Positive Selection in CD8+T-Cell Epitopes of Influenza Virus Nucleoprotein Revealed by a Comparative Analysis of Human and Swine Viral Lineages

ABSTRACTNumerous experimental studies have demonstrated that CD8+T cells contribute to immunity against influenza by limiting viral replication. It is therefore surprising that rigorous statistical tests have failed to find evidence of positive selection in the epitopes targeted by CD8+T cells. Here we use a novel computational approach to test for selection in CD8+T-cell epitopes. We define all epitopes in the nucleoprotein (NP) and matrix protein (M1) with experimentally identified human CD8+T-cell responses and then compare the evolution of these epitopes in parallel lineages of human and swine influenza viruses that have been diverging since roughly 1918. We find a significant enrichment of substitutions that alter human CD8+T-cell epitopes in NP of human versus swine influenza virus, consistent with the idea that these epitopes are under positive selection. Furthermore, we show that epitope-altering substitutions in human influenza virus NP are enriched on the trunk versus the branches of the phylogenetic tree, indicating that viruses that acquire these mutations have a selective advantage. However, even in human influenza virus NP, sites in T-cell epitopes evolve more slowly than do nonepitope sites, presumably because these epitopes are under stronger inherent functional constraint. Overall, our work demonstrates that there is clear selection from CD8+T cells in human influenza virus NP and illustrates how comparative analyses of viral lineages from different hosts can identify positive selection that is otherwise obscured by strong functional constraint.IMPORTANCEThere is a strong interest in correlates of anti-influenza immunity that are protective against diverse virus strains. CD8+T cells provide such broad immunity, since they target conserved viral proteins. An important question is whether T-cell immunity is sufficiently strong to drive influenza virus evolution. Although many studies have shown that T cells limit viral replication in animal models and are associated with decreased symptoms in humans, no studies have proven with statistical significance that influenza virus evolves under positive selection to escape T cells. Here we use comparisons of human and swine influenza viruses to rigorously demonstrate that human influenza virus evolves under pressure to fix mutations in the nucleoprotein that promote escape from T cells. We further show that viruses with these mutations have a selective advantage since they are preferentially located on the “trunk” of the phylogenetic tree. Overall, our results show that CD8+T cells targeting nucleoprotein play an important role in shaping influenza virus evolution.

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Immunogenicity of a Recombinant Influenza Virus Bearing Both the CD4+ and CD8+ T Cell Epitopes of Ovalbumin

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/497364 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Bruno Garulli ◽

Giuseppina Di Mario ◽

Ester Sciaraffia ◽

Yoshihiro Kawaoka ◽

Maria R. Castrucci

Keyword(s):

T Cells ◽

Influenza Virus ◽

T Cell ◽

Cell Epitope ◽

Cd8 T Cell ◽

Influenza Viruses ◽

T Cell Epitope ◽

T Cell Epitopes ◽

Additional Tool

Recombinant influenza viruses that bear the single immunodominant CD8+ T cell epitopeOVA257−264or the CD4+ T cell epitopeOVA323−339of the model antigen ovalbumin (OVA) have been useful tools in immunology. Here, we generated a recombinant influenza virus,WSN-OVAI/II, that bears both OVA-specific CD8+ and CD4+ epitopes on its hemagglutinin molecule. Live and heat-inactivatedWSN-OVAI/IIviruses were efficiently presented by dendritic cellsin vitroto OT-I TCR transgenic CD8+ T cells and OT-II TCR transgenic CD4+ T cells.In vivo,WSN-OVAI/IIvirus was attenuated in virulence, highly immunogenic, and protected mice from B16-OVA tumor challenge in a prophylactic model of vaccination. Thus,WSN-OVAI/IIvirus represents an additional tool, along with OVA TCR transgenic mice, for further studies on T cell responses and may be of value in vaccine design.

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IMMUNOLOGICAL RELATIONSHIP BETWEEN THE SWINE AND HUMAN INFLUENZA VIRUSES IN SWINE

Journal of Experimental Medicine ◽

10.1084/jem.66.2.151 ◽

1937 ◽

Vol 66 (2) ◽

pp. 151-168 ◽

Cited By ~ 11

Author(s):

Richard E. Shope

Keyword(s):

Influenza Virus ◽

Influenza Infection ◽

Swine Influenza Virus ◽

Swine Influenza ◽

Influenza Viruses ◽

Human Influenza ◽

Human Influenza Virus ◽

Immunological Relationship ◽

Cross Immunity ◽

The Cross

Swine recovered from infection with either swine influenza or swine influenza virus alone are usually not only immune but refractory to human influenza infection. Swine recovered from infection with a mixture of human influenza virus and H. influenzae suis are usually immune to swine influenza while those recovered from infection with human influenza virus alone are usually not immune to swine influenza. The possible mechanisms involved in the cross-immunity between the influenza viruses are discussed.

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NEUTRALIZATION TESTS WITH SERA OF CONVALESCENT OR IMMUNIZED ANIMALS AND THE VIRUSES OF SWINE AND HUMAN INFLUENZA

Journal of Experimental Medicine ◽

10.1084/jem.63.5.645 ◽

1936 ◽

Vol 63 (5) ◽

pp. 645-653 ◽

Cited By ~ 25

Author(s):

Thomas Francis ◽

Richard E. Shope

Keyword(s):

Influenza Virus ◽

Swine Influenza Virus ◽

Swine Influenza ◽

Influenza Viruses ◽

Human Influenza ◽

Human Influenza Virus ◽

Neutralizing Capacity ◽

Heterologous Virus

Human and swine influenza viruses were regularly neutralized by their homologous immune sera. However, the sera of animals convalescent from infection with either the swine or human influenza virus possessed little, if any, neutralizing capacity for the heterologous virus. Hyperimmunization of animals against swine influenza virus tended to increase the neutralizing capacity of their sera for human influenza virus, but in an inconstant fashion, whereas repeated inoculations with human influenza virus frequently resulted in sera with strong neutralizing activities against swine influenza virus. These observations serve to emphasize both the immunological distinctiveness and the interrelationships of swine and human influenza viruses.

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THE INFECTION OF MICE WITH SWINE INFLUENZA VIRUS

Journal of Experimental Medicine ◽

10.1084/jem.62.4.561 ◽

1935 ◽

Vol 62 (4) ◽

pp. 561-572 ◽

Cited By ~ 23

Author(s):

Richard E. Shope

Keyword(s):

Influenza Virus ◽

Virus Disease ◽

Swine Influenza Virus ◽

Swine Influenza ◽

Serial Passage ◽

Virus Infections ◽

Human Influenza ◽

Human Influenza Virus ◽

The Stability ◽

At Will

The experiments confirm the earlier observation of Andrewes, Laidlaw and Smith that the swine influenza virus is pathogenic for white mice when administered intranasally. Two field strains of the swine influenza virus were found to differ in their initial pathogenicity for mice. One strain was apparently fully pathogenic even in its 1st mouse passage while the other required 2 or 3 mouse passages to acquire full virulence for this species. Both strains, however, were initially infectious for mice, without the necessity of intervening ferret passages. There is no evidence that bacteria play any significant rôle in the mouse disease though essential in that of swine, and fatal pneumonias can be produced in mice by pure virus infections. Mice surviving the virus disease are immune to reinfection for at least a month. In mice the disease is not contagious though it is notably so in swine. The virus, while regularly producing fatal pneumonias when administered intranasally to mice, appears to be completely innocuous when given subcutaneously or intraperitoneally. Prolonged serial passage of the virus in mice does not influence its infectivity or virulence for swine or ferrets. It is a stable virus so far as its infectivity is concerned, and can be transferred at will from any one of its three known susceptible hosts to any other. In discussing these facts the stability of the swine influenza virus has been contrasted with the apparent instability of freshly isolated strains of the human influenza virus. Though the mouse is an un-natural host for the virus it is, nevertheless, useful for the study of those aspects of swine influenza which have to do with the virus only.

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Swine Influenza Virus PA and Neuraminidase Gene Reassortment into Human H1N1 Influenza Virus Is Associated with an Altered Pathogenic Phenotype Linked to Increased MIP-2 Expression

Journal of Virology ◽

10.1128/jvi.00087-15 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5651-5667 ◽

Cited By ~ 6

Author(s):

Daniel Dlugolenski ◽

Les Jones ◽

Elizabeth Howerth ◽

David Wentworth ◽

S. Mark Tompkins ◽

...

Keyword(s):

Influenza Virus ◽

Sialic Acid ◽

Influenza A ◽

H1n1 Virus ◽

Swine Influenza ◽

Influenza Viruses ◽

Pandemic H1n1 ◽

Human Influenza ◽

Swine Virus ◽

Virus Reassortment

ABSTRACTSwine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Coinfection of swine cells with 2009 pandemic H1N1 virus (huH1N1) and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2) resulted in a 23% reassortment rate that was independent of α2,3- or α2,6-sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine virus PA and neuraminidase (NA) into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early postinfection, resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza virus reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic H1N1 virus with endemic swine viruses and for reassortants to have increased pathogenicity linked to the swine virus NA and PA genes which are associated with increased pulmonary neutrophil trafficking that is related to MIP-2 expression.IMPORTANCEInfluenza A viruses can change rapidly via reassortment to create a novel virus, and reassortment can result in possible pandemics. Reassortments among subtypes from avian and human viruses led to the 1957 (H2N2 subtype) and 1968 (H3N2 subtype) human influenza pandemics. Recent analyses of circulating isolates have shown that multiple genes can be recombined from human, avian, and swine influenza viruses, leading to triple reassortants. Understanding the factors that can affect influenza A virus reassortment is needed for the establishment of disease intervention strategies that may reduce or preclude pandemics. The findings from this study show that swine cells provide a mixing vessel for influenza virus reassortment independent of differential sialic acid distribution. The findings also establish that circulating neuraminidase (NA) and PA genes could alter the pathogenic phenotype of the pandemic H1N1 virus, resulting in enhanced disease. The identification of such factors provides a framework for pandemic modeling and surveillance.

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Divergent Human-Origin Influenza Viruses Detected in Australian Swine Populations

Journal of Virology ◽

10.1128/jvi.00316-18 ◽

2018 ◽

Vol 92 (16) ◽

Cited By ~ 6

Author(s):

Frank Y. K. Wong ◽

Celeste Donato ◽

Yi-Mo Deng ◽

Don Teng ◽

Naomi Komadina ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Swine Influenza ◽

Influenza Viruses ◽

Antigenic Drift ◽

Human Origin ◽

Human Influenza ◽

Influenza A Viruses ◽

Data Set ◽

Antigenic Properties

ABSTRACTGlobal swine populations infected with influenza A viruses pose a persistent pandemic risk. With the exception of a few countries, our understanding of the genetic diversity of swine influenza viruses is limited, hampering control measures and pandemic risk assessment. Here we report the genomic characteristics and evolutionary history of influenza A viruses isolated in Australia from 2012 to 2016 from two geographically isolated swine populations in the states of Queensland and Western Australia. Phylogenetic analysis with an expansive human and swine influenza virus data set comprising >40,000 sequences sampled globally revealed evidence of the pervasive introduction and long-term establishment of gene segments derived from several human influenza viruses of past seasons, including the H1N1/1977, H1N1/1995, H3N2/1968, and H3N2/2003, and the H1N1 2009 pandemic (H1N1pdm09) influenza A viruses, and a genotype that contained gene segments derived from the past three pandemics (1968, reemerged 1977, and 2009). Of the six human-derived gene lineages, only one, comprising two viruses isolated in Queensland during 2012, was closely related to swine viruses detected from other regions, indicating a previously undetected circulation of Australian swine lineages for approximately 3 to 44 years. Although the date of introduction of these lineages into Australian swine populations could not be accurately ascertained, we found evidence of sustained transmission of two lineages in swine from 2012 to 2016. The continued detection of human-origin influenza virus lineages in swine over several decades with little or unpredictable antigenic drift indicates that isolated swine populations can act as antigenic archives of human influenza viruses, raising the risk of reemergence in humans when sufficient susceptible populations arise.IMPORTANCEWe describe the evolutionary origins and antigenic properties of influenza A viruses isolated from two separate Australian swine populations from 2012 to 2016, showing that these viruses are distinct from each other and from those isolated from swine globally. Whole-genome sequencing of virus isolates revealed a high genotypic diversity that had been generated exclusively through the introduction and establishment of human influenza viruses that circulated in past seasons. We detected six reassortants with gene segments derived from human H1N1/H1N1pdm09 and various human H3N2 viruses that circulated during various periods since 1968. We also found that these swine viruses were not related to swine viruses collected elsewhere, indicating independent circulation. The detection of unique lineages and genotypes in Australia suggests that isolated swine populations that are sufficiently large can sustain influenza virus for extensive periods; we show direct evidence of a sustained transmission for at least 4 years between 2012 and 2016.

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Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses

Journal of Virology ◽

10.1128/jvi.01254-16 ◽

2016 ◽

Vol 90 (24) ◽

pp. 11157-11167 ◽

Cited By ~ 15

Author(s):

Xiangjie Sun ◽

Hui Zeng ◽

Amrita Kumar ◽

Jessica A. Belser ◽

Taronna R. Maines ◽

...

Keyword(s):

Endothelial Cells ◽

Influenza Virus ◽

Virus Infection ◽

Avian Influenza ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Human Influenza ◽

Avian Influenza Viruses ◽

Human Influenza Virus ◽

Pulmonary Endothelial Cells

ABSTRACTA role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans.IMPORTANCEAvian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses, we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factors IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza virus strains, whereas avian influenza viruses overcome these restriction factors in this cell type.

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THE INCIDENCE OF NEUTRALIZING ANTIBODIES FOR HUMAN INFLUENZA VIRUS IN THE SERUM OF HUMAN INDIVIDUALS OF DIFFERENT AGES

Journal of Experimental Medicine ◽

10.1084/jem.63.5.655 ◽

1936 ◽

Vol 63 (5) ◽

pp. 655-668 ◽

Cited By ~ 40

Author(s):

Thomas Francis ◽

T. P. Magill

Keyword(s):

Influenza Virus ◽

Neutralizing Antibodies ◽

Swine Influenza Virus ◽

Swine Influenza ◽

The Other ◽

Human Influenza ◽

Complete Protection ◽

Human Influenza Virus ◽

Human Sera ◽

Epidemiological Significance

The results of mouse protection tests with 136 human sera and a strain of human influenza virus are described. After the 1st year of life, the sera of approximately half the individuals tested contained sufficient antibody to furnish complete protection to mice. A much higher percentage of the sera obtained from individuals recently convalescent from influenza exerted a completely protective effect. On the other hand, certain sera protected only partially under the conditions of the tests. The results have been compared with those obtained by Shope in tests done with the same sera against swine influenza virus. The possible epidemiological significance of the results is discussed.

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