Michael Frace

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.

Novel Epidemic Clones of Listeria monocytogenes, United States, 2011

We identified a novel serotype 1/2a outbreak strain and 2 novel epidemic clones of Listeria monocytogenes while investigating a foodborne outbreak of listeriosis associated with consumption of cantaloupe during 2011 in the United States. Comparative analyses of strains worldwide are essential to identification of novel outbreak strains and epidemic clones.

Characterization of novel Brucella strains originating from wild native rodent species in North Queensland, Australia.

ABSTRACT We report on the characterization of a group of seven novel Brucella strains isolated in 1964 from three native rodent species in North Queensland, Australia, during a survey of wild animals. The strains were initially reported to be Brucella suis biovar 3 on the basis of microbiological test results. Our results indicated that the rodent strains had microbiological traits distinct from those of B. suis biovar 3 and all other Brucella spp. To reinvestigate these rodent strains, we sequenced the 16S rRNA, recA, and rpoB genes and nine housekeeping genes and also performed multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA). The rodent strains have a unique 16S rRNA gene sequence compared to the sequences of the classical Brucella spp. Sequence analysis of the recA, rpoB, and nine housekeeping genes reveals that the rodent strains are genetically identical to each other at these loci and divergent from any of the currently described Brucella sequence types. However, all seven of the rodent strains do exhibit distinctive allelic MLVA profiles, although none demonstrated an amplicon for VNTR 07, whereas the other Brucella spp. did. Phylogenetic analysis of the MLVA data reveals that the rodent strains form a distinct clade separate from the classical Brucella spp. Furthermore, whole-genome sequence comparison using the maximal unique exact matches index (MUMi) demonstrated a high degree of relatedness of one of the seven rodent Brucella strains (strain NF 2653) to another Australian rodent Brucella strain (strain 83-13). Our findings strongly suggest that this group of Brucella strains isolated from wild Australian rodents defines a new species in the Brucella genus.

Virulence Factors Encoded by Legionella longbeachae Identified on the Basis of the Genome Sequence Analysis of Clinical Isolate D-4968

Legionella longbeachae causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of L. longbeachae-specific diagnostic tests. L. longbeachae displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to Legionella pneumophila, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems, L. longbeachae is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an L. longbeachae clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of L. pneumophila. The results revealed that the D-4968 genome is larger than the L. pneumophila genome and has a gene order that is different from that of the L. pneumophila genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding L. pneumophila Icm/Dot substrates have been found in the D-4968 genome. L. longbeachae encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the L. longbeachae Icm/Dot secretion system. In contrast to the L. pneumophila genome, the L. longbeachae D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of L. longbeachae to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of L. longbeachae may reflect adaptation of this species to life in soil.

The Phylogenetics and Ecology of the Orthopoxviruses Endemic to North America

The data presented herein support the North American orthopoxviruses (NA OPXV) in a sister relationship to all other currently described Orthopoxvirus (OPXV) species. This phylogenetic analysis reaffirms the identification of the NA OPXV as close relatives of “Old World” (Eurasian and African) OPXV and presents high support for deeper nodes within the Chordopoxvirinae family. The natural reservoir host(s) for many of the described OPXV species remains unknown although a clear virus-host association exists between the genus OPXV and several mammalian taxa. The hypothesized host associations and the deep divergence of the OPXV/NA OPXV clades depicted in this study may reflect the divergence patterns of the mammalian faunas of the Old and New World and reflect a more ancient presence of OPXV on what are now the American continents. Genes from the central region of the poxvirus genome are generally more conserved than genes from either end of the linear genome due to functional constraints imposed on viral replication abilities. The relatively slower evolution of these genes may more accurately reflect the deeper history among the poxvirus group, allowing for robust placement of the NA OPXV within Chordopoxvirinae. Sequence data for nine genes were compiled from three NA OPXV strains plus an additional 50 genomes collected from Genbank. The current, gene sequence based phylogenetic analysis reaffirms the identification of the NA OPXV as the nearest relatives of “Old World” OPXV and presents high support for deeper nodes within the Chordopoxvirinae family. Additionally, the substantial genetic distances that separate the currently described NA OPXV species indicate that it is likely that many more undescribed OPXV/NA OPXV species may be circulating among wild animals in North America.

Analysis of a unique Clostridium botulinum strain from the Southern hemisphere producing a novel type E botulinum neurotoxin subtype

BackgroundClostridium botulinum strains that produce botulinum neurotoxin type E (BoNT/E) are most commonly isolated from botulism cases, marine environments, and animals in regions of high latitude in the Northern hemisphere. A strain of C. botulinum type E (CDC66177) was isolated from soil in Chubut, Argentina. Previous studies showed that the amino acid sequences of BoNT/E produced by various strains differ by < 6% and that the type E neurotoxin gene cluster inserts into the rarA operon.ResultsGenetic and mass spectral analysis demonstrated that the BoNT/E produced by CDC66177 is a novel toxin subtype (E9). Toxin gene sequencing indicated that BoNT/E9 differed by nearly 11% at the amino acid level compared to BoNT/E1. Mass spectrometric analysis of BoNT/E9 revealed that its endopeptidase substrate cleavage site was identical to other BoNT/E subtypes. Further analysis of this strain demonstrated that its 16S rRNA sequence clustered with other Group II C. botulinum (producing BoNT types B, E, and F) strains. Genomic DNA isolated from strain CDC66177 hybridized with fewer probes using a Group II C. botulinum subtyping microarray compared to other type E strains examined. Whole genome shotgun sequencing of strain CDC66177 revealed that while the toxin gene cluster inserted into the rarA operon similar to other type E strains, its overall genome content shared greater similarity with a Group II C. botulinum type B strain (17B).ConclusionsThese results expand our understanding of the global distribution of C. botulinum type E strains and suggest that the type E toxin gene cluster may be able to insert into C. botulinum strains with a more diverse genetic background than previously recognized.

Malignant Transformation of Hymenolepis nana in a Human Host

Neoplasms occur naturally in invertebrates but are not known to develop in tapeworms. We observed nests of monomorphic, undifferentiated cells in samples from lymph-node and lung biopsies in a man infected with the human immunodeficiency virus (HIV). The morphologic features and invasive behavior of the cells were characteristic of cancer, but their small size suggested a nonhuman origin. A polymerase-chain-reaction (PCR) assay targeting eukaryotes identified Hymenolepis nana DNA. Although the cells were unrecognizable as tapeworm tissue, immunohistochemical staining and probe hybridization labeled the cells in situ. Comparative deep sequencing identified H. nana structural genomic variants that are compatible with mutations described in cancer. Invasion of human tissue by abnormal, proliferating, genetically altered tapeworm cells is a novel disease mechanism that links infection and cancer.

Progressive vaccinia: case description and laboratory-guided therapy with vaccinia immune globulin, ST-246 and CMX001

Progressive vaccinia (PV) is a rare but potentially lethal complication that develops in smallpox vaccine recipients with severely impaired cellular immunity. We describe a patient with PV who required treatment with vaccinia immune globulin and who received 2 investigational agents, ST-246 and CMX001. We describe the various molecular, pharmacokinetic, and immunologic studies that provided guidance to escalate and then successfully discontinue therapy. Despite development of resistance to ST-246 during treatment, the patient had resolution of PV. This case demonstrates the need for continued development of novel anti-orthopoxvirus pharmaceuticals and the importance of both intensive and timely clinical and laboratory support in management of PV.