Evidence for multiplication of the leafhopper-borne maize yellow stripe virus in its vector using ELISA and dot-blot hybridization.

Maize yellow stripe virus (MYSV) has several features in common with tenuiviruses, but is transmitted by a leafhopper, Cicadulina chinai (Cicadellidae, Hemiptera), rather than planthoppers (Delphacidae, Hemiptera). Herein, MYSV was shown to be propagatively transmitted like tenuiviruses. MYSV RNA was not detected in leafhoppers by dot blot hybridization one day following a 2-day acquisition access period (AAP), but was detected in single or groups of leafhoppers 5-20 days post-acquisition. Likewise, capsid protein of MYSV was not detected by ELISA in single leafhoppers until the third day after the beginning of a 1- or 3-day AAP, but subsequently, mean absorbance values (405 nm) increased gradually, reaching their highest levels 8-14 days post-acquisition. The percentage of ELISA-positive leafhoppers also increased during the same period. Unlike most tenuiviruses, transovarial transmission of MYSV was not detected in 600 C. chinai nymphs that hatched from eggs laid by females that had acquired MYSV from diseased plants. The implications of our findings for MYSV classification are discussed.

Lack of infection of vertebrate cells by the densovirus from the maize worm Mythimna loreyi (MlDNV).

Several densoviruses have been used successfully in biological control of pests in the tropics. The densovirus from Mythimna loreyi (MlDNV) could also be an important tool in biological control of important pests. However, safety concerns remain as previous reports suggested that densoviruses may infect and transform L cells (from mouse). In this study, we show using molecular-biology tools that neither L nor other vertebrate cells support replication or transcription of densovirus, either after infection or after transfection. Quantitative PCR indicated no increase of viral DNA due to replication in vertebrate cells, in contrast to that in insect LD652 cells. After transfection, both the NS and VP mRNAs could be detected in LD652 cells but not in L cells. Moreover, the viral genome was excised from the plasmid after transfection of the infectious clone in LD652 cells, indicative of viral NS protein production, in contrast to L cells. The viral genome was able to integrate in the host chromosome of L cells after transfection, but not after infection. However, no viral transcription could be detected after integration.

Genetic heterogeneity of Phthorimaea operculella granulovirus: restriction analysis of wild-type isolates and clones obtained in vitro.

Genetic heterogeneity of a wild-type granulovirus (Tunisia isolate) of the potato tuber moth Phthorimaea operculella (Phthorimaea operculella granulovirus, Phop GV) has been studied. The heterogeneity was indicated by the presence of several submolar fragments in the profiles obtained by use of several restriction endonucleases. It was also demonstrated by variations in the restriction profile of the wild-type Tunisia isolate that had underwent since 1991 in our laboratory numerous passages in vivo. A comparison of the Tunisia isolate used in Egypt in the biological control programme with other PhopGV isolates indicated that it could not be related to any of the 3 genotypes previously defined. Five clones obtained from the Tunisia isolate in vitro were further grown both in vitro and in vivo. The restriction analysis of these clones demonstrated that none of them was identical to the parental wild type virus and to any other PhopGV geographic isolates. Genotypic differences between the clones were also shown. A 19 kbp BamHI fragment absent in the original Tunisia isolate but present in its passages since 1995 at a submolar concentration, was always present at a molar concentration in its clones. The presence of this fragment reflects probably a selection of one or more variants present in the original isolate and its possible adaptation to the growth in our laboratory conditions.

Evidence for two small viruses persistently infecting established cell lines of Phthorimaea operculella, deriving from embryos of the potato tuber moth.

Two small viruses were isolated from established cell lines of P. operculella deriving from embryos. The first one probably related to the Nodaviridae family, is a 30 nm in diameter icosahedral virus, with a bisegmented RNA genome and a single polypeptide of 39 kilodaltons. The second one related to the Parvoviridae family, is a 25 nm in diameter icosahedral virus with a DNA genome and a capsid constituted of 4 polypeptides of respectively, 90,000; 64,000; 56,000 and 43,500 daltons. The two viruses probably chronically infect the cell lines and may be consider latent viruses.

Yield and activity of Autographa californica multicapsid nucleopolyhedrovirus and Phthorimaea operculella granulosis virus in cloned and uncloned cell lines of P. operculella.

Three selected uncloned Pop 2, Pop 3, Pop 4 and two cloned cell lines Pop cl1A and Pop cl2B were derived from the original cell line established from Phthorimaea operculella (ORS-Pop-93). Three new non-selected cell lines ORS-Pop-94A, ORS-Pop-94B and ORS-Pop-95 were also established from embryos of the same insect. Differences in morphology, growth rate and polypeptide profile were determined between these cell lines. All the cell lines were susceptible to the Autographa californica nucleopolyhedrovirus (AcMNPV). The cloned cell lines produced higher levels of AcMNPV (TCID-50 and PIB) than the parental cells and at the same rate as the Sf9 reference cell line. Substantial amounts of viral DNA were synthesized in the clone Pop cl 2B after infection with the granulosis virus of the potato tuber moth P. operculella (PTMGV) and a complete multiplication was obtained in the ORS-Pop-95 cell line. The comparison between Pop cell lines which support limited or complete replication of certain baculoviruses can offer insights into some of the molecular barriers which restrict the host range of these viruses. These cell lines with variable susceptibility to baculoviruses could also be used for in vitro recombinations, increasing their virus host range to be used for the control of this pest.

Multiplication of a granulosis virus isolated from the potato tuber moth in a new established cell line of Phthorimaea operculella.

A newly established cell line was obtained from the culture of embryonic cells of the potato tuber moth Phthorimaea operculella in low temperature conditions (19 degrees C) using modified Grace's medium supplemented with 10% fetal bovine serum. The population doubling time was about 80 h when cells were cultivated at 19 degrees C and 38 h at 27 degrees C. The cell line had a relatively homogeneous population consisting of various sized spherical cells. The cells were cultivated for more than 25 passages. Their polypeptidic profile was different from profiles of other P. operculella cell lines we previously described and from other lepidopteran cells. The new cell line was designated ORS-Pop-95. The complete replication of the potato tuber moth granulosis virus (PTM GV) was obtained in vitro by both viral infection and DNA transfection. PTM GV multiplied at a significant level during several passages of the cell line that was maintained at 19 degrees C. As long as the cells were maintained at 19 degrees C, virus multiplication could also be obtained at the same rate at 27 degrees C. To compare PTM GV multiplied both in vivo and in vitro, we used morphological identification, serological, DNA probe diagnosis and endonuclease digest profile analysis and confirmed the identity of the virus.

Multiplication of Spodoptera littoralis granulosis virus in a cell line established from Phthorimaea operculella.

A complete replication of the Spodoptera littoralis granulosis virus (SpliGV) was obtained, in vitro by both virus infection and DNA transfection in the ORS-Pop-95 (Pop-95) cell line established from embryonic cells of the potato tuber moth, Phthorimaea operculella. SpliGV multiplied significantly during several passages in Pop-95 cells at 19 degrees C. When the cells were infected and kept at 19 degrees C for the first 4 hrs and then at 27 degrees C for the rest of the experiment (20 days), the viral multiplication proceeded at the same rate. Comparison of SpliGV progenies, multiplied either in vivo or in vitro, using electron microscopy and restriction profile analysis, showed their identity.

Establishment of a cell line derived from embryos of the potato tuber moth phthorimaea operculella (Zeller).

A cell line from the main insect pest of potatoes in tropical and subtropical areas, Phthorimaea operculella (Zeller), was obtained from embryoculture. These cells were cultured in Grace's modified medium. The cell line, designated ORS-Pop-93, had a heterogeneous population consisting of spherical and spindle cells with great capacity to adhere and a doubling time of 40 h. They were subcultured for more than 60 passages. Their polypeptidic profile was different from profiles of other lepidopteran cell lines. The cell line supports the multiplication of the Autographa californica nuclear polyhedrosis virus.