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PubMed2005

RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor).

Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding.

Abdomen↗
PubMed1972

Comparison of the 3' termini of discrete segments of the double-stranded ribonucleic acid genomes of cytoplasmic polyhedrosis virus, wound tumor virus, and reovirus.

The 3' terminal nucleosides of the isolated components of double-stranded ribonucleic acids of reovirus, wound tumor virus, and cytoplasmic polyhedrosis virus were determined by labeling with tritiated sodium borohydride. All wound tumor virus and cytoplasmic polyhedrosis virus components appear to contain approximately equal amounts of U(OH) and C(OH) termini. Reovirus segments have essentially only C(OH) termini.

Adenosine↗
PubMedDate not supplied

Comparative studies of baculovirus granulins and polyhedrins.

Granulins and polyhedrins from five baculoviruses exhibit similar chemical and physical properties. Although similarities are demonstrated, it has been shown that each of the proteins is different and apparently specific to a given virus. The granulins and polyhedrins exhibit a major polypeptide component of an estimated molecular weight of 28,000. Alkaline protease activity has been detected in each preparation. N-terminal analyses reflect differences between granulins and polyhedrins studied: granulosis virus = Asx, and nuclear polyhedrosis virus = Glx. Two-dimensional high-voltage electrophoresis of highly purified granulin and polyhedrin preparations reveals relatedness as well as differences among the proteins as assessed by electrophoretic migration patterns.

Amino Acids↗
PubMed1993

Reverse transcriptase and protease activities of avian leukosis virus Gag-Pol fusion proteins expressed in insect cells.

Protease (PR)-defective avian leukosis virus particles display 300-fold-reduced levels of reverse transcriptase (RT) activity relative to wild-type particles. This observation suggests that during virion assembly RT is activated by proteolytic maturation of the Gag-Pol polyprotein precursor. To study the relationship between proteolytic cleavage and RT activation, we subjected PR-defective virion cores to digestion with purified viral PR and analyzed the structure of the major polypeptides produced as well as RT activity. Under conditions in which Gag precursors were fully matured, the RT domain was only incompletely released from the Gag-Pol precursor, remaining tethered to the upstream Gag domains PR or NC-PR. In the same reaction, RT activity was stimulated only three-fold, or 100-fold less than expected for a fully active RT. The poor activation suggested that the NC or PR domains could repress RT activity. To test this idea, we constructed recombinant baculoviruses expressing 19 different fusion proteins with upstream Gag or downstream Pol sequences attached to RT. Each protein was partially purified and assayed for its inherent RT activity. The results are consistent with the idea that Gag sequences can inhibit RT activity but indicate that the size of the Pol domain as well as the status of the PR domain (wild-type or mutant) also can profoundly influence activity. Several of the constructed Gag-Pol fusion proteins contained a wild-type PR domain. Some of these underwent intracellular PR-mediated processing, while others did not. All proteins in which the PR domain was preceded by upstream Gag sequences showed specific proteolysis. By contrast, all proteins initiated with a methionine placed one residue upstream of the natural N terminus of PR failed to show specific proteolysis. Amino-terminal sequencing of one such protein yielded the correct amino acid sequence and showed that the initiating methionine was not removed. One interpretation of these findings is that activation of PR requires the generation of the precise N terminus of the mature PR.

Animals↗
PubMed2004

A nucleopolyhedrovirus from Uranotaenia sapphirina (Diptera: Culicidae).

In this report we present data on biology, gross pathology, ultrastructure, and host range studies of a naturally occurring nucleopolyhedrovirus from the mosquito Uranotaenia sapphirina (UrsaNPV). Development of this virus was restricted to nuclei of epithelial cells in posterior midgut and distal gastric caecum. Occlusion bodies contained numerous singly enveloped rod-shaped virions. Early occlusion bodies were irregularly shaped and seemed to subsequently coalesce to form larger polyhedra. Mature occlusion bodies had a unique dumbbell shape, and lacked a polyhedron envelope and crystalline structure. Developmental and structural features of UrsaNPV were generally similar to other mosquito NPVs, with major differences in occlusion body shape and size. Transmission tests showed that only members of Uranotaenia (Ur. sapphirina and Ur. lowii) were susceptible to this virus. Transmission was facilitated by magnesium. Field collected Ur. sapphirina larvae had a relatively high rate of dual infections with UrsaNPV and UrsaCPV (cypovirus).

Animals↗
PubMed1982

Transmission efficiency of the sigma virus in natural populations of its host, Drosophila melanogaster.

A study of the viral samples collected in French natural populations of Drosophila melanogaster since 1969, indicates that natural populations include, as expected, both stabilized and non stabilized infected individuals. In agreement with previous observations made on other characters of the virus, the viral samples collected appear to be homogeneous for the efficiency of the hereditary transmission. However, this efficiency is greater than the average value observed with virus perpetuated in infected laboratory fly strains. One sample collected in Gabon and three in the U.S.A. appear to differ from the French samples for one at least of the traits studied in these experiments.

Animals↗
PubMed1989

Bombyx mori nuclear polyhedrosis virus-based vectors for expressing passenger genes in silkmoth cells under viral or cellular promoter control.

The polyhedrin gene of the nuclear polyhedrosis virus of the silkmoth Bombyx mori (BmNPV) has been subjected to deletion mutagenesis. A number of clones containing partially deleted polyhedrin genes were characterized and four clones containing limited deletions of the 5'-untranslated or 5'-flanking sequences of the gene were further analyzed with respect to polyhedrin promoter activity. The functional characterization of the deletion mutants was achieved through the insertion of a chloramphenicol acetyl transferase (CAT) gene (cat) into each deletion junction. The resultant cat constructs were introduced into the genome of BmNPV through homologous recombination and the effect of each deletion on the activity of the polyhedrin promoter was evaluated by measurements of CAT enzymatic activity in extracts of tissue culture cells infected with the corresponding recombinant BmNPVs as well as by primer extension assays. Removal of the entire leader region and eleven adjacent residues of the 5'-flanking sequences of the polyhedrin gene results in a dramatic decrease in promoter activity, which, however, remains detectable through CAT activity measurements. Elimination of an additional 30 nucleotides (nt) of the upstream sequences results in the complete inactivation of the polyhedrin promoter. The functional characterization of a deletion mutant lacking 41 nt of the 5'-flanking sequences has demonstrated that no functions necessary for viral infectivity, replication or assembly are disrupted by this deletion, since the corresponding recombinant viruses propagate in the cells with the same kinetics and to the same extent as wild-type BmNPV. As a result of the deletion mutagenesis, two classes of transfer vectors have become available. The first class can be used for introducing into the viral genome foreign nucleotide sequences under polyhedrin promoter control, while the second one can be used for obtaining recombinant viruses harboring foreign genetic material in an environment which is devoid of polyhedrin promoter activity.

Amino Acid Sequence↗
PubMed2006

Detection of viral sequences in semen of honeybees (Apis mellifera): evidence for vertical transmission of viruses through drones.

Honeybees (Apis mellifera) can be attacked by many eukaryotic parasites, and bacterial as well as viral pathogens. Especially in combination with the ectoparasitic mite Varroa destructor, viral honeybee diseases are becoming a major problem in apiculture, causing economic losses worldwide. Several horizontal transmission routes are described for some honeybee viruses. Here, we report for the first time the detection of viral sequences in semen of honeybee drones suggesting mating as another horizontal and/or vertical route of virus transmission. Since artificial insemination and controlled mating is widely used in honeybee breeding, the impact of our findings for disease transmission is discussed.

Animals↗
PubMed1990

Wild-type, but not mutant, human p53 proteins inhibit the replication activities of simian virus 40 large tumor antigen.

Murine p53 blocks many of the replication activities of simian virus 40 (SV40) large tumor antigen (T antigen) in vitro. As murine cells do not replicate SV40 DNA, it was of interest to determine how p53 from permissive human cells functions. Recombinant baculoviruses encoding either the wild-type form of human p53 or a mutant p53 cloned from a human tumor cell line were constructed, and p53 proteins were purified from infected insect cells. Surprisingly, we found that wild-type human p53 was as inhibitory to the ability of T antigen to mediate replication of an SV40 origin-containing (ori DNA) plasmid in vitro as was murine p53. Wild-type human p53 also blocked the DNA unwinding activity of T antigen, as did its murine counterpart. In contrast to murine and wild-type human p53, the mutant human p53 did not block ori DNA replication or DNA unwinding. Murine p53 formed a complex with mutant human p53 in vivo. Furthermore, mutant human p53 reduced the inhibition of SV40 ori DNA replication by murine p53 in vitro. These results provide a model for the way in which mutant p53 proteins can affect normal functions of p53.

Animals↗
PubMed2006

RNA interference directs innate immunity against viruses in adult Drosophila.

Innate immunity against bacterial and fungal pathogens is mediated by Toll and immune deficiency (Imd) pathways, but little is known about the antiviral response in Drosophila. Here, we demonstrate that an RNA interference pathway protects adult flies from infection by two evolutionarily diverse viruses. Our work also describes a molecular framework for the viral immunity, in which viral double-stranded RNA produced during infection acts as the pathogen trigger whereas Drosophila Dicer-2 and Argonaute-2 act as host sensor and effector, respectively. These findings establish a Drosophila model for studying the innate immunity against viruses in animals.

Animals↗
PubMed1975

Replication of nuclear polyhedrosis virus in a continuous cell culture of Spodoptera frugiperda: microscopy study of the sequence of events of the virus infection.

A microscopy study of the sequence of morphogenic events of Spodoptera frugiperda nuclear polyhedrosis virus infection of S. frugiperda cells is presented which orders the sequence of replication and establishes the time scale within which the events occur. The virus entered the cell by 1 h postinfection and was uncoated. The eclipse period was 9 h and the latent period was 12 h. Polyhedron formation was detected by 18 h postinfection and continued until the deposition of the polyhedron membrane was completed by 48 h postinfection. Aberrant morphogenic characteristics of virus repeatedly passaged in the cell culture were also recorded. Adsorption, envelope morphogenesis, and release mechanisms are discussed in light of other data on in vivo and in vitro baculovirus infections.

Adsorption↗
PubMed1975

Purification and properties of a nuclear polyhedrosis virus from Choristoneura fumiferana.

The polyhedral inclusion bodies of a nuclear polyhedrosis virus from the spruce budworm Choristoneura fumiferana were purified by fluorocarbon treatment, sucrose columns, sucrose density gradients, and equilibrium centrifugation. The preparation yields a single and homogeneous band in potassium tartrate gradients at a density of 1.193 g/ml. The virions were released from the polyhedra by alkali treatment and purified by either rate zonal or equilibrium centrifugation in sucrose gradients. The purified virions, which band at a density of 1.265-1.300 g/ml in sucrose, were infectious to the spruce budworm larvae and their mean LD50 was 1.09 +/- 0.1 mug of virus. However, viral nucleocapsids, released by a nonionic detergent, Nonidet P-40, had a density of 1.315 g/ml and were noninfectious by either per os inoculation or intrahemocoelic injection.

Animals↗
PubMed1978

Apparent replication of an unusual virus-like particle in both a parasitoid wasp and its host.

Nuclear inclusion bodies are found in the hemocytes of all tussock moth larvae parasitized by the braconid wasp Apanteles melanoscelus. These inclusion bodies represent the apparent site of replication of an unusual virus-like particle. Identical particles are observed in the nuclei of a small number of parasitoid calyx cells and are probably transmitted to host larvae during oviposition.

Animals↗
PubMed1980

Further studies on the use of protein A in immune electron microscopy for detecting virus particles.

The immune electron microscopic technique which involves pre-coating electron microscope grids with protein A before coating them with the specific antiserum, has been found suitable for detecting isometric insect and plant viruses. With the three virus-antibody combinations tested, the optimum antiserum dilution for protein A plus antiserum treatment was found to be 1:100 or less, whereas in the case of grids treated with antiserum alone it ranged from 1:1000 to 1:2000 although the titre of the antisera ranged from 1:512 to 1:4096. The increase in the number of particles on grids treated with protein A plus antiserum over those treated with antiserum alone, at each optimal antiserum dilution, was 25.7-fold (sugarcane mosaic virus), 2.1-fold (tobacco mosaic virus) and 1.6- and 2.4-fold (Erysimum latent virus--two different sap dilutions). Protein A plus antiserum-coated grids can be stored for up to 6 months at 4 degrees C, but not at room temperature or in a desiccator, and still retain about 25% of their activity, sufficient to detect any virus using the electron microscope. Antisera preserved in glycerol can be used successfully for detecting viruses by immune electron microscopy.

Immunologic Techniques↗
PubMed1990

Structural homology among four nodaviruses as deduced by sequencing and X-ray crystallography.

The genomic RNA2s of nodaviruses encode a single gene, that of protein alpha, the precursor of virion proteins beta and gamma. We compared the sequences of the RNA2s of the nodaviruses, black beetle virus (BBV), flock house virus, boolarra virus and nodamura virus, with the objective of identifying homologies in the primary and secondary structure of these RNAs and in the structure of their encoded protein. The sequences of the four RNAs were found to be similar, so that homologous regions relating to translation and RNA replication were readily identified. However, the overall, secondary structures in solution, deduced from calculations of optimal Watson-Crick base-pairing configurations, were very different for the four RNAs. We conclude that a particular, overall, secondary structure in solution within host cells is not required for virus viability. The partially refined X-ray structure of BBV (R = 26.4% for the current model) was used as a framework for comparing the structure of the encoded proteins of the four viruses. Mapping of the four protein sequences onto the BBV capsid showed many amino acid differences on the outer surface, indicating that the exteriors of the four virions are substantially different. Mapping in the beta-barrel region showed an intermediate level of differences, indicating that some freedom in choice of amino acid residues is possible there although the basic framework of the capsids is evidently conserved. Mapping onto the interior surface of the BBV capsid showed a high degree of conservation of amino acid residues, particularly near the protein cleavage site, implying that that region is nearly identical in all four virions and has an essential role in virion maturation, and also suggests that all four capsid interior surfaces have similar surfaces exposed to the viral RNA. Apart from a small portion of the C promoter, the amino terminus of the BBV protein (residues 1 to 60) is crystallographically disordered and the amino acid residues in that region are not well conserved. The disordered portion of the BBV protein clearly projects from the capsid inner surface into the interior of the virion, the region occupied by the viral RNA. In all four viruses, residues 1 to 60 had a high proportion of basic residues, suggesting a virus-specific interaction of the amino terminus with the virion RNA.

Amino Acid Sequence↗
PubMed1995

Characterization of a murine-human chimeric antibody with specificity for the pre-S2 surface antigen of hepatitis B virus expressed in baculovirus-infected insect cells.

We have produced and characterized a murine-human chimeric antibody with specificity for the pre-S2 surface antigen of hepatitis B virus (HBV) in baculovirus-infected insect cells. Recombinant baculovirus carrying the cDNA coding for the heavy or light chain of the chimeric antibody was constructed and co-infected into insect cells. The chimeric antibody (BV-S2) expressed in the cells was purified by an affinity chromatography on Protein A-Sepharose 4B column and characterized by N-terminal amino acid sequencing, affinity determination for pre-S2 peptide, endoglycosidase digestion and Clq binding assay, which were then compared with those of the chimeric antibody H69K that has the same amino acid sequence as BV-S2, but produced from transfected murine myeloma cells. The N-linked glycosylation of the BV-S2 antibody was also analyzed by culturing the baculovirus-infected cells in the presence of tunicamycin. The results showed that the BV-S2 was secreted following correct removal of the leader peptides, contained N-linked carbohydrate at the heavy chain, and had the same binding affinity and Clq binding ability as H69K, suggesting that the BV-S2 chimeric antibody is functional and thus may be useful in the prevention of HBV infection.

Animals↗
PubMed2006

Antigenic properties and diagnostic potential of African swine fever virus protein pp62 expressed in insect cells.

African swine fever (ASF) is an infectious and economically important disease of domestic pigs. The absence of vaccine renders the diagnostic test the only tool that can be used for the control of new outbreaks of the disease. At present, the enzyme-linked immunosorbent assay (ELISA) test is the most useful method for large-scale ASF serological studies, although false positives have been detected, mainly on poorly preserved sera. In order to improve the current diagnostic test available for ASF, we have studied the antigenic properties of the ASF virus polyprotein pp62 and its suitability for use in a novel ELISA. Two well-known antigenic proteins of ASF virus, p32 and p54, were also included in this study. These proteins were expressed in the baculovirus expression system and used as antigens in ASF serological tests. Our results indicate that the use of these recombinant proteins as antigens in the ELISAs improves the sensitivity and specificity obtained with the conventional diagnosis test used to detect antibodies against ASF virus. Furthermore, the use of polyprotein pp62 in an ELISA for testing poorly preserved sera allows performance of the diagnosis of ASF without the need to confirm the results by the immunoblot test. These features make pp62 one of the most interesting viral proteins to be used for serological ASF diagnosis.

African Swine Fever↗