JOURNAL OF VIROLOGY, Apr. 2004, p. 3244–3251
0022-538X/04/$08.00⫹0 DOI: 10.1128/JVI.78.7.3244–3251.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 7
Ancient Coevolution of Baculoviruses and Their Insect Hosts
Elisabeth A. Herniou,1,2* Julie A. Olszewski,1 David R. O’Reilly,1† and Jenny S. Cory2‡
Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, London
SW7 2AZ,1 and Molecular Ecology and Biocontrol Group, NERC Centre for Ecology and
Hydrology, Oxford OX1 3SR,2 United Kingdom
Received 23 July 2003/Accepted 10 December 2003
If the relationships between baculoviruses and their insect hosts are subject to coevolution, this should lead
to long-term evolutionary effects such as the specialization of these pathogens for their hosts. To test this
hypothesis, a phylogeny of the Baculoviridae, including 39 viruses from hosts of the orders Lepidoptera,
Diptera, and Hymenoptera, was reconstructed based on sequences from the genes lef-8 and ac22. The tree
showed a clear division of the baculoviruses according to the order of their hosts. This division highlighted the
need to reconsider the classification of the baculoviruses to include one or possibly two new genera. Furthermore, the specialization of distinct virus lineages to particular insect orders suggests ancient coevolutionary
interactions between baculoviruses and their hosts.
Coevolution, reciprocal evolution in interacting species
driven by natural selection (54), is a major driving factor in the
historical associations between pathogens and their hosts (13,
25, 59). Studies on the evolution of pathogen virulence and
host resistance have shown that within populations both pathogens and hosts are able to adapt in response to the interactions
(51, 59). However, there is much debate on how these microevolutionary scale changes can influence the patterns of speciation of the interacting species at macroevolutionary levels.
Coevolution need not necessarily lead to the cospeciation of
the interacting species. However, coevolutionary theories (54)
all support the hypothesis that the processes of coadaptations
would lead to a general trend of parasite specialization for
their hosts (53), regardless of the age of the association. Retroviruses and herpesviruses, with their vertebrate hosts, are
good examples of specialist pathogens for which coevolution
leading to a certain level of cocladogenesis within subfamilies
has been demonstrated (38–40).
The family Baculoviridae comprises a diverse group of arthropod-specific DNA viruses. They have been reported worldwide from over 600 host species (37), mostly from insects of the
order Lepidoptera but also from the orders Diptera, Hymenoptera, and the crustacean order Decapoda (6, 16). The family Baculoviridae is currently subdivided into two genera based
on several criteria, including the morphology of the occlusion
bodies (OBs) and on mechanisms of nucleocapsid envelopment in infected cells (6). The genus Nucleopolyhedrovirus
(NPV) is characterized by viruses forming polyhedral OBs,
each containing many virions formed within the nucleus (49),
whereas viruses of the genus Granulovirus (GV) typically produce ovoid OBs, with a single virion formed in the nucleocy-
toplasmic milieu (58). GVs have been described solely from
lepidopteran hosts, whereas NPVs have been isolated from a
wider range of arthropods. However, the taxonomic status of
nonlepidopteran baculoviruses is still uncertain (6). Baculovirus phylogenies have usually been based on individual gene
sequences. The polyhedrin/granulin (polh) gene, encoding the
major matrix protein of the OBs, has been the most widely
used (5, 7, 14, 34, 60), but other genes, such as DNA polymerase, egt, gp41, chitinase, cathepsin, and lef2, have also been
utilized (7, 8, 10, 12, 29, 30, 34, 42). In general, these studies
agree that the lepidopteran NPVs and GVs constitute distinct,
well-defined groups (7, 14, 23, 24, 60).
Almost all phylogenetic studies have been based on sequences from lepidopteran baculoviruses. Mostly because of
the rarity of the samples, little work has been done to try to
investigate the position of nonlepidopteran baculoviruses. Resolving the relationships between viruses isolated from Hymenoptera, Diptera, and Lepidoptera would greatly enhance our
understanding of the evolution of the virus family Baculoviridae. Early amino acid sequencing of the polyhedrin protein of
Neodiprion sertifer NPV (NeseNPV) showed that the polh sequence of this hymenopteran virus is quite divergent from that
of the lepidopteran viruses, including NPVs and GVs (50).
This result has been confirmed by determination of the complete DNA sequence of the gene (60). These phylogenies
based on the OB protein imply that the hymenopteran virus is
from an ancient lineage. More recently, phylogenetic analyses
based on the p74 and DNA polymerase genes, including
sequences from the dipteran virus Culex nigripalpus NPV
(CuniNPV), also showed that this virus is very divergent from
the lepidopteran viruses and that it is more ancestral (41).
Similar results were obtained based on complete genome phylogenetic analyses (24). There are currently no baculovirus
phylogenies including viruses from the three insect orders.
A comparison of nine complete genome sequences and their
study in an evolutionary framework highlighted the genes that
were most suitable for phylogenetic studies (23). Among them,
two genes conserved in all the baculovirus genomes were chosen for the present study to address the phylogenetic relationships within the Baculoviridae. The gene lef-8 encodes a sub-
* Corresponding author. Mailing address: Department of Biological
Sciences, Imperial College London, Silwood Park, Ascot Berkshire
SL5 7PY, United Kingdom. Phone: 44 2075942304. Fax: 44
2075942339. E-mail: e.herniou@imperial.ac.uk.
† Present address: Syngenta, Jealotts Hill International Research
Station, Bracknell, Berkshire RG42 6EY, United Kingdom.
‡ Present address: Laboratory of Virology, Wageningen University,
6909 PD Wageningen, The Netherlands.
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VOL. 78, 2004
INSECT BACULOVIRUS EVOLUTION
3245
TABLE 1. Baculoviruses included in this study
Virus namea
Host order
Abbreviation
Culex nigripalpus NPV
Gilpinia hercyniae NPV239
Gilpinia hercyniae NPVi7
Gilpinia hercyniae NPVk14
Neodiprion lecontei NPV
Neodiprion lecontei NPV726
Neodiprion sertifer NPV345
Neodiprion sertifer NPV411
Neodiprion sertifer NPV413
Neodiprion sertifer NPVk5
Neodiprion sertifer NPV-Virox
Abraxas grossulariata NPV112
Achaea janata GV835
Actias selene NPV47
Antheraea polyphemus NPV30
Autographa californica MNPV
Bombyx mori NPV
Bombyx mori NPV460
Choristoneura fumiferana MNPV
Cydia pomonella GV
Epiphyas postvittana MNPV
Harrisina brillians GVm2
Helicoverpa armigera SNPV
Helicoverpa zea SNPV
Helioconius erato NPV789
Lymantria dispar MNPV
Lymantria monacha NPVb4
Mamestra configurata NPVA
Mamestra configurata NPVB
Natada nararia GV254
Orgyia pseudotsugata MNPV
Orgyia pseudotsugata NPVb5
Phthorimaea operculella GV
Phthorimaea operculella GVc4
Pieris brassicae GV384
Pieris rapae GV95
Plodia interpunctella GVb3
Plutella xylostella GV
Rachiplusia ou MNPV
Spodoptera exigua MNPV
Spodoptera littoralis GV66
Spodoptera litura NPV
Trichoplusia ni NPVc2
Wiseana cervinata NPV344
Xestia c-nigrum GV
Diptera
Hymenoptera
Hymenoptera
Hymenoptera
Hymenoptera
Hymenoptera
Hymenoptera
Hymenoptera
Hymenoptera
Hymenoptera
Hymenoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
Lepidoptera
CuniNPV
GiheNPV239
GiheNPVi7
GiheNPVK14
NeleNPV
NeleNPV726
NeseNPV345
NeseNPV411
NeseNPV413
NeseNPVk5
NeseNPVv
AbgrNPV112
AcjaGV835
AcseNPV47
AnpoNPV30
AcMNPV
BmNPV
BmNPV460
ChMNPV
CpGV
EppoMNPV
HabrGVm2
HaSNPV
HzSNPV
HeerNPV789
LdMNPV
LymoNPVb4
MacoNPVA
MacoNPVB
NanaGV254
OpMNPV
OpNPVb5
PhopGV
PhopGVc4
PbGV384
PiraGV95
PiGVb3
PlxyGV
RoMNPV
SeMNPV
SpliGV66
SpltMNPV
TnNPVc2
WiceNPV344
XecnGV
Collection
source
GenBank
accession no.b
AF4033738
NERC,
J. Cory
J. Cory
B. Arif
NERC,
NERC,
NERC,
NERC,
J. Cory
NERC,
NERC,
NERC,
NERC,
NERC,
CEH
AY449800-779
CEH
CEH
CEH
CEH
CEH
CEH
CEH
CEH
CEH
NERC, CEH
P. Krell
B. Federici
NERC, CEH
J. Cory
NERC, CEH
J. Cory
J. Cory
NERC, CEH
NERC, CEH
J. Cory
NERC, CEH
NERC, CEH
NERC, CEH
AY449791-770
AY449785-765
AY449786
AY449781-761
AY449793-772
AY449789-768
AY449783-763
L22858
L33180
AY449788-767
AF512031
U53466
AY043265
AY449801-780
AF271059
AF334030
AY449792-771
AF081810
AY449796-775
AF467808
AY126275
AY449782-762
U75930
AY449797-776
AF499596
AY449799-778
AY449787-766
AY449794-773
AY449795-774
AF270937
AY145471
AF169823
AY449790-769
AF325155
AY449798-777
AY449784-764
AF162221
Reference
1
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a
Completely sequenced baculoviruses are shown in boldface type.
GenBank accession numbers of the sequences obtained in this study are indicated as lef-8-ac22; only the last three digits of the ac22 accessions are reported, as
they have the same starting code as the lef-8 accession numbers.
b
unit of the baculovirus RNA polymerase, and ac22 encodes a
per os infectivity factor (pif-2) (44). The polh gene was not
considered for this study primarily because CuniNPV does not
harbor a homologue of this gene (1). This suggests that other
divergent baculoviruses might not possess a homologue of polh
to encode their major OB protein. At the time of carrying out
the analysis, 18 sequences, including that of CuniNPV, were
available from the database for lef-8 and ac22. We supplemented this information with 22 novel sequences for these two
genes from lepidopteran and hymenopteran baculoviruses.
This allowed the reconstruction of phylogenetic trees including
baculoviruses isolated from hosts of the arthropod orders Lepidoptera, Hymenoptera, and Diptera to improve our under-
standing of the early evolution of the virus family Baculoviridae.
Traditionally, two competing evolutionary hypotheses have
been put forward to explain the current host distribution of the
baculoviruses (16). The first hypothesis states that baculoviruses could have evolved within one group of arthropods, such
as the Lepidoptera, and switched to other insect groups (48).
The second proposes that the association between baculoviruses and their hosts dates back to the origin of insects or even
arthropods and that they coevolved during evolutionary time
with the viruses colonizing the insect orders as they arose (by
cocladogenesis) (16). We propose to examine these two hypotheses in this study with the reconstruction of a baculovirus
3246
HERNIOU ET AL.
J. VIROL.
TABLE 2. Degenerate oligonucleotide primers used for amplification of a diverse range of baculoviruses
Gene
Sequencea
Oligonucleotide
Amino acid motifb
Size (bp)c
lef-8
L8F2
L8R2
gtaaaacgacggccagtNNNACNRCNGARGAYCC
aacagctatgaccatgMMNCCYTTYTGNCCRTG
XTAEDP
HGQKGV
450
ac22
Ac22F
Ac22R
gtaaaacgacggccagtGGWNNTGYATNSGNGARGAYCC
aacagctatgaccatgRTYNCCRCANTCRCANRMNCC
W(TSN)CI(AP)EDP
G(EVF)C(ED)CG(DN)
400
a
M13 primer sequences are in lowercase type (this part of the primer allows for the direct sequencing of PCR products), and degenerate baculovirus primers are
in uppercase type. R, A or G; Y, T or C; M, A or C; W, A or T; N, A, C, G, or T.
b
Amino acid sequence corresponding to the primer site (single letter code, X ⫽ any).
c
Expected size of the amplification product.
phylogeny including, for the first time, viruses from three distinct insect orders.
Furthermore, this study might shed new lights on the interrelationships between baculoviruses and question the phylogenetic validity of the present classification of the Baculoviridae,
which divides the family into two genera.
MATERIALS AND METHODS
Molecular sequences. The samples examined for this study belonged to the
historical insect virus collection held at the Natural Environment Research
Council (NERC), Centre for Ecology and Hydrology (CEH), Oxford, England.
They include nine isolates of hymenopteran baculoviruses from three sawfly host
species (Gilpinia hercyniae, Neodiprion lecontei, and N. sertifer) and 17 lepidopteran baculoviruses, including 9 NPVs and 8 GVs (Table 1). OBs were dissolved
in 10 mM NaOH (pH 12.5) for 15 min (modified from the method of Moser et
al. [41]). The DNA was then purified with the DNAeasy kit (Qiagen).
Degenerate primers (Table 2) were designed to amplify fragments of the genes
lef-8 and ac22 (pif-2). These primers included universal primer [M13(⫺20) and
M13R] tails to allow for direct sequencing of the PCR products. PCR amplifications were performed with Ready-to-Go PCR beads (Amersham Pharmacia)
under touchdown amplification cycles (95°C for 5 min; 94°C for 30 s, 55 to 43°C
for 20 s, and 72°C for 30 s [3 times], down 3°C after each third cycle, for 15 cycles;
94°C for 30 s, 60°C for 20 s, and 72°C for 45 s [20 times]; and 72°C for 5 min).
Successful amplifications were purified by using the Qiaquick PCR purification
kit (Qiagen). Direct cycle sequencing of the entire PCR fragments was performed in both directions by using M13(⫺20) and M13R universal primers with
the Big Dye terminator reaction mix (Applied Biosystems). The sequences were
run on a 3700 ABI automated sequencer. Chromatograms were checked, and
contiguous sequences were assembled in Sequencher 4.1 (Gene Codes Corporation). BLAST searches (3) were performed on all the new sequences to verify
authenticity before any phylogenetic analyses were undertaken.
Phylogenetic analyses. The DNA sequences obtained from the PCR fragments
were aligned in MacClade 4, based on amino acid coding (36), against sequences
of the same genes from 18 completely sequenced baculoviruses available from
the databases (Table 1). The alignments were trimmed to the size of the PCR
fragments. They have been deposited in TreeBASE (http://www.treebase.org)
under the accession numbers S1005 and M1697.
Maximum-likelihood (ML) analyses were performed in PAUP*, version
4.0b10 (52). Each alignment was analyzed by using a statistical model-fitting
approach implemented in MODELTEST, version 3.06, to choose between substitution models (45, 46). The selected models were used to calculate a tree by
using the neighbor-joining method under ML distances. This tree was then used
to start an ML heuristic search including branch swapping by nearest-neighbor
interchange to find shorter trees.
Bayesian phylogenetic analyses of the combined data set were conducted with
MrBayes, version 3.0b4 (26). Five Markov chains were run for 1 million generations, and the ML parameters were estimated for each gene partition in every
analysis. Trees were sampled every 100th generation; 1,000 trees obtained in the
early phase of the analysis were discarded before computing the consensus of the
remaining 9,001 trees to assess the posterior probability of each node.
The robustness of the tree topologies was also evaluated by bootstrap analysis
under the following conditions: ML heuristic searches with 100 replicates and
maximum-parsimony (MP) methods with 1,000 replicates. For the MP reconstructions, uninformative characters were excluded from the data matrices, the
trees were built by stepwise addition, and tree bisection reconnection branch
swapping was performed to find the best MP tree at each replication step.
Differences in tree topologies were assessed by using one-tailed Kishino-Hasegawa (KH) and Shimodaira-Hasegawa (SH) tests implemented for ML tree
scores in PAUP* (52).
RESULTS
Phylogenetic analyses. Phylogenetic analyses were performed to elucidate the relationships of the hymenopteran and
dipteran baculoviruses within the Baculoviridae. Two genes
were used in these analyses, ac22 (pif-2) and lef-8. In total,
sequences from 39 virus isolates were used (Table 1). Only
three new sawfly sequences were used in the phylogenetic
analyses, as sequences obtained from additional samples were
very similar (345 and 413) or identical (i7 and K14) to each
other between samples from the same host species. The lef-8
PCR fragments produced an alignment of 513 nucleotides,
which resulted in 74.8% parsimony informative and 18.5%
constant sites. For ac22, the 357-nucleotide-long alignment
resulted in 70% informative and 19% constant sites. A partition homogeneity test was performed in PAUP*. The resulting
P value (P ⫽ 0.27) showed that both data sets were congruent
and could be combined into one data set. Furthermore, assessment of the tree topologies obtain for both genes, with KH and
SH tests, showed that they were not significantly incongruent
(data not shown).
For the Lef-8 & Ac22 data set, the best-fit model of evolution selected by MODELTEST (45) was characterized by 9.4%
of invariable sites (I) and a gamma shape parameter (G ⫽
1.317%), which reflects the heterogeneity of variation rates
across sites, and the substitution model had variable transition
rates (A⬍⬎G ⫽ 2.08; T⬍⬎C ⫽ 2.82). The tree obtained from
the combined alignment (Fig. 1, T1) showed the lepidopteran
NPVs divided into two groups and the sawfly viruses with
CuniNPV clearly separated from the GVs. However, it also
showed that the GVs might be paraphyletic (i.e., split within
the tree). To test whether this was strongly supported by the
data, ML heuristic searches were performed again to find the
most likely tree (T2) under the constraint that the GVs should
be monophyletic (enforcing that the GVs should all derive
from a single common ancestor). To measure the significance
of the differences between T1 and T2, KH and SH tests were
performed. The constrained tree (T2) had a likelihood value
(⫺lnL ⫽ 19,508.2) only marginally lower than that of the best
tree (T1) (⫺lnL ⫽ 19,507.9; delta ⫽ 0.32). The KH and SH
tests, used to assess the differences between the topologies of
T1 and T2, showed that they were not significantly different
VOL. 78, 2004
INSECT BACULOVIRUS EVOLUTION
3247
DISCUSSION
FIG. 1. ML trees obtained from the Lef-8 & Ac22 data set. T1,
unconstrained tree; T2, GV monophyletic constraint tree. The trees
were found by a heuristic search starting with neighbor joining and
nearest-neighbor interchange branch swapping. Virus abbreviations
are shown in Table 1.
(KH: P ⫽ 0.416; SH: P ⫽ 0.975; P value showing a significant
difference with best tree, ⬍0.05). This indicates that T2 represents a hypothesis for the evolution of these viruses that is
equivalent to that presented by T1. Therefore, in view of the
present classification and phylogeny of the baculoviruses, we
believe that T2 represents a satisfactory hypothesis for the
phylogeny of the baculoviruses. Bayesian phylogenetic analysis
further confirmed this evolutionary hypothesis, as the majority
rule consensus tree derived from this analysis showed the
monophyly of the GVs. The topology of the Bayesian consensus tree was also found to be not significantly different from T1
or T2 by KH and SH tests (data not shown).
Robustness. The robustness of the phylogenies was evaluated by bootstrap analysis and analysis of Bayesian posterior
probability. The backbone of the T2 tree is fairly well supported (Fig. 2). It shows that the lepidopteran NPVs are well
separated from other baculoviruses, with separate group I
NPVs being strongly supported; although T2 shows a monophyletic group II, there is little support for this group from any
analysis. The separation of the nonlepidopteran baculoviruses
at the base of the tree is well supported by high Bayesian
posterior probability. The GVs as a whole are supported by
62% of the trees obtained in the Bayesian analysis. Within the
GVs, a group including Natada nararia GV, Harrisina brillians
GV, Pieris brassicae GV, Pieris rapae GV, Cydia pomonella GV,
Phthorimaea operculella GVs, and Plodia interpunctella GV
seems to detach within the group (Fig. 2). Terminal nodes
leading to closely related viruses generally have high bootstrap
and probability values with all methodologies of phylogenetic
reconstruction. This includes the hymenopteran viruses, which
strongly cluster together to the exclusion of other viruses.
Within this group, the relationships are also well defined (Fig.
2).
Should the Baculoviridae comprise more genera? The family
Baculoviridae is currently split into two genera, NPV and GV
(6). So far, viruses from nonlepidopteran hosts have been classified within the NPV genus because their morphology and
cytopathology fulfilled the criteria of this genus. However there
is no evidence of the monophyly of this genus. Prior to this
study, it was clear that the mosquito baculovirus CuniNPV is a
distant relative to the lepidopteran baculoviruses and could
represent a new genus (24, 41). There was also some indication
that the sawfly virus NeseNPV is distantly related to the lepidopteran baculoviruses (60).
The phylogenetic analyses described here include baculoviruses from hosts of the arthropod orders Lepidoptera, Diptera,
and Hymenoptera. They indicate that there are at least three,
and possibly four, distinct groups of baculoviruses (Fig. 3A).
The lepidopteran NPVs clearly form a discrete group, which is
distinct from the rest of the baculoviruses. The branch leading
to this group is quite long (l ⫽ 0.26) (Fig. 3A) and well supported by high bootstrap values and Bayesian posterior probabilities. The GVs, however, appear to be more genetically
diverse than the NPVs, and the branch leading to the group is
comparatively short (l ⫽ 0.03) and poorly supported (Fig. 2
and 3A). This suggests that the GVs are a much older group
than the lepidopteran NPVs, that the sampling of the GVs was
wider, or that both groups speciate at different speeds. Furthermore, the phylogeny shows that neither the dipteran virus
nor the hymenopteran viruses belong to either the GVs or the
lepidopteran NPVs. The branch separating the lepidopteran
from the nonlepidopteran baculoviruses is long (l ⫽ 0.36) (Fig.
3A) and well supported. The branch lengths between the mosquito virus and the sawfly viruses are also large (l ⫽ 0.88 and
0.54) (Fig. 3A). This suggests that hymenopteran and dipteran
baculoviruses probably belong to distinct and separate groups.
The members of the Baculoviridae appear to be clearly divided
according to the classification of their hosts.
In the past, baculoviruses (NPVs) have been reported from
a wide variety of nonlepidopteran insects including three families of Coleoptera, six families of Diptera, four families of
Hymenoptera, two families of Neuroptera, and one family of
Trichoptera (37). The taxonomic status of most of these viruses
remains uncertain. Most of them are rare, poorly characterized, correspond to isolates identified by light microscopy only,
and have been removed from the International Committee on
Taxonomy of Viruses (ICTV) baculovirus list for lack of molecular data.
Unsuccessful attempts were made to obtain sequences from
virus isolates from lacewings (Neuroptera, Chrysopa PV-330,
and Hemerobius NPV-318 and -440; NERC, CEH) and craneflies (Diptera, Tipula oleracea NPV-35 and ⫺281; NERC,
CEH) that had been classified as NPVs (22). These viruses
might be extremely divergent baculoviruses beyond the range
of our degenerate primers, but these results may also indicate
that they belong to other virus families yet to be identified. No
samples of crustacean baculoviruses were available for this
study. A baculovirus of the shrimp Peneaus monodon is still
included in the ICTV tentative baculovirus list (6). A recent
morphological description of Monodon baculovirus might correspond to this virus (47). However, the lack of molecular
3248
HERNIOU ET AL.
J. VIROL.
FIG. 2. Robustness of the Lef-8 & Ac22 ML tree (T2). Numbers in roman type (ML/MP ratio of ⬎50) indicate bootstrap scores obtained by
the ML method with 100 replicates. The second number, when present, indicates the score obtained by the MP method with 1,000 replicates.
Numbers in bold italic type indicate the Bayesian posterior probabilities of the nodes.
sequences for this virus still casts doubt on its affiliation with
the Baculoviridae, especially as another shrimp virus, the white
spot syndrome virus, formerly classified as a baculovirus, is in
the process of being classified to its own virus family, Nimaviridae, since sequences have become available (55, 56). Thus,
it was not possible to confirm the presence of baculoviruses in
the Crustacea, nor in insect orders other than Lepidoptera,
Diptera, and Hymenoptera.
From the unrooted tree (Fig. 3A), it is possible to envisage
a scenario where the GVs could have given rise to all the NPVs
(lepidopteran and nonlepidopteran). This would reconcile the
phylogeny with the present classification of the baculoviruses
into two genera. However, evidence from DNA polymerase
phylogenies (41) and comparative genomics studies including
GVs, NPVs, and CuniNPV genomes (24) showed that the GVs
and lepidopteran NPVs are more closely related to each other
than they are to the mosquito virus. This therefore reinforces
the idea that in a phylogenetic context the NPV genus might
not include viruses from a nonlepidopteran background.
Taxonomic proposals. If the ICTV was to consider using
phylogenetic concepts for the classification of baculoviruses,
this would require the genera to be monophyletic. This study
shows that under the present ICTV classification, the NPV
genus is polyphyletic. So from a phylogenetic perspective,
CuniNPV and the sawfly NPVs should be removed from the
NPV genus and classified under the unclassified baculovirus
VOL. 78, 2004
FIG. 3. Evolution of the Baculoviridae. (A) Phylogeny of the baculoviruses, highlighting four main groups of the unrooted tree (T2);
numbers indicate branch lengths in substitutions per site. (B) Relationships of the three arthropod orders infected by the baculoviruses
shown in panel A (57).
section. Our evidence would also support a taxonomic proposal to create one or two new genera of baculoviruses. The
number of new genera would depend on further evidence to
cluster together or keep apart the dipteran and hymenopteran
baculoviruses.
BLAST search results showed that NeseNPV345,
NeseNPV413, and NeseNPV726 are isolates of the species N.
sertifer NPV (taxonomic code 00.006.0.01.017.). The other virus
isolated from N. lecontei should still be classified as a separate
species, N. lecontei NPV (taxonomic code 00.006.0.01.323.).
Comparisons of phylogenetic distances and distinct host ranges
(15) suggest that NPVs of G. hercyniae are part of a third
distinct species.
Evolution of the Baculoviridae. Among baculovirologists, two
views are commonly held for the evolutionary origins of the
Baculoviridae (16). The first hypothesis proposes that the baculoviruses originated within the Lepidoptera, with subsequent
horizontal transmissions to other insect orders from lepidopteran virus clades (48). The second postulates that the origin of
baculoviruses dates back to the origin of arthropods, with the
cocladogenesis of the viruses and their hosts (16).
If the first hypothesis was true, a phylogeny including baculoviruses from different orders of hosts would not show any
clear clustering of baculoviruses according to host order. It
would show nonlepidopteran viruses, either in clusters or sin-
INSECT BACULOVIRUS EVOLUTION
3249
glets, arising within the NPVs or GVs, making lepidopteran
NPVs or GVs paraphyletic. The phylogeny of baculoviruses
including hosts from three different insect orders (Fig. 3A)
seems to reject this hypothesis, as viruses strongly cluster according to the order of insects from which they have been
isolated. We believe that our lepidopteran virus sampling is
diverse enough to address this question. However, this does
not exclude the discovery of a nonlepidopteran baculovirus
belonging to the lepidopteran NPVs or GVs.
The second hypothesis would lead to a phylogeny where the
relationships between groups of baculoviruses mirror the evolutionary relationships of insect orders, with the ages of the
different baculovirus lineages reflecting those of their hosts.
The orders Diptera and Lepidoptera are more closely related
to each other than to the Hymenoptera (Fig. 3B) (57). The
phylogeny obtained in this study could be consistent with the
phylogenetic host tracking of insect orders by baculoviruses, as
it is possible to root the tree on the hymenopteran baculovirus
branch (Fig. 3A). However, further evidence needs to be gathered before accepting the hypothesis, particularly the comparison of evolutionary rates between baculoviruses and their
hosts. From our data set, it is not possible to infer directly a
reliable rate of sequence evolution for the baculoviruses, particularly because the genes used in this study do not have any
homologues outside of the Baculoviridae.
A third scenario can be suggested for the origin of the
baculoviruses. We propose that ancestral baculoviruses were
probably able to horizontally infect hosts of different orders,
with ancient coevolution between the hosts and pathogens
then leading to the progressive specialization of different baculovirus lineages to hosts of different orders. According to this
hypothesis, a phylogeny of the baculoviruses would show a
clear separation of the viruses infecting different kinds of hosts
without necessarily reflecting the evolution of insect orders.
The phylogeny obtained in this study supports this hypothesis
(Fig. 3A).
The uncertainty of the position of the root in the baculovirus
phylogeny does not allow us to completely discard the second
hypothesis in favor of the third. Some elements of baculovirus
biology suggest that the dipteran baculovirus might belong to
the more ancestral lineage, thus favoring the third scenario.
The complete genome sequence of the mosquito virus
CuniNPV showed that this virus does not possess a polyhedrin
gene and that another protein is the major constituent of the
OBs (1, 41), whereas polyhedrin sequences have been obtained
from sawfly viruses (50, 60). Furthermore, lepidopteran and
hymenopteran sawfly larvae share similar feeding ecologies
and are often found in the same environment in the wild, i.e.,
terrestrial plants. They would thus be exposed to each others’
viruses, whereas mosquito larvae are aquatic. This suggests
that the lepidopteran and hymenopteran viruses could be more
closely related to each other than to the mosquito virus. However, the dipteran virus lineage might have undergone a nonorthologous gene displacement for its OB protein.
In terms of pathogenesis and tissue tropism, mosquito and
sawfly viruses are more similar to each other than to the lepidopteran GVs or NPVs. These viruses only infect midgut
epithelial cells (15, 16, 41), whereas GVs and lepidopteran
NPVs generally cause systemic infections, often infecting a
wide range of tissues. The restriction of infection to midgut
3250
HERNIOU ET AL.
epithelial cells has been proposed as an ancestral characteristic
of baculoviruses (16). Only one lepidopteran baculovirus has
been found to have a similar pathology, H. brillians GV (17).
However, phylogenies including this virus showed that this
virus is not basal to the GV group (Fig. 2) and, therefore, that
the restriction of infection to the midgut epithelial cells is not
an ancestral trait in lepidopteran baculoviruses (5). Thus, it is
not possible to conclude whether the mosquito and sawfly
viruses are more primitive or more derived than the lepidopteran baculoviruses based on the cell specificity of their infections. Although they were based on smaller taxon sets, previous phylogenetic studies suggested that they were more
ancestral than the lepidopteran NPVs or GVs (41, 50, 60).
The relationships of the deeper branches of the baculovirus
phylogenies might benefit in the near future from comparative
genomic analyses. If sawfly virus genomes were found to be
more similar to the lepidopteran baculoviruses, then the mosquito virus could remain at the base of the tree. If they share
more genomic features with CuniNPV, then the hymenopteran
and dipteran viruses could be grouped together to the exclusion of the lepidopteran baculoviruses. However, if CuniNPV
is more similar to the lepidopteran baculoviruses, then the
hymenopteran baculoviruses could be the more ancestral lineage. This last option would favor the second theory of early
cospeciation between the Baculoviridae and the Arthropoda, as
the baculovirus phylogeny would then reflect that of the order
of their hosts, although this would need to be correlated with
a comparative study of evolutionary rates between hosts and
pathogens.
Regardless of the position of the root of the baculovirus
tree, the phylogenetic separation of the viruses into groups
according to the hosts’ classification indicates that baculoviruses have been specialist pathogens of insects since the diversification of the family Baculoviridae. The selection pressure
exerted on baculoviruses by their different hosts has promoted
their specialization to the point where specific baculovirus lineages are specific to particular kinds of host insects, such as
larvae of Lepidoptera, Hymenoptera, and Diptera. These coevolutionary adaptations have constrained the range of possible hosts available to each virus lineage. Over time, this has
translated into the phylogenetic pattern that we observe today,
where the Baculoviridae from different insect orders belong to
different evolutionary lineages.
ACKNOWLEDGMENTS
We thank Basil Arif, Just Vlak, and Andy Purvis for discussion and
comments on the manuscript and Hilary Lauzon and Peter Krell for
sharing the sequences of N. lecontei NPV and Choristoneura fumiferana
MNPV.
The Natural Environment Research Council CASE studentship
award GT04/99/TS/142 supported E.A.H.
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