• Host range;
  • influenza;
  • interspecies transmission;
  • pandemic;
  • virus adaptation


  1. Top of page
  2. Abstract
  3. Introduction
  4. Biology of IAVs
  5. Molecular mechanism of host range restriction: receptor specificity and viral polymerase complex
  6. Molecular basis of pathogenicity: role of ha cleavage, NS1, and PB1-F2 proteins
  7. Evolutionary pathways and molecular mechanisms of IAVs involved in human adaptation
  8. Genetic markers
  9. Pandemic overview
  10. Conclusion
  11. Acknowledgements
  12. Conflict of interest
  13. References

Influenza A is a highly contagious respiratory virus in constant evolution and represents a threat to both veterinary and human public health. IA viruses (IAVs) originate in avian reservoirs but may adapt to humans, either directly or through the spillover to another mammalian species, to the point of becoming pandemic. IAVs must successfully be able to (i) transmit from animal to human, (ii) interact with host cells, and (iii) transmit from human to human. The mechanisms by which viruses evolve, cause zoonotic infections, and adapt to a new host species are indeed complex and appear to be a heterogeneous collection of viral evolutionary events rather than a single phenomenon. Progress has been made in identifying some of the genetic markers mainly associated with virulence and transmission; this achievement has improved our knowledge of how to manage a pandemic event and of how to identify IAVs with pandemic potential. Early evidence of emerging viruses and surveillance of animal IAVs is made possible only by strengthening the collaboration between the public and veterinary health sectors.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Biology of IAVs
  5. Molecular mechanism of host range restriction: receptor specificity and viral polymerase complex
  6. Molecular basis of pathogenicity: role of ha cleavage, NS1, and PB1-F2 proteins
  7. Evolutionary pathways and molecular mechanisms of IAVs involved in human adaptation
  8. Genetic markers
  9. Pandemic overview
  10. Conclusion
  11. Acknowledgements
  12. Conflict of interest
  13. References

IA viruses cause recurrent epidemics and global pandemics.[1] The emergence of a novel H1N1 swine-origin virus (H1N1 S-OIV) in 2009 and the ongoing occurrence of human cases of infection with avian H5N1 IAVs are only recent examples of the zoonotic and pandemic potential of IAVs.[2-4] Different mechanisms are believed to be able to transform an animal virus to a human pandemic strain, and these include a constellation of viral evolutionary events that are still to be thoroughly investigated.[5-7] By and large, swine and avian influenza viruses cause the greatest concerns for public health. Understanding the molecular evolution of IAVs in the animal reservoir and the mechanisms associated with interspecies transmission would improve our knowledge and prediction skills on relevant characteristics of zoonotic and pandemic influenza viruses.[8-10]

Biology of IAVs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biology of IAVs
  5. Molecular mechanism of host range restriction: receptor specificity and viral polymerase complex
  6. Molecular basis of pathogenicity: role of ha cleavage, NS1, and PB1-F2 proteins
  7. Evolutionary pathways and molecular mechanisms of IAVs involved in human adaptation
  8. Genetic markers
  9. Pandemic overview
  10. Conclusion
  11. Acknowledgements
  12. Conflict of interest
  13. References

IA viruses are members of the Orthomyxoviridae family.[11] On the basis of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), they currently cluster into 17 HA (H1–H17)[11, 12] and nine NA (N1–N9) subtypes.[11] IAVs consist of eight segmented, single-stranded RNA genomes of negative polarity, encoding 11 proteins: polymerase polypeptides PB1, PA, PB2 (polymerase complex), HA and NA, nucleocapsid protein (NP), matrix protein (M1), ionic channel protein (M2), non-structural protein 1 (NS1), nuclear export protein (NEP), and mitochondria-associated protein (PB1-F2).[11, 13] The HA glycoprotein is critical for binding to cellular host receptors and for the fusion of the viral and endosomal membranes.[11] Replication and transcription of viral RNAs are carried out by the three polymerase subunits PB1, PB2, and PA, and by the NP. Newly synthesized viral ribonucleoprotein (RNP) complexes are exported from the nucleus to the cytoplasm by the NEP and M1 and are assembled into virions at the plasma membrane.[14] The NA facilitates the virus release from infected cells by removing sialic acid (SA) from cellular and viral HA and NA proteins.[11] The NS1 protein is an interferon antagonist,[15] and the PB1-F2 protein is an important virulence factor that induces cell apoptosis.[16]

Recently, a novel PA-X fusion protein encoded in part from a + 1 frameshifted X open reading frame (X-ORF) in segment 3 of AI viruses has been described and has been associated with the immune response modulation in the mouse model.[17]

Molecular mechanism of host range restriction: receptor specificity and viral polymerase complex

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biology of IAVs
  5. Molecular mechanism of host range restriction: receptor specificity and viral polymerase complex
  6. Molecular basis of pathogenicity: role of ha cleavage, NS1, and PB1-F2 proteins
  7. Evolutionary pathways and molecular mechanisms of IAVs involved in human adaptation
  8. Genetic markers
  9. Pandemic overview
  10. Conclusion
  11. Acknowledgements
  12. Conflict of interest
  13. References

Receptor distribution

The receptor-binding site of the HA glycoprotein recognizes the SA bond attached to galactose (Gal) in either α2-3 or α2-6 linkage.[13, 18] IAVs recognize mainly two species of SAs, NeuAc (N-acetylneuraminic acid), and NeuGc (N-glycolylneuraminic acid), which are attached to galactose in SAα2-3Gal or SAα-2-6Gal linkages. For instance, avian viruses preferably recognize SAα2-3Gal linkages, which are mainly found in the intestine and respiratory epithelia of birds,[11, 19] whereas human influenza viruses recognize SAα2-6Gal linkages, which mainly populate the human upper respiratory tract (URT) epithelia.[20-22] However, toward the lower epithelial tract of humans, there is a relative increase in SAα2-3Gal expression,[21, 23] and this has been associated with severe pulmonary pathology observed in some cases of H5N1 infection.[24, 25]

Pigs are known to exhibit dual expression of both SA linkages in the respiratory tract;[26-28] however, recent studies indicate that receptor distribution is similar to that found in humans, suggesting that the classical “mixing vessel” hypothesis regarding the unique role played by pigs needs further discussion.[28, 29] Given similar receptor distribution, one might expect humans and pigs to have similar susceptibility to direct infection by IAVs. Additional factors may play a role in species differences however, including relative abundance of the preferred glycan topology (as discussed later), which might influence the viral binding kinetics and/or equilibrium shift.[30] In addition, the human upper respiratory tract contains many complex types of glycans,[31] and human bronchial mucus contains a mucin, a heavily glycosylated protein with attached α-2,3-linked oligosaccharides, which may bind avian IAVs and help prevent infection.[20]

Concerning other species, the presence of SAα2-6Gal in the alveoli of dogs, cats, tigers, pigs, and ferrets[32] and in the trachea of chickens and ducks has been reported,[33] while both types are present throughout the respiratory tract of pheasant, turkeys, and quails.[34]

Viral characteristics of receptor-binding site

The amino acid residues in the receptor-binding site of HA affect the virus host range.[35, 36] Glutamine (Q) at position 226 and glycine (G) at position 228 of H3 HA confer binding to SAα2-3Gal, while leucine (L) and serine (S) at these positions determine binding to SAα2-6Gal. For H1 strains, glutamic acid (E) and glycine (G) at positions 190 and 225 confer binding to SAα2-3Gal, whereas aspartic acid (D) at the same positions confers binding to SAα2-6Gal.[11, 37, 38]

Influenza virus–receptor interactions are more complex than the simple α2–3 versus α2–6 dichotomy on the host range restriction,[39] suggesting that glycan species (linked to SA) and their topology could also play an important role.[30] The human respiratory tract expresses only NeuAc, whereas NeuGc is present in other species.[40] For instance, avian, human, and swine IAVs exhibit preference for NeuAc rather than NeuGc. Interestingly, the abundant presence of NeuAc in swine trachea could render this species as the possible host for adaptation and/or intermediate virus reassortment in the creation of novel viruses for humans.[41] On the other hand, SA glycans are classified as having umbrella-like (long α2-6) and cone-like (α2-3 or short α2-6) structural topology and this may also influence virus–receptor affinity.[30, 31, 42] Recently, it has been demonstrated that human-adapted HAs bind with high affinity to umbrella-like topology SAs, whereas avian and swine HAs preferentially recognize cone-like topology.[31] These findings indicate that glycan composition and topological changes may also be important determinants in species-specific switch events.[30, 31]

Viral polymerase complex

Another determinant of host restriction is the IAV polymerase complex.[43-45] The amino acid residue 627 in the PB2 subunit regulates polymerase activity in a species-specific fashion.[46] The PB2 derived from human viruses mainly possesses lysine (K) at position 627 (PB2-K627), whereas glutamic acid (PB2-E627) predominates in avian viruses,[47-49] with the exception of most of the H5N1 clade 2.2 viruses and their descendants.[50] PB2-K627 correlates with enhanced polymerase activity, virus replication, transmission, and pathogenicity in mammals,[51, 52] as well as with a possible virus replication at 33°C (human URT temperature).[53] However, PB2-K627 is not obligatory for efficient infection or disease induction in mammals, as observed in some swine viruses, certain avian H5N1 isolates, and most notably in the H1N1 S-OIV.[50, 54] Indeed, engineering a 627K change into the H1N1 S-OIV did not result in increased virulence.[54, 55] In such cases, other residues within PB2 as T271A, Q591K, D701N, and S714R could contribute to viral adaptation and replication in mammalian cells through the increase in polymerase activity at relatively low temperatures.[56, 57] The amino acid at position 701 of PB2 has emerged as a determinant of virulence, facilitating the binding of PB2 to importin α (a cellular nuclear import factor) in mammalian cells.[58, 59]

Molecular basis of pathogenicity: role of ha cleavage, NS1, and PB1-F2 proteins

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biology of IAVs
  5. Molecular mechanism of host range restriction: receptor specificity and viral polymerase complex
  6. Molecular basis of pathogenicity: role of ha cleavage, NS1, and PB1-F2 proteins
  7. Evolutionary pathways and molecular mechanisms of IAVs involved in human adaptation
  8. Genetic markers
  9. Pandemic overview
  10. Conclusion
  11. Acknowledgements
  12. Conflict of interest
  13. References

The HA protein is synthesized as a precursor protein that is cleaved into two subunits (HA1 and HA2) by host cell proteases.[11, 13] This proteolytic cleavage is a prerequisite for fusion of the viral and endosomal membranes to release viral RNP to the cytoplasm.[60] Low pathogenic avian influenza viruses (LPAI) possess a cleavage site with a monobasic motif recognized by trypsin-like proteases, which confine viral replication to the respiratory and gastrointestinal tracts.[11, 61] In contrast, highly pathogenic avian influenza (HPAI) viruses possess a polybasic HA cleavage site cleavable by the ubiquitous furin, supporting the systemic replication.[62] This polybasic HA cleavage of HPAI viruses has originated from LPAI precursors by acquisition of a multibasic cleavage site (MBCS) under both in vitro[63, 64] and in vivo[64] experimental conditions in domestic poultry.

The NS1 protein is an interferon antagonist.[15] The majority of IAV NS1 proteins have a class 1 PDZ-binding motif at the C-terminus, and its truncation results in attenuation of the virulence in mice,[65] as well as in limited virus replication and enhanced type I IFN induction in human dendritic cells.[15] Additionally, NS1 has been associated with exacerbated pro-inflammatory cytokine production in humans.[66, 67] On the other hand, PB1-F2 is a small protein encoded by the +1 alternate ORF in the PB1 polymerase gene of some IAVs. This protein is thought to play a role as a virulence factor by compromising mitochondrial function and eventually leading to apoptosis,[16] and inhibiting the induction of type I interferon.[68] PB1-F2 has also been associated with the induction of severe pulmonary immunopathology and inflammation during primary viral infection, increasing the risk of secondary bacterial pneumonia.[69]

Evolutionary pathways and molecular mechanisms of IAVs involved in human adaptation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biology of IAVs
  5. Molecular mechanism of host range restriction: receptor specificity and viral polymerase complex
  6. Molecular basis of pathogenicity: role of ha cleavage, NS1, and PB1-F2 proteins
  7. Evolutionary pathways and molecular mechanisms of IAVs involved in human adaptation
  8. Genetic markers
  9. Pandemic overview
  10. Conclusion
  11. Acknowledgements
  12. Conflict of interest
  13. References


IA viruses evolve using different mechanisms. The most prominent is antigenic drift, a result of mutations introduced during replication of the viral genome by viral RNA polymerase, which lacks proofreading activity.[70, 71] The rate of mutation during replication of the influenza genome is about 1 nucleotide change for every copied genome.[72] Antigenic shift occurs through viral reassortment, which can result in the shuffling of entire gene segments.[73, 74] For transmission to humans, animal IAVs need to acquire the ability to recognize SAα2-6Glu as a prerequisite to igniting a pandemic.[75-77] Key mutations of HA at positions 138, 190, 194, 225, 226, and 228 (H3 numbering) affect receptor-binding preference of several subtypes including H2, H3, H4, and H9,[78-82] whereas the HAs from H1 human-adapted viruses bear changes at positions 190 and 225 (Table 1).[81]

Table 1. Principal amino acid mutations and signatures associated with interspecies transmission of influenza A viruses
  1. cSIAV, classical swine H1N1 viruses; HA, hemagglutinin; HPAI, highly pathogenic avian influenza; IAVs, IA viruses; SA, sialic acid; SIAV, swine Influenza viruses; S-OIV, swine-origin virus.




Viral strains with residues D190/D225 are human-specific, D190/G225 swine-specific and E190/G225 avian-specific.

Mutations in these residues cause a switch in receptor binding preference from α2-3 to α2-6 SA



European ‘avian-like’ swine H1N1

D225G/EEnhances receptor binding to dual hosts (pigs and humans)H1N1 S-OIV135,141

Enhances binding to SAα2-3Glu receptors

Associated with severe infection outcome in humans

H1N1 S-OIV142,154




Enhance binding to human-type receptors in vitroH5N1155
Q192HIncreases binding to SAα2-6Glu and virulence in miceH5N177



In H2 and H3 viruses 226Q and 228G correlate with binding to avian receptors; 226L and 228S correlate with the shift from avian to human receptor binding

Confers affinity for human-type receptors and infectivity to respiratory epithelial cells

H2 and H3

Avian H4N6

226LSignature which exhibits preferential binding to human-like SAα2-6Glu receptors. A key element for the successful infection of humans.H9N282
A143TIncreases viral attachment to human alveolar macrophagesH7N797
E391KAssociated with the fitness of the virusH1N1 S-OIV143,144
T160ARequired to sustain the avian virus transmission in guinea pig modelH5N1156
K193RDecreases binding to SAα2-3Glu or increases binding to SAα2-6GluH5N1157





Respiratory droplet transmission in ferrets








Airborne transmission between ferretsH5N1 genetically modified86





Enhancement of virulence in mouse modelH1N1 S-OIV129

Avian strains have 627E and human strains 627K signature.

Associated with increased transmission. Important determinant of host range

Increases polymerase activity in mammalian cells at relatively low temperatures



Determinant of host range. Increases transcription at a low temperatureH7N7 isolated from human FC97
Increases virulence in mammalsMouse-adapted H9N2103

Enhances the binding of PB2 to importin α1, increasing the level of PB2 in the nucleus in mammalian cells. Important role in the interspecies transmission of IAVs

Increases polymerase activity in mammal cells at relatively low temperatures

Some HPAI H5N1 strains51,92
Involved in mammalian adaptationEuropean ‘avian like’ swine H1N1107
Increases transmissibility of Influenza A viruses in guinea pig modelH5N151
Enhances the polymerase activity in mammalian cellsAvian- and mouse- adapted H7N758
S714REnhances the polymerase activity in mammalian cellsH7N758
K318RCorrelates with high pathogenicity in mice in the presence of additional mutationsH5N189

Enhances activity only at higher temperatures (37 and 39°C)

Contributes to avian polymerase adaptation to mammalian hosts


Enhances viral replication in human cells and involved in mammalian adaptation

Compensates the lack of PB2-E627K mutation in the S-OIV



G590SAssociated with mammalian pathogenicity and enhanced replicative ability in mammalsH1N1 S-OIV43
A684SAssociated with host shift from avian to swine and the subsequent transfer to humansAvian IAVs79
E158G/AAssociated in the adaptation of PB2 genes to mammals (mouse model)H1N1 S-OIV129
PB1L13PEnhances the activity of viral polymeraseH7N758
G375SAssociated with adaptation to a new species (swine to human)H1N1158



Increase virulence and polymerase activity in mouse modelH3N2 human isolates159
PAK615NEnhances activity of viral RNA polymerase and stimulates viral replication and pathogenicity in mouse modelH7N758
K356RAssociated with host shift from avian to swine and the subsequent transfer to humansAvian IAVs79




Multiple residues that contribute to the enhancement of avian polymerase activity in mammalian cells which is essential for mammalian host adaptationH1N1 S-OIV160
NPN319KIncreases binding to mammalian importin α-1 proteins and polymerase activity. Related to host range specificity.H7N759
V100IIncreases transmissibility among humansH1N1 S-OIV135


Segment 3

PA-XModulates virulence and host immune response in mouse modelIAVs17

Transmission of H5N1 HPAI virus from poultry to humans was first reported in Hong Kong in 1997.[83] From 2003 to 2012, 608 cases of H5N1 virus infections in humans and 359 deaths have been reported in 15 different countries ( Even if human-to-human transmission has been limited, H5N1 is believed to be a significant health threat due to “spillover” infections in humans associated with widespread infection in poultry populations.[84] The single mutation HA-Q192H in some H5N1 strains isolated from humans increased viral binding to SAα2-6Glu, correlating as well with an increased virulence in mice.[77]

After the literature search for this review was completed, two works describing potential molecular determinants of airborne transmission were published. Four mutations in the HA of a reassortant virus (N220K, Q222L, N154D, and T314I) possessing the HA from a H5N1 virus and the seven remaining gene segments from a H1N1 S-OIV have been described as important determinants of airborne transmission in ferrets.[85] Similarly, Herfst et al.[86] reported important amino acid substitutions in the HA of a HPAI H5N1 (T156A, N154K, H103Y) that also confer airborne transmission in ferrets.

However, mutations enhancing the binding to SAα2-6Glu are not in themselves sufficient for host switching and transmission, meaning that other virus factors may be involved.[10, 36, 87, 88] In this regard, the adaptation of the IAV polymerase to host factors is an important mechanism underlying interspecies transmission.[52, 88, 89] In addition to the PB2-E627K mutation present in some H5N1 strains,[90, 91] mutations such as PB2-T271A,[56] PB2-Q591K,[57] and PB2-D701N have been associated with elevated avian polymerase activity in human cells, replication, and transmissibility in guinea pigs and with an increased transport of PB2 into the nucleus of mammalian cells.[51, 92]

Prior to 2003, infection with H7 viruses was not considered a serious health threat, although some H7 outbreaks in poultry have been sporadically associated with conjunctivitis in humans.[93, 94] This could be linked to the presence of SAα2-3Glu linkages in corneal and conjunctival epithelial cells of the human eye.[21] During the H7N7 HPAI outbreak in poultry that occurred in the Netherlands in 2003, 86 people involved in a culling operation and three in-contact persons were infected, prompting a reevaluation of the human health risks attributed to this virus, even if the majority of these infections in humans resulted in self-limiting conjunctivitis with occasional mild respiratory illness.[93, 95, 96]

During the 2003 H7N7 Dutch outbreak, different mutations in the polymerase complex, HA, NA, and NS1 were found in viruses isolated from a fatal case when compared with strains isolated from conjunctivitis cases. Among these mutations, PB2-E627K was the main determinant of virus pathogenicity, whereas the HA-A143T mutation correlated with viral attachment to human alveolar macrophages.[97] Additionally, viruses from fatal cases presented the PB2-D701N, PB2-S714R, and PA-K615N mutations, which conferred increased polymerase activity in mammal cells at relatively low temperatures.[58, 97]

H9N2 LPAI viruses have become enzootic in domestic poultry populations of many Eurasian countries, causing sporadic human infections characterized by influenza-like symptoms.[98] H9N2 viruses have been isolated from pigs[99] and humans[100, 101] and are believed to be potential pandemic candidates.[82, 102] Molecular characterization of H9N2 viruses circulating in the Middle East and Asia has revealed that more than 70% of the viruses contained the HA-L226 signature, which modifies receptor preference to SAα2-6Glu linkages.[82] Along the same lines, Sorrell et al.[80] demonstrated that the combination of four key amino acid residues at the receptor-binding site of the HA (H183, A189, E190, and L226) in a chimeric virus carrying the surface proteins of avian H9N2 in a human H3N2 backbone is essential for transmission in ferrets. Additionally, the PB2-E627K mutation in mouse-adapted H9N2 viruses was correlated with increased virulence in mammals.[103]

To date, swine influenza viruses (SIAV) H1N1, H3N2, and H1N2 subtypes are circulating in swine all over the world.[104] Classical swine H1N1 viruses (cSIAV) presumably emerged from the 1918 pandemic, circulating, and reassorting with other viruses to give rise to the “triple reassortant” H3N2 SIAV.[105] Independently, an avian-like H1N1 SIAV emerged in Europe.[106] Phylogenetic analysis of different SIAVs showed that cSIAVs analyzed possess the HA-E190D mutation (H3 numbering), which is required to switch the host specificity. In addition, cSIAVs possess the avian signature HA-225G, whereas in the European lineage, this signature is variable (G225E or G225K). Interestingly, the European avian-like H1N1 lineage possesses the PB2-D701N that may play a role in mammalian adaptation.[107]


Because the genome of IAVs consists of eight separate RNA segments, coinfection of one host cell with two different strains can result in progeny viruses containing gene segments of both parental viruses.[108-110] Theoretically, there are 256 possible combinations of the eight gene segments between two viruses.[111] Swine are considered as the main candidates for generating reassortant viruses between human and avian IAVs.[112, 113] Available reports have demonstrated the isolation of whole avian IAVs in pigs,[114-116] meanwhile complete genomic analyses have confirmed the reassortment of swine, avian, and/or human viruses in pigs worldwide, as recently reported in China.[117] Importantly, swine are also capable of transmitting reassortant viruses to humans, as demonstrated during the last 2009 pandemic.[118-120]

H9N2 IAVs have become established worldwide in poultry and wild birds and have been occasionally transmitted to mammals including humans and pigs.[100, 117] The continuous circulation among different hosts has provided the conditions for the evolution and generation of multiple novel genotypes through reassortment events.[121] Fusaro et al.[82] reported significant inter- and intra-subtype reassortments associated with specific amino acid substitutions that are believed to result in increased transmissibility in mammals. To date, inter-subtype reassortments have been detected between H9N2, H5N1 HPAI, and H7N3 viruses in China[99] and Pakistan.[121-123] In vivo studies have demonstrated that a reassortant virus containing the surface glycoprotein genes from H9N2 and the six internal genes of a human H3N2 virus,[124] as well as a reassortant virus carrying the HA of H9N2 in the background of a H1N1 S-OIV,[125] were both able to replicate and be transmitted from ferret to ferret.

Among reassortment dynamics of internal IAV gene segments, an avian-origin PB1 segment is present both in the H2N2/57 and in the H3N2/68 pandemic strains. This suggests that the reassortment of polymerase subunit genes between mammalian and avian IAVs might play a role in interspecies transmission.[5, 10, 45] To test this hypothesis, Li et al.[44] studied the compatibility between avian H5N1 and human H1N1 polymerases, observing that recombinant viruses carrying the PB2-H1N1 and PB1-H5N1 had stronger polymerase activity in cell culture. Furthermore, a study demonstrated that in vivo coinfection with avian H5N1 and human H3N2 viruses of ferrets generated reassortant viruses containing genes from both progenitor viruses.[74]

Genetic markers

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biology of IAVs
  5. Molecular mechanism of host range restriction: receptor specificity and viral polymerase complex
  6. Molecular basis of pathogenicity: role of ha cleavage, NS1, and PB1-F2 proteins
  7. Evolutionary pathways and molecular mechanisms of IAVs involved in human adaptation
  8. Genetic markers
  9. Pandemic overview
  10. Conclusion
  11. Acknowledgements
  12. Conflict of interest
  13. References

Surveillance for genetic markers of adaptation could help in predicting the risk of an epidemic emergence.[9] Previous studies have reported up to 52 species-associated signatures that differentiate between avian and human IAVs.[47-49] Unfortunately, these methods did not take into account the phylogenetic relationship of the isolates and treated each sequence as an independent observation, resulting in an overestimation of statistical significance.[79, 126] Other studies reported 18 mortality markers in three pandemic strains,[127] 172 markers under selective pressure during avian-to-human switch,[79] and 68 conserved mutations in eight internal proteins.[128] In addition, 42 markers have been reported in mouse-adapted H9N2 viruses[103] and 10 in mouse-adapted H1N1 S-OIVs.[129] Although the identification of genetic markers is not a trivial task and mechanisms of viral adaptation in mammals are thought to be polygenic, a great number of the mutations identified to date involve the IAV polymerase complex genes.[49, 52, 57]

At present, the study of GC content in each gene segment has been referred to as a possible indicator of the evolutionary process, showing that avian-origin IAVs have a higher GC content than human-adapted viruses.[130] Similar changes in nucleotide composition with diminished GC content (compared with their putative ancestor) were also evident in swine-adapted IAVs.[107] However, the biological basis for these observations is still unclear and needs further investigation.

Pandemic overview

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biology of IAVs
  5. Molecular mechanism of host range restriction: receptor specificity and viral polymerase complex
  6. Molecular basis of pathogenicity: role of ha cleavage, NS1, and PB1-F2 proteins
  7. Evolutionary pathways and molecular mechanisms of IAVs involved in human adaptation
  8. Genetic markers
  9. Pandemic overview
  10. Conclusion
  11. Acknowledgements
  12. Conflict of interest
  13. References

To date, only viruses of the H1, H2, and H3 subtypes are known to have caused pandemics.[131] It has been estimated that there have been at least 13 pandemics in the last 500 years, including four virologically well-documented ones in the 20th century.[7, 10] Although the origin of the “Spanish” influenza pandemic (1917–1918) has not been fully resolved, it is thought that an avian-like H1N1 virus was involved.[105, 132, 133] Alternatively, it may have evolved in swine prior to its emergence.[1] Since then, there have been two major influenza pandemics (1957 and 1968) caused by H2N2 and H3N2 subtypes, respectively. Both strains originated by reassortment between the existing seasonal strain and an animal virus. The human viruses seem to have acquired three avian segments (HA, NA, and PB1), as in the case of the pandemic of 1957 and two avian segments (HA, PB1) in the case of the pandemic of 1968.[134] The other segments are believed to have been circulating in humans and pigs since the 1918 pandemic. Until 2009, H3N2 and H1N1 (reintroduced in 1977) were still circulating in the human population.[10]

In early April 2009, the H1N1 S-OIV emerged in Mexico and spread to the United States and then around the world, causing the World Health Organization to raise its pandemic alert from level 5 to level 6.[119, 135, 136] The H1N1 S-OIV derived its NA and M gene segments from the European avian-like H1N1 lineage and its remaining six gene segments from the North American swine H1N2 triple reassortant lineage.[135] The HA, NP, and NS genes segments were derived from cSIAV H1N1, while the polymerase gene segments PB2 and PA were derived from avian source and PB1 from a human seasonal H3N2. It was established that the virus had already been circulating in swine for more than 10 years[136] and that the transmission from pigs to humans had occurred several months before the index case. The H1N1 S-OIV has evolved rapidly, mutating and reassorting with other IAVs that are currently circulating.[137-140] The H1N1 S-OIV has the D190/D225 signature, supporting efficient transmissibility among humans, although some recent strains possess the HA-D225G/E mutation, which allow the viruses to have dual hosts (pigs and humans).[135, 141] In addition, the HA-D222G mutation has been involved in severe infection outcomes in humans.[142] On the other hand, the HA-E391K mutation could alter the salt bridge pattern and stability in a region of the HA oligomerization interface that is important for membrane fusion.[143, 144] It has been reported that H1N1 S-OIV lacks both PB2-E627K and PB2-D701N mutations.[54]

On June 8, 2011, the first case of coinfection with seasonal H3N2 and H1N1 S-OIV followed by in vivo reassortment in humans was reported in Canada. The phylogenetic analysis demonstrated that the reassortant virus consisted of HA and NA of H3N2 and the remaining genes of H1N1 S-OIV.[145] Human mixed infections of H1N1 S-OIVs and seasonal H3N2 viruses were reported in China,[146] while an infection with a triple reassortant SIAV H1N1 distinct from H1N1 S-OIV containing the HA and NA genes of seasonal H1N1 virus was detected in Canada.[147] On the other hand, H1N1 S-OIV is able to reinfect swine[148] and to reassort with other viruses circulating in swine herds, as reported to have occurred in Canada,[149] Hong Kong,[150] and China.[151] A study demonstrated that reassortant viruses containing the HA gene from a seasonal H1N1 on a H1N1 S-OIV background showed enhanced growth in cell culture.[152] However, limited compatibility among polymerase subunits from different IAVs must be considered as a restricting factor for reassortment.[153]


  1. Top of page
  2. Abstract
  3. Introduction
  4. Biology of IAVs
  5. Molecular mechanism of host range restriction: receptor specificity and viral polymerase complex
  6. Molecular basis of pathogenicity: role of ha cleavage, NS1, and PB1-F2 proteins
  7. Evolutionary pathways and molecular mechanisms of IAVs involved in human adaptation
  8. Genetic markers
  9. Pandemic overview
  10. Conclusion
  11. Acknowledgements
  12. Conflict of interest
  13. References

Avian IAVs have played an important role in the generation of the well-known H1, H2, and H3 human pandemics, and in all cases, at least one of eight segments was donated by these viruses to the pandemic strains. This proves that the medical and veterinary influenza communities are challenged with a virus that constantly changes through different mechanisms, as it adapts to different species and reassorts with other IAVs of avian and mammalian origin. This indicates the need to strengthen the collaboration in an interdisciplinary manner to identify new emerging viruses with pandemic potential.

Molecular determinants of host specificity and pathogenicity have been identified in most viral genes that encode viral surface glycoproteins, proteins involved in the viral genome replication and those that counteract the host immune response. Recent findings show that not only pigs but also humans and some gallinaceous avian species express both α2-3- and α2-6-linked receptors, facilitating possible reassortment events between mammals and avian viruses and probably extending the number of species that need to be considered as “mixing vessels.”

New IAV strains emerge through the accumulation of mutations, natural reassortment, and adaptation to their new host. Recent investigations have demonstrated that mutations in the receptor-binding site of the HA protein of avian IAVs may change the binding preference of these strains toward the human host; however, this factor is not sufficient for host switching and further transmission. In this regard, several studies have shown that adaptation of the IAV polymerase to host factors is one of the most important mechanisms, which highlights interspecies transmission. Therefore, the evolution and adaptation of IAVs are complex and polygenic, involving several viral genes and other unknown host factors.

H5N1 HPAI viruses are still to be considered as a significant threat for public health. In addition, some H9 avian IAVs have the ability to bind to α2-6 receptors, and the evidence of reassortment with other IAVs emphasizes their potential to emerge as possible pandemic strains. In the same way, H1N1 S-OIV is evolving rapidly and reassorting with other IAVs that are currently circulating. However, further investigations are needed to clarify the rules that govern the reassortment and the successful gene combination in IAVs, as wells as the human and host genes involved in modulation of IAV infections.

Several genetic markers in IAV genes have been reported as being associated with certain biological properties, such as receptor binding, host restriction and tropism, virulence and modulation of host immunity, as well as efficiency of replication and transmission. Some studies in animal models have shown the importance of individual virus proteins, such as the HA, NA, polymerase and non-structural proteins, and even point mutations within these molecules. However, they are polygenic, correlation between molecular markers and biological properties is not absolute, and the full understanding of their correlation with biological properties is yet to come.

A valuable means to identify strains of pandemic potential would be to strengthen the use of molecular methods to study IAV evolution, such as (i) large-scale genomic sequencing to improve the surveillance of mutations and gene constellations; (ii) bioinformatics analyses to study the spatio-temporal evolution dynamics to identify mutations under positive selection and protein structural prediction, and (iii) deep sequencing to monitor within-host viral population diversity.

In recent times, accessible databases have become available to support the growing pool of genetic data for IAVs, although epidemiological and ecological data should also be included to improve our understanding and establish new research studies regarding IAV emergence. Finally, the development of programs involving interdisciplinary teams to promote a broader collaboration to identify new emerging viruses could be an important approach to improve data collection and integrative analysis in future pandemics.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Biology of IAVs
  5. Molecular mechanism of host range restriction: receptor specificity and viral polymerase complex
  6. Molecular basis of pathogenicity: role of ha cleavage, NS1, and PB1-F2 proteins
  7. Evolutionary pathways and molecular mechanisms of IAVs involved in human adaptation
  8. Genetic markers
  9. Pandemic overview
  10. Conclusion
  11. Acknowledgements
  12. Conflict of interest
  13. References
  • 1
    Smith GJ, Bahl J, Vijaykrishna D et al. Dating the emergence of pandemic influenza viruses. Proc Natl Acad Sci USA 2009; 106:1170911712.
  • 2
    Capua I, Cattoli G. One flu for one health. Emerg Infect Dis 2010; 16:719.
  • 3
    Kandeel A, Manoncourt S, Abd el Kareem E et al. Zoonotic transmission of avian influenza virus (H5N1), Egypt, 2006–2009. Emerg Infect Dis 2010; 16:11011107.
  • 4
    Kayali G, Webby RJ, Ducatez MF et al. The epidemiological and molecular aspects of influenza H5N1 viruses at the human-animal interface in Egypt. PLoS ONE 2011; 6:e17730.
  • 5
    Munier S, Moisy D, Marc D, Naffakh N. Interspecies transmission, adaptation to humans and pathogenicity of animal influenza viruses. Pathol Biol 2010; 58:e59e68.
  • 6
    Belser JA, Maines TR, Tumpey TM, Katz JM. Influenza A virus transmission: contributing factors and clinical implications. Expert Rev Mol Med 2010; 12:e39. Review.
  • 7
    Morens DM, Taubenberger JK. Pandemic influenza: certain uncertainties. Rev Med Virol 2011; 21:262284.
  • 8
    Forrest HL, Webster RG. Perspectives on influenza evolution and the role of research. Anim Health Res Rev 2010; 11:318. Review.
  • 9
    Pepin KM, Lass S, Pulliam JR, Read AF, Lloyd-Smith JO. Identifying genetic markers of adaptation for surveillance of viral host jumps. Nat Rev Microbiol 2010; 8:802813. Review.
  • 10
    Klenk HD, Garten W, Matrosovich M. Molecular mechanisms of interspecies transmission and pathogenicity of influenza viruses: lessons from the 2009 pandemic. BioEssays 2011; 33:180188.
  • 11
    Wright PF, Neumann G, Kawaoka Y, Knipe DM, Howley PM (eds). Fields Virology: Orthomyxoviruses, 5th edn. Vol 1, Chapter 48. Philadelphia, PA: Lippincott, Williams & Wilkins, 2007; 16931740.
  • 12
    Tong S, Li Y, Rivailler P et al. A distinct lineage of influenza A virus from bats. Proc Natl Acad Sci USA 2012; 13:42694274.
  • 13
    Capua Ilaria, Alexander Dennis J. Avian Influenza and Newcastle Disease: A Field and Laboratory Guide. Milan, Italy: Springer-Verlag, 2009.
  • 14
    Neumann G, Brownlee GG, Fodor E, Kawaoka Y. Orthomyxovirus replication, transcription, and polyadenylation. Curr Top Microbiol Immunol 2004; 283:121143.
  • 15
    Haye K, Burmakina S, Moran T, García-Sastre A, Fernandez-Sesma A. The NS1 protein of a human influenza virus inhibits type I interferon production and the induction of antiviral responses in primary human dendritic and respiratory epithelial cells. J Virol 2009; 83:68496862.
  • 16
    McAuley JL, Chipuk JE, Boyd KL, Van De Velde N, Green DR, McCullers JA. PB1-F2 proteins from H5N1 and 20 century pandemic influenza viruses cause immunopathology. PLoS Pathog 2010; 6:e1001014.
  • 17
    Jagger BW, Wise HM, Kash JC et al. An overlapping protein-coding region in influenza A virus segment 3 modulates the host response. Science 2012; 13:199204.
  • 18
    Rogers GN, Paulson JC. Receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the H3 hemagglutinin based on species of origin. Virology 1983; 127:361373.
  • 19
    Pillai SP, Lee CW. Species and age related differences in the type and distribution of influenza virus receptors in different tissues of chickens, ducks and turkeys. Virol J 2010; 7:5.
  • 20
    Couceiro JN, Paulson JC, Baum LG. Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity. Virus Res 1993; 29:155165.
  • 21
    Shinya K, Ebina M, Yamada S, Ono M, Kasai N, Kawaoka Y. Avian flu: influenza virus receptors in the human airway. Nature 2006; 440:435436.
  • 22
    Shelton H, Ayora-Talavera G, Ren J et al. Receptor binding profiles of avian influenza virus hemagglutinin subtypes on human cells as a predictor of pandemic potential. J Virol 2011; 85:18751880.
  • 23
    Nicholls JM, Bourne AJ, Chen H, Guan Y, Peiris JS. Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses. Respir Res 2007; 8:73.
  • 24
    Kalthoff D, Globig A, Beer M. Highly pathogenic avian influenza as a zoonotic agent. Vet Microbiol 2010; 140:237245.
  • 25
    Piwpankaew Y, Monteerarat Y, Suptawiwat O, Puthavathana P, Uipresertkul M, Auewarakul P. Distribution of viral RNA, sialic acid receptor, and pathology in H5N1 avian influenza patients. APMIS 2010; 118:895902.
  • 26
    Ito T, Couceiro JN, Kelm S et al. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J Virol 1998; 72:73677373.
  • 27
    Bateman AC, Karamanska R, Busch MG, Dell A, Olsen CW, Haslam SM. Glycan analysis and influenza A virus infection of primary swine respiratory epithelial cells: the importance of NeuAc{alpha}2-6 glycans. J Biol Chem 2010; 285:3401634026.
  • 28
    Van Poucke SG, Nicholls JM, Nauwynck HJ, Van Reeth K. Replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution. Virol J 2010; 7:38.
  • 29
    Nelli RK, Kuchipudi SV, White GA, Perez BB, Dunham SP, Chang KC. Comparative distribution of human and avian type sialic acid influenza receptors in the pig. BMC Vet Res 2010; 6:4.
  • 30
    Xu D, Newhouse EI, Amaro RE et al. Distinct glycan topology for avian and human sialopentasaccharide receptor analogues upon binding different hemagglutinins: a molecular dynamics perspective. J Mol Biol 2009; 387:465491.
  • 31
    Chandrasekaran A, Srinivasan A, Raman R et al. Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin. Nat Biotechnol 2008; 26:107113.
  • 32
    Thongratsakul S, Suzuki Y, Hiramatsu H et al. Avian and human influenza A virus receptors in trachea and lung of animals. Asian Pac J Allergy Immunol 2010; 28:294301.
  • 33
    Kuchipudi SV, Nelli R, White GA, Bain M, Chang KC, Dunham S. Differences in influenza virus receptors in chickens and ducks: implications for interspecies transmission. J Mol Genet Med 2009; 3:143151.
  • 34
    Yu JE, Yoon H, Lee HJ et al. Expression patterns of influenza virus receptors in the respiratory tracts of four species of poultry. J Vet Sci 2011; 12:713.
  • 35
    Wiley DC, Skehel JJ. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu Rev Biochem 1987; 56:365394. Review.
  • 36
    Yassine HM, Lee CW, Gourapura R, Saif YM. Interspecies and intraspecies transmission of influenza A viruses: viral, host and environmental factors. Anim Health Res Rev 2010; 11:5372. Review.
  • 37
    Glaser L, Stevens J, Zamarin D et al. A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity. J Virol 2005; 79:1153311536.
  • 38
    Liu J, Stevens DJ, Haire LF et al. Structures of receptor complexes formed by hemagglutinins from the Asian influenza pandemic of 1957. Proc Natl Acad Sci USA 2009; 106:1717517180.
  • 39
    Nicholls JM, Chan RW, Russell RJ, Air GM, Peiris JS. Evolving complexities of influenza virus and its receptors. Trends Microbiol 2008; 16:149157.
  • 40
    Suzuki Y, Ito T, Suzuki T et al. Sialic acid species as a determinant of the host range of influenza A viruses. J Virol 2000; 74:1182511831.
  • 41
    Sriwilaijaroen N, Kondo S, Yagi H et al. N-glycans from porcine trachea and lung: predominant NeuAcα2-6Gal could be a selective pressure for influenza variants in favor of human-type receptor. PLoS ONE 2011; 6:e16302.
  • 42
    Viswanathan K, Chandrasekaran A, Srinivasan A, Raman R, Sasisekharan V, Sasisekharan R. Glycans as receptors for influenza pathogenesis. Glycoconj J 2010; 27:561570. Review.
  • 43
    Mehle A, Doudna JA. Adaptive strategies of the influenza virus polymerase for replication in humans. Proc Natl Acad Sci USA 2009; 106:2131221316.
  • 44
    Li OT, Chan MC, Leung CS et al. Full factorial analysis of mammalian and avian influenza polymerase subunits suggests a role of an efficient polymerase for virus adaptation. PLoS ONE 2009; 4:e5658.
  • 45
    Taubenberger JK, Kash JC. Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe 2010; 7:440451. Review.
  • 46
    Foeglein A, Loucaides EM, Mura M, Wise HM, Barclay WS, Digard P. Influence of PB2 host-range determinants on the intranuclear mobility of the influenza A virus polymerase. J Gen Virol 2011; 92:16501661.
  • 47
    Chen GW, Chang SC, Mok CK et al. Genomic signatures of human versus avian influenza A viruses. Emerg Infect Dis 2006; 12:13531360.
  • 48
    Finkelstein DB, Mukatira S, Mehta PK et al. Persistent host markers in pandemic and H5N1 influenza viruses. J Virol 2007; 81:1029210299.
  • 49
    Miotto O, Heiny A, Tan TW, August JT, Brusic V. Identification of human-to-human transmissibility factors in PB2 proteins of influenza A by large-scale mutual information analysis. BMC Bioinformatics 2008; 9:S18.
  • 50
    Chen H, Li Y, Li Z et al. Properties and dissemination of H5N1 viruses isolated during an influenza outbreak in migratory waterfowl in Western China. J Virol 2006; 80:59765983.
  • 51
    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.
  • 52
    Moncorgé O, Mura M, Barclay WS. Evidence for avian and human host cell factors that affect the activity of influenza virus polymerase. J Virol 2010; 84:99789986.
  • 53
    Scull MA, Gillim-Ross L, Santos C et al. Avian influenza virus glycoproteins restrict virus replication and spread through human airway epithelium at temperatures of the proximal airways. PLoS Pathog 2009; 5:e1000424.
  • 54
    Herfst S, Chutinimitkul S, Ye J et al. Introduction of virulence markers in PB2 of pandemic swine-origin influenza virus does not result in enhanced virulence or transmission. J Virol 2010; 84:37523758.
  • 55
    Jagger BW, Memoli MJ, Sheng ZM et al. The PB2-E627K mutation attenuates viruses containing the 2009 H1N1 influenza pandemic polymerase. MBio 2010; 1:19.
  • 56
    Bussey KA, Bousse TL, Desmet EA, Kim B, Takimoto T. PB2 residue 271 plays a key role in enhanced polymerase activity of influenza A viruses in mammalian host cells. J Virol 2010; 84:43954406.
  • 57
    Yamada S, Hatta M, Staker BL et al. Biological and structural characterization of a host-adapting amino acid in influenza virus. PLoS Pathog 2010; 6:111.
  • 58
    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 USA 2005; 102:1859018595.
  • 59
    Gabriel G, Klingel K, Otte A et al. Differential use of importin-α isoforms governs cell tropism and host adaptation of influenza virus. Nat Commun 2011; 2:156.
  • 60
    Rott R. The pathogenic determinant of influenza virus. Vet Microbiol 1992; 33:303310.
  • 61
    Foucault ML, Moules V, Rosa-Calatrava M, Riteau B. Role for proteases and HLA-G in the pathogenicity of influenza A viruses. J Clin Virol 2011; 51:155159.
  • 62
    Stieneke-Grober A, Vey M, Angliker H et al. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMOB J 1992; 11:24072414.
  • 63
    Stech O, Veits J, Weber S et al. Acquisition of a polybasic hemagglutinin cleavage site by a low-pathogenic avian influenza virus is not sufficient for immediate transformation into a highly pathogenic strain. J Virol 2009; 83:58645868.
  • 64
    Munster VJ, Schrauwen EJ, de Wit E et al. Insertion of a multibasic cleavage motif into the hemagglutinin of a low-pathogenic avian influenza H6N1 virus induces a highly pathogenic phenotype. J Virol 2010; 84:79537960.
  • 65
    Jackson D, Hossain MJ, Hickman D, Perez DR, Lamb RA. A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity. Proc Natl Acad Sci USA 2008; 105:43814386.
  • 66
    Malik PJS. Avian influenza viruses in humans. Rev Sci Tech Off Int Epizoot 2009; 28:161174.
  • 67
    Phung TT, Sugamata R, Uno K et al. Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells. Microbiol Immunol 2011; 55:874884.
  • 68
    Varga ZT, Ramos I, Hai R et al. The influenza virus protein PB1-F2 inhibits the induction of type I interferon at the level of the MAVS adaptor protein. PLoS Pathog 2011; 7:116.
  • 69
    McAuley JL, Hornung F, Boyd KL et al. Expression of the 1918 influenza A virus PB1-F2 enhances the pathogenesis of viral and secondary bacterial pneumonia. Cell Host Microbe 2007; 2:240249.
  • 70
    Chen R, Holmes EC. Avian influenza virus exhibits rapid evolutionary dynamics. Mol Biol Evol 2006; 23:23362341.
  • 71
    Both GW, Sleigh MJ, Cox NJ, Kendal AP. Antigenic drift in influenza virus H3 hemagglutinin from 1968 to 1980: multiple evolutionary pathways and sequential amino acid changes at key antigenic sites. J Virol 1983; 48:5260.
  • 72
    Drake JW. Rates of spontaneous mutation among RNA viruses. Proc Natl Acad Sci USA 1993; 90:41714175.
  • 73
    Dos Reis M, Hay AJ, Goldstein RA. Using non-homogeneous models of nucleotide substitution to identify host shift events: application to the origin of the 1918 ‘Spanish’ influenza pandemic virus. J Mol Evol 2009; 69:333345.
  • 74
    Jackson S, Van Hoeven N, Chen LM et al. Reassortment between avian H5N1 and human H3N2 influenza viruses in ferrets: a public health risk assessment. J Virol 2009; 83:81318140.
  • 75
    Ayora-Talavera G, Shelton H, Scull MA et al. Mutations in H5N1 influenza virus hemagglutinin that confers binding to human tracheal airway epithelium. PLoS ONE 2009; 4:111.
  • 76
    Yea C, McCorrister S, Westmacott G, Petric M, Tellier R. Early detection of influenza A (H5) viruses with affinity for the human sialic acid receptor by MALDI-TOF mass spectrometry based mutation detection. J Virol Methods 2011; 172:7277.
  • 77
    Watanabe Y, Ibrahim MS, Ellakany HF et al. Acquisition of human-type receptor binding specificity by New H5N1 influenza virus sublineages during their emergence in birds in Egypt. PLoS Pathog 2011; 7:119.
  • 78
    Bateman AC, Busch MG, Karasin AI, Bovin N, Olsen CW. Amino acid 226 in the hemagglutinin of H4N6 influenza virus determines binding affinity for alpha2, 6-linked sialic acid and infectivity levels in primary swine and human respiratory epithelial cells. J Virol 2008; 82:82048209.
  • 79
    Tamuri AU, Dos Reis M, Hay AJ, Goldstein RA. Identifying changes in selective constraints: host shifts in influenza. PLoS Comput Biol 2009; 5:e1000564.
  • 80
    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 USA 2009; 106:75657570.
  • 81
    Tambunan US, Ramdhan. Identification of sequence mutations affecting hemagglutinin specificity to sialic acid receptor in influenza A virus subtypes. Bioinformation 2010; 5:244249.
  • 82
    Fusaro A, Monne I, Salviato A et al. Phylogeography and evolutionary history of reassortant H9N2 viruses with potential human health implications. J Virol 2011; 85:84138421.
  • 83
    Subbarao K, Klimov A, Katz J et al. Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 1998; 279:393396.
  • 84
    Taubenberger JK, Morens DM. Pandemic influenza-including a risk assessment of H5N1. Rev Sci Tech 2009; 28:187202.
  • 85
    Imai M, Watanabe T, Hatta M et al. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 2012; 486:420428.
  • 86
    Herfst S, Schrauwen EJA, Linster M et al. Airborne transmission of influenza A/H5N1 virus between ferrets. Science 2012; 336:15341541.
  • 87
    Maines TR, Chen LM, Van Hoeven N et al. Effect of receptor binding domain mutations on receptor binding and transmissibility of avian influenza H5N1 viruses. Virology 2011; 413:139147.
  • 88
    Sakabe S, Ozawa M, Takano R, Iwastuki-Horimoto K, Kawaoka Y. Mutations in PA, NP, and HA of a pandemic (H1N1) 2009 influenza virus contribute to its adaptation to mice. Virus Res 2011; 158:124129.
  • 89
    Lycett SJ, Ward MJ, Lewis FI, Poon AF, Kosakovsky Pond SL, Brown AJ. Detection of mammalian virulence determinants in highly pathogenic avian influenza H5N1 viruses: multivariate analysis of published data. J Virol 2009; 83:99019910.
  • 90
    Maines TR, Lu XH, Erb SM et al. Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals. J Virol 2005; 79:1178811800.
  • 91
    Fornek JL, Gillim-Ross L, Santos C et al. 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; 83:1110211115.
  • 92
    Ping J, Dankar SK, Forbes NE et al. PB2 and hemagglutinin mutations are major determinants of host range and virulence in mouse-adapted influenza A virus. J Virol 2010; 84:1060610618.
  • 93
    Koopmans M, Wilbrink B, Conyn M et al. Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands. Lancet 2004; 363:587593.
  • 94
    Belser JA, Bridges CB, Katz JM, Tumpey TM. Past, present, and possible future human infection with influenza virus A subtype H7. Emerg Infect Dis 2009; 15:859865. Review.
  • 95
    Fouchier RA, Schneeberger PM, Rozendaal FW et al. Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proc Natl Acad Sci USA 2004; 101:13561361.
  • 96
    Belser JA, Blixt O, Chen LM et al. Contemporary North American influenza H7 viruses possess human receptor specificity: implications for virus transmissibility. Proc Natl Acad Sci USA 2008; 105:75587563.
  • 97
    De Wit E, Munster VJ, van Riel D et al. Molecular determinants of adaptation of highly pathogenic avian influenza H7N7 viruses to efficient replication in the human host. J Virol 2010; 84:15971606.
  • 98
    Butt AM, Siddique S, Idrees M, Tong Y. Avian influenza A (H9N2): computational molecular analysis and phylogenetic characterization of viral surface proteins isolated between 1997 and 2009 from the human population. Virol J 2010; 7:319.
  • 99
    Yu H, Zhou YJ, Li GX et al. Genetic diversity of H9N2 influenza viruses from pigs in China: a potential threat to human health? Vet Microbiol 2011; 149:254261.
  • 100
    Butt KM, Smith GJD, Chen H et al. Human infection with an Avian H9N2 influenza A virus in Hong Kong in 2003. J Clin Microbiol 2005; 43:57605767.
  • 101
    Lin YP, Shaw M, Gregory V et al. Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates. Proc Natl Acad Sci USA 2000; 97:96549658.
  • 102
    Sun Y, Pu J, Jiang Z et al. Genotypic evolution and antigenic drift of H9N2 influenza viruses in China from 1994 to 2008. Vet Microbiol 2010; 146:215225.
  • 103
    Wu R, Zhang H, Yang K et al. Multiple amino acid substitutions are involved in the adaptation of H9N2 avian influenza virus to mice. Vet Microbiol 2009; 138:8591.
  • 104
    Brockwell-Staats C, Webster RG, Webby RJ. Diversity of influenza viruses in swine and the emergence of a novel human pandemic influenza A (H1N1). Influenza Other Respi Viruses 2009; 3:207213. Review.
  • 105
    Taubenberger JK, Reid AH, Janczewski TA, Fanning TG. Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus. Philos Trans R Soc Lond B Biol Sci 2001; 356:18291839. Review.
  • 106
    Kuntz-Simon G, Madec F. Genetic and antigenic evolution of swine influenza viruses in Europe and evaluation of their zoonotic potential. Zoonoses Public Health 2009; 56:310325. Review.
  • 107
    Dunham EJ, Dugan VG, Kaser EK et al. Different evolutionary trajectories of European avian-like and classical swine H1N1 influenza A viruses. J Virol 2009; 83:54855494.
  • 108
    Muramoto Y, Takada A, Fujii K et al. Hierarchy among viral RNA (vRNA) segments in their role in vRNA incorporation into influenza A virions. J Virol 2006; 80:23182325.
  • 109
    Nelson MI, Viboud C, Simonsen L et al. Multiple reassortment events in the evolutionary history of H1N1 influenza A virus since 1918. PLoS Pathog 2008; 4:112.
  • 110
    Cong Y, Wang G, Guan Z et al. Reassortant between human-Like H3N2 and avian H5 subtype influenza A viruses in pigs: a potential public health risk. PLoS ONE 2010; 5:18.
  • 111
    Greenbaum BD, Li OT, Poon LL, Levine AJ, Rabadan R. Viral reassortment as an information exchange between viral segments. Proc Natl Acad Sci USA 2012; 109:33413346.
  • 112
    Kida H, Ito T, Yasuda J et al. Potential for transmission of avian influenza viruses to pigs. J Gen Virol 1994; 75:21832188.
  • 113
    Ma W, Lager KM, Vincent AL, Janke BH, Gramer MR, Richt JA. The role of swine in the generation of novel influenza viruses. Zoonoses Public Health 2009; 56:326337. Review.
  • 114
    Nidom CA, Takano R, Yamada S et al. Influenza A (H5N1) viruses from pigs, Indonesia. Emerg Infect Dis 2010; 16:15151523.
  • 115
    Hu Y, Liu X, Li S, Guo X, Yang Y, Jin M. Complete genome sequence of a novel H4N1 influenza virus isolated from a pig in Central China. J Virol 2012; 86:13879.
  • 116
    Wang N, Zou W, Yang Y et al. Complete genome sequence of an H10N5 avian influenza virus isolated from pigs in Central China. J Virol 2012; 86:1386513866.
  • 117
    Cong YL, Wang CF, Yan CM, Peng JS, Jiang ZL, Liu JH. Swine infection with H9N2 influenza viruses in China in 2004. Virus Genes 2008; 36:461469.
  • 118
    Girard MP, Tam JS, Assossou OM, Kieny MP. The 2009 A (H1N1) influenza virus pandemic: a review. Vaccine 2010; 28:48954902. Review.
  • 119
    Arias CF, Escalera-Zamudio M, Soto-Del Rio M de L, Cobian-Guemes AG, Isa P, Lopez S. Molecular anatomy of 2009 influenza virus A (H1N1). Arch Med Res 2009; 40:643654. Review.
  • 120
    Garten RJ, Davis CT, Russell CA et al. Antigenic and genetic characteristics of swine-origin 2009 A (H1N1) influenza viruses circulating in humans. Science 2009; 325:197201.
  • 121
    Dong G, Luo J, Zhang H et al. Phylogenetic diversity and genotypical complexity of H9N2 influenza A viruses revealed by genomic sequence analysis. PLoS ONE 2011; 6:19.
  • 122
    Abbas MA, Spackman E, Swayne DE et al. Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from poultry in Pakistan 1995–2004. Virol J 2010; 7:137.
  • 123
    Iqbal M, Yaqub T, Reddy K, McCauley JW. Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan containing NS genes similar to highly pathogenic H7N3 and H5N1 viruses. PLoS ONE 2009; 4:e5788.
  • 124
    Wan H, Sorrell EM, Song H et al. Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential. PLoS ONE 2008; 3:113.
  • 125
    Kimble JB, Sorrell E, Shao H, Martin PL, Perez DR. Compatibility of H9N2 avian influenza surface genes and 2009 pandemic H1N1 internal genes for transmission in the ferret model. Proc Natl Acad Sci USA 2011; 108:1208412088.
  • 126
    Dos Reis M, Tamuri AU, Hay AJ, Goldstein RA. Charting the host adaptation of influenza viruses. Mol Biol Evol 2011; 28:17551767.
  • 127
    Allen JE, Gardner SN, Vitalis EA, Slezak TR. Conserved amino acid markers from past influenza pandemic strains. BMC Microbiol 2009; 9:77.
  • 128
    Miotto O, Heiny AT, Albrecht R et al. Complete-proteome mapping of human influenza A adaptive mutations: implications for human transmissibility of zoonotic strains. PLoS ONE 2010; 5:113.
  • 129
    Ilyushina NA, Khalenkov AM, Seiler JP et al. Adaptation of pandemic H1N1 influenza viruses in mice. J Virol 2010; 84:86078616.
  • 130
    Greenbaum BD, Levine AJ, Bhanot G, Rabadan R. Patterns of evolution and host gene mimicry in influenza and other RNA viruses. PLoS Pathog 2008; 4:e1000079.
  • 131
    Tang JW, Shetty N, Lam TT, Hon KL. Emerging, novel, and known influenza virus infections in humans. Infect Dis Clin North Am 2010; 24:603617. Review.
  • 132
    Gammelin M, Altmüller A, Reinhardt U et al. Phylogenetic analysis of nucleoproteins suggests that human influenza A viruses emerged from a 19th-century avian ancestor. Mol Biol Evol 1990; 7:194200.
  • 133
    Guan Y, Vijaykrishna D, Bahl J, Zhu H, Wang J, Smith GJ. The emergence of pandemic influenza viruses. Protein Cell 2010; 1:913. Review.
  • 134
    Kawaoka Y, Krauss S, Webster RG. Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. J Virol 1989; 63:46034608.
  • 135
    Christman MC, Kedwaii A, Xu J, Donis RO, Lu G. Pandemic (H1N1) 2009 virus revisited: an evolutionary retrospective. Infect Genet Evol 2011; 11:803811.
  • 136
    Smith GJ, Vijaykrishna D, Bahl J et al. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 2009; 459:11221125.
  • 137
    Schrauwen EJ, Herfst S, Chutinimitkul S et al. Possible increased pathogenicity of pandemic (H1N1) 2009 influenza virus upon reassortment. Emerg Infect Dis 2011; 17:200208.
  • 138
    Li W, Shi W, Qiao H et al. Positive selection on hemagglutinin and neuraminidase genes of H1N1 influenza viruses. Virol J 2011; 8:183.
  • 139
    Hiromoto Y, Parchariyanon S, Ketusing N et al. Isolation of the pandemic (H1N1) 2009 virus and its reassortant with an H3N2 swine influenza virus from healthy weaning pigs in Thailand in 2011. Virus Res 2012; 169:175181.
  • 140
    Han JY, Park SJ, Kim HK et al. Identification of reassortant pandemic H1N1 influenza virus in Korean pigs. J Microbiol Biotechnol 2012; 22:699707.
  • 141
    Chen H, Wen X, To KK et al. Quasispecies of the D225G substitution in the hemagglutinin of pandemic influenza A(H1N1) 2009 virus from patients with severe disease in Hong Kong, China. J Infect Dis 2010; 201:15171521.
  • 142
    Kilander A, Rykkvin R, Dudman SG, Hungnes O. Observed association between the HA1 mutation D222G in the 2009 pandemic influenza A(H1N1) virus and severe clinical outcome, Norway 2009–2010. Euro Surveill 2010; 15:13.
  • 143
    Mak GC, Leung CK, Cheng KC, Wong KY, Lim W. Evolution of the haemagglutinin gene of the influenza A (H1N1) 2009 virus isolated in Hong Kong, 2009–2011. Euro Surveill 2011; 16:19807.
  • 144
    Maurer-Stroh S, Lee RT, Eisenhaber F, Cui L, Phuah SP, Lin RT. A new common mutation in the hemagglutinin of the 2009 (H1N1) influenza A virus. PLoS Curr 2010; 2:17.
  • 145
    Gubbay J. Reassortment following coinfection with seasonal H3N2 and pandemic (H1N1) 2009 viruses in Ontario, Canada. 2011. ProMED-mail post. Archive number: 20110609.1749.
  • 146
    Liu W, Li ZD, Tang F et al. Mixed infections of pandemic H1N1 and seasonal H3N2 viruses in 1 outbreak. Clin Infect Dis 2010; 50:13591365.
  • 147
    Bastien N, Antonishyn NA, Brandt K et al. Human infection with a triple-reassortant swine influenza A(H1N1) virus containing the hemagglutinin and neuraminidase genes of seasonal influenza virus. J Infect Dis 2010; 201:11781182.
  • 148
    Nagarajan K, Saikumar G, Arya RS, Gupta A, Somvanshi R, Pattnaik B. Influenza A H1N1 virus in Indian pigs and its genetic relatedness with pandemic human influenza A 2009 H1N1. Indian J Med Res 2010; 132:160167.
  • 149
    Nfon CK, Berhane Y, Hisanaga T et al. Characterization of H1N1 swine influenza viruses circulating in Canadian pigs in 2009. J Virol 2011; 85:86678679.
  • 150
    Poon LL, Mak PW, Li OT et al. Rapid detection of reassortment of pandemic H1N1/2009 influenza virus. Clin Chem 2010; 56:13401344.
  • 151
    Zhao G, Fan Q, Zhong L et al. Isolation and phylogenetic analysis of pandemic H1N1/09 influenza virus from swine in Jiangsu province of China. Res Vet Sci 2012; 93:125132.
  • 152
    Octaviani CP, Li C, Noda T, Kawaoka Y. Reassortment between seasonal and swine-origin H1N1 influenza viruses generates viruses with enhanced growth capability in cell culture. Virus Res 2011; 156:147150.
  • 153
    Octaviani CP, Goto H, Kawaoka Y. Reassortment between seasonal H1N1 and pandemic (H1N1) 2009 influenza viruses is restricted by limited compatibility among polymerase subunits. J Virol 2011; 85:84498452.
  • 154
    Chutinimitkul S, Herfst S, Steel J et al. Virulence-associated substitution D222G in the hemagglutinin of 2009 pandemic influenza A (H1N1) virus affects receptor binding. J Virol 2010; 84:1180211813.
  • 155
    Yamada S, Suzuki Y, Suzuki T et al. Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors. Nature 2006; 444:378382.
  • 156
    Gao Y, Zhang Y, Shinya K et al. Identification of amino acids in HA and PB2 critical for the transmission of H5N1 avian influenza viruses in a mammalian host. PLoS Pathog 2009; 5:111.
  • 157
    Stevens J, Blixt O, Chen LM, Donis RO, Paulson JC, Wilson IA. Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity. J Mol Biol 2008; 381:13821394.
  • 158
    Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature 2005; 437:889893.
  • 159
    Ping J, Keleta L, Forbes NE et al. Genomic and protein structural maps of adaptive evolution of human influenza A virus to increased virulence in the mouse. PLoS ONE 2011; 6:121.
  • 160
    Bussey KA, Desmet EA, Mattiacio JL et al. PA residues in the 2009 H1N1 pandemic influenza virus enhance avian influenza virus polymerase activity in mammalian cells. J Virol 2011; 85:70207028.