The 1918-like avian virus possessing these eight 1918-like avian viral segments was successfully recovered from 293T cells transfected with the plasmids required to generate this virus. The virus grew well in Madin-Darby canine kidney (MDCK) cells and embryonated chicken eggs (8.3 ± 0.1 and 8.6 ± 0.2 log 10 plaque-forming units (pfu)/ml at 24 hr postinfection, respectively), and its growth was comparable to that of the 1918 virus (8.8 ± 0.1 and 8.4 ± 0.2 log 10 pfu/ml in MDCK cells and eggs, respectively, at 24 hr postinfection).

To assess the risk of emergence of a 1918-like virus and to delineate the amino acid changes that are needed for such a virus to become transmissible via respiratory droplets in mammals, we attempted to generate an influenza virus composed of avian influenza viral segments that encoded proteins with high homology to the 1918 viral proteins. In particular, we selected the following genes to generate a 1918-like avian influenza virus (referred to as 1918-like avian virus) (see Supplemental Experimental Procedures ): the PB2 segment of A/blue-winged teal/Ohio/926/2002 (H3N8), the PB1 segment of A/blue-winged teal/Alberta (ALB)/286/77 (H3N6), the PA segment of A/pintail duck/ALB/219/77 (H1N1), the NP segment of A/blue-winged teal/Ohio/908/2002 (H1N1), the M segment of A/duck/Germany/113/95 (H9N2), the NS segment of A/canvasback duck/Alberta/102/76 (H3N6), the HA segment of A/pintail duck/ALB/238/79 (H1N1), and the NA segment of A/mallard/duck/ALB/46/77 (H1N1), as described in the Supplemental Information . The resulting virus differs by 8 (PB2), 6 (PB1), 20 (PB1-F2), 9 (PA), 7 (NP), 33 (HA), 31 (NA), 1 (M1), 5 (M2), 4 (NS1), and 0 (NS2) amino acids from the 1918 virus.

We first determined whether influenza A virus gene segments that encode proteins with high homology to the 1918 viral proteins exist in the avian influenza virus gene pool. We focused on amino acid sequence comparisons because our goal was to identify avian influenza viral proteins that closely resemble 1918 virus proteins in structure and function and may therefore allow the emergence of a 1918-like virus. To this end, we performed a sequence similarity search using BLAST ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) to identify the closest relatives to the 1918 viral proteins. Interestingly, for most viral proteins (except for hemagglutinin [HA], neuraminidase [NA], and PB1-F2), we found avian influenza virus proteins that differed from their 1918 counterparts by only a limited number of amino acids ( Table S1 ). For example, for polymerase basis 2 (PB2) we found one avian influenza PB2 protein that differed from 1918 PB2 by eight amino acids. Similarly, we found avian influenza PB1, polymerase acid (PA), nucleoprotein (NP), matrix protein 1 (M1), M2, nonstructural protein 1 (NS1), and NS2 proteins that closely resembled their 1918 counterparts. Most of the viruses from which these proteins were derived were isolated recently, suggesting that 95 years after the devastating 1918 pandemic, avian influenza virus genes encoding 1918-like proteins continue to circulate in nature. For the 1918 HA and NA proteins, we identified the closest avian H1 and N1 relatives, respectively. These proteins differed from the 1918 proteins by 33 and 31–33 amino acids, respectively, a finding that was expected due to the higher evolutionary rates in these genes relative to the other influenza viral genes.

The ferret is considered the best current model for influenza virus infection because infected animals exhibit symptoms that resemble those of humans infected with influenza A virus. Therefore, we next tested the pathogenicity of the 1918-like avian virus in ferrets. Ferrets (three animals per group) were intranasally inoculated with 10pfu/500 μl of the 1918-like avian virus, 1918 virus, or DK/ALB virus ( Figure S1 , available online). All animals infected with the 1918 virus became symptomatic; their body weights declined drastically (the mean maximum body weight loss was 17.5% ± 2.2%), and one of them died on day 8 postinfection ( Figure S1 A). By contrast, none of the ferrets infected with DK/ALB exhibited noticeable clinical signs (no appreciable body weight loss in any of the animals; Figure S1 C), whereas animals infected with the 1918-like avian virus became symptomatic and showed substantial body weight loss (the mean maximum body weight loss was 11.9% ± 3.9%; see Table 1 and Figure S1 B). Of the three viruses tested, the 1918 virus replicated the most efficiently in the upper and lower respiratory tract (p < 0.01; Figure 1 and Table S2 ). The 1918- and 1918-like avian virus-infected ferrets displayed numerous viral antigen-positive cells in tracheal, bronchial, bronchiolar, and glandular epithelial cells and necrotized changes in some trachea-bronchial glands on day 6 postinfection ( Figure 2 A), whereas the 1918-like avian virus-infected ferrets presented few antigen-positive cells on day 3 postinfection ( Figure S2 ). In the lungs, no significant differences in pathologic changes were detected between the 1918 and 1918-like avian virus groups ( Figures 2 A, 2B, and S2 ). In contrast, the DK/ALB infection caused only minimal to mild bronchitis or bronchiolitis with little viral antigen expression on days 3 and 6 postinfection ( Figures 2 A, 2B, and S2 ). Taken together with the results of recent studies that showed no appreciable clinical signs in ferrets infected with more commonplace avian influenza viruses of various subtypes (), the finding that the 1918-like avian influenza virus is of intermediate pathogenicity in mammals may suggest that the progenitor of the 1918 virus was an unusual avian influenza virus whose pathogenicity in mammals was higher than that of most avian influenza viruses.

(B) Pathological severity scores for infected ferrets. To represent comprehensive histological changes, respiratory tissue slides were evaluated by scoring pathological changes as described in the Supplemental Experimental Procedures . The sum of the pathologic scores for all five lung lobes was calculated for each ferret. The means ± SD from three ferrets are shown. Asterisks indicate virus pathological scores significantly different from that of the 1918-like avian virus (Dunnett’s test; p < 0.05). See also Figure S2

(A) Histopathological findings in virus-infected ferrets. Shown are representative pathological changes in tracheae and lungs of ferrets infected with 10 6 pfu of the indicated viruses on day 6 postinfection. Three ferrets per group were infected intranasally with 10 6 pfu of virus, and tissues were collected on day 6 after infection for pathological examination. No virus was detected from the lungs of the DK/ALB-infected ferrets. Left: H&E staining. Right: immunohistochemical staining for influenza viral antigen (NP). Scale bars, 100 μm.

Virus replication in respiratory organs of ferrets. Ferrets were intranasally infected with 10pfu of virus. Six animals per group were euthanized on days 3 and 6 postinfection for virus titration. Virus titers in nasal turbinates, tracheae, and lungs were determined by plaque assay in MDCK cells. Horizontal bars indicate the mean virus titers. Asterisks indicate virus titers significantly different from those of the 1918-like avian virus (p < 0.05;p < 0.01). See also Table S2

See also Figures S1 and S3 and Table S3 . For each pair of ferrets, one animal was intranasally inoculated with 10pfu of virus (0.5 ml) (virus-inoculated ferret), and 1 day later a naive ferret was placed in an adjacent cage (contact ferret). Virus-inoculated ferrets were monitored for 14 days to determine survival rates and body weight changes.

The 1918-like avian virus possessing the avian influenza viral segments that encoded proteins with high homology to the 1918 viral proteins described in the Supplemental Experimental Procedures was generated by using reverse genetics. The effectiveness of current vaccines and antiviral drugs against the 1918-like avian virus was examined (see Tables S4 and S5 ).

To assess the pathogenicity of the 1918-like avian virus, we first determined the mouse lethal dose 50 (MLD; the dose required to kill 50% of infected mice) values of the 1918-like avian influenza virus and authentic 1918 virus. As a control for the authentic avian influenza virus, we used A/duck/ALB/35/76 (H1N1; DK/ALB) because this avian strain was well characterized in a previous study (). The 1918-like avian influenza virus showed intermediate pathogenicity (with an MLDof 5.5 logpfu) compared with DK/ALB (with an MLDof 6.8 logpfu) and the 1918 virus (with an MLDof 2.7 logpfu). These data indicate that the avian influenza virus genes that were selected because of their close relationship with 1918 virus proteins not only cooperate at a functional level, but also support pathogenicity higher than that of an authentic avian influenza virus.

For an influenza virus to cause a pandemic, it must achieve efficient human-to-human transmission. Therefore, we tested transmissibility in ferrets of the authentic 1918 virus, as well as the 1918-like avian virus and its reassortants. Three animals were each intranasally inoculated with 10pfu of virus. On day 1 after infection, a naive ferret was housed in a cage adjacent to each of the infected ferrets. This setup prevented direct contact between animals but allowed the spread of influenza virus through respiratory droplets. Viral titers were determined in nasal washes collected from both the inoculated and contact ferrets every other day postinfection/contact (for up to 9 days). The 1918 virus was recovered from two of the three contact ferrets ( Figure 3 A), demonstrating respiratory droplet transmission of the 1918 virus. In contrast, the 1918-like avian and DK/ALB viruses failed to transmit among ferrets; no virus was detected in nasal washes collected from contact animals, although the inoculated animals did shed virus ( Figures 3 B and 3C). Similarly, no virus was recovered from contact animals for the 1918 PB2/Avian, 1918 HA/Avian, and 1918 PB2:HA/Avian virus groups ( Figures S4 A–S4C). However, virus was recovered from one of the three contact animals in the 1918(3P+NP):HA/Avian and two of three contact ferrets in the 1918 PB2:HA:NA/Avian virus group ( Figures S4 D and S4E), suggesting potential roles for the RNA replication complex, HA, and NA in virus transmission among ferrets. Taken together, our data suggest that the 1918 PB2 and HA genes confer enhanced pathogenicity and transmissibility to the 1918-like avian virus in ferrets.

Groups of three ferrets were infected intranasally with 10pfu of 1918 (A), 1918-like avian (B), DK/ALB (C), 1918-like avian PB2-627K (D), 1918-like avian PB2-627K:HA-89ED/190D/225D (E), and 1918-like avian PB2-627K/684D:HA-89ED/113N/190D/225D/265DV:PA-253M (F) viruses. After 1 day, a naive ferret (contact ferret) was placed in a cage adjacent to each infected ferret. Nasal washes were collected from infected ferrets on day 1 after inoculation and from contact ferrets on day 1 after cohousing and then every other day (for up to 9 days) for virus titration. The lower limit of detection is indicated by the horizontal dashed line. See also Figure S4 and Table S3

To identify the 1918 viral segments that are responsible for the intermediate pathogenicity of the 1918-like avian virus relative to the 1918 and authentic avian viruses, we examined the pathogenicity of reassortant viruses possessing 1918 viral genes in the genetic background of the 1918-like avian virus in ferrets. We focused on the HA and PB2 genes, which are known to play important roles in the adaptation of avian influenza viruses to mammals (). We generated 1918 PB2/Avian, 1918 HA/Avian, and 1918 PB2:HA/Avian viruses, which possess the PB2, HA, or PB2 and HA genes of the 1918 virus, respectively, and the remaining genes from the 1918-like avian virus ( Figure S3 ). We also created 1918 PB2:HA:NA/Avian and 1918(3P+NP):HA/Avian viruses, which possess the 1918 virus PB2, HA, and NA genes, or the 1918 virus PA, PB1, PB2, NP, and HA genes, respectively, and the remaining genes from the 1918-like avian virus ( Figure S3 ). All animals infected with these viruses became symptomatic; they lost appetite and body weight ( Table 1 and Figure S1 ). Two of three ferrets died upon infection with the 1918 HA/Avian, 1918 PB2:HA/Avian, and 1918(3P+NP):HA/Avian viruses ( Table 1 ). In the ferret tracheae, the mean virus titers of all reassortant viruses possessing the 1918 PB2 gene or the 1918 HA gene were higher than that of the 1918-like avian virus on day 3 postinfection ( Table S2 ), although these differences were not statistically significant. These results suggest that the 1918 PB2 and HA genes confer high pathogenicity to the 1918-like avian virus in the ferret model.

The 1918-like avian PB2-627K/684D:HA-89ED/113SN/190D/225D /265DV:PA-253M virus replicated in trachea and lungs more efficiently than the 1918-like avian virus (p < 0.05; Figure 1 and Table S2 ) and caused severe weight loss in the infected ferrets (maximum body weight loss was 21.0% ± 5.1%), although no infected animals died ( Table 1 ). The 1918-like avian PB2-627K/684D:HA-89ED/113SN/190D/225D/265DV:PA-253M infection caused more progressive inflammation in the lungs of ferrets on day 3 postinfection compared with that caused by the 1918-like avian virus (Dunnett’s test; p < 0.05; Figures 2 B and S2 ). The ferrets infected with this mutant virus presented numerous viral antigen-positive cells in tracheal, bronchial, bronchiolar, and glandular epithelial cells on days 3 and 6 postinfections ( Figures 2 A and S2 ). We then tested the transmissibility of this virus and found that two of the three contact ferrets were positive for virus between days 5 and 9 after contact; these animals were also seropositive ( Figures 3 F and S4 F and Table S3 ). Sequence analysis showed that the virus recovered from the contact animal for pair 1 possessed an additional I-to-T amino acid substitution at position 187 of HA ( Table 2 ), which is located at the receptor-binding site of HA ( Figure 4 A). For the pair 3 contact ferret, the recovered virus possessed an additional T-to-I mutation at position 232 of NP ( Table 2 ). Taken together, our results demonstrate that ten amino acid substitutions (E627K and A684D in PB2; E89D, S113N, I187T, E190D, G225D, and D265V in HA; V253M in PA; and T232I in NP) may be associated with efficient 1918-like avian virus transmission in ferrets.

(B–E) The receptor specificities of viruses possessing 1918 HA (B), 1918-like avian HA (C), 1918-like avian HA-190D/225D (D), and 1918-like avian HA-89D/113N/190D/225D (E) were assessed by using a glycan microarray containing a diverse library of α2,3- and α2,6-linked sialosides (). Viruses, directly labeled with biotin, were applied at 128 hemagglutination units/ml for 1 hr and, after washing, were incubated with Streptavidin-Alexa Fluor 647 (1 μg/ml) for 1 hr to detect bound virus. Error bars represent the SD calculated from six replicate spots of each glycan. A complete list of glycans is found in Table S6 . See also Figure S5

(A) Localization of amino acid changes identified in viruses recovered from ferrets in the transmission study. Shown is the 3D structure of the monomer of A/Brevig Mission/1/18 (H1N1) HA in complex with human receptor analogs (Protein Data Bank [PDB] ID code 2WRG). A close-up view of the globular head is also shown to the right. Mutations known to increase affinity to human-type receptors are shown in red (E190D and G225D). Mutations that emerged in HA during replication and/or transmission in ferrets are shown in green (E89D, S113N, I187T, and D265V). The amino acid changes at positions 89 and 113 are located close to an amino acid at position 110 (103 with H5 numbering) that was previously found to be associated with the transmissibility of an H5 virus (). Images were created with MacPyMOL ( http://www.pymol.org/ ).

We sequenced the virus isolated on days 5 and 9 postcontact from the nasal washes of the contact ferret in the 1918-like avian PB2-627K:HA-89ED/190D/225D virus group and found three additional mutations, HA-S113N, PB2-A684D, and PA-V253M ( Table 2 ). After propagating this virus in MDCK cells, the virus stock possessed the following mutations: PB2-627K, PB2-684D, PA-253M, HA-190D, HA-225D, HA-89E/D, HA-113S/N, and HA-265D/V (the mixed populations of amino acids were found at positions 89, 113, and 265) (designated as 1918-like avian PB2-627K/684D:HA-89ED/113SN/190D/225D/265DV:PA-253M) ( Table 2 and Figure S3 ).

HA and PB2 are known to play important roles in restricting viral transmission from avian species to humans (). The receptor-binding specificity of the HA protein is a major determinant of influenza viral host range (). E-to-D and G-to-D mutations at positions 190 and 225 of HA (H3 numbering), respectively, are essential for avian virus HAs of the H1 subtype to bind to human-type receptors (). In addition, Lys at position 627 of the PB2 protein is important for avian viruses to efficiently replicate in mammalian cells and at the lower temperatures of the upper human airway (). To test whether these mammalian-adapting mutations in HA and PB2 affect the replicative ability, pathogenicity, and transmissibility of the 1918-like avian virus, we attempted to generate mutant 1918-like reassortant avian viruses possessing these amino acid substitutions (i.e., 1918-like avian PB2-627K and 1918-like avian PB2-627K:HA-190D/225D viruses). 1918-like avian PB2-627K virus was generated upon inoculation of the supernatant of transfected 293T cells into embryonated chicken eggs ( Figure S3 ). We were unable to generate 1918-like avian PB2-627K:HA-190D/225D virus in embryonated chicken eggs, but we were able to obtain this virus after propagation of 293T cell supernatant in MDCK cells. However, this virus consisted of a mixed virus population possessing E or D at position 89 of HA (89ED) in addition to the PB2-627K and HA-190D/225D mutations (designated as 1918-like avian PB2-627K:HA-89ED/190D/225D; Figure S3 and Table 2 ). These mutant viruses replicated well in the nasal turbinates and tracheal tissues of ferrets ( Figure 1 and Table S2 ). Infection of ferrets with 1918-like avian PB2-627K and 1918-like avian PB2-627K:HA-89ED/190D/225D caused substantial body weight loss (10.3% ± 3.6% and 19.4% ± 9.2%, respectively; Table 1 ) and appreciable pathologic changes in the trachea and lungs of the infected animals ( Figures 2 A and S2 ). We tested the transmissibility of these viruses and found that no virus was recovered from contact ferrets for the 1918-like avian PB2-627K virus group. Interestingly, for the 1918-like avian PB2-627K:HA-89ED/190D/225D virus group, virus and seroconversion were detected for one of the three contact ferrets ( Table 1 Figures 3 E and S4 F, and Table S3 ), indicating its partial transmissibility in this animal model.

Virus isolated from the nasal washes of the contact ferret in this 1918-like avian PB2-627K:HA-89ED/190D/225D virus group was propagated in MDCK cells, and the resulting virus stock was sequenced.

f Virus isolated from the nasal washes of the contact ferret in this 1918-like avian PB2-627K:HA-89ED/190D/225D virus group was propagated in MDCK cells, and the resulting virus stock was sequenced.

The Effects of Amino Acid Changes in the HA of the Transmissible 1918-like Avian Viruses on Receptor-Binding Specificity and HA Stability