Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use.

resp, encoding amino acids 21-398 of Plasmodium falciparum circumsporozoite protein (CSP), under control of tacP was integrated into the chromosomal ΔaroC locus of attenuated ΔaroC, ΔaroD Salmonella typhi CVD 908. By immunoblot and ELISA, rCSP expression was greater from a multicopy plasmid than from the single chromosomal gene. CVD 908 Ω(ΔaroCI019:: _tacP-resp) was well tolerated by 10 volunteers who were fed two doses of 5 × 10⁷ organisms 8 days apart. Seven subjects excreted the vaccine strain for 1-3 days. All subjects developed serologic responses to O and H antigens of the live vector, whereas 3 vaccinees responded to the foreign antigen: I developed an 80-fold rise in serum anti-sporozoite antibody, another had a 4-fold rise in antibody to a recombinant portion of CSP (residues 309-345), while a third vaccinee developed CSP-specific CD8⁺ cytotoxic T lymphocyte activity. This is the first report of attenuated S. typhi eliciting a human serologic or a cytotoxic T lymphocyte response to a foreign protein, Improved foreign gene expression should enhance immunogenicity,

The Zika virus (ZIKV) epidemic in the Americas established ZIKV as a major public health threat and uncovered its association with severe diseases, including microcephaly. However, genetic epidemiology in some at-risk regions, particularly Central America and Mexico, remains limited. We report 61 ZIKV genomes from this region, generated using metagenomic sequencing with ZIKV-specific enrichment, and combine phylogenetic, epidemiological, and environmental data to reconstruct ZIKV transmission. These analyses revealed multiple independent ZIKV introductions to Central America and Mexico. One introduction, likely from Brazil via Honduras, led to most infections and the undetected spread of ZIKV through the region from late 2014. Multiple lines of evidence indicate biannual peaks of ZIKV transmission in the region, likely driven by varying local environmental conditions for mosquito vectors and herd immunity. The spatial and temporal heterogeneity of ZIKV transmission in Central America and Mexico challenges arbovirus surveillance and disease control measures.

Infection with dengue virus type-2 (DENV-2) begins with virus adherence to cell surface receptors. In endothelial cells (HMEC-1), a cell model for DENV-2 infection,α5β3 integrin has been identified as a putative receptor for the virus. Previous work had suggested that the actin cytoskeleton of HMEC-1 cells plays an important role in virus entry and infection. In the present work, fixed and living HMEC-1 cells expressing enhanced green fluorescent protein–actin were monitored for actin reorganization after virus inoculation, utilizing fluorescence and time lapse microscopy. Cell infection and production of infective viruses were quantified using an anti-E protein antibody and by measuring the p.f.u. ml⁻¹. Specific drugs that antagonize actin organization and regulate actin-signalling pathways were tested in viral adhesion and infection assays, as were the expression of dominant-negative Rac1 and Cdc42 proteins. Disorganization of actin precluded infection, while microtubule depolymerization had no effect. Activation of Rac1 and Cdc42 signalling, which occurs upon virus binding, induced reorganization of actin to form filopodia in the cellular periphery. Formation of filopodia was a requirement for virus entry and further cell infection. Expression of the dominant-negative proteins Rac1 and Cdc42 confirmed the role of these GTPases in the actin reorganization that is required to form filopodia. In addition, inhibition of the ATPase activity of myosin II greatly decreased infection, suggesting its participation in filopodial stability. We show here, for the first time, that internalization of DENV-2 into endothelial cells requires viral induction of dynamic filopodia regulated by Rac1 and Cdc42 cross-talk and myosin II motor activities.

Purpose: Rituximab [chimeric anti-CD20 monoclonal antibody], alone or combined with chemotherapy, is used in the treatment of non–Hodgkin's lymphoma (NHL). Rituximab binds to CD20 and inhibits intracellular survival/growth pathways leading to chemo/immunosensitization of tumor cells in vitro. The contribution of rituximab Fc-FcR interaction in signaling is not known. This study examined the role of Fc-FcR interactions in rituximab-induced signaling using rituximab (Fab')₂ fragments as well as rituximab devoid of the CH2 Fc-binding domain (CH2⁻).