Rapid Dissemination and Monopolization of Viral Populations in Mice Revealed Using a Panel of Barcoded Viruses

Abstract

The gastrointestinal tract presents a formidable barrier for pathogens to initiate infection. Despite this barrier, enteroviruses, including coxsackievirus B3 (CVB3), successfully penetrate the intestine to initiate infection and spread systemically prior to shedding in stool. However, the effect of the gastrointestinal barrier on CVB3 population dynamics is relatively unexplored, and the selective pressures acting on CVB3 in the intestine are not well characterized. To examine viral population dynamics in orally infected mice, we produced over 100 CVB3 clones harboring nine unique nucleotide "barcodes." Using this collection of barcoded viruses, we found diverse viral populations throughout each mouse within the first day postinfection, but by 48 h the viral populations were dominated by fewer than three barcoded viruses in intestinal and extraintestinal tissues. Using light-sensitive viruses to track replication status, we found that diverse viruses had replicated prior to loss of diversity. Sequencing whole viral genomes from samples later in infection did not reveal detectable viral adaptations. Surprisingly, orally inoculated CVB3 was detectable in pancreas and liver as soon as 20 min postinoculation, indicating rapid systemic dissemination. These results suggest rapid dissemination of diverse viral populations, followed by a major restriction in population diversity and monopolization in all examined tissues. These results underscore a complex dynamic between dissemination and clearance for an enteric virus.IMPORTANCE Enteric viruses initiate infection in the gastrointestinal tract but can disseminate to systemic sites. However, the dynamics of viral dissemination are unclear. In this study, we created a library of 135 barcoded coxsackieviruses to examine viral population diversity across time and space following oral inoculation of mice. Overall, we found that the broad population of viruses disseminates early, followed by monopolization of mouse tissues with three or fewer pool members at later time points. Interestingly, we detected virus in systemic tissues such as pancreas and liver just 20 min after oral inoculation. These results suggest rapid dissemination of diverse viral populations, followed by a major restriction in population diversity and monopolization in all examined tissues.

Keywords: coxsackievirus; dissemination; evolution; viral pathogenesis; viral population dynamics.

FIG 1

Construction of barcoded collection of CVB3 clones. (A) A random 9-nucleotide sequence was inserted into CVB3 5′ untranslated region (5′UTR) (nucleotide 709) between the internal ribosome entry site (IRES) and the start of coding sequence. (B) Workflow for production and analysis of virus barcode frequencies in a pool of 135 barcoded CVB3 clones. Different colors in plasmids and virions represent uniquely barcoded viruses. (C) Growth curves of a single representative barcoded CVB3 clone (barcode ATCGTACCA) and the wild type (WT) in HeLa cells (n = 4, 2-way analysis of variance [ANOVA], Sidak posttest, standard errors of the means [SEM] shown). (D) Sequencing ratios for each barcode in 4 cDNA replicates of CVB3 barcode virus stock.

FIG 2

Virus population dynamics in gastrointestinal tract and extraintestinal tissues over time. Ifnar^−/− mice were orally infected with 1 × 10⁹ PFU of the CVB3 barcode library and were dissected 7.5 hpi, 19 hpi, 48 hpi, or 72 hpi. The height of the bar corresponds to the titer of virus, and the height of the color bands is proportional to the frequency of a viral barcode within the sample, with total frequencies equaling 1. The horizontal dashed line represents the limit of detection for plaque assay, and bars with slanted lines represent results below the sequencing threshold.

FIG 3

Viral barcode diversity decreases over time and is lower in extraintestinal tissues. (A and B) Comparing changes in diversity over time, we quantified the total number of barcodes detected (A) and Shannon diversity (B) for the GI tract or for extraintestinal tissues (MLN, pancreas, and liver) combined. Data in panel B were analyzed by Kruskal-Wallis test with Dunn’s correction for multiple comparisons; only statistically significant results are shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (C and D) To directly compare GI versus extraintestinal sites, we analyzed the number of barcodes detected (C) and Shannon diversity (D). To obtain the data shown in panel D, we performed mixed-effects analysis with Sidak’s correction for multiple comparisons; only statistically significant results are shown.

FIG 4

Monopolization of viral populations occurs in the absence of detectable adaption. Sanger consensus sequencing of CVB3 whole genomes was performed for 48-hpi MLN and liver samples from four representative mice. Each line represents the genome of consensus sequence derived from each sample, and each “X” represents a consensus mutation at that site.

FIG 5

CVB3 in vivo replication dynamics. (A) Mice were orally infected with neutral red-labeled CVB3 barcoded viruses in the dark. Mice were dissected in the dark, and tissue samples were homogenized and either kept in the dark or exposed to light. Virus was quantified by plaque assay and then amplified for a single cycle in HeLa cells, and deep sequencing was performed on barcodes of progeny. In the example here, the blue virus had replicated in vivo, and therefore it was light insensitive and amplified in HeLa cells. (B) Viral titers in samples kept in the dark, representing both input/inoculum and replicated viruses. Whiskers show maximums to minimums; points labeled with numbers indicate mouse number. (C) Percentages of replicated virus (PFU from light-exposed samples divided by PFU of dark-exposed samples multiplied by 100). The lower dotted line indicates the limit of detection derived from plaque assay for light-exposed samples, and the upper dotted line indicates 100% replication within a population.

FIG 6

The level of nearly all light-insensitive virus clones recovered from mice is higher than background in neutral red-labeled virus stock. A small fraction of viruses within neutral red-labeled stock are light insensitive, and their presence can confound interpretation of results from subsequent mouse experiments. To rule out this effect, we quantified the fraction of light-insensitive viruses in our stock and compared it with the data from mouse experiments. We found that 1/4,000 PFU in neutral red-labeled virus inoculum was light insensitive. Assuming that (i) neutral red-labeled and unlabeled virions equally penetrated each tissue and (ii) no viral replication occurred, then 1/4,000 PFU from dark-treated tissues would represent light-insensitive inoculum PFU. This sets the background level for each sample. The light-treated virus titer was divided by this background level to obtain the fold signal over background. Values over 1 indicate that virus replication had occurred; the probability of bona fide viral replication increased exponentially as indicated on the y axis. Numbers indicate mouse identifiers.

FIG 7

Diverse viruses replicated prior to population monopolization. The ratio of viral barcodes within each sample is shown, with viral barcodes derived from virus that had replicated in vivo (light exposed) and total replicated-plus-unreplicated inoculum viral barcodes (dark samples). The crosshatch (#) symbol represents samples with results below the sequencing depth threshold.

FIG 8

Viral barcode diversity of viruses replicated in vivo. Data represent the number of viral barcodes in each tissue at each time point from samples exposed to light and amplified for a single cycle in HeLa cells. (A) Number of barcodes present of replicated virus in each sample. (B) Shannon diversity (H’) of replicated viral barcodes. Analyses were performed using 2-way ANOVA and Tukey multiple comparisons; only statistically significant results are shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 9

Orally inoculated CVB3 rapidly spreads to extraintestinal tissues in mice. (A) Ifnar^+/+ and Ifnar^−/− mice were orally inoculated with 1 × 10⁹ PFU CVB3, and tissues were collected at 20 min postinfection (mpi). Virus was quantified in homogenized tissues by plaque assay (n = 4). (B) Dissemination of CVB3 barcode collection at 20 mpi (n = 5). The horizontal dashed line represents the limit of detection for the plaque assay, and bars with slanted lines represent results below the sequencing threshold. (C) PVR Ifnar^−/− mice were orally inoculated with 1 × 10⁹ PFU poliovirus, and tissues were collected at 20 mpi. Virus was quantified in homogenized tissues by plaque assay (n = 13). (D) Comparison of CVB3 and PV extraintestinal tissue titers represented in panels A and C. (E) Dissemination of ³⁵S radiolabeled cysteine and methionine at 20 min after oral feeding.