Biomedical subjects

R Goldbach

Publications and source records attributed to R Goldbach.

At least 19 recordsLinked to original sources

PubMed2006

Molecular and biological comparison of two Tomato yellow ring virus (TYRV) isolates: challenging the Tospovirus species concept.

Two strains of Tomato yellow ring virus (TYRV, genus Tospovirus), one from tomato (referred to as TYRV-t) and the other from soybean and potato (denoted TYRV-s), collected from different geographical regions in Iran, were compared. Their genomic S RNA segments differed in size by 55 nucleotides. Comparison of the S RNA intergenic regions revealed the absence of a stretch of 115 nucleotides within the S RNA segment of TYRV-s and, conversely, of 56 nts in that of TYRV-t, apparently a stable genetic difference as it was also found in another isolate of TYRV-s collected from potato. Sequence comparison of the N protein ORFs revealed an identity of 92% between the N proteins of both strains, and the observed strong cross-reaction of TYRV-s in DAS-ELISA with a polyclonal antiserum directed against the TYRV-t N protein confirmed this high identity. Host range analysis revealed several differences, e.g. TYRV-s, but not TYRV-t, being able to systemically infect Nicotiana species, and TYRV-s being localised in tomato. The observed molecular and biological differences of both viruses call into question the currently used criteria for Tospovirus species demarcation.

Base Sequence↗

PubMed2006

Microsatellites reveal extensive geographical, ecological and genetic contacts between invasive and indigenous whitefly biotypes in an insular environment.

Human-mediated bioinvasions provide the opportunity to study the early stages of contact between formerly allopatric, divergent populations of a species. However, when invasive and resident populations are morphologically similar, it may be very difficult to assess their distribution in the field, as well as the extent of ecological overlap and genetic exchanges between invasive and resident populations. We here illustrate the use of data obtained from a set of eight microsatellite markers together with Bayesian clustering methods to document invasions in a group of major tropical pests, Bemisia tabaci, which comprises several morphologically indistinguishable biotypes with different agronomic impacts. We focus on the island of La Réunion, where an invasive biotype (B) has recently been introduced and now interacts with the resident biotype (Ms). The temporal and spatial distribution, host-plant range and genetic structure of both biotypes are investigated. We showed (i) that, without prior information, clustering methods separate two groups of individuals that can safely be identified as the B and Ms biotypes; (ii) that the B biotype has invaded all regions of the island, and showed no signs of genetic founder effect relative to the Ms biotype; (iii) that the B and Ms biotypes coexist in sympatry throughout most of their geographical ranges, although they tend to segregate into different host plants; and finally (iv) that asymmetrical and locus-specific introgression occurs between the two biotypes when they are in syntopy.

Animals↗

PubMed2005

Nucleotide sequence analyses of genomic RNAs of Peanut stunt virus Mi, the type strain representative of a novel PSV subgroup from China.

The complete nucleotide sequence of Peanut stunt virus strain Mi (PSV-Mi) from China was determined and compared to other viruses of the genus Cucumovirus. The tripartite genome of PSV-Mi encoded five open reading frames (ORFs) typical of cucumoviruses. Distance analyses of four ORFs indicated that PSV-Mi differed sufficiently in nucleotide sequence from other PSV strains of subgroups I and II to warrant establishment of a third subgroup of PSV.

Arachis↗

PubMed2005

A new silverleaf-inducing biotype Ms of Bemisia tabaci (Hemiptera: Aleyrodidae) indigenous to the islands of the south-west Indian Ocean.

Following the first detection of tomato yellow leaf curl virus (TYLCV) from Reunion (700 km east of Madagascar) in 1997 and the upsurge of Bemisia tabaci (Gennadius) on vegetable crops, two genetic types of B. tabaci were distinguished using RAPD-PCR and cytochrome oxidase I (COI) gene sequence comparisons. One type was assigned to biotype B and the other was genetically dissimilar to the populations described elsewhere and was named Ms, after the Mascarenes Archipelago. This new genetic type forms a distinct group that is sister to two other groups, one to which the B biotype is a member and one to which the Q biotype belongs. The Ms biotype is thought to be indigenous to the region as it was also detected in Mauritius, the Seychelles and Madagascar. Both B and Ms populations of B. tabaci induced silverleaf symptoms on Cucurbita sp., and were able to acquire and transmit TYLCV. Taken together these results indicate that the Ms genetic type should be considered a new biotype of B. tabaci.

Animals↗

PubMed2002

Genetic organisation of Iris yellow spot virus M RNA: indications for functional homology between the G(C) glycoproteins of tospoviruses and animal-infecting bunyaviruses.

The complete nucleotide sequence (4838 nucleotides) of Iris yellow spot virus (IYSV) M RNA indicates, typical for tospoviruses, the presence of two genes in ambisense arrangement. The vRNA ORF codes for the potential cell-to-cell movement (NSm) protein (34.8 kDa) and the vcRNA ORF for the viral glycoprotein (G1/G2) precursor (128.6 kDa). Multiple sequence alignment of the NSm and G1/G2 precursor proteins of IYSV with those of other tospoviruses, showed highest homologies to Peanut bud necrosis virus (PBNV) and Watermelon silver mottle virus (WSMV). The potential cell-to-cell movement protein of tospoviruses is highly conserved (40-70% identity), with the exception of the first 60 N terminal amino acids, a domain that clearly diverged. For the G1 and G2 viral glycoproteins, blast searches revealed a significant homology between the C-terminally located tospoviral G1 (G(C)) protein with the counterpart of the animal-infecting bunyaviruses, suggesting a functional homology for these proteins.

Amino Acid Sequence↗

PubMed2001

In vivo analysis of the TSWV cap-snatching mechanism: single base complementarity and primer length requirements.

Requirements for capped leader sequences for use during transcription initiation by tomato spotted wilt virus (TSWV) were tested using mutant alfalfa mosaic virus (AMV) RNAs as specific cap donors in transgenic Nicotiana tabacum plants expressing the AMV replicase proteins. Using a series of AMV RNA3 mutants modified in either the 5'-non-translated region or in the subgenomic RNA4 leader, sequence analysis revealed that cleaved leader lengths could vary between 13 and 18 nucleotides. Cleavage occurred preferentially at an A residue, suggesting a requirement for a single base complementarity with the TSWV RNA template, which could be confirmed by analyses of host mRNAs used in vivo as cap donors.

5' Untranslated Regions↗

PubMed2001

High sequence conservation among cucumber mosaic virus isolates from lily.

For classification of Cucumber mosaic virus (CMV) isolates from ornamental crops of different geographical areas, these were characterized by comparing the nucleotide sequences of RNAs 4 and the encoded coat proteins. Within the ornamental-infecting CMV viruses both subgroups were represented. CMV isolates of Alstroemeria and crocus were classified as subgroup II isolates, whereas 8 other isolates, from lily, gladiolus, amaranthus, larkspur, and lisianthus, were identified as subgroup I members. In general, nucleotide sequence comparisons correlated well with geographic distribution, with one notable exception: the analyzed nucleotide sequences of 5 lily isolates showed remarkably high homology despite different origins.

Capsid↗

PubMed2001

Identification and characterization of a novel tospovirus species using a new RT-PCR approach.

A novel tospovirus serologically distinct from all established tospovirus species was found in Thailand in Physalis minima L. The S RNA of this virus was cloned by a new RT-PCR approach revealing a nucleotide sequence of 3257 nucleotides. The ambisense RNA segment encoded a nonstructural protein (NSs) of 469 amino acids, with a predicted Mr of 53.2 kDa, and a nucleoprotein (N) of 279 amino acids and a Mr of 31.0 kDa, so far the largest N protein known for any tospovirus species. N protein sequence comparisons revealed closet relationship to the species Watermelon bud necrosis virus (58% identity), Watermelon silver mottle virus and Peanut bud necrosis virus (57%) and a distant relationship to Peanut yellow spot virus (23%) and Peanut chlorotic fanspot virus (22%).

Amino Acid Sequence↗

PubMed2001

Tomato spotted wilt virus glycoproteins exhibit trafficking and localization signals that are functional in mammalian cells.

The glycoprotein precursor (G1/G2) gene of tomato spotted wilt virus (TSWV) was expressed in BHK cells using the Semliki Forest virus expression system. The results reveal that in this cell system, the precursor is efficiently cleaved and the resulting G1 and G2 glycoproteins are transported from the endoplasmic reticulum (ER) to the Golgi complex, where they are retained, a process that could be blocked by tunicamycin. Expression of G2 alone resulted in transport to and retention in the Golgi complex, albeit less efficient, suggesting that G2 contains a Golgi retention signal. G1 alone was retained in the ER, irrespective of whether it contained the precursor's signal sequence or its own N-terminal hydrophobic sequence. Coexpression of G1 and G2 from separate gene constructs resulted in rescue of efficient G1 transport, as the proteins coaccumulated in the Golgi complex, indicating that their interaction is essential for proper targeting to this organelle. The results demonstrate that transport and targeting of the plant TSWV glycoproteins in mammalian BHK cells are strikingly similar to those of animal-infecting bunyavirus glycoproteins in mammalian cells. The observations are likely to reflect the dual tropism of TSWV, which replicates both in its plant host and in its animal (thrips) vector.

Amino Acid Sequence↗

PubMed2000

Mutational analysis of the cowpea mosaic virus movement protein.

Cowpea mosaic virus moves from cell-to-cell in a virion form through tubular structures that are assembled in modified plasmodesmata. Similar tubular structures are formed on the surface of protoplasts inoculated with cowpea mosaic virus. The RNA 2-encoded movement protein (MP) is responsible for the induction and formation of these structures. To define functional domains of the MP, an alanine-substitution mutagenesis was performed on eight positions in the MP, including two conserved sequence motifs, the LPL and D motifs. Results show that these two conserved motifs as well as the central region of the MP are essential for cell-to-cell movement. Several viruses carrying mutations in the N- or C-terminal parts of their MP retained infectivity on cowpea plants. Coexpression studies revealed that mutant MPs did not interfere with the activity of wild-type MP and could not mutually complement their defects.

Alanine↗

PubMed1999

Infection of barley protoplasts with rice hoja blanca tenuivirus. Brief report.

A barley protoplast system has been established that supports replication of Rice hoja blanca tenuivirus (RHBV). Following polyethylene glycol-mediated RHBV inoculation of barley protoplasts, newly synthesized viral RNAs and proteins could be detected. Time course analyses revealed de novo synthesis of genome length viral RNA4, as well as subgenomic-sized RNA4 molecules of both polarities. Two proteins, N and NS4, encoded by viral complementary RNA3 and viral RNA4 respectively, were detected by Western immunoblot analysis. The barley protoplast system thus constitutes a promising tool for in vivo studies of the sequential steps involved in the multiplication cycle of RHBV.

Hordeum↗

PubMed1999

Tissue tropism related to vector competence of Frankliniella occidentalis for tomato spotted wilt tospovirus.

The development of tomato spotted wilt tospovirus (TSWV) infection in the midgut and salivary glands of transmitting and non-transmitting thrips, Frankliniella occidentalis, was studied to elucidate tissue tropism and the virus pathway within the body of this vector. Immunohistological techniques used in this study showed that the midgut, foregut and salivary glands were the only organs in which virus accumulated. The first signals of infection, observed as randomly distributed fluorescent granular spots, were found in the epithelial cells of the midgut, mainly restricted to the anterior region. The virus subsequently spread to the circular and longitudinal midgut muscle tissues, a process which occurred late in the larval stage. In the adult stage, the infection occurred in the visceral muscle tissues, covering the whole midgut and foregut, and was abolished in the midgut epithelium. The infection of the salivary glands was first observed 72 h post-acquisition, and simultaneously in the ligaments connecting the midgut with these glands. The salivary glands of transmitting individuals appeared heavily or completely infected, while no or only a low level of infection was found in the glands of non-transmitting individuals. Moreover, the development of an age-dependent midgut barrier against virus infection was observed in second instar larvae and adults. The results show that the establishment of TSWV infection in the various tissues and the potential of transmission seems to be regulated by different barriers and processes related to the metamorphosis of thrips.

Animals↗

PubMed1999

Tomato spotted wilt virus particle morphogenesis in plant cells.

A model for the maturation of tomato spotted wilt virus (TSWV) particles is proposed, mainly based on results with a protoplast infection system, in which the chronology of different maturation events could be determined. By using specific monoclonal and polyclonal antisera in immunofluorescence and electron microscopy, the site of TSWV particle morphogenesis was determined to be the Golgi system. The viral glycoproteins G1 and G2 accumulate in the Golgi prior to a process of wrapping, by which the viral nucleocapsids obtain a double membrane. In a later stage of the maturation, these doubly enveloped particles fuse to each other and to the endoplasmic reticulum to form singly enveloped particles clustered in membranes. Similarities and differences between the maturation of animal-infecting (bunya)viruses and plant-infecting tospoviruses are discussed.

Animals↗

PubMed1999

Alfalfa mosaic virus RNAs serve as cap donors for tomato spotted wilt virus transcription during coinfection of Nicotiana benthamiana.

Tomato spotted wilt virus (TSWV) was shown to use alfalfa mosaic virus (AMV) RNAs as cap donors in vivo during a mixed infection in Nicotiana benthamiana. By use of nested reverse transcription-PCR, TSWV N and NSs mRNAs provided with capped leader sequences derived from all four AMV RNAs could be cloned and sequenced. The sequence specificity of the putative TSWV endonuclease involved is discussed.

Alfalfa mosaic virus↗

PubMed1998

Molecular characterization of tomato spotted Wilt virus defective interfering RNAs and detection of truncated L proteins.

Junction sites of 25 different defective interfering (DI) RNAs of tomato spotted wilt virus (TSWV) were characterized. The DI RNAs varied in size from 2.0 to 5.2 kilobases (kb) and contained a single internal deletion. The absence of DI RNAs smaller than 2 kb suggested a size constraint for the survival of TSWV DI RNAs. This hypothesis was reinforced by the finding of a dimeric DI RNA formed by two 1.6-long monomers linked head to tail. Three types of junction sites were found, one type originating from a simple deletion; the second type contained a few extra nucleotides of unknown origin; and the third type contained a stretch of three to five nucleotides, originally occurring at both sides of the deletion and of which one was deleted. In 19 of the 25 DI RNAs studied, the original reading frame was maintained, suggesting a selective preference of DI RNAs with translational potency. Truncated proteins encoded by these DI RNAs were indeed detected in the nucleocapsid preparations. Folding studies of the complete L RNA revealed that the calculated minimal energy of folding was at 16 degreesC lower than at 23 degrees, indicating a higher stability of this molecule at low temperatures. The results suggest an involvement of locally folded secondary structures in the process of deletion, rather than the requirement of certain sequences around the deletion point. The DI RNA generation in TSWV is essentially, as discussed, similar to the process of RNA recombination described in many viruses.

Base Sequence↗

PubMed1998

Postnatal development of muscarinic autoreceptors modulating acetylcholine release in the septohippocampal cholinergic system. I. Axon terminal region: hippocampus.

We studied the postnatal development of the release of acetylcholine (ACh) and of presynaptic, release-inhibiting muscarinic autoreceptors in the rat hippocampus. To this end, hippocampal slices (350 microns thick) from rats of various postnatal ages (postnatal day 3 [P3] to P16) were preincubated with [3H]choline and stimulated twice (S1, S2: 360 pulses, 2 ms, 3 Hz, 60 mA) during superfusion with physiological buffer containing hemicholinium-3 (10 microM). In parallel, the activities of hemicholinium-sensitive high-affinity choline uptake (HACU, in synaptosomes) and of choline acetyltransferase (ChAT, in crude homogenates) were determined as markers for the cholinergic ingrowth. In hippocampal slices preincubated with [3H]choline, the electrically evoked overflow of 3H at S1 increased from 0.11 (P3) to 0.81% of tissue 3H (P16), the latter value being still much lower than that of hippocampal slices from adult rats (2.89% of tissue 3H). Already at P3 the evoked overflow of 3H was Ca(2+)-dependent and sensitive to tetrodotoxin, indicating an action potential-evoked exocytotic mechanism of ACh release. The muscarinic agonist oxotremorine (1 microM) significantly inhibited the evoked ACh release in hippocampal slices with increasing effectivity from P4 to P16; no significant effect was detectable at P3. The ACh esterase inhibitor physostigmine and the muscarinic antagonist atropine (1 microM, each) exhibited significant inhibitory and facilitatory effects, respectively, only at P15-16. The specific activities of both hippocampal HACU (pmoles/mg protein/min) and ChAT (nmoles/mg protein/min) continuously increased from P3 to P16. It is concluded (1) that cholinergic nerve terminals arriving at the hippocampal formation during postnatal ingrowth are already endowed with the apparatus for action potential-induced, Ca(2+)-sensitive (exocytotic) ACh release; (2) that, in contrast, the expression of presynaptic muscarinic autoreceptors on these cholinergic axon terminals is delayed; and (3) that autoinhibition due to endogenous ACh develops even later, probably when the density of presynaptic terminals in the hippocampus and hence, the concentration of released ACh has reached a suprathreshold value.

Acetylcholine↗

PubMed1998

The emerging problem of tospovirus infection and nonconventional methods of control.

The emergence of tospoviruses as a significant problem in the cultivation of many crops around the world makes it vital to develop strategies to restrain these viruses. So far, only a few natural resistance genes suitable for introduction into plant breeding programs have been identified, prompting the exploitation of alternative ways of introducing virus resistance into crop plants, such as genetic modifications.

Animals↗

PubMed1997

Serological comparison of tospoviruses with polyclonal antibodies produced against the main structural proteins of tomato spotted wilt virus.

A new purification procedure for the tospoviruses of serogroups II and III was developed. SDS-polyacrylamide gel electrophoresis of purified tomato spotted wilt virus (TSWV; serogroup I), groundnut ringspot virus (GRSV: serogroup II), tomato chlorotic spot virus (TCSV; serogroup II) and impatiens necrotic spot virus (INSV; serogroup III) showed that the glycoprotein G2 of serogroup II members differs significantly in size from that of the serogroup I virus. Western immunoblot analysis using polyclonal antisera produced against purified glycoproteins TSWV-G1 and G2 as well as peptides of hydrophilic sequences of TSWV-G1 and G2 expressed in Escherichia coli demonstrated a higher homology amongst G1 of different serogroups than for G2. These results are supported by comparing the glycoprotein gene sequences of different serogroups.

Antibodies, Viral↗