Avian influenza virus in East Asia arose through a series of genetic reassortment events with other viruses

An avian influenza virus that has caused three major outbreaks among poultry and killed several people in East Asia over the past seven years arose through a series of genetic reassortment events with other viruses.

This study finding, by scientists from St. Jude Children's Research Hospital, the People's Republic of China, China's Special Administrative Region of Hong Kong, Thailand, Vietnam and Indonesia, is published in the July 8 online edition of Nature.

Reassortment is the swapping of genes when two or more viruses infect the same animal.

The researchers say that their study of the genetic makeup of H5N1 subtypes collected since 1997 traces the evolution of the virus into a dangerous pathogen through a series of reassortment events. Results of the study indicate that domestic ducks in southern China played a key role in the generation of this virus. The H5N1 virus forced health authorities to slaughter millions of chickens in order to prevent the spread of the disease, which can quickly wipe out poultry in open-air markets and farms and spread to other flocks.

The investigators warn that outbreaks of H5N1 in East Asian poultry populations must be rapidly and effectively controlled to prevent H5N1 from evolving into a virus that causes a human pandemic, or worldwide epidemic. By cleaning up open-air markets and regularly slaughtering infected birds, Hong Kong remained free of H5N1 outbreaks in poultry during the 2004 influenza crisis, according to Robert Webster, Ph.D., member of Infectious Diseases department and holder of the Rose Marie Thomas Chair at St. Jude, and Richard Webby, Ph.D., also of the department of Infectious Diseases at St. Jude.

"In order to reduce the ability of H5N1 to trigger another poultry epidemic, officials in East Asia must follow Hong Kong's lead," Webster said. "Otherwise, H5N1 will likely continue to infect birds and other animals and eventually could evolve into a dangerous human pathogen as well."

Webster and Webby are co-authors of the Nature report, which details genetic studies of the evolving H5N1 virus that caused the initial human outbreak in Hong Kong in 1997. The report traces the origins of the outbreaks of highly pathogenic H5N1 disease in Asian poultry that occurred in 2003 and 2004.

The researchers collected samples of the virus from poultry in Indonesia, Thailand and Vietnam, and from humans in Thailand and Vietnam. They then compared the eight genes carried by these viruses with the eight genes carried by samples of 253 H5N1 viruses collected from live poultry markets in Hong Kong and the Chinese provinces of Guangdong, Hunan and Yunnan between 2000 and 2004.

The study found that H5N1 viruses occurred only in ducks during 2000, but from 2001 on, the viruses spread to chickens, too. Furthermore, the various H5N1 viruses isolated over the years all contained two genes (HA and NA) derived from the same, older virus called Goose/Guangdong/1/96. The other six genes came from various influenza viruses through reassortment. HA and NA are two proteins on the surface of flu viruses that permit the virus to infect cells and to spread from animal to animal or from person to person.

The study also showed that a specific type of H5N1 called the Z genotype, although widely entrenched in poultry in southern China, is still adapting to these birds, suggesting that these viruses will continue to evolve through mutation or reassortment to achieve greater fitness in poultry species.

In addition, the researchers found that genotype Z viruses in Thailand, Vietnam and Indonesia carried a specific mutation in a protein called M2, known to cause resistance to a family of antiviral drugs used to treat human influenza. The mutation also developed in some genotype B, Y and Z+ viruses.

The fact that the M2 resistance mutation is arising in different viruses suggests that it can be readily acquired, according to the researchers. If this gene were passed on to a human flu virus during reassortment in an animal infected with both avian and human influenza viruses, the resulting virus would be resistant to an important family of drugs.

In fact, the common human influenza H3N2 has been found inside pigs in southern China, according to the St. Jude researchers. If H5N1 infects a pig harboring H3N2, these two viruses might swap genes. The resulting "recombinant" virus might be particularly dangerous to humans, depending on which gene or genes it acquired.

The report in Nature also notes other indications that H5N1 could evolve into a worldwide threat to humans: the outbreak of H5N1 in poultry in Asia in 2003 and 2004 was unprecedented in its geographical range, which showed the wide reach this virus already has.

"The transmission of H5N1 to even just a relatively few people was an ominous sign that it has the potential to adapt to humans," Webster said.

A key question left unanswered by the present study is whether the highly pathogenic H5N1 influenza virus is now being spread by wild migratory birds.

"Although these deadly viruses were isolated from dead migrating birds, we don't know if the birds are actually spreading H5N1," Webby said. "We must do further research to find out, since migratory birds could conceivably spread highly pathogenic H5N1 throughout the rest of Asia and into Europe and the Americas."

The key to preventing a human pandemic of H5N1 is the rapid and effective control of poultry infections, the St. Jude researchers say. And while this is a challenging task, the recent success of Hong Kong in avoiding H5N1 poultry outbreaks due to preventive measures taken demonstrates the wisdom of this approach.

The culling of hundreds of millions of poultry across East Asia reduced the threat of transmission of bird flu to humans, and possibly even prevented the outbreak of a human pandemic, the St. Jude researchers said. But that public health victory came at a significant cost to poultry farms. The question remaining is whether such farmers and their governments should bear this financial burden by themselves.

"If we consider H5N1 to be a global problem that could get much worse, perhaps the costs should be borne instead by the World Health Organization's global influenza program," Webster said. "The results of this study are a wake-up call for the world to provide the resources needed to prevent future outbreaks of H5N1 among poultry or to prepare for a human pandemic of a very dangerous virus."

This work was supported in part by the National Institutes of Health, The Wellcome Trust, the Ellison Foundation, the Li Ka Shing Foundation and the Research Grants Council of Hong Kong.

Other authors of the report are K. S. Li (Shantou University Medical College; Guangdon, P.R. China); Y. Guan, J. Wang, K. M. Xu, L. Duan, H. Chen, J.S. M. Peiris (Shantou University and The University of Hong Kong, Queen Mary Hospital; Hong Kong SAR P.R. China); G. J.D. Smith, L.L. M. Poon, K.Y. Yuen (The University of Hong Kong, Queen Mary Hospital; Hong Kong SAR, P.R. China); A. P. Rahaedjo, A.T. S. Estoepangeste (Universitas Airlangga; Surabaya, Indonesia); P. Puthavathana, P. Auewarakul (Sriraj Hospital; Bangkok, Thailand); C. Buranathai and A. Chaisingh (National Institute of Animal Health; Bangkok, Thailand); H.T. Long and N.T. H. Hanh (National Institute of Hygiene and Epidemiology; Hanoi, Vietnam); and W. Lim (Department of Health, Hong Kong SAR, P.R. China).

St. Jude Children's Research Hospital St. Jude Children's Research Hospital is internationally recognized for its pioneering work in finding cures and saving children with cancer and other catastrophic diseases. Founded by late entertainer Danny Thomas and based in Memphis, Tennessee, St. Jude freely shares its discoveries with scientific and medical communities around the world. No family ever pays for treatments not covered by insurance, and families without insurance are never asked to pay. St. Jude is financially supported by ALSAC, its fundraising organization.