Molecular quantitation of H9N2 avian influenza virus in various organs of broiler chickens using TaqMan real time PCR

Abstract

During the past decade, H9N2 low pathogenic avian influenza virus (LPAI) has caused considerable economic loss due to decreased production, increased mortality and the cost of vaccination in Iranian poultry industry. Because of widespread occurrence of this disease and the virus potential to mutate to highly-pathogenic (HP) form and transmission to humans, it is, therefore, imperative to understand the pathogenesis and properties of these viruses. In this study, a two step TaqMan real time PCR assay was performed for the quantitation of A/chicken/Iran/772/1998(H9N2) virus in various organs of broiler chickens at different days post inoculation (DPI). Forty 5-week-old commercial broiler chickens were inoculated with the virus. Five chickens were randomly selected on days 1, 3, 6 and 9 PI. Their trachea, lungs, spleen, kidneys, pancreas, blood and faeces were collected for virus detection. A PCR test was performed and the positive samples were used for quantitative real time PCR assay. The result of RT-PCR assay showed the presence of the virus in trachea (40%, 33%), lungs (20%, 66.6%) and spleen (20%, 50%) of infected chickens on days 3 and 6 PI, respectively. The virus was also detected in the kidneys of inoculated chickens on 3 (40%), 6 (60%) and 9 (100%) DPI. In faecal samples the virus was only detected on day 6 PI (83.3%). The molecular quantitation of AIV showed that the AIV titre in the trachea, lungs and spleen of chickens at 3 DPI is lower than the AIV titre at 6 DPI in these organs. The highest titre was observed in the faeces. The AIV titre in all organs of the birds which died at 6 DPI was higher than those of the same organs in the other experimental birds.

Matriptase, HAT, and TMPRSS2 Activate the Hemagglutinin of H9N2 Influenza A Viruses

“…Mortality has been associated with virus replication in various organs, including the trachea, lung, and kidney. In addition, coinfections with bacteria or infectious bronchitis virus (IBV) have been shown to exacerbate disease in chickens (23,(25)(26)(27). Moreover, some H9N2 strains cause significant morbidity and mortality in experimentally infected mice (11,20).…”

Antiviral activity of the oseltamivir and Melissa officinalis L. essential oil against avian influenza A virus (H9N2)

“…Then the upper liquid was removed and the precipitate was allowed to dry in room temperature. The pellet was dissolved in a final volume of 50 ll distilled water (DW) and stored at -70°C until used [21].…”

“…The cDNA was synthesized using AccuPowder TAMRA) used in this study as described previously [21]. The primers amplified a 104 bp fragment in the M1 gene of influenza A.…”

“…The concentration of the plasmid DNA was calculated with a spectrophotometer. Recombinant plasmids containing 450 bp fragment of matrix protein gene of influenza A, were serially diluted tenfold as a standard in the quantitative real-time PCR, The data and standard curves were obtained during target cDNA and recombinant plasmid amplification [21].…”

Monitoring viral RNA in infected cells with LNA flow-FISH

“…Antibodies are widely used to detect viruses and viral proteins (Cho et al 1996;Bentzen et al 2005;Dawson 2007;Rabenau et al 2007;Polage and Petti 2009), but due to their specificity, must be produced and calibrated for every target and are highly vulnerable to viral mutations. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR), microarrays, and enzymelinked immunosorbent assays (ELISAs) are other widely used methods to detect and quantify viruses (Steininger et al 2002;Wang et al 2002;Fox 2007;Leski et al 2009;Mosleh et al 2009). While these methods are highly sensitive, they each have shortcomings.…”