We applied, for the first time, next generation sequencing to perform whole genome virus population studies of potato virus Y (PVY), NTN strain, in plant samples. Using ultra deep Illumina sequencing, the diversities of two coexisting Potato virus Y sequence pools present within a plant were investigated: RNA isolated form viral particles and virus derived small interfering RNAs (the derivates of a plant RNA silencing mechanism). The mutational landscape of the within-host virus populations were highly similar between both pools. Sequencing of viral particle pool enhanced the efficiency of consensus viral genome sequence reconstruction. Non-homologous recombinations were commonly detected in viral particle pool, with a hotspot in 3` untranslated and coat protein region of the genome. The work was published in Journal of Virology, the most cited journal in the virology category.
COBISS.SI-ID: 3333711
The emergence of next-generation "deep" sequencing has enabled the study of virus populations with much higher resolutions. This new tool increases the possibility of observing mixed infections caused by combinations of plant viruses, which are likely to occur more frequently than previously thought. The biological impact of co-infecting viruses on their host has yet to be determined and fully understood, and the first step towards reaching this goal is the separation and purification of individual species. Ion-exchange monolith chromatography has been used successfully for the purification and concentration of different viruses, and number of them have been separated from plant homogenate or bacterial and eukaryotic lysate. Thus, the question remained as to whether different virus species present in a single sample could be separated. In this study, anion-exchange chromatography using monolithic supports was optimized for fast and efficient partial purification of three model plant viruses: Turnip yellow mosaic virus, Tomato bushy stunt virus, and Tobacco mosaic virus. The virus species, as well as two virus strains, were separated from each other in a single chromatographic experiment from an artificially mixed sample. Based on A260/280 ratios, we were able to attribute specific peaks to a certain viral morphology/structure (icosahedral or rod-shaped). This first separation of individual viruses from an artificially prepared laboratory mixture should encourage new applications of monolithic chromatographic supports in the separation of plant, bacterial, or animal viruses from all kinds of mixed samples.
COBISS.SI-ID: 3344463
Potato virus X is one of the most widespread viruses of potato. However, little is known about its diversity in its likely center of radiation, the Andean region of South America. To fill this gap, the strategy of Illumina deep sequencing of small RNAs was used to obtain complete or near complete genome sequence of PVX from symptomatically infected plant samples from Peru. A complete genome sequence of a representative of a new PVX phylogenetic lineage was obtained, which shows a high level of sequence dissimilarity to other completely sequenced isolates. One of the field samples was infected with the mixture of two PVX strains, which were efficiently discriminated using small RNA sequencing approach. The study confirms the utility of small RNAs deep sequencing for successful viral strain differentiation and discovery of new viral strains and indicates a high diversity of PVX in the Andean region of South America, a pattern which may be expected also for other potato pathogens.
COBISS.SI-ID: 3188559
A fluorescence-based real-time loop-mediated isothermal amplification (LAMP) assay for ‘Candidatus Phytoplasama solani’ (Bois noir phytoplasma; BNp) detection was developed and optimised for rapid laboratory and on-site BNp detection. This assay is highly specific, rapid and as sensitive as qPCR. It was validated according to European and Mediterranean Plant Protection Organisation recommendations. In addition, 286 grapevine leaf samples from the 2015 growing season were tested with this new real-time LAMP assay and an assay previously developed for detection of Flavescence dorée phytoplasma (FDp). These LAMP assays for detection of both BNp and FDp used without any DNA extraction step, which is a required step for qPCR analysis, were comparably effective to qPCR, and positive results were obtained in less than 35 min.
COBISS.SI-ID: 4088399
Raspberry leaf blotch virus (RLBV), a member of the genus Emaravirus, has recently been reported to be associated with raspberry leaf blotch disorder in Scotland. It was found in several samples of raspberry in England, Serbia, Finland and Bulgaria. Red raspberry plants with similar symptoms were found in Montenegro in 2011 and infection with RLBV was confirmed in symptomatic plants using RT-PCR. Two RNA5 amplicons were cloned and sequenced and compared with sequences in GenBank. BLAST analysis of Montenegrin RLBV nucleotide sequence showed 93% nucleotide (nt) identity with three available RLBV RNA5 sequences (HG738849, HG738846 and FR823303) from Bulgaria and the United Kingdom. The deduced protein sequence was identical to sequences from Bulgaria (CDJ26745) and showed 96.1% identity and 100% similarity to P5 sequence from the UK (CBZ42028). Our results show that the prevalence of RLBV in red raspberry in Montenegro is high. This finding will help to improve the quality of the increasing red raspberry production in Montenegro.
COBISS.SI-ID: 4779880