Biology subjects

Langsjoen, R. M.

Publications and source records attributed to Langsjoen, R. M..

5 recordsLinked to original sources

bioRxiv2024

Evidence for Powassan virus deletions and defective RNA in field collected ticks

Powassan virus (POWV) is a tick-borne flavivirus in the tick-borne encephalitis virus (TBEV) serogroup endemic to the United States, Canada, and parts of Russia. POWV remains an under-studied pathogen, despite the potential for serious and life-threatening neurologic complications following infection. While prior studies have characterized viral diversity due to single nucleotide polymorphisms, little is known about POWV recombination, defective RNAs (D-RNAs), and functional structural variants (SVs). Understanding POWV recombination in its natural vector can provide important insights into its replication and evolution. Thus, we analyzed POWV sequence data from 51 ticks collected from the Northeast United States to characterize deletion expression levels and patterns in naturally infected ticks, and we compared these results to single-passage isolates. We found that deletions were common in POWV RNA from ticks and that several areas of the genome were enriched for recombination junctions. Deletions were often associated with areas of microhomology. While most deletions were sample-specific, two major deletion archetypes were observed across multiple tick samples. The first consisted of small 19-50 base deletions in the methyltransferase domain of the ns5 RNA-dependent RNA-polymerase gene, resulting in a mixture of putative SVs and D-RNAs. The second consisted of approximately 1600 base deletions spanning the ns2a-ns3 genes, resulting in putative D-RNAs with abrogated viral protease function. Protease deletions were significantly enriched after one passage in baby hamster kidney cells despite a decrease in overall deletion expression. These results demonstrate the proclivity of POWV for recombination, with potential implications for immune evasion and persistence in ticks. IMPORTANCEPowassan virus is a tick-borne flavivirus that can cause serious, life-threatening neurological disease. Understanding how Powassan virus replicates and evolves within its tick vector may elucidate factors important in persistence, transmission, and human disease. Defective RNAs are replication-incompetent viral genomes generated through internal deletions, which have been associated with disease severity and persistent infection in other viruses but have not been described for Powassan virus. Here, we show that Powassan virus produces abundant defective RNAs in field-caught ticks, and that expression patterns of these defective RNAs changes after one passage in mammalian cells. Although the function of these defective RNAs remains unknown, this work establishes a critical framework for investigating the role of defective RNAs in Powassan virus replication and transmission.

genomics↗

bioRxiv2023

North American Powassan virus encompasses diverse in vitro phenotypes.

Powassan virus (POWV) is a tick-borne flavivirus which has resulted in increasing human cases over the past two decades. Despite high prevalence in ticks and evidence of broad distribution in North America, fewer than 50 human cases are detected annually with evidence of undetected asymptomatic infections. Experimental studies of the relationships between POWV genetic diversity and disease potential are currently lacking. In the present study, sixteen isolates originating from 13 locations in the United States and Canada were used to assess in vitro phenotypic diversity in human neuronal cells. Broad differences in replication and cytopathic ability were observed between isolates, even amongst those in the same sublineage. In vitro phenotype was not associated with geographic or temporal location and could not be associated with specific genotypes. These results support the observation that the North American POWV population may be highly genetically and phenotypically diverse. The degree to which in vitro phenotype reflects transmission and pathogenesis remains to be determined.

microbiology↗

bioRxiv2021

Mouse Adapted SARS-CoV-2 protects animals from lethal SARS-CoV challenge

The emergence of SARS-CoV-2 has resulted in a worldwide pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of COVID-19 disease have been hampered by the lack of robust mouse models. To overcome this barrier, we utilized a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARSCoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse-adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Utilizing this model, we demonstrate that SARS-CoV-2 infected mice are protected from lethal challenge with the original SARS-CoV, suggesting immunity from heterologous CoV strains. Together, the results highlight the utility of this mouse model for further study of SARS-CoV-2 infection and disease.

microbiology↗

bioRxiv2021

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called Tiled-ClickSeq, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

molecular biology↗

bioRxiv2020

Differential alphavirus defective RNA diversity between intracellular and encapsidated compartments is driven by subgenomic recombination events

Alphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective genomes featuring large deletions during replication. Defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity of infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated NGS libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and encapsidated P2 virus and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than encapsidated and specific populations of D-RNAs are differentially expressed between intracellular and encapsidated compartments. Importantly, these trends were likewise observed in a murine model of CHIKV 15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that are conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which was confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template. IMPORTANCEOur understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we show that specific populations of alphavirus D-RNAs arise de novo and that they are not packaged into virions, thus imposing a transmission bottleneck and impeding their prior detection. This raises important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection and whether their influence is constrained by packaging requirements. Further, during the course of these studies, we found a novel type of alphavirus D-RNA that is enriched intracellularly; dubbed subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between capsid/E3 and E1/3UTR regions and are common to chikungunya, Mayaro, Sindbis, and Aura viruses. These sgD-RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally may exist as subgenome-derived transcripts.

molecular biology↗