Replication-incompetent influenza A viruses that stably express a foreign gene

A biologically contained influenza A virus that stably expresses a foreign gene can be effectively traced, used to generate a novel multivalent vaccine and have its replication easily assessed, all while satisfying safety concerns regarding pathogenicity or reversion. This study generated a PB2-knockout (PB2-KO) influenza virus that harboured the GFP reporter gene in the coding region of its PB2 viral RNA (vRNA). Replication of the PB2-KO virus was restricted to a cell line stably expressing the PB2 protein. The GFP gene-encoding PB2 vRNA was stably incorporated into progeny viruses during replication in PB2-expressing cells. The GFP gene was expressed in virus-infected cells with no evidence of recombination between the recombinant PB2 vRNA and the PB2 protein mRNA. Furthermore, other reporter genes and the haemagglutinin and neuraminidase genes of different virus strains were accommodated by the PB2-KO virus. Finally, the PB2-KO virus was used to establish an improved assay to screen neutralizing antibodies against influenza viruses by using reporter gene expression as an indicator of virus infection rather than by observing cytopathic effect. These results indicate that the PB2-KO virus has the potential to be a valuable tool for basic and applied influenza virus research.

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Exploitation of Nucleic Acid Packaging Signals To Generate a Novel Influenza Virus-Based Vector Stably Expressing Two Foreign Genes

Journal of Virology ◽

10.1128/jvi.77.19.10575-10583.2003 ◽

2003 ◽

Vol 77 (19) ◽

pp. 10575-10583 ◽

Cited By ~ 131

Author(s):

Tokiko Watanabe ◽

Shinji Watanabe ◽

Takeshi Noda ◽

Yutaka Fujii ◽

Yoshihiro Kawaoka

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Fluorescent Protein ◽

Influenza Viruses ◽

Coding Region ◽

Influenza A Viruses ◽

Virion Incorporation ◽

Foreign Genes ◽

Green Fluorescent ◽

Genomic Regions

ABSTRACT At the final step in viral replication, the viral genome must be incorporated into progeny virions, yet the genomic regions required for this process are largely unknown in RNA viruses, including influenza virus. Recently, it was reported that both ends of the neuraminidase (NA) coding region are critically important for incorporation of this vRNA segment into influenza virions (Y. Fujii, H. Goto, T. Watanabe, T. Yoshida, and Y. Kawaoka, Proc. Natl. Acad. Sci. USA 100:2002-2007, 2003). To determine the signals in the hemagglutinin (HA) vRNA required for its virion incorporation, we made a series of deletion constructs of this segment. Subsequent analysis showed that 9 nucleotides at the 3′ end of the coding region and 80 nucleotides at the 5′ end are sufficient for efficient virion incorporation of the HA vRNA. The utility of this information for stable expression of foreign genes in influenza viruses was assessed by generating a virus whose HA and NA vRNA coding regions were replaced with those of vesicular stomatitis virus glycoprotein (VSVG) and green fluorescent protein (GFP), respectively, while retaining virion incorporation signals for these segments. Despite the lack of HA and NA proteins, the resultant virus, which possessed only VSVG on the virion surface, was viable and produced GFP-expressing plaques in cells even after repeated passages, demonstrating that two foreign genes can be incorporated and maintained stably in influenza A virus. These findings could serve as a model for the construction of influenza A viruses designed to express and/or deliver foreign genes.

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Cloning of the Chicken RNA Polymerase I Promoter and Use for Reverse Genetics of Influenza A Viruses in Avian Cells

Journal of Virology ◽

10.1128/jvi.79.21.13811-13816.2005 ◽

2005 ◽

Vol 79 (21) ◽

pp. 13811-13816 ◽

Cited By ~ 52

Author(s):

Pascale Massin ◽

Pierre Rodrigues ◽

Monica Marasescu ◽

Sylvie van der Werf ◽

Nadia Naffakh

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Reverse Genetics ◽

Promoter Sequence ◽

Influenza Viruses ◽

Rna Polymerase I ◽

Influenza A Viruses ◽

Virus Research ◽

Influenza Vaccine Production ◽

Polymerase I

ABSTRACT Reverse genetics techniques to rescue influenza viruses have thus far been based on the use of a human polymerase I (PolI) promoter to direct the synthesis of the eight viral RNAs. They can only be used on cells from primate origin due to the species specificity of the PolI promoter. Here we report the cloning of the chicken PolI promoter sequence and the generation of recombinant influenza virus upon transfection of bidirectional PolI/PolII plasmids in avian cells. Potential contributions of this new reverse genetics system in the fields of influenza virus research and influenza vaccine production are discussed.

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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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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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A Host-Restricted Self-Attenuated Influenza Virus Provides Broad Pan-Influenza A Protection in a Mouse Model

Frontiers in Immunology ◽

10.3389/fimmu.2021.779223 ◽

2021 ◽

Vol 12 ◽

Author(s):

Minjin Kim ◽

Yucheol Cheong ◽

Jinhee Lee ◽

Jongkwan Lim ◽

Sanguine Byun ◽

...

Keyword(s):

Influenza Virus ◽

Mouse Model ◽

Neutralizing Antibodies ◽

Influenza A ◽

Protective Efficacy ◽

Influenza Viruses ◽

Cross Protection ◽

Infection Model ◽

Wild Type ◽

Group 1

Influenza virus infections can cause a broad range of symptoms, form mild respiratory problems to severe and fatal complications. While influenza virus poses a global health threat, the frequent antigenic change often significantly compromises the protective efficacy of seasonal vaccines, further increasing the vulnerability to viral infection. Therefore, it is in great need to employ strategies for the development of universal influenza vaccines (UIVs) which can elicit broad protection against diverse influenza viruses. Using a mouse infection model, we examined the breadth of protection of the caspase-triggered live attenuated influenza vaccine (ctLAIV), which was self-attenuated by the host caspase-dependent cleavage of internal viral proteins. A single vaccination in mice induced a broad reactive antibody response against four different influenza viruses, H1 and rH5 (HA group 1) and H3 and rH7 subtypes (HA group 2). Notably, despite the lack of detectable neutralizing antibodies, the vaccination provided heterosubtypic protection against the lethal challenge with the viruses. Sterile protection was confirmed by the complete absence of viral titers in the lungs and nasal turbinates after the challenge. Antibody-dependent cellular cytotoxicity (ADCC) activities of non-neutralizing antibodies contributed to cross-protection. The cross-protection remained robust even after in vivo depletion of T cells or NK cells, reflecting the strength and breadth of the antibody-dependent effector function. The robust mucosal secretion of sIgA reflects an additional level of cross-protection. Our data show that the host-restricted designer vaccine serves an option for developing a UIV, providing pan-influenza A protection against both group 1 and 2 influenza viruses. The present results of potency and breadth of protection from wild type and reassortant viruses addressed in the mouse model by single immunization merits further confirmation and validation, preferably in clinically relevant ferret models with wild type challenges.

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International Laboratory Comparison of Influenza Microneutralization Assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) Influenza Viruses by CONSISE

Clinical and Vaccine Immunology ◽

10.1128/cvi.00278-15 ◽

2015 ◽

Vol 22 (8) ◽

pp. 957-964 ◽

Cited By ~ 28

Author(s):

Karen L. Laurie ◽

Othmar G. Engelhardt ◽

John Wood ◽

Alan Heath ◽

Jacqueline M. Katz ◽

...

Keyword(s):

Influenza Virus ◽

Incubation Time ◽

Influenza A ◽

Influenza Viruses ◽

Enzyme Linked Immunosorbent Assay ◽

Influenza A Viruses ◽

H5n1 Influenza ◽

The World ◽

International Laboratory ◽

The Impact

ABSTRACTThe microneutralization assay is commonly used to detect antibodies to influenza virus, and multiple protocols are used worldwide. These protocols differ in the incubation time of the assay as well as in the order of specific steps, and even within protocols there are often further adjustments in individual laboratories. The impact these protocol variations have on influenza serology data is unclear. Thus, a laboratory comparison of the 2-day enzyme-linked immunosorbent assay (ELISA) and 3-day hemagglutination (HA) microneutralization (MN) protocols, using A(H1N1)pdm09, A(H3N2), and A(H5N1) viruses, was performed by the CONSISE Laboratory Working Group. Individual laboratories performed both assay protocols, on multiple occasions, using different serum panels. Thirteen laboratories from around the world participated. Within each laboratory, serum sample titers for the different assay protocols were compared between assays to determine the sensitivity of each assay and were compared between replicates to assess the reproducibility of each protocol for each laboratory. There was good correlation of the results obtained using the two assay protocols in most laboratories, indicating that these assays may be interchangeable for detecting antibodies to the influenza A viruses included in this study. Importantly, participating laboratories have aligned their methodologies to the CONSISE consensus 2-day ELISA and 3-day HA MN assay protocols to enable better correlation of these assays in the future.

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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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Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

Journal of Virology ◽

10.1128/jvi.03099-14 ◽

2015 ◽

Vol 89 (7) ◽

pp. 3610-3618 ◽

Cited By ~ 50

Author(s):

Wenqian He ◽

Caitlin E. Mullarkey ◽

J. Andrew Duty ◽

Thomas M. Moran ◽

Peter Palese ◽

...

Keyword(s):

Influenza Virus ◽

Monoclonal Antibodies ◽

Virus Vaccine ◽

Neutralizing Antibodies ◽

Influenza A ◽

Human Peripheral Blood ◽

Influenza Virus Vaccine ◽

Influenza Viruses ◽

Broadly Neutralizing Antibodies ◽

Variable Regions

ABSTRACTCurrent influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferiorin vitrobut was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design.IMPORTANCEInfluenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the emergence of pandemic strains, a new class of broadly neutralizing antibodies has been recently discovered and may be the key to developing a “universal” influenza virus vaccine. While much has been learned about the biology of these antibodies, most studies have focused only on monoclonal antibodies of IgG subtypes. However, the study of monoclonal antibodies often fails to capture the complexity of antibody functions that occur during natural polyclonal responses. Here, we provide the first detailed analyses of the biological activity of these antibodies in polyclonal contexts, comparing both IgG and IgA isotypes isolated from human donors. The striking differences observed in the functional properties of broadly neutralizing antibodies in polyclonal contexts will be essential for guiding design of “universal” influenza virus vaccines and therapeutics.

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Resurrected ancestral influenza A viruses recapitulate the avian-to-mammalian adaptation of European avian-like swine influenza virus

10.21203/rs.3.rs-436907/v1 ◽

2021 ◽

Author(s):

Wen Su ◽

Rhodri Harfoot ◽

Yvonne Su ◽

Jennifer DeBeauchamp ◽

Udayan Joseph ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Influenza A ◽

Swine Influenza Virus ◽

Swine Influenza ◽

Polymerase Activity ◽

Influenza Viruses ◽

Mammalian Host ◽

Influenza A Viruses ◽

Sequence Reconstruction

Abstract The emergence of a pandemic influenza virus may be better anticipated if we better understand the evolutionary steps taken by avian influenza viruses as they adapt to mammals. We used ancestral sequence reconstruction to resurrect viruses representing initial adaptive stages of the European avian-like H1N1 virus as it transitioned from avian to swine hosts. We demonstrate that efficient transmissibility in pigs was gained through stepwise adaptation after 1983. These time-dependent adaptations resulted in changes in hemagglutinin receptor binding specificity and increased viral polymerase activity. An NP-R351K mutation under strong positive selection increased the transmissibility of a reconstructed virus. The stepwise-adaptation of a wholly avian influenza virus to a mammalian host suggests a window where targeted intervention may have highest impact. Successful intervention will, however, require strategic coordination of surveillance and risk assessment activities to identify these adapting viruses and guide pandemic preparedness resources.

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