Unique RNA 2 sequences of two Brazilian isolates of Pepper ringspot virus, a tobravirus Adriana Ribeiro Silva Batista • Cı ´cero Nicolini • Kelly Barreto Rodrigues • Fernando Lucas Melo • Raquel Medeiros Vasques • Mo ˆnica Alves de Mace ˆdo • Alice Kazuko Inoue-Nagata • Tatsuya Nagata Received: 28 January 2014 / Accepted: 1 April 2014 Ó Springer Science+Business Media New York 2014 Abstract Pepper ringspot virus (PepRSV) is a tobravirus reported only in Brazil. Here, the sequences of the com- plete RNA 2 segments and the 3 0 end of the RNA 1 genomic regions of two new isolates from tomato plants were analyzed. The main ORF encodes the CP gene as other tobraviruses and termed ORF 1 of RNA 2. The sec- ond ORF was found only in one of the new isolates, although this gene was absent in the type isolate, CAM (collected in the 1960’s). Interestingly, this ORF 2 gene did not show any nucleotide and amino acid sequence simi- larities with known 2b genes of tobraviruses, an essential gene of tobraviruses for nematodes-transmission. The 5 0 UTR sequence of RNA 2 segment of CAM isolate was previously reported showing two impaired direct repeats; however, the direct-repeats were absent in these new iso- lates. An additional ORF was predicted upstream of the CP gene. This putative protein possessed a transmembrane domain similar to the ORFN1 of RNA 2 of Tobacco rattle virus SYM isolate, although there was no sequence simi- larity. This is the first report on the diversity of the RNA 2 sequences of PepRSV. Keywords Virgaviridae Á Tobravirus Á TRV Á RNA 2 segment Á Recombination Introduction Currently, the genus Tobravirus consists of three distinct virus species, Tobacco rattle virus (TRV), Pea early- browning virus (PEBV), and Pepper ringspot virus (Pep- RSV) [1, 2]. TRV is reported to occur in Europe, North and Central America, New Zealand, Australia, China, and Japan; PEBV is present in Western Europe, especially in the Netherlands and UK; and PepRSV is restricted to Brazil [2–4]. Tobraviruses have a bi-segmented single- stranded RNA genome with positive polarity. The RNA 1 segment forms the longer particle with approximately 6.8 kb, and isolates of the same virus species share a high sequence identity [4, 5]. This genome fragment encodes the small and the large (by ribosome read-through) replicase subunit, the cell-to-cell movement protein (p29 or P1a) and the PTGS (post-transcriptional gene silencing) suppressor protein (p16 or P1b). In contrast to the RNA 1, RNA 2 shows considerable variability among the isolates [4, 6]. The tobravirus RNA 2 encodes the coat protein (CP) and generally one or two nonstructural proteins [7–10], but some TRV isolates are reported with an unusual RNA 2 coding strategy [11]. Tobraviruses are naturally transmitted by nematodes. There is evidence that one or more nonstructural proteins (2b and 2c), encoded in the RNA 2 segment (downstream of the CP gene), are involved in the nematode-transmission process [4, 8, 12, 13]. At least the presence of 2b protein is essential for the virus transmission by nematodes [4, 9, 14, 15]. In nature, many tobravirus RNA 2 molecules are recombinants with RNA 1, particularly in the 3 0 end, which Electronic supplementary material The online version of this article (doi:10.1007/s11262-014-1066-8) contains supplementary material, which is available to authorized users. A. R. S. Batista Á K. B. Rodrigues Á T. Nagata (&) Instituto de Cie ˆncias Biolo ´gicas, Universidade de Brası ´lia, Brası ´lia, DF, Brazil e-mail: tatsuya@unb.br C. Nicolini Á F. L. Melo Á R. M. Vasques Á T. Nagata Laborato ´rio de Virologia, Instituto de Cie ˆncias Biolo ´gicas, Universidade de Brası ´lia, Brası ´lia, DF, Brazil M. A. de Mace ˆdo Á A. K. Inoue-Nagata Embrapa Vegetables, Brası ´lia, DF, Brazil 123 Virus Genes DOI 10.1007/s11262-014-1066-8