Thanks to high-throughput sequencing technologies, genome sequencing has become a common component in nearly all aspects of viral research; thus, we are experiencing an explosion in both the number of available genome sequences and the number of institutions producing such data. However, there are currently no common standards used to convey the quality, and therefore utility, of these various genome sequences. Here, we propose five “standard” categories that encompass all stages of viral genome finishing, and we define them using simple criteria that are agnostic to the technology used for sequencing. We also provide genome finishing recommendations for various downstream applications, keeping in mind the cost-benefit trade-offs associated with different levels of finishing. Our goal is to define a common vocabulary that will allow comparison of genome quality across different research groups, sequencing platforms, and assembly techniques.
It has been suggested that four amino acids which are absolutely conserved in th nsP1 nonstructural proteins encoded by togaviruses and in the homologous proteins encoded by plant viruses in the Sindbis virus (SV) superfamily may constitute a "methyltransferase motif." In the Sindbis virus nsP1 protein (540 amino acids) these four amino acids are represented by His39, Arg91, Asp94, and Tyr249. Earlier, in assays of methyltransferase (MTase) activity generated in SV-infected cells, we had shown that amino acid changes at positions 87 and 88 of SV nsP1 resulted in a 10-fold lower Km for S-adenosyl methionine, the methyl donor in MTase reactions. Using site-directed mutagenesis we now report the expression of nsP1 in Escherichia coli, and in the infectious clone of Sindbis virus, Toto/1101, in which His39, Arg91, Asp94, and Tyr249 were changed one at a time to Ala. We also expressed nsP1 with C-terminal deletions of varying size, as well as with internal deletions in the C-terminal portion of the protein, in E. coli. Changing His39, Arg91, Asp94, or Tyr249 to Ala led to a loss of both MTase activity and viral infectivity; however, changing Ile369 to Val, a conservative change in the carboxy-terminal half of nsP1, had no effect on either MTase activity or viral infectivity. With respect to the deleted forms of nsP1, a carboxy-terminal deletion of 48 amino acids was still compatible with MTase activity in vitro. However, larger deletions including those in which the amino acids between positions 442 and 492 were deleted abolished MTase activity.
Symptomatic cytomegalovirus (CMV) disease has been the standard endpoint for clinical trials in organ transplant recipients. Viral load may be a more relevant endpoint due to low frequency of disease. We performed a meta-analysis and systematic review of the literature. We found several lines of evidence to support the validity of viral load as an appropriate surrogate end-point, including the following: (1) viral loads in CMV disease are significantly greater than in asymptomatic viremia (odds ratio, 9.3 95% confidence interval, 4.6-19.3); (2) kinetics of viral replication are strongly associated with progression to disease; (3) pooled incidence of CMV viremia and disease is significantly lower during prophylaxis compared with the full patient follow-up period (viremia incidence: 3.2% vs 34.3%; P < .001) (disease incidence: 1.1% vs 13.0%; P < .001); (4) treatment of viremia prevented disease; and (5) viral load decline correlated with symptom resolution. Based on the analysis, we conclude that CMV load is an appropriate surrogate endpoint for CMV trials in organ transplant recipients.
The NIH Virtual SARS-CoV-2 Antiviral Summit, held on November 6, 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for COVID-19, including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss SARS-CoV-2 targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.
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