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1.  Detection of Evolutionarily Distinct Avian Influenza A Viruses in Antarctica 
mBio  2014;5(3):e01098-14.
Distinct lineages of avian influenza viruses (AIVs) are harbored by spatially segregated birds, yet significant surveillance gaps exist around the globe. Virtually nothing is known from the Antarctic. Using virus culture, molecular analysis, full genome sequencing, and serology of samples from Adélie penguins in Antarctica, we confirmed infection by H11N2 subtype AIVs. Their genetic segments were distinct from all known contemporary influenza viruses, including South American AIVs, suggesting spatial separation from other lineages. Only in the matrix and polymerase acidic gene phylogenies did the Antarctic sequences form a sister relationship to South American AIVs, whereas distant phylogenetic relationships were evident in all other gene segments. Interestingly, their neuraminidase genes formed a distant relationship to all avian and human influenza lineages, and the polymerase basic 1 and polymerase acidic formed a sister relationship to the equine H3N8 influenza virus lineage that emerged during 1963 and whose avian origins were previously unknown. We also estimated that each gene segment had diverged for 49 to 80 years from its most closely related sequences, highlighting a significant gap in our AIV knowledge in the region. We also show that the receptor binding properties of the H11N2 viruses are predominantly avian and that they were unable to replicate efficiently in experimentally inoculated ferrets, suggesting their continuous evolution in avian hosts. These findings add substantially to our understanding of both the ecology and the intra- and intercontinental movement of Antarctic AIVs and highlight the potential risk of an incursion of highly pathogenic AIVs into this fragile environment.
Avian influenza viruses (AIVs) are typically maintained and spread by migratory birds, resulting in the existence of distinctly different viruses around the world. However, AIVs have not previously been detected in Antarctica. In this study, we characterized H11N2 viruses sampled from Adélie penguins from two geographically different sites in Antarctica and show that the segmented AIV genome diverged between 49 and 80 years ago from other AIVs, with several genes showing similarity and shared ancestry with H3N8 equine influenza viruses. This study provides the first insight into the ecology of AIVs in Antarctica and highlights the potential risk of an introduction of highly pathogenic AIVs into the continent.
PMCID: PMC4010832  PMID: 24803521
2.  Estimating the Fitness Advantage Conferred by Permissive Neuraminidase Mutations in Recent Oseltamivir-Resistant A(H1N1)pdm09 Influenza Viruses 
PLoS Pathogens  2014;10(4):e1004065.
Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.
Author Summary
Antimicrobial resistance is an increasing problem for the treatment of infectious diseases. In 2007–2008 human seasonal A(H1N1) influenza viruses rapidly acquired resistance to the most commonly used anti-influenza drug oseltamivir, via a H275Y amino acid mutation within the neuraminidase (NA) protein. In 2009 the oseltamivir sensitive A(H1N1)pdm09 virus (encoding NA 275H) emerged in the human population, rapidly replacing the oseltamivir resistant seasonal A(H1N1) virus. However, there is increasing concern that currently circulating A(H1N1)pdm09 viruses may similarly acquire oseltamivir resistance (via the NA H275Y mutation) and become widespread. Here we demonstrate that two novel amino acid changes present in virtually all recent A(H1N1)pdm09 viruses (NA V241I and N369K) enable the acquisition of the NA H275Y oseltamivir resistance mutation without compromising viral fitness. As such recent A(H1N1)pdm09 viruses are now one step closer to acquiring widespread oseltamivir resistance.
PMCID: PMC3974874  PMID: 24699865
3.  Practical Considerations for High-Throughput Influenza A Virus Surveillance Studies of Wild Birds by Use of Molecular Diagnostic Tests ▿  
Journal of Clinical Microbiology  2008;47(3):666-673.
Influenza A virus surveillance studies of wild bird populations are essential to improving our understanding of the role of wild birds in the ecology of low-pathogenic avian influenza viruses and their potential contribution to the spread of H5N1 highly pathogenic avian influenza viruses. Whereas the primary results of such surveillance programs have been communicated extensively, practical considerations and technical implementation options generally receive little attention. In the present study, the data obtained from 39,490 samples were used to compare the impacts of variables such as the sampling procedure, storage and transport conditions, and the choice of molecular and classical diagnostic tests on the outcome of the results. Molecular diagnostic tests allowed estimation of the virus load in samples, which has implications for the ability to isolate virus. Virus isolation in embryonated eggs was more sensitive than virus isolation in cell cultures. Storage and transport conditions had less of an impact on diagnostics by the use of molecular tests than by the use of classical approaches. These findings indicate that molecular diagnostic tests are more sensitive and more reliable than classical tests. In addition, molecular diagnostic tests facilitated analyses in real time and allowed the discrimination of H5 influenza viruses with low and high pathogenicities without the need for virus isolation. Critical assessment of the methods used in large surveillance studies like this will facilitate comparison of the results between studies. Moreover, the lessons learned from current large-scale influenza A virus surveillance activities could be valuable for other pathogen surveillance programs in the future.
PMCID: PMC2650931  PMID: 19109483
4.  Vaccination against Human Influenza A/H3N2 Virus Prevents the Induction of Heterosubtypic Immunity against Lethal Infection with Avian Influenza A/H5N1 Virus 
PLoS ONE  2009;4(5):e5538.
Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains.
Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza.
The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.
PMCID: PMC2678248  PMID: 19440239
5.  Clearance of influenza virus from the lung depends on migratory langerin+CD11b− but not plasmacytoid dendritic cells 
The Journal of Experimental Medicine  2008;205(7):1621-1634.
Although dendritic cells (DCs) play an important role in mediating protection against influenza virus, the precise role of lung DC subsets, such as CD11b− and CD11b+ conventional DCs or plasmacytoid DCs (pDCs), in different lung compartments is currently unknown. Early after intranasal infection, tracheal CD11b−CD11chi DCs migrated to the mediastinal lymph nodes (MLNs), acquiring co-stimulatory molecules in the process. This emigration from the lung was followed by an accumulation of CD11b+CD11chi DCs in the trachea and lung interstitium. In the MLNs, the CD11b+ DCs contained abundant viral nucleoprotein (NP), but these cells failed to present antigen to CD4 or CD8 T cells, whereas resident CD11b−CD8α+ DCs presented to CD8 cells, and migratory CD11b−CD8α− DCs presented to CD4 and CD8 T cells. When lung CD11chi DCs and macrophages or langerin+CD11b−CD11chi DCs were depleted using either CD11c–diphtheria toxin receptor (DTR) or langerin-DTR mice, the development of virus-specific CD8+ T cells was severely delayed, which correlated with increased clinical severity and a delayed viral clearance. 120G8+ CD11cint pDCs also accumulated in the lung and LNs carrying viral NP, but in their absence, there was no effect on viral clearance or clinical severity. Rather, in pDC-depleted mice, there was a reduction in antiviral antibody production after lung clearance of the virus. This suggests that multiple DCs are endowed with different tasks in mediating protection against influenza virus.
PMCID: PMC2442640  PMID: 18591406
6.  Spatial, Temporal, and Species Variation in Prevalence of Influenza A Viruses in Wild Migratory Birds 
PLoS Pathogens  2007;3(5):e61.
Although extensive data exist on avian influenza in wild birds in North America, limited information is available from elsewhere, including Europe. Here, molecular diagnostic tools were employed for high-throughput surveillance of migratory birds, as an alternative to classical labor-intensive methods of virus isolation in eggs. This study included 36,809 samples from 323 bird species belonging to 18 orders, of which only 25 species of three orders were positive for influenza A virus. Information on species, locations, and timing is provided for all samples tested. Seven previously unknown host species for avian influenza virus were identified: barnacle goose, bean goose, brent goose, pink-footed goose, bewick's swan, common gull, and guillemot. Dabbling ducks were more frequently infected than other ducks and Anseriformes; this distinction was probably related to bird behavior rather than population sizes. Waders did not appear to play a role in the epidemiology of avian influenza in Europe, in contrast to the Americas. The high virus prevalence in ducks in Europe in spring as compared with North America could explain the differences in virus–host ecology between these continents. Most influenza A virus subtypes were detected in ducks, but H13 and H16 subtypes were detected primarily in gulls. Viruses of subtype H6 were more promiscuous in host range than other subtypes. Temporal and spatial variation in influenza virus prevalence in wild birds was observed, with influenza A virus prevalence varying by sampling location; this is probably related to migration patterns from northeast to southwest and a higher prevalence farther north along the flyways. We discuss the ecology and epidemiology of avian influenza A virus in wild birds in relation to host ecology and compare our results with published studies. These data are useful for designing new surveillance programs and are particularly relevant due to increased interest in avian influenza in wild birds.
Author Summary
Significant gaps in our knowledge of the ecology of avian influenza in wild migratory birds have become apparent during recent outbreaks of H5N1 highly pathogenic avian influenza, in particular in relation to the risk of virus spread by wild birds. An eight-year surveillance study, which included more than 36,000 wild birds tested for low pathogenic avian influenza, provides new information on host species, prevalence, and temporal and geographical variation of avian influenza in wild migratory birds in Europe. Dabbling ducks harbored nearly all known influenza virus subtypes, with the exception of H13 and H16, which were found primarily in gulls. In contrast to American studies, waders did not play a role in the epidemiology of avian influenza in Europe. This study provides important information on the ecology and epidemiology of avian influenza A virus and could assist in the design of new surveillance studies for high and low pathogenic avian influenza in wild birds.
PMCID: PMC1876497  PMID: 17500589
7.  Mallards and Highly Pathogenic Avian Influenza Ancestral Viruses, Northern Europe 
Emerging Infectious Diseases  2005;11(10):1545-1551.
Surveillance studies in wild birds help generate prototypic vaccine candidates and diagnostic tests.
Outbreaks of highly pathogenic avian influenza (HPAI), which originate in poultry upon transmission of low pathogenic viruses from wild birds, have occurred relatively frequently in the last decade. During our ongoing surveillance studies in wild birds, we isolated several influenza A viruses of hemagglutinin subtype H5 and H7 that contain various neuraminidase subtypes. For each of the recorded H5 and H7 HPAI outbreaks in Europe since 1997, our collection contained closely related virus isolates recovered from wild birds, as determined by sequencing and phylogenetic analyses of the hemagglutinin gene and antigenic characterization of the hemagglutinin glycoprotein. The minor genetic and antigenic diversity between the viruses recovered from wild birds and those causing HPAI outbreaks indicates that influenza A virus surveillance studies in wild birds can help generate prototypic vaccine candidates and design and evaluate diagnostic tests, before outbreaks occur in animals and humans.
PMCID: PMC3366752  PMID: 16318694
avian influenza; influenza A virus; surveillance; wildlife; HPAI; pandemic; research
8.  Protection of Mice against Lethal Infection with Highly Pathogenic H7N7 Influenza A Virus by Using a Recombinant Low-Pathogenicity Vaccine Strain 
Journal of Virology  2005;79(19):12401-12407.
In 2003, an outbreak of highly pathogenic avian influenza occurred in the Netherlands. The avian H7N7 virus causing the outbreak was also detected in 88 humans suffering from conjunctivitis or mild respiratory symptoms and one person who died of pneumonia and acute respiratory distress syndrome. Here we describe a mouse model for lethal infection with A/Netherlands/219/03 isolated from the fatal case. Because of the zoonotic and pathogenic potential of the H7N7 virus, a candidate vaccine carrying the avian hemagglutinin and neuraminidase proteins produced in the context of the high-throughput vaccine strain A/PR/8/34 was generated by reverse genetics and tested in the mouse model. The hemagglutinin gene of the recombinant vaccine strain was derived from a low-pathogenicity virus obtained prior to the outbreak from a wild mallard. The efficacy of a classical nonadjuvanted subunit vaccine and an immune stimulatory complex-adjuvanted vaccine was compared. Mice receiving the nonadjuvanted vaccine revealed low antibody titers, lack of clinical protection, high virus titers in the lungs, and presence of virus in the spleen, liver, kidneys, and brain. In contrast, mice receiving two doses of the immune stimulatory complex-adjuvanted vaccine revealed high antibody titers, clinical protection, ∼1,000-fold reduction of virus titers in the lungs, and rare detection of the virus in other organs. This is the first report of an H7 vaccine candidate tested in a mammalian model. The data presented suggest that vaccine candidates based on low-pathogenicity avian influenza A viruses, which can be prepared ahead of pandemic threats, can be efficacious if an effective adjuvant is used.
PMCID: PMC1211531  PMID: 16160167

Results 1-8 (8)