MutaGAN: A sequence-to-sequence GAN framework to predict mutations of evolving protein populations.

The ability to predict the evolution of a pathogen would significantly improve the ability to control, prevent, and treat disease. Machine learning, however, is yet to be used to predict the evolutionary progeny of a virus. To address this gap, we developed a novel machine learning framework, named MutaGAN, using generative adversarial networks with sequence-to-sequence, recurrent neural networks generator to accurately predict genetic mutations and evolution of future biological populations. MutaGAN was trained using a generalized time-reversible phylogenetic model of protein evolution with maximum likelihood tree estimation. MutaGAN was applied to influenza virus sequences because influenza evolves quickly and there is a large amount of publicly available data from the National Center for Biotechnology Information's Influenza Virus Resource. MutaGAN generated 'child' sequences from a given 'parent' protein sequence with a median Levenshtein distance of 4.00 amino acids. Additionally, the generator was able to generate sequences that contained at least one known mutation identified within the global influenza virus population for 72.8 per cent of parent sequences. These results demonstrate the power of the MutaGAN framework to aid in pathogen forecasting with implications for broad utility in evolutionary prediction for any protein population.

Lassa Virus Infection: a Summary for Clinicians.

OBJECTIVES: This summary on Lassa virus (LASV) infection and Lassa fever disease (LF) was developed from a clinical perspective to provide clinicians with a condensed, accessible understanding of the current literature. The information provided highlights pathogenesis, clinical features, and diagnostics emphasizing therapies and vaccines that have demonstrated potential value for use in clinical or research environments. METHODS: We conducted an integrative literature review on the clinical and pathological features, vaccines, and treatments for LASV infection, focusing on recent studies and in vivo evidence from humans and/or non-human primates (NHPs), when available. RESULTS: Two antiviral medications with potential benefit for the treatment of LASV infection and 1 for post-exposure prophylaxis were identified, although a larger number of therapeutic candidates are currently being evaluated. Multiple vaccine platforms are in pre-clinical development for LASV prevention, but data from human clinical trials are not yet available. CONCLUSION: We provide succinct summaries of medical countermeasures against LASV to give the busy clinician a rapid reference. Although there are no approved drugs or vaccines for LF, we provide condensed information from a literature review for measures that can be taken when faced with a suspected infection, including investigational treatment options and hospital engineering controls.

Genomic diversity of SARS-CoV-2 during early introduction into the Baltimore-Washington metropolitan area.

The early COVID-19 pandemic was characterized by rapid global spread. In Maryland and Washington, DC, United States, more than 2500 cases were reported within 3 weeks of the first COVID-19 detection in March 2020. We aimed to use genomic sequencing to understand the initial spread of SARS-CoV-2 - the virus that causes COVID-19 - in the region. We analyzed 620 samples collected from the Johns Hopkins Health System during March 11-31, 2020, comprising 28.6% of the total cases in Maryland and Washington, DC. From these samples, we generated 114 complete viral genomes. Analysis of these genomes alongside a subsampling of over 1000 previously published sequences showed that the diversity in this region rivaled global SARS-CoV-2 genetic diversity at that time and that the sequences belong to all of the major globally circulating lineages, suggesting multiple introductions into the region. We also analyzed these regional SARS-CoV-2 genomes alongside detailed clinical metadata and found that clinically severe cases had viral genomes belonging to all major viral lineages. We conclude that efforts to control local spread of the virus were likely confounded by the number of introductions into the region early in the epidemic and the interconnectedness of the region as a whole.

Genomic Diversity of SARS-CoV-2 During Early Introduction into the United States National Capital Region.

BACKGROUND: The early COVID-19 pandemic has been characterized by rapid global spread. In the United States National Capital Region, over 2,000 cases were reported within three weeks of its first detection in March 2020. We aimed to use genomic sequencing to understand the initial spread of SARS-CoV-2, the virus that causes COVID-19, in the region. By correlating genetic information to disease phenotype, we also aimed to gain insight into any correlation between viral genotype and case severity or transmissibility. METHODS: We performed whole genome sequencing of clinical SARS-CoV-2 samples collected in March 2020 by the Johns Hopkins Health System. We analyzed these regional SARS-CoV-2 genomes alongside detailed clinical metadata and the global phylogeny to understand early establishment of the virus within the region. RESULTS: We analyzed 620 samples from the Johns Hopkins Health System collected between March 11-31, 2020, comprising 37.3% of the total cases in Maryland during this period. We selected 143 of these samples for sequencing, generating 114 complete viral genomes. These genomes belong to all five major Nextstrain-defined clades, suggesting multiple introductions into the region and underscoring the diversity of the regional epidemic. We also found that clinically severe cases had genomes belonging to all of these clades. CONCLUSIONS: We established a pipeline for SARS-CoV-2 sequencing within the Johns Hopkins Health system, which enabled us to capture the significant viral diversity present in the region as early as March 2020. Efforts to control local spread of the virus were likely confounded by the number of introductions into the region early in the epidemic and interconnectedness of the region as a whole.

Novel insights in genetic transformation of the probiotic yeast Saccharomyces boulardii.

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.

C1q binding to dengue virus decreases levels of infection and inflammatory molecules transcription in THP-1 cells.

Dengue virus infection elicits a spectrum of clinical presentations ranging from asymptomatic to severe disease. The mechanisms leading to severe dengue are not known, however it has been reported that the complement system is hyper-activated in severe dengue. Screening of complement proteins demonstrated that C1q, a pattern recognition molecule, can bind directly to dengue virus envelope protein and to whole dengue virus serotype 2. Incubation of dengue virus serotype 2 with C1q prior to infection of THP-1 cells led to decreased virus infectivity and modulation of mRNA expression of immunoregulatory molecules suggesting reduced inflammatory responses.

Association between magnitude of the virus-specific plasmablast response and disease severity in dengue patients.

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 d of illness, averaging 46% and reaching 87% of B cells, significantly greater than those seen in mild illness or primary infections. On average >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.

West Nile virus infection induces depletion of IFNAR1 protein levels.

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.

West Nile virus.

West Nile virus (WNV) is responsible for thousands of cases of morbidity and mortality in birds, horses, and humans. Epidemics were localized to Europe, Africa, the Middle East, and parts of Asia, and primarily caused a mild febrile illness in humans. In the late 1990s, the virus became more virulent and spread to North America. In humans, the clinical presentation ranges from asymptomatic, seen frequently, to encephalitis/paralysis and death, seen rarely. There is no FDA (Food and Drug Administration)-licensed vaccine for human use, and the only recommended treatment is supportive care. Often, there is a long recovery period. This article reviews the current literature summarizing the molecular virology, epidemiology, clinical manifestations, pathogenesis, diagnosis, treatment, immunology, and protective measures against WNV and WNV infections in humans.

Differential effects of mutations in NS4B on West Nile virus replication and inhibition of interferon signaling.

West Nile virus (WNV) is a human pathogen that can cause symptomatic infections associated with meningitis and encephalitis. Previously, we demonstrated that replication of WNV inhibits the interferon (IFN) signal transduction pathway by preventing the accumulation of phosphorylated Janus kinase 1 (JAK1) and tyrosine kinase 2 (Tyk2) (J. T. Guo et al., J. Virol. 79:1343-1350, 2005). Through a genetic analysis, we have now identified a determinant on the nonstructural protein 4B (NS4B) that controls IFN resistance in HeLa cells expressing subgenomic WNV replicons lacking the structural genes. However, in the context of infectious genomes, the same determinant did not influence IFN signaling. Thus, our results indicate that NS4B may be sufficient to inhibit the IFN response in replicon cells and suggest a role for structural genes, or as yet unknown interactions, in the inhibition of the IFN signaling pathway during WNV infections.

Relocalization of the Mre11-Rad50-Nbs1 complex by the adenovirus E4 ORF3 protein is required for viral replication.

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.

Distinct roles of the Adenovirus E4 ORF3 protein in viral DNA replication and inhibition of genome concatenation.

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.