Title: Characterisation of the faecal virome of captive and wild Tasmanian devils using virus-like particles metagenomics and meta-transcriptomics
Author: Mang Shi, Vanessa R Barrs, ...
University of Sydney,
Posted October 15, 2018.
Abstract
Background: The Tasmanian devil(袋獾,塔斯馬尼亞惡魔) is an endangered carnivorous(肉食的) marsupial(有袋類動物) threatened by devil facial tumour disease (DFTD,魔鬼面部腫瘤病). While research on DFTD has been extensive, little is known about the viruses present in devils, and whether any of these are of potential conservation relevance for this endangered species.
Methods: Using both metagenomics based on virus-like particle (VLP) enrichment and sequence-independent amplification (VLP metagenomics), and meta-transcriptomics based on bulk RNA sequencing, we characterised and compared the faecal viromes(病毒基因組) of captive and wild Tasmanian devils. 兩種方法：VLP metagenomics和meta-transcriptomics；比較了圈養和野生的袋獾的糞便病毒組。VLP metagenomics利用病毒的顆粒特性讓病毒富集；meta-transcriptomics：將所有的RNA都提取出來。
Results: A total of 54 devil faecal samples collected from captive (n = 2) and wild (n = 4) populations were processed for virome characterisation using both approaches. We detected many novel, highly divergent(高度趨異) viruses, including vertebrate(脊椎動物) viruses, bacteriophage(噬菌體) and other dietary associated plant and insect viruses. In total, 18 new vertebrate viruses, including novel sapelovirus(細小病毒), astroviruses(星狀病毒), bocaviruses(博卡病毒), papillomaviruses(乳頭瘤病毒) and gammaherpesvirus(伽馬皰疹病毒) were identified, as well as known mammalian pathogens(哺乳動物病原體) including rabbit haemorrhagic disease virus 2 (RHDV2,兔出血性病毒). Captive devils showed significantly lower levels of viral diversity than wild devils. Comparison of the two methodological approaches revealed substantial differences in the number and types of viruses detected, with meta-transcriptomics mainly identifying RNA viruses, and VLP metagenomics largely identifying DNA viruses.
Conclusion: This study has greatly expanded our knowledge of eukaryotic viruses in the Tasmanian devil and provides important baseline information that will contribute to the conservation and captive management of this endangered species. In addition, our results showed that a combination of VLP metagenomics and meta-transcriptomics may be a more comprehensive approach to virome characterisation than either method alone.
Outline:
1. Background
2. Materials and Methods
2.1 Sample collection
2.2 Meta-transcriptomics
2.3 VLP Metagenomics
3. Results
3.1 Overview of the devil virome
3.2 Detection of viruses previously identified in other mammalian hosts (Rabbit haemorrhagic disease virus and Torovirus)
3.3 Detection and characterisation of novel marsupial-associated viruses (Picornaviridae, Astroviridae, Reoviridae, Picobirnaviridae, Parvoviridae, Papillomaviridae, Herpesviridae, Polyomaviridae and Circoviridae)
3.4 Other viruses: plant and insect viruses, and bacteriophage
3.5 Virome ecology: comparison between devil populations
4. Discussion
5. Conclusion
正文摘錄：
Introduction: 兩種方法是目前studying viral metagenomics常用的方法，尚且沒有對兩種方法進行比較的研究。
To date, the most widely used method for studying viral metagenomics relies on the enrichment of virus-like particles (VLP) and subsequent sequence-independent amplification prior to sequencing [15-17]. Removal of non-viral genomic host and bacterial nucleic acids and enrichment of VLP is often necessary for the detection of low-titre viruses [17, 18]. More recently, the use of RNA sequencing of total non-ribosomal RNA from environmental samples gave rise to viral meta-transcriptomics, which has been successfully applied to characterise the viromes of diverse invertebrate and vertebrate species [13, 19, 20]. To our knowledge, no studies have been conducted to compare these two approaches, although doing so would allow us to understand the detection capabilities and biases associated with these different nucleic acid extraction and sample treatment methods.
Meta-transcriptomics
Total RNA extraction, library preparation and sequencing
VLP Metagenomics
VLP ENRICHMENT AND NUCLEIC ACID EXTRACTION
A second aliquot from each faecal sample was processed for the VLP metagenomics approach, as described previously with minor modifications
Discussion: 他們是第一個用兩種方法進行病毒研究的research。
We provide the first comprehensive characterisation of the faecal virome of an endangered marsupial species, the Tasmanian devil. Using both VLP metagenomics and meta-transcriptomics, we identified a huge diversity of viruses in the faeces of devils, including vertebrate viruses, bacteriophage and other eukaryotic viruses. Novel viruses identified as potentially marsupial-associated – including, sapelovirus, rotaviruses, picobirnaviruses, astrovirus, bocaviruses, papillomaviruses, herpesvirus and polyomaviruses – were detected and have greatly expanded our current knowledge of viruses that are found within this unique group of mammals.
Our comparison of VLP metagenomics and meta-transcriptomics approaches revealed marked differences in terms of the viruses detected. In general, VLP metagenomics mainly detected DNA viruses, while meta-transcriptomics detected both DNA and RNA viruses, although the DNA viruses detected with meta-transcriptomics were limited to those with relatively high abundance (Fig 2d). A high abundance level is often indicative of an active viral infection, during which DNA viruses are transcribed into RNA intermediates that can be readily detected by RNA sequencing [57]. Conversely, RNA viruses identified in meta-transcriptomic were rarely detected in VLP metagenomics, even if they were highly abundant based on the RSEM estimated counts.
無論存在什麼偏見，VLP對於病毒檢測仍然重要，因爲它可以檢測低丰度的DNA病毒。
Regardless of its known bias [58, 59], VLP metagenomics still holds merit for use in virome characterisation due to its ability to identify low abundance DNA viruses, which is especially relevant for dormant or non-active viruses.
meta-transcriptomics與VLP相比，具有非病毒特異性，並且它需要較少的樣本去富集，並且可以揭示樣品的整個轉錄組。
Compared to VLP metagenomics, meta-transcriptomics is non-viral specific, requires less sample processing, and reveals the entire transcriptome within a sample [20, 64]. Omitting the need for VLP enrichment and additional sample processing, the likelihood of biased detection is plausibly reduced in meta-transcriptomics.
兩種方法都沒有辦法單獨使用檢測出所有的病毒，但是兩種方法可以互爲補充。結合起來使用將是一個強大的工具。
Importantly, then, our results show that the taxonomic compositions of viral communities as revealed by VLP metagenomics and meta-transcriptomics were not interchangeable and neither of the approaches was able to detect all viruses present. However, these two approaches were complementary to one-another, and an integrated approach using both VLP metagenomics and meta-transcriptomics will prove to be a powerful tool for obtaining a complete overview of both the taxonomic and functional profiles of viral communities in a sample.
Conclusion:
we showed that a combination of VLP metagenomics and meta-transcriptomics may be a more comprehensive virome characterisation approach that will encompass both DNA and RNA viruses.