PROJECTS

Our laboratory studies the relationship between a virus and the organism it infects. Our goal is to understand how the different components of a virus population affect the infected organism and how this interaction influences the virus evolution and its maintenance in nature. Below is a summary of our current areas of interest and related projects.


Antiviral immunity

Our laboratory has systematically investigated the impact of defective viral genomes (DVGs) on the induction of the antiviral response. We have shown that during infection with many negative sense RNA viruses DVGs are the primary triggers of the antiviral response through the stimulation of the intracellular virus sensor molecules RIG-I and MDA5. We identified a specific RNA motif on a Sendai virus DVG of the copy-back type that is responsible for its strong immunostimulatory activity. This DVG is one of the strongest known natural stimulators of the antiviral response. Some of the questions that remain to be addressed are: How, when and where is this motif exposed to cellular sensors during infection? and are there similar strongly stimulatory motifs present in DVGs from other viruses?

Top: Cells infected with Sendai virus and stained using RNA FISH to distinguish full length viral genomes (orange) and DVGs (green), and IFA for IRF3. Bottom: predicted secondary structure of the hyperstimulatory motif present in a SeV DVG.

Defective viral genomes (DVGs) and the cell

DVGs are known to exist since the late 1940s. However, many fundamental questions related to their biology and function remain unanswered, including how DVGs affect the biology and function of the infected cell. Our data revealed unexpected heterogeneity in the content of DVGs and full-length viral genomes in different cells during infection. Some cells have a higher proportion of DVGs than full-length genomes, while others are enriched in full-length genomes. In addition, we observed that DVGs interact with the infected cell differently than the full-length genomes. These distinct interactions result in different cellular responses, including the formation of stress granules, the stimulation of the antiviral response, and cell survival. Remaining open questions in this area are: What determines the establishment of the DVG high and DVG low phenotypes? Are these phenotypes stable in time? Can cells change from a DVG high cell to a full-length genome-high phenotype? Are other physiological processes impacted by DVGs?


Full-length (FL) high and DVG high-cell stained by RNA FISH/IFA for FL genomes, DVGs and the mitochondrial marker Tom20 as indicated.

DVG biogenesis and function

One critical question left unanswered in the DVG field is what is the molecular mechanism that drive DVG generation? Our laboratory identified hotspots in the genome of the respiratory syncytial virus (RSV) that are frequently used to signal the polymerase reattachment to continue replication during the formation of DVGs of the copy-back type. This knowledge can be now used to generate viruses lacking certain copy-back DVGs, which can serve as useful tools to study the function of different DVGs. In addition, we discovered that in humans naturally infected with the respiratory syncytial virus, a handful of conserved DVG species predominates in most DVG+ individuals. Open questions are: What is the function of these highly predominant and conserved DVG that are generated during RSV infection? Are they essential for the virus maintenance in nature? Why are they conserved? Are there similarly conserved species in other viral infections?


Top: Schematic of copy-back DVG formation during replication of nsRNA viruses. Bottom: Hotspots in the RSV genome for the polymerase break (orange) and rejoin (blue) during copy-back DVG formation.

Predicting viral disease severity in humans

The respiratory syncytial virus (RSV) infects all children under the age of two and a large number of these children (~3%) are hospitalized with moderate to severe disease. About half of the hospitalized patients have life threatening conditions and/or develop chronic bronchiolitis, asthma or COPD. Currently, there are no means to predict the clinical outcome of RSV infection in order to optimize treatment of patients at high risk of developing severe disease. We found a strong correlation between detection of DVGs in respiratory secretions from RSV patients and the clinical severity of the disease. Importantly, the kinetics of DVG generation critically impacts their protective function in humans. Some open questions in this area are: Can we use DVGs as a disease prognosis tool in humans? Can DVGs predict disease severity in other viral infections?


Top right: Percentage of DVG+ and DVG- pediatric samples. Bottom: Correlation among relative amount of DVGs and expression of an antiviral gene

Host determinants of DVG generation

We and others have extensively shown that DVGs alter the course and pathogenesis of viral infections. Fast and strong production of DVGs generally results in a lower viral load and reduced inflammation, while slow or weak production of DVGs results in higher viral titers and delayed but robust pathogenic inflammatory responses, both in mice and humans. Our evidence suggests that the speed and amount of DVGs generated in humans varies independently of the virus, but which host factors modulate DVG generation is unknown. Projects in this area will use exciting and unique tools including respiratory organoids generated from the collaborative cross mice and unbiased genetic screens to identify host factors that determine the quality and quantity of DVG generation in mice and humans.


C57BL6 tracheal organoid stained as indicated

Virus dynamics, ecology and evolution

DVGs are part of the virus ecosystem. Little is known of the impact of DVGs on the dynamics of standard virus spread, transmission, or evolution. The ubiquitous presence of DVGs in most viral infections and their critical role in initiating host immune responses reveal a puzzling gap in our understanding of the determinants and evolutionary tradeoffs of virus recognition by the host immune system and raise the question: why do viruses retain production of immunostimulatory DVGs that may ultimately lead to their clearance? We are interested in exploring this question and in addressing how virus, host and DVGs affect each other. Are DVGs drivers or regulators of virus evolution? Do different environments within a host affect the virus/DVG dynamics? Does the immunological status affect viral evolution and DVG generation?

In addition, the impact of different types of DVG and other secondary viral products during infection is unknown, nor is how these viral products affect the ecology of other commensal or pathogenic microorganisms. Projects in this area will answer the following questions: How do copy-back DVGs relate and collaborate with other viral secondary products? How do they interact with other microbes during infection? Do they impact on the ecology and pathogenesis of other viruses, commensal bacteria and/or fungi?

RNA virus persistence and impact on chronic lung diseases

Increasing evidence supports the persistence of RNA viruses in different tissues long after the acute infection has been cleared. This evidence includes reports of persistent virus RNA in infections with Ebola virus, Zika virus, measles virus, parainfluenza viruses and respiratory syncytial virus. Persistent viruses are a continue source of virus and are associated with the development of chronic diseases, such as asthma and COPD, after parainfluenza or respiratory syncytial virus infections. It is unknown how these RNA viruses persist and whether there are means of eliminating this reservoir. We have established a mouse model to study the persistence of RNA viruses that are believed to be acute. Available projects in this area will answer the following questions: What are the mechanisms behind RNA virus persistence in vivo? What is the cellular reservoir for the persistence of respiratory viruses? How does a persistent viral infection impact the development of chronic diseases such as asthma? Are DVGs responsible for persistence in vivo as it has been observed in vitro?


Immunosuppression treatments reactivate SeV protein expression in cells from the lung parenchyma a month after the acute infection has been cleared

Vaccines and antivirals

A challenge for vaccine development is the absence of safe immunostimulatory molecules to induce protective responses against intracellular pathogens that can evade antibody detection, such as viruses. This type of immune response would ideally be a “type I” immune response including antigen-specific Th1 CD4+ T cells and cytotoxic CD8+ T cells. We have identified DVG-derived oligonucleotides (DDOs) as potential immunostimulatory molecules able to promote the development of protective type I immunity against viruses. DDOs contain the critical immunostimulatory motif that makes SeV DVGs one of the stronger natural immunostimulants. Our data show that in mice DDOs trigger a type I cellular immune response able to protect from challenge with a highly pathogenic virus. Available projects in this area will answer the following questions: Can we use DDOs to identify the minimal requirements for effective induction of type I immunity during vaccination? Can we use DDOs to make better vaccines / can we harness them as adjuvants or antivirals? Do DDOs work in larger animals? Can we optimize their safety and delivery?


Histology of lungs from C57BL/6 mice immunized with UV-inactivated IAV alone or adjuvanted with DDO or Alum and challenged with live IAV three weeks after boost immunization. Analysis was performed 10 days after challenge.