Ralf G. Dietzgen

Taxonomy of the order Mononegavirales: update 2016

In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.

Genetic Diversity Among Banana streak virus Isolates from Australia

ABSTRACT Banana streak virus (BSV) is an important pathogen of bananas and plantains (Musa spp.) throughout the world. We have cloned and sequenced part of the genomes of four isolates of BSV from Australia, designated BSV-RD, BSV-Cav, BSV-Mys, and BSV-GF. These isolates originated from banana cvs. Red Dacca, Williams, Mysore, and Goldfinger, respectively. All clones contained a sequence covering part of open reading frame III and the intergenic region of the badnavirus genome. The sequences were compared with those of other badnaviruses, including BSV-Onne, a previously characterized isolate from Nigeria. The BSV-RD sequence was virtually identical to that of BSV-Onne, differing by only two nucleotides over 1,292 bp. However, BSV-Cav, -Mys, and -GF were divergent in nucleotide sequence. Phylogenetic analyses using conserved sequences in the ribonuclease H domain revealed that all BSV isolates were more closely related to each other than to any other badnavirus. BSV-Cav was most closely related to BSV-Onne, and there was 95.1% identity between the two amino acid sequences. Other relationships between the BSV isolates were less similar, with sequence identities ranging from 66.4 to 78.2%, which is a magnitude comparable to the distance between some of the recognized badnavirus species. Immunocapture-polymerase chain reaction assays have been developed, allowing specific detection and differentiation of the four isolates of BSV.

Nucleotide sequence of one component of the banana bunchy top virus genome contains a putative replicase gene

One DNA component of the banana bunchy top virus (BBTV) genome was cloned and sequenced. This component is present as a circular, ssDNA in the virions and consists of 1111 nucleotides. It contains one large open reading frame (ORF) of 858 nucleotides in the virion sense; this ORF encodes a putative replicase based on the presence of a dNTP-binding motif (GGEGKT). Two smaller ORFs (249 and 366 nucleotides), in the complementary orientation, could not be assigned any obvious function. Neither of these ORFs had significant sequence homology with any known DNA plant virus gene or gene product. Computer analysis of this component-predicted a strong stem-loop structure in the virion sense putative untranslated region; a nonanucleotide sequence in the loop was nearly identical to the nonanucleotide invariant loop sequence of geminiviruses and coconut foliar decay virus. There is strong evidence that the genome of BBTV consists of more than one component because no ORF was found that would encode a protein the size of the BBTV coat protein. BBTV has some characteristics in common with geminiviruses but cannot be classified as one. Rather, BBTV probably belongs to an undescribed plant virus group which could also include subterranean clover stunt virus and coconut foliar decay virus.

The rhabdoviruses: biodiversity, phylogenetics, and evolution.

Rhabdoviruses (family Rhabdoviridae) include a diversity of important pathogens of animals and plants. They share morphology and genome organization. The understanding of rhabdovirus phylogeny, ecology and evolution has progressed greatly during the last 30 years, due to enhanced surveillance and improved methodologies of molecular characterization. Along with six established genera, several phylogenetic groups at different levels were described within the Rhabdoviridae. However, comparative relationships between viral phylogeny and taxonomy remains incomplete, with multiple representatives awaiting further genetic characterization. The same is true for rhabdovirus evolution. To date, rather simplistic molecular clock models only partially describe the evolutionary dynamics of postulated viral lineages. Ongoing progress in viral evolutionary and ecological investigations will provide the platform for future studies of this diverse family.

Detection of DNA and RNA plant viruses by PCR and RT-PCR using a rapid virus release protocol without tissue homogenization.

A simple, single-step plant tissue preparation protocol suitable for the detection of viruses by the polymerase chain reaction and reverse transcription-polymerase chain reaction is described. The effect of buffer components and pH, and the incubation temperature for the release of virus from plant material was evaluated. A small amount of plant tissue was heated in a solution containing 100 mM Tris-HCl, pH 7.4 or 8.4, 1 M KCl and 10 mM EDTA for 10 min at 95 degrees C and the supernatant used for enzymatic amplification. This protocol was suitable for the detection of both DNA and RNA viruses in a variety of plant species and tissues and reduced plant inhibitory factors which may interfere with PCR. The application of this method was demonstrated for the detection of banana bunchy top virus in banana leaves, root and corn, zucchini yellow mosaic potyvirus in squash leaves and lettuce necrotic yellows rhabdovirus in lettuce and Nicotiana glutinosa leaves.

Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana

A multiplex, immunocapture PCR (M-IC-PCR) was developed for the simultaneous detection of three viruses from crude sap extracts of banana and plantain (Musa spp.). A reverse transcription step was required for Banana bract mosaic virus and Cucumber mosaic virus, which have ssRNA genomes. The detection of Banana bunchy top virus (ssDNA genome) was not adversely affected by inclusion in this step. All the three viruses could be detected simultaneously from a mixed infection. Identification and detection of individual viruses was achieved through the visualisation of discretely sized PCR amplicons by gel electrophoresis. Alternatively, a colourimetric microplate detection system utilising digoxigenin-labelled virus-specific probes was used. The latter assay was up to five times more sensitive than detection by gel electrophoresis and between 25 and 625 times more sensitive than ELISA for the various viruses. Careful selection of PCR primers was necessary to ensure the detection of a wide range of virus isolates and to avoid detrimental interactions between heterologous templates and primers.

Purification, Characterization and Serological Detection of Virus-Like Particles Associated with Banana Bunchy Top Disease in Australia

Isometric virus-like particles, 18 nm in diameter, have been isolated from banana (Musa spp.) affected by bunchy top disease in Australia. Banana bunchy top disease-associated virus-like particles (BBTV) banded as a single component with buoyant density of 1.28 to 1.29 g/ml in Cs2SO4 and sedimented at about 46S in isokinetic sucrose density gradients. The A260/A280 of purified preparations was about 1.33. A single coat protein of Mr 20,500 was identified with antibodies to BBTV particles from Australia. Single-stranded DNA of about 1 kb as well as ssRNA smaller than 0.45 kb was also associated with the particles. A polyclonal antiserum to BBTV, suitable for use in ELISA, was prepared. Stability and antigenicity of purified BBTV was impaired by storage at pH greater than or equal to 8.5 and freezing at -20 degrees C without protectants. BBTV was detected by double antibody sandwich-ELISA with monoclonal and polyclonal antibodies, in field-infected banana plants, single aphids from an infective colony, and in experimentally aphid-inoculated banana plants. After transmission of BBTV particles by aphids from a banana bunchy top disease-affected to an uninfected banana plant, the disease was induced and BBTV was detected by ELISA in symptomatic leaves only. BBTV isolates from Australia, Taiwan, Peoples Republic of China, Tonga, Western Samoa and Hawaii were found to be serologically related, which suggests a common aetiology for the disease.

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the β-glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

Rhabdovirus accessory genes

Abstract The Rhabdoviridae is one of the most ecologically diverse families of RNA viruses with members infecting a wide range of organisms including placental mammals, marsupials, birds, reptiles, fish, insects and plants. The availability of complete nucleotide sequences for an increasing number of rhabdoviruses has revealed that their ecological diversity is reflected in the diversity and complexity of their genomes. The five canonical rhabdovirus structural protein genes (N, P, M, G and L) that are shared by all rhabdoviruses are overprinted, overlapped and interspersed with a multitude of novel and diverse accessory genes. Although not essential for replication in cell culture, several of these genes have been shown to have roles associated with pathogenesis and apoptosis in animals, and cell-to-cell movement in plants. Others appear to be secreted or have the characteristics of membrane-anchored glycoproteins or viroporins. However, most encode proteins of unknown function that are unrelated to any other known proteins. Understanding the roles of these accessory genes and the strategies by which rhabdoviruses use them to engage, divert and re-direct cellular processes will not only present opportunities to develop new anti-viral therapies but may also reveal aspects of cellar function that have broader significance in biology, agriculture and medicine.