The nucleopolyhedroviruses of Rachiplusia ou and Anagrapha falcifera are isolates of the same virus

Robert L. Harrison1 and Bryony C. Bonning1

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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The 7·8 kb EcoRI-G fragment of Rachiplusia ou multicapsid nucleopolyhedrovirus (RoMNPV), containing the polyhedrin gene, was cloned and sequenced. The sequence of the fragment was 92·3% identical to the sequence of the corresponding region in the Autographa californica (Ac)MNPV genome. A comparison of the EcoRI-G sequence with other MNPV sequences revealed that RoMNPV was most closely related to AcMNPV. However, the predicted amino acid sequence of RoMNPV polyhedrin shared more sequence identity with the polyhedrin of Orygia pseudotsugata MNPV. In addition, the RoMNPV sequence was almost completely identical (99·9%) to a previously published 6·3 kb sequence of Anagrapha falcifera MNPV (AfMNPV). The Eco RI and HindIII restriction fragment profiles of RoMNPV and AfMNPV also were nearly identical, with an additional EcoRI band detected in RoMNPV DNA. Bioassays of these viruses with three different hosts (the European corn borer, Ostrinia nubilalis H übner, the corn earworm, Helicoverpa zea Boddie, and the tobacco budworm, Heliothis virescens Fabricius) failed to detect any differences in the biological activities of RoMNPV and AfMNPV. These results indicate that RoMNPV and AfMNPV are different isolates of the same virus. The taxonomic relationship of Ro/AfMNPV and AcMNPV is discussed.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Baculoviruses are invertebrate-specific pathogens of the family Baculoviridae that have been isolated primarily from species of the insect order Lepidoptera. Members of this family in the genus Nucleopolyhedrovirus have been developed as foreign gene expression vectors (Jarvis, 1997 ) and biopesticides (Black et al., 1997 ; Van Beek & Hughes, 1998 ). The type species of this genus, the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), is one of a group of genetically similar viruses that includes NPVs isolated from Galleria mellonella L., Trichoplusia ni H übner and Rachiplusia ou Guenée (Jewell & Miller, 1980 ; Smith & Summers, 1979 , 1980 ).

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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Viruses, cells and insects.
AcMNPV strain C6 (Possee, 1986 ), RoMNPV strain R1 (Smith & Summers, 1980 ) and Anagrapha falcifera (Af)MNPV (Chen et al., 1996 ) were propagated in Spodoptera frugiperda cell lines (Vaughn et al., 1977 ) and titred by plaque assay. Sf21 cells were grown in Ex-Cell 405 medium (JRH Biosciences) supplemented with 3% foetal bovine serum (Intergen) and antibiotics (1 U/ml penicillin, 1 µg/ml streptomycin; Sigma). Sf9 cells were grown in TNM-FH medium (JRH Biosciences) which was also supplemented with 3% foetal bovine serum, antibiotics and 0·1% Pluronic F-68 (JRH Biosciences). Eggs of Ostrinia nubilalis as well as diet for these insects were obtained from the USDA/ARS Corn Insects and Crop Genetics Research Unit in Ames, IA, USA. Eggs of Heliothis virescens and Helicoverpa zea were obtained from the USDA/ARS Southern Insect Management Research Unit in Stoneville, MS, USA and diet for these species was obtained from BioServ (Frenchtown, NJ, USA) and Southland Products (Lake Village, AR, USA), respectively.

{blacksquare} 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 glycol–1 M NaCl. After pelleting by centrifugation, the BV was resuspended in 10 mM Tris–HCl–1 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 phenol–chloroform 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.

{blacksquare} 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.

{blacksquare} 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. Dose–mortality 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.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Sequence of the RoMNPV polh locus
The EcoRI-G fragment of RoMNPV contains the polh gene for this virus (Summers et al., 1980 ). This fragment was cloned and sequenced. Alignment of the 7781 nucleotide (nt) EcoRI-G sequence with the AcMNPV-C6 genomic sequence (Ayres et al., 1994 ) using the GCG BESTFIT program revealed 93·2% sequence identity with 32 gaps to nt 248–9318 of the AcMNPV sequence. With the exception of ORF 2, ctl and ORF 12, RoMNPV had the same array of genes found in AcMNPV in this region (Fig. 1). Consequently, we have adapted the same ORF numbering scheme used by Ayres et al. (1994) for AcMNPV, with ORF 1 corresponding to ptp. Approximately 1275 nt that in AcMNPV contains the ORF 2 and ctl ORFs were absent from the corresponding region in RoMNPV, and in their place was a 51 nt sequence with no significant similarity to other baculovirus sequences. The RoMNPV sequence which aligns with AcMNPV ORF 12 has a 5 nt insertion at codon position 16 which causes a frameshift and results in termination of the ORF at position 25. The EcoRI-G fragment aligns with the regions in AcMNPV containing most of the `homologous region' elements hr1 and all of hr1a, but much of the AcMNPV hr sequence in this region was not present in the RoMNPV sequence.



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Fig. 1. Physical map of restriction sites and genes on the RoMNPV-R1 EcoRI-G fragment and the corresponding region in AcMNPV-C6. Restriction sites are indicated (B, BamHI; E, EcoRI; H, HindIII) along with their nucleotide positions in the EcoRI-G and the AcMNPV-C6 genomic sequences. The positions of homologous regions (hrs) on the AcMNPV sequence are also indicated. ORFs in EcoRI-G are numbered in accordance with the standard established for AcMNPV (Ayres et al., 1994 ), with ptp designated as ORF1. Arrowheads indicate ORF orientation, and the AcMNPV ORF2 and ctl genes, which are absent from EcoRI-G, are represented with hatched arrows. The position of the AfMNPV sequence reported by Federici & Hice (1997) is indicated with a bar below the EcoRI-G map.

 
We also found that the EcoRI-G sequence was almost completely identical (99·9% identity with two gaps) to a 6289 nt sequence of the same region of AfMNPV (Federici & Hice, 1997 ). The AfMNPV sequence begins with an Eco RI site which is 152 nt downstream of the first EcoRI site in EcoRI-G and ends at nt 6437 of EcoRI-G (Fig. 1). The nucleotide sequence divergence between these viruses in this region consists of five 1 and 2 nt mismatches that occur within ptp, ORF2, lef2, and polh, and two 1 nt deletions that are present in intergenic regions of RoMNPV. The predicted amino acid sequences of RoMNPV ptp, ORF 3, ORF 5, polh, ORF 7 and pk1 are 100% conserved with the corresponding AfMNPV ORFs (Table 1), while RoMNPV ORF 2 has a serine-to- cysteine substitution at codon position 116 and lef2 has an arginine-to-alanine substitution at position 63 when compared to the AfMNPV sequences. The predicted amino acid sequence of RoMNPV ORF 9 lies outside the AfMNPV sequence.


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Table 1. Comparison of deduced amino acid sequences for genes in RoMNPV-R1 EcoRI-G

 
Comparison of the ORFs in EcoRI-G with homologous ORFs from AcMNPV, Orygia pseudotsugata (Op)MNPV and Bombyx mori (Bm)NPV revealed that RoMNPV appears to be most closely related to AcMNPV (Table 1). As described for AfMNPV (Federici & Hice, 1997 ), the RoMNPV polyhedrin amino acid sequence shares greater sequence identity with OpMNPV than with AcMNPV, suggesting that RoMNPV acquired its polh gene by recombination with a more distantly related baculovirus. A sequence identity search revealed that the polyhedrin of Epiphyas postvittana MNPV (Hyink et al., 1998 ) possessed the highest degree of sequence identity (97·1%) with RoMNPV polyhedrin.

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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Fig. 2. EcoRI and HindIII digests of AcMNPV-C6 (Ac), RoMNPV-R1 (Ro) and AfMNPV (Af) viral DNA. Size standards (indicated in kb) are the 1 kb ladder (1 kb, Gibco BRL) and HindIII fragments of phage {lambda} ({lambda}HIII, Promega). The arrow indicates an additional EcoRI band in RoMNPV DNA.

 
Lethal concentration (LC) bioassays were carried out to compare the biological activities of RoMNPV and AfMNPV. In our LC bioassays, RoMNPV and AfMNPV were equally virulent against larvae of Ostrinia nubilalis, Helicoverpa zea and Heliothis virescens (Table 2). Both viruses had significantly lower LC50s than AcMNPV against O. nubilalis and H. zea, which is consistent with previous studies showing that RoMNPV is more virulent against O. nubilalis than AcMNPV (Lewis & Johnson, 1982 ) and that AfMNPV is more effective against H. zea than AcMNPV (Hostetter & Puttler, 1991 ).


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Table 2. Dose–mortality response of neonate larvae infected with RoMNPV, AfMNPV and AcMNPV

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
In the process of assembling a transfer vector to make recombinant RoMNPV, we discovered that RoMNPV and the more recently described AfMNPV are different isolates of the same virus. AfMNPV infects a wide variety of agriculturally significant Lepidoptera (Hostetter & Puttler, 1991 ), and much work evaluating its potential as a biological control agent has been published. The differences between RoMNPV and AfMNPV are so minor that there is little reason to distinguish between the two viruses. In this case, AfMNPV should be referred to as RoMNPV, since the latter was originally isolated in 1960 from Rachiplusia ou.

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 ).


   Acknowledgments
 
The authors wish to thank Dr Max Summers (Texas A&M University) for providing RoMNPV-R1, Dr Suzanne Thiem (Michigan State University) for AfMNPV and Dr Don Jarvis (University of Wyoming) for Sf9 cells. This research was funded by a grant from the Illinois–Missouri Biotechnology Alliance (Project 96-3, awarded to B. C. Bonning). Journal Paper No. J-17983 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 3301, and supported by Hatch Act and State of Iowa funds.


   Footnotes
 
The GenBank accession number of the sequence reported in this paper is AF068270.


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Abstract
Introduction
Methods
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Discussion
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Received 20 April 1999; accepted 4 June 1999.