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  AVIAN FLU  -  Living with influenza

The epidemic has been spreading since 1997, but it was not until the summer of 2005 that the A H5N1 flu virus first hit the headlines. Since then the public has been kept well informed of the progress of a disease that is causing growing concern: multiple outbreaks on poultry farms in the Far East, with the widespread slaughter of birds; limited, but often fatal, cases of transmission to humans; and propagation of the virus among wild and migrating birds bringing the Asian epidemic to the EU’s borders. Most serious of all, this epizootic disease is raising the spectre of the appearance of a mutant virus that would be transmissible between humans. All of this begs the question as to what weapons are available to science and research to combat the threat. 

Type A flu virus
Type A flu virus
© Institut Pasteur
The infectious pulmonary disease that we call ‘flu’ has long plagued humankind. Mention of flu epidemics can be found as far back as the third century BC in a text by Hippocrates, the father of European medicine. From the 16th century, a study of historical archives indicates an average of three serious epidemics or pandemics (1) per century. Worst of all was the Spanish flu epidemic of 1918-19 that killed between 20 and 40 million people worldwide (see box). Two other very serious epidemics struck in 1857 and 1968, when the United States was hit particularly hard.

The extended flu family
After slaughtering the poultry, the barn is burned in the province of Ling An, in a region close to Ho Chi Minh City in Vietnam.© FAO
After slaughtering the poultry, the barn is burned in the province of Ling An, in a region close to Ho Chi Minh City in Vietnam.
Given the destruction caused, this contagious disease was the subject of extensive research throughout the 20th century, aided by developments in molecular biology applied to virology. Gradually, the contours of this vast family of influenza viruses were defined. In addition to the viruses that cause the traditional human flu, and that do their work every winter – although rarely proving fatal, except for the very frail – influenza viruses also affect large numbers of birds as well as pigs and other mammals.

In around 1930, two viruses, affecting man and pigs, were identified for the first time. During the 1940s, anti-flu vaccines were developed by growing the virus in chicken eggs and then inactivating it. These ‘inactivated viral strains’ trigger an immune reaction that prevents infection by the actual flu virus. However, this does not offer the population 100% protection. 

Three types of influenza
Today, a great many influenza viruses have been identified, classified into three major types: A, B and C.

The distinguishing feature of the influenza A flu viruses – the so-called orthomyxoviridae group – is that they possess two proteins, haemagglutines (H) and neuraminidases (N). There are 15 different haemagglutines (H1 to H15) and nine neuraminidases (N1 to N9). Each virus sub-type, defined by an ‘H(X)N(Y)’ combination, has its own specificities, in particular in regard to the infected hosts and the seriousness of the illness caused. But these influenza A viruses tend to mutate frequently and can also adapt to a new host with a specific pathogenicity. This explains why any flu epidemic among animals is now seen as potentially dangerous to man.

It has been established that all viruses regarded as highly pathogenic (HPAI) belong to the H5 and H7 sub-types. For its part, the H5N1 virus, which is at the origin of the growing alarm over the past two years, infects many bird species specifically and has also been found recently in some species of mammal. In principle, harmless for wild birds – especially the palmipeds which, no doubt, are the reservoir species – it kills gallinaceans in large numbers and can infect humans who are in direct contact with infected birds. To date very limited, these cases lead to serious flu symptoms with a high proportion of fatalities. 

More worrying is the possible mutation or recombination of the virus currently circulating in Asia. If two influenza A viruses infect the same host, they can exchange elements and give rise to a new recombined virus that could be even more virulent. This recombination could occur in pigs, which are sensitive to both bird and human flu viruses, and could lead to the appearance of a new virus that affects man and can be transmitted directly between humans.

Threats to humans
Although, back in 1997, H5N1 had already proved its ability to infect man, to date there have been no more than isolated cases which are always due to close contact with sick birds. Only poultry farmers, abattoir workers and vets are potentially at risk as they can inhale the virus present in the faeces or the expectorations of sick or dead birds. It is probable that the H5N1 virus is transmitted solely through inhalation, and that cooking the birds destroys it.

In South-East Asia, about 120 people have been infected with this virus since the start of the bird flu epidemic, and around 60 of them have died. So, while H5N1 presents a real danger for man, who has no immune defence against it, its power of infection remains very low given the relatively few cases in relation to the density of population in these regions and the close proximity between humans and animals. 

This means that there will be no epidemic unless this bird flu virus adapts to man, through mutation or recombination. To date, the international laboratories that are analysing the viruses taken from birds or sick humans have identified no ‘humanised’ strain. Consequently, the virus that could trigger an epidemic does not yet exist. 

Immediate lines of defence
A net to prevent migrating birds from landing at a poultry farm in Thailand. © FAO
A net to prevent migrating birds from landing at a poultry farm in Thailand.
The first defence is to try and eradicate the epidemic among birds. This implies two priorities. “First of all, there is a need to strengthen scientific systems for virological monitoring and immediate reporting to all the responsible regional, national and international health officials,” stresses Isabel Minguez Tudela of the DG Research’s Safety of Food Production Systems Unit. “In this field, under the Fifth and Sixth Framework Programmes the EU is supporting research on the collection and processing of epidemiological data among birds and pigs, as well as on the development of effective, reliable and rapid diagnostic tests.”

Subsequently, as soon as an outbreak of avian flu is reported, the animals concerned must be destroyed systematically, the pens disinfected and the farms quarantined in the regions affected. In this respect, the European Commission strengthened the warning and response procedures considerably during 2005. 

Also, in addition to vaccination research (see below), a number of projects are in progress or being negotiated under the Fifth and Sixth Framework Programmes to combat the disease in animals. These include research on pathogenesis, epizootic diagnosis and monitoring (Aviflu project), as well as the development of in situ diagnostic technologies (Lab on site), the collection of epizootic data (Healthy poultry) and the computerised management of the bird flu crisis (Fluaid). Two networks have also been set up: Epizone on the epizootic monitoring of influenza in pigs, and Virgil on combating resistance to ant-viral agents.

Traditional vaccines
Flu vaccines were first developed several decades ago. The technique used involves bringing together, in a fertilised chicken egg, the flu virus against which the vaccine is being developed and a viral strain developed in the laboratory that is harmless but multiplies very quickly. The two viruses exchange their genes and form a recombinant virus that has the multiplication capacity of the neutral strain plus the H and N genes that determine the pathogenicity of the virus responsible for the disease in man and animals. 

This new recombined strain is isolated and then sent to the vaccine manufacturers which multiply it in millions of fertilised poultry eggs. The viruses produced are then chemically inactivated and purified before entering the vaccine composition. This is a safe and inexpensive method, but it requires several months to produce large quantities of doses. This is why, in the spring of each year, the WHO defines the viral strains that are likely to be involved in the following winter’s flu epidemic, so as to have the human vaccine available by the autumn. 

“This vaccination weapon is already developed and available in helping to combat the spread of the disease in birds,” stresses Isabel Minguez Tudela. “However, in the short term we can only vaccinate a part of the contaminated birds at the many contamination sites in Asia. On the other hand, as the human virus that is likely to appear due to the Asian bird flu epidemic is not yet known, it is clearly impossible at that stage to produce the vaccine with which to combat it.” 

Inverse genetics
The development of new and more efficient ways of producing flu vaccines as an alternative to this traditional technique is now a research priority. One of the most promising avenues being explored is that of ‘inverse genetics’ in which the initial spontaneous recombination is replaced by the direct ‘manufacture’ of the desired virus. For this, the researchers extract RNA (the genetic material) from the pathogenic virus strain and convert its H and N genes into DNA, a more stable molecule than RNA which, consequently, is easier to work with.

Inverse genetics, cell culture, and the use of harmless viruses as vectors for haemagglutines: since the late 1990s, these technologies have been at the basis of research projects financed by the European Commission under the Fifth and Sixth Framework Programmes. First there is the Flupan project, which is working on the H7N1 strain with a view to developing vaccines for humans. Clinical trials are already being carried out on one vaccine. There is also the Novaflu project that is concentrating on developing vaccination technologies and is particularly interested in recombinant viruses. Meanwhile, the SARS/Flu project is using an inactivated human-type flu virus as a vector for antigens derived from the SARS virus so as to create a vaccine against the two diseases. Finally, Fluvacc is working on a nasal vaccine against flu.

New projects are set to be launched in 2006 following the targeted call for proposals for research projects on avian flu, issued by the Commission in December 2005. 

Attacking the new antiviral agents
Confirmation of the presence of the avian flu virus in the majority of domestic fowl in a given zone, Laboratory of the Centre of Investigation in the region of Yogyakarta, in Indonesia. © FAO
Confirmation of the presence of the avian flu virus in the majority of domestic fowl in a given zone, Laboratory of the Centre of Investigation in the region of Yogyakarta, in Indonesia.
While, since the time of Louis Pasteur and throughout the 20th century, vaccination has been the major weapon in combating pathogenic viruses, today there is a parallel interest in developing antiviral medicines that attack the pathogenic agents directly – a method traditionally little used in the case of influenza. It is the fight against Aids and, more recently, against the SARS corona virus that has highlighted this therapeutic approach. Antiviral agents can be an essential weapon when, for technical or socio-economic reasons, vaccination is not possible. Also, in the case of the critical emergence of a new viral pathogen, the situation demands a more immediate response.

At the forefront of medicine technologies, the Integrated Project Viral Enzymes Involved in Replication (Vizier) targets the mechanism by which the RNA viruses – including human and bird flu viruses – are replicated so as to discover the three-dimensional structure and thereby permit specific ‘designs’ that will prevent replication of the viral agents.

Nevertheless, one of the problems encountered by the antiviral approach is linked to the ability of flu viruses to mutate, leading to the emergence of resistance. The Vigilance Against Viral Resistance (Virgil) Network of Excellence is the first monitoring system able to follow the emergence and development of the phenomena of resistance to antiviral medicines. It concentrates on three virus families (influenza and hepatitis B and C) by applying processes whose flexibility will subsequently make it possible to tackle resistance problems encountered by many other viruses that are pathogenic for man.

The network includes four platforms. Virgil-Models is developing in vitro and in vivo modelling platforms that supply new tools both to understand the defence mechanisms developed by the viruses and to evaluate viral resistance by conducting tests on clinical samples. Virgil-Host is studying the genetic and immunological factors peculiar to patients who develop resistance to treatment. Virgil-Drugpharm and Virgil-Innotech are dedicated to pharmaceutical developments and innovations. Finally, Virgil-Impact is assessing, at European level, the social aspects of the phenomenon of antiviral resistance, in terms of morbidity, control and treatment costs as well as cost-benefits at the medical and health level.

(1) The term ’epidemic’ applies to the appearance of a large number of cases of a human contagious and transmissible disease in a relatively limited area, while ‘pandemic’ implies a very wide geographical area. An ‘epizootic disease’ is a pandemic that applies to animal species. 

  Etymology of a name  
  Influenza. An Italian word originating in the 15th century and meaning ‘flow of liquid’ or ‘influence’, hence its use to express belief in the impact of the stars on the appearance of epidemics. The word later came to designate, in all languages, human and animal pulmonary infections resulting from epi- or pandemic viral attacks. The synonym grippe is more commonly used in German, Spanish and French. In English, it is commonly abbreviated to ‘flu’.


  Explaining Spanish flu  
The gifted Austrian artist Egon Schiele who fell victim to Spanish flu in 1918, at the age of 28 – Self Portrait, 1915.
The gifted Austrian artist Egon Schiele who fell victim to Spanish flu in 1918, at the age of 28 – Self Portrait, 1915.
It took almost a century to resolve the viral mystery of what was without a doubt the greatest mortal pandemic in human history – one that killed tens of millions of people worldwide at the end of the First World War. The virus responsible was recently reconstituted by researchers in the United States from fragments preserved in the tissue of American soldiers who died of the illness at the time. It is a very pathogenic avian influenza of the H1N1 strain that mutated to adapt to man and become transmissible between humans. This precedent is a clear illustration behind the reason for the current fears surrounding H5N1, if such a mutation were to occur again – which is not yet the case.

  Early warning defeats SARS  
  Severe Acute Respiratory Syndrome (SARS), a potentially fatal atypical pneumonia, first appeared in China at the end of 2002. Travel meant that it spread quickly, first in Asia (Hong Kong and elsewhere) and then as far afield as Canada. In March 2003, the WHO issued a global alert and charged a network of laboratories with the task of identifying the agent responsible. After a false alarm that indicated it was H5N1 that had contaminated two patients, one month later the culprit was found. It was an unknown corona virus, the reservoir species for which remains uncertain. The civet, a small carnivore sold in Chinese markets, was initially thought to be the culprit, but some now believe that the virus was spread by bats.

In July 2003, the epidemic ended as a result of quarantining the affected regions. Although SARS had time to kill about 800 of its 8 000 victims, the potential pandemic was nipped in the bud in just a few months by means of containment measures, without any treatment or vaccine ever being used.


  The spread of H5N1  
  Discovered in South Africa in 1961, the H5N1 virus first infected the tern, a small migrating seabird, without any apparent ill effects. In 1997, it hit the headlines following a dramatic wave of deaths among domestic chicken in Hong Kong. Benign in its natural environment, it had become highly pathogenic, at least for gallinaceans. All poultry in the area of the major Chinese metropolis were slaughtered rapidly, which eradicated the epizootic disease for a while. Nevertheless, 18 people were contaminated, six of whom died. 

H5N1 reappeared at the end of 2003 on Korean and then Vietnamese poultry farms. Becoming endemic in South-East Asia, it resulted in the deaths of millions of domestic fowl, either directly or due to a policy of preventive slaughter. Its pathogenicity was also observed to affect various wild species of fowl.

This highly pathogenic strain consequently spread along the major routes taken by migrating birds. Moving from South-East Asia to Central Asia, H5N1 reached the Caspian Sea in autumn 2005 and then the Black Sea and Danube Delta. Isolated sites of mortality appeared in Turkey, Western Russia, Romania and Croatia. According to the WHO, FAO and OIE, the virus is likely to arrive soon in the Middle East and East Africa, and risks spreading to West and North Africa before moving up into Western Europe next spring. Wild birds will suffer a heavy toll. The impact on domestic birds will depend on the contact between farm birds and wild birds. The risks are much higher in Africa than Europe, where farm birds are generally caged or penned.


  European Commission

World organisation for animal health (OIE) [ http://www.oie.int/downld/AVIAN%20INFLUENZA/f_AI-Asia.htm ]

World Health Organisation (WHO) [ http://www.who.int/csr/disease/avian_influenza/en/index.html ]


  DG Research