Viruses and parasitism in insects.
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We determined the complete genome sequence of Himetobi P virus (HiPV), an insect picorna-like virus, which was isolated from the small brown planthopper, Laodelphax striatellus. The genome of HiPV consists of 9,275 nucleotides excluding the poly (A) tail, and contains two large open reading frames (ORFs), which were separated by a 176-nucleotide noncoding region. The deduced amino acid sequence of the first ORF contains core motifs of picornaviral helicase, protease, and RNA-dependent RNA polymerase. The capsid protein-coding region was mapped onto the second ORF by determining the N-terminal amino acid sequences of the capsid proteins. Subgenomic RNA for the capsid protein gene was not detected in the infected tissue. The capsid protein precursor gene of HiPV lacks an AUG initiation codon at the expected position and the upstream sequence of the gene is predicted to form several stem-loop structures, suggesting that the precursor is produced by internal ribosome entry site (IRES) mediated-translation, as occurs in Plutia stali intestine virus (PSIV). These characteristics of the HiPV genome are similar to those of a new group of RNA viruses consisting of Drosophila C virus (DCV), Rhopalosiphum padi virus (RhPV), and PSIV.
The hemagglutinin of influenza (fowl plague) virus was expressed in larvae of Heliothis virescens by using recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector. Animals were infected with the recombinant virus either by parenteral injection or by feeding. For oral uptake, recombinant virus occluded in polyhedra obtained from cultured Spodoptera frugiperda cells after coinfection with authentic AcNPV was used. Immunohistological analyses of infected animals revealed that the hemagglutinin was expressed only in those tissues that are also permissive for the replication of authentic AcNPV. These tissues included hypodermis, fat body, and tracheal matrix. After oral infection, hemagglutinin was also detected in individual gut cells. The amount of hemagglutinin synthesized in larvae after parenteral infection was 0.3% of the total protein, compared with 5% obtained in cultured insect cells. The hemagglutinin was transported to the cell surface and expressed in polarized cells only at the apical plasma membrane. It was processed by posttranslational proteolysis into the cleavage products HA1 and HA2. Oligosaccharides were attached by N-glycosidic linkages and were smaller than those found on hemagglutinin obtained from vertebrate cells. Hemagglutinin from larvae expressed receptor binding and cell fusion activities, but quantitation of the hemolytic capacity revealed that it was only about half as active as hemagglutinin from vertebrate or insect cell cultures. Chickens immunized with larval tissues containing hemagglutinin were protected from infection with fowl plague virus. These observations demonstrate that live insects are able to produce a recombinant membrane protein of vertebrate origin in biologically active form.
The molecular weight of Tipula iridescent virus, based on sedimentation and diffusion coefficients, was 5.51 x 10(8), with hydration of 0.57 g of water per g of virus. Deoxyribonucleic acid content, based on total inorganic phosphorus liberated, was 19 +/- 0.2%. At 260 mmu, the virus gave an uncorrected absorbance of 18.2 cm(2)/mg of virus and a light-scattering corrected absorbance of 9.8 cm(2)/mg of virus. Amino acid analyses of the virus protein revealed a remarkable similarity to Sericesthis iridescent virus. The possibility is discussed that the four iridescent insect viruses reported to date bear a strain relationship.
Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in trans from nonreplicating RNA. However, capsid protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.
We have determined the nucleotide sequence of sacbrood virus (SBV), which causes a fatal infection of honey bee larvae. The genomic RNA of SBV is longer than that of typical mammalian picornaviruses (8832 nucleotides) and contains a single, large open reading frame (179-8752) encoding a polyprotein of 2858 amino acids. Sequence comparison with other virus polyproteins revealed regions of similarity to characterized helicase, protease and RNA-dependent RNA polymerase domains; structural genes were located at the 5' terminus with non-structural genes at the 3' end. Picornavirus-like agents of insects have two distinct genomic organizations; some resemble mammalian picornaviruses with structural genes at the 5' end and non-structural genes at the 3' end, and others resemble caliciviruses in which this order is reversed; SBV thus belongs to the former type. Sequence comparison suggested that SBV is distantly related to infectious flacherie virus (IFV) of the silk worm, which possesses an RNA of similar size and gene order.
Apoptosis has been considered to be a host defense mechanism against viral infection in multicellular organisms. This is based on the findings that apoptogenic mutants of insect viruses cannot grow because infected host cells die by apoptosis. This suggests that the apoptotic response of host cells has a deleterious effect on virus infection. Thus, apoptosis is an important host defense mechanism that is capable of inhibiting viral replication during infection. However, in vitro studies indicated that apoptosis alone does not provide the same protection against viral infection in animal cells as it does in the insect cells. Still, most animal viruses have acquired a strategy to overcome host cell apoptosis. In addition, a varying degree of necrosis usually accompanies apoptosis, suggesting a possible contribution of necrosis to the host reactions against virus. To understand the physiological significance of apoptosis during animal virus infection, we have characterized viral growth and the cellular responses against virus infection in a wide variety of virus-cell interaction systems. Mainly based on our own works, we discuss the nature of apoptosis in the animal virus infection and verify its role as a host defense mechanism against virus infection.
There are several factors involved in the mechanical transmission of equine infectious anemia (EIA) virus by insects. Large hematophagous insects, especially tabanids, which feed from extravascular sites (ie, pool feeding) appear to be the most efficient vectors. The biology of the host-seeking and blood-feeding behavior of the vectors are important variables that have been overlooked in the mechanical transmission of pathogens like EIA virus. The biology, population levels, and diversity of the vectors, in addition to the clinical status and proximity of EIA virus-infected horses maintained with susceptible animals are all important variables that contribute to EIA virus transmission in nature.
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Polydnaviruses are an unusual group of insect viruses that have an obligate symbiotic association with certain parasitic wasps. These viruses are transmitted with the wasp egg during oviposition into lepidopteran insects, enabling the survival and development of the egg inside the host larvae. We report the three-dimensional structure of a novel polydnaviral cysteine-rich motif (cys-motif), identified as the carboxyl-terminal domain of a two cys-motif containing polydnaviral VHv1.1 gene product, abbreviated "C-term VHv1.1". This 65-residue domain was identified experimentally by limited proteolysis of the full-length protein and was subsequently cloned in a bacterial expression system for NMR studies. The C-term VHv1.1 3D structure was determined in solution by two-dimensional (1)H NMR spectroscopy. Calculation of the structure was based on a total of 300 upper distance restraints and 20 dihedral angle constraints, and resulted in an ensemble of 25 representative conformers with an average rmsd of 0.47 A from the mean structure for core backbone atoms. The protein core is made of a four beta-strand scaffold held together in a compact structure by three disulfide bonds, which form a cystine knot. The four beta-strands are arranged in an unusual configuration to form a triple-stranded beta-sheet and double-stranded beta-sheet. Comparison with other classes of cystine knots provides indication that C-term VHv1.1 represents a new and distinct cystine knot motif. This analysis provides a structural basis for interpretation of the genetic and amino acid sequence data classifying polydnavirus gene products as members of cysteine-rich protein families.
A series of cell lines unique in insect virus susceptibility pattern have been isolated from the ovaries of the gypsy moth (Lymantria dispar: Lepidoptera: Lymantriidae) on a synthetic medium with mammalian and avian serum supplementation. Growth curves showed the poorest growth occurring on peptone-based media with somewhat better growth on amino-acid-based media. The best growth was obtained with combined media. Serological study distinguished the present cell lines from one another and from cell lines derived from other insect species grown routinely in the same laboratory. Baculovirus susceptibility among the new lines varied from no response to a specific complete replication response upon challenge by the homologous (gypsy moth) nuclear polyhedrosis virus.
We report the genome sequence of Aleuroclava gordoniae-associated polerovirus (AgAP), identified from the whitefly Aleuroclava gordoniae. The 5,650-nt AgAP genome contains 6 open reading frames. Phylogenetic analysis places AgAP within the genus Polerovirus, which comprises plant-infecting viruses. This study provides a genomic resource for further investigation of virus-insect associations.
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Representatives of several families of insect viruses were tested for growth and pathogenicity in the olive fruit fly, Dacus oleae Gmel. The viruses included nuclear polyhedrosis viruses, an iridovirus, two picornaviruses, and Trichoplusia ni small RNA virus (a member of the Nudaurelia beta family), in addition to two naturally occurring viruses of the olive fruit fly. Two viruses, one of the two picornaviruses (cricket paralysis virus [CrPV] and the iridovirus (type 21 from Heliothis armigera), were found to replicate in adult flies. Flies which were fed on a solution containing CrPV for 1 day demonstrated a high mortality with 50% dying within 5 days and nearly 80% dying within 12 days of being fed. The virus was transmissible from infected to noninfected flies by fecal contamination. The CrPV which replicated in the infected flies was demonstrated to be the same as input virus by infection of Drosophila melanogaster cells and examination of the expressed viral proteins, immunoprecipitation of the virus purified from flies, and electrophoretic analysis of the structural proteins.
In view of the increasing and devastating damage by rhinoceros beetle (Oryctes rhinoceros) to coconut palms in the middle of last century, many efforts were made to find an efficient natural control factor against this pest, which could not be controlled by pesticides. The basic procedures of these monitoring programmes are outlined together with the final detection of a virus disease in oil palm estates in Malaysia in 1963. In extensive laboratory studies, the virus was isolated and identified as the first non-occluded, rod-shaped insect virus, morphologically resembling the baculoviruses. Infection experiments clarified the pathology, histopathology, and virulence of the virus and demonstrated that the virus was extremely virulent to larvae after peroral application. These findings encouraged the first pilot release of virus in 1967 in coconut plantations of Western Samoa where breeding sites were contaminated with virus. Surprisingly, the virus became established in the Samoan rhinoceros beetle populations and spread autonomously throughout the Western Samoan islands. As a consequence, there was a drastic decline of the beetle populations followed by a conspicuous recovery of the badly damaged coconut stands. This unexpected phenomenon could only be explained after it was shown that the adult beetle itself is a very active virus vector and thus was responsible for the efficient autodissemination of the virus. The functioning of the beetle as a 'flying virus factory' is due to the unique cytopathic process developing in the midgut after peroral virus infection. Pathological details of this process are presented. Because of the long-term persistence of the virus in the populations, rhinoceros beetle control is maintained. Incorporation of virus into integrated control measures and successful virus releases in many other countries are recorded.