ReviewRecent developments in the diagnosis of avian influenza
Introduction
Influenza A viruses have segmented, negative-strand RNA genomes and belong to the genus Influenzavirus A of the family Orthomyxoviridae (Wright et al., 2007). Influenza A viruses have been classified into subtypes according to their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and comprise 16 HA and 9 NA subtypes (H1–H16 and N1–N9, respectively; Palese and Shaw, 2007). Wild water birds, predominantly ducks, geese and shorebirds, are the natural reservoirs of all subtypes of influenza A viruses (Kida, Yanagawa, 1979, Webster et al, 1992). Avian influenza viruses (AIVs) sporadically infect poultry, resulting in either asymptomatic infection or clinical manifestations ranging from decline in egg production and mild respiratory disease to death.1
Influenza A viruses that infect chickens are categorised into two pathotypes based on their virulence in chickens: low pathogenic avian influenza virus (LPAIV) and highly pathogenic avian influenza virus (HPAIV). Among the 16 HA subtypes of AIV, HPAIV has been associated with only a small proportion of the H5 or H7 subtypes.2 The HA molecules of HPAIVs differ from those of LPAIVs in possessing multiple basic amino acids at the carboxyl-terminus of HA1 (Horimoto et al., 1995a). This series of basic amino acids at the endoproteolytic cleavage site is a motif that is recognised by ubiquitous intracellular proteases, allowing for systemic infection associated with high mortality in chickens (Horimoto et al, 1994, Steinhauer, 1999). In addition, some LPAIVs of the H5 and H7 subtypes can mutate into HPAIVs, and several mechanisms involved in the emergence of HPAIV from an LPAIV precursor have been documented. It was shown that LPAIV evolved into an HPAIV that caused a severe outbreak in Pennsylvania in 1983 through the removal of a carbohydrate side chain in the vicinity of the cleavage site (Kawaoka et al., 1984). The causative agents of the Mexican outbreaks from 1994 to 1995 were derived from LPAIV that had accumulated numerous basic residues at the cleavage site (Horimoto et al., 1995b), as were the viruses that caused the outbreaks in British Columbia, Chile and Taiwan (Hirst et al, 2004, Suarez et al, 2004, Soda et al, 2011).
The diagnosis of avian influenza begins with the detection of the causative agent because infections in birds can give rise to a wide variety of clinical signs that depend on the host and its immune status, viral strain, and the presence of any secondary exacerbating organisms and environmental conditions (Swayne and Halvorson, 2003). The isolation of viruses using embryonated chicken eggs is the most sensitive and reliable method for detecting infective viruses; however, the assay is not suitable for the high-throughput detection of a large number of clinical samples. Furthermore, classical laboratory diagnostic methods, including agar gel immunodiffusion (AGID; Beard, 1970), haemagglutination, haemagglutination inhibition (HI) and virus neutralization tests,3 are time consuming and require high-level biosafety laboratory. These methods have recently been replaced in certain situations by the direct detection of the influenza A virus genome. Here we review both classical and recently developed methods that are now applied to diagnose avian influenza throughout the world.
Section snippets
Virus isolation
Virus isolation is the gold standard for the diagnosis because of its high sensitivity for the detection of live viruses. It is often primarily used for the diagnosis of the index case to obtain virus isolates for further laboratory analyses. The biggest disadvantage of virus isolation is that it takes more time than other diagnostic techniques. It also requires embryonated chicken eggs (or cultured cells) and biosafety laboratory facilities. Methods for isolating AIVs as well as Newcastle
Genetic detection
Genetic detections of the influenza A virus from field clinical specimens are rapid and sensitive laboratory tests. The influenza A virus is a negative-sense, single-stranded RNA virus. Nucleic acid detection systems, including PCR-based methods and nucleic acid amplification under isothermal conditions, require the extraction of viral RNAs. Because these techniques and the necessary reagents are commonly used in facilities with other viral and bacterial diseases, and inactivation of the virus
Viral protein detection
Several antigen-capture immunoassay tests have been developed in recent years. Some were first developed for detecting influenza A viruses in humans, but have since been used for AIVs. Both immunochromatography and antigen-capture ELISA are widely used. The kit uses a monoclonal antibody against the nucleoprotein (NP) and should therefore be able to detect all influenza viruses. For the detection of viruses with a specific subtype, monoclonal antibodies against HA have also been used. Recently,
Conclusions
Since 2003, outbreaks of H5 highly pathogenic avian influenza viruses have led to a dramatic acceleration in the development of methods for viral diagnosis and surveillance. These methods were also important in the 2009 pandemic influenza and in H7N9 virus infections in humans in 2013 (Dawood et al, 2009, Gao et al, 2013). Technologies such as real-time RT-PCR, isothermal nucleic acid amplification, NGS and immunochromatography are contributing to simpler and more rapid diagnosis and typing of
Conflict of interest statement
None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
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