Fighting bird flu with RNAi

Scientists at CSIRO's Australian Animal Health Laboratory (AAHL) in Geelong have launched a potentially revolutionary research project aimed at reducing the threat of the lethal H5N1 avian influenza virus.

Dr John Lowenthal's AAHL research team will explore two different ways of protecting the US$300 billion global poultry industry against influenza - one therapeutic, the other prophylactic. Both will exploit the natural cellular anti-viral mechanism, RNA interference (RNAi).

The therapeutic option would involve delivering small RNA molecules to chickens in their drinking water or via an aerosol spray, priming the birds' own natural RNAi defences to recognise and destroy the virus.

The prophylactic option is to insert custom-designed RNAi transgenes into poultry, to protect birds against influenza. If successful this would confer protection against all current and future strains of influenza, including the deadly H5N1 strain that health authorities fear could spawn the first, disastrous human pandemic of the 21st century.

Lowenthal says if either approach works, it would still need to be examined by the federal Office of the Gene Technology Regulator, and subjected to public scrutiny, before it could be released.

The project will take at least three years to establish whether the technology can be used to protect chickens from avian influenza under laboratory conditions (proof-of-concept), and then up to two years more to determine whether the approach is practicable.

Advanced Technology Services Australia (ATSA), an Australian subsidiary of one of the world's leading poultry breeding companies, is backing the five-year pilot project. Lowenthal's team hopes to achieve proof-of-concept within three years.

The H5N1 'bird flu' has killed 172 people over the past four years and forced the slaughter of hundreds of millions of chickens across Asia and Europe. Virologists fear it could spawn a human pandemic strain. American virologist and Nobel laureate Professor Joshua Lederberg has described the influenza virus as the planet's most dangerous human pathogen.

The World Health Organisation has stated that the best way of reducing the risk of the next human flu pandemic is to get better control of the disease in poultry. "And that's exactly what we aim to do," Lowenthal says.

ddRNAi

At CSIRO's Horizons in Livestock Science conference in Surfer's Paradise in 2005, Cambridge University virologist Dr Laurence Tiley described how RNAi technology could protect the global poultry industry against influenza.

Tiley said the unique structure of the $300 billion poultry industry could facilitate replacement of the entire global poultry flock with influenza-resistant birds in as little as four years. Only half a dozen companies supply the elite breeding stock on which the world's commercial flocks are based, and two account for more than 50 per cent of production.

Lowenthal's team actually conceived the idea of using RNAi technology to develop influenza-resistant poultry in 2001, after CSIRO colleagues Dr Peter Waterhouse and Dr Ming-Bo Wang's invention of DNA-delivered (dd)RNAi technology raised the extraordinary possibility of protecting all the world's livestock species and crop plants against their major virus diseases.

With CSIRO's patented ddRNAi technology, researchers can custom-design genes that arm plant and animal cells to recognise and destroy specific viruses, with absolute precision.

Lowenthal says experiments in Australia and overseas have already established that ddRNAi completely blocks viral infections in laboratory plants and animals.

The technology allows researchers to design transgenes with small, embedded sequences that recognise and target the influenza virus's genetic code. These 'designer' transgenes would be inserted into the chromosomes of chickens, or other livestock species.

The RNAi transgenes would be designed to be "always switched on" in all the bird's tissues so that invading viruses can be instantly destroyed.

In contrast, the therapeutic approach of delivering small RNAi molecules to chickens via aerosols or their drinking water would confer transient protection during an influenza outbreak.

Like the deadly H1N1 strain that killed at least 40 million people in the 1918-19 Spanish Flu pandemic, the H5N1 bird flu is so virulent that it overwhelms the birds' normal RNAi and immune defences before they can mount a protective response.

Therapeutic RNAi could give birds 'breathing room' to develop their own natural RNAi response, and retain a molecular memory of the infection that would protect them in the event of any encounter with a new influenza strain.

The transgenic option programs the birds' cells to produce hairpin-shaped RNA molecules that are cleaved by cellular enzymes into small RNA fragments less than 23 nucleotides in length.

These 'targeting sequences' are taken up by tiny cellular structures called RISCs (RNA-Induced Silencing Complexes). They serve as templates that allow the RISC to attach to and destroy the corresponding sequence in the virus' genetic blueprint, preventing the virus replicating.

Target sequences

Lowenthal says AAHL researchers have already identified target sequences shared by all strains of the influenza virus. The virus has only eight genes, three of which are virtually identical across all strains because they are essential for its replication.

The other genes, including those encoding the haemagglutinin and neuraminidase proteins of the virus's coat, vary considerably from strain to strain. The influenza virus differs from most other viruses in that it constantly mutates and spawns new strains - an evolutionary strategy that prevents its animal hosts developing a protective immune response after infection.

Lowenthal says target sequences copied from the three highly conserved genes should confer broad-spectrum protection against influenza, making it much more difficult for the virus to mutate and evade the bird's RNAi defences.

Hairpin RNAi transgenes could be designed to confer simultaneous protection against several major poultry viruses, such as avian influenza, Marek's disease, and Newcastle disease. An outbreak of the latter disease, probably caused by migratory birds, forced NSW poultry farmers to slaughter seven million chickens in 1998.

US molecular geneticists Professor Craig Mello and Professor Andrew Fire were awarded last year's Nobel Prize for Medicine for discovering RNA interference in the nematode worm, Caenorhabditis elegans, in 1997.

In that same year, CSIRO's Waterhouse and Wang independently discovered RNAi in plants, and subsequently published the first detailed account of how RNAi works in plant cells.

CSIRO Plant Industry scientists have already used ddRNAi to develop prototype cereal varieties with resistance to Barley Yellow Dwarf Virus (BYDV) and a complex of closely related viruses.

BYDV defied decades of effort to breed resistant lines by conventional breeding and hybridisation. CSIRO has recently used chromosome manipulation to move a natural source of resistance from a near relative to wheat.

However the additional protection conferred by ddRNAi would make the protection stronger and more durable. Lowenthal says that there is very limited potential to breed influenza-resistant poultry with conventional methods.

By 2005, the AAHL team's research was sufficiently advanced for CSIRO to approach major poultry breeding companies to determine their interest in possible applications of the technology.

Although ATSA's German parent company was impressed with the potential of the technology, it delayed involvement because it was keen to ensure this research project was completely independent of any of the company's other operations and also because of uncertainty about consumer reactions.

But the SARS epidemic, and a spate of human deaths from the H5N1 bird flu - including a European veterinarian who had treated an infected duck - caused a change of heart this year.

"They accepted that the technology shows potential, and asked the CSIRO team to develop a research plan to demonstrate proof-of-concept," Lowenthal says. "The risk of a pandemic is sufficiently high to at least make the attempt - it's no longer just a commercial issue, it has spilled over into human health."

Multiple strains

The fast-mutating virus spawns multiple new strains every year, and Lowenthal says it is not yet feasible to produce a vaccine to protect against all animal and human strains. There are currently no effective vaccines for H5N1.

In the 1918-19 Spanish Flu pandemic, healthy young adults died within six hours of exhibiting the first signs of infection, literally drowning as their lungs filled with fluid.

The highly pathogenic H5N1 virus causes the immune system to release huge amounts of inflammatory molecules - a phenomenon called a cytokine storm - that cause lung cells to leak copious amounts of fluid. Healthy young adults are at particular risk because of their strong immune response.

"Even if we had a highly effective vaccine and could produce enough vaccine doses to vaccinate in the midst of an epidemic, this virus acts so rapidly and aggressively that the immune response can't cope and there would still be a huge death toll," Lowenthal says.

If this new RNAi approach is successful in poultry, it could help in reducing the risk of the next flu pandemic in humans.