FLU-LAB-NET - About Avian Influenza

Influenza viruses have segmented, negative sense, single strand RNA genomes and are placed in the family Orthomyxoviridae. At present the Orthomyxoviridae family consists of five genera: Influenzavirus A, Influenzavirus B, Influenzavirus C, Thogotovirus and Isavirus. Only viruses of the Influenzavirus A genus are known to infect birds and those isolated from birds are termed avian influenza (AI) viruses (AIVs).

All influenza A viruses have antigenically related nucleoprotein and matrix proteins, but they are further divided into subtypes based on antigenic relationships of the surface glycoproteins, haemagglutinin (H) and neuraminidase (N). To date 16 haemagglutinin subtypes have been recognised (H1-H16) and nine neuraminidase subtypes (N1-N9). Each virus has one H and one N antigen, apparently in any combination. All influenza A subtypes in the majority of possible combinations have been isolated from avian species.

There are strict rules for naming influenza A isolates. The name should show: (i) antigenic type; (ii) host of origin; (iii) geographical location; (iv) strain reference number; (v) year of isolation; and (vi) the H and N subtypes, e.g. A/turkey/England/199/79 (H7N7). Influenza A viruses infecting susceptible poultry can be divided into two groups on the basis of the severity of the disease they cause. The very virulent viruses cause highly pathogenic avian influenza [HPAI], often with rapid flock mortality reaching 100%, when death isn’t rapid the clinical signs shown by may be: cessation of egg laying, respiratory signs, rales, excessive lacrimation, sinusitis, oedema of the head and face, subcutaneous haemorrhage with cyanosis of the skin, particularly of the head and wattles, and diarrhoea, sometimes neurological signs may be present, none maybe regarded as pathognomonic. All other viruses cause a milder disease termed low pathogenic avian influenza [LPAI], these infections may be extremely mild in chickens and turkeys with only slight respiratory disease or egg production problems. However, sometimes exacerbation of infection of LPAI viruses in poultry, by other respiratory viruses or environmental conditions results in a severe disease with high mortality, especially in turkeys. Apart from some nephropathogenic H10 subtype viruses that may cause high mortality when inoculated intravenously, but not by other routes, to date HPAI viruses have been restricted to subtypes H5 and H7, although not all H5 and H7 viruses cause HPAI. Initially, HPAI viruses appear to arise by mutation in the gene coding for the haemagglutinin of LPAI viruses of H5 or H7 subtype after they have been introduced into poultry flocks. HPAI viruses are not necessarily virulent for all species of birds and the clinical severity seen in any host appears to vary with both bird species and virus strain.

Emergence of HPAI viruses

The haemagglutinin (HA) glycoprotein of influenza A viruses has two important functions that are required for the virus to be infectious. The HA first brings about attachment of a virus particle to binding to sialic acid receptors on the surface of a host cell and, after being taken in to an endosome, then brings about fusion between the host cell membrane and the virus membrane so that the viral genetic material is introduced into the cell. However, the HA glycoprotein is produced as a precursor, HA0, which requires post translational cleavage by host proteases before it is able to induce membrane fusion and virus particles become infectious.

Various studies have demonstrated that the amino acid motif at the cleavage site of the HA0 precursor protein is extremely important in facilitating its cleavage by different proteases and it is of primary importance in the virulence of the virus. The HA0 precursor proteins of avian influenza viruses of low virulence for poultry have a single arginine at the cleavage site and another basic amino acid [arginine or lysine] at position -3 or -4. These viruses are limited to cleavage by extracellular host proteases such as trypsin-like enzymes and thus restricted to replication at sites in the host where such enzymes are found, i.e. the respiratory and intestinal tracts.

In contrast HPAI viruses possess multiple basic amino acids at their HA0 cleavage sites either as a result of apparent insertion or apparent substitution and appear to be cleavable by a ubiquitous protease[s], probably one or more proprotein-processing subtilisin-related endoproteases of which furin is the leading candidate. These viruses are able to replicate throughout the bird, damaging vital organs and tissues which results in disease and death. It appears that HPAI viruses arise by mutation after LPAI viruses have been introduced into poultry. Several mechanisms may be responsible for the mutation resulting in the emergence of HPAI viruses.

Definitions for trade and control

In the Terrestrial Animal Health Code 2005 the World Organisation for Animal Health (OIE) revised the definition of notifiable avian influenza. Previously, OIE had required notification only for outbreaks of avian influenza (AI) in which the AI virus fulfilled a molecular or in vivo virulence criterion i.e. highly pathogenic AI (HPAI) viruses. In the new definition notifiable avian influenza (NAI) was extended to include H5 and H7 subtype viruses of low virulence (LPNAI) in addition to the highly virulent viruses (HPNAI). Similarly in the new European Union Directive control measures were extended to H5 and H7 subtype AI viruses that fulfilled neither molecular nor in vivo virulence criteria, although these are not as severe as control measures for HPAI viruses. The definitions given in the OIE 2005 Terrestrial Animal Health Code are:

“For the purposes of this Terrestrial Code, avian influenza in its notifiable form (NAI) is defined as an infection of poultry caused by any influenza A virus of the H5 or H7 subtypes or by any AI virus with an intravenous pathogenicity index (IVPI) greater than 1.2 (or as an alternative at least 75% mortality) as described below. NAI viruses can be divided into highly pathogenic notifiable avian influenza (HPNAI) and low pathogenicity notifiable avian influenza (LPNAI):

a) HPNAI viruses have an IVPI in 6-week-old chickens greater than 1.2 or, as an alternative, cause at least 75% mortality in 4-to 8-week-old chickens infected intravenously. H5 and H7 viruses which do not have an IVPI of greater than 1.2 or cause less than 75% mortality in an intravenous lethality test should be sequenced to determine whether multiple basic amino acids are present at the cleavage site of the precursor haemagglutinin molecule (HA0); if the amino acid motif is similar to that observed for other HPNAI isolates, the isolate being tested should be considered as HPNAI.

b) LPNAI are all influenza A viruses of H5 and H7 subtype that are not HPNAI viruses.”

The term low pathogenicity avian influenza (LPAI) is then used to define all infections caused by AI viruses that are not NAI viruses.

Epidemiology

Surveillance studies undertaken in the 1970s revealed that vast pools of influenza A viruses of all the various H and N subtype combinations exist in the feral bird population. Surveys of wild birds have resulted in the isolation of viruses from species representing all the major avian families throughout the world. However, the frequency and number of isolations from other species has been overshadowed by the presence of these viruses in waterfowl, especially ducks.

Analysis of the various published reports of surveillance studies indicates an overall isolation rate of about 11% from sampled birds. However, that isolation rate fell to about 2% if ducks and geese were excluded, and within the Order Anseriformes the large majority of viruses were isolated from birds of the genus Anas, particularly mallard ducks (Anas platyrhynchos). Isolation rates for ducks congregating on lakes prior to migration have been very high, usually in the range of 20%, although somewhat lower further along migratory routes. Nevertheless, this indicates the potential for the spread of influenza viruses throughout the world by migratory birds. Despite the predominance of AI viruses in waterfowl, studies suggest that waterfowl do not act as a reservoir for all avian influenza viruses. It seems likely that part of the influenza gene pool is maintained in shorebirds and gulls, from which the predominant number of isolated influenza viruses are of different subtypes to those isolated from ducks.

Until recently, HPAI viruses had been isolated rarely from free-living birds and when they had been isolated it was usually in the vicinity of outbreaks of HPAI in poultry or geographically and chronologically close to known outbreaks in poultry. However, the emergence of the Asian-lineage H5N1 HPAI virus in Southern China in 1996 and its establishment as an endemic disease in at least some sectors of the poultry industries in several SE Asian countries, where it continues to be perpetuated despite attempts at control, is entirely new in the epidemiology of AI viruses. The virus appeared to affect all sectors of the poultry populations in most of these countries, but its presence in free range commercial ducks, village poultry, live bird markets and fighting cocks seemed especially significant in the spread of the virus. Inevitably this HPAI virus spread to wild birds, probably on many occasions. While there is no evidence that the virus has become endemic in any species of wild bird, the spread of Asian-lineage H5N1 HPAI virus westwards from Eastern Asia and especially into Europe appears, in part at least, due to the movement of infected wild birds.

History

The history of avian influenza really begins with the recognition of HPAI then termed “fowl plague” (until 1981) as a distinct disease by Perroncito in Italy in 1878, although clearly outbreaks probably preceded that date. The causative organism of this disease was shown to be a virus as early as 1901 but it was not until 1955 that the relationship to mammalian influenza A viruses (first isolated in the 1930s) was demonstrated.

From the literature it would appear that HPAI was endemic in Italy for at least 50 years up to the last outbreaks of HPAI recorded in the mid 1930s. The virus was reported in Germany in 1890 and probably remained endemic into the 1930s. Writing in 1930, Todd & Rice considered HPAI to have occurred in Austria, Switzerland, France, Belgium, The Netherlands, England, Egypt, China, Japan, USA, Argentina and Brazil. In England outbreaks occurred in 1922 and 1929, but there did not appear to be much spread.

HPAI occurred in the USA in 1924-1925 and spread to nine eastern states. Spread appeared to be primarily as a result of the movement of birds and was especially associated with live bird markets. There were further outbreaks in 1929 and it was not clear whether the virus had remained since 1925 or there had been a new introduction. Very few reports describing HPAI outbreaks appeared in the literature from the mid-1930s to 1959, but authors often stated that outbreaks were not uncommon in Africa, Asia and Eastern Europe.

Since 1959, when the first reported HPAI outbreak caused by a virus of H5 subtype occurred, there have been 24 reports of the emergence of new HPAI viruses in poultry; 8 have been due to H5 viruses and 16 to H7 viruses. In about half of the 24 there has been little or no spread from the farm where the virus was first identified. Significant spread to numerous sites resulting in huge economic losses was recorded in Pennsylvania in 1983, Mexico in 1994, Pakistan in 1994, Italy in 1999/2000, The Netherlands 2003, Canada 2004 and of course unprecedented outbreaks and losses have been associated with the Asian H5N1 virus that since 1996 has spread throughout Asia and into Europe and Africa.

Human infections

Up to 1995 there had been only three recorded instances of AI viruses infecting humans, in 1959 virus was isolated from a patient in the USA suffering from hepatitis, and in 1977 in Australia and 1981 in the USA laboratory accidents had resulted in eye infections of laboratory staff presenting as conjunctivitis; interestingly all three of these were due to H7N7 subtype viruses.

However, since 1996 there have been regular reports of natural infections of humans with avian influenza viruses. The first natural infections of a human was detected in 1996 when a LPAI virus of H7N7 was isolated from a woman in England with conjunctivitis. Evidence of single human infections with LPAI H7N2 virus was obtained in the USA in 2002 and 2003. During the severe epizootic in poultry in The Netherlands in 2003 evidence of infection with the HPAI H7N7 virus responsible was obtained in 83 humans. These cases usually presented with conjunctivitis or mild flu-like illness, but there was one fatality. Two people were infected during the HPAI H7N3 outbreak in Canada in 2003 again with mild symptoms. However, while these infections may be important in view of the potential for the emergence of pandemic virus (see below) they have been overshadowed by the number and severity of human infections with the Asian HPAI H5N1 virus over the past ten years.

Since 1997 [to 25.07.07] there had been 339 confirmed cases in humans with 199 (58.7%) deaths. For all these infections transmission between humans appears to have occurred only on very rare, exceptional occasions and in nearly all reported cases of human infections with AI viruses there has been close association with infected birds or infective carcases.

However, many consider there is a much greater concern than the sickness and deaths seen so far for the human infections with AI viruses, which is possibility that they may lead to the emergence of an influenza pandemic. Influenza A infections in humans have been marked throughout history by serious pandemics every 10-50 years. In the 20th Century the sudden emergence of antigenically different strains in humans, termed antigenic shift, resulted in pandemics occurring on 4 occasions, 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) and 1977 (H1N1). By far the worst influenza pandemic for which there are accurate records was the one beginning in 1918. It has been estimated that during the pandemic more than 40 million people died. Because the viral RNA of influenza viruses is segmented, genetic reassortment can occur in mixed infections with different strains of influenza A viruses. This means that when two viruses infect the same cell, progeny viruses may inherit sets of RNA segments made up of combinations of segments identical to those of either of the parent viruses. Both the H2N2 1957 and the H3N2 1968 pandemic viruses differed from the prevailing viruses in the human population as a result of reassortment with avian influenza viruses. These reassortments brought about antigenic shift resulting in viruses with both surface glycoprotein genes from the avian virus for the 1957 virus and the important HA gene from the avian virus for the 1968 virus, but retained internal protein genes that allowed these viruses to be readily transmissible in the human population.

The current concern is that human infections with the Asian H5N1 virus (or any other AI virus) could lead to reassortment, antigenic shift and the emergence of a pandemic virus if the person was also infected with an influenza virus currently circulating in the population. The potential of these human infections with AI virus to result in a pandemic virus emerging has been added to by the determination of the entire genome of the 1918 pandemic virus and the suggestion that this virus emerged by a complete avian virus adapting to humans without reassortment.

Author: Dennis Alexander. October 2007