Extracellular Loops of the Treponema pallidum FadL Orthologs TP0856 and TP0858 Elicit IgG Antibodies and IgG+-Specific B-Cells in the Rabbit Model of Experimental Syphilis.

The resurgence of syphilis in the new millennium has called attention to the importance of a vaccine for global containment strategies. Studies with immune rabbit serum (IRS) indicate that a syphilis vaccine should elicit antibodies (Abs) that promote opsonophagocytosis of treponemes by activated macrophages. The availability of three-dimensional models for Treponema pallidum's (Tp) repertoire of outer membrane proteins (OMPs) provides an architectural framework for identification of candidate vaccinogens with extracellular loops (ECLs) as the targets for protective Abs. Herein, we used Pyrococcus furiosus thioredoxin (PfTrx) as a scaffold to display Tp OMP ECLs to interrogate sera and peripheral blood mononuclear cells (PBMCs) from immune rabbits for ECL-specific Abs and B cells. We validated this approach using a PfTrx scaffold presenting ECL4 from BamA, a known opsonic target. Using scaffolds displaying ECLs of the FadL orthologs TP0856 and TP0858, we determined that ECL2 and ECL4 of both proteins are strongly antigenic. Comparison of ELISA and immunoblot results suggested that the PfTrx scaffolds present conformational and linear epitopes. We then used the FadL ECL2 and ECL4 PfTrx constructs as "hooks" to confirm the presence of ECL-specific B cells in PBMCs from immune rabbits. Our results pinpoint immunogenic ECLs of two newly discovered OMPs, while advancing the utility of the rabbit model for circumventing bottlenecks in vaccine development associated with large-scale production of folded OMPs. They also lay the groundwork for production of rabbit monoclonal Abs (MAbs) to characterize potentially protective ECL epitopes at the atomic level. IMPORTANCE Recent identification and structural modeling of Treponema pallidum's (Tp) repertoire of outer membrane proteins (OMPs) represent a critical breakthrough in the decades long quest for a syphilis vaccine. However, little is known about the antigenic nature of these ß-barrel-forming OMPs and, more specifically, their surface exposed regions, the extracellular loops (ECLs). In this study, using Pyrococcus furiosus thioredoxin (PfTrx) as a scaffold to display Tp OMP ECLs, we interrogated immune rabbit sera and peripheral blood mononuclear cells for the presence of antibodies (Abs) and circulating rare antigen-specific B cells. Our results pinpoint immunogenic ECLs of two newly discovered OMPs, while advancing the utility of the rabbit model for surveying the entire Tp OMPeome for promising OMP vaccinogens. This work represents a major advancement toward characterizing potentially protective OMP ECLs and future vaccine studies. Additionally, this strategy could be applied to OMPs of nonspirochetal bacterial pathogens.

Genome Sequence of Escherichia coli Isolated from an Adult in Kibera, an Urban Informal Settlement in Nairobi, Kenya.

An Escherichia coli strain (sequence type 636) was isolated from an adult residing in an urban informal settlement in Nairobi, Kenya, and was sequenced using the Illumina MiSeq platform. The draft genome was 5,075,726 bp, with a Col(BS512) plasmid plus aph(6)-Id, blaTEM-1B, and dfrA7 genes, which encode kanamycin, ampicillin, and trimethoprim resistance proteins, respectively.

Evolutionary dynamics of ten novel Gamma-PVs: insights from phylogenetic incongruence, recombination and phylodynamic analyses.

BACKGROUND: Human papillomaviruses (HPVs) are genetically diverse, belonging to five distinct genera: Alpha, Beta, Gamma, Mu and Nu. All papillomaviruses have double stranded DNA genomes that are thought to evolve slowly because they co-opt high-fidelity host cellular DNA polymerases for their replication. Despite extensive efforts to catalogue all the HPV species that infect humans, it is likely that many still remain undiscovered. Here we use the sequences of ten novel Gammapapillomaviruses (Gamma-PVs) characterized in previous studies and related HPVs to analyse the evolutionary dynamics of these viruses at the whole genome and individual gene scales. RESULTS: We found statistically significant incongruences between the phylogenetic trees of different genes which imply gene-to-gene variation in the evolutionary processes underlying the diversification of Gamma-PVs. We were, however, only able to detect convincing evidence of a single recombination event which, on its own, cannot explain the observed incongruences between gene phylogenies. The divergence times of the last common ancestor (LCA) of the Alpha, Beta, Mu, Nu and Gamma genera was predicted to have existed between 49.7-58.5 million years ago, before splitting into the five main lineages. The LCA of the Gamma-PVs at this time was predicted to have existed between 45.3 and 67.5 million years ago: approximately at the time when the simian and tarsier lineages of the primates diverged. CONCLUSION: Consequently, we report here phylogenetic tree incongruence without strong evidence of recombination.

Evolutionary analyses of Sindbis virus strains isolated from mosquitoes in Kenya.

Sindbis virus (SINV) is a mosquito borne virus maintained in nature in a mosquito-bird cycle, with human outbreaks known to occur in Northern Europe and parts of Africa. We analyzed five SINV strains isolated in Kenya from five different mosquito species and geographic locations between 2007 and 2013. Phylogenetic relationships and evolutionary inferences were performed using maximum likelihood and Bayesian phylogenetic inference approaches. Selection analyses were carried out based on the virus envelope glycoproteins (E1, E2) and non-structural protein (nsP4) genes. Phylogenetic analysis revealed that all the Kenyan SINV isolates belonged to genotype 1 with selection analyses suggesting that SINV E1, E2 and nsP4 protein encoding genes were predominantly evolving under negative selection.

Phylogenetic Exploration of Nosocomial Transmission Chains of 2009 Influenza A/H1N1 among Children Admitted at Red Cross War Memorial Children's Hospital, Cape Town, South Africa in 2011.

Traditional modes of investigating influenza nosocomial transmission have entailed a combination of confirmatory molecular diagnostic testing and epidemiological investigation. Common hospital-acquired infections like influenza require a discerning ability to distinguish between viral isolates to accurately identify patient transmission chains. We assessed whether influenza hemagglutinin sequence phylogenies can be used to enrich epidemiological data when investigating the extent of nosocomial transmission over a four-month period within a paediatric Hospital in Cape Town South Africa. Possible transmission chains/channels were initially determined through basic patient admission data combined with Maximum likelihood and time-scaled Bayesian phylogenetic analyses. These analyses suggested that most instances of potential hospital-acquired infections resulted from multiple introductions of Influenza A into the hospital, which included instances where virus hemagglutinin sequences were identical between different patients. Furthermore, a general inability to establish epidemiological transmission linkage of patients/viral isolates implied that identified isolates could have originated from asymptomatic hospital patients, visitors or hospital staff. In contrast, a traditional epidemiological investigation that used no viral phylogenetic analyses, based on patient co-admission into specific wards during a particular time-frame, suggested that multiple hospital acquired infection instances may have stemmed from a limited number of identifiable index viral isolates/patients. This traditional epidemiological analysis by itself could incorrectly suggest linkage between unrelated cases, underestimate the number of unique infections and may overlook the possible diffuse nature of hospital transmission, which was suggested by sequencing data to be caused by multiple unique introductions of influenza A isolates into individual hospital wards. We have demonstrated a functional role for viral sequence data in nosocomial transmission investigation through its ability to enrich traditional, non-molecular observational epidemiological investigation by teasing out possible transmission pathways and working toward more accurately enumerating the number of possible transmission events.

Whole genome phylogenetic investigation of a West Nile virus strain isolated from a tick sampled from livestock in north eastern Kenya.

BACKGROUND: West Nile virus (WNV) has a wide geographical distribution and has been associated to cause neurological disease in humans and horses. Mosquitoes are the traditional vectors for WNV; however, the virus has also been isolated from tick species in North Africa and Europe which could be a means of introduction and spread of the virus over long distances through migratory birds. Although WNV has been isolated in mosquitoes in Kenya, paucity of genetic and pathogenicity data exists. We previously reported the isolation of WNV from ticks collected from livestock and wildlife in Ijara District of Kenya, a hotspot for arbovirus activity. Here we report the full genome sequence and phylogenetic investigation of their origin and relation to strains from other regions. METHODS: A total of 10,488 ticks were sampled from animal hosts, classified to species and processed in pools of up to eight ticks per pool. Virus screening was performed by cell culture, RT-PCR and sequencing. Phylogenetic analysis was carried out to determine the evolutionary relationships of our isolate. RESULTS: Among other viruses, WNV was isolated from a pool of Rhipicephalus pulchellus sampled from cattle, sequenced and submitted to GenBank (Accession number: KC243146). Comparative analysis with 27 different strains revealed that our isolate belongs to lineage 1 and clustered relatively closely to isolates from North Africa and Europe, Russia and the United States. Overall, Bayesian analysis based on nucleotide sequences showed that lineage 1 strains including the Kenyan strain had diverged 200 years ago from lineage 2 strains of southern Africa. Ijara strain collected from a tick sampled on livestock was closest to another Kenyan strain and had diverged 20 years ago from strains detected in Morocco and Europe and 30 years ago from strains identified in the USA. CONCLUSION: To our knowledge, this is the first characterized WNV strain isolated from R. pulchellus. The epidemiological role of this tick in WNV transmission and dissemination remains equivocal but presents tick verses mosquito virus transmission has been neglected. Genetic data of this strain suggest that lineage 1 strains from Africa could be dispersed through tick vectors by wild migratory birds to Europe and beyond.