The dsRNA Virus Papaya Meleira Virus and an ssRNA Virus Are Associated with Papaya Sticky Disease

Abstract

Papaya sticky disease, or “meleira”, is one of the major diseases of papaya in Brazil and Mexico, capable of causing complete crop loss. The causal agent of sticky disease was identified as an isometric virus with a double stranded RNA (dsRNA) genome, named papaya meleira virus (PMeV). In the present study, PMeV dsRNA and a second RNA band of approximately 4.5 kb, both isolated from latex of papaya plants with severe symptoms of sticky disease, were deep-sequenced. The nearly complete sequence obtained for PMeV dsRNA is 8,814 nucleotides long and contains two putative ORFs; the predicted ORF1 and ORF2 display similarity to capsid proteins and RdRp's, respectively, from mycoviruses tentatively classified in the family Totiviridae. The sequence obtained for the second RNA is 4,515 nucleotides long and contains two putative ORFs. The predicted ORFs 1 and 2 display 48% and 73% sequence identity, respectively, with the corresponding proteins of papaya virus Q, an umbravirus recently described infecting papaya in Ecuador. Viral purification in a sucrose gradient allowed separation of particles containing each RNA. Mass spectrometry analysis indicated that both PMeV and the second RNA virus (named papaya meleira virus 2, PMeV2) were encapsidated in particles formed by the protein encoded by PMeV ORF1. The presence of both PMeV and PMeV2 was confirmed in field plants showing typical symptoms of sticky disease. Interestingly, PMeV was detected alone in asymptomatic plants. Together, our results indicate that sticky disease is associated with double infection by PMeV and PMeV2.

Structural Analysis and Whole Genome Mapping of a New Type of Plant Virus Subviral RNA: Umbravirus-Like Associated RNAs

“…Another group of coat protein-dependent replicons are similar to tlaRNAs in their coding capacity (ORFs 1 and 2) but their RdRps are reported to be more closely related to those of umbraviruses ( Figure 1 ). As with umbraviruses, these umbravirus-like associated RNAs (ulaRNAs) generate their RdRp by a -1PRF event [ 15 , 16 , 17 ]. ulaRNAs infecting papaya and babaco in Brazil, Ecuador and Mexico are larger in size than tlaRNAs, with the size difference mainly due to the expanded length of their 3′ UTRs.…”

An Umbra-related Virus Found in Vasconcellea X Heilbornii (Caricaceae)

“…Umbraviruses have 4 -4.2 kb genomes containing four open reading frames (ORFs) but lack a capsid protein (CP) gene, which makes them dependent on a 'helper' virus, usually from the Luteoviridae, for genome encapsidation and plant-to-plant transmission [1,2]. In recent years, several viral RNA sequences discovered from different hosts have been regarded as "umbralike" viruses based on sequence homology to the RNA-dependent-RNA-polymerase (RdRp) [3][4][5][6]. However, biological and genomic differences warrant a further examination to determine whether these viruses should be included within the Umbravirus or belong to a distinct genus of umbra-related viruses.…”

“…However, biological and genomic differences warrant a further examination to determine whether these viruses should be included within the Umbravirus or belong to a distinct genus of umbra-related viruses. For example, papaya virus Q (PpVQ) and papaya meleira virus 2 (PMeV-2), two umbra-like viruses found in papaya plantings of Ecuador and Brazil, respectively, possess a ~ 4.4 kb genome containing two nonoverlapping ORFs (ORF1 and ORF2) followed by an 1,800-nt-long non-coding region (NCR) [3,4]. While no evidence for a luteovirid helper virus was found for PpVQ [7], a double-stranded RNA (dsRNA) virus, known as papaya meleira virus (PMeV), was found as the helper virus for PMeV-2 [4].…”

“…For example, papaya virus Q (PpVQ) and papaya meleira virus 2 (PMeV-2), two umbra-like viruses found in papaya plantings of Ecuador and Brazil, respectively, possess a ~ 4.4 kb genome containing two nonoverlapping ORFs (ORF1 and ORF2) followed by an 1,800-nt-long non-coding region (NCR) [3,4]. While no evidence for a luteovirid helper virus was found for PpVQ [7], a double-stranded RNA (dsRNA) virus, known as papaya meleira virus (PMeV), was found as the helper virus for PMeV-2 [4]. Interestingly, a similar umbra-like virus was reported in commercial papaya plants from Mexico and referred to as papaya meleira virus -Mexican variant (PMeV-Mx) [5].…”

RNase If -treated quantitative PCR for dsRNA quantitation of RNAi trait in genetically modified crops

“…Using the monoclonal dsRNA-specific mouse antibody, the dsRNA may be detected via immunoblot or immunohistochemistry [ 18 , 19 ]. Moreover, studies in viral biology use high (e.g., 96 °C) and low temperature (e.g., 70 °C) to denature the dsRNA and ssRNA, respectively [ 20 ]. Recently, a hybridization-based method using the QuantiGene technology (Affymetrix) has been reported for effective dsRNA quantitation [ 21 ].…”