Passion Fruit Green Spot Virus Genome Harbors a New Orphan ORF and Highlights the Flexibility of the 5′-End of the RNA2 Segment Across Cileviruses

Abstract

Passion fruit green spot and passion fruit sudden death are two reportedly distinct viral diseases that recurrently affect passion fruit (Passiflora spp.) groves in Brazil. Here we used a systematic approach that interconnects symptoms, transmission electron microscopy, RT-PCR detection assays followed by Sanger sequencing, and highthroughput sequencing of the RNA of affected passion fruit plants to gain insights about these diseases. Our data confirmed not only the involvement of cileviruses in these two pathologies, as previously suggested, but also that these viruses belong to the same tentative species: passion fruit green spot virus (PfGSV). Results revealed that PfGSV has a positive-sense RNA genome split into two molecules of approximately 9 kb (RNA1) and 5 kb (RNA2), which share about 50-70% nucleotide sequence identity with other viruses in the genus Cilevirus. Genome sequences of five PfGSV isolates suggest that they have more conserved RNA1 (<5% of nucleotide sequence variability) compared to RNA2 (up to 7% of variability) molecules. The highest nucleotide sequence divergence among PfGSV isolates and other cileviruses is in the genomic segment covering from the 5-end of the RNA2 until the 5-end of the open reading frame (ORF) p61, which includes the ORF p15 and the intergenic region. This genomic stretch also harbors a novel orphan ORF encoding a 13 kDa protein presenting a cysteine-rich domain. High variability of 5end of the RNA2 in cileviruses is discussed in an evolutionary context assuming that they share putative common ancestors with unclassified arthropod-infecting single-strand positive RNA viruses, including mosquito-specific viruses of the group Negevirus (clades Nelorpivirus and Sandwavirus), and other viruses in the family Kitaviridae.

Virome Analysis of Aphid Populations That Infest the Barley Field: The Discovery of Two Novel Groups of Nege/Kita-Like Viruses and Other Novel RNA Viruses

“…Members of these two proposed genera, together with two proposed taxa of arthropod-restrictive viruses, "Nelorpivirus" and "Sandewavirus, " which all have non-segmented genomes, could be classified into a novel viral family or be assigned to the family Kitaviridae. However, it is to too early to establish a family for these viruses or to assign them into the Kitaviridae, because there is no reliable statistical support for these lineages within the currently available phylogenetic trees (Kallies et al, 2014;Nunes et al, 2017;Kondo et al, 2019;Ramos-González et al, 2020; Figure 3). The result of molecular phylogenetics indicate that the two major lineages of kitaviruses (cile/higreviruses and bulunerviruses) are not monophyletic, which all have two or multi-segmented RNA genomes and separately encode their replicase (RdRp) and SP24 family protein on the different segments (Locali-Fabris et al, 2006;Melzer et al, 2013;Hao et al, 2018), suggesting that genome segmentation possibly occurred independently during the evolution of kitaviruses.…”

“…Based on phylogenetic analysis, this taxon could be separated into two groups at the genus level, namely "Nelorpivirus" and "Sandewavirus" (Kallies et al, 2014). Negeviruses are distantly related to members of three plant virus genera, Cilevirus, Higrevirus, and Blunervirus (Vasilakis et al, 2013;Kallies et al, 2014;Nunes et al, 2017;Ramos-González et al, 2020). These genera have recently been assigned as the members of the family Kitaviridae (Walker et al, 2019).…”

Sequence Relationships of RNA Helicases and Other Proteins Encoded by Blunervirus RNAs Highlight Recombinant Evolutionary Origin of Kitaviral Genomes

“…The family Kitaviridae includes a small number of positive-stranded RNA plant viruses. Interestingly, this family name is not derived from the name of the type virus member, as usually, but from the family name of Dr. Elliot Watanabe Kitajima, a prominent virologist participating in kitavirus studies (Ramos-González et al, 2020;Quito-Avila et al, in press). Members of the kitavirus genera Blunervirus, Cilevirus, and Higrevirus produce in planta bacilliform (or near spherical) virus particles and have multipartite genomes composed of two, three, or four RNAs for cile-, higre-and bluner-viruses, respectively.…”

“…Members of the kitavirus genera Blunervirus, Cilevirus, and Higrevirus produce in planta bacilliform (or near spherical) virus particles and have multipartite genomes composed of two, three, or four RNAs for cile-, higre-and bluner-viruses, respectively. In addition to common particle morphology, most viruses share some important biological peculiarities, such as lack of systemic movement (Ramos-González et al, 2020;Quito-Avila et al, in press).…”

“…Molecular phylogenetic analyses of the RNA-dependent RNA polymerase (RdRp) domain or concatenated sequences of methyl transferase (MT), replicative RNA helicase (HEL), and RdRp unambiguously showed that kitaviruses are most closely related to members of family Virgaviridae [this family name is also not derived from the name of the type virus, like Bromoviridae, but from Latin word virga (rod), as all viruses in this family are rod-shaped] followed by those of the family Bromoviridae (Quito-Avila et al, 2013;Nunes et al, 2017;Hao et al, 2018;Ramos-González et al, 2020). Accordingly, cileviruses and blunerviruses encode a putative movement protein (MP) with sequence similarity to the MP of Bromoviridae (Hao et al, 2018;Quito-Avila et al, in press).…”