Avian influenza issues analysed
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March 2006
Dr RONALD R CUTLER CSci FIBMS, a principal lecturer in infectious diseases and pathology at the School of Health and Bioscience, University of East London, profiles the avian influenza pathogen and advises against complacency.
The name “influenza” originated in 15th Century Italy, where an epidemic was attributed to the “influence of the stars”, although the disease predates this time. The first pandemic fitting the clinical description of influenza was in 1580. At least four pandemics of influenza occurred in the 19th Century and three in the 20th Century.1,2
Today, humanity again worries about the potential of a new human flu pandemic. In 1918–19, more people died of influenza in a single year than in the four year reign (1347 to 1351) of the Black Death (bubonic plague). The Spanish flu or “La Grippe” was a global disaster.3 In the past 300 years, 10 pandemics have been identified, but Spanish flu was the most devastating with an estimated 20–40 million people killed.
Our most recent viral candidate, avian influenza A (H5N1) may have such deadly potential.4,5 A new influenza pandemic has been predicted for some years, as influenza viruses are uniquely capable of causing recurrent epidemics and global pandemics. Each year, the global death toll from influenza-related disease is over a million. So, why is this virus so deadly?
Influenza viruses have two features that enable them to be successful pathogens: an ability to circulate in animals and spread to man; and to undergo rapid and unpredictable antigenic change. Studies on influenza A viruses have revealed speciesspecific lineages for viral genes and genes that have crossed species barriers. Aquatic birds have been shown to be the source of all influenza A viruses, and avian influenza viruses have been transmitted to a wide range of other species including pigs, horses, cats and sea mammals (Fig. 1).
CLASSIFICATION AND STRUCTURE OF THE ORTHOMYXOVIRIDAE
Negative-strand RNA viruses are classified into seven families. Influenza belongs to the family Orthomyxoviridae (Greek: orthos, straight or correct; myxa, mucus), which has four genera: influenza virus A, B, and C, and Thogotovirus. Genera are classed on antigenic differences in their nucleoprotein (NP) and matrix (M1) proteins. All avian influenza viruses are classified as group A.
Subtyping is based primarily on the antigenicity of the two main surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA). Influenza C viruses lack NA but contain instead a haemagglutinin-esterase fusion protein (HEF).6,7 Orthomyxoviruses contain a segmented, linear negative-sense (complementary to messenger RNA [mRNA]), single-stranded RNA. There are eight RNA segments in influenza A and B, and seven in influenza C.
Influenza A viruses are classified into subtypes based on their HA and NA molecules. There are 15 recognised HA subtypes (H1–H15) and nine NA subtypes (N1–N9). The full nomenclature for each new isolate should include the type of virus, the host of origin (except for human), the geographical site of isolation, the type strain number (if available) and the year of isolation. The antigenic description of the HA and NA is given in parentheses. For example, a type strain isolated from a turkey in Toronto in 1966 may be called A/Turkey/Ontario/ 7732/66 (H5N9). The original animal source often relates to the viral type, all HA types however are found in birds, with humans the second most common host (Table 1).8,9
The physical structure of all influenza A viruses is similar. The virions or virus particles are enveloped and can be either spherical or filamentous in form. In clinical isolates that have undergone limited passages in eggs or tissue culture, there are more filamentous than spherical particles, whereas passaged laboratory strains consist mainly of spherical virions (Fig. 2).
Covering each influenza A virion are surface projections of about 500 spikes.10 The HA spike is rod-shaped and protrudes from the envelope, allowing the virus to attach specifically to host-cell surfaces, and is responsible for the viruses' haemagglutinin activity. The NA spike also protrudes from the envelope and is topped by a mushroom-shaped tetramer. This is a sialidase that promotes the release of the virus particles after budding.11
CLINICAL MANIFESTATIONS AND PATHOGENESIS
Clinical manifestations
In man, symptoms range from mild respiratory disease with rhinitis or pharyngitis to viral or secondary bacterial pneumonia leading to death. During an epidemic, however, rates of asymptomatic infection in patients can be as great as those with symptomatic infection and this will help the spread of the disease.
Early symptoms in adults are a high fever (38–40°C, usually peaking in 24 hours), chills, headache, sore throat and a dry cough. Pyrexia can last for up to five days, while the cough and malaise may persist for weeks. In an examination of 27 H5N1 infections in patients from Vietnam, Thailand and Hong Kong, all patients had fevers >38°C, 23 had a cough and 21 had a sore throat. In all cases, lower respiratory tract manifestations can develop early in the course of the illness, and progression to respiratory failure is associated with acute respiratory distress syndrome (ARDS).12
In the past, H5N1 was considered to be non-pathogenic for humans. However, in 1997, 18 humans were infected in Hong Kong and six of them died. The H5N1 virus was linked to this outbreak through a live bird market and area farms. In 2003, in The Netherlands, there were 83 confirmed cases in humans but this time only one death. Another subtype, H7N7, in South- East Asia (Indonesia, Vietnam, Thailand and Cambodia) in 2004–05 produced 118 cases with 61 deaths.12
In birds, especially free flying aquatic birds, there is a worldwide distribution and they act as a reservoir. Clinical symptoms in birds develop in three to 14 days, there is a significant reduction in egg production and there are neurological signs with depression, anorexia and ruffled feathers. The combs may be swollen and cyanotic and there may be incidences of sudden death. Influenza is clinically indistinguishable from virulent Newcastle disease and further tests are necessary for definitive diagnosis.5,6,13,14
PATHOGENESIS
Virus particles are inoculated as an aerosol into the respiratory tract where they replicate, causing desquamation of ciliated and mucus-secreting cells. The viral neuraminidase may facilitate access to the cell surface by liquifying the mucus cover. Adherence to the cilia may also be a mechanism whereby influenza virus resists removal by mucociliary action. Virulence factors of avian influenza A (H5N1) include a highly cleavable haemagglutinin that is activated by host cellular proteases and a substitution in NS1 (Table 2) that gives increased resistance to interferon and enhanced replication.
Avian and human HAs have different abilities to bind to other forms of sialic acid.15 Avian HAs generally bind poorly to the sialic acid receptors in the human respiratory tract. These receptor affinities act as a barrier to cross-species infection. An avian virus must adapt its HA binding before it can replicate and spread efficiently in humans.
After binding to sialic acid-containing receptors the virus is endocytosed and fuses with the vesicle membrane. Transcription and replication of the genome occurs in the nucleus. Viral proteins are synthesised and helical nucleocapsids form and associate with M1/M2 and the protein-lined membranes containing HA and NA glycoproteins. The virus buds from the plasma membrane and moves on to infect other cells (Fig. 3).12
In birds, two types of strains have been identified. Low pathogenic avian influenza (LPAI), which includes subtypes H1–H15, and highly pathogenic avian influenza (HPAI), which involves some H5 or H7 subtypes. In infections with HPAI strains, morbidity and mortality can approach 100% in commercial poultry flocks. Death normally occurs two to 12 days after the first signs of illness. The infection is spread by aerosol, shared drinking water, faeces or fomites. Virus is found in respiratory secretions and in very high concentrations (107–109 particles/g) in bird faeces.6,13,14,16–18
As in man, the pathogenicity of H5 and H7 influenza A viruses for poultry is determined by the amino acids at the HA cleavage site. Cleavability influences tissue specificity and is a major determinant of pathogenicity for H5 and H7 viruses.
EMERGENCE OF NEW VIRUSES
In addition to the virulence factors described above, influenza A viruses continually evolve, changing antigenicity often with increased environmental stability. There are two main ways that mutations occur and these are antigenic drift and antigenic shift.12,18–25
Influenza A is designed for continuous evolution. It has highly variable antigenic domains, situated at the outer end of the spike glycoproteins. This permits variability without affecting the function or the assembly of the virion.11
In antigenic drift, small point mutations occur during the normal virus replication process. This type of mutation does not usually result in major changes in virulence but produces genetic variation among influenza viruses. The lack of proofreading among RNA polymerases contributes to replication errors of the order of 1 in 104 bases, which contrasts with the higher replication fidelity of DNA polymerases (ie errors of 1 in 109 bases per replication cycle). RNA virus replication therefore results in a mixed population, with many variants.18 Although most of these are not viable, some will be potentially advantageous and become dominant under the right selective pressures.
In contrast, antigenic shift results in major changes in genetic material. This occurs when cells are infected with two different strains of influenza virus at the same time. This co-infection may involve viruses from different species (human, avian, swine or equine). Movement of genes from one virus to another is possible, due to the segmented RNA. The presence of eight separate segments is an advantage, as it allows a “mix and match” process to occur. This antigenic shift then creates a new hybrid virus, which is potentially pathogenic to a different species (Table 3).
HISTORY OF HUMAN AND AVIAN INFLUENZA
Naturally, due to its importance to man, the history of human influenza can be traced further back in time than can avian influenza. However, identifying the epicentre of influenza pandemics and links to animal and avian sources of the disease is very important for future planning to combat influenza pandemics.
As the first human influenza virus was not isolated until 1932, evidence prior to that of influenza epidemics and pandemics has been provisionally identified from observational data. Influenza outbreaks occur without warning, with sudden onset of fever, muscle pains and prostration. Hippocrates (412 BC) is often quoted as reporting the first influenza outbreak but it is unsubstantiated. Prior to 1700, the data are difficult to interpret, although the first influenza pandemic agreed by several authors was in 1580. This spread from Asia and Africa and infected the whole of Europe over a six-month period.2,8,17
Since 1700, pandemics have been identified as occurring in regular cycles of around 30–60 years (Fig. 4), although the deaths in the 1918–19 pandemic were far greater in number than those previously recorded. There has been some controversy as to the original source of the outbreak. Was it in Spain (hence the name Spanish flu) or in the USA, where an outbreak was documented in an army camp in 1918?
The most recent hypothesis points to an outbreak in 1916 in a huge military camp in Northern France. Here, the mixture of overcrowding, stress, gas attacks and open markets with live pigs, ducks, geese and horses produced conditions in which the transfer of avian influenza A virus could occur. This outbreak showed the same pathology that characterised patients in the 1918 outbreak.26,27 The trigger for the 1918 pandemic may have been the return of millions of soldiers to their homelands around the world. This would also fit with the emergence of the first outbreak in the USA, occurring later in an American army camp.2
Several factors, such as age and nutritional status, can affect susceptibility to influenza infections. The effect of external agents on the lungs is also important, and recent evidence has shown exposure to air pollutants, such as diesel exhaust, can effect respiratory virus infections in rodent models. This would also fit with the hypothesis suggested above relating to the emergence of the 1918 pandemic.1,2,26–29
There is historical evidence that can link influenza carriage in animals to occurrence in man. Fowl plague, the disease we now recognise as avian flu, was first described in Italy in 1878. The causative agent was isolated from a chicken in 1902, but was not finally identified as a member of an influenza A group, A/Chicken/Bresica/1902 (H7N7), until 1955.
Two types of avian strains have been identified: highly virulent types (H5 and H7) and avirulent types. Non-pathogenic strains have been isolated from a wide variety of birds, including wild, captive and caged birds and domestic ducks, chickens and turkeys. The highly pathogenic strains (H5 and H7) were both identified originally in chickens.6,13,30,31
The H5N1 subtype currently causing such problems was first identified over 40 years ago and was classified as A/Chicken/Scotland/ 59 (H5N1). It was identified again in 1963 in England, although without genetic subtyping it is not possible to identify how close these two strains actually were.6,13,32
Influenza A and its subtypes have been found in over 90 species of wild birds, all of which were apparently healthy. Wild ducks are the most frequent carriers of a large variety of influenza viruses. Wild waterfowl are a natural reservoir of influenza A viruses and can carry them over large distances and excrete huge numbers of virions in faeces. Viruses causing highly lethal disease are restricted to H5 and H7 subtypes (Table 4).
Highly pathogenic strains have no natural reservoir but may emerge by mutation when a wild bird introduces the virus into poultry, where it mutates to a lethal version of the original strain. Prior to 2004, outbreaks of highly pathogenic strains were relatively rare, but 10 of the 24 outbreaks in the past 46 years have occurred since 1997, six of which have involved H7 strains and four have involved H5 strains, although the H5N1 outbreak of 2004 was by far the largest outbreak.2,6,22,24,25,33–35
DIAGNOSTICS
Rapid diagnostic tests are used increasingly as they yield results in a clinically relevant time frame (approximately 30 minutes). However, the reference standard for diagnosis of influenza remains virus culture.
Most of the rapid influenza tests are >70% sensitive and >90% specific compared with virus culture. Most tests with positive results correctly identify infection, but up to 30% of negative test results may be false negatives. The predictive values of influenza tests depend on the level of influenza activity in the community; exposure of the patient to a contagious person; susceptibility of the patient; sensitivity and specificity of the tests; and adequacy of specimen collection.
Inappropriate and inadequate specimens will yield false-negative results. Tests are most reliable when there is known influenza activity in the community and when they are performed on patients who have signs and symptoms consistent with influenza (e.g. fever, cough, sore throat, muscle aches, headache and malaise). However, not all patients manifest typical signs and symptoms. Nasopharyngeal and nasal specimens (swab, aspirate, wash) are preferred over other samples for diagnostic testing, as they contain higher quantities of detectable virus. Specimens should be collected within the first three to four days of illness.
Assays available for the diagnosis of influenza A virus infections include virus culture, rapid antigen detection methods, and polymerase chain reaction (PCR) and real-time PCR assays.36
Virus culture
Virus culture can produce results in two to 10 days. Both shell-vial and standard cell culture methods are used to detectinfluenza viruses. Positive cultures may or may not exhibit cytopathic effects in cell culture systems. It is necessary to further identify the virus by immunofluorescence of cell cultures or haemagglutination-inhibition (HI) assay of the cell culture medium supernatant.
Rapid antigen detection methods
Rapid antigen detection methods include point-of-care (POC) tests, immunofluorescence assays and enzyme immunoassays, with results obtainable in 15–30 minutes. Point-of-care tests are commercially available.37–39
Immunofluorescence assays are a sensitive method for diagnosis of influenza A and B virus infections and five other clinically important respiratory viruses, and are used widely.40–43 Enzyme immunoassays mainly target influenza A nucleoprotein.36
Polymerase chain reaction and real-time PCR assays
Primer sets specific for the haemagglutinin (HA) gene of currently circulating influenza A/H1, A/H3 and B viruses are used widely. Results are available in a few hours, either from clinical swabs or infected cell cultures.13,36,44–46
Specimens that give a positive result using any the above approaches for influenza A virus and are suspected as avian influenza infection must be tested further and verified by a designated WHO H5 reference laboratory.
TREATMENT AND CONTROL
At present there is no available human vaccine for avian flu and production of a new vaccine may not begin until the disease shows significant human-tohuman transmission. The problems in developing human influenza vaccines relate to antigenic changes that take place in influenza viruses, which require new strains to be used to develop new vaccines each year. Also, there are other drawbacks to vaccination: it is expensive and provides no cross protection between all 15 H subtypes. Currently, in the USA, an inactivated H5 recombinant vaccine is being licensed for emergency use in HPAI outbreaks.
Influenza vaccine development is not reliable in helping to quell viral pandemics. Annual worldwide vaccine usage is over 300 million doses, but this requires more than six months and 350 million chicken eggs to meet demand. These constraints and other factors, such as the microbial contamination of vaccines, means that influenza vaccine technology is unable to respond quickly to pandemic threats.
Detection of a new strain in the human population may render subsequent vaccine development too slow a process on which to rely for protection. Current vaccines (killed virus) contain H1 and H3 components but they lack other HA components.
Influenza vaccine development
At present, two types of influenza vaccine are available and these could be used as a basis for the development of new vaccines. A trivalent inactivated influenza vaccine (TIV), administered intramuscularly, contains three inactivated viruses: type A (H1N1 and H3N2) and type B. There is also a live attenuated influenza vaccine (LAIV), which is approved for use in the USA. The latter is administered as a nasal spray and contains the same three viruses as does the TIV variant. The live attenuated influenza viruses in LAIV are temperature-sensitive, so they do not replicate effectively at core body temperature (38–39°C). The viruses are also cold-adapted and replicate effectively in the mucosa of the nasopharynx.38,47
Antiviral agents
Only four drugs are available for the treatment or prophylaxis of influenza. The adamantanes (amantadine and rimantadine) interfere with viral uncoating inside the cell. They are effective only against influenza A and are also associated with toxic effects and the rapid emergence of drug-resistant variants.
The neuraminidase inhibitors, (zanamivir [Relenza] and oseltamivir [Tamiflu]) interfere with the release of progeny influenza virus from infected host cells. This prevents infection of new host cells and halts the spread of infection in the respiratory tract. As replication of influenza virus in the respiratory tract reaches a peak 24–72 hours after the onset of illness, drugs such as the neuraminidase inhibitors, which act at the stage of viral replication, must be administered as early as possible. In contrast to the adamantanes, the neuraminidase inhibitors show little toxicity and are less able to promote the development of drugresistant strains.48
Neuraminidase inhibitors reduce the length of illness but this effect relies on early administration after the onset of symptoms. A one- to two-day reduction of symptoms has been demonstrated with zanamivir or oseltamivir, if given 36–48 hours after onset, or a three- to four-day reduction with oseltamivir, if given six to 12 hours after onset. However, in a controlled study on high-risk elderly populations, it was found that the prophylactic use of oseltamivir led to a 92% reduction in the incidence of laboratoryconfirmed influenza.49
Animal controls
There is clear evidence of transfer of avian influenza from wild birds to poultry flocks and of transfer from infected poultry to man. In the absence of fully effective antivirals and vaccination programmes, biosecurity is currently of prime importance in the control of avian flu in animals (and man). Controls include: • Controlling human traffic and transfer of fomites. • Reducing the introduction of new birds into flock. • Avoiding open-range rearing in waterfowl-prevalent areas. • Educating the poultry industry. • Making sure there is a prompt response to any AI outbreak.
In infected farms it is important to eliminate insects and mice, depopulate flocks and destroy carcasses, remove manure down to bare concrete, highpressure spray to clean equipment and surfaces, and spray with residual disinfectant.
There should also be a ban on the import of live chickens and other poultry products from countries affected with “bird flu”. Major issues identified from the current outbreaks are that persons working in close contact with infected poultry are at risk, as are those who eat raw, infected produce. These risks can be reduced by taking the following precautions: • Wear masks and gloves when handling poultry. • Clean kitchen surfaces and utensils before and after use. • Cook chicken until boiling temperature is reached. • Control human traffic into poultry houses.
FUTURE CONSIDERATIONS
It is necessary to identify how influenza viruses pass from animals to humans and how often this occurs. Does low-level exposure occur all the time in people who work with infected animals?
Clearly, preparations for a potential influenza pandemic must be undertaken and should include: • Continued surveillance efforts, both in humans (particularly in South East Asia) and animal (avian) species, must be stepped up and reinforced. • Continued research into vaccine design, development and rapid scale-up production methods is essential. • Other efforts could include the stockpiling of antiviral drugs, and a greater commitment by governments to an early-warning system. • Development of new antiviral drugs as prophylactic agents.
Global cooperation, with expanded surveillance and an agreed research plan, is required in order to manage the risk of avian flu. Countries must be willing to share knowledge and expertise.
When dealing with poultry, there should be modified production and distribution methods, with a stricter enforcement system. This must apply also to the transport of wild birds and the quarantine procedures.
In humans, there is a need for better investigation of cluster outbreaks and contact tracking. There should also be more targeted use of antivirals and continued research into vaccines and new antivirals.
In addition, WHO research guidelines50 emphasises the following specific research needs: • To understand the potential of H5N1 to re-assort by undertaking studies to mimic re-assortment under controlled conditions. • To clarify the role of animal influenzas in the emergence of pandemics by gathering further data on the prevalence of H5N1 in animals. • To improve knowledge on human disease, looking at incubation periods, virus excretion, factors affecting disease outcomes and the effectiveness of different treatments. • To improve the efficiency of the antigen content needed to produce vaccines, and to maximise the use of limited amounts of available antigen to enhance manufacture.
Avian flu (H5N1) in its current form may be “strictly for the birds”, but we cannot afford to be complacent when dealing with such an adaptable pathogen.
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