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PubMed2003

Rice stripe virus 23.9 K protein aggregates and forms inclusion bodies in cultured insect cells and virus-infected plant cells.

The genome of Rice stripe virus (RSV, genus Tenuivirus) contains seven open reading frames (ORFs). Little is known about the products of four of these ORFs, including the 23.9 K protein encoded by the virus-sense ORF of RNA3. Western blotting revealed that the 23.9 K protein was synthesized in the host plant and also in the planthopper vector of RSV. Using a baculovirus vector, the 23.9 K protein was expressed, both unfused and fused with red-shifted green fluorescent protein, in Spodoptera frugiperda cells. Inclusion bodies were observed by light microscope in cells expressing fused or unfused proteins. Inclusion bodies in cells expressing the fused protein fluoresced under blue light. By immunoelectron microscopy, electron-dense inclusion bodies in cells expressing the unfused protein were specifically labeled with 23.9 K protein antiserum. Moreover, electron-dense masses labeled with 23.9 K protein antiserum were observed in virus-infected wheat tissue by electron microscopy. This paper thus demonstrates that RSV 23.9 K protein can aggregate in vivo and form inclusion bodies in infected plant tissue.

Animals↗
PubMed2005

Insect contribution to horizontal transmission of Reticuloendotheliosis virus.

The involvement of insects in Reticuloendotheliosis virus (REV) transmission was examined by testing insects trapped at commercial farms and by controlled feeding experiments using mosquitoes, Culex pipiens L. and house flies, Musca domestica L. We established sensitive methods of REV detection, including reverse transcription-polymerase chain reaction (PCR) for REV-LTR and REV-gag genes, REV antigenemia measurements by enzyme-linked immunosorbent assay, and virus isolation in tissue cultures. A variety of blood-sucking species of insects were trapped at farms with infected poultry and tested, but none were positive. To rule out the possibility of PCR inhibition by insect RNA, spiking experiments were conducted and no interference was observed. Because Cx. pipiens mosquitoes were trapped frequently at farms, we performed feeding experiments with mosquito females fed on a REV-containing tissue culture medium and chicken blood mixture. Virus was detected in the mosquitoes up to 5 h postfeeding, compared with 96 h in the feeding mixture, indicating that Cx. pipiens can only harbor REV for a short period. House flies were suspected to be involved in the virus transmission because they frequently were trapped on positive farms. In contrast to mosquitoes, REV was harbored within the house fly digestive tract for up to 72 h and could infect chickens, as demonstrated by seroconversion and by detection of viral gag-sequence in the cloaca. The current study is supportive for the role of house flies as a mechanical vector of REV among poultry.

Animals↗
PubMed2002

Molecular anatomy of Chilo iridescent virus genome and the evolution of viral genes.

Chilo iridescent virus (CIV) or Insect iridescent virus 6 (IIV-6) is the type species of the genus iridovirus, a member of the Iridoviridae family. CIV is highly pathogenic for a variety of insect larvae and this implicates a possible use as a biological insecticide. CIV progeny and assembly occur in the cytoplasm of the infected cell and accumulate in the fatbody of the infected insects. Since the discovery of CIV in 1966, many attempts were made to elucidate the viral genome structure and the amino acid sequences of different viral gene products. The elucidation of the coding capacity and strategy of CIV was the first step towards understanding the underlying mechanisms of viral infection, replication and virus-host interaction. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The coding capacity of the CIV genome was determined by the analysis of the complete DNA nucleotide sequence consisting of 212,482 bp that represent 468 open reading frames encoding for polypeptides ranging from 40 to 2432 amino acid residues. The analysis of the coding capacity of the CIV genome revealed that 50% (234 ORFs) of all identified ORFs (468 ORFs) were non-overlapping. The identification of several putative viral gene products including a DNA ligase and a viral antibiotic peptide is a powerful tool for the investigation of the phylogenetic relatedness of this evolutionary and ecologically relevant eukaryotic virus.

Amino Acid Sequence↗
PubMed1994

Susceptibility of the Sf9 insect cell line to infection with adventitious viruses.

Sf9, the insect cell line commonly used for gene expression by recombinant baculovirus (BV), can be infected by St. Louis encephalitis (SLE) virus, a flavivirus, resulting in a persistent, productive, and cytopathic infection, while retaining the ability to be infected with a recombinant baculovirus (rBV). We now demonstrate using double immunofluorescence that single cells are dually infected with SLE virus and rBV. Fourteen additional viruses including additional flaviviruses, other arbovirus classes, vesicular stomatitis virus (VSV), and herpes simplex virus, type 1 (HSV-1) failed to produce a cytopathic effect (CPE) in Sf9 cells. Plaque assays indicated infectious virus was present for several weeks post-inoculation for Yellow fever (YF), Dengue types 1 and 2 (DEN-1 and DEN-2), Gumbo limbo (GL), Eastern equine encephalomyelitis virus (EEE), Western equine encephalomyelitis virus (WEE), HSV-1, and VSV viruses. For HSV-1, GL, EEE, WEE and VSV, but not for YF, DEN-1 or DEN-2 viruses, this could be attributed solely to survival in the Sf9 cell culture media. Of the 14 viruses tested, only HSV-1 could be detected after 2 weeks in serum-free media. The data indicate that several viruses which are pathogenic for humans are stable for long periods of time at 27 degrees C in the serum-containing media used for cultivation of Sf9 cells. YF, DEN-1 and DEN-2 viruses may replicate in Sf9 cells at extremely low levels. This suggests that adventitious agents which do not produce obvious CPE or interfere with rBV infection or recombinant protein expression could contaminate Sf9 cell cultures or media.

Animals↗
PubMed1989

Overexpression and assembly of the herpes simplex virus type 1 helicase-primase in insect cells.

Herpes simplex virus type 1 (HSV-1) encodes a helicase-primase that consists of three polypeptides encoded by the UL5, UL8, and UL52 genes (Crute, J.J., Tsurumi, T., Zhu, L., Weller, S.K., Olivo, P.D., Challberg, M.D., Mocarski, E.S., and Lehman, I.R. (1989) Proc. Natl. Acad, Sci, U.S.A. 86, 2186-2189). To obtain sufficient quantities of the enzyme for study, we have overexpressed the three genes using the baculovirus expression system. We find that the fully active enzyme can be assembled in vivo by triply infecting Spodoptera frugiperda SF9 cells with a baculovirus recombinant for each gene. The recombinant enzyme which we have purified to near homogeneity from the insect cells has a molecular weight of 270,000 and is composed of the three polypeptides encoded by the UL5, UL8, and UL52 genes. The enzyme possesses DNA-dependent ATPase, DNA-dependent GTPase, DNA helicase, and DNA primase activities that are essentially identical to the enzyme isolated from HSV-1-infected cells.

Animals↗
PubMed1990

Expression and characterization of a functional human immunodeficiency virus envelope glycoprotein in insect cells.

Recombinant baculoviruses were used to express the gp160 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) and a truncated variant designated gp160(t) which lacks a transmembrane domain. Glycosylation, proteolytic cleavage, secretion, and biological activities of gp160 and gp160(t) 160(t) were studied in Spodoptera frugiperda cells. Both proteins were rapidly glycosylated and initially were found to be totally endo-beta-N-acetyl-D-glucosaminidase H (endo-H) sensitive. However, partial resistance to endo-H was gradually acquired by both molecules. gp160 was found to remain cell-associated, whereas gp160(t) was secreted into the culture medium in large amounts. A fraction of gp160 and gp160(t) appeared to be proteolytically cleaved, and a cleavage product corresponding in size to gp120 was identified in the culture medium. gp160(t) was found to interact specifically with CD4 receptors without any requirement for proteolytic cleavage. The gp160 protein was shown to be expressed on the surface of S. frugiperda cells by direct immunofluorescence. These surface molecules were biologically active, as demonstrated by their ability to induce syncytium formation when cocultivated with HeLa T4 cells.

Acetylglucosaminidase↗
PubMed1990

The oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector.

The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (SF) cell cultures using a baculovirus vector. To elucidate the structure of the carbohydrate side chains, radioactively labeled oligosaccharides were liberated by treatment with endoglucosaminidase H and glycopeptidase F. Sequential degradation with exoglycosidases and chromatographic analyses revealed the presence of oligomannosidic side chains, predominantly of the structures Man5-9GlcNAc2, and the truncated oligosaccharide cores Man3GlcNAc2 and Man3[Fuc]GlcNAc2. Polyacrylamide gel electrophoresis of endoglycosidase-treated hemagglutinin showed that most side chains of the HA1 subunit are truncated, whereas the HA2 subunit has one oligomannosidic and one truncated oligosaccharide. Comparison of these results with the glycosylation pattern of hemagglutinin obtained from vertebrate cells allowed a tentative allocation of the oligosaccharides to individual glycosylation sites. The results indicate that SF cells have the capacity to trim N-glycans to trimannosyl cores and to further process these by the addition of fucose. Thus, the complex oligosaccharides found on hemagglutinin from vertebrate hosts are replaced on hemagglutinin derived from insect cells by small truncated side chains.

Animals↗
PubMed1996

The production of cricket paralysis virus in suspension cultures of insect cell lines.

The yields of cricket paralysis virus from two insect cell lines propagated in suspension culture were examined. Although Trichoplusia ni (TN368) cells produced more virus per cell than Drosophila line 2 (DL2) cells, the smaller DL2 cells reached a higher cell density. Thus the difference in production between TN368 and DL2 cultures was minimal. In mixed infections of Flock House virus and cricket paralysis virus (CrPV), CrPV was the predominant virus produced despite being present at a much lower multiplicity of infection.

Animals↗
PubMed1992

Expression of processed envelope protein of hepatitis C virus in mammalian and insect cells.

The putative envelope protein of hepatitis C virus (HCV) was expressed in insect cells by using a baculovirus expression vector and in monkey COS cells under the control of exogenous promoters. The expressed envelope proteins, identified by immunoblot analysis using sera from patients with chronic HCV infection, were a series of glycoproteins of 35 to 24 kDa (gp35-24) in insect cells and a single species of glycoprotein of 35 kDa (gp35) in monkey cells. The size difference of these proteins was due to the different degrees of glycosylation. The envelope proteins expressed in these cells were produced by common specific cleavage from the precursor protein, and cleavage positions of the envelope protein were mapped at about amino acids 190 and 380. The gp35-24 proteins expressed in insect cells were used for detection of antibody against HCV envelope protein in patient sera. The results showed that (i) the antibody is detected in 2 to 17% of various patients with hepatitis C, (ii) three patients were apparently cured after acquiring the antienvelope antibody, and (iii) in sera of patients with more than a 20-year history of infection, the antibody sometimes coexisted with HCV. These results suggest that the antienvelope antibody is neutralizing only in limited number of patients with hepatitis C.

Animals↗
PubMed1993

Insect-transmitted vertebrate viruses: alphatogaviruses.

Alphatogaviruses, of which Sindbis virus (SV) is the prototype, replicate to high titer in the laboratory both in mosquito cells and in vertebrate cells. By studying the replication of SV in mosquito cells as well as in vertebrate cells, we were able to obtain several viral mutants which have novel phenotypes and have contributed to our basic knowledge of this virus family. These include three host range mutants: SVAP15/21 which replicates normally in mosquito cells but is restricted in vertebrate cells and SVCL35 and SVCL58, which are restricted in mosquito cells but replicate normally in vertebrate cells. As well, two other mutants are described here: SVLM21, which can replicate in methionine-starved mosquito cells and SVMPA, which can replicate in mosquito cells treated with mycophenolic acid or ribavirin. The causal mutations of both SVLM21 and SVMPA are within the sequence encoding the nonstructural protein nsPl; these and other findings have enabled us to associate the capping and methylation of the viral mRNAs with the nsPl protein. Our work serves to emphasize that it is both worthwhile and important to study the replication of arthropod-borne viruses in cells derived from the arthropod host as well as in cells derived from the vertebrate host.

Aedes↗
PubMed1988

Bluetongue virus tubules made in insect cells by recombinant baculoviruses: expression of the NS1 gene of bluetongue virus serotype 10.

Bluetongue virus (BTV) forms tubules in mammalian cells. These tubules appear to be composed of only one type of protein, NS1, a major nonstructural protein of the virus. To obtain direct evidence for the origin of the tubules, the complete M6 gene of BTV serotype 10 was inserted into the baculovirus transfer vector pAcYM1, so that it was under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus. After cotransfection of Spodoptera frugiperda cells with wild-type A. californica nuclear polyhedrosis virus DNA in the presence of recombinant transfer vector DNA, polyhedrin-negative baculoviruses were recovered. When S. frugiperda cells were infected with one of the derived recombinant viruses, a protein similar in size and antigenic properties to the authentic BTV NS1 protein was made (representing ca. 50% of the stained cellular proteins). The protein reacted with BTV antibody and formed numerous tubular structures in the cytoplasm of S. frugiperda cells. The tubular structures have been purified to homogeneity from infected-cell extracts by gradient centrifugation. By enzyme-linked immunosorbent assay, the recombinant virus antigen has been used to identify antibodies to five United States BTV serotypes in infected sheep sera, indicating the potentiality of the expressed protein as a group-reactive antigen in the diagnosis of BTV infections.

Animals↗