Molecular characterisation of subgenomic single-stranded and double-stranded DNA forms isolated from plants infected with tomato golden mosaic virus.

A subgenomic single-stranded DNA present in particles of the geminivirus, tomato golden mosaic virus, has been shown by electron microscope heteroduplex mapping and Southern hybridisation analysis to consist of circular molecules, ca. 1.2 kb in size, derived from the smaller of the two genomic DNA components, DNA B, by deletion of open reading frame (ORF) BR1 and the C-terminal portion of ORF BL1. A covalently closed circular, supercoiled, double-stranded form of the subgenomic DNA has been isolated from virus-infected plants and cloned into pEMBL9. Analysis of the sequence of 22 clones across the deletion boundaries revealed only four different deletion boundaries, derived from four different left hand borders and three different right hand borders. Each border was within a region of 11 nucleotides and gave rise to a narrow size range (1248-1261 nucleotides) for the population of 22 subgenomic DNAs. However apparently smaller subgenomic DNAs were sometimes formed when plants were inoculated with cloned subgenomic DNA, or a construct derived from a subgenomic DNA in which a neomycin phosphotransferase gene had been inserted, together with the genomic DNA components. Mechanisms to account for the size, specificity and formation of the subgenomic DNA are discussed.     

Olive latent virus-1, an isometric virus with a single RNA species isolated from olive in Apulia, Southern Italy

A mechanically transmissible virus isolated from some olive trees growing in Apulia, Southern Italy is described under the name of olive latent virus-1 (OLV–1). Virus particles (c. 30 nm in diameter) sedimented as a single component (11 IS) and contained a major single-stranded RNA species which when denatured had a size of about 4100 nucleotides and up to seven smaller RNAs. When centrifuged to equilibrium either in CsCl or CS2SO4 gradients, virus preparations were homogeneous. In polyacrylamide gel electrophoresis, about 50% of virus coat protein preparations formed a band of mol. wt 32 000 (32K protein) but two other bands of mol. wt 36 000 (36K protein) and 65 000 (65K protein) and some diffuse bands were also observed. The 32K protein, its probable dimer (65K protein) and diffuse bands are regarded as components of virus coat protein, whereas the 36K protein is probably a contaminant from the host plant. Virus particles were stabilised by divalent cations, pH-dependent bonds and salt links between protein and RNA. EDTA-treated virions were sensitive to ribonuclease and SDS. OLV-1 was serologically unrelated to four tombusviruses, seven sobemoviruses or putative sobemoviruses, and seven other single component isometric plant viruses.     

Molecular cloning and characterisation of the two DNA components of tomato golden mosaic virus.

We report the molecular cloning of the tomato golden mosaic virus (TGMV) genome in the E. coli plasmid pAT 153. The results of this work conclusively show that TGMV DNA consists of two components (designated A and B) of almost, but not exactly, the same size. Four different recombinant plasmids are described, two containing component A in opposite orientation and two containing component B in opposite orientation. Southern blot analysis revealed little sequence homology between A and B and showed both components to be equally represented in viral and intracellular DNA forms. Detailed restriction maps of the cloned DNAs are presented, and a comparison of these with digests of intracellular viral dsDNA indicates that the former are full-length faithful copies of TGMV DNA. This is the first report of the cloning of a geminivirus genome.     

Molecular characterisation of equid alphaherpesvirus 1 strains isolated from aborted fetuses in Poland

Background

Equid alphaherpesvirus 1 (EHV-1) is one of the main infectious causative agents of abortion in mares and can also be associated with stillbirth, neonatal foal death, rhinopneumonitis in young horses and a neurological disorder called equine herpesvirus myeloencephalopathy (EHM). The neuropathogenicity of the virus was shown to be significantly higher in EHV-1 strains that carry a single nucleotide point (SNP) mutation in the ORF30, which encodes a catalytic subunit of viral DNA polymerase (ORF30 D₇₅₂). Another gene, ORF68 is frequently used for phylogenetic analysis of EHV-1.

Methods

27 EHV-1 strains isolated from aborted equine fetuses in Poland, collected between 1993 and 2017, were subjected to PCR targeting the open reading frames (ORFs) 30 and 68 of the EHV-1 genome. PCR products obtained were sequenced and SNPs were analyzed and compared to sequences available in GenBank.

Results

None of the analyzed sequences belonged to the ORF30 D₇₅₂neuropathogenic genotype: all EHV-1 belonged to the non-neuropathogenic variant N₇₅₂. On the basis of ORF68 sequences, the majority of EHV-1 sequences (76.9%) cannot be assigned to any of the known groups; only six sequences (23.1%) clustered within groups II and IV.

Conclusions

EHV-1 strains obtained from abortion cases belong to the non-neuropathogenic genotype. Many EHV-1 ORF68 sequences have similar SNPs to those already described in Poland, but a clear geographical distribution was not observed. A single particular ORF68 sequence type was observed in strains isolated from 2001 onwards.

     

Molecular characterisation of cDNA clones representing pectin esterase isozymes from tomato

Two pectin esterase cDNA clones representing different isozymes with ca. 95% homology were isolated from an early ripening tomato fruit cDNA library. Both clones were longer than previously published sequences, and the encoded proteins possessed extended (229–233 amino acid) putative N-terminal extensions. In addition, the mRNA species corresponding to the two clones showed differential levels of expression in fruit.     

The full-length genome analysis of a street rabies virus strain isolated in Yunnan province of China

The epidemic of rabies has rapidly increased and expanded in Yunnan province in recent years.In order to further analyze and understand the etiological reasons for the rapid expansion of rabies in Yunn...     

Molecular characterisation of Xanthomonas strains isolated from aroids in Mauritius

Mauritius is one of the largest world producers of Anthurium cut flowers but outbreaks of bacterial blight have never been reported on the island. This work was about the characterisation and identification of bacterial strains isolated from Anthurium andreanum, Dieffenbachia maculata and Aglaonema simplex in Mauritius. Fifteen strains, that showed the morphological properties of Xanthomonas on conventional media, were tested on two semi-selective media (Esculin-trehalose and cellobiose-starch). ELISA tests using a panel of monoclonal antibodies were carried out and three out of 15 strains reacted with a Xanthomonas-specific monoclonal antibody (MAb XII). Analysis using four sets of ribosomal primers revealed that the same three Mauritius strains shared conserved PCR products with reference xanthomonads including virulent strains of Xanthomonas axonopodis pv. dieffenbachiae (Xad). BIOLOG tests and the Sherlock Microbial Identification system (MIDI) identified these three new strains at the species level as X. axonopodis. The complementary tests that were carried out clearly confirmed that the three strains are xanthomonads and, moreover, a DNA probe which showed specificity to Xad strains suggested that the three Mauritius strains are non-virulent forms of the pathogen causing Anthurium blight.     

我国2009年新分离乙脑病毒全基因组序列特征研究

本研究对我国2009年新分离的两株乙脑病毒进行全基因组序列测定和分析,以了解病毒全基因组分子特征。通过RT-PCR和核苷酸序列测定方法获得病毒全基因组序列,采用ClustalX、DNASTAR、MEGA等生物学软件完成核苷酸序列及氨基酸序列分析和系统进化分析等。研究结果显示,新分离两株乙脑病毒YN0911和YN0967株基因组全长均为10 965个核苷酸,编码3 432个氨基酸。这2株乙脑病毒之间核苷酸同源性为98.7%,氨基酸同源性为99.8%。与国际乙脑病毒流行株相比,核苷酸同源性为83.5%～98.9%,氨基酸同源性为94.8%～99.7%。与乙脑病毒疫苗株SA14-14-2相比,在E蛋白有13个氨基酸差异位点,但都位于抗原关键位点之外。这2株病毒在3′UTR区域存在11nt缺失。基于C/PrM区段、E基因、全基因组系统进化分析结果均显示新分离2株乙脑病毒为G I乙脑病毒,并且和越南、四川、贵州、广西以往的分离株遗传进化关系较近。本研究提示我国新分离的2株乙脑病毒均为G I乙脑病毒,决定病毒毒力的关键氨基酸位点未见明显变化。     

Molecular characterisation of Neoparamoeba strains isolated from gills of Scophthalmus maximus

Small subunit ribosomal RNA gene sequences were determined for 5 amoeba strains of the genus Neoparamoeba Page, 1987 that were isolated from gills of Scophthalmus maximus (Linnaeus, 1758). Phylogenetic analyses revealed that 2 of 5 morphologically indistinguishable strains clustered with 6 strains identified previously as N. pemaquidensis (Page, 1970). Three strains branched as a clade separated from N. pemaquidenis and N. aestuarina (Page, 1970) clades. Our analyses suggest that these 3 strains could be representatives of an independent species. In a more comprehensive eukaryotic tree, strains belonging to Neoparamoeba spp. formed a monophyletic group with a sister-group relationship to Vannella anglica Page, 1980. They did not cluster with Gymnamoebae of the families Hartmannellidae, Flabellulidae, Leptomyxidae or Amoebidae presently available in GenBank.     

Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples

Long RT-PCR (LRP) amplification of RNA templates is sometimes difficult compared to long PCR of DNA templates. Among RNA templates, hepatitis C virus (HCV) represents an excellent example to challenge the potential of LRP technology due to its extensive secondary structures and its difficulty to be readily cultured in vitro. The only source for viral genome amplification is clinical samples in which HCV is usually present at low titers. We have created a comprehensive optimization protocol that allows robust amplification of a 9.1 kb fragment of HCV, followed by efficient cloning into a novel vector. Detailed analyses indicate the lack of potential LRP-mediated recombination and the preservation of viral diversity. Thus, our LRP protocol could be applied for the amplification of other difficult RNA templates and may facilitate RNA virus research such as linked viral mutations and reverse genetics.     

Complete genome sequence of lymphocystis disease virus isolated from China

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.     

Molecular Characterization of a Cucumber Bulgarian latent virus Isolate from Iran

Cucumber Bulgarian latent virus (CBLV) was first reported from cucumber in Bulgaria in 2003 and has been assigned to the genus Tombusvirus. Ten years after the first and only report of CBLV, an isolate from a cucumber sample collected in Iran was characterized. Its complete genomic sequence was determined and analysed. Except for the coat protein, CBLV shows the highest sequence identities to the isolates of other species of the genus Tombusvirus. However, sequence comparison and phylogenetic analyses based on the coat protein (CP) revealed that CBLV is more closely related to the genus Aureusvirus rather than to the isolates of the genus Tombusvirus. The sequence identities to some aureusviruses are above the species demarcation threshold value, demonstrating that CBLV is an unusual tombusvirus species. This suggests that it is necessary to review the CP threshold value for species demarcation in the genus Aureusvirus. In addition, CBLV has an intermediate genome size compared to other tombus‐ and aureusviruses. Several polyclonal antisera raised against different tombus‐ and aureusviruses were used to assess the serological relation to CBLV. The ELISA results indicate that CBLV is not serologically related to any of those tested.     

Molecular characterisation and origin of the Coffea arabica L. genome

Restriction fragment length polymorphism (RFLP) markers were used in combination with genomic in situ hybridisation (GISH) to investigate the origin of the allotetraploid species Coffea arabica (2n = 44). By comparing the RFLP patterns of potential diploid progenitor species with those of C. arabica, the sources of the two sets of chromosomes, or genomes, combined in C. arabica were identified. The genome organisation of C. arabica was confirmed by GISH using simultaneously labelled total genomic DNA from the two putative genome donor species as probes. These results clearly suggest that C. arabica is an amphidiploid formed by hybridisation between C. eugenioides and C. canephora, or ecotypes related to these diploid species. Our results also indicate low divergence between the two constituent genomes of C. arabica and those of its progenitor species, suggesting that the speciation of C. arabica took place relatively recently. Precise localisation in Central Africa of the site of the speciation of C. arabica, based on the present distribution of the coffee species, appears difficult, since the constitution and extent of tropical forest has varied considerably during the late Quaternary period. Received: 6 June 1998 / Accepted: 10 November 1998     

Complete genome characterisation of a novel 26th bluetongue virus serotype from Kuwait

Bluetongue virus is the "type" species of the genus Orbivirus, family Reoviridae. Twenty four distinct bluetongue virus (BTV) serotypes have been recognized for decades, any of which is thought to be capable of causing "bluetongue" (BT), an insect-borne disease of ruminants. However, two further BTV serotypes, BTV-25 (Toggenburg orbivirus, from Switzerland) and BTV-26 (from Kuwait) have recently been identified in goats and sheep, respectively. The BTV genome is composed of ten segments of linear dsRNA, encoding 7 virus-structural proteins (VP1 to VP7) and four distinct non-structural (NS) proteins (NS1 to NS4). We report the entire BTV-26 genome sequence (isolate KUW2010/02) and comparisons to other orbiviruses. Highest identity levels were consistently detected with other BTV strains, identifying KUW2010/02 as BTV. The outer-core protein and major BTV serogroup-specific antigen "VP7" showed 98% aa sequence identity with BTV-25, indicating a common ancestry. However, higher level of variation in the nucleotide sequence of Seg-7 (81.2% identity) suggests strong conservation pressures on the protein of these two strains, and that they diverged a long time ago. Comparisons of Seg-2, encoding major outer-capsid component and cell-attachment protein "VP2" identified KUW2010/02 as 26th BTV, within a 12th Seg-2 nucleotype [nucleotype L]. Comparisons of Seg-6, encoding the smaller outer capsid protein VP5, also showed levels of nt/aa variation consistent with identification of KUW2010/02 as BTV-26 (within a 9th Seg-6 nucleotype - nucleotype I). Sequence data for Seg-2 of KUW2010/02 were used to design four sets of oligonucleotide primers for use in BTV-26, type-specific RT-PCR assays. Analyses of other more conserved genome segments placed KUW2010/02 and BTV-25/SWI2008/01 closer to each other than to other "eastern" or "western" BTV strains, but as representatives of two novel and distinct geographic groups (topotypes). Our analyses indicate that all of the BTV genome segments have evolved under strong purifying selection.     

Olive latent ringspot virus, a newly recognised virus infecting olive in Italy

A manually transmissible virus, for which the name olive latent ringspot virus (OLRV) is proposed, was isolated from a symptomless olive tree. The virus was mechanically transmitted to test plants. Purified preparations of OLRV contained three classes of isometric particle, c. 28 nm in diameter, with sedimentation coefficients of 525 (T), 975 (M) and 1325 (B) and containing 0, 30 and 43% nucleic acid respectively. At equilibrium in CsCl gradients, the buoyant densities of T and M components were 1–29 and 1–43 g/cm³ respectively, whereas B component separated into two sub-components with buoyant densities of 1–51 g/cm³ (BJ and 1–52 g/cm³ (B₂). Particle preparations contained two species of single-stranded RNA with mol. wt 1–40 times 10⁶ and 2–65 times 10⁶, both necessary for infectivity. The coat protein of OLRV, dissociated under strong denaturing conditions, separated into four components in polyacrylamide gel electrophoresis. Over 75% of the protein was found in a band with mol. wt 57 600, but all four components were recognised as oligomers of a monomeric form with mol. wt 14 300. OLRV was serologically unrelated to 26 different isometric plant viruses including 17 recognised nepoviruses. Its properties strongly indicate that it is a hitherto undescribed member of the nepovirus group.     

Artichoke latent virus: characterisation, ultrastructure and geographical distribution

An isolate of artichoke latent virus (ALV-I) obtained from a symptomless artichoke plant in Southern Italy was characterised and compared with ALV isolates from other countries. ALV occurs in California and throughout the western part of the Mediterranean basin but of Mediterranean countries east of Italy, it was found only in Israel and Turkey. ALV-I was readily transmissible by inoculation of sap to a moderate range of hosts, was transmitted in a non-persistent manner by Aphis fabae, Brachicaudus cardui and Myzus persicae, but was not seed transmitted. The virus has flexuous rod-shaped particles measuring c. 12 nm × 746 nm with a sedimentation coefficient of 145 S and a buoyant density of 1·31 g/cm³. The particles contain single stranded RNA with a mol. wt of 3 × 10⁶ and protein composed of a single polypeptide species with a mol. wt of 33 000. Cylindrical cytoplasmic inclusions consisting of pinwheels and laminated aggregates were present in cells of naturally and artificially infected plants. ALV isolates from different geographical origin were indistinguishable from ALV-I biologically, morphologically, serologically and ultrastructurally. These properties place ALV in the Potyvirus group, but it was serologically unrelated to 12 other potyviruses 10 of which occur commonly in Italy.     

Molecular characterization of Alternaria alternata population isolated from Upper Egyptian tomato fruits

Alternaria alternata is the most common fungal pathogen of tomatoes in Upper Egypt. Morphological identification of this fungus is challenging; therefore, this study searched for new classification tools based on molecular techniques. Using a dilution plating method, 67 strains of A. alternata were isolated from 34 samples of rotten tomato fruits representing the Giza 80 and Edkawy cultivars. The collected strains were identified using the amplification products of the internal transcribed spacer (ITS) region, glyceraldehyde 3‐phosphate dehydrogenase (Gpd) and Alt a1, which is a gene involved in the production of most of the allergens produced by A. alternata. The screening revealed that A. alternata constituted more than half of the total fungi recovered from rotten tomatoes in this study. According to the phylogenetic analysis using these three loci, the collected strains clustered in accordance with the host cultivar type from which they had been isolated. Specific gene random primer polymerase chain reaction (SGRP‐PCR) techniques indicated that the A. alternata population in the tested region has a high genetic diversity. The pathogenicity test showed that most of the A. alternata isolates (67.2%) were highly pathogenic, and no correlation was found between the phylogenetic analysis and pathogenicity. In addition, the influence of the fungicide Disan 80% on the collected strains showed significant differences that were attributed to the source of isolation.     

Molecular evolution of the hepatitis B virus genome

The hepatitis B virus (HBV) has a circular DNA genome of about 3,200 base pairs. Economical use of the genome with overlapping reading frames may have led to severe constraints on nucleotide substitutions along the genome and to highly variable rates of substitution among nucleotide sites. Nucleotide sequences from 13 complete HBV genomes were compared to examine such variability of substitution rates among sites and to examine the phylogenetic relationships among the HBV variants. The maximum likelihood method was employed to fit models of DNA sequence evolution that can account for the complexity of the pattern of nucleotide substitution. Comparison of the models suggests that the rates of substitution are different in different genes and codon positions; for example, the third codon position changes at a rate over ten times higher than the second position. Furthermore, substantial variation of substitution rates was detected even after the effects of genes and codon positions were corrected; that is, rates are different at different sites of the same gene or at the same codon position. Such rates after the correction were also found to be positively correlated at adjacent sites, which indicated the existence of conserved and variable domains in the proteins encoded by the viral genome. A multiparameter model validates the earlier finding that the variation in nucleotide conservation is not random around the HBV genome. The test for the existence of a molecular clock suggests that substitution rates are more or less constant among lineages. The phylogenetic relationships among the viral variants were examined. Although the data do not seem to contain sufficient information to resolve the details of the phylogeny, it appears quite certain that the serotypes of the viral variants do not reflect their genetic relatedness. Correspondence to: Z. Yang