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PubMed1994

Flock house virus: a simple model for studying persistent infection in cultured Drosophila cells.

Flock house virus (FHV), isolated from twenty Drosophila melanogaster cell lines, persistently infected with the virus, were examined during successive serial passages by plaque assay and sequence analysis. No phenotypic or genotypic changes in the virus were observed during the establishment of persistent infection, suggesting that it was a cellular modification that led to the first step in establishing the persistent state. Once this state was initiated, the virus was relieved of the need for a functional coat protein to propagate itself and mutations began to accumulate selectively in RNA2, the gene for the coat protein. These changes were manifested by a gradual drift to a smaller plaque population. The replicase activity, coded by RNA1, remained unaltered.

Animals↗
PubMed2002

Amino acid substitutions within the Cys-rich domain of the tobacco etch potyvirus HC-Pro result in loss of transmissibility by aphids.

We examined the role of several amino acid residues located at the N-terminus of the tobacco etch potyvirus (TEV) helper component-proteinase (HC-Pro) in virus transmissibility by aphids. Site-directed mutagenesis resulted in changes affecting amino acids that appear highly conserved among a number of potyviruses. The TEV HC-Pro amino acid residues Gly343, Val345, Ala346, Ile348, Pro355, Lys358, and Ile359 were arranged within a Cys-rich domain in a region dispensable for TEV infectivity. Two HC-Pro mutants (TEV-P355R and -K358N) exhibited a drastically reduced rate of aphid transmission whereas other mutants (TEV-G343D, -V345E, -A346H, -I348D, and -P355L) were completely unable to be aphid transmitted. In contrast, the I359M mutation had no effect on aphid transmissibility of TEV. This lack of transmissibility did not appear to be due to large differences in the amounts of both coat protein (CP) and HC-Pro in infected tobacco plants. Our results indicated that these amino acid residues likely play a highly conserved role in aphid transmission among potyviruses.

Amino Acid Sequence↗
PubMed2005

Absence of a coding region for the helper component-proteinase in the genome of cucumber vein yellowing virus, a whitefly-transmitted member of the Potyviridae.

The complete nucleotide sequence of isolates of Cucumber vein yellowing virus (CVYV) has been determined. The viral genome comprises 9734 nucleotides, excluding a 3'-terminal poly(A) sequence. The genome of CVYV has a 5'-non coding and a 3' non coding region of respectively 67 and 240 nucleotides. The RNA of CVYV encodes a single polyprotein of 3148 amino acid residues and has a deduced genome organization and motifs typical for a member of the family Potyviridae. However, CVYV is atypical because it lacks a coding sequence region for the putative helper-component as well as conserved helper-component-proteinase motifs which may account for its vector relations. All the present coding regions were compared to those from several members of the Potyviridae family. CVYV is most closely related to Sweetpotato mild mottle virus confirming its assignation to the genus Ipomovirus, despite similarities with tritimoviruses.

Animals↗
PubMed1999

A comparison of six cypovirus isolates by cross-hybridisation of their dsRNA genome segments.

Genetic relationships between the genome segments of six cypovirus (CPV) isolates were analysed by RNA cross-hybridisation. These included three type 1 viruses and single isolates of types 2, 5 and 12, which collectively are identical to those previously compared by serology and electrophoresis [Mertens et al. (1989), J Gen Virol 70: 173-185]. Since only genome segment 10 of three cypovirus types and segments 8 and 9 of a single virus strain (of type 1) have currently been sequenced, this initial study provides some additional information on sequence variation/similarity in each of the ten genome segments. The RNA of the type 1 viruses showed high levels of cross-hybridisation. Significant but much lower levels of cross-hybridisation were detected between type 1 and the related type 12 CPV. However, only very low levels of cross-hybridisation were detected between the other pairs of viruses. Apart from evidence of a slightly higher level of sequence similarity between the largest segments, the RNA sequence appeared to vary uniformly across the whole genome. There was no evidence for any type specific RNA sequences restricted to individual genome segment(s). The sequence variation, reflected in the levels of RNA sequence similarity and cross hybridisation, correlates well with serological data, showing large differences between CPV types and supports the continued use of electropherotype as one of the 'species parameters' for the classification of cypoviruses.

Animals↗
PubMed1985

The 3' end of large ribosomal subunit RNA from mosquito mitochondria: homogeneity of transcribed moieties.

3' Terminal sequences of mosquito mitochondrial 16 S ribosomal RNA, which is post-transcriptionally polyadenylated, have been examined with the aim of determining the degree of homogeneity of transcribed moieties. 3' End-labeled samples were subjected to oligonucleotide fingerprint analysis and to ladder gel analysis after primary and secondary nuclease digestion; and products of reverse transcriptase reactions were characterized using 16 S RNA as template and selected oligodeoxynucleotides as primers. The results indicated a remarkable degree of homogeneity compared to homologous mammalian mitochondrial systems, and suggested differences in modes of expression of insect, versus mammalian, mitochondrial genomes.

Aedes↗
PubMed2004

Aedes aegypti genomics.

The mosquito, Aedes aegypti, is the primary, worldwide arthropod vector for the yellow fever and dengue viruses. As it is also one of the most tractable mosquito species for laboratory studies, it has been and remains one of the most intensively studied arthropod species. This has resulted in the development of detailed genetic and physical maps for Ae. aegypti and considerable insight into its genome organization. The research community is well-advanced in developing important molecular tools that will facilitate a whole genome sequencing effort. This includes generation of BAC clone end sequences, physical mapping of selected BAC clones and generation of EST sequences. Whole genome sequence information for Ae. aegypti will provide important insight into mosquito chromosome evolution and allow for the identification of genes and gene function. These functions may be common to all mosquitoes or perhaps unique to individual species, possibly specific to host-seeking and blood-feeding behaviors, as well as the innate immune response to pathogens encountered during blood-feeding. This information will be invaluable to the global effort to develop novel strategies for preventing arthropod-borne disease transmission.

Aedes↗
PubMed2005

Mosquito immunity against Plasmodium.

Understanding the molecular mechanisms of the innate immune responses of Anopheles gambiae against Plasmodium parasites is of great importance for current efforts to develop novel strategies for malaria disease control. The parasite undergoes substantial stage-specific losses during its development in the mosquito, which in some cases lead to complete refractoriness of the mosquito against the parasite. The underlying genetics of refractoriness are complex and multifactorial. Completion of the genome sequence of An. gambiae 2 years ago, together with the development of DNA microarrays in this species and the extension of the RNAi technique to adult mosquitoes, has allowed comparative and functional genomic approaches of the mosquito innate immune system. A variety of factors were shown to negatively affect the development of Plasmodium parasites in the mosquito, in some cases leading to complete transmission blockage. In addition, mosquito factors have been identified that play positive roles and are required for successful transmission of the parasite. These findings indicate a highly complex interplay between parasite and vector. Research is continuing to identify new factors involved in this interaction and to decipher the interplay of these molecules and their regulation.

Animals↗
PubMed2004

Genetically variable nucleopolyhedroviruses isolated from spatially separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) in Orkney.

Here we report a lepidopteran system in which a pathogen is both abundant and genotypically variable. Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles to investigate the prevalence of a pathogen, O. brumata Nucleopolyhedrovirus (OpbuNPV), within the natural system. Virus was recorded in 11 of the 13 winter moth populations sampled, with two populations suffering mortality due to virus at levels of 50%. The virus genome from 200 single insect isolations was investigated for variation using restriction endonuclease digests. Twenty-six variants of OpbuNPV were detected using SalI. The polyhedrin gene of the virus was partially sequenced, allowing the relationship between the 26 variants to be portrayed as a cladogram. The phylogenetic relationship between OpbuNPV and other known baculovirus polyhedrin gene sequences was also established. The discovery of virus at such high prevalence is discussed with reference to occurrence and genetic variation of pathogens in other lepidopteran host populations. This study shows encouraging results for further studies into the role of pathogens in the regulation of host insect populations.

Animals↗
PubMed2006

Detection and quantitation of Solenopsis invicta virus in fire ants by real-time PCR.

A quantitative real-time PCR (QPCR) method was developed to detect and quantify the amount of Solenopsis invicta virus (SINV) infecting individual ants of S. invicta. The two-step method utilized a gene-specific oligonucleotide primer targeting the SINV RNA-dependent RNA polymerase (RdRp) for cDNA synthesis. Dithiothreitol used in the cDNA synthesis step was found to significantly decrease the detection sensitivity for SINV RdRp and was therefore omitted. SINV RdRp cDNA was then quantified by QPCR using SYBR Green dye and a standard curve generated from SINV RdRp plasmid clones. A standard curve was successfully constructed from clones of the SINV RdRp region. A strong linear relationship [r2=0.998; y=(-3.63+/-0.37)x+(39.19+/-1.33)] between C(T) and starting SINV RdRp copy number was observed within a dynamic range of 5-5 x 10(6) copies. SINV RdRp copy number was determined with the optimized QPCR method in individual S. invicta ants taken from an infected field colony. Worker ants exhibited the highest RdRp copy number (2.1 x 10(9) copies/worker ant) and pupae exhibited the lowest (4.2 x 10(2) copies/pupa). Mean RdRp copy number was lowest in early larvae and pupae. Overall, SINV RdRp copy number increased through larval development, sharply declined during pupation, then sharply increased in adults.

Animals↗
PubMed2006

Evaluation of a real-time two-step RT-PCR assay for quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-infected bee tissues and in life stages of a symptomatic colony.

A two-step real-time RT-PCR assay, based on TaqMan technology using a fluorescent probe (FAM-TAMRA) was developed to quantify Chronic bee paralysis virus (CBPV) genome in bee samples. Standard curves obtained from a CBPV control RNA and from a plasmid containing a partial sequence of CBPV showed that this assay provided linear detection over a 7-log range (R(2)>0.99) with a limit of detection of 100 copies, and reliable inter-assay and intra-assay reproducibility. Standardisation including RNA purification and cDNAs synthesis was also validated. The CBPV TaqMan methodology was first evaluated by quantifying the CBPV genomic load in bee samples from an experimental infection obtained by topical application. Up to 1.9 x 10(10) CBPV copies per segment of insect body (head, thorax and abdomen) were revealed whereas a lower CBPV genomic load was detected in dissected organs such as mandibular and hypopharyngeal glands, brain and alimentary canal (up to 7.2 x 10(6) CBPV copies). The CBPV genomic loads in different categories of bees from a hive presenting the trembling symptoms typical of Chronic paralysis were then quantified. Significantly higher CBPV loads were found in guard, symptomatic and dead bees (up to 1.9 x 10(13) CBPV copies) than in forager, drones and house bees (up to 3.4 x 10(6) CBPV copies). The results obtained for symptomatic or dead bees support the correlation between high CBPV genomic load and pathology expression. Moreover, the high CBPV genomic load revealed in guard bees highlights the possible pivotal role played by this category of bees in CBPV infection.

Animal Husbandry↗
PubMed2006

The procyclin-associated genes of Trypanosoma brucei are not essential for cyclical transmission by tsetse.

EP and GPEET procyclins are the major surface glycoproteins of Trypanosoma brucei in the midgut of tsetse flies (Glossina spp.). The procyclin genes are located at the beginning of polycistronic transcription units and are followed by at least one procyclin-associated gene (PAG). The EP/PAG1 locus on one copy of chromosome X begins with the three genes EP1, EP2 and PAG1; the end of this unit has not been characterized previously. The EP/PAG2 locus on the other copy of chromosome X contains the same procyclin genes followed by PAG2 and PAG4. Here we show that the EP/PAG1 locus in AnTat1.1 has to be extended by three more PAGs, which we named PAG5, PAG2* and PAG4. The EP/PAG2 locus most likely evolved from the EP/PAG1 locus by deletion of a fragment from within PAG1 to PAG2*. The procyclin loci on the two copies of chromosome VI are indistinguishable, and contain the genes GPEET, EP3, PAG3 and GRESAG2.1. The mRNA levels of PAG1, PAG2 and PAG3 are transiently increased during differentiation of bloodstream forms to procyclic forms. Unexpectedly, procyclic forms of a PAG knockout clone lacking all eight PAGs in the procyclin loci were transmissible by Glossina morsitans. Furthermore, the deletion mutant could still establish midgut infections when competing with a tagged clone with the full complement of PAGs. Cyclical transmission was also possible when tsetse flies were infected with bloodstream forms of the deletion mutant, demonstrating that the PAGs are not essential for the differentiation of bloodstream to procyclic forms in vivo.

Animals↗
PubMed2005

The glucosinolate-myrosinase system in an ecological and evolutionary context.

Functional analysis of natural variation in the model species Arabidopsis thaliana has enabled the cloning of many glucosinolate biosynthesis and hydrolysis genes. Variation in these genes is central to understanding the ecological role of the glucosinolate-myrosinase defense system, and allows us to dissect the evolutionary and ecological forces that shape polymorphism at underlying loci. These same genes are also variable in other crucifer species, suggesting the presence of recurring selection, possibly mediated by insects. By utilizing the genomic tools available in A. thaliana to investigate these loci fully, it might be possible to generate detailed evolutionary or ecological models to apply to other species.

Animals↗
PubMed2006

A virus between families: nucleotide sequence and evolution of Strawberry latent ringspot virus.

Several clones of golden ginger mint (Mentha x gracilis, 'Variegata') were found infected with Strawberry latent ringspot virus (SLRSV). The virus was purified and cloned and the complete nucleotide sequence of a mint isolate was obtained. RNA 1 consists of 7,496 nucleotides excluding the poly-A tail and encodes a polyprotein with signature enzymatic motifs found in other picorna-like plant viruses. RNA 2 consists of 3,842 nucleotides excluding the poly-A tail, encoding a polyprotein that is processed to a putative movement protein and the two coat proteins of the virus. A satellite RNA of 1,117 nucleotides was associated with this isolate encoding for a putative protein of 31 kDa. Phylogenetic analysis revealed that SLRSV shares characteristics with members of the Cheravirus, Fabavirus, Comovirus and Sadwavirus genera indicative of the uniqueness of SLRSV. The close relationship of SLRSV with these genera led to the examination of aphid and beetle transmission of the virus with, however, negative results.

Animals↗