A library of 135 barcoded coxsackievirus B3 clones shows that orally inoculated virus reaches systemic tissues within 20 minutes and replicates as a diverse population, after which fewer than three variants come to dominate every tissue in the animal without any detectable adaptive mutation.
McCune BT, Lanahan MR, tenOever BR, Pfeiffer JK. Rapid Dissemination and Monopolization of Viral Populations in Mice Revealed Using a Panel of Barcoded Viruses. Journal of Virology. 2020. Volume 94, issue 2, article e01590-19.
DOI 10.1128/jvi.01590-19. PMID 31666382. PMCID PMC6955244.
Attribution note. This study was led by the Pfeiffer laboratory at the University of Texas Southwestern, where all mouse work was carried out and to whose corresponding author correspondence is addressed. Benjamin tenOever is the third of four authors. The paper carries no author contributions statement, so the specific tenOever contribution cannot be stated from the text.
A library of 135 barcoded coxsackievirus B3 clones shows that orally inoculated virus reaches systemic tissues within 20 minutes and replicates as a diverse population, after which fewer than three variants come to dominate every tissue in the animal without any detectable adaptive mutation.
The gastrointestinal tract restricts what passes from the lumen into the body, and enteric viruses must cross it to spread systemically. How that barrier shapes viral population structure over time had not been resolved, because earlier studies of enteric virus bottlenecks sampled late, at disease onset, and so could not distinguish a restriction imposed on entry from one imposed after replication. This study builds 135 clones of coxsackievirus B3 carrying unique nine nucleotide barcodes in the 5' untranslated region, mixes them at equal titer, and inoculates interferon alpha beta receptor deficient mice orally, sampling gastrointestinal and extraintestinal tissues from 20 minutes to 72 hours. All 135 barcodes were recoverable from the upper gastrointestinal tract at 7.5 and 19 hours, while by 48 and 72 hours generally three or fewer barcodes made up most of the population in every tissue sampled, with the same small set shared across tissues within an animal and differing between animals. Neutral red labeling, which renders inoculum virions light sensitive while progeny are not, showed that diverse viruses had replicated before diversity was lost, so the collapse is not explained by only a few inoculum members replicating. Whole-genome consensus sequencing of later samples found no mutation that would account for the takeover. Virus was detectable in pancreas, liver and mesenteric lymph nodes 20 minutes after oral inoculation of receptor deficient mice, and this was not seen in wild-type mice or with poliovirus.
RNA virus populations are diverse because the polymerase lacks proofreading, and that diversity can aid replication and spread within a host. Diversity is lost through selection or through stochastic bottlenecks. Barcoded viruses, carrying short unique sequences that are otherwise neutral, had been used to quantify such changes for plant viruses, human immunodeficiency virus, hepatitis C virus, Zika virus, influenza virus and poliovirus, often revealing founder effects in which a small subpopulation seeds a new tissue.
The Pfeiffer laboratory had previously shown that the gastrointestinal tract limits dissemination of poliovirus and reduces population diversity in tissues after oral inoculation, and had attributed the loss to combined bottlenecks between mouth and gut and between gut and blood. Those experiments sampled at disease onset, generally 72 hours or later, so they lacked the temporal resolution needed to say when diversity is lost or whether the surviving members are the only ones that ever replicated. Coxsackievirus B3 spreads by the fecal-oral route and replicates in extraintestinal tissues before shedding. Immunocompetent mice are infectable but do not succumb, while mice lacking the interferon alpha beta receptor support high replication and disease, which had established them as the tractable model for oral enterovirus infection.
How does the population structure of an orally acquired enteric virus change across tissues and over time, and is the loss of diversity in systemic tissues caused by restricted entry, by selection of adapted variants, or by events occurring after replication.
The design separates questions that a single late time point confounds. Barcodes are placed at a single site in the 5' untranslated region between the internal ribosome entry site and the start codon, so that the 135 clones are otherwise isogenic and any change in their relative frequencies reflects population processes rather than encoded differences. Each clone is grown separately and the library assembled from equal plaque-forming unit amounts, so the starting distribution is known rather than assumed, and replicate sequencing of the stock establishes that the readout is even and reproducible. Time is then sampled from 20 minutes to 72 hours, and tissues are split into those directly exposed to lumenal virus and those reached only after systemic spread, so that spatial and temporal structure can be read together. Two diversity metrics are used, the raw count of barcodes and the Shannon index, the latter sensitive to evenness as well as richness.
Two additional strategies address the ambiguity of the frequency data. Neutral red labeling makes inoculum virions light sensitive, so that light exposure of a tissue homogenate followed by amplification in HeLa cells reports only the barcodes that replicated in the animal, which distinguishes a restriction on replication from a restriction imposed afterwards. Whole-genome consensus sequencing of the dominant populations tests whether adaptation explains the takeover. Finally, a 20 minute time point, poliovirus as a comparator enterovirus, wild-type versus receptor deficient mice, and orally administered radiolabeled amino acids together test whether rapid appearance in systemic tissues is a property of this virus, of enteroviruses in general, or of the barrier itself.
The study does not establish a mechanism for either of its two main observations, and the discussion frames both as open questions.
For monopolization, the data exclude two candidate explanations and leave two others open. Selection on a barcode is argued against because the dominant barcodes differ between animals. Selection on an adaptive mutation is argued against by consensus sequencing, with the acknowledged caveat that low-frequency variants would not be seen. Restriction of replication to a few inoculum members is excluded by the light-sensitivity experiments, since diverse viruses demonstrably replicated first. The authors offer two alternatives without deciding between them, that all the populations descend from one or two infectious events, or that populations in separate tissues disseminate, intermingle, and can be subsumed by an invading population. They list as open objectives how a subpopulation comes to dominate a tissue and a whole animal, what the anatomical source of the monopolizing population is, and whether clearance is what enables monopolization.
For rapid dissemination, no route is demonstrated. The authors raise passage across the intestinal epithelium or through M cells giving access to the lymphatics and then the portal vein and bloodstream, and note that other mechanisms may contribute. Because radiolabeled amino acids also appeared systemically within 20 minutes, they suggest the route may not be specific to viruses. The dependence on interferon receptor status is an observation rather than an explained effect. The authors note that interferon gamma has been reported to affect gastrointestinal permeability but that a role for interferon alpha and beta in rapid viral dissemination has not been described, and they identify the contribution of type I interferon signaling to barrier permeability as a subject for future work. Notably, the radiolabel reached systemic tissues equally in both mouse genotypes while virus did not, so barrier permeability alone does not account for the genotype difference.
The combination of a barcoded library with a replication-reporting label separates two things that frequency data alone cannot distinguish, which members of a population arrived somewhere and which members replicated there. Applying that combination across a time course changes the interpretation of enteric virus bottlenecks. Earlier work sampling at disease onset supported a picture in which barriers restrict which viruses get through. Here the restriction is shown to occur after broad dissemination and after broad replication, which reframes the loss of diversity as a postreplication event, plausibly involving clearance, rather than as a gate at the intestinal barrier.
The 20 minute observation makes the timing of enteric virus dissemination a tractable question. Appearance in pancreas and liver that fast is too rapid for replication to be involved, is not shared by poliovirus, and depends on interferon receptor status, which identifies a specific and testable set of variables. The barcoded coxsackievirus B3 library itself is a reusable resource for population studies in this system.
The tenOever laboratory has used barcoded virus libraries to measure population bottlenecks, and Varble and colleagues in 2014 reported that influenza A virus transmission bottlenecks are defined by infection route and recipient host, a study with tenOever as senior author that is cited here as one of the precedents for the approach. The laboratory has separately used libraries of marked or engineered viruses, including barcode libraries as drift controls, in its own in vivo screening work.
Category 3 synthesis. Read beside Varble and colleagues in 2014 on influenza transmission bottlenecks and the barcode control arm of Varble and colleagues in 2013, this paper places the tenOever contribution within a recurring methodological interest in reading population structure from sequence-tagged virus libraries passaged through animals, applied here to a different virus, a different route and a different laboratory's disease model. This connection is synthesis across papers and is not claimed in the article itself.
Nearly all in vivo work uses mice lacking the interferon alpha beta receptor, chosen because immunocompetent mice do not develop disease after oral coxsackievirus B3 infection. The authors state that examining population dynamics in wild-type mice and other immunodeficient strains is future work. Since the rapid dissemination phenotype was absent in wild-type mice, the possibility that the interferon status of the host also shapes the monopolization dynamics is not excluded by anything reported here.
The inoculum is a single very high dose, one times ten to the ninth plaque-forming units delivered by pipette into the mouth, so the population dynamics observed apply to that dose and route and not necessarily to natural exposure. Only one virus strain, coxsackievirus B3 H3, is characterized in depth, with poliovirus used only for the 20 minute comparison and in a different mouse background carrying the poliovirus receptor transgene, which weakens the comparison.
The evidence against adaptation is consensus sequencing of eight samples from two tissues at one time point, and the authors note that consensus sequencing cannot detect low-frequency mutations within a population, so selection on a minority variant is not excluded. Barcode assignment has an acknowledged technical floor, since 13 percent of the library lies within one nucleotide of another member and thirteen barcodes contain an ATG, with five of those out of frame, although all were well represented in the stock. Barcode insertion itself reduced replication at one point in the HeLa growth curve. The replication readout depends on a background correction, since roughly one in 4,000 plaque-forming units of the neutral red stock is already light insensitive, and the interpretation assumes labeled and unlabeled virions penetrate tissues equally. The colon and stool discrepancy at 7.5 hours is explained by inference about unsampled gastrointestinal sites rather than by measurement.
Finally, neither central observation is mechanistically resolved. The source of the monopolizing population, the reason a subpopulation takes over an entire animal, the anatomical route of 20 minute dissemination, and the basis of its dependence on interferon receptor status are all stated by the authors as unresolved.
Tagged coxsackieviruses fed to mice reached the liver within twenty minutes and replicated as a diverse population, yet within two days two or three variants dominated everywhere.
A library of 135 otherwise identical coxsackieviruses, each carrying a short sequence tag, was fed to mice. The full population spread through the gut within hours, and a dye-based label showed that many members genuinely replicated. Even so, by two days almost every tissue in each animal was dominated by three or fewer tags, with no mutation to explain it. Virus also reached pancreas and liver twenty minutes after feeding.
How the intestinal barrier shapes enteric virus populations had been studied only at late time points, which cannot separate restricted entry from later loss. Here 135 coxsackievirus B3 clones carrying unique nine nucleotide tags were pooled and given orally to interferon receptor deficient mice. All 135 were recovered from the upper gastrointestinal tract at 7.5 and 19 hours, but by 48 and 72 hours generally three or fewer dominated every tissue sampled, with the dominant set shared within an animal and differing between animals. Neutral red labeling, which distinguishes inoculum from progeny virions by light sensitivity, showed that diverse members had replicated before diversity collapsed, and consensus sequencing found no adaptive mutation, though low-frequency variants would escape detection. Virus reached pancreas, liver and mesenteric lymph nodes within 20 minutes, not seen in wild-type mice or with poliovirus. The routes behind both rapid spread and monopolization remain undetermined.
lab-led for Varble 2014. McCune 2020 is collaborative and led by the Pfeiffer laboratory at the University of Texas Southwestern, where all mouse work was done, and Muñoz-Moreno 2019 is collaborative and led by the García-Sastre laboratory, with the tenOever contribution in both being the barcoded library method and supervision
No explicit publication relationship was recorded in the reviewed graph.