Taqman real-time PCR detects Avipoxvirus DNA in blood of Hawai'i 'amakihi (Hemignathus virens)

Abstract

Background: Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawai'i, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds.

Methodology/principal findings: Here, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawai'i 'amakihi (Hemignathus virens) with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawai'i but were observed in two individuals in this study.

Conclusions/significance: We anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus.

Figure 1. Real-time amplification of a serially diluted known positive sample.

PCR base line subtracted curve fit data shows amplification of the Avipoxvirus 4b core protein gene from a 1∶2 serial dilution of first reaction PCR product using gDNA from Avipoxvirus culture lysate as template. The threshold for this reaction was 79.0 rfu.