Assessment of the antiviral properties of recombinant porcine SP-D against various influenza A viruses in vitro

The emergence of influenza viruses resistant to existing classes of antiviral drugs raises concern and there is a need for novel antiviral agents that could be used therapeutically or prophylacticaly. Surfactant protein D (SP-D) belongs to the family of C-type lectins which are important effector molecules of the innate immune system with activity against bacteria and viruses, including influenza viruses. In the present study we evaluated the potential of recombinant porcine SP-D as an antiviral agent against influenza A viruses (IAVs) in vitro. To determine the range of antiviral activity, thirty IAVs of the subtypes H1N1, H3N2 and H5N1 that originated from birds, pigs and humans were selected and tested for their sensitivity to recombinant SP-D. Using these viruses it was shown by hemagglutination inhibition assay, that recombinant porcine SP-D was more potent than recombinant human SP-D and that especially higher order oligomeric forms of SP-D had the strongest antiviral activity. Porcine SP-D was active against a broad range of IAV strains and neutralized a variety of H1N1 and H3N2 IAVs, including 2009 pandemic H1N1 viruses. Using tissue sections of ferret and human trachea, we demonstrated that recombinant porcine SP-D prevented attachment of human seasonal H1N1 and H3N2 virus to receptors on epithelial cells of the upper respiratory tract. It was concluded that recombinant porcine SP-D holds promise as a novel antiviral agent against influenza and further development and evaluation in vivo seems warranted.     

Preferential HLA usage in the influenza virus-specific CTL response

To study whether individual HLA class I alleles are used preferentially or equally in human virus-specific CTL responses, the contribution of individual HLA-A and -B alleles to the human influenza virus-specific CTL response was investigated. To this end, PBMC were obtained from three groups of HLA-A and -B identical blood donors and stimulated with influenza virus. In the virus-specific CD8(+) T cell population, the proportion of IFN-gamma- and TNF-alpha-producing cells, restricted by individual HLA-A and -B alleles, was determined using virus-infected C1R cells expressing a single HLA-A or -B allele for restimulation of these cells. In HLA-B*2705- and HLA-B*3501-positive individuals, these alleles were preferentially used in the influenza A virus-specific CTL response, while the contribution of HLA-B*0801 and HLA-A*0101 was minor in these donors. The magnitude of the HLA-B*0801-restricted response was even lower in the presence of HLA-B*2705. C1R cells expressing HLA-B*2705, HLA-A*0101, or HLA-A*0201 were preferentially lysed by virus-specific CD8(+) T cells. In contrast, the CTL response to influenza B virus was mainly directed toward HLA-B*0801-restricted epitopes. Thus, the preferential use of HLA alleles depended on the virus studied.     

Recognition of homo- and heterosubtypic variants of influenza A viruses by human CD8+ T lymphocytes

In the present study, the recognition of epitope variants of influenza A viruses by human CTL was investigated. To this end, human CD8(+) CTL clones, specific for natural variants of the HLA-B*3501-restricted epitope in the nucleoprotein (NP(418-426)), were generated. As determined in (51)Cr release assays and by flow cytometry with HLA-B*3501-peptide tetrameric complexes, CTL clones were found to be specific for epitopes within one subtype or cross-reactive with heterosubtypic variants of the epitope. Using eight natural variants of the epitope, positions in the 9-mer important for T cell recognition and involved in escape from CTL immunity were identified and visualized using multidimensional scaling. It was shown that positions 4 and 5 in the 9-mer epitope were important determinants of T cell specificity. The in vivo existence of CD8(+) cells cross-reactive with homo- and heterosubtypic variants of the epitope was further confirmed using polyclonal T cell populations obtained after stimulation of PBMC with different influenza A viruses. Based on the observed recognition patterns of the clonal and polyclonal T cell populations and serology, it is hypothesized that consecutive infections with influenza viruses containing different variants of the epitope select for cross-reactive T cells in vivo.     

Introduction of Virulence Markers in PB2 of Pandemic Swine-Origin Influenza Virus Does Not Result in Enhanced Virulence or Transmission

In the first 6 months of the H1N1 swine-origin influenza virus (S-OIV) pandemic, the vast majority of infections were relatively mild. It has been postulated that mutations in the viral genome could result in more virulent viruses, leading to a more severe pandemic. Mutations E627K and D701N in the PB2 protein have previously been identified as determinants of avian and pandemic influenza virus virulence in mammals. These mutations were absent in S-OIVs detected early in the 2009 pandemic. Here, using reverse genetics, mutations E627K, D701N, and E677G were introduced into the prototype S-OIV A/Netherlands/602/2009, and their effects on virus replication, virulence, and transmission were investigated. Mutations E627K and D701N caused increased reporter gene expression driven by the S-OIV polymerase complex. None of the three mutations affected virus replication in vitro. The mutations had no major impact on virus replication in the respiratory tracts of mice and ferrets or on pathogenesis. All three mutant viruses were transmitted via aerosols or respiratory droplets in ferrets. Thus, the impact of key known virulence markers in PB2 in the context of current S-OIVs was surprisingly small. This study does not exclude the possibility of emergence of S-OIVs with other virulence-associated mutations in the future. We conclude that surveillance studies aimed at detecting S-OIVs with increased virulence or transmission should not rely solely on virulence markers identified in the past but should include detailed characterization of virus phenotypes, guided by genetic signatures of viruses detected in severe cases of disease in humans.The new H1N1 swine-origin influenza virus (S-OIV) recently emerged to cause the first influenza pandemic in 40 years (2). The S-OIV presumably emerged from pigs, as its genome was shown to consist of six gene segments of “triple-reassortant” swine viruses and two of “Eurasian lineage” swine viruses (9). The start of the S-OIV pandemic has been relatively mild, with a clinical spectrum ranging from mild upper respiratory tract illness to sporadic cases of severe pneumonia leading to acute respiratory distress syndrome (22). As of 15 November 2009, worldwide, more than 206 countries have reported laboratory-confirmed cases of S-OIV infection, including over 6,770 deaths (32).In previous influenza pandemics, such as the Spanish influenza pandemic of 1918 and the Hong Kong influenza pandemic of 1968, a first wave of cases of relatively mild illnesses was followed by more severe subsequent waves (29). The reason for this increased severity has remained largely unknown, but one possible explanation could be that the pandemic viruses required further adaptation to the human host, resulting in the emergence of viruses that were more virulent than those of the first wave. Such adaptive changes could occur by gene reassortment between cocirculating influenza A viruses or by mutation.In the past decade, determinants of influenza A virus virulence have been mapped using reverse genetics with a variety of pandemic, epidemic, and zoonotic influenza viruses. Mutations affecting virulence and host range have frequently been mapped to hemagglutinin (HA) and neuraminidase (NA) in relation to their interaction with sialic acids, the virus receptors on host cells (11, 18, 30). Nonstructural protein 1 (NS1) has been implicated in the virulence of highly pathogenic avian influenza (HPAI) virus H5N1 and the 1918 H1N1 virus, as the NS1 proteins of these viruses were shown to act as strong antagonists of the interferon pathways (10, 25). Furthermore, the polymerase genes, in particular the PB2 gene, have been shown to be important determinants of virulence in the HPAI H5N1 and H7N7 viruses and of transmission in the 1918 H1N1 virus (11, 21, 31). One of the most commonly identified virulence markers to date is E627K in PB2. The glutamic acid (E) residue is found generally in avian influenza viruses, while human viruses have a lysine (K), and this mutation has been described as a determinant of the host range in vitro (28). When avian viruses lacking the E627K substitution were passaged in mice, the viruses acquired the mutation spontaneously upon a single passage (15, 17). In the HPAI H5N1 and H7N7 viruses, E627K was shown to be the prime determinant of pathogenesis in mice (11, 21, 23). Given that all human and many zoonotic influenza viruses of the last century contained 627K (1), it was surprising that the S-OIV had 627E.Additionally, the aspartate (D)-to-asparagine (N) mutation at position 701 of PB2, which was shown to compensate for the absence of E627K, has also not been detected in S-OIV (27). This D701N mutation has previously been shown to expand the host range of avian H5N1 to mice and humans (3, 15) and to increase virus transmission in guinea pigs (27). Thus, S-OIV was the first known human pandemic virus with 627E and 701D, and it has been speculated that S-OIV could mutate into a more virulent form by acquiring one of these mutations, or both.On 8 May 2009, the detection of another mutation in the PB2 gene of S-OIV, an E-to-glycine (G) mutation at position 667, was reported (http://www.promedmail.org/pls/apex/f?p=2400:1000, archive no. 20090508.1722). It has previously been suggested that the E667G substitution in PB2 of HPAI H5N1 virus was under positive selection and possibly played a role in sustainable transmission in humans (14).On 28 September 2009, detection of the E627K mutation in PB2 of S-OIVs of two individuals in the Netherlands was reported (http://www.promedmail.org/pls/apex/f?p=2400:1000, archive no. 20090928.3394) and raised concern about the possible enhanced replication of the S-OIV in humans, possibly associated with increased virulence. To date, the D701N mutation in PB2 has not been reported in any of the S-OIVs sequenced, and additional viruses with mutation E627K have not been recorded, either. In contrast, viruses with E677G have been reported from the United States, Canada, Germany, the United Kingdom, Norway, and France, according to the public sequence databases.Here, the effects of the E627K, D701N, and E677G mutations in the PB2 genes of S-OIVs was investigated using genetically engineered influenza viruses based on a prototype S-OIV, A/Netherlands/602/2009. Polymerase activity was measured in minigenome assays in human 293T cells, virus replication was analyzed in Madin-Darby Canine kidney (MDCK) cells, virulence was tested in mouse and ferret models, and transmission by aerosols or respiratory droplets was tested in ferrets. In contrast to the earlier assumptions based on experience with other influenza A viruses, S-OIVs with E627K, D701N, or E677G in PB2 did not show a marked increase in virulence or transmission compared to the wild-type virus.     

On the reproducibility of microcosm experiments - different community composition in parallel phototrophic biofilm microcosms

Phototrophic biofilms were cultivated simultaneously using the same inoculum in three identical flow-lane microcosms located in different laboratories. The growth rates of the biofilms were similar in the different microcosms, but denaturing gradient gel electrophoresis (DGGE) analysis of both 16S and 18S rRNA gene fragments showed that the communities developed differently in terms of species richness and community composition. One microcosm was dominated by Microcoleus and Phormidium species, the second microcosm was dominated by Synechocystis and Phormidium species, and the third microcosm was dominated by Microcoleus- and Planktothrix- affiliated species. No clear effect of light intensity on the cyanobacterial community composition was observed. In addition, DGGE profiles obtained from the cultivated biofilms showed a low resemblance with the profiles derived from the inoculum. These findings demonstrate that validation of reproducibility is essential for the use of microcosm systems in microbial ecology studies.     

Mating factor linkage and genome evolution in basidiomycetous pathogens of cereals

Sex in basidiomycete fungi is controlled by tetrapolar mating systems in which two unlinked gene complexes determine up to thousands of mating specificities, or by bipolar systems in which a single locus (MAT) specifies different sexes. The genus Ustilago contains bipolar (Ustilago hordei) and tetrapolar (Ustilago maydis) species and sexual development is associated with infection of cereal hosts. The U. hordei MAT-1 locus is unusually large (approximately 500 kb) and recombination is suppressed in this region. We mapped the genome of U. hordei and sequenced the MAT-1 region to allow a comparison with mating-type regions in U. maydis. Additionally the rDNA cluster in the U. hordei genome was identified and characterized. At MAT-1, we found 47 genes along with a striking accumulation of retrotransposons and repetitive DNA; the latter features were notably absent from the corresponding U. maydis regions. The tetrapolar mating system may be ancestral and differences in pathogenic life style and potential for inbreeding may have contributed to genome evolution.     

Iron availability increases the pathogenic potential of Salmonella typhimurium and other enteric pathogens at the intestinal epithelial interface

Recent trials have questioned the safety of untargeted oral iron supplementation in developing regions. Excess of luminal iron could select for enteric pathogens at the expense of beneficial commensals in the human gut microflora, thereby increasing the incidence of infectious diseases. The objective of the current study was to determine the effect of high iron availability on virulence traits of prevalent enteric pathogens at the host-microbe interface. A panel of enteric bacteria was cultured under iron-limiting conditions and in the presence of increasing concentrations of ferric citrate to assess the effect on bacterial growth, epithelial adhesion, invasion, translocation and epithelial damage in vitro. Translocation and epithelial integrity experiments were performed using a transwell system in which Caco-2 cells were allowed to differentiate to a tight epithelial monolayer mimicking the intestinal epithelial barrier. Growth of Salmonella typhimurium and other enteric pathogens was increased in response to iron. Adhesion of S. typhimurium to epithelial cells markedly increased when these bacteria were pre-incubated with increasing iron concentration (P = 0.0001), whereas this was not the case for the non-pathogenic Lactobacillus plantarum (P = 0.42). Cellular invasion and epithelial translocation of S. typhimurium followed the trend of increased adhesion. Epithelial damage was increased upon incubation with S. typhimurium or Citrobacter freundii that were pre-incubated under iron-rich conditions. In conclusion, our data fit with the consensus that oral iron supplementation is not without risk as iron could, in addition to inducing pathogenic overgrowth, also increase the virulence of prevalent enteric pathogens.     

Avian influenza a virus in wild birds in highly urbanized areas

Avian influenza virus (AIV) surveillance studies in wild birds are usually conducted in rural areas and nature reserves. Less is known of avian influenza virus prevalence in wild birds located in densely populated urban areas, while these birds are more likely to be in close contact with humans. Influenza virus prevalence was investigated in 6059 wild birds sampled in cities in the Netherlands between 2006 and 2009, and compared with parallel AIV surveillance data from low urbanized areas in the Netherlands. Viral prevalence varied with the level of urbanization, with highest prevalence in low urbanized areas. Within cities virus was detected in 0.5% of birds, while seroprevalence exceeded 50%. Ring recoveries of urban wild birds sampled for virus detection demonstrated that most birds were sighted within the same city, while few were sighted in other cities or migrated up to 2659 km away from the sample location in the Netherlands. Here we show that urban birds were infected with AIVs and that urban birds were not separated completely from populations of long-distance migrants. The latter suggests that wild birds in cities may play a role in the introduction of AIVs into cities. Thus, urban bird populations should not be excluded as a human-animal interface for influenza viruses.     

Genome comparison of barley and maize smut fungi reveals targeted loss of RNA silencing components and species-specific presence of transposable elements

Ustilago hordei is a biotrophic parasite of barley (Hordeum vulgare). After seedling infection, the fungus persists in the plant until head emergence when fungal spores develop and are released from sori formed at kernel positions. The 26.1-Mb U. hordei genome contains 7113 protein encoding genes with high synteny to the smaller genomes of the related, maize-infecting smut fungi Ustilago maydis and Sporisorium reilianum but has a larger repeat content that affected genome evolution at important loci, including mating-type and effector loci. The U. hordei genome encodes components involved in RNA interference and heterochromatin formation, normally involved in genome defense, that are lacking in the U. maydis genome due to clean excision events. These excision events were possibly a result of former presence of repetitive DNA and of an efficient homologous recombination system in U. maydis. We found evidence of repeat-induced point mutations in the genome of U. hordei, indicating that smut fungi use different strategies to counteract the deleterious effects of repetitive DNA. The complement of U. hordei effector genes is comparable to the other two smuts but reveals differences in family expansion and clustering. The availability of the genome sequence will facilitate the identification of genes responsible for virulence and evolution of smut fungi on their respective hosts.     

Characterization of the active microbiotas associated with honey bees reveals healthier and broader communities when colonies are genetically diverse

Recent losses of honey bee colonies have led to increased interest in the microbial communities that are associated with these important pollinators. A critical function that bacteria perform for their honey bee hosts, but one that is poorly understood, is the transformation of worker-collected pollen into bee bread, a nutritious food product that can be stored for long periods in colonies. We used 16S rRNA pyrosequencing to comprehensively characterize in genetically diverse and genetically uniform colonies the active bacterial communities that are found on honey bees, in their digestive tracts, and in bee bread. This method provided insights that have not been revealed by past studies into the content and benefits of honey bee-associated microbial communities. Colony microbiotas differed substantially between sampling environments and were dominated by several anaerobic bacterial genera never before associated with honey bees, but renowned for their use by humans to ferment food. Colonies with genetically diverse populations of workers, a result of the highly promiscuous mating behavior of queens, benefited from greater microbial diversity, reduced pathogen loads, and increased abundance of putatively helpful bacteria, particularly species from the potentially probiotic genus Bifidobacterium. Across all colonies, Bifidobacterium activity was negatively correlated with the activity of genera that include pathogenic microbes; this relationship suggests a possible target for understanding whether microbes provide protective benefits to honey bees. Within-colony diversity shapes microbiotas associated with honey bees in ways that may have important repercussions for colony function and health. Our findings illuminate the importance of honey bee-bacteria symbioses and examine their intersection with nutrition, pathogen load, and genetic diversity, factors that are considered key to understanding honey bee decline.