Two viruses from Stylosanthes guianensis may represent a new genus within Potyviridae.

Forage crops occupy large areas of tropical pastures for cattle feeding in Brazil. The use of stylos (Stylosanthes spp.) in these pastures, which are leguminous shrubs, has increased in the country due to their outstanding nutritional value and for being an efficient and alternative source for nitrogen fixation in the soil. In recent years, virus-like mosaic symptoms on S.guianensis leaves have often been observed in the field, indicating possible virus-like pathogen infections. In an effort to identify the causal agent, virus semi-purification protocol was performed using symptomatic S. guianensis leaves collected at EMBRAPA Beef Cattle Research Center. Total RNA extracted from this semi-purified preparation was submitted to high-throughput sequencing, which revealed complete genome sequences of novel viruses of the family Potyviridae. These viruses, tentatively named stylo mosaic-associated virus 1 (StyMaV-1) and stylo mosaic-associated virus 2 (StyMaV-2), shared 73 % CP aa identity and 77 % polyprotein aa identity with each other and, after that, being closest related to blackberry virus Y, genus Brambyvirus (only 41 % CP aa identity). Based on ICTV genus demarcation criteria, StyMaV-1 and StyMaV-2 represent new species of a new genus within the family Potyviridae. StyMaV-1 and StyMaV-2 are also not efficiently transmitted to other plant species by mechanical inoculation.

An isolate of sweet potato chlorotic stunt virus from Brazil with a distinct genome organization.

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae), is an economically important pathogen of sweet potato. In the present work, the nucleotide sequences of two RNA segments of SPCSV (isolate SPCSV-UNB-01) were determined by MiSeq Illumina sequencing of samples of sweet potato plants grafted onto Ipomoea setosa. A comparative analysis of the genome organization of SPCSV-UNB-01 and other SPCSV sequences showed that RNA1 was lacking p22, and p5.1 and that p5.2. was absent in RNA2, indicating a unique genomic pattern. SPCSV-UNB-01 contained longer p6 and p5 regions, with little similarity to orthologous sequences. Sequence comparison did not reveal any previously identified functional domains within these open reading frames (ORFs). No recombination or rearrangement events were detected. Phylogenetic analysis suggested the possibility of separate entries of SPCSV into South America based on the genetic distance between SPCSV-UNB-01 and the Peruvian isolate m2-47. Samples from northeastern Brazil (State of Pernambuco) were positive for SPCSV when tested using specific primers for the major coat protein (CP) gene. This is the first full-length genome sequence of SPCSV-UNB-01 from Brazil.

A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector.

Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host-virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants.