Runting-stunting syndrome (RSS), also known as malabsorption syndrome, is an infectious poultry disease affecting the intestinal tract of broilers at a very young age. RSS leads to retarded growth, feathering abnormality, diarrhea and other enteric problems, resulting in significant economic losses in the poultry industry.
A number of studies have implicated RNA and DNA viruses as the etiologic agents. Over the years, various viruses including reovirus, rotavirus, chicken astrovirus (CAstV) and avian nephritis virus (ANV) have been detected from clinically affected birds. It is still not clear which of the viruses or a combination of them is the causative agent, as the same viruses have been also detected in healthy flocks. One of the major challenges studies face is the limited quantity of extracted DNA/RNA.
In the past, RT-PCR has been used to identify and characterize viruses from the gut content of clinically affected flocks. However, the conventional PCR approach was limited by small sample sizes, and amplification of the viruses using in vitro cell culture techniques was challenging (1). Viral cultivation and Sanger sequencing methods are also difficult and time-consuming, highlighting the need for a fresh approach to characterizing the viruses potentially responsible for this disease.
Next-generation sequencing (NGS) emergence, cost-effectiveness and commercial availability has drastically changed virus discovery and research. NGS has several major advantages over traditional sequencing. Firstly, it can give a more comprehensive picture of the virus on whole genome level. NGS can also detect multiple viruses from heterogeneous samples with high sensitivity, making it an ideal approach to identify and characterize the set of viruses harbored in the enteric tracts of broiler flocks. Moreover, NGS enables deep sequencing, which allows a comprehensive comparative metagenomic analysis. The challenge often faced here is that the amount of viral RNA/DNA starting material is often limited and insufficient for sequencing.
To overcome this challenge, there are various whole genome amplification (WGA) and whole transcriptome amplification (WTA) technologies available. These technologies are either PCR-based or PCR-free. The two PCR-based amplification technologies are degenerative oligo-primer PCR (DOP-PCR) and multiple annealing and looping based amplification cycles (MALBAC). QIAGEN’s REPLI-g multiple displacement amplification (MDA) technology is a non-PCR-based DNA amplification technique enabling superior performance in genome coverage, uniformity and accuracy. In a comparison study led by Hou et al (2), REPLI-g MDA technology was shown to be superior to other methods for variant calling and to provide highly uniform amplification across the entire genome, with minimal locus bias and minimized mutation rates during amplification.
Find out more about why MDA technology is the preferred choice for these researchers. Read the review.
Which approach would you take to amplify limited amounts of viral RNA/DNA for your studies?
Recently, Devaney et al. gave an excellent example of how identifying a variety of enteric viruses and viral communities from small samples can move animal disease research forward (3). In the study, Devaney et al. used NGS to characterize the viral communities present in samples of 2- to 3-week old broiler chickens affected and unaffected by RSS. NGS requires amounts of material in the nanogram range, so the researchers used the REPLI-g Cell WGA & WTA Kit to amplify the samples. They concluded that the viral communities associated with the RSS-affected samples were also found in unaffected healthy samples, suggesting that certain strains within these families may be constituents of a healthy flock gut virome.
This new study is just one of many fascinating examples of the application of NGS and metagenomics analyses in research into disease etiology and environmental microbiology. It’s an exciting time – we will potentially see the revelation of many diseases’ secrets, putting researchers in a strong position to find new ways to combat them.
1. Todd, D. et al. (2010) Development and application of an RT-PCR test for detecting avian nephritis virus. Avian Pathol. 39, 207. Link
2. Hou, Y. et al. (2015) Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing. Gigascience 4, 37. Link
- 3. Devaney, R. et al. (2016) A metagenomic comparison of endemic viruses from broiler chickens with runting stunting syndrome and from normal birds. Avian Pathol. 23, 1. [Epub ahead of print] Link