scholarly journals Influenza B Virus Requires BM2 Protein for Replication

2004 ◽  
Vol 78 (11) ◽  
pp. 5576-5583 ◽  
Author(s):  
Masato Hatta ◽  
Hideo Goto ◽  
Yoshihiro Kawaoka
Keyword(s):  
Reverse Genetics ◽  
Mdck Cells ◽  
Open Reading Frame ◽  
Initiation Codon ◽  
Influenza B Virus ◽  
Influenza B ◽  
M Gene ◽  
Reading Frame ◽  
Sialidase Activity ◽  
B Virus

ABSTRACT The BM2 protein of influenza B virus functions as an ion channel, which is suggested to be important for virus uncoating in endosomes of virus-infected cells. Because direct support for this function is lacking, whether BM2 plays an essential role in the viral life cycle remains unknown. We therefore attempted to generate BM2 knockout viruses by reverse genetics. Mutant viruses possessing M segments with the mutated initiation codon of BM2 protein at the stop-start pentanucleotide were viable and still expressed BM2. The introduction of multiple stop codons and a one-nucleotide deletion downstream of the stop-start pentanucleotide, in addition to disablement of the BM2 initiation codon, failed to generate viable mutant viruses, but the mutant M segments still expressed proteins that reacted with the BM2 peptide antiserum. To completely abolish BM2 expression, we generated a mutant M gene whose BM2 open reading frame was deleted. Although this mutant was not able to replicate in normal MDCK cells, it did replicate in a cell line that we established which constitutively expresses BM2. Furthermore, a virus possessing the mutant M gene lacking the BM2 open reading frame and a mutant NA gene containing the BM2 open reading frame instead of the NA open reading frame underwent multiple cycles of replication in MDCK cells, with exogenous sialidase used to supplement the deleted viral sialidase activity. These findings demonstrate that the BM2 protein is essential for influenza B virus replication.

scholarly journals Region Required for Protein Expression from the Stop-Start Pentanucleotide in the M Gene of Influenza B Virus

2009 ◽  
Vol 83 (11) ◽  
pp. 5939-5942 ◽  
Author(s):  
Masato Hatta ◽  
Candice K. Kohlmeier ◽  
Yasuko Hatta ◽  
Makoto Ozawa ◽  
Yoshihiro Kawaoka
Keyword(s):  
Protein Expression ◽  
Stop Codon ◽  
Initiation Codon ◽  
Influenza B Virus ◽  
Influenza B ◽  
Coding Region ◽  
M Gene ◽  
Coding Sequences ◽  
B Virus ◽  
Efficient Expression

ABSTRACT Segment 7 of influenza B virus encodes two proteins, M1 and BM2. BM2 is expressed from a stop-start pentanucleotide, in which the BM2 initiation codon overlaps with the M1 stop codon. Here, we demonstrate that 45 nucleotides of the 3′ end of the M1 coding region, but not the 5′ end of the BM2 coding region, are sufficient for the efficient expression of the downstream protein. Placing these 45 nucleotides and the stop-start pentanucleotide in between the coding sequences induced the expression of at least three noninfluenza proteins, suggesting the utility of this system for expressing multiple proteins from one mRNA.

scholarly journals Influenza B Virus NS1-Truncated Mutants: Live-Attenuated Vaccine Approach

2008 ◽  
Vol 82 (21) ◽  
pp. 10580-10590 ◽  
Author(s):  
Rong Hai ◽  
Luis Martínez-Sobrido ◽  
Kathryn A. Fraser ◽  
Juan Ayllon ◽  
Adolfo García-Sastre ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Influenza Viruses ◽  
Influenza B Virus ◽  
Influenza B ◽  
Type I ◽  
Live Attenuated Vaccine ◽  
Live Virus ◽  
B Virus

ABSTRACT Type B influenza viruses can cause substantial morbidity and mortality in the population, and vaccination remains by far the best means of protection against infections with these viruses. Here, we report the construction of mutant influenza B viruses for potential use as improved live-virus vaccine candidates. Employing reverse genetics, we altered the NS1 gene, which encodes a type I interferon (IFN) antagonist. The resulting NS1 mutant viruses induced IFN and, as a consequence, were found to be attenuated in vitro and in vivo. The absence of pathogenicity of the NS1 mutants in both BALB/c and C57BL/6 PKR−/− mice was confirmed. We also provide evidence that influenza B virus NS1 mutants induce a self-adjuvanted immune response and confer effective protection against challenge with both homologous and heterologous B virus strains in mice.

scholarly journals A Reverse Genetics Approach for Recovery of Recombinant Influenza B Viruses Entirely from cDNA

2002 ◽  
Vol 76 (22) ◽  
pp. 11744-11747 ◽  
Author(s):  
David Jackson ◽  
Andrew Cadman ◽  
Thomas Zurcher ◽  
Wendy S. Barclay
Keyword(s):  
Amino Acid ◽  
Influenza A ◽  
Amino Acid Change ◽  
Reverse Genetics ◽  
Single Gene ◽  
Neuraminidase Inhibitor ◽  
Influenza B Virus ◽  
Influenza B ◽  
B Virus

ABSTRACT The recovery of recombinant influenza A virus entirely from cDNA was recently described (9, 19). We adapted the technique for engineering influenza B virus and generated a mutant bearing an amino acid change E116G in the viral neuraminidase which was resistant in vitro to the neuraminidase inhibitor zanamivir. The method also facilitates rapid isolation of single-gene reassortants suitable as vaccine seeds and will aid further investigations of unique features of influenza B virus.

scholarly journals Universal Influenza B Virus Genomic Amplification Facilitates Sequencing, Diagnostics, and Reverse Genetics

2014 ◽  
Vol 52 (5) ◽  
pp. 1330-1337 ◽  
Author(s):  
B. Zhou ◽  
X. Lin ◽  
W. Wang ◽  
R. A. Halpin ◽  
J. Bera ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Influenza B Virus ◽  
Influenza B ◽  
Genomic Amplification ◽  
B Virus

scholarly journals Mechanism of Uncoating of Influenza B Virus in MDCK Cells: Action of Chloroquine

1983 ◽  
Vol 64 (5) ◽  
pp. 1149-1156 ◽  
Author(s):  
M. Shibata ◽  
H. Aoki ◽  
T. Tsurumi ◽  
Y. Sugiura ◽  
Y. Nishiyama ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Influenza B Virus ◽  
Influenza B ◽  
B Virus

Efficient influenza B virus propagation due to deficient interferon-induced antiviral activity in MDCK cells

Vaccine ◽  
2011 ◽  
Vol 29 (41) ◽  
pp. 7125-7129 ◽  
Author(s):  
Timo Frensing ◽  
Claudius Seitz ◽  
Bjoern Heynisch ◽  
Corinna Patzina ◽  
Georg Kochs ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Mdck Cells ◽  
Influenza B Virus ◽  
Influenza B ◽  
Virus Propagation ◽  
B Virus

scholarly journals Influenza B Virus BM2 Protein Is a Crucial Component for Incorporation of Viral Ribonucleoprotein Complex into Virions during Virus Assembly

2004 ◽  
Vol 78 (20) ◽  
pp. 11007-11015 ◽  
Author(s):  
Masaki Imai ◽  
Shinji Watanabe ◽  
Ai Ninomiya ◽  
Masatsugu Obuchi ◽  
Takato Odagiri
Keyword(s):  
Influenza A ◽  
Reverse Genetics ◽  
Virus Assembly ◽  
Growth Cycle ◽  
Host Cells ◽  
Influenza B Virus ◽  
Influenza B ◽  
Ribonucleoprotein Complex ◽  
B Virus ◽  
Knockout Virus

ABSTRACT Influenza B virus contains four integral membrane proteins in its envelope. Of these, BM2 has recently been found to have ion channel activity and is considered to be a functional counterpart to influenza A virus M2, but the role of BM2 in the life cycle of influenza B virus remains unclear. In an effort to explore its function, a number of BM2 mutant viruses were generated by using a reverse genetics technique. The BM2ΔATG mutant virus synthesized BM2 at markedly lower levels but exhibited similar growth to wild-type (wt) virus. In contrast, the BM2 knockout virus, which did not produce BM2, did not grow substantially but was able to grow normally when BM2 was supplemented in trans by host cells expressing BM2. These results indicate that BM2 is a required component for the production of infectious viruses. In the one-step growth cycle, the BM2 knockout virus produced progeny viruses lacking viral ribonucleoprotein complex (vRNP). The inhibited incorporation of vRNP was regained by trans-supplementation of BM2. An immunofluorescence study of virus-infected cells revealed that distribution of hemagglutinin, nucleoprotein, and matrix (M1) protein of the BM2 knockout virus at the apical membrane did not differ from that of wt virus, whereas the sucrose gradient flotation assay revealed that the membrane association of M1 was greatly affected in the absence of BM2, resulting in a decrease of vRNP in membrane fractions. These results strongly suggest that BM2 functions to capture the M1-vRNP complex at the virion budding site during virus assembly.

scholarly journals Chimeric Influenza A Viruses with a Functional Influenza B Virus Neuraminidase or Hemagglutinin

2003 ◽  
Vol 77 (17) ◽  
pp. 9116-9123 ◽  
Author(s):  
Astrid Flandorfer ◽  
Adolfo García-Sastre ◽  
Christopher F. Basler ◽  
Peter Palese
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
Type A ◽  
Influenza B Virus ◽  
Influenza B ◽  
Type B ◽  
Wild Type ◽  
B Virus

ABSTRACT Reassortment of influenza A and B viruses has never been observed in vivo or in vitro. Using reverse genetics techniques, we generated recombinant influenza A/WSN/33 (WSN) viruses carrying the neuraminidase (NA) of influenza B virus. Chimeric viruses expressing the full-length influenza B/Yamagata/16/88 virus NA grew to titers similar to that of wild-type influenza WSN virus. Recombinant viruses in which the cytoplasmic tail or the cytoplasmic tail and the transmembrane domain of the type B NA were replaced with those of the type A NA were impaired in tissue culture. This finding correlates with reduced NA content in virions. We also generated a recombinant influenza A virus expressing a chimeric hemagglutinin (HA) protein in which the ectodomain is derived from type B/Yamagata/16/88 virus HA, whereas both the cytoplasmic and the transmembrane domains are derived from type A/WSN virus HA. This A/B chimeric HA virus did not grow efficiently in MDCK cells. However, after serial passage we obtained a virus population that grew to titers as high as wild-type influenza A virus in MDCK cells. One amino acid change in position 545 (H545Y) was found to be responsible for the enhanced growth characteristics of the passaged virus. Taken together, we show here that the absence of reassortment between influenza viruses belonging to different A and B types is not due to spike glycoprotein incompatibility at the level of the full-length NA or of the HA ectodomain.

scholarly journals Analysis of the desialidation process of the haemagglutinin protein of influenza B virus: the host-dependent desialidation step

2002 ◽  
Vol 83 (7) ◽  
pp. 1729-1734 ◽  
Author(s):  
C. Luo ◽  
E. Nobusawa ◽  
K. Nakajima
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Influenza B Virus ◽  
Influenza B ◽  
Dependent Manner ◽  
Cos Cells ◽  
Trans Golgi Network ◽  
B Virus ◽  
Golgi Network ◽  
Ha Protein

It was reported previously that haemadsorption by the haemagglutinin (HA) protein of influenza B virus required that the protein must undergo desialidation. When MDCK and COS cells were infected with influenza B/Kanagawa/73 virus in the presence of a neuraminidase (NA) inhibitor, Zanamivir, haemadsorption on MDCK cells was inhibited but that on COS cells was not. The activity of the NA protein of the two types of infected cells was similar and both were inhibited by Zanamivir in a dose-dependent manner. A comparison of the desialidation of the HA protein was made on MDCK and COS cells in the presence of bacterial NA and both cells were found to have similar sensitivity. On the accumulation of the HA and NA proteins in the trans-Golgi network of MDCK cells by means of low-temperature treatment, desialidation of the HA protein in the presence of Zanamivir was detected by two-dimensional gel electrophoresis. Because this agent was reported to be unable to penetrate cells, these data suggest that, in MDCK cells, desialidation of the HA protein occurs on the cell surface but, in COS cells, the HA and NA proteins might accumulate in the trans-Golgi network, thus allowing NA desialidation before their migration to the cell surface.

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