Zoonoses and Public Health

REVIEW ARTICLE

Avian Influenza – Diagnosis D. J. Alexander Virology Department, Veterinary Laboratories Agency Weybridge, Addlestone, Surrey, UK

Impacts • To review the characterization of avian influenza viruses. • To provide diagnostic methodology. • To provide guidelines on molecular, virological and serological diagnosis. Keywords: Avian influenza; diagnosis Correspondence: D. J. Alexander. Virology Department, Veterinary Laboratories Agency Weybridge, Addlestone, Surrey, UK. Tel.: +44 1932 357466; Fax: +44 1932 357239; E-mail: [email protected] Received for publication September 5, 2007 doi: 10.1111/j.1863-2378.2007.01082.x

Summary The diagnosis of avian influenza (AI) virus infections, even highly pathogenic AI (HPAI), represents a considerable challenge due to the lack of pathognomonic or specific clinical signs and their variation in different avian hosts plus the marked antigenic variation amongst influenza A viruses. Conventional laboratory techniques involve the isolation, identification and characterization (including virulence estimates) of the virus. While this has proven successful in the past and remains the method of choice, for at least the initial outbreak, the delays associated with conventional diagnosis are often considered unacceptable for the application of control measures, especially stamping out policies, and there is an overwhelming demand for rapid results. More and more, molecular biological techniques are being used and in particular reverse transcriptasepolymerase chain reaction (RT-PCR) and real-time RT-PCR technologies are being employed for rapid diagnosis. In this paper, clinical signs, the molecular basis for virulence of AI viruses, international definitions, conventional diagnosis and the use of molecular techniques are reviewed and discussed.

Introduction Influenza viruses are segmented, negative strand RNA viruses that are placed in the family Orthomyxoviridae in three genera: Influenzavirus A, B and C. Only influenza A viruses have been reported to cause natural infections of birds. Type A influenza viruses are further divided into subtypes based on the antigenic relationships in the surface glycoproteins haemagglutinin (H) and neuraminidase (N). At present, 16 H subtypes have been recognized (H1–H16) and nine neuraminidase subtypes (N1–N9). Each virus has one H and one N antigen, apparently in any combination, all subtypes and the majority of possible combinations have been isolated from avian species. Although influenza viruses have been isolated from a large number of bird species (Stallknecht, 1998), the genetic pool for all AI viruses is primarily in aquatic birds, which are responsible for the perpetuation of these viruses in nature (Alexander, 2000). Phylogenetic studies (Rohm et al., 1995; Banks et al., 2000a,b) of AI viruses show that lineages and clades of isolates are more related 16

to geographical and temporal parameters than the host from which they were isolated and there is no distinction between wild and domestic bird isolates. Influenza A viruses infecting poultry can be divided into two distinct groups on the basis of the severity of the disease they cause. The severe form of avian influenza (AI) termed highly pathogenic (HPAI), at one time known as ‘fowl plague’, is one of the two most feared diseases of poultry and other birds. This is not only because flock mortality can reach 100%, but also the economic impact of trading restrictions and embargoes placed on infected areas. There have been 25 reported primary isolates of such viruses from domestic poultry since 1959. The scale of the ensuing epidemics has ranged from infections limited to a single house on a turkey farm seen with A/turkey/England/50-92/91 (H5N1) to the epidemic resulting from the progenitor virus A/goose/Guandong/1/ 96 (H5N1), which emerged in 1996 and since then its descendents have spread throughout Asia and into Europe and Africa becoming endemic in poultry in some countries.

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The very virulent viruses causing HPAI have been restricted to subtypes H5 and H7, although not all viruses of these subtypes cause HPAI. All other viruses cause a much milder disease consisting primarily of mild respiratory disease, depression and egg production problems in laying birds, these are termed low pathogenicity AI (LPAI) viruses. Sometimes other infections or environmental conditions may cause exacerbation of influenza infections leading to much more serious disease. For example, in outbreaks of LPAI in Italy in 1999, high mortality was often recorded in young turkeys, reaching 97% in one flock (Capua et al., 2000). The great variation in antigenicity and the lack of classical clinical signs, even for HPAI means that, while they may serve as a suspicion, full diagnosis of AI or HPAI cannot be based on the disease observed, nor demonstration of infection and laboratory diagnosis is essential. The need to distinguish between LPAI and HPAI infections means that an understanding of virulence and definitions of the different categories of disease are essential. Molecular Basis of Virulence An understanding of the molecular basis of the difference in virulence between HPAI and LPAI viruses has proved especially important in the diagnosis of AI and has allowed rapid diagnostic molecular-based techniques to be employed in detecting and predicting the virulence phenotype. The haemagglutinin glycoprotein for influenza viruses has two important functions that are imperative for the infectivity of the virus. First, it brings about attachment to host cell and then fusion between the host cell membrane and the virus membrane so that the viral genetic material is introduced into the host cell. This glycoprotein is produced as a precursor, HA0, which requires posttranslational cleavage by host proteases before it is able to induce membrane fusion and virus particles become infectious (Rott, 1992). The HA0 precursor proteins of LPAI viruses have a single arginine at the cleavage site and another at position -3 or -4. These viruses are limited to cleavage only by certain host proteases such as trypsinlike enzymes and are thus restricted to replication at sites in the host where such enzymes are found, i.e. the respiratory and intestinal tracts. The HA0 proteins of HPAI viruses possess multiple basic amino acids (arginine and lysine) at their HA0 cleavage sites either as a result of apparent insertion or apparent substitution (Vey et al., 1992; Wood et al., 1993; Senne et al., 1996) 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 (StienekeGrober et al., 1992). These viruses are able to replicate

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throughout the bird, damaging vital organs and tissues, which results in disease and death (Rott, 1992). For example, all H7 subtype LPAI viruses have the amino acid motif at the HA0 cleavage site of either -PEIPKGR*GLF- or -PENPKGR*GLF-, whereas examples of cleavage site amino acid motifs for HPAI H7 viruses are: -PEIPKKKKR*GLF-, PETPKRKRKR*GLF-, -PEIPKKR EKR*GLF-, -PETPKRRRR*GLF-, -PEIPKGSRVRR*GLF-. The Chile 2002 (Suarez et al., 2004) and the Canada 2004 (Pasick et al., 2005) H7N3 HPAI viruses show distinct and unusual cleavage site amino acid sequences of PEKPKTCSPLSRCRETR*GLF and PENPKQAYRKRMTR* GLF respectively. These viruses appear to have arisen as a result of a recombination event between the HA gene and nucleoprotein gene and matrix gene respectively resulting in an insertion at the HA0 cleavage site of 11 amino acids for the Chile virus and seven amino acids for the Canadian virus. Current theories suggest that AI subtype H5 and H7 viruses of high virulence emerge from viruses of low virulence by mutation (Garcia et al., 1996; Perdue et al., 1998) although there must be more than one mechanism by which this occurs. This is supported by phylogenetic studies of H7 subtype viruses, which indicate that HPAI viruses do not constitute a separate phylogenetic lineage or lineages, but appear to arise from non-pathogenic strains (Rohm et al., 1995; Banks et al., 2000a) and the in vitro selection of mutants virulent for chickens from an avirulent H7 virus (Li et al., 1990). It appears that such mutations occur only after the viruses have moved from their natural wild bird host to poultry. However, the mutation to virulence is unpredictable and may occur very soon after introduction to poultry, or after the LPAI virus has circulated for several months. Clinical Signs Low pathogenic avian influenza The severity of the disease produced by viruses inducing little or no disease in chickens infected experimentally and without multiple basic amino acids at the HA0 cleavage site (LPAI viruses) is greatly influenced by: the strain of virus, the species and age of host, the immune status of the host against the virus and particularly the presence of other infectious agents such as: Pasteurella spp, Newcastle disease viruses (including vaccine strains), avian pneumovirus, infectious bronchitis virus, Escherichia coli and Mycoplasma spp, immunodeficiency conditions and environmental factors (such as excess ammonia, dust, hot or cold temperatures). At one extreme, the disease seen may be inapparent or slight. For example, Alexander and Spackman (1981) reported that an LPAI infection in a turkey laying flock

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resulted in only transient mild respiratory signs and 2% white-shelled eggs. Other LPAI outbreaks occurring in turkeys at about the same time produced 20–40% egg production drops and respiratory disease with low but significant mortality. At the other extreme, infections with LPAI viruses may be associated with severe disease and with high mortality. In outbreaks in chickens in Alabama in 1975 with a LPAI virus of H4N8 subtype up to 69% mortality was recorded in infected flocks (Johnson et al., 1977). In 1995 major outbreaks caused by LPAI viruses of H7N3 subtype affected turkeys in Utah USA and was associated with significant mortality especially in young birds, with about 40% mortality in 0- to 4-week-old birds (Halvorson et al., 1998). In most cases, mortality was associated with dual infections with E. coli or Pasteurella multocida. During the LPAI H7N1 infections in Italy in 1999 turkeys were particularly affected. In turkeys reared for meat the severity of the clinical and post mortem disease varied considerably, clinical signs were dominated by respiratory distress with mortality ranging from 5% to 97% depending on the age of the affected birds (Capua et al., 2000). In young meat birds, the signs were usually sufficiently severe to result in 40– 97% mortality in infected flocks. In turkey breeders, a milder form of the same clinical condition was observed that consisted of rales, coughing, swelling of the infraorbital sinuses and a febrile condition associated with loss of appetite. Egg production dropped by 30–80% during the acute phase, but partially recovered to subnormal levels within 3 weeks from the onset of the disease. Mortality rates ranged from 5% to 20% (Capua et al., 2000). Equally serious problems have been reported in recent years associated with widespread outbreaks of viruses of H9N2 subtype particularly in Pakistan and Iran, but also in the Middle East and Asian countries through to China. Highly pathogenic avian influenza Often the first signs of HPAI in flocks of chickens or turkeys, especially birds not in cages, are the sudden onset of high mortality in the flock, which may approach 100% within a few days. Clinical signs that may be associated with high mortality are: 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, occasionally neurological signs may be present. Usually, these signs are most marked in birds that take some time to die. It should always be borne in mind that 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 (Alexander et al., 1978, 18

1986). In particular ducks, although readily infected in experiments with HPAI viruses rarely show clinical signs as a result of these infections. Definitions The lack of pathognomonic clinical signs, the considerable antigenic variation, both into 16 H subtypes and within those subtypes and the fact that, to date, two subtypes H5 and H7 have been shown to be responsible for HPAI but not all H5 and H7 subtype viruses cause HPAI offers a severe challenge for the accurate and rapid diagnosis of AI. In particular careful, specific definitions are required for statutory control and trade purposes and diagnostic methods that allow detection and differentiation of the different types of AI virus within the definitions must be employed. In the Terrestrial Animal Health Code 2005, the World Organisation for Animal Health (OIE, 2005) revised the definition of notifiable AI (NAI). Previously, OIE had required notification only for outbreaks of AI in which the AI virus fulfilled a molecular or in vivo virulence criterion, i.e. HPAI viruses. In the new definition, 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 (CEC, 2006a) 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.

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(b) LPNAI are all influenza A viruses of H5 and H7 subtype that are not HPNAI viruses. The term LPAI is then used to define all infections caused by AI viruses that are not NAI viruses. As a consequence diagnostic methods must allow detection of and distinction between infections of birds with HPNAI viruses, LPNAI viruses and LPAI viruses. Two further points that have important affects on diagnosis are clear from the definition[s]. The first is that sequencing of the HA0 cleavage site cannot confirm that a virus is not HPNAI/HPAI an in vivo test is required. The second that H5 and H7 viruses with HA0 cleavage site amino acid sequences similar to that observed in another HPNAI virus are HPNAI viruses regardless of their virulence for chickens. To date, there have been four AI viruses isolated that have low virulence for chickens in in vivo tests, but have multiple basic amino acids at the HA0 cleavage site and are therefore HPNAI/HPAI viruses (Londt et al., 2007). Laboratory diagnosis In the diagnostic manuals of the EU (CEC 2006a,b) and OIE (Alexander, 2005), the primary method of diagnosis recommended is to use conventional virological techniques, but, more and more, molecular biological techniques are being used and many laboratories now have the capacity to employ reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RRT-PCR) technologies for rapid diagnosis. The best application of these tests may be for rapidly identifying subsequent outbreaks once the primary infected premises has been detected and the virus characterized. In addition, extremely rapid antigen detection methods have been developed and in some cases are available as commercial kits. These methods will be mentioned briefly in this review, more detailed reviews have been published recently (Brown, 2006; Suarez et al., 2007). Conventional laboratory diagnosis The conventional techniques to be used for the diagnosis of AI are described in detail in the EU (CEC 2006a,b) and OIE (Alexander, 2005) diagnostic manuals. In summary, these consist of isolation and characterization of the infecting AI virus using the following techniques: Virus isolation Suspensions in antibiotic solution of tracheal and cloacal swabs (or faeces) taken from live birds, or of faeces and pooled samples of organs from dead birds, are inoculated into the allantoic cavity of 9- to 11-day-old embryonated

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fowls’ eggs. The eggs are incubated at 35–37C for 4–7 days. The allantoic fluid of any eggs containing dead or dying embryos as they arise, and all eggs at the end of the incubation period, are tested for the presence of haemagglutinating activity. Fluids that give a negative reaction should be passaged into at least one further batch of eggs. Virus identification If a haemagglutinating agent is isolated the presence of influenza A virus can be confirmed by an immunodiffusion test between concentrated virus and an antiserum to the nucleocapsid or matrix antigens, both of which are common to all influenza A viruses. Most laboratories will have antiserum specific for Newcastle disease virus (avian paramyxovirus type 1), and in view of its widespread occurrence and almost universal use as a live vaccine in poultry, it is best to evaluate its presence by haemagglutination inhibition (HI) tests. Subtype identification of influenza A viruses is usually done using polyclonal chicken antisera raised against a battery of intact influenza viruses. Neuraminidase identification is carried out using a similar approach. Subtype identification by these techniques is labour intensive and requires maintenance of large stocks of antisera and other reagents. It is, therefore usually beyond the scope of most diagnostic laboratories not specializing in influenza viruses. However, National and Regional Laboratories should be in a position to identify influenza viruses of H5 and H7 subtype, and assess the virulence of such viruses either by in vivo or in vitro tests in line with international definitions. Assessment of pathogenicity As discussed above the definition of AI for which statutory measures will be applied currently involves an assessment of the virulence of the AI virus for chickens. In the EU, the IVPI test is used for this assessment, which is carried out as follows. 1. Fresh infective allantoic fluid with a HA titre >1/16 (>24 or >4 log2 when expressed as the reciprocal) is diluted 1/10 in sterile isotonic saline. 2. 0.1 ml of the diluted virus is injected intravenously into each of ten 6-week-old SPF chickens. 3. Birds are examined at 24-h intervals for 10 days. At each observation, each bird is scored 0 if normal, 1 if sick, 2 if severely sick, 3 if dead. (The judgement of sick and severely sick birds is a subjective clinical assessment. Normally, ‘sick’ birds would show one of the following signs and ‘severely sick’ more than one of the following signs: respiratory involvement,

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depression, diarrhoea, cyanosis of the exposed skin or wattles, oedema of the face and/or head, nervous signs. Dead individuals must be scored as 3 at each of the remaining daily observations after death). 4. The IVPI is the mean score per bird per observation over the 10-day period. An index of 3.00 means that all birds died within 24 h, an index of 0.00 means that no bird showed any clinical sign during the 10-day observation period. In addition to the H5 and H7 HPAI viruses some nephropathogenic H10 subtype viruses may cause high mortality when inoculated intravenously (Swayne and Alexander, 1994) and give IVPI values >1.2. In the current definitions, viruses of H5 and H7 subtype also need to be sequenced at the HA0 cleavage site to demonstrate the presence of absence of multiple basic amino acids. A variety of strategies and techniques have been used successfully to sequence the nucleotides at that portion of the HA gene coding for the cleavage site region of the haemagglutinin of H5 and H7 subtypes of AI, enabling the amino acids there to be deduced. The most commonly used method has been RT-PCR, using oligonucleotide primers complementing areas of the gene either side of the cleavage site coding region, followed by cycle sequencing of the cDNA produced (Wood et al., 1993). Various stages in the procedure can be facilitated using commercially available kits and automatic sequencers. Developing techniques for AI diagnosis At present, the conventional isolation and virus characterization techniques for the diagnosis of AI remain the method of choice, for at least the initial diagnosis of AI infections. However, conventional methods tend to be costly, labour intensive and slow and for the application of control measures, especially stamping out policies, there is an overwhelming demand for rapid results. The past 10 years or so has seen enormous developments and improvements in molecular and other diagnostic techniques, many of these have been applied to the diagnosis of AI infections. Antigen detection A number of antigen capture immunoassay tests have been developed in recent years. Some have been developed for detecting all influenza A viruses in humans, but have been used for AI; with the spread of Asian lineage HPAI H5N1 some have been developed that detect viruses of H5 or H5 and H7 subtypes in birds (Ryan-Poirier et al., 1992; Davison et al., 1998; Cattoli et al., 2004). The commercially available Directigen Flu A kit (Becton 20

Dickinson Microbiology Systems, Franklin Lakes, NJ, USA) has been the most widely used for detecting the presence of influenza A viruses in poultry (Slemons and Brugh, 1998), particularly in the USA. The kit uses a monoclonal antibody against the nucleoprotein and should therefore be able to detect any influenza A virus. Although it was developed to detect virus in mammalian infections it has been successfully applied to detecting viruses in poultry and other birds, although there may be some variation in the sensitivity for different specimens. The main advantage of this test and many of the other antigen detection kits is that they can demonstrate the presence of AI within 15 min. The disadvantages are that subtype identification is not achieved, sensitivity may be low so that a flocks rather than individuals need to be sampled and the kits are expensive. Direct RNA detection Although, as demonstrated by the EU and OIE definitions of HPAI, molecular techniques have been used in the diagnosis of AI for some time, recently there have been developments in their application for detection and characterization of AI virus directly from clinical specimens from infected birds. Reverse transcriptase-polymerase chain reaction techniques on clinical specimens can, with the correctly defined primers, result in rapid detection and subtype (at least of H5 and H7) identification, plus a cDNA product that could be used for nucleotide sequencing (Suarez, 1998; Starick et al., 2000; Munch et al., 2001). Results obtained by Koch (2003) indicated that care should be taken in clinical specimens used as while tracheal samples from infected birds showed high sensitivity and specificity relative to virus isolation, RT-PCR tests on faecal samples lacked sensitivity. This technique was used with success during the 2003 HPAI outbreaks in the Netherlands. Modifications on the use of RT-PCR have been applied to reduce the time for both identification of virus subtype and sequencing. For example, Spackman et al. (2002) used a ‘real time’ single step RT-PCR primer/fluorogenic hydrolysis probe system to allow detection of AI viruses and determination of subtype H5 or H7. The authors concluded that the test performed well relative to virus isolation and offered a cheaper and much more rapid alternative. Modifications on the straightforward RT-PCR method of detection of viral RNA have been designed to reduce the effect of inhibitory substances in the sample taken, the possibility of contaminating nucleic acids and the time taken to produce a result. For example, nucleic acid sequence-based amplification (NASBA) with electrochemiluminescent detection (NASBA/ECL) is a continuous

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isothermal reaction in which specialized thermocycling equipment is not required. NASBA assays have been developed for the detection of AI virus subtypes H7 and H5 in clinical samples within 6 h (Collins et al., 2002; Ko et al., 2003). RT-PCR with product detection by enzymelinked immunosorbent assay (ELISA) has also been used for the detection of RNA coding for nucleoprotein (Dybkaer et al., 2004). Although ring trials conducted recently in the EU identified H5 and H7 conventional RT-PCR protocols that were sufficiently sensitive to amplify directly from extracted clinical specimens, at least from swabs obtained from HPAI infected poultry (Slomka et al., 2007a), RRT-PCR, usually based around the hydrolysis probe or ‘TaqMan’ method for generation of the target-specific fluorescence signal, has become the method of choice for at least partial diagnosis directly from clinical specimens in many laboratories. The method offers rapid results, with sensitivity and specificity comparable to virus isolation and these are ideal qualities for AI outbreak management, where the speed with which an unequivocal diagnosis can be is crucial for decision making by the relevant veterinary authority. In addition, RRT-PCR systems can be designed to operate in a 96-well format, and thus combined with high-throughput robotic RNA extraction from specimens (Agu¨ero et al., 2007). The approach to diagnosis using RRT-PCR adopted in most laboratories has been based on initial generic detection of AIV in clinical specimens, primarily by initially targeting the matrix (M) gene, which is highly conserved for all type A influenza viruses, followed by specific RRT-PCR testing for H5 and H7 subtype viruses. For example, this approach was used for diagnostic and surveillance specimens in the recent HPAI H5N1 outbreak in England (Irvine et al., 2007). For subtype identification, primers used in TaqMan RRT-PCRs are targeted at the HA2 region as this is relatively well conserved within the haemagglutinin genes of the H5 and H7 subtypes, and has served as the target region for H5 and H7. Spackman et al. (2002) demonstrated specific detection of these subtypes but cautioned that their H5 and H7 primer/probe sequences had been designed for the detection of North American H5 and H7 isolates and might not be suitable for all H5 and H7 isolates. This has proved the case. Slomka et al. (2007b) described modification of the H5 oligonucleotide sequences used by Spackman et al. (2002) to enable the detection of this Asian lineage HPAI H5N1 AI virus and other Eurasian H5 AI viruses that have been isolated within the past decade in both poultry and wild birds. This validated Eurasian H5 RRT-PCR has proved valuable in the investigation of many H5N1 HPAI clinical specimens submitted to International Reference Laboratories from Europe, Africa and Asia since autumn 2005 (Slomka et al., 2007b).

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One of the problems with rapidly emerging new tests is that methods and protocols may be developed and reported without the test being properly validated. This has been addressed for some of the RRT-PCR protocols (Slomka et al., 2007b; Suarez et al., 2007). In the European Union National Reference Laboratories have collaborated to define and validate protocols that can be recommended for use within the European Union (Slomka et al., 2007a,b). Real-time reverse transcriptase-polymerase chain reaction protocols have been described that amplify regions across the cleavage site of the HA0 gene. This may result in useful tests for specific viruses. For example, Hoffmann et al. (2007) have described an RRT-PCR test specific for the Asian HPAI H5N1 Quinghai clade 2.2 viruses that represents a rapid means of determining the pathotype for this subgroup of H5N1 HPAI viruses without sequencing. Future developments In the short term, the main thrust for laboratories both nationally and internationally appears to be the use of RRT-PCR tests for the very rapid diagnosis of AI. In particular, agreement on the validation and the use of controls appears to be the priority (Suarez et al., 2007). In the longer term, the advances in recent years suggest that it is highly likely that within a very short time molecular based technology will have developed sufficiently to allow rapid ‘flock-side’ tests for the detection of the presence of AI virus, specific subtype and virulence markers. The extent to which such tests are employed in the diagnosis of AI, will depend very much on the agreement on and adoption of definitions of statutory infections for control and trade purposes. References Agu¨ero, M., E. San Miguel, A. Sa´nchez, C. Go´mez-Tejedor, and M. A. Jime´nez-Clavero, 2007: A Fully automated procedure for the high-throughput detection of avian influenza virus by real-time reverse transcription–polymerase chain reaction. Avian Dis. 51, 235–241. Alexander, D. J., 2000: A review of avian influenza in different bird species. Proceedings of the ESVV Symposium on Animal Influenza Viruses, Gent 1999. Vet. Microbiol. 74, 3–13. Alexander, D. J., 2005: Chapter 2.07.12 Avian Influenza. Manual for Diagnostic Tests and Vaccines for Terrestrial Animals, 5th edn. World Organisation for Animal Health, Paris, France. Available at: http://www.oie.int/eng/normes/ MMANUAL/A_00037.htm (accessed 12th August 2007). Alexander, D. J., and D. Spackman, 1981: Characterization of influenza A viruses isolated from turkeys in England during March–May 1979. Avian Pathol. 10, 281–293.

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