edition of the Guide for the Care and Use of Laboratory Animals, National Research Council, 2013. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
Twenty female ferrets weighing 0.75-1 kg were housed in groups of 3 and 2 animals per vaccine group. Before vaccination, subjects were anesthetized by i.m. injection with ketamine-acepromazine-xylazine (KAX) cocktail and had transponder chips (BioMedic Data Systems, Seaford, DE) implanted subcutaneously for animal identification and temperature monitoring. For procedures, animals were anesthetized with KAX and vaccinated with ~ 1 × 107 PFU by i.m. injection on day -28 (Figure 2A). Animals were inoculated intranasally (i.n.) with ~ 5 × 103 pfu of NiVM in 1 ml of Dulbecco’s minimal essential medium (DMEM) (Sigma-Aldrich, St Louis, MO) 28 days after vaccination (Figure 2A, *). Animals were anesthetized for clinical examination including temperature, respiration quality, blood collection, and on days 0, 6, and 21 p.c. Before and after challenge animals were assessed daily for weight, temperature, and scored on a scale of 0 of 9 for clinical observations based on coat appearance, social behavior, and provoked behavior; animals scoring 7 or greater were euthanized per IACUC protocol. Subjects in the vaccine cohorts were euthanized at the study endpoint on day 22 p.c. whereas the subjects in Group 1 had to be euthanized according to approved humane end points on day 7 or 8 p.c. All other subjects survived until the end of the study.
Measurement of serum or plasma NiV F and G specific antibodies
Ferret serum collected at indicated time points was tested for IgG antibodies against NiV F and G using previously developed multiplexed microsphere assays . 96-well filter plates were primed with PBS. Test sera were diluted in PBS at 1:10 for pre vaccination time points and 1:10,000 for time points after vaccination. Biotinylated goat anti-ferret IgG and streptavidin-phycoerythrin (strep-PE) were also diluted in PBS. Coupled microspheres (sG-HeV, sG-NiV, sF-Hev, sF-NiV) were prepared by sonication for 1 minute followed by vortex mixing for 1 minute each and then diluted in PBS. Priming liquid was removed from plates using a Bio-Plex Pro II Wash Station (Bio-Rad, Hercules, CA) and 100 μL containing 1500 of each coupled microsphere was added to each well. The microsphere mixture was removed by vacuum, 100 μL of diluted test sera was added to appropriate wells and incubated at room temperature (RT) for 30 minutes while shaking in the dark. Diluted test samples were removed by vacuum and 100 μL of diluted biotinylated goat anti-ferret (1:500) (Pierce, ThermoScientific, Rockford, IL) was added to each well and incubated as previously described above. Liquid was removed by vacuum and 100 μL of strep-PE (1:1000) (Qiagen, Valencia, CA) was added to each well and again incubated for 30 minutes. All liquid was removed from plates with a vacuum manifold and washed twice with 300 μL PBS, removing liquid between wash steps. Finally, 125 μL of PBS was added to each well and incubated for 2 minutes as described above. Samples were assayed for mean fluorescence intensity (MFI) across at least a 100 bead region performed on the BioPlex-200 machine and analyzed using Bio-Plex Manager Software (v 6.1) (Bio-Rad). MFI and the standard deviation (s.d.) of fluorescence intensity across 100 beads were determined for each sample and plotted.
NiVM serum neutralization assays
PRNTs were determined using a conventional serum neutralization assay. Briefly, sera were serially diluted twofold, and incubated with ~ 100 pfu of NiV for 1 hour at 37°C. Virus and antibodies were then added to individual wells of 6-well plates of confluent Vero cell monolayers. Plates were stained with neutral red 2 days after infection and plaques were counted 24 hours after staining. The 50% neutralization titer (PRNT50) was determined as the serum dilution at which there was a 50% reduction in plaque counts versus control wells.
Specimen collection and processing in NiV-infected ferrets
Blood was collected and placed in MiniCollect EDTA tubes or serum tubes (Greiner Bio One, Monroe, NC). Immediately following sampling, 100 μl of blood was added to 600 μl of AVL viral lysis buffer (Qiagen) for RNA extraction. For tissues, approximately 100 mg was stored in 1 ml RNAlater (Qiagen) for 7 days to stabilize RNA. RNAlater was completely removed, and tissues were homogenized in 600 μl RLT buffer (Qiagen) in a 2-ml cryovial using a tissue lyser (Qiagen) and stainless steel beads. The tissues sampled included right lung upper lobe, right lung middle lobe, right lung lower lobe, left lung upper lobe, left lung middle lobe, left lung lower lobe, liver, spleen, kidney, adrenal gland, pancreas, and brain (frontal cortex). All blood samples were inactivated in AVL viral lysis buffer, and tissue samples were homogenized and inactivated in RLT buffer prior to removal from the BSL-4 laboratory. Subsequently, RNA was isolated from blood and swabs using the QIAamp viral RNA kit (Qiagen) and from tissues using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions supplied with each kit.
Hematology and serum biochemistry
Prior to the study, baseline blood and sera were collected via the anterior vena cava from all 20 ferrets. On days -28, 0, 6, and 21 blood was collected from all animals. Complete blood counts of total white blood cell counts, white blood cell differentials, red blood cell counts, platelet counts, hematocrit values, total hemoglobin concentrations, mean cell volumes, mean corpuscular volumes, and mean corpuscular hemoglobin concentrations were analyzed from blood collected in MiniCollect EDTA tubes (Greiner Bio One) using a Hemavet HV950FS instrument per manufacturer’s instructions (Drew Scientific, Oxford, CT). Serum analysis of blood chemistries was performed using a VetScan classic analyzer and comprehensive diagnostic profile rotors measuring of albumin (ALB), amylase, alanine aminotransferase (ALT), alkaline phosphatase (ALP), calcium, glucose, total protein, total bilirubin (TBIL), blood urea nitrogen (BUN), creatinine (CRE), phosphorus, sodium, and total protein (Abaxis, Union City, CA). All blood and serum samples were processed and analyzed immediately after collection.
Histopathology and immunohistochemistry
Necropsy was performed on all subjects. Tissue samples of all major organs were collected for histopathologic and immunohistochemical examination and were immersion-fixed in 10% neutral buffered formalin for at least 21 days in BSL-4. Subsequently, formalin was changed; specimens were removed from BSL-4, processed in BSL-2 by conventional methods and embedded in paraffin and sectioned at 5 μm thickness. For immunohistochemistry, specific anti-NiV immunoreactivity was detected using an anti-NiV N protein rabbit primary antibody (kindly provided by Dr. Christopher Broder) at a 1:5000 dilution for 30 minutes. The tissue sections were processed for immunohistochemistry using the Dako Autostainer (Dako, Carpinteria, CA). Secondary antibody used was biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at 1:200 for 30 minutes followed by Dako LSAB2 streptavidin-HRP (Dako) for 15 minutes. Slides were developed with Dako DAB chromagen (Dako) for 5 minutes and counterstained with hematoxylin for one minute. Non-immune rabbit IgG was used as a negative staining control.
Detection of NiV load
RNA was isolated from blood or tissues and analyzed using primers/probe targeting the N gene and intergenic region between N and P of NiV for quantitative real-time PCR (qRT-PCR) with the probe used here being 6-carboxyfluorescein (6FAM)-5′ CGT CAC ACA TCA GCT CTG ACG A 3′-6 carboxytetramethylrhodamine (TAMRA) (Life Technologies, Carlsbad, CA). NiV RNA was detected using the CFX96 detection system (Bio-Rad) in One-step probe qRT-PCR kits (Qiagen) with the following cycle conditions: 50°C for 10 minutes, 95°C for 10 seconds, and 40 cycles of 95°C for 10 seconds and 59°C for 30 seconds. Threshold cycle (CT) values representing NiV genomes were analyzed with CFX Manager Software, and data are shown as genome equivalents (GEq). To create the GEq standard, RNA from NiV challenge stocks was extracted and the number of NiV genomes was calculated using Avogadro’s number and the molecular weight of the NiV genome. Virus titration was performed by plaque assay with Vero cells from all serum and control tissue samples. Briefly, increasing 10-fold dilutions of the samples were adsorbed to Vero cell monolayers in duplicate wells (200 μl); the limit of detection was 25 pfu/ml.