Complete genome organization of American hop latent virus and its relationship to carlaviruses

The complete genomic sequence of American hop latent virus (AHLV; genus Carlavirus) was determined. The genome consists of 8,601 nucleotides plus a 3′-polyadenylate tail. The genome encompasses six potential open reading frames (ORF) in the positive sense, and their organization is typical of other carlaviruses. Analysis of the coat protein coding sequence at both the nucleic acid level and the amino acid level indicates that AHLV is only remotely related to the other carlaviruses known to infect common hop. Polyclonal antibodies were produced against the bacterially expressed coat protein of AHLV. These antibodies differentiated between AHLV and other carlaviruses of hop

Complete nucleotide sequences and genome organization of a cherry isolate of cherry leaf roll virus

The complete nucleotide sequence of cherry leaf roll virus (CLRV, genus Nepovirus) from a naturally infected cherry tree (Prunus avium cv. Bing) in North America was determined. RNA1 and RNA2 consist of 7,893 and 6,492 nucleotides, respectively, plus a poly-(A) tail. Each RNA encodes a single potential open reading frame. The first 657 nucleotides of RNA1 and RNA2 are 99% identical and include the 5′-UTR and the first 214 deduced amino acids of the polyproteins following the first of two in-frame start codons. Phylogenetic analysis reveals close relationships between CLRV and members of subgroup C of the genus Nepovirus

A new and distinct species in the genus Caulimovirus exists as an endogenous plant pararetroviral sequence in its host, Dahlia variabilis

Viruses in certain genera in family Caulimoviridae were shown to integrate their genomic sequences into their host genomes and exist as endogenous pararetroviral sequences (EPRV). However, members of the genus Caulimovirus remained to be the exception and are known to exist only as episomal elements in the infected cell. We present evidence that the DNA genome of a new and distinct Caulimovirus species, associated with dahlia mosaic, is integrated into its host genome, dahlia ( Dahlia variabilis). Using cloned viral genes as probes, Southern blot hybridization of total plant DNA from dahlia seedlings showed the presence of viral DNA in the host DNA. Fluorescent in situ hybridization using labeled DNA probes from the D10 genome localized the viral sequences in dahlia chromosomes. The natural integration of a Caulimovirus genome into its host and its existence as an EPRV suggests the co-evolution of this plant–virus pathosystem

Evaluation of the promoter activity of caulimoviruses associated with Dahlia spp by transient expression of the beta-glucoronidase gene (GUS)

One of the most common promoters used for constitutive gene expression was derived from a plant virus. The 35S promoter from Cauliflower mosaic virus (CaMV) drives high levels of transgene expression; directs constitutive expression and displays no tissue-specificity. Due to these properties, the CaMV 35S promoter is widely applied in expressing foreign genes in plants. However, some limitations including insufficient promoter activity or strong down-regulation necessitated the exploration of more efficient promoters from other caulimoviruses. Dahlia mosaic virus (DMV) and two new caulimoviruses tentatively designated as Dahlia common mosaic virus (DCMV) and an endogenous sequence (DMV-D10) have been reported from dahlia (Dahlia variabilis) plants. Not much information is known about the promoters from these caulimoviruses and they represent a potential source for new promoters. Based on sequence comparisons and promoter prediction programs, we identified the putative 35S promoter in DMV, DCMV and DMV-10 from cultivated and wild dahlia species. The intergenic regions containing the putative 35S promoter were separately cloned into pCAMBIA1281Z, a binary vector. Constructs were delivered into Agrobacterium tumefaciens by electroporation and agroinfiltrations were done into Nicotiana tabacum, N. benthamiana and Verbesina encelioides. The activity and strength of the putative promoters from DMV, DCMV and DMV-D10 was determined by GUS assays. Preliminary results from qualitative GUS assays demonstrated that DMV, DCMV and DMV-D10 promoter activity is similar to the one showed by 35S promoter of CaMV. Transient expression showed stronger activity in N. benthamiana leaf tissue in comparison to N. tabacum leaf tissue for all constructs. Deletion analysis of the 3' and 5' end of the promoter region in order to establish the optimal boundaries for maximal promoter activity is underway. Quantitative GUS assays are also in progress. Since promoter selection has become increasingly important for successful gene transfer and expression of transgenes in plants, the findings could provide important information on new promoters for gene expression in plants

Genetic diversity among endogenous plant pararetroviral sequences from geographically diverse sources of dahlia (Dahlia spp).

Two distinct caulimoviruses, Dahlia mosaic virus (DMV) and Dahlia common mosaic virus (DCMV), and an endogenous plant pararetroviral sequence (DvEPRS) were reported in Dahlia spp. DvEPRS, previously referred to as DMV-D10, was originally isolated in the US from the cultivated Dahlia variabilis, and has been also found in New Zealand, Lithuania and Egypt as well as in wild dahlia species growing in their natural habitats in Mexico. Here we report the complete genome sequences of three new DvEPRS isolates from a Lithuanian cultivar (7159 nt), a New Zealander cultivar (7156 nt) and from the wild dahlia species, D. rupicola (7133 nt). The three have the structure and organization typical of a caulimovirus species and showed identities between 71 and 97% at the nucleotide level (nt) among various open reading frames (ORFs) when compared to those of DvEPRS. To better understand the genetic diversity, DvEPRS from cultivated and wild dahlia species were compared. A total of 7 full-length sequences were used for phylogenetic analyses, mutation frequencies, potential recombination events, selection and fitness as evolutionary evidences for genetic diversity. Phylogentic analyses showed one clade of all DvEPRS indicating a lack of clustering by geographical origin. When the various DvEPRS were grouped into two taxa, no difference was observed between those from cultivated and wild dahlia species. Assessment of population genetic parameters found strong negative selection for all ORFs, with the replicase region more variable than other ORFs. Identification of potential recombination events involving parents from different lineages provided strong evolutionary evidence for genetic diversity among various DvEPRS. This study contributes to an increased understanding of molecular population genetics and evolutionary pathways of these reverse transcribing viral elements

Rapid, Highly Sensitive and Quantitative Molecular Assays for Investigating Virus-Host and Virus-Virus Interactions using Negative-Stranded RNA Viruses as a Model System.

A highly sensitive method was developed and used for determining virus levels and to study the movement and distribution of viruses in infected plants

Seasonal dynamics of thrips (Thrips tabaci) (Thysanoptera: Thripidae) transmitters of iris yellow spot virus: a serious viral pathogen of onion bulb and seed crops

Thrips-transmitted Iris yellow spot virus (IYSV) is an important economic constraint to the production of bulb and seed onion crops in the United States and many other parts of the world. Because the virus is exclusively spread by thrips, the ability to rapidly detect the virus in thrips vectors would facilitate studies on the role of thrips in virus epidemiology, and thus formulation of better vector management strategies. Using a polyclonal antiserum produced against the recombinant, Escherichia coli-expressed nonstructural protein coded by the small (S) RNA of IYSV, an enzyme linked immunosorbent assay was developed for detecting IYSV in individual as well as groups of adult thrips. The approach enabled estimating the proportion of potential thrips transmitters in a large number of field-collected thrips collected from field-grown onion plants. Availability of a practical and inexpensive test to identify viruliferous thrips would be useful in epidemiological studies to better understand the role of thrips vectors in outbreaks of this economically important virus of onion