Biomedical subjects

Yoshihiro Kawaoka

Publications and source records attributed to Yoshihiro Kawaoka.

At least 19 recordsLinked to original sources

PubMed2024

In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system.

The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E.coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E.coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventional E.coli cloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method enables the pathogenicity analysis and vaccine development using genetically modified viruses, and it is expected to allow for faster analysis of newly emerging variants than ever before. Furthermore, its application to other RNA viruses is also expected.

Genome, Viral↗

PubMed2007

Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus.

The 1918 influenza pandemic was unusually severe, resulting in about 50 million deaths worldwide. The 1918 virus is also highly pathogenic in mice, and studies have identified a multigenic origin of this virulent phenotype in mice. However, these initial characterizations of the 1918 virus did not address the question of its pathogenic potential in primates. Here we demonstrate that the 1918 virus caused a highly pathogenic respiratory infection in a cynomolgus macaque model that culminated in acute respiratory distress and a fatal outcome. Furthermore, infected animals mounted an immune response, characterized by dysregulation of the antiviral response, that was insufficient for protection, indicating that atypical host innate immune responses may contribute to lethality. The ability of influenza viruses to modulate host immune responses, such as that demonstrated for the avian H5N1 influenza viruses, may be a feature shared by the virulent influenza viruses.

Animals↗

PubMed2007

Mapping of the VP40-binding regions of the nucleoprotein of Ebola virus.

Expression of Ebola virus nucleoprotein (NP) in mammalian cells leads to the formation of helical structures, which serve as a scaffold for the nucleocapsid. We recently found that NP binding with the matrix protein VP40 is important for nucleocapsid incorporation into virions (T. Noda, H. Ebihara, Y. Muramoto, K. Fujii, A. Takada, H. Sagara, J. H. Kim, H. Kida, H. Feldmann, and Y. Kawaoka, PLoS Pathog. 2:e99, 2006). To identify the region(s) on the NP molecule required for VP40 binding, we examined the interaction of a series of NP deletion mutants with VP40 biochemically and ultrastructurally. We found that both termini of NP (amino acids 2 to 150 and 601 to 739) are essential for its interaction with VP40 and for its incorporation into virus-like particles (VLPs). We also found that the C terminus of NP is important for nucleocapsid incorporation into virions. Of interest is that the formation of NP helices, which involves the N-terminal 450 amino acids of NP, is dispensable for NP incorporation into VLPs. These findings enhance our understanding of Ebola virus assembly and in so doing move us closer to the identification of targets for the development of antiviral compounds to combat Ebola virus infection.

Binding Sites↗

PubMed2007

Proteolytic processing of the Ebola virus glycoprotein is not critical for Ebola virus replication in nonhuman primates.

Enveloped viruses often require cleavage of a surface glycoprotein by a cellular endoprotease such as furin for infectivity and virulence. Previously, we showed that Ebola virus glycoprotein does not require the furin cleavage motif for virus replication in cell culture. Here, we show that there are no appreciable differences in disease progression, hematology, serum biochemistry, virus titers, or lethality in nonhuman primates infected with an Ebola virus lacking the furin recognition sequence compared to those infected with wild-type virus. We conclude that glycoprotein cleavage by subtilisin-like endoproteases is not critical for Ebola virus infectivity and virulence in nonhuman primates.

Amino Acid Motifs↗

PubMed2007

Efficient vagina-to-lower respiratory tract immune trafficking in a murine model of influenza A virus infection.

Effective vaccination strategies for infectious diseases take into account the induction, long-term maintenance and recall of memory T-cell populations. To understand the immunological cross-talk within the mucosal compartments, we compared intranasal to vaginal immunization and demonstrated that vaginal infection of BALB/c mice with influenza A virus provides protective mucosal immunity against both homosubtypic and heterosubtypic virus challenge in the respiratory tract. We found that, prior to the viral challenge, in vaginally primed mice, antigen-specific CD8+ T cells were not detected in the lung airways and levels of serum antibodies were lower than those observed in intranasally immunized mice. However, following pulmonary challenge, NP147-specific CD8+ T cells were recruited and amplified in vaginally primed mice to the same extent as those in intranasally primed mice. Thus, the long-term memory immune response elicited by vaginal immunization with influenza virus is efficiently recalled and offers reasonable protection against infection in the respiratory tract.

Administration, Intranasal↗

PubMed2006

Rapid and simple detection of Ebola virus by reverse transcription-loop-mediated isothermal amplification.

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. Rapid identification of the virus is required to prevent spread of the infection. In this study, we developed and evaluated a one-step simple reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for the rapid detection of Zaire ebolavirus (ZEBOV), the most virulent species of EBOV, targeting the trailer region of the viral genome. The assay could detect 20 copies of the artificial ZEBOV RNA in 26 min with a real time-monitoring detection, and also detect 10(-3) FFU of the cell-culture propagated viruses. The reaction time needed to detect 10(4) FFU of ZEBOV was only 20 min. In addition, the assay was highly specific for ZEBOV. The RT-LAMP assay developed in this study is rapid, simple, highly specific, and sensitive for the detection of ZEBOV, and so may be an effective diagnostic tool. Furthermore, as this technique does not require sophisticated instrumentation, it seems very suitable for diagnosis in the field or laboratories in Ebola outbreak areas such as Central Africa.

Animals↗

PubMed2006

Interactions between T cells responding to concurrent mycobacterial and influenza infections.

CD4(+) T cells are central in mediating granuloma formation and limiting growth and dissemination of mycobacterial infections. To determine whether T cells responding to influenza infection can interact with T cells responding to Mycobacterium bovis bacille Calmette-Guérin (BCG) infection and disrupt granuloma formation, we infected mice containing two monoclonal T cell populations specific for the model Ags pigeon cytochrome c (PCC) and hen egg lysozyme (HEL). These mice were chronically infected with PCC epitope-tagged BCG (PCC-BCG) and acutely infected with HEL epitope-tagged influenza virus (HEL-flu). In these mice, PCC-BCG infection is much more abundant in the liver than the lung, whereas HEL-flu infection is localized to the lung. We observe that both T cells have access to both inflammatory sites, but that PCC-specific T cells dominate the PCC-BCG inflammatory site in the liver, whereas HEL-specific T cells dominate the HEL-flu inflammatory site in the lung. Influenza infection, in the absence of an influenza-specific T cell response, is able to increase the activation state and IFN-gamma secretion of PCC-BCG-specific T cells in the granuloma. Activation of HEL-specific T cells allows them to secrete IFN-gamma and contribute to protection in the granuloma. Ultimately, infection with influenza has little effect on bacterial load, and bacteria do not disseminate. In summary, these data illustrate complex interactions between T cell responses to infectious agents that can affect effector responses to pathogens.

Animals↗

PubMed2006

Lower clinical effectiveness of oseltamivir against influenza B contrasted with influenza A infection in children.

BACKGROUND: Recently, many Japanese physicians have claimed that oseltamivir is less effective in children with influenza B virus infection. This study assesses the effectiveness of oseltamivir against influenza A (H3N2) and influenza B in children on the basis of the duration of febrile illness. METHODS: We used oseltamivir to treat 127 children with influenza A (H3N2; mean age, 6.97 years [range, 1-15 years]) and 362 children with influenza B (mean age, 5.16 years [range, 1-15 years]) in outpatient clinics. The duration of fever after the start of oseltamivir therapy was compared in the influenza A group and the influenza B group. RESULTS: The mean duration of fever after the start of oseltamivir therapy was significantly greater in the influenza B group than in the influenza A (H3N2) group (2.18 days vs. 1.31 days, respectively; P<.001). The difference was marked in young children (1-5 years old; 2.37 days for the influenza B group vs. 1.42 days for the influenza A group) but was not significant among older children (11-15 years old). The 50% inhibitory concentration of oseltamivir against influenza B virus was 75.4+/-41.7 nmol/L and was substantially higher than that for type A (H3N2) virus (0.3+/-0.1 nmol/L). Only 3 (1.6%) of 192 influenza B viruses were resistant to oseltamivir. CONCLUSIONS: Oseltamivir is much less effective against influenza B virus infection in young children, probably because of the low sensitivity of influenza B viruses to oseltamivir. The effectiveness of oseltamivir against influenza B is influenced by age and host immunity. A few oseltamivir-resistant influenza B strains were isolated before the start of oseltamivir therapy.

Adolescent↗

PubMed2006

Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors.

H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-alpha2,6-galactose (SAalpha2,6Gal), whereas the latter prefer those ending in SAalpha2,3Gal (refs 3-6). A conversion from SAalpha2,3Gal to SAalpha2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.

Animals↗

PubMed2006

Contributions of two nuclear localization signals of influenza A virus nucleoprotein to viral replication.

The RNA genome of influenza A virus, which forms viral ribonucleoprotein complexes (vRNPs) with viral polymerase subunit proteins (PA, PB1, and PB2) and nucleoprotein (NP), is transcribed and replicated in the nucleus. NP, the major component of vRNPs, has at least two amino acid sequences that serve as nuclear localization signals (NLSs): an unconventional NLS (residues 3 to 13; NLS1) and a bipartite NLS (residues 198 to 216; NLS2). Although both NLSs are known to play a role in nuclear transport, their relative contributions to viral replication are poorly understood. We therefore investigated their contributions to NP subcellular/subnuclear localization, viral RNA (vRNA) transcription, and viral replication. Abolishing the unconventional NLS caused NP to localize predominantly to the cytoplasm and affected its activity in vRNA transcription. However, we were able to create a virus whose NP contained amino acid substitutions in NLS1 known to abolish its nuclear localization function, although this virus was highly attenuated. These results indicate that while the unconventional NLS is not essential for viral replication, it is necessary for efficient viral mRNA synthesis. On the other hand, the bipartite NLS, whose contribution to the nuclear transport of NP is limited, was essential for vRNA transcription and NP's nucleolar accumulation. A virus with nonfunctional NLS2 could not be generated. Thus, the bipartite NLS, but not the unconventional NLS, of NP is essential for influenza A virus replication.

Animals↗

PubMed2006

Protective efficacy of neutralizing antibodies against Ebola virus infection.

Ebola virus causes lethal hemorrhagic fever in humans and nonhuman primates, but no effective antiviral compounds are available for the treatment of this infection. The surface glycoprotein (GP) of Ebola virus is an important target of neutralizing antibodies. Although passive transfer of GP-specific antibodies has been evaluated in mouse and guinea pig models, protection was achieved only by treatment shortly before or after virus challenge. Using these animal models, we evaluated the protective efficacy of two monoclonal antibodies whose epitopes are distinct from those of the antibodies tested by others. Treatment of mice with these antibodies 2 days after challenge completely protected most of the animals; even treatment 3 or 4 days after challenge was partially effective. Although antibody treatment in the guinea pig model was not as effective as in the mouse model, single-dose treatment of guinea pigs 1 day before, or 1 or 2 days after challenge did protect some animals. Interestingly, the protective effects seen in these animal models did not correlate with the in vitro neutralizing activity of the antibodies, suggesting different mechanisms of the neutralization by these antibodies. These results underscore the potential therapeutic utility of monoclonal antibodies for postexposure treatment of Ebola virus infections.

Animals↗

PubMed2006

Strategies for developing vaccines against H5N1 influenza A viruses.

Recent outbreaks of highly pathogenic avian influenza A virus (H5N1 subtype) infections in poultry and humans (through direct contact with infected birds) have raised concerns that a new influenza pandemic might occur in the near future. Effective vaccines against H5N1 virus are, therefore, urgently needed. Reverse-genetics-based inactivated vaccines have been prepared according to World Health Organization (WHO) recommendations and are now undergoing clinical evaluation in several countries. Here, we review the current strategies for the development of H5N1 influenza vaccines, and future directions for vaccine development.

Animals↗

PubMed2006

Avian flu: influenza virus receptors in the human airway.

Although more than 100 people have been infected by H5N1 influenza A viruses, human-to-human transmission is rare. What are the molecular barriers limiting human-to-human transmission? Here we demonstrate an anatomical difference in the distribution in the human airway of the different binding molecules preferred by the avian and human influenza viruses. The respective molecules are sialic acid linked to galactose by an alpha-2,3 linkage (SAalpha2,3Gal) and by an alpha-2,6 linkage (SAalpha2,6Gal). Our findings may provide a rational explanation for why H5N1 viruses at present rarely infect and spread between humans although they can replicate efficiently in the lungs.

Animals↗

PubMed2006

Architecture of ribonucleoprotein complexes in influenza A virus particles.

In viruses, as in eukaryotes, elaborate mechanisms have evolved to protect the genome and to ensure its timely replication and reliable transmission to progeny. Influenza A viruses are enveloped, spherical or filamentous structures, ranging from 80 to 120 nm in diameter. Inside each envelope is a viral genome consisting of eight single-stranded negative-sense RNA segments of 890 to 2,341 nucleotides each. These segments are associated with nucleoprotein and three polymerase subunits, designated PA, PB1 and PB2; the resultant ribonucleoprotein complexes (RNPs) resemble a twisted rod (10-15 nm in width and 30-120 nm in length) that is folded back and coiled on itself. Late in viral infection, newly synthesized RNPs are transported from the nucleus to the plasma membrane, where they are incorporated into progeny virions capable of infecting other cells. Here we show, by transmission electron microscopy of serially sectioned virions, that the RNPs of influenza A virus are organized in a distinct pattern (seven segments of different lengths surrounding a central segment). The individual RNPs are suspended from the interior of the viral envelope at the distal end of the budding virion and are oriented perpendicular to the budding tip. This finding argues against random incorporation of RNPs into virions, supporting instead a model in which each segment contains specific incorporation signals that enable the RNPs to be recruited and packaged as a complete set. A selective mechanism of RNP incorporation into virions and the unique organization of the eight RNP segments may be crucial to maintaining the integrity of the viral genome during repeated cycles of replication.

Animals↗

PubMed2006

Human trachea primary epithelial cells express both sialyl(alpha2-3)Gal receptor for human parainfluenza virus type 1 and avian influenza viruses, and sialyl(alpha2-6)Gal receptor for human influenza viruses.

We reported previously that the dominant receptors of influenza A and B viruses, and human and murine respiroviruses, were sialylglycoproteins and gangliosides containing monosialo-lactosamine type I-and II-residues, such as sialic acid-alpha2-3(6)-Galbeta1-3(4)-GlcNAcbeta1-. In addition, the Siaalpha2-3Gal linkage was predominantly recognized by avian and horse influenza viruses, and human parainfluenza virus type 1 (hPIV-1), whereas the Siaalpha2-6Gal linkage was mainly recognized by human influenza viruses (Paulson JC in "The Receptors'' [Conn M Ed] 2, 131-219 (1985); Suzuki Y, Prog Lipid Res 33, 429-57 (1994); Ito T, J Virol 73, 6743-51 (2000); Suzuki Y, J Virol 74, 11825-31 (2000); Suzuki T, J. Virol 75, 4604-4613 (2001); Suzuki Y, Biol. Pharm. Bull. 28, 399-408 (2005)). To clarify the distribution of influenza virus receptors on the human bronchial epithelium cell surface, we investigated a primary culture of normal human bronchial epithelial (NHBE) cells using two types of lectin (MAA and SNA), which recognize sialyl linkages (alpha2-3 and alpha2-6), using fluorescence-activated cell-sorting analysis. The results showed that both alpha2-3- and alpha2-6-linked Sias were expressed on the surface of primary human bronchial epithelial cells. The cells infected by hPIV-1 bound to MAA, confirming that cells targeted by hPIV-1 have alpha2-3-linked oligosaccharides. We also compared the ability of hPIV-1 and human influenza A virus to infect primary human bronchial epithelial cells pre-treated with Siaalpha2-3Gal-specific sialidase from Salmonella typhimurium. No difference was observed in the number of sialidase pre-treated and non-treated cells infected with human influenza A virus, which binds to Siaalpha2-6Gal-linked oligosaccharides. By contrast, the number of cells infected with hPIV-1 decreased significantly upon sialidase treatment. Thus, cultured NHBE cells showed both alpha2-3-linked Sias recognized by hPIV-1 and avian influenza virus receptors, and alpha2-6-linked Sias recognized by human influenza virus receptors.

Animals↗

PubMed2006

Highly pathogenic H5N1 influenza virus causes coagulopathy in chickens.

Severe hemorrhage at multiple organs is frequently observed in chickens infected with highly pathogenic avian influenza (HPAI) A viruses. In this study we examined whether HPAI virus infection leads to coagulation disorder in chickens. Pathological examinations showed that the fibrin thrombi were formed in arterioles at the lung, associated with the viral antigens in endothelial cells of chickens infected intravenously with HPAI virus. Hematological analyses of peripheral blood collected from the chickens revealed that coagulopathy was initiated at early stage of infection when viral antigens were detected only in the endothelial cells and monocytes/macrophages. Furthermore, gene expression of the tissue factor, the main initiator of blood coagulation, was upregulated in the spleen, lung, and brain of HPAI virus-infected chickens. These results suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens.

Animals↗

PubMed2006

[Influenza pandemic--current crisis].

In most cases, influenza is not fatal, even without treatment. Moreover, vaccination and antivirals have reduced influenza-related mortality in recent years. However, the direct transmission of avian influenza viruses to humans with lethal outcomes in Hong Kong in 1997 was a potent reminder of the devastating potential of the disease. Currently, H5N1 avian influenza viruses are circulating in many Asian countries, and the human death toll continues to rise as the virus spreads to European countries as well. Since the beginning of the outbreak in Asia, more than 120 cases have been confirmed and the mortality rate has been no less than 50%. Current vaccines for H3N2 and H1N1 viruses, of course, have no effect on infection by H5N1 viruses. In addition, H5N1 viruses that are resistant to the antiviral drugs amantadine and oseltamivir have emerged. Fortunately, a virus that is capable of efficient transmission among humans has not emerged. However, it is not a matter of if, but when, such a virus will appear. Here, we review the current situation of avian influenza and pandemic preparedness.

Animals↗

PubMed2006

The cytoplasmic tail of the influenza A virus M2 protein plays a role in viral assembly.

The viral replication cycle concludes with the assembly of viral components to form progeny virions. For influenza A viruses, the matrix M1 protein and two membrane integral glycoproteins, hemagglutinin and neuraminidase, function cooperatively in this process. Here, we asked whether another membrane protein, the M2 protein, plays a role in virus assembly. The M2 protein, comprising 97 amino acids, possesses the longest cytoplasmic tail (54 residues) of the three transmembrane proteins of influenza A viruses. We therefore generated a series of deletion mutants of the M2 cytoplasmic tail by reverse genetics. We found that mutants in which more than 22 amino acids were deleted from the carboxyl terminus of the M2 tail were viable but grew less efficiently than did the wild-type virus. An analysis of the virions suggested that viruses with M2 tail deletions of more than 22 carboxy-terminal residues apparently contained less viral ribonucleoprotein complex than did the wild-type virus. These M2 tail mutants also differ from the wild-type virus in their morphology: while the wild-type virus is spherical, some of the mutants were filamentous. Alanine-scanning experiments further indicated that amino acids at positions 74 to 79 of the M2 tail play a role in virion morphogenesis and affect viral infectivity. We conclude that the M2 cytoplasmic domain of influenza A viruses plays an important role in viral assembly and morphogenesis.

Amino Acid Sequence↗