An integrated protein localization and interaction map for Potato yellow dwarf virus, type species of the genus Nucleorhabdovirus

The genome of Potato yellow dwarf virus (PYDV; Nucleorhabdovirus type species) was determined to be 12,875 nucleotides (nt). The antigenome is organized into seven open reading frames (ORFs) ordered 3'-N-X-P-Y-M-G-L-5', which likely encode the nucleocapsid, phospho, movement, matrix, glyco and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. The ORFs are flanked by a 3' leader RNA of 149 nt and a 5' trailer RNA of 97 nt, and are separated by conserved intergenic junctions. Phylogenetic analyses indicated that PYDV is closely related to other leafhopper-transmitted rhabdoviruses. Functional protein assays were used to determine the subcellular localization of PYDV proteins. Surprisingly, the M protein was able to induce the intranuclear accumulation of the inner nuclear membrane in the absence of any other viral protein. Finally, bimolecular fluorescence complementation was used to generate the most comprehensive protein interaction map for a plant-adapted rhabdovirus to date.     

Complete genome sequence of a Watermelon silver mottle virus isolate from China

The complete genome of a Watermelon silver mottle virus (WSMoV) (genus Tospovirus, family Bunyaviridae) isolate (WSMoV-GZ) from Guangdong province, China was sequenced. The genomes of WSMoV-GZ contained 3,603, 4,909, and 8,914 nt of small (S), medium (M), and large (L) RNA segments, respectively, and had a genomic organization characteristic of members of the genus Tospovirus. The amino acid sequence of the nucleocapsid (N) protein, S RNA-encoded nonstructural (NSs) protein, M RNA-encoded nonstructural (NSm) protein, Gn/Gc glycoprotein precursor, and RNA-dependent RNA polymerase (RdRp) protein showed 94.3–97.5 % identity with those of other WSMoV isolates. Phylogenetic analysis showed that the N protein of WSMoV-GZ was clustered together with those of the WSMoV isolates. The full sequence of WSMoV-GZ provides a reference genome for comparison with other tospoviruses.     

Complete genome sequence of a novel monopartite begomovirus infecting sweet potato in China

The complete genome sequence of a new monopartite begomovirus isolate SC-1 was obtained from sweet potato samples in Sichuan province, China. The viral genome consists of 2,764 nucleotides (nt) and encodes two open reading frames (ORFs) called AV1 and AV2 genes in the viral-sense strand and four ORFs (AC1–AC4) in the complementary-sense strand. Sequence comparisons revealed that it shared the highest level of nt sequence identity (81.2 %) with Sweet potato leaf curl Georgia virus (AF326775). Phylogenetic analysis showed that the SC-1 genome was in a separate clade from other 29 begomovirus isolates. Thus, the SC-1 isolate is a novel species according to the demarcation criteria of species in the genus Begomovirus, for which the name “Sweet potato leaf curl China Sichuan Virus” (SPLCCSV) is proposed. Recombination analysis suggests that SPLCCSV has sequences derived from recombination between Sweet potato leaf curl virus (SPLCV) isolate GZ01 (JX286653) and SPLCV isolate Merremia N4 (DQ644563).     

The complete genome sequence of Lamium mild mosaic virus, a member of the genus Fabavirus

Lamium mild mosaic virus (LMMV) is the only one of the five members of the genus Fabavirus for which there are no nucleotide sequence data. In this study, the complete genome sequence of LMMV was determined and compared with the available complete genome sequences of other members of the genus Fabavirus. The genome was the largest of the genus but maintained the typical organization, with RNA 1 of 6080 nucleotides (nt), RNA 2 of 4065 nt, and an unusually long 3′ untranslated region in RNA 2 of 603 nt. Phylogenetic analysis of the amino acid sequences of the protease-polymerase (Pro-Pol) region and the two coat proteins confirmed that LMMV belongs to a distinct species within the genus Fabavirus.     

Complete nucleotide sequence of a putative new cytorhabdovirus infecting lettuce

Summary The full-length nucleotide sequence of the genomic RNA of a new cytorhabdovirus infecting lettuce was determined. Six open reading frames were found in the antigenomic sequence of the 12,926-nt negative-sense viral RNA genome. The genomic organisation was similar to that of lettuce necrotic yellows virus (LNYV), the type member of the genus Cytorhabdovirus: 3′-N-P-3-M-G-L-5′, where N is the capsid protein gene, P the putative phosphoprotein gene, 3 a gene coding for a putative protein of unknown function, M the putative matrix protein gene, G the glycoprotein gene, and L the putative polymerase gene. Amino acid sequence comparison with the corresponding sequences of other rhabdoviruses revealed the closest relationship to LNYV, with identities ranging from 41% for the matrix proteins and 65% for the L polymerase proteins. These results indicate that this virus may be a member of a new cytorhabdovirus species, for which the name Lettuce yellow mottle virus (LYMoV) is proposed. Correspondence: Thierry Wetzel, RLP Agroscience, AlPlanta – Institute for Plant Research, Breitenweg 71, 67435 Neustadt an der Weinstrasse, Germany     

Kotonkan and Obodhiang viruses: African ephemeroviruses with large and complex genomes

Kotonkan virus (KOTV) and Obodhiang virus (OBOV) are rhabdoviruses that were isolated from arthropods in Africa and formerly classified as lyssaviruses. KOTV causes clinical bovine ephemeral fever in cattle; the ecology and pathogenicity of OBOV is poorly understood. In this paper, we report the complete genome sequences of KOTV and OBOV, their gene expression profiles, and their serological and phylogenetic relationships to other rhabdoviruses. The 15,870 nt KOTV genome (3′-l-N-P-M-G-G_NS-α1-α2-β-γ-δ-L-t-5′) is similar to that of bovine ephemeral fever virus but encodes an additional protein (δ) that shares homology with the pleckstrin homology domain of coactivator-associated arginine methyltransferase. The 14,717 nt OBOV genome (3′-l-N-P-M-G-G_NS-α1-α2-β-L-t-5′) is similar to that of Adelaide River virus from which it is distinguishable serologically. In each virus, all ORFs, except α1 and α2, are transcribed as monocistronic mRNA. Genetic and serological data indicate that KOTV and OBOV should be classified as new species in the genus Ephemerovirus.     

A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family

Summary.  A universal primer (Sprimer: 5^′-GGX AAY AAY AGY GGX CAZ CC-3^′, X = A, G, C or T; Y = T or C; Z = A or G), designed from the consensus sequences that code for the conserved sequence GNNSGQP in the NIb region of members of the family Potyviridae, was used to amplify by RT-PCR the 3′-terminal genome regions from infected plant samples representing 21 different viruses in the family. Sequencing of some of the fragments (c. 1.7 kb) showed that the type strain (ATTC PV-107) of Oat necrotic mottle virus is not a distinct species in the genus Rymovirus, but is synonymous with Brome streak mosaic virus (genus Tritimovirus) and that Celery mosaic virus is a distinct member of the genus Potyvirus not closely related to any other sequenced species. Potyviruses infecting crops in China were also investigated, showing that viruses on cowpea and maize in Hangzhou, Zhejiang province were respectively Bean common mosaic virus and Sugarcane mosaic virus and that one on garlic in Nanjing, Jiangsu province was Onion yellow dwarf virus. Fragments were also sequenced from Chinese isolates of Lettuce mosaic virus and Soybean mosaic virus (from Hangzhou), Turnip mosaic virus (2 different isolates from Zhejiang province) and RNA1 of Wheat yellow mosaic virus (from Rongcheng, Shandong province). Received June 30, 2000 Accepted September 28, 2000     

Establishment of three new genera in the family Geminiviridae: Becurtovirus,Eragrovirus and Turncurtovirus

The family Geminiviridae includes plant-infecting circular single-stranded DNA viruses that have geminate particle morphology. Members of this family infect both monocotyledonous and dicotyledonous plants and have a nearly global distribution. With the advent of new molecular tools and low-cost sequencing, there has been a significant increase in the discovery of new geminiviruses in various cultivated and non-cultivated plants. In this communication, we highlight the establishment of three new genera (Becurtovirus, Eragrovirus and Turncurtovirus) to accommodate various recently discovered geminiviruses that are highly divergent and, in some cases, have unique genome architectures. The genus Becurtovirus has two viral species, Beet curly top Iran virus (28 isolates; leafhopper vector Circulifer haematoceps) and Spinach curly top Arizona virus (1 isolate; unknown vector), whereas the genera Eragrovirus and Turncurtovirus each have a single assigned species: Eragrostis curvula streak virus (6 isolates; unknown vector) and Turnip curly top virus (20 isolates; leafhopper vector Circulifer haematoceps), respectively. Based on analysis of all of the genome sequences available in public databases for each of the three new genera, we provide guidelines and protocols for species and strain classification within these three new genera.     

Molecular characterization of the largest and smallest genome segments,S1 and S12, of <Emphasis Type="Italic">Rice gall dwarf virus</Emphasis>

The nucleotide sequences of segments S1 and S12 of a Chinese isolate of Rice gall dwarf virus (RGDV) were determined. This provides the first complete sequences of these segments. The complete sequence of S1, the largest genome segment of RGDV, was 4,505 nucleotides in length and was predicted to encode a large protein of 1,458 amino acids with a calculated molecular mass of nearly 166.2 kDa. The protein was related to that encoded by S1 of Rice dwarf virus (RDV; 50% identity and 67% similarity) and (to a lesser extent) to some large proteins of other reoviruses. It appears to be an RNA-dependent RNA polymerase (RdRp) and is probably present in particles as a minor core protein. S12, the smallest genome segment of RGDV, was 853 nucleotides in length, encoding a single major protein of 206 amino acids with a calculated molecular mass of nearly 23.6 kDa. This protein, though a little larger than those of RDV S11 and Wound tumor virus (WTV) S12 in size, showed some similarity to them, especially in the conserved N-terminal region and may have RNA-binding properties. Despite having a common host plant, RDV and RGDV were not more closely related to one another than either of them was to WTV. Phylogenetic analysis of the RdRp showed that members of the genus Phytoreovirus were more closely related to those of the genus Rotavirus than to any other genus within the family Reoviridae. The nucleotide sequence data reported in this article have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers DQ333946 and DQ494209.     

Detection and differentiation of bovine herpesvirus 1 and 5 using a multiplex real-time polymerase chain reaction

This study investigated the suitability of a two step real-time RT-PCR melting curve analysis as a tool for the detection and discrimination of nine species in the plant virus family Luteoviridae, being Soybean dwarf virus [SbDV], Bean leafroll virus [BLRV], Beet chlorosis virus [BChV], Beet mild yellowing virus [BMYV], Beet western yellows virus [BWYV], Cereal yellow dwarf virus-RPV [CYDV-RPV], Cucurbit aphid-borne yellows virus [CABYV], Potato leafroll virus [PLRV] and Turnip yellows virus [TuYV]. Melting temperature and shape of the melting peak were analysed for 68 bp and 148 bp coat protein gene amplicons using SYBR^® GreenER™ fluorescent dye. Specific melting peaks with unique melting temperature were observed for the various species of the family Luteoviridae using the 68 bp amplicon, but not with the 148 bp amplicon. Due to the high variability of sequences for some members of this family, different melting temperatures were also observed between different isolates of the species CYDV-RPV and TuYV. Nevertheless, discrimination between species was achieved for SbDV, BLRV, BChV, BMYV, CABYV and either PLRV or BWYV. Melting curve analysis, in this study, is a faster and more discriminatory alternative to gel electrophoresis of end-point PCR products for the detection of Luteoviridae infection.     

Revisiting the classification of curtoviruses based on genome-wide pairwise identity

Members of the genus Curtovirus (family Geminiviridae) are important pathogens of many wild and cultivated plant species. Until recently, relatively few full curtovirus genomes have been characterised. However, with the 19 full genome sequences now available in public databases, we revisit the proposed curtovirus species and strain classification criteria. Using pairwise identities coupled with phylogenetic evidence, revised species and strain demarcation guidelines have been instituted. Specifically, we have established 77 % genome-wide pairwise identity as a species demarcation threshold and 94 % genome-wide pairwise identity as a strain demarcation threshold. Hence, whereas curtovirus sequences with >77 % genome-wide pairwise identity would be classified as belonging to the same species, those sharing >94 % identity would be classified as belonging to the same strain. We provide step-by-step guidelines to facilitate the classification of newly discovered curtovirus full genome sequences and a set of defined criteria for naming new species and strains. The revision yields three curtovirus species: Beet curly top virus (BCTV), Spinach severe surly top virus (SpSCTV) and Horseradish curly top virus (HrCTV).     

Nine novel DNA components associated with the foorkey disease of large cardamom: Evidence of a distinct babuvirus species in Nanoviridae

Foorkey disease is a serious constraint to the production of large cardamom (Amomum subulatum, family Zingiberaceae). The disease is characterized by profuse proliferation of excessive stunted shoots, which makes the clump totally unproductive. The disease has been known in India since 1936 but the complete genome of the virus had not yet been characterized. In a preliminary study, an associated virus tentatively named as Cardamom bushy dwarf virus (CBDV) was identified based on the partial sequence of a single DNA component (DNA-R). In the present study, a high incidence (37.2–39.3%) of foorkey was recorded in certain plantations in the Darjeeling hills located at lower altitudes (300–1380 m) and CBDV was detected in several field samples by PCR. Nine novel DNA components were isolated and characterized from foorkey affected plants. CBDV contained six major DNA components (DNA-R, -S, -M, -C, -N and -U3) similar to the integral genome components known for the members of the genus Babuvirus in the family Nanoviridae. Additional components, satellite Rep (DNA-sRep1) and unknown components (DNA-Uf1 and -Uf2) were also identified. The size of the genome components ranged from 1028 to 1127. The sequence identity and phylogeny based on the individual components as well as overall genome (59.8–62% identity) distinguished CBDV from the two existing babuvirus species, Banana bunchy top virus and Abaca bunchy top virus. CBDV is the first distinct babuvirus species that affects plant species outside family Musaceae. This study shows further diversity in the genus Babuvirus.     

<Emphasis Type="Italic">Pepo aphid</Emphasis>-<Emphasis Type="Italic">borne yellows virus</Emphasis>: a new species in the genus <Emphasis Type="Italic">Polerovirus</Emphasis>

Pepo aphid-borne yellows virus (PABYV) has been proposed as a putative representative of a new species in the genus Polerovirus in the family Luteoviridae. The genomes of two South African (SA) isolates of cucurbit-infecting PABYV were described in this record. Total RNA, extracted from a pattypan (Cucurbita pepo L.) and a baby marrow (C. pepo L.) leaf samples, was subjected to next-generation sequencing (NGS) on the HiSeq Illumina platform. Sanger sequencing was subsequently used to authenticate the integrity of PABYV’s genome generated from de novo assembly of the NGS data. PABYV genome of SA isolates consists of 5813 nucleotides and displays an organisation typical of poleroviruses. Genome sequence comparisons of the SA PABYV isolates to other poleroviruses support the classification of PABYV as a new species in the genus Polerovirus. Recombination analyses showed that PABYV and Cucurbit aphid-borne yellows virus (CABYV) shared the same ancestor for the genome part situated between breaking points. Phylogenetic analyses of the RNA-dependent RNA polymerase and the coat protein genes showed that SA PABYV isolates shared distant relationship with CABYV and Suakwa aphid-borne yellows virus. Based on our results, we propose that PABYV is a distinct species in the genus Polerovirus.     

A natural M RNA reassortant arising from two species of plant- and insect-infecting bunyaviruses and comparison of its sequence and biological properties to parental species

Reassortment allows multicomponent viruses to exchange genome segments, a process well-documented in the vertebrate- and arthropod-infecting members of the family Bunyaviridae but not between distinct species of the plant- and insect-infecting members of the genus Tospovirus. Genome sequence comparisons of a virus causing severe tospovirus-like symptoms in Florida tomato with Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV) demonstrated that reassortment has occurred, with the large (L) and small (S) RNAs coming from GRSV and the medium (M) RNA coming from TCSV (i.e. L_GM_TS_G). Neither parental genotype is known to occur in the U.S. suggesting that L_GM_TS_G was introduced as a reassortant. L_GM_TS_G was transmitted by western flower thrips (Frankliniella occidentalis [Pergande]), and was not able to overcome the Sw5 resistance gene of tomato. Our demonstration of reassortment between GRSV and TCSV suggests caution in defining species within the family Bunyaviridae based on their ability to reassort.     

Complete sequence analysis of rice transitory yellowing virus and its comparison to rice yellow stunt virus

Rice transitory yellowing virus (RTYV), a member of the genus Nucleorhabdovirus, is closely related to or synonymous with rice yellow stunt virus (RYSV). To clarify the relationship between RTYV and RYSV, we determined the nucleotide sequence of the RTYV genome. The RTYV genome consists of 14,029 nucleotides. The overall nucleotide identity between RTYV and RYSV was 98.5%, and the deduced amino acid sequence identities between the seven genes in RTYV and RYSV ranged from 82.3 to 99.7%. The sequence information from RTYV revealed that these two viruses should be categorized as members of the same species rather than distinct species.     

Phylogeny, classification and evolutionary insights into pestiviruses

The genus Pestivirus comprises four established species: Bovine viral diarrhoea viruses 1 (BVDV-1) and 2 (BVDV-2), Border disease virus (BDV), and Classical swine fever virus (CSFV); and a tentative species, Pestivirus of giraffe. Additional pestiviruses have been identified and suggested for recognition as novel subgroups/species. To achieve a reliable phylogeny as the basis for classification of pestiviruses, a molecular dataset of 56 pestiviruses and 2089 characters, comprising the 5′UTR, complete N^pro and E2 gene regions was analysed by Maximum likelihood and Bayesian approach. An identical, robust tree topology was inferred, where seven well-supported monophyletic clades and two highly divergent lineages were identified. Dating most recent common ancestor was estimated for major pestivirus lineages and their evolutionary histories were revealed. Accordingly, a new proposal is presented for the classification of pestiviruses into nine species: BVDV-1, BVDV-2, BVDV-3 (atypical bovine pestiviruses), Pestivirus of giraffe, CSFV, BDV, Tunisian sheep virus (TSV; previously termed “Tunisian isolates”), Antelope and Bungowannah.