H7亚型禽流感病毒与禽类和人类的关系

2013年在中国首次发生了H7N9亚型流感病毒感染人事件,已经证实H7N9型禽流感是一种新型禽流感,是全球首次发现感染人类的新亚型流感病毒,以往这种病毒只在野生鸟类存在和传播。H7N9型禽流感病毒属于H7亚型中的一种,全球感染人的H7亚型病毒主要分为两大支系,即北美支系和欧亚支系,感染人的流感亚型也主要集中在H7N7,H7N3,H7N2等亚型上。为了清晰的了解H7亚型病毒的来龙去脉,本文重点讨论了A亚型流感病毒的宿主分布、H7亚型病毒感染禽类和人类的历史、H7亚型病毒的生物学特性以及未来研究展望。     

Histone H1 subtype synthesis in neurons and neuroblasts.

Rat cerebral cortex neurons contain the five histone H1 subtypes H1a-e and the subtype H1 zero present in other mammalian somatic tissues. The four subtypes H1a-d decay exponentially during postnatal development and are partially or totally replaced by H1e that becomes the major H1 subtype in adults. H1 zero accumulates in a period restricted to neuronal terminal differentiation. Here we study the synthesis of the H1 subtypes in cortical neurons and their neuroblasts by in vivo labeling with [14C]lysine. The subtype synthesis pattern of neuroblasts has been determined by labeling gravid rats during the period of proliferation of cortical neurons and synthesis in neurons has been studied by postnatal labeling. The subtype H1a is synthesized in neuroblasts but not in neurons and is therefore rapidly removed from neuronal chromatin. The synthesis of H1b and H1d is much lower in neurons than in neuroblasts so that these subtypes are replaced to a large extent during postnatal development. H1c is synthesized at levels much higher than the other subtypes both in neurons and neuroblasts, but its very high turnover, about one order of magnitude faster than that of H1e in neurons, favors its partial replacement during postnatal development. Comparison of the synthesis rates of H1 zero in newborn and 30-day-old rats shows that the accumulation of H1 zero in differentiating neurons is due to an increased level of synthesis.     

H1 subtype expression during cell proliferation and growth arrest

H1 histone subtype genes differ in their expression patterns during the different stages of the cell cycle interphase. While the group of replication-dependent H1 histone subtypes is synthesized during S phase, the replacement histone subtype H1.0 is also expressed replication-independently in non-proliferating cells. The present study is the first report about the analysis of the cell cycle-dependent expression of all five replication-dependent H1 subtypes, the replacement histone H1.0 and the ubiquitously expressed subtype H1x. The expression of these H1 histone subtypes in HeLa cells was analysed on mRNA level by quantitative real-time RT-PCR as well as on protein level by immunoblotting. We found that after arrest of HeLa cells in G1 phase by treatment with sodium butyrate, the mRNA levels of all replication-dependently expressed H1 subtypes decreased, but to very different extent. During S phase the individual replication-dependently expressed H1 subtypes show similar kinetics regarding their mRNA levels. However, the variations in their protein amounts partially differ from the respective RNA levels which especially applies to histone H1.3. In contrast, the mRNA as well as the protein level of H1x remained nearly unchanged in G1 as well as during S phase progression. The results of the present study demonstrate that the cell cycle-dependent mRNA and protein expression of various H1 subtypes is differentially regulated, supporting the hypothesis of a functional heterogeneity.     

G1 phase-dependent nucleolar accumulation of human histone H1x.

BACKGROUND INFORMATION: H1 histones are a protein family comprising several subtypes. Although specific functions of the individual subtypes could not be determined so far, differential roles are indicated by varied nuclear distributions as well as differential expression patterns of the H1 subtypes. Although the group of replication-dependent H1 subtypes is synthesized during S phase, the replacement H1 subtype, H1 degrees , is also expressed in a replication-independent manner in non-proliferating cells. Recently we showed, by protein biochemical analysis, that the ubiquitously expressed subtype H1x is enriched in the micrococcal nuclease-resistant part of chromatin and that, although it shares common features with H1 degrees , its expression is differentially regulated, since, in contrast to H1 degrees , growth arrest or induction of differentiation did not induce an accumulation of H1x. RESULTS: In the present study, we show that H1x exhibits a cell-cycle-dependent change of its nuclear distribution. This H1 subtype showed a nucleolar accumulation during the G(1) phase, and it was evenly distributed in the nucleus during S phase and G(2). Immunocytochemical analysis of the intranucleolar distribution of H1x indicated that it is located mainly in the condensed nucleolar chromatin. In addition, we demonstrate that the amount of H1x protein remained nearly unchanged during S phase progression, which is in contrast to the replication-dependent subtypes. CONCLUSION: These results suggest that the differential localization of H1x provides a mechanism for a control of H1x activity by means of shuttling between nuclear subcompartments instead of a controlled turnover of the protein.     

Changes in H1 complement in differentiating rat-brain cortical neurons

Neuronal nuclei have a low H1 content. A stoichiometry of 0.47 molecule/nucleosome, on average, is calculated for rat brain cortical neurons by comparing its H1 content with that of liver nuclei. The H1 fraction of rat cerebral cortex neurons has been resolved into five subtypes, H1a--e, that have the same mobility as the unphosphorylated H1 forms of other rat tissues. The subtypes H1a--d decay exponentially during postnatal development and are substituted to different extents by H1e. The higher replacement rate is shown by H1a with an apparent half-lifetime of about 5 days. The corresponding values for H1b, H1c and H1d are 11, 21 and 15 days. Several conclusions can be drawn from the observation of postnatal changes in H1 subtype proportions. The low H1 content of neuronal nuclei does not imply the presence of notable peculiarities in subtype composition or in subtype substitution pattern. There is turnover of H1 in differentiating neurons once cell proliferation and DNA replication have ceased. The relative rates of synthesis and/or degradation of the subtypes differ in germinal cells and in neurons. Comparison with previous results on H1 degrees accumulation also shows that in cortical neurons the regulation of the subtypes H1a--e differs from that of H1 degrees.     

Nucleosome Interaction Surface of Linker Histone H1c Is Distinct from That of H10

The fully organized structure of the eukaryotic nucleosome remains unsolved, in part due to limited information regarding the binding site of the H1 or linker histone. The central globular domain of H1 is believed to interact with the nucleosome core at or near the dyad and to bind at least two strands of DNA. We utilized site-directed mutagenesis and in vivo photobleaching to identify residues that contribute to the binding of the globular domain of the somatic H1 subtype H1c to the nucleosome. As was previously observed for the H1⁰ subtype, the binding residues for H1c are clustered on the surface of one face of the domain. Despite considerable structural conservation between the globular domains of these two subtypes, the locations of the binding sites identified for H1c are distinct from those of H1⁰. We suggest that the globular domains of these two linker histone subtypes will bind to the nucleosome with distinct orientations that may contribute to higher order chromatin structure heterogeneity or to differences in dynamic interactions with other DNA or chromatin-binding proteins.     

Normal spermatogenesis in mice lacking the testis-specific linker histone H1t

H1 histones bind to linker DNA and nucleosome core particles and facilitate the folding of chromatin into a more compact structure. Mammals contain seven nonallelic subtypes of H1, including testis-specific subtype H1t, which varies considerably in primary sequence from the other H1 subtypes. H1t is found only in pachytene spermatocytes and early, haploid spermatids, constituting as much as 55% of the linker histone associated with chromatin in these cell types. To investigate the role of H1t in spermatogenesis, we disrupted the H1t gene by homologous recombination in mouse embryonic stem cells. Mice homozygous for the mutation and completely lacking H1t protein in their germ cells were fertile and showed no detectable defect in spermatogenesis. Chromatin from H1t-deficient germ cells had a normal ratio of H1 to nucleosomes, indicating that other H1 subtypes are deposited in chromatin in place of H1t and presumably compensate for most or all H1t functions. The results indicate that despite the unique primary structure and regulated synthesis of H1t, it is not essential for proper development of mature, functional sperm.     

Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin

Background  

Histone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1° for long chromatin fragments (30–35 nucleosomes) in physiological salt (0.14 M NaCl) at constant stoichiometry.     

Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls

In wild aquatic birds and poultry around the world, influenza A viruses carrying 15 antigenic subtypes of hemagglutinin (HA) and 9 antigenic subtypes of neuraminidase (NA) have been described. Here we describe a previously unidentified antigenic subtype of HA (H16), detected in viruses circulating in black-headed gulls in Sweden. In agreement with established criteria for the definition of antigenic subtypes, hemagglutination inhibition assays and immunodiffusion assays failed to detect specific reactivity between H16 and the previously described subtypes H1 to H15. Genetically, H16 HA was found to be distantly related to H13 HA, a subtype also detected exclusively in shorebirds, and the amino acid composition of the putative receptor-binding site of H13 and H16 HAs was found to be distinct from that in HA subtypes circulating in ducks and geese. The H16 viruses contained NA genes that were similar to those of other Eurasian shorebirds but genetically distinct from N3 genes detected in other birds and geographical locations. The European gull viruses were further distinguishable from other influenza A viruses based on their PB2, NP, and NS genes. Gaining information on the full spectrum of avian influenza A viruses and creating reagents for their detection and identification will remain an important task for influenza surveillance, outbreak control, and animal and public health. We propose that sequence analyses of HA and NA genes of influenza A viruses be used for the rapid identification of existing and novel HA and NA subtypes.     

Characterisation of human histone H1x

The members of the H1 histone family can be classified into three groups, which are the main class subtypes expressed in somatic cells, the developmental- and tissue-specific subtypes, and the replacement subtype H1(o). Until now, the subtype H1x was not classified, since it has not yet been thoroughly examined. The results of this study show that H1x shares similarities but also exhibits slight differences in its biochemical behaviour in comparison to the main class H1 histones. In HeLa cells it is located in the nucleus and partially associated with nucleosomes. Nevertheless, it is, like H1(o), mainly located in chromatin regions that are not affected by micrococcal nuclease digestion. Further common features of H1x and the replacement histone H1(o) are that the genes of both subtypes are solitarily located and give rise to polyadenylated mRNA. However, comparison of the inducibility of their expression revealed that their genes are regulated differentially.     

Analysis of Synonymous Codon Usage Bias in 09H1N1

A novel subtype of influenza A virus 09H1N1 has rapidly spread across the world. Evolutionary analyses of this virus have revealed that 09H1N1 is a triple reassortant of segments from swine, avian and human influenza viruses. In this study, we investigated factors shaping the codon usage bias of 09H1N1 and carried out cluster analysis of 60 strains of influenza A virus from different subtypes based on their codon usage bias. We discovered that more preferentially used codons of 09H1N1 are A-ended or U-ended...     

Isolation and characterisation of five histone H1 subtypes from adult rat liver

A procedure is described for quantitative purification of H10 and five H1-1 subtypes--named H1-1a to e--from adult rat liver by reverse-phase high-pressure liquid chromatography. Milligram amounts of each fraction have been obtained. The H1-1a subtype shows a very high lysine content (34%) and H1-1d subtype has an amino-acid composition close to that of H10, but its electrophoretic mobility is different. Salt dependent folding of these subtypes has been studied by circular dichroism. In the presence of 2 or 10 mM sodium phosphate buffers at pH 7.5, H1-1a shows the lowest alpha-helix content. In phosphate-buffer containing 1 M NaCl the number of residues in alpha-helix for all the subtypes rises to 9-10%. Partial cleavage of these subtypes by endoproteinase Glu-C produce three main peptides arising from C-terminal domains. The interaction of the H1-1 subtypes with 196 basepairs linear DNA, purified from rat liver chromatin by high-pressure ion-exchange liquid chromatography, has for consequences a modification of the patterns of digestion: partial proteolysis of the H1-1a and H1-1b subtypes shows differences in the presence or in absence of DNA; on the contrary, H1-1c and H1-1d seem to have the same organization. So these subtypes may play a role in the differential packing of specific region of chromatin.     

G1 phase-dependent nucleolar accumulation of human histone H1x

Background information: H1 histones are a protein family comprising several subtypes. Although specific functions of the individual subtypes could not be determined so far, differential roles are indicated by varied nuclear distributions as well as differential expression patterns of the H1 subtypes. Although the group of replication‐dependent H1 subtypes is synthesized during S phase, the replacement H1 subtype, H1°, is also expressed in a replication‐independent manner in non‐proliferating cells. Recently we showed, by protein biochemical analysis, that the ubiquitously expressed subtype H1x is enriched in the micrococcal nuclease‐resistant part of chromatin and that, although it shares common features with H1°, its expression is differentially regulated, since, in contrast to H1°, growth arrest or induction of differentiation did not induce an accumulation of H1x. Results: In the present study, we show that H1x exhibits a cell‐cycle‐dependent change of its nuclear distribution. This H1 subtype showed a nucleolar accumulation during the G₁ phase, and it was evenly distributed in the nucleus during S phase and G₂. Immunocytochemical analysis of the intranucleolar distribution of H1x indicated that it is located mainly in the condensed nucleolar chromatin. In addition, we demonstrate that the amount of H1x protein remained nearly unchanged during S phase progression, which is in contrast to the replication‐dependent subtypes. Conclusion: These results suggest that the differential localization of H1x provides a mechanism for a control of H1x activity by means of shuttling between nuclear subcompartments instead of a controlled turnover of the protein.     

Selection of DNA aptamers that bind to influenza A viruses with high affinity and broad subtype specificity

Many cases of influenza are reported worldwide every year. The influenza virus often acquires new antigenicity, which is known as antigenic shift; this results in the emergence of new virus strains, for which preexisting immunity is not found in the population resulting in influenza pandemics. In the event a new strain emerges, diagnostic tools must be developed rapidly to detect the novel influenza strain. The generation of high affinity antibodies is costly and takes time; therefore, an alternative detection system, aptamer detection, provides a viable alternative to antibodies as a diagnostic tool. In this study, we developed DNA aptamers that bind to HA1 proteins of multiple influenza A virus subtypes by the SELEX procedure. To evaluate the binding properties of these aptamers using colorimetric methods, we developed a novel aptamer-based sandwich detection method employing our newly identified aptamers. This novel sandwich enzyme-linked aptamer assay successfully detected the H5N1, H1N1, and H3N2 subtypes of influenza A virus with almost equal sensitivities. These findings suggest that our aptamers are attractive candidates for use as simple and sensitive diagnostic tools that need sandwich system for detecting the influenza A virus with broad subtype specificities.     

Differences in Patient Age Distribution between Influenza A Subtypes

Since the spring of 1977, two subtypes of influenza A virus (H3N2 and H1N1) have been seasonally infecting the human population. In this work we study the distribution of patient ages within the populations that exhibit the symptomatic disease caused by each of the different subtypes of seasonal influenza viruses. When the publicly available extensive information is pooled across multiple geographical locations and seasons, striking differences emerge between these subtypes. We report that the symptomatic flu due to H1N1 is distributed mainly in a younger population relative to H3N2. (The median age of the H3N2 patients is 23 years while H1N1 patients are 9 years old.) These distinct characteristic spectra of age groups, possibly carried over from previous pandemics, are consistent with previous reports from various regional population studies and also findings on the evolutionary dynamics of each subtype. Moreover, they are relevant to age-related risk assessments, modeling of epidemiological networks for specific age groups, and age-specific vaccine design. Recently, a novel H1N1 virus has spread around the world. Preliminary reports suggest that this new strain causes symptomatic disease in the younger population in a similar fashion to the seasonal H1N1 strains.     

Calculated flexibility of histone proteins correlate with mammalian histone H1 subtype.

We have calculated the polypeptide flexibility index for mammalian histone H1 sequences obtained from the National Center for Biotechnology Information Histone Sequence Database. This database contains over 1000 histone protein entries, from various species, compiled from SWISS_PROT, PIR, the Protein Data Bank (PDB), and CDS translations from GenBank. Histone H1 proteins were analyzed because of their critical role in chromatin structure and gene expression. Flexibility calculations revealed that histone subtype H1.0, which accumulates during terminal differentiation, has the highest flexibility index of all mammalian H1 subtypes. Other mammalian H1 subtypes had lower flexibility indices, including the human H1.2 subtype whose mRNA contains both a hairpin loop sequence and a poly(A) addition sequence. Histone mRNAs containing both of these structures have been shown to be expressed prior to and after terminal differentiation, yet these proteins do not necessarily accumulate in the chromatin of terminally differentiated cells. H1.2 and the H1.t have the lowest flexibility index (most ridged) of all human H1 subtypes. All human H1 proteins of the replication dependent subtypes have intermediate values for their flexibility indices.     

Subtype cross-reactive, infection-enhancing antibody responses to influenza A viruses.

Antibody-dependent enhancement of the uptake of influenza A virus by Fc receptor-bearing cells was analyzed by using virus strains of the three human influenza A virus subtypes, A/PR/8/34 (H1N1), A/Japan/305/57 (H2N2), and A/Port Chalmers/1/73 (H3N2). Immune sera obtained from mice following primary infection with an H1N1, H2N2, or H3N2 subtype virus neutralized only virus of the same subtype; however, immune sera augmented the uptake of virus across subtypes. Immune sera from H1N1-infected mice augmented uptake of the homologous (H1N1) and H2N2 viruses. Antisera to the H2N2 virus augmented the uptake of virus of all subtypes (H1N1, H2N2, or H3N2). Antisera to the H3N2 virus augmented the uptake of the homologous (H3N2) and H2N2 viruses. These results show that subtype cross-reactive, nonneutralizing antibodies augment the uptake of influenza A virus strains of different subtypes. Antibodies to neuraminidase may contribute to the enhanced uptake of viruses of a different subtype, because N2-specific monoclonal antibodies augmented the uptake of both A/Japan/305/57 (H2N2) and A/Port Chalmers/1/73 (H3N2) viruses.     

Influenza-A viruses in ducks in northwestern Minnesota: fine scale spatial and temporal variation in prevalence and subtype diversity

Waterfowl from northwestern Minnesota were sampled by cloacal swabbing for Avian Influenza Virus (AIV) from July-October in 2007 and 2008. AIV was detected in 222 (9.1%) of 2,441 ducks in 2007 and in 438 (17.9%) of 2,452 ducks in 2008. Prevalence of AIV peaked in late summer. We detected 27 AIV subtypes during 2007 and 31 during 2008. Ten hemagglutinin (HA) subtypes were detected each year (i.e., H1, 3-8, and 10-12 during 2007; H1-8, 10 and 11 during 2008). All neuraminidase (NA) subtypes were detected during each year of the study. Subtype diversity varied between years and increased with prevalence into September. Predominant subtypes during 2007 (comprising ≥5% of subtype diversity) included H1N1, H3N6, H3N8, H4N6, H7N3, H10N7, and H11N9. Predominant subtypes during 2008 included H3N6, H3N8, H4N6, H4N8, H6N1, and H10N7. Additionally, within each HA subtype, the same predominant HA/NA subtype combinations were detected each year and included H1N1, H3N8, H4N6, H5N2, H6N1, H7N3, H8N4, H10N7, and H11N9. The H2N3 and H12N5 viruses also predominated within the H2 and H12 subtypes, respectively, but only were detected during a single year (H2 and H12 viruses were not detected during 2007 and 2008, respectively). Mallards were the predominant species sampled (63.7% of the total), and 531 AIV were isolated from this species (80.5% of the total isolates). Mallard data collected during both years adequately described the observed temporal and spatial prevalence from the total sample and also adequately represented subtype diversity. Juvenile mallards also were adequate in describing the temporal and spatial prevalence of AIV as well as subtype diversity.     

Synthesis and degradation of H1 histone subtypes in mouse lymphoma L5178Y cells

H1 histone of mouse lymphoma L5178Y was fractionated into five subtypes, I-V, by Bio-Rex 70 column chromatography. The rates of synthesis of subtypes III and V were higher than those of I, II, and IV, as determined by the measurement of [3H]lysine incorporation. The degradation of the subtype was estimated assuming first order kinetics; subtypes III and V had half-lives of 18 h and 25 h, respectively, and the three other subtypes all had half-lives of 63 h. The syntheses of these subtypes during the cell cycle were examined using synchronized cultures. The syntheses of subtypes I, II, and IV started at the beginning of S phase, whereas those of III and V started in mid-S phase. The syntheses of III and V were at least 1.5-2 times more rapid than those of I, II, and IV, and their active synthesis was accompanied by their rapid degradation. The five subtypes of H1 were further characterized in relation to phosphorylation. Each showed characteristic differences in its synthetic pattern or phosphorylation, and we concluded that each H1 subtype has its own specific function at least in the process of replication of chromatin.