scholarly journals Effect of the PB2 and M Genes on the Replication of H6 Influenza Virus in Chickens

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Hiroichi Ozaki ◽  
Yi Guan ◽  
Malik Peiris ◽  
Robert Webster ◽  
Ayato Takada ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Genome ◽  
Mdck Cells ◽  
Genome Segment ◽  
Influenza Viruses ◽  
Surface Glycoprotein ◽  
Limited Information ◽  
Aquatic Birds ◽  
M Proteins ◽  
Efficient Replication

H6 subtype influenza viruses are commonly isolated from wild aquatic birds. However, limited information is available regarding H6 influenza virus isolated from chickens. We compared the viral genome segment between A/chicken/Hong Kong/W312/97 (H6N1), which was able to grow in chicken trachea, and A/duck/Shantou/5540/01 (H6N2), which was isolated from wild aquatic duck, to explore the factors for effective replication in chicken. When chickens were inoculated with 7+1 reassortants (W312 background), the replication of viruses with PB2 and M genes derived from the duck strain was significantly reduced. Chimeras of PB2 and M proteins, encoding the C-terminal region of the PB2 protein and the M2 protein from W312, were required for efficient replication in canine-derived (MDCK) cells and in chicken trachea. These results indicate that host range may be determined by some types of internal proteins such as PB2 and M2, as well as by surface glycoprotein like hemagglutinin.

scholarly journals Prediction, dynamics, and visualization of antigenic phenotypes of seasonal influenza viruses

2016 ◽  
Vol 113 (12) ◽  
pp. E1701-E1709 ◽  
Author(s):  
Richard A. Neher ◽  
Trevor Bedford ◽  
Rodney S. Daniels ◽  
Colin A. Russell ◽  
Boris I. Shraiman
Keyword(s):  
Influenza Virus ◽  
Seasonal Influenza ◽  
Sequence Data ◽  
Influenza Viruses ◽  
Surface Glycoprotein ◽  
Amino Acid Substitutions ◽  
Model Output ◽  
Virus Population ◽  
Web Browser ◽  
Antigenic Properties

Human seasonal influenza viruses evolve rapidly, enabling the virus population to evade immunity and reinfect previously infected individuals. Antigenic properties are largely determined by the surface glycoprotein hemagglutinin (HA), and amino acid substitutions at exposed epitope sites in HA mediate loss of recognition by antibodies. Here, we show that antigenic differences measured through serological assay data are well described by a sum of antigenic changes along the path connecting viruses in a phylogenetic tree. This mapping onto the tree allows prediction of antigenicity from HA sequence data alone. The mapping can further be used to make predictions about the makeup of the future A(H3N2) seasonal influenza virus population, and we compare predictions between models with serological and sequence data. To make timely model output readily available, we developed a web browser-based application that visualizes antigenic data on a continuously updated phylogeny.

scholarly journals Characterization of a Porcine Lung Epithelial Cell Line Suitable for Influenza Virus Studies

2001 ◽  
Vol 75 (19) ◽  
pp. 9517-9525 ◽  
Author(s):  
Sang Heui Seo ◽  
Olga Goloubeva ◽  
Richard Webby ◽  
Robert G. Webster
Keyword(s):  
Influenza Virus ◽  
Epithelial Cell ◽  
Cell Line ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Lung Epithelial Cell ◽  
Clinical Samples ◽  
Epithelial Cell Line ◽  
Porcine Endogenous Retrovirus ◽  
Lung Epithelial

ABSTRACT We established a porcine lung epithelial cell line designated St. Jude porcine lung cells (SJPL) and demonstrated that all tested influenza A and B viruses replicated in this cell line. The infectivity titers of most viruses in SJPL cells were comparable to or better than those in MDCK cells. The propagation of influenza viruses from clinical samples in SJPL cells did not lead to antigenic changes in the hemagglutinin molecule. The numbers of both Sia2-3Gal and Sia2-6Gal receptors on SJPL cells were greater than those on MDCK cells. Influenza virus infection of SJPL cells did not lead to apoptosis, as did infection of MDCK cells. No porcine endogenous retrovirus was detected in SJPL cells, and in contrast to MDCK cells, SJPL cells did not cause tumors in nude mice.

scholarly journals Reemerging H5N1 Influenza Viruses in Hong Kong in 2002 Are Highly Pathogenic to Ducks

2004 ◽  
Vol 78 (9) ◽  
pp. 4892-4901 ◽  
Author(s):  
Katharine M. Sturm-Ramirez ◽  
Trevor Ellis ◽  
Barry Bousfield ◽  
Lucy Bissett ◽  
Kitman Dyrting ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Hong Kong ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Neurological Dysfunction ◽  
Aquatic Birds ◽  
Antigenic Analysis ◽  
Highly Pathogenic ◽  
H5n1 Influenza

ABSTRACT Waterfowl are the natural reservoir of all influenza A viruses, which are usually nonpathogenic in wild aquatic birds. However, in late 2002, outbreaks of highly pathogenic H5N1 influenza virus caused deaths among wild migratory birds and resident waterfowl, including ducks, in two Hong Kong parks. In February 2003, an avian H5N1 virus closely related to one of these viruses was isolated from two humans with acute respiratory distress, one of whom died. Antigenic analysis of the new avian isolates showed a reactivity pattern different from that of H5N1 viruses isolated in 1997 and 2001. This finding suggests that significant antigenic variation has recently occurred among H5N1 viruses. We inoculated mallards with antigenically different H5N1 influenza viruses isolated between 1997 and 2003. The new 2002 avian isolates caused systemic infection in the ducks, with high virus titers and pathology in multiple organs, particularly the brain. Ducks developed acute disease, including severe neurological dysfunction and death. Virus was also isolated at high titers from the birds' drinking water and from contact birds, demonstrating efficient transmission. In contrast, H5N1 isolates from 1997 and 2001 were not consistently transmitted efficiently among ducks and did not cause significant disease. Despite a high level of genomic homology, the human isolate showed striking biological differences from its avian homologue in a duck model. This is the first reported case of lethal influenza virus infection in wild aquatic birds since 1961.

scholarly journals Primary Swine Respiratory Epithelial Cell Lines for the Efficient Isolation and Propagation of Influenza A Viruses

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Victoria Meliopoulos ◽  
Sean Cherry ◽  
Nicholas Wohlgemuth ◽  
Rebekah Honce ◽  
Karen Barnard ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Influenza A ◽  
Vaccine Development ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Clinical Samples ◽  
Respiratory Epithelial Cells ◽  
Respiratory Epithelial

ABSTRACT Influenza virus isolation from clinical samples is critical for the identification and characterization of circulating and emerging viruses. Yet efficient isolation can be difficult. In these studies, we isolated primary swine nasal and tracheal respiratory epithelial cells and immortalized swine nasal epithelial cells (siNEC) and tracheal epithelial cells (siTEC) that retained the abilities to form tight junctions and cilia and to differentiate at the air-liquid interface like primary cells. Critically, both human and swine influenza viruses replicated in the immortalized cells, which generally yielded higher-titer viral isolates from human and swine nasal swabs, supported the replication of isolates that failed to grow in Madin-Darby canine kidney (MDCK) cells, and resulted in fewer dominating mutations during viral passaging than MDCK cells. IMPORTANCE Robust in vitro culture systems for influenza virus are critically needed. MDCK cells, the most widely used cell line for influenza isolation and propagation, do not adequately model the respiratory tract. Therefore, many clinical isolates, both animal and human, are unable to be isolated and characterized, limiting our understanding of currently circulating influenza viruses. We have developed immortalized swine respiratory epithelial cells that retain the ability to differentiate and can support influenza replication and isolation. These cell lines can be used as additional tools to enhance influenza research and vaccine development.

scholarly journals Cleavage of Influenza Virus Hemagglutinin by Airway Proteases TMPRSS2 and HAT Differs in Subcellular Localization and Susceptibility to Protease Inhibitors

2010 ◽  
Vol 84 (11) ◽  
pp. 5605-5614 ◽  
Author(s):  
Eva Böttcher-Friebertshäuser ◽  
Catharina Freuer ◽  
Frank Sielaff ◽  
Sarah Schmidt ◽  
Markus Eickmann ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Subcellular Localization ◽  
Protease Inhibitors ◽  
Transcriptional Activation ◽  
Mdck Cells ◽  
New Drugs ◽  
Surface Glycoprotein ◽  
Virus Spread ◽  
Virus Surface ◽  
Proteolytic Activation

ABSTRACT Proteolytic cleavage of the influenza virus surface glycoprotein hemagglutinin (HA) by host cell proteases is crucial for infectivity and virus spread. The proteases HAT (human airway trypsin-like protease) and TMPRSS2 (transmembrane protease serine S1 member 2) known to be present in the human airways were previously identified as proteases that cleave HA. We studied subcellular localization of HA cleavage and cleavage inhibition of seasonal influenza virus A/Memphis/14/96 (H1N1) and pandemic virus A/Hamburg/5/2009 (H1N1) in MDCK cells that express HAT and TMPRSS2 under doxycycline-induced transcriptional activation. We made the following observations: (i) HA is cleaved by membrane-bound TMPRSS2 and HAT and not by soluble forms released into the supernatant; (ii) HAT cleaves newly synthesized HA before or during the release of progeny virions and HA of incoming viruses prior to endocytosis at the cell surface, whereas TMPRSS2 cleaves newly synthesized HA within the cell and is not able to support the proteolytic activation of HA of incoming virions; and (iii) cleavage activation of HA and virus spread in TMPRSS2- and HAT-expressing cells can be suppressed by peptide mimetic protease inhibitors. The further development of these inhibitors could lead to new drugs for influenza treatment.

scholarly journals Antiviral activity of novel oseltamivir derivatives against some influenza virus strains.

2014 ◽  
Vol 61 (3) ◽  
Author(s):  
Janusz Kocik ◽  
Marcin Kołodziej ◽  
Justyna Joniec ◽  
Magdalena Kwiatek ◽  
Michał Bartoszcze
Keyword(s):  
Influenza Virus ◽  
Antiviral Activity ◽  
Mdck Cells ◽  
Antiviral Effect ◽  
In Vitro Cytotoxicity ◽  
Influenza Viruses ◽  
H3n2 Virus ◽  
High Concentration ◽  
Virus Dose

The aim of this study was to investigate the in vitro cytotoxicity of oseltamivir derivatives and determine their activity against A/H1N1/PR/8/34 and A/H3N2/HongKong/8/68 - strains of influenza virus. Antiviral activity of these compounds was determined by using two methods. MTT staining was used to assess the viability of MDCK cells infected with influenza viruses and treated with various concentrations of drugs. In parallel, the effect of drugs on viral replication was assessed using the hemagglutination test. The most toxic compounds were: OS-64, OS-35, OS-29, OS-27 and OS-25, whereas OS-11, OS-20 and OS-23 were the least toxic ones. Statistically significant antiviral effect at a higher virus dose was shown by compounds: OS-11, OS-20, OS-27, OS-35, and OS-64. H3N2 virus was sensitive to 10-times lower concentrations of OS-11 and OS-35 than H1N1. At a lower infection dose, the antiviral activity was observed for OS-11, OS 27, OS-35 and OS-20. OS-64 turned out to be effective only at a high concentration. OS-23 showed no antiviral effect.

Phylogenetic and variability study on all known hemagglutinin subtypes of influenza A virus

2017 ◽  
Vol 13 (3) ◽  
Author(s):  
Rafał Filip ◽  
Jacek Leluk
Keyword(s):  
Phylogenetic Analysis ◽  
Influenza Virus ◽  
Life Cycle ◽  
Influenza A Virus ◽  
Influenza A ◽  
Current Knowledge ◽  
Influenza Viruses ◽  
Surface Glycoprotein ◽  
Long Period ◽  
Variability Study

AbstractHemagglutinin (HA) is a surface glycoprotein found in influenza viruses. This particle plays two crucial functions in the viral life cycle: it allows for the attachment of the virus into the host cell and participates in the fusion of the virus and host membranes. There are 18 different subtypes of HA. Recently, the H17 and H18 strains have been discovered whose hosts were bats. The evolution of these two strains had most likely occurred in isolation for a long period of time. This work presents the phylogenetic analysis and study on mutational variability based on sequences from all 18 currently known HA strains belonging to influenza virus type A. The results have been presented regarding the current knowledge about influenza. The classical software (Clustal, PHYLIP, and ConSurf) as well as original applications (SSSSg and Consensus Constructor) have been used in this research.

scholarly journals Equine and Canine Influenza H3N8 Viruses Show Minimal Biological Differences Despite Phylogenetic Divergence

2015 ◽  
Vol 89 (13) ◽  
pp. 6860-6873 ◽  
Author(s):  
Kurtis H. Feng ◽  
Gaelle Gonzalez ◽  
Lingquan Deng ◽  
Hai Yu ◽  
Victor L. Tse ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Severe Disease ◽  
Biological Properties ◽  
Sialic Acids ◽  
Influenza Viruses ◽  
Host Cells ◽  
Canine Influenza ◽  
Biological Differences

ABSTRACTThe A/H3N8 canine influenza virus (CIV) emerged from A/H3N8 equine influenza virus (EIV) around the year 2000 through the transfer of a single virus from horses to dogs. We defined and compared the biological properties of EIV and CIV by examining their genetic variation, infection, and growth in different cell cultures, receptor specificity, hemagglutinin (HA) cleavage, and infection and growth in horse and dog tracheal explant cultures. Comparison of sequences of viruses from horses and dogs revealed mutations that may be linked to host adaptation and tropism. We prepared infectious clones of representative EIV and CIV strains that were similar to the consensus sequences of viruses from each host. The rescued viruses, including HA and neuraminidase (NA) double reassortants, exhibited similar degrees of long-term growth in MDCK cells. Different host cells showed various levels of susceptibility to infection, but no differences in infectivity were seen when comparing viruses. All viruses preferred α2-3- over α2-6-linked sialic acids for infections, and glycan microarray analysis showed that EIV and CIV HA-Fc fusion proteins bound only to α2-3-linked sialic acids. Cleavage assays showed that EIV and CIV HA proteins required trypsin for efficient cleavage, and no differences in cleavage efficiency were seen. Inoculation of the viruses into tracheal explants revealed similar levels of infection and replication by each virus in dog trachea, although EIV was more infectious in horse trachea than CIV.IMPORTANCEInfluenza A viruses can cross species barriers and cause severe disease in their new hosts. Infections with highly pathogenic avian H5N1 virus and, more recently, avian H7N9 virus have resulted in high rates of lethality in humans. Unfortunately, our current understanding of how influenza viruses jump species barriers is limited. Our aim was to provide an overview and biological characterization of H3N8 equine and canine influenza viruses using various experimental approaches, since the canine virus emerged from horses approximately 15 years ago. We showed that although there were numerous genetic differences between the equine and canine viruses, this variation did not result in dramatic biological differences between the viruses from the two hosts, and the viruses appeared phenotypically equivalent in most assays we conducted. These findings suggest that the cross-species transmission and adaptation of influenza viruses may be mediated by subtle changes in virus biology.

scholarly journals 993. Combining Key Residues of the Russian and US Live-Attenuated Influenza Viruses for a More Attenuated Virus

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S294-S295
Author(s):  
Andrew Smith ◽  
Laura Rodriguez-Garcia ◽  
Maya El Ghouayel ◽  
Luis Martinez-Sobrido ◽  
Stephen Dewhurst
Keyword(s):  
United States ◽  
Influenza Virus ◽  
Growth Curve ◽  
Mdck Cells ◽  
The United States ◽  
Polymerase Activity ◽  
Influenza Viruses ◽  
Renilla Luciferase ◽  
Temperature Sensitive

Abstract Background The Live Attenuated Influenza Virus (LAIV) used in the United States is based on the cold-passaged A/AnnArbor/6/60 strain (AA). An alternative LAIV (Len), developed from the cold-passaged A/Leningrad/134/17/57 strain, has also been used in some countries outside the United States. Recent concerns with the efficacy and safety of the current US LAIV warrant the development of an improved LAIV. Methods We used in vitro minireplicon and multicycle viral growth assays to analyze the combined effects of polymerase mutations from LAIV (AA) and LAIV (Len) on the phenotype of PR8. Mini-replicon assays were performed in HEK-293T cells with firefly luciferase under the control of the influenza virus NP promoter; we controlled for cell density with a constitutively active Renilla luciferase. Multicycle growth curve experiments were performed at 33°C, 37°C, and 39°C in MDCK cells with an m.o.i. of 0.001. Mean values for triplicate infections at 12, 24, 48, and 72 hours were plotted as TCID50/mL. Results Control experiments showed replication of PR8 (AA) and PR8 (Len) in MDCK cells was significantly decreased as compared with WT PR8 at 37°C and 39°C at 24–48 hour time points, but not at 33C (the temperature of nasal passages). We found that polymerase activity was up to 3 logs more temperature-sensitive (ts) at 37°C and 39°C with the combined Len and AA mutations using the mini-replicon assay. In the growth curve experiments, the combined Len and AA mutations conferred up to a 4-log decrease in replication levels at 37°C as compared with PR8 (Len) and an even greater decrease compared with PR8 (AA). Conclusion Our findings suggest combining the AA and Len LAIV polymerase mutations decreases LAIV replication at body temperature (37°C), as compared with either LAIV alone. This could be useful in developing an improved LAIV that is safer in vulnerable hosts (e.g., children under the age of 2 who may be vulnerable to wheezing), while also permitting dose escalation that might result in greater efficacy. Minireplicon assay. Polymerase activity of combination mutants. Multicycle replication kinetics of combination mutants (red) against PR8 Len (black) at 33C (solid), 37C (dashed) and 39C (dotted). Disclosures All authors: No reported disclosures.

scholarly journals Optimizing Viral Protein Yield of Influenza Virus Strain A/Vietnam/1203/2004 by Modification of the Neuraminidase Gene

2009 ◽  
Vol 83 (9) ◽  
pp. 4023-4029 ◽  
Author(s):  
Joan E. Adamo ◽  
Teresa Liu ◽  
Falko Schmeisser ◽  
Zhiping Ye
Keyword(s):  
Influenza Virus ◽  
Mdck Cells ◽  
Significant Loss ◽  
Protein Yield ◽  
Surface Glycoprotein ◽  
High Yield ◽  
Virus H5n1 ◽  
Reassortant Viruses ◽  
The Relationship ◽  
Na Gene

ABSTRACT The preparation of high-yield prepandemic influenza virus H5N1 strains has presented a challenge to both researchers and vaccine manufacturers. The reasons for the relatively low yield of the H5N1 strains are not fully understood, but it might be partially dependent on the interactions between the hemagglutinin (HA) or neuraminidase (NA) surface glycoprotein and other influenza virus proteins. In this study, we have constructed chimeras between the A/Puerto Rico/8/34 (PR8) NA gene and the A/Vietnam/1203/2004 (VN1203) NA gene that have resulted in an increase in the virus yield of the reassortant viruses without a significant loss of NA activity. By combining the amino terminus of NA from the PR8 strain with the carboxy terminus of NA from VN1203, the surface epitopes unique to the H5N1 VN1203 NA glycoprotein are maintained. This reassortant virus had a higher titer and total protein yield in eggs, grew to a higher titer, produced large plaques on MDCK cells, and retained NA activity. This work describes a novel recombinant technique designed to increase the yields of vaccine candidates for the production of pandemic influenza virus vaccines. The relationship between the infectivity and protein yield of the reassortants also is discussed.

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