Thien Ho

Development of a virus detection and discovery pipeline using next generation sequencing

Next generation sequencing (NGS) has revolutionized virus discovery. Notwithstanding, a vertical pipeline, from sample preparation to data analysis, has not been available to the plant virology community. We developed a degenerate oligonucleotide primed RT-PCR method with multiple barcodes for NGS, and constructed VirFind, a bioinformatics tool specifically for virus detection and discovery able to: (i) map and filter out host reads, (ii) deliver files of virus reads with taxonomic information and corresponding Blastn and Blastx reports, and (iii) perform conserved domain search for reads of unknown origin. The pipeline was used to process more than 30 samples resulting in the detection of all viruses known to infect the processed samples, the extension of the genomic sequences of others, and the discovery of several novel viruses. VirFind was tested by four external users with datasets from plants or insects, demonstrating its potential as a universal virus detection and discovery tool.

Epidemiology of Blackberry yellow vein associated virus

Blackberry yellow vein disease is one of the most important diseases of blackberry in the United States. Several viruses are found associated with the symptomology but Blackberry yellow vein associated virus (BYVaV) appears to be the most prevalent of all, leading to the need for a better understanding of its epidemiology. Efficient detection protocols were developed using end-point and quantitative reverse-transcription polymerase chain reaction. A multi-state survey was performed on wild and cultivated blackberry to assess the geographical distribution of the virus. Two whitefly species, Trialeurodes abutilonea and T. vaporariorum, were identified as vectors and 25 plant species were tested as potential BYVaV hosts. The information obtained in this study can be used at multiple levels to better understand and control blackberry yellow vein disease.

The evolution of emaraviruses is becoming more complex: seven segments identified in the causal agent of Rose rosette disease

There are few examples of a plant disease as devastating as rose rosette, a disorder that could lead to total loss for the nursery industry and rosarians alike. Although described over 75 years ago, the causal agent of rose rosette remains elusive. Utilizing the bottleneck created during vector transmission and large scale sequencing it was determined that the causal agent of the disease is rose rosette virus (RRV), a member of the genus Emaravirus. The genome structure of emaraviruses displays significant fluidity and for this reason the genome composition of RRV was revisited, leading to the discovery of three additional segments, one of which is predicted to be bicistronic.

A new ophiovirus is associated with blueberry mosaic disease.

Blueberry mosaic disease (BMD) was first described more than 60 years ago and is caused by a yet unidentified graft transmissible agent. A combination of traditional methods and next generation sequencing disclosed the presence of a new ophiovirus in symptomatic plants. The virus was detected in all BMD samples collected from several production areas of North America and was thus named blueberry mosaic associated virus. Phylogenetic analysis, supported by high bootstrap values, places the virus within the family Ophioviridae. The genome organization resembles that of citrus psorosis virus, the type member of the genus Ophiovirus. The implications of this discovery in BMD control and blueberry virus certification schemes are also discussed.

A novel emaravirus is associated with redbud yellow ringspot disease.

Yellow ringspot is the only virus-like disease reported in redbud (Cercis spp.) with symptoms including vein clearing, chlorotic ringspots and oak-leaf pattern. A putative new emaravirus was present in all trees displaying typical yellow ringspot symptoms and the name redbud yellow ringspot associated virus is proposed. The virus genome is composed of at least five RNA segments. Two coding regions were studied to determine isolate diversity with results pointing to a homogeneous virus population. Host range was evaluated using graft transmission and by testing species found in close proximity to infected trees. Mite transmission with Aculops cercidis, the predominant species found in redbud trees in the epicenter of the disease, was evaluated but was not found to be a vector of the virus. Based on this study and the accumulated knowledge on emaravirus evolution we propose that speciation is allopatric, with vectors being a major component of the process.

Next-generation sequencing of elite berry germplasm and data analysis using a bioinformatics pipeline for virus detection and discovery.

Berry crops (members of the genera Fragaria, Ribes, Rubus, Sambucus, and Vaccinium) are known hosts for more than 70 viruses and new ones are identified continually. In modern berry cultivars, viruses tend to be asymptomatic in single infections and symptoms only develop after plants accumulate multiple viruses. Most certification programs are based on visual observations. Infected, asymptomatic material may be propagated in the nursery system and shipped to farms where plants acquire additional viruses and develop symptoms. This practice may result in disease epidemics with great impact to producers and the natural ecosystem alike. In this chapter we present work that allows for the detection of known and discovery of new viruses in elite germplasm, having the potential to greatly reduce virus dispersal associated with movement of propagation material.

Detection of Strawberry necrotic shock virus using conventional and TaqMan(®) quantitative RT-PCR.

Graft-indexing of an advanced selection from the University of Florida strawberry breeding program produced virus-like symptoms on Fragaria vesca. However; RT-PCR testing of the material did not detect the presence of any of 16 strawberry virus species or members of virus groups for which strawberries are routinely indexed. Large scale sequencing of the material revealed the presence of an isolate of Strawberry necrotic shock virus. The nucleotide sequence of this isolate from Florida shows a significant number of base changes in the annealing sites of the primers compared to the primers currently in use for the detection of SNSV thereby explaining the most probable reason for the inability to detect the virus in the original screening. RT-PCR and Taqman(®) qPCR assays were developed based on conserved virus sequences identified in this isolate from Florida and other sequences for SNSV currently present in GenBank. The two assays were applied successfully on multiple samples collected from several areas across the United States as well as isolates from around the world. Comparison between the RT-PCR and the qPCR assays revealed that the qPCR assay is at least 100 times more sensitive than conventional PCR.

Epidemiology of Blackberry chlorotic ringspot virus

The pollen- and seed-borne ilarviruses pose a substantial threat to many specialty crops, including berries, rose, and tree fruit, because there are no efficient control measures other than avoidance. The case of Blackberry chlorotic ringspot virus (BCRV) is of particular interest because the virus has been found to be an integral part of blackberry yellow vein disease and is widespread in rose plants affected by rose rosette disease. This study provides insight into the epidemiology of BCRV, including incidence in blackberry and rose; host range, with the addition of apple as a host of the virus; and seed transmission that exceeded 50% in rose. Sensitive detection protocols that can be used to avoid dissemination of infected material through nurseries and breeding programs were also developed.

Evidence of sympatric speciation of elderberry carlaviruses.

Five new carlaviruses infecting elderberry were characterized and tentatively named as elderberry virus A-E (ElVA-ElVE). Their genome organization is similar to that of other carlaviruses with size ranging from 8540 to 8628 nucleotides, excluding the polyadenylated tails. ElVA, ElVB and ElVD share a common ancestor as do ElVC and ElVE, indicating that speciation may be sympatric with all viruses having emerged in elderberry. Analyses of the carlavirus conserved domains indicate that the 2-oxoglutarate and Fe(II)-dependent oxygenase motifs are reliable indicators of virus phylogenetic classification with recombination playing a significant role in the evolution of the genus. A universal RT-PCR assay that detects all the elderberry carlaviruses and potentially other members of the genus has been developed. This tool can be used for research and regulatory purposes as elderberry cultivation is rapidly expanding to new areas where the viruses may be absent.

Genome sequence and detection of peach rosette mosaic virus

Peach rosette mosaic disease was first described in the 1940s affecting peach and plum. It was later determined that peach rosette mosaic virus (PRMV) is the causal agent of the disease. PRMV, a member of the genus Nepovirus, infects several perennial crops including stone fruit, grape and blueberry as well as several weed species found in orchards around the world. The molecular characterization of the virus is limited to partial genome sequences making it difficult to develop reliable and sensitive molecular detection tests; the reason that detection is routinely performed using ELISA with antibodies risen against a single virus isolate. Given the potential economic impact of the virus and the modes of transmission which, in addition to nematodes, include seed we studied PRMV in more depth using a modified dsRNA extraction protocol to obtain the virus genome. We determined the full nucleotide sequence and developed a protocol that detects conserved regions present in RNA 1 and RNA 2, making it an excellent alternative to the detection protocols used today.