Showing papers on "Influenza A virus published in 2003"

Mortality Associated With Influenza and Respiratory Syncytial Virus in the United States

Abstract: Context Influenza and respiratory syncytial virus (RSV) cause substantial morbidity and mortality. Statistical methods used to estimate deaths in the United States attributable to influenza have not accounted for RSV circulation. Objective To develop a statistical model using national mortality and viral surveillance data to estimate annual influenza- and RSV-associated deaths in the United States, by age group, virus, and influenza type and subtype. Design, Setting, and Population Age-specific Poisson regression models using national viral surveillance data for the 1976-1977 through 1998-1999 seasons were used to estimate influenza-associated deaths. Influenza- and RSV-associated deaths were simultaneously estimated for the 1990-1991 through 1998-1999 seasons. Main Outcome Measures Attributable deaths for 3 categories: underlying pneumonia and influenza, underlying respiratory and circulatory, and all causes. Results Annual estimates of influenza-associated deaths increased significantly between the 1976-1977 and 1998-1999 seasons for all 3 death categories (P Conclusions Mortality associated with both influenza and RSV circulation disproportionately affects elderly persons. Influenza deaths have increased substantially in the last 2 decades, in part because of aging of the population, underscoring the need for better prevention measures, including more effective vaccines and vaccination programs for elderly persons.

Ecological and immunological determinants of influenza evolution

Abstract: In pandemic and epidemic forms, influenza causes substantial, sometimes catastrophic, morbidity and mortality. Intense selection from the host immune system drives antigenic change in influenza A and B, resulting in continuous replacement of circulating strains with new variants able to re-infect hosts immune to earlier types. This ‘antigenic drift’1 often requires a new vaccine to be formulated before each annual epidemic. However, given the high transmissibility and mutation rate of influenza, the constancy of genetic diversity within lineages over time is paradoxical. Another enigma is the replacement of existing strains during a global pandemic caused by ‘antigenic shift’—the introduction of a new avian influenza A subtype into the human population1. Here we explore ecological and immunological factors underlying these patterns using a mathematical model capturing both realistic epidemiological dynamics and viral evolution at the sequence level. By matching model output to phylogenetic patterns seen in sequence data collected through global surveillance2, we find that short-lived strain-transcending immunity is essential to restrict viral diversity in the host population and thus to explain key aspects of drift and shift dynamics.

RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription

Abstract: Influenza A virus causes widespread infection in the human respiratory tract, but existing vaccines and drug therapy are of limited value. Here we show that short interfering RNAs (siRNAs) specific for conserved regions of the viral genome can potently inhibit influenza virus production in both cell lines and embryonated chicken eggs. The inhibition depends on the presence of a functional antisense strand in the siRNA duplex, suggesting that viral mRNA is the target of RNA interference. However, siRNA specific for nucleocapsid (NP) or a component of the RNA transcriptase (PA) abolished the accumulation of not only the corresponding mRNA but also virion RNA and its complementary RNA. These siRNAs also broadly inhibited the accumulation of other viral, but not cellular, RNAs. The findings reveal that newly synthesized NP and PA proteins are required for influenza virus transcription and replication and provide a basis for the development of siRNAs as prophylaxis and therapy for influenza infection in humans.

Birds, Migration and Emerging Zoonoses: West Nile Virus, Lyme Disease, Influenza A and Enteropathogens

Abstract: Wild birds are important to public health because they carry emerging zoonotic pathogens, either as a reservoir host or by dispersing infected arthropod vectors. In addition, bird migration provides a mechanism for the establishment of new endemic foci of disease at great distances from where an infection was acquired. Birds are central to the epidemiology of West Nile virus (WNV) because they are the main amplifying host of the virus in nature. The initial spread of WNV in the U.S. along the eastern seaboard coincided with a major bird migration corridor. The subsequent rapid movement of the virus inland could have been facilitated by the elliptical migration routes used by many songbirds. A number of bird species can be infected with Borrelia burgdorferi, the etiologic agent of Lyme disease, but most are not competent to transmit the infection to Ixodes ticks. The major role birds play in the geographic expansion of Lyme disease is as dispersers of B. burgdorferi-infected ticks. Aquatic waterfowl are asymptomatic carriers of essentially all hemagglutinin and neuraminidase combinations of influenza A virus. Avian influenza strains do not usually replicate well in humans, but they can undergo genetic reassortment with human strains that co-infect pigs. This can result in new strains with a marked increase in virulence for humans. Wild birds can acquire enteropathogens, such as Salmonella and Campylobacter spp., by feeding on raw sewage and garbage, and can spread these agents to humans directly or by contaminating commercial poultry operations. Conversely, wild birds can acquire drug-resistant enteropathogens from farms and spread these strains along migration routes. Birds contribute to the global spread of emerging infectious diseases in a manner analogous to humans traveling on aircraft. A better understanding of avian migration patterns and infectious diseases of birds would be useful in helping to predict future outbreaks of infections due to emerging zoonotic pathogens.

Newcastle disease virus (NDV)-based assay demonstrates interferon-antagonist activity for the NDV V protein and the Nipah virus V, W, and C proteins.

Abstract: We have generated a recombinant Newcastle disease virus (NDV) that expresses the green fluorescence protein (GFP) in infected chicken embryo fibroblasts (CEFs). This virus is interferon (IFN) sensitive, and pretreatment of cells with chicken alpha/beta IFN (IFN-α/β) completely blocks viral GFP expression. Prior transfection of plasmid DNA induces an IFN response in CEFs and blocks NDV-GFP replication. However, transfection of known inhibitors of the IFN-α/β system, including the influenza A virus NS1 protein and the Ebola virus VP35 protein, restores NDV-GFP replication. We therefore conclude that the NDV-GFP virus could be used to screen proteins expressed from plasmids for the ability to counteract the host cell IFN response. Using this system, we show that expression of the NDV V protein or the Nipah virus V, W, or C proteins rescues NDV-GFP replication in the face of the transfection-induced IFN response. The V and W proteins of Nipah virus, a highly lethal pathogen in humans, also block activation of an IFN-inducible promoter in primate cells. Interestingly, the amino-terminal region of the Nipah virus V protein, which is identical to the amino terminus of Nipah virus W, is sufficient to exert the IFN-antagonist activity. In contrast, the anti-IFN activity of the NDV V protein appears to be located in the carboxy-terminal region of the protein, a region implicated in the IFN-antagonist activity exhibited by the V proteins of mumps virus and human parainfluenza virus type 2.

Caspase 3 activation is essential for efficient influenza virus propagation

Abstract: Apoptosis is a hallmark event observed upon infection with many viral pathogens, including influenza A virus. The apoptotic process is executed by a proteolytic system consisting of a family of cysteinyl proteases, termed caspases. Since the consequences of apoptosis induction and caspase activation for the outcome of an influenza virus infection are not clear, we have addressed this issue by interfering with expression or function of a major virus-induced apoptosis effector, caspase 3. Surprisingly, influenza virus propagation was strongly impaired in the presence of an inhibitor that blocks caspase 3 and in cells where caspase 3 was partially knocked down by small interfering RNAs. Consistent with these findings, poor replication efficiencies of influenza A viruses in cells deficient for caspase 3 could be boosted 30-fold by ectopic expression of the protein. Mechanistically, the block in virus propagation appeared to be due to retention of the viral RNP complexes in the nucleus, preventing formation of progeny virus particles. Our findings indicate that caspase 3 activation during the onset of apoptosis is a crucial event for efficient influenza virus propagation.

Overexpression of the α-2,6-Sialyltransferase in MDCK Cells Increases Influenza Virus Sensitivity to Neuraminidase Inhibitors

Abstract: No reliable cell culture assay is currently available for monitoring human influenza virus sensitivity to neuraminidase inhibitors (NAI). This can be explained by the observation that because of a low concentration of sialyl-alpha2,6-galactose (Sia[alpha2,6]Gal)-containing virus receptors in conventional cell lines, replication of human virus isolates shows little dependency on viral neuraminidase. To test whether overexpression of Sia(alpha2,6)Gal moieties in cultured cells could make them suitable for testing human influenza virus sensitivity to NAI, we stably transfected MDCK cells with cDNA of human 2,6-sialyltransferase (SIAT1). Transfected cells expressed twofold-higher amounts of 6-linked sialic acids and twofold-lower amounts of 3-linked sialic acids than parent MDCK cells as judged by staining with Sambucus nigra agglutinin and Maackia amurensis agglutinin, respectively. After transfection, binding of a clinical human influenza virus isolate was increased, whereas binding of its egg-adapted variant which preferentially bound 3-linked receptors was decreased. The sensitivity of human influenza A and B viruses to the neuraminidase inhibitor oseltamivir carboxylate was substantially improved in the SIAT1-transfected cell line and was consistent with their sensitivity in neuraminidase enzyme assay and with the hemagglutinin (HA) receptor-binding phenotype. MDCK cells stably transfected with SIAT1 may therefore be a suitable system for testing influenza virus sensitivity to NAI.

A Recombinant Influenza A Virus Expressing an RNA-Binding-Defective NS1 Protein Induces High Levels of Beta Interferon and Is Attenuated in Mice

Abstract: Previously we found that the amino-terminal region of the NS1 protein of influenza A virus plays a key role in preventing the induction of beta interferon (IFN-β) in virus-infected cells. This region is characterized by its ability to bind to different RNA species, including double-stranded RNA (dsRNA), a known potent inducer of IFNs. In order to investigate whether the NS1 RNA-binding activity is required for its IFN antagonist properties, we have generated a recombinant influenza A virus which expresses a mutant NS1 protein defective in dsRNA binding. For this purpose, we substituted alanines for two basic amino acids within NS1 (R38 and K41) that were previously found to be required for RNA binding. Cells infected with the resulting recombinant virus showed increased IFN-β production, demonstrating that these two amino acids play a critical role in the inhibition of IFN production by the NS1 protein during viral infection. In addition, this virus grew to lower titers than wild-type virus in MDCK cells, and it was attenuated in mice. Interestingly, passaging in MDCK cells resulted in the selection of a mutant virus containing a third mutation at amino acid residue 42 of the NS1 protein (S42G). This mutation did not result in a gain in dsRNA-binding activity by the NS1 protein, as measured by an in vitro assay. Nevertheless, the NS1 R38AK41AS42G mutant virus was able to replicate in MDCK cells to titers close to those of wild-type virus. This mutant virus had intermediate virulence in mice, between those of the wild-type and parental NS1 R38AK41A viruses. These results suggest not only that the IFN antagonist properties of the NS1 protein depend on its ability to bind dsRNA but also that they can be modulated by amino acid residues not involved in RNA binding.

Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses.

Abstract: Recombination is increasingly seen as an important means of shaping genetic diversity in RNA viruses. However, observed recombination frequencies vary widely among those viruses studied to date, with only sporadic occurrences reported in RNA viruses with negative-sense genomes. To determine the extent of homologous recombination in negative-sense RNA viruses, phylogenetic analyses of 79 gene sequence alignments from 35 negative-sense RNA viruses (a total of 2154 sequences) were carried out. Powerful evidence was found for recombination, in the form of incongruent phylogenetic trees between different gene regions, in only five sequences from Hantaan virus, Mumps virus and Newcastle disease virus. This is the first report of recombination in these viruses. More tentative evidence for recombination, where conflicting phylogenetic trees were observed (but were without strong bootstrap support) and/or where putative recombinant regions were very short, was found in three alignments from La Crosse virus and Puumala virus. Finally, patterns of sequence variation compatible with the action of recombination, but not definitive evidence for this process, were observed in a further ten viruses: Canine distemper virus, Crimean-Congo haemorrhagic fever virus, Influenza A virus, Influenza B virus, Influenza C virus, Lassa virus, Pirital virus, Rabies virus, Rift Valley Fever virus and Vesicular stomatitis virus. The possibility of recombination in these viruses should be investigated further. Overall, this study reveals that rates of homologous recombination in negative-sense RNA viruses are very much lower than those of mutation, with many viruses seemingly clonal on current data. Consequently, recombination rate is unlikely to be a trait that is set by natural selection to create advantageous or purge deleterious mutations.

Influenza A and B Virus Infections in Children

Abstract: To obtain data on the clinical manifestations of infection, the age distribution, and the underlying conditions of children with influenza severe enough to lead to hospital referral, we performed a retrospective study of children treated at Turku University Hospital (Turku, Finland) in 1980-1999. Influenza A or B antigen was detected in the nasopharyngeal aspirates of 683 of the 15,420 children studied. The median age of children with influenza A was 2.0 years (n=544), and that of children with influenza B was 4.2 years (n=139) (P<.001). One-fourth of the children had an underlying medical condition. High fever, cough, and rhinorrhea were the most frequently recorded symptoms. Acute otitis media developed in 24% of the children, and pneumonia developed in 9% of the children. The study shows that the majority of patient hospitalizations for pediatric influenza involve previously healthy infants and young children. Laboratory confirmation of influenza is particularly important for children because the clinical presentation of the infection is less characteristic than that seen in adults.

Characterization of H9 Subtype Influenza Viruses from the Ducks of Southern China: a Candidate for the Next Influenza Pandemic in Humans?

Abstract: A current view of the emergence of pandemic influenza viruses envisages a gene flow from the aquatic avian reservoir to humans via reassortment in pigs, the hypothetical “mixing vessel.” Understanding arising from recent H5N1 influenza outbreaks in Hong Kong since 1997 and the isolation of avian H9N2 virus from humans raises alternative options for the emergence of a new pandemic virus. Here we report that H9N2 influenza viruses established in terrestrial poultry in southern China are transmitted back to domestic ducks, in which the viruses generate multiple reassortants. These novel H9N2 viruses are double or even triple reassortants that have amino acid signatures in their hemagglutinin, indicating their potential to directly infect humans. Some of them contain gene segments that are closely related to those of A/Hong Kong/156/97 (H5N1/97, H5N1) or A/Quail/Hong Kong/G1/97 (G1-like, H9N2). More importantly, some of their internal genes are closely related to those of novel H5N1 viruses isolated during the outbreak in Hong Kong in 2001. This study reveals a two-way transmission of influenza virus between terrestrial and aquatic birds that facilitates the generation of novel reassortant H9N2 influenza viruses. Such reassortants may directly or indirectly play a role in the emergence of the next pandemic virus.

Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-PCR assay.

Abstract: The clinical presentation of infections caused by the heterogeneous group of the respiratory viruses can be very similar. Thus, the implementation of virological assays that rapidly identify the most important viruses involved is of great interest. A new multiplex reverse transcription nested-polymerase chain reaction (RT-PCR) assay that is able to detect and type different respiratory viruses simultaneously is described. Primer sets were targeted to conserved regions of nucleoprotein genes of the influenza viruses, fusion protein genes of respiratory syncytial viruses (RSV), and hexon protein genes of adenoviruses. Individual influenza A, B, and C viruses, RSV (A and B), and a generic detection of the 48 serotypes of adenoviruses were identified and differentiated by the size of the PCR products. An internal amplification control was included in the reaction mixture to exclude false-negative results due to sample inhibitors and/or extraction failure. Detection levels of 0.1 and 0.01 TCID50 of influenza A and B viruses and 1-10 molecules of cloned amplified products of influenza C virus, RSV A and B, and adenovirus serotype 1 were achieved. The specificity was checked using specimens containing other respiratory viruses and no amplified products were detected in any case. A panel of 290 respiratory specimens from the 1999-2000 and 2000-2001 seasons was used to validate the assay. Accurately amplifying RNA from influenza and RSV prototype strains and DNA from all adenovirus serotypes demonstrates the use of this method for both laboratory routine diagnosis and surveillance of all these viruses.

Influenza type A in humans, mammals and birds: Determinants of virus virulence, host-range and interspecies transmission

Abstract: The virulence of a virus is determined by its ability to adversely affect the host cell, host organism or population of host organisms. Influenza A viruses have been responsible for four pandemics of severe human respiratory disease this century. Avian species harbour a large reservoir of influenza virus strains, which can contribute genes to potential new pandemic human strains. The fundamental importance of understanding the role of each of these genes in determining virulence in birds and humans was dramatically emphasised by the recent direct transmission of avian influenza A viruses to humans, causing fatal infection but not community spread. An understanding of the factors involved in transmission between avian and mammalian species should assist in the development of better surveillance strategies for early recognition of influenza A virus strains having human pandemic potential, and possibly in the design of anti-viral strategies.

Comparison of the Directigen Flu A+B test, the QuickVue influenza test, and clinical case definition to viral culture and reverse transcription-PCR for rapid diagnosis of influenza virus infection

Abstract: The diagnostic performances of the clinical case definition of influenza virus infection based on the combination of fever and cough and of two rapid influenza diagnostic tests, the Directigen Flu A+B test (Directigen; BD Diagnostic Systems, Sparks, Md.) and the QuickVue influenza test (QuickVue; Quidel, San Diego, Calif.), were compared to those of viral culture and an in-house reverse transcription (RT)-PCR during the 2000-2001 flu season. Two hundred consecutive nasopharyngeal aspirates were analyzed from 192 patients, including 122 adults and 70 children. Viral culture identified influenza virus A in 16 samples and influenza virus B in 55 samples, whereas RT-PCR identified influenza virus A in 21 samples and influenza virus B in 64 samples. When RT-PCR was used as the reference standard, the likelihood ratios for a positive test were 40.0 for Directigen, 8.6 for QuickVue, and 1.4 for the combination of fever and cough, whereas the likelihood ratios for a negative test were 0.22, 0.16, and 0.48, respectively. Our study suggests that (i) the poor specificity (35 to 58%) and the poor positive predictive value (41 to 60%) of the clinical case definition of influenza preclude its use for prediction of influenza virus infections during epidemics, especially when infection control decision making in the hospital setting is considered; (ii) Directigen has a higher diagnostic yield than QuickVue but is associated with a larger number of invalid results; (iii) the sensitivities of the rapid diagnostic tests are significantly lower with samples from adults than with samples from children, with the rates of false-negative results reaching up to 29%; and (iv) RT-PCR detects more cases of influenza than viral culture, and this greater accuracy makes it a more useful reference standard.

The origin of the 1918 pandemic influenza virus: a continuing enigma.

Abstract: Influenza A virus is a major public health threat, killing more than 30,000 per year in the USA alone, sickening millions and inflicting substantial economic costs. Novel influenza virus strains emerge periodically to which humans have little immunity, resulting in devastating pandemics. The 1918 pandemic killed nearly 700,000 Americans and 40 million people worldwide. Pandemics in 1957 and 1968, while much less devastating than 1918, also caused tens of thousands of deaths in the USA. The influenza A virus is capable of enormous genetic variability, both by continuous, gradual mutation and by reassortment of gene segments between viruses. Both the 1957 and 1968 pandemic strains are thought to have originated as reassortants, in which one or both human-adapted viral surface proteins were replaced by proteins from avian influenza virus strains. Analyses of the surface proteins of the 1918 pandemic strain, however, suggest that this strain may have had a different origin. The haemagglutinin gene segment of the virus may have come directly from an avian source different from those currently circulating. Alternatively, the virus, or some of its gene segments, may have evolved in an intermediate host before emerging as a human pathogen. Determining whether pandemic influenza virus strains can emerge via different pathways will affect the scope and focus of surveillance and prevention efforts.

Role of Quail in the Interspecies Transmission of H9 Influenza A Viruses: Molecular Changes on HA That Correspond to Adaptation from Ducks to Chickens

Abstract: H9 influenza viruses have become endemic in land-based domestic poultry in Asia and have sporadically crossed to pigs and humans. To understand the molecular determinants of their adaptation to land-based birds, we tested the replication and transmission of several 1970s duck H9 viruses in chickens and quail. Quail were more susceptible than chickens to these viruses, and generation of recombinant H9 viruses by reverse genetics showed that changes in the HA gene are sufficient to initiate efficient replication and transmission in quail. Seven amino acid positions on the HA molecule corresponded to adaptation to land-based birds. In quail H9 viruses, the pattern of amino acids at these seven positions is intermediate between those of duck and chicken viruses; this fact may explain the susceptibility of quail to duck H9 viruses. Our findings suggest that quail provide an environment in which the adaptation of influenza viruses from ducks generates novel variants that can cross the species barrier.

Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes.

Abstract: At the final step in viral replication, the viral genome must be incorporated into progeny virions, yet the genomic regions required for this process are largely unknown in RNA viruses, including influenza virus. Recently, it was reported that both ends of the neuraminidase (NA) coding region are critically important for incorporation of this vRNA segment into influenza virions (Y. Fujii, H. Goto, T. Watanabe, T. Yoshida, and Y. Kawaoka, Proc. Natl. Acad. Sci. USA 100:2002-2007, 2003). To determine the signals in the hemagglutinin (HA) vRNA required for its virion incorporation, we made a series of deletion constructs of this segment. Subsequent analysis showed that 9 nucleotides at the 3' end of the coding region and 80 nucleotides at the 5' end are sufficient for efficient virion incorporation of the HA vRNA. The utility of this information for stable expression of foreign genes in influenza viruses was assessed by generating a virus whose HA and NA vRNA coding regions were replaced with those of vesicular stomatitis virus glycoprotein (VSVG) and green fluorescent protein (GFP), respectively, while retaining virion incorporation signals for these segments. Despite the lack of HA and NA proteins, the resultant virus, which possessed only VSVG on the virion surface, was viable and produced GFP-expressing plaques in cells even after repeated passages, demonstrating that two foreign genes can be incorporated and maintained stably in influenza A virus. These findings could serve as a model for the construction of influenza A viruses designed to express and/or deliver foreign genes.

Prolonged shedding of multidrug-resistant influenza A virus in an immunocompromised patient.

Abstract: To the Editor: Immunocompromised patients can persistently shed influenza, increasing the potential for resistance to antiviral drugs and for nosocomial transmission.1,2 We report the case of a 23-...

Codon bias and frequency-dependent selection on the hemagglutinin epitopes of influenza A virus.

Abstract: Although the surface proteins of human influenza A virus evolve rapidly and continually produce antigenic variants, the internal viral genes acquire mutations very gradually. In this paper, we analyze the sequence evolution of three influenza A genes over the past two decades. We study codon usage as a discriminating signature of gene- and even residue-specific diversifying and purifying selection. Nonrandom codon choice can increase or decrease the effective local substitution rate. We demonstrate that the codons of hemagglutinin, particularly those in the antibody-combining regions, are significantly biased toward substitutional point mutations relative to the codons of other influenza virus genes. We discuss the evolutionary interpretation and implications of these biases for hemagglutinin's antigenic evolution. We also introduce information-theoretic methods that use sequence data to detect regions of recent positive selection and potential protein conformational changes.

Pathogenic and Antigenic Properties of Phylogenetically Distinct Reassortant H3N2 Swine Influenza Viruses Cocirculating in the United States

Abstract: Swine influenza is an acute respiratory disease caused by type A influenza viruses. Before 1998, swine influenza virus isolates in the United States were mainly of the classical H1N1 lineage. Since then, phylogenetically distinct reassortant H3N2 viruses have been identified as respiratory pathogens in pigs on U.S. farms. The H3N2 viruses presently circulating in the U.S. swine population are triple reassortants containing avian-like (PA and PB2), swine-like (M, NP, and NS), and human-like (HA, NA, and PB1) gene segments. Recent sequence data show that the triple reassortants have acquired at least three distinct H3 molecules from human influenza viruses and thus form three distinct phylogenetic clusters (I to III). In this study we analyzed the antigenic and pathogenic properties of viruses belonging to each of these clusters. Hemagglutination inhibition and neutralization assays that used hyperimmune sera obtained from caesarian-derived, colostrum-deprived pigs revealed that H3N2 cluster I and cluster III viruses share common epitopes, whereas a cluster II virus showed only limited cross-reactivity. H3N2 viruses from each of the three clusters were able to induce clinical signs of disease and associated lesions upon intratracheal inoculation into seronegative pigs. There were, however, differences in the severity of lesions between individual strains even within one antigenic cluster. A correlation between the severity of disease and pig age was observed. These data highlight the increased diversity of swine influenza viruses in the United States and would indicate that surveillance should be intensified to determine the most suitable vaccine components.