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. 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).
Table 1. Principal amino acid mutations and signatures associated with interspecies transmission of influenza A viruses
|HA|| || |
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/E||Enhances receptor binding to dual hosts (pigs and humans)||H1N1 S-OIV||135,141|
Enhances binding to SAα2-3Glu receptors
Associated with severe infection outcome in humans
| ||Enhance binding to human-type receptors in vitro||H5N1||155|
|Q192H||Increases binding to SAα2-6Glu and virulence in mice||H5N1||77|
| || |
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
|226L||Signature which exhibits preferential binding to human-like SAα2-6Glu receptors. A key element for the successful infection of humans.||H9N2||82|
|A143T||Increases viral attachment to human alveolar macrophages||H7N7||97|
|E391K||Associated with the fitness of the virus||H1N1 S-OIV||143,144|
|T160A||Required to sustain the avian virus transmission in guinea pig model||H5N1||156|
|K193R||Decreases binding to SAα2-3Glu or increases binding to SAα2-6Glu||H5N1||157|
| ||Respiratory droplet transmission in ferrets|| ||85|
| ||Airborne transmission between ferrets||H5N1 genetically modified||86|
| ||Enhancement of virulence in mouse model||H1N1 S-OIV||129|
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 temperature||H7N7 isolated from human FC||97|
|Increases virulence in mammals||Mouse-adapted H9N2||103|
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 strains||51,92|
|Involved in mammalian adaptation||European ‘avian like’ swine H1N1||107|
|Increases transmissibility of Influenza A viruses in guinea pig model||H5N1||51|
|Enhances the polymerase activity in mammalian cells||Avian- and mouse- adapted H7N7||58|
|S714R||Enhances the polymerase activity in mammalian cells||H7N7||58|
|K318R||Correlates with high pathogenicity in mice in the presence of additional mutations||H5N1||89|
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
|G590S||Associated with mammalian pathogenicity and enhanced replicative ability in mammals||H1N1 S-OIV||43|
|A684S||Associated with host shift from avian to swine and the subsequent transfer to humans||Avian IAVs||79|
|E158G/A||Associated in the adaptation of PB2 genes to mammals (mouse model)||H1N1 S-OIV||129|
|PB1||L13P||Enhances the activity of viral polymerase||H7N7||58|
|G375S||Associated with adaptation to a new species (swine to human)||H1N1||158|
| ||Increase virulence and polymerase activity in mouse model||H3N2 human isolates||159|
|PA||K615N||Enhances activity of viral RNA polymerase and stimulates viral replication and pathogenicity in mouse model||H7N7||58|
|K356R||Associated with host shift from avian to swine and the subsequent transfer to humans||Avian IAVs||79|
| ||Multiple residues that contribute to the enhancement of avian polymerase activity in mammalian cells which is essential for mammalian host adaptation||H1N1 S-OIV||160|
|NP||N319K||Increases binding to mammalian importin α-1 proteins and polymerase activity. Related to host range specificity.||H7N7||59|
|V100I||Increases transmissibility among humans||H1N1 S-OIV||135|
| ||PA-X||Modulates virulence and host immune response in mouse model||IAVs||17|
Transmission of H5N1 HPAI virus from poultry to humans was first reported in Hong Kong in 1997. From 2003 to 2012, 608 cases of H5N1 virus infections in humans and 359 deaths have been reported in 15 different countries (http://www.who.int/influenza/human_animal_interface/EN_GIP_20120810CumulativeNumberH5N1cases.pdf). 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. 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.
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. Similarly, Herfst et al. 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, PB2-Q591K, 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. 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. 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. H9N2 viruses have been isolated from pigs 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. Along the same lines, Sorrell et al. 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.
To date, swine influenza viruses (SIAV) H1N1, H3N2, and H1N2 subtypes are circulating in swine all over the world. 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. Independently, an avian-like H1N1 SIAV emerged in Europe. 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.
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. 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. 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. Fusaro et al. 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 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, as well as a reassortant virus carrying the HA of H9N2 in the background of a H1N1 S-OIV, 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. 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.