In-vitro cell growth and manipulations
As the infectivity of the viruses in this work was higher in a
Mustela vison (mink) lung (Mv1 Lu) cell line (validated at the Midwest Research Institute) than in the more commonly used Madin Darby canine kidney (MDCK) cell line used for influenza virus work (data to be presented elsewhere), Mv1 Lu cells were used to obtain viral titers. The Mv1 Lu cells were propagated in Modified Eagle's Medium with Earle's salts (EMEM) supplemented with L-Alanyl-L-Glutamine (GlutaMAX™, Invitrogen Corp., Carlsbad, CA), antibiotics [PSN; penicillin, streptomycin, neomycin (Invitrogen Corp.)], pyruvate (Invitrogen Corp.), non-essential amino acids (Invitrogen Corp.), and 10% (v/v) gamma-irradiated fetal bovine serum (HyClone, Pittsburgh, PA). The cells were negative by PCR for the presence of mycoplasma DNA using a Takara PCR Mycoplasma Detection kit (Takara Bio, USA, Thermo Fisher). Influenza viruses were grown in Mv1 Lu cells in serum-free EMEM otherwise supplemented as previously described plus L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated mycoplasma- and extraneous virus-free trypsin (Worthington Biochemical Company, Lakewood, NJ) in 5% CO
2 at 37°C (H5N1) or 35°C (seasonal viruses). The TPCK-trypsin used for this work had higher specific activity than TPCK-trypsin acquired elsewhere and therefore used at a final concentration 0.1 μg/ml. Virus preparations were harvested when cytopathic effects (CPE) typical for influenza viruses were ≥ 80% [
40]. The 50% tissue culture infectious dose (TCID
50) were calculated by the Reed-Muench method [
41].
Ferrets and their Pre-qualification for Studies
Studies were performed using descented, spayed 3-month-old female ferrets (0.5 - 0.9 kg) (Triple F Farms, Sayre, PA) that were housed individually in HEPA-filtered (inlet and exhaust) ventilated individual cages (Allentown, Inc., Allentown, NJ). The animals lacked signs of epizootic catarrhal enteritis, and were negative by microscopy for enteric protozoans such as
Eimeria and
Isospora species using fecasol, a sodium nitrate fecal flotation solution (EVSCO Pharmaceuticals, Buena, NJ). The ferrets were seronegative by a hemagglutination inhibition (HAI) assay [
43] to circulating influenza B viruses and H1N1, H3N2, and the H5N1 influenza A viruses. Prior to performance of the HAI assay, the ferret sera were treated overnight with receptor destroying enzyme (RDE) (Denka Seiken USA, Inc., Campbell, CA) at 37°C to inactivate non-specific HAI activity, then heated at 56°C for 60 minutes to inactivate remaining RDE activity and complement proteins.
Room conditions for all work included 12 hr. light cycles, and an average relative humidity at 30% within a room temperature range between 64°and 84°F (17.8°to 28.9°C). The animals were fed pelleted ferret food (Triple F Farms) and watered ad libitum, and housed and maintained under applicable laws and guidelines such as the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, National Academic Press, 1996) and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 89-544 and Subsequent Amendments), and with appropriate approvals from the Midwest Research Institute Animal Care and Use Committee. Body temperatures were measured twice daily via subcutaneously implantable programmable temperature transponders (model IPTT-300, Bio Medic Data Systems, Seaford, DE) implanted in the neck.
Intranasal inoculation studies
Procedures based on those of Zitzow
et al. [
22] were used. Briefly, twelve ferrets were used for each virus study: nine (n = 9) for virus infection, three (n = 3) for non-infected controls. Ferrets were anesthesized by intramuscular administration of ketamine HCl (25 mg/kg)-xylazine (2 mg/kg)-atropine (0.05 mg/kg), and instilled with selected doses of viruses in isotonic phosphate buffered saline (PBS) with 0.5 % purified bovine serum albumin (to stabilize the viruses) and antibiotics. Fifty μl of virus suspension was instilled into each nostril (100 μl of virus suspension/ferret). Two ferrets each were inoculated IN with 10
4, 10
3 and 10
2 TCID
50, and three ferrets each with 10
1 TCID
50 of virus (TCID
50 values determined in Mv1 Lu)
. A back-titration was performed on the virus doses to verify viral titers per dose. Three animals served as controls and received IN doses of a 1:30 dilution of sterile, non-inoculated chicken allantoic fluid in PBS. All the animals were caged individually and weighed once daily for the duration of the study. Body temperatures were recorded twice daily from conscious animals that were stimulated and active for at least five minutes (as there is a relatively large variance in the resting and active temperatures of ferrets). A temperature increase ≥1.4°C over baseline was considered significant; the baseline was the average temperature for the entire group over the pre-dose observation period.
Nasal washes and rectal swab specimens were collected at 3 and 5 days post-inoculation with virus. Clinical signs including sneezing (before anesthesia), inappetence, dyspnea, and level of activity were assessed daily for the duration of the study (8 - 10 days). Inappetence was judged through visual observation of the food remaining in the feeder and spilled within the surrounding area. A scoring system (relative inactivity index [RII]) based on that described by Reuman
et al. [
44] and as used by Govorkova
et al.[
19] and Zitzow
et al.[
22] was used to assess the activity level as follows: 0, alert and playful; 1, alert but playful only when stimulated;
2, alert but not playful when stimulated; and 3, neither alert nor playful when stimulated. They were also monitored daily for nasal and ocular discharge, neurological dysfunction, and semi-solid or liquid stools. Neurologic dysfunction was defined as development of motor dysfunction (including paralysis or posterior paresis), convulsions, ataxia, seizures, and depression. Ferrets with > 25% loss of body weight or with neurologic dysfunction were anesthetized by intramuscular administration of ketamine HCl (25 mg/kg)-xylazine (2 mg/kg)-atropine (0.05 mg/kg), then euthanized with Beuthanasia-D Special (sodium pentobarbital and phenytoin sodium) or equivalent (Euthasol) via the jugular vein.
Exposure system and generation of aerosols
A nose-only bioaerosol inhalation exposure system (NBIES) assembled in a Class III IsoGARD
® Glovebox (The Baker Company, Sanford, ME) in an ABSL3+ laboratory was used for this work [
36] (Figure
1). A nose-only system was chosen for this study over whole-body and other exposure routes because: (a) it minimizes infection by non-inhalation routes, (b) reduces requirements for post-exposure decontamination of animals (such as by wiping exterior of conscious animal with bleach), (c) minimizes potential contamination of animal housing areas, (d) lessens contamination risks for animal care personnel, and (e) permits testing at high virus concentrations while minimizing quantities of starting material. The latter consideration is important for cradle to grave work with select agents, wherein experiments are preferentially designed to utilize small amounts of agent.
Ferret restraint tubes with push rods (prototypes built for this work by CH Technologies, USA, Westwood, NJ) (Figure
2) were used along with a model 3314 Aerodynamic Particle Sizer
® (APS) Spectrometer (TSI Inc. St. Paul, MN). The APS is used to measure the aerosol size distribution in the test atmosphere and is operated with Aerosol Instrument Manager software, release version 8.0.0.0 (TSI, Inc.) run in a Dell Latitude D600 computer. A 3-jet BioAerosol Nebulizing Generator (BANG), (BGI Inc., Waltham, MA) was used (CH Technologies). The BANG is a low flow, low dead space nebulizer designed to operate in the range of 1 to 4 liters per minute with a pumped fluid (recirculated) flow that features minimal sample utilization. It was chosen over other nebulizers as the most appropriate generation device for the aerosolization of influenza virus; considerations included: minimal potential damage to agent, reduced clumping of virus, uniformity of droplet size distribution, and efficiency (the amount of virus that needs to be prepared is much smaller than that required by similar aerosol generators).
The exposure system contains sampling ports that are tapped for: (a) measurement of aerosol particle size, and (b), sample collection to assess live-agent aerosol concentration. Up to three animals were exposed per experiment [
36]. An exposure time of 10 minutes was used [
36]. Total flow through the inhalation system was 5 liters per min during the exposures created by the BANG. The metrics for using the BANG generation devices in association with the inhalation system was previously described [
36]. Exposure concentration expressed in TCID
50/ml was determined by sampling of the aerosol stream using two model 7531 midget impingers (AGI; Ace Glass Incorporated, Vineland, NJ) connected in series.
The dynamic air flow through the aerosol delivery ports on the system exceeds 3× the total ventilation volume of all animals exposed. Influenza virus is mixed with a non-toxic vehicle (sterile PBS solution with 0.5% purified BSA fraction V) to help maintain viability of the virus and act as a vehicle to generate the test aerosol. The saline solution is well characterized and its acute inhalation toxicity known; it does not cause an acute inflammatory response or stimulate excess mucus secretion leading to increased mucociliary clearance. Thus, the ferrets remain susceptible to challenge infection when the saline solution is inhaled in the quantities used in this work (J. Lednicky, unpublished). The exposure system is operated dynamically at negative pressure.
Prior to live-agent work, the aerosol system was characterized to assess individual parameters, including exposure port to port aerosol homogeneity, aerosol concentration ramp up, concentration stability and decline, sample measurement, exposure location to exposure location variation, and sampling system collection efficiencies [
36].
Calculations for aerosol transmission studies
The presented dose D (defined as the inhaled dose estimated from the multiplication of the aerosol concentration and the total volume of air breathed in by the animal) is estimated from the ferret respiratory rate and duration of aerosol exposure. By convention used in aerobiology, , where R refers to respiration rate, C refers to the concentration of aerosolized agent, f(t) = % of agent deposited in the lungs, and t
exp
= exposure duration time. When the following assumptions are made: a constant minute volume (Vm) for R(t), a constant live-agent aerosol concentration (integrated air sample determined concentration for C(t), 100% deposition for f(t), and t(exp) is fixed at the time of exposure, then: D = R × C × t
exp
.
The ferret respiratory minute volume (V
m), defined as the volume of air inhaled or exhaled over a minute, was estimated using Guyton's formula [
45], where BW = body weight in gr, and the volume calculated in ml:
Ferrets in this work ranged from about 500 to 900 gr. For a 500 gr ferret, log10BW3/4 = 0.75 × log10500 = 2.02. The antilog of 2.02 = 105.7; therefore, Vm = 2.10 × 105.7 = 222.0 ml/min (0.22 L/min). Similarly, for a 900 gr ferret, Vm = 345.1 ml/min.
Since most of the ferrets were close to 500 gr, an
approximate V
m value of 0.2 L/min was used for this work. The V
m value of 0.2 L/min used in this work was consistent with estimates obtained by multiplying the ferret tidal volume (V
t) expressed in ml × the breathing rate (BR) of conscious ferrets expressed as breaths/minute (bpm). By definition, V
t = the volume of air inspired or expired with each normal breath, whereas BR = number of breaths/minute (bpm) for a conscious ferret. For ferrets, V
t = 6.06 ± 0.30 ml, and BR = 33 - 36 bpm [
46,
47].
Using an average Vt value of 6.06 ml and an average BR of 34.5 bpm, Vm = 209.01 ml/min = 0.21 L/min.
Precision in calculations of aerosol concentrations and estimates of the number of viruses inhaled per experiment depend largely on the collection efficiency/efficacy of the impinger(s). Therefore, the impinger system must first be characterized to establish operational parameters determined to obtain the required
D. Systems similar to ours are often designed with a single impinger and are operated with the assumption that > 90% of the aerosolized microorganisms are entrained during sampling of the aerosol flow through the impinger. If the true efficiency is < 90%, a significant undercount of the aerosol concentration can result, and this causes both an underestimate of the inhaled dose and an overestimate of virulence (since the number of organisms to cause an infection is undercounted). Moreover, the collection fluid in the impinger must maintain the aerosolized agent in a viable (infectious) manner and quantification should be for viable agent. Otherwise, quantification of aerosolized agent based solely on biochemical or immunological assays (such as PCR or ELISA) may confound understanding by measuring both live and inactivated agents. The NBIES was designed with a dual impinger arrangement based on our previous experience: aerosolized VN/04 is not collected with high efficiency with one impinger alone under the conditions we used, whereas some seasonal influenza viruses can be (data not shown). The collection fluid (PBS + 0.5% w/v purified BSA fraction V) was validated for this work (data not shown). The lengthy steps and procedures to determine impinger collection efficiency will be presented elsewhere. After establishing conditions resulting in > 90% collection of live agent at the impingers, calculations based on (theoretical) 100% efficacy of aerosol dissemination are derived to set operating parameters:
(1)
Assuming 100% efficiency, the quantity of aerosolized virus particles (VP) for a given
C
s
is calculated as:
(2)
The conc. of virus in impinger A is determined for a given C
s
(3)
The conc. of virus in impinger B is determined for the same C
s
in step 2
(4)
The volume sampled by both impingers (V
i
) is calculated for t
exp
(for this work, 1 L/min × 10 min = 10 L)
(5)
Assuming even dissemination by the system, the apparent concentration of virus (
C
app
) in the aerosol stream is calculated as:
(6)
The volume disseminated by the system (
V
s
) is calculated as:
(7)
At 100% efficiency, the concentration of VP in the aerosol stream (C
aero
) is: VP/V
s
(8)
The true efficiency (expressed as %) of the system is: C
app
/C
aero
× 100
Once the above are established, calculations typically used in aerobiology can be made. The concentration of virus in the aerosol stream, calculated from the virus collected in impingers 1 and 2, where
Qagi 1+2is the collection flow rate in L/min through impingers (
agi) 1 and 2, is:
The spray factor (
SF), defined as the ratio of aerosol concentration to starting concentration, is a unitless measure used to predict aerosol concentration for a starting solution. A
SF is calculated for a range of starting concentrations using the same nebulizer and flows designed for the aerosol challenge; as:
where
C
neb
= concentration of starting solution in the BANG reservoir. An average spray factor
SF
avg
is then determined from a range of virus concentrations. The predicted respiratory volume during exposure (
V
e
) is calculated as:
The aerosol concentration (C
aero
) needed to attain D is calculated as: V
e
C
aero
= D
The starting concentration
C
s
is then calculated from the value calculated for
SF
avg
as:
The system displacement volume,
V
tot
, which is the volume of aerosolized material leaving the nebulizer/unit time, was approximated using the formula below, where
d refers to inner diameter of the tube/cylinder and
l is the length:
The velocity of air at the animals' nose (the exposure port aerosol flow velocity) was calculated as:
Finally, the number of system (total) air changes was calculated as:
Aerosol exposure studies
Virus was diluted to the appropriate concentration in aerosol vehicle (PBS + 0.5% BSA fraction V), to which was added antifoam 0.25% (v/v) molecular-grade antifoam agent B (Sigma-Aldrich, Inc., St. Louis, MO). After mixing, 4 ml of virus + antifoam was placed in the reservoir. Similarly, 10 ml of PBS + 0.5% BSA fraction V but with 0.5.% (v/v) antifoam agent B was placed into each impinger. Conscious ferrets were used for inhalation studies. As for the intranasal inoculation studies, ferrets were exposed to aerosolized viruses to attain delivered doses of 101, 102, 103, or 104 infectious virus particles over a 10 minute exposure period. All work was performed expeditiously to minimize stress; animals were moved in and out of the ferret restraint tubes relatively quickly. Just prior to exposure, the animals were loaded into ferret restraint tubes and quickly transported to the Class III glovebox housing the NBIES. The tubes were affixed onto designated inhalation ports, the aerosol generated, and the animals exposed according to experimental design.
Upon completion, the tubes were disengaged and placed in a transport bucket. The bucket was sealed, its outsides decontaminated, removed from the glovebox, and transported within the ABSL-3 suite to an animal room, where the tubes were removed within a BSC. The ferrets were then removed from the tubes and placed in cages (1 animal/cage) within the BSC, and the cages thereafter stacked in racks. Following aerosol exposure, cage-side observations including evaluation of mortality, moribundity, general health and morbidity were performed once daily during the pre-clinical stage and twice daily (at approximately 8-hour intervals) after symptoms of influenza had developed. Weight and temperature were determined once daily.
Necropsy
All procedures were performed in an ABSL3+ laboratory. For scheduled necropsies, animals were anesthetized then humanely euthanized as described above by trained technicians. After confirming death, the animals were prosected within a Class II A2 BSC. Following gross evaluation, tissues and organs were collected in this order spleen, kidneys, intestines, liver, heart, lungs, brain. To reduce hazards, a rotary saw was not used to excise the whole brains from skulls. Instead, Dean bone side-cutting forceps were used (Robosz Surgical Instruments Company, Gaithersburg, MD), and the skull cut from back to front along the medial suture lines using long (> 5 mm) cutting strokes. Noteworthy, fragmentation and production of airborne bone chips were common when other cutting tools were used, especially with short (< 2 mm) cutting strokes.