A/Mexico/4108/2009 (H1N1) (Mex4108) was isolated from a clinical specimen (kindly provided by the Centers for Disease Control and Prevention, Influenza Branch) obtained from a 4-year-old boy from Mexico who was mildly ill. A/Mexico/InDRE4487/2009 (H1N1) (Mex4487) was isolated from the bronchial aspirate of a 26-year-old man from a family cluster of three confirmed severe cases in Mexico (20
). A/Kawasaki/UTK-4/2009 (H1N1) (Kaw) was isolated from a human and represents a recent seasonal virus (17
). The viruses were grown in MDCK cells, harvested at a cytopathogenic effect of 3+ (>70% rounded off and detached cells) for virus stock preparation, and titrated by using a 50% tissue culture infective dose (TCID50
) assay on MDCK cells. The Mex4108, Mex4487, and Kaw infections were done with MDCK passage numbers 3, 2, and 2, respectively. All infectious work with the SOIVs was done under enhanced BSL3 conditions in the Integrated Research Facility of the Rocky Mountain Laboratories (RML), National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH).
In vitro infection (A549 cells).
Freshly seeded monolayer of A549 cells were infected with the different H1N1 virus isolates with 104 TCID50/100 μl/well. Following an adsorption of 1 h at 37°C, unbound viruses were washed away, and 1 ml of minimal essential medium (MEM) supplemented with 0.35 μg of TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone)-trypsin/ml was added to the cultures. The cells were incubated at 37°C, and supernatants were collected at 12, 24, 48, and 72 h postinfection. Virus titration was performed by using a TCID50 assay on MDCK cells.
In vivo infection (cynomolgus macaques).
Twenty-one cynomolgus macaques (Macaca fascicularis
), 4 to 15 years old, weighing 3.0 to 8.7 kg, were evenly distributed with regard to age and weight over the different groups and infected with either Mex4108 (n
= 6), Mex4487 (n
= 9), or Kaw (n
= 6) under anesthesia through a combination of intratracheal (4 ml), intranasal (0.5 ml per nostril), conjunctival (0.5 ml per eyelid), and oral (1 ml) routes with a suspension containing 106
/ml (the total infectious dose was 7 × 106
). The animals were monitored daily for clinical signs using an approved scoring sheet (fever, posture, respiration, feces/urine, food intake, recumbence, attitude, and skin turgor). On days −2, 0, 1, 3, 6, 8, 11, and 14 postinfection the animals were examined under anesthesia, at which point X-ray, pulse rate, blood pressure, temperature, and respiration rate were taken, and each animal was bled for blood chemistry analysis, differential blood count, virology, and cytokine profiles. Two (Mex4108) or three (Mex4487, Kaw) animals were euthanized and necropsied on days 1, 6, and 14 postinfection (Kaw only on days 1 and 6) with collection of clinical specimens from nasal mucosa, oral mucosa, conjunctiva, tonsils, cervical lymph nodes, trachea, bronchi, right and left lungs (upper, middle, and caudal), hilar lymph nodes, heart, liver, spleen, pancreas, jejunum, transverse colon, kidney, adrenal gland, and mesenteric lymph nodes. All animal experiments were approved by the RML Institutional Animal Care and Use Committee (IACUC) and performed according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) by certified staff in an AAALAC-approved facility.
Radiographic imaging and gross pathology scoring.
X-ray images were scored blindly according to the following scheme: grade 0, normal examination; grade 1, mild interstitial pulmonary infiltrates; grade 2, moderate interstitial pulmonary infiltrates, possibly including partial cardiac border effacement and small areas of pulmonary consolidation; and grade 3, pulmonary consolidation as primary lung pathology, often seen as progression from grade 2 lung pathology. The percentage of affected lung area was assessed in all lung lobes during necropsy by macroscopic examination.
Hematology and serum biochemistry.
The total white blood cell (WBC) count, lymphocyte, platelet, reticulocyte, and red blood cell counts, hemoglobin, hematocrit values, mean cell volume, mean corpuscular volume, and mean corpuscular hemoglobin concentrations were determined from EDTA blood with the HemaVet 950FS+ laser-based hematology analyzer (Drew Scientific, Waterbury, CT). Plasma biochemistry was analyzed from heparin blood by using the blood chemistry analyzer iSTAT1 (Abbott Point of Care, Princeton, NJ). Urea nitrogen (BUN), glucose, chloride, sodium, potassium, hematocrit, hemoglobin, pH, PCO2, TCO2, base excess (BEecf), and anion gap values were determined by using an EC8+ cartridge. Creatinine values were evaluated by using Crea cartridges.
Plasma cytokine analysis.
Concentrations of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, gamma interferon, interleukin-1β (IL-1β), IL-4, IL-5, IL-6, IL-8, IL-17, monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 1α in plasma of animals was determined on days 0, 1, 3, 6, 8, 11, and 14 postinfection by using a non-human primate cytokine Milliplex map kit (Millipore Corp., Billerica, MA) as described by the manufacturer. Samples were read by using a Bio-Plex 200 system (Bio-Rad, Hercules, CA).
Tissue samples were placed in RNAlater (Ambion) for subsequent RNA extraction (RNeasy kit; Qiagen). Tissue homogenates (10% [wt/vol]) were prepared in MEM-bovine serum albumin. Debris was pelleted by centrifugation (2,000 × g, 5 min), and virus titers were determined in 10-fold dilutions of supernatant by standard TCID50 assay on MDCK cells, in triplicate for each dilution. Virus titers were similarly determined in blood and swab suspensions.
Histopathology and immunohistochemistry.
Animal tissues were fixed in 10% phosphate-buffered formalin. Fixed tissues were processed by conventional methods, embedded in paraffin, cut into 5-μm-thick sections, and stained with standard hematoxylin and eosin. Slides for immunohistochemistry were stained by using the Discovery XT and DAB map kit from Ventana Medical Systems (VMS), Tucson, AZ. The polyclonal SOIV (H1N1) hemagglutinin (HA) antibody (ProSci., Inc., Poway, CA) was diluted in antibody dilution buffer (VMS) at 1:500, followed by incubation for 32 min. Next, a biotinylated secondary of goat anti-rabbit SS Link (Biogenex, San Ramon, CA) was incubated for 32 min, followed by enzyme conjugate, and diaminobenzidine. Slides were counterstained with hematoxylin, dehydrated, cleared in xylene, and coverslipped.
Microarray analysis was performed using Agilent rhesus macaque 4×44K microarrays using the manufacturer's one-color analysis protocol. TaqMan was used to analyze lung samples for the presence of viral RNA. Because viral RNA was not detected in the sample available for microarray from animal 2 in the Mex4487 group, this animal was not included in subsequent transcriptional analyses. Gene expression in individual samples was quantified relative to gene expression from a pooled sample of lung RNA from seven uninfected cynomolgus macaques (five females and two males; ages, 7.5 to 12.9 years; 2.6 to 6.2 kg). Differential gene expression between the groups was identified at day 1 postinfection using a one-way Textbook analysis of variance (ANOVA; P
< 0.01) using Rosetta Resolver (Rosetta Biosoftware). To filter gene sequence that changed minimally between treatment groups, gene sets from the ANOVAs were then filtered to include genes that changed as least 2-fold between any two of the treatment groups. Functional analysis of statistically significant gene expression changes was performed by using ingenuity pathway analysis (IPA; Ingenuity Systems), which analyzes gene expression data in the context of known biological response and regulatory networks. A Fisher exact test was used to determine the probability that each biological function assigned to that data set was due to chance alone. Heat maps for gene expression were created by using Spotfire DecisionSite 9.1.1. All gene expression data described here are publicly available at http://viromics.washington.edu