The biology of picobirnaviruses, highly abundant RNA viruses in human enteric viromes

About This Grant

SUMMARY Picobirnaviruses (PBVs) are small double-stranded, bisegmented RNA viruses that are found in humans, other mammals and birds. PBVs are among the most commonly detected RNA viruses in the human enteric tract, and they have been linked to human diseases. For example, PBV is associated with type 1 diabetes and detection of PBVs is predictive of the development of severe graft-versus-host-disease in hematopoietic stem cell transplant recipients. These observations raise a key question as to whether PBVs play causal roles in these diseases. However, since no PBV culture or animal model exists, it is currently impossible to experimentally test disease causality. The lack of any PBV isolate is the rate-limiting step in further characterization of the basic biology of PBVs and their role in pathogenic outcomes. Key to overcoming this technical barrier is the understanding of the type of host organism(s) PBVs infect. Dogma asserts that PBVs are human and animal- infecting viruses, but definitive proof of this is lacking. Instead, recent studies support the hypothesis that PBVs are RNA phages that infect bacteria. For example, there is high prevalence of bacterial ribosome binding sites (Shine-Dalgarno) preceding PBV ORFs, a feature characteristic of most phages. In addition, many PBVs encode proteins that can lyse bacteria (lysins) a property necessary for phages to egress from their host bacteria. Critically, in preliminary data we demonstrate that PBV3 can be cultured anaerobically in a stool-derived bacterial community, establishing the first in vitro culture for any PBV. Furthermore, some antibiotic treatments completely block PBV3 growth, and PBV3 RNA and conserved bacterial 16S rRNA co-localize in the same bacteria from in vitro cultures. To identify the hosts of PBVs, we will optimize a bacterial single-cell RNA-sequencing (scRNAseq) approach for co-detection of phage and host RNA in individual bacteria from complex communities. In parallel, specific PBV-targeted FISH- and antibody-FACS approaches will be used to purify and identify PBV infected bacteria. As a key step towards obtaining pure PBV isolates, we will harness germ-free mice to propagate PBVs in vivo. As shown in our preliminary data, germ-free mice gavaged with PBV-containing human stool specimens serve as a vessel to propagate PBVs in vivo. This innovative approach will generate renewable quantities of infectious PBVs and their host for in vitro isolation efforts. Guided by this information and results from scRNAseq, antibody-, and FISH-based assays, we will isolate PBVs by infecting bacterial monocultures of the candidate hosts. Importantly, lytic RNA phage proteins have been coined “protein antibiotics”. PBV-encoded bacterial lysins provide a unique opportunity to characterize new lytic phage proteins and mechanisms that could provide an alternative to traditional antibiotics for treatment of bacterial infections. The overall goals are to: (1) Identify the bacterial hosts of PBVs; (2) establish PBV culture systems in vivo and isolate PBVs in vitro; (3) Define mechanisms of action of PBV-encoded bacterial lysins.