Appearances Can Be Deceptive: Revealing a Hidden Viral Infection with Deep Sequencing in a Plant Quarantine Context

Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share >91% genome-wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non-intercepted virus pathogens passing through plant quarantine stations.

[1]  R. Frutos,et al.  Nucleotide sequence evidence for three distinct sugarcane streak mastreviruses , 2014, Archives of Virology.

[2]  P. Simmonds,et al.  Bioinformatic and Physical Characterizations of Genome-Scale Ordered RNA Structure in Mammalian RNA Viruses , 2008, Journal of Virology.

[3]  R. Briddon,et al.  Evidence that dicot-infecting mastreviruses are particularly prone to inter-species recombination and have likely been circulating in Australia for longer than in Africa and the Middle East. , 2013, Virology.

[4]  Alvaro G. Hernandez,et al.  Deep Sequencing of Small RNAs in Tomato for Virus and Viroid Identification and Strain Differentiation , 2012, PloS one.

[5]  Y. Qi,et al.  A Structured Viroid RNA Serves as a Substrate for Dicer-Like Cleavage To Produce Biologically Active Small RNAs but Is Resistant to RNA-Induced Silencing Complex-Mediated Degradation , 2007, Journal of Virology.

[6]  C. Llave,et al.  High-throughput sequencing of Hop stunt viroid-derived small RNAs from cucumber leaves and phloem. , 2010, Molecular plant pathology.

[7]  Forest Rohwer,et al.  Laboratory procedures to generate viral metagenomes , 2009, Nature Protocols.

[8]  A. Poon,et al.  Evidence of Pervasive Biologically Functional Secondary Structures within the Genomes of Eukaryotic Single-Stranded DNA Viruses , 2013, Journal of Virology.

[9]  Guoan Shen,et al.  Ecogenomics: using massively parallel pyrosequencing to understand virus ecology , 2010, Molecular ecology.

[10]  M. Madkour,et al.  Sensitive detection of the Egyptian species of sugarcane streak virus by PCR-probe capture hybridization (PCR-ELISA) and its complete nucleotide sequence. , 2001, Journal of Virological Methods.

[11]  Reinhard Simon,et al.  Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. , 2009, Virology.

[12]  A. Tuplin,et al.  Detection of genome-scale ordered RNA structure (GORS) in genomes of positive-stranded RNA viruses: Implications for virus evolution and host persistence. , 2004, RNA.

[13]  Vincent Moulton,et al.  RDP3: a flexible and fast computer program for analyzing recombination , 2010, Bioinform..

[14]  P. Mullineaux,et al.  Bidirectional transcription of maize streak virus DNA and identification of the coat protein gene. , 1985, Nucleic acids research.

[15]  K. Dupuis,et al.  Rapid Identification of Known and New RNA Viruses from Animal Tissues , 2008, PLoS pathogens.

[16]  Ben Berkhout,et al.  HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome , 2005, Nucleic acids research.

[17]  Jinmin Ma,et al.  Sequence Recombination and Conservation of Varroa destructor Virus-1 and Deformed Wing Virus in Field Collected Honey Bees (Apis mellifera) , 2013, PloS one.

[18]  O. Gascuel,et al.  Estimating maximum likelihood phylogenies with PhyML. , 2009, Methods in molecular biology.

[19]  E. Delwart,et al.  From orphan virus to pathogen: the path to the clinical lab. , 2011, Current opinion in virology.

[20]  Olivier Voinnet,et al.  Induction and suppression of RNA silencing: insights from viral infections , 2005, Nature Reviews Genetics.

[21]  M. Breitbart,et al.  Discovery of a novel mastrevirus and alphasatellite-like circular DNA in dragonflies (Epiprocta) from Puerto Rico. , 2013, Virus research.

[22]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[23]  Roger A. C. Jones,et al.  Plant virus emergence and evolution: origins, new encounter scenarios, factors driving emergence, effects of changing world conditions, and prospects for control. , 2009, Virus research.

[24]  R. Brlansky,et al.  A Case Study on Discovery of Novel Citrus Leprosis Virus Cytoplasmic Type 2 Utilizing Small RNA Libraries by Next Generation Sequencing and Bioinformatic Analyses , 2016 .

[25]  E. Holmes,et al.  Deep sequencing reveals persistence of intra- and inter-host genetic diversity in natural and greenhouse populations of zucchini yellow mosaic virus. , 2012, The Journal of general virology.

[26]  E. Rybicki,et al.  Comparative analysis of Panicum streak virus and Maize streak virus diversity, recombination patterns and phylogeography , 2009, Virology Journal.

[27]  J. Harding,et al.  Discovery of Sclerotinia sclerotiorum Hypovirulence-Associated Virus-1 in Urban River Sediments of Heathcote and Styx Rivers in Christchurch City, New Zealand , 2013, Genome Announcements.

[28]  W. Lipkin.  Microbe Hunting , 2010, Microbiology and Molecular Biology Reviews.

[29]  M. Breitbart,et al.  Exploring the viral world through metagenomics. , 2011, Current opinion in virology.

[30]  A. Gray,et al.  NASP: a parallel program for identifying evolutionarily conserved nucleic acid secondary structures from nucleotide sequence alignments , 2011, Bioinform..

[31]  Jonathan E. Allen,et al.  Ultra-Deep Sequencing of Intra-host Rabies Virus Populations during Cross-species Transmission , 2013, PLoS neglected tropical diseases.

[32]  Robert C. Edgar,et al.  MUSCLE: a multiple sequence alignment method with reduced time and space complexity , 2004, BMC Bioinformatics.

[33]  M. Roossinck.  The good viruses: viral mutualistic symbioses , 2011, Nature Reviews Microbiology.

[34]  D. Posada.  jModelTest: phylogenetic model averaging. , 2008, Molecular biology and evolution.

[35]  K. Mayer,et al.  Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes. , 2009, Virology.

[36]  B. Roe,et al.  Evidence for novel viruses by analysis of nucleic acids in virus-like particle fractions from Ambrosia psilostachya. , 2008, Journal of virological methods.

[37]  M. V. D. van der Heijden,et al.  The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. , 2008, Ecology letters.

[38]  Kate E. Jones,et al.  Global trends in emerging infectious diseases , 2008, Nature.

[39]  V. Mäkinen,et al.  Detection of Viruses in Sweetpotato from Honduras and Guatemala Augmented by Deep-Sequencing of Small-RNAs. , 2012, Plant disease.

[40]  A. Geering,et al.  Australian monocot-infecting mastrevirus diversity rivals that in Africa. , 2012, Virus research.

[41]  P. Hugenholtz,et al.  From deep sequencing to viral tagging: Recent advances in viral metagenomics , 2013, BioEssays : news and reviews in molecular, cellular and developmental biology.

[42]  David Posada,et al.  ProtTest: selection of best-fit models of protein evolution , 2005, Bioinform..

[43]  Darren P. Martin,et al.  A genome-wide pairwise-identity-based proposal for the classification of viruses in the genus Mastrevirus (family Geminiviridae) , 2013, Archives of Virology.

[44]  A. ElSayed,et al.  Sugarcane yellow leaf virus introduction and spread in Hawaiian sugarcane industry: Retrospective epidemiological study of an unnoticed, mostly asymptomatic plant disease , 2010, European Journal of Plant Pathology.

[45]  D. Golino,et al.  Deep sequencing analysis of RNAs from a grapevine showing Syrah decline symptoms reveals a multiple virus infection that includes a novel virus. , 2009, Virology.

[46]  P. Froussard.  rPCR: a powerful tool for random amplification of whole RNA sequences. , 1993, PCR methods and applications.

[47]  A. Varsani,et al.  Novel myco-like DNA viruses discovered in the faecal matter of various animals. , 2013, Virus research.

[48]  F. Rohwer,et al.  Metagenomics and future perspectives in virus discovery , 2012, Current Opinion in Virology.

[49]  Peter Daszak,et al.  Emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers. , 2004, Trends in ecology & evolution.