Journal of Virological Methods,
153
38 (1992) 153-166 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00
VIRMET
01340
A simple vacuum dot-blot hybridisation assay for the detection of Drosophila A and C viruses in single Drosophila Peter D. Christian Population and Molecular
Genetics Group, Research School of Biological Sciences, National University, Canberra, Australia
(Accepted
14 January
Australian
1992)
Summary Specific cDNA clones were constructed from the single stranded RNA genome of Australian isolates of both Drosophila A and C viruses. These clones were used to develop a nucleic acid hybridisation assay capable of detecting reliably 3.9 ng of DAV and 19.3 ng of DCV virus particles, respectively. The sensitivity of the assays were largely unaffected by soluble host material. Single Drosophila naturally infected or artificially inoculated with DAV or DCV were found to contain in excess of 130 ng of virus. The results presented here demonstrate that the vacuum dot-blotting protocol and the hybridisation assays developed are capable of detecting DAV and DCV in single Drosophila and may therefore be applied to the study of DAV and DCV in natural Drosophila communities. Drosophila C virus; Drosophila A virus; Dot-blot
hybridisation
assay
Introduction Three small picorna-like viruses have been isolated from laboratory and natural populations of Drosophila melanogaster: Drosophila P virus (DPV) (Plus and Duthoit, 1969), Drosophila C virus (DCV) (Jousset et al., 1972) and Drosophila A virus (DAV) (Plus et al., 1975). Biochemical and biophysical Correspondence to: 260 1, Australia.
P.D. Christian,
CSIRO,
Division
of Entomology,
P.O. BOX 1700, Canberra,
ACT
154
characterisations of these three viruses (Brun and Plus, 1980) have shown that DCV shares many of the characteristics of vertebrate picornaviruses in the genus Enterovirus. While DAV and DPV show many of the characteristics of picornaviruses they do not fit into any of the four existing genera (Matthews, 1982). Serological studies have shown that DPV is related to iota virus of D. immigrans (Jousset et al., 1972) and that DCV has partial identity with cricket paralysis virus (CrPV) (Plus et al., 1978). Although exhibiting partial serological identity, DCV and CrPV show little homology at the nucleotide level (King et al., 1984). To date, studies on the distribution of viruses in natural Drosophila populations have relied upon the serial passage of homogenates from wildcaught flies followed by electron microscopic and serological identification of the viruses (Plus et al., 1975). This approach has two major limitations. First, it is time consuming and unsuitable for screening large numbers of individual flies. Second, a virus-free reference stock of D. melanogaster must be maintained, requiring expensive isolation facilities. Routine screening of D. melanogaster stocks derived from Australian populations revealed the presence of either DAV or DCV in most lines. To study further the effects of these viruses on infected laboratory stocks and their distribution in natural Drosophila populations, a sensitive technique capable of screening large numbers of individual flies for both viruses was required. This paper reports the construction of cDNA clones from the RNA genomes of DAV and DCV and the development of simple DNA-RNA dot-blot hybridisation assays for detection of these viruses. These are the first such dot-blot hybridisation assays reported for the detection of picorna-like viruses in insects.
Materials and Methods Insects
Stocks of D. melanogaster were established from flies caught at Huonville, Tasmania (HV), Ellis Beach, North Queensland (EB) and Coff s Harbour, New South Wales (CH). All stocks were maintained routinely at 22°C on a wheatgerm-maize meal media (2.5% w/v wheatgerm, 5.0% w/v maize meal, 0.6% w/v dried yeast, 5.0% w/v glucose, 1.6% w/v sucrose, 1.O% w/v agar, 0.055% v/v propionic acid, 0.55% v/v orthophosphoric acid). A virus-free stock of D. melanogaster was established from the CH stock (referred to as CH-VF) as described by Brun and Plus (1980). Briefly, females were aged for at least 20 days, transferred to fresh media and allowed to lay eggs for 12-16 h. The eggs were collected from the surface of the media and treated for 10 min with 3% w/v sodium hypochlorite in sterile insect saline (SIS) (0.6% w/v NaCl, 0.04% w/v KCl, 0.024% w/v Ca&l, 0.02% w/v NaHCOs). After treatment, the eggs were washed in several changes of SIS, transferred to moist filter paper and placed on fresh media. Stocks established
155
from eggs treated in this fashion were maintained from other Drosophila stocks.
at 22°C in a room isolated
Viruses DCVo (strain Ouarzazarte, Morocco) was donated by Dr N. Plus, (Station de Recherches Cytopathologiques, St. Christol-les-Ales, France) and passaged in vivo in CH-VF flies as described by Plus et al. (1975). CrPVsEE (strain from the honeybee Apis mellzjkra, Canberra, Australia) was supplied by Dr D. Anderson (CSIRO, Division of Entomology, Canberra, Australia) and passaged in penultimate instar Galleria mellonella as described by Scotti (1976). Beet western yellows virus (BWYV) RNA was provided by A. McKenzie (Research School of Biological Sciences, Canberra, Australia). Virus purification DAV virions were purified from the HV and CH laboratory stocks and DCV from the EB stock. Briefly, groups of 50 flies were frozen and homogenised in 10 times their own weight of SIS. The homogenate was clarified by centrifugation at 16000 x g for 10 min and the supernatant diluted 1:9 with SIS. The diluted extract was then sterilised by filtration through a 45-pm filter and injected into flies from the same stock from which the homogenate was derived. Injection was by a modification of the method described by L’HCritier (1952). Each fly received 0.5 ,~l of the sterile homogenate injected into the abdominal cavity. After injection, flies were allowed to recover before being transferred onto standard wheatgerm-maize meal media. After 3 days, flies were transferred daily and the cadavers collected and stored at - 30°C. All flies surviving after 10 days were killed and stored at -30°C. Groups of 30&400 flies injected by the method described above, or CH-VF flies inoculated with a sterile homogenate of DCVo, were homogenised in 10 times their own weight of 0.05 M phosphate buffer, pH 7.4 (PB). The homogenate was diluted to a final volume of 13 ml and debris removed by centrifugation at 12000 x g for 10 min at 4°C. Virus was sedimented from the supernatant by centrifugation at 110 000 x g for 3 h at 4°C and resuspended overnight in 1 ml of PB. Viral suspensions were clarified by centrifugation for 1 min at 16000 x g and layered onto l&40% w/v continuous sucrose gradients in PB. After centrifugation at 70000 x g for 2 h (4”(Z), virus bands were harvested, diluted with PB and the virus sedimented by centrifugation at 110 000 x g for 3 h at 4°C. Pellets were resuspended overnight in 1 ml of PB and stored at -30°C until needed. Alternatively, virus prepared for purification of viral RNA was resuspended overnight in 2 ml of RNA extraction buffer (0.01 M Tris-HCl, pH 7.4, 0.01 M KCl, 1.5 mM MgC12, 0.2% w/v SDS). CrPVBEE was purified by the procedure of Scotti (1976). The concentration of RNA in purified virus preparations was estimated by measuring ODz+
156
Isolation of viral RNA
Viral RNA was isolated according to the method of Both and Air (1979). Briefly, the viral suspension, in 2 ml of RNA extraction buffer, was incubated at 56°C for 20 min in the presence of 2 mg/ml proteinase K. NaCl was added to a final concentration of 0.15 M and the suspension extracted with 5 ml of water-saturated phenol at 56°C for 5 min. After extraction with 5 ml of chloroform at room temperature for 20 min, the RNA was precipitated twice with ethanol to remove final traces of phenol. cDNA
synthesis and cloning
Viral cDNA synthesis was performed as described by Gubler and Hoffman (1983) and the cDNAs cloned into the BamHT site of pBR322 after the addition of BamHI linkers. Plasmids containing virus-related inserts were identified by colony hybridisation (Maniatis et al., 1982) using 32P-labelled cDNA probes. Plasmid DNA was isolated by the method of Holmes and Quigley (1981) or purified in larger quantities through CsCl gradients (Maniatis et al., 1982). The size of the virally related inserts was estimated by electrophoresis through 1% agarose gels in TAE buffer (0.04 M Tris-acetate, pH 8.0, 0.002 M EDTA). Virus-related inserts were subcloned into the BamHI site of M13mp18 and transformed into E. coli strain JMlOl for the synthesis of single-stranded probes. Dot-blot hybridisation procedures
1 ~1 samples of purified viral RNA or virus were applied directly to HybondN membranes (Amersham, UK) and allowed to air-dry at room temperature. The RNA was then bound to the membrane by baking at 80°C for 4 h. Alternatively, 150 ~1 samples of viral RNA or virus in 0.01% diethylpyrocarbonate (DEPC) were applied under vacuum to Hybond-N that had been pretreated by soaking in 12 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate). Membranes were then air-dried and baked as described above. Prehybridisation was carried out overnight at 45°C in buffer containing 40% v/v deionised formamide, 1 mM EDTA, 1% w/v SDS, 5 x Denhardt’s solution and 10 mM Tris-HCl, pH 8.0. Hybridisation was then allowed to roceed at 45°C overnight in the presence of fresh buffer containing either 3’P-labelled cDNA or strand-specific probes. Following hybridisation, membranes were washed twice in 2 x SSC and once in 0.1 x SSC, 0.1% SDS at 45°C and airdried before autoradiography at -70°C using Kodak XAR X-ray film and intensifying screens. Autoradiography was used routinely for a period of 72 h. Antisera and serological
techniques
Reference antisera against DAV FR (strain Fort Lamy, France), DCVz (strain Zaragoza, Yugoslavia) and DPV were kindly provided by Dr. N. Plus. Antisera against CrPV uuu was prepared in New Zealand white rabbits. Antigen-antibody complexes formed between DAVi.rv and anti-DAVFa serum and DCVEB and anti-DCVZ serum were used to produce specific antisera
157
against DAV (termed anti-DAVop) and DCV (termed anti-DCVGp), respectively, by the method of Hornitzky and Taylor (1983). Antisera were titrated against homologous virus preparations using immunoosmophoresis (Scotti and Wigley, 1982). Double-diffusion in agar was performed as described by Mansi (1958). Immunoelectrophoresis (IE) was carried out in 0.75% agarose (Sea-Kern, USA) gels in 100 mM Tris-HCl, pH 7.0, 50 mM EDTA (Crowle, 1961). In some cases, virus was transferred from agarose gels to Hybond-N membranes for 4 h in 20 x SSC. Membranes were then baked at 80°C for 4 h prior to hybridisation as described above.
Results Characterisation
of virus stocks
The available anti-sera were used to determine the purity of the DAVHv, DCVnn, DCVo and CrPVBEE preparations. Reciprocal end-point dilutions for each virus preparation, determined by immmunoosmophoresis, are summarised in Table 1. These data suggested that DAVHv and DCVo were homogenous preparations, while DCV nB was contaminated with a small amount of DAV. Identification
of cloned fragments
Seven and four plasmids were selected for further characterisation from the cDNA clones constructed from DAV and DCV viral RNA, respectively. All selected plasmids contained virus-derived inserts of greater then 300 bp which hybridised strongly to cDNA probes synthesised from their respective viral RNAs. Restriction enzyme analysis and cross-hybridisation studies revealed that the seven cDNA clones derived from DAV HV preparations comprised two distinct overlapping groups representing a total of 1,800 bases of viral RNA. The four TABLE 1 Reciprocal determined
end-point dilutions of DAV “v, DCVaa, by immunoosmophoresis
Antiserum
DCVo
and CrPVaEE virus preparations
Virus DAVHV
DCVEB
DCVo
CrPVBEE
Anti-DAVFa Anti-DAVGp
16 16
2 2
nr nr
nr nr
Anti-DCVZ Anti-DCVGp
nr’ nr
64 64
32 32
nr nr
Anti-CrPVaEE
nr
16
16
64
‘nr. no reaction.
158
cDNA clones from DCVnn preparations comprised a single overlapping group representing approximately 1,600 bases of the viral genome. This region has been located subsequently to within 400 bases of the viral poly(A) tail (unpublished results). Two non-overlapping clones, covering approximately 1,100 bases of the viral genome, were selected for each virus and subcloned into Ml3mpl8 for synthesis of strand-specific probes. The DAV strand-specific probe did not hybridise to CrPV RNA or total nucleic acid isolated from Drosophila free of known viruses (Fig. 1). However, it did hybridise to the DCVnn RNA preparation, albeit with approximately 25 fold lower intensity. This result confirmed the serological observations (Table 1) that DCVnn was contaminated with a small amount of DAV. Like the strand-specific DAV probe, the DCV probe did not hybridise to CrPV RNA or to total nucleic acid from virus-free flies (Fig. 1B). Nevertheless, the DCV probe did hybridise very weakly to the DAV”v RNA, suggesting that DAVHv preparation was contaminated by DCV. This result contrasts with that from the serological tests (Table 1) which suggested that DAVrtv preparation was free of DCV contamination. However, the very weak signal obtained from the DAVHv RNA preparation (about 125-fold lower than that from DCVnn) is consistent with the fact that the contamination could not be detected by the less sensitive immunoosmophoresis technique. As the RNA preparations used to prepare cDNA clones were found to contain RNA from two virus types it was necessary to ensure that the clones used to synthesise probes were derived from a single virus and were specific for that virus. Immunoelectrophoresis revealed a difference in the relative mobilities of DAVHv (rf = 1.28), DCVnn (t-f = 0.65) and DCVo (rf= 0.39) when compared to a xylene cyan01 standard. To utilise this property in
H
R
E
G
W
‘H
‘G
W
Fig. 1. A. Hybridisation of DAV strand-specific probes to RNA purified from DAVn, (H), CrPV (R), DCVna (E), BWYV (W) purified RNAs and total Drosophila nucleic acid (G). B. Hybridisation of DCV strand-specific probes to RNA purified from DAV us (H), CrPV (R), DCVna (E), BWYV (W) purified RNAs and total Drosophila nucleic acid (G). 5-fold serial dilutions of 30 ng of each nucleic acid were made from the top of each blot and applied directly to hybridisation membranes.
159
determining the specificity of the probes, samples of DAVHv, DCVna and DCVo were subjected to electrophoresis and transferred to Hybond-N. Membranes were then hybridised to one or other of the strand-specific probes (Fig. 2). The DAV strand-specific probe was only able to detect RNA in a position that corresponded to the known electrophoretic mobility of DAV (Fig. 2A). Conversely, the strand-specific DCV probe was only able to detect RNA in positions that corresponded to the known electrophoretic mobilities of DCVEB and DCVo (Fig. 2B). This demonstrated that both the DAV and DCV strand-specific probes were derived from a single virus type and were specific to that virus type. To confirm further this observation, five virus isolates were prepared from laboratory stocks known to contain both DAV and DCV. Each preparation was subjected to the vacuum dot-blot hybridisation assay using strand-specific probes. The hybridisation end-point for each isolate was consistent with the serological end-points measured by immunoosmophoresis (data not shown). Sensitivity
of RNA and virus detection
Hybridisation of strand-specific probes to dot-blots of purified viral RNA that had been applied directly to the nylon hybridisation membranes detected reliably as little as 240 pg of either DAV or DCV RNA (Fig. 3). The detection sensitivity was the same when purified viral RNA was applied to the hybridisation membrane under vacuum. In contrast to the results obtained with purified viral RNA, when purified virus samples were applied to the membrane either directly or under vacuum, and hybridised to strand-specific probes, only 1.2 ng of DAV RNA (3.9 ng of virus; 2.9 x lo8 particles) or 8 ng of DCV RNA (19.5 ng of virus; 1.45 x lo9 particles) were detected. This suggests that the viral RNA was much less readily available for hybridisation when applied to the hybridisation membrane in its nucleoprotein form i.e., as intact virus particles rather than naked RNA.
Fig. 2. A. Hybridisation of DAV strand-specific probes to DAV HV (H), DCVE~ (E) and DCVo (0) B. Hybridisation of DCV strand-specific probes DAVH~ (H), DCVE~ (E) and DCVo (0). Viral preparations were separated by electrophoresis and transferred to hybridisation membrane. The position at which the samples were loaded (0) and the migration position of the xylene cyanol (+) standard are indicated.
160
Fig. 3. A. Hybridisation of DAV strand-specific probes to purified DAVH~ (VH) and DAVH~ viral RNA (RH) applied directly to the hybridisation membrane. B. Hybridisation of DCV strand-specific probes to DCV purified DCV,a (VE) and DCVaa viral RNA (RE) applied directly to the hybridisation membrane. 5-fold serial dilutions of 30 ng of each nucleic acid were made from the top of each blot.
Furthermore, the data also suggests that viral RNA from intact DCV particles is much less readily available for hybridisation than RNA from intact DAV. Because the detection sensitivity for DAV and DCV did not depend upon the method by which the virus was applied to the hybridisation membrane, vacuum application was chosen for use in further investigations. This method has the advantage that it allows relatively large volumes (up to 150 ~1) of each sample to be applied to the membranes. Furthermore, by loading two thicknesses of membrane into the vacuum dot-blotting apparatus it was found that duplicate filters could be produced. Samples transferred to two thicknesses of membrane in this way showed no appreciable loss of hybridisation from the lower of the membranes. Since there was a considerable reduction in the amount of RNA made available for hybridisation when virus was in its nucleoprotein form (Fig. 3), several different homogenisation buffers and post-homogenisation treatments were tested to try to optimise the amount of viral RNA made available for hybridisation. Grinding in insect saline, 15 x SSC or RNA extraction buffer did not improve the sensitivity of DAV or DCV detection. None of the postgrinding treatments tested, including incubation with proteinase K and extraction with organic solvents, improved the detection sensitivity, and some severely reduced the amount of viral RNA made available for hybridisation (data not shown). To test the effect of the presence of host material on hybridisation sensitivity, extracts of virus-free flies (CH-VF line) were added to 2-fold dilutions of purified virus and the samples subjected to dot-blot hybridisation assay (Fig.
161
Fig. 4. Vacuum dot-blot showing the effect of increasing amounts of host extract on the detection of purified DAVHv using DAV strand-specific probe. Serial 2-fold dilutions of purified virus containing the equivalent of 16 ng of RNA were made from the top of the blot. Each series of dilutions contain the equivalent of either 0.1 (a), 1.0 (b), 2.0 (c) or 5.0 (d) times the soluble extract from a single virus-free Drosophila.
4). The results were similar for both DAV and DCV (results are shown only for DAV). Only high concentrations, the equivalent of the soluble material from 5 flies, reduced the amount of virus which could be detected relative to a dilution series containing no host extract (Fig. 4). Therefore, the soluble extract from a single fly appears to have no appreciable effect on the detection sensitivity of DAV and DCV in the vacuum dot-blot hybridisation assay described. Detection of DAV and DCV in single flies Purified DAV and DCV were neutralised (1: 1) with anti-DCVGp and antiDAVG~ sera respectively, diluted 1:9 with SIS and filter sterilised. Groups comprising live sets of 25 D. melanogaster from the CH-VF stock were then injected with one of these sterile homogenates, each fly receiving 6 x lo5 virus particles. A control group of five sets of flies were injected with sterile SIS. One set of flies from each of the two test groups and the control group were harvested, and fresh cadavers collected, at 0 (immediately after injection), 2, 5, 8 and 11 days after injection. After freezing at -30°C individual flies were homogenised in 0.01% DEPC and transferred to replicate filters under vacuum for hybridisation to either DAV or DCV strand-specific probes. Table 2 shows that immediately after injection (0 days), no flies from either the test or control groups had detectable levels of either DAV or DCV. At 2 days post injection, 67% and 79% of surviving flies from the two test groups had detectable levels of DAV and DCV, respectively. At other times after injection, all surviving flies in each of the test groups had detectable levels of virus (Table 2). Neither DAV
162 TABLE 2 Detection frequency of DAV and DCV by nucleic acid hybridisation groups of D. melanogaster Inoculum
Virus assayed
in experimentally-inoculated
Days after inoculation 0
2
5
8
DAV DCV
0.00 0.00
0.67 0.00
1.00 0.00
1.oo
1.00
0.00
0.00
DCV
DAV DCV
0.00 0.00
0.00 0.79
0.00 1.00
0.00 1.00
0.00 1.00
SIS
DAV DCV
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
DAV
11
or DCV was detectable in control group flies collected at any time after injection. This demonstrates that, in the case of flies artificially inoculated with either DAV or DCV, virus could readily be detected in individual flies. In a second experiment, the ability of the assays to detect DAV and DCV in flies from persistently infected laboratory stocks was tested. Flies less than 2 days of age were taken from laboratory stocks known to be persistently infected with either DAV (HV) or DCV (EB). Four sets of 100 flies from each stock were placed on fresh media and transferred every 4 days, at which time dead flies were counted and collected. Surviving flies from one group of each of the two stocks were harvested at 0, 6, 12 and 30 days. After freezing at -30°C individual flies were homogenised and subjected to the dot-blot hybridisation assay. The results from this experiment are summarised in Table 3. In contrast to the results obtained when flies were inoculated with virus (see Table 2), the frequency of DAV detection in surviving flies from persistently infected stocks (HV and CH) is much lower between days 0 and 6, rising slowly after this time to a frequency of 1.00 at day 30. Despite the apparently slow replication of DAV in these stocks, all cadavers collected during the course of TABLE 3 Detection frequency of DAV and DCV by nucleic acid hybridisation laboratory stocks of D. melanogaster Stock
Virus assayed
assay in persistently
Days after emergence 0
6
12
30
HV
DAV DCV
0.00 0.00
0.34 0.00
0.82 0.00
1.00 0.00
CH
DAV DCV
0.00 0.00
0.10 0.00
0.47 0.00
1.00 0.00
EB
DAV DCV
0.00 0.00
0.00 0.05
0.00 0.03
0.00 0.06
infected
163
the experiment were found to contain virus. In the line persistently infected with DCV (EB) the results were quite different from those obtained with DAV. At all times through the experiment the frequency of DCV infection was found to be low in the surviving flies. However, as with the DAV infected stocks, all the cadavers collected during the course of the experiment were found to contain virus. When compared to control amounts of purified virus, infected flies in both test groups, whether collected as surviving flies or cadavers, were always found to contain in excess of 130 ng of virus (10” particles) (data not shown). This demonstrates that naturally infected flies do replicate both DAV and DCV to levels that are detectable by the assays described.
Discussion The work reported here was initiated as part of a study to investigate the distribution of viruses in natural Drosophila communities in Australia. As part of this work, I wished to assess the frequency of virus infection within these communities. Consequently, an assay was needed that was specific for a particular virus, and applicable to the screening of large numbers of individual Drosophila. These requirements indicated two possibilities, either a nucleic acid hybridisation assay, or a serologically based assay, e.g., enzyme linked immunosorbant assay (ELISA). Initial screening of laboratory populations derived from wild-caught Australian Drosophila revealed the presence of more than one virus in most stocks. Serological characterisation of these viruses proved them to be DAV and DCV. It is not unusual to find in laboratory populations of insects, populations infected with more than one virus, e.g., Drosophila (Plus et al., 1975), honeybees A. mellifera (Anderson and Gibbs, 1988), pine emperor moth Nuduurelia cupensis (Juckes, 1970) and citrus red mite Panonychus citri (Reed and Desjardins, 1978). Although the presence of more than one virus was not itself a problem, the presence of DCV was - particularly in the choice of an assay system. Because of the close serological relationship between DCV and CrPV (Plus et al., 1978) and the fact that CrPV grows readily in Drosophila, the specificity of a serological based technique, and its ability to differentiate between DCV and CrPV was questioned. As it had previously been shown that although DCV and CrPV were serologically related they shared no homology at the nucleotide level (King et al., 1984), it was decided that a nucleic acid hybridisation procedure would offer the sensitivity and specificity required in the screening of individual Drosophila. Using the vacuum dot-blot procedure described it was possible to detect reliably 5.2 ng and 19.3 ng of purified DAV and DCV virus particles, respectively. The presence of the soluble extract from a single Drosophila did not affect these detection levels which were achieved using a simple virusextraction method, with no requirement for concentration or subsequent
164
treatment of the crude extract. Consequently, preparation time for each sample is relatively short. Further time can be saved by using two thicknesses of the hybridisation membrane simultaneously to produce replicate filters, and by using a mechanical homogeniser that fits directly into a microcentrifuge tube. Using the above techniques it has proven possible to prepare and vacuum dotblot up to 700 individual Drosophila per day. The major apparent limitation in the reported assay procedure is the relative reduction in the amount of RNA made available for hybridisation when the virus is in its nucleoprotein form. This reduction amounts to 5-fold and 25-fold decrease in DAV and DCV detection sensitivities, respectively, when compared to those for purified viral RNA. Despite this limitation, the results obtained from screening single flies that had either been inoculated with the viruses or removed from persistently infected laboratory stocks, indicate that this may not be as great a problem as first suggested. In both of the above instances, the amount of detectable DAV and DCV was in excess of 130 ng. This amount is well above the lower detection limit for both DAV and DCV. The assays reported here offer the opportunity to screen large numbers of individual Drosophila for the presence of DAV or DCV. For the first time these assays will allow the frequency of DAV and DCV occurrence in natural Drosophila populations and communities to be rapidly assessed, thereby gaining further insights into the geographic distribution and ecology of these viruses. Acknowledgements
The author wishes to thank Dr. R.J. Russell and Dr. J.G. Oakeshott for critical reading of the manuscript. Financial support for this work was provided by an Australian National University Postgraduate Scholarship and The Research School of Biological Sciences, Australian National University. References Anderson, D.L. and Gibbs, A.J. (1988) Inapparent infections and their interactions in pupae of the honeybee (&is mellifera Linnaeus) in Australia. J. Gen. Virol. 69, 1617-1625. Both, G.W. and Air, G.M. (1979) Nucleotide sequence coding for the N-terminal region of the matrix protein of influenza virus. Eur. J. Biochem. 96, 363-372. Brun, G. and Plus, N. (1980) The viruses of Drosophila. In: M. Ashburner and T.R.F. Wright (Eds), The biology and genetics of Drosophila, Vol. 3a, Academic Press, New York, pp. 625-702. Crowle, A.J. (1961) Immunodiffusion. Academic Press, New York. Gubler, U. and Hoffman, B.J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269. Holmes, D.S. and Quigley, M. (1981) A rapid boiling method for the preparation of bacterial plasmids. Analyt. B&hem. 114, 193-197. Hornitzky, M.A.Z. and Taylor, V.E. (1983) Preparation of specific antisera to honeybee viruses by immunization with agar gel precipitates. J. Apicultural Res. 22, 261-263. Jousset, F-X., Plus, N., Croizier, G. and Thomas, M. (1972) Existence chez Drosophila de deux
165 groups de picomavirus de propittts serologiques et biologiques differentes. C.R. Acad. Sci. (Paris) (Ser. D) 275, 3043-3045. Juckes, I.R.M. (1970) Viruses of the pine emperor moth. Bulletin of the South African Society of Plant Pathology and Microbiology 4, 18. King, L.A., Massalski, P.R., Cooper, J.I. and Moore, N.F. (1984) Comparison of the genome RNA sequence homology between cricket paralysis virus and strains of Drosophila C virus by complementary hybridization analysis. J. Gen. Virol. 65, 1193-l 196. L’HCritier, P. (1952) A convenient device for injecting large numbers of flies. Drosophila Information Service 26, 131. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York. Mansi, W. (1958) Slide gel-diffusion precipitation test. Nature (London) 181, 1289. Matthews, R.E.F. (1982) Classification and nomenclature of viruses. Intervirology 17, I-200. Plus, N. and Duthoit, J.L. (1969) Un noveau virus de Drosophila melunoguster, le virus P. CR. Acad. Sci. (Paris) (Ser. D) 268, 2313.-2315. Plus, N., Croizier, G., Jousset, F-X. and David, J. (1975) Picornaviruses of laboratory and wild Drosophila melunogaster: geographical distribution and serotypic composition. Ann. Microbial. (Inst. Pasteur) 126A, 107-l 17. Plus, N., Croizer, G., Reinganum, C. and Scotti, P.D. (1978) Cricket paralysis virus and Drosophila C virus: serological analysis and comparison of capsid polypeptides and host range. J. Invertebr. Pathol. 3 I, 296302. Reed, D.K. and Desjardins, P.R. (1978) Isometric virus-like particles from the citrus red mite, Panonychus citri. J. Invertebr. Pathol. 31, 188-193. Scotti, P.D. (1976) Cricket paralysis virus replicates in cultured Drosophila cells. Intervirology 6, 333-342. Scotti, P.D. and Wigley, P.J. (1982) Factors affecting the use of immunoosmophoresis for the detection of two insect riboviruses. J. Virol. Methods 4, 129-137.
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38 (1992) 153-166 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00
VIRMET
01340
A simple vacuum dot-blot hybridisation assay for the detection of Drosophila A and C viruses in single Drosophila Peter D. Christian Population and Molecular
Genetics Group, Research School of Biological Sciences, National University, Canberra, Australia
(Accepted
14 January
Australian
1992)
Summary Specific cDNA clones were constructed from the single stranded RNA genome of Australian isolates of both Drosophila A and C viruses. These clones were used to develop a nucleic acid hybridisation assay capable of detecting reliably 3.9 ng of DAV and 19.3 ng of DCV virus particles, respectively. The sensitivity of the assays were largely unaffected by soluble host material. Single Drosophila naturally infected or artificially inoculated with DAV or DCV were found to contain in excess of 130 ng of virus. The results presented here demonstrate that the vacuum dot-blotting protocol and the hybridisation assays developed are capable of detecting DAV and DCV in single Drosophila and may therefore be applied to the study of DAV and DCV in natural Drosophila communities. Drosophila C virus; Drosophila A virus; Dot-blot
hybridisation
assay
Introduction Three small picorna-like viruses have been isolated from laboratory and natural populations of Drosophila melanogaster: Drosophila P virus (DPV) (Plus and Duthoit, 1969), Drosophila C virus (DCV) (Jousset et al., 1972) and Drosophila A virus (DAV) (Plus et al., 1975). Biochemical and biophysical Correspondence to: 260 1, Australia.
P.D. Christian,
CSIRO,
Division
of Entomology,
P.O. BOX 1700, Canberra,
ACT
154
characterisations of these three viruses (Brun and Plus, 1980) have shown that DCV shares many of the characteristics of vertebrate picornaviruses in the genus Enterovirus. While DAV and DPV show many of the characteristics of picornaviruses they do not fit into any of the four existing genera (Matthews, 1982). Serological studies have shown that DPV is related to iota virus of D. immigrans (Jousset et al., 1972) and that DCV has partial identity with cricket paralysis virus (CrPV) (Plus et al., 1978). Although exhibiting partial serological identity, DCV and CrPV show little homology at the nucleotide level (King et al., 1984). To date, studies on the distribution of viruses in natural Drosophila populations have relied upon the serial passage of homogenates from wildcaught flies followed by electron microscopic and serological identification of the viruses (Plus et al., 1975). This approach has two major limitations. First, it is time consuming and unsuitable for screening large numbers of individual flies. Second, a virus-free reference stock of D. melanogaster must be maintained, requiring expensive isolation facilities. Routine screening of D. melanogaster stocks derived from Australian populations revealed the presence of either DAV or DCV in most lines. To study further the effects of these viruses on infected laboratory stocks and their distribution in natural Drosophila populations, a sensitive technique capable of screening large numbers of individual flies for both viruses was required. This paper reports the construction of cDNA clones from the RNA genomes of DAV and DCV and the development of simple DNA-RNA dot-blot hybridisation assays for detection of these viruses. These are the first such dot-blot hybridisation assays reported for the detection of picorna-like viruses in insects.
Materials and Methods Insects
Stocks of D. melanogaster were established from flies caught at Huonville, Tasmania (HV), Ellis Beach, North Queensland (EB) and Coff s Harbour, New South Wales (CH). All stocks were maintained routinely at 22°C on a wheatgerm-maize meal media (2.5% w/v wheatgerm, 5.0% w/v maize meal, 0.6% w/v dried yeast, 5.0% w/v glucose, 1.6% w/v sucrose, 1.O% w/v agar, 0.055% v/v propionic acid, 0.55% v/v orthophosphoric acid). A virus-free stock of D. melanogaster was established from the CH stock (referred to as CH-VF) as described by Brun and Plus (1980). Briefly, females were aged for at least 20 days, transferred to fresh media and allowed to lay eggs for 12-16 h. The eggs were collected from the surface of the media and treated for 10 min with 3% w/v sodium hypochlorite in sterile insect saline (SIS) (0.6% w/v NaCl, 0.04% w/v KCl, 0.024% w/v Ca&l, 0.02% w/v NaHCOs). After treatment, the eggs were washed in several changes of SIS, transferred to moist filter paper and placed on fresh media. Stocks established
155
from eggs treated in this fashion were maintained from other Drosophila stocks.
at 22°C in a room isolated
Viruses DCVo (strain Ouarzazarte, Morocco) was donated by Dr N. Plus, (Station de Recherches Cytopathologiques, St. Christol-les-Ales, France) and passaged in vivo in CH-VF flies as described by Plus et al. (1975). CrPVsEE (strain from the honeybee Apis mellzjkra, Canberra, Australia) was supplied by Dr D. Anderson (CSIRO, Division of Entomology, Canberra, Australia) and passaged in penultimate instar Galleria mellonella as described by Scotti (1976). Beet western yellows virus (BWYV) RNA was provided by A. McKenzie (Research School of Biological Sciences, Canberra, Australia). Virus purification DAV virions were purified from the HV and CH laboratory stocks and DCV from the EB stock. Briefly, groups of 50 flies were frozen and homogenised in 10 times their own weight of SIS. The homogenate was clarified by centrifugation at 16000 x g for 10 min and the supernatant diluted 1:9 with SIS. The diluted extract was then sterilised by filtration through a 45-pm filter and injected into flies from the same stock from which the homogenate was derived. Injection was by a modification of the method described by L’HCritier (1952). Each fly received 0.5 ,~l of the sterile homogenate injected into the abdominal cavity. After injection, flies were allowed to recover before being transferred onto standard wheatgerm-maize meal media. After 3 days, flies were transferred daily and the cadavers collected and stored at - 30°C. All flies surviving after 10 days were killed and stored at -30°C. Groups of 30&400 flies injected by the method described above, or CH-VF flies inoculated with a sterile homogenate of DCVo, were homogenised in 10 times their own weight of 0.05 M phosphate buffer, pH 7.4 (PB). The homogenate was diluted to a final volume of 13 ml and debris removed by centrifugation at 12000 x g for 10 min at 4°C. Virus was sedimented from the supernatant by centrifugation at 110 000 x g for 3 h at 4°C and resuspended overnight in 1 ml of PB. Viral suspensions were clarified by centrifugation for 1 min at 16000 x g and layered onto l&40% w/v continuous sucrose gradients in PB. After centrifugation at 70000 x g for 2 h (4”(Z), virus bands were harvested, diluted with PB and the virus sedimented by centrifugation at 110 000 x g for 3 h at 4°C. Pellets were resuspended overnight in 1 ml of PB and stored at -30°C until needed. Alternatively, virus prepared for purification of viral RNA was resuspended overnight in 2 ml of RNA extraction buffer (0.01 M Tris-HCl, pH 7.4, 0.01 M KCl, 1.5 mM MgC12, 0.2% w/v SDS). CrPVBEE was purified by the procedure of Scotti (1976). The concentration of RNA in purified virus preparations was estimated by measuring ODz+
156
Isolation of viral RNA
Viral RNA was isolated according to the method of Both and Air (1979). Briefly, the viral suspension, in 2 ml of RNA extraction buffer, was incubated at 56°C for 20 min in the presence of 2 mg/ml proteinase K. NaCl was added to a final concentration of 0.15 M and the suspension extracted with 5 ml of water-saturated phenol at 56°C for 5 min. After extraction with 5 ml of chloroform at room temperature for 20 min, the RNA was precipitated twice with ethanol to remove final traces of phenol. cDNA
synthesis and cloning
Viral cDNA synthesis was performed as described by Gubler and Hoffman (1983) and the cDNAs cloned into the BamHT site of pBR322 after the addition of BamHI linkers. Plasmids containing virus-related inserts were identified by colony hybridisation (Maniatis et al., 1982) using 32P-labelled cDNA probes. Plasmid DNA was isolated by the method of Holmes and Quigley (1981) or purified in larger quantities through CsCl gradients (Maniatis et al., 1982). The size of the virally related inserts was estimated by electrophoresis through 1% agarose gels in TAE buffer (0.04 M Tris-acetate, pH 8.0, 0.002 M EDTA). Virus-related inserts were subcloned into the BamHI site of M13mp18 and transformed into E. coli strain JMlOl for the synthesis of single-stranded probes. Dot-blot hybridisation procedures
1 ~1 samples of purified viral RNA or virus were applied directly to HybondN membranes (Amersham, UK) and allowed to air-dry at room temperature. The RNA was then bound to the membrane by baking at 80°C for 4 h. Alternatively, 150 ~1 samples of viral RNA or virus in 0.01% diethylpyrocarbonate (DEPC) were applied under vacuum to Hybond-N that had been pretreated by soaking in 12 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate). Membranes were then air-dried and baked as described above. Prehybridisation was carried out overnight at 45°C in buffer containing 40% v/v deionised formamide, 1 mM EDTA, 1% w/v SDS, 5 x Denhardt’s solution and 10 mM Tris-HCl, pH 8.0. Hybridisation was then allowed to roceed at 45°C overnight in the presence of fresh buffer containing either 3’P-labelled cDNA or strand-specific probes. Following hybridisation, membranes were washed twice in 2 x SSC and once in 0.1 x SSC, 0.1% SDS at 45°C and airdried before autoradiography at -70°C using Kodak XAR X-ray film and intensifying screens. Autoradiography was used routinely for a period of 72 h. Antisera and serological
techniques
Reference antisera against DAV FR (strain Fort Lamy, France), DCVz (strain Zaragoza, Yugoslavia) and DPV were kindly provided by Dr. N. Plus. Antisera against CrPV uuu was prepared in New Zealand white rabbits. Antigen-antibody complexes formed between DAVi.rv and anti-DAVFa serum and DCVEB and anti-DCVZ serum were used to produce specific antisera
157
against DAV (termed anti-DAVop) and DCV (termed anti-DCVGp), respectively, by the method of Hornitzky and Taylor (1983). Antisera were titrated against homologous virus preparations using immunoosmophoresis (Scotti and Wigley, 1982). Double-diffusion in agar was performed as described by Mansi (1958). Immunoelectrophoresis (IE) was carried out in 0.75% agarose (Sea-Kern, USA) gels in 100 mM Tris-HCl, pH 7.0, 50 mM EDTA (Crowle, 1961). In some cases, virus was transferred from agarose gels to Hybond-N membranes for 4 h in 20 x SSC. Membranes were then baked at 80°C for 4 h prior to hybridisation as described above.
Results Characterisation
of virus stocks
The available anti-sera were used to determine the purity of the DAVHv, DCVnn, DCVo and CrPVBEE preparations. Reciprocal end-point dilutions for each virus preparation, determined by immmunoosmophoresis, are summarised in Table 1. These data suggested that DAVHv and DCVo were homogenous preparations, while DCV nB was contaminated with a small amount of DAV. Identification
of cloned fragments
Seven and four plasmids were selected for further characterisation from the cDNA clones constructed from DAV and DCV viral RNA, respectively. All selected plasmids contained virus-derived inserts of greater then 300 bp which hybridised strongly to cDNA probes synthesised from their respective viral RNAs. Restriction enzyme analysis and cross-hybridisation studies revealed that the seven cDNA clones derived from DAV HV preparations comprised two distinct overlapping groups representing a total of 1,800 bases of viral RNA. The four TABLE 1 Reciprocal determined
end-point dilutions of DAV “v, DCVaa, by immunoosmophoresis
Antiserum
DCVo
and CrPVaEE virus preparations
Virus DAVHV
DCVEB
DCVo
CrPVBEE
Anti-DAVFa Anti-DAVGp
16 16
2 2
nr nr
nr nr
Anti-DCVZ Anti-DCVGp
nr’ nr
64 64
32 32
nr nr
Anti-CrPVaEE
nr
16
16
64
‘nr. no reaction.
158
cDNA clones from DCVnn preparations comprised a single overlapping group representing approximately 1,600 bases of the viral genome. This region has been located subsequently to within 400 bases of the viral poly(A) tail (unpublished results). Two non-overlapping clones, covering approximately 1,100 bases of the viral genome, were selected for each virus and subcloned into Ml3mpl8 for synthesis of strand-specific probes. The DAV strand-specific probe did not hybridise to CrPV RNA or total nucleic acid isolated from Drosophila free of known viruses (Fig. 1). However, it did hybridise to the DCVnn RNA preparation, albeit with approximately 25 fold lower intensity. This result confirmed the serological observations (Table 1) that DCVnn was contaminated with a small amount of DAV. Like the strand-specific DAV probe, the DCV probe did not hybridise to CrPV RNA or to total nucleic acid from virus-free flies (Fig. 1B). Nevertheless, the DCV probe did hybridise very weakly to the DAV”v RNA, suggesting that DAVHv preparation was contaminated by DCV. This result contrasts with that from the serological tests (Table 1) which suggested that DAVrtv preparation was free of DCV contamination. However, the very weak signal obtained from the DAVHv RNA preparation (about 125-fold lower than that from DCVnn) is consistent with the fact that the contamination could not be detected by the less sensitive immunoosmophoresis technique. As the RNA preparations used to prepare cDNA clones were found to contain RNA from two virus types it was necessary to ensure that the clones used to synthesise probes were derived from a single virus and were specific for that virus. Immunoelectrophoresis revealed a difference in the relative mobilities of DAVHv (rf = 1.28), DCVnn (t-f = 0.65) and DCVo (rf= 0.39) when compared to a xylene cyan01 standard. To utilise this property in
H
R
E
G
W
‘H
‘G
W
Fig. 1. A. Hybridisation of DAV strand-specific probes to RNA purified from DAVn, (H), CrPV (R), DCVna (E), BWYV (W) purified RNAs and total Drosophila nucleic acid (G). B. Hybridisation of DCV strand-specific probes to RNA purified from DAV us (H), CrPV (R), DCVna (E), BWYV (W) purified RNAs and total Drosophila nucleic acid (G). 5-fold serial dilutions of 30 ng of each nucleic acid were made from the top of each blot and applied directly to hybridisation membranes.
159
determining the specificity of the probes, samples of DAVHv, DCVna and DCVo were subjected to electrophoresis and transferred to Hybond-N. Membranes were then hybridised to one or other of the strand-specific probes (Fig. 2). The DAV strand-specific probe was only able to detect RNA in a position that corresponded to the known electrophoretic mobility of DAV (Fig. 2A). Conversely, the strand-specific DCV probe was only able to detect RNA in positions that corresponded to the known electrophoretic mobilities of DCVEB and DCVo (Fig. 2B). This demonstrated that both the DAV and DCV strand-specific probes were derived from a single virus type and were specific to that virus type. To confirm further this observation, five virus isolates were prepared from laboratory stocks known to contain both DAV and DCV. Each preparation was subjected to the vacuum dot-blot hybridisation assay using strand-specific probes. The hybridisation end-point for each isolate was consistent with the serological end-points measured by immunoosmophoresis (data not shown). Sensitivity
of RNA and virus detection
Hybridisation of strand-specific probes to dot-blots of purified viral RNA that had been applied directly to the nylon hybridisation membranes detected reliably as little as 240 pg of either DAV or DCV RNA (Fig. 3). The detection sensitivity was the same when purified viral RNA was applied to the hybridisation membrane under vacuum. In contrast to the results obtained with purified viral RNA, when purified virus samples were applied to the membrane either directly or under vacuum, and hybridised to strand-specific probes, only 1.2 ng of DAV RNA (3.9 ng of virus; 2.9 x lo8 particles) or 8 ng of DCV RNA (19.5 ng of virus; 1.45 x lo9 particles) were detected. This suggests that the viral RNA was much less readily available for hybridisation when applied to the hybridisation membrane in its nucleoprotein form i.e., as intact virus particles rather than naked RNA.
Fig. 2. A. Hybridisation of DAV strand-specific probes to DAV HV (H), DCVE~ (E) and DCVo (0) B. Hybridisation of DCV strand-specific probes DAVH~ (H), DCVE~ (E) and DCVo (0). Viral preparations were separated by electrophoresis and transferred to hybridisation membrane. The position at which the samples were loaded (0) and the migration position of the xylene cyanol (+) standard are indicated.
160
Fig. 3. A. Hybridisation of DAV strand-specific probes to purified DAVH~ (VH) and DAVH~ viral RNA (RH) applied directly to the hybridisation membrane. B. Hybridisation of DCV strand-specific probes to DCV purified DCV,a (VE) and DCVaa viral RNA (RE) applied directly to the hybridisation membrane. 5-fold serial dilutions of 30 ng of each nucleic acid were made from the top of each blot.
Furthermore, the data also suggests that viral RNA from intact DCV particles is much less readily available for hybridisation than RNA from intact DAV. Because the detection sensitivity for DAV and DCV did not depend upon the method by which the virus was applied to the hybridisation membrane, vacuum application was chosen for use in further investigations. This method has the advantage that it allows relatively large volumes (up to 150 ~1) of each sample to be applied to the membranes. Furthermore, by loading two thicknesses of membrane into the vacuum dot-blotting apparatus it was found that duplicate filters could be produced. Samples transferred to two thicknesses of membrane in this way showed no appreciable loss of hybridisation from the lower of the membranes. Since there was a considerable reduction in the amount of RNA made available for hybridisation when virus was in its nucleoprotein form (Fig. 3), several different homogenisation buffers and post-homogenisation treatments were tested to try to optimise the amount of viral RNA made available for hybridisation. Grinding in insect saline, 15 x SSC or RNA extraction buffer did not improve the sensitivity of DAV or DCV detection. None of the postgrinding treatments tested, including incubation with proteinase K and extraction with organic solvents, improved the detection sensitivity, and some severely reduced the amount of viral RNA made available for hybridisation (data not shown). To test the effect of the presence of host material on hybridisation sensitivity, extracts of virus-free flies (CH-VF line) were added to 2-fold dilutions of purified virus and the samples subjected to dot-blot hybridisation assay (Fig.
161
Fig. 4. Vacuum dot-blot showing the effect of increasing amounts of host extract on the detection of purified DAVHv using DAV strand-specific probe. Serial 2-fold dilutions of purified virus containing the equivalent of 16 ng of RNA were made from the top of the blot. Each series of dilutions contain the equivalent of either 0.1 (a), 1.0 (b), 2.0 (c) or 5.0 (d) times the soluble extract from a single virus-free Drosophila.
4). The results were similar for both DAV and DCV (results are shown only for DAV). Only high concentrations, the equivalent of the soluble material from 5 flies, reduced the amount of virus which could be detected relative to a dilution series containing no host extract (Fig. 4). Therefore, the soluble extract from a single fly appears to have no appreciable effect on the detection sensitivity of DAV and DCV in the vacuum dot-blot hybridisation assay described. Detection of DAV and DCV in single flies Purified DAV and DCV were neutralised (1: 1) with anti-DCVGp and antiDAVG~ sera respectively, diluted 1:9 with SIS and filter sterilised. Groups comprising live sets of 25 D. melanogaster from the CH-VF stock were then injected with one of these sterile homogenates, each fly receiving 6 x lo5 virus particles. A control group of five sets of flies were injected with sterile SIS. One set of flies from each of the two test groups and the control group were harvested, and fresh cadavers collected, at 0 (immediately after injection), 2, 5, 8 and 11 days after injection. After freezing at -30°C individual flies were homogenised in 0.01% DEPC and transferred to replicate filters under vacuum for hybridisation to either DAV or DCV strand-specific probes. Table 2 shows that immediately after injection (0 days), no flies from either the test or control groups had detectable levels of either DAV or DCV. At 2 days post injection, 67% and 79% of surviving flies from the two test groups had detectable levels of DAV and DCV, respectively. At other times after injection, all surviving flies in each of the test groups had detectable levels of virus (Table 2). Neither DAV
162 TABLE 2 Detection frequency of DAV and DCV by nucleic acid hybridisation groups of D. melanogaster Inoculum
Virus assayed
in experimentally-inoculated
Days after inoculation 0
2
5
8
DAV DCV
0.00 0.00
0.67 0.00
1.00 0.00
1.oo
1.00
0.00
0.00
DCV
DAV DCV
0.00 0.00
0.00 0.79
0.00 1.00
0.00 1.00
0.00 1.00
SIS
DAV DCV
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
DAV
11
or DCV was detectable in control group flies collected at any time after injection. This demonstrates that, in the case of flies artificially inoculated with either DAV or DCV, virus could readily be detected in individual flies. In a second experiment, the ability of the assays to detect DAV and DCV in flies from persistently infected laboratory stocks was tested. Flies less than 2 days of age were taken from laboratory stocks known to be persistently infected with either DAV (HV) or DCV (EB). Four sets of 100 flies from each stock were placed on fresh media and transferred every 4 days, at which time dead flies were counted and collected. Surviving flies from one group of each of the two stocks were harvested at 0, 6, 12 and 30 days. After freezing at -30°C individual flies were homogenised and subjected to the dot-blot hybridisation assay. The results from this experiment are summarised in Table 3. In contrast to the results obtained when flies were inoculated with virus (see Table 2), the frequency of DAV detection in surviving flies from persistently infected stocks (HV and CH) is much lower between days 0 and 6, rising slowly after this time to a frequency of 1.00 at day 30. Despite the apparently slow replication of DAV in these stocks, all cadavers collected during the course of TABLE 3 Detection frequency of DAV and DCV by nucleic acid hybridisation laboratory stocks of D. melanogaster Stock
Virus assayed
assay in persistently
Days after emergence 0
6
12
30
HV
DAV DCV
0.00 0.00
0.34 0.00
0.82 0.00
1.00 0.00
CH
DAV DCV
0.00 0.00
0.10 0.00
0.47 0.00
1.00 0.00
EB
DAV DCV
0.00 0.00
0.00 0.05
0.00 0.03
0.00 0.06
infected
163
the experiment were found to contain virus. In the line persistently infected with DCV (EB) the results were quite different from those obtained with DAV. At all times through the experiment the frequency of DCV infection was found to be low in the surviving flies. However, as with the DAV infected stocks, all the cadavers collected during the course of the experiment were found to contain virus. When compared to control amounts of purified virus, infected flies in both test groups, whether collected as surviving flies or cadavers, were always found to contain in excess of 130 ng of virus (10” particles) (data not shown). This demonstrates that naturally infected flies do replicate both DAV and DCV to levels that are detectable by the assays described.
Discussion The work reported here was initiated as part of a study to investigate the distribution of viruses in natural Drosophila communities in Australia. As part of this work, I wished to assess the frequency of virus infection within these communities. Consequently, an assay was needed that was specific for a particular virus, and applicable to the screening of large numbers of individual Drosophila. These requirements indicated two possibilities, either a nucleic acid hybridisation assay, or a serologically based assay, e.g., enzyme linked immunosorbant assay (ELISA). Initial screening of laboratory populations derived from wild-caught Australian Drosophila revealed the presence of more than one virus in most stocks. Serological characterisation of these viruses proved them to be DAV and DCV. It is not unusual to find in laboratory populations of insects, populations infected with more than one virus, e.g., Drosophila (Plus et al., 1975), honeybees A. mellifera (Anderson and Gibbs, 1988), pine emperor moth Nuduurelia cupensis (Juckes, 1970) and citrus red mite Panonychus citri (Reed and Desjardins, 1978). Although the presence of more than one virus was not itself a problem, the presence of DCV was - particularly in the choice of an assay system. Because of the close serological relationship between DCV and CrPV (Plus et al., 1978) and the fact that CrPV grows readily in Drosophila, the specificity of a serological based technique, and its ability to differentiate between DCV and CrPV was questioned. As it had previously been shown that although DCV and CrPV were serologically related they shared no homology at the nucleotide level (King et al., 1984), it was decided that a nucleic acid hybridisation procedure would offer the sensitivity and specificity required in the screening of individual Drosophila. Using the vacuum dot-blot procedure described it was possible to detect reliably 5.2 ng and 19.3 ng of purified DAV and DCV virus particles, respectively. The presence of the soluble extract from a single Drosophila did not affect these detection levels which were achieved using a simple virusextraction method, with no requirement for concentration or subsequent
164
treatment of the crude extract. Consequently, preparation time for each sample is relatively short. Further time can be saved by using two thicknesses of the hybridisation membrane simultaneously to produce replicate filters, and by using a mechanical homogeniser that fits directly into a microcentrifuge tube. Using the above techniques it has proven possible to prepare and vacuum dotblot up to 700 individual Drosophila per day. The major apparent limitation in the reported assay procedure is the relative reduction in the amount of RNA made available for hybridisation when the virus is in its nucleoprotein form. This reduction amounts to 5-fold and 25-fold decrease in DAV and DCV detection sensitivities, respectively, when compared to those for purified viral RNA. Despite this limitation, the results obtained from screening single flies that had either been inoculated with the viruses or removed from persistently infected laboratory stocks, indicate that this may not be as great a problem as first suggested. In both of the above instances, the amount of detectable DAV and DCV was in excess of 130 ng. This amount is well above the lower detection limit for both DAV and DCV. The assays reported here offer the opportunity to screen large numbers of individual Drosophila for the presence of DAV or DCV. For the first time these assays will allow the frequency of DAV and DCV occurrence in natural Drosophila populations and communities to be rapidly assessed, thereby gaining further insights into the geographic distribution and ecology of these viruses. Acknowledgements
The author wishes to thank Dr. R.J. Russell and Dr. J.G. Oakeshott for critical reading of the manuscript. Financial support for this work was provided by an Australian National University Postgraduate Scholarship and The Research School of Biological Sciences, Australian National University. References Anderson, D.L. and Gibbs, A.J. (1988) Inapparent infections and their interactions in pupae of the honeybee (&is mellifera Linnaeus) in Australia. J. Gen. Virol. 69, 1617-1625. Both, G.W. and Air, G.M. (1979) Nucleotide sequence coding for the N-terminal region of the matrix protein of influenza virus. Eur. J. Biochem. 96, 363-372. Brun, G. and Plus, N. (1980) The viruses of Drosophila. In: M. Ashburner and T.R.F. Wright (Eds), The biology and genetics of Drosophila, Vol. 3a, Academic Press, New York, pp. 625-702. Crowle, A.J. (1961) Immunodiffusion. Academic Press, New York. Gubler, U. and Hoffman, B.J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269. Holmes, D.S. and Quigley, M. (1981) A rapid boiling method for the preparation of bacterial plasmids. Analyt. B&hem. 114, 193-197. Hornitzky, M.A.Z. and Taylor, V.E. (1983) Preparation of specific antisera to honeybee viruses by immunization with agar gel precipitates. J. Apicultural Res. 22, 261-263. Jousset, F-X., Plus, N., Croizier, G. and Thomas, M. (1972) Existence chez Drosophila de deux
165 groups de picomavirus de propittts serologiques et biologiques differentes. C.R. Acad. Sci. (Paris) (Ser. D) 275, 3043-3045. Juckes, I.R.M. (1970) Viruses of the pine emperor moth. Bulletin of the South African Society of Plant Pathology and Microbiology 4, 18. King, L.A., Massalski, P.R., Cooper, J.I. and Moore, N.F. (1984) Comparison of the genome RNA sequence homology between cricket paralysis virus and strains of Drosophila C virus by complementary hybridization analysis. J. Gen. Virol. 65, 1193-l 196. L’HCritier, P. (1952) A convenient device for injecting large numbers of flies. Drosophila Information Service 26, 131. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York. Mansi, W. (1958) Slide gel-diffusion precipitation test. Nature (London) 181, 1289. Matthews, R.E.F. (1982) Classification and nomenclature of viruses. Intervirology 17, I-200. Plus, N. and Duthoit, J.L. (1969) Un noveau virus de Drosophila melunoguster, le virus P. CR. Acad. Sci. (Paris) (Ser. D) 268, 2313.-2315. Plus, N., Croizier, G., Jousset, F-X. and David, J. (1975) Picornaviruses of laboratory and wild Drosophila melunogaster: geographical distribution and serotypic composition. Ann. Microbial. (Inst. Pasteur) 126A, 107-l 17. Plus, N., Croizer, G., Reinganum, C. and Scotti, P.D. (1978) Cricket paralysis virus and Drosophila C virus: serological analysis and comparison of capsid polypeptides and host range. J. Invertebr. Pathol. 3 I, 296302. Reed, D.K. and Desjardins, P.R. (1978) Isometric virus-like particles from the citrus red mite, Panonychus citri. J. Invertebr. Pathol. 31, 188-193. Scotti, P.D. (1976) Cricket paralysis virus replicates in cultured Drosophila cells. Intervirology 6, 333-342. Scotti, P.D. and Wigley, P.J. (1982) Factors affecting the use of immunoosmophoresis for the detection of two insect riboviruses. J. Virol. Methods 4, 129-137.
