Kallikrein-Related Peptidase 5 Contributes to H3N2 Influenza Virus Infection in Human Lungs

ABSTRACT Hemagglutinin (HA) of influenza virus must be activated by proteolysis before the virus can become infectious. Previous studies indicated that HA cleavage is driven by membrane-bound or extracellular serine proteases in the respiratory tract. However, there is still uncertainty as to which proteases are critical for activating HAs of seasonal influenza A viruses (IAVs) in humans. This study focuses on human KLK1 and KLK5, 2 of the 15 serine proteases known as the kallikrein-related peptidases (KLKs). We find that their mRNA expression in primary human bronchial cells is stimulated by IAV infection. Both enzymes cleaved recombinant HA from several strains of the H1 and/or H3 virus subtype in vitro, but only KLK5 promoted the infectivity of A/Puerto Rico/8/34 (H1N1) and A/Scotland/20/74 (H3N2) virions in MDCK cells. We assessed the ability of treated viruses to initiate influenza in mice. The nasal instillation of only the KLK5-treated virus resulted in weight loss and lethal outcomes. The secretion of this protease in the human lower respiratory tract is enhanced during influenza. Moreover, we show that pretreatment of airway secretions with a KLK5-selective inhibitor significantly reduced the activation of influenza A/Scotland/20/74 virions, providing further evidence of its importance. Differently, increased KLK1 secretion appeared to be associated with the recruitment of inflammatory cells in human airways regardless of the origin of inflammation. Thus, our findings point to the involvement of KLK5 in the proteolytic activation and spread of seasonal influenza viruses in humans. IMPORTANCE Influenza A viruses (IAVs) cause acute infection of the respiratory tract that affects millions of people during seasonal outbreaks every year. Cleavage of the hemagglutinin precursor by host proteases is a critical step in the life cycle of these viruses. Consequently, host proteases that activate HA can be considered promising targets for the development of new antivirals. However, the specific proteases that activate seasonal influenza viruses, especially H3N2 viruses, in the human respiratory tract have remain undefined despite many years of work. Here we demonstrate that the secreted, extracellular protease KLK5 (kallikrein-related peptidase 5) is efficient in promoting the infectivity of H3N2 IAV in vitro and in vivo. Furthermore, we found that its secretion was selectively enhanced in the human lower respiratory tract during a seasonal outbreak dominated by an H3N2 virus. Collectively, our data support the clinical relevance of this protease in human influenza pathogenesis.

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Broadly Reactive H2 Hemagglutinin Vaccines Elicit Cross-Reactive Antibodies in Ferrets Preimmune to Seasonal Influenza A Viruses

mSphere ◽

10.1128/msphere.00052-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Z. Beau Reneer ◽

Amanda L. Skarlupka ◽

Parker J. Jamieson ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Recombinant Proteins ◽

Human Population ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Wild Type ◽

Influenza A Viruses ◽

Global Pandemic

ABSTRACT Influenza vaccines have traditionally been tested in naive mice and ferrets. However, humans are first exposed to influenza viruses within the first few years of their lives. Therefore, there is a pressing need to test influenza virus vaccines in animal models that have been previously exposed to influenza viruses before being vaccinated. In this study, previously described H2 computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) vaccines (Z1 and Z5) were tested in influenza virus “preimmune” ferret models. Ferrets were infected with historical, seasonal influenza viruses to establish preimmunity. These preimmune ferrets were then vaccinated with either COBRA H2 HA recombinant proteins or wild-type H2 HA recombinant proteins in a prime-boost regimen. A set of naive preimmune or nonpreimmune ferrets were also vaccinated to control for the effects of the multiple different preimmunities. All of the ferrets were then challenged with a swine H2N3 influenza virus. Ferrets with preexisting immune responses influenced recombinant H2 HA-elicited antibodies following vaccination, as measured by hemagglutination inhibition (HAI) and classical neutralization assays. Having both H3N2 and H1N1 immunological memory regardless of the order of exposure significantly decreased viral nasal wash titers and completely protected all ferrets from both morbidity and mortality, including the mock-vaccinated ferrets in the group. While the vast majority of the preimmune ferrets were protected from both morbidity and mortality across all of the different preimmunities, the Z1 COBRA HA-vaccinated ferrets had significantly higher antibody titers and recognized the highest number of H2 influenza viruses in a classical neutralization assay compared to the other H2 HA vaccines. IMPORTANCE H1N1 and H3N2 influenza viruses have cocirculated in the human population since 1977. Nearly every human alive today has antibodies and memory B and T cells against these two subtypes of influenza viruses. H2N2 influenza viruses caused the 1957 global pandemic and people born after 1968 have never been exposed to H2 influenza viruses. It is quite likely that a future H2 influenza virus could transmit within the human population and start a new global pandemic, since the majority of people alive today are immunologically naive to viruses of this subtype. Therefore, an effective vaccine for H2 influenza viruses should be tested in an animal model with previous exposure to influenza viruses that have circulated in humans. Ferrets were infected with historical influenza A viruses to more accurately mimic the immune responses in people who have preexisting immune responses to seasonal influenza viruses. In this study, preimmune ferrets were vaccinated with wild-type (WT) and COBRA H2 recombinant HA proteins in order to examine the effects that preexisting immunity to seasonal human influenza viruses have on the elicitation of broadly cross-reactive antibodies from heterologous vaccination.

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Pathogenesis and Transmission of Genetically Diverse Swine-Origin H3N2 Variant Influenza A Viruses from Multiple Lineages Isolated in the United States, 2011–2016

Journal of Virology ◽

10.1128/jvi.00665-18 ◽

2018 ◽

Vol 92 (16) ◽

Cited By ~ 7

Author(s):

Xiangjie Sun ◽

Joanna A. Pulit-Penaloza ◽

Jessica A. Belser ◽

Claudia Pappas ◽

Melissa B. Pearce ◽

...

Keyword(s):

United States ◽

Respiratory Tract ◽

Influenza A ◽

Seasonal Influenza ◽

The United States ◽

Influenza Viruses ◽

Upper Respiratory Tract ◽

Pandemic Preparedness

ABSTRACTWhile several swine-origin influenza A H3N2 variant (H3N2v) viruses isolated from humans prior to 2011 have been previously characterized for their virulence and transmissibility in ferrets, the recent genetic and antigenic divergence of H3N2v viruses warrants an updated assessment of their pandemic potential. Here, four contemporary H3N2v viruses isolated during 2011 to 2016 were evaluated for their replicative ability in bothin vitroandin vivoin mammalian models as well as their transmissibility among ferrets. We found that all four H3N2v viruses possessed similar or enhanced replication capacities in a human bronchial epithelium cell line (Calu-3) compared to a human seasonal influenza virus, suggestive of strong fitness in human respiratory tract cells. The majority of H3N2v viruses examined in our study were mildly virulent in mice and capable of replicating in mouse lungs with different degrees of efficiency. In ferrets, all four H3N2v viruses caused moderate morbidity and exhibited comparable titers in the upper respiratory tract, but only 2 of the 4 viruses replicated in the lower respiratory tract in this model. Furthermore, despite efficient transmission among cohoused ferrets, recently isolated H3N2v viruses displayed considerable variance in their ability to transmit by respiratory droplets. The lack of a full understanding of the molecular correlates of virulence and transmission underscores the need for close genotypic and phenotypic monitoring of H3N2v viruses and the importance of continued surveillance to improve pandemic preparedness.IMPORTANCESwine-origin influenza viruses of the H3N2 subtype, with the hemagglutinin (HA) and neuraminidase (NA) derived from historic human seasonal influenza viruses, continue to cross species barriers and cause human infections, posing an indelible threat to public health. To help us better understand the potential risk associated with swine-origin H3N2v viruses that emerged in the United States during the 2011-2016 influenza seasons, we use bothin vitroandin vivomodels to characterize the abilities of these viruses to replicate, cause disease, and transmit in mammalian hosts. The efficient respiratory droplet transmission exhibited by some of the H3N2v viruses in the ferret model combined with the existing evidence of low immunity against such viruses in young children and older adults highlight their pandemic potential. Extensive surveillance and risk assessment of H3N2v viruses should continue to be an essential component of our pandemic preparedness strategy.

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Efficient Inhibition of Avian and Seasonal Influenza A Viruses by a Virus-Specific Dicer-Substrate Small Interfering RNA Swarm in Human Monocyte-Derived Macrophages and Dendritic Cells

Journal of Virology ◽

10.1128/jvi.01916-18 ◽

2018 ◽

Vol 93 (4) ◽

Cited By ~ 2

Author(s):

Miao Jiang ◽

Pamela Österlund ◽

Veera Westenius ◽

Deyin Guo ◽

Minna M. Poranen ◽

...

Keyword(s):

Dendritic Cells ◽

Influenza Virus ◽

Small Interfering Rna ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Influenza A Viruses ◽

Primary Monocyte ◽

Prevention And Treatment ◽

Interfering Rna

ABSTRACTInfluenza A viruses (IAVs) are viral pathogens that cause epidemics and occasional pandemics of significant mortality. The generation of efficacious vaccines and antiviral drugs remains a challenge due to the rapid appearance of new influenza virus types and antigenic variants. Consequently, novel strategies for the prevention and treatment of IAV infections are needed, given the limitations of the presently available antivirals. Here, we used enzymatically produced IAV-specific double-stranded RNA (dsRNA) molecules andGiardia intestinalisDicer for the generation of a swarm of small interfering RNA (siRNA) molecules. The siRNAs target multiple conserved genomic regions of the IAVs. In mammalian cells, the produced 25- to 27-nucleotide-long siRNA molecules are processed by endogenous Dicer into 21-nucleotide siRNAs and are thus designated Dicer-substrate siRNAs (DsiRNAs). We evaluated the efficacy of the above DsiRNA swarm at preventing IAV infections in human primary monocyte-derived macrophages and dendritic cells. The replication of different IAV strains, including avian influenza H5N1 and H7N9 viruses, was significantly inhibited by pretransfection of the cells with the IAV-specific DsiRNA swarm. Up to 7 orders of magnitude inhibition of viral RNA expression was observed, which led to a dramatic inhibition of IAV protein synthesis and virus production. The IAV-specific DsiRNA swarm inhibited virus replication directly through the RNA interference pathway although a weak induction of innate interferon responses was detected. Our results provide direct evidence for the feasibility of the siRNA strategy and the potency of DsiRNA swarms in the prevention and treatment of influenza, including the highly pathogenic avian influenza viruses.IMPORTANCEIn spite of the enormous amount of research, influenza virus is still one of the major challenges for medical virology due to its capacity to generate new variants, which potentially lead to severe epidemics and pandemics. We demonstrated here that a swarm of small interfering RNA (siRNA) molecules, including more than 100 different antiviral RNA molecules targeting the most conserved regions of the influenza A virus genome, could efficiently inhibit the replication of all tested avian and seasonal influenza A variants in human primary monocyte-derived macrophages and dendritic cells. The wide antiviral spectrum makes the virus-specific siRNA swarm a potentially efficient treatment modality against both avian and seasonal influenza viruses.

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Community-Level Differences in the Microbiome of Healthy Wild Mallards and Those Infected by Influenza A Viruses

mSystems ◽

10.1128/msystems.00188-16 ◽

2017 ◽

Vol 2 (1) ◽

Cited By ~ 20

Author(s):

Holly H. Ganz ◽

Ladan Doroud ◽

Alana J. Firl ◽

Sarah M. Hird ◽

Jonathan A. Eisen ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Respiratory Tract ◽

Influenza A ◽

Seasonal Influenza ◽

Detailed Examination ◽

Influenza Viruses ◽

Morbidity And Mortality ◽

Significant Morbidity ◽

Influenza A Viruses ◽

Health And Disease

ABSTRACT Seasonal influenza causes 3 to 5 million severe illnesses and 250,000 to 500,000 human deaths each year. While pandemic influenza viruses emerge only periodically, they can be devastating—for example, the 1918 H1N1 pandemic virus killed more than 20 million people. IAVs infect the respiratory tract and cause significant morbidity and mortality in humans. In contrast, IAVs infect the gastrointestinal tract of waterfowl, producing little pathology. Recent studies indicated that viruses can alter the microbiome at the respiratory and gastrointestinal mucosa, but there are no reports of how the microbiota of the natural host of influenza is affected by infection. Here we find that the mallard microbiome is altered during IAV infection. Our results suggest that detailed examination of humans and animals infected with IAVs may reveal individualized microbiome profiles that correspond to health and disease. Moreover, future studies should explore whether the altered microbiome facilitates maintenance and transmission of IAVs in waterfowl populations. Waterfowl, especially ducks and geese, are primary reservoirs for influenza A viruses (IAVs) that evolve and emerge as important pathogens in domestic animals and humans. In contrast to humans, where IAVs infect the respiratory tract and cause significant morbidity and mortality, IAVs infect the gastrointestinal tract of waterfowl and cause little or no pathology and are spread by fecal-oral transmission. For this reason, we examined whether IAV infection is associated with differences in the cloacal microbiome of mallards (Anas platyrhyncos), an important host of IAVs in North America and Eurasia. We characterized bacterial community composition by sequencing the V4 region of 16S rRNA genes. IAV-positive mallards had lower species diversity, richness, and evenness than IAV-negative mallards. Operational taxonomic unit (OTU) cooccurrence patterns were also distinct depending on infection status. Network analysis showed that IAV-positive mallards had fewer significant cooccurring OTUs and exhibited fewer coassociation patterns among those OTUs than IAV-negative mallards. These results suggest that healthy mallards have a more robust and complex cloacal microbiome. By combining analytical approaches, we identified 41 bacterial OTUs, primarily representatives of Streptococcus spp., Veillonella dispar, and Rothia mucilaginosa, contributing to the observed differences. This study found that IAV-infected wild mallards exhibited strong differences in microbiome composition relative to noninfected mallards and identified a concise set of putative biomarker OTUs. Using Random Forest, a supervised machine learning method, we verified that these 41 bacterial OTUs are highly predictive of infection status. IMPORTANCE Seasonal influenza causes 3 to 5 million severe illnesses and 250,000 to 500,000 human deaths each year. While pandemic influenza viruses emerge only periodically, they can be devastating—for example, the 1918 H1N1 pandemic virus killed more than 20 million people. IAVs infect the respiratory tract and cause significant morbidity and mortality in humans. In contrast, IAVs infect the gastrointestinal tract of waterfowl, producing little pathology. Recent studies indicated that viruses can alter the microbiome at the respiratory and gastrointestinal mucosa, but there are no reports of how the microbiota of the natural host of influenza is affected by infection. Here we find that the mallard microbiome is altered during IAV infection. Our results suggest that detailed examination of humans and animals infected with IAVs may reveal individualized microbiome profiles that correspond to health and disease. Moreover, future studies should explore whether the altered microbiome facilitates maintenance and transmission of IAVs in waterfowl populations.

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SARS-CoV-2 and Influenza A virus Co-infections in Ferrets

Journal of Virology ◽

10.1128/jvi.01791-21 ◽

2021 ◽

Author(s):

Ying Huang ◽

Amanda L. Skarlupka ◽

Hyesun Jang ◽

Uriel Blas-Machado ◽

Nathan Holladay ◽

...

Keyword(s):

Influenza Virus ◽

Direct Contact ◽

Human Population ◽

Influenza A ◽

Seasonal Influenza ◽

Severe Disease ◽

Influenza Viruses ◽

Single Infection ◽

Influenza A Viruses ◽

Severe Morbidity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and seasonal influenza viruses are co-circulating in the human population. However, only a few cases of viral co-infection with these two viruses have been documented in humans with some people having severe disease and others mild disease. In order to examine this phenomenon, ferrets were co-infected with SARS-CoV-2 and human seasonal influenza A viruses (IAVs) (H1N1 or H3N2) and were compared to animals that received each virus alone. Ferrets were either immunologically naïve to both viruses or vaccinated with the 2019-2020 split-inactivated influenza virus vaccine. Co-infected naive ferrets lost significantly more body weight than ferrets infected with each virus alone and induced more severe inflammation in both the nose and lungs than ferrets single-infected with each virus. Co-infected naïve animals had predominantly higher IAV titers than SARS-CoV-2 titers, and IAVs efficiently transmitted to the co-housed ferrets by direct contact. Comparatively, SARS-CoV-2 failed to transmit to the ferrets that co-housed with co-infected ferrets by direct contact. Moreover, vaccination significantly reduced IAVs virus titers and shortened the viral shedding, but did not completely block influenza virus direct contact transmission. Notably, vaccination significantly ameliorated the influenza associated disease by protecting vaccinated animals from severe morbidity after IAV single infection or IAV and SARS-CoV-2 co-infection, suggesting that seasonal influenza virus vaccination is pivotal to prevent severe disease induced by IAVs and SARS-CoV-2 co-infection during the COVID-19 pandemic. Importance Influenza A viruses cause severe morbidity and mortality during each influenza virus season. The emergence of SARS-CoV-2 infection in the human population offers the opportunity to potential co-infections of both viruses. The development of useful animal models to asses pathogenesis, transmission, and viral evolution of these viruses as the co-infect a host is of critical importance for the development of vaccines and therapeutics. The ability to prevent the most severe effects of viral co-infections can be studied using effect co-infection ferret models described in this report.

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Broadly Reactive H2 Hemagglutinin Vaccines Elicit Cross-Reactive Antibodies in Ferrets Pre-Immune to Seasonal Influenza A Viruses

10.1101/2021.02.17.431747 ◽

2021 ◽

Author(s):

Z. Beau Reneer ◽

Amanda S. Skarlupka ◽

Parker J. Jamieson ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Recombinant Proteins ◽

Human Population ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Morbidity And Mortality ◽

Influenza A Viruses ◽

Global Pandemic

AbstractInfluenza vaccines have traditionally been tested in naïve mice and ferrets. However, humans are first exposed to influenza viruses within the first few years of their lives. Therefore, there is a pressing need to test influenza virus vaccines in animal models that have been previously exposed to influenza viruses before being vaccinated. In this manuscript, previously described H2 computationally optimized broadly reactive antigen (COBRA) HA vaccines (Z1, Z5) were tested in influenza virus ‘pre-immune’ ferret models. Ferrets were infected with historical, seasonal influenza viruses to establish pre-immunity. These pre-immune ferrets were then vaccinated with either COBRA H2 HA recombinant proteins or WT H2 HA recombinant proteins in a prime-boost regimen. A set of naïve pre-immune or non pre-immune ferrets were also vaccinated to control of the effects of the multiple different pre-immunities. All of the ferrets were then challenged with a swine H2N3 influenza virus. Ferrets with pre-existing immune responses influenced recombinant H2 HA elicited antibodies following vaccination as measured by HAI and classical neutralization assays. Having both H3N2 and H1N1 immunological memory regardless of the order of exposure significantly decreased viral nasal wash titers and completely protected all ferrets from both morbidity and mortality, including the mock vaccinated ferrets in the group. While the vast majority of the pre-immune ferrets were protected from both morbidity and mortality across all of the different pre-immunities, the Z1 COBRA HA vaccinated ferrets had significantly higher antibody titers and recognized the highest number H2 influenza viruses in a classical neutralization assay compared to the other H2 HA vaccines.ImportanceH1N1 and H3N2 influenza viruses have co-circulated in the human population since 1977. Nearly every human alive today has antibodies and memory B and T cells against these two subtypes of influenza viruses. H2N2 influenza viruses caused the 1957 global pandemic and people born after 1968 have never been exposed to H2 influenza viruses. It is quite likely that a future H2 influenza virus could transmit within the human population and start a new global pandemic, since the majority of people alive today are immunologically naïve to viruses of this subtype. Therefore, an effective vaccine for H2 influenza viruses should be tested in an animal model with previous exposure to influenza viruses that have circulated in humans. Ferrets were infected with historical influenza A viruses to more accurately mimic the immune responses in people who have pre-existing immune responses to seasonal influenza viruses. In this study, pre-immune ferrets were vaccinated with WT and COBRA H2 recombinant HA proteins in order to examine the effects of pre-existing immunity to seasonal human influenza viruses have on the elicitation of broadly cross-reactive antibodies from heterologous vaccination.

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Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses

Scientific Reports ◽

10.1038/s41598-020-79590-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

James D. Allen ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Next Generation ◽

Virus Infections ◽

Wild Type ◽

Influenza Hemagglutinin ◽

H3n2 Influenza

AbstractWhile vaccines remain the best tool for preventing influenza virus infections, they have demonstrated low to moderate effectiveness in recent years. Seasonal influenza vaccines typically consist of wild-type influenza A and B viruses that are limited in their ability to elicit protective immune responses against co-circulating influenza virus variant strains. Improved influenza virus vaccines need to elicit protective immune responses against multiple influenza virus drift variants within each season. Broadly reactive vaccine candidates potentially provide a solution to this problem, but their efficacy may begin to wane as influenza viruses naturally mutate through processes that mediates drift. Thus, it is necessary to develop a method that commercial vaccine manufacturers can use to update broadly reactive vaccine antigens to better protect against future and currently circulating viral variants. Building upon the COBRA technology, nine next-generation H3N2 influenza hemagglutinin (HA) vaccines were designed using a next generation algorithm and design methodology. These next-generation broadly reactive COBRA H3 HA vaccines were superior to wild-type HA vaccines at eliciting antibodies with high HAI activity against a panel of historical and co-circulating H3N2 influenza viruses isolated over the last 15 years, as well as the ability to neutralize future emerging H3N2 isolates.

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Influenza: An Emerging and Re-emerging Public Health Threat in Nepal

Annals of Clinical Chemistry and Laboratory Medicine ◽

10.3126/acclm.v3i2.20737 ◽

2018 ◽

Vol 3 (2) ◽

pp. 1-2

Author(s):

Bishnu Prasad Upadhyay

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Winter Season ◽

Emerging Infectious Diseases ◽

Influenza Viruses ◽

Influenza B ◽

Influenza A Viruses ◽

Influenza A H1n1 ◽

New Variant ◽

Seasonal Epidemics

Influenza virus type A and B are responsible for seasonal epidemics as well as pandemics in human. Influenza A viruses are further divided into two major groups namely, low pathogenic seasonal influenza (A/H1N1, A/H1N1 pdm09, A/H3N2) and highly pathogenic influenza virus (H5N1, H5N6, H7N9) on the basis of two surface antigens: hemagglutinin (HA) and neuraminidase (NA). Mutations, including substitutions, deletions, and insertions, are one of the most important mechanisms for producing new variant of influenza viruses. During the last 30 years; more than 50 viral threat has been evolved in South-East Asian countriesof them influenza is one of the major emerging and re-emerging infectious diseases of global concern. Similar to tropical and sub-tropical countries of Southeast Asia; circulation of A/H1N1 pdm09, A/H3N2 and influenza B has been circulating throughout the year with the peak during July-November in Nepal. However; the rate of infection transmission reach peak during the post-rain and winter season of Nepal.

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Plasticity of the Influenza Virus H5 HA Protein

mBio ◽

10.1128/mbio.03324-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Huihui Kong ◽

David F. Burke ◽

Tiago Jose da Silva Lopes ◽

Kosuke Takada ◽

Masaki Imai ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Mammalian Cells ◽

Influenza A ◽

Viral Antigen ◽

Influenza Viruses ◽

Influenza A Viruses ◽

Highly Pathogenic ◽

Antigenic Properties ◽

Ha Protein

ABSTRACT Since the emergence of highly pathogenic avian influenza viruses of the H5 subtype, the major viral antigen, hemagglutinin (HA), has undergone constant evolution, resulting in numerous genetic and antigenic (sub)clades. To explore the consequences of amino acid changes at sites that may affect the antigenicity of H5 viruses, we simultaneously mutated 17 amino acid positions of an H5 HA by using a synthetic gene library that, theoretically, encodes all combinations of the 20 amino acids at the 17 positions. All 251 mutant viruses sequenced possessed ≥13 amino acid substitutions in HA, demonstrating that the targeted sites can accommodate a substantial number of mutations. Selection with ferret sera raised against H5 viruses of different clades resulted in the isolation of 39 genotypes. Further analysis of seven variants demonstrated that they were antigenically different from the parental virus and replicated efficiently in mammalian cells. Our data demonstrate the substantial plasticity of the influenza virus H5 HA protein, which may lead to novel antigenic variants. IMPORTANCE The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants.

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Influenza A Virus Inhibits RSV Infection via a Two-Wave Expression of IFIT Proteins

Viruses ◽

10.3390/v12101171 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1171

Author(s):

Yaron Drori ◽

Jasmine Jacob-Hirsch ◽

Rakefet Pando ◽

Aharona Glatman-Freedman ◽

Nehemya Friedman ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Influenza Viruses ◽

Mass Spectrometry Analysis ◽

Influenza A Viruses ◽

Rsv Infection ◽

Syncytial Virus ◽

Mouse Lungs

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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