Adenovirus replication is controlled by the relocalization or modification of nuclear protein complexes, including promyelocytic leukemia protein (PML) nuclear domains and the Mre11-Rad50-Nbs1 (MRN) DNA damage machinery. In this study, we demonstrated that the E4 ORF3 protein effects the relocalization of both PML and MRN proteins to similar structures within the nucleus at early times after infection. These proteins colocalize with E4 ORF3. Through the analysis of specific viral mutants, we found a direct correlation between MRN reorganization at early times after infection and the establishment of viral DNA replication domains. Further, the reorganization of MRN components may be uncoupled from the ability of E4 ORF3 to rearrange PML. At later stages of infection, components of the MRN complex disperse within the nucleus, Nbs1 is found within viral replication centers, Rad50 remains localized with E4 ORF3, and Mre11 is degraded. The importance of viral regulation of the MRN complex is underscored by the complementation of E4 mutant viruses in cells that lack Mre11 or Nbs1 activity. These results illustrate the importance of nuclear organization in virus growth and suggest that E4 ORF3 regulates activities in both PML nuclear bodies and the MRN complex to stimulate the viral replication program.

Overview Since its isolation in Uganda in 1937, West Nile virus (WNV) has been responsible for thousands of cases of morbidity and mortality in birds, horses, and humans. Historically, epidemics were localized to Europe, Africa, the Middle East, and parts of Asia, and primarily caused a mild febrile illness in humans. However, in the late 1990’s, the virus became more virulent and expanded its geographical range to North America. In humans, the clinical presentation ranges from asymptomatic (approximately 80% of infections) to encephalitis/paralysis and death (less than 1% of infections). There is no FDA-licensed vaccine for human use, and the only recommended treatment is supportive care. Individuals that survive infection often have a long recovery period. This article will review the current literature summarizing the molecular virology, epidemiology, clinical manifestations, pathogenesis, diagnosis, treatment, immunology, and protective measures against WNV and WNV infections in humans.

Adenovirus early proteins E4 ORF3 and E4 ORF6 have complementary functions during viral infection.Both proteins facilitate efficient viral DNA replication, late protein expression, and prevention of concatenation of viral genomes. Additionally, E4 ORF6 is involved in the shutoff of the host cell protein synthesis through its interaction with the E1B 55K protein. This complex also leads to the degradation of p53. A unique function of E4 ORF3 is the reorganization of nuclear structures known as PML oncogenic domains (PODs). The function of these domains is unclear, but PODs have been implicated in a number of important cellular processes, including transcriptional regulation, apoptosis, transformation, and response to interferon. The goal of this study was to determine the functional significance of the reorganization of PODs by E4 ORF3. Point mutations were made in the E4 ORF3 gene. These mutants were recombined into a virus lacking E4 ORF6 and expressed under the control of the natural virus E4 promoter. The panel of mutant viruses was used to investigate the role of E4 ORF3 during the course of the viral infection program. One of the mutant viruses exhibited aberrant reorganization of PODs and had a severe defect in viral DNA replication, thus leading to a dramatic decrease in virus production. A number of mutants accumulated viral DNA and infectious virus particles to wild-type levels but showed significant viral genome concatenation. These data show that E4 ORF3 is a multifunctional protein and that a specific rearrangement of nuclear PML domains is coupled to efficient viral DNA replication. This function is distinct from the role of E4 ORF3 in the regulation of virus genome concatenation via inhibition of cellular double-strand break repair.

Dengue is a globally expanding disease caused by infection with dengue virus (DENV) that ranges from febrile illness to acute disease with serious complications. Secondary infection predisposes individuals to more severe disease, and B lymphocytes may play a role in this phenomenon through production of Ab that enhance infection. To better define the acute B cell response during dengue, we analyzed peripheral B cells from an adult Brazilian hospital cohort with primary and secondary DENV infections of varying clinical severity. Circulating B cells in dengue patients were proliferating, activated and apoptotic relative to individuals with other febrile illnesses. Severe secondary DENV infection was associated with extraordinary peak plasmablast frequencies between 4 and 7 days of illness, averaging 46% and reaching 87% of B cells, significantly greater than those seen in mild illness or primary infections. On average more than 70% of IgG-secreting cells in individuals with severe secondary DENV infection were DENV-specific. Plasmablasts produced Ab that cross-reacted with heterotypic DENV serotypes but with a 3-fold greater reactivity to DENV-3, the infecting serotype. Plasmablast frequency did not correlate with acute serum neutralizing Ab titers to any DENV serotype regardless of severity of disease. These findings indicate that massive expansion of DENV-specific and serotype cross-reactive plasmablasts occurs in acute secondary DENV infection of adults in Brazil which is associated with increasing disease severity.

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evolution of the virus but also the fundamental mechanisms by which control measures affected its epidemic spread. These efforts complement the information provided by the rapidly growing public databases of SARS-CoV-2 sequences by focusing the collection of genomic data in settings where we can access extensive current and past clinical data to investigate fundamental questions about this evolving virus's changing relationship with human health. MethodsData availability. Raw nanopore and Illumina data are deposited at SRA (BioProject PRJNA629390). Consensus sequences are deposited at GISAID and Genbank (MT509452-MT509493, and MT646048-MT646120) under BioProject PRJNA650037 (accession numbers available in Supplemental Table 3).Specimens and patient data. Clinical specimens used for genetic characterization were remnant nasopharyngeal swabs available at the completion of standard of care testing at the Johns Hopkins Hospital clinical virology laboratory. In total, 143 samples were selected for analysis based on their distribution throughout March 2020 and representation of the range of disease severity observed during this period. During this period, automated patient metadata extraction was limited to the date a sample was confirmed positive; all other data required patient chart reviews. Samples were sequenced in 2 phases, with the first phase enriched for patients admitted to the ICU (14 of 55 samples collected March 11-21), and the second a convenience sample that captured as many samples as possible for sequencing, irrespective of disease severity or ICU admission (10 of 88 samples collected March 13 -31).Clinical data analysis. Data including patient demographics, symptoms, comorbidities, COVID-19 exposure, recent travel history, and results of chest imaging at presentation were abstracted from the electronic medical record (EMR). COVID-19 treatment (medication, supplemental oxygen, and invasive mechanical ventilation) and outcomes (home observation without inpatient admission, discharge after admission, ongoing admission, and death) were also abstracted from the EMR. Race as self-reported by the patient and documented in the EMR was collected in prespecified categories. Patients who reported (a) contact with an individual known to be COVID-19-infected or (b) high-risk exposure (e.g., healthcare worker) were classified as COVID-19-exposed. Comorbidities were assessed based on diagnoses in the EMR (i.e., diabetes, obesity, or alcohol use disorder) and further categorized for lung disease (e.g., asthma, COPD), cardiac disease (e.g., valvular heart disease, arrhythmias, hypertension), and immunocompromised (e.g., HIV positive, hematologic malignancy, solid organ transplant).Nucleic acid extraction. Automated nucleic acid extraction was performed using either the NucliSENS easy-Mag or eMAG instruments (bioMérieux) using software version 2.1.0.1. easyMag or eMAG lysis buffer (2 mL) was added to 500 μL of aliquoted viral transport media in a biosafety cabinet in either a BSL-3 or BSL-2 facility using BSL-3 biosafe...

Saccharomyces boulardii (S. boulardii) is a probiotic yeast related to Saccharomyces cerevisiae (S. cerevisiae) but with distinct genetic, taxonomic and metabolic properties. S. cerevisiae has been used extensively in biotechnological applications. Currently, many strains are available, and multiple genetic tools have been developed, which allow the expression of several exogenous proteins of interest with applications in the fields of medicine, biofuels, the food industry, and scientific research, among others. Although S. boulardii has been widely studied due to its probiotic properties against several gastrointestinal tract disorders, very few studies addressed the use of this yeast as a vector for expression of foreign genes of interest with biotechnological applications. Here we show that, despite the similarity of the two yeasts, not all genetic tools used in S. cerevisiae can be applied in S. boulardii. While transformation of the latter could be obtained using a commercial kit developed for the former, consequent screening of successful transformants had to be optimized. We also show that several genes frequently used in genetic manipulation of S. cerevisiae (e.g., promoters and resistance markers) are present in S. boulardii. Sequencing revealed a high rate of homology (> 96%) between the orthologs of the two yeasts. However, we also observed some of them are not eligible to be targeted for transformation of S. boulardii. This work has important applications toward the potential of this probiotic yeast as an expression system for genes of interest.

Productive virus infection requires evasion, inhibition, or subversion of innate immune responses. West Nile virus (WNV), a human pathogen that can cause symptomatic infections associated with meningitis and encephalitis, inhibits the interferon (IFN) signal transduction pathway by preventing phosphorylation of Janus kinases and STAT transcription factors. Inhibition of the IFN signal cascade abrogates activation of IFN-induced genes, thus attenuating an antiviral response. We investigated the mechanism responsible for this inhibition and found that WNV infection prevents accumulation of the IFN-α receptor subunit 1 (IFNAR1). The WNV-induced depletion of IFNAR1 was conserved across multiple cell types. Our results indicated that expression of WNV nonstructural proteins resulted in activated lysosomal and proteasomal protein degradation pathways independent of the unfolded protein response (UPR). Furthermore, WNV infection did not induce serine phosphorylation, a modification on IFNAR1 that precedes its natural turnover. These data demonstrate that WNV infection results in a reduction of IFNAR1 protein through a non-canonical protein degradation pathway, and may participate in the inhibition of the IFN response.