High-throughput single-molecule analysis of the influenza A genome structure and assembly

The influenza (flu) virus is the microscopic pathogen that causes flu in humans and animals. We propose to use special microscopy methods to understand the structure of the genetic material of the flu virus, and how interactions between the genetic material can become important in flu pandemics.

The genetic material, or genome, of the flu virus is made of RNA (a long chain of molecules that encodes genetic information) and is divided into eight individual segments. The natural host of the flu virus is wild birds; however, in a process called 'reassortment', different strains of the virus can swap RNA segments in a way that allows us to infect other host organisms, including humans. Reassortment events result in the generation of entirely new virus strains, to which humans have never previously been exposed. When there is no existing immunity in the human population to a novel influenza virus, reassortment can cause deadly worldwide pandemics, such as the Spanish flu pandemic in 1918, which was responsible for over 50 million deaths worldwide, and the 'swine flu' pandemic in 2009. Whilst H5N1 'bird flu' strains haven't yet reassorted to the extent that they can reliably infect humans and cause a pandemic, outbreaks in birds have been devastating for the poultry industry since millions of birds have been culled to reduce virus spread, resulting in huge economic losses.

Despite flu being one of the best-studied viruses, we are still unsure how exactly the genomic segments contact each other inside virus particles. Much of our knowledge of this process has relied on data obtained using methods that measure the properties of millions of molecules (or particles) in one go, and therefore report on the averaged properties of all of the molecules. We plan to use advanced 'single-molecule techniques', which allow us to study one molecule (or particle) at a time; this allows us to see details unique to each molecule (or particle) that may be impossible to see using traditional analytical or biological methods.

We plan to perform our single-molecule analysis by using many small pieces of fluorescent DNA, that will bind all the way along the virus gene segments, to detect the presence and map the structure of the gene segments at very high resolution. Further, the fluorescent DNAs will not be able to bind to sections of the genome that interact with other genomic segments (as these areas will not be accessible due to the close contacts between segments), thereby allowing us to build up a picture of crucial RNA-RNA interactions within virus particles. To image the virus particles as they are being detected by the small DNA pieces, we will use a specialised "single-molecule fluorescence" microscope, which is carefully designed to allow the detection and monitoring of individual fluorescent molecules present in a detection zone (as opposed to conventional microscopes that require thousands or millions of molecules to be present in a detection zone).

We anticipate that our research will help the scientific community to better understand how the genome segments of the flu virus interact and cause pandemics, and help in efforts to control and stop its spread. Our discoveries should also help us understand and control other viruses that cause danger to humans and animals, and contribute towards development of new diagnostic tests for rapid and sensitive detection of influenza and other pathogenic viruses.

Influenza viruses are important animal and human pathogens that can cause severe disease. The influenza virus genome is comprised of eight RNA segments packaged together in a single virus particle (virion) as eight ribonucleoprotein particles (RNPs). Co-infection of the same host with different influenza strains can lead to reassortment of gene segments from different strains into virus particles, resulting in the generation of novel viruses that can cause devastating pandemics.

There is increasing evidence for a selective packaging mechanism that uses RNA-RNA contacts between the viral segments to ensure the incorporation of a complete set of RNPs into a particle. Further, high-resolution structures of virions suggest the presence of a specific structural arrangement of segments within viral particles. However, since the RNA-RNA contacts were initially identified in ensemble experiments involving billions of particles, they cannot be correlated with each other or attributed to a specific RNP arrangement.

We will close the present knowledge gap by studying the fidelity of selective segment packaging and the spatial organisation of RNPs within single influenza virus particles; we will also correlate with presence of key inter-segment contacts with the structure of the genome in single virions. To this end, we will apply cutting-edge super-resolution and single-molecule fluorescence imaging to large populations of single virions outside cells, as well as clustered RNPs inside infected cells; these efforts will build on our extensive experience with imaging and analysing viruses. From these large datasets, we will gain unprecedented access to the structural variability of the influenza virus genome, and lay the foundation for evaluating the possibility of future influenza pandemics based on the viral genome structure. Our methods will be general and applicable on other RNA viruses, and help elucidate variable nucleotide structures in all domains of life.

The influenza virus was responsible for one of the deadliest pandemics in human history and frequently causes catastrophic losses in agriculture worldwide; in particular, avian influenza has been economically devastating for food industries such as the poultry industry, resulting in the culling of millions of birds and huge economic losses. As result, there is a pressing need to understand the fundamental mechanisms of this deadly pathogen. Our high-throughput single-molecule approach should reveal rich mechanistic detail about the influenza virus genome structure and assembly, and enable new ways to combat influenza viral disease. Our work will provide the foundation that will help scientists to understand viral reassortment and to better predict and prepare for future pandemics. Via our collaboration with colleagues at the Pirbright Institute, we will contribute to the understanding of the genome structural organisation in avian influenza viruses and its impact on pathogenicity and cross-species transmission. Since our methodology is general, it can be adapted to the study of many viruses affecting humans or animals.

Our work will be instrumental in developing anti-viral drugs against influenza. The correct packaging of RNA segments into newly made virus particles is essential for virus replication in cells, and RNA-RNA contacts appear to play an important role in genome packaging; disrupting these RNA contacts is thus an attractive target for drug design. The structural information we will obtain should also help in silico drug screening for antivirals.

Our studies will contribute to the development of rapid and sensitive diagnostic tests for influenza virus infections, allowing identification of dangerous strains and prescription of appropriate treatment; this capability should improve the health of animal and human populations by minimizing the spread of infections, and result in significant savings since it can limit unnecessary use of antivirals and antibiotics.

Our results will be communicated through academic channels such as scholarly journals, research conferences, and university websites, as well as non-academic channels, such as media interviews, social media announcements, press releases, and short videos produced for the industry and the public. To maximise our impact and help form a network of researchers interested in single-molecule approaches to virology, we will organise an international meeting on this topic for academic, industrial, and national-lab researchers. We will also use our single-molecule studies of influenza, coupled with the use of a compact commercial microscope, and motivated by a real-world health challenge, to perform public engagement in schools and science festivals.

Finally, the researcher co-investigator involved in this work will gain important transferable skills desirable in interdisciplinary academic and industrial environments involving demanding bioassay development, image analysis, and data processing; the researcher will have many opportunities to develop their independence, and acquire the necessary experience and track record for successful career progression.