Wave 2 strains of atypical Vibrio cholerae El Tor caused the 2009-2011 cholera outbreak in Papua New Guinea

Vibrio cholerae is the causative agent of cholera, a globally important human disease for at least 200 years. In 2009-2011, the first recorded cholera outbreak in Papua New Guinea (PNG) occurred. We conducted genetic and phenotypic characterization of 21 isolates of V. cholerae, with whole-genome sequencing conducted on 2 representative isolates. The PNG outbreak was caused by an atypical El Tor strain harbouring a tandem repeat of the CTX prophage on chromosome II. Whole-genome sequence data, prophage structural analysis and the absence of the SXT integrative conjugative element was indicative that the PNG isolates were most closely related to strains previously isolated in South-East and East Asia with affiliations to global wave 2 strains. This finding suggests that the cholera outbreak in PNG was caused by an exotic (non-endemic) strain of V. cholerae that originated in South-East Asia

Wave 2 strains of atypical Vibrio cholerae El Tor caused the 2009-2011 cholera outbreak in Papua New Guinea.

Vibrio cholerae is the causative agent of cholera, a globally important human disease for at least 200 years. In 2009-2011, the first recorded cholera outbreak in Papua New Guinea (PNG) occurred. We conducted genetic and phenotypic characterization of 21 isolates of V. cholerae, with whole-genome sequencing conducted on 2 representative isolates. The PNG outbreak was caused by an atypical El Tor strain harbouring a tandem repeat of the CTX prophage on chromosome II. Whole-genome sequence data, prophage structural analysis and the absence of the SXT integrative conjugative element was indicative that the PNG isolates were most closely related to strains previously isolated in South-East and East Asia with affiliations to global wave 2 strains. This finding suggests that the cholera outbreak in PNG was caused by an exotic (non-endemic) strain of V. cholerae that originated in South-East Asia

Investigating locally relevant risk factors for Campylobacter infection in Australia: Protocol for a case-control study and genomic analysis

Introduction The CampySource project aims to identify risk factors for human Campylobacter infection in Australia. We will investigate locally relevant risk factors and those significant in international studies in a case-control study. Case isolates and contemporaneous isolates from food and animal sources will be sequenced to conduct source attribution modelling, and findings will be combined with the case-control study in a source-assigned analysis. Methods and analysis The case-control study will include 1200 participants (600 cases and 600 controls) across three regions in Australia. Cases will be recruited from campylobacteriosis notifications to health departments. Only those with a pure and viable Campylobacter isolate will be eligible for selection to allow for whole genome sequencing of isolates. Controls will be recruited from notified cases of influenza, frequency matched by sex, age group and geographical area of residence. All participants will be interviewed by trained telephone interviewers using a piloted questionnaire. We will collect Campylobacter isolates from retail meats and companion animals (specifically dogs), and all food, animal and human isolates will undergo whole genome sequencing. We will use sequence data to estimate the proportion of human infections that can be attributed to animal and food reservoirs (source attribution modelling), and to identify spatial clusters and temporal trends. Source-assigned analysis of the case-control study data will also be conducted where cases are grouped according to attributed sources. Ethics and dissemination Human and animal ethics have been approved. Genomic data will be published in online archives accompanied by basic metadata. We anticipate several publications to come from this study

Establishment, Immortalisation and Characterisation of Pteropid Bat Cell Lines

BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study

Exploring the Zoonotic Potential of Mycobacterium avium Subspecies paratuberculosis through Comparative Genomics

A comparative genomics approach was utilised to compare the genomes of Mycobacterium avium subspecies paratuberculosis (MAP) isolated from early onset paediatric Crohn's disease (CD) patients as well as Johne's diseased animals. Draft genome sequences were produced for MAP isolates derived from four CD patients, one ulcerative colitis (UC) patient, and two non-inflammatory bowel disease (IBD) control individuals using Illumina sequencing, complemented by comparative genome hybridisation (CGH). MAP isolates derived from two bovine and one ovine host were also subjected to whole genome sequencing and CGH. All seven human derived MAP isolates were highly genetically similar and clustered together with one bovine type isolate following phylogenetic analysis. Three other sequenced isolates (including the reference bovine derived isolate K10) were genetically distinct. The human isolates contained two large tandem duplications, the organisations of which were confirmed by PCR. Designated vGI-17 and vGI-18 these duplications spanned 63 and 109 open reading frames, respectively. PCR screening of over 30 additional MAP isolates (3 human derived, 27 animal derived and one environmental isolate) confirmed that vGI-17 and vGI-18 are common across many isolates. Quantitative real-time PCR of vGI-17 demonstrated that the proportion of cells containing the vGI-17 duplication varied between 0.01 to 15% amongst isolates with human isolates containing a higher proportion of vGI-17 compared to most animal isolates. These findings suggest these duplications are transient genomic rearrangements. We hypothesise that the over-representation of vGI-17 in human derived MAP strains may enhance their ability to infect or persist within a human host by increasing genome redundancy and conferring crude regulation of protein expression across biologically important regions

Molecular Characterization of a 21.4 Kilobase Antibiotic Resistance Plasmid from an α-Hemolytic Escherichia coli O108:H- Human Clinical Isolate

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes

Host shifts and molecular evolution of H7 avian influenza virus hemagglutinin

Evolutionary consequences of host shifts represent a challenge to identify the mechanisms involved in the emergence of influenza A (IA) viruses. In this study we focused on the evolutionary history of H7 IA virus in wild and domestic birds, with a particular emphasis on host shifts consequences on the molecular evolution of the hemagglutinin (HA) gene. Based on a dataset of 414 HA nucleotide sequences, we performed an extensive phylogeographic analysis in order to identify the overall genetic structure of H7 IA viruses. We then identified host shift events and investigated viral population dynamics in wild and domestic birds, independently. Finally, we estimated changes in nucleotide substitution rates and tested for positive selection in the HA gene. A strong association between the geographic origin and the genetic structure was observed, with four main clades including viruses isolated in North America, South America, Australia and Eurasia-Africa. We identified ten potential events of virus introduction from wild to domestic birds, but little evidence for spillover of viruses from poultry to wild waterbirds. Several sites involved in host specificity (addition of a glycosylation site in the receptor binding domain) and virulence (insertion of amino acids in the cleavage site) were found to be positively selected in HA nucleotide sequences, in genetically unrelated lineages, suggesting parallel evolution for the HA gene of IA viruses in domestic birds. These results highlight that evolutionary consequences of bird host shifts would need to be further studied to understand the ecological and molecular mechanisms involved in the emergence of domestic bird-adapted viruses

Transcriptional Responses of Leptospira interrogans to Host Innate Immunity: Significant Changes in Metabolism, Oxygen Tolerance, and Outer Membrane

Leptospirosis is an important tropical disease around the world, particularly in humid tropical and subtropical countries. As a major pathogen of this disease, Leptospira interrogans can be shed from the urine of reservoir hosts, survive in soil and water, and infect humans through broken skin or mucous membranes. Recently, host adaptability and immune evasion of L. interrogans to host innate immunity was partially elucidated in infection or animal models. A better understanding of the molecular mechanisms of L. interrogans in response to host innate immunity is required to learn the nature of early leptospirosis. This study focused on the transcriptome of L. interrogans during host immune cells interaction. Significant changes in energy metabolism, oxygen tolerance and outer membrane protein profile were identified as potential immune evasion strategies by pathogenic Leptospira during the early stage of infection. The major outer membrane proteins (OMPs) of L. interrogans may be regulated by the major OmpR specific transcription factor (LB333). These results provide a foundation for further studying the pathogenesis of leptospirosis, as well as identifying gene regulatory networks in Leptospira spp

Transient Expression of Hemagglutinin Antigen from Low Pathogenic Avian Influenza A (H7N7) in Nicotiana benthamiana

The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species

In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

Background: Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin. Methodology/Principal Findings: We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin. Conclusions/Significance: PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico)Fundacao Butantan, BrazilFAPESP (Brazil