Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 May 1.
Published in final edited form as: J Gen Virol. 2009 Dec 16;91(Pt 5):1284–1289. doi: 10.1099/vir.0.018143-0

Role of host-specific amino acids in the pathogenicity of avian H5N1 influenza viruses in mice

Jin Hyun Kim 1, Masato Hatta 1, Shinji Watanabe 1, Gabriele Neumann 1, Tokiko Watanabe 1,*, Yoshihiro Kawaoka 1,2,3,4,5,6,*
PMCID: PMC2878586  NIHMSID: NIHMS181412  PMID: 20016035

SUMMARY

Recent large-scale sequence analyses revealed ‘signature’ amino acids at specific positions in viral proteins that distinguish human influenza viruses from avian viruses. To determine the role of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses, we generated mutant viruses possessing signature amino acids in the PB2, PA, and NP of human influenza isolates (‘human-like amino acids’) in the genetic background of an avian H5N1 virus, and tested their pathogenicity in mice. We found that some of these mutants exhibited enhanced pathogenicity in mice, suggesting the involvement of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses in mammals.

H5N1 influenza outbreaks in poultry have affected many countries in Asia, Europe, and Africa (Cooper et al., 1999; Deng et al., 2006; Gabriel et al., 2005; Itoh et al., 2009; Maines et al., 2005; Shaw et al., 2002; Sorrell et al., 2009). Continued circulation of H5N1 viruses in birds provides ample opportunity for them to be transmitted to humans. Indeed, H5N1 viruses have overcome host species barriers and infected humans sporadically in several countries, with 436 confirmed human cases of H5N1 infection and 262 fatalities as of 1 July 2009 (http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_07_01/en/index.html). Nonetheless, H5N1 viruses have not caused a pandemic in humans yet because of their inefficient human-to-human transmission (Maines et al., 2006; Yen et al., 2007). By contrast, the recently emerged swine-origin H1N1 2009 influenza virus has transmitted very efficiently among humans, causing the first influenza pandemic of the 21st century (Dawood et al., 2009; Itoh et al., 2009; Munster et al., 2009).

Recently, large-scale sequence analyses revealed ‘signature’ amino acids at specific positions in viral proteins that distinguish human influenza viruses from avian influenza viruses (Chen et al., 2006; Finkelstein et al., 2007; Shaw et al., 2002). These host lineage-specific amino acids were mainly found in components of the viral RNA polymerase complex, such as PB2, PA, and NP, which is essential for viral genome replication (Deng et al., 2006; Klumpp et al., 1997). It is likely that these amino acids contribute to the host range restriction of influenza viruses (Gabriel et al., 2005; Scholtissek et al., 1985); however, their biological significance remains to be established, with the exception of the amino acids at positions 627 and 701 of PB2 whose importance in virulence have been demonstrated in a rodent model (Hatta et al., 2001; Li et al., 2005; Shinya et al., 2004; Steel et al., 2009). Therefore, to understand the biological role of the host-specific amino acids of the viral RNA polymerase complex of avian H5N1 viruses in a mammalian host, we generated a series of mutant viruses possessing human-like amino acids in PB2, PA and/or NP in a genetic background of an avian H5N1 virus and examined their pathogenicity in a mouse model. We found that some of these amino acid changes conferred enhanced pathogenicity in mice, suggesting a potential role for these host lineage-specific amino acids in pathogenicity in mammals.

Identification of host lineage-specific amino acids that distinguish human from avian virus isolates

To identify host lineage-specific amino acids that distinguish human influenza isolates from avian isolates, we first compared the sequences of avian and human viruses of various subtypes. Sequence data were obtained from the Influenza Sequence Database (ISD) operated by Los Alamos National Laboratory (https://flu.lanl.gov/) (Macken et al., 2001). Based on our analysis and other data (Chen et al., 2006; Finkelstein et al., 2007; Shaw et al., 2002), we identified 75 amino acids that are conserved in either human or avian influenza viruses, which we designated as ‘human-like’ or ‘avian-like’ amino acids, respectively (Supplementary table 1).

Host lineage-specific amino acids were mostly found in the viral RNA polymerase complex components, PB2, PA and NP (Supplementary table 1), which is consistent with the findings of others (Chen et al., 2006; Finkelstein et al., 2007; Shaw et al., 2002). We then examined the residues at these ‘signature’ amino acid positions in the sequences of the PB2, PA and NP proteins of avian viruses that had been isolated from humans, including H5N1 viruses isolated from humans, A/Hong Kong/1774/99 (H3N2) virus, which is an avian-like swine virus isolated from a human (Gregory et al., 2001), and A/Wisconsin/10/98 (H1N1) virus, which is a triple reassortant virus isolated from a human whose PA and PB2 genes are of avian origin, while PB1 gene is of human origin and the 5 remaining genes are of a classical swine virus (Cooper et al., 1999; Olsen, 2002). We also included A/Brevig Misson/1/18 (H1N1; the 1918 pandemic virus), which is known to share many amino acids with avian viruses (Taubenberger et al., 1997; Taubenberger et al., 2005) in this analysis. For these viruses, the presence of ‘human-like’ amino acids at any of the ‘signature’ positions would suggest the acquisition of that human-like amino acid during replication in the new host. For example, two human H5N1 isolates, A/Hong Kong/482/97 and A/Hong Kong/483/97, encode Asn at position 9 of PB2 instead of Asp, which is conserved in avian viruses at this position (Table 1), suggesting a role for this residue in avian virus adaptation in humans.

Table 1.

Comparison between avian- and human-like amino acids in the PB2, PA and NP proteins of influenza A viruses*.

Protein Amino acid position Avian-like amino acid Human-like amino acid Human isolates originated from avian viruses possessing a human-like amino acid or those possessing the corresponding gene segment from an avian virus
PB2 9 D N A/Hong Kong/482/97 (H5N1), A/Hong Kong/483/97 (H5N1)
199 A S A/Brevig Misson/1/1918 (H1N1), Several Hong Kong isolates in 1997 (H5N1)
368 R K A/Hong Kong/1774/99(H3N2)
627 E K Many H5N1 isolates (i.e., A/Hong Kong/483/97, A/Hong Kong/213/03, A/Vietnam/1203/04)
PA 142 K N
E† 		A/Hong Kong/156/97 (H5N1)§, A/Vietnam/1203/04 (H5N1)
421 S I A/Vietnam/1203/04 (H5N1), A/Vietman/1204/04 (H5N1)
NP 33 V I A/Brevig Misson/1/1918 (H1N1), A/Hong Kong/1774/99 (H3N2), A/Wisconsin/10/98 (H1N1), many 2006 European isolates (H5N1)
100 R V
I‡ 		A/Wisconsin/10/98 (H1N1) A/Brevig Misson/1/1918 (H1N1)
283 L P A/Brevig Misson/1/1918 (H1N1), A/Hong Kong/156/97 (H5N1)§
357 Q K A/Brevig Misson/1/1918 (H1N1), A/Wisconsin/10/98 (H1N1), A/Thailand/5(KK494)/04 (H5N1)
Open in a new tab
*

We analyzed protein sequences in The Influenza Sequence Database and identified 75 host-specific amino acids conserved in human and avian influenza viruses. For this study, we selected 12 amino acids, as described in the text.

†

Two H5N1 human isolates (A/Hong Kong/156/97 and A/Veitnam/1203/2004) encode Glu at position 142 of PA, whereas most human influenza viruses possess Asn at this position.

‡

A/Brevig Misson/1/1918 (1918 pandemic) virus encodes Ile at position 100 of NP, whereas most human isolates possess Val at this position.

§

A mixed population of avian- and human-like amino acids was observed at position 142 of PA and 283 of NP for A/HK/156/97 virus in the Influenza Sequence Database.

For position 142 of PA and position 100 of NP, we also tested Glu and Ile, respectively, in the genetic background of an avian virus, since we found that two H5N1 human isolates (A/Hong Kong/156/97 and A/Veitnam/1203/2004) encode Glu at position 142 of PA and that the 1918 pandemic virus encodes Ile at position 100 of NP, suggesting roles for these residues in viral adaptation in humans. Based on this approach, we chose 12 out of the 75 human-like amino acids (4 in PB2, 3 in PA and 5 in NP; Table 1) to test their contribution to avian virus replication in a mammalian host. In this study, we used a mouse model because the biological significance of the amino acids at positions 627 and 701 of PB2 has been established in this model (Hatta et al., 2001; Li et al., 2005; Shinya et al., 2004).

Generation of mutant viruses possessing a single human-like amino acid in their PA, PB2, or NP proteins in the genetic background of an avian H5N1 virus

To examine the role of host lineage-specific amino acids in the pathogenicity of avian H5N1 viruses, we generated a series of mutant viruses possessing the test human-like amino acids in their PB2, PA or NP in the genetic background of the avian H5N1 virus A/chicken/Vietnam/NCVD5/2003 (H5N1; VD5), which was kindly provided by the Centers for Disease Control and Prevention. We selected this virus because of its low pathogenicity in mice despite possessing a virulent type HA (Hatta et al., 2007). All experiments with live viruses and with transfectants generated by reverse genetics were performed in a biosafety level 3 containment laboratory approved for such use by the Centers for Disease Control and Prevention and by the U.S. Department of Agriculture.

After transfection of 293T cells with plasmids for generating influenza virus (Neumann et al., 1999), virus in the supernatant was harvested at 18 hours post-transfection to minimize virus replication in mammalian cells, and then amplified in 10-day-old embryonated eggs to make virus stock. Most mutants were successfully rescued and grew well in eggs, although three NP mutant viruses, VD5-NP-100I, VD5-NP-100V and VD5-NP-283P were not viable (Table 2).

Table 2.

Biological properties of mutant VD5 viruses with human-like amino acids in their PB2, PA or NP genes.

Virus Mutations
 Titers of stock virus grown in eggs (PFU/ml)* MLD50(PFU)†
PB2 PA NP
VD5 2.6 × 107 > 106
VD5-PB2-9N D9N 1.8 × 108 8.9 × 105
VD5-PB2-199S A199S 6.6 × 107 3.3 × 105
VD5-PB2-368K R368K 1.9 × 108 > 106
VD5K E627K 2.6 × 108 41
VD5-PA-142N K142N 1.6 × 108 2.5 × 105
VD5-PA-142E K142E 3.3 × 108 > 106
VD5-PA-421I S421I 4.9 × 107 5.7 × 105
VD5-NP-33I V33I 9.1 × 107 > 106
VD5-NP-100V R100V - ND
VD5-NP-100I R100I - ND
VD5-NP-283P L283P - ND
VD5-NP-357K Q357K 3.0 × 108 > 106
VD5K-PB2-9N E627K/D9N 3.9 × 108 11
VD5K-PB2-199S E627K/A199S 4.3 × 108 6.8
VD5K-PB2-368K E627K/R368K 3.7 × 108 1.0
VD5K-PA-142N E627K K142N 6.0 × 108 1.7
VD5K-PA-142E E627K K142E 3.4 × 108 5.4
VD5K-PA-421I E627K S421I 2.6 × 108 7.3
VD5K-NP-33I E627K V33I 2.5 × 108 41
VD5K-NP-100V E627K R100V 5.8 × 108 > 106
VD5K-NP-100I E627K R100I 4.1 × 108 > 106
VD5K-NP-283P E627K L283P 5.0 × 108 4.5 × 103
VD5K-NP-357K E627K Q357K 2.8 × 108 0.8
Open in a new tab
*

Mutant viruses possessing human-like amino acids in the genetic background of VD5 generated by reverse genetics as described previously (Neumann et al., 1999). Rescued viruses were amplified in eggs and their virus titers were determined by plaque assays in MDCK cells. -, virus was not rescued.

†

To determine the dose lethal to 50% of mice (MLD50), we infected 6-week-old female Balb/c mice intranasally with 50μl of serial 10-fold dilutions of virus and observed them for 2 weeks. ND, not determined.

Pathogenicity of mutant VD5 viruses possessing a single human-like amino acid in PA, PB2 or NP in mice

To determine the effect of introducing a human-like amino acid into an avian H5N1 virus on its pathogenicity in mice, we examined the dose lethal to 50% of mice (MLD50). Based on the MLD50s, we categorized the mutant viruses into three groups (Table 2). The first group included four mutants, PB2-368K, PA-142E, NP-33I and NP-357K, whose MLD50s were more than 106 PFU. The second group comprised PB2-9N, PB2-199S, PA-142N and PA-421I, which showed intermediate pathogenicity (MLD50s > 105 PFU, but < 106 PFU). The third group contained PB2-627K whose MLD50 was 41 PFU, representing a significant increase in pathogenicity in mice, as has been previously reported (Fornek et al., 2009; Hatta et al., 2001; Hatta et al., 2007; Maines et al., 2005). Thus, the following substitutions of avian-like amino acids with the respective human-like amino acids increased the pathogenicity of the VD5 virus in mice: PB2-9N, PB2-199S, PA-142N, PA-421I, and PB2-627K.

Generation of VD5 viruses possessing a second human-like amino acid mutation in addition to PB2-627K

To examine whether additional mutations were introduced into the mutant viruses during replication in mice, we collected lungs from 16 dead mice infected with the mutant viruses and attempted to isolate virus with MDCK cells for sequencing. We successfully isolated and sequenced the entire genomes of 12 viruses. We confirmed that all of these viruses possessed the human-like amino acid that was originally introduced. Interestingly, we found that 6 of the 12 viruses had the PB2-627K mutation in addition to the original human-like amino acid (data not shown). These data suggest that avian H5N1 viruses with a human-like amino acid may require the PB2-627K mutation to replicate efficiently in mice and that the MLD50s of the mutant viruses described above represent the sum of the effect of the intended human-like amino acids and the PB2-627K substitution that was introduced during replication in mice.

To examine the combined effect of introducing selected human-like amino acids and PB2-627K on pathogenicity in mice, we generated viruses possessing these two mutations (Table 2; VD5 with the PB2-627K mutation was designated as ‘VD5K’). All of the mutant viruses, including three NP mutants that were not rescued without the PB2-627K mutation, were successfully rescued and replicated well in eggs. Titers of all stock viruses with double mutations were greater than 108 PFU (Table 2), suggesting that these host-specific signature amino acids are compatible in the genetic background of an avian H5N1 virus.

Pathogenicity in mice of VD5 possessing a second human-like amino acid mutation in addition to PB2-627K

To examine whether the combination of a human-like amino acid and PB2-627K contributes to pathogenicity in mice, we calculated MLD50s of viruses possessing such mutations (Table 2). Two NP mutants, VD5K-NP-100I and VD5K-NP-100V, were significantly attenuated and did not kill any mice, suggesting that the human-like amino acids of NP at position 100 have a negative effect on the pathogenicity in mice, even though the viruses could replicate well in eggs. VD5K-NP-283P was also attenuated and its MLD50 value was 100-fold higher than that of VD5K, whereas the pathogenicity of VD5K-NP-33I was comparable to that of VD5K in mice. In contrast, all of PA and PB2 mutants, as well as the NP-357K mutant, had increased pathogenicity in mice: the MLD50 values of VD5K-PB2-368K, VD5K-PA-142N, VD5K-PA-142E and VD5K-NP-357K were reduced approximately 41, 24, 7.6 and 51 times, respectively (Table 2). Two human-like amino acids, PB2-368K and NP-357K, showed no change in pathogenicity when they were introduced into VD5 alone, suggesting that these two human-like amino acids affect viral pathogenicity in mice only when they co-exist with PB2-627K.

Growth in mouse organs of VD5 mutant viruses with human-like amino acids

To examine the effect of these mutations on virus growth in mice, we collected organs from infected mice on days 1 and 3 post-infection for virus titration. No virus was recovered from mice infected with 100 PFU of a single human-like amino acid mutation (data not shown). In contrast, all of the VD5K mutant viruses that possessed PB2-627K and an additional human-like amino acid replicated well in mouse lungs and their lung titers were comparable to those of the VD5K-infected mice (Table 3). Interestingly, lung titers of mice infected with VD5K-PA-142E virus were significantly higher on day 1 post-infection than those of mice infected with other VD5K mutants. These results suggest a potential role for Glu at position 142 of PA, which occurs in two H5N1 human isolates from 1997 and 2004, in efficient replication and high virulence in mice in combination with PB2-627K.

Table 3.

Growth properties in mice of mutant VD5 viruses possessing human-like amino acids.

Virus Days post-infection Virus Titer (mean log10 PFU ± SD/g) in: *
Lung Nasal turbinates Brain
VD5 1 - - -
3 - - -
VD5K 1 4.7 ± 0.9 - -
3 7.0 ± 0.4 3.0 ± 0.7 -
VD5K-PB2-9N 1 4.9 ± 0.5 - -
3 7.0 ± 0.2 2.9, 6.0 -
VD5K-PB2-199S 1 4.8 ± 0.3 - -
3 7.1 ± 0.3 2.2 -
VD5K-PB2-368K 1 3.9 ± 0.3 - -
3 6.9 ± 0.6 3.9 -
VD5K-PA-142E 1 7.0 ± 0.3† 2.3 -
3 7.5 ± 0.1 3.7, 4.4 1.4
VD5K-PA-142N 1 5.7 ± 0.5 - -
3 7.2 ± 0.4 5.7, 5.2 -
VD5K-PA-421I 1 4.7 ± 0.4 - -
3 6.8 ± 0.1 - -
VD5K-NP-33I 1 4.0 ± 0.3 - -
3 6.3 ± 0.1 4.8 -
VD5K-NP-357K 1 4.6 ± 0.1 - -
3 6.2 ± 0.2 2.2 -
Open in a new tab
*

Mice (3 per group) were infected intranasally with 100 PFU of virus. Organs were collected from infected mice on days 1 and 3 post-infection for virus titration. Individual titers were recorded when virus was not recovered from all three animals.

†

The virus titer in lungs of mice infected with VD5K-PA-142E was significantly higher than that of VD5K-infected mice on day 1 post-infection as determined by Student’s t-test (p<0.02).

-, Virus was not recovered (detection limit for lung and nasal turbinates is < 1.6 log10 PFU/g and for brain is <1.1 log10 PFU/g).

In this study, to examine the role of host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses in a mammalian host, we generated mutant viruses that possessed conserved amino acids in the PB2, PA, and/or NP of human influenza isolates (‘human-like amino acids’) in a genetic background of an avian H5N1 virus of low pathogenicity in mice, and tested their pathogenicity in a mouse model. We found that some of the mutants possessing human-like amino acids acquired increased pathogenicity in mice (Table 2), suggesting roles for host lineage-specific amino acids, in addition to the amino acid at position 627 of PB2 (Hatta et al., 2001), in the pathogenicity of H5N1 avian influenza viruses in mammals.

Our sequence analyses revealed that two H5N1 human isolates (A/Hong Kong/156/97 and A/Veitnam/1203/2004) encode Glu at position 142 of PA (Table 1), whereas the PA of most avian and human viruses possesses Lys and Asn at position 142, respectively (Macken et al., 2001). We demonstrated that the Lys-to-Glu mutation at position 142 of PA resulted in enhanced replication and pathogenicity in mice when combined with the PB2-627K mutation (Table 2 and 3), suggesting a potential role of PA-142E in adaptation and pathogenicity of H5N1 viruses in a mammalian host. The mechanism by which PA-142E supports increased replication in mice at an early time point (day 1; Table 3) is not clear. However, one possibility is that this substitution may cause conformational changes in the PA protein, affecting the structure of the N-terminal residues of the PA subunit, which were recently shown to be involved in the promoter binding (Kashiwagi et al., 2009) and contain the cap-snatching endonuclease active site (Dias et al., 2009).

We were unable to rescue three of the NP mutants in the VD5 background (Table 2). However, in the presence of the PB2-627K mutation, viruses with these NP mutations were viable. We also showed that some of the human-like amino acid mutants (i.e., PB2-R368K, PA-K142E and NP-Q357K) enhanced pathogenicity in mice only in combination with PB2-627K. These results highlight the critical contribution of PB2-627K to avian virus replication in mammals, as well as the contribution of other human-like amino acids identified in this study.

An H5N1 pandemic in humans has not yet occurred, but we can assume that the gradual acquisition of a number of amino acid changes in viral proteins, which are important for host restriction (i.e., HA, polymerase proteins and NP [Gabriel et al., 2005; Harvey et al., 2004; Matrosovich et al., 2004; Scull et al., 2009]), would promote adaptation of H5N1 viruses to humans and make an H5N1 pandemic more likely. To be fully prepared for such an event, it is important to strengthen worldwide surveillance to keep track of these amino acid changes in H5N1 viruses.

Supplementary Material

Supplemental Table

Acknowledgments

We thank Susan Watson for editing the manuscript and Krisna Wells and Martha McGregor for excellent technical assistance. We thank the Centers for Disease Control and Prevention for A/chicken/Vietnam/NCVD5/2003 virus. This work was supported, in part, by U.S. National Institute of Allergy and Infectious Diseases Public Health Service research grants, by a grant-in-aid for Specially Promoted Research and by a contract research fund for the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases from the Ministry of Education, Culture, Sports, Science and Technology, by ERATO (Japan Science and Technology Agency), and by grants-in-aid from the Ministry of Health, Labor, Welfare of Japan.

Footnotes

Publisher's Disclaimer: This is an author manuscript that has been accepted for publication in Journal of General Virology, copyright Society for General Microbiology, but has not been copy-edited, formatted or proofed. Cite this article as appearing in Journal of General Virology. This version of the manuscript may not be duplicated or reproduced, other than for personal use or within the rule of 'Fair Use of Copyrighted Materials' (section 17, Title 17, US Code), without permission from the copyright owner, Society for General Microbiology. The Society for General Microbiology disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it by any other parties. The final copy-edited, published article, which is the version of record, can be found at http://vir.sgmjournals.org, and is freely available without a subscription.

References

  1. Chen GW, Chang SC, Mok CK, Lo YL, Kung YN, Huang JH, Shih YH, Wang JY, Chiang C, Chen CJ, Shih SR. Genomic signatures of human versus avian influenza A viruses. Emerg Infect Dis. 2006;12:1353–1360. doi: 10.3201/eid1209.060276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cooper L, Olsen C, Xu K, Klimov A, Cox N, Subbarao K. Molecular characterization of human influenza A viruses bearing swine-like hemagglutinin genes. Virus Evolution Workshop; Ardmore, OK. 1999. [Google Scholar]
  3. Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, Garten RJ, Gubareva LV, Xu X, Bridges CB, Uyeki TM. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med. 2009;360:2605–2615. doi: 10.1056/NEJMoa0903810. [DOI] [PubMed] [Google Scholar]
  4. Deng T, Sharps JL, Brownlee GG. Role of the influenza virus heterotrimeric RNA polymerase complex in the initiation of replication. J Gen Virol. 2006;87:3373–3377. doi: 10.1099/vir.0.82199-0. [DOI] [PubMed] [Google Scholar]
  5. Dias A, Bouvier D, Crepin T, McCarthy AA, Hart DJ, Baudin F, Cusack S, Ruigrok RW. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature. 2009;458:914–918. doi: 10.1038/nature07745. [DOI] [PubMed] [Google Scholar]
  6. Finkelstein DB, Mukatira S, Mehta PK, Obenauer JC, Su X, Webster RG, Naeve CW. Persistent host markers in pandemic and H5N1 influenza viruses. J Virol. 2007;81:10292–10299. doi: 10.1128/JVI.00921-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fornek JL, Gillim-Ross L, Santos C, Carter V, Ward JM, Cheng LI, Proll S, Katze MG, Subbarao K. A single amino acid substitution in a polymerase protein of an H5N1 influenza virus is associated with systemic infection and impaired T cell activation in mice. J Virol. 2009;83:11102–11115. doi: 10.1128/JVI.00994-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gabriel G, Dauber B, Wolff T, Planz O, Klenk HD, Stech J. The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc Natl Acad Sci U S A. 2005;102:18590–18595. doi: 10.1073/pnas.0507415102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gregory V, Lim W, Cameron K, Bennett M, Marozin S, Klimov A, Hall H, Cox N, Hay A, Lin YP. Infection of a child in Hong Kong by an influenza A H3N2 virus closely related to viruses circulating in European pigs. J Gen Virol. 2001;82:1397–1406. doi: 10.1099/0022-1317-82-6-1397. [DOI] [PubMed] [Google Scholar]
  10. Harvey R, Martin AC, Zambon M, Barclay WS. Restrictions to the adaptation of influenza a virus h5 hemagglutinin to the human host. J Virol. 2004;78:502–507. doi: 10.1128/JVI.78.1.502-507.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hatta M, Gao P, Halfmann P, Kawaoka Y. Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science. 2001;293:1840–1842. doi: 10.1126/science.1062882. [DOI] [PubMed] [Google Scholar]
  12. Hatta M, Hatta Y, Kim JH, Watanabe S, Shinya K, Nguyen T, Lien PS, Le QM, Kawaoka Y. Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice. PLoS Pathog. 2007;3:1374–1379. doi: 10.1371/journal.ppat.0030133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Itoh Y, Shinya K, Kiso M, Watanabe T, Sakoda Y, Hatta M, Muramoto Y, Tamura D, Sakai-Tagawa Y, et al. In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses. Nature. 2009;460:1021–1025. doi: 10.1038/nature08260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kashiwagi T, Leung BW, Deng T, Chen H, Brownlee GG. The N-terminal region of the PA subunit of the RNA polymerase of influenza A/HongKong/156/97 (H5N1) influences promoter binding. PLoS One. 2009;4:e5473. doi: 10.1371/journal.pone.0005473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klumpp K, Ruigrok RW, Baudin F. Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure. EMBO J. 1997;16:1248–1257. doi: 10.1093/emboj/16.6.1248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Li Z, Chen H, Jiao P, Deng G, Tian G, Li Y, Hoffmann E, Webster RG, Matsuoka Y, Yu K. Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model. J Virol. 2005;79:12058–12064. doi: 10.1128/JVI.79.18.12058-12064.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Macken C, Lu H, Goodman J, Boykin L. The value of a database in surveillance and vaccine selection. In: Osterhaus ADME, Cox N, Hampson AW, editors. Options for the Control of Influenza IV. Amsterdam: Elsevier Science; 2001. pp. 103–106. [Google Scholar]
  18. Maines TR, Chen LM, Matsuoka Y, Chen H, Rowe T, Ortin J, Falcon A, Nguyen TH, Mai le Q, et al. Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model. Proc Natl Acad Sci U S, A. 2006;103:12121–12126. doi: 10.1073/pnas.0605134103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maines TR, Lu XH, Erb SM, Edwards L, Guarner J, Greer PW, Nguyen DC, Szretter KJ, Chen LM, et al. Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals. J Virol. 2005;79:11788–11800. doi: 10.1128/JVI.79.18.11788-11800.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD. Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. J Virol. 2004;78:12665–12667. doi: 10.1128/JVI.78.22.12665-12667.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Munster VJ, de Wit E, van den Brand JM, Herfst S, Schrauwen EJ, Bestebroer TM, van de Vijver D, Boucher CA, Koopmans M, et al. Pathogenesis and transmission of swine-origin 2009 A(H1N1) influenza virus in ferrets. Science. 2009;325:481–483. doi: 10.1126/science.1177127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neumann G, Watanabe T, Ito H, Watanabe S, Goto H, Gao P, Hughes M, Perez DR, Donis R, Hoffmann E, Hobom G, Kawaoka Y. Generation of influenza A viruses entirely from cloned cDNAs. Proc Natl Acad Sci U S A. 1999;96:9345–9350. doi: 10.1073/pnas.96.16.9345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Olsen CW. The emergence of novel swine influenza viruses in North America. Virus Res. 2002;85:199–210. doi: 10.1016/s0168-1702(02)00027-8. [DOI] [PubMed] [Google Scholar]
  24. Scholtissek C, Burger H, Kistner O, Shortridge KF. The nucleoprotein as a possible major factor in determining host specificity of influenza H3N2 viruses. Virology. 1985;147:287–294. doi: 10.1016/0042-6822(85)90131-x. [DOI] [PubMed] [Google Scholar]
  25. Scull MA, Gillim-Ross L, Santos C, Roberts KL, Bordonali E, Subbarao K, Barclay WS, Pickles RJ. Avian Influenza virus glycoproteins restrict virus replication and spread through human airway epithelium at temperatures of the proximal airways. PLoS Pathog. 2009;5:e1000424. doi: 10.1371/journal.ppat.1000424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shaw M, Cooper L, Xu X, Thompson W, Krauss S, Guan Y, Zhou N, Klimov A, Cox N, et al. Molecular changes associated with the transmission of avian influenza a H5N1 and H9N2 viruses to humans. J Med Virol. 2002;66:107–114. doi: 10.1002/jmv.2118. [DOI] [PubMed] [Google Scholar]
  27. Shinya K, Hamm S, Hatta M, Ito H, Ito T, Kawaoka Y. PB2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of Hong Kong H5N1 influenza A viruses in mice. Virology. 2004;320:258–266. doi: 10.1016/j.virol.2003.11.030. [DOI] [PubMed] [Google Scholar]
  28. Sorrell EM, Wan H, Araya Y, Song H, Perez DR. Minimal molecular constraints for respiratory droplet transmission of an avian-human H9N2 influenza A virus. Proc Natl Acad Sci U S A. 2009;106:7565–7570. doi: 10.1073/pnas.0900877106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Steel J, Lowen AC, Mubareka S, Palese P. Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N. PLoS Pathog. 2009;5:e1000252. doi: 10.1371/journal.ppat.1000252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–1796. doi: 10.1126/science.275.5307.1793. [DOI] [PubMed] [Google Scholar]
  31. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–893. doi: 10.1038/nature04230. [DOI] [PubMed] [Google Scholar]
  32. Yen HL, Lipatov AS, Ilyushina NA, Govorkova EA, Franks J, Yilmaz N, Douglas A, Hay A, Krauss S, et al. Inefficient transmission of H5N1 influenza viruses in a ferret contact model. J Virol. 2007;81:6890–6898. doi: 10.1128/JVI.00170-07. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Table
NIHMS181412-supplement-Supplemental_Table.doc (177KB, doc)

RESOURCES