Department of Entomology and Interdepartmental Genetics Program, Iowa State University, Ames, IA 50011, USA1
Author for correspondence: Bryony Bonning.Fax +1 515 294 5957. e-mail bbonning{at}iastate.edu
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Abstract |
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Introduction |
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Rachiplusia ou (Ro)MNPV was first isolated in 1960 during an epizootic in the mint looper, Rachiplusia ou, in Indiana (Paschke & Hamm, 1961 ; Paschke & Sweet, 1966
). Restriction enzyme digest and nucleic acid hybridization studies show that RoMNPV is closely related to AcMNPV (Jewell & Miller, 1980
; Smith & Summers, 1980
, 1982
). Homologous recombination between the genomes of these two viruses has been observed during co-transfection and co-infection of cell lines and insects, further underscoring the degree of nucleotide sequence identity between these viruses (Croizier et al ., 1988
; Summers et al., 1980
). Although a restriction map of RoMNPV has been assembled (Smith & Summers, 1980
; Summers et al., 1980
), there are no published reports of RoMNPV gene sequences.
We are developing recombinant clones of RoMNPV for control of the European corn borer, Ostrinia nubilalis, a major agricultural pest. As a preliminary step towards this goal, we cloned and sequenced an RoMNPV restriction fragment containing the polyhedrin (polh ) gene. Here we report the analysis of this sequence and consider implications for the classification of this virus as a species separate and distinct from AcMNPV.
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Methods |
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Viral DNA isolation and restriction digest.
Sf9 cells were infected with NPVs at an m.o.i. of 1. Budded virus (BV) was harvested at 5 days post-infection. BV was precipitated by overnight incubation on ice with an equal volume of 20% polyethylene glycol1 M NaCl. After pelleting by centrifugation, the BV was resuspended in 10 mM TrisHCl1 mM EDTA pH 8·0 and incubated for 3 h at 37 °C with 1% SDS and 1 mg/ml proteinase K. Viral DNA was purified by phenolchloroform extraction and ethanol precipitation. Five µg of viral DNAs was digested with restriction enzymes for 3 h, and restriction fragments were separated by electrophoresis on a 0·8% agarose gel. The gel was stained with ethidium bromide and photographed under UV illumination.
DNA sequencing.
The plasmid pUC19M (Clontech) is a variant of pUC19 in which the EcoRI site has been substituted with an EcoRV site. An EcoRI site was inserted into pUC19M by digesting with SalI, filling in the termini with Klenow fragment, and attaching EcoRI adaptors (Promega) to the blunt ends. This plasmid, called pUC19M-RI, was used to clone the RoMNPV-R1 EcoRI-G fragment, as well as overlapping KpnI and BamHI subfragments of EcoRI-G. Nested unidirectional deletions of the subfragments were created by the method of Henikoff (1984) and sequenced using M13 forward and reverse primers by automated dideoxy terminator sequencing (Sanger et al., 1977
) at the Iowa State University DNA Sequencing and Synthesis Facility. Compilation of overlapping sequences and analysis of the final assembled sequence of EcoRI-G were carried out with the programs of the Genetics Computer Group Wisconsin package (version 9.0; Devereux et al., 1984
) and the Baylor College of Medicine Search Launcher web page (http://kiwi.imgen.bcm.tmc.edu:8088/search-launcher/launcher.html). The GenBank accession number of the RoMNPV EcoRI-G sequence is AF068270.
Insect bioassays.
Viral occlusions were prepared from cadavers of virus-killed H . virescens by a standard method (O'Reilly et al ., 1992 ). Lethal concentration bioassays were conducted using the droplet feeding method of Hughes & Wood (1981)
with five different concentrations of occlusions and 35 larvae per dose. Dosemortality relationships were analysed by probit analysis using the POLO program (Russell et al., 1977
). Statistical analysis of LC50s was carried out by the lethal dose ratio comparison method of Robertson & Preisler (1992)
. All bioassays were repeated at least three times.
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Results |
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Restriction digest and bioassay analysis
Restriction digests of RoMNPV and AfMNPV DNA yielded nearly identical fragment patterns, confirming that these viruses are the same (Fig. 2). An additional band mi grating between the 7 and 8 kb markers was present in the RoMNPV Eco RI fragment pattern (Fig. 2
).
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Discussion |
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It is possible that the original isolate of RoMNPV contained a virus that is different and distinct from AfMNPV and that we have selectively amplified an AfMNPV contaminant in our stock of RoMNPV-RI. However, Smith & Summers (1980) worked with two separate stocks of RoMNPV and found that all the clones derived from these stocks yielded identical EcoRI fragment patterns. This fragment pattern matched that obtained by Jewell & Miller (1980)
, who worked with an RoMNPV stock that was a few passages removed from the original isolate of Paschke & Sweet (1966)
and that had undergone three more passages before analysis of viral DNA. The EcoRI fragment patterns of our stock of RoMNPV-R1 exactly matched the fragment patterns obtained by these other two groups. It seems unlikely, then, that our RoMNPV is a selectively amplified AfMNPV contaminant.
There has been some controversy over whether RoMNPV/AfMNPV should be classified as a variant of AcMNPV or as a separate species (Smith & Summers, 1980 ; Volkman et al., 1995
; Federici & Hice, 1997
). Federici & Hice (1997)
proposed that Ro/AfMNPV should be classified as a variant of AcMNPV. However, there are differences between these viruses that argue against this classification.
(1) Restriction fragment differences. It has been stated that the differences between AcMNPV and RoMNPV/AfMNPV are of the same magnitude as those existing among other viruses regarded as AcMNPV variants, such as Trichoplusia ni (Tn) and Galleria mellonella (Gm) MNPVs (Federici & Hice, 1997 ). However, this is not the case. Smith & Summers (1979)
found that TnMNPV and GmMNPVs had over 90% of restriction fragments with identical or highly similar mobilities to AcMNPV restriction fragments. This degree of relatedness was similar to that found for other AcMNPV variants (Smith & Summers, 1979
), and also to Spodoptera exempta MNPV (Brown et al., 1984
). RoMNPV, in contrast, only had 35 of 60 fragments (58·3%) co-migrate with AcMNPV fragments. Smith & Summers (1980)
concluded that while TnMNPV and GmMNPV should be considered variants of AcMNPV, RoMNPV is more distantly related.
(2) Sequence divergence. Although the EcoRI-G nucleotide sequence identity with AcMNPV was 93·2%, 32 gaps were required to produce this alignment. For a DNA virus, this indicates that a small but significant degree of sequence divergence has taken place. Although RoMNPV and AcMNPV can undergo recombination with each other, recombination has also been observed between AcMNPV and Cydia pomonella granulovirus, which share little nucleotide sequence identity (Crook et al., 1993 ). Hence, it is uncertain to what extent the occurrence of recombination can serve as a criterion in the classification of baculovirus species.
(3) Missing hr elements and ORFS. Baculovirus genomes have `homologous regions' (hrs) that function as viral transcriptional enhancers and DNA replication origins (Possee & Rohrmann, 1997 ). In AcMNPV, each hr consists of two to eight repeats of an imperfect palindromic sequence with an EcoRI site at the centre of the palindrome. The Ro/AfMNPV genome has five regions with sequence similarity to AcMNPV hr4L, but none of these map to the EcoRI-G fragment (Chen et al., 1996
). Although we found single copies of the hr1 and hr1a palindromes in the RoMNPV EcoRI-G sequence, much of the rest of the hr sequences were missing.
In addition, an intact AcMNPV ORF 12 homologue was not found in RoMNPV. Although the function of ORF 12 is unknown, its absence from the genomes of OpMNPV and BmNPV (Ahrens et al., 1997 ; Possee & Rohrmann, 1997
) suggests that it does not play an essential role in the nucleopolyhedrovirus life-cycle.
A large segment containing AcMNPV ORF 2 and ctl is also missing from the EcoRI-G region, another feature shared by OpMNPV and BmNPV. Federici & Hice (1997) did not detect hybridization of a probe containing ORF 2 and ctl sequences to AfMNPV DNA under low-stringency conditions, indicating that Ro/AfMNPV does not contain these genes or that they are present but with highly diverged sequences. Multiple ORF 2- and ctl-like sequences are present in the genomes of OpMNPV, BmNPV and Lymantria dispar MNPV (Ahrens et al., 1997
; Possee & Rohrmann, 1997
; Kuzio et al., 1999
).
(4) Host-range differences. Federici & Hice (1997) state that variability in biological activities is common among virus variants and is not used as a criterion in species demarcation. However, there is nothing in the polythetic species concept, endorsed by the International Committee on the Taxonomy of Viruses (ICTV) for the definition of a virus species (Mayo & Pringle, 1998
), that precludes the use of variability in biological activities against different hosts as one of the polythetic criteria. Ro/AfMNPV and AcMNPV infect many of the same species, but there are multiple differences in their host ranges. One of the distinguishing characteristics of AfMNPV is its greater virulence against Helicoverpa zea (Hostetter & Puttler, 1991
) when compared to AcMNPV. Differences in the susceptibilities of another eight host species to AfMNPV and AcMNPV were also observed by Hostetter & Puttler (1991)
. In addition, Ro/AfMNPV infects species not susceptible to AcMNPV, such as the tobacco hornworm (Manduca sexta L.) and the navel orangeworm (Amyelois transitella Walker) (Hostetter & Puttler, 1991
; Vail et al., 1993
).
In conclusion, it appears that the group of NPVs closely related to AcMNPV can be divided into two separate and distinct lineages. One lineage contains AcMNPV, TnMNPV, GmMNPV and Spodoptera exempta MNPV. The other lineage contains RoMNPV. The taxonomic classification of these baculovirus lineages (either as a single species or as separate species) needs to be clarified. This will require the establishment of polythetic criteria for demarcation of baculovirus species (Van Regenmortel et al., 1997 ), as published recently for reoviruses of the genus Coltivirus (Attoui et al., 1998
).
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Acknowledgments |
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Footnotes |
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References |
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Received 20 April 1999;
accepted 4 June 1999.