Viruses.

H7N7 influenza viruses A/Netherlands/33/03 (CC) and A/Netherlands/219/03 (FC) were isolated from patients during the H7N7 outbreak in The Netherlands in 2003 (20) and passaged twice in embryonated chicken eggs. cDNA of influenza virus A/Chicken/Netherlands/03010132/03 was provided by G. Koch (Central Veterinary Institute at Wageningen UR, The Netherlands). Recombinant H7N7 influenza viruses were generated by reverse genetics as described previously (17). To this end, transient calcium phosphate-mediated transfections of 293T cells were performed as described previously (17) with plasmids carrying gene segments of the CC and FC viruses. The supernatant of the transfected cells was harvested at 48 h after transfection and was used to inoculate embryonated chicken eggs and MDCK cells. Wild-type recombinant FC and CC virus, all 60 possible reassortants thereof, and mutant viruses were produced. For efficient rescue of all recombinant and mutant viruses, 5 μg of a pCAGGS plasmid expressing PB2 of the CC or FC virus was added to the transfection mixture of 293T cells, and Vibrio cholerae neuraminidase (VCNA) (Sigma Aldrich, Zwijndrecht, The Netherlands) was added to the supernatants of 293T and MDCK cells at a concentration of 1 mU/ml. Reassortant and mutant viruses consisting of seven gene segments of influenza virus A/PR/8/34 and one gene segment of the CC or FC virus were produced using a previously described reverse genetics system for influenza virus A/PR/8/34 (17). The genotypes of recombinant viruses were confirmed by sequencing prior to use.

Plasmids.

The gene segments of the CC and FC viruses were amplified by reverse transcription-PCR (RT-PCR) and cloned in the BsmBI site of a modified version of plasmid pHW2000 (17, 27, 42). Gene segments 1, 2, 3, and 5 of the CC and FC viruses were also cloned in the KpnI and NheI sites of the eukaryotic expression plasmid pCAGGS. Plasmid pCAGGS was kindly provided by A. Garcia-Sastre (Mount Sinai School of Medicine, New York, NY).

For the construction of plasmids containing single, double, or triple nucleotide substitutions in PB2, HA, and NA, a QuikChange Multi site-directed mutagenesis kit (Qiagen, Venlo, The Netherlands) was used according to the instructions of the manufacturer.

For construction of a viral RNA-like reporter plasmid, a firefly luciferase open reading frame was PCR amplified using primers extended with the noncoding regions (NCRs) of segment 8 of the CC virus. This insert was cloned in negative sense orientation under the control of a bacteriophage T7 RNA polymerase promoter (18).

All plasmids were sequenced using a Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Nieuwerkerk a/d Ijssel, The Netherlands) and a 3130XL Genetic Analyser (Applied Biosystems), according to the instructions of the manufacturer.

Replication kinetics.

Multistep replication kinetics were generated by inoculating MDCK, QT6, A549, or 293T cells at a multiplicity of infection (MOI) of 0.01 50% tissue culture infective dose (TCID₅₀) per cell. One hour after inoculation, at time zero, cells were washed once with phosphate-buffered saline (PBS) and fresh infection medium was placed on the cells. Supernatants were sampled at 6, 12, 24, and 48 h after inoculation, and virus titers in these supernatants were determined as described below.

Virus titrations.

Virus titrations were performed by end point titration in MDCK cells as described previously (17). MDCK cells were inoculated with 10-fold serial dilutions of culture supernatants. One hour after inoculation, cells were washed once with PBS and grown in 200 μl of infection medium, consisting of EMEM (Cambrex, Heerhugowaard, the Netherlands) supplemented with 4% bovine serum albumin (BSA), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 1.5 mg/ml sodium bicarbonate (Cambrex), 10 mM HEPES (Cambrex), nonessential amino acids (MP Biomedicals), and 20 μg/ml trypsin (Cambrex). Three days after inoculation, the supernatants of infected cell cultures were tested for agglutinating activity using turkey erythrocytes as an indicator of infection of the cells. Infectious titers were calculated from five replicates by the Spearman-Karber method (30).

Virus histochemistry.

Virus histochemistry using the CC-HA 143T and FC-HA 143A viruses was performed as described previously (13, 57). After concentration and purification of virus stocks using sucrose gradients, viruses were inactivated by dialysis against 0.1% formalin and labeled with an equal volume of 0.1 mg/ml of fluorescein isothiocyanate (FITC) (Sigma-Aldrich, Zwijndrecht, The Netherlands).

Formalin-fixed, paraffin-embedded tissues were deparaffinized with xylene and rehydrated with alcohol. FITC-labeled influenza viruses (50 to 100 hemagglutination units) were incubated with the tissues overnight at 4°C. The FITC label was detected with peroxidase-labeled rabbit anti-FITC (Dako, Heverlee, Belgium). Tissues were counterstained with hematoxylin and embedded in glycerol-gelatin (Merck, Amsterdam, The Netherlands). All tissues were from individuals without histological lesions or evidence of respiratory tract infection at the time of death. Tissues from three individuals were analyzed.

Minigenome assay.

Minigenome assays were performed by transfecting 293T or QT6 cells with 5 μg of pCAGGS expression plasmids encoding PB2, PB1, PA, and NP; 5 μg of the firefly luciferase reporter; 10 μg of pAR3132 expressing T7 RNA polymerase (19); and 0.2 μg of a Renilla luciferase expression plasmid (pRL; Promega, Leiden, The Netherlands) as an internal control. At 24 h after transfection, luminescence was assayed using a Dual-Glo luciferase assay system (Promega) according to the instructions of the manufacturer. Luminescence was measured in a Lumistar Galaxy apparatus (BMG Labtech GmbH, Offenburg, Germany). Relative light units (RLU) were calculated as the ratio of firefly and Renilla luciferase luminescences. The relative luminescence was calculated as a percentage of the maximum RLU in each experiment. Plasmid pEGFP-N1 (Clontech, BD Biosciences, Amsterdam, The Netherlands) was transfected in parallel, and the percentage of fluorescent cells was measured in a FACSCalibur (Becton Dickinson) flow cytometer, confirming that the transfection efficiency ranged from 95 to 100% for 293T and QT6 cells in all experiments.

Real-time RT-PCR.

RNA was isolated using a Magnapure LC system with the Magnapure LC total nucleic acid isolation kit (Roche Diagnostics, Almere, The Netherlands), and influenza A virus was detected using a real time RT-PCR assay targeting the matrix gene as described previously (41).

EM.

Transfected 293T cells were fixed for transmission electron microscopy (EM) in 4% formaldehyde and 1% glutar(di)aldehyde and postfixed in 1% osmium tetroxide. After embedding in epoxy resin, thin sections were prepared, stained with 6% saturated uranyl acetate and lead citrate, and examined with a Philips Morgagni 268D electron microscope.

A single substitution in HA affects replication and attachment.

There were three amino acid differences between the HAs of the CC and FC viruses: I13S, A143T, and K416R (Table ​(Table1).1). The contribution of HA of the FC virus to more efficient virus replication than for HA of the CC virus was studied by comparing the FC virus and FC-CC HA virus replication kinetics. The FC-CC HA virus replicated less efficiently than the FC virus (Fig. ​(Fig.2),2), indicating a contribution of the amino acid substitutions in FC HA to the efficient virus replication of the FC virus compared to the CC virus. This finding was confirmed using the CC virus genome as the backbone (data not shown). To determine which of the three substitutions in HA of the FC virus was responsible for enhanced replication, three mutant HA genes were generated, consisting of the CC HA gene with either the I13S, A143T, or K416R substitution. We produced FC viruses with each of these three HAs (FC-CC HA I13S, FC-CC HA A143T, and FC-CC HA K416R) and studied the replication kinetics of these mutant viruses. As shown in Fig. ​Fig.2,2, the A143T substitution was solely responsible for the enhanced replication kinetics observed with HA of the FC virus. This observation was confirmed using recombinant A/PR/8/34 viruses with CC HA I13S, CC HA A143T, and CC HA K416R (data not shown). Thus, although differences in replication kinetics were small, the FC HA gene, and in particular the A143T mutation in HA, increased virus replication, irrespective of the genetic backbone of the virus.

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FIG. 2.

Replication kinetics of viruses with the H7 HAs in MDCK cells. MDCK cells were inoculated at an MOI of 0.01 TCID₅₀/cell with the FC (•), FC-CC HA (), FC-CC HA I13S (□), FC-CC HA A143T (), and FC-CC HA K416R (○) viruses. Supernatants were harvested at 6, 12, 24, and 48 h after inoculation and titrated by end point dilution in MDCK cells. Geometric mean titers were calculated from two independent experiments; the cutoff value was used for negative samples. Error bars indicate standard deviations. The cutoff value of the virus titration assay is indicated by a dotted line.

The A143T substitution introduces a potential glycosylation site at position 141, near the receptor binding site, that may affect the receptor binding specificity or affinity of HA. To determine whether the A143T substitution altered receptor binding of HA, two viruses with mutant HAs were generated: the CC virus with a T at position 143 of HA (CC-HA143T) and the FC virus with an A at position 143 of HA (FC-HA143A). The patterns of attachment of these viruses to human lung tissues were studied and compared to the attachment patterns of the CC and FC viruses using virus histochemistry methods described previously (13, 42, 57). As shown in Fig. ​Fig.3,3, the attachment pattern of the FC-HA143A virus was comparable to that of the CC virus, and the attachment pattern of the CC-HA143T virus was similar to that described previously for the FC virus (42). In the bronchioles, the CC-HA143T virus attached more abundantly to nonciliated cuboidal epithelial cells than the FC-HA143A virus. In the alveoli, both the FC-HA143A and CC-HA143T viruses attached to type II pneumocytes, but only the CC-HA143T virus attached to alveolar macrophages. The FC-HA143A virus also attached to type I pneumocytes, whereas the CC-HA143T virus hardly did (Fig. ​(Fig.3).3). Thus, the A143T substitution in HA of the FC virus caused a subtle change in virus attachment pattern to human lung tissue and increased the replication efficiency of this virus in MDCK cells.

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FIG. 3.

Patterns of attachment of the CC-HA143T and FC-HA143A viruses to tissues of the human lower respiratory tract. Attachment to bronchioles (left panels) and alveoli (right panels) is shown. For comparison, attachment of the CC and FC viruses to these tissues is displayed, as shown before (42). Virus attachment is shown in red. The insets in the right panels show the pattern of virus attachment to alveolar macrophages. The depicted panels reflect the attachment pattern in the whole tissue section as much as possible, but small differences between the single panels and the overall view may exist. Arrows indicate attachment to type I pneumocytes; arrowheads indicate attachment to type II pneumocytes. (Reprinted from reference 42 with permission of the publisher.)

Substitutions in PB2 and PA affect polymerase activity in human and avian cells.

To compare the polymerase activities of the CC and FC viruses, we used a T7 RNA polymerase promoter-based minigenome assay to study the effect of amino acid substitutions on polymerase activity in cells of mammalian and avian origin (18). The reporter minigenome consisted of the firefly luciferase open reading frame flanked by the NCRs of gene segment 8 of the CC virus. Furthermore, we used a plasmid constitutively expressing Renilla luciferase as an internal control to standardize transfection efficiency and overall protein expression levels.

In 293T cells, the polymerase complex of the FC virus induced higher levels of expression of the luciferase reporter than the CC virus (Fig. ​(Fig.4A).4A). A CC virus polymerase complex with PB2 of the FC virus yielded higher luciferase reporter levels than the CC virus polymerase complex (Fig. ​(Fig.4A).4A). Conversely, a polymerase complex of the FC virus with PB2 of the CC virus yielded lower luciferase expression than the wild-type FC virus polymerase complex, demonstrating that PB2 was largely responsible for the differences in polymerase activity between the CC and FC viruses. One of the five amino acid differences between the PB2s of the CC and FC viruses, glutamic acid to lysine at position 627 (E627K), was shown to be the main determinant of pathogenicity of the FC virus in a mouse model (42) and was described in in vitro studies as a determinant of host range (53) and to enable transcription at a low temperature (38). Therefore, two mutant PB2 genes were generated: CC PB2 with a lysine at position 627 (CC E627K) and FC PB2 with a glutamic acid at position 627 (FC K627E). The expression levels of the luciferase minigenome reporter induced by a polymerase complex with CC E627K were comparable to those induced by the wild-type FC PB2 protein, regardless of the origin of the rest of the polymerase complex (Fig. ​(Fig.4A).4A). When the polymerase complexes contained the FC K627E, the reverse pattern was observed and the expression levels of the luciferase minigenome reporter were comparable to those induced by the wild-type CC PB2 protein (Fig. ​(Fig.4A).4A). Thus, a lysine residue at position 627 of PB2 had a large positive effect on the polymerase activity in 293T cells.

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FIG. 4.

Polymerase activities and replication kinetics of H7N7 viruses. (A and B) Minigenome assays were performed by transfecting 293T (A) or QT6 (B) cells with plasmids encoding the polymerase proteins and NP of the CC and FC viruses or mutants thereof. After transfection, cells were incubated 24 h at 33°C (white bars), 37°C (gray bars), or 41°C (black bars). Luminescence of a firefly luciferase reporter was standardized using a plasmid constitutively expressing Renilla luciferase protein. Relative luminescence was calculated as the percentage relative light units (firefly luciferase/Renilla luciferase) of the maximum in each experiment. Averages and standard deviations from three independent experiments are shown. Arrowheads indicate the effect of PA on polymerase activity in the context of the FC virus PB2, as explained in the text. (C to E) Replication kinetics were determined by infecting 293T (C), QT6 (D), or MDCK (E) cells at an MOI of 0.01 TCID₅₀/cell with the CC (triangles), FC (circles), and CC-FC PB2 (squares in panels C and D) or CC-FC PA (diamonds in panel E) virus. Supernatants were collected at 6, 12, 24, and 48 h after inoculation and titrated by end point dilution in MDCK cells. Filled symbols, 37°C; open symbols, 33°C. Geometric mean titers were calculated from two independent experiments; the cutoff value was used for negative samples. Error bars indicate standard deviations. The cutoff value of the virus titration assay is indicated by a dotted line. The replication kinetics shown for the CC and FC viruses in QT6 and MDCK cells are identical to those shown in Fig. ​Fig.11.

Because the effect of PB2 E627K has previously been suggested to be dependent on temperature, the minigenome assay was performed at two additional temperatures: 33°C, representing the temperature in the upper respiratory tract of humans, and 41°C, the temperature of the intestinal tract of birds. At 33°C, the overall expression levels of the luciferase minigenome reporter were higher than those at 37°C, irrespective of the polymerase complex used. At 41°C, expression levels of the luciferase minigenome reporter were comparable to those at 37°C (Fig. ​(Fig.4A).4A). Regardless of the temperature at which the assay was performed, the luciferase minigenome reporter expression induced by a polymerase complex with a lysine at position 627 of PB2 was higher than that induced by a polymerase complex with a glutamic acid at this position.

In QT6 cells, differences in expression of the luciferase reporter by the CC or FC virus polymerase complex were relatively small compared to those in 293T cells. The expression level of the reporter minigenome was slightly but significantly higher with the FC virus polymerase complex than with the CC virus polymerase complex, again due to PB2 of the FC virus. However, there were no significant differences in reporter expression levels with PB2 with either a glutamic acid or lysine at position 627 (Fig. ​(Fig.4B).4B). Also, there was no clear effect of temperature on expression levels of the minigenome reporter (Fig. ​(Fig.4B4B).

To confirm these results with the minigenome assays, in particular the unexpected lack of an effect of temperature on the polymerase activity, replication kinetics of the CC, FC, and CC-FC PB2 viruses were determined in 293T and QT6 cells at 33°C and 37°C. In 293T and QT6 cells, all three viruses replicated faster at 33°C than at 37°C (Fig. 4C and D). In all cases, the FC virus replicated faster than the CC virus and the CC-FC PB2 virus displayed kinetics approaching those of the FC virus. As observed in the minigenome assay, differences in replication efficiency between the three viruses and between the two temperatures were much smaller in QT6 cells than in 293T cells.

Although exchanging PA alone had no effect on the levels of luciferase reporter expressed by the polymerase complexes of the CC and FC viruses (data not shown), there was a difference in the expression of the luciferase reporter with FC PB2 in the context of the FC versus the CC polymerase complex (Fig. ​(Fig.4A,4A, arrowheads). At the amino acid level, the only difference between these polymerase complexes was in PA. PA F666L increased reporter expression levels in the context of the FC virus polymerase complex but not in the context of the CC virus polymerase complex. In theory, silent nucleotide changes in PB1 (n = 2), PA (n = 4), or NP (n = 1) could also explain the difference in the activities of the CC and FC polymerase complexes in the presence of FC PB2, but replication kinetics of a reassortant CC virus with PA of the FC virus showed an increased replication efficiency compared to the CC virus in MDCK cells (Fig. ​(Fig.4E),4E), thus excluding the possibility that silent changes in PB1 or NP contributed to the increased polymerase activity seen in the background of the FC polymerase complex.

There are no differences in modulation of the innate immune response by NS1.

There were two amino acid differences (R126K and V137I) in NS1 of the FC virus compared to the CC virus (Table ​(Table1).1). To test whether these substitutions in NS1 contributed to the efficient replication of the FC virus, two reassortant viruses consisting of seven gene segments of influenza virus A/PR/8/34 and NS of the CC or FC virus (PR8-CC NS and PR8-FC NS) were produced. Of note, there are no differences in NS2 of the CC virus and the FC virus at the nucleotide and protein levels. These two viruses displayed identical growth kinetics in MDCK cells, indicating that the substitutions in NS1 did not have an effect on virus replication (data not shown). To test whether the two NS1 proteins differed in their ability to counteract the induction of type I interferon (IFN) by the host, we used a firefly luciferase reporter construct under the control of an interferon-stimulated response element (ISRE) that is induced in the presence of type I IFN. Supernatants collected 24 h after infection of 293T cells with PR8-CC NS or PR8-FC NS were UV inactivated and subsequently transferred to cells transfected with the ISRE-luciferase reporter (pISRE-Luc; Clontech) and a constitutively expressed Renilla luciferase. No differences in reporter expression were observed between the virus with CC NS or FC NS; both viruses completely blocked the induction of type I IFN (data not shown).

Four substitutions independently affect NA function.

For this study, all 60 possible reassortant viruses consisting of all possible combinations of the gene segments of the CC and FC viruses were produced. By doing so, we found that all viruses containing PB2 or NA of the FC virus, or both, could be rescued without problems, whereas viruses containing PB2 and NA of the CC virus could not be, irrespective of the origin of the other six gene segments. Upon overexpression of PB2 through the addition of an extra plasmid expressing PB2 of the CC virus to the transfection mixture and trans-complementation of NA through the addition of Vibrio cholerae neuraminidase (VCNA) to the culture medium, viruses with PB2 and NA of the CC virus could also be rescued (data not shown). From these data it was concluded that besides PB2, NA also had a large impact on virus production from human 293T cells in vitro. To clarify the role of NA in the replication kinetics of the CC and FC viruses, we constructed a CC virus with NA of the FC virus and vice versa (CC-FC NA and FC-CC NA). The replication kinetics of these reassortant viruses and the wild-type viruses were determined in MDCK cells. The origin of NA had a large effect on the replication efficiency of these H7N7 viruses (Fig. ​(Fig.5A).5A). The CC-FC NA virus replicated faster and to higher titers than the CC virus. Conversely, the FC-CC NA virus replicated slower and to lower titers than the FC virus. The addition of VCNA to culture medium resulted in a modest increase in replication of the CC and FC-CC NA viruses, but replication was not restored to levels observed for the syngeneic viruses with the FC NA (Fig. ​(Fig.5B).5B). The increased replication efficiency of the CC and FC-CC NA viruses upon addition of exogenous NA indicated that the neuraminidase activity of NA of the CC virus was low. Low NA activity may result in aggregation of virus particles and hence in a relatively low infectious virus titer despite high genome copy numbers. Therefore, the number of virus genome copies present in the samples used for Fig. 5A and B were determined by quantitative RT-PCR and compared to the infectious titers of the same samples. In CC and FC virus samples with a comparable number of genome copies (depicted as cycle threshold [C_T] values in Fig. ​Fig.5C),5C), infectious virus titers were more than 10-fold lower in CC virus samples than in FC virus samples. When VCNA was added to the culture medium, the infectious virus titer in CC virus samples was higher than that without VCNA for the same C_T value. VCNA had no discernible effect on virus titers for the FC virus, suggesting that its NA gene was fully functional.

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FIG. 5.

Effect of NA on replication of H7N7 virus. Supernatants of MDCK cells inoculated with an MOI of 0.01 TCID₅₀/cell of the CC (triangles), FC (circles), CC-FC NA (squares), and FC-CC NA (diamonds) viruses in the absence (open symbols) (A) or presence (closed symbols) (B) of 1 mU/ml VCNA were harvested at 6, 12, 24, and 48 h after inoculation and titrated by end point dilution in MDCK cells. Cycle threshold values of the samples taken at 6, 12, and 24 h were determined using a real-time RT-PCR (C) and plotted against the virus titers shown in panels A and B. Geometric mean titers were calculated from two independent experiments; the cutoff value was used for negative samples. Error bars indicate standard deviations. The cutoff value of the virus titration assay is indicated by a dotted line.

To confirm the aggregation of virus particles with NA of the CC virus, virus budding was analyzed by electron microscopy (EM). To minimize intrinsic differences in virus formation between the CC and FC viruses, the FC virus was compared to an FC-CC NA reassortant. 293T cells transfected with eight plasmids carrying the FC-CC NA or FC virus genome were harvested at 12 h after transfection, when virus particles were budding from these cells. Aggregation of CC virus particles on the cell surface was clearly observed by EM, whereas FC virus particles were dispersed around the cell (Fig. ​(Fig.6).6). The addition of VCNA to culture medium after transfection resulted in normal release of FC-CC NA virus from 293T cells (data not shown).

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FIG. 6.

Electron microscopy of H7N7 viruses budding from 293T cells. 293T cells were transfected with plasmids encoding the FC-CC NA or FC virus. At 12 h after transfection, cells were fixed and virus budding was assessed using electron microscopy.

To determine which of the four amino acid substitutions in the FC virus NA caused the efficient release of virus particles from the host cell, we produced four mutant NA genes consisting of the CC NA with one of the four substitutions and tried to rescue these in the context of the CC virus. We were unable to rescue the virus containing the T442A substitution (M3) in three independent attempts (Table ​(Table2).2). The other three mutant viruses could be rescued, but all replicated to lower titers than the wild-type CC virus. Therefore, all possible double and triple mutant NAs were constructed. Two double mutant viruses containing the T442A substitution in combination with N308S (M6) and A346V (M8) could not be rescued in three independent attempts. Only three of the double and triple mutant viruses (M7, M10, and M13) were rescued with similar efficiency as the CC virus, but none replicated to similar titers as the CC-FC NA or FC virus. From the single and triple mutant viruses, it was concluded that each of the four individual substitutions resulted in decreased NA function of both the CC and the FC NAs. Therefore, all four amino acid substitutions appear to be required independently for the efficient functioning of the N7 NA.

TABLE 2.

Effect of amino acid substitutions in NA on rescue of the CC virus^a

Virus	Amino acid at positionb:				Rescue successc	Virus titerd (10 log TCID50/ml)
	308	346	442	458
CC	N	A	T	P	+++	2.7-5.9
Single mutants
M1	S	A	T	P	+++	1.7-3.3
M2	N	V	T	P	++	1.9-4.3
M3	N	A	A	P	−
M4	N	A	T	S	+++	0.7-3.7
Double mutants
M5	S	V	T	P	+	0.9
M6	S	A	A	P	−
M7	S	A	T	S	+++	3.7-5.3
M8	N	V	A	P	−
M9	N	V	T	S	++	3.3-6.1
M10	N	A	A	S	+++	2.5-5.9
Triple mutants
M11	S	A	A	S	+	2.1
M12	N	V	A	S	++	1.1-6.1
M13	S	V	T	S	+++	3.5-4.1
M14	S	V	A	P	+	0.7
CC-FC NA	S	V	A	S	+++	4.5-7.3
FC	S	V	A	S	+++	7.5-7.9

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^a293T cells were transfected with plasmids encoding the wild-type CC or FC virus or the CC virus with mutant NA as indicated and a plasmid expressing PB2 of the CC virus; VCNA was added to the culture medium. 293T supernatants were harvested at 48 h after transfection and used to inoculate MDCK cells, without the addition of VCNA to the culture medium. MDCK supernatants were harvested after approximately 4 days and used to determine infectious virus titer as described in Materials and Methods.

^bMutated amino acid positions are indicated in bold.

^cThree independent rescue attempts were performed; each + indicates one successful virus rescue attempt, and − indicates that the virus could not be rescued at all.

^dRange of virus titers in MDCK cell supernatants of successful virus rescue attempts only.

We thank Guus Koch and Sylvia Pritz-Verschuren at CVI, Lelystad, for providing us with A/Chicken/Netherlands/03010132/03; Adolfo Garcia-Sastre for plasmid pCAGGS; and John Dunn for plasmid pAR3132. We thank Ton de Jong, Frank van der Panne, Theo Bestebroer, Monique Spronken, Brigitte van Uden, and Bernadette van den Hoogen for excellent technical assistance.

This study was funded in part by EU FP6 program RiviGene (no. SSPE-CT-2005-022639) and contract NIAID-NIH HHNSN266200700010C.