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Showing papers on "Influenza A virus published in 2002"


Journal ArticleDOI
TL;DR: The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation in embryonated chicken eggs and hemagglutinin subtyping by hemagGLutination inhibition (HI) assay.
Abstract: A real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype-specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 103 to 104 gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI.

1,531 citations


Journal ArticleDOI
TL;DR: The H5N1/97 viruses induced much higher gene transcription of proinflammatory cytokines than did H3N2 or H1N1 viruses, particularly TNF alpha and interferon beta, which may contribute to the unusual severity of human H 5N1 disease.

895 citations


Journal ArticleDOI
TL;DR: It is shown that lethal H5N1 influenza virus, unlike other human, avian and swine influenza viruses, are resistant to the antiviral effects of interferons and tumor necrosis factor α, and the nonstructural (NS) gene of H5n1 viruses is associated with this resistance.
Abstract: The H5N1 influenza viruses transmitted to humans in 1997 were highly virulent, but the mechanism of their virulence in humans is largely unknown. Here we show that lethal H5N1 influenza viruses, unlike other human, avian and swine influenza viruses, are resistant to the antiviral effects of interferons and tumor necrosis factor alpha. The nonstructural (NS) gene of H5N1 viruses is associated with this resistance. Pigs infected with recombinant human H1N1 influenza virus that carried the H5N1 NS gene experienced significantly greater and more prolonged viremia, fever and weight loss than did pigs infected with wild-type human H1N1 influenza virus. These effects required the presence of glutamic acid at position 92 of the NS1 molecule. These findings may explain the mechanism of the high virulence of H5N1 influenza viruses in humans.

659 citations


Journal ArticleDOI
TL;DR: Reactive hemophagocytic syndrome was the most characteristic pathologic finding and might have contributed to the lymphopenia, liver dysfunction, and abnormal clotting profiles that were observed among patients with severe infection.
Abstract: The first outbreak of avian influenza A(H5N1) virus in humans occurred in Hong Kong in 1997. Infection was confirmed in 18 individuals, 6 of whom died. Infections were acquired by humans directly from chickens, without the involvement of an intermediate host. The outbreak was halted by a territory-wide slaughter of more than 1.5 million chickens at the end of December 1997. The clinical spectrum of H5N1 infection ranges from asymptomatic infection to fatal pneumonitis and multiple organ failure. Reactive hemophagocytic syndrome was the most characteristic pathologic finding and might have contributed to the lymphopenia, liver dysfunction, and abnormal clotting profiles that were observed among patients with severe infection. Rapid diagnosis with the use of reverse-transcription polymerase chain reaction and monoclonal antibody-based immunofluorescent assay were of great clinical value in the management of the outbreak. The experience of the H5N1 outbreak in Hong Kong underscores the importance of continuous surveillance of influenza virus strains in humans and in other animal species.

452 citations


Journal ArticleDOI
19 Aug 2002-Vaccine
TL;DR: These findings demonstrate that the eight-plasmid system allows the rapid and reproducible generation of reassortant influenza A viruses for use in the manufacture of vaccines.

443 citations


Journal ArticleDOI
TL;DR: Data suggest that PAFr-independent pathways are operative in the model, prompting further study of receptor interactions during pneumonia and bacteremia, and the model of lethal synergism will be a useful tool for exploring this and other mechanisms underlying viral-bacterial interactions.
Abstract: A lethal synergism exists between influenza virus and pneumococcus, which likely accounts for excess mortality from secondary bacterial pneumonia during influenza epidemics. Characterization of a mouse model of synergy revealed that influenza infection preceding pneumococcal challenge primed for pneumonia and led to 100% mortality. This effect was specific for viral infection preceding bacterial infection, because reversal of the order of administration led to protection from influenza and improved survival. The hypothesis that influenza up-regulates the platelet-activating factor receptor (PAFr) and thereby potentiates pneumococcal adherence and invasion in the lung was examined in the model. Groups of mice receiving CV-6209, a competitive antagonist of PAFr, had survival rates similar to those of control mice, and lung and blood bacterial titers increased during PAFr inhibition. These data suggest that PAFr-independent pathways are operative in the model, prompting further study of receptor interactions during pneumonia and bacteremia. The model of lethal synergism will be a useful tool for exploring this and other mechanisms underlying viral-bacterial interactions.

410 citations


Journal ArticleDOI
TL;DR: The results suggest that the cellular response to influenza A virus infection in human lung cells is significantly influenced by the sequence of theNS1 gene, demonstrating the importance of the NS1 protein in regulating the host cell response triggered by virus infection.
Abstract: The NS1 protein of influenza A virus contributes to viral pathogenesis, primarily by enabling the virus to disarm the host cell type IFN defense system. We examined the downstream effects of NS1 protein expression during influenza A virus infection on global cellular mRNA levels by measuring expression of over 13,000 cellular genes in response to infection with wild-type and mutant viruses in human lung epithelial cells. Influenza A/PR/8/34 virus infection resulted in a significant induction of genes involved in the IFN pathway. Deletion of the viral NS1 gene increased the number and magnitude of expression of cellular genes implicated in the IFN, NF-κB, and other antiviral pathways. Interestingly, different IFN-induced genes showed different sensitivities to NS1-mediated inhibition of their expression. A recombinant virus with a C-terminal deletion in its NS1 gene induced an intermediate cellular mRNA expression pattern between wild-type and NS1 knockout viruses. Most significantly, a virus containing the 1918 pandemic NS1 gene was more efficient at blocking the expression of IFN-regulated genes than its parental influenza A/WSN/33 virus. Taken together, our results suggest that the cellular response to influenza A virus infection in human lung cells is significantly influenced by the sequence of the NS1 gene, demonstrating the importance of the NS1 protein in regulating the host cell response triggered by virus infection.

392 citations


Journal ArticleDOI
TL;DR: It is demonstrated that influenza virus may enter and infect HeLa cells that are unable to take up ligands by clathrin-mediated endocytosis, and is believed to be the first conclusive analysis of virus entry via such a non-clathrin -dependent, non-caveola-dependent endocytic pathway.
Abstract: Influenza virus has been described to enter host cells via clathrin-mediated endocytosis. However, it has also been suggested that other endocytic routes may provide additional entry pathways. Here we show that influenza virus may enter and infect HeLa cells that are unable to take up ligands by clathrin-mediated endocytosis. By overexpressing a dominant-negative form of the Eps15 protein to inhibit clathrin-mediated endocytosis, we demonstrate that while transferrin uptake and Semliki Forest virus infection were prevented, influenza virus could enter and infect cells expressing Eps15Δ95/295. This finding is supported by the successful infection of cells with influenza virus in the presence of chemical treatments that block endocytosis, namely, chlorpromazine and potassium depletion. We show also that influenza virus may infect cells incapable of uptake by caveolae. Treatment with the inhibitors nystatin, methyl-β-cyclodextrin, and genistein, as well as transfection of cells with dominant-negative caveolin-1, had no effect on influenza virus infection. By combining inhibitory methods to block both clathrin-mediated endocytosis and uptake by caveolae in the same cell, we demonstrate that influenza virus may infect cells by an additional non-clathrin-dependent, non-caveola-dependent endocytic pathway. We believe this to be the first conclusive analysis of virus entry via such a non-clathrin-dependent pathway, in addition to the traditional clathrin-dependent route.

378 citations


Journal ArticleDOI
TL;DR: Virus carrying H274Y NA enzyme selected in vivo has reduced sensitivity to oseltamivir carboxylate, and pathogenicity of the mutant virus is significantly compromised in ferret, compared to the corresponding wild type virus.

371 citations


Journal ArticleDOI
TL;DR: The early detection of H5N1 viruses in the retail, live poultry markets led to preemptive intervention before the occurrence of human disease, but these newly emerging, highly pathogenic H5n1 viruses provide cause for pandemic concern.
Abstract: Although A/Hong Kong/156/97 (H5N1/97)-like viruses associated with the "bird flu" incident in Hong Kong SAR have not been detected since the slaughter of poultry in 1997, its putative precursors continue to persist in the region. One of these, Goose/Guangdong/1/96 (H5N1 Gs/Gd)-like viruses, reassorted with other avian viruses to generate multiple genotypes of H5N1 viruses that crossed to chickens and other terrestrial poultry from its reservoir in geese. Whereas none of these recent reassortants had acquired the gene constellation of H5N1/97, these events provide insight into how such a virus may have been generated. The recent H5N1 reassortants readily infect and kill chicken and quail after experimental infection, and some were associated with significant mortality of chickens within the poultry retail markets in Hong Kong. Some genotypes are lethal for mice after intra-nasal inoculation and spread to the brain. On this occasion, the early detection of H5N1 viruses in the retail, live poultry markets led to preemptive intervention before the occurrence of human disease, but these newly emerging, highly pathogenic H5N1 viruses provide cause for pandemic concern.

367 citations


Journal ArticleDOI
TL;DR: It is demonstrated that both H5N1 viruses were highly virulent in the outbred ferret model, unlike the differential pathogenicity documented in inbred BALB/c mice.
Abstract: Highly pathogenic avian influenza A H5N1 viruses caused outbreaks of disease in domestic poultry and humans in Hong Kong in 1997. Direct transmission of the H5N1 viruses from birds to humans resulted in 18 documented cases of respiratory illness, including six deaths. Here we evaluated two of the avian H5N1 viruses isolated from humans for their ability to replicate and cause disease in outbred ferrets. A/Hong Kong/483/97 virus was isolated from a fatal case and was highly pathogenic in the BALB/c mouse model, whereas A/Hong Kong/486/97 virus was isolated from a case with mild illness and exhibited a low-pathogenicity phenotype in mice. Ferrets infected intranasally with 107 50% egg infectious doses (EID50) of either H5N1 virus exhibited severe lethargy, fever, weight loss, transient lymphopenia, and replication in the upper and lower respiratory tract, as well as multiple systemic organs, including the brain. Gastrointestinal symptoms were seen in some animals. In contrast, weight loss and severe lethargy were not noted in ferrets infected with 107 EID50 of two recent human H3N2 viruses, although these viruses were also isolated from the brains, but not other extrapulmonary organs, of infected animals. The results demonstrate that both H5N1 viruses were highly virulent in the outbred ferret model, unlike the differential pathogenicity documented in inbred BALB/c mice. We propose the ferret as an alternative model system for the study of these highly pathogenic avian viruses.

Journal ArticleDOI
19 Aug 2002-Vaccine
TL;DR: This hypothesis provides a different way to view and to probe the enigma of pandemic influenza, and suggests that wild viruses of man acquire a new HA ligand of avian origin more frequently in nature than the authors are presently aware.

Journal ArticleDOI
TL;DR: Though the capacity to mediate the CD8+-T-cell effector function is broadly preserved in the absence of concurrent CD4+- T-cell help, both the maintenance and recall of memory are compromised and the clearance of residual virus is delayed.
Abstract: The primary influenza A virus-specific CD8(+)-T-cell responses measured by tetramer staining of spleen, lymph node, and bronchoalveolar lavage (BAL) lymphocyte populations were similar in magnitude for conventional I-A(b+/+) and CD4(+)-T-cell-deficient I-A(b-/-) mice. Comparable levels of virus-specific cytotoxic-T-lymphocyte activity were detected in the inflammatory exudate recovered by BAL following challenge. However, both the size of the memory T-cell pool and the magnitude of the recall response in the lymphoid tissues (but not the BAL specimens) were significantly diminished in mice lacking the CD4(+) subset. Also, the rate of virus elimination from the infected respiratory tract slowed at low virus loads following challenge of naive and previously immunized I-A(b-/-) mice. Thus, though the capacity to mediate the CD8(+)-T-cell effector function is broadly preserved in the absence of concurrent CD4(+)-T-cell help, both the maintenance and recall of memory are compromised and the clearance of residual virus is delayed. These findings are consistent with mathematical models that predict virus-host dynamics in this, and other, models of infection.

Journal ArticleDOI
TL;DR: More-intensive poultry exposure, such as butchering and exposure to ill poultry, was associated with having anti-H5 antibody, and an increased risk for avian influenza infection from occupational exposure is suggested.
Abstract: In 1997, outbreaks of highly pathogenic influenza A (H5N1) among poultry coincided with 18 documented human cases of H5N1 illness. Although exposure to live poultry was associated with human illness, no cases were documented among poultry workers (PWs). To evaluate the potential for avian-to-human transmission of H5N1, a cohort study was conducted among 293 Hong Kong government workers (GWs) who participated in a poultry culling operation and among 1525 PWs. Paired serum samples collected from GWs and single serum samples collected from PWs were considered to be anti-H5 antibody positive if they were positive by both microneutralization and Western blot testing. Among GWs, 3% were seropositive, and 1 seroconversion was documented. Among PWs, approximately 10% had anti-H5 antibody. More-intensive poultry exposure, such as butchering and exposure to ill poultry, was associated with having anti-H5 antibody. These findings suggest an increased risk for avian influenza infection from occupational exposure.

Journal ArticleDOI
TL;DR: A method for predicting future dominant HA amino acid sequences is introduced and its potential relevance to vaccine choice is discussed, as well as the relationship between cluster structure and the primary antibody-combining regions of the HA protein.
Abstract: Continual mutations to the hemagglutinin (HA) gene of influenza A virus generate novel antigenic strains that cause annual epidemics. Using a database of 560 viral RNA sequences, we study the structure and tempo of HA evolution over the past two decades. We detect a critical length scale, in amino acid space, at which HA sequences aggregate into clusters, or swarms. We investigate the spatio-temporal distribution of viral swarms and compare it to the time series of the influenza vaccines recommended by the World Health Organization. We introduce a method for predicting future dominant HA amino acid sequences and discuss its potential relevance to vaccine choice. We also investigate the relationship between cluster structure and the primary antibody-combining regions of the HA protein.

Journal ArticleDOI
TL;DR: The M2 ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle.
Abstract: The amantadine-sensitive ion channel activity of influenza A virus M2 protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2 protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2 ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656-5662, 2001). An M2 protein containing a deletion in the transmembrane (TM) domain (M2-del(29-31)) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del(29-31) virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2 TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2 residue N31 is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del(29-31) virus. We found that the M2-del(29-31) virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del(29-31) virus in 4 days after cocultivation of a 100:1 ratio of M2-del(29-31) virus to wt virus, respectively. We conclude that the M2 ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle.

Journal ArticleDOI
TL;DR: Structural and sequence comparisons suggest that HA subtypes may have originated by diversification of properties that affected the metastability of HAs required for their membrane fusion activities in viral infection.
Abstract: There are 15 subtypes of influenza A virus (H1–H15), all of which are found in avian species. Three caused pandemics in the last century: H1 in 1918 (and 1977), H2 in 1957 and H3 in 1968. In 1997, an H5 avian virus and in 1999 an H9 virus caused outbreaks of respiratory disease in Hong Kong. We have determined the three-dimensional structures of the haemagglutinins (HAs) from H5 avian and H9 swine viruses closely related to the viruses isolated from humans in Hong Kong. We have compared them with known structures of the H3 HA from the virus that caused the 1968 H3 pandemic and of the HA–esterase–fusion (HEF) glycoprotein from an influenza C virus. Structure and sequence comparisons suggest that HA subtypes may have originated by diversification of properties that affected the metastability of HAs required for their membrane fusion activities in viral infection.

Journal ArticleDOI
TL;DR: TNF-α showed strong antiviral activity against avian, swine, and human influenza viruses, and the antiviral effect of TNF- α was greater than that of gamma or alpha interferon.
Abstract: Previous studies have associated influenza virus-induced expression of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), with influenza pathogenesis in the human respiratory tract and have suggested that alpha and beta interferons are the first cytokines recruited to counteract such infection. However, we report here that TNF-α has powerful anti-influenza virus activity. When infected with influenza virus, cultured porcine lung epithelial cells expressed TNF-α in a dose-dependent manner. Expression of TNF-α was induced only by replicating virus. TNF-α showed strong antiviral activity against avian, swine, and human influenza viruses, and the antiviral effect of TNF-α was greater than that of gamma or alpha interferon. These findings suggest that TNF-α serves as the first line of defense against influenza virus infection in the natural host.

Journal ArticleDOI
TL;DR: Data indicate that the IFN antagonistic NS1 protein of influenza A viruses has IFN-dependent antiapoptotic potential.
Abstract: Wild-type (WT) influenza A/PR/8/34 virus and its variant lacking the NS1 gene (delNS1) have been compared for their ability to mediate apoptosis in cultured cells and chicken embryos. Cell morphology, fragmentation of chromatin DNA, and caspase-dependent cleavage of the viral NP protein have been used as markers for apoptosis. Another marker was caspase cleavage of the viral M2 protein, which was also found to occur in an apoptosis-specific manner. In interferon (IFN)-competent host systems, such as MDCK cells, chicken fibroblasts, and 7-day-old chicken embryos, delNS1 virus induced apoptosis more rapidly and more efficiently than WT virus. As a consequence, delNS1 virus was also more lethal for chicken embryos than WT virus. In IFN-deficient Vero cells, however, apoptosis was delayed and developed with similar intensity after infection with both viruses. Taken together, these data indicate that the IFN antagonistic NS1 protein of influenza A viruses has IFN-dependent antiapoptotic potential.

Journal ArticleDOI
TL;DR: The results demonstrate that the eight-plasmid system can be used for the generation of high yields of influenza B virus vaccines expressing current HA and NA glycoproteins from either of the two lineages of influenza A virus.
Abstract: Influenza B virus causes a significant amount of morbidity and mortality, yet the systems to produce high yield inactivated vaccines for these viruses have lagged behind the development of those for influenza A virus. We have established a plasmid-only reverse genetics system for the generation of recombinant influenza B virus that facilitates the generation of vaccine viruses without the need for time consuming coinfection and selection procedures currently required to produce reassortants. We cloned the eight viral cDNAs of influenza B/Yamanashi/166/98, which yields relatively high titers in embryonated chicken eggs, between RNA polymerase I and RNA polymerase II transcription units. Virus was detected as early as 3 days after transfection of cocultured COS7 and Madin-Darby canine kidney cells and achieved levels of 10(6)-10(7) plaque-forming units per ml of cell supernatant 6 days after transfection. The full-length sequence of the recombinant virus after passage into embryonated chicken eggs was identical to that of the input plasmids. To improve the utility of the eight-plasmid system for generating 6 + 2 reassortants from recently circulating influenza B strains, we optimized the reverse transcriptase-PCR for cloning of the hemagglutinin (HA) and neuraminidase (NA) segments. The six internal genes of B/Yamanashi/166/98 were used as the backbone to generate 6 + 2 reassortants including the HA and NA gene segments from B/Victoria/504/2000, B/Hong Kong/330/2001, and B/Hawaii/10/2001. Our results demonstrate that the eight-plasmid system can be used for the generation of high yields of influenza B virus vaccines expressing current HA and NA glycoproteins from either of the two lineages of influenza B virus.

Journal ArticleDOI
TL;DR: The high degree of susceptibility of quail to Go/Gd (H5N1)-like viruses and the continued circulation of H6N1 and H9N2 viruses in quail support the hypothesis that quail were the host of origin of the H5N 1/97 virus.
Abstract: The H5N1 influenza virus, which killed humans and poultry in 1997, was a reassortant that possibly arose in one type of domestic poultry present in the live-poultry markets of Hong Kong. Given that all the precursors of H5N1/97 are still circulating in poultry in southern China, the reassortment event that generated H5N1 could be repeated. Because A/goose/Guangdong/1/96-like (H5N1; Go/Gd) viruses are the proposed donors of the hemagglutinin gene of the H5N1 virus, we investigated the continued circulation, host range, and transmissibility of Go/Gd-like viruses in poultry. The Go/Gd-like viruses caused weight loss and death in some mice inoculated with high virus doses. Transmission of Go/Gd-like H5N1 viruses to geese by contact with infected geese resulted in infection of all birds but limited signs of overt disease. In contrast, oral inoculation with high doses of Go/Gd-like viruses resulted in the deaths of up to 50% of infected geese. Transmission from infected geese to chickens occurred only by fecal contact, whereas transmission to quail occurred by either aerosol or fecal spread. This difference is probably explained by the higher susceptibility of quail to Go/Gd-like virus. The high degree of susceptibility of quail to Go/Gd (H5N1)-like viruses and the continued circulation of H6N1 and H9N2 viruses in quail support the hypothesis that quail were the host of origin of the H5N1/97 virus. The ease of transmission of Go/Gd (H5N1)-like viruses to land-based birds, especially quail, supports the wisdom of separating aquatic and land-based poultry in the markets in Hong Kong and the need for continued surveillance in the field and live-bird markets in which different types of poultry are in contact with one another.

Journal ArticleDOI
TL;DR: Protective immunity to influenza A viruses is mediated primarily by serum IgG and mucosal IgA antibodies directed against the HA and NA, making it necessary that these two antigens are derived from the newly emerged antigenic variant virus.
Abstract: THE COLD-ADAPTED (ca) influenza A and B viruses developed by Maassab show great promise for use as a live attenuated virus vaccine to protect against the upper and lower respiratory tract disease caused by the influenza viruses (8,52,58). Since influenza A and B viruses undergo continuous antigenic changes, the live attenuated virus vaccines, like the licensed influenza virus subunit vaccines, will need to be updated frequently to be able to protect against the newly emerged epidemic antigenic variants of influenza A and B viruses. There are 15 antigenically distinct hemagglutinins (H1–H15) and nine antigenically distinct neuraminidases (N1–N9) in influenza A viruses that infect birds or mammals (98). Currently, two antigenic subtypes of influenza A virus (H1N1 and H3N2) co-circulate with influenza B virus in humans. A trivalent ca vaccine is therefore needed to protect against each of these three influenza viruses. The influenza A viruses possess a single-stranded, negative-sense RNA genome in eight segments that encode three polymerase proteins (98) (PB1, PB2, and PA); four membrane-associated proteins (the hemagglutinin [HA] glycoprotein, which mediates attachment and penetration; the neuraminidase [NA] protein, which mediates release of virus from the infected cell; the membrane protein [M], which lines the inner surface of the viral membrane and plays an essential role in virion structure and assembly, and the M2 protein, which forms an H1 ion channel that is required for release of the nucleocapsid following viral penetration of the host cell); a nucleocapsid protein (NP); a nonstructural protein (NS1) that functions as an interferon antagonist; a nuclear-export structural protein (NEP); and a newly described PB1-F2 protein (for influenza A viruses) that induces apoptosis in macrophages and lymphocytes (12). The genome of the influenza B viruses is organized similarly, but the NA gene contains a second open reading frame that encodes a second protein, NB, and the M gene does not encode an M2 protein (50). The NB protein of influenza B virus is thought to have a similar function as the M2 protein of influenza A virus (50). The segmented nature of the influenza virus genome permits the exchange of viral genes between two viruses co-infecting the same cell (Fig. 1). Such reassortment occurs between two influenza A viruses or two influenza B viruses but not between influenza A and B viruses. Protective immunity to influenza A viruses is mediated primarily by serum IgG and mucosal IgA antibodies directed against the HA and NA, making it necessary that these two antigens are derived from the newly emerged antigenic variant virus (98) (Fig. 1). Heterosubtypic immunity, that is, resistance to infection with an influenza A virus conferred by previ-

Journal ArticleDOI
TL;DR: Inf influenza A virus nonstructural NS1 protein is identified as the first viral protein that antagonizes virus- and dsRNA-induced activation of the stress response-signaling pathway mediated through Jun N-terminal kinase.
Abstract: The influenza A virus nonstructural NS1 protein is known to modulate host cell gene expression and to inhibit double-stranded RNA (dsRNA)-mediated antiviral responses. Here we identify NS1 as the first viral protein that antagonizes virus- and dsRNA-induced activation of the stress response-signaling pathway mediated through Jun N-terminal kinase.

Journal ArticleDOI
TL;DR: In the absence of antigenically matched hemagglutinin-based vaccines, DNA vaccination with conserved influenza genes may provide a useful first line of defense against a rapidly spreading pandemic virus.
Abstract: Influenza vaccination practice, which is based on neutralizing antibodies, requires being able to predict which viral strains will be circulating. If an unexpected strain, as in the 1997 H5N1 Hong Kong outbreak, or even a pandemic emerges, appropriate vaccines may take too long to prepare. Therefore, strategies based on conserved influenza antigens should be explored. We studied DNA vaccination in mice with plasmids expressing conserved nucleoprotein (NP) and matrix (M) from an H1N1 virus. After vaccination, mice were challenged with A/H5N1 viruses of low, intermediate, and high lethality. A/NP+A/M DNA vaccination reduced replication of A/Hong Kong/486/97 (HK/486), a nonlethal H5N1 strain, and protected against lethal challenge with more virulent A/Hong Kong/156/97 (HK/156). After HK/156 exposure, mice survived rechallenge with A/Hong Kong/483/97 (HK/483), although the DNA vaccination alone protected poorly against this highly virulent strain. In the absence of antigenically matched hemagglutinin-based vaccines, DNA vaccination with conserved influenza genes may provide a useful first line of defense against a rapidly spreading pandemic virus.

Journal ArticleDOI
TL;DR: It is concluded that loss of antiinflammatory properties of HDL after influenza infection in mice is associated with increased arterial macrophage traffic that can be prevented by administration of D-4F.
Abstract: Background— We reported that HDL loses its antiinflammatory properties during acute influenza A infection in mice, and we hypothesized that these changes might be associated with increased traffick...

Journal ArticleDOI
TL;DR: The isolation of a highly pathogenic H5N1 influenza virus from poultry indicates that such viruses are still circulating in China and may present a risk for transmission of the virus to humans.
Abstract: Since the 1997 H5N1 influenza virus outbreak in humans and poultry in Hong Kong, the emergence of closely related viruses in poultry has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur In May 2001, an avian H5N1 influenza A virus was isolated from duck meat that had been imported to South Korea from China Phylogenetic analysis of the hemagglutinin (HA) gene of A/Duck/Anyang/AVL-1/01 showed that the virus clustered with the H5 Goose/Guandong/1/96 lineage and 1997 Hong Kong human isolates and possessed an HA cleavage site sequence identical to these isolates Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens The pathogenesis of DK/Anyang/AVL-1/01 virus in Pekin ducks was further characterized and compared with a recent H5N1 isolate, A/Chicken/Hong Kong/3175/01, and an H5N1 1997 chicken isolate, A/Chicken/Hong Kong/220/97 Although no clinical signs of disease were observed in H5N1 virus-inoculated ducks, infectious virus could be detected in lung tissue, cloacal, and oropharyngeal swabs The DK/Anyang/AVL-1/01 virus was unique among the H5N1 isolates in that infectious virus and viral antigen could also be detected in muscle and brain tissue of ducks The pathogenesis of DK/Anyang/AVL-1/01 virus was characterized in BALB/c mice and compared with the other H5N1 isolates All viruses replicated in mice, but in contrast to the highly lethal CK/HK/220/97 virus, DK/Anyang/AVL-1/01 and CK/HK/3175/01 viruses remained localized to the respiratory tract DK/Anyang/AVL-1/01 virus caused weight loss and resulted in 22 to 33% mortality, whereas CK/HK/3175/01-infected mice exhibited no morbidity or mortality The isolation of a highly pathogenic H5N1 influenza virus from poultry indicates that such viruses are still circulating in China and may present a risk for transmission of the virus to humans

Journal ArticleDOI
TL;DR: Results indicate that antibodies to M2e, especially in combination with cell-mediated immune responses, exacerbate disease and clinical signs after infection should be observed closely in further studies using M2 e as an immunogen and caution should be exercised in using M 2e in humans.
Abstract: In mice, vaccines inducing antibodies to the extracellular domain of the M2 protein (M2e) can confer protection to influenza A virus infection. Unlike the surface glycoproteins, haemagglutinin and neuraminidase, this domain of M2 is highly conserved and is therefore a potential broad-spectrum immunogen. In this study, the protection conferred by vaccines inducing antibodies to M2e was evaluated in a challenge model for swine influenza in pigs. A protein resulting from the fusion between M2e and the hepatitis B virus core protein (M2eHBc), with or without adjuvant, was evaluated. In addition, a DNA construct expressing a fusion protein between M2e and influenza virus nucleoprotein (M2eNP) was evaluated to see if the broad-spectrum protection conferred by antibodies could be further enhanced by T helper cells and cytotoxic T cells. All vaccines induced an antibody response against M2e, and the M2eNP DNA vaccine additionally induced an influenza virus-specific lymphoproliferation response. However, after challenge with a swine influenza virus (H1N1), no protection was observed in the vaccinated groups compared with the non-vaccinated control group. On the contrary, vaccinated pigs showed more severe clinical signs than the control pigs. The M2eNP DNA-vaccinated pigs showed the most severe clinical signs and three out of six pigs died on days 1 and 2 post-challenge. These results indicate that antibodies to M2e, especially in combination with cell-mediated immune responses, exacerbate disease. Thus, clinical signs after infection should be observed closely in further studies using M2e as an immunogen and caution should be exercised in using M2e in humans.

Journal ArticleDOI
TL;DR: The presence of H6N1 viruses in poultry markets in Hong Kong that possess seven of the eight genes of the A/Hong Kong/156/97 (H5N1) virus raises the following fundamental questions relevant to influenza pandemic preparedness: could the pathogenic H5N 1 virus reemerge and could the H6n1 viruses directly cross the species barrier to mammals?
Abstract: The A/teal/Hong Kong/W312/97 (H6N1) influenza virus and the human H5N1 and H9N2 influenza viruses possess similar genes encoding internal proteins, suggesting that H6N1 viruses could become novel human pathogens. The molecular epidemiology and evolution of H6 influenza viruses were characterized by antigenic and genetic analyses of 29 H6 influenza viruses isolated from 1975 to 1981 and 1997 to 2000. Two distinct groups were identified on the basis of their antigenic characteristics. Phylogenetic analysis revealed that all H6N1 viruses isolated from terrestrial poultry in 1999 and 2000 are closely related to A/teal/Hong Kong/W312/97 (H6N1), and the nucleotide sequences of these viruses and of A/Hong Kong/156/97 (H5N1) were more than 96% homologous. The hemagglutinin (HA) of the 1999 and 2000 terrestrial viruses does not have multiple basic amino acids at the site of cleavage of HA1 to HA2; however, a unique insertion of aspartic acid in HA1 between positions 144 and 145 (H3 numbering) was found. The neuraminidase of these terrestrial H6N1 viruses has a deletion of 19 amino acids characteristic of A/Hong Kong/156/97 (H5N1). Evolutionary analysis suggested that these H6N1 viruses coevolved with A/quail/Hong Kong/G1/97-like H9N2 viruses and became more adapted to terrestrial poultry. These terrestrial 1999 and 2000 A/teal/Hong Kong/W312/97 (H6N1)-like viruses, along with the H9N2 viruses, could have been involved in the genesis of the pathogenic H5N1 influenza viruses of 1997. The presence of H6N1 viruses in poultry markets in Hong Kong that possess seven of the eight genes of the A/Hong Kong/156/97 (H5N1) virus raises the following fundamental questions relevant to influenza pandemic preparedness: could the pathogenic H5N1 virus reemerge and could the H6N1 viruses directly cross the species barrier to mammals?

Journal ArticleDOI
TL;DR: Swine virus seropositivity was significantly associated with being a farm owner or a farm family member, living on a farm, or entering the swine barn >4 days/week.
Abstract: We evaluated seropositivity to swine and human H1 influenza viruses in 74 swine farm owners, employees, their family members, and veterinarians in rural south-central Wisconsin, compared with 114 urban Milwaukee, Wisconsin, residents. The number of swine farm participants with positive serum hemagglutination-inhibition (HI) antibody titers > or = 40 to swine influenza viruses (17/74) was significantly higher (p or = 4 days/week. Because pigs can play a role in generating genetically novel influenza viruses, swine farmers may represent an important sentinel population to evaluate the emergence of new pandemic influenza viruses.

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TL;DR: Findings provide evidence of a close relationship between the generation of IL‐8 and symptoms during acute community‐acquired infections with rhinovirus or influenza A, and for RSV and parainfluenza infections, factors in addition to IL‐9 production appear to contribute to thegeneration of clinical symptoms.
Abstract: Both virus-mediated damage to airway tissues and induction of pro-inflammatory cytokines such as interleukin-8 (IL-8) could contribute to symptom severity during viral respiratory infections in children. To test the hypothesis that IL-8 contributes to the pathogenesis of respiratory symptoms during naturally acquired respiratory viral infections in children, nasal wash samples collected from infants with acute viral infections (n = 198) or from healthy uninfected infants (n = 31) were analysed for IL-8. Nasal wash IL-8 was positively related to age in uninfected children (rs = 0.36, p influenza, p < 0.05). Finally, there were significant correlations between nasal wash IL-8 levels and symptom scores during infections with rhinovirus (rs = 0.56, p < 0.001) or influenza A (rs = 0.45, p < 0.05), but not with parainfluenza virus or RSV. These findings provide evidence of a close relationship between the generation of IL-8 and symptoms during acute community-acquired infections with rhinovirus or influenza A. In contrast, for RSV and parainfluenza infections, factors in addition to IL-8 production appear to contribute to the generation of clinical symptoms.