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Growth of nuclear polyhedrosis virus in larvae of the cabbage moth, Mamestra brassicae L.

Growth of nuclear polyhedrosis virus (NPV) in 5 larval instars of cabbage moth, Mamestra brassicae, has been quantified using 2 methods. Numbers of polyhedra were estimated by light microscope counts while concentrations of virus protein antigen were estimated using ELISA. Virus growth was rapid initially but slowed during its later stages, although ELISA protein concentrations decreased once a peak had been reached. There was a linear correlation between polyhedral counts and virus protein during the initial growth phase. Maximum polyhedral production ranged from 2 x 107 (first instar) to 3.4 x 109 (fifth instar) and could be correlated directly to increasing larval weight. Using ELisa, virus antigen was detectable at least 24 hours before polyhedra were observed under the light microscope. Productivity ratios ranged from 83,500 in the first instar to 1352 in the fifth instar.

Animals↗

A capsid-associated protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata: genetic location, sequence, transcriptional mapping, and immunocytochemical characterization.

Two lambda gt11 clones containing overlapping DNA inserts encoding portions of a structural protein gene from Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) were identified by their immunoreactivity with polyclonal antisera produced against purified polyhedra-derived virus. Sequence analysis of a 3.6-kb region of the baculovirus genome (map units 69.1-71.6) from which the lambda gt11 inserts originated revealed an open reading frame of 1872 nt (624 amino acids) encoding a predicted protein of 70.6 kDa. Northern blot, primer extension, and 3' S1 analysis of this ORF indicated that an mRNA of approximately 2100 nt was transcribed from this gene. The mRNA appears to initiate from a late promoter/mRNA start site consensus sequence GTAAG and is expressed at late times postinfection. A gene fusion containing the C-terminal 368 amino acids of the gene was constructed using a bacterial trpE expression vector. Rabbit antiserum made against the purified fusion protein reacted with a protein of 87 kDa on Western blots of infected cell extracts at 24 hr p.i. and thereafter. The p87 protein was shown to be a component of both budded and polyhedra-derived virus and purified capsids. Immunofluorescence analysis indicated that p87 is expressed late in infection and concentrated in infected cell nuclei.

Amino Acid Sequence↗

Identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus.

Monoclonal antibodies with specificity for the abundant envelope surface glycoprotein (gp67) of Autographa californica nuclear polyhedrosis virus (AcMNPV) were used to screen a lambda gt11 expression library of AcMNPV DNA fragments. The gp67 gene was mapped to the left end of the EcoRI H fragment in a right-to-left orientation on the consensus map of AcMNPV. A 2.1-kilobase transcript which hybridized to the region was first detected in cell extracts at 2 h postinfection; it peaked in abundance at 18 h postinfection and thereafter was present at lower levels. The nucleotide sequence of the region was determined, and a 1,590-nucleotide open reading frame flanked by an AT-rich sequence was identified that could encode a polypeptide with 529 amino acid residues (molecular mass of 60,167 daltons). Computer analysis indicated that the peptide possesses two hydrophobic regions near the N and C termini as well as six potential N-linked glycosylation sites. We suggest that following cleavage of a signal peptide, the polypeptide undergoes further processing and becomes anchored at its C terminus in the virus envelope. The final seven amino acid residues at the C terminus contain basic amino acids and may have a role in virion assembly.

Amino Acid Sequence↗

Characterization of nucleic acids associated with Arkansas bee virus.

Arkansas bee virus (ABV) is a 30-nm isometric virus composed of one major species of polypeptide (mol. wt. 43 X 10(3] and one species of single-stranded RNA (mol. wt. 1.8 X 10(6]. The size of the genomic RNA and the lack of evidence for encapsidation or synthesis of a second smaller RNA species exclude further consideration of this virus for inclusion with the physicochemically similar viruses of the Nodaviridae. Several independent isolations of ABV were made from bees, and in each case it was associated in mixed infection with a picorna-like virus termed Berkeley bee picornavirus (BBPV). No evidence for replicative dependence, serological relationship, or genome relatedness by complementary DNA hybridization was obtained between ABV and BBPV.

Animals↗

Female characteristics in the Drosophila melanogaster-sigma virus system in natural populations from Languedoc (southern France).

The Drosophila melanogaster-sigma virus system has been extensively analysed for some years in Languedoc. The sigma virus, endemically present in natural populations of the fly, is not contagious but vertically transmitted. The frequency of infected flies dramatically increased from 1983 to 1988 in Languedoc and the increase was clearly correlated with some viral characteristics. From 1988 onwards, this frequency steadily decreased. Three zones were identified in Languedoc, from North to South, in each of which the virus evolved differently. Transovarial transmission and other parameters on the female side in the host virus system were investigated in samples collected in 1992. There were differences in values between the three zones. The contribution of these parameters to the observed differential frequencies of infected flies is discussed.

Alleles↗

Characterization of a non-occluded baculovirus (subgroup C) from the field cricket, Gryllus rubens.

A non-occluded baculovirus was isolated from nymphs of the field cricket, Gryllus rubens. SDS-polyacrylamide gel electrophoresis revealed the presence of 6 major and 11 minor polypeptides in these particles. Restriction endonuclease analysis indicated that the genome, 87.0 +/- 1.8 kilobase pairs, was a closed circular DNA molecule. DNA-DNA hybridization in low strigency conditions revealed no homology with the genomes of Oryctes baculovirus or Autographa california NPV. The virus replicated in nuclei of fat body cells, and was transmitted per os to a small proportion of first instar G. rubens nymphs.

Animals↗

Purification of viruses by centrifugation in gradients of inert substances.

By using a reorienting gradient centrifuge rotor cut from a block of Nylon and fitted with eight septae, it was possible to separate the components of the haemolymph of the mollusc Turbo sarmaticus into three fractions in a sucrose gradient held in the bowl of the rotor. The fractions were (108 and 98)S, 44S and 16-22S. The success of the experiment was due to the large differences in the sedimentation coefficients of the components. When the rotor was applied to the natural mixture of the five viruses of the caterpillars of Nudaurelia cytheria only the main component could be isolated in a pure state. The viruses were separated by isopycnic centrifugation in "self formed" caesium chloride gradients, using a Beckman Model E analytical centrifuge in which a separation cell fitted with a centerpiece with two perforated partitions was used.

Animals↗

Triatoma virus pathogenicity in laboratory colonies of Triatoma infestans (Hemiptera:Reduviidae).

In a survey of wild populations of Triatoma infestans (Klug) in Argentina, 10% were infected with Triatoma virus (TrV). The virus also was detected in a laboratory colony 18 mo after being established, with infection rates up to 100%. Mortality rate was 97.6% in nymphs and the molting process was inhibited, thereby increasing development time. Because the virus was detected in colony nymphs. TrV may be transmitted vertically. However, the higher infection rate in the colony compared with natural populations also indicates other route(s) of transmission.

Animals↗

Deformed wing virus is not related to honey bees' aggressiveness.

Guards of Cyprian honey bee colonies, Apis mellifera cypria, display a great defensive behaviour against hornets' attacks. The deformed wing virus (DWV) and the kakugo virus (KV) genomes are very similar, but unlike KV, the presence of DWV is not related to honey bees' aggressiveness. This discrepancy is further discussed.

Aggression↗

Isolation of large and small plaque variants of the Autographa californica nuclear polyhedrosis virus. Brief report.

Variants of the Autographa californica nuclear polyhedrosis virus which produce large (1.4 mm) or small (0.5 mm) plaques on Spodoptera littoralis cells have been isolated. Yields of extracellular virus and polyhedra by the large plaque variant were six-fold higher and 140-fold lower, respectively, than those obtained with the small plaque variant. However the two variants could not be distinguished when Spodoptera frugiperda cells were used.

Animals↗

Two related viral genes are located on a single superhelical DNA segment of the multipartite Campoletis sonorensis virus genome.

Campoletis sonorensis virus (CsV) (Polydnaviridae) is a large eucaryotic DNA virus with a structurally complex genome consisting of 28 or more superhelical (SH) DNA segments. Little is known of the relationship between different SH DNAs, but some SH DNAs do cross hybridize, indicating a relatedness between certain SH DNAs. In a previous study of viral expression in parasitized Heliothis virescens larvae, several partially homologous viral mRNAs were also identified (G. W. Blissard, S. B. Vinson, and M. D. Summers, 1986, J. Virol. 57, 318-327). To study the organization of the viral genome and the relationship between two partially homologous viral mRNAs, we analyzed cDNA clones and a cloned SH DNA segment of the CsV genome. Of the two CsV mRNAs examined (1.6 and 1.0 kb), both mRNAs were abundant at 48 hr after parasitization and the 1.6-kb mRNA was detected as early as 2 hr after parasitization. Nucleotide sequence analyses of cDNA clones representing the two partially homologous CsV mRNAs (1.6 and 1.0 kb) show that the two CsV mRNAs share five regions of imperfect homology (68 to 88%) which include a large part of each mRNA. These data indicate that the two mRNAs are transcribed from two separate but closely related CsV genes. Comparison of predicted amino acid sequences shows that the two related viral genes encode proteins with divergent amino acid sequences. Northern and Southern hybridization analyses using cloned cDNAs as probes showed that one CsV mRNA (1.6 kb) is homologous to CsV SH DNAs W, R, and M, while the other mRNA (1.0 kb) shows strong homology only to SH DNA W. By cloning and Southern hybridization mapping of the 15.8-kbp SH DNA W, we demonstrate that the genes for the related 1.6- and 1.0-kb mRNAs are located on different regions of this single 15.8-kbp SH DNA. Alignment of nucleotide sequences from a cloned viral genomic DNA and a cDNA demonstrates that the CsV gene encoding the 1.6-kb mRNA is a spliced gene containing at least two introns. Conservation of splice junctions between the two mRNAs suggests that the 1.0-kb mRNA is also spliced. These data represent the detailed analysis of two closely related CsV mRNAs abundantly expressed in parasitized H. virescens larvae, the first observation of related viral genes in a eucaryotic DNA virus, the demonstration of splicing in the Polydnaviridae, and the cloning and mapping of one of the largest SH DNA segments of the CsV genome.

Base Sequence↗

Mapping and molecular characterization of a functional thymidine kinase from Amsacta moorei entomopoxvirus.

A thymidine kinase (TK) gene from the entomopoxvirus of Amsacta moorei (AmEPV) has been identified, mapped, cloned, and sequenced. The AmEPV TK was shown to be biologically functional as cloning of the gene into a TK-derivative of the orthopoxvirus vaccinia creates a TK+ virus. The gene has been localized to a 1.5-kb EcoRI-Q DNA fragment which maps to the far left end of the viral genome. Sequence analysis reveals an open reading frame (ORF) of 182 amino acids potentially encoding a polypeptide of 21.2 kDa. Amino acid homology comparisons indicate that the gene is most closely related to the TKs of a variety of poxviruses (approximately 45%) and less so to the TKs of vertebrates (approximately 40%). The TK from African swine fever virus (ASF) showed the least homology (31.4%) to the AmEPV TK gene, suggesting that these two viruses are not closely related although ASF shares some biological features of poxviruses, and both ASF and AmEPV can replicate within arthropod hosts.

Amino Acid Sequence↗

Detection and purification of a recombinant human B lymphotropic virus (HHV-6) in the baculovirus expression system by limiting dilution and DNA dot-blot hybridization.

A recombinant virus Ac373-HB was detected and purified from the transfection mixture of wild type and recombinant virus in the baculovirus expression system using a combination of limiting dilution and DNA dot-blot hybridization. This method allows for a quick and convenient way of detection and purification of recombinant virus without the need to use a plaque purification step. It is generally applicable to other expression systems besides the baculovirus system described here.

B-Lymphocytes↗

Assessment of the human and ecological hazards of microbial insecticides.

A comprehensive account is given of the evolution of registration guidelines and safety testing procedures for microbial insecticidal agents. Particular emphasis is given to the use of viruses for pest control and the various guidelines developed to assess their possible hazards. The likely gains and risks associated with using viruses are discussed. Several meetings have been held in the last 10 years to assess the hazards of virus insecticides. Some of these meetings have produced recommendations, some have developed guidelines for safety testing. These meetings are reviewed. The various guidelines developed for safety testing are critically evaluated and the UK Registration Criteria for Biological Agents used as Pesticides are reproduced in full. Examples of viruses that have been safety tested and registered for use are given and the criteria used are described. Conclusions are drawn on the merits of the guidelines presently available and the likely future development of safety testing schemes is considered.

Animals↗

Rifampin inhibition of the occluded virus form of a nuclear polyhedrosis virus.

Rifampin at concentrations toxic to noninfected cells but not to infected cells is a selective inhibitor of occluded virus of the group A Baculoviridae (nuclear polyhedrosis virus). However, the titer of nonoccluded virus is not affected. Rifampin blocks occlusion until late in the replication cycle (14 to 16 h), and its effects are reversible. Modes of action of polyhedral inclusion body production are unknown.

Inclusion Bodies↗

Separation of the plus and minus strands of cytoplasmic polyhedrosis virus and human reovirus double-stranded genome RNAs by gel electrophoresis.

The complementary strands of most of the genome double-stranded RNA segments of insect cytoplasmic polyhedrosis virus (CPV) and human reovirus are separated for the first time by agarose gel electrophoresis in in the presence of 7 M urea. CPV (+) strands and most reovirus (-) strands migrate faster than the corresponding strands of opposite polarity. Glyoxal treatment, which modifies guanine residues and prevents G-C basepairing, results in a loss of strand resolution and concomitantly a significant decrease in electrophoretic mobilities. Reovirus mRNAs synthesized in vitro with ITP substituted for GTP show similar decreased electrophoretic mobilities as the glyoxalated mRNAs. These results clearly indicate that the basis for (+) and (-) strand resolution is the presence of secondary structure formed mainly by G-C(U) base-pairs that are maintained during gel electrophoresis in the presence of 7 M urea. When the plus and minus strands of CPV genomes were separated and compared for protein synthesizing activity, it was found that only the plus strands were able to form stable 80S ribosome-RNA initiation complexes in wheat germ cell-free extracts.

Base Composition↗

A new small RNA virus persistently infecting an established cell line of Galleria mellonella, induced by a heterologous infection.

A persistent infection in a Galleria mellonella cell line was revealed when infected with a maize stem borer picorna-like virus isolated on Sesamia cretica (MSBV). The new virus, completely different from the MSBV, is designated as G. mellonella cell line virus (GmclV), induces spectacular cytopathic effects, and is also considered efficient in vivo. The GmclV is a 29-nm-diameter isometric virus, with single-strand RNA of 2.9 x 10(6) Da molecular weight with a poly(A) tract. Its capsid is constituted of only two major polypeptides, of 34,500 and 32,500 Da, and no minor bands could be detected. The characteristics of the GmclV do not permit us to classify it with assurance. Even though it has not yet been identified as a picornavirus, it can be classified in the small RNA virus group of the Picornaviridae. G. mellonella represents a very interesting model, owing to the fact that two different persistent viruses belonging to the same family were isolated in vivo and in vitro, to further the understanding of the general phenomenon of persistency and induction.

Animals↗