Reassortment between Avian H5N1 and Human H3N2 Influenza Viruses in Ferrets: a Public Health Risk Assessment

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1 JOURNAL OF VIROLOGY, Aug. 2009, p Vol. 83, No X/09/$ doi: /jvi Reassortment between Avian H5N1 and Human H3N2 Influenza Viruses in Ferrets: a Public Health Risk Assessment Sara Jackson, Neal Van Hoeven, Li-Mei Chen, Taronna R. Maines, Nancy J. Cox, Jacqueline M. Katz, and Ruben O. Donis* Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia Received 14 March 2009/Accepted 28 May 2009 This study investigated whether transmissible H5 subtype human-avian reassortant viruses could be generated in vivo. To this end, ferrets were coinfected with recent avian H5N1 (A/Thailand/16/04) and human H3N2 (A/Wyoming/3/03) viruses. Genotype analyses of plaque-purified viruses from nasal secretions of coinfected ferrets revealed that approximately 9% of recovered viruses contained genes from both progenitor viruses. H5 and H3 subtype viruses, including reassortants, were found in airways extending toward and in the upper respiratory tract of ferrets. However, only parental H5N1 genotype viruses were found in lung tissue. Approximately 34% of the recovered reassortant viruses possessed the H5 hemagglutinin (HA) gene, with five unique H5 subtypes recovered. These H5 reassortants were selected for further studies to examine their growth and transmissibility characteristics. Five H5 viruses with representative reassortant genotypes showed reduced titers in nasal secretions of infected ferrets compared to the parental H5N1 virus. No transmission by direct contact between infected and naïve ferrets was observed. These studies indicate that reassortment between H5N1 avian influenza and H3N2 human viruses occurred readily in vivo and furthermore that reassortment between these two viral subtypes is likely to occur in ferret upper airways. Given the relatively high incidence of reassortant viruses from tissues of the ferret upper airway, it is reasonable to conclude that continued exposure of humans and animals to H5N1 alongside seasonal influenza viruses increases the risk of generating H5 subtype reassortant viruses that may be shed from upper airway secretions. Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused devastating outbreaks in avian species during the past decade. After emerging in the Guangdong province of China in 1996, H5N1 viruses have extended their geographic distribution from Asia into Europe and Africa (45, 51). Sporadic transmission of H5N1 viruses from infected birds to humans has resulted in over 380 laboratory-confirmed infections and a case fatality rate of 60% since 2003 (48). Currently circulating H5N1 viruses lack the ability to undergo efficient and sustained transmission among humans although instances of limited human-to-human transmission have been reported (13, 41). If H5N1 viruses were to acquire genetic changes that confer efficient transmissibility among humans, then another pandemic would likely occur. The pandemics of 1957 and 1968 highlight the importance of genetic reassortment between avian and human influenza viruses as a mechanism for the generation of human pandemic strains (15, 46, 47). The structural separation of the influenza virus genome into eight independent genes allows formation of hybrid progeny viruses during coinfections. The 1957 H2N2 and 1968 H3N2 pandemic viruses acquired the hemagglutinin (HA) and PB1 genes, with or without the neuraminidase (NA) * Corresponding author. Mailing address: Influenza Division, NCIRD, CCID, Centers for Disease Control and Prevention, Mail Stop G-16, 1600 Clifton Road, Atlanta, GA Phone: (404) Fax: (404) rvd6@cdc.gov. S.J. and N.V.H. contributed equally to this work. Present address: Solvay Pharmaceuticals, Inc., 901 Sawyer Road, Marietta, GA, Supplemental material for this article may be found at Published ahead of print on 3 June gene, respectively, from an avian virus progenitor (14, 33). The remaining genes of these pandemic reassortants were derived from a contemporary human virus (14, 33). The host species in which such human pandemic strains were generated by reassortment between human and avian viruses is not known. However, coinfection of the same cell with both human and avian viruses must have occurred, even though human and avian influenza viruses have preferences for different sialic acid receptor structures present on cell surface glycoproteins and glycolipids (20, 30). The HA of human viruses preferentially binds (2,6)-linked sialic acids while that of avian viruses preferentially bind (2,3)-linked sialic acids (3, 12). Cells possessing both of these receptors could support coinfection of avian and human viruses, leading to reassortment. Human respiratory tract epithelial cells can possess surface glycans with (2,3)- and (2,6)-linked sialic acids and as such represent a potential host for the generation of avian-human reassortant viruses (24, 35). The general distribution of surface (2,3)- and (2,6)-linked sialic acids varies among cells of the human upper and lower respiratory tracts, which are anatomically separated by the larynx. Recent studies have shown that (2,3)-linked sialic acids are present in tissues of the human lower respiratory tract (i.e., lung alveolar cells) (24, 35) as well as tissues of the human upper respiratory tract (24). Consistent with these findings, HPAI H5N1 viruses have been shown to attach to and infect tissues belonging to the lower respiratory tract (i.e., trachea, bronchi, and lung) (5, 25, 35, 40, 42, 43) as well as tissues belonging to the upper respiratory tract (i.e., nasopharyngeal, adenoid, and tonsillar) (25). Glycans with (2,6)-linked sialic acids are more widespread on epithelial cells of the upper airways than lung alveoli (24, 35). In accor- 8131

2 8132 JACKSON ET AL. J. VIROL. dance, human seasonal influenza viruses preferentially attach to and infect cells of the upper respiratory tract (6, 25, 35, 43). If cells with both types of receptors are present in the human respiratory tract, simultaneous infection of a person with both human and avian viruses could generate reassortant viruses. Although viruses derived by reassortment between avian H5N1 and human H3N2 progenitors have been generated in vitro (17), reassortment between these avian and human strains in a coinfected mammalian host has not been shown. Furthermore, our knowledge of the genetic and phenotypic repertoire of such reassortants generated in vivo and their potential for transmission to uninfected hosts is limited (2, 17). In the present study, we used the ferret model to better understand the generation of reassortant viruses in a host coinfected with contemporary avian (H5N1) and human (H3N2) viruses and the extent to which such reassortants replicate and transmit from animal to animal. The domestic ferret (Mustela putoris) serves as an ideal small-animal model for influenza because ferrets are susceptible to human and avian influenza viruses, including HPAI H5N1 viruses, and reflect the relative transmissibility of human and avian influenza viruses in humans (9, 17, 18, 31, 36, 39, 53). Our study revealed that coinfection of ferrets reproducibly generated reassortant viruses that could be recovered from tissues within and extending toward the upper respiratory tract. Although H5 reassortant viruses were recovered from the upper airways, they displayed no transmissibility to contact ferrets, suggesting that additional functional changes are required for these viral subtypes to become pandemic within human populations. MATERIALS AND METHODS Viruses. The derivation of reverse genetics-generated A/Thailand/16/04 (Thai16) (H5N1) and A/Wyoming/3/03 (Wyom03) (H3N2) viruses was performed as described previously using standard reverse genetics procedures (22, 23). All experiments using A/Thailand/16/04, Thai16, and H5 subtype reassortant viruses were conducted under biosafety level 3 containment ( /OD/ohs/biosfty/bmbl5/bmbl5toc.htm) including enhancements required by the U.S. Department of Agriculture and Select Agent Program (29), available at: Coinfection of ferrets. Male Fitch ferrets, 6 to 12 months of age (Triple F Farms, Sayre, PA), were used for all experiments. Prior to inoculation, all animals were confirmed to be serologically negative for circulating seasonal influenza viruses (influenza A virus H1N1, H3N2, and influenza B virus) by hemagglutination inhibition (HI) assay. For all experiments, ferrets were housed in cages contained within a Duo-Flo Bioclean mobile clean room (Lab Products, Seaford, DE). Temperature and weight data were taken daily for approximately 3 days prior to infection. Temperatures were measured using a subcutaneous implantable temperature transponder (BioMedic Data Systems, Seaford, DE). A baseline blood specimen was collected on the day prior to infection. For coinfection studies, three ferrets were inoculated intranasally with PFU of Thai16 and PFU of Wyom03 in a total volume of 1 ml using previously described methods (18). Nasal washes were collected from all ferrets every 24 h, beginning 1 day postinfection and continuing for 7 days. Any animal losing 25% of its day 0 body weight, exhibiting neurological symptoms, or determined to be in a moribund state was humanely euthanized. Tissue extractions were performed using previously described protocols (18), with trachea removed first, followed by lungs, bronchi, and nasal turbinates. Transmission experiments. Respiratory droplet and contact transmission experiments were carried out as described previously (17). Two or three pairs of inoculated and contact animals were evaluated for each experiment. Ferrets were inoculated intranasally with 10 6 PFU of parental wild-type (Wyom03 or Thai16) or reassortant viruses in a total volume of 1 ml, using phosphate-buffered saline as the diluent. Approximately 24 h following infection, contact was established. For respiratory droplet experiments, one naïve ferret was individually placed in a cage adjacent to inoculated animals such that contact animals were singly housed and could exchange air and respiratory droplets only through a perforated wall between cages. It should be noted that the use of the phrase respiratory droplet transmission throughout this article refers to transmission in the absence of direct or indirect contact between animals and does not imply knowledge of the diameter of virus-containing particles. For direct contact transmission experiments, a naïve contact animal was placed in the same cage with each inoculated ferret. Clinical signs of infection were monitored daily in all ferrets for at least 14 days postinfection or contact. Virus replication in inoculated and contact animals was determined by titration of nasal washes collected every 48 h following inoculation or contact. Nasal washing was performed by instillation of 1 ml of phosphate-buffered saline containing 100 g/ml penicillin-streptomycin (Gibco), 100 g/ml gentamicin (Gibco), and 1% bovine serum albumin (Gibco) into the nostrils, and liquid runoff was collected into a sterile petri dish. Nasal washes were immediately frozen on dry ice and stored at 80 C until titer determination. Real-time RT-PCR. RNA standards for quantitative real-time reverse transcriptase PCR (RT-PCR) were made by in vitro transcription of HA (H3 and H5) cdna templates using a Riboprobe System-T7 (Promega) in vitro transcription kit. cdna templates for RNA transcription were generated by reverse-transcription of Thai16 RNA or Wyom03 RNA using a One-Step RT-PCR kit (Qiagen). Copy numbers of H3 and H5 RNA were determined for nucleic acids that were isolated from ferret nasal fluid and respiratory tract tissue by MagNA Pure LC robotics and either a Total Nucleic Acid Kit (Roche) or RNA Isolation Kit III-Tissue Kit (Roche), respectively. QuantiTech (Qiagen) kit chemistry was used for real-time RT-PCR reactions for H3 and H5 RNA quantification. Both H3 and H5 primers and probes used in this assay were specific for Wyom03 or Thai16 RNA, respectively (data not shown). Primers and reaction conditions are available upon request. H3 (FAM-CAGGATCACATATGGGGCCTGTCCCA G-BHQ1, where FAM is 6-carboxyfluorescein and BHQ is Black Hole quencher) and H5 (Cy5-AcTCcAcTTaTtTcCTcTcT-BHQ3, where lowercase letters were locked nucleic acid [LNA] nucleotides) probes were used to detect amplified nucleic acids. Quantitative real-time PCR was done using an Mx3005P Real- Time PCR System and corresponding software (Stratagene). Genotyping. One-Step RT-PCR (Qiagen) was used to determine the Wyom03- or Thai16-based origin of each gene in plaque-purified virus populations. Primers were designed to hybridize specifically to Wyom03- or Thai16-derived HA, NA, PB1, PB2, PA, nucleoprotein (NP), M, or NS genes at appropriate annealing temperatures. Multiplex reactions containing Wyom03- and Thai16-specific primers for each gene were performed in order to simultaneously detect both Wyom03- and Thai16-derived genes of different amplicon sizes. Primer sequences and reaction conditions are available upon request. RT-PCR products were resolved and visualized on 2%, 96-well E-Gels (Invitrogen). Cell and virus growth. MDCK and A549 cells were cultured at 37 C under 5% CO 2 using standard tissue culture procedures. MDCK cells were grown in high glucose Dulbecco s modified Eagle medium (DMEM) supplemented with antibiotics (100 U/ml penicillin and 100 g/ml streptomycin) and 10% fetal bovine serum (Invitrogen). A549 cells were grown in F-12 Kaighn s medium supplemented with antibiotics and 10% fetal bovine serum (Invitrogen). To determine viral growth curves, monolayers of MDCK or A549 cells were infected at a multiplicity of infection (MOI) of for 1 h at 37 C. MDCK monolayers were overlaid with DMEM supplemented with 2.5% bovine serum albumin fraction V, antibiotics, and 1 g/ml TPCK (tosylsulfonyl phenylalanyl chloromethyl ketone)-treated trypsin and then placed at 37 C. A549 monolayers were overlaid with F-12 Kaighn s medium supplemented as DMEM but without trypsin and then placed at 37 C. At each time point, cells were harvested into medium, and the cell suspension was frozen. Cell debris was removed from the thawed suspension by centrifugation at 2,500 RPM for 5 min. Virus yields were determined on MDCK cells by plaque assay. All plaque assays were preformed on monolayers of MDCK cells using standard procedures and an agarose (0.8%) solution containing modified Eagle medium (Invitrogen), 2.5% bovine serum albumin fraction V (Invitrogen), antibiotics, 0.25 g/ml amphotericin B (Fungizone; Invitrogen), and 1 g/ml TPCK-treated trypsin (Sigma-Aldrich). After 48 to 72 h of growth at 37 C, plaques were visualized by crystal violet staining or by overlaying agarose with 100 l of 5 mg/ml methylthiazolyldiphenyl-tetrazolium bromide (MTT) (Sigma- Aldrich) for 2 to 3 h. Plaque assays for viral growth curve experiments were performed in duplicate at three different 10-fold dilutions to ensure accurate titers. Careful visual inspection of plaques stained with MTT was used to ensure isolation of plaques with variable sizes when virus samples from ferrets were analyzed. Approximately one-half of each plaque aspirate was resuspended in DMEM and then used to inoculate and grow clonal virus populations in tube cultures containing a monolayer of MDCK cells. Cultures were incubated at 37 C until cytopathic effects were observed. Centrifugation was performed to remove

3 VOL. 83, 2009 IN VIVO REASSORTMENT OF INFLUENZA VIRUSES IN FERRETS 8133 homologous to the parental Thai16 strain were analyzed using horse red blood cells. RESULTS FIG. 1. Replication and respiratory droplet transmission of parental Wyom03 and Thai16 reverse genetics-generated viruses in ferrets. (A) Two ferrets were inoculated with 10 6 PFU Wyom03 virus. On day 1 postinoculation, two uninfected ferrets were separately exposed to the two infected ferrets housed in adjacent cages. Infectious virus (log 10 PFU/ml) recovered from nasal washes of inoculated (days 1 to 7) and contact (days 5 to 15) animals is shown. There was no infectious virus recovered from contact animals on days 1 and 3 (not shown). (B) Three uninfected ferrets were separately exposed to three ferrets inoculated with 10 6 PFU Thai16 virus and then analyzed as in panel A. In both panels A and B, black, gray, or white bars indicate corresponding inoculated and contact pairs. A horizontal line indicates the limit of detection (10 2 ). cell debris from viral stocks. Each clonal virus population was plaque purified three times prior to further in vitro and in vivo characterization. HI assays. Specific antibodies to Wyom03 or Thai16 HA present in ferret serum were determined by HI analysis as previously described (38). For Wyom03 virus experiments, serum was tested against virus bearing the homologous HA using turkey red blood cells. For all H5 HA virus experiments, antibodies to virus Ferret treatment group Replication and transmissibility of parental H3N2 and H5N1 influenza viruses in the ferret model. A/Wyoming/3/03 (H3N2) and A/Thailand/16/04 (H5N1) are recent influenza virus isolates of human and avian origin, respectively, and as such were chosen as representative strains for this risk assessment study. Reverse genetics-derived Thai16 and Wyom03 viruses were used throughout the study in order to minimize the genetic variability of viruses originating from clinical specimens. In humans, H3N2 viruses typically have low virulence/ high transmissibility characteristics, whereas HPAI H5N1 viruses generally possess properties of high virulence/low transmissibility. Consistent with this high virulence, A/Thailand/16/04 was isolated from a fatal human infection and is reported to be lethal in ferrets (18). To ascertain the reciprocal lethality and transmissibility properties of the Wyom03 and Thai16 reverse genetics viruses used here, infection and transmission studies were performed in the ferret model (Fig. 1). As expected, the Wyom03 human virus replicated efficiently after intranasal inoculation of ferrets, reaching a mean maximum virus titer of PFU/ml in nasal secretions on day 1 postinfection (p.i.) (Fig. 1A). The virus was transmitted efficiently by respiratory droplet to two contact animals that were individually caged and separately exposed to inoculated ferrets. These contact animals both shed infectious virus (Fig. 1A) and seroconverted by the end of the experiment (Table 1). Clinical signs associated with Wyom03 virus infection of ferrets were generally mild; animals exhibited no significant weight loss ( 5%) and had a mean relative inactivity index (RII) of 1.3 (Table 1) (28, 54). The Thai16 virus also replicated efficiently and was shed in the nasal secretions of ferrets, achieving mean peak virus titers of PFU/ml on day 3 p.i. (Fig. 1B). Clinical signs of influ- TABLE 1. Clinical and serological responses of ferrets inoculated with virus and of naïve ferrets exposed to inoculated ferrets Virus Subtype % Weight loss Clinical profile No. of ferrets positive/total no. of ferrets RII b HI titer Seroconversion No. of ferrets positive/total no. of ferrets Inoculated Wyom03 H3N2 5 (0/2) 1.3 1,280 (2/2) Thai16 H5N (3/3) c (1/3) Thai16:Wyom03-NA H5N2 6 (2/3) (3/3) Thai16:Wyom03-NA-NS-NP-M H5N2 5 (0/3) (0/3) Thai16:Wyom03-NA-NS H5N2 5.6 (3/3) (3/3) Thai16:Wyom03-NS-M H5N1 7.7 (1/3) (3/3) Thai16:Wyom03-PB1 H5N1 8 (2/3) ,280 (3/3) Exposed a Wyom03 H3N2 10 (1/2) 1.0 1,280 (2/2) Thai16 H5N1 5 (0/3) (0/3) Thai16:Wyom03-NA H5N2 5 (0/3) (0/3) Thai16:Wyom03-NA-NS-NP-M H5N2 5 (0/3) (0/3) Thai16:Wyom03-NA-NS H5N2 5 (0/3) (0/3) Thai16:Wyom03-NS-M H5N1 5 (0/3) (0/3) Thai16:Wyom03-PB1 H5N1 5 (0/3) (0/3) a Naïve ferrets were exposed to Wyom03- or Thai16-infected ferrets by an indirect respiratory droplet transmission mechanism (see text). A direct contact mechanism was used to determine reassortant transmission (see text) and to further analyze Thai16 transmission (data not shown). b Virus-induced lethargy is described using the RII, as previously described (54). c Only one animal survived and was tested.

4 8134 JACKSON ET AL. J. VIROL. enza virus infection in Thai16 virus-inoculated animals were more severe than those observed in Wyom03 virus-infected animals. Thai16 virus-infected ferrets had an average maximum weight loss of 14.4% and a mean RII of 1.5 (Table 1), and two of three inoculated ferrets died by day 6 p.i. It should be noted that the overall RII score for Thai16 virus-infected ferrets in this experiment is lower than expected due to collection of clinical data from only one surviving ferret after day 6 p.i. Despite efficient replication of Thai16 virus in inoculated ferrets, no respiratory droplet transmission was detected (Fig. 1B); no virus was isolated from nasal washes of the contact ferrets at any time during the experiment, and the contact animals did not seroconvert (Table 1). To determine if Thai16 virus could be transmitted by direct contact, naïve ferrets were introduced into the same cage as ferrets inoculated with Thai16 virus 24 h earlier. As with the respiratory droplet experiment, no transmission of Thai16 virus was detected either by viral shedding or seroconversion of contact animals (data not shown). These results indicate that human Wyom03 virus exhibits low virulence and high transmissibility in ferrets and confirm that avian Thai16 virus exhibits high virulence with low transmissibility in ferrets. Thus, the biological properties of the reverse genetics seasonal human virus and the HPAI H5N1 virus in ferrets were consistent with those previously described in humans for H3N2 and HPAI H5N1 strains, respectively (17, 18). The virulence and transmissibility properties of the Wyom03 and Thai16 viruses in ferrets met the phenotypic requirements for this study, in which we hypothesized that in vivo reassortment could generate a novel transmissible H5 subtype virus. Isolation of H5 subtype reassortant viruses following parental Wyom03 and Thai16 virus coinfection of ferrets. Although reassortment between human H3N2 and avian H5N1 viruses has been achieved in vitro, their potential for reassortment in vivo is not known (2, 17). To determine whether a transmissible H5 subtype reassortant virus could arise in a coinfected mammalian host, three ferrets were inoculated simultaneously by the intranasal route with PFU of each of the Wyom03 (H3N2) and Thai16 (H5N1) viruses. Coinfected animals exhibited clinical signs similar to those observed during single infection with Thai16 virus, suggesting that the coinoculated Wyom03 virus did not interfere with Thai16 virus replication or induction of severe disease. The average maximum weight loss in coinfected animals was 15.4%, and the mean RII was 1.3. Virus was shed in nasal washes of coinfected ferrets for up to 7 days p.i. (Fig. 2A). Five days after inoculation, one animal was euthanized due to severe illness, and viral titers in surviving animals declined thereafter. To investigate the replication kinetics and efficiency of viruses possessing either Wyom03 H3 HA or Thai16 H5 HA in the upper respiratory tract of coinfected ferrets, nasal wash specimens collected daily were analyzed by quantitative realtime RT-PCR assays specific for each HA subtype (H3 or H5). On day 1, RNA copies of Wyom03 HA exceeded Thai16 HA by 70- to 250-fold (Fig. 2B). However, by days 2 to 7, this difference decreased to three- to sevenfold due to an increase in Thai16 HA levels as well as a decline in Wyom03 HA levels. These results suggest that viruses with HA genes of human and avian origin replicated efficiently in coinfected ferrets and that FIG. 2. Replication of Thai16 and Wyom03 viruses in ferrets and the emergence of reassortant genotypes after coinfection. (A) Infectious virus (log 10 PFU/ml) recovered from nasal secretions on days 1 to 7 postcoinoculation with Wyom03 and Thai16 in three ferrets. (B) Concentration of H3 and H5 HA RNA in the same ferret nasal secretions as in panel A on days 1 to 7 postcoinoculation. (C) Genotypes (Wyom03, Thai16, or reassortant) of plaque-purified viruses (n 20) isolated on days 1 to 7 from the same nasal secretions as in panel A and B of three coinfected ferrets. Percentages are shown for the Wyom03, Thai16, and reassortant genotype viruses. Viruses that were incompletely purified by plaquing were designated as dual genotype. Detailed genotype information is shown in Table 1 and in the Table S1 in the supplemental material. Note that the asterisk indicates that the ferret was euthanized due to severe illness, and no infectious virus was recovered from nasal washes from ferret 3 on day 7. A horizontal line in graphs A and B indicates the limit of detection (10 2 ). the H3 and H5 HA RNA molecules were present in similarly abundant quantities in nasal secretions for most of the infectious period. To determine if reassortant viruses were present in upper respiratory tract of coinfected animals, plaque-purified viruses were isolated from nasal secretions for genetic analysis. Twenty individual plaques were isolated from each nasal wash specimen collected daily (days 1 through 7 p.i.) to sample the genotypes of viruses in the three coinfected ferrets throughout the infectious period. Viral genomes were analyzed by a multiplex RT-PCR assay in order to establish the progenitor of each of the eight gene segments (Wyom03 or Thai16) by simultaneous detection and identification of Wyom03- and Thai16-derived genes. Nasal secretions were diluted to concentrations that generated well-separated plaques in order to ensure clonality of the harvested virus. Based on the origins of the eight genes, plaque-purified viruses were characterized as having a Wyom03 or Thai16 wild-type genotype if they contained all eight genes from only one parental virus, and they were characterized as a reassortant genotype if they contained

5 VOL. 83, 2009 IN VIVO REASSORTMENT OF INFLUENZA VIRUSES IN FERRETS 8135 H5 HA reassortant TABLE 2. H5 HA reassortant genotypes recovered from coinfected ferrets Origin of the indicated influenza virus gene segment a HA NA PB1 PB2 PA NP M NS No. of reassortant isolates (total no. of viruses examined) in: Ferret 1 (140) Ferret 2 (100) Thai16:Wyom03-NA T W T T T T T T Thai16:Wyom03-NA-NP-M-NS T W T T T W W W Thai16:Wyom03-NA-NS T W T T T T T W Thai16:Wyom03-M-NS T T T T T T W W Thai16:Wyom03-PB1 T T W T T T T T a W, gene derived from Wyom03 parental virus; T, gene derived from Thai16 parental virus. Ferret 3 (120) genes of both Wyom03 and Thai16 virus origin (Fig. 2C). Any plaques that contained the same genes of both parental origins were identified through the multiplex RT-PCR assay and noted as having a dual genotype. Among the samples analyzed, Wyom03 virus was found in all three animals at the highest incidence on day 1, declined in abundance on days 2 and 3, and then increased slightly thereafter (Fig. 2C). In contrast, Thai16 virus was present at low levels on day 1 but increased in abundance through day 5. The proportion of Thai16 virus declined at later time points, coincident with a resurgence of Wyom03 virus (Fig. 2C, days 4 to 7). Analysis of a total of 360 virus plaques from nasal secretions of three ferrets collected daily for 7 days (20 plaques per ferret per day) after infection revealed the most frequent genotypes within this sample. A feasible sample size (20 viruses per day for 7 days from three ferrets) was chosen to make a preliminary assessment for the proportions of reassortant viruses isolated from ferrets and was not intended to statistically reflect the proportion of reassortant or parental genotypes for all viruses present within the infected ferrets. However, the proportions of parental genotypes were isolated in a kinetically similar pattern to the replication of each parental virus in single virus-infected animals during transmission experiments (Fig. 1). Viruses of a Wyom03 genotype were isolated at high frequency during the first 2 days of coinfection, declined on days 3 and 4, and reemerged on day 5. In contrast, viruses with a Thai16 genotype could be isolated consistently from days 2 to 6 in coinfected animals. Reassortant viruses were isolated from the nasal secretions of all coinfected ferrets (32 reassortants from a total of 360 isolated viruses), suggesting an ecologically significant and reproducible yield (9%) of reassortant viruses from coinfected animals. Importantly, at least 34% of the recovered reassortant viruses possessed the H5 HA (11 of the 32 reassortant viruses). This group of reassortant viruses comprised five distinct genotypes (Table 2) and have been denoted as Thai16:Wyom03- [gene], specifying only those genes derived from the Wyom03 virus; all other genes were derived from the Thai16 virus. H5 subtype viruses with the same genotype were found in two ferrets (ferrets 1 and 3 in Table 2) and were recovered multiple times from the same ferret (ferret 1 in Table 2), suggesting that viruses possessing these H5 genotypes may replicate efficiently in these animals and in mammalian cells. Eight unique reassortant genotypes with H3 HA genes were also recovered from nasal secretions (see Table S1 in the supplemental material). Because H3 subtype viruses lack pandemic potential as a result of widespread immunity in the human population, these reassortant viruses were not further analyzed. Characterization of H5 subtype reassortant viruses in vitro. To assess the relative replication efficiency of H5N1 avianhuman virus reassortants, five viruses representing unique reassortant genotypes were studied in two mammalian cell models. The replication kinetics of each reassortant with H5 HA was examined after infection at a low MOI (0.001) in human alveolar lung epithelial cells (A549) and canine kidney (MDCK) cells. In A549 cells, all reassortants replicated with slower kinetics than that of parental Thai16 virus, which multiplied very efficiently, reaching a peak titer of PFU/ml within 24 h (Fig. 3A). Although slower in replication than the parental Thai16 virus, all reassortants grew to relatively high maximum titers of to PFU/ml within 48 h. Parental Wyom03 virus achieved a peak titer of PFU/ml, indicating efficient replication in a single cycle of infection in A549 cells. Multicycle replication of this virus in A549 cells was not studied due to trypsin toxicity. Similar growth kinetics was observed in MDCK cells (Fig. 3B) in which all reassortant viruses grew to maximum titers of to PFU/ml within 48 h. Again, the replication kinetics of the five reassortants was slower than that of the parental Thai16 virus, which rapidly achieved a titer of PFU/ml within 24 h. Reassortants possessing the H5 subtype replicated more efficiently than the parental Wyom03 virus within the first 24 h, even with the inclusion of trypsin in the assay. At 24 h p.i., Wyom03 virus had reached a titer of only PFU/ml while all five reassortants had a titer of at least PFU/ml. At subsequent time points, parental Wyom03 virus titers continued to increase while the growth of H5 bearing reassortants slowed. Collectively, the production of abundant progeny virus by the five reassortants in both A549 and MDCK cell types indicates that they were sufficiently fit to replicate in mammalian and human lung-derived cells, albeit with slower kinetics than the parental Thai16 virus. Replication and transmissibility of H5 subtype reassortant viruses in the ferret model. Efficient and sustained transmission of viruses in the population is considered to be an essential property of a pandemic virus. However, avian H5N1 viruses isolated from humans lack this property. To determine whether acquisition of genes from Wyom03 virus conferred transmissibility to H5 subtype reassortant genotypes, we next examined the replication and transmissibility of five unique reassortant viral genotypes in ferrets using the direct contact transmission model. Three ferrets were inoculated intranasally with 10 6 PFU of virus, a dose that was previously determined to be sufficient for high-level replication of both parental viruses in ferrets (Fig. 1). Peak titers achieved in the nasal

6 8136 JACKSON ET AL. J. VIROL. FIG. 3. Replication kinetics of recovered H5 subtype reassortants in A549 and MDCK cells. Infectious virus yields (log 10 PFU/ml) in A549 (A) and MDCK (B) cells infected with reassortant, Thai16, and Wyom03 viruses (MOI of 0.001) harvested at 1 to 48 h postinoculation. Infectious virus from panels A and B was quantified on MDCK cells by plaque assay performed in duplicate at three different 10-fold dilutions to ensure accurate titers. NA, Thai16:Wyom03-NA; NA-NP-M-NS, Thai16:Wyom03-NA-NP-M-NS; NA-NS, Thai16:Wyom03-NA-NS; NS-M, Thai16: Wyom-NS-M; PB1, Tha16:Wyom03-PB1;Thai, Thai16 parental;wyom, Wyom03 parental. The limit of detection was 10 for both panels A and B. washes of inoculated animals were 10-fold (Thai16:Wyom03- PB1) to 100-fold (Thai16:Wyom03-NA, Thai16:Wyom03-NA- NP-M-NS, Thai16:Wyom03-M-NS) lower than the peak titers observed in ferrets infected with the parental Thai16 virus (Fig. 4). One reassortant (Thai16:Wyom03-NA-NP-M-NS) was isolated only from nasal washes at an early time point postinoculation and did not elicit seroconversion of inoculated animals (Table 1). Consistent with the decreased replication of these viruses in ferrets, the clinical symptoms observed were less severe than those observed in animals infected with Thai16 virus (Table 1). To assess direct contact transmission of reassortants, an immunologically naïve animal was placed in the same cage with each inoculated animal 1 day after inoculation. None of the reassortant viruses transmitted to contact animals; virus was not detected in nasal washes from any contact animals at any time point, and seroconversion was not observed (Table 1). The reduced replication as well as the low virulence and transmissibility of reassortants possessing the H5 subtype in ferrets suggests that these reassortants lack critical properties required for a virus to be of immediate public health concern. Distribution of Wyom03, Thai16, and reassortant genotypes in the respiratory tract of ferrets. Previous studies suggest that FIG. 4. Replication of recovered H5 subtype reassortant viruses in ferrets. Three ferrets (indicated by black, gray, white bars) were separately infected with each reassortant virus, designated by the Wyom03-derived gene(s) within their genotype. A control group was infected with parental Thai16 virus. Infectious virus (log 10 PFU/ml) recovered from nasal washes of each infected ferret on days 1 to 9 postinoculation was determined by plaque assay on MDCK cells. A horizontal line indicates the limit of detection (10 2 ). NA, Thai16: Wyom03-NA; NA-NP-M-NS, Thai16:Wyom03-NA-NP-M-NS; NA-NS, Thai16:Wyom03-NA-NS; M-NS, Thai16:Wyom-M-NS; PB1, Tha16: Wyom03-PB1.

7 VOL. 83, 2009 IN VIVO REASSORTMENT OF INFLUENZA VIRUSES IN FERRETS 8137 both the upper and lower respiratory tracts of humans can support the replication of HPAI H5N1 viruses (16, 17, 25, 35, 40, 43), whereas H3N2 replication may occur primarily toward the upper airway epithelium (6, 25, 35, 42, 43). To identify specific anatomical sites in which reassortant viruses could be recovered, we next investigated the tissue distribution of both parental and reassortant genotype viruses in ferret upper and lower respiratory tract tissues, which were anatomically distinguished by the larynx. Three ferrets were examined for the presence of reassortant viruses in tissues of the upper airways (i.e., nasal turbinate) and tissues of the lower airways (i.e., trachea, bronchi, and alveolar lung) following coinfection with PFU of each of the Wyom03 and Thai16 viruses by intranasal inoculation. Tissues were collected on day 5 p.i. because the number of reassortants isolated from nasal washes was highest at this time in previous experiments (Fig. 2C). Infectious virus was detected in all tissues examined, but the highest virus titers were observed in nasal turbinates (Fig. 5A). The anatomical distribution of viruses possessing H3 or H5 HA was established by performing Wyom03 and Thai16 HA subtype-specific quantitative real-time RT-PCR on tissue homogenates prepared from coinfected ferrets (Fig. 5B). In nasal turbinates and bronchi, both H3 and H5 HA genes were present in high quantities (10 6 to 10 9 copies/ml) although the H3 gene was 2- to 20-fold more abundant. Comparably high levels of H5 HA were also detected in trachea and lung tissues during coinfection (Fig. 5B). In contrast, little or no H3 HA RNA was detected in lung tissues of coinfected animals (Fig. 5B). Moreover, in all (three of three) lung tissue samples, abundant H5 but no H3 RNA was detected after coinfection even though the assay was sufficiently sensitive to detect as few as 100 copies of either gene (data not shown). The H3 gene was detected in the trachea of only one animal, and the H5 gene was detected in the trachea of only two animals, which could be potentially due to the relatively minimal number of cells in the trachea available for infection. Together, these results suggest that during coinfection of ferrets with Wyom03 and Thai16 viruses, viruses possessing the H3 gene are abundant in tissues within and extending toward the upper airway (i.e., nasal turbinate bronchi, and trachea) at this time p.i., while those possessing the H5 gene are abundant throughout the respiratory tract. This relative distribution of H5 and H3 genes in respiratory tract tissues during coinfection is similar to that observed during single infection with either Thai16 or Wyom03 viruses alone, where H5 RNA can be found throughout the respiratory tract, and H3 RNA can be found only in tissues extending toward and within the upper respiratory tract but not in the lung (data not shown). To investigate where reassortant viruses could be recovered from tissues along the respiratory tract, plaque-purified viruses were isolated from nasal turbinates (20 plaques per three ferrets), lung tissues (20 plaques per three ferrets), the trachea (20 plaques per two ferrets), and bronchi (40 plaques per three ferrets). Isolated viruses were then genotyped by multiplex RT-PCR. In nasal turbinates, viruses with Thai16, Wyom03, and reassortant genotypes were recovered in similar proportions to those found in ferret nasal secretions on day 5 after inoculation (Fig. 5C and 2C). Three previously unidentified reassortant genotypes were recovered from nasal turbinates, and a single reassortant virus was also isolated from bronchi FIG. 5. Recovery of viruses with parental or reassortant genotype from ferret respiratory tract after coinfection. (A) Total infectious virus (log 10 PFU/ml) recovered from nasal turbinate (nasal turb.), trachea, bronchi, or lung tissue of three ferrets (labeled 1 to 3 on the x axis) on day 5 postcoinoculation with Wyom03 and Thai16. (B) Concentration of H3 and H5 HA RNA genes recovered from the same tissues of the same three ferrets in panel A on day 5 postcoinoculation. (C) Viral genotypes (Wyom03, Thai16, and Reassortant) of plaquepurified viruses (n 20) isolated on day 5 from the same tissues of the same three ferrets in panels A and B. Percentages are shown for the Wyom03, Thai16, and reassortant genotype viruses. Viruses that could not be conclusively genotyped due to incomplete plaque purification were designated as dual genotype. Detailed genotype information can be found in Table S1 in the supplemental material. A horizontal line in graphs A and B indicates the limit of detection (10 2 ). The asterisk indicates that no infectious viruses could be isolated from this tissue. tissues (see Table S1 in the supplemental material). Only Thai16 genotype viruses were recovered from lung tissue. Similarly, all viruses recovered from tracheal tissue possessed a Thai16 genotype although low levels of H3 RNA could be detected in this tissue from one coinfected animal. Taken together, the real-time RT-PCR data and the genotyping results suggest that reassortant viruses are likely to be found in airways extending toward and within the upper respiratory tract (i.e., nasal turbinate, bronchi, and trachea) but not in the lungs of ferrets coinfected with avian H5N1 and human H3N2 parental strains. DISCUSSION Highly pathogenic avian H5N1 viruses have been evolving rapidly, extending both their geographic distribution and host range since 1996 (45, 49 51). H5N1 continues to infect hu-

8 8138 JACKSON ET AL. J. VIROL. mans, often at the same time that H1N1 and H3N2 influenza virus epidemics occur within a community (reviewed in reference 26). Reassortment of H5N1 gene segments with those of circulating human influenza viruses is a potential mechanism by which H5N1 viruses could become transmissible and then spread among humans. However, it is not known whether coinfection of a host could result in reassortment between an avian H5N1 virus and a human seasonal influenza virus strain. This risk assessment study has shown that reassortant viruses generated from recent avian H5N1 and human H3N2 viruses can be recovered from the upper respiratory tract of a mammalian host species. After coinfection, 9% (32 reassortants viruses per 360 viruses isolated) of viruses recovered from ferret nasal secretions were reassortants. Importantly, over 34% (11 H5 subtype reassortant viruses per 32 reassortant viruses) of reassortant viruses recovered possessed the avian H5 HA, comprising five unique genotypes. Taken together, these data indicate that recovery of H3 and H5 subtype reassortant viruses from nasal secretions is a likely event in ferrets that are exposed simultaneously to both viruses. All H5 subtype reassortant viruses examined replicated less efficiently than the Thai16 parental virus in human alveolar lung epithelial (A549) and canine kidney (MDCK) cell cultures. Nevertheless, titers of 10 6 PFU/ml or greater were achieved in either cell type within 48 h, indicating their ability to replicate in relevant cells of animal and human origin. The reduced viral replication compared to the parental Thai16 virus seen both in vitro and in vivo may be a result of decreased virus fitness caused by functional incompatibilities between reassorted genes. H5N2 subtype reassortants, with the exception of Thai16:Wyom03-NA-NP-M-NS, replicated relatively poorly in the two cell lines compared to parental Thai16 virus, a finding that is consistent with previous studies showing that optimal replication in vitro requires balanced receptor attachment (HA) and release (NA) activities for efficient multicycle replication (11, 44). Nevertheless, these same H5N2 reassortants replicated with considerably high efficiency in ferrets, suggesting that the requirements for virion surface protein compatibility are host dependent. On the other hand, H5 subtype reassortants that possess the Wyom03 M protein replicated efficiently in vitro but not in vivo. Impaired in vivo replication of HA and M gene reassortant viruses has been attributed to disruption of assembly and budding pathways or inadequate ph regulation by M2 in the trans Golgi pathway (4, 34; reviewed in references 21 and 32) although other genes could also play a significant role (1). Overall, the poor correlation between the in vivo and in vitro replication of reassortants suggests that different factors may govern fitness of reassortant viruses in vitro and in vivo and emphasizes the need for verification of in vitro viral fitness assays using animal models. All subtype H5 reassortants analyzed in this study failed to transmit efficiently between infected and naïve contact ferrets. None of the recovered hybrid genotypes with an H5 HA possessed the virulence of parental Thai16 virus and/or the transmissibility of parental Wyom03 virus. Previous studies in ferrets have shown that decreased in vivo replication levels are correlated with reduced transmission efficiency (10, 17, 52). However, it has also recently been shown that even a reverse genetics-generated reassortant virus with the HA and NA from a transmissible H3N2 human virus and the remaining genes from an H5N1 virus did not transmit efficiently in a ferret model (17). In addition, a recent study has established a correlation between influenza virus transmissibility and the preferential binding of HA to structurally distinct (2,6)-linked sialic acid receptors (37, 39). Taken together, the evidence suggests that transmissibility of H5N1 viruses is multifactorial. The tissue tropism of avian and human influenza viruses in humans has been correlated with the expression of surface receptors that may determine the primary site of coinfection and subsequent reassortment (24, 35). Studies have shown that HPAI H5N1 viruses may target cells throughout the human respiratory tract (i.e., nasopharynx, adenoid, tonsil, trachea, bronchi, and lung) (5, 25, 35, 40, 42, 43), where (2,3)-linked sialic acids are available (24, 35), while human viruses may target cells primarily found toward the human upper respiratory airways (24, 35), where there may be a higher abundance of (2,6)-linked sialic acids (6). The available evidence suggests that the distribution of glycans with (2,6)- and (2,3)- linked sialic acids in human respiratory tract epithelia is similar to that of ferrets. If this assumption is correct, avian H5N1 and human seasonal influenza viruses are predicted to coinfect cells within the upper airways of ferrets. The anatomical sites from which H5 subtype and H3 subtype viruses were recovered in our study are consistent with this hypothesis (42, 43). H3 reassortant viruses were recovered twice as frequently (66%; 21 H3 subtype reassortant viruses per 32 reassortant viruses) as H5 reassortant viruses (34%; 11 H5 subtype reassortant viruses per 32 reassortant viruses) from ferret nasal secretions. The preponderance of H3 subtype reassortants may be explained by the abundance of (2,6) receptors for these viruses in the upper respiratory tract of ferrets. Conversely, the delayed nasal shedding of H5 subtype viruses compared to that of H3 viruses may reflect the lower (2,3) glycan receptor availability as well as other factors such as optimal temperature for replication (7, 8, 19). The results of this study suggest that airways in and toward the upper respiratory tract of ferrets are permissive for coinfection and subsequent reassortment between Wyom03 and Thai16 viruses. The undetectable levels of H3 HA RNA and viruses in lung tissue are consistent with the lack of reassortment in this tissue and with previous study reports (17) although strain-to-strain variability among human H3N2 viruses with respect to infection of the lower respiratory tract of ferrets has been demonstrated (27). On the basis of these results and the parallels between influenza virus infections in the ferret model and in humans, we postulate that the site with the highest propensity for reassortment between H5N1 and H3N2 viruses in coinfected humans is located in areas toward the upper airway. However, additional studies will be required to more fully describe the strain-specific tissue tropism of both avian and human viruses in the ferret and to definitively identify the coinfected tissues and cells that serve as the source of reassortant viruses. In summary, we analyzed the potential generation of an H5 influenza virus with pandemic properties by in vivo reassortment of an H5N1 HPAI with a human influenza virus. Although such studies have the acknowledged potential to generate a highly pathogenic and transmissible H5 subtype virus, these experiments address an important question of immediate public health concern as H5N1 subtype viruses continue to

9 VOL. 83, 2009 IN VIVO REASSORTMENT OF INFLUENZA VIRUSES IN FERRETS 8139 infect humans. Furthermore, all experiments described in this study were performed under controlled biosafety level 3 laboratory conditions, with specific additional safety precautions taken when reassortant viruses and animals infected with such highly pathogenic viruses were handled. Using the ferret model, we have found a relatively high incidence of HPAI H5N1 and human H3N2 reassortant viruses after coinfection, indicating that susceptible mammalian hosts can support the formation of new reassortant viruses generated from these subtypes. Further study of the anatomical distribution of parental and reassortant viruses from various tissues of the respiratory tract suggests that generation of reassortant viruses most likely occurred in airway epithelia toward and within the upper respiratory tract of ferrets. When characterized further, H5 subtype reassortant viruses recovered from coinfected ferrets showed decreased fitness and lack of transmissibility in this model, which reduces their current potential threat to human health. Nevertheless, since H5N1 viruses continue to circulate and evolve in poultry, it remains possible for an H5 reassortant virus, which has acquired key, as yet ill-defined determinants of transmissibility, to arise among humans and spread via shedding from the upper respiratory tract. Controlled animal studies such as those described here with newly emergent H5N1 and H3N2 viruses as well as those of additional subtypes are required to continually analyze the risk for genetically drifted strains to generate highly pathogenic and transmissible reassortant viruses. These studies are warranted prior to the uncontrolled emergence of such reassortant viruses in nature, where they may pose an imminent pandemic threat to both animal and human populations. Vaccination and surveillance for seasonal influenza virus in areas where H5N1 is endemic remain important public health measures to minimize the risk of the emergence of such a reassortant pandemic strain. ACKNOWLEDGMENTS This research was supported in part by the National Vaccine Program Office, Department of Health and Human Services, agreement 04FED Sara Jackson and Neal Van Hoeven received financial support for this work from the Oak Ridge Institute of Science and Education, Oak Ridge, TN. We acknowledge colleagues from National Institute of Health, Ministry of Public Health, Thailand, for their contribution of the original H5N1 virus used for reverse genetics and Sunchai Payungporn for his assistance with real-time RT-PCR. We thank the Scientific Resources Program for real-time PCR probe synthesis and the Animal Resources Branch for excellent animal care. 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