Publications (23)203.13 Total impact
-
Article: Synergistic effect of the PDZ and the p85β-binding domains of the NS1 protein in virulence of an avian H5N1 influenza A virus.
[show abstract] [hide abstract]
ABSTRACT: The influenza A virus NS1 protein affects virulence through several mechanisms including the host's innate immune response and various signaling pathways. Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype continue to evolve through reassortment and mutations. Our recent phylogenetic analysis identified a group of HPAI H5N1 viruses with two characteristic mutations in NS1: the avian virus-type PDZ domain-binding motif ESEV (which affects virulence) was replaced with ESKV, and NS1-138F (which is highly conserved among all influenza A viruses and may affect the activation of the PI3K/Akt signaling pathway) was replaced with NS1-138Y. Here, we show that an HPAI H5N1 virus (A/duck/Hunan/69/2004) encoding NS1-ESKV and NS1-138Y was confined to the respiratory tract of infected mice, whereas a mutant encoding NS1-ESEV and NS1-138F caused systemic infection and killed mice more efficiently. Mutation of either one of these sites had small effects on virulence. In addition, we found that the amino acid at NS1-138 affected not only the induction of the PI3K/Akt pathway, but also the interaction of NS1 with cellular PDZ domain proteins. Similarly, the mutation in the PDZ domain-binding motif of NS1 altered its binding to cellular PDZ domain proteins and affected Akt phosphorylation. These findings suggest a functional interplay between the mutations at NS1-138 and NS1-229 that results in a synergistic effect on influenza virulence.Journal of Virology 02/2013; · 5.40 Impact Factor -
Article: Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets.
[show abstract] [hide abstract]
ABSTRACT: Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus-comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus-that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures.Nature 06/2012; 486(7403):420-8. · 36.28 Impact Factor -
Article: Characterization in vitro and in vivo of pandemic (H1N1) 2009 influenza viruses isolated from patients.
[show abstract] [hide abstract]
ABSTRACT: The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. Molecular evolutionary analyses of the 2009 pandemic influenza A H1N1 [A(H1N1)pdm09] virus revealed two major clusters, cluster I and cluster II. Although the pathogenicity of viruses belonging to cluster I, which became extinct by the end of 2009, has been examined in a nonhuman primate model, the pathogenic potential of viruses belonging to cluster II, which has spread more widely in the world, has not been studied in this animal model. Here, we characterized two Norwegian isolates belonging to cluster II, namely, A/Norway/3568/2009 (Norway3568) and A/Norway/3487-2/2009 (Norway3487), which caused distinct clinical symptoms, despite their genetic similarity. We observed more efficient replication in cultured cells and delayed virus clearance from ferret respiratory organs for Norway3487 virus, which was isolated from a severe case, compared with the efficiency of replication and time of clearance of Norway3568 virus, which was isolated from a mild case. Moreover, Norway3487 virus to some extent caused more severe lung damage in nonhuman primates than did Norway3568 virus. Our data suggest that the distinct replicative and pathogenic potentials of these two viruses may result from differences in their biological properties (e.g., the receptor-binding specificity of hemagglutinin and viral polymerase activity).Journal of Virology 06/2012; 86(17):9361-8. · 5.40 Impact Factor -
Article: Functional analysis of conserved motifs in influenza virus PB1 protein.
[show abstract] [hide abstract]
ABSTRACT: The influenza virus RNA polymerase complex is a heterotrimer composed of the PB1, PB2, and PA subunits. PB1, the catalytic core and structural backbone of the polymerase, possesses four highly conserved amino acid motifs that are present among all viral RNA-dependent RNA polymerases. A previous study demonstrated the importance of several of these conserved amino acids in PB1 for influenza polymerase activity through mutational analysis. However, a small number of viruses isolated in nature possesses non-consensus amino acids in one of the four motifs, most of which have not been tested for their replicative ability. Here, we assessed the transcription/replication activities of 25 selected PB1 mutations found in natural isolates by using minireplicon assays in human and avian cells. Most of the mutations tested significantly reduced polymerase activity. One exception was mutation K480R, observed in several pandemic (H1N1) 2009 viruses, which slightly increased polymerase activity relative to wild-type. However, in the background of the pandemic A/California/04/2009 (H1N1) virus, this mutation did not affect virus titers in cell culture. Our results further demonstrate the functional importance of the four conserved PB1 motifs in influenza virus transcription/replication. The finding of natural isolates with non-consensus PB1 motifs that are nonfunctional in minireplicon assays suggests compensatory mutations and/or mixed infections which may have 'rescued' the inactive PB1 protein.PLoS ONE 01/2012; 7(5):e36113. · 4.09 Impact Factor -
Article: Conserved host response to highly pathogenic avian influenza virus infection in human cell culture, mouse and macaque model systems.
[show abstract] [hide abstract]
ABSTRACT: Understanding host response to influenza virus infection will facilitate development of better diagnoses and therapeutic interventions. Several different experimental models have been used as a proxy for human infection, including cell cultures derived from human cells, mice, and non-human primates. Each of these systems has been studied extensively in isolation, but little effort has been directed toward systematically characterizing the conservation of host response on a global level beyond known immune signaling cascades. In the present study, we employed a multivariate modeling approach to characterize and compare the transcriptional regulatory networks between these three model systems after infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Using this approach we identified functions and pathways that display similar behavior and/or regulation including the well-studied impact on the interferon response and the inflammasome. Our results also suggest a primary response role for airway epithelial cells in initiating hypercytokinemia, which is thought to contribute to the pathogenesis of H5N1 viruses. We further demonstrate that we can use a transcriptional regulatory model from the human cell culture data to make highly accurate predictions about the behavior of important components of the innate immune system in tissues from whole organisms. This is the first demonstration of a global regulatory network modeling conserved host response between in vitro and in vivo models.BMC Systems Biology 11/2011; 5:190. · 3.15 Impact Factor -
Article: Cross-reactive T cells are involved in rapid clearance of 2009 pandemic H1N1 influenza virus in nonhuman primates.
[show abstract] [hide abstract]
ABSTRACT: In mouse models of influenza, T cells can confer broad protection against multiple viral subtypes when antibodies raised against a single subtype fail to do so. However, the role of T cells in protecting humans against influenza remains unclear. Here we employ a translational nonhuman primate model to show that cross-reactive T cell responses play an important role in early clearance of infection with 2009 pandemic H1N1 influenza virus (H1N1pdm). To "prime" cellular immunity, we first infected 5 rhesus macaques with a seasonal human H1N1 isolate. These animals made detectable cellular and antibody responses against the seasonal H1N1 isolate but had no neutralizing antibodies against H1N1pdm. Four months later, we challenged the 5 "primed" animals and 7 naive controls with H1N1pdm. In naive animals, CD8+ T cells with an activated phenotype (Ki-67+ CD38+) appeared in blood and lung 5-7 days post inoculation (p.i.) with H1N1pdm and reached peak magnitude 7-10 days p.i. In contrast, activated T cells were recruited to the lung as early as 2 days p.i. in "primed" animals, and reached peak frequencies in blood and lung 4-7 days p.i. Interferon (IFN)-γ Elispot and intracellular cytokine staining assays showed that the virus-specific response peaked earlier and reached a higher magnitude in "primed" animals than in naive animals. This response involved both CD4+ and CD8+ T cells. Strikingly, "primed" animals cleared H1N1pdm infection significantly earlier from the upper and lower respiratory tract than the naive animals did, and before the appearance of H1N1pdm-specific neutralizing antibodies. Together, our results suggest that cross-reactive T cell responses can mediate early clearance of an antigenically novel influenza virus in primates. Vaccines capable of inducing such cross-reactive T cells may help protect humans against severe disease caused by newly emerging pandemic influenza viruses.PLoS Pathogens 11/2011; 7(11):e1002381. · 9.13 Impact Factor -
Article: Avian-type receptor-binding ability can increase influenza virus pathogenicity in macaques.
[show abstract] [hide abstract]
ABSTRACT: The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. An Asp-to-Gly change at position 222 of the receptor-binding protein hemagglutinin (HA) correlates with more-severe infections in humans. The amino acid at position 222 of HA contributes to receptor-binding specificity with Asp (typically found in human influenza viruses) and Gly (typically found in avian and classic H1N1 swine influenza viruses), conferring binding to human- and avian-type receptors, respectively. Here, we asked whether binding to avian-type receptors enhances influenza virus pathogenicity. We tested two 2009 pandemic H1N1 viruses possessing HA-222G (isolated from severe cases) and two viruses that possessed HA-222D. In glycan arrays, viruses possessing HA-222D preferentially bound to human-type receptors, while those encoding HA-222G bound to both avian- and human-type receptors. This difference in receptor binding correlated with efficient infection of viruses possessing HA-222G, compared to those possessing HA-222D, in human lung tissue, including alveolar type II pneumocytes, which express avian-type receptors. In a nonhuman primate model, infection with one of the viruses possessing HA-222G caused lung damage more severe than did infection with a virus encoding HA-222D, although these pathological differences were not observed for the other virus pair with either HA-222G or HA-222D. These data demonstrate that the acquisition of avian-type receptor-binding specificity may result in more-efficient infection of human alveolar type II pneumocytes and thus more-severe lung damage. Collectively, these findings suggest a new mechanism by which influenza viruses may become more pathogenic in mammals, including humans.Journal of Virology 09/2011; 85(24):13195-203. · 5.40 Impact Factor -
Article: Replication-incompetent influenza A viruses that stably express a foreign gene.
[show abstract] [hide abstract]
ABSTRACT: A biologically contained influenza A virus that stably expresses a foreign gene can be effectively traced, used to generate a novel multivalent vaccine and have its replication easily assessed, all while satisfying safety concerns regarding pathogenicity or reversion. This study generated a PB2-knockout (PB2-KO) influenza virus that harboured the GFP reporter gene in the coding region of its PB2 viral RNA (vRNA). Replication of the PB2-KO virus was restricted to a cell line stably expressing the PB2 protein. The GFP gene-encoding PB2 vRNA was stably incorporated into progeny viruses during replication in PB2-expressing cells. The GFP gene was expressed in virus-infected cells with no evidence of recombination between the recombinant PB2 vRNA and the PB2 protein mRNA. Furthermore, other reporter genes and the haemagglutinin and neuraminidase genes of different virus strains were accommodated by the PB2-KO virus. Finally, the PB2-KO virus was used to establish an improved assay to screen neutralizing antibodies against influenza viruses by using reporter gene expression as an indicator of virus infection rather than by observing cytopathic effect. These results indicate that the PB2-KO virus has the potential to be a valuable tool for basic and applied influenza virus research.Journal of General Virology 08/2011; 92(Pt 12):2879-88. · 3.36 Impact Factor -
Article: Host regulatory network response to infection with highly pathogenic H5N1 avian influenza virus.
[show abstract] [hide abstract]
ABSTRACT: During the last decade, more than half of humans infected with highly pathogenic avian influenza (HPAI) H5N1 viruses have died, yet virus-induced host signaling has yet to be clearly elucidated. Airway epithelia are known to produce inflammatory mediators that contribute to HPAI H5N1-mediated pathogenicity, but a comprehensive analysis of the host response in this cell type is lacking. Here, we leveraged a system approach to identify and statistically validate signaling subnetworks that define the dynamic transcriptional response of human bronchial epithelial cells after infection with influenza A/Vietnam/1203/2004 (H5N1, VN1203). Importantly, we validated a subset of transcripts from one subnetwork in both Calu-3 cells and mice. A more detailed examination of two subnetworks involved in the immune response and keratinization processes revealed potential novel mediators of HPAI H5N1 pathogenesis and host response signaling. Finally, we show how these results compare to those for a less virulent strain of influenza virus. Using emergent network properties, we provide fresh insight into the host response to HPAI H5N1 virus infection and identify novel avenues for perturbation studies and potential therapeutic interventions for fatal HPAI H5N1 disease.Journal of Virology 08/2011; 85(21):10955-67. · 5.40 Impact Factor -
Article: The nucleoprotein and matrix protein segments of H5N1 influenza viruses are responsible for dominance in embryonated eggs.
[show abstract] [hide abstract]
ABSTRACT: Since their emergence in 1996 in southern China, highly pathogenic H5N1 avian influenza viruses have spread widely and continue to circulate in some countries. Genetic reassortment has created multiple H5N1 virus lineages, some of which are dominant in nature. However, the mechanism by which certain H5N1 influenza virus lineages (or genotypes) become dominant in avian species remains unknown. Here, we used competitive inoculation and genetic analysis of the resultant viruses to show that the nucleoprotein (NP) and matrix protein (M) segments of Fujian-like viruses (clade 2.3.4), which became predominant in southern China in mid-2006, are responsible for viral dominance in embryonated eggs. We further found that specific residues in the NP and M proteins play key roles in conferring this viral dominance; specifically, a glutamic acid at position 66 in M2 was conserved among the Fujian-like viruses. These results suggest roles for these viral proteins in influenza virus dominance.Journal of General Virology 04/2011; 92(Pt 7):1645-9. · 3.36 Impact Factor -
Article: Integrative deep sequencing of the mouse lung transcriptome reveals differential expression of diverse classes of small RNAs in response to respiratory virus infection.
[show abstract] [hide abstract]
ABSTRACT: We previously reported widespread differential expression of long non-protein-coding RNAs (ncRNAs) in response to virus infection. Here, we expanded the study through small RNA transcriptome sequencing analysis of the host response to both severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza virus infections across four founder mouse strains of the Collaborative Cross, a recombinant inbred mouse resource for mapping complex traits. We observed differential expression of over 200 small RNAs of diverse classes during infection. A majority of identified microRNAs (miRNAs) showed divergent changes in expression across mouse strains with respect to SARS-CoV and influenza virus infections and responded differently to a highly pathogenic reconstructed 1918 virus compared to a minimally pathogenic seasonal influenza virus isolate. Novel insights into miRNA expression changes, including the association with pathogenic outcomes and large differences between in vivo and in vitro experimental systems, were further elucidated by a survey of selected miRNAs across diverse virus infections. The small RNAs identified also included many non-miRNA small RNAs, such as small nucleolar RNAs (snoRNAs), in addition to nonannotated small RNAs. An integrative sequencing analysis of both small RNAs and long transcripts from the same samples showed that the results revealing differential expression of miRNAs during infection were largely due to transcriptional regulation and that the predicted miRNA-mRNA network could modulate global host responses to virus infection in a combinatorial fashion. These findings represent the first integrated sequencing analysis of the response of host small RNAs to virus infection and show that small RNAs are an integrated component of complex networks involved in regulating the host response to infection. IMPORTANCE: Most studies examining the host transcriptional response to infection focus only on protein-coding genes. However, mammalian genomes transcribe many short and long non-protein-coding RNAs (ncRNAs). With the advent of deep-sequencing technologies, systematic transcriptome analysis of the host response, including analysis of ncRNAs of different sizes, is now possible. Using this approach, we recently discovered widespread differential expression of host long (>200 nucleotide [nt]) ncRNAs in response to virus infection. Here, the samples described in the previous report were again used, but we sequenced another fraction of the transcriptome to study very short (about 20 to 30 nt) ncRNAs. We demonstrated that virus infection also altered expression of many short ncRNAs of diverse classes. Putting the results of the two studies together, we show that small RNAs may also play an important role in regulating the host response to virus infection.mBio 01/2011; 2(6). · 5.31 Impact Factor -
Article: Reassortment between seasonal and swine-origin H1N1 influenza viruses generates viruses with enhanced growth capability in cell culture.
[show abstract] [hide abstract]
ABSTRACT: One year after the emergence of the 2009 influenza (H1N1) pandemic, most cases have been relatively mild; however, the possibility of enhanced viral growth ability by reassortment with seasonal viruses cannot be overlooked. Here, we show that reassortant viruses containing a seasonal H1 HA and swine-origin NA and M genes have enhanced virus growth over their wild-type parental viruses. The emergence of such viruses in nature could, therefore, represent a threat.Virus Research 12/2010; 156(1-2):147-50. · 2.94 Impact Factor -
Article: Reassortment between avian H5N1 and human H3N2 influenza viruses creates hybrid viruses with substantial virulence.
[show abstract] [hide abstract]
ABSTRACT: The spread of avian H5N1 influenza viruses around the globe has become a worldwide public health concern. To evaluate the pathogenic potential of reassortant viruses between currently cocirculating avian H5N1 and human H3N2 influenza viruses, we generated all the 254 combinations of reassortant viruses between A/chicken/South Kalimantan/UT6028/06 (SK06, H5N1) and A/Tokyo/Ut-Sk-1/07 (Tok07, H3N2) influenza viruses by reverse genetics. We found that the presence of Tok07 PB2 protein in the ribonucleoprotein (RNP) complex allowed efficient viral RNA transcription in a minigenome assay and that RNP activity played an essential role in the viability and replicative ability of the reassortant viruses. When the pathogenicity of 75 reassortant H5 viruses was tested in mice, 22 were more pathogenic than the parental SK06 virus, and three were extremely virulent. Strikingly, all 22 of these viruses obtained their PB2 segment from Tok07 virus. Further analysis showed that Tok07 PB1 alone lacked the ability to enhance the pathogenicity of the reassortant viruses but could do so by cooperating with Tok07 PB2. Our data demonstrate that reassortment between an avian H5N1 virus with low pathogenicity in mice and a human virus could result in highly pathogenic viruses and that the human virus PB2 segment functions in the background of an avian H5N1 virus, enhancing its virulence. Our findings highlight the importance of surveillance programs to monitor the emergence of human H5 reassortant viruses, especially those containing a PB2 segment of human origin.Proceedings of the National Academy of Sciences 02/2010; 107(10):4687-92. · 9.68 Impact Factor -
Article: Biological and structural characterization of a host-adapting amino acid in influenza virus.
[show abstract] [hide abstract]
ABSTRACT: Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the protein's interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals.PLoS Pathogens 01/2010; 6(8):e1001034. · 9.13 Impact Factor -
Article: In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.
[show abstract] [hide abstract]
ABSTRACT: Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.Nature 08/2009; 460(7258):1021-5. · 36.28 Impact Factor -
Article: In vitro and in vivo characterization of new swine-origin H1N1 influenza virusesnear-final version
[show abstract] [hide abstract]
ABSTRACT: Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine virusesNature 07/2009; 460(7258):1021-1025. · 36.28 Impact Factor -
Article: Recombinant fowlpox virus vector-based vaccine completely protects chickens from H5N1 avian influenza virus.
[show abstract] [hide abstract]
ABSTRACT: With the widespread presence of influenza virus H5N1 in poultry and wildlife species, particularly migrating birds, vaccination has become an important control strategy for avian influenza (AI). In this study, the immune efficacy and hemagglutination inhibition (HI) antibody responses induced by a recombinant fowlpox virus (FPV) vector-based rFPV-HA-NA vaccine was evaluated in SPF and commercial chickens. Four-week old SPF chickens vaccinated with one dose of vaccine containing 2 x 10(3) plaque forming units (PFU) of virus were completely protected from H5N1 AI virus 1 week after vaccination, and protective immunity lasted for at least 40 weeks. Two-week old commercial layer chickens were vaccinated with the rFPV-HA-NA vaccine and boosted with the same dose of vaccine following an interval of 18 weeks. The HI antibody titers higher than 4log2 lasted for 52 weeks after the booster immunization. We also examined the efficacy of the rFPV-HA-NA vaccine in SPF chickens administrated by different routes. The results showed that effective application of rFPV-HA-NA vaccine in poultry may be restricted to wing-web puncture, intramuscular or subcutaneous injection. These results demonstrate that the rFPV-HA-NA vaccine is effective in the prevention of infection of H5N1 AI virus.Antiviral research 01/2009; 81(3):234-8. · 3.61 Impact Factor -
Article: Compatibility among polymerase subunit proteins is a restricting factor in reassortment between equine H7N7 and human H3N2 influenza viruses.
[show abstract] [hide abstract]
ABSTRACT: Reassortment is an important driving force for influenza virus evolution, and a better understanding of the factors that affect this process could improve our ability to respond to future influenza pandemics and epidemics. To identify factors that restrict the generation of reassortant viruses, we cotransfected human embryonic kidney cells with plasmids for the synthesis of viral RNAs of both A/equine/Prague/1/56 (Prague; H7N7) and A/Yokohama/2017/03 (Yokohama; H3N2) viruses together with the supporting protein expression plasmids. Of the possible 256 genotypes, we identified 29 genotypes in 120 randomly plaque-picked reassortants examined. Analyses of these reassortants suggested that the formation of functional ribonucleoprotein (RNP) complexes was a restricting factor, a finding that correlated with the activities of RNP complexes composed of different combinations of the proteins from the two viruses, as measured in a minigenome assay. For at least one nonfunctional RNP complex (i.e., Prague PB2, Prague PB1, Yokohama PA, and Prague NP), the lack of activity was due to the inability of the three polymerase subunit proteins to form a heterotrimer. Adaptation of viruses possessing a gene encoding a chimera of the PA proteins of the two viruses and the remaining genes from Prague virus resulted in compensatory mutations in the PB2 and/or PA protein. These results indicate substantial incompatibility among the gene products of the two test viruses, a critical role for the RNP complex in the generation of reassortant viruses, and a functional interaction of PB2 and PA.Journal of Virology 10/2008; 82(23):11880-8. · 5.40 Impact Factor -
Article: H5N1 influenza marker vaccine for serological differentiation between vaccinated and infected chickens.
[show abstract] [hide abstract]
ABSTRACT: Using plasmid-based reverse genetics, we generated a molecularly altered virus, H5N1/PR8-5B19, containing modified HA and NA genes from A/Goose/Guangdong/1/96 (GS/GD/1/96). In the H5N1/PR8-5B19 virus, the HA cleavage site was modified to resemble that of low-pathogenic avian strains and a portion of the NA stalk region was replaced by the immunodominant 5B19 epitope of the S2 glycoprotein of murine hepatitis virus (MHV). H5N1/PR8-5B19 is not lethal to embryonated eggs or chickens. Chickens immunized with the H5N1/PR8-5B19 inactivated vaccine produced high levels of HI antibody and a measurable antibody response against the MHV 5B19 epitope, and were fully protected against subsequent challenge with different highly pathogenic H5N1 avian influenza viruses. H5N1/PR8-5B19 is therefore an attractive marker vaccine candidate, eliciting a strong, protective antibody response and enabling serological discrimination between vaccinated and wild-type virus-infected chickens.Biochemical and Biophysical Research Communications 08/2008; 372(2):293-7. · 2.48 Impact Factor -
Article: Single-amino-acid mutation in the HA alters the recognition of H9N2 influenza virus by a monoclonal antibody.
[show abstract] [hide abstract]
ABSTRACT: We explored the molecular basis of antigenic variation by comparing two H9N2 subtype avian influenza viruses, A/Chicken/Shandong/6/96 (CK/SD/6) and A/Chicken/Guangxi/10/99 (CK/GX/10), that react differently to a monoclonal antibody C/B3. To assess the genetic basis for this antigenic difference, we used reverse genetics to generate a series of chimera and mutants of these two viruses. We found that a single-amino-acid substitution of asparagine for serine at position 145 (S145N) in the HA protein prevents the reaction of CK/SD/6 virus with C/B3. Substitution of serine for asparagine at the same position (N145S) enables the CK/GX/10 to react with C/B3 in hemaglutinin inhibition, immunofluorescence and neutralization assays. We further demonstrated that the amino acid N145 in the H9 HA protein is glycosylated. Our results provide experimental evidence that the glycosylation of HA oligosaccharide attachment sites implicated in antibody binding could have a role in antigenic variation.Biochemical and Biophysical Research Communications 07/2008; 371(1):168-71. · 2.48 Impact Factor
Top co-authors
Top Journals
Institutions
-
2009–2013
-
University of Wisconsin, Madison
- • Department of Pathobiological Sciences
- • School of Veterinary Medicine
Madison, MS, USA -
Shiga University of Medical Science
- Department of Pathology
Ōtsu-shi, Shiga-ken, Japan
-
-
2008–2011
-
The University of Tokyo
- Institute of Medical Science
Tokyo, Tokyo-to, Japan
-
-
2005–2008
-
Harbin Veterinary Research Institute
Harbin, Heilongjiang Sheng, China
-
