Seafood-Associated Outbreak of ctx-Negative Vibrio mimicus Causing Cholera-Like Illness, Florida, USA.

Vibrio mimicus caused a seafood-associated outbreak in Florida, USA, in which 4 of 6 case-patients were hospitalized; 1 required intensive care for severe diarrhea. Strains were ctx-negative but carried genes for other virulence determinants (hemolysin, proteases, and types I-IV and VI secretion systems). Cholera toxin-negative bacterial strains can cause cholera-like disease.

Molecular Basis of the Toxigenic Vibrio cholerae O1 Serotype Switch from Ogawa to Inaba in Haiti.

Toxigenic Vibrio cholerae O1 serotype Ogawa was introduced involuntarily into Haiti in October 2010, and virtually all of the clinical strains isolated during the first 5 years of the epidemic were Ogawa. Inaba strains were identified intermittently prior to 2015, with diverse mutations resulting in a common phenotype. In 2015, the percentage of clinical infections due to the Inaba serotype began to rapidly increase, with Inaba supplanting Ogawa as the dominant serotype during the subsequent 4 years. We investigated the molecular basis of the serotype switch and confirmed that all Inaba strains had the same level of mRNA expression of the wbeT genes, as well as the same translation levels for the truncated WbeT proteins in the V. cholerae Inaba isolates. Neither wbeT gene expression levels, differential mutations, or truncation size of the WbeT proteins appeared to be responsible for the successful Inaba switch in 2015. Our phylodynamic analysis demonstrated that the V. cholerae Inaba strains in Haiti evolved directly from Ogawa strains and that a significant increase of diversifying selection at the population level occurred at the time of the Ogawa-Inaba switch. We conclude that the emergence of the Inaba serotype was driven by diversifying selection, independent of the mutational pattern in the wbeT gene. IMPORTANCE Our phylodynamic analysis demonstrated that Vibrio cholerae Inaba strains in Haiti evolved directly from Ogawa strains. Our results support the hypothesis that after an initial Ogawa-dominated epidemic wave, V. cholerae Inaba was able to become the dominant strain thanks to a selective advantage driven by ongoing diversifying selection, independently from the mutational pattern in the wbeT gene.

Emergence and Evolutionary Response of Vibrio cholerae to Novel Bacteriophage, Democratic Republic of the Congo1.

Cholera causes substantial illness and death in Africa. We analyzed 24 toxigenic Vibrio cholerae O1 strains isolated in 2015-2017 from patients in the Great Lakes region of the Democratic Republic of the Congo. Strains originating in southern Asia appeared to be part of the T10 introduction event in eastern Africa. We identified 2 main strain lineages, most recently a lineage corresponding to sequence type 515, a V. cholerae cluster previously reported in the Lake Kivu region. In 41% of fecal samples from cholera patients, we also identified a novel ICP1 (Bangladesh cholera phage 1) bacteriophage, genetically distinct from ICP1 isolates previously detected in Asia. Bacteriophage resistance occurred in distinct clades along both internal and external branches of the cholera phylogeny. This bacteriophage appears to have served as a major driver for cholera evolution and spread, and its appearance highlights the complex evolutionary dynamic that occurs between predatory phage and bacterial host.

Development of a Monoclonal Antibody to a Vibriophage as a Proxy for Vibrio cholerae Detection.

Cholera is an acute watery, diarrheal disease that causes high rates of morbidity and mortality without treatment. Early detection of the etiologic agent of toxigenic Vibrio cholerae is important to mobilize treatment and mitigate outbreaks. Monoclonal antibody (mAb) based rapid diagnostic tests (RDTs) enable early detection in settings without laboratory capacity. However, the odds of an RDT testing positive are reduced by nearly 90% when the common virulent bacteriophage ICP1 is present. We hypothesize that adding a mAb for the common, and specific, virulent bacteriophage ICP1 as a proxy for V. cholerae to an RDT will increase diagnostic sensitivity when virulent ICP1 phage is present. In this study, we used an in-silico approach to identify immunogenic ICP1 protein targets that were conserved across disparate time periods and locations. Specificity of targets to cholera patients with known ICP1 was determined, and specific targets were used to produce mAbs in a murine model. Candidate mAbs to the head protein demonstrated specificity to ICP1 by Enzyme linked immunosorbent assay (ELISA) and an ICP1 phage neutralization assay. The limit of detection of the final mAb candidate for ICP1 phage particles spiked into cholera stool matrix was 8 × 105 PFU by Western blotting analysis. This mAb will be incorporated into a RDT prototype for evaluation in a future diagnostic study to test the guiding hypothesis behind this study.

Analysis of the Origin and Dissemination of HIV-1 Subtype C in Bulgaria.

HIV-1 subtype C is the most abundant strain of HIV-1 infections worldwide and was found in the first known patients diagnosed with HIV/AIDS in Bulgaria in 1986. However, there is limited information on the molecular-epidemiological characteristics of this strain in the epidemic of the country. In this study, we analyze the evolutionary history of the introduction and dissemination of HIV-1 subtype C in Bulgaria using global phylogenetic analysis, Bayesian coalescent-based approach, and molecular clock methods. All available samples with HIV-1 subtype C from individuals diagnosed with HIV/AIDS between 1986 and 2017 were analyzed. Men and women were equally represented, and 24.3% of patients reported being infected abroad. The global phylogenetic analysis indicated multiple introductions of HIV-1 subtype C from various countries of the world. The reconstruction of a Bayesian time-scaled phylogenies showed that several Bulgarian strains segregated together in clusters, while others were intermixed in larger clades containing strains isolated from both European and non-European countries. The time-scale of HIV-1 subtype C introductions in Bulgaria demonstrates the early introduction of these viruses in the country. Our in-depth phylogenetic and phylogeographic analyses are compatible with a scenario of multiple early introductions in the country followed by limited local distribution in the subsequent years. HIV-1 subtype C was introduced in the early years of the epidemic, originating from different countries of the world. Due to the comprehensive measures for prevention and control in the early years of the epidemic in Bulgaria, HIV-1 subtype C was not widely disseminated among the general population of the country.

Toxigenic Vibrio cholerae evolution and establishment of reservoirs in aquatic ecosystems.

The spread of cholera in the midst of an epidemic is largely driven by direct transmission from person to person, although it is well-recognized that Vibrio cholerae is also capable of growth and long-term survival in aquatic ecosystems. While prior studies have shown that aquatic reservoirs are important in the persistence of the disease on the Indian subcontinent, an epidemiological view postulating that locally evolving environmental V. cholerae contributes to outbreaks outside Asia remains debated. The single-source introduction of toxigenic V. cholerae O1 in Haiti, one of the largest outbreaks occurring this century, with 812,586 suspected cases and 9,606 deaths reported through July 2018, provided a unique opportunity to evaluate the role of aquatic reservoirs and assess bacterial transmission dynamics across environmental boundaries. To this end, we investigated the phylogeography of both clinical and aquatic toxigenic V. cholerae O1 isolates and show robust evidence of the establishment of aquatic reservoirs as well as ongoing evolution of V. cholerae isolates from aquatic sites. Novel environmental lineages emerged from sequential population bottlenecks, carrying mutations potentially involved in adaptation to the aquatic ecosystem. Based on such empirical data, we developed a mixed-transmission dynamic model of V. cholerae, where aquatic reservoirs actively contribute to genetic diversification and epidemic emergence, which underscores the complexity of transmission pathways in epidemics and endemic settings and the need for long-term investments in cholera control at both human and environmental levels.

UBASH3A Regulates the Synthesis and Dynamics of TCR-CD3 Complexes.

The TCR-CD3 complex is a multicomponent membrane receptor, the expression of which is tightly regulated in thymocytes, as well as in mature T cells both at steady state and upon stimulation. In this study, we report novel roles for UBASH3A in TCR-CD3 synthesis and turnover. UBASH3A is a negative regulator of T cell function and plays a broad role in autoimmunity. We show that modulation of UBASH3A levels in unstimulated Jurkat cells leads to altered amounts of total cellular CD3 chains and of cell-surface TCR-CD3 complexes; in contrast, UBASH3A does not affect the level of cell-surface CD28, an important T cell costimulatory receptor. Upon TCR engagement, UBASH3A enhances the downmodulation of cell-surface TCR-CD3. Mass spectrometry and protein-protein interaction studies uncover novel associations between UBASH3A and components of several cellular pathways involved in the regulation of TCR-CD3 turnover and dynamics, including endoplasmic reticulum-associated protein degradation, cell motility, endocytosis, and endocytic recycling of membrane receptors. Finally, we demonstrate that the SH3 domain of UBASH3A mediates its binding to CBL-B, an E3 ubiquitin ligase that negatively regulates CD28-mediated signaling and, hence, T cell activation. In summary, this study provides new mechanistic insights into how UBASH3A regulates T cell activation and contributes to autoimmunity. The interaction between UBASH3A and CBL-B may synergistically inhibit T cell function and affect risk for type 1 diabetes, as both genes have been shown to be associated with this autoimmune disease.

Detection and phylogenetic characterization of arbovirus dual-infections among persons during a chikungunya fever outbreak, Haiti 2014.

In the context of recent arbovirus epidemics, questions about the frequency of simultaneous infection of patients with different arbovirus species have been raised. In 2014, a major Chikungunya virus (CHIKV) epidemic impacted the Caribbean and South America. As part of ongoing screening of schoolchildren presenting with acute undifferentiated febrile illness in rural Haiti, we used RT-PCR to identify CHIKV infections in 82 of 100 children with this diagnosis during May-August 2014. Among these, eight were infected with a second arbovirus: six with Zika virus (ZIKV), one with Dengue virus serotype 2, and one with Mayaro virus (MAYV). These dual infections were only detected following culture of the specimen, suggesting low viral loads of the co-infecting species. Phylogenetic analyses indicated that the ZIKV and MAYV strains differ from those detected later in 2014 and 2015, respectively. Moreover, CHIKV and ZIKV strains from co-infected patients clustered monophyletically in their respective phylogeny, and clock calibration traced back the common ancestor of each clade to an overlapping timeframe of introduction of these arboviruses onto the island.

Evidence for Mother-to-Child Transmission of Zika Virus Through Breast Milk.

Zikavirus (ZIKV) is an emerging viral pathogen that continues to spread throughout different regions of the world. Herein we report a case that provides further evidence that ZIKV transmission can occur through breastfeeding by providing a detailed clinical, genomic, and virological case-based description.

Detection of Alphacoronavirus vRNA in the Feces of Brazilian Free-Tailed Bats (Tadarida brasiliensis) from a Colony in Florida, USA.

Bats are natural reservoirs of coronaviruses and other viruses with zoonotic potential. Florida has indigenous non-migratory populations of Brazilian free-tailed bats (Tadarida brasiliensis) that mostly roost in colonies in artificial structures. Unlike their counterparts in Brazil and Mexico, the viruses harbored by the Florida bats have been underexplored. We report the detection of an alphacoronavirus RNA-dependent RNA polymerase (RdRp) gene sequence in the feces of two of 19 different T. brasiliensis that were capture/release bats that had been evaluated for overall health. The RdRp sequence is similar but not identical to previously detected sequences in the feces of two different species of bats (T. brasiliensis and Molossus molossus) in Brazil. In common with the experience of others doing similar work, attempts to isolate the virus in cell cultures were unsuccessful. We surmise that this and highly related alphacoronavirus are carried by Brazilian free-tailed bats living in a wide eco-spatial region. As various coronaviruses (CoVs) that affect humans emerged from bats, our study raises the question whether CoVs such as the one detected in our work are yet-to-be-detected pathogens of humans and animals other than bats.

UBASH3A Mediates Risk for Type 1 Diabetes Through Inhibition of T-Cell Receptor-Induced NF-κB Signaling.

Although over 40 type 1 diabetes (T1D) risk loci have been mapped in humans, the causative genes and variants for T1D are largely unknown. Here, we investigated a candidate gene in the 21q22.3 risk locus-UBASH3A, which is primarily expressed in T cells where it is thought to play a largely redundant role. Genetic variants in UBASH3A have been shown to be associated with several autoimmune diseases in addition to T1D. However, the molecular mechanism underlying these genetic associations is unresolved. Our study reveals a previously unrecognized role of UBASH3A in human T cells: UBASH3A attenuates the NF-κB signal transduction upon T-cell receptor (TCR) stimulation by specifically suppressing the activation of the IκB kinase complex. We identify novel interactions of UBASH3A with nondegradative polyubiquitin chains, TAK1 and NEMO, suggesting that UBASH3A regulates the NF-κB signaling pathway by an ubiquitin-dependent mechanism. Finally, we show that risk alleles at rs11203203 and rs80054410, two T1D-associated variants in UBASH3A, increase UBASH3A expression in human primary CD4+ T cells upon TCR stimulation, inhibiting NF-κB signaling via its effects on the IκB kinase complex and resulting in reduced IL2 gene expression.

Complete Genome Sequences of Identical Zika virus Isolates in a Nursing Mother and Her Infant.

Complete genome sequences were obtained for Zika viruses isolated from the breast milk of a Venezuelan patient and her child, who was exclusively breastfeeding at the time. These sequences are the first to be reported from a presumptive autochthonous postnatal transmission case from mother to child in Venezuela.

Mississippi River Plume Enriches Microbial Diversity in the Northern Gulf of Mexico.

The Mississippi River (MR) serves as the primary source of freshwater and nutrients to the northern Gulf of Mexico (nGOM). Whether this input of freshwater also enriches microbial diversity as the MR plume migrates and mixes with the nGOM serves as the central question addressed herein. Specifically, in this study physicochemical properties and planktonic microbial community composition and diversity was determined using iTag sequencing of 16S rRNA genes in 23 samples collected along a salinity (and nutrient) gradient from the mouth of the MR, in the MR plume, in the canyon, at the Deepwater Horizon wellhead and out to the loop current. Analysis of these datasets revealed that the MR influenced microbial diversity as far offshore as the Deepwater Horizon wellhead. The MR had the highest microbial diversity, which decreased with increasing salinity. MR bacterioplankton communities were distinct compared to the nGOM, particularly in the surface where Actinobacteria and Proteobacteria dominated, while the deeper MR was also enriched in Thaumarchaeota. Statistical analyses revealed that nutrients input by the MR, along with salinity and depth, were the primary drivers in structuring the microbial communities. These results suggested that the reduced salinity, nutrient enriched MR plume could act as a seed bank for microbial diversity as it mixes with the nGOM. Whether introduced microorganisms are active at higher salinities than freshwater would determine if this seed bank for microbial diversity is ecologically significant. Alternatively, microorganisms that are physiologically restricted to freshwater habitats that are entrained in the plume could be used as tracers for freshwater input to the marine environment.

Zika and Chikungunya virus co-infection in a traveller returning from Colombia, 2016: virus isolation and genetic analysis.

INTRODUCTION: Zikavirus (ZIKV) and Chikungunyavirus (CHIKV) can share the same mosquito vector, and co-infections by these viruses can occur in humans. While infections with these viruses share commonalities, CHIKV is unique in causing arthritis and arthralgias that may persist for a year or more. These infections are commonly diagnosed by RT-PCR-based methods during the acute phase of infection. Even with the high specificity and sensitivity characteristic of PCR, false negatives can occur, highlighting the need for additional diagnostic methods for confirmation. CASE PRESENTATION: On her return to the USA, a traveller to Colombia, South America developed an illness consistent with Zika, Chikungunya and/or Dengue. RT-PCR of her samples was positive only for ZIKV. However, arthralgias persisted for months, raising concerns about co-infection with CHIKV or Mayaro viruses. Cell cultures inoculated with her original clinical samples demonstrated two types of cytopathic effects, and both ZIKV and CHIKV were identified in the supernatants. On phylogenetic analyses, both viruses were found to be related to strains found in Colombia. CONCLUSION: These findings highlight the need to consider CHIKV co-infection in patients with prolonged rheumatological symptoms after diagnosis with ZIKV, and the usefulness of cell culture as an amplification step for low-viremia blood and other samples.

Effects of a ciliate protozoa predator on microbial communities in pitcher plant (Sarracenia purpurea) leaves.

The aquatic communities found within the water filled leaves of the pitcher plant, Sarracenia purpurea, have a simple trophic structure providing an ideal system to study microscale interactions between protozoan predators and their bacterial prey. In this study, replicate communities were maintained with and without the presence of the bactivorous protozoan, Colpoda steinii, to determine the effects of grazing on microbial communities. Changes in microbial (Archaea and Bacteria) community structure were assessed using iTag sequencing of 16S rRNA genes. The microbial communities were similar with and without the protozoan predator, with>1000 species. Of these species, Archaea were negligible, with Bacteria comprising 99.99% of the microbial community. The Proteobacteria and Bacteroidetes were the most dominant phyla. The addition of a protozoan predator did not have a significant effect on microbial evenness nor richness. However, the presence of the protozoan did cause a significant shift in the relative abundances of a number of bacterial species. This suggested that bactivorous protozoan may target specific bacterial species and/or that certain bacterial species have innate mechanisms by which they evade predators. These findings help to elucidate the effect that trophic structure perturbations have on predator prey interactions in microbial systems.