Identification and characterization of privet leaf blotch‐associated virus, a novel idaeovirus

Abstract

A novel virus has been identified by next-generation sequencing (NGS) in privet (Ligustrum japonicum L.) affected by a graft-transmissible disease characterized by leaf blotch symptoms resembling infectious variegation, a virus-like privet disease with an unclear aetiology. This virus, which has been tentatively named 'privet leaf blotch-associated virus' (PrLBaV), was absent in non-symptomatic privet plants, as revealed by NGS and reverse transcription-polymerase chain reaction (RT-PCR). Molecular characterization of PrLBaV showed that it has a segmented genome composed of two positive single-stranded RNAs, one of which (RNA1) is monocistronic and codes for the viral replicase, whereas the other (RNA2) contains two open reading frames (ORFs), ORF2a and ORF2b, coding for the putative movement (p38) and coat (p30) proteins, respectively. ORF2b is very probably expressed through a subgenomic RNA starting with six nucleotides (AUAUCU) that closely resemble those found in the 5'-terminal end of genomic RNA1 and RNA2 (AUAUUU and AUAUAU, respectively). The molecular signatures identified in the PrLBaV RNAs and proteins resemble those of Raspberry bushy dwarf virus (RBDV), currently the only member of the genus Idaeovirus. These data, together with phylogenetic analyses, are consistent with the proposal of considering PrLBaV as a representative of the second species in the genus Idaeovirus. Transient expression of a recombinant PrLBaV p38 fused to green fluorescent protein in leaves of Nicotiana benthamiana, coupled with confocal laser scanning microscopy assays, showed that it localizes at cell plasmodesmata, strongly supporting its involvement in viral movement/trafficking and providing the first functional characterization of an idaeovirus encoded protein.

The first phlebo‐like virus infecting plants: a case study on the adaptation of negative‐stranded RNA viruses to new hosts

“…Specific primers (Table S3) were designed into putative viral contigs to generate overlapping cDNA by RT‐PCR. RNA preparation, RT‐PCR solutions and conditions were similar to those reported previously (Navarro et al , ), except that the polymerase extension step ranged from 1 to 3 min, depending on the size of the expected PCR product. The termini of the viral genomic RNAs were determined by RACE.…”

“…Three primer pairs (Bunya‐cit.1F/Bunya‐cit.4R; CG.15/CG.20; CG.18/CG.19) (Table S3) were designed to generate PCR amplicons of 485, 292 and 154 nt from regions of the CCGaV genome coding for the RdRP (RNA1), nucleocapsid and MP proteins (RNA2), respectively. Total nucleic acids (100 ng) were extracted and reverse transcribed as reported previously (Navarro et al , ). PCR amplification was performed using 2 µL of the cDNA reaction and 1.25 units of GoTaq polymerase (Promega) in a reaction volume of 25 µL, containing a final concentration of 0.2 µ m of each primer.…”

A Negative-Stranded RNA Virus Infecting Citrus Trees: The Second Member of a New Genus Within the Order Bunyavirales

“…The primer pair Ka-1 (5′-TCCTGATGAAGTCTTAAGATCGC-3′) and Ka-3 (5′-TTGCAGTAGTGAGAAGGGAGT-3′) was designed to amplify a cDNA fragment of 620 nt of CiVA RNA2 ( Supplementary Table S1 ). TNA (100 ng) were extracted and reverse-transcribed as reported previously ( Navarro et al, 2017 ). An aliquot (2 μl) of the cDNA reaction was used for PCR amplification performed in a reaction volume of 25 μl containing 1.25 units of GoTaq polymerase (Promega, Madison, WI, United States) and a final concentration of 0.2 μM for each primer.…”

Blackcurrant Leaf Chlorosis Associated Virus: Evidence of the Presence of Circular RNA during Infections

“…BCLCaV genomic components include: (1) RNA-1 that is monocistronic with a single open reading frame (ORF1) that encodes the replicase complex (M r 197 kDa); (2) a bicistronic RNA-2 that encodes a putative movement protein (MP, ORF2a, M r 38.8 kDa) and the putative coat protein (CP, ORF2b, M r 30 kDa) of the virus, with ORFs that overlap by a single adenine (A) nucleotide (nt), representing the third position of an opal stop codon of ORF2a and the first position of the start codon of ORF2b; (3) a subgenomic form of RNA-2 (putative RNA-3) that contains ORF2b, the putative CP coding region; and (4) a concatenated form of RNA-2 that consists of an inverted RNA-3 conjoined to the full-length RNA-2 [ 1 , 2 ]. Navarro et al [ 3 ] recently described a novel virus in privet, privet leaf blotch-associated virus (PrLBaV) and proposed that PrLBaV be recognized also as a species in the genus Idaeovirus .…”

“…RBDV, PrLBaV, and BCLCaV are closely related phylogenetically; they possess similar genome organizations expressing similar proteins of similar sizes and their 3′ terminal nucleotide (nt) sequences form typical stem loops and are terminated by a 3′ poly(C) tail [ 1 , 2 , 3 , 7 ], supporting classification of the latter two viruses as members of the genus Idaeovirus . There are certain peculiarities associated with these viruses.…”