Complete genome sequence of a novel bromovirus infecting elderberry (Sambucus nigra L.) in the Czech Republic.

The genus Bromovirus currently contains six species whose members have relatively narrow host ranges. In the present work, a new bromovirus infecting elderberry (Sambucus nigra L.) is reported. dsRNA was purified and sequenced by next-generation sequencing, and with minimal additional completion by Sanger sequencing, the full tripartite genome was obtained. RNA1 is 3241 nt long and contains ORF1 (1a protein), RNA2 is 2810 nt long and contains ORF2 (2a protein), and RNA3 is 2244 nt long and contains ORF3a (movement protein) and ORF3b (coat protein, CP), separated by an intercistronic poly(A) stretch. Proteins 1a and 2a showed highest sequence identity (69.9% and 69.4%) to the corresponding proteins of melandrium yellow fleck virus. The coat protein showed highest sequence identity (67.9%) to that of brome mosaic virus. The genome shows a typical bromovirus organisation comprising of all the conserved protein domains within the genus. Phylogenetic analysis supports the assignment of this virus as a new member of the genus Bromovirus, for which the name "sambucus virus S" (SVS) is proposed.

Molecular characterization of the complete genomes of two new field isolates of Cowpea chlorotic mottle virus, and their phylogenetic analysis.

Cowpea chlorotic mottle virus (CCMV, family Bromoviridae) is found worldwide and has been used as a model virus for a long time, but no data is available about the genetic diversity of field isolates. Recently, two new field isolates (Car1 and Car2) of CCMV obtained from cowpea showed distinct phenotypic symptoms when inoculated to cowpea. CCMV-Car1 induced severe mosaic and interveinal chlorosis, while CCMV-Car2 produced mild mottling and leaf rolling. Both isolates produced asymptomatic infection in Nicotiana benthamiana. The complete genome of both isolates was amplified by reverse transcription-polymerase chain reaction using specific primers against the CCMV sequences available in the GenBank database, cloned and sequenced. Both nucleotide and amino acid sequences were compared between the newly sequenced CCMV isolates and the three previously characterized CCMV strains (T, M1, and R). Phylogenetic analysis of the RNA 1 sequence showed that CCMV-Car1 was in a separate branch from the rest of the CCMV isolates while CCMV-Car2 grouped together with CCMV-R. On the basis of RNA 2 and RNA 3 sequences, two major groupings were obtained. One group included CCMV-Car1 and CCMV-Car2 isolates while the other contained CCMV-T, CCMV-M1, and CCMV-R strains. Recombination programs detected a potential recombination event in the RNA 1 sequence of CCMV-Car2 isolate but not in RNA 2 and RNA 3 sequences. The results showed that both mutations and recombination have played an important role in the genetic diversity of these two new isolates of CCMV.

The interplay between capsid dynamics and pathogenesis in tripartite bromoviruses.

Infectious virus capsids or virions are considered static structures and undergo various conformational transitions to replicate and infect a wide range of eukaryotic cells. Therefore, virus capsids must be stable enough to overcome the physicochemical environment and flexible enough to reorganize their biologically relevant surface peptides for optimal interaction with the host machinery. Although viral capsid fluctuations, referred to as dynamics or breathing, have been well studied in RNA viruses pathogenic to animals, such information is limited among plant viruses. However, more recent attempts have been made in characterizing the capsid dynamics in the plant virus genus bromovirus characterized by having a tripartite, positive-sense RNA genome. Using the available research data on the genus bromovirus members, this review is focused on updating the readers on the interrelationships between the viral capsid dynamics and host-pathogen interactions.

A simple technique for separation of Cowpea chlorotic mottle virus from Cucumber mosaic virus in natural mixed infections.

A simple technique was developed to separate Cowpea chlorotic mottle virus (CCMV) from Cucumber mosaic virus (CMV) in natural mixed infections. Sap from cowpea leaves infected naturally with a mixture of CCMV and CMV was inoculated mechanically on the first tri-foliolate leaf of cowpea seedlings. Both inoculated and non-inoculated upper leaves were sampled 3 or 8 days post-inoculation and tested by reverse transcription polymerase chain reaction (RT-PCR) using primers specific to CCMV and CMV. RT-PCR analysis showed the presence of only CCMV in the inoculated leaf and both viruses in the non-inoculated systemically infected upper leaves. Total RNA from the inoculated leaves positive to CCMV only was further confirmed upon re-inoculation to cowpea seedlings. Typical CCMV symptoms were produced within 1 week and RT-PCR analysis showed only the presence of CCMV in both inoculated and non-inoculated systemically infected upper leaves. Systemically infected upper leaves of the same plants were used for CCMV purification. RT-PCR analysis of the purified virion and RNA extracted from the virion further confirmed the absence of CMV contamination. To our knowledge, this is the first report of a method separating CCMV directly from mixed infections with CMV in cowpea.

Structure of Cowpea mottle virus: a consensus in the genus Carmovirus.

Cowpea mottle virus (CPMoV) is a T = 3 virus that belongs to Carmovirus genus of the Tombusviridae family. Here, we report the crystal structure of CPMoV determined to a resolution of 7.0 angstroms. The structures and sequences of three Carmoviruses, CPMoV, Turnip crinkle virus (TCV), and Carnation mottle virus (CarMV) have been compared to TBSV from the Tombusvirus genus. CPMoV, TCV, and CarMV all have a deletion in betaC strand in the S domain relative to TBSV that may be distinctive to the genus. Although CPMoV has an elongated C-terminus like TBSV, it does not interact with the icosahedrally related P domain as observed in TBSV. In CPMoV, the termini of A and B interact with the icosahedrally related shell domains of A and C, respectively, to form a chain of interactions around the 5-fold axes. The C subunit terminus does not, however, interact with the B subunit because of quasi-equivalent differences in the P domain orientations.