Small RNA NGS Revealed the Presence of Cherry Virus A and Little Cherry Virus 1 on Apricots in Hungary

Abstract

Fruit trees, such as apricot trees, are constantly exposed to the attack of viruses. As they are propagated in a vegetative way, this risk is present not only in the field, where they remain for decades, but also during their propagation. Metagenomic diagnostic methods, based on next generation sequencing (NGS), offer unique possibilities to reveal all the present pathogens in the investigated sample. Using NGS of small RNAs, a special field of these techniques, we tested leaf samples of different varieties of apricot originating from an isolator house or open field stock nursery. As a result, we identified Cherry virus A (CVA) and little cherry virus 1 (LChV-1) for the first time in Hungary. The NGS results were validated by RT-PCR and also by Northern blot in the case of CVA. Cloned and Sanger sequenced viral-specific PCR products enabled us to investigate their phylogenetic relationships. However, since these pathogens have not been described in our country before, their role in symptom development and modification during co-infection with other viruses requires further investigation.

High-Throughput Sequencing Assists Studies in Genomic Variability and Epidemiology of Little Cherry Virus 1 and 2 infecting Prunus spp. in Belgium

“…The presence of CVA, PDV and PrVF was also confirmed by conventional RT-PCR with specific primers and validated by Sanger sequencing (Tables S1–S3, and ref. [10,42,43,44].…”

Recent Advances on Detection and Characterization of Fruit Tree Viruses Using High-Throughput Sequencing Technologies

“…These molecules can be isolated from the infected plants, subjected to HTS, and used to reconstruct viral genomes. Virus-derived siRNAs have been used to detect known and unknown plant viruses in fruit trees by HTS [ 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ]. The advantage of this approach is that siRNAs can be used to detect RNA and DNA viruses, and also, to detect integrated endogenous viral elements (EVEs), as long as they are transcribed.…”

“…More specifically, several variants of PBNSPaV were retrieved from a project of sequencing four Prunus samples of unclear or unknown etiology [ 100 ], while a first isolate from sweet cherry was sequenced in China [ 101 ]. Recently, isolates of LChV1 were sequenced from peach in South Korea [ 102 ], apricot in Hungary [ 41 ], and sweet cherry in China [ 103 ]. Full genome sequences from isolates of CVA were obtained from apricot in Japan [ 104 ] and Hungary [ 41 ], and from sweet cherry in China [ 105 ].…”

“…Recently, isolates of LChV1 were sequenced from peach in South Korea [ 102 ], apricot in Hungary [ 41 ], and sweet cherry in China [ 103 ]. Full genome sequences from isolates of CVA were obtained from apricot in Japan [ 104 ] and Hungary [ 41 ], and from sweet cherry in China [ 105 ]. In a wild cherry tree from Slovakia, together with a multiple viral infection, Glasa et al [ 9 ] found two divergent isolates of CVA (differing by 14% at the nucleotide level) and gave some evidence on the recombinant nature of these isolates and for the presence of “non-cherry” group CVA isolates in cherry host plants.…”

“…Moreover, the high number of different reads obtained from the HTS run makes possible the reconstruction of the full genome sequences of a virus species, which can be subsequently used to perform genetic variability studies. Complete viral genomes or big fragments of them have been obtained using either siRNAs [ 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ], dsRNA [ 40 , 43 , 44 , 45 , 46 , 48 , 49 , 50 , 71 , 106 ], or total RNA [ 9 , 40 , 49 , 57 , 76 ] template from a multitude of different fruit tree plants. Where HTS excels, however, is the identification of genetic variants of a virus species that often co-exist in the same perennial host.…”