Institute for Medical Virology, University of Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, Federal Republic of Germany1
Federal Biological Research Center for Agriculture and Forestry, Institute for Biological Control, Heinrichstrasse 243, D-64287 Darmstadt, Federal Republic of Germany2
Robert-Koch-Institute, Nordufer 20, 13353 Berlin, Federal Republic of Germany3
Author for correspondence: Gholamreza Darai. Fax +49 6221 564104. e-mail g.darai{at}urz.uni-heidelberg.de
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Abstract |
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Introduction |
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Iridoviruses are subdivided into four genera: Iridovirus, Chloriridovirus, Lymphocystivirus and Ranavirus (van Regenmortel et al., 2000 ). The type species for the genus Iridovirus is Insect iridescent virus type 6 (IIV-6 or the recognized synonym Chilo iridescent virus). IIV-6 was isolated from the stem-boring lepidopteran Chilo suppressalis (rice-stem borer; Fukaya & Nasu, 1966
). Recently, we succeeded in identifying the primary structure and coding strategy of the complete genome of IIV-6 (Jakob et al., 2001
).
The cricket iridovirus isolate (CrIV) was isolated in 1996 from Gryllus campestris L. and Acheta domesticus L. (both Orthoptera, Gryllidae) (Kleespies et al., 1999 ). It was found that the DNA restriction pattern of CrIV DNA is distinct from that of IIV-6 (Kleespies et al., 1999
) but seems to be identical to the restriction patterns of the CrIV isolate described by Just & Essbauer (2001)
. Host range studies revealed that CrIV can be transmitted perorally to orthopteran species, resulting in characteristic symptoms and fatal disease (Kleespies et al., 1999
). The orthopteran species include Gryllus bimaculatus L. (Orthoptera, Gryllidae) and the African migratory locust Locusta migratoria migratorioides (Reiche & Fairmaire) (Orthoptera, Acrididae), which represent important insect pests in some developing countries. Furthermore, CrIV is lethal for first instars of the cockroaches Blattella germanica L. and Blatta orientalis L. (both Orthoptera, Blattidae) (Kleespies et al., 1999
).
A common feature of CrIV and IIV-6 is the ability to grow in CF-124 (Lepidoptera, Choristoneura fumiferana) cell cultures. Cell lysis, rounding and hypertrophy of the usually bipolar cells were observed within 24 h post-infection.
The molecular biology and genomic features of IIV-6 are well documented (Cerutti et al., 1981 ; Cerutti & Devauchelle, 1980
, 1982
, 1985
, 1990
; Delius et al., 1984
; Schnitzler et al., 1987
; Soltau et al., 1987
; Fischer et al., 1988a
, b
, 1990
; Sonntag & Darai, 1992
, 1995
; Handermann et al., 1992
; Stohwasser et al., 1993
; Sonntag et al., 1994a
, b
; Bahr et al., 1997
; Tidona & Darai, 1997
, 2000
; Tidona et al., 1998
; Müller et al., 1998
, 1999
; Jakob et al., 2001
).
The present study is a comparative analysis of the host range and genomic properties of CrIV and IIV-6.
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Methods |
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Host range studies.
To analyze host range infection, experiments were performed using a modification of the method described by Kleespies et al. (1999) . In the present work, host range experiments were conducted separately with the two iridoviruses, CrIV and IIV-6, together with 19 different orthopteran and lepidopteran species. The following species were tested for susceptibility: crickets, Acheta domesticus L., Gryllus campestris L., Gryllus bimaculatus L., Gryllus assimilis Fabricius and Gryllotalpa gryllotalpa L. (a mole cricket); locusts and grasshoppers: Locusta migratoria migratorioides (Reiche & Fairmaire), Schistocerca gregaria Forsk
l and Tachycines asynamorus Adel.; cockroaches: Blattella germanica L., Blatta orientalis L., Periplaneta americana L., Pycnoscelus surinamensis L. and Leucophaea maderae L.; and butterflies: Spodoptera exigua Hbn., Spodoptera littoralis Boisd., Heliothis armigera Hbn., Agrotis segetum Schiff., Galleria mellonella F. and Lymantria dispar L. Peroral transmission studies were carried out with various nymphal/larval instars and/or adults. For this purpose, virus contaminated food-like pieces of carrot (crickets and cockroaches), pieces of apple (Tachycines asynamorus and Grylus assimilis), wheat germ (locusts), oak leaves (Lymantria dispar) or different diets (all other lepidopteran larvae) were offered. The food was contaminated with a virus suspension of 2·2x1011 particles/ml CrIV or IIV-6 and was provided for 1 day. Virus concentration was determined by direct particle count, as described by Geister & Peters (1963)
. To avoid cannibalism in each test, 20 nymphs, larvae or adults were placed singly in beakers of different size depending on the size of the insect (diameter 6·5 or 7·5 cm, height 4·5 or 9·5 cm). The control group comprised 10 nymphs, larvae or adults and each species was fed with uncontaminated food. All specimens were incubated at a day/night regime of 25/20 °C. Bioassays were repeated two times and mortality was recorded daily up to 50 days post-inoculation. For verifying the causal agent, fat body preparations from dead specimens were investigated by negative-staining electron microscopy. In the same way, surviving insects were diagnosed for the presence or absence of virus infection.
Electron microscopy.
To study and compare fine structures and morphogenesis of the two viruses, series of fat body samples of diseased animals were dissected and fixed in 2·5% glutaraldehyde. Specimens were prepared for thin section transmission electron microscopy (TEM) following published procedures (Gelderblom et al., 1987 ; Yan et al., 2000
). Briefly, after post-fixation with 1% osmium tetroxide for 2 h, samples were treated with 0·1% tannic acid for 2 h followed by en bloc-staining with 2% uranyl acetate for 2 h and stepwise dehydration in increasing concentrations of ethanol. Samples were embedded in Epon. Ultrathin sections were obtained using a Leica Ultracut S microtome, post-stained with lead citrate and evaluated under a ZEISS EM 10 A TEM.
Measurements of virus particles were performed on negatives taken at 40000 magnification using a 10x magnifying glass. The average diameters of IIV-6 and CrIV were determined by measuring 100 particles in two directions, i.e. the long and short axes.
DNA isolation.
DNA isolation, incubation and electrophoresis were carried out as described previously (Schnitzler et al., 1987 ; Soltau et al., 1987
; Fischer et al., 1988a
, b
; Kleespies et al., 1999
).
PCR.
Oligonucleotide primers were synthesized with an Oligo 1000 M DNA Synthesizer (Beckman). PCR was performed using 0·5 fmol of the individual template DNA in 100 µl volumes containing 80 mM TrisHCl pH 8·9, 20 mM (NH4)2SO4, 5 mM MgCl2, 12·5 nmol of each dNTP, 5 pmol of each primer and 2·5 units Taq DNA polymerase (Applied Biosystems). A total of 35 cycles was run on an automated temperature cycling reactor (GeneE) under cycling conditions of 96 °C for 30 s, 55 °C for 1 min and 72 °C for 2 min per cycle.
DNA sequencing.
DNA templates and PCR products were prepared as described previously (Tidona & Darai, 1997 ). PCR products of the viral genomic DNA were automatically sequenced (Smith et al., 1986
) with a 373A Extended DNA sequencer using the DyeDeoxy Terminator Taq cycle sequencing technique (Applied Biosystems). Sequencing reactions were performed as described previously (Tidona & Darai, 1997
). The nucleotide sequences obtained from individual sequencing reactions were assembled using the Sequence Navigator software, version 1·0 (Applied Biosystems).
Computer-assisted analysis.
Nucleotide and amino acid sequences were compiled and analyzed using the OMIGA V2 (Oxford Molecular) and Vector NTI Suite 6 (InforMax) and PC/GENE program release 6·85 (Intelligenetics). Protein alignments were generated using the CLUSTAL program (Higgins & Sharp, 1988 ).
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Results |
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To determine the dimensions of CrIV and IIV-6 in ultrathin sections, the diameters of the capsids were measured. CrIV showed a long (apex-to-apex) axis of 145±6 nm and a short axis of 131±5 nm, while IIV-6 measured 142·5± 6 nm and 126·5±5 nm, respectively. The subtle differences observed here are insignificant and point to identical sizes. When compared in detail (Fig. 1), CrIV and IIV-6 did not show any apparent morphological differences.
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Discussion |
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Peroral transmission tests were conducted instead of infecting the insects by injection (Ohba, 1975 ; Anthony & Comps, 1991
; Carter, 1973a
, b
, 1974
; Smith, 1976
), because this method is irrelevant, both in nature and for potential biological control. It was found that CrIV and IIV-6 have a broad and identical orthopteran host range. The same results were found when Galleria mellonella, a member of Lepidoptera, was infected. However, infection tests with several noctuids failed. A common feature of the species tested is the fact that they are known to cause severe damage in different cultures, e.g. sugar cane or cotton. As far as the host range of IIV-6 is concerned, Fukuda (1971)
succeeded in perorally infecting 13 species of mosquitoes and Ohba (1975)
infected 65 species of Lepidoptera by injection, eight species of Coleoptera and two species (Diptera and Orthoptera) of Hymenoptera.
The average identity of several viral genes and their gene products that where investigated was found to be 95·98 and 95·18% at the nucleotide and amino acid level, respectively. These data and the results obtained from host range studies led to the conclusion that CrIV and IIV-6 are not different species within the Iridoviridae family. CrIV must be considered to be a variant and/or a novel strain of IIV-6.
In light of the biological relevance of these viruses for pest control, the results of the present study are of particular importance, as both virus isolates are able to infect different species of Orthoptera, such as the two important pest insects in some developing countries, the African migratory locust Locusta migratoria migratorioides and the desert locust Schistocerca gregaria.
Although IIV-6 also infects predatory and beneficial insects/invertebrates, including the silkworm Bombyx mori (Ohba, 1975 ), it could provide the basis for, or be a model for, the development of a biological insecticide for pest control. Bioassays with Locusta migratoria migratorioides in the laboratory have already delivered promising results (Kleespies et al., 1999
). Also, the virus susceptibility of first instars to three cockroach species (Blattella germanica, Blatta orientalis and Periplaneta americana) is of practical interest. Therefore, further bioassays are planned with different stages of the virus-susceptible pest insects under special climatic conditions to elucidate the control potential of the two virus isolates.
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Acknowledgments |
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References |
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Received 27 July 2001;
accepted 15 October 2001.