Targeting genomic SARS-CoV-2 RNA with siRNAs allows efficient inhibition of viral replication and spread.

A promising approach to tackle the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. So far it is unclear, which viral replication steps can be efficiently inhibited with siRNAs. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, and thereby terminate replication before start of transcription and prevent virus-induced cell death. Coronaviruses replicate via negative sense RNA intermediates using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5' and 3' end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting common sequences shared by all viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs that targeted open reading frame 1 (ORF1) which only exists in gRNA. In contrast, siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs leading to an impaired antiviral efficacy. Verifying the translational relevance of these findings, we show that a chemically modified siRNA that targets a highly conserved region of ORF1, inhibited SARS-CoV-2 replication ex vivo in explants of the human lung. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with specifically targeting gRNA, might be key for high antiviral efficacy.

Prolonged norovirus infections correlate to quasispecies evolution resulting in structural changes of surface-exposed epitopes.

In this study, we analyzed norovirus (NoV) evolution in sequential samples of six chronically infected patients. The capsid gene was amplified from stool samples, and deep sequencing was performed. The role of amino acid flexibility in structural changes and ligand binding was studied with molecular dynamics (MD) simulations. Concentrations of capsid-specific antibodies increased in sequential sera. Capsid sequences accumulated mutations during chronic infection, particularly in the surface-exposed antigenic epitopes A, D, and E. The number of quasispecies increased in infections lasting for >1 month. Interestingly, high genetic complexity and distances were followed by ongoing NoV replication, whereas lower genetic complexity and distances preceded cure. MD simulation revealed that surface-exposed amino acid substitutions of the P2 domain caused fluctuation of blockade epitopes. In conclusion, the capsid protein accumulates numerous mutations during chronic infection; however, only those on the protein surface change the protein structure substantially and may lead to immune escape.

Reply to the Letter of Charre et al. "Mis-Genotyping of Some Hepatitis D Virus Genotype 2 and 5 Sequences Using HDVdb".

We thank Charre and colleagues for spotting the mis-annotation of sequences in our database, which was caused by human error [...].

Global Occurrence of Clinically Relevant Hepatitis B Virus Variants as Found by Analysis of Publicly Available Sequencing Data.

Several viral factors impact the natural course of hepatitis B virus (HBV) infection, the sensitivity of diagnostic tests, or treatment response to interferon-α and nucleos(t)ide analogues. These factors include the viral genotype and serotype but also mutations affecting the HBV surface antigen, basal core promoter/pre-core region, or reverse transcriptase. However, a comprehensive overview of the distribution of HBV variants between HBV genotypes or different geographical locations is lacking. To address this, we performed an in silico analysis of publicly available HBV full-length genome sequences. We found that not only the serotype frequency but also the majority of clinically relevant mutations are primarily associated with specific genotypes. Distinct mutations enriched in certain world regions are not explained by the local genotype distribution. Two HBV variants previously identified to confer resistance to the nucleotide analogue tenofovir in vitro were not identified, questioning their translational relevance. In summary, our work elucidates the differences in the clinical manifestation of HBV infection observed between genotypes and geographical locations and furthermore helps identify suitable diagnostic tests and therapies.

HDVdb: A Comprehensive Hepatitis D Virus Database.

Hepatitis D virus (HDV) causes the most severe form of viral hepatitis, which may rapidly progress to liver cirrhosis and hepatocellular carcinoma (HCC). It has been estimated that 15-20 million people worldwide are suffering from the chronic HDV infection. Currently, no effective therapies are available to treat acute or chronic HDV infection. The remarkable sequence variability of the HDV genome, particularly within the hypervariable region has resulted in the provisional classification of eight major genotypes and various subtypes. We have developed a specialized database, HDVdb (http://hdvdb.bio.wzw.tum.de/), which contains a collection of partial and complete HDV genomic sequences obtained from the GenBank and from our own patient cohort. HDVdb enables the researchers to investigate the genetic variability of all available HDV sequences, correlation of genotypes to epidemiology and pathogenesis. Additionally, it will contribute in understanding the drug resistant mutations and develop effective vaccines against HDV infection. The database can be accessed through a web interface that allows for static and dynamic queries and offers integrated generic and specialized sequence analysis tools, such as annotation, genotyping, primer prediction, and phylogenetic analyses.

The Global Hepatitis B Virus Genotype Distribution Approximated from Available Genotyping Data.

Hepatitis B virus (HBV) is divided into nine genotypes, A to I. Currently, it remains unclear how the individual genotypes contribute to the estimated 250 million chronic HBV infections. We performed a literature search on HBV genotyping data throughout the world. Over 900 publications were assessed and data were extracted from 213 records covering 125 countries. Using previously published HBV prevalence, and population data, we approximated the number of infections with each HBV genotype per country and the genotype distribution among global chronic HBV infections. We estimated that 96% of chronic HBV infections worldwide are caused by five of the nine genotypes: genotype C is most common (26%), followed by genotype D (22%), E (18%), A (17%) and B (14%). Genotypes F to I together cause less than 2% of global chronic HBV infections. Our work provides an up-to-date analysis of global HBV genotyping data and an initial approach to estimate how genotypes contribute to the global burden of chronic HBV infection. Results highlight the need to provide HBV cell culture and animal models that cover at least genotypes A to E and represent the vast majority of global HBV infections to test novel treatment strategies.

ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing.

Chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) is a new technology to study genome-wide long-range chromatin interactions bound by protein factors. Here we present ChIA-PET Tool, a software package for automatic processing of ChIA-PET sequence data, including linker filtering, mapping tags to reference genomes, identifying protein binding sites and chromatin interactions, and displaying the results on a graphical genome browser. ChIA-PET Tool is fast, accurate, comprehensive, user-friendly, and open source (available at http://chiapet.gis.a-star.edu.sg).

An oestrogen-receptor-alpha-bound human chromatin interactome.

Genomes are organized into high-level three-dimensional structures, and DNA elements separated by long genomic distances can in principle interact functionally. Many transcription factors bind to regulatory DNA elements distant from gene promoters. Although distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Here we describe the development of a new strategy, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) for the de novo detection of global chromatin interactions, with which we have comprehensively mapped the chromatin interaction network bound by oestrogen receptor alpha (ER-alpha) in the human genome. We found that most high-confidence remote ER-alpha-binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ER-alpha functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes.