Avian Influenza Virus (AIV)
Pathogenicity refers to the ability of an organism to harm its host by causing disease. The avian influenza virus, subtype H7, exists as one of two pathotypes, highly pathogenic (HPAIV) and low pathogenic (LPAIV). Even LPAIV carries a threat – it is known to give rise to the highly pathogenic pathotype. This results from spontaneous insertional mutations within the hemagglutinin protein cleavage site (HACS). Sporadic outbreaks of LPAIV H7 are continuously reported across Europe, with some exhibiting zoonotic transmission. For these reasons, it is important to diagnose AIV cases and distinguish between the two H7 pathotypes as soon as possible, enabling suitable control measures to be implemented.
Current Methods for Distinguishing Pathotypes
Determining the type of HACS is critical for differentiating between the two H7 pathotypes. This is currently performed either by determining the intravenous pathogenicity index using experimentally inoculated chickens, or nucleotide sequence analysis of the HACS site. More recently, studies have shown the benefits of using RT-qPCR methods in diagnosing various AIV subtypes. The specificity and sensitivity of these techniques, combined with the widespread availability of PCR technology, provides a great alternative to current methods.
RT-qPCR proved to be a useful, sensitive and highly specific alternative
In the study, primers were selected that targeted a short fragment of the HACS region. Closer analysis found that the two H7 pathotypes can be distinguished by a single G to A mutation in the HACS region. Therefore, a probe was designed which placed this critical nucleotide position centrally within a probe. This enabled RT-qPCR to provide a clear distinction between H7 LPAIV and HPAIV.
For validation of the assays, viral RNA from reference H7 LPAIV and HPAIV was used. None of the HPAIV positive samples cross reacted in the LPAIV RT-qPCR assay, meaning that total specificity was present. Various concentration combinations of the two pathotypes were also used, to test the ability of the assay to detect H7 HPAIV and LPAIV RNA mixtures. It was found that the Cq values reflected the concentration of the RNA species in that particular mixture. This will be useful for studying the generation of HPAIV from its LPAIV precursor.
“This pathotype-specific RT-qPCR proved to be a useful, sensitive and highly specific alternative to nucleotide sequence analysis for the characterisation of LPAIV and HPAIV H7 viruses of European origin.”