Cellular Immune Recognition of Influenza A Viruses in Equines: in vitro and in vivo Studies on the Immunogenicity of Equine Influenza Viruses

Influenza viruses and respiratory syncytial virus (RSV) are respiratory viruses that primarily circulate worldwide during the autumn and winter seasons. Seasonal surveillance has shown that RSV infection generally precedes influenza. However, in the last four winter seasons (2016–2020) an overlap of the morbidity peaks of both viruses was observed in Israel, and was paralleled by significantly lower RSV infection rates. To investigate whether the influenza A virus inhibits RSV, human cervical carcinoma (HEp2) cells or mice were co-infected with influenza A and RSV. Influenza A inhibited RSV growth, both in vitro and in vivo. Mass spectrometry analysis of mouse lungs infected with influenza A identified a two-wave pattern of protein expression upregulation, which included members of the interferon-induced protein with the tetratricopeptide (IFITs) family. Interestingly, in the second wave, influenza A viruses were no longer detectable in mouse lungs. In addition, knockdown and overexpression of IFITs in HEp2 cells affected RSV multiplicity. In conclusion, influenza A infection inhibits RSV infectivity via upregulation of IFIT proteins in a two-wave modality. Understanding the immune system involvement in the interaction between influenza A and RSV viruses will contribute to the development of future treatment strategies against these viruses.

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Attenuation and immunogenicity in mice of temperature-sensitive influenza viruses expressing truncated NS1 proteins

Journal of General Virology ◽

10.1099/vir.0.80991-0 ◽

2005 ◽

Vol 86 (10) ◽

pp. 2817-2821 ◽

Cited By ~ 36

Author(s):

Ana M. Falcón ◽

Ana Fernandez-Sesma ◽

Yurie Nakaya ◽

Thomas M. Moran ◽

Juan Ortín ◽

...

Keyword(s):

Influenza A ◽

Influenza Viruses ◽

Wild Type Virus ◽

Temperature Sensitive ◽

Ns1 Protein ◽

Wild Type ◽

Influenza A Viruses ◽

Lethal Challenge

It was previously shown that two mutant influenza A viruses expressing C-terminally truncated forms of the NS1 protein (NS1-81 and NS1-110) were temperature sensitive in vitro. These viruses contain HA, NA and M genes derived from influenza A/WSN/33 H1N1 virus (mouse-adapted), and the remaining five genes from human influenza A/Victoria/3/75 virus. Mice intranasally infected with the NS1 mutant viruses showed undetectable levels of virus in lungs at day 3, whereas those infected with the NS1 wild-type control virus still had detectable levels of virus at this time. Nevertheless, the temperature-sensitive mutant viruses induced specific cellular and humoral immune responses similar to those induced by the wild-type virus. Mice immunized with the NS1 mutant viruses were protected against a lethal challenge with influenza A/WSN/33 virus. These results indicate that truncations in the NS1 protein resulting in temperature-sensitive phenotypes in vitro correlate with attenuation in vivo without compromising viral immunogenicity, an ideal characteristic for live attenuated viral vaccines.

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Influenza A and B viruses with reduced baloxavir susceptibility display attenuated in vitro fitness but retain ferret transmissibility

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1916825117 ◽

2020 ◽

Vol 117 (15) ◽

pp. 8593-8601 ◽

Cited By ~ 8

Author(s):

Jeremy C. Jones ◽

Philippe Noriel Q. Pascua ◽

Thomas P. Fabrizio ◽

Bindumadhav M. Marathe ◽

Patrick Seiler ◽

...

Keyword(s):

Influenza A ◽

Clinical Trial Data ◽

Drug Susceptibility ◽

Influenza Viruses ◽

Endonuclease Activity ◽

Influenza A Viruses ◽

Complex Activity ◽

Minimal Impact

Baloxavir marboxil (BXM) was approved in 2018 for treating influenza A and B virus infections. It is a first-in-class inhibitor targeting the endonuclease activity of the virus polymerase acidic (PA) protein. Clinical trial data revealed that PA amino acid substitutions at residue 38 (I38T/F/M) reduced BXM potency and caused virus rebound in treated patients, although the fitness characteristics of the mutant viruses were not fully defined. To determine the fitness impact of the I38T/F/M substitutions, we generated recombinant A/California/04/2009 (H1N1)pdm09, A/Texas/71/2017 (H3N2), and B/Brisbane/60/2008 viruses with I38T/F/M and examined drug susceptibility in vitro, enzymatic properties, replication efficiency, and transmissibility in ferrets. Influenza viruses with I38T/F/M substitutions exhibited reduced baloxavir susceptibility, with 38T causing the greatest reduction. The I38T/F/M substitutions impaired PA endonuclease activity as compared to that of wild-type (I38-WT) PA. However, only 38T/F A(H3N2) substitutions had a negative effect on polymerase complex activity. The 38T/F substitutions decreased replication in cells among all viruses, whereas 38M had minimal impact. Despite variable fitness consequences in vitro, all 38T/M viruses disseminated to naive ferrets by contact and airborne transmission, while 38F-containing A(H3N2) and B viruses failed to transmit via the airborne route. Reversion of 38T/F/M to I38-WT was rare among influenza A viruses in this study, suggesting stable retention of 38T/F/M genotypes during these transmission events. BXM reduced susceptibility-associated mutations had variable effects on in vitro fitness of influenza A and B viruses, but the ability of these viruses to transmit in vivo indicates a risk of their spreading from BXM-treated individuals.

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Multi-spectral fluorescent reporter influenza viruses (Color-flu) as powerful tools for in vivo studies

Nature Communications ◽

10.1038/ncomms7600 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 65

Author(s):

Satoshi Fukuyama ◽

Hiroaki Katsura ◽

Dongming Zhao ◽

Makoto Ozawa ◽

Tomomi Ando ◽

...

Keyword(s):

Influenza A ◽

Fluorescent Proteins ◽

Virus Antigen ◽

Influenza Viruses ◽

In Vivo Studies ◽

Live Cells ◽

Influenza A Viruses ◽

Fluorescent Reporter ◽

Viral Spread

Abstract Seasonal influenza A viruses cause annual epidemics of respiratory disease; highly pathogenic avian H5N1 and the recently emerged H7N9 viruses cause severe infections in humans, often with fatal outcomes. Although numerous studies have addressed the pathogenicity of influenza viruses, influenza pathogenesis remains incompletely understood. Here we generate influenza viruses expressing fluorescent proteins of different colours (‘Color-flu’ viruses) to facilitate the study of viral infection in in vivo models. On adaptation to mice, stable expression of the fluorescent proteins in infected animals allows their detection by different types of microscopy and by flow cytometry. We use this system to analyse the progression of viral spread in mouse lungs, for live imaging of virus-infected cells, and for differential gene expression studies in virus antigen-positive and virus antigen-negative live cells in the lungs of Color-flu-infected mice. Collectively, Color-flu viruses are powerful tools to analyse virus infections at the cellular level in vivo to better understand influenza pathogenesis.

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Inhibition of Influenza Virus Infection by a Novel Antiviral Peptide That Targets Viral Attachment to Cells

Journal of Virology ◽

10.1128/jvi.01678-06 ◽

2006 ◽

Vol 80 (24) ◽

pp. 11960-11967 ◽

Cited By ~ 98

Author(s):

Jeremy C. Jones ◽

Elizabeth A. Turpin ◽

Hermann Bultmann ◽

Curtis R. Brandt ◽

Stacey Schultz-Cherry

Keyword(s):

Influenza A ◽

Signal Sequence ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Influenza A Viruses ◽

Viral Attachment ◽

H5n1 Influenza ◽

Eb Peptide

ABSTRACT Influenza A viruses continue to cause widespread morbidity and mortality. There is an added concern that the highly pathogenic H5N1 influenza A viruses, currently found throughout many parts of the world, represent a serious public health threat and may result in a pandemic. Intervention strategies to halt an influenza epidemic or pandemic are a high priority, with an emphasis on vaccines and antiviral drugs. In these studies, we demonstrate that a 20-amino-acid peptide (EB, for entry blocker) derived from the signal sequence of fibroblast growth factor 4 exhibits broad-spectrum antiviral activity against influenza viruses including the H5N1 subtype in vitro. The EB peptide was protective in vivo, even when administered postinfection. Mechanistically, the EB peptide inhibits the attachment to the cellular receptor, preventing infection. Further studies demonstrated that the EB peptide specifically binds to the viral hemagglutinin protein. This novel peptide has potential value as a reagent to study virus attachment and as a future therapeutic.

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PRESENCE OF RESPIRATORY VIRUSES IN EQUINES IN BRAZIL

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652014000300002 ◽

2014 ◽

Vol 56 (3) ◽

pp. 191-195

Author(s):

Dalva Assunção Portari Mancini ◽

Aparecida Santo Pietro Pereira ◽

Rita Maria Zucatelli Mendonça ◽

Adelia Hiroko Nagamori Kawamoto ◽

Rosely Cabette Barbosa Alves ◽

...

Keyword(s):

Influenza A ◽

Respiratory Viruses ◽

Influenza Viruses ◽

Influenza A Viruses ◽

Equine Influenza ◽

Hemagglutination Inhibition Test ◽

Parainfluenza Viruses ◽

Significant Difference ◽

Antibody Titers ◽

The Difference

Equines are susceptible to respiratory viruses such as influenza and parainfluenza. Respiratory diseases have adversely impacted economies all over the world. This study was intended to determine the presence of influenza and parainfluenza viruses in unvaccinated horses from some regions of the state of São Paulo, Brazil. Blood serum collected from 72 equines of different towns in this state was tested by hemagglutination inhibition test to detect antibodies for both viruses using the corresponding antigens. About 98.6% (71) and 97.2% (70) of the equines responded with antibody protective titers (≥ 80 HIU/25µL) H7N7 and H3N8 subtypes of influenza A viruses, respectively. All horses (72) also responded with protective titers (≥ 80) HIU/25µL against the parainfluenza virus. The difference between mean antibody titers to H7N7 and H3N8 subtypes of influenza A viruses was not statistically significant (p > 0.05). The mean titers for influenza and parainfluenza viruses, on the other hand, showed a statistically significant difference (p < 0.001). These results indicate a better antibody response from equines to parainfluenza 3 virus than to the equine influenza viruses. No statistically significant differences in the responses against H7N7 and H3N8 subtypes of influenza A and parainfluenza 3 viruses were observed according to the gender (female, male) or the age (≤ 2 to 20 years-old) groups. This study provides evidence of the concomitant presence of two subtypes of the equine influenza A (H7N7 and H3N8) viruses and the parainfluenza 3 virus in equines in Brazil. Thus, it is advisable to vaccinate equines against these respiratory viruses.

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Viruses harness YxxØ motif to interact with host AP2M1 for replication: A vulnerable broad-spectrum antiviral target

Science Advances ◽

10.1126/sciadv.aba7910 ◽

2020 ◽

Vol 6 (35) ◽

pp. eaba7910

Author(s):

Shuofeng Yuan ◽

Hin Chu ◽

Jingjing Huang ◽

Xiaoyu Zhao ◽

Zi-Wei Ye ◽

...

Keyword(s):

Broad Spectrum ◽

Influenza A ◽

Host Protein ◽

Influenza A Viruses ◽

Protein Protein Interaction ◽

Enterovirus A71 ◽

Evolutionarily Conserved ◽

Immunodeficiency Virus

Targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current COVID-19. Here, we found a common YxxØ-motif of multiple viruses that exploits host AP2M1 for intracellular trafficking. A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza A viruses (IAVs), Zika virus (ZIKV), human immunodeficiency virus, and coronaviruses including MERS-CoV and SARS-CoV-2. YxxØ mutation, AP2M1 depletion, or disruption by ACA causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of IAV nucleoprotein and diminished endoplasmic reticulum localization of ZIKV-NS3 and enterovirus-A71-2C proteins, thereby suppressing viral replication. Our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target.

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Genetically and Antigenically Divergent Influenza A(H9N2) Viruses Exhibit Differential Replication and Transmission Phenotypes in Mammalian Models

Journal of Virology ◽

10.1128/jvi.00451-20 ◽

2020 ◽

Vol 94 (17) ◽

Author(s):

Jessica A. Belser ◽

Xiangjie Sun ◽

Nicole Brock ◽

Claudia Pappas ◽

Joanna A. Pulit-Penaloza ◽

...

Keyword(s):

Influenza A ◽

Human Infection ◽

Influenza Viruses ◽

Human Populations ◽

Human Respiratory Tract ◽

Live Bird Markets ◽

Human Infections ◽

Live Bird

ABSTRACT Low-pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Cocirculation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses. IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world and for over 20 years have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported, and it was found to exhibit properties of adaptation to humans and, importantly, it shows similarities to strains isolated from the live bird markets of Vietnam.

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The influenza A virus nucleoprotein gene controls the induction of both subtype specific and cross-reactive cytotoxic T cells.

Journal of Experimental Medicine ◽

10.1084/jem.160.2.552 ◽

1984 ◽

Vol 160 (2) ◽

pp. 552-563 ◽

Cited By ~ 119

Author(s):

A R Townsend ◽

J J Skehel

Keyword(s):

T Cells ◽

Influenza A ◽

Cytotoxic T Cells ◽

Target Cells ◽

Spleen Cells ◽

Influenza A Viruses ◽

Group A ◽

Selection Of

Using genetically typed recombinant influenza A viruses that differ only in their genes for nucleoprotein, we have demonstrated that repeated stimulation in vitro of C57BL/6 spleen cells primed in vivo with E61-13-H17 (H3N2) virus results in the selection of a population of cytotoxic T lymphocytes (CTL) whose recognition of infected target cells maps to the gene for nucleoprotein of the 1968 virus. Influenza A viruses isolated between 1934 and 1979 fall into two groups defined by their ability to sensitize target cells for lysis by these CTL: 1934-1943 form one group (A/PR/8/34 related) and 1946-1979 form the second group (A/HK/8/68 related). These findings complement and extend our previous results with an isolated CTL clone with specificity for the 1934 nucleoprotein (27, 28). It is also shown that the same spleen cells derived from mice primed with E61-13-H17 virus in vivo, but maintained in identical conditions by stimulation with X31 virus (which differs from the former only in the origin of its gene for NP) in vitro, results in the selection of CTL that cross-react on target cells infected with A/PR/8/1934 (H1N1) or A/Aichi/1968 (H3N2). These results show that the influenza A virus gene for NP can play a role in selecting CTL with different specificities and implicate the NP molecule as a candidate for a target structure recognized by both subtype-directed and cross-reactive influenza A-specific cytotoxic T cells.

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Use of influenza A viruses expressing reporter genes to assess the frequency of double infections in vitro

Journal of General Virology ◽

10.1099/vir.0.042671-0 ◽

2012 ◽

Vol 93 (8) ◽

pp. 1645-1648 ◽

Cited By ~ 9

Author(s):

R. Bodewes ◽

N. J. Nieuwkoop ◽

R. J. Verburgh ◽

R. A. M. Fouchier ◽

A. D. M. E. Osterhaus ◽

...

Keyword(s):

Influenza A ◽

Single Cells ◽

Reporter Genes ◽

Influenza Viruses ◽

Influenza A Viruses ◽

Double Infection ◽

Madin Darby Canine Kidney ◽

Darby Canine Kidney ◽

Canine Kidney

Exchange of gene segments between mammalian and avian influenza A viruses may lead to the emergence of potential pandemic influenza viruses. Since co-infection of single cells with two viruses is a prerequisite for reassortment to take place, we assessed frequencies of double-infection in vitro using influenza A/H5N1 and A/H1N1 viruses expressing the reporter genes eGFP or mCherry. Double-infected A549 and Madin–Darby canine kidney cells were detected by confocal microscopy and flow cytometry.

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