Biology subjects

Sagan, S. M.

Publications and source records attributed to Sagan, S. M..

5 recordsLinked to original sources

bioRxiv2024

Within-host genetic diversity of SARS-CoV-2 across animal species

Infectious disease transmission to different host species makes eradication very challenging and expands the diversity of evolutionary trajectories taken by the pathogen. Since the beginning of the ongoing COVID-19 pandemic, SARS-CoV-2 has been transmitted from humans to many different animal species, within which viral variants of concern could potentially evolve. Previously, using available whole genome consensus sequences of SARS-CoV-2 from four commonly sampled animals (mink, deer, cat, and dog) we inferred similar numbers of transmission events from humans to each animal species. Using a genome-wide association study (GWAS), we identified 26 single nucleotide variants (SNVs) that tend to occur in deer - more than any other animal - suggesting a high rate of viral adaptation to deer. The reasons for this rapid adaptive evolution remain unclear, but within-host evolution - the ultimate source of the viral diversity that transmits globally - could provide clues. Here we quantify intra-host SARS-CoV-2 genetic diversity across animal species and show that deer harbor more intra-host SNVs (iSNVs) than other animals, providing a larger pool of genetic diversity for natural selection to act upon. Mixed infections involving more than one viral lineage are unlikely to explain the higher diversity within deer. Rather, a combination of higher mutation rates, longer infections, and species-specific selective pressures are likely explanations. Combined with extensive deer-to-deer transmission, the high levels of within-deer viral diversity help explain the apparent rapid adaptation of SARS-CoV-2 to deer.

genomics↗

bioRxiv2022

Zooanthroponotic transmission of SARS-CoV-2 and host-specific viral mutations revealed by genome-wide phylogenetic analysis

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a generalist virus, infecting and evolving in numerous mammals, including captive and companion animals, free-ranging wildlife, and humans. Transmission among non-human species poses a risk for the establishment of SARS-CoV-2 reservoirs, makes eradication difficult, and provides the virus with opportunities for new evolutionary trajectories, including selection of adaptive mutations and emergence of new variant lineages. Here we use publicly available viral genome sequences and phylogenetic analysis to systematically investigate transmission of SARS-CoV-2 between human and non-human species and to identify mutations associated with each species. We found the highest frequency of animal-to-human transmission from mink, compared with lower transmission from other sampled species (cat, dog, and deer). Although inferred transmission events could be limited by sampling biases, our results provide a useful baseline for further studies. Using genome-wide association studies, no single nucleotide variants (SNVs) were significantly associated with cats and dogs, potentially due to small sample sizes. However, we identified three SNVs statistically associated with mink and 26 with deer. Of these SNVs, [~][2/3] were plausibly introduced into these animal species from local human populations, while the remaining [~][1/3] were more likely derived in animal populations and are thus top candidates for experimental studies of species-specific adaptation. Together, our results highlight the importance of studying animal-associated SARS-CoV-2 mutations to assess their potential impact on human and animal health. ImportanceSARS-CoV-2, the causative agent of COVID-19, can infect many animal species, making eradication difficult because it can be reseeded from different reservoirs. When viruses replicate in different species, they may be faced with different evolutionary pressures and acquire new mutations, with unknown consequences for transmission and virulence in humans. Here we analyzed SARS-CoV-2 genome sequences from cats, dogs, deer, and mink to estimate transmission between each of these species and humans. We found several transmission events from humans to each animal, but relatively few detectable transmissions from animals back to humans, with the exception of mink. We also identified three mutations more likely to be found in mink than humans, and 26 in deer. These mutations could help the virus adapt to life in these different species. Ongoing surveillance of SARS-CoV-2 from animals will be important to understand their potential impacts on both human and animal health.

genomics↗

bioRxiv2022

A highly sensitive strand-specific multiplex RT-qPCR assay for quantitation of Zika virus replication

Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is widely used to quantify viral RNA genomes for diagnostics and research, yet conventional RT-qPCR protocols are unable to accurately distinguish between the different viral RNA species that exist during infection. Here we show that false-priming and self-priming occur during reverse transcription with several published Zika virus (ZIKV) primer sets. We developed a RT-qPCR assay using tagged primers and thermostable reverse transcriptase, which greatly reduced the occurrence of nonspecific cDNA products. Furthermore, we optimized the assay for use in multiplex qPCR which allows for simultaneous quantitative detection of positive-strand and negative-strand ZIKV RNA along with an internal control from both human and mosquito cells. Importantly, this assay is sensitive enough to study early stages of virus infection in vitro. Strikingly, using this assay, we detected ZIKV negative-strand RNA as early as 3 h post-infection in mammalian cell culture, at a time point prior to the onset of positive-strand RNA synthesis. Overall, the strand-specific RT-qPCR assay developed herein is a valuable tool to quantify ZIKV RNA and to study viral replication dynamics during infection. The application of these findings has the potential to increase accuracy of RNA detection methods for a variety of viral pathogens. HighlightsO_LISelf-primed cDNA is amplified by widely-used ZIKV qPCR primer sets C_LIO_LIUse of tagged primers and thermostable RT increases strand-specificity for RT-qPCR C_LIO_LIMultiplexed qPCR allows for simultaneous quantitation of (+) and (-) strand viral RNAs, and an internal control C_LIO_LIStrand-specific RT-qPCR can detect fewer than one copy of viral RNA per cell in human and mosquito cells C_LI

microbiology↗

bioRxiv2022

The cellular poly(rC)-binding protein 2 prevents the early steps of hepatitis C virus virion assembly

Poly(rC)-binding protein 2 (PCBP2) was previously shown to bind to the hepatitis C virus (HCV) genome; however, its precise role in the viral life cycle remained unclear. Herein, we found that PCBP2 does not directly affect viral entry, translation, genome stability, replication, or virion egress. Rather, our data suggests that endogenous PCBP2 normally limits virion assembly, thereby indirectly promoting translation and replication by increasing the translating/replicating pool of viral RNAs. Additionally, we found that an alternative RNA conformation (SLIIalt) was important for efficient virion assembly, but functions in a PCBP2-independent manner. The latter may explain why the Japanese fulminant hepatitis 1 isolate is able to produce infectious particles in cell culture, while other HCV isolates are lost in translation. Taken together, our results suggest that PCBP2 and SLIIalt independently modulate HCV genome packaging and alter the balance of viral RNAs in the translating/replicating pool and those engaged in virion assembly. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2021

Poly(rC)-binding protein 1 limits hepatitis C virus assembly and egress

The hepatitis C virus (HCV) co-opts numerous cellular elements - including proteins, lipids, and microRNAs - to complete its viral life cycle. The cellular RNA-binding protein, poly(rC)-binding protein 1 (PCBP1), was previously reported to bind to the 5 untranslated region (UTR) of the HCV genome; however, its importance in the viral life cycle has remained unclear. Herein, we sought to clarify the role of PCBP1 in the HCV life cycle. Using the HCV cell culture (HCVcc) system, we found that knockdown of endogenous PCBP1 resulted in an overall decrease in viral RNA accumulation, yet resulted in an increase in extracellular viral titers. To dissect PCBP1s specific role in the HCV life cycle, we carried out assays for viral entry, translation, genome stability, RNA replication, as well as virion assembly and secretion. We found that PCBP1 knockdown did not directly affect viral entry, translation, RNA stability, or RNA replication, but resulted in an overall increase in infectious particle secretion. This increase in virion secretion was evident even when viral RNA synthesis was inhibited, and blocking virus secretion could partially restore the viral RNA accumulation decreased by PCBP1 knockdown. We therefore propose a model where endogenous PCBP1 normally limits virion assembly and secretion, which increases viral RNA accumulation in infected cells by preventing the departure of viral genomes packaged into virions. Overall, our findings improve our understanding of how cellular RNA-binding proteins influence viral genomic RNA utilization during the HCV life cycle.

microbiology↗