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PubMed1990

Characterization of the DA26 gene in a hypervariable region of the Autographa californica nuclear polyhedrosis virus genome.

A region of the baculovirus Autographa californica nuclear polyhedrosis virus genome that is frequently found to be altered after serial passage of the virus in cell culture was characterized. Sequence analysis of this region of the genome in wild-type and mutant viruses revealed that some of the mutations affected a 675 bp open reading frame, designated DA26. The DA26 gene was disrupted both by deletion and by insertion of sequences that resembled transposable elements. Northern blot analysis of DA26 showed that it was expressed very early after infection. DA26-specific transcripts could be detected after the 1 h viral adsorption period upon infection of cultured Trichoplusia ni cells. These transcripts were mapped by nuclease protection assays. A recombinant virus was constructed in which DA26 was disrupted by insertion of the Escherichia coli lacZ gene. This virus was viable in both T. ni and Spodoptera frugiperda cells and analysis of the kinetics of protein synthesis revealed no differences between wild-type and recombinant viruses. The disruption of DA26 also did not interfere with the ability of the virus to infect T. ni or S. frugiperda larvae.

Amino Acid Sequence↗
PubMed2005

Intricate transmission routes and interactions between picorna-like viruses (Kashmir bee virus and sacbrood virus) with the honeybee host and the parasitic varroa mite.

Viral diseases of honeybees are a major problem in apiculture, causing serious economic losses worldwide, especially in combination with varroa mites. To increase understanding of the relationship among viruses, mites and colony decline, the tripartite relationships among bees, viruses [Kashmir bee virus (KBV) and sacbrood virus (SBV)] and varroa mites have been investigated systematically. To develop an antibody-based test for KBV, two structural recombinant proteins were purified for polyclonal-antibody production. By using ELISA and RT-PCR, the presence of KBV and SBV was studied comparatively in different developmental stages and castes of bees. The results demonstrated that KBV may persist as a viral genome with extremely low levels of viral-capsid proteins and that KBV and SBV can co-infect honeybees. This study indicated the presence of KBV and SBV RNAs in both queens and eggs by RT-PCR, suggesting a route of transovarial transmission. Horizontal transmission is also very likely among adult bees and from adult workers to larvae through contaminated food resources, because both viruses have been detected in all developmental stages and food sources (brood food, honey, pollen and royal jelly). Furthermore, it was demonstrated that mites were another possible route of horizontal transmission, as both viruses were detected in mites and their saliva. This study, for the first time, detected co-occurrence of viruses in varroa, further underlining the importance of the mites in vectoring different bee viruses. Therefore, these results indicated that multiple infection routes exist for honeybee viral diseases.

Animals↗
PubMedDate not supplied

[Physico-chemical characteristics of the DNA of the nuclear polyhedrosis virus of the moth Porthetria dispar L].

DNA preparations were obtained after dissolving the inclusion bodies, polyhedra virus particles, from the purified bundle virus of Porthetria dispar L. nuclear polyhedrosis. The DNA molecules in the preparations obtained are of different conformation and separate within the CsCl density gradient in the presence of ethidium bromide into supercoiled catenated and relaxed circular molecules (with the admixture of linear molecules). The circular DNA was studied by electron microscopy. The size of virus genome according to the data of reassociation kinetics of DNA is about 100 MD. Estimated on the basis of the values of buoyant density (p) and the melting temperature (Tmelt.) the content of guanine-cytosine pairs (GC pairs) in the viral DNA varies from 61 up to 65 mol%, and in the insect cell DNA--from 38 up to 40 mol%. The viral and cellular DNA are distinctly separated by centrifugation within the CsCl density gradient.

Animals↗
PubMed1975

[Study of the vertical transmission and horizontal transmission of "Drosophila melanogaster" and "Drosophila immigrans" picornavirus (author's transl)].

The contamination of drosophila eggs, of larvae of the 3 instars and of adults, was studied using several strains of P and C viruses of D. melanogaster and of iota virus of D. immigrans. The infected adults contaminated other flies if they were rich in viral particles and if the contact was long enough. Infection of the adults occurred in the presence of concentrated viral suspensions. The larvae were easily infected when they grew in contaminated media; the more sensitive stage was the first instar. Transovarian transmission was observed only in naturally infected flies propagating viruses of serotype I or III. C viruses were not hereditarily transmitted. Persistence of the Picornaviruses of drosophila populations can be explained by the additive effects of the 3 mechanisms of contamination.

Animals↗
PubMed1976

[Electron microscopy studies of the early stages of mosquito cell infection in vitro by the chilo iridescent virus and the infectious bovine rhinotracheitis (author's transl)].

The early stage of the viral infection in vitro of mosquito cells were studied by electron microscopy using a microculture technique. We demonstrate in this paper that two viruses: iridescent (CIV) and herpetic (Infectious bovine rhinotracheitis) can enter by viropexis and be uncoated in several lines of mosquito cells.

Aedes↗
PubMed2005

Isolation and identification of a new tetravirus from Dendrolimus punctatus larvae collected from Yunnan Province, China.

In this study, Dendrolimus punctatus tetravirus (DpTV) has been identified as a new member of the genus Omegatetravirus of the family Tetraviridae that may be related serologically to Nudaurelia capensis omega virus (NomegaV). DpTV particles are isometric, with a diameter of about 40 nm and a buoyant density of 1.281 g cm(-3) in CsCl. The virus has two capsid proteins (of 62 500 and 6800 Da) and two single-stranded RNA molecules (RNA1 and RNA2), which are 5492 and 2490 nt long, respectively. RNA1 has a large open reading frame (ORF) encoding a polypeptide of 180 kDa; RNA2 contains two partially overlapping ORFs encoding polypeptides of 17 and 70 kDa. The 180 kDa protein, which contains consensus motifs of a putative methyltransferase, helicase and RNA-dependent RNA polymerase, shows significant similarity to those of other tetraviruses. The 17 kDa protein is a PEST (Pro/Glu/Ser/Thr) protein of unknown function. The 70 kDa protein is the coat protein precursor and is predicted to be cleaved at an Asn-Phe site located after residue 570. The 70 kDa protein shows 86 and 66 % identity to its homologues in NomegaV and Helicoverpa armigera stunt virus, respectively. Secondary-structure analysis revealed that the RNAs of DpTV have tRNA-like structures at their 3' termini.

Animals↗
PubMed1978

A new host, Simulium argyreatum Meig., for the cytoplasmic polyhedrosis virus of blackflies in Czechoslovakia.

The cytoplasmic polyhedrosis virus (CPV) from the midgut of blackfly larvae is recorded from a population of Simulium argyreatum in Czechoslovakia. A 7.7% infection of 2766 sampled larvae was manifested by white spots in their body. 85% of the white-spotted larvae were infected with CPV when sampled in June. In August 9% of 1377 larvae had white spots but only 10.6% of them were with CPV. Out of 823 collected pupae, only 3 imperfect pupae had the virus in apparent form. Other infections producing white spots in larvae were due to the microsporidia Plistophora multispora, P. simulii, P. debaisieuxi, Thelohania fibrata, the fungus Coelomycidium simulii and the mermithids.

Animals↗
PubMed1972

A cytoplasmic polyhedrosis virus in midgut cells of Anopheles stephensi and in the sporogonic stages of Plasmodium berghei yoelii.

Although it has been known for some time that nuclear and cytoplasmic polyhedrosis viruses may infect the larval stages of mosquitos capable of transmitting mammalian malaria this paper reports for the first time the presence of a cytoplasmic polyhedrosis virus in adult Anopheles stephensi. The virus was shown to be present in sporogonic stages of Plasmodium berghei yoelii with which the mosquitos were infected. It is possible that other viruses may affect both vector and malaria parasites. Further studies are required of viral infections in mosquitos as a possible method of biological control.

Animals↗
PubMedDate not supplied

Development of an antibody-based assay for determination of baculovirus titers in 10 hours.

The baculovirus expression system has been used to produce large amounts of biologically active proteins by infecting insect cells with a recombinant baculovirus expressing the target protein. For an efficient expression of the target protein, it is necessary to infect insect cells with an adequate amount of virus. However, current methods are time-consuming and either have technical difficulties or are limited as a result of virus expression mechanism using a reporter gene. A novel method is developed to yield virus titers in 10 h that is easy to perform using 96-well plates and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immunostaining. The titer is determined by counting foci produced as a result of infection of the virus under a fluorescent microscope. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post-infection time of 4 h. Therefore, 10 h was enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer. Titers determined using this immunological assay are comparable, both in value and validity, to those obtained using a traditional method, provided that the stocks have titers above 10(3) pfu/mL.

Animals↗
PubMed1983

Persistent baculovirus infections: Spodoptera frugiperda NPV and Autographa californica NPV in Spodoptera frugiperda cells.

Establishment of a persistent infection of Spodoptera frugiperda nuclear polyhedrosis virus (NPV) in Spodoptera frugiperda (S.f.) cells occurred in three phases: the first phase was characterised by high levels of cell infection and death, the second phase by decreasing cell infection levels leading to the final phase where less than one per cent of the cells were infected during any subculture. The virus persisted at this level of infection provided the cells were maintained by regular subculturing and incubated at the optimum growth temperature of 27 degrees C. Because of the low proportion of cells infected, cultures of virus-free cells could be selected ('cured') by dilution of the persistent infection without the use of viral antiserum. Unlike the parent S.f. cells, cultures of cured cells were partially resistant to infection with S. frugiperda NPV or infection with an unrelated baculovirus Autographa californica NPV. A. californica NPV, which is cytolytic for the parent S.f. cell line, established a persistent infection in the cured cells. The establishment pattern was similar to that previously found for S. frugiperda NPV and only one to five per cent of the cells were infected at equilibrium. Cured cells from the A. californica NPV persistent infection were highly resistant to infection with both S. frugiperda NPV and A. californica NPV. All attempts to find a viral interference phenomenon to explain the resistance of the cured cells were unsuccessful. All cell types adsorbed virus equally well. Slower growth of S.f. cells cured from the persistent A. californica NPV infection is the only difference so far observed between any of the S.f. cell types.

Animals↗
PubMed1988

Association of Autographa californica nuclear polyhedrosis virus (AcMNPV) with the nuclear matrix.

Nuclear matrices from uninfected Spodoptera frugiperda cells and those infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) were isolated and their protein constituents were compared. Proteins were characterized according to size and several different antibodies to Drosophila nuclear proteins were employed in an attempt to identify the proteins comprising this nuclear substructure. Three species of lamins were identified as major constituents of the nuclear matrix of Spodoptera cells. Two DNA-binding proteins having molecular weights of 54 and 36 kDa were also identified as components of the nuclear matrix of uninfected cells. Infection resulted in a superimposition of viral proteins upon the nuclear matrix of the host cell. Polyhedrin, the basic viral DNA-binding protein (p6.9), and the major capsid protein of AcMNPV were identified immunologically as components of the nuclear matrix fraction of infected cells. Infection also resulted in the increased association of cellular histones with the nuclear matrix. DNA-binding assays demonstrated histones and p6.9 were the predominant DNA-binding proteins associated with the nuclear matrix of infected cells. Nuclear matrices from uninfected cells and cells infected with AcMNPV for 10 and 24 hr were examined using transmission electron microscopy. Morphologically, the nuclear matrix of the uninfected cell consists of the outer nuclear lamina (including nuclear pore complexes), an internal fibrogranular protein constituent, and a residual nucleolar structure. Numerous viral capsids were observed associated with the nuclear matrix in cells infected with either wild-type AcMNPV or a polyhedrin-deletion mutant by 10 hr p.i. The capsids appeared to be attached in an end-on association with the internal fibrogranular protein network of the nuclear matrix. The matrix-associated capsids were similar in width and length to those packaged within the polyhedra. In addition to the capsids, polyhedra in various stages of maturation were seen at 24 hr following infection of the cells with the wild-type virus. The nuclear matrix of the infected cell appears to play an important role in baculovirus assembly.

Animals↗
PubMed1990

Transcription of the baculovirus polyhedrin gene reduces the levels of an antisense transcript initiated downstream.

A late 3.2-kilobase (kb) RNA initiated approximately 2 kb downstream of the 3' end of the Autographa californica nuclear polyhedrosis virus polyhedrin-coding sequence and traversed the polyhedrin gene in an antisense direction. This RNA was sense RNA for the two open reading frames flanking the polyhedrin gene. A mutant virus, vXpoly, which differs from wild-type virus only at the essential RNA initiation site in the polyhedrin promoter, exhibited higher levels of the 3.2-kb RNA than did wild-type virus during the polyhedrin transcriptional phase. Thus, transcription of the polyhedrin gene down regulates the levels of this 3.2-kb RNA.

Animals↗
PubMed1986

Restriction endonuclease activity induced by PBCV-1 virus infection of a Chlorella-like green alga.

An enzyme was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1 which exhibits type II restriction endonuclease activity. The enzyme recognized the sequence GATC and cleaved DNA 5' to the G. Methylation of deoxyadenosine in the GATC sequence inhibited enzyme activity. In vitro the enzyme cleaved host Chlorella nuclear DNA but not viral DNA because host DNA contains GATC and PBCV-1 DNA contains GmATC sequences. PBCV-1 DNA is probably methylated in vivo by the PBCV-1-induced methyltransferase described elsewhere (Y. Xia and J. L. Van Etten, Mol. Cell. Biol. 6:1440-1445). Restriction endonuclease activity was first detected 30 to 60 min after viral infection; the appearance of enzyme activity required de novo protein synthesis, and the enzyme is probably virus encoded. Appearance of enzyme activity coincided with the onset of host DNA degradation after PBCV-1 infection. We propose that the PBCV-1-induced restriction endonuclease participates in host DNA degradation and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.

Base Sequence↗
PubMed1993

St Louis encephalitis virus establishes a productive, cytopathic and persistent infection of Sf9 cells.

The Sf9 cell line, commonly used for gene expression by recombinant baculovirus, has been productively infected by St Louis encephalitis (SLE) virus, a flavivirus. SLE viral infection produced a c.p.e. in the Sf9 cells characterized by giant cells and the presence of 10-fold fewer cells in the infected cultures after the first week of infection compared with uninoculated control cultures. Infected Sf9 cells expressed SLE viral antigens, and intracellular virus particles were observed by electron microscopy. Titres of cell-associated SLE virus rose slightly over an 8 week period, whereas titres of cell-free virus remained stable, suggesting that SLE virus establishes a productive and persistent infection of Sf9 cells. The SLE virus produced by the Sf9 cells could be neutralized by SLE virus-immune mouse ascitic fluid, and no evidence of escape mutants was detected. Sf9 cells persistently infected with SLE virus could be superinfected with a recombinant baculovirus and expressed recombinant antigen. The successful infection of Sf9 cells by SLE virus represents the first report of production of c.p.e. by SLE virus in insect cells under routine cell culture conditions and of the infection of Sf9 cells by a human pathogen.

Animals↗
PubMed2002

Development of infectious transcripts and genome manipulation of Black queen-cell virus of honey bees.

The South African isolate of Black queen-cell virus (BQCV), a honey bee virus, was previously found to have an 8550 nucleotide genome excluding the poly(A) tail. Its genome contained two ORFs, a 5'-proximal ORF encoding a putative replicase protein and a 3'-proximal ORF encoding a capsid polyprotein. Long reverse transcription (RT)-PCR was used to produce infectious transcripts for BQCV and to manipulate its genome. Primers were designed for the amplification of the complete genome, the in vitro transcription of infectious RNA and PCR-directed mutagenesis. An 18-mer antisense primer was designed for RT to produce full-length single-stranded cDNA (ss cDNA). Unpurified ss cDNA from the RT reaction mixture was used directly as a template to amplify the full genome by long high-fidelity PCR. The SP6 promoter sequence was introduced into the sense primer to transcribe RNA directly from the amplicon. RNA was transcribed in vitro with and without the presence of a cap analogue and injected directly into bee pupae, which were then incubated for 8 days. In vitro transcripts were infectious but the presence of a cap analogue did not increase the amount of virus recovered. A single base mutation abolishing an EcoRI restriction site was introduced by fusion-PCR, to distinguish viral particles recovered from infectious transcripts from wild-type virus (wtBQCV). Mutant virus (mutBQCV) and wtBQCV were indistinguishable by electron microscopy and Western blot analysis. The EcoRI restriction site was present in wtBQCV and not in mutBQCV.

Animals↗
PubMed1990

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response.

Animals↗
PubMed1984

Comparison of the virus-associated polymerases of cytoplasmic polyhedrosis virus types 1 and 2.

A detailed comparison was made of the virus-associated polymerase activities of cytoplasmic polyhedrosis virus (CPV) types 1 and 2 which had previously been shown to differ in their response to the methyl donor S-adenosyl-L-methionine (AdoMet). While the type 1 CPV polymerase was approximately twice as active as the type 2 CPV enzyme in the presence of AdoMet, temperature, pH and divalent cation optima of the two enzymes were similar. Both viruses synthesized in vitro single-stranded RNA copies of only one strand of the double-stranded RNA genome. In addition, each RNA segment of both viruses was transcribed in approximately equal amounts by weight. The results suggest that most features of CPV polymerase activity are highly conserved, even among CPV types which show substantial antigenic and biochemical differences.

Insect Viruses↗
PubMed1997

Infection of the gonads of the SimES strain of Drosophila simulans by the hereditary reovirus DSV.

The S character of Drosophila simulans SimES-st strain undergoes a non-mendelian transmission. It has been postulated that a virus, Drosophila S virus (DSV), could be the causative agent. Electron microscopy analysis of gonads of flies showing a strong S phenotype revealed the presence of virus in or near the germ cells. The S character transmission rate is greater in females than that in males. Similarly, the level of infection by DSV is higher in ovaries than that in testes. Flies treated at a nonpermissive temperature do not present the S phenotype and appear to be cured from the virus. This information, taken together with previous work, makes the hypothesis that DSV is the causative agent of the S phenotype more than likely.

Animals↗