United States Department of Agriculture, Agricultural Research Service, Center for Medical, Agricultural, and Veterinary Entomology, 1600/1700 SW 23rd Drive, Gainesville, FL 32604, USA
USDA/ARS Plum Island Animal Disease Center, Plum Island, New York, USA2
Sequencing Core Facility of the ICBR, University of Florida, Gainesville, Florida, USA3
Author for correspondence: Bettina Moser. Fax +1 352 374 5922. e-mail bmoser{at}gainesville.usda.ufl.edu
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Abstract |
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Introduction |
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Methods |
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Infection of larvae for histopathology.
Groups of 100 3 to 4-day-old C. quinquefasciatus or C. nigripalpus larvae were exposed to 20 LE virus in 100 ml of water with 10 mM MgCl2 plus 0·02% alfalfa and potbelly pig chow mixture (2:1). Groups without the addition of the virus served as controls. Exposed and unexposed larvae were removed at 30 min, 1, 2, 4, 8, 12, 13, 14, 15, 16, 18, 24, 32 and 48 h p.i. for histological studies.
Histopathology.
The larvae of C. quinquefasciatus and C. nigripalpus have a relatively clear cuticle allowing the infected cells of the midgut to be detected with a dissecting microscope (Fig. 1a, b
). Midguts were dissected from virus-infected and uninfected larvae in Ringers solution (Becnel, 1997
). Cytopathological effects were determined by examination of mounted midguts by phase-contrast microscopy. Midguts were prepared for ultrastructural examination by primary fixation in 2·5% glutaraldehyde for 2 h, post-fixed in 2% osmium tetroxide, dehydrated in an ethanol series and embedded in EponAraldite. Thin sections, stained in uranyl acetate and lead citrate, were photographed at 75 kV. Suspensions of virions, released by alkaline treatment, were negatively stained in 1% phosphotungstic acid on coated grids and photographed at 75 kV.
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Virus OB purification.
OBs were purified from 5-day-old (48 h p.i.) C. quinquefasciatus larvae, infected as described above. Larvae were ground with a Tekmar Tissuemizer in 0·1% aqueous SDS and filtered through polyester to remove large parts. The filtrate was spun at 1470 g for 5 min and the supernatant filtered through a 0·45 µm hydrophobic filter. The filtrate was further purified by differential centrifugation on a continuous Ludox gradient (Undeen & Alger, 1971 ) or 30% Ludox (30 min at 16320 g). The OBs banded at a density of about 1·141·18 g/ml on the continuous gradient and pelleted in 30% Ludox. They were washed in 0·1 mM NaOH, pH 10·0 (twice) followed by deionized water (twice) and stored at 4 °C. The OB concentration was estimated spectrophotometrically by establishing a standard curve at an OD of 260 nm. OBs were quantified initially with a PetroffHauser counting chamber and darkfield microscopy optics. The OD260 was measured for different concentrations, and a regression analysis done on the data. The resulting regression equation was used to calculate the OB concentration based on OD260.
Protein sequencing.
A pure OB suspension (5·4x109 OBs/µl) was boiled in sample loading buffer (4xbuffer: 250 mM TrisHCl, pH 6·9, 12% SDS, 20% -mercaptoethanol, 40% glycerol) for 5 min to solubilize proteins. The proteins were separated on a standard SDSPAGE gel (5% stacking gel, 10% separating gel) (Laemmli, 1970
) of dimensions 11x18x0·75 cm overnight at 20 V and transferred to a PVDF membrane using standard Western blotting procedures. The N-terminal sequences of a major protein band, of size approximately 29 kDa, was determined by Edman degradation chemistry at the University of Floridas Protein Chemistry Core Facility. Protein comparisons with entries in the GenBank, EMBL, PIR and SWISS-PROT databases were performed with BLAST (Altschul et al., 1990
).
Viral DNA preparation.
Culex nigripalpus nucleopolyhedrovirus (CuniNPV) DNA was isolated directly from purified OBs. Because OBs do not dissolve readily at pH<12 (unpublished results), we modified the standard alkali release procedure for lepidopteran baculoviruses. Briefly, the OB suspension was incubated for 5 min at pH 12·0 (raised pH to 12·0 with 0·01 M NaOH, pH 12·0), neutralized with 1/10 vol. of 1 M TrisHCl, pH 8·0) and digested with Proteinase K (2 µg/µl) in 1% SDS and 200 mM -mercaptoethanol overnight at 56 °C. The following day, a standard phenolchloroform extraction and ethanol precipitation were performed (Sambrook et al., 1989
).
Genome size estimate.
Viral DNA was sized by pulsed-field gel electrophoresis and restriction enzyme analysis. Approximately 3 µg of viral DNA was restricted with XbaI, SpeI or BglII. The resulting fragments were separated by agarose gel electrophoresis (0·6% agarose gel in 1xTAE buffer, 1·5 V/cm, 4 °C, 30 h) and visualized by UV transillumination.
Shotgun cloning and sequencing.
Viral DNA was restricted with PstI, HindIII or EcoRI; the resulting fragments were ligated into dephosphorylated plasmid pUC19 and grown in E. coli DH5 cells. Recombinant clones were selected by blue-white screening (Sambrook et al., 1989
). Recombinant plasmid DNA was purified with the QiaPrep miniprep kit and restricted with the appropriate enzyme to estimate the size of the inserts. Additionally, dot-blots of the recombinant clones were probed with 32P-labelled or DIG-labelled viral DNA to confirm that viral DNA was cloned. DNA templates were sequenced at the University of Florida DNA Sequencing Core Laboratory using dideoxy chain terminator sequencing chemistries and an ABI PRISM 377 automated DNA sequencer. The sequences were assembled with Sequencher software.
Nucleotide sequence analysis.
The sequence composition was determined with the Wisconsin Genetics Computer Group (GCG) software (Wisconsin Package version 10.0). DNA and protein comparisons with entries in the GenBank, EMBL, PIR and SWISS-PROT databases were performed with BLAST (Altschul et al., 1990 ). Two genes, a gene involved in DNA replication (dnapol) and a structural gene (p74), were selected for phylogenetic analysis.
A multiple amino acid sequence alignment of a conserved region of the DNA polymerase gene (Bulach et al., 1999 ) from eleven lepidopteran baculoviruses, one lepidopteran ascovirus, two entomopoxviruses (one from Orthoptera, one from Lepidoptera) and CuniNPV (see Fig. 6
) was performed with GCG PileUp, CLUSTAL W (1.7) (Thompson et al., 1994
) and PepTool (Wishart et al., 1997
) computer programs and edited visually. Alignment files were analysed with PAUP 4* beta version 4.0b4a (Swofford, 1999
) by distance and parsimony methods. A heuristic search was performed to find the most parsimonious tree (rooted and unrooted). Bootstrap analysis was done to place confidence estimates on the groups contained in the optimal rooted tree. A multiple amino acid sequence alignment of nine lepidopteran baculovirus p74 proteins and the CuniNPV p74 protein was created and analysed in a similar fashion.
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Results |
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Gross pathology
Virus infection affected development, behaviour and appearance of the C. quinquefasciatus and C. nigripalpus larvae. By 24 h p.i., infected larvae were stunted in size when compared to unexposed individuals. Larvae were feeding actively through about 48 h p.i. but by 72 h p.i. larvae were lethargic and often remained suspended at the water surface. By 48 h p.i., nuclei of most cells in the gastric caeca and posterior stomach appeared opaque to white in colour due to the proliferation of OBs (Fig. 1a, b
). Cells in the anterior stomach rarely supported virus development (Fig. 1b
). Death of the larvae usually occurred by 7296 h p.i. at which time most nuclei in the posterior stomach and gastric caeca were infected (Fig. 1b
).
Histopathology
Virus development, restricted to specific regions and cell types in the larval mosquito midgut, occurred primarily in resorbing/secreting cells found only in the proximal portions of the gastric caeca and the posterior stomach. Regenerative cells were infected while cells of the cardia were not infected. Cytopathological effects were first detected by 12 h p.i. in nuclei of epithelial cells in the gastric caeca and anterior portion of the posterior stomach (Fig. 2a, b
). Healthy midgut epithelial cells were characterized by distinct areas of cytoplasmic organelles and a nucleus with a dense, centrally located nucleolus (Fig. 2a
, c
). The first signs of infection were a rounding of the cell and a more granular and dense appearance of the cytoplasm (Fig. 2d
). The centrally located nucleolus disappeared and heterochromatin accumulated along the inner margins of the nuclear envelope (Fig. 2d
). This was rapidly followed by hypertrophy of nuclei and the formation of distinct regions of heterochromatin throughout the nucleus (Fig. 2b
, e
). By 14 h p.i., highly refractile groups of OBs accumulated along the outer margins of the infected nuclei (Fig. 2f
, g
), frequently accompanied by the accumulation of large cytoplasmic vacuoles. It often appeared that large single OBs were formed (Fig. 2g
, h
), due to the inability to resolve individual OBs within these regions. The nuclear membrane remained intact throughout virogenesis. By 2448 h p.i., the nuclei of most of the cells of the gastric caeca and posterior stomach were filled with OBs (Fig. 1b
).
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The initial signs of virus activity were cytoplasmic vacuolization, nuclear hypertrophy and margination of heterochromatin along the inner margins of the nuclear envelope (Fig. 3a). The early appearance of the virogenic stroma was as a loosely granular material dispersed throughout the nucleus (Fig. 3a
). Empty capsids and nucleocapsids were located throughout the nucleoplasm and there was little or no evidence for nucleocapsid envelopment or occlusion (Fig. 3b
, c
). Rather, naked nucleocapsids budded through the nuclear envelope either singly or in groups (Fig. 3b
, c
). Once in the cytoplasm, transport vesicles (formed by the membranes of the nuclear envelope) released the nucleocapsids of the BV. BV accumulated at cell junctions, along the basement membrane and near the microvilli but we did not observe BV passing through any of these membranes (data not shown).
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Heat stability
After heat treatment of the viral suspension at 24 °C, the virus infection rate was 89·5±3·3% (n=6) and there was no significant difference between the crude or purified virus. Temperatures below 50 °C minimally affected virus infectivity. After heat treatment of the viral suspension at 50 °C, the infection rate was reduced to 6·4±3·3% (n=6) and at 55 °C it was 0·0% (n=4).
Protein sequences
To further characterize this virus and determine its relationship to other baculoviruses, a major band of 29·7 kDa that we predicted might be the OB protein was isolated from purified OBs by SDSPAGE (data not shown) and subjected to N-terminal sequence analysis. The N-terminal sequence, APQVK PRYRY AVAIT NHMDT, did not match any of the CuniNPV genome sequences or entries in the main databases using BLAST. It also showed no homology to the polyhedrin protein sequence of Neodiprion sertifer (Ns)NPV [published in Zanotto et al. (1993) with the permission of Robert D. Possee, (Oxford, UK)] or the N-terminal amino acid sequence of Tipula paludosa (Tp)NPV (Rohrmann et al., 1981
). These sequences are not in any of the major databases.
Genome size estimate
Based on restriction enzyme analysis and pulsed-field gel electrophoresis (results not shown), we estimated the CuniNPV genome size to be approximately 105110 kbp. The BglII digest yielded five DNA fragments with estimated sizes of 6·2, 16·0, 23·0, 26·0 and 38·0 kbp; SpeI digested the DNA into seven fragments of approximately 1·8, 2·6, 9·9, 18·0, 21·0, 25·0 and 31·0 kbp; XbaI digestion produced eight DNA fragments with estimated sizes of 3·5, 4·0, 4·8, 10·0, 11·8, 12·2, 25·0 and 33·5 kbp. A submolar band of 16·0 kbp in the XbaI digest may indicate the presence of more than one virus genotype.
Sequence data
To date we have sequenced approximately 33 kbp of the viral genome. The GC content of this sequence was 52%. We identified 96 CuniNPV potential ORFs of greater than 50 amino acids. Fifteen ORFs (16% of the predicted ORFs) were unique to CuniNPV and only eleven ORFs had a significant sequence similarity to the following lepidopteran baculovirus amino acid sequences: DNA polymerase, lef-1, lef-4, p47, p74, odv-e56, hypothetical 23·0 kDa protein, bro-a, p33, hypothetical 42·1 kDa viral capsid associated protein, and the product of Xestia c-nigrum granulovirus (XnGV) ORF33. These sequences, several of which are considered to be unique to baculoviruses, clearly define CuniNPV as a member of the family Baculoviridae. The ORFs for p74 and p47 were overlapping. Orientations, sizes of the predicted ORFs and putative proteins are detailed in Table 1
.
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The DNA polymerase sequences showed considerable divergence (data not shown). Within the NPVs, sequence differences ranged from 059%, the GV sequence differed from the NPVs by about 62%, and the CuniNPV sequence differed from both the NPVs and the GV by approximately 75%. The Spodoptera ascovirus (SAV) and Melanoplus sanguinipes entomopoxvirus (MsEPV) sequences diverged more than 85% from the others. CuniNPV was basal to all the other baculoviruses in the DNA polymerase protein phylogenetic tree (Fig. 7a). Likewise, the p74 proteins showed considerable divergence (data not shown), ranging from 8·5 to 48% within the NPVs, 58 to 61% between the NPVs and XnGV, 63 to 66% between CuniNPV and the NPVs and 71% between CuniNPV and XnGV. CuniNPV was basal to all the other known baculoviruses in the p74 protein phylogenetic analysis (Fig. 7b
).
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Discussion |
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The only mosquito baculovirus that has been studied in any detail (AesoNPV) was isolated from Aedes sollicitans in Louisiana (Clark et al., 1969 ). Its pathology and morphology were investigated in several different mosquito hosts (Federici & Lowe, 1972
; Federici & Anthony, 1972
; Federici, 1980
, Stiles et al., 1983
). The baculovirus reported here as well as all other reported mosquito baculoviruses are restricted to larval midgut epithelial cells but there are differences in cell specificity. CuniNPV appears to selectively infect only the resorbing/secreting cells of the gastric caeca and the posterior stomach (Clements, 1992
). The cells of the cardia are not infected and the cells of the anterior stomach are rarely infected. In AesoNPV the cardia, gastric caeca and the entire stomach supported virus development.
In studies with AesoNPV it was concluded that there was only a single replication cycle leading to the production of ODVs (Federici, 1980 ; Stiles et al., 1983
). There was no evidence for the presence of BV or lateral transmission of the virus within the midgut. The cytopathology, virion formation and structure of the OBs for CuniNPV were similar to that reported for AesoNPV (Federici & Lowe, 1972
; Federici & Anthony, 1972
; Federici, 1980
; Stiles et al., 1983
). This sequence of events involved: hypertrophy of nuclei, fragmentation of the nucleolus followed by margination of heterochromatin to the nuclear envelope and the formation of the virogenic stroma. Rod-shaped nucleocapsids were formed within this stroma and finally enveloped and occluded. The production of OBs was similar in the CuniNPV and AesoNPV with a few notable differences. Both this virus and AesoNPV produced small OBs. These coalesced in AesoNPV to form large rugose ellipsoids (47 µm) and finally large smooth-surfaced spindles (up to 20 µm; Federici & Anthony, 1972
). Although it did appear in fresh preparations of CuniNPV that OBs coalesced to form larger OBs this was not confirmed with EM observations. At no point in development were OBs larger than approximately 0·5 µm observed in this new baculovirus. Another baculovirus from the mosquito Wyeomyia smithii also produced small OBs similar to this new virus (Hall & Fish, 1974
). Those authors also observed that OBs appeared to coalesce in intact nuclei when viewed at the light level but could not confirm this with EM studies.
In insect baculoviruses, the primary site of infection is the midgut epithelium, where BV is produced that spreads the infection to other host tissues (Federici, 1997 ). We showed that spread of CuniNPV within the midgut apparently occurred by production of BV. Certain other NPVs from Hymenoptera, Thysanura, Trichoptera and Diptera (Federici, 1997
) and one lepidopteran GV (Federici & Stern, 1990
) also primarily infect the hosts midgut epithelium but the mechanisms by which the virus spreads from cell to cell in the midgut are not known. We surmise that a mechanism similar to the one described for CuniNPV involving BV is operating to disseminate the infection within the midgut.
We have presented evidence that virogenesis of this mosquito baculovirus involved two types of virions, ODV and BV. The first phase was initiated by ODVs that entered midgut cells through the plasmalemma of the microvilli. This initial replicative phase produced BV that disseminated the virus to other midgut cells. The nuclei of cells infected by BV may first produce additional BV for lateral transmission or more likely enter directly into the production of ODVs. This developmental cycle was similar to the cycles reported for other baculoviruses (Federici, 1997 ) except that the CuniNPV infections were confined to midgut tissues.
Unlike lepidopteran baculoviruses the OBs of CuniNPV were extremely resistant to dissolution at alkaline pH<12·0. Federici & Anthony (1972) studied the chemical behaviour of OBs of AesoNPV. They reported that OBs rapidly dissolved when treated with 0·1 or 0·03 M NaOH (corresponding to pH values of approximately 13·0 and 12·3). They did not study the behaviour of OBs at lower alkaline pH.
The OB proteins of baculoviruses have been used extensively in phylogenetic analyses. The known polyhedrin and granulin baculovirus proteins are the predominant proteins in the NPVs and GVs and are approximately 29 kDa in size (Funk et al., 1997 ). By inference, we speculated that the 29·7 kDa protein band, which was a predominant polypeptide, was the CuniNPV OB protein. The only polyhedrin sequence information for a dipteran baculovirus is that of TpNPV and studies on the relatedness of dipteran and lepidopteran OB proteins are limited (Guelpa et al., 1977
; Rohrmann et al., 1981
; Rohrmann, 1986
). The N-terminal sequence of TpNPV showed no homology to the N-terminal sequence of the 29·7 kDa CuniNPV protein. Mosquito baculovirus OBs are adapted to persist in aquatic environments (C. nigripalpus is a freshwater mosquito, A. sollicitans a saltwater species), and it is not unexpected that these OB proteins would show low sequence homology to the granulins and polyhedrins of terrestrial insects.
We have sequenced approximately one-third of the CuniNPV genome and detected 96 ORFs of 50 amino acids or more, including several genes considered to be unique to baculoviruses. Its GC content of 52% was similar to that of Orgyia pseudotsugata (Op)MNPV (55%) and Lymantria dispar (Ld)MNPV (58%). In comparison, the GC content of Autographa californica (Ac)MNPV (41%) and Bombyx mori (Bm)NPV (40%) is much lower. On the assumption that this sequence information is representative of the whole genome of CuniNPV it can be inferred that it shares relatively few sequence homologies and a low degree of gene conservation with the other members of the family Baculoviridae (11/96 ORFs=11%).
We have identified four genes that are related to those in the baculovirus late expression factor (LEF) category (Lu & Miller, 1997 ): dnapol, lef1, lef4 and p47. A number of domains of the dnapol gene, involved in DNA replication, are highly conserved among different organisms. Among the baculoviruses, the dnapol ORF was longest in CuniNPV (3417 nt). lef1 has been sequenced from seven lepidopteran baculoviruses and is essential for transient DNA replication in AcMNPV and OpMNPV. It has four conserved domains, three of which are also found in DNA primase genes, suggesting that lef1 may be a baculovirus primase gene (Lu et al., 1997
). It is located adjacent to the egt gene in Choristoneura fumiferana (Cf)MNPV, AcMNPV, OpMNPV, LdMNPV and Busura supressaria (Busu)NPV, but not in Spodoptera exigua (Se)MNPV and the putative egt gene of CuniNPV. XnGV apparently does not have an egt gene.
lef4 and p47 are equimolar subunits of a DNA-dependent RNA polymerase that is required for transcription of viral late genes (Guarino et al., 1998 ) and their presence in CuniNPV suggest that the virus, like the other baculoviruses, encodes its own RNA polymerase. lef4 has been sequenced from AcMNPV, BmNPV, LdMNPV, OpMNPV, SeMNPV and XnGV. p47 has been sequenced from these viruses and also from BusuNPV and CfNPV. In CuniNPV the ORFs of p47 and p74 overlapped, an unusual arrangement not found in any of the other baculoviruses studied so far. Promoter analysis of p74 and p47 revealed a possible p74 late promoter motif, TAAG, at positions -15 to -12 upstream of the p74 start codon. A possible p47 start codon and promoter motifs were located at the 3' end of the p74 ORF with the following nucleotide coordinates: 19321936 (conserved sequence motif CGTGC), 19501954 (putative p47 early promoter motif TATAA) and 19851987 (start codon). The unconventional CGTGC promoter motif (Friesen, 1997
) is positioned at the RNA start site of p47 and other genes. In the granulovirus XnGV, the ORFs of these two genes do not overlap but are immediately adjacent to each other. In the nucleopolyhedroviruses AcMNPV, BmNPV, CfMNPV, LdMNPV, OpMNPV and SeMNPV, the ORFs for p47 and p74 neither overlap nor are located next to each other (this information is not available for BusuNPV). The p74 gene encodes a structural protein associated with occluded viral envelopes (Funk et al., 1997
). It is located adjacent to p10 in most of the NPVs including SeMNPV, AcMNPV, CfMNPV and OpMNPV but not in BusuNPV, Spodoptera litura (Splt)NPV and XnGV. A p10 gene has not been identified on the CuniNPV sequence fragment that contains the p74 gene.
The odv-e56 gene encodes a structural protein of occluded viral envelopes (Funk et al., 1997 ). Its ORF is adjacent to the ORF of ie-1 in AcMNPV, BmNPV, CfMNPV, LdMNPV and OpMNPV, but not in SeMNPV and XnGV where the two ORFs are distant from each other. odv-e56 did not appear to neighbour the putative ie-1 in CuniNPV.
The study of gene arrangements in the phylogenetic analyses of baculoviruses in addition to gene homology has been discussed (Hu et al., 1998 ; IJkel et al., 1999
; Chen et al., 1999
). Clearly, CuniNPV appeared to have a distinct gene arrangement and is highly divergent from the other known baculoviruses not only in gene homologies but also in gene order.
The CuniNPV DNApol and p74 amino acid sequence analysis suggested that CuniNPV is a member of the family Baculoviridae yet forming a clade separate from the lepidopteran NPVs and GVs. DNApol and p74 tree topologies among the NPVs and GV appeared to be similar to previously published trees based on analysis of the DNA polymerase gene (Bulach et al., 1999 ), polyhedrin gene (Zanotto et al., 1993
), lef2 gene (Chen et al., 1999
), egt gene (Clarke et al., 1996
) and gp64 gene (Liu & Maruniak, 1999
). As discussed in Bulach et al. (1999)
, the DNA polymerase gene may be better suited for phylogenetic analysis than the polyhedrin gene because it has more informative characters and appropriate outgroup taxa are available.
Traditionally, the mosquito baculoviruses have been placed in the genus Nucleopolyhedrovirus. Federici & Lowe (1972) suggested placement of the baculovirus from A. sollicitans (AesoNPV) into a group separate from the NPVs and GVs, distinguished by the fusiform OBs (although based on our observations the use of fusiform OBs as a distinctive characteristic for the mosquito baculoviruses is not appropriate). The collective data available thus far from CuniNPV suggest that it is a member of a new genus within the family Baculoviridae but more information is needed to resolve the taxonomic status of CuniNPV.
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Concluding remarks |
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Like NPVs, the OBs are found exclusively in the nuclei of infected cells but unlike NPVs they are globular, not polyhedral (Blissard et al., 2000 ), in shape. They are similar in size (average diameter 400 nm) to GV OBs. Each OB typically contains four, sometimes up to eight, virions as opposed to GVs where each OB contains one or two, rarely more virions. The CuniNPV gene order is distinct, and phylogenetic analysis of the p74 and DNA polymerase polypeptides placed the CuniNPV into a separate taxon basal to the NPVs and GV.
The data show that CuniNPV is a baculovirus with unusual characteristics. Pending additional data, we have elected to retain it in the genus Nucleopolyhedrovirus but suggest that it may represent a new genus within the family Baculoviridae.
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Acknowledgments |
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Footnotes |
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References |
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Received 6 June 2000;
accepted 26 September 2000.