To generate the collection of human-avian reassortant viruses for this study, we first developed plasmid-based reverse genetics (rg) systems for the two parental viruses; A/Wyoming/3/2003 (subtype H3N2) (WY03) and A/Thailand/16/2004 (H5N1) (TH04) [17]. The parental WY03 virus showed α2,6 linked SA receptor specificity [18], replicated to high titers in MDCK cell culture, and was avirulent in mice (data not shown). The TH04 virus showed α2,3 receptor specificity [8], replicated efficiently in MDCK cells and was highly virulent for mice [17]. Virus recovery from plasmid DNA transfections was evaluated by quantitative plaque analysis at 72 hours (h) post-transfection; herein referred to as rescue efficiency. Cell cultures transfected with WY03 and TH04 rg plasmids yielded >10⁷ plaque-forming units (pfu)/ml of progeny virus, termed rgWY03 and rgTH04, which formed 4–5 mm diameter plaques, comparable to those of parental wildtype (wt) viruses (Figure 1). A wide range of virus yields and plaque diameters were obtained for each of the 63 H5N1 human-avian reassortant (rH5N1) plasmid transfections. In order to categorize the in vitro properties of each reassortant, rH5N1 genotypes were segregated into 4 phenotypic groups, according to their rescue efficiencies (Figures 1 and 2): (1) rH5N1 genotypes with wt or near-wt replication efficiency. Twenty-eight rH5N1 viruses (cell culture phenotype group 1) consistently yielded ≥10⁶ pfu/ml in the transfected cell cultures (Figure 1), which represented rescue efficiencies similar to those of rgWY03 and rgTH04. Most of the cell culture group 1 viruses formed ∼2–4 mm plaques in diameter (Figure 1). The efficient in vitro growth phenotypes of nearly one-half of the rH5N1 viruses in the group revealed a high frequency of functional compatibility between avian and human virus genes. (2) rH5N1 genotypes with moderate cell culture replication impairment. Fourteen rH5N1 viruses (22% of the rH5N1 genotypes) had rescue efficiencies between 10⁴ and 10⁶ pfu/ml (cell culture phenotype group 2), and most of these viruses formed 1–3 mm plaques (Figure 2). (3) rH5N1 genotypes with severe cell culture replication impairment. Eight reassortants (13% of the rH5N1 genotypes) yielded ∼10²–10⁴ pfu/ml from transfected cell cultures, with plaque size ranging from 0.5–2 mm (cell culture phenotype group 3 in Figure 2). (4) Non-viable or marginally viable rH5N1 genotypes. Thirteen rH5N1 viruses (∼20% of rH5N1 genotypes) yielded <100 pfu/ml from transfected cell cultures (cell culture phenotype group 4 in Figure 2). Their rescue efficiencies were 5 log₁₀ pfu/ml lower than their rg parent viruses. The severe replication defects of viruses in this group may reflect structural or functional incompatibilities in avian-human viral RNA and/or protein complexes. Collectively, these categories guided our rationale for excluding reassortants with severe replication defects from further in vivo studies.

Notably, the severely impaired rH5N1 viruses in group 4 (Figure 2) were all characterized by the association of the nucleoprotein (NP) gene from WY03 virus with matrix (M) and/or nonstructural (NS) genes derived from TH04 virus. For example, the single gene reassortant r5 (group 4), which carried the NP from WY03 and the five other internal genes from TH04 had a rescue efficiency of <10² pfu/ml. However, replacement of the TH04 NS gene with the WY03 NS in the same background increased rescue efficiency to ∼10⁴ pfu/ml (r5/8 virus, group 2, Figure 2), which was significantly higher than r5 (P≤0.0001). Further introduction of the WY03 M segment into this gene constellation restored the rescue efficiency and plaque size of the reassortant virus (r5/7/8, group 1, Figure 1) to nearly wt level (P≤0.0001). In contrast, introduction of polymerase complex genes did not improve replication (r5 replication is similar to r1/5, r2/5, r3/5, and r1/2/3/5; P>0.9) (Figure 2). Conversely, only 6 out of 28 rH5N1 viruses (group 1) that replicated efficiently had NP of human origin, in every case along with human NS (Figure 1). These observations suggest that the NP gene of WY03 origin preferentially interacts with M and NS genes of the same origin for optimal replication. In contrast, the NP gene of the TH04 avian virus appears to be more compatible with the M or NS of heterologous origin (e.g., r1/2/3/7/8 virus replication was similar to r1/2/3/7 or r1/2/3/8; P = 1.0). Although not all viruses with human NP and avian M or NS were severely impaired, they generally displayed significantly reduced replication, suggesting that avian M and/or NS may not be incorporated into seasonal human H3N2 viruses in the absence of avian NP.

Another remarkable gene incompatibility was noted with the r2/3/5/7/8 virus, bearing TH04 PB2 and the remaining five internal genes from WY03 virus (Figure 2, group 3). This reassortant virus formed tiny plaques (0.5 mm diameter) and had a very low rescue efficiency. This defect was repaired by providing a PA gene of avian virus origin; i.e., the rescue efficiency of r2/3/5/7/8 was significantly lower than r2/5/7/8 (Figure 1, group 1) (P<0.0001), suggesting a functional interaction of TH04 PB2 with the cognate avian PA gene. This finding suggests that reassortment of avian PB2 genes with human viruses may be linked to co-incorporation of the avian PA gene.

A set of 38 rH5N1 viruses with cell culture replication efficiencies comparable to those of parental viruses, or with only modest reductions, were chosen for study in a BALB/c mouse model to assess their virulence in a mammalian host. The plasmid-derived rgTH04 virus was highly virulent for mice, as indicated by a very low intranasal 50% mouse infectious dose (MID₅₀ = 10^1.5 pfu) and lethal dose (LD₅₀ = 10^1.8 pfu) (Figure 3). This virus replicated to high titers (>10⁷ pfu/ml) in lungs by day 4 following an intranasal inoculation of 10⁴ pfu and caused >19% body weight loss. Viral replication was also detected at systemic sites, such as brain and spleen, recapitulating the outcome of infection with the wt TH04 isolate [17]. In contrast, replication of the rgWY03 virus in mice was very inefficient as evidenced by an MID₅₀ of 10⁶ pfu and an LD₅₀ of >10⁶ pfu (determination of MID₅₀ for rgWY/03 required additional doses of 10⁵ and 10⁶ pfu to detect virus in tissues; data not shown). However, the reassortant virus bearing HA and NA from TH04 and the remaining genes from WY03 virus (r1/2/3/5/7/8) replicated efficiently (MID₅₀ of 10^1.8 pfu and titer of 10⁷ pfu/ml in the lung), suggesting that the HA and/or NA from WY03 lack appropriate interaction with receptors or other host factors in the mouse respiratory tract [19]. Most importantly, the internal genes from WY03 mediated efficient viral replication of r1/2/3/5/7/8 virus in mice validating the BALB/c mouse as a useful model to evaluate the influence of human/avian internal gene combinations on the virulence phenotypes of rH5N1 viruses.

Three rH5N1 viruses were highly virulent for mice, with an LD₅₀<10³ pfu (Figure 3, group A1). Each of these rH5N1 had an MID₅₀ of ≤10^1.5 pfu, replicated to high titers in the lung (≥10^6.5 pfu/ml), and caused >17% weight loss by 6–7 days post-infection (dpi) on average. The virulence of these viruses was comparable to that of wt TH04. In addition, the high frequency of virus detection in the spleen and brain of mice indicated systemic spread of these viruses, resembling infection with wt TH04. The ten viruses in group A2 were moderately virulent, with a mean LD₅₀ value that was significantly different from that of the highly virulent group A1 viruses (P<0.001). The remaining 25 rH5N1 viruses in groups B1 and B2 exhibited low virulence phenotypes in mice with LD₅₀ values >10⁴ pfu. However, five rH5N1viruses (Figure 3, virulence group B1) caused significant transient weight loss (>16%), clinical signs, such as ruffled haircoat and lethargy, and three viruses (r1/3/7/8, r1/2/3/8, r1/5/7/8) each caused mortality in a single mouse infected at 10⁴ pfu, suggesting potential for increased virulence at higher virus inoculums (data not shown). The other 20 rH5N1 viruses (Figure 3, virulence group B2) caused subclinical infections in mice, with minor weight loss (<15%). These viruses spread to the spleen and /or brain sporadically and their pulmonary replication capacity ranged from substantially efficient to nil. Although many rH5N1 viruses with high rescue efficiencies and large plaque phenotypes also displayed highly virulent phenotypes in mice, several rH5N1 viruses belonging to virulence group B2 (i.e., r2/7/8 and r1/3/8) had high rescue efficiencies but did not replicate well in mice. This finding highlights the limitations of inferring in vivo virulence properties solely from efficient in vitro replication characteristics.

Interestingly, r1/3/5/7/8, one of the most virulent rH5N1 among the 38 reassortants inoculated into mice had a gene constellation resembling that of the pandemic viruses from 1957 and 1968. In 1957, HA, NA and PB1 genes from an avian H2N2 virus were introduced into the circulating human H1N1 virus and caused the so-called “Asian flu” pandemic, whereas the 1968 “Hong Kong” pandemic virus incorporated the HA and PB1 genes from an avian donor [3]. In this study, a virus carrying HA, NA and PB1 of avian origin and the remaining genes from a human virus, namely r1/3/5/7/8, was highly virulent for mice (LD₅₀ = 10^2.5 pfu). In contrast, a reassortant virus (r1/2/3/5/7/8) with all the internal genes from WY03 virus, including PB1, caused minimal mortality and had a significantly different LD₅₀ (1.3 log₁₀ pfu increase; P<0.001), suggesting that the PB1 of contemporary H5N1 viruses can reassort into circulating H3N2 viruses and increase their virulence for mice.

Efficient viral replication at the lower temperature of the upper respiratory tract is thought to be essential for droplet transmission of influenza virus between humans. Avian influenza viruses with a PB2 polymerase bearing glutamic acid at position 627 instead of lysine have decreased replication at 33°C in mammalian cells [20],[21],[22]. Although both WY03 and TH04 viruses have lysine at position 627 in PB2, it is not known whether new avian and human gene constellations would compromise viral replication at lower temperature. To address this question, we determined reassortant viral titers in the nasal turbinates collected at 4 dpi. We found that in general, rH5N1 viruses replicated less efficiently in nasal turbinates than in lungs, as reported previously [21]. Interestingly, some reassortants (i.e., r2/7, r2/7/8, and r3/7/8) showed extremely low replication in nasal turbinates despite considerable titers in lungs (Figure 3). These reassortants would be expected to lack efficient transmissibility by generation of nasal secretion droplets.

Although mice are regarded as a useful mammalian model for studying the replication of HPAI viruses, the species differences between humans and mice mandate studies with models from the target species to complement the data. The epithelial cells of the respiratory tract are the primary targets of influenza infection. Therefore, we used in vitro differentiated HTBE cultures to evaluate the replication potential of the rH5N1 viruses in humans [23]. HTBE cells were infected with rgWY03 and rgTH04 viruses, or each of 38 rH5N1 viruses that were previously analyzed for virulence in mice. We quantified virus progeny released into the apical side of the pseudostratified epithelium because budding of HPAI H5N1 virus in the HTBE model remains polarized (data now shown). As shown in Figure 4A, both rgTH04 and rgWY03 parental viruses replicated efficiently in the HTBE cells. The rapid rise of WY03 virus titers to 10⁸ pfu/ml at 32 h post-infection was consistent with the efficient spread of human viruses in HTBE cells, as described previously [23]. The plateau in WY03 virus production may be caused by virus-induced cell death, first noted at 40 h post-infection. In contrast, HTBE cells infected with rgTH04 virus showed no cytopathology and virus progeny increased steadily throughout the 56 h infection. The majority of rH5N1 viruses produced ≥10⁴ pfu/ml by 24 h post-infection, and the growth kinetics were similar to parental rgTH04 or slightly delayed (e.g., r1/2/3/5/7/8 in Figure 4B and data not shown for others). In comparison, four rH5N1 viruses, r/3, r2/3, r3/8, r3/7/8, replicated substantially less efficiently in the HTBE cells (Figure 4, C and D). Interestingly, these four viruses also replicated poorly in mice; had MID₅₀ values of ≥4 log₁₀ pfu and caused minimal weight loss (Figure 3). These results supported the virulence data provided by the mouse model.

To study the mechanisms underlying the differences in the replication phenotypes of certain rH5N1, we exploited a mini-genome reporter assay which dissects the function of the viral ribonucleoprotein (RNP) complex from the rest of the viral gene products [24],[25]. The 16 possible RNP combinations of PB2, PB1, PA and NP from either the TH04 or WY03 viruses were studied at 33°C or 37°C, to recapitulate the temperatures of the upper and lower respiratory tract, as reported previously [20]. Another panel of RNP combinations with A/Vietnam/1203/2004 (VN04) viral genes replacing TH04 genes was also analyzed in parallel to extend the results for other H5N1 viruses. The human RNP was almost equally active at 33°C and 37°C, whereas the avian RNP activity was substantially reduced at 33°C despite the presence of lysine at position 627 of PB2, in both TH04 and VN04 backgrounds (Figure 5). The RNP constituted by PB1 and PA from WY03 virus and PB2 from TH04 (or VN04) virus resulted in extremely low polymerase activities at 33°C and 37°C (Figure 5A and B, RNP denoted by asterisks). Although the RNP complexes carrying PB2 and PB1 from TH04 and PA from WY03 virus showed partially reduced polymerase activity, a similar combination derived from VN04 and WY03 viruses showed a more pronounced loss of replication activity (Figure 5A and B, RNP denoted by arrows). The reduced polymerase activities of these gene constellations were consistent with the low viral titer from lungs and nasal turbinates of mice infected with reassortant viruses r3/7/8, r3/8, r3 and r2/3 (Figure 3). Interestingly, the polymerase activity of the RNP with PB1 from TH04 and the other proteins from WY03 was comparable to that of the wt WY03 RNP. These findings indicated that the increased mouse virulence attributed to avian PB1 in the WY03 genetic background (r1/3/5/7/8) may not directly result from stimulation of the polymerase activities of the RNP. Alternative hypotheses to reconcile these observations would include an in vivo role for PB1 in RNP function, a possible modulation of host cell function by PB1-F2, or unknown interactions of PB1 with the remaining 4 genes absent in this assay: HA, NA, M and NS.

Although H5N2 subtype viruses have been isolated from poultry in North America and Asia for many years [26], it is not clear whether the N2 derived from contemporary human H3N2 virus can support the efficient replication of a reassortant virus bearing the HA from circulating H5N1 virus. Balanced HA and NA activities are critical for efficient influenza virus infection and replication in various hosts. The HA of contemporary H5N1 viruses has retained a strong preference for α2,3 linked SA [7],[8]. In contrast, the NA derived from H3N2 human seasonal isolates has adapted over time to acquire α2,6 SA specificity [27],[28]. To evaluate the H5N2 reassortant, the NA of TH04 was replaced with the N2 from WY03 virus by reverse genetics. This virus, termed r6 (H5N2), was virtually identical to wt TH04 in rescue efficiency and replication in HTBE cells (Figure 1 and Figure 4B). In addition, this reassortant was highly virulent in mice, with an LD₅₀ comparable to the parental TH04 virus (Figure 3). These results suggested that the NA activity from circulating H3N2 viruses can functionally support the activity of the H5 HA and promote H5N2 virus spread in the mammalian host.

A/Thailand/16/2004 (TH04) and A/Vietnam/1203/2004 (VN04) H5N1 viruses and A/Wyoming/3/2003 (WY03) H3N2 virus obtained from the WHO Global Influenza Surveillance Network were provided by Alexander Klimov (CDC, Atlanta, USA). Madin-Darby canine kidney (MDCK) and human lung carcinoma (A549) cells were obtained from the American Type Culture Collection and propagated in Dulbecco's Modification of Eagle's Medium with 10% fetal bovine serum. Viral infectivity was determined by plaque assay on MDCK cells as described [41]. Reassortant viruses containing any segment derived from the H5N1 virus were generated in compliance with the Institutional Biosafety Committee and NIH Guidelines for Research Involving Recombinant DNA Molecules. Viruses were handled in biosafety level 3 containment, including enhancements required by the U.S. Department of Agriculture and the Select Agents program http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm.

RT-PCR amplicons of the eight viral genes from WY03 and TH04 viruses were cloned into a dual-promoter plasmid for influenza A reverse genetics [42]. Virus rescue was performed by plasmid DNA transfection into co-cultured 293T/MDCK cells [42]. Culture medium from the transfected cells was harvested at 72 h and analyzed by plaque assay on MDCK monolayers. The plaque count and diameter were recorded as a measure of the virus rescue efficiency from plasmid DNA. DNA transfection of each genotype was performed at least three times independently. WY03 and TH04 rg plasmid sets were included as controls during each reassortant rescue to evaluate experimental variation. Viruses with H5 HA were propagated in 10–11 days old embryonated chicken eggs. The H3N2 virus was propagated in MDCK cells in the presence of 1 μg/ml TPCK-treated trypsin. Following propagation, the full genomes of reassortant viruses were sequenced to confirm presence of parental virus sequence.

A549 cells cultured in 24-well tissue culture plates were co-transfected with pPol1-NS-Renilla (100 ng) encoding a reporter mini-genome under transcriptional control of the human RNA polymerase I, pSV-Luc (200 ng) encoding firefly luciferase under SV40 virus RNA polymerase II promoter control, and four plasmids expressing viral PB2, PB1, PA, NP (50 ng each) from the strain of interest. Twenty-four hours after the transfection, the cell lysates were harvested and further diluted to perform the dual luciferase assay according to the manufacturer's protocol (Promega). The influenza polymerase catalytic activity derived from the Renilla luciferase plasmid (pPol1-NS-Renilla) was corrected to account for well-to-well differences in transfection efficiency using the firefly luciferase activity values from pSV-Luc plasmid.

Groups of 6-8 week old female BALB/c mice (Jackson Laboratories, Bar Harbor, ME) were placed under light anesthesia and inoculated intranasally with 50 μl of serial 10-fold dilutions of infectious virus in PBS. For reassortant viruses tested, 10⁴ pfu of virus was the highest dose used to infect mice; for WY03 virus, 10⁶ pfu of virus was tested. Three mice from each group were euthanized at 4 days post-infection (dpi) and nasal turbinates, lungs, spleens, and brains were harvested, immediately frozen on dry ice, and stored at −80°C until further processing. Whole tissues were thawed, homogenized in 1 ml of cold PBS, and clarified by centrifugation (2,200×g) at 4°C. Virus titers of homogenates were determined by plaque assay in MDCK cells. Five additional mice in each group were monitored daily for clinical signs for 14 dpi. Mice that lost more than 25% of their body weight were euthanized humanely. The fifty percent mouse infectious dose (MID₅₀) and fifty percent lethal dose (LD₅₀) were calculated and expressed as the pfu value corresponding to 1 MID₅₀ or LD₅₀. Animal studies were conducted per approved Institutional Animal Care and Use Committee protocols.

Statistically significant differences of rescue efficiencies of avian-human reassortants in cell culture were determined by F-test adjusted for multiple comparisons. LD₅₀ and MID₅₀ values were calculated using the method of Reed and Muench [43]. Statistically significant differences between LD₅₀ values of viruses in virulence group A1 and A2 were determined by comparing groups A1 and A2 to TH04 WT and group A1 to group A2 using an analysis of variance performed by an F test for multiple comparisons.

Growth and differentiation of primary human tracheobronchial epithelial cells were performed as described previously [23],[30],[44]. Briefly, primary cells (passage level 3) were seeded in porous membrane inserts (Corning, 4.5 μm, 12 mm diameter) at the density of 5×10⁴ cell/cm². Three days after seeding the cells, the medium from the apical side was removed and the confluent monolayers were cultured at an air-liquid interface. The medium from the basal compartment was replaced daily, and the in vitro differentiation of primary cells was achieved after 4–6 weeks. Differentiated cells with trans-epithelial electrical resistance of ≥300Ω cm² were used in our study. Kinetic analysis of reassortant virus growth was performed after infection of the monolayer at a multiplicity of infection (moi) of 0.02 pfu/cell as described [23],[30]; apically released virus was harvested at the appropriate times and analyzed by plaque assay.

The GenBank (http://www.ncbi.nlm.nih.gov/sites/entrez) accession numbers for the genes described in this paper are: EU268216 (A/Thailand/16/2004, PB2 gene), EU268217 (A/Thailand/16/2004, PB1 gene), EU268218 (A/Thailand/16/2004, PA gene), EU268219 (A/Thailand/16/2004, HA gene), EU268220 (A/Thailand/16/2004, NP gene), EU268221 (A/Thailand/16/2004, NA gene), EU268222 (A/Thailand/16/2004, M gene), EU268223 (A/Thailand/16/2004, NS gene), EU268224 (A/Wyoming/03/2003, PB2 gene), EU268225 (A/Wyoming/03/2003, PB1 gene), EU268226 (A/Wyoming/03/2003, PA gene), EU268227 (A/Wyoming/03/2003, HA gene), EU268228 (A/Wyoming/03/2003, NP gene), EU268229 (A/Wyoming/03/2003, NA gene), EU268230 (A/Wyoming/03/2003, M gene), EU268231 (A/Wyoming/03/2003, NS gene).