MicroRNA-Based Attenuation of Influenza Virus across Susceptible Hosts

ABSTRACTInfluenza A virus drives significant morbidity and mortality in humans and livestock. Annual circulation of the virus in livestock and waterfowl contributes to severe economic disruption and increases the risk of zoonotic transmission of novel strains into the human population, where there is no preexisting immunity. Seasonal vaccinations in humans help prevent infection and can reduce symptoms when infection does occur. However, current vaccination regimens available for livestock are limited in part due to safety concerns regarding reassortment/recombination with circulating strains. Therefore, inactivated vaccines are used instead of the more immunostimulatory live attenuated vaccines. MicroRNAs (miRNAs) have been used previously to generate attenuated influenza A viruses for use as a vaccine. Here, we systematically targeted individual influenza gene mRNAs using the same miRNA to determine the segment(s) that yields maximal attenuation potential. This analysis demonstrated that targeting of NP mRNA most efficiently ablates replication. We further increased the plasticity of miRNA-mediated attenuation of influenza A virus by exploiting a miRNA, miR-21, that is ubiquitously expressed across influenza-susceptible hosts. In order to construct this targeted virus, we used CRISPR/Cas9 to eliminate the universally expressed miR-21 from MDCK cells. miR-21-targeted viruses were attenuated in human, mouse, canine, and avian cells and drove protective immunity in mice. This strategy has the potential to enhance the safety of live attenuated vaccines in humans and zoonotic reservoirs.IMPORTANCEInfluenza A virus circulates annually in both avian and human populations, causing significant morbidity, mortality, and economic burden. High incidence of zoonotic infections greatly increases the potential for transmission to humans, where no preexisting immunity or vaccine exists. There is a critical need for new vaccine strategies to combat emerging influenza outbreaks. MicroRNAs were used previously to attenuate influenza A viruses. We propose the development of a novel platform to produce live attenuated vaccines that are highly customizable, efficacious across a broad species range, and exhibit enhanced safety over traditional vaccination methods. This strategy exploits a microRNA that is expressed abundantly in influenza virus-susceptible hosts. By eliminating this ubiquitous microRNA from a cell line, targeted viruses that are attenuated across susceptible strains can be generated. This approach greatly increases the plasticity of the microRNA targeting approach and enhances vaccine safety.

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Functional Comparison of Mx1 from Two Different Mouse Species Reveals the Involvement of Loop L4 in the Antiviral Activity against Influenza A Viruses

Journal of Virology ◽

10.1128/jvi.01744-15 ◽

2015 ◽

Vol 89 (21) ◽

pp. 10879-10890 ◽

Cited By ~ 14

Author(s):

Judith Verhelst ◽

Jan Spitaels ◽

Cindy Nürnberger ◽

Dorien De Vlieger ◽

Tine Ysenbaert ◽

...

Keyword(s):

Influenza Virus ◽

Antiviral Activity ◽

Influenza A Virus ◽

Mus Musculus ◽

Influenza A ◽

Common Mechanism ◽

Mus Spretus ◽

Influenza A Viruses ◽

Content Type ◽

Mouse Species

ABSTRACTThe interferon-inducedMx1gene is an important part of the mammalian defense against influenza viruses.Mus musculusMx1 inhibits influenza A virus replication and transcription by suppressing the polymerase activity of viral ribonucleoproteins (vRNPs). Here, we compared the anti-influenza virus activity of Mx1 fromMus musculusA2G with that of its ortholog fromMus spretus. We found that the antiviral activity ofM. spretusMx1 was less potent than that ofM. musculusMx1. Comparison of theM. musculusMx1 sequence with theM. spretusMx1 sequence revealed 25 amino acid differences, over half of which were present in the GTPase domain and 2 of which were present in loop L4. However, thein vitroGTPase activity of Mx1 from the two mouse species was similar. Replacement of one of the residues in loop L4 inM. spretusMx1 by the corresponding residue of A2G Mx1 increased its antiviral activity. We also show that deletion of loop L4 prevented the binding of Mx1 to influenza A virus nucleoprotein and, hence, abolished the antiviral activity of mouse Mx1. These results indicate that loop L4 of mouse Mx1 is a determinant of antiviral activity. Our findings suggest that Mx proteins from different mammals use a common mechanism to inhibit influenza A viruses.IMPORTANCEMx proteins are evolutionarily conserved in vertebrates and inhibit a wide range of viruses. Still, the exact details of their antiviral mechanisms remain largely unknown. Functional comparison of theMxgenes from two species that diverged relatively recently in evolution can provide novel insights into these mechanisms. We show that bothMus musculusA2G Mx1 andMus spretusMx1 target the influenza virus nucleoprotein. We also found that loop L4 in mouse Mx1 is crucial for its antiviral activity, as was recently reported for primate MxA. This indicates that human and mouse Mx proteins, which have diverged by 75 million years of evolution, recognize and inhibit influenza A viruses by a common mechanism.

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In Vitro System for Modeling Influenza A Virus Resistance under Drug Pressure

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01385-09 ◽

2010 ◽

Vol 54 (8) ◽

pp. 3442-3450 ◽

Cited By ~ 19

Author(s):

Ashley N. Brown ◽

James J. McSharry ◽

Qingmei Weng ◽

Elizabeth M. Driebe ◽

David M. Engelthaler ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Mdck Cells ◽

Influenza Virus Infection ◽

Infection Model ◽

Virus Infections ◽

Drug Pressure

ABSTRACT One of the biggest challenges in the effort to treat and contain influenza A virus infections is the emergence of resistance during treatment. It is well documented that resistance to amantadine arises rapidly during the course of treatment due to mutations in the gene coding for the M2 protein. To address this problem, it is critical to develop experimental systems that can accurately model the selection of resistance under drug pressure as seen in humans. We used the hollow-fiber infection model (HFIM) system to examine the effect of amantadine on the replication of influenza virus, A/Albany/1/98 (H3N2), grown in MDCK cells. At 24 and 48 h postinfection, virus replication was inhibited in a dose-dependent fashion. At 72 and 96 h postinfection, virus replication was no longer inhibited, suggesting the emergence of amantadine-resistant virus. Sequencing of the M2 gene revealed that mutations appeared at between 48 and 72 h of drug treatment and that the mutations were identical to those identified in the clinic for amantadine-resistant viruses (e.g., V27A, A30T, and S31N). Interestingly, we found that the type of mutation was strongly affected by the dose of the drug. The data suggest that the HFIM is a good model for influenza virus infection and resistance generation in humans. The HFIM has the advantage of being a highly controlled system where multiplicity parameters can be directly and accurately controlled and measured.

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Influenza Viruses in Mice: Deep Sequencing Analysis of Serial Passage and Effects of Sialic Acid Structural Variation

Journal of Virology ◽

10.1128/jvi.01039-19 ◽

2019 ◽

Vol 93 (23) ◽

Cited By ~ 6

Author(s):

Brian R. Wasik ◽

Ian E. H. Voorhees ◽

Karen N. Barnard ◽

Brynn K. Alford-Lawrence ◽

Wendy S. Weichert ◽

...

Keyword(s):

Influenza Virus ◽

Sialic Acid ◽

Influenza A Virus ◽

Deep Sequencing ◽

Influenza A ◽

Natural Host ◽

H1n1 Pandemic ◽

Influenza A Viruses ◽

Canine Influenza Virus ◽

Canine Influenza

ABSTRACT Influenza A viruses have regularly jumped to new host species to cause epidemics or pandemics, an evolutionary process that involves variation in the viral traits necessary to overcome host barriers and facilitate transmission. Mice are not a natural host for influenza virus but are frequently used as models in studies of pathogenesis, often after multiple passages to achieve higher viral titers that result in clinical disease such as weight loss or death. Here, we examine the processes of influenza A virus infection and evolution in mice by comparing single nucleotide variations of a human H1N1 pandemic virus, a seasonal H3N2 virus, and an H3N2 canine influenza virus during experimental passage. We also compared replication and sequence variation in wild-type mice expressing N-glycolylneuraminic acid (Neu5Gc) with those seen in mice expressing only N-acetylneuraminic acid (Neu5Ac). Viruses derived from plasmids were propagated in MDCK cells and then passaged in mice up to four times. Full-genome deep sequencing of the plasmids, cultured viruses, and viruses from mice at various passages revealed only small numbers of mutational changes. The H3N2 canine influenza virus showed increases in frequency of sporadic mutations in the PB2, PA, and NA segments. The H1N1 pandemic virus grew well in mice, and while it exhibited the maintenance of some minority mutations, there was no clear evidence for adaptive evolution. The H3N2 seasonal virus did not establish in the mice. Finally, there were no clear sequence differences associated with the presence or absence of Neu5Gc. IMPORTANCE Mice are commonly used as a model to study the growth and virulence of influenza A viruses in mammals but are not a natural host and have distinct sialic acid receptor profiles compared to humans. Using experimental infections with different subtypes of influenza A virus derived from different hosts, we found that evolution of influenza A virus in mice did not necessarily proceed through the linear accumulation of host-adaptive mutations, that there was variation in the patterns of mutations detected in each repetition, and that the mutation dynamics depended on the virus examined. In addition, variation in the viral receptor, sialic acid, did not affect influenza virus evolution in this model. Overall, our results show that while mice provide a useful animal model for influenza virus pathology, host passage evolution will vary depending on the specific virus tested.

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Aloe vera and its Components Inhibit Influenza A Virus-Induced Autophagy and Replication

The American Journal of Chinese Medicine ◽

10.1142/s0192415x19500678 ◽

2019 ◽

Vol 47 (06) ◽

pp. 1307-1324 ◽

Cited By ~ 7

Author(s):

Jang-Gi Choi ◽

Heeeun Lee ◽

Young Soo Kim ◽

Youn-Hwan Hwang ◽

You-Chang Oh ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Matrix Protein ◽

Mdck Cells ◽

Aloe Vera ◽

Post Treatment ◽

M2 Protein ◽

Mrna Synthesis ◽

Catechin Hydrate

Aloe vera ethanol extract (AVE) reportedly has significant anti-influenza virus activity, but its underlying mechanisms of action and constituents have not yet been completely elucidated. Previously, we have confirmed that AVE treatment significantly reduces the viral replication of green fluorescent protein-labeled influenza A virus in Madin-Darby canine kidney (MDCK) cells. In addition, post-treatment with AVE inhibited viral matrix protein 1 (M1), matrix protein 2 (M2), and hemagglutinin (HA) mRNA synthesis and viral protein (M1, M2, and HA) expressions. In this study, we demonstrated that AVE inhibited autophagy induced by influenza A virus in MDCK cells and also identified quercetin, catechin hydrate, and kaempferol as the active antiviral components of AVE. We also found that post-treatment with quercetin, catechin hydrate, and kaempferol markedly inhibited M2 viral mRNA synthesis and M2 protein expression. A docking simulation suggested that the binding affinity of quercetin, catechin hydrate, and kaempferol for the M2 protein may be higher than that of known M2 protein inhibitors. Thus, the inhibition of autophagy induced by influenza virus may explain the antiviral activity of AVE against H1N1 or H3N2. Aloe vera extract and its constituents may, therefore, be potentially useful for the development of anti-influenza agents.

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Role of the PB2 627 Domain in Influenza A Virus Polymerase Function

Journal of Virology ◽

10.1128/jvi.02467-16 ◽

2017 ◽

Vol 91 (7) ◽

Cited By ~ 13

Author(s):

Benjamin E. Nilsson ◽

Aartjan J. W. te Velthuis ◽

Ervin Fodor

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Influenza A Virus ◽

Influenza A ◽

Influenza A Viruses ◽

Viral Polymerase ◽

Virus Rna ◽

Rna Genome ◽

Transcription And Replication

ABSTRACT The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627 domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome. IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this report, we characterize the role of the 627 domain in polymerase function and offer insights into the replication mechanism of influenza A viruses.

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Origins and Evolutionary Dynamics of H3N2 Canine Influenza Virus

Journal of Virology ◽

10.1128/jvi.03395-14 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5406-5418 ◽

Cited By ~ 38

Author(s):

Henan Zhu ◽

Joseph Hughes ◽

Pablo R. Murcia

Keyword(s):

Influenza Virus ◽

North American ◽

Influenza A ◽

Influenza Viruses ◽

Animal Origin ◽

Influenza A Viruses ◽

Canine Influenza Virus ◽

Zoonotic Infections ◽

Avian Origin ◽

Canine Influenza

ABSTRACTInfluenza A viruses (IAVs) are maintained mainly in wild birds, and despite frequent spillover infections of avian IAVs into mammals, only a small number of viruses have become established in mammalian hosts. A new H3N2 canine influenza virus (CIV) of avian origin emerged in Asia in the mid-2000s and is now circulating in dog populations of China and South Korea, and possibly in Thailand. The emergence of CIV provides new opportunities for zoonotic infections and interspecies transmission. We examined 14,764 complete IAV genomes together with all CIV genomes publicly available since its first isolation until 2013. We show that CIV may have originated as early as 1999 as a result of segment reassortment among Eurasian and North American avian IAV lineages. We also identified amino acid changes that might have played a role in CIV emergence, some of which have not been previously identified in other cross-species jumps. CIV evolves at a lower rate than H3N2 human influenza viruses do, and viral phylogenies exhibit geographical structure compatible with high levels of local transmission. We detected multiple intrasubtypic and heterosubtypic reassortment events, including the acquisition of the NS segment of an H5N1 avian influenza virus that had previously been overlooked. In sum, our results provide insight into the adaptive changes required by avian viruses to establish themselves in mammals and also highlight the potential role of dogs to act as intermediate hosts in which viruses with zoonotic and/or pandemic potential could originate, particularly with an estimated dog population of ∼700 million.IMPORTANCEInfluenza A viruses circulate in humans and animals. This multihost ecology has important implications, as past pandemics were caused by IAVs carrying gene segments of both human and animal origin. Adaptive evolution is central to cross-species jumps, and this is why understanding the evolutionary processes that shape influenza A virus genomes is key to elucidating the mechanisms underpinning viral emergence. An avian-origin canine influenza virus (CIV) has recently emerged in dogs and is spreading in Asia. We reconstructed the evolutionary history of CIV and show that it originated from both Eurasian and North American avian lineages. We also identified the mutations that might have been responsible for the cross-species jump. Finally, we provide evidence of multiple reassortment events between CIV and other influenza viruses (including an H5N1 avian virus). This is a cause for concern, as there is a large global dog population to which humans are highly exposed.

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Long-term survival of influenza A viruses in aquatic invertebrate zoocultures

Veterinariya, Zootekhniya i Biotekhnologiya ◽

10.36871/vet.zoo.bio.202103005 ◽

2021 ◽

Vol 1 (3) ◽

pp. 34-41

Author(s):

S. L. Nesterchuk ◽

◽

V. A. Ostapenko ◽

Keyword(s):

Influenza Virus ◽

Daphnia Magna ◽

Influenza A Virus ◽

Influenza A ◽

The Body ◽

Aquatic Invertebrate ◽

Human Influenza ◽

Influenza A Viruses ◽

Term Survival ◽

Chicken Embryos

In experiments to infect aquatic invertebrates in the zooculture, we used influenza A viruses, namely, to infect crustaceans Daphnia magna Straus, 1826 – human influenza virus, Hong Kong strain 1569/79 (H3N2), and to infect molluscs Anodonta cygnea Linné, 1758 – influenza virus A birds, Strain Rostok 1/34 (Hav1Neq1) – the so-called true bird plague virus. As a result of a series of experiments, found that influenza A viruses persist in the water for no more than 3 days, while in the gills and mantle of molluscs the virus is isolated on chicken embryos for at least another 35 days after contact with virus-containing water (a total of 70 individuals were studied). From the body Daphnia magna, to isolate the human influenza A virus on chicken embryos was possible within 14 days after infection through water (examined 6,800 individuals), by the method of immunofluorescence the influenza virus was determined in the intestines of crustaceans during the entire period of observation – 70 days from the time of infection. Influenza A viruses do not have a harmful effect on crustaceans or molluscs, infected animals also develop and reproduce, as well as individuals of control groups. Interesting is the fact that we have established the possibility of the loss of agglutination of red blood cells of chickens as a result of the reproduction of the human influenza A virus in the body of invertebrate Daphnia magna, which indicates a change in the viral protein hemagglutinin. The use of aquatic invertebrate zooculture can help in the study of the circulation of influenza A viruses in nature, as well as in the study of the variability of influenza A viruses.

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Interleukin-15 Is Critical in the Pathogenesis of Influenza A Virus-Induced Acute Lung Injury

Journal of Virology ◽

10.1128/jvi.02030-09 ◽

2010 ◽

Vol 84 (11) ◽

pp. 5574-5582 ◽

Cited By ~ 32

Author(s):

Risa Nakamura ◽

Naoyoshi Maeda ◽

Kensuke Shibata ◽

Hisakata Yamada ◽

Tetsuo Kase ◽

...

Keyword(s):

T Cells ◽

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Influenza Virus Infection ◽

Severe Pneumonia ◽

Influenza A Viruses ◽

Highly Pathogenic ◽

Interleukin 15

ABSTRACT Highly pathogenic influenza A viruses cause acute severe pneumonia to which the occurrence of “cytokine storm” has been proposed to contribute. Here we show that interleukin-15 (IL-15) knockout (KO) mice exhibited reduced mortality after infection with influenza virus A/FM/1/47 (H1N1, a mouse-adapted strain) albeit the viral titers of these mice showed no difference from those of control mice. There were significantly fewer antigen-specific CD44+ CD8+ T cells in the lungs of infected IL-15 KO mice, and adoptive transfer of the CD8+ T cells caused reduced survival of IL-15 KO mice following influenza virus infection. Mice deficient in β2-microglobulin by gene targeting and those depleted of CD8+ T cells by in vivo administration of anti-CD8 monoclonal antibody displayed a reduced mortality rate after infection. These results indicate that IL-15-dependent CD8+ T cells are at least partly responsible for the pathogenesis of acute pneumonia caused by influenza A virus.

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Isolation and Characterization of a Distinct Influenza A Virus from Egyptian Bats

Journal of Virology ◽

10.1128/jvi.01059-18 ◽

2018 ◽

Vol 93 (2) ◽

Cited By ~ 8

Author(s):

Ahmed Kandeil ◽

Mokhtar R. Gomaa ◽

Mahmoud M. Shehata ◽

Ahmed N. El Taweel ◽

Sara H. Mahmoud ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Mdck Cells ◽

Genomic Analysis ◽

Cross Reactivity ◽

Influenza Viruses ◽

Avian Host ◽

Influenza A Viruses ◽

Species Barrier ◽

Isolation And Characterization

ABSTRACT Recently, two genetically distinct influenza viruses were detected in bats in Guatemala and Peru. We conducted influenza A virus surveillance among four bat species in Egypt. Out of 1,202 swab specimens, 105 were positive by real-time PCR. A virus was successfully isolated in eggs and propagated in MDCK cells in the presence of N-tosyl-l-phenylalanine chloromethyl ketone-treated trypsin. Genomic analysis revealed that the virus was phylogenetically distinct from all other influenza A viruses. Analysis of the hemagglutinin gene suggested a common ancestry with other H9 viruses, and the virus showed a low level of cross-reactivity with serum raised against H9N2 viruses. Bats were seropositive for the isolated viruses. The virus replicated in the lungs of experimentally infected mice. While it is genetically distinct, this virus shares several avian influenza virus characteristics suggesting a more recent avian host origin. IMPORTANCE Through surveillance, we isolated and characterized an influenza A virus from Egyptian fruit bats. This virus had an affinity to avian-like receptors but was also able to infect mice. Our findings indicate that bats may harbor a diversity of influenza A viruses. Such viruses may have the potential to cross the species barrier to infect other species, including domestic birds, mammals, and, possibly, humans.

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Sulphatide binds to human and animal influenza A viruses, and inhibits the viral infection

Biochemical Journal ◽

10.1042/bj3180389 ◽

1996 ◽

Vol 318 (2) ◽

pp. 389-393 ◽

Cited By ~ 57

Author(s):

Takashi SUZUKI ◽

Ayako SOMETANI ◽

Yasuhiro YAMAZAKI ◽

Goh HORIIKE ◽

Yukiko MIZUTANI ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Sialic Acid Residue ◽

Influenza A Viruses ◽

Human Influenza Virus ◽

Viral Binding ◽

Overlay Assay ◽

Virus Isolates ◽

Darby Canine Kidney

We found, by using a virus overlay assay, that influenza A virus isolates bind to sulphatide (HSO3-Galβ1 → 1´Cer), which has no sialic acid residue, and that the infection of Madin–Darby canine kidney cells with the human influenza virus A/Memphis/1/71 (H3N2) is inhibited by sulphatide. A/Memphis/1/71 (H3N2) causes obvious haemagglutination and low-pH haemolysis of asialoerythrocytes reconstituted with sulphatide. All influenza A virus isolates from the species of animals so far tested bound to sulphatide. The sulphatide-binding specificity of the isolates was different from the viral sialyl-linkage specificity. Influenza A virus isolates also bound to galactosyl ceramide (GalCer; Galβ1 → 1´Cer), as well as sulphatide, in the virus overlay assays. In contrast, the influenza virus did not bind to N-deacyl, a derivative of sulphatide, glucosyl ceramide or the other neutral glycolipids tested. These results indicate that the linkage of galactose, or sulphated galactose, to ceramide is important for viral binding.

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