|
Type Species |
(CPV-1) |
Cypovirus virions have only a single capsid shell with surface spikes. Virions have transcriptase and capping enzymes that are active without particle modification. They can retain RNA polymerase activity despite particle disruption into ten distinct RNA protein complexes and consequently transcriptase activity in vitro is entirely resistant to repeated freeze-thawing. These complexes each represent a single genome segment and a transcriptase complex. The transcriptase activity may show very pronounced dependence on the presence of S-adenosyl-L-methionine or related compounds. Virus particles may be multiply or singly occluded by a virus coded polyhedrin protein to form “polyhedra” within the cytoplasm of infected cells. Cypoviruses only infect and are pathogenic for arthropods of particular species or a particular range of species.
Virus particles have a singled shelled capsid (55-69 nm in diameter) with icosahedral symmetry and hollow surface spikes at the vertices (about 20 nm in length and 15-23 nm wide) and a central compartment about 35 nm in diameter. Cypovirus virions are structurally equivalent to the core particles of other genera within the family Reoviridae, particularly those genera containing viruses with ‘spiked’ cores (Orthoreovirus, Aquareovirus, and Oryzavirus) (Figs. 14 and 15). Virus particles may also be occluded by a crystalline matrix of polyhedrin protein forming a polyhedral inclusion body (Fig. 16). These polyhedra have a symmetry (e.g., cubic, icosahedral, or irregular) which is dependent on both the virus strain (polyhedrin sequence) and the host. The polyhedrin protein appears to be arranged as a face-centred cubic lattice with centre to centre spacing varying between 4.1 and 7.4 nm.
Physicochemical and Physical Properties
The virion Mr is about 5.4 
107. The buoyant density in CsCl is 1.44 g/cm3 (virions), approximately 1.30 g/cm3 for empty particles, and 1.28 g/cm3 for polyhedra. The S20W is approximately 420S for virions and 260S for empty particles. Polyhedra vary considerably in size and Mr and do not have a single characteristic S value. Polyhedra may occlude many virus particles or only single particles. Large empty polyhedra (apparently containing no virions) have also been observed.
Cypoviruses retain infectivity for several weeks at -15°C, 5°C, or 25°C. The virus retains full enzymatic activity (dsRNA dependent ssRNA polymerase and capping activity) after repeated freeze-thawing (up to sixty cycles). However, it appears likely that this results in the breakdown of the virus particle into ten active and distinct enzyme/template complexes. Each complex contains one genome segment and a complete transcriptase complex, derived from the virion capsid and including one of the “spike” structures from the vertices of the icosahedron. Within the family Reoviridae, the ability to retain enzyme function despite particle breakdown may be unique to the cypoviruses. Cations have relatively little effect on the virus structure. Heat treatment of virions at 60°C for 1 hr leads to degradation and release of genomic RNA. Virus particles are relatively resistant to treatment with trypsin, chymotrypsin, ribonuclease A, deoxyribonuclease, or phospholipase. Virion enzyme functions also show some resistance to treatment with proteinase K. However, this may reflect the retention of enzyme activities despite particle disruption, particularly during the early stages of digestion.
Cypovirus particles are resistant to detergents such as sodium deoxycholate (0.5-1%) but are disrupted by 0.5-1% SDS, which releases the genomic dsRNA. One or two fluorocarbon treatments have little effect on virus infectivity, however treatment with ethanol leads to release of RNA from virions. Viruses and polyhedra are readily inactivated by UV-irradiation. It has been reported that UV also releases the dsRNA template from individual genome segment/transcriptase complexes. Polyhedra remain infectious for years at temperatures below 20°C. Virions can be released from polyhedra by treatment with carbonate buffer at pH greater than 10.5. As in permissive insects' mid guts, this pH treatment completely dissolves the polyhedral protein matrix. This process is partly due to increased solubility of polyhedrin at high pH but is also aided by alkaline activated proteases associated with polyhedra.
Polyhedra (but not virions) contain significant amounts of adenylate-rich oligonucleotides. Cypovirus particles contain 10 dsRNA genome segments, with estimated Mr that varies from 0.42 to 3.7 106 and a total genome Mr which varies from 19.3 to 22.0 106 (estimated by electrophoretic analyses). Currently only the sequence of genome segment 8, 9 and 10 of CPV-1 and segment 10 of CPV-5 and Choristoneura fumiferana cypovirus (CfCPV) have been fully analyzed. These genome segments are 1,328 bp, 1,186 bp, 942 bp, 883 bp and 1,171 bp in length (respectively). These sizes are considerably larger than those previously calculated from electrophoretic analyses, confirming suggestions that many of the Mr values previously published for cypovirus genome segments may be under estimated (Table 12). However, because primary sequence of dsRNA can significantly affect migration during PAGE, fully accurate Mr determinations will require complete sequence analysis of each of the viral genome segments.
The pattern of size distribution of the genome segments varies widely between different cypoviruses (e.g., the smallest dsRNA has an estimated Mr which varies between 0.42 and 0.79 106). These size differences have formed a basis for the recognition and classification of 14 distinct species (electropherotypes) of cypoviruses (14 distinct patterns of dsRNA migration, which differ significantly in the migration of at least three genome segments, as analyzed by electrophoresis using 1% agarose or 3% SDS-PAGE). The genome segment migration patterns of types 1, 12 and 14 have some overall similarity, although in each case at least 3 segments show significant migrational differences during agarose gel electrophoresis.
The termini of the coding strands are common for the different genome segments of Cypovirus 1 members, but differ from those reported for Cypovirus 5 members (Table 11). CfCPV-7, which shows high levels of overall sequence variation when compared to either Cypovirus 1 or 5 viruses (Fig. 17) and is therefore likely to be a different species, is reported to have a similar 5
- but different 3-ends to Cypovirus 5. These data indicate that different cypovirus species (electropherotypes) have different conserved RNA terminal sequences.
Cypovirus particles generally contain five distinct proteins, 2-3 with Mr of more than 100 103. For BmCPV-1 the structural proteins are Mr = 146, 138, 125, 70 and 31 103. Polyhedra also contain a 25-37 103 polyhedrin protein (Mr = 28.5 103 for BmCPV-1) that constitutes about 95% of the polyhedra protein dry weight. Due to the high level of variation between different cypoviruses it is unlikely that their homologous proteins can be identified simply by their migration order during PAGE.
The polyhedrin protein is glycosylated.
Cypoviruses are not known to contain any lipids in either virus particle or polyhedra.
Genome Organization and Replication
For BmCPV-1 the coding assignments are indicated in Table 13. The origin of the Mr 31 103 structural protein is not known; it may represent a product of post translational processing. The cognate genes of other cypoviruses are not known. The large variations in the sizes of genome segments between most cypoviruses (apart from CPV-1, 12 and 14) indicate that these assignments will not apply to other species. Genome segment coding assignments have been published for CPV-1 and 2. The data generated and sequencing studies indicate that in many cases polyhedrin may be encoded by the smallest segment.
Unlike orthoreoviruses, cell entry and initiation of cypovirus replication in insect cells does not require modification of the virions for activation of the core-associated enzymes. Uptake appears to be a relatively inefficient process in cell cultures, which can be very significantly improved by the use of liposomes. Virus replication and assembly occur in the host cell cytoplasm, although there is some evidence that viral RNA synthesis can occur within the nucleus. Replication is accompanied by the formation of viroplasm (or virogenic stroma) within the cytoplasm. Viroplasms contain large amounts of virus proteins and virus particles. How genome segments are selected for packaging and assembly into progeny particles is not known. The importance of the terminal regions in this process is indicated by the packaging and transcription of a mutant segment 10 of a CPV-1 that contained only 121 bp from the 5-end and 200 bp from the 3-end. Particles are occluded within polyhedra apparently at the periphery of the virogenic stroma, from about 15 hr post-infection. The polyhedrin protein is produced late in infection and in large excess compared to the other viral proteins. How polyhedrin synthesis is regulated is not known.
Many virus particles remain non-occluded. Lysis of the infected cells appears to be limited in cell cultures preventing the release of both virions and polyhedra. However, spread of infection may occur by cell contact. Lysis of infected gut cells which are sloughed off, may occur in infected host insects, allowing non occluded particles to spread the infection between cells within an individual host. Polyhedra serve to spread viruses between hosts.
Serological cross-comparisons of cypovirus structural and polyhedrin proteins support the use of genomic dsRNA electropherotype as one of the species parameters for the genus Cypovirus. Virus isolates within a single electropherotype exhibit high levels of antigenic cross-reaction (in both polyhedrin and virion structural proteins), as well as efficient cross-hybridization of denatured genomic RNA, even under high stringency conditions. In contrast there is evidence of little or no serological cross-reaction between viruses representing different electropherotypes. Exceptions are Cypovirus 1 and 12, which show low level serological cross-reactions but these viruses also have some overall similarity in their electropherotype pattern and show a low level of cross-hybridization of their genome segments. Cypovirus 14 also shows some similarity in its RNA electropherotype pattern to both Cypovirus 1 and 12. It may therefore also show some antigenic relationship and RNA sequence homology with these viruses.
Cypoviruses have only been isolated from arthropods. Attempts to infect vertebrates, or vertebrate cell lines, have failed. In addition, cypovirus replication is inhibited at 35°C. Even susceptible insect larvae treated with the virus fail to develop infections at 35°C. Cypoviruses are normally transmitted by ingestion of polyhedra on contaminated food materials. The polyhedra dissolve within the high pH environment of the insect gut releasing the occluded virus particles, which then infect the cells lining the gut wall. Virus infection in larvae is generally restricted to the columnar epithelial cells of the midgut, although goblet cells may also become infected. Cypovirus replication in the fat body has been reported. In larvae, the virus infection spreads throughout the midgut region. In some species the entire gut is occasionally infected. The production of very large numbers of polyhedra give the gut a characteristically creamy-white appearance. In infected cells the endoplasmic reticulum is progressively degraded, mitochondria enlarge and the cytoplasm becomes highly vacuolated. In most cases the nucleus shows few pathological changes. An exception is a cypovirus strain which produces inclusion bodies within the nucleus. In the later stages of infection cellular hypertrophy is common and microvillae are reduced or completely absent. Very large numbers of polyhedra are released by cell lysis into the gut lumen and excreted. The gut pH is lowered during infection and this prevents dissolution of progeny polyhedra in the gut fluid.
The majority of cypovirus infections produce chronic disease, often without extensive larval mortality. Consequently, many individuals reach the adult stage even though heavily diseased. However, cypovirus infections produce symptoms of starvation due to changes in the gut cell structure and reduced adsorptive capacity. Infected larvae stop feeding as early as two days post-infection. Larval body size and weight are often reduced and diarrhea is common. The larval stage of the host can be significantly increased (about by 1.5 times the normal generation time). The size of infected pupae is frequently reduced and the majority of diseased adults are malformed. They may not emerge correctly, and may be flightless. Infected females may exhibit a reduced egg laying capacity.
Virus can be transmitted on the surface of eggs, producing high levels of infection in the subsequent generation. However, no transovarial transmission has been observed provided the egg surface is disinfected. The infectious dose increases dramatically in the later larval instars. Different virus strains vary significantly in virulence. Larvae can recover from cypovirus infection, possibly because the gut epithelium has considerable regenerative capacity and because infected cells are shed at each larval moult.
List of Species Demarcation Criteria in the Genus
Cypoviruses are currently classified within 14 species that were initially characterized by their distinctive dsRNA electropherotype patterns. Cross-hybridization analyses of the dsRNA, limited comparative RNA sequence data (currently available for genome segment 10 only) and serological comparisons of cypovirus proteins have confirmed the validity of this classification. However, only a few cypoviruses have been analyzed using these methods.
The system of nomenclature currently used to identify different cypovirus isolates takes account of both the dsRNA electropherotype and the host species from which the virus was originally isolated (e.g., Bombyx mori cypovirus 1, BmCPV-1). The relationships, between different cypoviruses within a single electropherotype, or with other cypovirus types, are not fully understood at the molecular level. However, sequence analyses of genome segment 10 from three geographically distinct isolates of CPV-5 and comparisons of segment 10 from isolates of CPV-1, have shown very high levels of homology (>98%) within a single species. In contrast, comparisons of unrelated types (BmCPV-1 and CfCPV) showed only low levels of sequence homology in genome segment 10 (20-23%). Cross-hybridization studies of the genomic RNA from different cypoviruses isolates (including different isolates of CPV-1 or 5) have confirmed that the RNA sequence is highly conserved within each species. These studies also demonstrated that although there may be slightly higher conservation in the largest genome segments, the level of variation is relatively uniform across the whole genome.
Within the family Reoviridae, the prime determinant for inclusion of virus isolates within a single virus species is their ability to exchange genetic information by reassortment of their genome segments during co-infection, thereby generating viable progeny virus strains. There is no direct evidence concerning genome segment reassortment between different cypovirus isolates. However, evidence of similarity and therefore of the genetic compatability required for reassortment can be provided by other methods.
Members of a single Cypovirus species may be identified by:
1. |
Their ability to exchange genetic material by genome segment reassortment during dual infections, thereby producing viable progeny virus strains. |
2. |
Similar electrophoretic migration of at least 7 genome segments, as analysed using either an agarose, or a low percentage (3%) polyacrylamide gel system. Viruses of different species will have significant migrational differences in at least three genome segments. |
3. |
High levels of serological cross-reaction by ELISA or AGID (e.g., using polyclonal antisera to purified virions or polyhedrin proteins). Different but more closely related species (types) may show low levels of serological cross reaction (e.g., CPV-1 and 12). |
4. |
A high degree of sequence conservation (estimated >80%). |
5. |
Cross-hybridization of genome segments under high stringency conditions (designed to detect >90% homology). (Northern or dot blots, with probes made from viral RNA or cDNA.) |
6. |
Current limited evidence suggests that the conserved terminal sequences are likely to be the same within an electropherotype (species) but may be different between different species. The similarity between more closely related species (e.g., CPV-1, 12 and 14) is unknown. |
Below is provided a list of some of the lepidopteran cypoviruses for which the RNA electropherotypes have been deduced. In addition to many other lepidopteran cypoviruses that have been described (but are otherwise uncharacterized), there are dipteran and hymenopteran cypoviruses. One isolate from a freshwater daphnid has been reported. In total, more than 230 cypoviruses have been described, however the total number of species is unknown.
Official virus species names are in italics. Tentative virus species names, alternative names ( ), strains or serotypes are not italicized. Virus names, genome sequence accession numbers [ ], and assigned abbreviations ( ) are:
|
Cypovirus 1 |
(CPV-1) | |
|
Bombyx mori cypovirus 1 |
Seg 8: [AB016436], Seg 9: [AF061199] |
(BmCPV-1) |
|
Dendrolimus spectabilis cypovirus 1 |
Seg 10: [D37768] |
(DsCPV-1) |
|
Lymantria dispar cypovirus 1 |
(LdCPV-1) | |
|
Cypovirus 2 |
(CPV-2) | |
|
Aglais urticae cypovirus 2 |
(AuCPV-2) | |
|
Agraulis vanillae cypovirus 2 |
(AvaCPV-2) | |
|
Arctia caja cypovirus 2 |
(AcCPV-2) | |
|
Arctia villica cypovirus 2 |
(AviCPV-2) | |
|
Boloria dia cypovirus 2 |
(BdCPV-2) | |
|
Dasychira pudibunda cypovirus 2 |
(DpCPV-2) | |
|
Eriogaster lanestris cypovirus 2 |
(ElCPV-2) | |
|
Hyloicus pinastri cypovirus 2 |
(HpCPV-2) | |
|
Inachis io cypovirus 2 |
(IiCPV-2) | |
|
Lacanobia oleracea cypovirus 2 |
(LoCPV-2) | |
|
Malacosoma neustria cypovirus 2 |
(MnCPV-2) | |
|
Mamestra brassicae cypovirus 2 |
(MbCPV-2) | |
|
Operophtera brumata cypovirus 2 |
(ObCPV-2) | |
|
Papilio machaon cypovirus 2 |
(PmCPV-2) | |
|
Phalera bucephala cypovirus 2 |
(PbCPV-2) | |
|
Pieris rapae cypovirus 2 |
(PrCPV-2) | |
|
Cypovirus 3 |
(CPV-3) | |
|
Anaitis plagiata cypovirus 3 |
(ApCPV-3) | |
|
Arctia caja cypovirus 3 |
(AcCPV-3) | |
|
Danaus plexippus cypovirus 3 |
(DpCPV-3) | |
|
Gonometa rufibrunnea cypovirus 3 |
(GrCPV-3) | |
|
Malacosoma neustria cypovirus 3 |
(MnCPV-3) | |
|
Operophtera brumata cypovirus 3 |
(ObCPV-3) | |
|
Phlogophera meticulosa cypovirus 3 |
(PmCPV-3) | |
|
Pieris rapae cypovirus 3 |
(PrCPV-3) | |
|
Spodoptera exempta cypovirus 3 |
(SexmCPV-3) | |
|
Cypovirus 4 |
(CPV-4) | |
|
Actias selene cypovirus 4 |
(AsCPV-4) | |
|
Antheraea mylitta cypovirus 4 |
(AmCPV-4) | |
|
Antheraea pernyi cypovirus 4 |
(ApCPV-4) | |
|
Cypovirus 5 |
(CPV-5) | |
|
Euxoa scandens cypovirus 5 |
Seg 10: [J04338] |
(EsCPV-5) |
|
Heliothis armigera cypovirus 5 |
(HaCPV-5) | |
|
Orgyia pseudosugata cypovirus 5 |
(OpCPV-5) | |
|
Spodoptera exempta cypovirus 5 |
(SexmCPV-5) | |
|
Tichoplusia ni cypovirus 5 |
(TnCPV-5) | |
|
Cypovirus 6 |
(CPV-6) | |
|
Aglais urticae cypovirus 6 |
(AuCPV-6) | |
|
Agrochola helvolva cypovirus 6 |
(AhCPV-6) | |
|
Agrochola lychnidis cypovirus 6 |
(AlCPV-6) | |
|
Anaitis plagiata cypovirus 6 |
(ApCPV-6) | |
|
Anti xanthomista cypovirus 6 |
(AxCPV-6) | |
|
Biston betularia cypovirus 6 |
(BbCPV-6) | |
|
Eriogaster lanestris cypovirus 6 |
(E1CPV-6) | |
|
Lasiocampa quercus cypovirus 6 |
(LqCPV-6) | |
|
Cypovirus 7 |
(CPV-7) | |
|
Mamestra brassicae cypovirus 7 |
(MbCPV-7) | |
|
Noctua pronuba cypovirus 7 |
(NpCPV-7) | |
|
Cypovirus 8 |
(CPV-8) | |
|
Abraxas grossulariata cypovirus 8 |
(AgCPV-8) | |
|
Heliothis armigera cypovirus 8 |
(HaCPV-8) | |
|
Malacosoma disstria cypovirus 8 |
(MdCPV-8) | |
|
Nudaurelia cytherea cypovirus 8 |
(NcCPV-8) | |
|
Phlogophora meticulosa cypovirus 8 |
(PmCPV-8) | |
|
Spodoptera exempta cypovirus 8 |
(SexmCPV-8) | |
|
Cypovirus 9 |
(CPV-9) | |
|
Agrotis segetum cypovirus 9 |
(AsCPV-9) | |
|
Cypovirus 10 |
(CPV-10) | |
|
Aporophyla lutulenta cypovirus 10 |
(AlCPV-10) | |
|
Cypovirus 11 |
(CPV-11) | |
|
Heliothis armigera cypovirus 11 |
(HaCPV-11) | |
|
Heliothis zea cypovirus 11 |
(HzCPV-11) | |
|
Lymantria dispar cypovirus 11 |
(LdCPV-11) | |
|
Mamestra brassicae cypovirus 11 |
(MbCPV-11) | |
|
Pectinophora gossypiella cypovirus 11 |
(PgCPV-11) | |
|
Pseudaletia unipuncta cypovirus 11 |
(PuCPV-11) | |
|
Spodoptera exempta cypovirus 11 |
(SexmCPV-11) | |
|
Spodoptera exigua cypovirus 11 |
(SexgCPV-11) | |
|
Cypovirus 12 |
(CPV-12) | |
|
Autographa gamma cypovirus 12 |
(AgCPV-12) | |
|
Mamestra brassicae cypovirus 12 |
(MbCPV-12) | |
|
Pieris rapae cypovirus 12 |
(PrCPV-12) | |
|
Spodoptera exempta cypovirus 12 |
(SexmCPV-12) | |
|
Cypovirus 13 |
(CPV-13) | |
|
Polistes hebraeus cypovirus 13 |
(PhCPV-13) | |
|
Cypovirus 14 |
(CPV-14) | |
|
Heliothis armigera cypovirus 14 (‘A’ strain) |
(HaCPV-14) |
Tentative Species in the Genus
|
Heliothis armigera cypovirus (‘B’ strain) |
(HaCPV-B) | |
|
Choristoneura fumiferana cypovirus |
Seg 10: [U95954] |
(CfCPV) |
Phylogenetic Relationships within the Genus
See Fig. 17.
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