Lori A. Rowe

A distinct lineage of influenza A virus from bats

Influenza A virus reservoirs in animals have provided novel genetic elements leading to the emergence of global pandemics in humans. Most influenza A viruses circulate in waterfowl, but those that infect mammalian hosts are thought to pose the greatest risk for zoonotic spread to humans and the generation of pandemic or panzootic viruses. We have identified an influenza A virus from little yellow-shouldered bats captured at two locations in Guatemala. It is significantly divergent from known influenza A viruses. The HA of the bat virus was estimated to have diverged at roughly the same time as the known subtypes of HA and was designated as H17. The neuraminidase (NA) gene is highly divergent from all known influenza NAs, and the internal genes from the bat virus diverged from those of known influenza A viruses before the estimated divergence of the known influenza A internal gene lineages. Attempts to propagate this virus in cell cultures and chicken embryos were unsuccessful, suggesting distinct requirements compared with known influenza viruses. Despite its divergence from known influenza A viruses, the bat virus is compatible for genetic exchange with human influenza viruses in human cells, suggesting the potential capability for reassortment and contributions to new pandemic or panzootic influenza A viruses.

A hybrid approach for the automated finishing of bacterial genomes

Advances in DNA sequencing technology have improved our ability to characterize most genomic diversity. However, accurate resolution of large structural events is challenging because of the short read lengths of second-generation technologies. Third-generation sequencing technologies, which can yield longer multikilobase reads, have the potential to address limitations associated with genome assembly. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at >99.9% accuracy. Complex regions with clinically relevant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 cholera reference strain, we obtained 14 scaffolds of greater than 1 kb for the experimental data and 8 scaffolds of greater than 1 kb for the simulated data, which allowed us to correct several errors in contigs assembled from the short-read data alone. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly.

Comparative Genomics of Vibrio cholerae from Haiti, Asia, and Africa

A strain from Haiti shares genetic ancestry with those from Asia and Africa.

Evolutionary dynamics of Vibrio cholerae O1 following a single-source introduction to Haiti

ABSTRACT Prior to the epidemic that emerged in Haiti in October of 2010, cholera had not been documented in this country. After its introduction, a strain of Vibrio cholerae O1 spread rapidly throughout Haiti, where it caused over 600,000 cases of disease and >7,500 deaths in the first two years of the epidemic. We applied whole-genome sequencing to a temporal series of V. cholerae isolates from Haiti to gain insight into the mode and tempo of evolution in this isolated population of V. cholerae O1. Phylogenetic and Bayesian analyses supported the hypothesis that all isolates in the sample set diverged from a common ancestor within a time frame that is consistent with epidemiological observations. A pangenome analysis showed nearly homogeneous genomic content, with no evidence of gene acquisition among Haiti isolates. Nine nearly closed genomes assembled from continuous-long-read data showed evidence of genome rearrangements and supported the observation of no gene acquisition among isolates. Thus, intrinsic mutational processes can account for virtually all of the observed genetic polymorphism, with no demonstrable contribution from horizontal gene transfer (HGT). Consistent with this, the 12 Haiti isolates tested by laboratory HGT assays were severely impaired for transformation, although unlike previously characterized noncompetent V. cholerae isolates, each expressed hapR and possessed a functional quorum-sensing system. Continued monitoring of V. cholerae in Haiti will illuminate the processes influencing the origin and fate of genome variants, which will facilitate interpretation of genetic variation in future epidemics. IMPORTANCE Vibrio cholerae is the cause of substantial morbidity and mortality worldwide, with over three million cases of disease each year. An understanding of the mode and rate of evolutionary change is critical for proper interpretation of genome sequence data and attribution of outbreak sources. The Haiti epidemic provides an unprecedented opportunity to study an isolated, single-source outbreak of Vibrio cholerae O1 over an established time frame. By using multiple approaches to assay genetic variation, we found no evidence that the Haiti strain has acquired any genes by horizontal gene transfer, an observation that led us to discover that it is also poorly transformable. We have found no evidence that environmental strains have played a role in the evolution of the outbreak strain. Vibrio cholerae is the cause of substantial morbidity and mortality worldwide, with over three million cases of disease each year. An understanding of the mode and rate of evolutionary change is critical for proper interpretation of genome sequence data and attribution of outbreak sources. The Haiti epidemic provides an unprecedented opportunity to study an isolated, single-source outbreak of Vibrio cholerae O1 over an established time frame. By using multiple approaches to assay genetic variation, we found no evidence that the Haiti strain has acquired any genes by horizontal gene transfer, an observation that led us to discover that it is also poorly transformable. We have found no evidence that environmental strains have played a role in the evolution of the outbreak strain.

Characterization of the Bacterial Communities of Life Stages of Free Living Lone Star Ticks ( Amblyomma americanum )

The lone star tick (Amblyomma americanum) is an abundant and aggressive biter of humans, domestic animals, and wildlife in the southeastern-central USA and an important vector of several known and suspected zoonotic bacterial pathogens. However, the biological drivers of bacterial community variation in this tick are still poorly defined. Knowing the community context in which tick-borne bacterial pathogens exist and evolve is required to fully understand the ecology and immunobiology of the ticks and to design effective public health and veterinary interventions. We performed a metagenomic survey of the bacterial communities of questing A. americanum and tested 131 individuals (66 nymphs, 24 males, and 41 females) from five sites in three states. Pyrosequencing was performed with barcoded eubacterial primers targeting variable 16S rRNA gene regions 5–3. The bacterial communities were dominated by Rickettsia (likely R. amblyommii) and an obligate Coxiella symbiont, together accounting for 6.7–100% of sequences per tick. DNAs from Midichloria, Borrelia, Wolbachia, Ehrlichia, Pseudomonas, or unidentified Bacillales, Enterobacteriaceae, or Rhizobiales groups were also detected frequently. Wolbachia and Midichloria significantly co-occurred in Georgia (p<0.00001), but not in other states. The significance of the Midichloria-Wolbachia co-occurrence is unknown. Among ticks collected in Georgia, nymphs differed from adults in both the composition (p = 0.002) and structure (p = 0.002) of their bacterial communities. Adults differed only in their community structure (p = 0.002) with males containing more Rickettsia and females containing more Coxiella. Comparisons among adult ticks collected in New York and North Carolina supported the findings from the Georgia collection despite differences in geography, collection date, and sample handling, implying that the differences detected are consistent attributes. The data also suggest that some members of the bacterial community change during the tick life cycle and that some sex-specific attributes may be detectable in nymphs.

Genome Sequences for Five Strains of the Emerging Pathogen Haemophilus haemolyticus

We report the first whole-genome sequences for five strains, two carried and three pathogenic, of the emerging pathogen Haemophilus haemolyticus. Preliminary analyses indicate that these genome sequences encode markers that distinguish H. haemolyticus from its closest Haemophilus relatives and provide clues to the identity of its virulence factors.

Insights into the environmental reservoir of pathogenic Vibrio parahaemolyticus using comparative genomics

Vibrio parahaemolyticus is an aquatic halophilic bacterium that occupies estuarine and coastal marine environments, and is a leading cause of seafood-borne food poisoning cases. To investigate the environmental reservoir and potential gene flow that occurs among V. parahaemolyticus isolates, the virulence-associated gene content and genome diversity of a collection of 133 V. parahaemolyticus isolates were analyzed. Phylogenetic analysis of housekeeping genes, and pulsed-field gel electrophoresis, demonstrated that there is genetic similarity among V. parahaemolyticus clinical and environmental isolates. Whole-genome sequencing and comparative analysis of six representative V. parahaemolyticus isolates was used to identify genes that are unique to the clinical and environmental isolates examined. Comparative genomics demonstrated an O3:K6 environmental isolate, AF91, which was cultured from sediment collected in Florida in 2006, has significant genomic similarity to the post-1995 O3:K6 isolates. However, AF91 lacks the majority of the virulence-associated genes and genomic islands associated with these highly virulent post-1995 O3:K6 genomes. These findings demonstrate that although they do not contain most of the known virulence-associated regions, some V. parahaemolyticus environmental isolates exhibit significant genetic similarity to clinical isolates. This highlights the dynamic nature of the V. parahaemolyticus genome allowing them to transition between aquatic and host-pathogen states.

Genome Structural Diversity among 31 Bordetella pertussis Isolates from Two Recent U.S. Whooping Cough Statewide Epidemics.

Pertussis, or whooping cough, is the most poorly controlled vaccine-preventable bacterial disease in the United States, which has experienced a resurgence for more than a decade. Once viewed as a monomorphic pathogen, B. pertussis strains circulating during epidemics exhibit diversity visible on a genome structural level, previously undetectable by traditional sequence analysis using short-read technologies. For the first time, we combine short- and long-read sequencing platforms with restriction optical mapping for single-contig, de novo assembly of 31 isolates to investigate two geographically and temporally independent U.S. pertussis epidemics. These complete genomes reshape our understanding of B. pertussis evolution and strengthen molecular epidemiology toward one day understanding the resurgence of pertussis. ABSTRACT During 2010 and 2012, California and Vermont, respectively, experienced statewide epidemics of pertussis with differences seen in the demographic affected, case clinical presentation, and molecular epidemiology of the circulating strains. To overcome limitations of the current molecular typing methods for pertussis, we utilized whole-genome sequencing to gain a broader understanding of how current circulating strains are causing large epidemics. Through the use of combined next-generation sequencing technologies, this study compared de novo, single-contig genome assemblies from 31 out of 33 Bordetella pertussis isolates collected during two separate pertussis statewide epidemics and 2 resequenced vaccine strains. Final genome architecture assemblies were verified with whole-genome optical mapping. Sixteen distinct genome rearrangement profiles were observed in epidemic isolate genomes, all of which were distinct from the genome structures of the two resequenced vaccine strains. These rearrangements appear to be mediated by repetitive sequence elements, such as high-copy-number mobile genetic elements and rRNA operons. Additionally, novel and previously identified single nucleotide polymorphisms were detected in 10 virulence-related genes in the epidemic isolates. Whole-genome variation analysis identified state-specific variants, and coding regions bearing nonsynonymous mutations were classified into functional annotated orthologous groups. Comprehensive studies on whole genomes are needed to understand the resurgence of pertussis and develop novel tools to better characterize the molecular epidemiology of evolving B. pertussis populations. IMPORTANCE Pertussis, or whooping cough, is the most poorly controlled vaccine-preventable bacterial disease in the United States, which has experienced a resurgence for more than a decade. Once viewed as a monomorphic pathogen, B. pertussis strains circulating during epidemics exhibit diversity visible on a genome structural level, previously undetectable by traditional sequence analysis using short-read technologies. For the first time, we combine short- and long-read sequencing platforms with restriction optical mapping for single-contig, de novo assembly of 31 isolates to investigate two geographically and temporally independent U.S. pertussis epidemics. These complete genomes reshape our understanding of B. pertussis evolution and strengthen molecular epidemiology toward one day understanding the resurgence of pertussis.

Whole genome sequence and comparative genomics of the novel lyme borreliosis causing pathogen, Borrelia mayonii

Borrelia mayonii, a Borrelia burgdorferi sensu lato (Bbsl) genospecies, was recently identified as a cause of Lyme borreliosis (LB) among patients from the upper midwestern United States. By microscopy and PCR, spirochete/genome loads in infected patients were estimated at 105 to 106 per milliliter of blood. Here, we present the full chromosome and plasmid sequences of two B. mayonii isolates, MN14-1420 and MN14-1539, cultured from blood of two of these patients. Whole genome sequencing and assembly was conducted using PacBio long read sequencing (Pacific Biosciences RSII instrument) followed by hierarchical genome-assembly process (HGAP). The B. mayonii genome is ~1.31 Mbp in size (26.9% average GC content) and is comprised of a linear chromosome, 8 linear and 7 circular plasmids. Consistent with its taxonomic designation as a new Bbsl genospecies, the B. mayonii linear chromosome shares only 93.83% average nucleotide identity with other genospecies. Both B. mayonii genomes contain plasmids similar to B. burgdorferi sensu stricto lp54, lp36, lp28-3, lp28-4, lp25, lp17, lp5, 5 cp32s, cp26, and cp9. The vls locus present on lp28-10 of B. mayonii MN14-1420 is remarkably long, being comprised of 24 silent vls cassettes. Genetic differences between the two B. mayonii genomes are limited and include 15 single nucleotide variations as well as 7 fewer silent vls cassettes and a lack of the lp5 plasmid in MN14-1539. Notably, 68 homologs to proteins present in B. burgdorferi sensu stricto appear to be lacking from the B. mayonii genomes. These include the complement inhibitor, CspZ (BB_H06), the fibronectin binding protein, BB_K32, as well as multiple lipoproteins and proteins of unknown function. This study shows the utility of long read sequencing for full genome assembly of Bbsl genomes, identifies putative genome regions of B. mayonii that may be linked to clinical manifestation or tissue tropism, and provides a valuable resource for pathogenicity, diagnostic and vaccine studies.

The Establishment and Diversification of Epidemic-Associated Serogroup W Meningococcus in the African Meningitis Belt, 1994 to 2012

Meningococcal disease (meningitis and bloodstream infections) threatens millions of people across the meningitis belt of sub-Saharan Africa. A vaccine introduced in 2010 protects against Africa’s then-most common cause of meningococcal disease, N. meningitidis serogroup A. However, other serogroups continue to cause epidemics in the region—including serogroup W. The rapid identification of strains that have been associated with prior outbreaks can improve the assessment of outbreak risk and enable timely preparation of public health responses, including vaccination. Phylogenetic analysis of newly sequenced serogroup W strains isolated from 1994 to 2012 identified two groups of strains linked to large epidemics in Burkina Faso, one being descended from a strain that caused an outbreak during the Hajj pilgrimage in 2000. We find that applying whole-genome sequencing to meningococcal disease surveillance collections improves the discrimination among strains, even within a single nation-wide epidemic, which can be used to better understand pathogen spread. ABSTRACT Epidemics of invasive meningococcal disease (IMD) caused by meningococcal serogroup A have been eliminated from the sub-Saharan African so-called “meningitis belt” by the meningococcal A conjugate vaccine (MACV), and yet, other serogroups continue to cause epidemics. Neisseria meningitidis serogroup W remains a major cause of disease in the region, with most isolates belonging to clonal complex 11 (CC11). Here, the genetic variation within and between epidemic-associated strains was assessed by sequencing the genomes of 92 N. meningitidis serogroup W isolates collected between 1994 and 2012 from both sporadic and epidemic IMD cases, 85 being from selected meningitis belt countries. The sequenced isolates belonged to either CC175 (n = 9) or CC11 (n = 83). The CC11 N. meningitidis serogroup W isolates belonged to a single lineage comprising four major phylogenetic subclades. Separate CC11 N. meningitidis serogroup W subclades were associated with the 2002 and 2012 Burkina Faso epidemics. The subclade associated with the 2012 epidemic included isolates found in Burkina Faso and Mali during 2011 and 2012, which descended from a strain very similar to the Hajj (Islamic pilgrimage to Mecca)-related Saudi Arabian outbreak strain from 2000. The phylogeny of isolates from 2012 reflected their geographic origin within Burkina Faso, with isolates from the Malian border region being closely related to the isolates from Mali. Evidence of ongoing evolution, international transmission, and strain replacement stresses the importance of maintaining N. meningitidis surveillance in Africa following the MACV implementation. IMPORTANCE Meningococcal disease (meningitis and bloodstream infections) threatens millions of people across the meningitis belt of sub-Saharan Africa. A vaccine introduced in 2010 protects against Africa’s then-most common cause of meningococcal disease, N. meningitidis serogroup A. However, other serogroups continue to cause epidemics in the region—including serogroup W. The rapid identification of strains that have been associated with prior outbreaks can improve the assessment of outbreak risk and enable timely preparation of public health responses, including vaccination. Phylogenetic analysis of newly sequenced serogroup W strains isolated from 1994 to 2012 identified two groups of strains linked to large epidemics in Burkina Faso, one being descended from a strain that caused an outbreak during the Hajj pilgrimage in 2000. We find that applying whole-genome sequencing to meningococcal disease surveillance collections improves the discrimination among strains, even within a single nation-wide epidemic, which can be used to better understand pathogen spread.