GM flu-resistant chooks solution to pandemic threat - British scientist

It would take only four years to completely replace today's farmed chickens with genetically modified breeds fully resistant to infection to avian influenza, according to a visiting British molecular virologist.

Dr Laurence Tiley, of Cambridge University's Centre for Veterinary Science, told the annual CSIRO Horizons in Livestock Science conference in Surfer's Paradise that the tools already exist to make poultry resistant to influenza infection, thus eliminating the most likely source of a devastating pandemic of human influenza.

Tiley described several molecular techniques that had been developed to proof-of-concept stage in cellular systems.

He said the emergence of the H5N1 'bird flu' in Hong Kong in 1997 had come as a 'rude awakening', because it provided evidence that an avian influenza could directly infect humans without first undergoing genetic recombination with human 'flu viruses in pigs - the 'mixing vessel' hypothesis.

He said that while transmission to humans remained relatively infrequent, and there was still no proof of human-to-human transmission, the 50 per cent mortality rate in the outbreaks since 1997 had the research community 'waiting and dreading.'

The latest models were estimating that a H5N1 pandemic could cost the global economy $166 billion dollars, assuming a low rate of infection and mortality- the 1918-19 Spanish Flu pandemic had a mortality rate of only 2 per cent, and if the next pandemic was similar in impact, the economic costs would be "truly staggering".

Tiley said that that the rapid slaughter and disposal of massive numbers of chickens was a serious challenge.

The successful containment of the 2003 outbreak of the H7N7 avian influenza strain in the Netherlands had required the slaughter of 30 million chickens to contain it. The H5N1 epidemic in Asia had seen 150 million chickens slaughtered across south-east Asia, with no impact on the virus' spread.

Contact in Asia between free-ranging poultry and water contaminated with virus-laden faeces from shorebirds, the primary vector for influenza, was the most common route of infection.

Shorebirds harboured low-pathogenicity virus strains, but upon entering chickens or other bird species, the virus is not well adapted to its new hosts, and high levels of natural variation are subject to rapid selection for new variants that can reproduce in the new host, and "jump species."

Tiley said the notion that virulent strains of the virus would kill infected migratory waterbirds, limiting the spread of potential pandemic strains, was unduly optimistic. Even though mortality rates in waterbirds reached 95 per cent, there had been large die-offs of chickens and wild waterbirds in regions far way in Kazakhstan and the Caucasus region.

"There is a realistic expectation that the virus will migrate on a global scale, so how do we protect ourselves?" Tiley asked.

"We can't kill all the shorebirds, or stop raising chickens. But improved farming practices would be a good way to start - we could ensure that farmers don't keep chickens, ducks and pigs in the same environment.

"We should also prevent the practice of selling poultry live at markets.

"Vaccinating poultry is a double-edged sword, because you don't get fully sterile immunity - you just push down the virus to a sub-clinical level. It helps producers, but its no way to stop an epidemic, because the virus is still circulating in poultry flocks."

He said Mexico's poultry industry had suffered a major epidemic of avian influenza more than a decade ago, and had still not eliminated the virus, despite an intensive vaccination campaign.

"The way forward may be to engineer influenza resistance into chickens by selective breeding or transgenesis," Tiley said.

He described three strategies for developing 'flu-resistant chickens - one based on a natural anti-viral protein, Mx, another using RNAi transgenes to destroy the virus' RNA-encoded genetic blueprint, and a molecular 'decoy' technique based on highly conserved genetic elements of the virus itself.

The Mx protein is naturally expressed by many vertebrates in response to a broad range of viral infections - especially RNA-type viruses. It strongly inhibits infection by suppressing transcription and viral replication.

But the Mx protein appears to be inactive in most modern chicken breeds. Tiley said that if functional Mx gene is introduced into cultured avian cells, it is strongly protective. It prevents replication of the virus' genetic blueprint by knocks down viral polymerase activity.

Humans are strong Mx expressers, but they still get the 'flu. Tiley said the virus has evolved a defence: it blocks Mx expression by suppressing the interferon response."

Engineering chickens with an "always-on" Mx gene might be detrimental, so the strategy would be to make expression of the gene contingent on influenza infection.

The influenza virus suppresses the host's interferon response with a protein called Ns1. Another protective strategy would be to engineer chickens with RNAi transgene to suppress expression of the Ns1 gene. A potential problem with this approach was that it would overload the cell's RNA-induced silencing complexes (RISCS), which are essential to a range of natural gene-regulation processes.

But experiments in mouse, and transgenic chicken cells, had shown that it was not difficult to introduce an RNAi transgene to knock down Ns1 expression by the virus.

The third approach involved introducing a transgene that would produce 'molecular decoys' to keep the virus' RNA polymerase molecules from transcribing other viral genes.

The virus has a segmented genome, and in every major type and strain of the influenza virus, the 5' and 3' binding sites at the end of the segments are identical in sequence.

Experiments have shown that transgene coding for a hairpin molecule consisting solely of the highly conserved polymerase binding motifs, linked by a small non-coding RNA "hinge", acts as a decoy, tying up polymerase molecules and disrupting viral replication.

Tiley said that, again, the gene would be activated by the virus, so I was active only during infection.

Delivering any of these transgenes into chickens was a challenge, but recent advances had solved most of the technical problems involved. Neutered lentiviruses would be used to deliver the transgenes safely into chicken embryonic stem cells.

"Two well-characterised, isoallelic transgenic chickens [chickens with identical anti-influenza transgenes at identical loci on a chromosome pair] could be expanded to replace the entire broiler chicken population in four years," Tiley said.

"Why stop at influenza? If you do it, why not do the same for Marek's disease and Newcastle disease?"

The obstacle, Tiley said, was public opposition to genetically modified organisms - attitudes vary between nations, but the UK is most hostile to transgenic crops and animals."

"Public education is vital - it's very easy to alarm people, and much harder to allay their fears."