Emergence and pandemic potential of swine-origin h1n1 influenza virus

Besides the ongoing transmission of highly pathogenic avian H5N1 influenza viruses to humans, avian influenza A viruses were transmitted to pigs in Europe in , to horses in China, and to seals. Moreover, an avian influenza virus reassorted with a human virus in pigs and transmitted from there to humans. Human infections with avian or swine influenza viruses have been reported but have typically been self-limiting. It is in this context that the recently emerged H1N1 viruses, which are of swine origin and transmit among humans, raise great concern over an imminent pandemic.

In North American pig populations, classical swine H1N1 virus dominated for nearly six decades. For the prevention and control of influenza virus infections, both vaccines and antiviral drugs are available.

Nonetheless, the global community is likely not well prepared for a pandemic: antiviral drugs may not be in sufficient supply and the virus may acquire resistance to the available antiviral drugs. On the other hand, the production of a vaccine to a newly emerging strain would take months — during which time a virus could spread globally and substantially strain health care systems and the global economy.

Two classes of antiviral drugs — ion channel inhibitors and neuraminidase inhibitors — are currently licensed for use against influenza A viruses.

Adamantanes i. The S-OIV are also resistant to ion channel inhibitors Two neuraminidase inhibitors — oseltamivir and zanamivir — are currently licensed. Neuraminidase inhibitors interfere with the enzymatic activity of the NA protein, which is critical for the efficient release of newly synthesized viruses from infected cells. In clinical trials, the emergence of resistance to NA inhibitors was rare, and oseltamivir-resistant influenza viruses were attenuated in vitro and in vivo. Hence, the dissemination of these viruses was not considered a major issue, despite the frequent use of the drug in some countries.

Recently, the rate of oseltamivir-resistant H1N1 influenza viruses in the US has increased from 0. Similar numbers have been reported for other countries. Equally alarming, oseltamivir-resistant H5N1 viruses have been reported 77 , Oseltamivir-resistant human H1N1 viruses may have emerged in immunocompromised patients where prolonged replication 79 - 81 may have resulted in the selection of mutations that increase the fitness of oseltamivir-resistant viruses.

The S-OIV are sensitive to neuraminidase inhibitors when tested in vitro in enzymatic assays Recent structural data provide an explanation for oseltamivir-resistance 82 and suggest strategies for the design of improved compounds.

In clinical settings, resistance to zanamivir has been reported only once for an influenza B virus isolated from an immunocompromised child Several experimental antiviral drugs that target the NA or polymerase proteins are currently in different stages of development. Peramivir, an NA inhibitor that was developed through structure-based design 84 , is active in vitro tests against viruses of all 9 NA subtypes including highly pathogenic H5N1 viruses. Phase II clinical trials are currently underway to assess the efficacy of intramuscularly administered peramivir against seasonal influenza.

CS, a pro-drug of the novel NA inhibitor R 85 , is a long-acting neuraminidase inhibitor that was found to be effective in phase II clinical trials against seasonal influenza. We also found that CS has prophylactic and therapeutic efficacy against highly pathogenic H5N1 influenza viruses unpublished.

T acts as a nucleoside analogue that interferes with the polymerase activity of influenza A, B, and C viruses, but also other RNA viruses It protected mice against infection with highly pathogenic H5N1 viruses our unpublished findings.

Phase II clinical trials for use of T against seasonal influenza viruses have been completed in Japan, and phase III clinical trials are scheduled. In addition, monoclonal antibodies to the HA are being developed for treatment of influenza virus infections. In mice, some antibodies demonstrated prophylactic and therapeutic efficacy against lethal challenge with H5N1 virus 87 , suggesting monoclonal antibody treatment as an alternative strategy to treat influenza virus infections.

Seasonal influenza vaccines include human influenza A viruses of the H1N1 and H3N2 subtypes, and an influenza B virus. These vaccines need to be updated every years to account for mutations in the HA and NA proteins of circulating viruses antigen drift. Inactivated vaccines have been used for many decades. The allantoic fluid of embryonated, virus-infected chicken eggs is purified and concentrated by zonal centrifugation or column chromatography and inactivated with formalin or beta-propiolactone.

Treatment with detergents or ether and removal of vRNP complexes leads to split or subunit vaccines that are administered intramuscularly or subcutaneously. The development of improved influenza virus vaccines is thus clearly warranted.

A live attenuated influenza virus vaccine is now licensed in the US for healthy individuals age 2-to These viruses are then reassorted with currently circulating wild-type strains to generate seed viruses that possess the HA and NA genes of the circulating wild-type viruses in the background of the ts , ca , and att phenotypes. Live attenuated vaccines elicit both humoral and cellular immune responses, and are therefore believed to be superior to inactivated vaccines.

In fact, in infants and young children, live attenuated influenza vaccine provides better protection than inactivated vaccine Currently, only egg-based vaccines are licensed for use in the US. In case of a pandemic, however, eggs may be in short supply. By contrast, cell cultures are highly controllable systems that can be scaled-by up for the mass production of vaccines, including those to highly pathogenic H5N1 viruses.

The purity and immunogenicity of influenza vaccines produced in Madin-Darby canine kidney MDCK or African green monkey kidney Vero cells match those of vaccines produced in embryonated chicken eggs. Cell culture-based influenza vaccines have been approved for use in humans in Europe. Recently, particular emphasis has been given to the development of vaccines to highly pathogenic H5N1 viruses. These viruses kill chicken embryos. Propagation of these viruses in eggs therefore results in low yields.

To modify these viruses for efficient growth in embryonated chicken eggs and safe handling by production staff, reverse genetics technologies 5 were used to replace the multibasic HA cleavage site with an avirulent-type HA cleavage site 45 , a known virulence factor.

Clinical testing of an H5N1 vaccine candidate suggested low immunogenicity 89 , prompting the addition of adjuvants to vaccine candidates. In fact, aluminum hydroxide 90 , 91 or oil-in-water emulsions such as MF59 92 , 93 or AS03 94 resulted in significant antigen-sparing effects. Adjuvanted vaccines also seem to induce broader immune responses, which may be a critical advantage with the emergence of new clades and subclades of H5N1 viruses.

Similarly, the NA and modified HA genes of highly pathogenic avian H5N1 viruses were combined with the remaining genes derived from the live attenuated influenza A virus However, once H5N1 viruses are widespread in human populations, use of a live attenuated H5N1 vaccine, which is efficacious in nonhuman primates 96 , may be considered to overcome the low immunogenicity of inactivated vaccines. Several novel vaccine approaches are now in various stages of development.

Various virus-like particles VLPs expressing the HA and NA proteins, some in combination with the M1 and M2 proteins, have been tested for their antigenicity and protective efficacy. Also, vector approaches have been pursued. In one example, replication-incompetent adenoviruses expressing an H5 HA protected mice against challenge with homologous and heterologous H5N1 viruses 99 , In alternative approaches, Newcastle disease and fowlpox viruses have been explored as vector systems; however, these systems have not been approved for use in humans.

Although much has been learned about influenza viruses, key questions still remain unanswered: what factors determine interspecies transmission, reassortment, and human-to-human transmission — factors that have accounted for past pandemics and will be critical in the emergence of new pandemic viruses. Thus, careful monitoring of the S-OIV during the upcoming winter season in the southern hemisphere is of critical importance to detect more virulent variants, should they arise. In addition, large-scale sequencing efforts, bioinformatics analyses, and the ability to experimentally test predictions with recombinant viruses will eventually provide insight into key features for the emergence of pandemic viruses.

In the meantime, the development of improved and novel antiviral drugs and vaccines will be critical to control influenza virus outbreaks. We apologize to our colleagues whose critical contributions to influenza virus research could not be cited due to the number of references permitted.

We thank Krisna Wells for editing the manuscript. We also thank Makoto Ozawa and others in our laboratories who contributed to the data cited in this review. National Center for Biotechnology Information , U.

Author manuscript; available in PMC May Find articles by Yoshihiro Kawaoka. Author information Copyright and License information Disclaimer. Copyright notice. The publisher's final edited version of this article is available at Nature.

See other articles in PMC that cite the published article. Abstract Influenza viruses cause annual epidemics and occasional pandemics that have claimed the lives of millions. Influenza A viruses Influenza A viruses belong to the family Orthomyxoviridae. Open in a separate window. Schematic diagram of the influenza viral life cycle Following receptor-mediated endocytosis, the viral ribonucleoprotein vRNP complexes are released into the cytoplasm and subsequently transported to the nucleus, where replication and transcription take place.

Influenza pandemics Influenza A viruses cause recurrent epidemics and global pandemics. Highly pathogenic H5N1 influenza viruses The infection of 18 individuals in Hong Kong in with highly pathogenic avian influenza viruses of the H5N1 subtype, which resulted in 6 fatalities 17 , 18 , marked the first reported fatal infections of humans with avian influenza viruses.

Outbreak of swine-origin H1N1 viruses Epidemiological data now indicate that an outbreak of influenza-like respiratory illness started in the Mexican town of La Gloria, Veracruz, in mid February of 31 Table 1. Table 1 Timeline of swine-origin H1N1 virus outbreak see also Genesis of swine-origin H1N1 influenza viruses In the late 's, reassortment between human H3N2, North American avian, and classical swine viruses resulted in triple reassortant H3N2 and H1N2 swine viruses that have since circulated in North American pig populations.

Role of HA in viral pathogenicity Influenza virus pathogenicity is multigenic, and the determinants of pathogenicity may differ among animal species. Receptor distribution on host cells Influenza virus host specificity can be explained in part by the difference in receptor binding specificity for human and avian influenza viruses. HA receptor specificity The differences in receptor-binding specificity of human and avian viruses are determined by the amino acid residues in the HA receptor-binding pocket.

Role of PB2 in pathogenicity and host specificity Recently, the viral replication complex has been recognized as an important contributor to viral pathogenicity, likely by affecting viral growth. Interspecies transmission Wild waterfowl are the natural reservoir of influenza A viruses. Prevention and control For the prevention and control of influenza virus infections, both vaccines and antiviral drugs are available.

Antiviral drugs Two classes of antiviral drugs — ion channel inhibitors and neuraminidase inhibitors — are currently licensed for use against influenza A viruses. The Future Although much has been learned about influenza viruses, key questions still remain unanswered: what factors determine interspecies transmission, reassortment, and human-to-human transmission — factors that have accounted for past pandemics and will be critical in the emergence of new pandemic viruses.

Induction of apoptosis 74 HA Single basic a. Multiple basic a. Single basic a. Acknowledgements We apologize to our colleagues whose critical contributions to influenza virus research could not be cited due to the number of references permitted.

Reference List 1. Kobasa D, et al. Aberrant innate immune response in lethal infection of macaques with the influenza virus. Predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness. J Infect Dis. Neumann G, et al. Generation of influenza A viruses entirely from cloned cDNA. Tumpey TM, et al. Characterization of the reconstructed Spanish influenza pandemic virus.

Kash JC, et al. Genomic analysis of increased host immune and cell death responses induced by influenza virus. Fatal outcome of human influenza A H5N1 is associated with high viral load and hypercytokinemia. Enhanced virulence of influenza A viruses with the haemagglutinin of the pandemic virus. Existing antivirals are effective against influenza viruses with genes from the pandemic virus.

Pathogenicity and immunogenicity of influenza viruses with genes from the pandemic virus. Watanabe T, et al. Viral RNA polymerase complex promotes optimal growth of virus in the lower respiratory tract of ferrets. Sci U. Van Hoeven N, et al. Human HA and polymerase subunit PB2 proteins confer transmission of an avian influenza virus through the air.

Geiss GK, et al. Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza.

McAuley JL, et al. Expression of the influenza A virus PB1-F2 enhances the pathogenesis of viral and secondary bacterial pneumonia. Cell Host. Recent human influenza A H1N1 viruses are closely related genetically to strains isolated in Subbarao K, et al. Characterization of an avian influenza A H5N1 virus isolated from a child with a fatal respiratory illness. Claas EC, et al. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus.

Smith GJ, et al. Emergence and predominance of an H5N1 influenza variant in China. Several reports indicate that highly pathogenic avian H5N1 viruses replicate mainly in the lower respiratory tract of humans 8 , 24 , and that the virus load correlates with the outcome of the infection 8.

Human H5N1 infections cause severe pneumonia and lymphopenia 24 - 26 , and are characterized by high levels of cytokines and chemokines 8 , 27 , 28 , a finding that was also confirmed in in vitro studies 29 , The induction of hypercytokinemia and hyperchemokinemia may thus be associated with the level of virus replication 8. Epidemiological data now indicate that an outbreak of influenza-like respiratory illness started in the Mexican town of La Gloria, Veracruz, in mid February of 31 Table 1.

By the end of April, international spread and clusters of human-to-human transmission prompted the WHO to elevate the pandemic alert from phase 3 to phase 4, and shortly after, to phase 5 human-to-human spread in at least two countries, and signs of an imminent pandemic.

In Mexico, substantial social distancing measures were implemented. Moreover, massive campaigns were undertaken to educate the public about precautionary hygiene measures. As of May 21, , 41 countries have reported 11, cases, including 85 deaths. Most cases outside Mexico and the US have been caused by travelers from Mexico. The majority of infections seem to be mild and do not require hospitalization Careful monitoring will be necessary during the following months i.

Timeline of swine-origin H1N1 virus outbreak see also Data on the genetic composition of the virus became available soon after viral isolation from the initial cases These viruses do not possess markers associated with high pathogenicity see the following sections on the role of viral proteins in pathogenicity for more details. In the late 's, reassortment between human H3N2, North American avian, and classical swine viruses resulted in triple reassortant H3N2 and H1N2 swine viruses that have since circulated in North American pig populations.

A triple reassortant swine virus reassorted with a Eurasian avian-like swine virus, resulting in the S-OIV that are now circulating in humans. Influenza virus pathogenicity is multigenic, and the determinants of pathogenicity may differ among animal species.

However, the HA protein plays an important role in expressing high pathogenicity in many animal species. It mediates the binding of the virus to host cells and the subsequent fusion of the viral and endosomal membranes for vRNP release into the cytoplasm. These functions assign a critical role to HA in the viral life cycle.

Influenza virus host specificity can be explained in part by the difference in receptor binding specificity for human and avian influenza viruses. The receptor-binding specificity of human and avian influenza viruses suggests that avian influenza viruses need to acquire the ability to recognize human-type receptors in order to cause a pandemic.

Indeed, the earliest isolates of the , , and pandemics possessed HA that, although of avian origin, recognized human-type receptors. However, studies revealed avian-type receptors on human epithelial cells that line the respiratory bronchiole and the alveolar walls, but human-type receptors on human epithelial cells in nasal mucosa, paranasal sinuses, pharynx, trachea, and bronchi 36 , Another study, however, showed the ex vivo infection of upper respiratory organs with an H5N1 avian virus Still, the finding of avian-type receptors in human lungs explains the severe pneumonia seen in humans with highly pathogenic avian H5N1 viruses.

The differences in receptor-binding specificity of human and avian viruses are determined by the amino acid residues in the HA receptor-binding pocket. Gln at position and Gly at position of H2 and H3 HAs confer binding to avian-type receptors, while Leu and Ser at these positions determine binding to human-type receptors. For H1 HAs, amino acids at position and H3 numbering determine receptor binding specificity. Two viruses that differ in receptor recognition were circulating during the pandemic: one recognizing only human-type receptors that transmits efficiently among ferrets, and one recognizing both avian- and human-type receptors that transmits inefficiently in this animal Interestingly, some S-OIV isolates possess an amino acid substitution at position or that has been found in H5N1 viruses isolated from humans and has been shown to affect receptor binding our unpublished data.

These mutations may thus reflect viral adaptation in humans, an assumption that needs to be tested. For H5N1 viruses, amino acid changes at positions , , , , and H3 numbering have been identified in human isolates and confer human-type receptor recognition 41 - Experimental changes at positions and Gln-to-Leu and Gly-to-Ser, respectively , but not at position Glu-to-Asp , resulted in the recognition of human-type receptors in addition to avian-type receptors 44 ; however, the respective amino acid changes at positions and have not been detected in human H5N1 virus isolates.

HA cleavability is determined by the amino acid sequence at the cleavage site. By contrast, highly pathogenic H5 and H7 viruses possess multiple basic amino acids at the HA cleavage site This motif is recognized by ubiquitous proteases, such as furin and PC6, and leads to systemic infections. For several outbreaks in poultry, increased pathogenicity of avian influenza viruses has been linked to the acquisition of multibasic HA cleavage sites, a finding that underscores the significance of the HA cleavage motif for virulence.

Recently, the viral replication complex has been recognized as an important contributor to viral pathogenicity, likely by affecting viral growth. The amino acid at position of the PB2 protein was first described by Subbarao et al. Hatta et al. Viruses with lysine at this position were pathogenic in mice, whereas those with glutamic acid were nonpathogenic in these animals Lysine at position of PB2 is now recognized as a determinant of viral pathogenicity in several mammalian species.

Several studies have addressed the mechanism by which PBLys affects virulence. The amino acid change does not affect tissue tropism in mice but viral replicative ability.

Collectively, these findings suggest that PBLys allows efficient replication not only in the lower, but also in the upper respiratory tract of mammals, a feature that may facilitate transmission. In fact, replacement of PBLys with Glu reduced the transmissibility of human influenza viruses in a guinea pig model The underlying mechanism and the amino acids in PB2 that are critical for this function remain to be determined.

In addition to PB2, other components of the replication complex may contribute to viral pathogenicity as well A recent study also suggested that the replication complex, particularly the PB1 protein, contributes to the virulence of the pandemic virus in ferrets Structural data are now becoming available for the viral polymerase complex which may help in the interpretation of mutational analyses; in fact, two studies 57 , 58 showed that PBLys is part of a basic groove that is disrupted upon replacement with Glu.

Recently obtained structural data are expected to help in the identification of domains that are critical for the biological functions of NS1. Innate immune responses are stimulated upon the recognition of a pathogen by a pathogen recognition receptor PRR. The direct contribution of NS1 to these signaling events is currently not known.

The NS1 proteins of H5N1 viruses confer resistance to the antiviral effects of IFN and are associated with high levels of proinflammatory cytokines 27 , 28 , 30 , 65 , 66 ; the resulting cytokine imbalance likely contributes to the high mortality of H5N1 virus infections in humans. Several amino acids in NS1 have now been shown to affect virulence 65 , 67 , However, available data suggest that these amino acid changes affect virulence in a strain-specific manner, while a multibasic HA cleavage sequence and PBLys seem to be universal determinants of viral pathogenicity.

The four C-terminal amino acids of NS1 form a PDZ ligand domain motif that was identified by large-scale sequence analysis Introduction of the PDZ ligand domains of highly pathogenic H5N1 viruses or the pandemic virus into an otherwise human virus conferred slightly increased virulence in mice This increase in virulence was not paralleled by increased IFN production. The biological significance of this finding is currently unknown.

The length of PB1-F2 of swine influenza viruses differs depending on their origin; classical swine viruses possess truncated PB1-F2 proteins of amino acids, while Eurasian avian-like swine viruses possess full length PB1-F2 proteins amino acids. PB1-F2 induces apoptosis, likely by interaction with two mitochondrial proteins 71 , 72 , enhances inflammation in mice, and increases the frequency and severity of secondary bacterial infections It may also affect virulence by interacting with the PB1 protein to retain it in the nucleus for efficient viral replication A recent study demonstrated that the amino acid at position 66 of PB1-F2 affects the pathogenicity of an H5N1 virus in mice Wild waterfowl are the natural reservoir of influenza A viruses.

Besides the ongoing transmission of highly pathogenic avian H5N1 influenza viruses to humans, avian influenza A viruses were transmitted to pigs in Europe in , to horses in China, and to seals. Moreover, an avian influenza virus reassorted with a human virus in pigs and transmitted from there to humans. Human infections with avian or swine influenza viruses have been reported but have typically been self-limiting. It is in this context that the recently emerged H1N1 viruses, which are of swine origin and transmit among humans, raise great concern over an imminent pandemic.

In North American pig populations, classical swine H1N1 virus dominated for nearly six decades. For the prevention and control of influenza virus infections, both vaccines and antiviral drugs are available.

Nonetheless, the global community is likely not well prepared for a pandemic: antiviral drugs may not be in sufficient supply and the virus may acquire resistance to the available antiviral drugs.

On the other hand, the production of a vaccine to a newly emerging strain would take months — during which time a virus could spread globally and substantially strain health care systems and the global economy. Two classes of antiviral drugs — ion channel inhibitors and neuraminidase inhibitors — are currently licensed for use against influenza A viruses.

Adamantanes i. The S-OIV are also resistant to ion channel inhibitors Two neuraminidase inhibitors — oseltamivir and zanamivir — are currently licensed. Neuraminidase inhibitors interfere with the enzymatic activity of the NA protein, which is critical for the efficient release of newly synthesized viruses from infected cells. In clinical trials, the emergence of resistance to NA inhibitors was rare, and oseltamivir-resistant influenza viruses were attenuated in vitro and in vivo.

Hence, the dissemination of these viruses was not considered a major issue, despite the frequent use of the drug in some countries. Recently, the rate of oseltamivir-resistant H1N1 influenza viruses in the US has increased from 0. Similar numbers have been reported for other countries.

Equally alarming, oseltamivir-resistant H5N1 viruses have been reported 77 , Oseltamivir-resistant human H1N1 viruses may have emerged in immunocompromised patients where prolonged replication 79 - 81 may have resulted in the selection of mutations that increase the fitness of oseltamivir-resistant viruses.

The S-OIV are sensitive to neuraminidase inhibitors when tested in vitro in enzymatic assays Recent structural data provide an explanation for oseltamivir-resistance 82 and suggest strategies for the design of improved compounds. In clinical settings, resistance to zanamivir has been reported only once for an influenza B virus isolated from an immunocompromised child Several experimental antiviral drugs that target the NA or polymerase proteins are currently in different stages of development.

Peramivir, an NA inhibitor that was developed through structure-based design 84 , is active in vitro tests against viruses of all 9 NA subtypes including highly pathogenic H5N1 viruses. Phase II clinical trials are currently underway to assess the efficacy of intramuscularly administered peramivir against seasonal influenza. CS, a pro-drug of the novel NA inhibitor R 85 , is a long-acting neuraminidase inhibitor that was found to be effective in phase II clinical trials against seasonal influenza.

We also found that CS has prophylactic and therapeutic efficacy against highly pathogenic H5N1 influenza viruses unpublished. T acts as a nucleoside analogue that interferes with the polymerase activity of influenza A, B, and C viruses, but also other RNA viruses It protected mice against infection with highly pathogenic H5N1 viruses our unpublished findings.

Phase II clinical trials for use of T against seasonal influenza viruses have been completed in Japan, and phase III clinical trials are scheduled. In addition, monoclonal antibodies to the HA are being developed for treatment of influenza virus infections.

In mice, some antibodies demonstrated prophylactic and therapeutic efficacy against lethal challenge with H5N1 virus 87 , suggesting monoclonal antibody treatment as an alternative strategy to treat influenza virus infections. Seasonal influenza vaccines include human influenza A viruses of the H1N1 and H3N2 subtypes, and an influenza B virus. These vaccines need to be updated every years to account for mutations in the HA and NA proteins of circulating viruses antigen drift.

Inactivated vaccines have been used for many decades. The allantoic fluid of embryonated, virus-infected chicken eggs is purified and concentrated by zonal centrifugation or column chromatography and inactivated with formalin or beta-propiolactone. Treatment with detergents or ether and removal of vRNP complexes leads to split or subunit vaccines that are administered intramuscularly or subcutaneously.

The development of improved influenza virus vaccines is thus clearly warranted. A live attenuated influenza virus vaccine is now licensed in the US for healthy individuals age 2-to These viruses are then reassorted with currently circulating wild-type strains to generate seed viruses that possess the HA and NA genes of the circulating wild-type viruses in the background of the ts , ca , and att phenotypes.

Live attenuated vaccines elicit both humoral and cellular immune responses, and are therefore believed to be superior to inactivated vaccines. In fact, in infants and young children, live attenuated influenza vaccine provides better protection than inactivated vaccine Currently, only egg-based vaccines are licensed for use in the US. In case of a pandemic, however, eggs may be in short supply. By contrast, cell cultures are highly controllable systems that can be scaled-by up for the mass production of vaccines, including those to highly pathogenic H5N1 viruses.

The purity and immunogenicity of influenza vaccines produced in Madin-Darby canine kidney MDCK or African green monkey kidney Vero cells match those of vaccines produced in embryonated chicken eggs. Cell culture-based influenza vaccines have been approved for use in humans in Europe. Recently, particular emphasis has been given to the development of vaccines to highly pathogenic H5N1 viruses.

These viruses kill chicken embryos. Propagation of these viruses in eggs therefore results in low yields. To modify these viruses for efficient growth in embryonated chicken eggs and safe handling by production staff, reverse genetics technologies 5 were used to replace the multibasic HA cleavage site with an avirulent-type HA cleavage site 45 , a known virulence factor.

Clinical testing of an H5N1 vaccine candidate suggested low immunogenicity 89 , prompting the addition of adjuvants to vaccine candidates.

In fact, aluminum hydroxide 90 , 91 or oil-in-water emulsions such as MF59 92 , 93 or AS03 94 resulted in significant antigen-sparing effects. Adjuvanted vaccines also seem to induce broader immune responses, which may be a critical advantage with the emergence of new clades and subclades of H5N1 viruses.

Similarly, the NA and modified HA genes of highly pathogenic avian H5N1 viruses were combined with the remaining genes derived from the live attenuated influenza A virus However, once H5N1 viruses are widespread in human populations, use of a live attenuated H5N1 vaccine, which is efficacious in nonhuman primates 96 , may be considered to overcome the low immunogenicity of inactivated vaccines.

Several novel vaccine approaches are now in various stages of development. Various virus-like particles VLPs expressing the HA and NA proteins, some in combination with the M1 and M2 proteins, have been tested for their antigenicity and protective efficacy. Also, vector approaches have been pursued. In one example, replication-incompetent adenoviruses expressing an H5 HA protected mice against challenge with homologous and heterologous H5N1 viruses 99 , In alternative approaches, Newcastle disease and fowlpox viruses have been explored as vector systems; however, these systems have not been approved for use in humans.

Although much has been learned about influenza viruses, key questions still remain unanswered: what factors determine interspecies transmission, reassortment, and human-to-human transmission — factors that have accounted for past pandemics and will be critical in the emergence of new pandemic viruses. Thus, careful monitoring of the S-OIV during the upcoming winter season in the southern hemisphere is of critical importance to detect more virulent variants, should they arise.

In addition, large-scale sequencing efforts, bioinformatics analyses, and the ability to experimentally test predictions with recombinant viruses will eventually provide insight into key features for the emergence of pandemic viruses.

In the meantime, the development of improved and novel antiviral drugs and vaccines will be critical to control influenza virus outbreaks. We apologize to our colleagues whose critical contributions to influenza virus research could not be cited due to the number of references permitted. We thank Krisna Wells for editing the manuscript. We also thank Makoto Ozawa and others in our laboratories who contributed to the data cited in this review. Read article at publisher's site DOI : PLoS Pathog , 17 12 :e, 06 Dec Amitai A.

Acta Pharmacol Sin , 06 Dec Emerg Infect Dis , 27 12 , 01 Dec To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. Schnitzler SU , Schnitzler P. Virus Genes , 39 3 , 07 Oct J Virol , 86 18 , 27 Jun Free to read. Influenza Other Respir Viruses , 4 1 , 01 Jan Annu Rev Microbiol , , 01 Jan Cited by: 28 articles PMID: The H1N1 influenza virus lacks the properties associated with growth in eggs.

Prior to the discovery of the H1N1 influenza virus, this particular combination of gene segments from North American and Eurasian swine had never been detected before in a single influenza virus and this new virus is different from the influenza viruses that normally circulate in North American and Eurasian pigs. It is not known when reassortment occurred to create the H1N1 influenza virus. Testing of the virus suggests that this reassortment event may have occurred years prior to the first reports of H1N1 influenza infection in people.

Pigs can be infected by influenza viruses found in birds and other animals as well as people. Therefore, pigs represent a mixing vessel in which influenza viruses from different species can swap genes.

For example, in a setting where people and animals are in close contact, pigs can be infected by influenza viruses found in pigs, poultry or humans — sometimes at the same time. However, in the late s, a series of reassortment events occurred between influenza viruses found in pigs, humans and birds. As a result, swine influenza viruses with genes from humans, North American pigs and birds have existed in many parts of the world for around 10 years prior to H1N1 flu.

This is very unlikely. Each of the gene segments within the H1N1 influenza virus have been found in pigs for more than 10 years prior to the beginning of the H1N1 influenza outbreak.

In addition, a Nature study showed that reassortment between influenza viruses found in North American and Eurasia pigs had already occurred at least once naturally in the 5 years prior to the identification of H1N1 flu. Also, the H1N1 influenza virus does not have adaptations consistent with viruses grown in laboratories.

For more information, see reasons 1, 2 and 3 above. We know that reassortment occurs frequently in nature. Fortunately, reassortment rarely results in a virus with pandemic potential, though it has done so at least twice in the 20th century.

The influenza viruses that caused the and pandemics contained a mixture of gene segments from human and avian influenza viruses. What is clear from genetic analysis of the viruses that caused these past pandemics is that reassortment gene swapping occurred to produce novel influenza viruses that caused the pandemics.