Putative gene promoter sequences in the chlorella viruses

Three short (7 to 9 nucleotides) highly conserved nucleotide sequences were identified in the putative promoter regions (150 bp upstream and 50 bp downstream of the ATG translation start site) of three members of the genus Chlorovirus, family Phycodnaviridae. Most of these sequences occurred in similar locations within the defined promoter regions. The sequence and location of the motifs were often conserved among homologous ORFs within the Chlorovirus family. One of these conserved sequences (AATGACA) is predominately associated with genes expressed early in virus replication.     

A high capacity Alphavirus heterologous gene delivery system

A novel replication competent Sindbis virus based gene delivery vector has been developed for the introduction of genetic cargo into cell lines in vitro and potentially, animal models in vivo. This delivery system expands the previous uses of Sindbis virus as a gene delivery system in that no replicons are required and the resulting cargo containing virus particles are infectious. The heterologous vector is based on a morphological mutant in C, Ser180/Gly183 which produces larger than the normal size T = 4 virus particles of 70 nm in size. This mutant produced particles up to 205 nm in size equal to a triangulation number of 36. It was postulated that because the Ser180/Gly183 mutant was capable of assembling such large particles, that increasing the size of the RNA genome incorporated into this mutant capsid protein would favor the assembly of larger than T = 4 wild type sized virions. The first generation prototype larger vehicle, described here, carries a ~ 18 kb cDNA insert, however it is conceivable that RNA as large as 32 kb could be transcribed and packaged. The large variant produces a high virus titer of ~ 10⁹ pfu/ml from either mammalian or insect cells in culture. Multiple passages of the virus show no loss of the inserted genetic material.     

Sequence Divergence of Four Soilborne Sugarbeet-Infecting Viruses

Soilborne viruses are among the most harmful pathogens of sugarbeet (Beta vulgaris L.ssp. vulgaris) but most of them lack information on genetic variability due to paucity of sequence data. Only one isolate of Beet soil borne virus (BSBV; genus Pomovirus), Beet virus Q (BVQ; genus Pomovirus) and Beet soil borne mosaic virus (BSBMV; genus Benyvirus) has been characterised for the coat protein (CP) gene. In this study, the CP gene sequences of three isolates each of BSBV and Beet necrotic yellow vein virus (BNYVV; genus Benyvirus) (France, Germany and USA), two isolates of BVQ (France and Germany), and one isolate of BSBMV (USA) were determined. Phylogenetic analyses including sequences from databanks indicated that the French BNYVV isolate of this study belongs to so-called P-type, the American isolate to A-type and the German isolate to B-type. The CP genes of the three BSBV isolates characterised in this study and the one available from databank were highly identical (98.4–99.0% at nucleotide level; one variable amino acid). The BSBMV isolate studied here differed from the previously characterised isolate for five nucleotides and four amino acids in the CP region. The two BVQ isolates characterised in this study contained three additional nucleotides resulting in an additional amino acid residue (arginine) at CP position 86, as compared to the only isolate available in databank.     

Unique RNA 2 sequences of two Brazilian isolates of Pepper ringspot virus,a tobravirus

Pepper ringspot virus (PepRSV) is a tobravirus reported only in Brazil. Here, the sequences of the complete RNA 2 segments and the 3′ 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 second 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 similarities with known 2b genes of tobraviruses, an essential gene of tobraviruses for nematodes-transmission. The 5′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 isolates. 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 similarity. This is the first report on the diversity of the RNA 2 sequences of PepRSV.     

The core region of the coat protein gene is highly useful for establishing the provisional identification and classification of begomoviruses

Summary.  Polymerase chain reaction (PCR) was applied to detect and establish provisional identity of begomoviruses through amplification of a ∼ 575 bp fragment of the begomoviral coat protein gene (CP), referred to as the ‘core’ region of the CP gene (core CP). The core CP fragment contains conserved and unique regions, and was hypothesized to constitute a sequence useful for begomovirus classification. Virus relationships were predicted by distance and parsimony analyses using the A component (bipartite viruses) or full genome (monopartite viruses), CP gene, core CP, or the 200 5′-nucleotides (nt) of the CP. Reconstructed trees and sequence divergence estimates yielded very similar conclusions for all sequence sets, while the CP 5′-200 nt was the best strain discriminator. Alignment of the core CP region for 52 field isolates with reference begomovirus sequences permitted provisional virus identification based on tree position and extent of sequence divergence. Geographic origin of field isolates was predictable based on phylogenetic separation of field isolates examined here. A ‘closest match’ or genus-level identification could be obtained for previously undescribed begomoviruses using the BLAST program to search a reference core CP database located at our website and/or in GenBank. Here, we describe an informative molecular marker that permits provisional begomovirus identification and classification using a begomoviral sequence that is smaller than the presently accepted, but less accessible CP sequence. Received September 26, 2000/Accepted January 26, 2001     

Variability in the coat protein gene of Papaya ringspot virus isolates from multiple locations in India

Summary. The coat protein (CP) sequences of eleven Papaya ringspot virus (PRSV) isolates originating from different locations in India were determined, analysed and compared with the sequences of other isolates of PRSV. The virus isolates from India exhibited considerable heterogeneity in the CP sequences. The CP-coding region varied in size from 840–858 nucleotides, encoding protein of 280–286 amino acids. Comparative sequence analysis revealed that the PRSV isolates originating from India were divergent up to 11%. Though the PRSV isolates were differentiated in to two clusters, yet the sequence variation could not be correlated with the geographical origin of the isolates. Implication of the sequence variation in the coat protein derived transgenic resistance in papaya is discussed.     

Sequence diversity of NS(M) movement protein of tospoviruses

Summary.  In order to determine the diversity of the movement protein (NS_M) among tospoviruses, the NS_M genes of five distinct tospovirus species occurring in Brazil (Tomato chlorotic spot virus, Groundnut ring spot virus, Chrysanthemum stem necrosis virus, Zucchini lethal chlorosis virus and Iris yellow spot virus) were cloned, sequenced and compared with NS_M sequences of other available tospoviruses. The ‘D-motif’, a conserved region present in the majority of ‘30K superfamily’ virus movement proteins, is present in all NS_M amino acid sequences available. In addition to the ‘D-motif’, a conserved phospholipase A2 motif was found. The NS_M amino acid sequence comparisons among tospovirus species revealed several conserved regions located in the internal part of the protein and diverse domains mainly located in the amino-terminus. Prediction of secondary structure showed similar patterns among all NS_M proteins analyzed. Considering the geographical prevalence and phylogenetic analysis of N and NS_M proteins, tospoviruses were tentatively clustered in ‘American’ and ‘Eurasian’ groups. Both phylogenetic trees may reflect the natural evolution of tospovirus species within distinct ecological niches. The sequence information obtained in this work would facilitate functional analysis of NS_M during the tospovirus infection process. Received July 18, 2000 Accepted February 28, 2001     

Identification of a Naturally Occurring Recombinant Isolate of <Emphasis Type="Italic">Sugarcane Mosaic Virus</Emphasis> Causing Maize Dwarf Mosaic Disease

The complete nucleotide sequence of a potyvirus causing severe maize dwarf mosaic disease in Shaanxi province, northwestern China was determined (GenBank accession No. AY569692). The full genome is 9596 nucleotides in length excluding the 3 -terminal poly (A) sequence. It contains a large open reading frame (ORF) flanked by a 149 nt 5-untranslated region (UTR) and a 255 nt 3-UTR. The putative polyprotein encoded by this large ORF comprises of 3063 amino acid residues. Sequence comparisons and phylogenetic analyses showed that this potyvirus is an isolate of Sugarcane mosaic virus (SCMV). The entire sequences shared identities of 89.6–97.6 % and 79.3–93.3% with 9 sequenced SCMV isolates at the nucleotide and deduced amino acid levels, respectively. But it showed much lower identities with Maize dwarf mosaic virus (MDMV), Sorghum mosaic virus (SrMV) and Johnsongrass mosaic virus (JGMV) isolates. The putative coat protein sequence is identical to that of a Chinese maize isolate SCMV-HZ. However, partition comparisons and phylogenetic profile analyses of the viral nucleotide sequences indicated that it is a recombinant isolate of SCMV. The recombination sites are located within the 6K1 and CI coding regions.     

Complete Nucleotide Sequence of the RNA-2 of Grapevine Deformation and Grapevine Anatolian Ringspot Viruses

The nucleotide sequence of RNA-2 of Grapevine Anatolian ringspot virus (GARSV) and Grapevine deformation virus (GDefV), two recently described nepoviruses, has been determined. These RNAs are 3753 nt (GDefV) and 4607 nt (GARSV) in size and contain a single open reading frame encoding a polyprotein of 122 kDa (GDefV) and 150 kDa (GARSV). Full-length nucleotide sequence comparison disclosed 71–73% homology between GDefV RNA-2 and that of Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV), and 62–64% homology between GARSV RNA-2 and that of Grapevine chrome mosaic virus (GCMV) and Tomato black ring virus (TBRV). As previously observed in other nepoviruses, the 5 non-coding regions of both RNAs are capable of forming stem-loop structures. Phylogenetic analysis of the three proteins encoded by RNA-2 (i.e. protein 2A, movement protein and coat protein) confirmed that GDefV and GARSV are distinct viruses which can be assigned as definitive species in subgroup A and subgroup B of the genus Nepovirus, respectively. The nucleotide sequences data reported in this paper were submitted to the GenBank database and given the accession numbers AY291207 for the Grapevine Anatolian ringspot virus and AY291208 for the Grapevine deformation virus.     

Molecular Analysis of Fiji Disease Virus Segments 2, 4 and 7 Completes the Genome Sequence

The complete nucleotide sequences of Fiji disease virus (FDV) genome segments S2, S4 and S7 were determined. This now completes the sequencing of all ten dsRNA genome segments of the Fijivirus type member, FDV, which comprises a total of 29339 nt. FDV S2, S4 and S7 comprised 3820, 3568 and 2194 nt, respectively. S2 and S4 each contained a single open reading frame (ORF), which encoded putative proteins of 137 and 133 kDa, respectively, while S7 contained two ORFs, which encoded putative proteins of 42 and 37 kDa. The putative amino acid sequences of FDV S2 and S4 showed most similarity to the gene products of Rice black-streaked dwarf virus (RBSDV) S2 and RBSDV S3, respectively. The putative amino acid sequences of FDV S7 ORF I and II showed most similarity to Maize rough dwarf virus (MRDV) S6 ORF I and RBSDV S7 ORF II, respectively. Phylogenetic analyses showed that FDV was most closely related to the group 2 fijiviruses.     

The complete nucleotide sequence of the RNA2 of the crinivirus tomato infectious chlorosis virus: isolates from North America and Europe are essentially identical

The complete nucleotide sequences of the RNA2 of two isolates of Tomato infectious chlorosis virus (TICV, genus Crinivirus, family Closteroviridae) from the United States and Spain, respectively, were determined. The sequences of both isolates were found to be nearly identical. TICV RNA2 consisted of 7,914 nucleotides in both isolates and contains eight open reading frames that encompass the Closteroviridae hallmark gene array represented by a heat shock protein 70 family homologue, a protein of 59 kDa, the major coat protein, and a divergent copy of the coat protein. Phylogenetic analysis suggested that TICV is most similar to Lettuce infectious yellows virus (LIYV), the type species of the genus Crinivirus.     

Apolipophorin-III in Galleria mellonella potentiates hemolymph lytic activity

Heat-inactivated serum of non-immune Galleria mellonella larvae enhanced the lytic activity of larval cell-free hemolymph against Micrococcus lysodeikticus. The increase in bacterial lysis was due to a 17.2 kDa protein known previously to bind to bacterial lipopolysaccharides. The protein enhanced the lytic activity of insect cell-free hemolymph and hen lysozyme in vitro and insect hemolymph in vivo. The hydrophobic protein, which adhered to M. lysodeikticus, was identified by its amino acid sequence homology as apolipophorin-III. The titer of apolipophorin-III in 200–250 mg last instar larvae was 8.7 mg/ml of hemolymph. Apolipophorin-III did not bind to lysozyme. A possible mode of action of apolipophorin-III with lysozyme in the insect is proposed.     

The Molecular Variability Analysis of the RNA 3 of Fifteen Isolates of Prunus Necrotic Ringspot Virus Sheds Light on the Minimal Requirements for the Synthesis of its Subgenomic RNA

The nucleotide sequences of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus (PNRSV) varying in the symptomatology they cause in six different Prunus spp. were determined. Analysis of the molecular variability has allowed, in addition to study the phylogenetic relationships among them, to evaluate the minimal requirements for the synthesis of the subgenomic RNA in Ilarvirus genus and their comparison to other members of the Bromoviridae family. Computer assisted comparisons led recently to Jaspars (Virus Genes 17, 233–242, 1998) to propose that a hairpin structure in viral minus strand RNA is required for subgenomic promoter activity of viruses from at least two, and possibly all five, genera in the family of Bromoviridae. For PNRSV and Apple mosaic virus two stable hairpins were proposed whereas for the rest of Ilarviruses and the other four genera of the Bromoviridae family only one stable hairpin was predicted. Comparative analysis of this region among the fifteen PNRSV isolates characterized in this study revealed that two of them showed a 12-nt deletion that led to the disappearance of the most proximal hairpin to the initiation site. Interestingly, the only hairpin found in these two isolates is very similar in primary and secondary structure to the one previously shown in Brome mosaic virus to be required for the synthesis of the subgenomic RNA. In this hairpin, the molecular diversity was concentrated mostly at the loop whereas compensatory mutations were observed at the base of the stem strongly suggesting its functional relevance. The evolutionary implications of these observations are discussed.