The Effect of Vaccination on the Evolution and Population Dynamics of Avian Paramyxovirus-1

Newcastle Disease Virus (NDV) is a pathogenic strain of avian paramyxovirus (aPMV-1) that is among the most serious of disease threats to the poultry industry worldwide. Viral diversity is high in aPMV-1; eight genotypes are recognized based on phylogenetic reconstruction of gene sequences. Modified live vaccines have been developed to decrease the economic losses caused by this virus. Vaccines derived from avirulent genotype II strains were developed in the 1950s and are in use globally, whereas Australian strains belonging to genotype I were developed as vaccines in the 1970s and are used mainly in Asia. In this study, we evaluated the consequences of attenuated live virus vaccination on the evolution of aPMV-1 genotypes. There was phylogenetic incongruence among trees based on individual genes and complete coding region of 54 full length aPMV-1 genomes, suggesting that recombinant sequences were present in the data set. Subsequently, five recombinant genomes were identified, four of which contained sequences from either genotype I or II. The population history of vaccine-related genotype II strains was distinct from other aPMV-1 genotypes; genotype II emerged in the late 19^th century and is evolving more slowly than other genotypes, which emerged in the 1960s. Despite vaccination efforts, genotype II viruses have experienced constant population growth to the present. In contrast, other contemporary genotypes showed population declines in the late 1990s. Additionally, genotype I and II viruses, which are circulating in the presence of homotypic vaccine pressure, have unique selection profiles compared to nonvaccine-related strains. Collectively, these data show that vaccination with live attenuated viruses has changed the evolution of aPMV-1 by maintaining a large effective population size of a vaccine-related genotype, allowing for coinfection and recombination of vaccine and wild type strains, and by applying unique selective pressures on viral glycoproteins.     

Neutral Genomic Microevolution of a Recently Emerged Pathogen,Salmonella enterica Serovar Agona

Salmonella enterica serovar Agona has caused multiple food-borne outbreaks of gastroenteritis since it was first isolated in 1952. We analyzed the genomes of 73 isolates from global sources, comparing five distinct outbreaks with sporadic infections as well as food contamination and the environment. Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s. Homologous recombination with other serovars of S. enterica imported 42 recombinational tracts (360 kb) in 5/143 nodes within the genealogy, which resulted in 3,164 additional SNPs. In contrast to this paucity of genetic diversity, Agona is highly diverse according to pulsed-field gel electrophoresis (PFGE), which is used to assign isolates to outbreaks. PFGE diversity reflects a highly dynamic accessory genome associated with the gain or loss (indels) of 51 bacteriophages, 10 plasmids, and 6 integrative conjugational elements (ICE/IMEs), but did not correlate uniquely with outbreaks. Unlike the core genome, indels occurred repeatedly in independent nodes (homoplasies), resulting in inaccurate PFGE genealogies. The accessory genome contained only few cargo genes relevant to infection, other than antibiotic resistance. Thus, most of the genetic diversity within this recently emerged pathogen reflects changes in the accessory genome, or is due to recombination, but these changes seemed to reflect neutral processes rather than Darwinian selection. Each outbreak was caused by an independent clade, without universal, outbreak-associated genomic features, and none of the variable genes in the pan-genome seemed to be associated with an ability to cause outbreaks.     

Molecular Characterization of the Recently Emerged Poultry Pathogen Ornithobacterium rhinotracheale by Multilocus Sequence Typing

Ornithobacterium rhinotracheale (ORT) is an economically important bacterial pathogen of turkeys and chickens worldwide. Since its first detection, a variety of typing methods have been used to gain basic knowledge about the bacterial population structure, an issue that still needs to be addressed. Serological characterization revealed at least 18 different serotypes (A-R) with ORT of serotype A to be predominate among poultry. This study aimed to establish a multilocus sequence typing (MLST) scheme for ORT that could easily be used by other laboratories and allows for worldwide comparison of sequence data. For this purpose, 87 ORT strains from different poultry hosts, geographical origins, years of isolation and serotypes were included in the analysis to identify correlations. Fourteen different sequence types (ST) were found. The most common ST1 was identified in 40 ORT strains from turkeys and chickens on 4 continents and in 3 different European countries. Together with ST9, both STs represented over three quarters (77%) of ORT strains used in the MLST analysis and included strains of frequently cross-reacting ORT serotypes A, E and I. Nine STs were only represented by one ORT strain and might indicate possible avian host, disease or serotype-specific relationships. In contrast, discrepancies between serotype and phylogenetic relatedness were clearly demonstrated by ORT strains that belonged to identical serotypes but differed in their ST. The overall identified low genetic diversity among strains isolated from turkeys and chickens independent of host and geographical origins suggests that ORT has only recently been introduced into domestic poultry and dispersed worldwide.     

Rates of Molecular Evolution of Primates

This review considers the history and the current state of the debatable issue of variation in accumulation rates of mutational substitutions in the phylogeny of humans and other primates. Arguments pro and contra the hypothesis on gradually decreasing tempo of molecular evolution of humans and apes are discussed. It is shown that data on proteins and (nuclear and mitochondrial) DNA confirm this hypothesis. The conclusion is drawn that the total rate of mutation accumulation is determined by a number of interacting factors. The primary of these factors in most cases (including that of hominids) is generation time, which is closely associated with the number of germ cell divisions. At the same time, fixation rates of nonsynonymous substitutions are also affected by various forms of selection.     

Leukocyte Profiles in Wild House Finches with and without Mycoplasmal Conjunctivitis,a Recently Emerged Bacterial Disease

Leukocyte profiles (relative numbers of white blood cell types) have been used by a growing number of ecological studies to assess immune function and stress in wild birds. House Finches (Carpodacus mexicanus) in eastern North America are susceptible to an eye disease caused by the bacterium Mycoplasma gallisepticum, providing the opportunity to examine whether leukocyte profiles are associated with infection status and other host characteristics. In this study, we compared blood smears from 297 wild House Finches with and without conjunctivitis to examine whether leukocyte profiles were associated with the presence and severity of mycoplasmal conjunctivitis. We also evaluated the influence of age, sex, and molt on leukocyte profiles in both diseased and nondiseased birds. Of 243 House Finches of known age and sex sampled, 30% showed clinical signs of mycoplasmal conjunctivitis. House Finches with conjunctivitis had significantly higher heterophil to lymphocyte (H/L) ratios and harbored greater numbers and proportions of heterophils and monocytes than nondiseased birds. Leukocyte profiles of noninfected birds did not differ significantly with respect to sex, but young birds had significantly greater numbers of lymphocytes and total white blood cells than adults. Molting birds had significantly more eosinophils than nonmolting birds. Finally, House Finches with the most severe outward signs of conjunctivitis showed the most dramatic leukocyte changes relative to noninfected individuals, and increasing H/L ratios and monocytes in diseased birds were paralleled in a subset of birds that were recaptured during the study period. These results are consistent with patterns observed in domestic poultry and suggest that understanding patterns of leukocyte differentials in this host-pathogen system could improve our understanding of innate immunity and infectious disease risk in other wild passerines.     

Pharmacodynamics,Population Dynamics,and the Evolution of Persistence in Staphylococcus aureus

When growing populations of bacteria are confronted with bactericidal antibiotics, the vast majority of cells are killed, but subpopulations of genetically susceptible but phenotypically resistant bacteria survive. In accord with the prevailing view, these “persisters” are non- or slowly dividing cells randomly generated from the dominant population. Antibiotics enrich populations for pre-existing persisters but play no role in their generation. The results of recent studies with Escherichia coli suggest that at least one antibiotic, ciprofloxacin, can contribute to the generation of persisters. To more generally elucidate the role of antibiotics in the generation of and selection for persisters and the nature of persistence in general, we use mathematical models and experiments with Staphylococcus aureus (Newman) and the antibiotics ciprofloxacin, gentamicin, vancomycin, and oxacillin. Our results indicate that the level of persistence varies among these drugs and their concentrations, and there is considerable variation in this level among independent cultures and mixtures of independent cultures. A model that assumes that the rate of production of persisters is low and persisters grow slowly in the presence of antibiotics can account for these observations. As predicted by this model, pre-treatment with sub-MIC concentrations of antibiotics substantially increases the level of persistence to drugs other than those with which the population is pre-treated. Collectively, the results of this jointly theoretical and experimental study along with other observations support the hypothesis that persistence is the product of many different kinds of errors in cell replication that result in transient periods of non-replication and/or slowed metabolism by individual cells in growing populations. This Persistence as Stuff Happens (PaSH) hypothesis can account for the ubiquity of this phenomenon. Like mutation, persistence is inevitable rather than an evolved character. What evolved and have been identified are genes and processes that affect the frequency of persisters.     

Molecular Dating of HIV-1 Subtype C from Bangladesh

Bangladesh has an overall low HIV prevalence of <0.1% in the general population and <1% among key affected populations, but it is one of few Asian countries that has yet to reverse the epidemic. In order to do this, it is important to understand the transmission dynamics in this country. The aim of this study was to investigate the phylogenetic relationships of HIV-1 subtype C strains from Bangladesh and related strains from other countries, and thereby clarify when and from where subtype C was introduced in the country and how it subsequently spread within Bangladesh. The phylogenetic analysis included 118 Bangladeshi gag sequences and 128 sequences from other countries and was performed using the BEAST package. Our analysis revealed that the vast majority of Bangladeshi sequences (97/118, 82%) fall into a large regional cluster of samples from Bangladesh, India, China and Myanmar, which dates back to the early 1960’s. Following its establishment in the region, this strain has entered Bangladesh multiple times from around 1975 and onwards, but extensive in-country transmission could only be detected among drug users and not through sexual transmission. In addition, there have been multiple (at least ten) introductions of subtype C to Bangladesh from outside this region, but no extensive spread could be detected for any of these. Since many HIV-infections remain undetected while asymptomatic, the true extent of the transmission of each strain remains unknown, especially among hard to reach groups such as clients of sex workers and returning migrants with families.     

Origin and Population Dynamics of a Novel HIV-1 Subtype G Clade Circulating in Cape Verde and Portugal

The human immunodeficiency virus type 1 (HIV-1) subtype G is the most prevalent and second most prevalent HIV-1 clade in Cape Verde and Portugal, respectively; but there is no information about the origin and spatiotemporal dispersal pattern of this HIV-1 clade circulating in those countries. To this end, we used Maximum Likelihood and Bayesian coalescent-based methods to analyze a collection of 578 HIV-1 subtype G pol sequences sampled throughout Portugal, Cape Verde and 11 other countries from West and Central Africa over a period of 22 years (1992 to 2013). Our analyses indicate that most subtype G sequences from Cape Verde (80%) and Portugal (95%) branched together in a distinct monophyletic cluster (here called G_CV-PT). The G_CV-PT clade probably emerged after a single migration of the virus out of Central Africa into Cape Verde between the late 1970s and the middle 1980s, followed by a rapid dissemination to Portugal a couple of years later. Reconstruction of the demographic history of the G_CV-PT clade circulating in Cape Verde and Portugal indicates that this viral clade displayed an initial phase of exponential growth during the 1980s and 1990s, followed by a decline in growth rate since the early 2000s. Our data also indicate that during the exponential growth phase the G_CV-PT clade recombined with a preexisting subtype B viral strain circulating in Portugal, originating the CRF14_BG clade that was later disseminated to Spain and Cape Verde. Historical and recent human population movements between Angola, Cape Verde and Portugal probably played a key role in the origin and dispersal of the G_CV-PT and CRF14_BG clades.     

Molecular versus Taxonomic Rates of Evolution in Planktonic Foraminifera

Neogene planktonic foraminifera are among the most widely used microfossils in the study of tempo and mode of evolution. Comparisons of taxonomic rates between the two major clades in this group have shown that the nonspinose globorotaliids have undergone a significantly more rapid evolutionary turnover than the spinose globigerinids (S. M. Stanleyet al., 1988,Paleobiology14, 235–249). In order to test if similar fluctuations are observed in molecular data, we have used different methods to calculate absolute and relative rates of substitutions based on 16 partial SSU rDNA sequences from representatives of both groups. According to our data, rates of substitution are relatively constant within the globigerinids with a mean value of 4.3 subst./site/10⁹years, but vary in the globorotaliid clade with three species having a rate of about 1 subst./site/10⁹years and two species evolving much faster with rates of more than 7 subst./site/10⁹years. Assuming that the fast rates result from recent accelerations, the globorotaliids have basically much slower molecular evolutionary rates than the globigerinids, in opposition to the fossil data.     

Population Dynamics and Growth Rates of Endosymbionts During Diaphorina citri (Hemiptera,Liviidae) Ontogeny

The infection density of symbionts is among the major parameters to understand their biological effects in host–endosymbionts interactions. Diaphorina citri harbors two bacteriome-associated bacterial endosymbionts (Candidatus Carsonella ruddii and Candidatus Profftella armatura), besides the intracellular reproductive parasite Wolbachia. In this study, the density dynamics of the three endosymbionts associated with the psyllid D. citri was investigated by real-time quantitative PCR (qPCR) at different developmental stages. Bacterial density was estimated by assessing the copy number of the 16S rRNA gene for Carsonella and Profftella, and of the ftsZ gene for Wolbachia. Analysis revealed a continuous growth of the symbionts during host development. Symbiont growth and rate curves were estimated by the Gompertz equation, which indicated a negative correlation between the degree of symbiont–host specialization and the time to achieve the maximum growth rate (t*). Carsonella densities were significantly lower than those of Profftella at all host developmental stages analyzed, even though they both displayed a similar trend. The growth rates of Wolbachia were similar to those of Carsonella, but Wolbachia was not as abundant. Adult males displayed higher symbiont densities than females. However, females showed a much more pronounced increase in symbiont density as they aged if compared to males, regardless of the incorporation of symbionts into female oocytes and egg laying. The increased density of endosymbionts in aged adults differs from the usual decrease observed during host aging in other insect–symbiont systems.     

The Rates of Molecular Evolution in Rodent and Primate Mitochondrial DNA

A higher rate of molecular evolution in rodents than in primates at synonymous sites and, to a lesser extent, at amino acid replacement sites has been reported previously for most nuclear genes examined. Thus in these genes the average ratio of amino acid replacement to synonymous substitution rates in rodents is lower than in primates, an observation at odds with the neutral model of molecular evolution. Under Ohta's mildly deleterious model of molecular evolution, these observations are seen as the consequence of the combined effects of a shorter generation time (driving a higher mutation rate) and a larger effective population size (resulting in more effective selection against mildly deleterious mutations) in rodents. The present study reports the results of a maximum-likelihood analysis of the ratio of amino acid replacements to synonymous substitutions for genes encoded in mitochondrial DNA (mtDNA) in these two lineages. A similar pattern is observed: in rodents this ratio is significantly lower than in primates, again consistent only with the mildly deleterious model. Interestingly the lineage-specific difference is much more pronounced in mtDNA-encoded than in nuclear-encoded proteins, an observation which is shown to run counter to expectation under Ohta's model. Finally, accepting certain fossil divergence dates, the lineage-specific difference in amino acid replacement-to-synonymous substitution ratio in mtDNA can be partitioned and is found to be entirely the consequence of a higher mutation rate in rodents. This conclusion is consistent with a replication-dependent model of mutation in mtDNA. Received: 24 September 1999 / Accepted: 18 September 2000     

一株H5N2亚型鹦鹉源禽流感病毒分子进化分析

2005年在广东进行流行病学调查时分离到一株鹦鹉源禽流感病毒,经鉴定为H5N2亚型禽流感病毒(A/Parrot/Guangdong/268/2005)。该毒株的HA裂解位点附近的氨基酸序列为RETRGLF,只含有一个碱性氨基酸,符合低致病性禽流感病毒的HA裂解位点附近氨基酸序列的分子特征;与H5N2亚型禽流感代表毒株相比,该毒株HA和NA基因的糖基化位点、HA基因的受体结合位点编码区、NA基因的耐药性位点均未发生变异。将该毒株全基因组序列与GenBank已公布的19株H5N2亚型禽流感病毒株的相应序列进行比较分析并绘制系统进化树后发现:其与低致病性禽流感毒株A/Pheasant/NJ/1355/1998(H5N2)-like的亲缘关系最近,位于以A/Chicken/Pennsylvania/1/1983(H5N2)为代表的美洲进化分支。     

Genome Sequence of a Recently Emerged,Highly Transmissible,Multi-Antibiotic- and Antiseptic-Resistant Variant of Methicillin-Resistant Staphylococcus aureus,Sequence Type 239 (TW)

The 3.1-Mb genome of an outbreak methicillin-resistant Staphylococcus aureus (MRSA) strain (TW20) contains evidence of recently acquired DNA, including two large regions (635 kb and 127 kb). The strain is resistant to a wide range of antibiotics, antiseptics, and heavy metals due to resistance genes encoded on mobile genetic elements and also mutations in housekeeping genes.A 2-year outbreak of a highly transmissible methicillin-resistant Staphylococcus aureus (MRSA) strain (designated TW) in an intensive care unit (ICU) in London was recently reported (12). Acquisition of TW MRSA was four times more likely to be associated with bacteremia than was acquisition of other commonly found MRSA strains [>95% epidemic (E)MRSA-15 or EMRSA-16]. TW MRSA was also significantly more frequently isolated from vascular access device cultures but less frequently from carriage sites (anterior nares, axilla, and perineum), suggesting that TW differs in its colonization capacity from other MRSA strains. TW was initially defined by its extended antibiotic resistance pattern, being resistant to penicillin, methicillin, erythromycin, ciprofloxacin, gentamicin, neomycin, trimethoprim, and tetracycline (12). TW also had elevated minimum bactericidal concentrations (MBCs) for chlorhexidine and was resistant to a chlorhexidine-based antiseptic protocol effective against other MRSA strains in the ICU (6). TW20 (strain 0582) was a representative bacteremic isolate cultured on 21 October 2003 (12).Multilocus sequence typing (MLST) identified TW20 as sequence type 239 (ST239), an international health care-associated (HA) MRSA lineage prevalent in Asia (19, 38), South America (2, 37), and Eastern Europe (5, 33), which includes EMRSA-1, -4, -7, and -11 and the Brazilian, Portuguese, Hungarian, and Viennese clones (24). To investigate the genetic basis for increased resistance and transmissibility, the TW20 genome was completely sequenced, assembled, and finished and annotated as described previously (16, 25). The final finished genome (10) assembly contained 64,087 capillary reads, giving an average coverage of 13.3. At 3,075,806 bp, the TW20 genome is the largest S. aureus genome sequenced thus far. It consists of a single chromosome of 3,043,210 bp in size (Fig. ​(Fig.1)1) and 2 plasmids (pTW20_1 and pTW20_2), of 29,585 bp and 3,011 bp.Open in a separate windowFIG. 1.Schematic circular diagram of the S. aureus TW20 chromosome. Key for the circular diagram (outer to inner): outer colored segments on the gray outer ring represent genomic islands and horizontally acquired DNA (see the key in the figure); scale (in Mb); annotated CDSs colored according to predicted function are shown on a pair of concentric circles, representing both coding strands; S. aureus reciprocal Fasta matches shared with the S. aureus strains: MRSA252, (accession number {"type":"entrez-nucleotide","attrs":{"text":"BX571856","term_id":"49240382","term_text":"BX571856"}}BX571856) (16), MSSA476 (accession number {"type":"entrez-nucleotide","attrs":{"text":"BX571857","term_id":"49243355","term_text":"BX571857"}}BX571857) (16), MW2 (accession number {"type":"entrez-nucleotide","attrs":{"text":"BA000033","term_id":"47118312","term_text":"BA000033"}}BA000033) (4), N315 (accession number {"type":"entrez-nucleotide","attrs":{"text":"BA000018","term_id":"47118324","term_text":"BA000018"}}BA000018) (20), Mu50 (accession number {"type":"entrez-nucleotide","attrs":{"text":"BA000017","term_id":"47208328","term_text":"BA000017"}}BA000017) (20), Mu3 (accession number {"type":"entrez-nucleotide","attrs":{"text":"AP009324","term_id":"156720466","term_text":"AP009324"}}AP009324) (23), COL (accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000046","term_id":"57284222","term_text":"CP000046"}}CP000046) (13), NCTC8325 (accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000253","term_id":"87201381","term_text":"CP000253"}}CP000253) (14), USA3000 FPR3757 (accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000255","term_id":"87125858","term_text":"CP000255"}}CP000255) (11), JH9 (accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000703","term_id":"147739516","term_text":"CP000703"}}CP000703) (22), Newman (accession number {"type":"entrez-nucleotide","attrs":{"text":"AP009351","term_id":"150373012","term_text":"AP009351"}}AP009351) (3), and RF122 (accession number {"type":"entrez-nucleotide","attrs":{"text":"AJ938182","term_id":"82655308","term_text":"AJ938182"}}AJ938182) (15); regions of the chromosome derived from a CC8 ancestor (light green) or the CC30 ancestor (brown). Color coding for TW20 CDS functions: dark blue, pathogenicity/adaptation; black, energy metabolism; red, information transfer; dark green, surface associated; cyan, degradation of large molecules; magenta, degradation of small molecules; yellow, central/intermediary metabolism; pale green, unknown; pale blue, regulators; orange, conserved hypothetical; brown, pseudogenes; pink, phage and IS elements; gray, miscellaneous.TW20 belongs to clonal complex 8 (CC8), which contains strains NCTC8325 (ST8) (14), Newman (ST8) (3), USA300 (ST8) (11), and COL (ST250) (13). Comparative genomic analysis with these strains by reciprocal Fasta analysis (36) revealed that between 83.7 and 82.7% of protein coding sequences (CDSs) in the TW20 chromosome have reciprocal matches with CC8 members. The highest numbers of matches in any sequenced S. aureus strain, however, was with MRSA 252 (85.9% of CDSs). MRSA 252 (ST36) belongs to CC30 and is a representative of EMRSA-16 that has been a dominant MRSA clone in United Kingdom hospitals for more than 10 years (16). In comparison to CC8, most of the additional matches to MRSA 252 are to CDSs in horizontally acquired mobile genetic elements (MGEs) rather than to orthologous CDSs. A significant component of the S. aureus genome is derived from MGEs that contribute to the accessory genome (21). In the TW20 genome, 16.2% of the CDSs (12.6% of the total genomic DNA) are found in MGEs (Fig. ​(Fig.1).1). Both TW20 and MRSA 252 are representatives of successful hospital-associated MRSA lineages and have large accessory genomes that contain many of the CDSs associated with drug resistance.Methicillin resistance is conferred by a mecA gene on a type III staphylococcal cassette chromosome (SCC) mec element (SCCmecIII). TW20 has a composite SCC region of two SCC elements, SCCmercury and SCCmecIII, identical in structure to the type III SCCmec region found by Ito et al. (18) in an isolate from New Zealand in 1985. The SCCmecIII region is present in a part of the chromosome hypothesized to have been transferred from CC30 into a CC8 background as part of a large block of DNA (26). The approximate boundaries of the recombination were identified from pairwise comparisons of the TW20 chromosome with MRSA 252 (CC30) and USA300 TCH1516 (CC8). A marked shift in DNA percent identity of approximately 1 percentage point was observed across the approximate recombination breakpoints (data not shown), demonstrating that 635 kb (∼20.6% of the TW20 chromosome; SATW20_26800 to SATW20_03960) may have been transferred from a CC30 donor. This transfer event also contributes to the high level of reciprocal Fasta matches between TW20 and MRSA252 (ST36).The origins of SCCmecIII in the TW20 genome are unclear, since SCCmecIII has not been found in the CC30 lineage. Each of the SCC elements contains further MGEs: SCCmercury contains Tn554, encoding a streptomycin 3′-adenylyltransferase and an erythromycin resistance protein, ErmA1, and SCCmec contains an integrated plasmid, pT181, and ΨTn554, containing cadmium resistance CDSs. In addition to Tn554 and ΨTn554 in the SCCmec region, the TW20 chromosome contains an additional Tn554 and a Tn552 transposon, encoding the β-lactamase BlaZ, within an integrative conjugative element (ICE) (31).Further resistance determinants are found on plasmid pTW20_1. Importantly, it carries a gene encoding an antiseptic resistance protein, QacA, that confers resistance to antiseptics such as cationic biocides, quaternary ammonium salts, and diamidines via an export-mediated mechanism (29). In addition, part of the plasmid is highly similar (98 to 100% DNA identity) to the mer operon of the SCCmercury region found on the chromosome (Fig. ​(Fig.2).2). pTW20_1 also contains a homologue of the gene encoding the cadmium-transporting ATPase CadA, found in ΨTn554 of SCCmec. This region in pTW20_1 is bordered by IS431 elements, as it is in the chromosomal copy of SCCmercury. Notably, upstream of the SCCmercury mer operon, there is a CDS that encodes a putative NADH-binding protein. A fragment homologous to the 3′ end of this CDS is also present on pTW20_1 upstream of the mer operon and is truncated by an IS431 element. The absence of the 5′ region of this CDS on pTW20_1 suggests that this region, including the mer operon, may have arisen on the plasmid by recombination between chromosomal and plasmid IS431 elements. It is therefore possible that IS431-mediated recombination plays a role in the evolution of the SCC region.Open in a separate windowFIG. 2.Comparative analysis of the TW20 plasmid pTW20_1 with the mer operon of the TW20 SCC region. Pairwise comparisons of the TW20 SCC region containing the mer operon from the TW20 chromosome (top) with the TW20 plasmid pTW20_1 (bottom) using the Artemis Comparison Tool (ACT) (9) are shown. The colored bars separating each sequence (red and blue) represent matches identified by BlastN (1); red lines link matches in the same orientation, and blue lines link matches in the reverse orientation. CDSs associated with metal and drug resistance are marked, as are IS431 elements. Colored bars at the top of the figure indicate parts of the sequence found in the SCCmercury (blue) and SCCmec (green) elements, including ΨTn554 (yellow), that make up this region.Two other drug resistance genes, encoding a tetracycline resistance protein, TetM, and a trimethoprim-resistant dihydrofolate reductase, DfrG, are found in a 31.3-kb region (Tn5801-like), similar to transposons/ICE found in the genomes of S. aureus strains Mu50 (20) and Mu3 (23) and Streptococcus agalactiae strain COH1 (35). In comparison to the Tn5801 elements in Mu50 and Mu3, the TW20 element contains an additional four CDSs, including dfrG, in the central region of the element.There are three prophage within the TW20 genome, two of which are similar to those previously found in sequenced S. aureus genomes: φSa1(TW20) is 43.3 kb in size, is integrated within the 5′ region of a lipase gene, and does not carry CDSs with homology to known virulence factors; φSa3(TW20) is 44.7 kb in size, is integrated in the phospholipase C gene, and carries the staphylococcal complement inhibitor SCIN (28), staphylokinase (30), and enterotoxin A (7) genes associated with virulence. Genes for two other enterotoxins, enterotoxins K and Q, are carried on a Staphylococcus aureus pathogenicity island (SaPI), SaPI1.At 127.2 kb, the third prophage, φSPβ-like(TW20), is markedly larger than the other two and does not display similarity with other S. aureus prophage. φSPβ-like(TW20) exhibits extended similarity with the φSPß-like region in the Staphylococcus epidermidis RP62a genome (13) (Fig. ​(Fig.3).3). Comparison of the two sequences reveals a region of sequence divergence and rearrangement in the center of the prophage. In φSPβ-like(TW20), this region contains CDSs associated with aminoglycoside resistance and streptothricin resistance (Fig. ​(Fig.3).3). In addition, φSPβ-like(TW20) contains a CDS that may have a role in promoting persistence of TW20 in the hospital setting. S. aureus possesses many surface-anchored proteins with the LPxTG motif, which bind host molecules (27). SATW20_21850 encodes an LPxTG motif surface-anchored protein that does not have orthologs in any of the genomes of the other sequenced S. aureus strains currently available. A highly similar CDS (95.1% amino acid identity), sesI (8), is present in the S. epidermidis φSPβ-like region (Fig. ​(Fig.3).3). A recent study by Söderquist et al. found that sesI was absent from normal S. epidermidis flora of healthy individuals without any health care association but was found in approximately 50% of clinical isolates causing invasive infections, leading them to suggest that this gene was a potential marker of invasive capacity (32). The presence of an LPxTG motif surface-anchored protein on an MGE in TW20 suggests that this strain has augmented its array of this family of functionally important proteins through a recent acquisition event and therefore this LPxTG motif surface-anchored protein may not be widely distributed in related strains. Genome sequencing of a global collection of ST239 strains revealed only 7% (3/42) of isolates were positive for orthologs of this CDS (14a). Work is under way to survey the wider distribution of this gene in the S. aureus population and investigate the function of the encoded protein.Open in a separate windowFIG. 3.Comparative analysis of φSPβ-like(TW20) prophage with the S. epidermidis RP62a φSPß-like prophage. Pairwise BlastN comparison of the S. aureus TW20 prophage φSPβ-like(TW20) region from the TW20 chromosome (top) with the S. epidermidis RP62a φSPß-like prophage region from the RP62a chromosome (bottom) (13) displayed in ACT is shown. The extent of the φSPβ-like(TW20) prophage in the TW20 sequence, which extends from SATW20_20290 to SATW20_21850, is marked by the pink horizontal bar.Evidence of adaptation to survive in a health care environment is also found in the core genome. Several housekeeping genes have alleles associated with antibiotic resistance. The TW20 DNA gyrase subunit A (GyrA) contains a leucine residue at position 84. The more-widespread residue in S. aureus GyrA proteins is serine, suggesting this is the plesiomorphic amino acid at this position. In vitro studies have demonstrated that substitution of Ser84Leu generates resistance to quinolones in S. aureus (34). TW20 exhibits low-level resistance to mupirocin. The TW20 isoleucyl-tRNA synthetase contains a phenylalanine residue at position 588. The substitution of Val588Phe has been shown to confer chromosomal low-level mupirocin resistance in S. aureus without significantly affecting fitness (17).In conclusion, genomic analysis of TW20 provides evidence of its adaptation to survive in a health care setting through acquisition of drug and antiseptic resistance genes carried on MGEs, large chromosomal insertions, and point mutations in housekeeping genes. The large size of the TW20 genome reflects the ability of the ST239 lineage to undergo prolonged and continuing evolution to adapt to the hospital environment. Further studies are under way to elucidate the components of the genome that promote transmission and interaction with the host.     

人口动力学中非线性发展方程解的爆破现象

讨论描述人口发展规律的一类非线性发展方程具有第三类非线性边界条件的混合问题．在已知函数满足某些假设条件下,证明了其解在有限时间内爆破．     

Chinese and Global Distribution of H9 Subtype Avian Influenza Viruses

H9 subtype avian influenza viruses (AIVs) are of significance in poultry and public health, but epidemiological studies about the viruses are scarce. In this study, phylogenetic relationships of the viruses were analyzed based on 1233 previously reported sequences and 745 novel sequences of the viral hemagglutinin gene. The novel sequences were obtained through large-scale surveys conducted in 2008-2011 in China. The results revealed distinct distributions of H9 subtype AIVs in different hosts, sites and regions in China and in the world: (1) the dominant lineage of H9 subtype AIVs in China in recent years is lineage h9.4.2.5 represented by A/chicken/Guangxi/55/2005; (2) the newly emerging lineage h9.4.2.6, represented by A/chicken/Guangdong/FZH/2011, has also become prevalent in China; (3) lineages h9.3.3, h9.4.1 and h9.4.2, represented by A/duck/Hokkaido/26/99, A/quail/Hong Kong/G1/97 and A/chicken/Hong Kong/G9/97, respectively, have become globally dominant in recent years; (4) lineages h9.4.1 and h9.4.2 are likely of more risk to public health than others; (5) different lineages have different transmission features and host tropisms. This study also provided novel experimental data which indicated that the Leu-234 (H9 numbering) motif in the viral hemagglutinin gene is an important but not unique determinant in receptor-binding preference. This report provides a detailed and updated panoramic view of the epidemiological distributions of H9 subtype AIVs globally and in China, and sheds new insights for the prevention of infection in poultry and preparedness for a potential pandemic caused by the viruses.     

Amino-Acid Co-Variation in HIV-1 Gag Subtype C: HLA-Mediated Selection Pressure and Compensatory Dynamics

Background

Despite high potential for HIV-1 genetic variation, the emergence of some mutations is constrained by fitness costs, and may be associated with compensatory amino acid (AA) co-variation. To characterize the interplay between Cytotoxic T Lymphocyte (CTL)-mediated pressure and HIV-1 evolutionary pathways, we investigated AA co-variation in Gag sequences obtained from 449 South African individuals chronically infected with HIV-1 subtype C.

Methodology/Principal Findings

Individuals with CTL responses biased toward Gag presented lower viral loads than individuals with under-represented Gag-specific CTL responses. Using methods that account for founder effects and HLA linkage disequilibrium, we identified 35 AA sites under Human Leukocyte Antigen (HLA)-restricted CTL selection pressure and 534 AA-to-AA interactions. Analysis of two-dimensional distances between co-varying residues revealed local stabilization mechanisms since 40% of associations involved neighboring residues. Key features of our co-variation analysis included sites with a high number of co-varying partners, such as HLA-associated sites, which had on average 55% more connections than other co-varying sites.

Conclusions/Significance

Clusters of co-varying AA around HLA-associated sites (especially at typically conserved sites) suggested that cooperative interactions act to preserve the local structural stability and protein function when CTL escape mutations occur. These results expose HLA-imprinted HIV-1 polymorphisms and their interlinked mutational paths in Gag that are likely due to opposite selective pressures from host CTL-mediated responses and viral fitness constraints.