A molecular taxonomy for cricket paralysis virus including two new isolates from Australian...

Arch Virol (1996) 141:1509-1522

_Archives

Vi rology © Springer-Verlag 1996 Printed in Austria

A molecular taxonomy for cricket paralysis virus including two new

isolates from Australian populations of Drosophila (Diptera: Drosophilidae)

K. N. Johnson and P. D. Christian

Botany and Zoology Department, Australian National University, Canberra, Australia and CSIRO, Division of Entomology, Canberra, Australia

Accepted March 13, 1996

Summary. Two new isolates of cricket paralysis virus, TAR and SIM, are described that were originally isolated from laboratory colonies of Drosophila melanogaster and Drosophila simulans respectively. Using a combination of biological, serological and molecular characters it was possible to distinguish the SIM isolate from all other isolates and it is thus described as a new strain; CrPVsIM. The TAR isolate however, was indistinguishable from the CrPV reference isolate CrPVvIc/Gm/D2E/Gm/D22 ( Teleogryllus commodus, Victoria, Australia, 1968). The molecular characters used in the present study were obtained by combining PCR and restriction endonuclease digestion of the amplified fragments. This work demonstrates that such molecular characters, when used in combination with others, provide a powerful set of taxonomic characters for classifying CrPV isolates and strains and assessing their genetic relatedness.

Introduction

The picornavirus cricket paralysis virus (CrPV) is one of the most commonly isolated and widely distributed viruses of insects that has small (less than 60 nm in diameter) non-occluded virions and an RNA genome. A recent taxonomic revision of the existing nine isolates recognised six distinct strains and one further taxon containing an additional two isolates [4]. Despite its previous isolation from a wide range of insects, there has been no reported isolation of CrPV from any Dipteran species [22]. This is of special interest as all isolates of CrPV replicate well in Drosophila melanogaster cell lines [20] and adult flies [17]. The tephritid fruit-flies Dacus oIeae and Ceratitis capitata are also permissive hosts for CrPV [12, 17].

The frequency with which Drosophila are maintained in laboratories makes it surprising that there have been no reports of CrPV being isolated from

1510 K.N. Johnson and P. D. Christian

them. This is not simply due to lack of research on Drosophila viruses as there have been several small viruses with RNA genomes described from a range of species [1, 22].

In the process of screening laboratory populat ions of Drosophila for Drosophila C virus (DCV), we isolated two viruses that were able to replicate in Drosophila cells but failed to react with DCV antiserum. As CrPV does not normal ly react with the antisera being used [41, it appeared that the viruses isolated were either novel isolates of DCV or new isolates of CrPV. In this paper we report on the characterisation of these isolates and describe a simple and rapid method, using the polymerase chain reaction (PCR), that amplifies specific CrPV sequences and aids in the rapid identification of CrPV isolates.

Materials and methods

Virus isolates

Samples of the nine previously described isolates of CrPV [4] were passaged once in honeybees (Apis meIIifera), and two previously undescribed samples (TAR and SIM) were propagated in Drosophila melanogaster Line 2 (DL2) cells. The latter two samples were obtained from populations of Drosophila melanogaster from Tamar Valley (Tasmania) and Drosophila simulans from Canberra (Australian Capital Territory) that had been maintained in the laboratory for approximately 18 months. All samples were purified as described by Scotti (1976). Nomenclature for all virus isolates and strains follows that of Christian and Scotti [4] and is summarised along with the appropriate abbreviations in Table 1.

Ceil lines

Spodoptera frugiperda (Sf9) [26] cells were maintained in Schneider's media (Sigma Cell Culture, St Louis, Missouri, U.S.A.) supplemented with 10% foetal calf serum (FCS).

Table 1. List of abbreviations and passage histories for isolates used in the current study. Nomenclature follows that of Christian and Scotti [4-1

Abbreviation Original isolate Strain designation and passage history

ARK BEE BRK GRA KKH KTA NEU SIM TAR VIC WHP

PseudopIusia incIudens, Arkansas, USA Apis mellifera, Canberra, Australia Acheta domestica, Georgia, USA Antherea eucaIyptii, Canberra, Australia TeleogryIIus commodus, Kaikohe, New Zealand TeleoorylIus commodus, Kaitaia, New Zealand Teleogryllus commodus, Canberra, Australia D. simuIans, Canberra, Australia D. meIanogaster, Tamar Valley, Australia Teleogryllus commodus, Victoria, Australia Pteronemobius spp., Wharepapa, New Zealand

CrPVtARK/BRKjD2pp2/D22/Am CrP VBEFj Am/D 2 2 /Am CrPV~ARKmRKI/D2pp2/D22/Am CrPVGRA/D2ppZ/D2Z/Am CrPVKKH/D2pp2/D22/Am CrPVKTA/D2ppZ/D22/Am CrPVvio/D2pp2/D22/Am CrPV/Ds~/D22 CrPV/Dm/D22 CrPVvwjGm/D22/Gm/D22/Am CrPVwnp/D2pp2/D22/Am

~Ds Drosophila simulans

Molecular characterisation of cricket paralysis virus 1511

Lines derived from Agallia constricta (AC5G) (McIntosh, unpubl.), Anticarsia gemmatalis (BCIRL-AG-AM) [10], Cydia pomonella (CP-169) [8], Pieris rapae (PR-5) (Quhou and McIntosh, unpublished), Plutetla xylosteIla (BCIRL-PX2-HNV3) [2], Spodopterafrugiperda (IPLB-Sf21) [27], Spodoptera ornithogalIi and Trichoplusia ni (TN-CL1) [8] were maintained in TC199MK media [7] containing 10% FCS. Aedes albopictus [24] and Helicoverpa zea (BCIRL-Hz-AM1) [9] cells were maintained in TC199MK media supplemented with 10% FCS, 0.2% w/v Yeastolate (Difco Laboratories, Detroit, Michigan, U.S.A.) and 0.2% w/v trehalose (Sigma Cell Culture, St Louis, Missouri, U.S.A.). This latter media is referred to as TC199MK +. Aedes aegypti [16] and Drosophila melanogster Line 2 (DL2) [19] lines were maintained in both Schneider's and TC199MK + media as described above.

For in vitro host range studies, cells were sub-cultured in 50 gl of media into 96 well tissue culture plates at a cell density such that they would reach confluence in 7 days under normal maintenance conditions. Fifty microlitres of virus suspension was added to the cells to give a final concentration of 1 x 107 infectious units/ml; viruses had previously been titrated using a tissue culture infectious dose 50 (TCIDs0) as described by Scotti [21]. Cells were scored every day for seven days for visible cytopathic effect (C.P.E.).

RNA extraction

Viral genomic RNA was isolated using a method based on the acid phenol/guanidinium method described by Chomczynski and Sacchi [3]. Virus samples were diluted with 500 ~tl of lysis solution (4M guanidinium thiocyanate, 25mM sodium citrate, pH 7; 0.5% sarcosyl, 0.1 M 2-mercaptoethanol). After the addition of 100 ~tl of 2M sodium acetate, pH 4.0 the virus sample was extracted with 400 gl acid phenol and 100 gl of chloroform. An equal volume of isopropanol was added to the aqueous phase and RNA was precipitated at 4 °C. After centrifugation the RNA-containing pellet was washed with 70% ethanol and resuspended in diethyl pyrocarbonate (DEPC)-treated water.

Cloning of CrPVsequences

RNA was purified from the isolate CrPVvIc/Gm/D22/Gm/D22/Am (Teleogryllus commodus, Victoria, Australia) and cDNA synthesised using random primers and a cDNA synthesis kit from GIBCO BRL (Life Technologies Inc., Washington DC). After second strand synthesis, fragments of double-stranded DNA were concentrated and salts removed using a Big 101 Mermaid Kit (Big 101, Vista, California, USA) prior to ligation into the Sinai site of plasmid vector pBluescript SK-.

Hybridisation

For hybridisation studies 1 gl of each virus sample was spotted onto Hybond-N membranes (Amersham, UK), air dried and then baked at 80 °C for 4 hours. 3;p-labelled probes were synthesised from cloned CrPV sequences by random priming using the Boehringer Mannheim (Mannheim, Germany) Random-Priming Kit following the manufacturers instructions. Membranes were pre-hybridised at 65°C in buffer containing 5 x SSC (1 x SSC = 150mM NaC1, 15raM sodium citrate, pH 7.0), 5 x Denhardt's solution (1 x Denhardt's solution = 0.1 g/1 Ficoll (Type 400, Pharmacia, Uppsala, Sweden), 0.1 g/1 BSA (Fraction V, Sigma-Aldrich Chemical Co., Castle Hill, New South Wales, Australia), 0,1 g/1 polyvinyl pyrrolidine, 0.5% sodium dodecyl sulphate (SDS) and 100 gg/ml salmon sperm DNA. Following hybridisation, the filters were washed twice in 2 x SSC, 0.1% SDS and twice in 1 x SSC, 0.1% SDS at 65 °C before autoradiography.


Serology

All five antisera used in the present study were raised in New Zealand White rabbits. Anti-VIC E23/5 was raised against a VIC isolate [CrPV/Gm (TelogryIlus commodus, Victoria, Australia, 1968)] and was obtained from blood taken later from the same rabbit used to generate anti-VIC E23/4 serum [17]. Anti-BEE A/1 was raised against a BEE isolate of CrPV [CrPV/Gm (Apis meIlifera, Canberra, Australia, 1982)] and anti-BEE A/2 was a later bleed from the same rabbit used to generate anti-BEE. Anti-BRK R3/2, was raised against a BRK isolate [CrPV/D2pp2/D22 (Acheta domesticus, Georgia, USA)]. The final serum, anti-VIC C1, was raised against the reference isolate of CrPV, [CrPVvm/Gm/D22/Gm/D22 (TeleogrylIus commodus, Victoria, Australia, 1968)], that had been passaged an additional time in honeybees (Apis mellifera). Antisera were titrated against homologous virus preparations using immunoosmophoresis [23]. Double-diffusion in agar tests were carried out as described by Mansi [13], using the reference isolate, CrPVvlc/Gm/D22/Gm/D2 a (Teleogryllus commodus, Victoria, Australia, 1968) as a standard.

Amplification of Cr P V sequences

DNA fragments were amplified from CrPV viral RNAs in a two step procedure. Firstly, 75 pmol of the specific oligonucleotide (CrPV-2: 5'-ATCTCCAAGCGCACGCATAA-3') was annealed to 1 gg of viral RNA in total volume of 20 gl. This oligonucleotide was designed to be complementary to a nucleotide sequence towards the 3' end of the published viral RNA sequence [5]. cDNA was then synthesised at 37 °C for 90min in the presence of 50 mM Tris pH 8.3, 75 mM potassium chloride, 3 mM magnesium chloride, 10 mM DTT, 0.5 mM dATP, dCTP, dGTP, dTTP using 10-15 units of reverse transcriptase (murine Moloney leukaemia virus reverse transcriptase, Promega Corporation, Madison, Wisconsin, USA; avian myeloblastosis virus reverse transcriptase, Pharmacia Biotech, Uppsala, Sweden). The reverse transcriptase was then inactivated by heating at 80 °C for 10min.

In the second stage of the procedure, a 1272bp fragment was amplified using the polymerase chain reaction (PCR). For this reaction 50pmol of a second specific oligonucleotide (CrPV-I: 5'-GGTTACGTGACTAATGCTTT-3') was added to one quarter of the cDNA reaction mix from the first stage of the procedure. This second primer was from a 20 base pair sequence toward the 5' end of King et at.'s [5] published sequence.

Initial PCR conditions were determined using RNA and cDNA prepared fi'om a purified VIC isolate [CrPVvIc/Gm/D22/Gm/D22 (Teleogryllus commodus, Victoria, Australia, 1968)]. The standard conditions for these reactions were 80 °C while the Taq polymerase added; then 30 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min. The PCR was done in the presence of 10mM Tris-HC1, pH8.3; 50mM potassium chloride, 1.5mM magnesium chloride, 0.1% Triton-X. The reaction mix was overlaid with mineral oil and equilibrated at 80 °C before adding 0.5 gl (1U) of Taq polymerase (Boehringer Mannheim, (Mannheim, Germany). All PCR reactions were carried out in a FTS-1 thermal sequencer (Corbett Research, Victoria, Australia).

Under the above conditions, PCR was used to amplify DNA fragments fi'om 8 of the other 10 isolates. To generate DNA fragments from cDNA templates synthesised from genomic RNA of the BRK and ARK isolates, the primer annealing temperature had to be lowered by 10 °C to 45 °C. The DNA fragments generated from all isolates were of the size predicted from the published nucleic acid sequence [5].


Restriction digestion of amplified sequences

Restriction endonuclease digestion of unpurified PCR products (5 gl in a 20 gl reaction volume) was carried out as per the manufacturers instructions. Restriction fragments were separated by electrophoresis through 2% agarose gels in Tris/acetate/EDTA (TAE) buffer. Fragments were visualised under UV light after staining with ethidium bromide.

Since many restriction sites were expected for the enzymes DdeI, Hinfl, Sau96I and Sau 3A, PCR products were extracted with phenol/chloroform and precipitated in the presence of 0.3 M sodium acetate and two volumes of ethanol. After centrifugation pellets were washed with 70% ethanol and resuspended in 30-50 t~1 of sterile water. One microliter of this DNA was then digested in a 5ul reaction volume as per manufacturers instructions. The digested DNA was then end-labelled with 32p in the presence of 5 mM Tris-HC1, 5mM magnesium chloride, 25 mM sodium chloride, 0.5mM dithioerythritol, lmM dCTP, lmM dGTP, lmM dTTP, ~.32p dATP or c~-32p dCTP, using 0.4U of the Klenow fragment ofDNA polymerase I (Boehringer Mannheim, Mannheim, Germany). Samples were incubated at room temperature for 10 minutes before adding 3 ~tl 0.3% bromophenol blue, 0.3% xylene cyanol, 10mM EDTA, 97.5% deionised formamide. After denaturation at 95 °C samples were loaded on 14cm × 20cm 4-8% acrylamide gels and subjected to electrophoresis in Tris/borate/EDTA (TBE) buffer [11] at 700V until the bromophenol blue was 2-4 cm from the base of the gel. The gels were fixed and dried before being autoradiographed.

The sizes of fragments were estimated using the Getfrag program. In these estimations reference to a 1Kbp ladder (Gibco BRL, Gaithersberg, Maryland, USA) for those samples run on agarose gels and to pBluescript II digested with the restriction endonuclease, Hinfl, azp-labelled and treated in the same way as the amplified DNA fragments for samples run on acrylamide gels.

Calculation of genetic distances between CrP V isolates

Using restriction fragment data, two different measures of the similarity/genetic distance between the CrPV isolates were derived. The first of these, the similarity measure F, was calculated from the following formula:

F = 2nxv/n x + nv [14], Equation 21

nxv is the number of shared fragments between two isolates X and Y and n x and n v are the number of fragments in X and Y respectively.

In the second instance, the distance measured a (the average number of nucleotide substitutions per site) was derived. Unlike the measure F which is calculated from restriction fragment data, d is generated from mapped restriction site data. To derive suitable maps for each of the isolates, a preliminary map for the enzymes used in the current study was deduced from the sequence data of King et al. [5]. Assuming a minimum number of changes i.e. acquisition/loss of restriction sites, maps were then generated for each of the CrPV isolates under study. These map data were then analysed using the RESTSITE program of Nei and Miller [15] to generate the measure d.

Results

Hybridisation

F o r hybr id isa t ion experiments, 3 /p- label led probes were synthesised by random-pr iming of two CrPVvlc clones that conta in 6.0 K b p of the viral genome (located within 300b.p. of the 3' terminus of the viral g e n o m e - F . Weyt s

1514 K . N . J o h n s o n a n d P. D. C h r i s t i a n

unpublished). Samples of all eleven CrPV isolates were found to hybridise strongly with this probe, which did not, even under the low stringency conditions employed, hybridise to DCV o genomic RNA. This result confirmed that all of the isolates tested were CrPV as previously defined [4].

Serology

All of the isolates were analysed using immunoosmophoresis and double-diffusion. In immunoosmophoresis, all isolates other than ARK and BRK formed precipitin lines with anti-VIC E23/5, anti-BEE A/l, anti-BEE A/2, anti-VIC C1, and anti-BRK R3/2. ARK and BRK isolates reacted with only the anti-BRK R3/2 serum. Each isolate was compared by double-diffusion in agar to the reference isolate, CrPVvIc/Gm/D22/Gm/D22 (Teleogryllus commodus, Victoria, Australia) [41. Only the anti-VIC C1 and anti-BRK R3/2 sera were able to distinguish between any of the isolates. In reactions with anti-VIC C1, BEE, SIM and WHP all showed reactions of partial identity with the reference isolate while all other samples showed reactions of identity. In reactions with anti-BRK R3/2, ARK and BRK showed partial identity with the reference isolate, whereas all other isolates showed reactions of identity. The results of the discriminating serological tests are shown in Table 2.

Table 2. B e h a v i o u r of C r P V i so la tes in fou r insec t cell l i n e - m e d i a c o m b i n a t i o n s a n d d o u b l e - d i f f u s i o n

tes ts in aga r

In v i t ro h o s t r a n g e tes ts

Cell l ine a n d m e d i a

A. ae9 a A. ae 9 C. pore A. con Sch b T C 1 9 9 M K + T C 1 9 9 M K T C 1 9 9 M K

Sero log ica l tes ts

A n t i - s e r u m

V I C - C 1 BRK R2/3

Virus A R K . . . . N R c P

B E E + - - + P I

B R K . . . . N R P

G R A + - - + I I

K K H - - - + I I

K T A + + - + I I

N E U + - - + I I

S I M + + - + P I

S I M - - - + I I

T A R -- - - - + I I

W H P + + + + P I

aCell l ines are a b b r e v i a t e d ; A. ae9 Aedes aegypti, C. pore Cydia pomonella, A. con Agallia constricta bSch S c h n e i d e r ' s m e d i a

° N R N o reac t ion , P r e a c t i o n of pa r t i a l ident i ty ; I r e a c t i o n of i d e n t i t y


In vitro host range studies

Of the fifteen combinations of cell lines and media tested only four showed differential responses to the eleven CrPV isolates tested, and these are summarised in Table 2. Of the remaining eleven cell lines, the Anticarsia gemmatatis, Drosophila melanogaster Line 2 (maintained in both Schneider's and TC199MK+ media), Pieris rapae, Plutella xylostella, Spodoptera ornitho(jalli and Trichoplusia ni lines showed a C.P.E. when inoculated with all eleven of the CrPV isolates. In all of these lines, with the exception of the Pieris rapae line, cell lysis was observed albeit of varying amounts. In the case of the P. rapae line however, no cell lysis was observed and the only apparent symptoms of infection was an unusual growth pattern of the cells but after harvesting and lysis of the cells an increased titre of virus was observed. The Aedes albopictus, Helicoverpa zea, Sf9, and Sf21 cell lines did not show any C.P.E. with any of the eleven isolates.

Restriction analysis of amplicons

The DNA fragments generated by PCR were digested with a total of 20 restriction endonucleases. The enzymes BoII, EcoRI, EcoRV, NdeI, PvuI and XhoI did not hydrolyse any of the fragments generated by PCR from the viral cDNAs. The remaining 14 enzymes were found to generate up to four distinct haplotypes amongst the eleven isolates, a representative restriction profile is shown in Fig. 1. The sizes of the restriction fragments observed in each of these haplotypes are shown in Table 3. The composite genotype generated for each of the isolates using all enzymes is shown in Table 4.

Relationships arnongst CrPV isolates

Using the restriction data summarised in Table 3, the measures 1-F and d were calculated, and used to construct phenograms using either the UPGMA method of Sneath and Sokal [25] or the neighbour-joining method of Saitou and Nei [18]. Both analyses produced exactly the same branching pattern and this is

Fig. 1. Profile of fragments produced by restriction endonuclease digestion with HaeIII of PCR generated fragments from CrPV isolates. Restriction fragments were loaded in the following order. 1 DNA size marker; 2 undigested PCR fragment; 3 VIC; 4 BEE; 5 GRA;

6 KKH; 7 KTA; 8 NEU; 9 SIM; I0 TAR; I1 WHP; i2 ARK; t3 BRK


Table 3. Sizes of restriction fragments generated with each enzyme for each of the haplotypes identified for that enzyme

Haplotype

A B C D

AtuI 89, 298, 325, 538 CIaI 550, 691 1313 DdeI 202, 294, 385, 472 44, 202, 238, 365, 472 104, 185, 202, 365, DraI 124, 316, 940 1313 472 HaeII 592, 742 1338 HaeIII 1 283 370, 950 143, 370, 774 HindIII 1 338 23,243, 1 018 Itinfl 60, 225,253, 281,401 60, 104, 225, 253, 271 60, 104, 108, 124, 253

281 271,281 PstI 277,1056 RsaI 413,820 165,282,820 Sau3AI 594,740 173,378,740 Sau96I 42,171,222,259,501 42,222,388,501 ScaI 1335 327,1018 XbaI 130, t135 1275

365,384,413 26,535,594

1109,143

60,71,777,493,519

110,142,165,189,489 150,238,312,414

Table 4. Haplotypes derived from each isolate, ibr each enzyme digest from which more than one haplotype was observed across the isolates tested. Haplotypes present in an isolate in submolar

proportions are shown in brackets

Haplotype

ARK BEE BRK GRA KKH KTA NEU SIM TAR VIC WHP

AIuI A A A A A A A A A A A ClaI B A B A A A A A A A A DdeI A A A B a B(A) A A C A A A DraI B A B A A A A A A A A HaeII B A B A A A A A A A A HaeIII C A D A A A A B A A A H indIII B A B A A A A A A A A H infl D B D C C A A B A A B PstI A A A A A A A A A A A RsaI D A D A A B(A) A A A A C Sau3AI D A D B(A) B(A) A A A A A C Sau96I A A A B b B A A A A A A ScaI B A B A A A A A A A A XbaI A A A A A A A A A A B

"An extra fragment was observed in DdeI digests of the amplicon derived from GRA cDNA. This 496 bp band was not found to correspond to fragments present in any other haplotye

bThe profile generated from a Sau96I digest of the GRA derived amplicon showed the 259 bp fragment of haplotype A but lack the 171 bp fragment (see Table 3)


,-,--ARK

BRK

I I I I I I I I I

~ BEE

SIM ,,,,i,,

KTA

NEU

TAR

VIC

- I ¸ I I I I t" I F

WHP

GRA

KKH

0.07s 0.064 0.05a o.04a 0.0a2 o.o2~ 0.0~1 o.000

Fig. 2. A phenogram generated using the UPGMA method [25], displaying similarities among 11 CrPV isolates based on PCR-RFLP analysis. The distance measure used to

construct the phenogram was d [15]

presented in Figure 2. To test the robustness of the phenogram, the data were jackknifed by systematically removing each of the taxa in turn and repeating the analyses. This treatment produced no phenograms that were in conflict with the branching pattern presented in Fig. 2.

Discussion

The two new isolates, TAR and SIM that are described in this paper, were first observed during the routine screening of laboratory populations of Drosophila for Drosophila C and A viruses. The two viruses were originally isolated in a passage of homogenates from laboratory Drosophila populations through a virus-free Drosophila stock. After storing the isolates for several years at - 2 0 °C we passaged them through DL2 cells and produced a visible C.P.E. However, subsequent studies demonstrated that both isolates did not react with DCV antiserum, but showed strong hybridisation to 32P-labelled CrPV probes. These isolates therefore appeared to represent the first isolation of CrPV from a dipteran host.

Serological and biological characters

To characterise the TAR and SIM isolates we examined a suite of serological and biological characters similar to those previously used for CrPV [4]. From the data provided in Table 2 it is clear that the serogroups we were able to recognise differ substantially from those previously described [4]. However, this is not surprising as the antisera used in the current study were not those previously used. For instance, antisera VIC E23/5 used in the current study was produced


from a later bleeding of the same rabbit used to generate VIC E23/4 [4, 17]. In the current study antisera VIC E23/5 produced a reaction of identity between all isolates in double-diffusion experiments whereas antisera VIC E23/4 had previously been observed to produce reactions of partial identity between GRA, KKH and WHP when compared to VIC. Nevertheless, we could identify three distinct serogroups. The first of these serogroups comprise ARK and BRK, and are distinguished by their reaction of partial identity against the reference isolate in reactions with anti-BRK R2/3 serum and their inability to react with anti-VIC C1 serum. The second serogroup comprises BEE, SIM and WHP, all of which give a reaction of partial identity against the reference virus in reactions with anti-VIC C1 serum. The third group comprises the remaining six viruses all of which were indistinguishable from the reference isolate in all double-diffusion tests performed.

Within these serogroups some further separations can be made on the basis of the other characters examined. In the second serogroup, BEE could be differentiated from SIM and WHP by its inability to cause a C.P.E. in Aedes aegypti cells grown in T C t 9 9 M K + media, and SIM could be separated from WHP by its inability to replicate in the Cydia pomonella cells. In the third serogroup, KKH, TAR and VIC are characterised by their inability to produce a C.P.E. in Cydia pomoneIla cells or in Aedes aegypti cells maintained in either Schneider's or TC199MK + media. Of the remaining three isolates, GRA and NEU could not be separated by in vitro host range characters whereas KTA could be separated from these two isolates by virtue of its ability to cause a C.P.E.in Aedes aegypti cells maintained in TC 199MK + media. BRK and ARK could not be further separated by reference to any of the above in vitro host range characters.

Molecular characters

As the serological and biological characters were unable to separate all of the CrPV isolates into the discrete strains previously identified [4], we used a combination PCR-RFLP technique to attempt to differentiate between isolates. Using this technique, unique genotypes could be assigned to all isolates with the exception of two groups, one comprising VIC, NEU and TAR, and another comprising GRA and KKH.

Although unable to equivocally separate strains of CrPV from each other the PCR-RFLP approach does have several advantages over serological and biological characters. First, it gives an indication of the overall level of nucleotide sequence divergence between isolates. In the current study these estimates of nucleotide divergence (number of nucteotide substitutions per base-d) were derived from mapped restriction site data, but can also be estimated from fragment data alone [ 15]. Generally we observed estimated nucleotide divergen- ces in the range of 0-8 %, with two distinct clusters of of isolates- one comprising ARK and BRK and the other comprising the remaining nine isolates. ARK and BRK differ by about 0.3% but differ from all of the other isolates by about 6-8%.


It is encouraging that these groupings corroborate those already established for CrPV, in particular the strong demarcation between ARK/BRK and all other isolates [41. This is of particular interest as ARK and BRK were isolated in North America whereas all remaining isolates are Australasian, suggesting that there are major biogeographical differences within the CrPV species. Another advantage of the PCR-RFLP technique is that it estimates the genetic variation in a particular preparation and/or isolate.

In the current study, some of the isolates showed complex profiles with some enzymes, suggesting the presence of more than one haplotype. In most instances, the second presumptive haplotype was present in only minor amounts e.g. the HaeIII digest of the BEE fragment (see Fig. 1). From the PCR-RFLP study it is therefore apparent that many of the previously recognised strains of CrPV are in fact genetically mixed.

The Drosophila isolates TAR and SIM

The two new isolates that we describe from Drosophila populations came from populations of D. melanogaster (TAR) and of D. simulans (SIM). With the combination of characters that we employed, the SIM isolate could be separated from all other isolates but the TAR isolate proved to be indistinguishable from the existing VIC isolate. The latter may well have been a contaminant of the Tamar Valley laboratory stock from which it was isolated as the stock had been established for about 18 months prior to the isolation of TAR. Notwithstanding this possibility, its isolation from the laboratory population demonstrates that CrPV can exist as a persistent infection in D. melanogaster in much the same way as DAV, DCV and DPV are able to persist in laboratory populations. However, it remains to be seen whether CrPV can be isolated from natural populations of Drosophila.

The SIM isolate is clearly separable by molecular and biological characteris- tics from all other CrPV isolates. Interestingly, the SIM isolate appears from the PCR-RFLP data to be most closely related to the BEE isolate, which was isolated from honeybees in Canberra. This suggests that a cluster of closely related isolates may be present in a number of different species in the same geographic locality. However, it is also apparent that more than one cluster may be present in one locality as the GRA and NEU isolates (both isolated from the Canberra region-see Table 1), do not cluster with BEE and SIM (see Fig. 2).

Taxonomic implications

When the molecular data generated in the present study are combined with the serological and biological characters, we are able to separate all of the isolates examined into discrete taxa, except TAR and VIC. So, we therefore propose:

1) that the SIM isolate be accorded the status of a strain and be termed CrPVsIM (D. simulans, Canberra, Australia, 1985)

2) that the TAR isolate be recognised as an isolate of CrPVvic and called CrPVvic (D. melanogaster, Tamar Valley/Canberra, Australia, 1985)


3) that the NEU isolate be recognised as a distinct strain of CrPV, rather than an isolate of CrPVwc, and be termed CrPVNE~ (T. commodus, Canberra, Australia)

Although the molecular technique that we used was able to distinguish between ARK and BRK- which no previous study has been able to achieve [4 ] - we do not feel that it would be appropriate to separate these isolates at this time because we do not feel that genotypic homogeneity is the only criterion by which a strain of CrPV should be defined and that there should be other biological, serological or biophysical bases for establishing a strain. Such is the case with VIC and NEU, where the current study was able to distinguish between them on the basis of their ability to produce a C.P.E. in Aedes aegypti cells, even though no molecular characters could separate them.

From a taxonomic viewpoint, the characters obtained in the current study by the PCR-RFLP technique provides, for the first time, a set of characters to delineate CrPV strains and isolates that does not rely (necessarily) upon comparison to a large array of known standards. Christian and Scotti [4] in their revision of CrPV relied heavily upon biological and serological characters, and comparison across all available isolates when delineating strains and isolates. They raised concerns over the use of such serological and biological characters when extrapolating from one study to another- a feature borne out by the current study. For instance, in the current study we observed a reaction of identity between KKH and VIC with anti-BEE A1 serum, whereas studies carried out two years previously with the same antiserum but a slightly earlier passage of the virus had recorded a reaction of partial identity. Similar problems may also arise with biological characters such as the use of in vitro host ranges. In these studies parameters such as cell passage number, media composition and media quality vary with little information on the effect that this may have on the characters under study. For instance, in the current study we observed quite considerable differences in the susceptibility of Aedes aegypti cells to the CrPV isolates depending on the media in which they were maintained.

The serological and biological characters previously employed have been useful in constructing a taxonomy for CrPV and (more importantly) defining a set of reference isolates. However, as the range of available isolates increases such characters become more unwieldy and time-consuming to use. The molecular characters described in this paper are the first step in updating our taxonomic technology for this wide-ranging virus species. With the higher order taxonomy of CrPV being recently questioned [6] more molecular data is obviously needed. It is hoped that this will in turn have benefits for research on the specific taxonomy and evolution/biogeography of this very interesting and unusual insect virus.

References

1. Brun G, Plus N (1980) The viruses of Drosophila. In: Ashburner M, Wright TRF (eds) The biology and genetics of Drosophila, Vol 3a. Academic Press, New York, pp 625-702


2. Chen Q, McIntosh AH, Ignoffo CM (1983) Establishment of a new cell line from the pupae of PluteIla xylostella J Centr China Teach Coll 3:99-107

3. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159

4. Christian PD, Scotti PD (1994) A suggested taxonomy and nomenclature for Cricket paralysis virus and Drosophila C virus complex. J Invertebr Pathol 63:157-162

5. King LA, Pullin JSK, Stanway G, Almond JW, Moore NF (1987) Cloning of the genome of an insect picornavirus using a cDNA: RNA hybrid method: sequence of the 3' end. Virus Res 6:331-344

6. Koonin EV, Gorbalenya AE (1992) An insect picornavirus may have a genome organisa- tion similar to that of caliciviruses. FEBS Lett 297:81-86

7. McIntosh AH, Maramorosch K, Rechtoris C (1973) Adaptation of an insect cell line (Agatlia constricta) in a mammalian cell culture medium. In vitro 8:375-378

8. McIntosh AH, Rechtoris C (1974) Insect cells: colony formation and cloning in agar medium. In vitro 10:1-5

9. McIntosh AH, Ignoffo CM (1981) Replication and infectivity of the single-embedded nuclear polyhedrosis virus, Baculovirus heliothis, in homologous cell lines. J Invertebr Pathol 37:258-264

10. McIntosh AH, Ignoffo CM (1989) Replication of Autographa californica nuclear polyhedrosis virus in five lepidopteran cell lines. J Invertebr Pathol 54:97-102

11. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbour Laboratory Press, New York

12. Manousis T, Moore NF (1987) Cricket paralysis virus, a potential control agent for the olive fruit fly, Dacus oleae Gmal. Appl Environ Microbiol 53:142-148

13. Mansi W (1958) Slide gel-diffusion precipitation test. Nature 18 i: 1 289 14. Nei M, Li WH (1979) Mathematical model for studying genetic variation in terms of

restriction endonucleases. Proc Natl Acad Sci USA 76:5 269-5 273 15. Nei M, Miller JC (1990) A simple method for estimating average number of nucleotide

substitutions within and between populations from restriction data. Genetics 125: 873-879

16. Peleg J (1968) Growth of arboviruses in monolayers from subcultured mosquito embryo cells. Virology 35:317-319

17. Plus N, Scotti PD (1984) The biological properties of eight different isolates of cricket paralysis virus. Ann Virol Inst Pasteur 135E: 257-268

18. Saitou N, Nei M (i987) The neighbor-joining method: a new method for reconstructing phylogenetic tress. Mol Biol Evol 4:406 425

19. Schneider I (1972) Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morph 27:353-365

20. Sc•tti PD ( •976) Cricket para•ysis virus rep•icates in cu•tured Dr•s•phila ce••s. Intervir••- ogy 6:333-342

21. Scotti PD (1977) End-point dilution and plaque assay methods for titration of cricket paralysis virus in cultured Drosophila cells. J Gen Virol 35:393-396

22. Scotti PD, Longworth JF, Plus N, Croizier G, Reinganum C (1981) The biology and ecology of strains of an insect small RNA virus complex. Adv Virus Res 26:117- 143

23. Scotti PD, Wigley P (1982) Factors affecting the use of immunoosmophoresis for the detection of two insect riboviruses. J Virol Methods 4:129-137

24. Singh KRP (1967) Cell cultures derived from larvae of Aedes albopictus Skuse and Aedes aegypti (L.) Curr Sci 36:506-508

25. Sneath PHA, Sokal RR (1973) Numerical taxonomy. Freeman, San Francisco

1522 K.N. Johnson and P. D. Christian: Molecular characterisation of CrPV

26. Summers MD, Smith GG (1987) A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agric Exp Stat Bull No. 1 555

27. Vaughn JL, Goodwin RH, Tompkins GJ, McCawley P (t977) Establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In vitro 13: 213-217

Authors' address: Dr. P. D. Christian, CSIRO, Division of Entomology, P.O. Box 1700, Canberra, ACT 2601, Australia.

Received October 27, 1995

A molecular taxonomy for cricket paralysis virus including two new isolates from Australian...

Documents

Transcript of A molecular taxonomy for cricket paralysis virus including two new isolates from Australian...