scholarly journals Shortening the unstructured, interdomain region of the non-structural protein NS1 of an avian H1N1 influenza virus increases its replication and pathogenicity in chickens

2014 ◽  
Vol 95 (6) ◽  
pp. 1233-1243 ◽  
Author(s):  
Sascha Trapp ◽  
Denis Soubieux ◽  
Hélène Marty ◽  
Evelyne Esnault ◽  
Thomas W. Hoffmann ◽  
...  
Keyword(s):  
Rna Binding ◽  
H1n1 Influenza ◽  
Genetically Engineered ◽  
Influenza Viruses ◽  
Epithelial Cell Line ◽  
General Effect ◽  
Structural Protein ◽  
H5n1 Influenza

Currently circulating H5N1 influenza viruses have undergone a complex evolution since the appearance of their progenitor A/Goose/Guangdong/1/96 in 1996. After the eradication of the H5N1 viruses that emerged in Hong Kong in 1997 (HK/97 viruses), new genotypes of H5N1 viruses emerged in the same region in 2000 that were more pathogenic for both chickens and mice than HK/97 viruses. These, as well as virtually all highly pathogenic H5N1 viruses since 2000, harbour a deletion of aa 80–84 in the unstructured region of the non-structural (NS) protein NS1 linking its RNA-binding domain to its effector domain. NS segments harbouring this mutation have since been found in non-H5N1 viruses and we asked whether this 5 aa deletion could have a general effect not limited to the NS1 of H5N1 viruses. We genetically engineered this deletion in the NS segment of a duck-origin avian H1N1 virus, and compared the in vivo and in vitro properties of the WT and NSdel8084 viruses. In experimentally infected chickens, the NSdel8084 virus showed both an increased replication potential and an increased pathogenicity. This in vivo phenotype was correlated with a higher replicative efficiency in vitro, both in embryonated eggs and in a chicken lung epithelial cell line. Our data demonstrated that the increased replicative potential conferred by this small deletion was a general feature not restricted to NS1 from H5N1 viruses and suggested that viruses acquiring this mutation may be selected positively in the future.

scholarly journals In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses

Nature ◽  
2009 ◽  
Vol 460 (7258) ◽  
pp. 1021-1025 ◽  
Author(s):  
Yasushi Itoh ◽  
Kyoko Shinya ◽  
Maki Kiso ◽  
Tokiko Watanabe ◽  
Yoshihiro Sakoda ◽  
...  
Keyword(s):  
H1n1 Influenza ◽  
Influenza Viruses ◽  
In Vivo Characterization

scholarly journals N-linked glycosylation plays an important role in budding of NA protein and virulence of influenza viruses

2020 ◽  
Author(s):  
Danqi Bao ◽  
Ruixue Xue ◽  
Min Zhang ◽  
Chenyang Lu ◽  
Tianxin Ma ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Replication ◽  
Immune Escape ◽  
H1n1 Influenza ◽  
Influenza Viruses ◽  
H1n1 Influenza Virus ◽  
Protein Loss ◽  
Knock Out

Neuraminidase (NA) has multiple functions in the life cycle of influenza virus, especially in the late stage of virus replication. Both of Hemagglutinin (HA) and NA are highly glycosylated proteins. N-linked glycosylation (NLG) of HA has been reported to contribute to immune escape and virulence of influenza viruses. However, the function of NLG of NA remains largely unclear. In this study, we found that NLG is critical for budding ability of NA. Tunicamycin treatment or NLG knock-out significantly inhibited the budding of NA. Further studies showed that the NLG knock-out caused attenuation of virus in vitro and in vivo. Notably the NLG at 219 position plays an important role in budding, replication, and virulence of H1N1 influenza virus. To explore the underlying mechanism, unfolded protein response (UPR) was determined in NLG knock-out NA overexpressed cells, which showed that the mutant NA was mainly located in ER, and the UPR markers BIP and p-eIF2α were upregulated, and XBP1 was downregulated. All the results indicated that NLG knock-out NA was stacked in ER and triggered UPR, which might shut down the budding process of NA. Overall, the study shed light on the function of NLG of NA in virus replication and budding. IMPORTANCE NA is a highly glycosylated protein. Nevertheless, how the NLG affects the function of NA protein remains largely unclear. In this study, we found that NLG plays important roles in budding and Neuraminidase activity of NA protein. Loss of NLG attenuated viral budding and replication. Especially the 219 NLG site mutation significantly attenuated the replication and virulence of H1N1 influenza virus in vitro and in vivo, which suggested that NLG of NA protein is a novel virulence marker for influenza viruses.

scholarly journals Duck enteritis virus UL21 is a late gene and encodes a protein that interacts with pUL16

2019 ◽  
Author(s):  
Linjiang Yang ◽  
Mingshu Wang ◽  
Chunhui Zeng ◽  
Yong Shi ◽  
Anchun Cheng ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Rna Binding ◽  
Gene Interaction ◽  
Duck Enteritis Virus ◽  
Late Gene ◽  
Structural Protein ◽  
Virus Transport ◽  
Enteritis Virus

Abstract Background pUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited. Methods In our study, recombinant pUL21 was expressed using an pET-32c (+) vector in Escherichia coli BL21 cells induced with 0.4 mM isopropyl β-D-thiogalactoside for 8 h at 30°C. The antibody used for the indirect immunofluorescence (IFA) and western blotting (WB) analysis were prepared. Pharmacological inhibition, WB and quantitative reverse transcription PCR (RT-qPCR) were performed. A coimmunoprecipitation (CO-IP) assay was conducted to test the interaction between pUL21 and pUL16. Results We verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. Conclusions The DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21. Keywords: Duck enteritis virus, UL21, UL16, late gene, interaction

scholarly journals Structure and Sequence Determinants Governing the Interactions of RNAs with Influenza A Virus Non-Structural Protein NS1

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 947
Author(s):  
Alan Wacquiez ◽  
Franck Coste ◽  
Emmanuel Kut ◽  
Virginie Gaudon ◽  
Sascha Trapp ◽  
...  
Keyword(s):  
Influenza A ◽  
Rna Binding ◽  
3D Structure ◽  
Genetically Engineered ◽  
Phenotypic Characterization ◽  
Sequence Motifs ◽  
Structural Protein ◽  
Specific Sequence ◽  
Rna Interaction

The non-structural protein NS1 of influenza A viruses is an RNA-binding protein of which its activities in the infected cell contribute to the success of the viral cycle, notably through interferon antagonism. We have previously shown that NS1 strongly binds RNA aptamers harbouring virus-specific sequence motifs (Marc et al., Nucleic Acids Res. 41, 434–449). Here, we started out investigating the putative role of one particular virus-specific motif through the phenotypic characterization of mutant viruses that were genetically engineered from the parental strain WSN. Unexpectedly, our data did not evidence biological importance of the putative binding of NS1 to this specific motif (UGAUUGAAG) in the 3′-untranslated region of its own mRNA. Next, we sought to identify specificity determinants in the NS1-RNA interaction through interaction assays in vitro with several RNA ligands and through solving by X-ray diffraction the 3D structure of several complexes associating NS1′s RBD with RNAs of various affinities. Our data show that the RBD binds the GUAAC motif within double-stranded RNA helices with an apparent specificity that may rely on the sequence-encoded ability of the RNA to bend its axis. On the other hand, we showed that the RBD binds to the virus-specific AGCAAAAG motif when it is exposed in the apical loop of a high-affinity RNA aptamer, probably through a distinct mode of interaction that still requires structural characterization. Our data are consistent with more than one mode of interaction of NS1′s RBD with RNAs, recognizing both structure and sequence determinants.

scholarly journals Cross-Protection of Chicken Immunoglobulin Y Antibodies against H5N1 and H1N1 Viruses Passively Administered in Mice

2011 ◽  
Vol 18 (7) ◽  
pp. 1083-1090 ◽  
Author(s):  
Michael G. Wallach ◽  
Richard J. Webby ◽  
Fakhrul Islam ◽  
Stephen Walkden-Brown ◽  
Eva Emmoth ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza Pandemic ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Antigenic Drift ◽  
Virus Infectivity ◽  
Lethal Challenge ◽  
H5n1 Influenza

ABSTRACTInfluenza viruses remain a major threat to global health due to their ability to undergo change through antigenic drift and antigenic shift. We postulated that avian IgY antibodies represent a low-cost, effective, and well-tolerated approach that can easily be scaled up to produce enormous quantities of protective antibodies. These IgY antibodies can be administered passively in humans (orally and intranasally) and can be used quickly and safely to help in the fight against an influenza pandemic. In this study, we raised IgY antibodies against H1N1, H3N2, and H5N1 influenza viruses. We demonstrated that, using whole inactivated viruses alone and in combination to immunize hens, we were able to induce a high level of anti-influenza virus IgY in the sera and eggs, which lasted for at least 2 months after two immunizations. Furthermore, we found that by use ofin vitroassays to test for the ability of IgY to inhibit hemagglutination (HI test) and virus infectivity (serum neutralization test), IgYs inhibited the homologous as well as in some cases heterologous clades and strains of viruses. Using anin vivomouse model system, we found that, when administered intranasally 1 h prior to infection, IgY to H5N1 protected 100% of the mice against lethal challenge with H5N1. Of particular interest was the finding that IgY to H5N1 cross-protected against A/Puerto Rico/8/34 (H1N1) bothin vitroandin vivo. Based on our results, we conclude that anti-influenza virus IgY can be used to help prevent influenza virus infection.

scholarly journals Antibodies directed towards neuraminidase N1 control disease in a mouse model of influenza

2017 ◽  
pp. JVI.01584-17 ◽  
Author(s):  
E.R. Job ◽  
M. Schotsaert ◽  
L.I. Ibañez ◽  
A. Smet ◽  
T. Ysenbaert ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Antiviral Activity ◽  
Influenza A ◽  
Conformational Epitope ◽  
Influenza Viruses ◽  
Lethal Challenge ◽  
H5n1 Influenza ◽  
Antigenic Properties

There is increasing evidence to suggest that antibodies directed towards influenza A virus (IAV) neuraminidase (NA) are an important correlate of protection against influenza in humans. Moreover, the potential of NA-specific antibodies to provide broader protection than conventional hemagglutinin (HA) antibodies has been recognized. Herein, we describe the isolation of two monoclonal antibodies, N1-7D3 and N1-C4, directed towards the N1 NA. N1-7D3 binds to a conserved linear epitope in the membrane distal, carboxy-terminal part of the NA and reacted with the NA of seasonal H1N1 isolates ranging from 1977 till 2007 the 2009 H1N1pdm virus as well as A/Vietnam/1194/04 (H5N1). However, N1-7D3 lacked NA inhibition (NI) activity and the ability to protect BALB/c mice against a lethal challenge with a range of H1N1 viruses. Conversely, N1-C4 bound to a conformational epitope that is conserved between two influenza subtypes, the 2009 H1N1pdm and H5N1 IAV and displayed potentin vitroantiviral activity mediating both NI and plaque size-reduction. Moreover, N1-C4 could provide heterosubtypic protection in BALB/c mice against a lethal challenge with 2009 H1N1pdm or H5N1 virus. Glutamic acid residue 311 in the NA was found to be critical for the NA binding and antiviral activity of monoclonal antibody N1-C4. Our data provide further evidence on cross-protective epitopes within the N1 subtype and highlight the potential of NA as an important target for vaccine and therapeutic approaches.ImportanceInfluenza remains a world-wide burden to public health. As such the development of new and novel vaccines and therapeutics against influenza virus is crucial. Human challenge studies have recently highlighted the importance of antibodies directed towards the viral neuraminidase (NA) as an important correlate of reduced influenza-associated disease severity. Furthermore, there is evidence that anti-NA antibodies can provide broader protection than antibodies towards the viral hemagglutinin. Here we describe the isolation and detailed characterization of two N1 NA-specific monoclonal antibodies. One of these monoclonal antibodies broadly binds N1 type NAs and the second one displays NAI, in vitro and in vivo anti-viral activity against 2009 H1N1pdm and H5N1 influenza viruses. These two new anti-NA antibodies contribute to our understanding of the antigenic properties and protective potential of the influenza NA antigen.

scholarly journals Oseltamivir-Ribavirin Combination Therapy for Highly Pathogenic H5N1 Influenza Virus Infection in Mice

2008 ◽  
Vol 52 (11) ◽  
pp. 3889-3897 ◽  
Author(s):  
Natalia A. Ilyushina ◽  
Alan Hay ◽  
Neziha Yilmaz ◽  
Adrianus C. M. Boon ◽  
Robert G. Webster ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza Virus Infection ◽  
Antiviral Effect ◽  
Influenza Viruses ◽  
Oseltamivir Carboxylate ◽  
Highly Pathogenic ◽  
H5n1 Influenza ◽  
Pathogenic H5n1

ABSTRACT We studied the effects of a neuraminidase inhibitor (oseltamivir) and an inhibitor of influenza virus polymerases (ribavirin) against two highly pathogenic H5N1 influenza viruses. In vitro, A/Vietnam/1203/04 virus (clade 1) was highly susceptible to oseltamivir carboxylate (50% inhibitory concentration [IC50] = 0.3 nM), whereas A/Turkey/15/06 virus (clade 2.2) had reduced susceptibility (IC50 = 5.5 nM). In vivo, BALB/c mice were treated with oseltamivir (1, 10, 50, or 100 mg/kg of body weight/day), ribavirin (37.5, 55, or 75 mg/kg/day), or the combination of both drugs for 8 days, starting 4 h before virus inoculation. Monotherapy produced a dose-dependent antiviral effect against the two H5N1 viruses in vivo. Three-dimensional analysis of the drug-drug interactions revealed that oseltamivir and ribavirin interacted principally in an additive manner, with several exceptions of marginal synergy or marginal antagonism at some concentrations. The combination of ribavirin at 37.5 mg/kg/day and oseltamivir at 1 mg/kg/day and the combination of ribavirin at 37.5 mg/kg/day and oseltamivir at 10 mg/kg/day were synergistic against A/Vietnam/1203/04 and A/Turkey/15/06 viruses, respectively. These optimal oseltamivir-ribavirin combinations significantly inhibited virus replication in mouse organs, prevented the spread of H5N1 viruses beyond the respiratory tract, and abrogated the cytokine response (P < 0.01). Importantly, we observed clear differences between the efficacies of the drug combinations against two H5N1 viruses: higher doses were required for the protection of mice against A/Turkey/15/06 virus than for the protection of mice against A/Vietnam/1203/04 virus. Our preliminary results suggest that oseltamivir-ribavirin combinations can have a greater or lesser antiviral effect than monotherapy, depending on the H5N1 virus and the concentrations used.

scholarly journals Genomic Polymorphism of the Pandemic A (H1N1) Influenza Viruses Correlates with Viral Replication, Virulence, and Pathogenicity In Vitro and In Vivo

PLoS ONE ◽  
2011 ◽  
Vol 6 (6) ◽  
pp. e20698 ◽  
Author(s):  
Lili Xu ◽  
Linlin Bao ◽  
Jianfang Zhou ◽  
Dayan Wang ◽  
Wei Deng ◽  
...  
Keyword(s):  
Viral Replication ◽  
H1n1 Influenza ◽  
Influenza Viruses ◽  
Genomic Polymorphism
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