Ioannis E. Tzanetakis

The complete nucleotide sequence and genome organization of tomato chlorosis virus

Summary.The crinivirus tomato chlorosis virus (ToCV) was discovered initially in diseased tomato and has since been identified as a serious problem for tomato production in many parts of the world, particularly in the United States, Europe and Southeast Asia. The complete nucleotide sequence of ToCV was determined and compared with related crinivirus species. RNA 1 is organized into four open reading frames (ORFs), and encodes proteins involved in replication, based on homology to other viral replication factors. RNA 2 is composed of nine ORFs including genes that encode a HSP70 homolog and two proteins involved in encapsidation of viral RNA, referred to as the coat protein and minor coat protein. Sequence homology between ToCV and other criniviruses varies throughout the viral genome. The minor coat protein (CPm) of ToCV, which forms part of the “rattlesnake tail” of virions and may be involved in determining the unique, broad vector transmissibility of ToCV, is larger than the CPm of lettuce infectious yellows virus (LIYV) by 217 amino acids. Among sequenced criniviruses, considerable variability exists in the size of some viral proteins. Analysis of these differences with respect to biological function may provide insight into the role crinivirus proteins play in virus infection and transmission.

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.

A discovery 70 years in the making: characterization of the Rose rosette virus.

Rose rosette was first described in the early 1940s and it has emerged as one of the most devastating diseases of roses. Although it has been 70 years since the disease description, the rosette agent is yet to be characterized. In this communication, we identify and characterize the putative causal agent of the disease, a negative-sense RNA virus and new member of the genus Emaravirus. The virus was detected in 84/84 rose rosette-affected plants collected from the eastern half of the USA, but not in any of 30 symptomless plants tested. The strong correlation between virus and disease is a good indication that the virus, provisionally named Rose rosette virus, is the causal agent of the disease. Diversity studies using two virus proteins, p3 and p4, demonstrated that the virus has low diversity between isolates as they share nucleotide identities ranging from 97 to 99%.

Survey for Viruses of Grapevine in Oregon and Washington

Grapevines (Vitis spp.) in Washington and Oregon were surveyed for the prevalence of key grapevine viruses. Samples collected from 1,522 vines in Washington were tested for Rupestris stem pitting associated virus (RSPaV), Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Tomato ringspot virus (ToRSV), and Grapevine leafroll associated virus-3 (GLRaV-3). Tests were also conducted for GLRaV-1 and -2 on 420 samples from Washington. Two hundred forty samples collected from wine grape vineyards in Oregon were tested for GLRaV-1, -2, and -3, and an additional 2,880 samples were collected from 40 vineyards known to have high populations of Xiphinema americanum nematodes. The latter were tested for ArMV, ToRSV, and GFLV. GLRaV-1, -2, and -3 were detected in 2.6, 0.2, and 6.5% of the Washington samples and in 3.0, 0.4, and 4.4% of the Oregon samples. RSPaV was detected in 4.6% of the samples from Washington. No ToRSV, ArMV, or GFLV was detected in any of the samples from Oregon or Washington. Transmission of field isolates of GLRaV-3 from Washington by the grape mealybug also was demonstrated.

Viruses and Virus Diseases of Rubus

Blackberry and raspberry are members of the family Rosaceae. They are classified in the genus Rubus, which comprises hundreds of species and has a center of origin in the Far East. Rubus is divided into 15 subgenera with blackberries classified in the Rubus (formerly Eubatus) and raspberries in the Idaeobatus subgenera. Rubus species are propagated vegetatively and are subject to infection by viruses during development, propagation, and fruit production stages. Reports of initial detection and symptoms of more than 30 viruses, virus-like diseases, and phytoplasmas affecting Rubus spp. were reviewed more than 20 years ago. Since the last review on Rubus viruses, significant progress has been made in the molecular characterization of many of the viruses that infect Rubus spp. Currently, reverse transcription-polymerase chain reaction detection methods are available for most of the viruses known to infect Rubus. The goals of this article are to update the knowledge on previously characterized viruses of Rubus, highlight recently described viruses, review the virus-induced symptoms, describe the advances made in their detection, and discuss our knowledge about several virus complexes that cause serious diseases in Rubus. Virus complexes have been identified recently as the major cause of diseases in blackberries and raspberries.

Identification and Detection of a Virus Associated with Strawberry Pallidosis Disease

The etiology of pallidosis, a disease of strawberry identified more than 45 years ago, remains unknown. We report a putative agent of the disease, a virus belonging to the Crinivirus genus of the Closterovirideae family. A sensitive reverse transcription-polymerase chain reaction (RTPCR) test has been developed. Polyclonal antibodies that can be used to detect the virus in petiole tissue blots were developed using a recombinant virus coat protein. The nucleotide sequences of regions of the viral genome that encode the heat shock protein 70 homolog and the major coat protein were obtained. Alignments of the major coat protein show that the virus isolated from strawberry plants positive for pallidosis is most closely related to Cucumber yellows virus (syn. Beet pseudo-yellows virus) and Cucurbit yellow stunt disorder virus, members of the Crinivirus genus.

Blueberry latent virus: An amalgam of the Partitiviridae and Totiviridae

A new, symptomless virus was identified in blueberry. The dsRNA genome of the virus, provisionally named Blueberry latent virus (BBLV), codes for two putative proteins, one without any similarities to virus proteins and an RNA-dependent RNA polymerase. More than 35 isolates of the virus from different cultivars and geographic regions were partially or completely sequenced. BBLV, found in more than 50% of the material tested, has high degree of homogeneity as isolates show more than 99% nucleotide identity between them. Phylogenetic analysis clearly shows a close relationship between BBLV and members of the Partitiviridae, although its genome organization is related more closely to members of the Totiviridae. Transmission studies from three separate crosses showed that the virus is transmitted very efficiently by seed. These properties suggest that BBLV belongs to a new family of plant viruses with unique genome organization for a plant virus but signature properties of cryptic viruses including symptomless infection and very efficient vertical transmission.

Complete nucleotide sequence of a strawberry isolate of Beet pseudoyellows virus.

In our effort to identify the causal agent(s) of strawberry pallidosis we found a single pallidosis positive plant that did not give any amplicons after RT-PCR using primer sets representing multiple regions of Strawberry pallidosis associated virus (SPaV) genome and failed to react with antibodies directed against the recombinant coat protein (CP) of SPaV. DsRNA extracted from this plant showed a similar pattern to that of SPaV indicating that another crinivirus may infect strawberry. Sequence analysis of multiple cDNA clones corresponding to the heat shock 70 homolog gene (HSP70h) of the unknown virus indicated that it was Beet pseudoyellows virus (BPYV). Analysis of the complete nucleo- tide sequence of BPYV-strawberry revealed that this isolate has several distinct features when compared to Cucumber yellows virus (CuYV), a cucumber strain of BPYV, including an entire ORF not found in CuYV.

A new method for extraction of double-stranded RNA from plants

The occurrence of high molecular weight double-stranded RNA (dsRNA) in plants is associated with the presence of RNA viruses. DsRNA is stable, can be extracted easily from the majority of plant species and provides an excellent tool for characterization of novel viruses that are recalcitrant to purification. Several protocols have been developed for dsRNA purification, the majority of which are based on extraction with phenol and chloroform. We have developed a protocol for dsRNA extraction based on a lithium salts buffer that does not require organic solvents other than alcohols. The method yields comparable amount of dsRNA to protocols described previously and yields consistently dsRNA from Vaccinium hosts that have been recalcitrant to dsRNA purification using traditional protocols. The quality of the dsRNA purified is such that it can be used for downstream enzymatic reactions including reverse transcription-polymerase chain reaction and cloning.

An Integrated Badnavirus Is Prevalent in Fig Germplasm

Fig mosaic occurs worldwide and is the most common and important viral disease of fig. In the quest to identify the causal agent of the disease, several new viruses have been identified, including a new DNA virus, the subject of this communication. Phylogenetic analysis placed the virus, provisionally named Fig badnavirus-1 (FBV-1), in the genus Badnavirus, family Caulimoviridae. The experimental host range of FBV-1 was evaluated and the virus was mechanically transmitted to several herbaceous hosts. FBV-1 was detected in the National Clonal Germplasm Repository fig collection and additional samples from Arkansas, California, Florida, Michigan, Ohio, Oregon, and South Carolina, suggesting its wide distribution in the United States. Further tests revealed the presence of FBV-1 in seedlings and meristem tissue culture plants. Forty-four isolates were used in a study evaluating the population structure of the virus in the United States. Evidence that FBV-1 is integrated in the fig genome is presented and discussed.