Rebecca A. Halpin

Whole-Genome Sequence Analysis of Pseudomonas syringae pv. phaseolicola 1448A Reveals Divergence among Pathovars in Genes Involved in Virulence and Transposition

Pseudomonas syringae pv. phaseolicola, a gram-negative bacterial plant pathogen, is the causal agent of halo blight of bean. In this study, we report on the genome sequence of P. syringae pv. phaseolicola isolate 1448A, which encodes 5,353 open reading frames (ORFs) on one circular chromosome (5,928,787 bp) and two plasmids (131,950 bp and 51,711 bp). Comparative analyses with a phylogenetically divergent pathovar, P. syringae pv. tomato DC3000, revealed a strong degree of conservation at the gene and genome levels. In total, 4,133 ORFs were identified as putative orthologs in these two pathovars using a reciprocal best-hit method, with 3,941 ORFs present in conserved, syntenic blocks. Although these two pathovars are highly similar at the physiological level, they have distinct host ranges; 1448A causes disease in beans, and DC3000 is pathogenic on tomato and Arabidopsis. Examination of the complement of ORFs encoding virulence, fitness, and survival factors revealed a substantial, but not complete, overlap between these two pathovars. Another distinguishing feature between the two pathovars is their distinctive sets of transposable elements. With access to a fifth complete pseudomonad genome sequence, we were able to identify 3,567 ORFs that likely comprise the core Pseudomonas genome and 365 ORFs that are P. syringae specific.

Viral genome sequencing by random priming methods

BackgroundMost emerging health threats are of zoonotic origin. For the overwhelming majority, their causative agents are RNA viruses which include but are not limited to HIV, Influenza, SARS, Ebola, Dengue, and Hantavirus. Of increasing importance therefore is a better understanding of global viral diversity to enable better surveillance and prediction of pandemic threats; this will require rapid and flexible methods for complete viral genome sequencing.ResultsWe have adapted the SISPA methodology [1–3] to genome sequencing of RNA and DNA viruses. We have demonstrated the utility of the method on various types and sources of viruses, obtaining near complete genome sequence of viruses ranging in size from 3,000–15,000 kb with a median depth of coverage of 14.33. We used this technique to generate full viral genome sequence in the presence of host contaminants, using viral preparations from cell culture supernatant, allantoic fluid and fecal matter.ConclusionThe method described is of great utility in generating whole genome assemblies for viruses with little or no available sequence information, viruses from greatly divergent families, previously uncharacterized viruses, or to more fully describe mixed viral infections.

Genome sequence of Synechococcus CC9311: Insights into adaptation to a coastal environment.

Coastal aquatic environments are typically more highly productive and dynamic than open ocean ones. Despite these differences, cyanobacteria from the genus Synechococcus are important primary producers in both types of ecosystems. We have found that the genome of a coastal cyanobacterium, Synechococcus sp. strain CC9311, has significant differences from an open ocean strain, Synechococcus sp. strain WH8102, and these are consistent with the differences between their respective environments. CC9311 has a greater capacity to sense and respond to changes in its (coastal) environment. It has a much larger capacity to transport, store, use, or export metals, especially iron and copper. In contrast, phosphate acquisition seems less important, consistent with the higher concentration of phosphate in coastal environments. CC9311 is predicted to have differences in its outer membrane lipopolysaccharide, and this may be characteristic of the speciation of some cyanobacterial groups. In addition, the types of potentially horizontally transferred genes are markedly different between the coastal and open ocean genomes and suggest a more prominent role for phages in horizontal gene transfer in oligotrophic environments.

Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.

Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.

Temporally structured metapopulation dynamics and persistence of influenza A H3N2 virus in humans.

Populations of seasonal influenza virus experience strong annual bottlenecks that pose a considerable extinction risk. It has been suggested that an influenza source population located in tropical Southeast or East Asia seeds annual temperate epidemics. Here we investigate the seasonal dynamics and migration patterns of influenza A H3N2 virus by analysis of virus samples obtained from 2003 to 2006 from Australia, Europe, Japan, New York, New Zealand, Southeast Asia, and newly sequenced viruses from Hong Kong. In contrast to annual temperate epidemics, relatively low levels of relative genetic diversity and no seasonal fluctuations characterized virus populations in tropical Southeast Asia and Hong Kong. Bayesian phylogeographic analysis using discrete temporal and spatial characters reveal high rates of viral migration between urban centers tested. Although the virus population that migrated between Southeast Asia and Hong Kong persisted through time, this was dependent on virus input from temperate regions and these tropical regions did not maintain a source for annual H3N2 influenza epidemics. We further show that multiple lineages may seed annual influenza epidemics, and that each region may function as a potential source population. We therefore propose that the global persistence of H3N2 influenza A virus is the result of a migrating metapopulation in which multiple different localities may seed seasonal epidemics in temperate regions in a given year. Such complex global migration dynamics may confound control efforts and contribute to the emergence and spread of antigenic variants and drug-resistant viruses.

PB2 Residue 158 Is a Pathogenic Determinant of Pandemic H1N1 and H5 Influenza A Viruses in Mice

ABSTRACT Influenza A viruses are human and animal pathogens that cause morbidity and mortality, which range from mild to severe. The 2009 H1N1 pandemic was caused by the emergence of a reassortant H1N1 subtype (H1N1pdm) influenza A virus containing gene segments that originally circulated in human, avian, and swine virus reservoirs. The molecular determinants of replication and pathogenesis of H1N1pdm viruses in humans and other mammals are poorly understood. Therefore, we set out to elucidate viral determinants critical to the pathogenesis of this novel reassortant using a mouse model. We found that a glutamate-to-glycine substitution at residue 158 of the PB2 gene (PB2-E158G) increased the morbidity and mortality of the parental H1N1pdm virus. Results from mini-genome replication assays in human cells and virus titration in mouse tissues demonstrated that PB2-E158G is a pathogenic determinant, because it significantly increases viral replication rates. The virus load in PB2-E158G-infected mouse lungs was 1,300-fold higher than that of the wild-type virus. Our data also show that PB2-E158G had a much stronger influence on the RNA replication and pathogenesis of H1N1pdm viruses than PB2-E627K, which is a known pathogenic determinant. Remarkably, PB2-E158G substitutions also altered the pathotypes of two avian H5 viruses in mice, indicating that this residue impacts genetically divergent influenza A viruses and suggesting that this region of PB2 could be a new antiviral target. Collectively, the data presented in this study demonstrate that PB2-E158G is a novel pathogenic determinant of influenza A viruses in the mouse model. We speculate that PB2-E158G may be important in the adaptation of avian PB2 genes to other mammals, and BLAST sequence analysis identified a naturally occurring human H1N1pdm isolate that has this substitution. Therefore, future surveillance efforts should include scrutiny of this region of PB2 because of its potential impact on pathogenesis.

Deep Sequencing Reveals Mixed Infection with 2009 Pandemic Influenza A (H1N1) Virus Strains and the Emergence of Oseltamivir Resistance

Mixed infections with seasonal influenza A virus strains are a common occurrence and an important source of genetic diversity. Prolonged viral shedding, as observed in immunocompromised individuals, can lead to mutational accumulation over extended periods. Recently, drug resistance was reported in immunosuppressed patients infected with the 2009 pandemic influenza A (H1N1) virus within a few days after oseltamivir treatment was initiated. To better understand the evolution and emergence of drug resistance in these circumstances, we used a deep sequencing approach to survey the viral population from an immunosuppressed patient infected with H1N1/2009 influenza and treated with neuraminidase inhibitors. This patient harbored 3 genetic variants from 2 phylogenetically distinct viral clades of pandemic H1N1/2009, strongly suggestive of mixed infection. Strikingly, one of these variants also developed drug resistance de novo in response to oseltamivir treatment. Immunocompromised individuals may, therefore, constitute an important source of genetic and phenotypic diversity, both through mixed infection and de novo mutation.

Mixed Infection and the Genesis of Influenza Virus Diversity

ABSTRACT The emergence of viral infections with potentially devastating consequences for human health is highly dependent on their underlying evolutionary dynamics. One likely scenario for an avian influenza virus, such as A/H5N1, to evolve to one capable of human-to-human transmission is through the acquisition of genetic material from the A/H1N1 or A/H3N2 subtypes already circulating in human populations. This would require that viruses of both subtypes coinfect the same cells, generating a mixed infection, and then reassort. Determining the nature and frequency of mixed infection with influenza virus is therefore central to understanding the emergence of pandemic, antigenic, and drug-resistant strains. To better understand the potential for such events, we explored patterns of intrahost genetic diversity in recently circulating strains of human influenza virus. By analyzing multiple viral genome sequences sampled from individual influenza patients we reveal a high level of mixed infection, including diverse lineages of the same influenza virus subtype, drug-resistant and -sensitive strains, those that are likely to differ in antigenicity, and even viruses of different influenza virus types (A and B). These results reveal that individuals can harbor influenza viruses that differ in major phenotypic properties, including those that are antigenically distinct and those that differ in their sensitivity to antiviral agents.

Molecular Epidemiology of A/H3N2 and A/H1N1 Influenza Virus during a Single Epidemic Season in the United States

To determine the spatial and temporal dynamics of influenza A virus during a single epidemic, we examined whole-genome sequences of 284 A/H1N1 and 69 A/H3N2 viruses collected across the continental United States during the 2006–2007 influenza season, representing the largest study of its kind undertaken to date. A phylogenetic analysis revealed that multiple clades of both A/H1N1 and A/H3N2 entered and co-circulated in the United States during this season, even in localities that are distant from major metropolitan areas, and with no clear pattern of spatial spread. In addition, co-circulating clades of the same subtype exchanged genome segments through reassortment, producing both a minor clade of A/H3N2 viruses that appears to have re-acquired sensitivity to the adamantane class of antiviral drugs, as well as a likely antigenically distinct A/H1N1 clade that became globally dominant following this season. Overall, the co-circulation of multiple viral clades during the 2006–2007 epidemic season revealed patterns of spatial spread that are far more complex than observed previously, and suggests a major role for both migration and reassortment in shaping the epidemiological dynamics of human influenza A virus.

Genome Stability of Lyme Disease Spirochetes: Comparative Genomics of Borrelia burgdorferi Plasmids

Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33–40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi ∼900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short ≤20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.