Brain tropism acquisition: The spatial dynamics and evolution of a measles virus collective infectious unit that drove lethal subacute sclerosing panencephalitis

Abstract

It is increasingly appreciated that pathogens can spread as infectious units constituted by multiple, genetically diverse genomes, also called collective infectious units or genome collectives. However, genetic characterization of the spatial dynamics of collective infectious units in animal hosts is demanding, and it is rarely feasible in humans. Measles virus (MeV), whose spread in lymphatic tissues and airway epithelia relies on collective infectious units, can, in rare cases, cause subacute sclerosing panencephalitis (SSPE), a lethal human brain disease. In different SSPE cases, MeV acquisition of brain tropism has been attributed to mutations affecting either the fusion or the matrix protein, or both, but the overarching mechanism driving brain adaptation is not understood. Here we analyzed MeV RNA from several spatially distinct brain regions of an individual who succumbed to SSPE. Surprisingly, we identified two major MeV genome subpopulations present at variable frequencies in all 15 brain specimens examined. Both genome types accumulated mutations like those shown to favor receptor-independent cell-cell spread in other SSPE cases. Most infected cells carried both genome types, suggesting the possibility of genetic complementation. We cannot definitively chart the history of the spread of this virus in the brain, but several observations suggest that mutant genomes generated in the frontal cortex moved outwards as a collective and diversified. During diversification, mutations affecting the cytoplasmic tails of both viral envelope proteins emerged and fluctuated in frequency across genetic backgrounds, suggesting convergent and potentially frequency-dependent evolution for modulation of fusogenicity. We propose that a collective infectious unit drove MeV pathogenesis in this brain. Re-examination of published data suggests that similar processes may have occurred in other SSPE cases. Our studies provide a primer for analyses of the evolution of collective infectious units of other pathogens that cause lethal disease in humans.

Fig 1. Robust MeV replication and transcription in two brain specimens.

(A, B, left panels) Methylene blue stained RNA gels comparing the integrity of RNA extracted from SSPE brain specimens to that of HeLa cells uninfected or infected with a MeV vaccine strain. (A, B, right panels) Northern blots of the gels probed using (A) a probe detecting positive strand MeV N (monocistronic) and N-P (dicistronic) mRNAs or (B) a probe detecting negative sense genomic RNA). (C) Pie chart showing the number of reads that aligned to MeV genome, human genome (release #38) and unaligned reads in specimen SSPE1 and SSPE2. (D) MeV genome coverage plot showing the positive (blue line) and negative (red line) strand reads in specimen SSPE1. x-axis shows schematic of MeV genome in negative sense orientation and y-axis represents reads per nucleotide.

Fig 2. Frequency and genomic location of positions at variance between the reference genome and the SSPE1 (top) and SSPE2 (bottom) sequences.

x-axis: MeV genome location. y-axis: allele frequency. Nucleotide variants detected at nearly 100% frequency are shown in yellow, those detected at 60–75% in blue, those at 25–40% in red and those at other frequencies in grey. Variants shown in black are candidate neuropathogenesis drivers. Dots represent A to G and U to C transitions that may have been introduced by ADAR1 editing [78,79], triangles represent other transitions and squares represent transversions.

Fig 3. CG1 and CG2 replicate in the same cells and occasionally form spatially segregated replication centers.

In situ hybridization with CG1 (red) and CG2 (green) specific probes in temporal lobe tissue. Nuclei are counterstained with DAPI (blue). Red box highlights the area shown on the right.

Fig 4. Distribution of MeV plus and minus reads in brain specimens.

MeV genome coverage plot showing positive (blue line) and negative (red line) strand reads. x-axis: MeV genome; y-axis reads per nucleotide on a logarithmic scale. Pie charts show the ratio of positive (blue) and negative (red) strand reads.

Fig 5. Frequency of G1 and G2 mutations and two potential neuropathogenesis driver mutations in all brain specimens.

X-axis: brain specimens; y-axis; frequencies of G1 mutations (blue), G2 mutations (red) and all other mutations (grey). Black circles highlight F-Q527* mutations and black squares highlight M-F50S mutations.

Fig 6. Identification of a spatially restricted G1 subpopulation in frontal cortex 2.

For each panel x-axis: MeV genome location; y-axis: allele frequency. SNVs attributed to G-01 are shown in light blue and linked with a line. SNVs attributed to G-01b are shown in dark blue and linked with a line. SNVs attributed to G-01a are shown in black and linked with a line. SNVs attributed to G2 are shown in dark red and linked with a line. All other SNVs are shown in grey. SNVs are defined relative to BA.

Fig 7. Spatial dynamics of G1 and G2 subpopulations in the brain.

(A, left panel) Phylogenetic tree of G1, G2, and their descendants. (A, right panel): location of mutations attributed to the Brain Ancestor, G-01, G-FC2, G1 and its descendants (top), and G2 and its descendants (bottom). Crosses represent A to G and U to C transitions, vertical ticks represent other mutations. (B) Brain drawing with superimposed pie charts indicating the frequencies of G1 and G2 descendants. Area of pie chart sectors reflects the frequency of each cluster that are colored according to the key on the right. Large, intermediate, or small pies represent specimens with >13%, 5–13% or less than 5% MeV reads, respectively. C, cortex; L, lobe; U, upper; Int, internal; TN, towards nucleus. Brain image is from BioRender.

Fig 8. Hypothetical reconstruction of the evolution of a MeV collective infectious unit in a human brain.

X-axis: time. Y-axis: population size. Cartoon illustrating hypothesized MeV brain expansion over time, including the development of G1 (red), G2 (blue), G-FC2 (black) subpopulations, transit among brain regions, and modulation of F tail truncation. We do not illustrate the simultaneous process of viral diversification forming the G1 and G2 descendant subclusters, or H I8T mutational dynamics.