Three asymptomatic animal infection models of hemorrhagic fever with renal syndrome caused by hantaviruses
Three asymptomatic animal infection models of hemorrhagic fever with renal syndrome caused by hantaviruses
Casey C. PerleyID 0 1
Rebecca L. Brocato 0 1
Steven A. Kwilas 0 1
Sharon Daye 1
Alicia Moreau 1
Donald K. Nichols 1
Kelly S. Wetzel 1
Joshua Shamblin 0 1
Jay W. Hooper 0 1
0 Virology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Ft. Detrick, Maryland, United States of America, 2 Pathology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Ft. Detrick, Maryland, United States of America, 3 Molecular and Translational Sciences Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID) , Ft. Detrick, Maryland , United States of America
1 Editor: Alexander N Freiberg, University of Texas Medical Branch at Galveston , UNITED STATES
Hantaan virus (HTNV) and Puumala virus (PUUV) are rodent-borne hantaviruses that are the primary causes of hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia. The development of well characterized animal models of HTNV and PUUV infection is critical for the evaluation and the potential licensure of HFRS vaccines and therapeutics. In this study we present three animal models of HTNV infection (hamster, ferret and marmoset), and two animal models of PUUV infection (hamster, ferret). Infection of hamsters with a ~3 times the infectious dose 99% (ID99) of HTNV by the intramuscular and ~1 ID99 of HTNV by the intranasal route leads to a persistent asymptomatic infection, characterized by sporadic viremia and high levels of viral genome in the lung, brain and kidney. In contrast, infection of hamsters with ~2 ID99 of PUUV by the intramuscular or ~1 ID99 of PUUV by the intranasal route leads to seroconversion with no detectable viremia, and a transient detection of viral genome. Infection of ferrets with a high dose of either HTNV or PUUV by the intramuscular route leads to seroconversion and gradual weight loss, though kidney function remained unimpaired and serum viremia and viral dissemination to organs was not detected. In marmosets a 1,000 PFU HTNV intramuscular challenge led to robust seroconversion and neutralizing antibody production. Similarly to the ferret model of HTNV infection, no renal impairment, serum viremia or viral dissemination to organs was detected in marmosets. This is the first report of hantavirus infection in ferrets and marmosets.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: This work was supported in part by the
Postgraduate Research Participation Program at
USAMRIID administered by the Oak Ridge Institute
for Science and Education through an interagency
agreement between the U.S. DOE and U.S. Army
Medical Research and Material Command
(USAMRMC) (DE-SC0014664, Dr. Steven Kwilas),
and by the Military Infectious Disease Research
Hantaviruses are negative-sense RNA viruses transmitted to humans from small animal hosts.
Different viral species are associated with one of two disease syndromes: hemorrhagic fever
with renal syndrome (HFRS), or hantavirus pulmonary syndrome (HPS) [
]. Hantaan virus
Program (MIDRP) Program Area T (Dr. Jay
Hooper). There was no additional external funding
received for this study. Opinions, interpretations,
conclusions, and recommendations are ours and
are not necessarily endorsed by the U.S. Army or
the Department of Defense. No competing interests
declared. The funders had no role in study design,
data collection and analysis, decision to publish or
preparation of the manuscript.
(HTNV), primarily found in Asia, is among the most prevalent HFRS-causing hantaviruses
with a case fatality rate of between 1?15% [
]. Puumala virus (PUUV) causes most HFRS
cases in Europe, though its case fatality rate is lower at <1% [
]. There are currently no FDA
licensed vaccines or therapeutics for either HFRS or HPS .
The Syrian hamster (Mesocricetus auratus) is the typical animal used to model hantavirus
infection and disease. Andes virus (ANDV), an HPS-causing hantavirus, causes lethal disease
in immunocompetent hamsters [
], while numerous other HPS-causing hantaviruses
including Sin Nombre Virus (SNV) and Choclo virus cause lethal disease in hamsters
immunosuppressed with dexamethasone and cyclophosphamide [
]. In contrast to HPS-causing
hantaviruses, exposure of hamsters to HFRS-causing hantaviruses such as HTNV, PUUV,
Dobrava (DOBV) and Seoul (SEOV) leads to asymptomatic infection, despite viral
dissemination, even when immunosuppressed (Hooper Lab, unpublished data) [
]. In these studies
hamsters were exposed to high doses of HTNV and PUUV, far exceeding the infectious dose
99% (ID99) for the virus. Development and characterization of a uniformly infective, low-dose
challenge model, enhances the hamster model?s usefulness in vaccine and therapeutic testing.
In this report we present a low-dose hamster infection model for both HTNV and PUUV
Ferrets (Mustela putorius furo) have become a popular animal model for a number of
respiratory pathogens including influenza [
], coronavirus [
], Nipah virus [
], due to the similarity in lung physiology to humans. In addition, they have recently
been described as a disease model of two hemorrhagic fever viruses, Bundibugyo virus and
Ebola virus [
], supporting viral replication without prior adaptation. Most
hantavirusrelated human disease occurs by aerosolized transmission of the virus from the excreta or
secreta of infected rodents [
], a model of viral infection for which the ferret is well suited.
In this study we demonstrate that ferrets are capable of being infected by high titers of HTNV
and PUUV, though aside from gradual weight loss infected animals exhibit no clinical
symptoms or impaired renal function.
It has been established that infection of rhesus macaques (Macaca mulatta) with
HFRScausing hantaviruses (DOBV, SEOV, HTNV, and PUUV) leads to asymptomatic infection
and seroconversion [
], while infection of cynomolgus macaques (Macaca fascicularis) with
PUUV leads to a mild disease characterized by lethargy, mild proteinuria and hematuria, and
kidney pathology, similar to mild HFRS in humans [
]. However, the macaques? large size
and cost limits their usefulness in therapeutic studies, especially when test article availability is
limited, as is often the case in passive transfer studies. The common marmoset (Callithrix
jacchus) is becoming more popular for infectious disease studies. Its genetic similarity to humans,
cost, relative safety, and small size make it an attractive alternative to traditional non-human
primate species [
]. Marmosets have been used as a disease model for other viral agents
including Dengue virus [
], Hepatitis C virus [
], influenza virus [
], Lassa fever virus [
orthopox viruses [
], Rift Valley Fever virus [
], Eastern Equine Encephalitis virus [
and filoviruses [
]. In this study we demonstrate that exposure of marmosets to HTNV leads
to asymptomatic infection characterized by high levels of neutralizing antibodies. This is the
first report of hantavirus infection in marmosets.
Medical countermeasures are products including biologics (e.g., vaccines and antibodies)
and small molecule drugs that can be used to prevent or combat infectious disease outbreaks.
This study presents three animal models of HTNV infection, and two models of PUUV
infection that can be used to evaluate the efficacy of medical countermeasure that are intended to
prevent or mitigate infection (e.g., vaccines) by these viruses through induction of sterile
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Materials and methods
Viruses, cells and medium
HTNV strain 76?118 [
], PUUV strain K27 [
], and PUUV strains Beaumont, and Seloignes
(gifts of Piet Maes, Leuven, Beligium) were propagated in Vero E6 cells (Vero C1008, ATCC
CRL 1586) in T-150 flasks and cEMEM media (Eagle0s minimal essential medium with Earle0s
salts (EMEM) containing 10% heat inactivated fetal bovine serum, 200 ?M glutamine, 1%
non-essential amino acids, 10 mM HEPES pH 7.4; and antibiotics [penicillin (100 U/ml),
amphotericin B (250 ?g/ml), and gentamicin (50 mg/ml)]. Virus was collected from
infectedmonolayer supernatants. Cell debris was removed by low speed centrifugation (2500 rpm in a
table top centrifuge). HTNV and PUUV strain K27 were twice plaque purified according to
published methods [
]. Virus stocks were aliquoted and stored at -60?C or colder. Virus
identity has been confirmed by sequencing of the stocks.
Animal research was conducted under an IACUC approved protocol at USAMRIID (USDA
Registration Number 51-F-00211728 & OLAW Assurance Number A3473-01) in compliance
with the Animal Welfare Act and other federal statutes and regulations relating to animals
and experiments involving animals. The facility where this research was conducted is fully
accredited by the Association for Assessment and Accreditation of Laboratory Animal Care,
International and adheres to principles stated in the Guide for the Care and Use of Laboratory
Animals, National Research Council, 2011.
Female Syrian hamsters 6?8 wks of age (Envigo, Indianapolis, IN) were anesthetized by
inhalation of vaporized isoflurane using an IMPAC 6 veterinary anesthesia machine. Once
anesthetized, animals were injected with the indicated concentration of virus diluted in PBS.
Intramuscular (i.m.) injections (in the caudal thigh) consisted of 0.2 ml delivered with a 1ml
syringe with a 25-gauge, 5/8 in needle. Intranasal (i.n.) instillation consisted of 50 ?l total
volume delivered as 25 ?l per nare with a plastic pipette tip. Blood sampling from the vena cava
occurred under previously stated methods of anesthesia, and was limited to 7% of a hamster?s
total blood volume per week. At time of arrival animals were randomized into experimental
groups. Animals were housed in small animal pans, not exceeding four animals to a pan, in a
climate and humidity controlled animal biosafety level 3 (ABSL-3) with a 12-hour light/dark
cycle. Animals had pelleted food and water provided ad libitum. Enrichment in the form of
toys, nesting material and supplemental treats was provided. Humane endpoint conditions
were established as decreased mobility (inability to obtain food and water) and subdued
response to stimulation, and animals were monitored daily during the experiment. As infected
animals did not become ill, these criteria were not met and animals were euthanized by
terminal blood collection from the heart after administration of Ketamine?acepromazine?xylazine
(KAX)(USAMRIID, Fort Detrick MD) and prior to intracardiac injection with pentobarbital
sodium (USAMRIID) at the end of the study. Due to lack of illness no pain relief, aside from
anesthesia during procedures, was required.
Hamster sample size justification
Groups of 10 hamsters were used to determine the infectious dose 50% (ID50) and 99% (ID99).
Numerical simulations using SAS Probit indicate that a minimum of 3 groups spanning
infection rates 0?100% with 10 animals per group yield stable estimates of the values with
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confidence intervals reaching out to 0.7log10 (approximately 5-fold) under monotome
assumptions of response profiles for intermediate doses. As our initial selection of dosages
did not meet the desired infection rate span, some dosage groups were repeated leading to 20
hamsters per group. In the serial euthanasia study three hamsters per group were used. This
is the minimum number required to provide collection of sufficient samples for detection of
antibodies and viral kinetics in tissues. For each experiment pre-sera from animals served as
Adult, female neutered and descented ferrets (Marshall Farms, North Rose, NY), were
anesthetized by inhalation of vaporized isoflurane using an IMPAC 6 veterinary anesthesia machine,
or i.m. injection of Telazol (Zoetis, Parsippany, NJ). Injections (i.m. and i.n.) and blood
sampling were conducted under the same condition as hamsters. Intraperitoneal (i.p.) injections
consisted of 1 ml delivered with a 3 mL syringe and a 23-guage needle. Microchips (BMDS,
Seaford, DE) were used to identify and ascertain temperature during ferret experiments. In the
first ferret challenge study faulty chips lead to inaccurate temperature readings and were only
used for identification purposes. Animals were randomized upon receipt into experimental
groups. Ferrets were socially housed in metal caging, two to a cage, with sight lines to
additional animals in the study, in a climate and humidity controlled ABSL-3 with 12/hour light
and dark cycles. Each cage had a nesting box with bedding material, and numerous tubes and
shelfs for play. Ferrets had access to pelleted food supplemented with treats and potable water,
through an automated watering system. Enrichment in the form of manipulada (tubes, balls,
mirrors) and food was provided. Animals were observed daily by trained personnel in addition
to general husbandry assessments. Humane euthanasia criteria, defined as both dyspnea, loss
of mobility (to obtain food and water) and >20% weight loss. At the end of the experiment,
terminal blood samples were collected from the heart after administration of KAX and prior to
intracardiac injection with pentobarbital sodium. No pain relief, aside from anesthesia during
procedures, was used.
Ferret sample size justification
When disease occurs independently in each of four ferrets with 50% probability, the
experiment will have odds about 9:1 in favor of producing at least one diseased ferret. Conversely
failure to observe any diseased ferret in a group of four will yield a 95% confidence interval
extending from 0?50%. That is, with 95% confidence it will be admitted that the true disease
rate may be 50% or less. For this reason groups of four ferrets were used for the experiments.
For each experiment pre-sera from animals served as a negative control.
Adult marmosets weighing over 300g were anesthetized by inhalation of vaporized isoflurane
using an IMPAC 6 veterinary anesthesia machine. Once anesthetized, animals were injected
with the indicated concentration of virus diluted in PBS. I.m. injections (in the caudal thigh)
consisted of 0.2 ml delivered with a 1ml syringe with a 25-gauge, 5/8 in needle. Blood sampling
from a femoral vein occurred under previously stated methods of anesthesia, and were limited
to 7% of each marmoset?s total blood volume per week. At time of euthanasia, terminal blood
samples were collected from the heart after anesthetization by i.m. injection of Telazol and
prior to intracardiac administration of pentobarbital sodium. Animals were housed in
containment as previously described [
]. In brief, animals were singly housed in metal cages
meeting current standards in a climate and humidity controlled room. Animals were fed
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pelleted food supplemented with fruits and treats daily, and were provided potable water
through an automatic watering system ad libitum. Enrichment in the form of manipulada
(i.e. toys, metal mirrors), foraging devices, treats, and fruit were provided daily. Animals were
observed daily by trained personnel in addition to general husbandry assessments. Animals
were observed daily by trained personnel in addition to general husbandry assessments.
Animals found moribund (defined as labored breathing, decreased food consumption, persistent
prostration and moderate unresponsiveness) would be euthanized under humane endpoint
criteria, however, as animals did not become ill during the study this criteria was not met. No
additional pain relief, aside from anesthesia during procedures, was necessary. All work was
performed in an ABSL-3 laboratory.
Marmoset sample size justification
The marmoset study requires a sample size of 3 for adequate power to determine if the
incidence of seroconversion is significantly greater than that which would be expected in the
population. This sample size will allow the experimenter to detect seroconversion in at least 2 of 3
animals (66%) versus the expected population constant of <1% at a 95% confidence level
using a one-tailed binomial test for proportions.
The enzyme-linked immunosorbent assay (ELISA) used to detect nucleocapsid protein (N)
specific antibodies (N-ELISA) was described previously [
]. Species-specific secondary
antibodies were used at the following concentrations: peroxide-labeled anti-hamster (1:10,000)
(Sera Care, Gaithersburg, MD), peroxide-labeled anti-ferret (1:5,000) (Sigma Aldrich,
St. Louis, MO), and alkaline phosphatase conjugated anti-monkey (1:1,000) (MilliporeSigma,
St. Louis, MO). Assays using peroxide labeled antibodies were developed with TMB microwell
peroxidase substrate (Sera Care) at an absorbance of 450 nm; assays using alkaline phosphatase
conjugated antibodies were developed with p-nitrophenyl phosphate (PNPP) (ThermoFisher
Scientific, Waltham, MA) at 405 nm. A sample was considered positive if its peak optical
density (OD) value was greater than either 0.025 or the background value (the average of three
negative controls + 3 times their standard deviation), whichever was higher. The specific OD
sum is the summation of all values greater than background and represents the area under the
Plaque Reduction Neutralization test (PRNT)
PRNT assays were performed as previously described with minor modifications [
HTNV-infected monolayers were fixed 7 days post-infection, while PUUV-infected
monolayers were fixed 10 days post infection by 2 mL of 10% formalin per well. Immunostaining was
performed as previously described [
]. All sera samples were assayed in duplicate beginning
at a 1:20 final dilution. PRNT50 values represent the reciprocal dilution at which the serum
neutralizes 50% of the virus.
Isolation of RNA and real time RT-PCR
Approximately 200 mg of organ tissue was homogenized in 1.0 mL of TRIzol (ThermoFisher,
Waltham, MA) reagent using M tubes on the gentleMACS (Miltenyi Biotec,Auburn, CA)
dissociation system on the RNA setting. RNA was extracted from TRIzol per manufacturer?s
protocol. A Nanodrop 8000 was used to determined RNA concentration, which was then raised to
either 100 ng/?L or 1,000 ng/?L in UltraPure distilled water (Thermofisher). Real-time PCR
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was conducted on a BioRad CFX thermal cycler using either an Invitrogen Power SYBR Green
RNA-to-Ct one-step kit (Thermofisher) or Brilliant II QRT-PCR 1 -Step Master Mix (Agilent,
Santa Clara, CA) according to the manufacturer?s protocols. For spiked assays the master
mix was spiked with either HTNV or PUUV viral RNA prior to addition to the samples. For
HTNV, primer sequences were 594F 5'-AAG CAT GAA GGC AGA AGA GAT -3' and
830R 5'-TAG TCC CTG TTT GTT GCA GG-3'. Cycling conditions were 30 min at 48?C,
10 min at 95?C, followed by 40 cycles of 15 sec at 95?C and 45 sec at 60?C. Data acquisition
occurs following the annealing step [
]. For PUUV, primer sequences were 181F 5'-AGG
CAA CAA ACA GTG TCA GCA-3' and 334R 5'-GCA TTC ACA TCA AGG ACA TTT
CCA TA-3' with a FAM-conjugated probe 278 5? 5'-6-FAM-CTG ACC CGA CTG GGA
TTG AAC CTG ATG-BHQ-1-3'. Cycling conditions were 30 min at 48?C, 10 min at 95?C,
followed by 40 cycles of 15 sec at 95?C and 1 min at 60?C [
Cell culture amplification of infectious virus from urine
T-25 flasks of one week old Vero E6 cells were infected with 50 ?L of urine plus an additional
450 ?L of cEMEM media. After a 1 hr adsorption at 37?C with 5% CO2, the volume was raised
to 3.5 mL. On Day 4 post infection supernatant was collected and frozen down, 500 ?L of
which was used to infected fresh Vero E6 cells at a later time point. After a 1 hr adsorption at
37?C with 5% CO2, the volume was raised to 3.5 mL. On days 7, 11, 14, 17, 21 and 28 1.2 mL of
culture supernatant was collected and frozen down. The volume of cEMEM in the flask was
raised to 3.5 mL with fresh media.
Approximately 200 ?g of organ tissue were homogenized in 1 mL of cEMEM media using M
tubes on the gentleMACS dissociation system on the RNA setting. Plaque assays using urine,
sera, or organ homogenate were then performed beginning at the 1:10 dilution as described in
] with minor modifications. For spiked plaque assays the protocol was identical except
for equivalent amounts of virus being spiked into either media alone (control), or the 1:10?
1:1,000 dilution of organ homogenate. HTNV-infected monolayers were fixed 7 days
postinfection, while PUUV-infected monolayers were fixed 10 days post infection by 2 mL of 10%
formalin per well. Immunostaining was performed as previously described .
Post mortem procedures
Following euthanasia, necropsies were performed. Samples were collected aseptically for the
virology studies described above. For the hamsters and ferrets, samples of the following were
collected: heart, lung, liver, spleen, kidney, brain, and urine. In addition, ferrets had samples of
intestine, adrenal gland, pituitary gland, and eye were collected. Samples of the following were
collected from the marmosets: heart, lung, liver, spleen, kidney, intestine, and brain. After the
virology samples had been collected, all major internal organs in each animal were also
sampled for histology.
Preparation of tissues for histology and immunohistochemistry
Tissues were fixed in 10% neutral-buffered formalin for 21 days. Tissues were then trimmed,
processed under vacuum through increasing concentrations of alcohols, and embedded in
paraffin. Paraffin embedded tissue sections of 5?6 ?m were cut and mounted on glass slides,
stained with hematoxylin and eosin (H&E), and mounted under a glass coverslip for routine
histologic evaluation. The paraffin-embedded tissues used for producing the H&E-stained
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histology slides were also utilized for immunohistochemistry (IHC) studies in the hamsters.
Immunolocalization of HTNV in tissues was performed with an immunoperoxidase
procedure (horseradish peroxidase EnVision system; Dako) according to the manufacturer?s
directions. The primary antibody was an anti-HTNV nucleocapsid rabbit polyclonal antibody
diluted 1:3,500 (ferret) or 1:5,000 (hamster) (BEI Resources, Manassas, VA). After
deparaffinization and peroxidase blocking, tissue sections were pretreated with proteinase K for 6 min at
room temperature, rinsed, and then covered with primary antibody and incubated at room
temperature for 30 min. They were rinsed, and then the peroxidase-labeled polymer
(secondary antibody) was applied for 30 min. Slides were rinsed, and a substrate-chromogen solution
(3,3?-diaminobenzidine; Dako, Santa Clara, CA) was applied for 5 min. The
substrate-chromogen solution was rinsed off the slides, and the slides were stained with hematoxylin and rinsed.
The sections were dehydrated and cleared with xyless, and then a coverslip was placed.
Immunosuppression with cyclophosphamide (Cyp)
On the indicated days, anesthetized ferrets were injected i.p. with water soluble Cyp (Baxter
Health Care Corporation, Deerfield, IL) with the indicated dosages per body weight of drug
diluted in sterile phosphate-buffered saline (PBS), pH 7.4. In the first experiment ferrets were
administered a loading dose of 30 mg/kg on Day 41 post infection, with maintenance doses of
30 mg/kg administered every other day until euthanasia. In the second experiment, ferrets
were administered a loading dose of 30 mg/kg on Day -1, and a maintenance dose of 10 mg/kg
on Day 1, 3, 11, and 13. Administration of Cyp was discontinued between days 3 and 11 due to
secondary infection. To combat the infection (rapid onset of fever and weight loss), ferrets
were treated with 5mg/kg i.m. enrofloxicin(Norbrook Laboratories, Overland Park, KS) twice
daily per veterinarian instructions starting on Day 4. Beginning on Day 11 with the
resumption of immunosuppression ferrets were treated prophylactically with 10 mg/kg i.m.
enrofloxicin once daily).
Blood samples of 0.5 mL were collected in lithium heparin capillary blood collection tubes and
analyzed using an Advia 120 hematology analyzer (Software version 22.214.171.124-MS). Per
manufacturer?s recommendation, the dog setting was used for the complete blood count (CBC) and the
guinea pig setting was used for the white blood cell differential (WBC) in hamsters. For ferrets,
the dog setting was used for both the CBC and WBC.
Pseudovirion neutralization assay (PsVNA)
The PsVNA used to detect neutralizing antibodies in sera was described previously [
This is a replication-restricted, recombinant vesicular stomatitis virus (rVSV ?G) expressing
luciferase, which is pseudotyped with the Hantaan glycoprotein. First, heat-inactivated sera
was diluted 1:10, followed by five-fold serial dilutions that were mixed with an equal volume of
Eagle0s minimum essential medium with Earle0s salts and 10% fetal bovine sera containing
4000 fluorescent focus units of Hantaan pseudovirions. This mixture was incubated overnight
at 4?C. Following this incubation, 50 ?l was inoculated onto Vero cell monolayers in a clear
bottom, black-walled 96-well plate in duplicate. Plates were incubated at 37?C for 18?24 hr.
The media was discarded and cells were lysed according to the luciferase kit protocol
(Promega, Madison, WI). A Tecan M200 Pro was used to acquire luciferase data. The values were
graphed using GraphPad Prism (version 7) and used to calculate the percent neutralization
normalized to cells alone and pseudovirions alone as the minimum and maximum signals,
respectively. The percent neutralization values for duplicate serial dilutions were plotted. Fifty
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percent PsVNA (PsVNA50) titers were interpolated from 4-parameter curves, and geometric
mean titers were calculated.
Blood was collected in serum separator tube, and spun at 500x g to isolate sera. Ferret sera was
analyzed on the Piccolo comprehensive metabolic panel, and marmoset sera was analyzed on
the Piccolo general chemistry 13 panel per manufacturer?s instructions (Abaxis Global
Diagnostics, Union City, CA).
Urine was expressed from anesthetized ferrets and analyzed by urinalysis regent strips (VWR,
A Bayesian probit model was used to estimate 95% highest posterior density intervals for a
50% and 95% infectious dose calculation. Student?s t-test and Mann-Whitney tests were used
to compare white blood cell levels pre- and post-Cyp administration. P-values of <0.05 were
considered significant. Standard deviation of data was assessed to ensure data was normally
distributed prior to use of Student?s t-test. Analyses were conducted using GraphPad Prism
(version 7); Bayesian analyses were performed using SAS.
Calculation of ID50 and ID99 for hamster model of HTNV and PUUV
We have previously demonstrated that Syrian hamsters are capable of being infected by
HFRScausing hantaviruses, but they do not develop any signs of clinical disease [
]. To develop
standard models of HTNV and PUUV infection for future evaluation of vaccines and medical
countermeasures, groups of between 10 and 20 hamsters were exposed to serial ten-fold
dilutions of either HTNV or PUUV by either the i.m. or i.n. route (from 2?20,000 PFU HTNV or
0.2?20,000 PFU PUUV). Between 28?35 days post infection, hamsters were terminally bled
with infection status monitored by N-ELISA titers (Fig 1). From these data the doses required
to infect 50% (ID50) and 99% (ID99) were calculated (Table 1).
Refinement of low-dose HTNV and PUUV infection hamster models
To further characterize a low-dose standard hamster infection model for HFRS-causing
hantaviruses a hamster serial sacrifice study was performed. Hamsters were challenged with either
10 PFU (~3 ID99) HTNV i.m., 500 PFU (~1.5 ID99) HTNV i.n., 1,000 PFU (~1 ID99) PUUV
i.m., or 1,000 PFU (~1.5 ID99) PUUV i.n. On various days post infection, groups of three
hamsters were euthanized to monitor viral and serological parameters.
Seroconversion occurred, at least partially, by Day 17 post HTNV infection and Day 24
post PUUV infection. Seroconversion on Day 28 post HTNV i.m. infection was incomplete,
though viral genome was recovered from all hamsters euthanized that day indicating a
productive HTNV infection occurred (Fig 2). To confirm seroconversion, all hamsters euthanized on
days 17, 24, and 28 were assayed for neutralizing antibodies by PRNT regardless of N-ELISA
seroconversion status. All HTNV infected hamsters with N-ELISA titers had neutralizing
antibodies as measured by PRNT50, with all but one having full neutralization of virus at a 1:20
dilution of sera. (S1A and S1B Fig). Similarly, all PUUV hamsters with N-ELISA titers had
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Fig 1. HTNV and PUUV infect hamsters in a dose dependent manner. Syrian hamsters were challenged with varying
concentrations of either HTNV (A) or PUUV (B) through the i.m. (left) or i.n. (right) route. Between 28 and 35 days post infection
hamsters were terminally bled and N-ELISA endpoint titers (log10) were used to determine infection status. The mean titer is
displayed for each group, and the limit of detection (2log10) is depicted as a dashed line.
neutralizing antibodies as measured by PRNT50, though three of the five PUUV i.n. challenged
hamsters did not have complete neutralization of the virus at a 1:20 dilution of sera (S1C and
S1D Fig). Two of three PUUV i.m. challenged hamsters that had not seroconverted on Day 17
post infection had low levels of neutralizing antibodies, while none of the PUUV i.n.
challenged hamsters that were seronegative by N-ELISA had neutralizing antibodies. For both
HTNV and PUUV infected hamsters, infection via the i.m. route lead to a more robust
neutralizing antibody response than then i.n.route.
The kinetics of HTNV and PUUV infection in the heart, lung, liver, spleen, kidney, and
brain were monitored by both RT-PCR and plaque assay (Figs 3 and 4). HTNV infected by
either the i.m. or i.n. route resulted in a persistent infection. High levels of viral genome were
detected in the heart, lung, kidney and brain of HTNV infected hamsters beginning on either
Day 11 or 17 post infection (Fig 3A, 3B, 3E and 3F). Infection of the kidney and brain was
found in all examined hamsters beginning on either Day 17 or 24 post infection, while the
high titers of viral genome detected in the heart and lung were present in only one or two
hamsters at each time point. Low levels of viral genome were detected between days 11 and 24 post
infection in the liver of HTNV i.m. but not i.n. infected hamsters (Fig 3C). Hardly any viral
genome was detected in the spleen (Fig 3D). To confirm the lack of viral genome in the spleen
was not due to the presence of inhibitors all spleen samples from i.m. and i.n. infected
hamsters were spiked with HTNV prior to RT-PCR. No significant inhibition of the spiked RNA
was noted, indicating that HTNV infection does not result in virus dissemination to the spleen
(S2A and S2B Fig).
The detection of PUUV in organs was transient after i.m. infection, occurring between Day
11?17 for the heart, liver, kidney and brain, with no virus detected in the spleen (Fig 3A, 3C,
3E and 3F). Viral genome was more persistent in the lung where it was detected in at least one
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Fig 2. Kinetics of seroconversion in low dose hamster models. Syrian hamsters were infected either 10 PFU HTNV i.
m., 500 PFU HTNV i.n., 1000 PFU PUUV i.m., or 1000 PFU PUUV i.n. Hamsters were terminally bled at various
points post infection and N-ELISA endpoint titers (log10) were used to determine infection status. The mean titer
(A-D), and specific OD sum ? standard error of the mean (SEM) (E-F) for the N-ELISA is displayed for each group.
The limit of detection (2log10) for the N-ELISA is depicted as a dashed line. The specific OD sum represents the area
under the titer curve.
of three hamsters on/after Day 11 post infection (Fig 3B). A small amount of viral genome
detected in the brain of a PUUV i.n. infected hamster, 28 days post challenge, is the only viral
genome detected in any organ at any time point post PUUV i.n. infection (Fig 3F).
Serum viremia was detected in hamsters challenged with HTNV i.m. between 11 and 28
days post infection, though the presence of virus was sporadic except for Day 24. Serum
viremia was only detected in two hamsters challenged with HTNV i.n., one on Day 11 and one on
Day 17. No serum viremia was detected in hamsters challenged with PUUV by either route
(Fig 3G). Similarly, the presence of viral genome was sporadically detected in the urine of
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Fig 3. Viral genome is detectable in the organs and sera of infected hamsters. Syrian hamsters were infected either
10 PFU HTNV i.m., 500 PFU HTNV i.n., 1000 PFU PUUV i.m., or 1000 PFU PUUV i.n. Hamsters were euthanized at
various points post infection and heart (A), lung (B), liver (C), spleen (D), kidney (E), brain (F), sera (G), and urine
(H) were evaluated by RT-PCR for the presence of viral genome. The mean ? SEM is displayed at each time point. The
dashed line indicates the limit of detection (1log10).
11 / 30
Fig 4. Infectious virus was isolated from the organs of HTNV i.m. infected animals. Syrian golden hamsters were
infected with 10 PFU HTNV i.m. and sacrificed at various time points post infection. Heart (A), lung (B), liver (C),
spleen (D), kidney (E), and brain (F) from the time range where the organ was RT-PCR positive were sampled by
plaque assay for the presence of infectious virus. The mean ? SEM is displayed at each time point. The limit of
detection (1.7 log10) is depicted as a dashed line.
HTNV infected hamsters between days 17?28, but was not detected in PUUV challenged
hamsters (Fig 3H).
In HTNV i.m. infected hamsters, infectious virus was first detected in the liver beginning at
Day 11, and in the lung, liver, spleen, and kidney at Day 17 post infection (Fig 4B, 4C, 4D and
4E). With the exception of the kidney, in which infectious virus was recovered from every
hamster after 17 days post infection, infectious virus was recovered from the lung, liver, brain
and spleen in only a portion of hamsters at each time point. No infectious virus was detected
in the heart at any time point (Fig 4A). Virus recovery from HTNV i.n. infected hamsters was
markedly lower, with infectious virus being detected only in the kidney on Day17 and 28 post
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infection (Fig 4E). No infectious virus was recovered from the organ of any PUUV infected
hamster (Fig 4).
To confirm that the lack of infectious virus in the spleen was not due to the presence of
inhibitors select samples were spiked with HTNV virus prior to a plaque assay. No significant
inhibition of the spiked RNA was noted, confirming the RT-PCR results that that HTNV
infection does not result in viral dissemination to the spleen (S2C and S2D Fig).
No infectious virus was detected in the urine for any hamster tested, even those that were
RT-PCR positive for HTNV viral genome. In human ANDV infected patients with ANDV
antigen positive urine, samples had to be cultured in Vero E6 cells for between 16?22 days
post infection before infectious virus was detected [
]. Three HTNV RT-PCR positive and
three HTNV RT-PCR negative urine samples were cultured in Vero E6 cells, with supernatant
collected on various days post infection for evaluation by RT-PCR (S3A Fig). All samples were
HTNV RT-PCR negative for virus in the supernatant of infected cells at Day 4 post infection,
the first time point examined. All three RT-PCR positive urine samples yielded infectious virus
(two on Day 15, one on Day 32), while two of the RT-PCR negative urine samples also yielded
infectious virus (on Day 18) (S3B Fig) as measured by the conversion of cell supernatant to
HTNV RT-PCR positive. All samples were evaluated by plaque assay on Day 32 post infection.
These findings were confirmed, all samples whose cell supernatant became RT-PCR positive
post infection were positive for infectious virus by plaque assay. The one sample whose cell
supernatant did not convert to RT-PCR positive, remained negative for infectious virus by
plaque assay (S3C Fig).
Patients infected with HFRS hantaviruses exhibit leukocytosis and thrombocytopenia
during infection [
]. At every time point post infection, EDTA-treated whole blood from
HTNV and PUUV exposed hamsters was evaluated to determine if changes in white blood cell
count or platelets occurred. No changes were observed (S4 Fig).
To further characterize disease, tissue sections from HTNV i.m. infected hamsters were
analyzed by IHC and H&E to assess viral localization and any pathologic changes (S5 Fig). No
significant histopathological findings were noted in the kidney or brain. Splenic follicular
lymphoid hyperplasia and hepatic extramedullary hematopoiesis in the liver were each noted in
three hamsters. Both are seen in animals from later time points in the study (days 17?28) and
likely represent a reaction to HTNV infection, though other unidentified antigenic stimuli
cannot be ruled out. Five hamsters between days 4 and 17 post infection exhibited a minimal
to mild inflammation of the pericardium, characterized by a mixed lymphoplasmacytic
histiocytic and neutrophilic infiltrate. Occasional macrophages in the inflammatory infiltrate within
the pericardium are immunopositive suggesting a possible association with HTNV; however,
no evidence of cardiac tissue injury is associated with the presence of HTNV antigen.
Beginning on Day 4 post infection 66% (12/18) hamsters exhibit minimal (likely subclinical)
respiratory lesions consisting of interstitial neutrophilic and histiocytic infiltrates in the lungs, with
44% (8/18) also exhibiting minimal amounts of alveolar edema. An additional hamster had
minimal alveolar edema but not pulmonary infiltrates. Such findings suggest a response to
antigenic stimulus and the presence of immunopositive endothelial cells, pneumocytes and
alveolar macrophages suggest a response to HTNV infection.
Immunohistochemistry staining was uniformly negative in all tissues on Day 1 post
challenge. Between Day 4 and 11 post challenge minimal antigen was sporadically detected in
macrophages, pneumocytes and endothelial cells. By Day 17 post challenge mild to moderate levels
of antigen were observed in endothelial cells and choroid ependymal cells in the brain, in
endothelial cells, macrophages and pneumocytes in the lung, and endothelial cells in the
kidney. Minimal to mild levels of immunopositivity were found in endothelial cells and
macrophages in the heart, and in endothelial cells, hepatocytes and Kupffer cells in the liver.
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0: no cells in section are immunopositive (negative); 1+: < 10% of cells in section are immunopositive (minimal); 2+: 11?25% of cells in section are immunopositive
(mild); 3+: 26?50% of cells in section are immunopositive (moderate); 4+: 50?75% of cells in section are immunopositive (marked); 5+: >75% of cells in section are
positive (severe). e = endothelial cell, c = choroid ependymal cell, m = macrophage, h = hepatocyte, k = Kupffer cell, l = lymphocyte, p = pneumocyte.
Surprisingly, given the lack of viral genome and infectious virus recovered, mild to moderate
amounts of immunopositive macrophages, lymphocytes and endothelial cells were found in
the spleen (Table 2, S5 Fig).
Ferret model of HTNV and PUUV infection
No published studies detail if ferrets are susceptible to hantavirus infection. To examine this,
four ferrets were exposed to either 2,000 PFU HTNV or PUUV K27 i.n. No seroconversion
occurred within 35 days post infection. The same animals were re-exposed to either 200,000
PFU HTNV, 94,000 PFU PUUV Beaumont (a human PUUV isolate) or 164,000 PFU PUUV
Seloignes (a vole PUUV isolate) by i.m. Prior to the re-exposure one of the seronegative ferrets
in the HTNV group was removed for health concerns (rapid weight loss) unrelated to the
study, and it?s cage mate was subsequently removed for behavioral health reasons before the
completion of the study. Data from those two ferrets are not shown. As soon as three days post
infection ferrets began to lose weight with HTNV infected ferrets losing between 5?12% of
peak body weight as did PUUV infected ferrets (Fig 5A?5C). By Day 35 post infection all
animals had developed antibodies against all strains of the virus as measured by N-ELISA assay
(Fig 5D?5F). Neutralizing antibody development began as early as Day 14 post infection and
all ferrets developed neutralizing antibodies by Day 28 post infection (Fig 5G?5L). EDTA
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Fig 5. Ferrets are susceptible to HTNV and PUUV infection. Ferrets were challenged with either 2,000 PFU HTNV i.n., or 2,000
PFU PUUV i.n. (low dose challenge arrow) and did not seroconvert. The same animals were subsequently challenged with either
200,000 PFU HTNV (A,D,G,J), 94,000 PFU PUUV Beaumont (B,E,H,K), or 164,000 PFU of PUUV Seloignes (C,F,I,L) i.m. on day 0,
and immunosuppressed with 30 mg/kg cyclophosphamide on day 41. The daily weight (g) for each ferret is displayed (A-C).
Seroconversion of ferrets was monitored by N-ELISA specific OD sum (D-F), and neutralizing antibody production by PsVNA50
(G-I) and PRNT50 (J-L). The dashed line represents the limit of detection for the PRNT (20) and PsVNA (20) assay.
treated blood was analyzed weekly for evidence of leukocytosis and thrombocytopenia; none
was observed (S6 Fig) nor was serum viremia detected by RT-PCR (S7A Fig).
Syrian hamsters infected with SNV do not develop lethal disease, unless immunosuppressed
]. On Day 42 post infection infected ferrets were administered 30 mg/kg Cyp. Within two
days post administration the total white blood cell, lymphocyte and neutrophil count had
decreased significantly (S8 Fig), and was almost zero seven days post administration. The
immunosuppressed ferrets rapidly lost weight, became lethargic, with occasional vomiting and
diarrhea. Animals were euthanized between 3 and 7 days post administration of Cyp having
met humane endpoint criteria.
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The organs of infected ferrets were analyzed for viral load by RT-PCR and for the presence
of infectious virus by plaque assay. The lung, liver, spleen, intestine and urine of HTNV
infected ferrets were negative for viral genome, with small amounts (<2 log10) detected in the
heart and spleen of a single animal (S7B Fig). The heart, lung, kidney, intestine and urine of
PUUV infected ferrets were negative for viral genome, though small amounts were detected in
the liver (2/4) and spleen (1/4) of PUUV infected ferrets (S7C and S7D Fig). Viral genome was
spiked into the assay to confirm that the lack of signal was not due to the presence of
inhibitors. All organs except for the intestine (4/6) had no inhibition of RT-PCR product. Similarly,
no infectious virus was found in the organ of any ferret by plaque assay, despite spiked-in virus
exhibiting no significant inhibition (S9 Fig).
To confirm that immunosuppression of uninfected ferrets did not result in rapid weight
loss, four healthy ferrets were immunosuppressed with a loading dose of 30 mg/kg Cyp
followed by 10 mg/kg Cyp maintenance doses every other day (S10A Fig). Five days post
immunosuppression ferrets exhibited rapid weight loss, fever, and lethargy due to secondary
infection (S10B & S10C Fig). Immunosupression was discontinued and 5 mg/kg enrofloxicin
(a broad spectrum antibiotic) was given twice daily for a week. During this time, ferrets began
to gain weight and their fever diminished. On Day 11 immunosupression resumed for two
doses with prophylactic enrofloxicin given once daily. Even with prophylactic antibiotics two
ferrets spiked fevers within a few days post the second round of immunosuppressive treatment,
though they did not lose weight (S10B & S10C Fig). Based on these results the rapid weight
loss and clinical signs observed upon immunosuppression of HTNV and PUUV infected
ferrets was most likely due to secondary infection. Due to the inability to completely manage
secondary infection with prophylactic antibiotic treatment, no further immunosuppression
studies were carried out in ferrets.
To refine the HFRS-causing hantavirus ferret infection model four ferrets were challenged
with 94,000 PFU PUUV Beaumont i.m. on Day 0. Weight and temperature were recorded
daily, while twice weekly blood draws and urine collection was used to monitor kidney
function. As with the pilot experiment, ferrets slowly lost between 7?11% of peak body weight,
recapitulating our previous findings (Fig 6A). No elevated temperatures were observed
(Fig 6B). Ferrets developed a robust antibody response beginning on Day 10 post infection
(Fig 6C and 6D). Neutralizing antibodies developed early as Day 14 post infection, and
were present in all ferrets by Day 28 post infection, as measured by PsVNA and PRNT (Fig
6E and 6F).
Despite the slow weight loss no signs of renal impairment were observed. Proteinuria and
hematuria are hallmarks of PUUV infection occurring in between 94?100% (proteinuria) and
58?85% (hematuria) of human clinical cases [
]. No prolonged proteinuria or hematuria was
observed (Fig 7A and 7B) in infected ferrets. Similarly, blood urea nitrogen and creatinine
levels in the sera, both of which are elevated due to kidney failure in PUUV patients [
remained unchanged in PUUV infected ferrets (Fig 7C and 7D) [
]. No infectious virus
or viral genome was detected in the brain, heart, lung, liver, spleen, kidney, intestine, or eye
(S11 and S12 Figs). No changes in other serologic or urologic parameters were noted (S13 and
No gross pathological changes or significant lesions associated with PUUV infection were
noted in the ferrets (S15 Fig). In the lungs, one ferret had mild neutrophilic and histiocytic
inflammation centered on the bronchioles and expanded alveolar septa. Given that the
inflammation was centered around bronchioles and not the vasculature, it is unlikely to be
in response to PUUV infection. A number of other common or age-associated lesions were
observed in the ferrets. Two of four ferrets had proliferative cortical cells in either the adrenal
capsule or adrenal cortex that were likely clinically silent as they lack clinical signs consistent
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Fig 6. PUUV infected ferrets experience weight loss, but no other clinical signs of infection. Ferrets were infected with 94,000
PFU PUUV Beaumont i.m. and sera was collected twice weekly. Weight (A), temperature (B), N-ELISA specific OD sum (C),
N-ELISA titer (log10), (D) PsVNA50 titers (E), and PRNT50 titers (F). The dotted line represents the limit of dectection for the PRNT
assay (20), the PsVNA assay (20) and the N-ELISA (2log10).
Fig 7. PUUV infected ferrets do not exhibit signs of kidney impairment. Ferrets were infected with 94,000 PFU
PUUV Beaumont i.m. Blood and urine were collected twice weekly for analysis of kidney function by assaying
proteinuria (A), hematuria (B), blood urea nitrogen (C), and creatinine (D) levels. Shaded gray areas represent the
normal range [
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Fig 8. Marmosets are susceptible to HTNV infection. Three marmosets were infected with 1,000 PFU HTNV i.m. Sera was
collected weekly to monitor seroconversion. N-ELISA titer (log10) (A) N-ELISA specific OD sum (B) PRNT50 (C) and PsVNA50
titers(D) are displayed. Serum viremia was assayed by RT-PCR (E). The limit of detection for N-ELISA titer (2log10), PRNT (20),
PsVNA (20) and RT-PCR (1.0log10) is depicted by the dotted line.
with adrenal?associated endocrinopathy. Additionally, alveolar mineralization was noted in
all four ferrets as was eosinophilic and lymphoplasmacytic enteritis, and hepatitis. Two ferrets
had fibromyxomatous degeneration of the atrioventricular valve. The spleen, brain, kidney,
and pituitary gland were normal in all ferrets examined.
Marmoset model of HTNV infection
As with ferrets, there is no literature on the susceptibility of marmosets to hantavirus infection.
To test this, three male marmosets were exposed to 1,000 PFU HTNV i.m. Blood was collected
weekly to measure seroconversion, serum viremia, as well as serum parameters relating to
renal function. All three animals seroconverted by Day 21 post infection (Fig 8A and 8B)
despite displaying no clinical signs of illness. Neutralizing antibody production began around
the same time, and was robust by Day 30 post infection (14,866?221,557 by PsVNA50 and
10,240?20,480 by PRNT50) (Fig 8C and 8D). Due to low volumes of blood drawn at each time
point serum viremia could not be examined for each individual animal, however a pool of sera
from all three animals was evaluated at each time point post infection. Low levels of serum
viremia were detected between days 14 and 28 post infection by RT-PCR (Fig 8G). As with the
ferret infection model of HFRS-causing hantavirus, no renal injury as measured by changes
noted in blood urea nitrogen or serum creatinine were observed over the course of infection,
nor were changes in any of the other serum parameters monitored (S16 Fig).
Animals were euthanized on Day 30 post infection to examine organs for viral
dissemination. No infectious virus or viral genome was detected in the heart, lung, liver, spleen, kidney,
intestine or brain (S17 and S18 Figs). Lymphoid hyperplasia was noted in all three animals by
histology, though the location and intensity varied between the spleen, various lymph nodes
and gut associated lymphoid tissue. Such hyperplasia is indicative of a response to antigenic
stimulation and was most likely caused by the viral challenge. Mild to moderate congestion
18 / 30
was also seen in the lungs of each marmoset; however, this was an acute change and was most
likely associated with terminal anesthesia and euthanasia. No other significant histological
lesions were noted in any of the three animals (S19 Fig). Due to the negative virology results
and the lack of significant histologic changes, IHC to detect the presence of HTNV antigen in
tissues was not performed.
To date, the use of adult animal models to evaluate anti-HTNV and anti-PUUV medical
counter measures has been limited. Recombinant protein, vaccinia virus-vectored, and DNA
vaccines have been tested in the high dose HTNV hamster model [
9, 37, 50
]. Additionally, the
ability of passively administered neutralizing antibodies has been evaluated in the high-dose
hamster model of HTNV and PUUV and in PUUV challenged cynomolgus macaques PUUV
]. Both of these models have limitations; the size of the macaque requires large
quantities of passive transfer material, and the high dose of the hamster model, with challenge doses
of ~650 ID99, could require prohibitively large volumes of test article to neutralize the high
initial dose. Suckling mice, which present with a disseminated disease not reminiscent of HFRS,
have been used to evaluate HTNV therapeutics as well [
]. In this paper we present three
adult animal models of HFRS-causing hantavirus infection than can be used for future
evaluation of therapeutics, biologics and vaccines.
Low-dose hamster model of HFRS-hantavirus infection
The ID50?s for HTNV and PUUV determined in this report are similar to the lethal dose 50%
(LD50) calculated for SNV and ANDV, <3 PFU via the i.m. route [
6, 7, 41
]. Also similar is that
the challenge dose of ANDV required to infect/kill 50% of hamsters by the i.n. or intragastric
route is ~10?30 fold higher [
]. Previous reports also demonstrate that PUUV is capable of
infecting hamsters by the intragastric route, with an ID50 of >10,000, making it a much less
effective route of infection [
]. Despite similarities in their ID50?s, the ID99?s of HTNV and
PUUV greatly diverge, with ~200 times less HTNV required to infect hamsters via the i.m.
route, and ~2 fold less required to infect via the i.n. route (Table 1). The lower ID99 doses,
coupled with viral persistence in HTNV infected animals as opposed to viral clearance (Fig 3),
suggest that HTNV is more infectious than PUUV in the hamster. The mechanism for this
difference between these closely related hantaviruses remains unknown.
Lethal infection of hamsters with ANDV leads to extensive organ dissemination, with
infectious virus recovered in the lung, liver, kidney, spleen and heart [
]. Asymptomatic infection
of hamsters with SNV repeatedly passaged through hamsters has a similar organ distribution
]. The organ distribution of both those viruses is similar to the low dose HTNV hamster
model, with two notable exceptions. First, the HTNV model has low, transient levels of virus
in the liver and hardly any virus in the spleen (Figs 3 and 4). This dissimilarity between the
models led us to confirm the lack of virus in the spleen was not due to the presence of
inhibitors by spiking either infectious virus or viral RNA into samples prior to evaluation (S2 Fig).
Second, in the ANDV model, the presence of virus was determined by plaque assay, indicating
the virus was infectious and replication competent. In the HTNV low dose hamster model,
while there is detection of high levels of viral genome (and in the case of the HTNV i.m.
model, by pathology) by RT-PCR, recovery of infectious virus is sporadic, typically occurring
at low levels, in only a few hamsters per time point (Figs 3 and 4, Table 1). The discrepancy
between RT-PCR/pathology and plaque assay is notable. The hamsters in this study were not
perfused, and given the appearance of neutralizing antibodies as early as day XX post infection,
the presence of these antibodies could be impairing out ability to recover live virus via the
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plaque assay. It is also possible that viral packaging is somehow impaired in the hamster,
leading to a larger amounts of nucleocapsid protein and viral genome than live virus. Further
studies need to be undertaken to elucidate.
The low dose HTNV hamster model also mimics the infection pattern of the virus in its host
species, the striped field mouse (Apodemus agrarius), with viral genome being detected in the
lung, liver and kidney but not the spleen (the heart and brain were not examined) [
In contrast to the HTNV low dose model, the organ distribution of virus in the PUUV low
dose model is transient. In PUUV i.m. infected hamsters virus is detectable by RT-PCR around
Day 11 post infection, with the virus being cleared from all organs except the lung by Day 24.
No infectious virus was recovered at any time point examined. Even less virus was detected in
the PUUV i.n. model; only a small amount in the brain of one hamster on Day 28 post
infection. In neither case was serum viremia observed (Figs 3 and 4). This is most similar to the
SNV models involving low passage virus in immunocompetent hamsters: the virus is
transiently detected in the lung by PCR, and then sporadically found in organs 12 to 14 days post
infection using immunohistochemistry [
]. The distribution of PUUV in the hamster
differs somewhat from its host species the bank vole, where it is found to persist in the lung,
spleen and kidney, and was not detected in the heart or the brain .
Seroconversion of hamsters post viral exposure remains the best way to measure infection,
and should be a considered the primary endpoint for efficacy studies. Though the PRNT assay
was slightly more sensitive than the N-ELISA assay, detecting neutralizing antibodies in all
animals with N-ELISA titers, and in two PUUV animals that did not have N-ELISA titers, the
increased time, sample, and biosafety requirements necessary for a PRNT assay make the
N-ELISA a better choice (Fig 2, S1 Fig). For a 10 PFU HTNV i.m. challenge, given lower titer
and specific OD sum values as compared with higher challenge doses, and the fact that one
hamster with significant viral genome in its organs at Day 28 post infection (7.1 log10 in the
brain, and 6.8 log10 in the kidney) did not seroconvert, waiting until Day 35 post infection to
monitor seroconversion is advisable (Figs 1?3). Viral load in the brain, kidney, and lungs as
measured by RT-PCR need to be evaluated at Day 35 post low-dose challenge to determine
their usefulness as secondary endpoints. Recovery of infectious virus in any organ, and viremia
are too sporadic to serve as proxy markers for infection.
Ferret infection model
The ferret has been used as an experimental model for numerous hemorrhagic fever viruses,
and respiratory viruses [
], though no published reports exist examining its susceptibility
to hantavirus infection. In comparison to the hamster, the ferret is far more resistant to HTNV
and PUUV infection. Exposure of ferrets to 2,000 PFU i.n. (greater than the ID99 for both
viruses in hamsters), failed to result in a productive infection and seroconversion. Instead i.m.
challenge doses of ~100,000?200,000 PFU were needed (Fig 5). Initially ferrets were exposed
to PUUV K27, a commonly used laboratory isolate that has been in cell culture for over 15
years. Repeated passaging of hantavirus is known to cause mutations [
]. In contrast
PUUV Beaumont and Seloignes are relatively recent isolates, with no more than 3 passages in
cell culture post isolation. These strains were used for all subsequent experiments to maximize
the likelihood of PUUV to cause disease by eliminating possible attenuation of the laboratory
strain of the virus. Despite the high challenge dose and use of recent isolates, no elevation in
white blood cells was observed over the course of the experiment, no pathology or organ
burden was detected at the conclusion of the experiment, and the N-ELISA specific OD sum,
PRNT50, and PsVNA50 titers remained low (Figs 6 and 8 and S9 Fig). This outcome is almost
identical to that of Marburg and Ravn virus infection in ferrets, where the development of
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neutralizing antibody titers was the only sign that productive infection occurred . Previous
experiments in hamsters, have demonstrated that 2x105 PFU of gamma-irradiated ANDV and
SNV are not sufficient to cause seroconversion, and neither is 1x104 gamma-irradiated PFU
]. Thus the seroconversion observed in ferrets, though low, is not likely to be due
to a reaction to the large quantity of antigen administered, but to a productive infection.
PUUV antigen, viral genome, or infectious virus has been found in the brain, pituitary
gland, lung, heart, liver, kidney, spleen, cerebrospinal fluid, and gastrointestinal tract of
human patients with clinical symptoms of NE, though the pattern of viral dissemination varies
between individuals [
]. Acute kidney injury and vision disturbances including blurred
vision, myopic shift, and lens thickening, while pulmonary involvement including pleural
effusion and vascular congestion, and renal failure occurred less frequently [
]. Given the
lack of high neutralizing antibody titers, which could have aided in viral clearance, the lack of
viral genome in any of the ferret organs examined is rather surprising (S6, S7, S11 and S12
Figs). Furthermore, the lack of viral antigen and pathology in the organs tested suggest either a
transient infection cleared prior to Day 35 post infection, levels of virus so low as to be
undetectable by the tests used, or a viral reservoir outside of the organs tested.
The lack of detectable virus is most surprising given the gradual weight loss infected
animals exhibit (Figs 5 and 6). The animals gained weight until ~3 days post challenge, at which
point a gradual weight loss occurs, regardless of if HTNV or PUUV was the challenge virus.
While weight loss is a feature of other ferret models of infectious diseases, the pattern we
observed was unique: ferrets infected with morbillivirus, avian influenza and filoviruses rapidly
lose weight during the first week to two weeks post infection, while infection with severe acute
respiratory syndrome virus results in no significant weight loss [
16, 17, 75?77
In the hamster, infection with SNV is asymptomatic unless the hamster is
immunosuppressed. When ferrets were immunosuppressed on Day 42 post infection, rapid weight loss
and lethargy ensured (Fig 5). Given that these clinical signs were also observed in unchallenged
control animals (S10 Fig), this could likely be the result of a secondary infection or drug
toxicity. The use of Cyp is well documented in ferrets, primarily given at a high dose as an emetic,
and no dosage for long term immunosuppression was found [
]. The dosages used in this
study (between 10?30 mg/kg) successfully reduced white blood cell levels, in ferrets and
demonstrate that Cyp can be used to induce long-term immunosuppression, if antibiotics are
given to control for secondary infection (S8 Fig).
Despite not being able to detect infectious virus or viral genome in the kidney, we
hypothesized that the weight loss we observed could be due to kidney failure. Individuals with HFRS
exhibit proteinuria and hematuria, both of which can indicate kidney damage [
Additionally, serum blood urea nitrogen and creatinine levels are both elevated in HFRS patients
and provide a second way to measure kidney function [
46, 47, 73
]. Decreased platelet levels
also characterize clinical HFRS in humans, impairing coagulation [
46, 47, 73
]. In a second
experiment designed to monitor kidney parameters, PUUV infected ferrets exhibited the same
gradual weight loss that characterized the first experiment. However, no prolonged signs of
clinical kidney failure were observed: blood urea nitrogen and creatinine levels did not
dramatically increase over the five week study period. Only one animal exhibited proteinuria
(day 35), and two exhibited hematuria (one on Day 4, and one on Day 21) (Fig 7). Similarly to
parameters monitoring renal failure, no thrombocytopenia occurred in PUUV infected ferrets
(S9 and S13 Figs). The cause of the weight loss remains undetermined.
Though susceptible to both HTNV and PUUV, the ferret has limited usefulness for studies
involving medical countermeasure efficacy testing. The ferret?s large mass, even as an
adolescent, makes the amount of test article needed also prohibitively large. The large challenge dose
required for infection could potentially obscure the protective effect of drugs of vaccines, due
21 / 30
to the overwhelming amount of virus administered. Moreover, although the animals are
infected the resultant neutralizing antibody titers are small, resulting in potential sensitivity
issues with the model. Husbandry and handling of the animals under ABSL-3 procedures is
also substantially more difficult than hamsters, and they lack the genetic similarity to humans
that marmosets possess.
Marmoset infection model
In this study we have demonstrated the susceptibility of marmosets to HTNV infection.
Marmosets represent an attractive model for testing vaccines and therapeutics against
HFRS-causing hantaviruses due to genetic similarities to humans and small size. Also, the model has a
simple read-out of infection, i.e. robust antibody production as measured by N-ELISA and
PsVNA, making the determination of protection by vaccine or passive transfer material,
straightforward. Further optimization of the model, namely to determine the ID99, could
prove important as a 1,000 PFU challenge dose could be excessively high and prohibit
therapeutic effects of candidate medical countermeasures.
Overall the marmoset model is more similar to the ferret HFRS-causing hantavirus
infection model than the hamster, though there are key differences. Like the ferret, no significant
pathological abnormalities were noted, and no signs of renal failure were observed (S16 and
S19 Figs). Serum chemistry values do not differ from the normal range with the exception of
albumin, total bilirubin, and amylase. While these values fell outside the normal range, they
did not change over the course of infection indicating the problem may lie in the reference
values used. The Piccolo general chemistry panel used to evaluate the parameters is optimized for
human testing, and therefore may be less than optimal for evaluating the marmoset, especially
those parameters. Additionally, no infectious virus or viral genome was recovered from any
organ at Day 30 post infection [
]. This is not surprising, given the high levels of
neutralizing antibodies present as early as 21 days post infection (Fig 8). Unlike the ferret, however,
marmosets develop exceptionally high neutralizing antibody titers (10,240?20,480 by PRNT50
and 14,866?221,557 by PsVNA50), and display low-level serum viremia between two and
four weeks post infection (Fig 8). The serum viremia is significantly lower than in hamsters
infected with HTNV, where some animals displayed RT-PCR titers of >7log10, and in
hamsters infected with ANDV, where infectious virus titers prior to death are > 6log10 .
Despite not exhibiting clinical signs of disease, the model?s robust antibody response (as
measured by PRNT, PsVNA and N-ELISA) make it a useful tool for evaluating vaccines and
pre-or post-exposure therapeutics.
This paper has explored the use of three laboratory animal species as possible infection and
disease models for HFRS-causing hantaviruses: the hamster, the ferret, and the marmoset.
These models, especially the hamster model and marmoset model, will be useful for evaluating
medical countermeasures with the potential to induce sterile immunity. The marmosets
should be particularly useful for the evaluation of passively transferred protective human
antibodies because of the relative genetic similarities between species in the Order Primates, and
the small size of marmosets, allowing testing with smaller volumes of material than would be
required for larger species such as macaques.
S1 Fig. Seroconversion in low dose hamster models. Syrian hamsters were infected either 10
PFU HTNV i.m. (A), 500 PFU HTNV i.n. (B), 1000 PFU PUUV i.m. (C), or 1000 PFU PUUV
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i.n. (D). No sera was available for one hamster in the Day 28 HTNV i.m. group. Hamsters
sacrificed on days 17?28 had their sera screened for neutralizing activity by PRNT. The mean
titer is displayed for each group, and the limit of detection for the assay (20) is depicted by a
S2 Fig. Lack of viral RNA in the spleen of HTNV infected animals is not due to inhibitors.
Syrian hamsters were infected either 10 PFU HTNV i.m. or 500 PFU HTNV i.n. Hamsters
were terminally bled at various points post infection. Samples were evaluated with and without
the addition of exogenous viral genome (by RT-PCR) (A&B) and randomly selected negative
samples were evaluated with and without infectious virus by at the 1:10 dilution by plaque
assay (C&D). The mean ? the SEM is shown for each group and the limit of detection for each
(RT-PCR LOD = 1 log10; Plaque assay = 50 (1.7 log10) plaques) is displayed as a dashed line.
S3 Fig. Repeated cell culture passaging can result in detection of infectious virus in the
urine. Syrian hamsters were infected either 10 PFU HTNV i.m. or 500 PFU HTNV i.n.
Hamsters were euthanized and urine was collected at various points post infection. Three RT-PCR
positive, and three RT-PCR negative urine samples underwent amplification by cell culture.
(A) Schematic of urine amplification strategy. Red arrow indicates infection of Vero E6 cells,
purple arrow indicates sample collection. On Day 4 supernatant was collected and frozen, and
used to infect fresh Vero E6 cells at a later date. (B) Presence of viral genome over the course
of amplification as tested by RT-PCR. (C) Pre- and post-amplification plaque assay results
with the mean titer is displayed for each group as a solid line. The limit of detection for each
(RT-PCR LOD = 1 log10; Plaque assay = 1.1 log10) is displayed as a dashed line. (POS) is virus
spiked into water (B) or media (D) to serve as a positive control.
S4 Fig. HTNV and PUUV infection do not lead to changes in hematological parameters.
Syrian hamsters were infected either 10 PFU HTNV i.m., 500 PFU HTNV i.n., 1000 PFU
PUUV i.m., or 1000 PFU PUUV i.n. Whole blood was collected at the time of euthanasia and
evaluated for white blood cell count (A), red blood cell count (B), hematocrit (C), platelets
(D), neutrophils (E), lymphocytes (F), monocytes (G), eosinophils (H), basophils (I). The
gray box indicates the normal range of hamsters as determined by uninfected control
S5 Fig. Viral dissemination to organs in HTNV i.m. infected hamsters as detected by IHC.
Hamsters were infected with 10 PFU HTNV i.m and sacrificed at various time points post
infection. Heart, lung, liver, spleen, kidney and brain tissue were fixed in formalin, sectioned,
and stained by IHC to identify HTNV viral antigen. Representative images of organs from
normal and Day 28 are shown. Pictures at 400x magnification.
S6 Fig. HTNV or PUUV infection does not cause any changes in white blood cell levels.
Ferrets were challenged with either 200,000 PFU HTNV, 94,000 PFU PUUV Beaumont, or
164,000 PFU of PUUV Seloignes i.m. Whole blood was drawn weekly post infection and
evaluated for White blood cell count (A), platelets (B), neutrophils (C), lymphocytes (D), monocytes
(E), eosinophils (F), basophils (G). The gray box represents the average range of values for
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S7 Fig. No appreciable viral genome was detected in HTNV and PUUV infected ferrets.
Ferrets were challenged with either 200,000 PFU HTNV, 94,000 PFU PUUV Beaumont, or
164,000 PFU of PUUV Seloignes i.m. on Day 0, and immunosuppressed with 30 mg/kg Cyp
on Day 41. Sera was collected weekly and assayed for serum viremia by RT- PCR (A). At time
of euthanasia, heart, lung, liver, spleen, kidney, intestine and urine (except #7) were assayed by
RT-PCR for the presence of viral genome. No appreciable genome was recovered in ferrets
infected with HTNV (B), PUUV Beaumont (C) or PUUV Seloignes (D). Virus was spiked into
the samples to confirm no inhibitor was present. The limit of detection for RT-PCR is 1 log10
and is represented by the dashed line. (POS) is virus spiked into water to serve as a control.
S8 Fig. Immunosuppression with Cyp decreases ferret white blood cell counts. Ferrets were
challenged with either 200,000 PFU HTNV, 94,000 PFU PUUV Beaumont, or 164,000 PFU of
PUUV Seloignes i.m. and immunosuppressed with 30 mg/kg Cyp every other day beginning
on Day 41. Whole blood was drawn from ferrets to evaluate white blood count (WBC) (A),
lymphocyte count (B) and neutrophil count (C) prior to, two days post, and seven days post
Cyp administration (if alive). Line depicting mean is shown. Student t-test or Mann?Whitney
test was used to compare values between pre-Cyp and Cyp day 2 depending on the standard
deviation of the groups being compared. As only two ferrets survived until Cyp Day 7 no
statistics are possible. P<0.0001.
S9 Fig. No infectious virus was recovered from the organs of HTNV or PUUV infected
ferrets. Ferrets were challenged with either 200,000 PFU HTNV, 94,000 PFU PUUV Beaumont,
or 164,000 PFU of PUUV Seloignes i.m. on Day 0, and immunosuppressed with 30 mg/kg Cyp
on Day 41. Heart, lung, liver, spleen, kidney, and intestine were collected and assayed by
plaque assay for the presence of infectious virus (A). To confirm lack of virus recovered was not
due to inhibitors, virus was spiked into serial dilutions of organ homogenate to confirm no
inhibitor was present (B). For a standard plaque assay the limit of detection, 1.7 log10, is
depicted as a dashed line in (A). In (B) the dashed line is amount of HTNV plaques obtained
when spiked into media rather than organ homogenate.
S10 Fig. Immunosuppression of uninfected ferrets leads to rapid weight loss and secondary
bacterial infection. Uninfected ferrets were administered 10mg/kg Cyp, and the antibiotic
enrofloxicin, according to the schedule in (A). Weight (B) and temperature (C) are shown. b.i.d
indicates antibody was administered twice daily, and q.d indicates antibiotic was administered daily.
S11 Fig. PUUV infected ferrets had no infectious virus in the organs. Ferrets were infected
with 94,000 PFU PUUV Beaumont i.m. Heart, lung, liver, spleen, kidney, intestine, brain, eye,
and adrenal gland were collected on day 35 post infection and assayed for infectious virus by
plaque assay (A). Virus was spiked in to confirm no inhi bitor was present (B). For a standard
plaque assay the limit of detection, 1.7 log10, is depicted as a dashed line in (A). In (B) the
mean ? SEM is depicted in all spiked groups and the dashed line is amount of HTNV plaques
obtained when spiked into media rather than organ homogenate.
S12 Fig. PUUV infected ferrets had no viral genome in their organs. Ferrets were infected
with 94,000 PFU PUUV Beaumont i.m. Heart (A), lung (B), liver (C), spleen (D), kidney (E),
intestine (F), brain (G), and eye (H), were collected on Day 35 post infection and assayed for
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viral genome by RT-PCR. Viral genome was spiked in to confirm no inhibitor was present.
The mean ? SEM is depicted and the limit of detection, 1 log10, is shown as a dashed line. (NS)
not spiked, (S) spiked and (POS) virus spiked into water.
S13 Fig. No blood chemistry changes occurred in ferrets infected with PUUV. Ferrets were
infected with 94,000 PFU PUUV Beaumont i.m. Sera was collected twice weekly for blood
chemistry analysis. Sodium (A), potassium (B), total CO2 (C), chlorine (D), calcium (E),
glucose (F), albumin (G), total protein (H), ALP (I), ALT (J), AST (K), total bilirubin (L). Shaded
gray areas represent normal range (all reference values except tC02 from [
], tC02 from [
S14 Fig. Urinalysis remained normal after PUUV infection of ferrets. Ferrets were infected
with 94,000 PFU PUUV Beaumont i.m. Urine was collected twice weekly for urinalysis.
Leukocytes (A), nitrite (B), urobilinogen (C), pH (D), specific gravity (E), ketone (F), bilirubin (G),
and glucose (H).
S15 Fig. Organs from PUUV infected ferrets do not display virus-associated pathology.
Ferrets were euthanized 35 days post a 94,000 PUUV Beaumont i.m. challenge. Heart, lung,
liver, kidney, spleen, small intestine, adrenal gland, pituitary gland, cerebellum and cerebrum
were evaluated by H&E. Pictures taken at 10x magnification. ND, no data.
S16 Fig. No changes in HTNV infected marmoset blood chemistry occurred. Three
marmosets were infected with 1,000 PFU HTNV i.m. Sera was collected weekly for blood chemistry
analysis. Glucose (A), GGT (B), amylase (C), calcium (D), ALT (E), AST (F), ALP (G), total
bilirubin (H), total protein (I) albumin (J), blood urea nitrogen (K), and creatinine (L) were
measured. Gray shaded area represents reference values (all reference values except GGT and
bilirubin from [
], reference values for GGT and bilirubin from [
S17 Fig. Marmoset do not have infectious virus in their organs after HTNV infection.
Three marmosets were infected with 1,000 PFU HTNV i.m. On Day 30 post infection organs
were harvested, and the presence of infectious virus was determined by plaque assay (A).
To confirm no inhibitors were present, virus was spiked into samples (B). For a standard
plaque assay the limit of detection, 1.7 log10, is depicted as a dashed line in (A). In (B) the
mean ? SEM is displayed for all spiked groups, and the dashed line is amount of HTNV
plaques obtained when spiked into media rather than organ homogenate.
S18 Fig. Marmoset do not have viral genome in their organs after HTNV infection. Three
marmosets were infected with 1,000 PFU HTNV i.m. On Day 30 organs were harvested, and
the presence of viral genome was determined by RT-PCR. To confirm no inhibitors were
present, viral genome was spiked into samples. Heart (A), lung (B), liver (C), spleen (D), kidney
(E), intestine (F), and brain (G) were collected. The mean ? SEM is shown for the not spiked
(NS) and spiked (S) groups and the limit of detection, 1 log10, is depicted as a dashed line.
(POS) is viral genome spiked into water.
S19 Fig. Organs collected from HTNV infected marmosets do not display virus-associated
pathology. Marmosets were euthanized 30 days post 1,000 PFU HTNV i.m. challenge. Heart,
25 / 30
lung, liver, kidney, spleen, small intestine, adrenal gland, pituitary gland, cerebrum, and
cerebellum from normal and infected animals were stained by H&E for gross pathological changes.
Representative images in this Fig are taken from all three animals. Pictures at 10x
We thank the USAMRIID Veterinary Medical Division and Pathology Division, specifically
Simon Long and Kevin Zeng, for technical assistance. This work was supported in part by the
Postgraduate Research Participation Program at USAMRIID administered by the Oak Ridge
Institute for Science and Education through an interagency agreement between the U.S. DOE
and U.S. Army Medical Research and Material Command (USAMRMC) (DE-SC0014664, Dr.
Steven Kwilas), and by the Military Infectious Disease Research Program (MIDRP) Program
Area T (Dr. Jay Hooper). There was no additional external funding received for this study.
Opinions, interpretations, conclusions, and recommendations are ours and are not necessarily
endorsed by the U.S. Army or the Department of Defense. No competing interests declared.
The funders had no role in study design, data collection and analysis, decision to publish or
preparation of the manuscript.
Conceptualization: Casey C. Perley, Rebecca L. Brocato, Jay W. Hooper.
Formal analysis: Casey C. Perley, Rebecca L. Brocato, Steven A. Kwilas, Sharon Daye, Alicia
Moreau, Donald K. Nichols, Jay W. Hooper.
Funding acquisition: Casey C. Perley, Rebecca L. Brocato, Jay W. Hooper.
Investigation: Casey C. Perley, Rebecca L. Brocato, Steven A. Kwilas, Sharon Daye, Alicia
Moreau, Donald K. Nichols, Kelly S. Wetzel, Joshua Shamblin, Jay W. Hooper.
Methodology: Casey C. Perley, Rebecca L. Brocato.
Project administration: Jay W. Hooper.
Writing ? original draft: Casey C. Perley.
Writing ? review & editing: Rebecca L. Brocato, Steven A. Kwilas, Sharon Daye, Alicia
Moreau, Donald K. Nichols, Kelly S. Wetzel, Joshua Shamblin, Jay W. Hooper.
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27 / 30
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