Susceptibility to and transmission of H5N1 and H7N1 highly pathogenic avian influenza viruses in bank voles (Myodes glareolus)

Viruses

The two HPAI isolates used for this study were H7N1 A/ostrich/Italy/2332/2000 (Os/H7N1)
[GenBank: PB2:DQ991309, PB1:DQ991310, PA:DQ991311, HA:DQ991312, NP:DQ991313, NA:DQ991314,
MA:DQ991315, NS: DQ991316], isolated during the Italian epidemic of 1999–2000 33], and H5N1 A/turkey/Turkey/1/2005 (Tk/H5N1) [GenBank: PB2: EF619975, PB1: EF619976,
PA: EF619979, HA: EF619980, NP: EF619977, NA: EF619973, MA: EF619978, NS: EF619974].
The two viruses were replicated in the allantoic cavity of 9- to 11-days old embryonated-specific
pathogen-free (SPF) hen eggs according to OIE guidelines 34], and allantoic fluids were titrated to calculate the 50% Embryo Infectious Dose (EID₅₀) using the Reed and Muench formula 35].

Animals

Eighty five (male and female) bank voles (Myodes glareolus) of 4–6 weeks of age (13–16 g body weight) were obtained from a colony originally
derived from the Istituto Superiore di Sanità (ISS, Rome, Italy). Prior to each experiment,
all animals were left for acclimatization to the local environment for at least 1 week.
Bank voles were housed in mouse cages with standard rodent feed and water ad libitum, and were weekly supplemented with grain seeds. Cages were prepared with a 3–4 cm
bedding layer, and animals were provided with sufficient nesting material and environmental
enrichments in order to express their species-specific behaviour (digging and burrowing).
All in vivo experiments, approved by the IZSVe Ethics Committee (CE.IZSVE.24/2014)
and authorized by the Italian Ministry of Health (Decree N.180/2011-B), were performed
in containment facilities (BSL-3) and in accordance with the relevant legislation
on the use of animals for scientific purposes 36].

Study design

Serology

Prior to challenge, blood was collected from all voles (tail vein) and sera were tested
by means of a competitive commercial anti-nucleoprotein (NP) influenza ELISA (ID-screen®,
IDVet, Montpellier, France) and through the Haemagglutination Inhibition (HI) test
according to the WHO procedure used for mammal sera 37], using the challenge viruses as antigens (naïve sera from animals of each experimental
group were tested only with the corresponding challenge virus). The same tests were
used to evaluate the seroconversion on convalescent sera.

Nasal shedding experiment

Since no preliminary information existed regarding the infectivity of HPAI viruses
in bank voles, the challenge dose of each selected virus was established as ten times
the 50% Mouse Lethal Dose (MLD₅₀) as for previous experiments in BALB/c mice 38].

To evaluate the occurrence of nasal shedding, two groups of 12 male and female bank
voles each (groups Os/H7N1 and Tk/H5N1) were inoculated intranasally, under light
anesthesia (50 mg/kg ketamine and 4 mg/kg xylazine, intraperitoneally), with 50 ?L
of PBS-diluted allantoic fluid containing 10 times the MLD₅₀ calculated in Balb/C mice, equivalent to 10^3.75 and 10^4.4 EID₅₀/0.1 mL of the H7N1 and H5N1 viruses, respectively. Three voles were used as negative
controls and were mock-infected with 50 ?L of sterile phosphate-buffered saline (PBS)-diluted
allantoic fluid.

On a daily basis, challenged animals were monitored for the onset of clinical signs.
Animals reaching the humane endpoint (weight loss greater than 20% and/or severe depression
and respiratory distress) were euthanized. On days 3, 5, 7 and 9 post-infection (pi),
three subjects were randomly selected and euthanized to collect organs. Brain, lungs
and trachea tissues were collected from each animal along with a nasal wash (using
400 ?L of sterile PBS). On day 3 post-inoculation, all control voles were also euthanized
for collection of samples. Brain and nasal washes were tested by quantitative real
time reverse transcriptase polymerase chain reaction (qRRT-PCR), while whole lungs
and trachea were paraffin-embedded for histopathological and immunohistochemical (IHC)
examination. Virus isolation in SPF embryonated hen eggs was attempted from all qRRT-PCR
positive samples.

Pathogenicity and contact transmission experiment

Two groups of 24 female voles each were used for the transmission experiments; each
group was randomly selected after weaning and reared together in a proper size cage
(2065 cm² floor area). For each isolate, the experimental group included 12 infected voles
(groups I-Os/H7N1 and I-Tk/H5N1) and 12 sentinels. The voles of each infected group
were challenged by the intranasal route, as previously described, with 50 ?L of PBS-diluted
isolate. Twenty-four hours later, infected voles were moved into a clean cage hosting
a group of 12 serologically naïve sentinels. Contemporaneously, 10 mock-infected voles
were used as controls.

On a daily basis, all animals were observed for the onset of clinical signs and for
mortality, and body weight was monitored on days 2, 4, 7 and 9 pi. Significance in
body weight changes was calculated by the Student’s t-test, and a P value less than 0.05 (p??0.05) was considered statistically significant. On days 3 and 4 pi, post-contact
(pc) and post-inoculation, two animals per group (infected, sentinels and control,
respectively) were randomly selected and euthanized to collect nasal washes and different
organs including lungs, trachea, brain, spleen, kidney and intestine. On day 30 pi/29
pc, all remaining voles were humanly euthanized, and convalescent sera were collected
to determine seroconversion.

RRT-PCR and qRRT-PCR

Sample preparation. One hundred milligrams of tissues were homogenized in 200 ?L of lysis buffer of
the commercial kit Nucleospin RNA (Macherey Nagel, Düren, Germany), using the TissueLyser
II (Qiagen, Germantown, USA) and following a homogenization program of 30 Hz for 3 min.
The homogenates were clarified by centrifugation and tested by means of RRT-PCR.

RNA extraction. Viral RNA was extracted from 100 ?L of clarified supernatant from tissue samples
or from nasal washes, using the commercial kit Nucleospin RNA II and following the
manufacturer’s instructions. Viral RNA was eluted in 60 ?L of RNAse-free water and
stored at ?80 °C after the addition of 1 ?L of RNasin Plus RNase Inhibitor (Promega®,
Madison, USA).

One–Step RRT–PCR and qRRT–PCR. The extracted RNA was amplified using the published primers and probe from Spackman
et al. 39], targeting the conserved matrix (M) gene of the IA virus. Five microlitres of RNA
were added to the reaction mixture, composed of 300 nM of forward and reverse primers
(M25F and M124-R, respectively) and 100 nM of fluorescent label probe (M?+?64). The
amplification reaction was performed in a final volume of 25 ?L using the commercial
QuantiTect Multiplex RT-PCR kit (Qiagen, Hilden, Germany). The RRT-PCR was performed
using the following protocol: 20 min at 50 °C and 15 min at 95 °C, followed by 40 cycles
at 94 °C for 45 s and 60 °C for 45 s. According to the internal validation trails,
the samples with a threshold cycle (Ct) higher than 35 were considered negative; however,
all Ct values obtained for each sample were reported in the results.

The same one-step RRT-PCR protocol was used for the qRRT-PCR analysis. The synthetic
RNA used as reference standard was created in vitro by means of the MegaScript® T7
kit (Invitrogen, CA, USA) from the H7N1 (A/Ostrich/Italy/2332/2000) and H5N1 (A/turkey/Turkey/1/2005)
HPAI strains.

Virus isolation

Nasal washes (around 200 ?L) were diluted in 400 ?L of PBS containing antibiotics
(10 000 IU/mL of penicillin, 10 mg/mL of streptomycin, 5000 UI/mL of nystatin and
250 mg/mL of gentamycin sulphate), while tissue samples (100 mg) were homogenized
using sterile polypropylene micropestles (Sigma-Aldrich, Hamburg, Germany) in 700 ?L
of PBS containing antibiotics, and then clarified by centrifugation. Processed samples
were propagated into the allantoic cavities of 9 to 11 days-old SPF embryonated hen
eggs according to the OIE guidelines 34].

Histopathology and Immunohistochemistry

After 24 h, the lungs and trachea collected from the infected and mock-infected voles
were fixed in 10% neutral phosphate-buffered formalin, paraffin-embedded, and stained
with hematoxylin-and-eosin method for histopathological examination. The immunohistochemistry
(IHC) was carried out by an automated immunostainer (Autostainerâ„¢ Link 48 Dako, Italy).
Briefly, the antigen retrieval was performed with Proteinase K (Dako, Italy) for 3 min
at room temperature (RT). Endogenous peroxidases were neutralized by incubating the
sections with the EnVision FLEX Peroxidase-Blocking Reagent (Dako, Italy) for 10 min
at RT. Sections were incubated with a primary monoclonal antibody against IA virus
nucleoprotein (Clone 1331, BIODESIGN International, USA), applied at 1:500 dilution
for 10 min at RT. The EnVision FLEX/HRP (Dako, Carpinteria, CA, USA) and the EnVision
FLEX Substrate Buffer EnVision FLEX DAB+ were used as the detection system and chromogen,
respectively. Sections were then counterstained with the EnVision FLEX Hematoxylin
(Dako, Italy). The specificity of the immunostaining was verified by incubating some
sections with PBS instead of the specific primary antibody.