Materials and MethodsCells and Viru

Materials and Methods
Cells and Viruses.MDCKcells were maintained in Eagle’sminimum
essential medium supplemented with 10% FBS. Stocks of the
influenza viruses Pan
99 (H3N2), A
Hong Kong
8
68 (H3N2),
A
WSN
33 (H1N1), A
Texas
36
91 (H1N1), avian influenza
A
Vietnam
1203
04 (H5N1), and the 1918 virus (24) were prepared
by inoculation of 10-day-old embryonated hens’ eggs or
MDCK cells followed by incubation at 37°C for 48 h for human
viruses or at 37°C for 24 h for the avian virus. All experiments
involving the 1918 and A
Vietnam
1203
04 viruses were performed
under strict biosafety level 3 conditions (35).All guinea pigs
were seronegative to Pan
99 virus on arrival, and those inoculated
or secondarily infected were found to seroconvert.
Animals. Eight-week-old female BALB
c mice and female Hartley
strain guinea pigs weighing 300–350 g were obtained from
Charles River Laboratories. Animals were allowed free access to
food and water and kept on a 12-h light
dark cycle. During
guinea pig transmission experiments, strict measures were followed
to prevent aberrant cross-contamination between cages.
Sentinel animals were handled before inoculated animals, gloves
were changed between animal handlings, and work surfaces were
sanitized after each use.
Mouse Transmission Experiments. Mice were anesthetized with
Avertin (tribromoethanol; Sigma) before infections. Fifty microliters
of infectious virus diluted in PBS was given intranasally.
Twenty-four hours later, three naı¨ve mice were placed in the
same cage with seven inoculated mice. Duplicate cages were set
up for each virus to monitor for (i) virus replication in the
respiratory tract and (ii) clinical signs and seroconversion at 21
days p.i. For virus determination, three inoculated or contact
mice were killed on day 4 p.i.; lung and nose tissues were
collected, immediately frozen on dry ice, and stored at 70°C
until processing. Frozen tissues were later thawed, homogenized
in 1 ml of cold PBS, and clarified by centrifugation (at 2,200 g) at 4°C. Clarified tissue homogenates were titrated for virus
infectivity in eggs. Weight loss was determined on alternate days
for all mice for 21 days p.i.
Infection and Monitoring of Guinea Pigs. Before infection, guinea
pigs were anesthetized with a mixture of ketamine (30 mg
kg)
and xylazine (2 mg
kg) administered intramuscularly. For intranasal
infections, Pan
99 virus stock was diluted in PBS
containing 100 units
ml penicillin, 100 g
ml streptomycin, and
0.3% BSA (PBS-PS-BA). A 300-l volume of inoculum containing
102 or 103 pfu was instilled into the nostrils (150 l on
each side). Body temperature was measured twice daily from 2
days before infection up to 14 days p.i. by using s.c. implanted
telemetric transponders from BioMedic Data Systems (Maywood,
NJ). Body weight was measured once daily.
Collection of Guinea Pig Nasal Wash Samples and Lung Tissue. Nasal
washing was performed by instilling a total of 1ml of PBS-PS-BA
into the nostrils and allowing it to drain onto a sterile Petri dish.
Samples were collected in 1.5-ml tubes and centrifuged for 5 min
at 2,000  g and 4°C; supernatants were stored at 80°C before
analysis by plaque assay. For the collection of lungs, animals
were killed through i.p. injection of 100 mg
kg sodium pentobarbital
(Nembutal; Ovation Pharmaceuticals, Deerfield, IL).
Extracted lung tissue was weighed, homogenized in PBS, and
subjected to centrifugation at 12,000  g and 4°C for 10 min to
pellet debris. Supernatants were stored at 80°C before analysis
by plaque assay.
Quantification of Viral Titers. Virus titers were determined by
plaque assay of 10-fold serial dilutions on MDCK cells or by
limiting dilution in embryonated chicken eggs, starting from an
initial dilution of 1:10 in PBS. The limit of virus detection was
101.2 50% egg infectious dose
ml or 10 pfu
ml.
This work was supported by National Institutes of Health Grants P01
AI58113, U54 AI057158, and UC19AI-062623 (to A.G.-S. and P.P.) and
R01 AI18998-25 (to P.P.). P.P. is a senior fellow of the Ellison Medical
Foundation. S.M. was supported by Sunnybrook Health Sciences Centre,
University of Toronto (Toronto)