Cells and Tissues
Madin-Darby canine kidney (MDCK) cells and MDCK cells overexpressing Siaα2,6 Gal (MDCK/AX4)32,33 were maintained in Eagle’s minimal essential medium (MEM) containing 5% newborn calf serum (NCS) and antibiotics. Human epithelial HeLa cells were maintained in MEM containing 10% FBS and antibiotics. Adenocarcinomic human alveolar basal epithelial (A549) cells were maintained in a 1:1 mixture of Dulbecco’s modified essential medium (DMEM) and Ham’s F12 (DMEM/F12, Gibco) medium with 10% fetal calf serum (FCS) and antibiotics. Chicken fibroblast (DF-1) cells were grown in Dulbecco’s modified essential medium (DMEM) with 10% FCS and antibiotics. Cells were maintained at 37 °C or 39 °C (DF-1) in 5% CO2. Paraffin-embedded tissue sections of the human trachea and lung were purchased from US Biomax.
Oropharyngeal swabs were collected from household poultry as part of routine and targeted surveillance activities in different localities of Egypt (Supplementary Table S1). Swabs were placed in transport medium [phosphate-buffered saline with antibiotics], transported to the laboratory on ice, and stored at −80 °C. All samples were amplified in embryonated chicken eggs, and virus stock titers were determined by means of plaque assays in MDCK cells. Virus stocks were stored at −80 °C. The full genomic sequences of the amplified surveillance samples and viruses isolated from exposed animals were determined by Sanger sequencing. The sequence data were submitted to Genbank (Accession numbers LC106039 - LC106110).
Human and avian control viruses used in this study included A/Kawasaki/173/2001 (K173, H1N1)11, A/Vietnam/1203/2004 (VN1203, H5N1)11, A/California/04/2009 (H1N1)34, A/Kawasaki/UTK-4/2009 (H1N1)34, and A/Kawasaki/UTK-23/2008 (H1N1)34.
Viruses were diluted 10-fold in MEM containing 0.3% BSA. Confluent monolayers of MDCK cells were washed with MEM containing 0.3% BSA, infected with diluted viruses, and incubated for 30–60 min at 37 °C. After the virus inoculum was removed, the cells were washed with MEM containing 0.3% BSA and overlayed with a 1:1 mixture of 2x MEM/0.6% BSA and 2% agarose containing 0.5–1 μg/ml Tosylsulfonyl Phenylalanyl Chloromethyl Ketone (TPCK)-trypsin. Plates were incubated at 37 °C for 48–72 h before virus plaques were counted; titers were calculated by using the method of Reed and Munch35.
Hemagglutination (HA) Assay
Viruses (50 μl) were serially diluted two-fold with 50 μl of PBS in a U-well microtiter plate. Fifty microliters of 0.5% (vol/vol) of chicken red blood cells (CRBC) or turkey red blood cells (TRBCs) were added to each well. The plates were incubated at room temperature and hemagglutination was evaluated after 45 min. The HA titers were calculated as the highest dilution at which complete agglutination was observed.
Hemagglutination Inhibition (HI) Assay
To detect hemagglutination inhibition activity, serum samples were treated with receptor-destroying enzyme (RDE; Denka Seiken Co., Ltd) at 37 °C for 16–20 h, followed by RDE inactivation at 56 °C for 30–60 min. The RDE-treated sera were then serially diluted two-fold in PBS, mixed with an amount of virus equivalent to eight hemagglutination units, and incubated at room temperature for 30–60 min. After the addition of 50 μl of 0.5% (vol/vol) CRBC or TRBC, the resulting mixture was gently mixed and incubated at 4 °C or room temperature for 30–45 min. HI titers were recorded as the inverse of the highest antibody dilution that inhibited 8 HA units of virus.
Virus Neutralization Assay
Viral neutralization assays were performed by using the methodology outlined in the WHO Manual on Animal Influenza Diagnosis and Surveillance with the following modifications. Briefly, sera were treated with RDE at 37 °C for 18–20 h, followed by RDE inactivation at 56 °C for 30–60 min. Fifty microliters of virus (100 tissue culture infectious dose 50) was incubated with 50 μl of two-fold serial dilutions of RDE-treated sera for 30 min at 37 °C, and the mixtures were added to confluent MDCK cells in 96-well microplates, and incubated for 1 h at 37 °C. After the inoculum was removed, the cells were incubated with MEM containing 0.3% BSA and 0.75 μg/ml TPCK-trypsin at 37 °C for 48–72 h. Viral cytopathic effects were observed under an inverted microscope and virus neutralization titers were calculated as described in the WHO manual.
The following human serum samples were obtained from the NIH Biodefense and Emerging Infections Research Resources Repository (BEI Resources), NIAID, NIH: Polyclonal anti-monovalent influenza subvirion vaccine rgA/Vietnam/1203/2004 (H5N1), low titer pool, NR-4110, and high titer pool, NR-4109; and human reference antiserum to influenza A/Indonesia/05/2005 (H5N1), low titer, NR-33667, medium titer, NR-33668, and high titer, NR-33669. Human serum samples were also collected from volunteers who had received the alum-adjuvanted, inactivated whole H5N1 virus vaccine A/Egypt/N03072/2010 (H5N1; IDCDC-RG 29) under a research protocol approved by the Research Ethics Review Committee of the Institute of Medical Science, University of Tokyo (approval number 25-58-1205). Informed consent was obtained from all subjects. All methods were carried out in accordance with the “Ethical Guidelines for Medical and Health Research Involving Human Subjects” from Ministry of Education, Culture, Sports, Science and Technology, Japan. Samples were treated with RDE, heat-inactivated, and tested in HI and viral neutralization assays. The viruses indicated in Supplementary Tables S13 and S14 served as antigens.
Human A549 and avian DF-1 cells were transfected with plasmids for the expression of Giza virus PB2, PB1, PA, and NP proteins. To test the PB2-A84T and -D146G mutations, the wild-type PB2 protein expression plasmid was substituted with plasmids expressing Dakahlia PB2-A84T or Giza PB2-D146G, respectively (note that the Dakahlia and Giza PB2 genes differ by two synonymous nucleotide replacements, but encode identical PB2 proteins). Cells were also transfected with pPol-I-NP(0)Luc2(0) (for A549 cells) or pPol-IGG-NP(0)Fluc(0) (for DF-1 cells), which express the firefly luciferase reporter protein from a virus-like RNA transcribed by the human or avian polymerase I promoter, respectively. Plasmid pRL-TK (Promega, Madison, WI) served as an internal control for the dual-luciferase assay. Transfected cells were incubated for 24 h at 33 °C and 37 °C (human A549 cells), or at 39 °C (avian DF-1 cells). Luciferase activity was measured by using the Dual-Glo luciferase assay system (Promega) on a Glomax microplate luminometer (Promega) according to the manufacturer’s instructions. The results of two experiments (both carried out in duplicate) were compared using one-way ANOVA, followed by Tukey’s Post-hoc test. We considered the results significant at p < 0.05.
Solid-Phase Binding Assay
Viruses were amplified in MDCK cells. Virus-containing supernatant was centrifuged at low speed for 15 min to remove cell debris, and then laid over a cushion of 30% sucrose in PBS. After ultracentrifugation at 96,174 × g for 90 min at 4 °C, the virus pellet was resuspended in PBS containing glycerol and stored at −80 °C. The concentration of the viruses was determined by means of HA assays with 0.5% (vol/vol) chicken red blood cells.
For the binding assay, microtiter plates (Nunc) were incubated with the sodium salts of sialylglycopolymers [poly-L-glutamic acid backbones containing N-acetylneuraminic acid linked to galactose through either an α2,3 (Neu5Acα2,3Galβ1,4GlcNAcβ1-pAP) or an α2,6 (Neu5Acα2,6Galβ1,4GlcNAcβ1-pAP) bond]36 in PBS at 4 °C overnight. After removing the glycopolymers, we blocked the plates with 150 μl of PBS containing 4% BSA at room temperature for 1 h, and then washed them four times with cold PBS. Influenza virus equivalent to 32 HA units (in PBS) was then added to the plates and incubated overnight at 4 °C. After washing the plates as described above, we incubated them for 2 h at 4 °C with rabbit polyclonal anti-H5N1 (VN1203) or ferret polyclonal anti-H1N1 (K173) antibodies, respectively. After additional washes as described above, the plates were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Zymed) antiserum or anti-ferret IgG (Bethyl Laboratories) for 2 h at 4 °C. After several additional washes, the plates were incubated with O-phenylenediamine (Sigma) in citrate-phosphate buffer containing 0.03% H2O2 for 10 min at room temperature. The reaction was stopped with 50 μl of 1 M HCl, and absorbance was determined at 490 nm by using an optical plate reader (Mark microplate reader model 680; BioRad).
Tissue binding assay
Viruses were amplified in MDCK/AX4 cells. Virus-containing supernatants were collected from infected cells and centrifuged at 1,462 × g for 15 min to remove cell debris. Viruses were inactivated by incubating them with 0.1% β-propiolactone (final concentration) for at least 16 h at 4 °C. Virus supernatant was laid over a cushion of 30% sucrose in PBS and ultracentrifuged at 96,174 × g for 90 min at 4 °C; virus pellet was resuspended in PBS and stored at −80 °C. Virus concentrations were determined by using hemagglutination assays with 0.5% (vol/vol) TRBCs. To assess tissue binding, we deparaffinized and rehydrated paraffin-embedded normal human trachea and lung (US Biomax). The rehydrated sections were blocked with carbo-free blocking solution (Vector) and TNB blocking buffer (Perkin Elmer). Next, we added the equivalent of 8 HA units of fluorescein-5-isothiocyanate (FITC)-labeled virus to the tissue sections and incubated them at 4 °C overnight. The slides were washed five times with cold PBS, and subsequently incubated with an HRP-conjugated rabbit anti-FITC antibody (Dako) for 30 min at room temperature. After additional washes, the slides were incubated with AEC (3-amino-9-ethyl-carbozole) substrate-chromogen (Dako) for 15 min at room temperature. The slides were then rinsed with water, counterstained with Mayer’s hematoxylin (Sigma Aldrich) for 3 min at room temperature, and rinsed again with water. Finally, coverslips were mounted by using Shandon Immu-Mount (Thermo Scientific). We examined all tissue sections by using a high resolution camera (AxioCam HRc) mounted on a microscope (Zeiss, Axio Imager. A2).
Experimental infection of ferrets
Six-month-old female ferrets (Triple F Farms), which were serologically negative by HI assay for currently circulating human influenza viruses, were used in this study. Under anaesthesia, six ferrets per group were intranasally inoculated with 106 PFU (0.5 ml) of A/duck/Giza/15292 S/2015 (H5N1), A/duck/Dakahlia/1536CAG/2015 (H5N1), or A/duck/Cairo/1578CA/2015 (H5N1) virus. Three ferrets per group were euthanized on days 3 and 6 post-infection for virological and pathological examinations. The virus titres in various organs were determined by use of plaque assays in MDCK cells.
All experiments with ferrets were performed in accordance with the Science Council of Japan’s Guidelines for Proper Conduct of Animal Experiments and guidelines set by the Institutional Animal Care and Use Committee at the University of Wisconsin-Madison. The protocol was approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (approval number PA15-02) and the Animal Care and Use Committee of the University of Wisconsin-Madison (protocol number V00806). For more information, see ‘Biosafety Statement’.
Ferret Transmission Study
Six-month-old female ferrets (Triple F Farms) were first serologically tested for exposure to currently circulating human influenza viruses by using the HI assay. Two-to-six ferrets per transmission group were anaesthetized intramuscularly with ketamine and xylazine (5–30 mg and 0.2–6 mg/kg of body weight, respectively) in the studies carried out at University of Tokyo (i.e., studies 1–3), or with ketamine and dexmedetomidine (4–5 mg/kg and 0.01–0.04 mg/kg, respectively) in the study carried out at the University of Madison (i.e., study 4); animals were then inoculated intranasally with 106 PFU (500 μl) of virus. For ferrets anesthetized with ketamine and dexmedetomidine, atipamezole was used to shorten recovery time from anesthesia. The infected ferrets were housed in transmission cages that prevent direct and indirect contact between animals but allow spread of influenza virus through the air (Showa Science). Twenty-four hours later, one naïve ferret was placed in a cage adjacent to each inoculated ferret (exposed ferrets). All animals were assessed daily for clinical signs and symptoms and changes in body weight. Nasal washes were collected from infected and exposed animals on day 1 after infection or exposure, respectively, and then every other day. Virus titers in nasal washes were determined by means of plaque assays.
Mouse Virulence Studies
To determine the dose required to kill 50% of infected mice (MLD50), six-week-old female BALB/c mice (Jackson Laboratory, Bar Harbor, ME) (3 mice/group) were anesthetized with isoflurane and inoculated intranasally with 10-fold serially diluted virus (from 10−1 to 105 PFU) in a 50-μl volume. Changes in body weight and mortality were recorded daily for 14 days. Mice were euthanized if they lost more than 25% of their initial body weight The MLD50 was calculated by using the method of Reed and Muench35. To determine viral titers in the organs of infected animals, 6 mice/group were infected intranasally with 103 PFU of virus. Three mice in each group were euthanized on days 3 and 6 post-infection, respectively. Lungs, nasal turbinates, kidneys, spleens, and brains were collected for virus titration by use of plaque assays in MDCK cells. The data shown are the mean virus titers ± standard deviation.
All experiments with mice were performed in accordance with the guidelines set by the Institutional Animal Care and Use Committee of the University of Wisconsin-Madison, which also approved the protocol (protocol number V00806).
Excised tissues of animal organs were preserved in 10% phosphate-buffered formalin, processed for paraffin embedding, and cut into 3-μm-thick sections. One section from each tissue sample was stained using a standard hematoxylin-and-eosin procedure; another one was stained with a mouse monoclonal antibody for type A influenza virus nucleoprotein (NP) antigen (prepared in our laboratory) that reacts comparably with all of the viruses tested in this study. Specific antigen–antibody reactions were visualized with 3,3′-diaminobenzidine tetrahydrochloride staining by using the DAKO Envision detection system (DAKO Cytomation, Copenhagen, Denmark).
Neuraminidase Inhibition Assay
Diluted viruses were mixed with different concentrations of oseltamivir carboxylate (the active form of oseltamivir), zanamivir, laninamivir, or peramivir. Samples were incubated for 30 min at 37 °C, followed by the addition of methylumbelliferyl-N-acetylneuraminic acid (Sigma, St Louis, MO) as a fluorescent substrate. After incubation for 1 h at 37 °C, the reaction was stopped with the addition of sodium hydroxide in 80% ethanol. The fluorescence of the solution was measured at an excitation wavelength of 360 nm and an emission wavelength of 465 nm, and the 50% inhibitory concentration (IC50) was calculated.
HA gene sequences of highly pathogenic H5Nx influenza viruses of the goose/Guangdong lineage were downloaded from GISAID (Supplementary Table S19) and Genbank during the week of November 18, 2015; sequences from laboratory mutants were excluded. We required that the sequence included at least 75% of the HA coding region. This selection process resulted in a dataset of 5,848 sequences, including the HA sequence from A/chicken/Scotland/1959 for an out-group. Sequences were aligned using CLUSTALW37, then edited manually as needed. RAxML38 on CIPRES39 was used to infer a phylogeny for the final dataset, using the GTR model of evolution with a gamma model of rate heterogeneity; 100 bootstrap replicates under this model were used to assess topological certainty. The final tree was displayed in Archaeopteryx40.
Because this study involved the characterization of natural influenza viruses, it does not fall under the pause on gain-of-function research announced by the US Government on October 17, 2014.
All experiments were approved by the respective committees at the University of Tokyo and by the University of Wisconsin-Madison’s Institutional Biosafety Committee (IBC). When virus transmission was detected, the University of Wisconsin’s Alternate Responsible Official (ARO) and Institutional Contact for Dual Use Research (ICDUR) was contacted and a risk assessment was performed. All practices and procedures used for additional experiments followed the requirements of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules for working with mammalian-transmissible H5N1 viruses. The ARO/ICDUR was kept informed of the research results. This manuscript was reviewed by the University of Wisconsin-Madison Dual Use Research of Concern (DURC) Subcommittee in accordance with the United States Government September 2014 DURC Policy, which concluded that the studies described herein do not constitute DURC since the natural virus isolates were not modified or sequentially passed in our laboratory. In addition, the University of Wisconsin-Madison Biosecurity Task Force regularly reviews the research, policies, and practices of research conducted with pathogens of high consequence at the institution. This task force has a diverse skill set and provides support in the areas of biosafety, facilities, compliance, security, law, and health. Members of the Biosecurity Task Force are in frequent contact with the principal investigator and laboratory personnel to provide oversight and assure biosecurity.
All experiments with HPAI H5N1 viruses were performed in enhanced biosafety level 3 laboratories at the University of Tokyo (Tokyo, Japan), which are approved for such use by the Ministry of Agriculture, Forestry and Fisheries, Japan, or in biosafety level 3 agricultural (BSL-3Ag) laboratories at the University of Wisconsin-Madison approved for such use by the Centers for Disease Control and Prevention (CDC) and Animal and Plant Health Inspection Service (APHIS). Ferret transmission studies were conducted in enhanced BSL-3 containment at the University of Tokyo by PhD-level scientists who are highly experienced in such studies. Mouse virulence studies were conducted in BSL-3Ag at the University of Wisconsin-Madison, also by scientists who have several years of experience working with highly pathogenic influenza viruses and performing animal studies with such viruses. In vitro experiments were conducted in Class II biological safety cabinets and transmission experiments were conducted in HEPA-filtered ferret isolators. Staff working in enhanced BSL-3 and BSL-3Ag wear disposable overalls and powered air-purifying respirators. The enhanced BSL-3 facility at the University of Tokyo includes controlled access, exit through a shower change room, effluent decontamination, negative air-pressure, double-door autoclaves, HEPA-filtered supply and exhaust air, and airtight dampers on ductwork connected to the animal cage isolators and biosafety cabinets. The structure is pressure-decay tested regularly. All personnel complete biosafety and BSL-3 training before participating in BSL-3-level experiments. Refresher training is scheduled on a regular basis. Select Agent virus inventory, secured behind two physical barriers, is checked regularly. Virus inventory is submitted once a year to the Ministry of Agriculture, Forestry and Fisheries, Japan
The BSL-3Ag facility at University of Wisconsin-Madison was designed to exceed the standards outlined in Biosafety in Microbiological and Biomedical Laboratories (5th edition; http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf
The 1918 Spanish Influenza Pandemic
Dr. Taubenberger stated that the Spanish flu, an H1N1 strain, killed 40-50 million people worldwide during 1918-1919, including almost 700,000 people in the United States (1-2 percent of those infected died). At the conclusion of the pandemic, this strain lost much of its virulence but persisted globally, circulating and causing typical seasonal flu. (See Figure 3-3.)
In the mid-1990s, Taubenberger’s research group and others investigated the Spanish flu using genetic and virologic techniques to explore the virus’s origin, pathogenicity, and transmissibility in experimental models. The research was motivated by questions such as: How did the strain evolve and adapt to humans? Could the mutations be used to improve current surveillance strategies? Why was the strain pathogenic? Could data generated as the result of the research be used to develop new therapies and vaccines?
Taubenberger’s team recovered viral RNA fragments from two formalin-fixed samples from autopsy collections in Washington, DC and London and one frozen, unfixed sample from Alaska and used RT-PCR to sequence the fragments. Taubenberger observed that both the public and the scientific community were aware of this research as it proceeded and noted that, in comparison with the reception of the research of Drs. Fouchier and Kawaoka, the research caused little alarm.2 Researchers rebuilt cDNAs, cloned the cDNAs into bacterial plasmids, and, in high containment facilities, reconstructed the virus’s complete genome by reverse genetics.3 Taubenberger’s team also constructed novel viruses that contained one or more of the genes from the original 1918 strain. In 1997, the first genetic sequences were published, and in 2005, the entire genome was published with the details about the methods used to reconstruct the virus.
Taubenberger stated that the research was subject to several forms of regulatory oversight. The research was funded by the Armed Forces Institute of Pathology, the Departments of Defense and Veterans Affairs, the American Registry of Pathology, and the National Institutes of Health,
2 While not discussed explicitly at the workshop, there has been significant discussion about the way that certain H5N1 research results were conveyed and subsequently interpreted by the press. See, for example, Katherine Harmon, “What Will the Next Influenza Pandemic Look Like?,” Scientific American, September 19, 2011, http://www.scientificamerican.com/article.cfm?id=next-influenza-pandemic.; Deborah MacKenzie, “Five Easy Mutations to Make Bird Flu a Lethal Pandemic,” New Scientist 2831 (September 26, 2011): 3.; Editorial, “An Engineered Doomsday,” New York Times, January 7, 2012, http://www.nytimes.com/2012/01/08/opinion/sunday/an-engineered-doomsday.html?_r=0.; Peter M. Sandman, “Science versus Spin: How Ron Fouchier and Other Scientists Miscommunicated about the Bioengineered Bird Flu Controversy,” June 7, 2012, http://www.psandman.com/articles/Fouchier.htm.
3 The genome of the 1918 influenza virus was assembled at Mount Sinai School of Medicine (laboratories of Drs. Basler, García-Sastre, and Palese) and the infectious virus was rescued at the Centers for Disease Control and Prevention (laboratory of Dr. Tumpey).