A measles virus collective infectious unit that caused lethal human brain disease includes many locally restricted and few widespread copy-back defective genomes

During virus replication in cultured cells, copy-back defective viral genomes (cbDVG) can arise. CbDVG are powerful inducers of innate immune responses in vitro, but their occurrence and impact on natural infections of human hosts remain poorly defined. We asked whether cbDVG were generated in the brain of a patient who succumbed to subacute sclerosing panencephalitis (SSPE) about 20 years after acute measles virus (MeV) infection. Previous analyses of 13 brain specimens of this patient indicated that a collective infectious unit (CIU) drove lethal MeV spread. In this study, we identified 276 replication-competent cbDVG species, each present in over 100 copies in the brain. Six species were detected in multiple forebrain locations, implying that they travelled long-distance with the CIU. The cbDVG to full-length genomes ratio was often close to 1 (0.6-1.74). Most cbDVG were 324-2000 bases in length, corresponding to 2-12% of the full-length genome; all are predicted to have complementary terminal sequences. If improperly encapsidated, these sequences have the potential to form double-stranded structures that can induce innate immune responses. To assess this, we examined the transcriptome of all brain specimens. Several interferon- and inflammatory response genes were upregulated, but upregulation levels did not correlate with cbDVG levels in the specimens. Thus, the CIU that drove MeV pathogenesis in this brain includes, in addition to two complementary full-length genome populations, many locally restricted and few widespread cbDVG species. The widespread cbDVG species may have been positively selected but how they impacted pathogenesis remains to be determined. Overall design: RNA was extracted from frozen brain specimens using Trizol extraction protocol. The concentration and integrity of the RNA were assessed on an Agilent Bioanalyzer DNA 100 chip (Agilent). cDNA library prep was conducted using Illumina TruSeq Stranded Total RNA Sample Prep Kit (Illumina) according to the manufacturer's protocol, which depletes ribosomal RNA. DNA fragmentation of 150 bp and two paired end sequencing (2 x 150) of each library was performed on an Illumina MiSeq or HiSeq4000 platform. Direct RNA library preparation was based on Oxford Nanopore Technologies (ONT) SQK-RNA004 kit and used 1 ug of RNA from brain specimens in 8.5?µL volume. For MeV cbDVG RNA sequencing, we synthesized a cbDVG specific oligo B to form part of the reverse transcriptase adapter (RTA), which has 12 nucleotides complementary to the conserved 3' end of MeV antigenome and cbDVG. This oligoB-MeV-cbDVG (5' to 3') GAGGCGAGCGGTCAATTTTCCTAAGAGCAAGAAGAAGCCaccagacaaagc was combined with oligo A in a 1:1 ratio at 1.4 µM in buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl) by heating to 95°C for 2 minutes and letting them cool down slowly (0.1°C/sec). This mixture was used as a direct replacement to the RTA supplied in the ONT SQK-RNA004 kit. Adapter ligated MeV-cbCVG RNA was directly sequenced on the GridION ONT sequencer using MinION RNA flow cells (FLO-MIN004RA). All libraries were sequenced in separate flow cells using the high-accuracy base-calling model and the default run settings for direct RNA sequencing.