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An isometric virus of the potato tuber moth Tecia solanivora (Povolny) (Lepidoptera: Gelechiidae) has a tri-segmented RNA genome
Journal of Invertebrate Pathology 99 (2008) 204–211
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An isometric virus of the potato tuber moth Tecia solanivora (Povolny) (Lepidoptera: Gelechiidae) has a tri-segmented RNA genome Jean-Louis Zeddam a,b,c,*, Katerine Orbe c, Xavier Léry a,b,d, Olivier Dangles a,b,c, Stéphane Dupas a,b,c, Jean-François Silvain c a
IRD, UR 072, Laboratoire Evolution, Génomes et Spéciation, UPR 9034, CNRS, 91198 Gif-sur-Yvette cedex, France Université Paris-Sud 11, 91405 Orsay Cedex, France c Pontificia Universidad Católica del Ecuador, Facultad de Ciencias Naturales y Biológicas, Whymper 442 y Coruña, Quito, Ecuador d Station de Pathologie comparée, 30380 Saint-Christol-les-Alès, France b
a r t i c l e
i n f o
Article history: Received 8 March 2008 Accepted 11 June 2008 Available online 19 June 2008 Keywords: Small RNA virus Phthorimaea operculella Symmetrischema plaesiosema Lepidoptera Gelechiidae Host specificity Potato pest Biological control
a b s t r a c t A small isometric virus has been isolated from larvae of the Guatemala potato tuber moth, Tecia solanivora (Povolny), collected in Ecuador. It was designated the Anchilibi virus (AnchV). The non-enveloped viral particles have an estimated diameter of 32 ± 2 nm. Three major proteins were found in virions, with estimated sizes of 102.0 ± 2.1, 95.8 ± 2.0 and 92.4 ± 1.5 kDa for AnchV as determined by polyacrylamide gel electrophoresis. After denaturing agarose gel electrophoresis, the genome of AnchV appeared to be a tri-segmented single-stranded RNA with fragment sizes of 4.1 ± 0.2, 2.8 ± 0.2 and 1.65 ± 0.2 kb. In addition to a high virulence towards its original host, AnchV also caused high mortality in larvae of two other potato tuber moth species, Phthorimaea operculella (Zeller) and Symmetrischema (tangolias) plaesiosema (Turner). Electron microscopy confirmed that AnchV replication occurs in the cell cytoplasm, mainly in vesicles. Several important characteristics exhibited by this virus differ from those reported for known families of insect viruses. Thus, AnchV might be member of a new taxonomic group. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Tecia solanivora (Povolny) (Lepidoptera, Gelechiidae) is one of three main potato tuber moth species, along with Phthorimaea operculella (Zeller) and Symmetrischema (tangolias) plaesiosema (Turner). T. solanivora (CAB International, 2000) was first described from Central America (Povolny, 1973). Recent genetic findings confirmed that Guatemala, may be inside the area of origin of the species (Puillandre et al., 2007). In the last decades, this species entered into different countries (King and Saunders, 1984; Torres, 2003), mainly through potato trade (Herrera, 1998). It successively invaded Costa Rica in 1970 (Povolny, 1973), Panama in 1973 (Niño, 2004), Venezuela in 1983 (Salazar and Escalante, 1984), Colombia in 1985 (Rincón and López-Avila, 2004) and, finally Ecuador in 1996 (Gallegos et al., 1997). It was also reported in the Canary Islands in 2000 leading to a ban on the importation of potatoes from the archipelago to Continental Europe (EPPO, 2006). The mining
* Corresponding author. Address: Pontificia Universidad Católica del Ecuador, Facultad de Ciencias Naturales y Biológicas, Whymper 442 y Coruña, Quito, Ecuador. Fax: +593 22504020. E-mail address: [email protected] (J.-L. Zeddam). 0022-2011/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.06.007
larvae of T. solanivora cause severe damage (up to 100% in some cases) to tubers both in the field and in traditional storage facilities (Niño, 2004). After its initial introduction, the species rapidly became a major potato pest in the Andean region (Pollet et al., 2003). An integrated pest management (IPM) strategy for T. solanivora was developed and promoted by different institutions (Raman, 1988; López and Barreto, 2003). This IPM package is very similar to the one used for the control of P. operculella as it relies on a dozen of similar practices (Das et al., 1992). The main component of this IPM package is a biopesticide that is applied to the surface of the tubers in farm storage. The biopesticide is formulated using the Phthorimaea operculella granulovirus or PhopGV, (Reed, 1969; Raman et al., 1987; von Arx et al., 1987; Vickers et al., 1991; Lagnaoui et al., 1996; Taha et al., 2000; Sporleder et al., 2008) which is also able to infect T. solanivora larvae (Zeddam et al., 1994). However, initial trials showed that T. solanivora larvae were much less susceptible to available PhopGV isolates than P. operculella larvae. Thus, we examined wild T. solanivora populations of Ecuador in order to find more efficient biological control agents which could be used as a substitute for PhopGV. This work reports the preliminary characterization of an isometric virus isolated from T. solanivora, which was named Anchilibi virus (AnchV) and exhibited overall characteristics that are distinct from those of other insect viruses.
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2. Materials and methods 2.1. Larvae collection and rearing Potatoes infested by larvae of T. solanivora were collected in 2003 and 2004 from traditional farm storage from different regions of Ecuador in the provinces of Carchi, Cotopaxi, Tungurahua, Bolivar and Cañar. Some infested potatoes were also obtained from traditional markets in Riobamba, Saquisili and Salcedo (province of Cotopaxi). Each collection was maintained separately at the Pontificia Universidad Católica del Ecuador (PUCE) insect unit in Quito until emergence of adults. Dead larvae and larvae diagnosed as diseased were kept for virus screening. Massive mortality was observed among larvae infesting potatoes collected in 2003 from a farm storage located in Anchilibi in the province of Cotopaxi. In 2004, larvae present in tubers bought from a potato market in Riobamba (province of Chimborazo) pupated but many of these pupae were later found dead. Both dead larvae and pupae were examined by different methods (in particular, electron microscopy) for the presence of pathogenic micro-organisms. The virus found in larvae was named Anchilibi virus (AnchV) while the non-infectious (see below) virions found in pupae were named Shyris virus-like particles (ShyVLP). 2.2. Virus multiplication and cross-infection tests A healthy colony of T. solanivora was maintained at 15–20 °C in a separate rearing unit and was used for bioassays and virus multiplication. Adults were fed 10% sucrose in distilled water (dH2O) and larvae provided with potato tubers of the variety Leona blanca. For virus propagation, 2nd- and 3rd-instar larvae of the natural host T. solanivora were immersed for 5 s in a homogenate of virusinfected individuals. Homogenates were prepared at the concentration of 10 larva-equivalents/mL (L-Eq/mL) for AnchV. Ten infected pupae per milliliter were used in the case of Shy-VLP. For both AnchV and Shy-VLP, cross-infections were also carried with larvae of P. operculella and S. tangolias using the same protocol. As an alternate protocol, 4 ll of Shy-VLP homogenate containing 1 mg/ml streptomycin sulfate were injected into 3rd-instar larvae of T. solanivora using a Hamilton 10 ll 701N syringe (Sigma–Aldrich, MO, USA). Controls were immersed in or injected with a homogenate of healthy larvae or pupae. In all cases, the presence of the virus was assessed by electron microscopy or by SDS–PAGE to demonstrate the presence of viral proteins.
To avoid damage to virus particles during gradient centrifugation, AnchV was also purified using either NaCl–polyethylene glycol (PEG) precipitation (Detroy and Still, 1974) or Viraffinity (Biotech Support Group, NJ, USA), a water insoluble elastomeric polyelectrolyte. Briefly, 1=4 volume of Viraffinity was gently mixed with a filtered homogenate (in 0.5 PBS, pH 6.5) of AnchV-infected larvae. After 5 min incubation at room temperature, the mixture was centrifuged 10 min at 1000g. The supernatant was discarded. The pellet was rinsed with 0.5 PBS, pH 6.5 and centrifuged again. This step was repeated three times. Finally, the virus was separated from Viraffinity beads by adding 20 ll of 0.5 PBS, pH 8.5. After a 10 min centrifugation at 1000g, the virus-containing supernatant was stored at 4 °C. TM
TM
TM
2.4. Transmission electron microscopy Virus suspensions were deposited onto 200-mesh grids, stained with 2% phosphotungstic acid (Brenner and Horne, 1959) and then were examined with a Zeiss EM 10 CR transmission electron microscope with an 80 keV acceleration voltage. Virus-infected larvae were fixed for 2 h in glutaraldehyde and then post-fixed in osmium tetroxide, both at concentrations of 2% in cacodylate buffer (pH 7.4, 0.1 M). Tissues were dehydrated in a series of graded acetone solutions (20–100%), embedded in Epon, and cut in 0.1 lm slices with an LKB ultratome. Finally, sections of the insects were double stained in 2% uranyl acetate and lead citrate (Reynolds, 1963) before being examined as described above. 2.5. SDS–polyacrylamide gel electrophoresis (SDS PAGE) of proteins and protein microsequencing Ten microliter virus samples were heat denaturated in 2 loading buffer (80 mM Tris/HCl, pH 6.8; 10% Mercaptoethanol; 2% SDS; 10% Glycerol; 0.01% Bromophenol blue) and loaded onto SDS– PAGE gels (5% stacking gel and 12.5% separating gel) (Laemmli, 1970). Proteins were separated by electrophoresis for 3 h at 75 V and stained with Coomassie Brilliant Blue. Weights were estimated by comparison with protein weight markers using the software PhotoCapt version 10.01 (Vilber Lourmat, France). Viral proteins separated by SDS–PAGE were transferred onto an ABI ProBlot polyvinylidene difluoride (PVDF) membrane (Applied Biosystems Inc., California, USA) using a Z340502 semidry blotter (Sigma, MO, USA). The transfer was performed at 0.8 mA/cm2 of gel using a Tris–Borate buffer (50 mM Tris, 5 mM sodium borate, pH 8.5) following the protocol of the provider. Edman microsequencing of the viral proteins transferred to the PVDF membrane was performed at the Protein Chemistry Laboratory, University of Texas Medical Branch at Galveston, TX, USA.
2.3. Virus purification 2.6. Extraction and electrophoresis of viral nucleic acid Infected larvae were homogenized in a buffer using a PolytronÒ PT 1600E homogenizer (Kinematica, Germany). Buffers tested were 0.5 PBS (1.6 mM Na2HPO4, 0.25 mM KH2PO4, 0.65 mM KCl, 67.5 mM NaCl), TE (10 mM Tris, 1 mM EDTA), TEN (10 mM Tris, 1 mM EDTA, 100 mM NaCl) or RNALater (25 mM sodium citrate, 10 mM EDTA, 5.3 M ammonium sulfate), all at pH 7.0. Insect fragments and debris were eliminated by centrifugation at 10,000g for 20 min at 10 °C. The supernatant was layered onto a 15–45% w/v sucrose (or glycerol) gradient prepared with the same buffer and centrifuged at 100,000g for 2 h at 10 °C. The band corresponding to the virus was collected with a Pasteur pipette, diluted three times in 0.5 PBS and then pelleted at 100,000g for 2.5 h. Finally, the pellet was resuspended in 100 ll of 0.5 PBS. Alternatively, the virus was sometimes directly pelleted (100,000g for 2.5 h) from the supernatant without the gradient step.
Viral nucleic acids were extracted from viral solution using the SV Total RNA Isolation System (Promega, WI, USA). Viral RNAs were heat-denatured and separated on a 1% agarose formaldehyde-containing gel prepared with MOPS buffer (20 mM MOPS, 8 mM sodium acetate, 1 mM EDTA, pH 7.0) as described in Maniatis et al., (1982). After Syber Safe (Promega, USA) staining, the gel was photographed using a UV transilluminator and the sizes of RNA bands were determined by comparison with RNA molecular weight markers using the software Photocapt version 10.01. 2.7. Bioassays Four groups of 60 T. solanivora first-instar larvae from the PUCE virus-free colony were placed on tubers at an infestation ratio of 10
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larvae per 50–70 g tuber. The tubers had been previously immersed in a viral solution at a concentration of 10 L-Eq/mL. Individuals which died within the first 24 h were assumed to have been injured during their manipulation and were eliminated. Mortality was registered daily after the first 24 h. Abbott’s corrected mortality (Abbott, 1925) was calculated using the data from the control (2 60 larvae placed on tubers treated with only 0.5 PBS). Before use, the tubers, of the variety Chaucha, were disinfected with 2% sodium hypochlorite, extensively washed with dH2O and air-dried. Larvae were randomly chosen from the dead and individually checked by SDS–PAGE for the presence of AnchV. Alternatively, two groups of 60 T. solanivora first-instar larvae were directly immersed in the 10 L-Eq/mL viral solution and then placed on non-contaminated tubers. The rest of the bioassay was carried out as above. In the case of P. operculella and S. tangolias, two groups of 50 first-instar larvae were immersed in the 10 L-Eq/mL viral solution and processed as described above for T. solanivora. 3. Results 3.1. Disease symptoms and cross-infection experiments AnchV-infected larvae exhibited slower movements and less feeding activity. They were paralyzed 1 or 2 days after infection. Some dying larvae (and pupae) turned black, mainly in the anterior part of their body, and all were completely black when dead (Fig. 1). The cadavers dehydrated in less than 24 h. Diseased larvae that survived infection had a significant increase in the duration of their larval stages compared to the control population. When almost all individuals from the control had pupated or even had emerged as adults, virus-surviving individuals were still in their larval stage. The disease did not block these individuals at a particular stage of their development but only retarded them. Cross-infections experiments showed AnchV was able to multiply on the two re-
lated potato tuber moth species P. operculella and S. tangolias. All dead individuals analyzed contained large amounts of AnchV proteins and viral particles. Moreover, symptoms produced by the AnchV infection were similar for larvae of the three species tested, T. solanivora, P. operculella and S. plaesiosema. Shy-VLP was found in dead pupae in which no other symptoms were noticed in particular, no change in color was observed. Trials to orally infect larvae with a viral solution were unsuccessful. The same negative result was obtained when larvae were injected with the virus using a micro-injector. Similarly, oral contamination of P. operculella and S. plaesiosema larvae with Shy-VLP was unsuccessful. Because of the impossibility to get an infection using this biological material, the structures observed by TEM were considered as virus-like particles rather than mature virions. 3.2. Transmission electron microscopy For both AnchV and Shy-VLP, large numbers of particles were found in crude extracts of dead or moribund larvae examined by electron microscopy. Negatively stained preparations of AnchV revealed non-occluded, non-enveloped, 32 ± 2 nm in diameter, icosahedral particles (Fig. 2a). Some of the virions exhibited a white core. The large number of presumed capsomers corresponding to particle degradation demonstrated that the conditions for the processing of the virus-infected individuals were not optimal. Similar results (data not shown) were obtained when using other classical buffers, TE, TEN or RNALater. This degradation occurred despite using NaCl-complemented buffers exhibiting characteristics close to the parameters we measured for the hemolymph of potato tuber moth larvae, a neutral pH and an osmolarity of about 300 milliosmoles. Shy-VLP were structurally similar to AnchV virions except that their white core was much less obvious (Fig. 2b). Their diameter was also of approximately 32 ± 2 nm. Because it was impossible to multiply Shy-VLP, no biological material was available for electron microscopy observations of infected cells in larvae. Similarly to what was observed in the case of AnchV preparations, large numbers of free presumed capsomers were noticed in Shy-VLPdead pupae after their homogenization. Electron microscope examination of ultra-thin sections of AnchV-infected cells showed intracytoplasmic aggregates of isometric virus particles located inside vesicles (Fig. 3). These vesicles apparently have a single-layer membrane. Viral particles were also observed free in the cytoplasm but in lower amounts than the vesicle-contained viral particles. This could be corresponding to a later stage of the infection in which the surrounding membrane has disappeared. No paracrystalline arrays were observed, even in heavily infected cells. 3.3. Density gradient centrifugation Even using large groups (>200 individuals) of AnchV-infected larvae, it was impossible to get significant amount of virus by centrifugation on sucrose or glycerol gradients. The viral particles were destroyed during the process, probably due to changes in osmotic pressure. Direct pelleting of the viral particles without the gradient step, allowed recovery of some biological material although more than 90% of the virions were lost as estimated by SDS–PAGE comparison of viral proteins of starting homogenate and final concentrate. NaCl–PEG precipitation allowed the recovery of AnchV virions but other contaminant proteins were also present as seen after SDS–PAGE (data not shown). It was possible to get small quantities of highly purified AnchV using Viraffinity , a water insoluble polyelectrolyte that has been designed for the purification of nonenveloped viruses while preserving infectivity. However, in both TM
Fig. 1. Healthy (left) and Anchilibi virus-dead (right) larvae of Tecia solanivora. (Bar equivalent to 2 mm).
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Fig. 2. Transmission electron microscopy of negatively stained viral particles isolated from Tecia solanivora (crude preparations). (A) Anchilibi virus (Top left corner: Close-up on virions). (B) Shyris virus. EmpVP: empty viral particles; CoVP: core-exhibiting viral particles, Caps: isolated presumed capsomers. Bar equivalent to 100 nm (A and B) or 50 nm (Insert).
Fig. 3. Transmission electron microscopy of an ultra-thin section in an AnchV-infected Tecia solanivora larva infected by Anchilibi virus (VP: viral particles; Mb: unilayer membrane surrounding virus aggregate; Mit: mitochondria; Bar equivalent to 0.3 lm).
cases, SDS–PAGE analysis demonstrated that the loss of virus was very high, in the same order as during gradient purification. Once again, this might be due to sub-optimal conditions of pH or osmolarity encountered by the virions during the purification process.
viral particles also have 3 major proteins (Fig. 4, lane 4) but their molecular weights were different of those of AnchV, 40.4 ± 2.0, 37.7 ± 2.0 and 33.4 ± 2.0 kDa, respectively. The second protein was significantly more abundant than the other two.
3.4. Electrophoresis of virus proteins
3.5. Agarose gel electrophoresis of viral nucleic acids
The AnchV exhibited three main proteins of respectively, 102.0 ± 2.1; 95.8 ± 2.0 and 92.4 ± 1.5 kDa in SDS–PAGE (Fig. 4). Mean sizes were calculated from 14 gel runs. In some cases, the proportional abundance of these three proteins was variable between individual larvae. Several adults emerged from larvae which survived exposure to AnchV were found to be infected and exhibited the same protein pattern as larvae. However, some other AnchV-infected adults collected before they died only had either the two higher weight proteins or the two lower ones (Fig. 4, lane 3). Microsequencing of AnchV proteins did not return an amino acid sequence. Apparently, the three proteins had an N-terminal modification which precluded Edman degradation procedure. Shy-VLP
Three bands (termed L, M and S) were identified by agarose gel electrophoresis of the viral nucleic acids extracted from either AnchV or Shy-VLP (Fig. 5). Treatment of the samples with ribonuclease A in 300 mM NaCl prior to electrophoresis resulted in the disappearance of the bands whereas, the addition of RNAse-free deoxyribonuclease did not eliminate the bands (data not shown). From their electrophoretic mobility in agarose gel, the molecular weights of the three AnchV RNAs were estimated to be 4.1 ± 0.2, 2.8 ± 0.2 and 1.65 ± 0.2 kb, respectively. Surprisingly, those of Shy-VLP RNAs had identical sizes. In the case of AnchV, the relative intensity of the three bands slightly varied between different RNA extractions. This raises the
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3.6. Bioassays Most of the first-instar larvae of T. solanivora exposed to AnchV died within 4 days (Fig. 6). This occurred in both treatments, i.e. larvae fed virus-treated tubers (81.0 ± 2.0% mortality) or larvae directly immersed in the larval homogenate (82.0 ± 8.0% mortality). Despite the mortality rates were almost identical, the limited number of replicates (2) for immersion treatment meant that no statistical comparison could be made between the two treatments. The virulence of the pathogen was obvious considering that, first, only a small amount of the viral solution was deposited on each tuber and, secondly, the period of virus acquisition was reduced as, in general, the mining neonates rapidly entered their galleries following emergence. Using the immersion protocol, AnchV also caused high mortality in P. operculella (54.0 ± 4.0%) and S. plaesiosema (58.5 ± 3.0%) larvae albeit lower than in T. solanivora. Because of the limited numbers of repetitions (2 replicates/species), it was not possible to determine whether the final mortality rates were statistically different between P. operculella and S. plaesiosema larvae contaminated following the immersion method. Identically, more bioassays would be necessary to establish whether T. solanivora is really more susceptible to AnchV than the two latter species. 4. Discussion
Fig. 4. SDS–polyacrylamide gel of viral particles. (A) Lane MW: protein molecular weight marker; Lane AV: Anchilibi virus; (B) Lane MW: protein molecular weight marker; Lane SV: Shyris virus; (C) Lanes Ad1 to Ad3: Anchilibi virus from alive infected adults).
Fig. 5. Agarose gel electrophoresis of formaldehyde-denaturated viral genomes ((A) Lane MW: RNA molecular weight marker with bands of 9.49, 7.46, 4.40, 2.37 and 1.35 kb, respectively; Lane AV: Anchilibi virus; (B) Lane MW: RNA molecular weight marker with bands of 9.49, 7.46, 4.40, 2.37 and 1.35 kb, respectively; Lane SV: Shyris virus).
question of whether the three RNAs were encapsidated together in the same virion or in different viral particles. This phenomenon was not noticed in the case of the RNAs of Shy-VLP.
Insect viruses are presently distributed among 24 taxonomic families, two unassigned genera (Iflavirus and Tenuivirus) and a number of unclassified viruses (Fauquet et al., 2005) which can exhibit almost all types of genome structure: monopartite or pluripartite genomes of single or double-stranded nucleic acids which can be either DNA or RNA. More than half of the groups of invertebrate viruses (13 families and the two genera) exhibit ssRNA genomes. Among them, only the Bunyaviridae are presently known to have a tri-segmented ssRNA viral genome (Fauquet et al., 2005). Characteristics of members of the Bunyaviridae are quite different from those of AnchV and Shy-VLP. Structurally, virions of Bunyaviridae are much larger (80–120 nm in diameter), enveloped and display glycoprotein spikes. The sizes of the virion proteins, which vary among the five genera of the family Bunyaviridae, are quite different from those of AnchV or Shy-VLP. The differences are also obvious when considering the sizes of the three genomic RNAs. In the Bunyaviridae the smaller one (S) ranges between 0.9–3 kb, the medium one (M) between 3–5 kb and the larger one (L) between 6–12 kb. Thus, it is unlikely that AnchV or Shy-VLP could be assigned to Bunyaviridae. Moreover, even more differences exist between AnchV and Shy-VLP and the other groups of insect viruses. Considering that the insects are by far the most numerous group of eukaryotes (several millions of species) and that only about half of the known viruses (around 7000 species) were isolated from insect hosts, it is reasonable to expect to find new types of genomic organization in entomoviruses. The results of the study presented here provide such evidence. AnchV and Shy-VLP were isolated from the same host species and share most of their characteristics as the size and organization of their RNA genome fragments or the size and structure of their viral (or virus-like) particles. The more striking difference was found in their respective protein patterns. While AnchV exhibited high molecular weight capsid proteins (around 90–100 kDa), Shy-VLP only presented proteins in the range 30–40 kDa. Considering this, the most parsimonious hypothesis of the nature of ShyVLP is that they are non-infectious particles of AnchV that have undergone some form of post-mortem degradation in the host that also results in loss of infectivity. These degradations might be due to the fact that viral particles stayed almost two weeks inside their dead hosts in the rearing facility (15–20 °C) before these individu-
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% Cumulative corrected mortality
100 90 80 70 60 A
50
B
40
C
30
D
20 10 0 2
3
4
Days after infection Fig. 6. Time-mortality response of first-instar larvae of the three potato tuber moth species infected with Anchilibi virus (at a concentration of 10 larva-equivalents/L). (A) T. solanivora larvae fed surface-contaminated tubers. (B) T. solanivora larvae contaminated by a 5-s immersion in the virus solution. (C) P. operculella larvae contaminated by a 5s immersion in the virus solution. (D) S. plaesiosema larvae contaminated by a 5-s immersion in the virus solution. (Final mortality rate in controls: A = 7%; B = 7%; C = 6%; D = 8%).
als were diagnosed as dead and then processed. Also, a different processing of capsid proteins could have been occurred in pupae (the source of Shy-VLP) compared to larvae. These alterations would be related to the incapacity for Shy-VLP to multiply in larvae during contamination trials. Indeed, the entry of viruses in host cell generally implies the interactions between cellular receptors and viral proteins located on the capsid surface. Sequence data would be able to definitively establish whether AnchV and Shy-VLP belong to the same virus species or not. There is no known insect virus exhibiting three capsid proteins with sizes between 102 and 92 kDa. Considering the size of the heaviest AnchV capsid protein (more than 100 kDa corresponding to at least 3000 encoding bases), it is obvious that the capsid coding gene can only be located on the L fragment since the others are smaller than this minimum 3 kb size required. Similarly, the presence of the two other proteins of about 95.8 and 92.4 kDa could be explained by the processing of the 102 kDa protein. This assumption is supported by the fact that the proportions of the three proteins were not constant but showed differences in their molarity according to the virus batches analyzed. Also, in some AnchV-infected adults, only two of the three bands were distinguishable on the SDS–PAGE gels. This indicates that the capsid protein processing in adults and larvae may be somewhat different, depending on protease activity and specificity of the insect instar. Probably because of their small sizes, the two small fragments (5–7 kDa) from the processing were not detected on gels. Proteolytic cleavage of a capsid precursor is known from different ssRNA insect virus families: Nodaviridae, Picornaviridae, Tetraviridae, Dicistroviridae (Ball and Johnson, 1998; Fauquet et al., 2005). This strategy can lead to a change in the ratio between proteins (the precursor and its derived products can be more or less abundant according to the efficiency of the proteolytic cleavage). Because of size considerations, it is probable that the replicase gene is located on fragments M or S; the former being the best candidate. Considering the similar sizes of the three RNA fragments in both viruses, it is tempting to assume that, in Shy-VLP, the L fragment might also bear the capsid gene(s). Similarly to AnchV, the capsid proteins could perhaps be generated through the cleavage of a precursor although the cumulative size of the nucleotide sequences necessary for separately coding the three proteins would not exceed the total size of the largest Shy-VLP fragment (L). The molecular weights of Shy-VLP proteins are close to those of insect picorna-like viruses (Christian and Scotti, 1998; Fauquet et al.,
2005). However, these viruses exhibit monopartite ssRNA genomes of 8–10 kb in size, which is quite different from what was found for AnchV and Shy-VLP. Three genera (Bromovirus, Cucumovirus and Ilarvirus) of the plant-infecting and insect-transmitted Begomoviridae family share several characteristics with AnchV and Shy-VLP. First, they exhibit icosahedral virions of 26–35 nm of diameter. Similarly to AnchV and Shy-VLP, they have tri-partite ssRNA genomes whose total length is approximately 8 kb. However, an important difference is that Begomoviridae have a unique capsid protein of 20–24 kDa (Fauquet et al., 2005). As most of the AnchV and Shy-VLP viral particles were damaged when centrifuged on sucrose or glycerol gradients, it was not possible to evidence the presence of distinct virus bands which could be related to the existence of particles exhibiting different densities. So, more investigations will be necessary to establish whether the three RNAs are encapsidated in the same viral particle. However, in the case of AnchV, the molar ratios of the three RNAs were not constant which suggest the existence of virions with different RNA contents. This was not observed for Shy-VLP perhaps because of a reduced number of batches analyzed. It is known that some virus groups encapsidate subgenomic RNAs of viral origin (for example, members of the insect-infecting Betatetravirus) or non viral RNAs as cellular mRNAs, tRNAs or rRNAs (Levin and Seidman, 1979; Adkins and Hunter, 1981; Jiang et al., 1993; Rumenapf et al., 1995; Berkowitz et al., 1996; Rulli et al., 2007). Such a situation could not be excluded in the case of AnchV or/and Shy-VLP but only complete sequencing of the viral genomes will permit to establish whether subgenomic or cellular RNAs are encapsidated in their virions. Genome-fragmented viruses can present a reassortment of genome fragments during replication in the same infected cell (Holland et al., 1982; Lin et al., 2004; Tomohisa et al., 2005; Chare and Holmes, 2006; Hay et al., 2001). This mechanism allows for large-scale evolutionary jumps. Considering this, it is possible that AnchV and Shy-VLP RNA fragments have different phylogenetic origins. Only sequence data will provide unequivocal information on the possible relationships that exist between AnchV or ShyVLP and the known groups of viruses. AnchV appeared as a fast-acting and virulent pathogen against the three species of potato tuber moths. Complementary trials should be carried out in order to establish whether AnchV is more active against T. solanivora. If it were the case, this might be related
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to the fact that AnchV was originally isolated from this species and thus, could be more adapted to its original host than to the two alternate host species tested. However, the three species are sympatric in different areas of Ecuador and are sometimes found in the same potato batches. According to the host range of the virus as determined through laboratory bioassays, it is highly probable that individuals belonging to any of the three potato tuber moth species are infected by AnchV in the wild. Works are underway to confirm this hypothesis. The presence of large amounts of AnchV in apparently healthy adults points to a possible vertical transmission of this pathogen. This would help to a better dissemination of AnchV because flying adults offer a much higher capacity of dispersion than larvae. A particular problem encountered during the study is that AnchV particles presented a marked susceptibility to degradation. The collection of AnchV-infected larvae before their death greatly enhanced the quantity of biologically active particles recovered. Degradation might be caused by proteases and other molecules (and, especifically, polyphenols) released after the lysis of cells (Wilson et al., 2001; Shelby and Popham, 2006). So, early collection before death could limit this phenomenon. On the other hand, important losses which occurred during centrifugation on sucrose gradients were probably due to osmotic pressure changes. Specific experiments should be carried out to solve this problem. This would allow for the preparation of the large amounts of purified virus necessary for further studies, as well as, the analysis of the sedimentation pattern of the virus in a sucrose gradient. This might provide valuable data to determine whether the three RNA segments are all present in the same viral particle. Finally, it is highly probable that both AnchV and Shy-VLP belong to a new taxonomic group of insect viruses as even in prokaryotes, plants and vertebrates, no tri-partite ssRNA virus exhibits all the other characteristics of AnchV and Shy-VLP. Apart of its academic interest, AnchV could offer promising perspectives as a biocontrol agent. Potato is an essential staple food for poor-resource families of the Andean highlands. The high level of damage caused by potato tuber moth larvae threaten the food security of these farmers, as well as, jeopardize their health (Yanggen et al., 2003) due to an increase in chemical insecticide application. All biological control agents with potential to be used in pest management programs should receive attention. In particular, AnchV seems to offer promising opportunities because of its ability to infect the three potato tuber moth species present in the same area. In comparison, PhopGV, which is currently used for the control of P. operculella in Peru, Bolivia and for control of T. solanivora in Venezuela and Colombia (Niño and Notz, 2000; Raman et al., 1987; Das et al., 1992; López and Barreto, 2003) is unable to provide an adequate control of S. tangolias which could limit its use in some areas. Obviously, toxicological tests should first be carried out in order to establish that the entomopathogen does not present a threat to non-target species including vertebrates. Then, trials will determine its real potential for pest control under potato storage conditions. The prevailing environmental conditions (no rain or UV radiation, temperatures generally in the range 5–15 °C) in the storage areas could be favorable to the use of an AnchV-based biopesticide. Acknowledgments The authors gratefully acknowledge two anonymous reviewers. They are also indebted to Dr. Marc Ravallec (Université de Montpellier II, France) and Dr. Yasuji Amano (Instituto Izquieta Perez, Guayaquil, Ecuador) for technical assistance in electron microscopy and Miss. Daniela Chevasco (PUCE) for technical assistance in gel electrophoresis and bioassays. They also acknowledge Dr.
Keil Clifford (PUCE) for his comments and help with the writing of this article. This work was partly funded by the McKnight Foundation, USA and by projects Ecos-Norte and Ecofor from France. References Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. Adkins, B., Hunter, T., 1981. Identification of a packaged cellular mRNA in virions of Rous sarcoma virus. J. Virol. 39, 471–480. Ball, L.A., Johnson, K.L., 1998. Nodaviruses of insects. In: Miller, L.K., Ball, L.A. (Eds.), The Insect Viruses. Plenum Publishing Company, NY, USA, pp. 225–267). Berkowitz, R., Fisher, J., Goff, S.P., 1996. RNA packaging. Curr. Top. Microbiol. Immunol. 214, 177–218. Brenner, S., Horne, W., 1959. A negative staining method for high resolution electron microscopy of viruses. Biochem. Biophys. Acta 34, 103–110. Christian, P.D., Scotti, P.D., 1998. The picorna-like viruses of insects. In: Miller, L.K., Ball, L.A. (Eds.), The Insect Viruses. 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Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa
An isometric virus of the potato tuber moth Tecia solanivora (Povolny) (Lepidoptera: Gelechiidae) has a tri-segmented RNA genome Jean-Louis Zeddam a,b,c,*, Katerine Orbe c, Xavier Léry a,b,d, Olivier Dangles a,b,c, Stéphane Dupas a,b,c, Jean-François Silvain c a
IRD, UR 072, Laboratoire Evolution, Génomes et Spéciation, UPR 9034, CNRS, 91198 Gif-sur-Yvette cedex, France Université Paris-Sud 11, 91405 Orsay Cedex, France c Pontificia Universidad Católica del Ecuador, Facultad de Ciencias Naturales y Biológicas, Whymper 442 y Coruña, Quito, Ecuador d Station de Pathologie comparée, 30380 Saint-Christol-les-Alès, France b
a r t i c l e
i n f o
Article history: Received 8 March 2008 Accepted 11 June 2008 Available online 19 June 2008 Keywords: Small RNA virus Phthorimaea operculella Symmetrischema plaesiosema Lepidoptera Gelechiidae Host specificity Potato pest Biological control
a b s t r a c t A small isometric virus has been isolated from larvae of the Guatemala potato tuber moth, Tecia solanivora (Povolny), collected in Ecuador. It was designated the Anchilibi virus (AnchV). The non-enveloped viral particles have an estimated diameter of 32 ± 2 nm. Three major proteins were found in virions, with estimated sizes of 102.0 ± 2.1, 95.8 ± 2.0 and 92.4 ± 1.5 kDa for AnchV as determined by polyacrylamide gel electrophoresis. After denaturing agarose gel electrophoresis, the genome of AnchV appeared to be a tri-segmented single-stranded RNA with fragment sizes of 4.1 ± 0.2, 2.8 ± 0.2 and 1.65 ± 0.2 kb. In addition to a high virulence towards its original host, AnchV also caused high mortality in larvae of two other potato tuber moth species, Phthorimaea operculella (Zeller) and Symmetrischema (tangolias) plaesiosema (Turner). Electron microscopy confirmed that AnchV replication occurs in the cell cytoplasm, mainly in vesicles. Several important characteristics exhibited by this virus differ from those reported for known families of insect viruses. Thus, AnchV might be member of a new taxonomic group. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Tecia solanivora (Povolny) (Lepidoptera, Gelechiidae) is one of three main potato tuber moth species, along with Phthorimaea operculella (Zeller) and Symmetrischema (tangolias) plaesiosema (Turner). T. solanivora (CAB International, 2000) was first described from Central America (Povolny, 1973). Recent genetic findings confirmed that Guatemala, may be inside the area of origin of the species (Puillandre et al., 2007). In the last decades, this species entered into different countries (King and Saunders, 1984; Torres, 2003), mainly through potato trade (Herrera, 1998). It successively invaded Costa Rica in 1970 (Povolny, 1973), Panama in 1973 (Niño, 2004), Venezuela in 1983 (Salazar and Escalante, 1984), Colombia in 1985 (Rincón and López-Avila, 2004) and, finally Ecuador in 1996 (Gallegos et al., 1997). It was also reported in the Canary Islands in 2000 leading to a ban on the importation of potatoes from the archipelago to Continental Europe (EPPO, 2006). The mining
* Corresponding author. Address: Pontificia Universidad Católica del Ecuador, Facultad de Ciencias Naturales y Biológicas, Whymper 442 y Coruña, Quito, Ecuador. Fax: +593 22504020. E-mail address: [email protected] (J.-L. Zeddam). 0022-2011/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.06.007
larvae of T. solanivora cause severe damage (up to 100% in some cases) to tubers both in the field and in traditional storage facilities (Niño, 2004). After its initial introduction, the species rapidly became a major potato pest in the Andean region (Pollet et al., 2003). An integrated pest management (IPM) strategy for T. solanivora was developed and promoted by different institutions (Raman, 1988; López and Barreto, 2003). This IPM package is very similar to the one used for the control of P. operculella as it relies on a dozen of similar practices (Das et al., 1992). The main component of this IPM package is a biopesticide that is applied to the surface of the tubers in farm storage. The biopesticide is formulated using the Phthorimaea operculella granulovirus or PhopGV, (Reed, 1969; Raman et al., 1987; von Arx et al., 1987; Vickers et al., 1991; Lagnaoui et al., 1996; Taha et al., 2000; Sporleder et al., 2008) which is also able to infect T. solanivora larvae (Zeddam et al., 1994). However, initial trials showed that T. solanivora larvae were much less susceptible to available PhopGV isolates than P. operculella larvae. Thus, we examined wild T. solanivora populations of Ecuador in order to find more efficient biological control agents which could be used as a substitute for PhopGV. This work reports the preliminary characterization of an isometric virus isolated from T. solanivora, which was named Anchilibi virus (AnchV) and exhibited overall characteristics that are distinct from those of other insect viruses.
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2. Materials and methods 2.1. Larvae collection and rearing Potatoes infested by larvae of T. solanivora were collected in 2003 and 2004 from traditional farm storage from different regions of Ecuador in the provinces of Carchi, Cotopaxi, Tungurahua, Bolivar and Cañar. Some infested potatoes were also obtained from traditional markets in Riobamba, Saquisili and Salcedo (province of Cotopaxi). Each collection was maintained separately at the Pontificia Universidad Católica del Ecuador (PUCE) insect unit in Quito until emergence of adults. Dead larvae and larvae diagnosed as diseased were kept for virus screening. Massive mortality was observed among larvae infesting potatoes collected in 2003 from a farm storage located in Anchilibi in the province of Cotopaxi. In 2004, larvae present in tubers bought from a potato market in Riobamba (province of Chimborazo) pupated but many of these pupae were later found dead. Both dead larvae and pupae were examined by different methods (in particular, electron microscopy) for the presence of pathogenic micro-organisms. The virus found in larvae was named Anchilibi virus (AnchV) while the non-infectious (see below) virions found in pupae were named Shyris virus-like particles (ShyVLP). 2.2. Virus multiplication and cross-infection tests A healthy colony of T. solanivora was maintained at 15–20 °C in a separate rearing unit and was used for bioassays and virus multiplication. Adults were fed 10% sucrose in distilled water (dH2O) and larvae provided with potato tubers of the variety Leona blanca. For virus propagation, 2nd- and 3rd-instar larvae of the natural host T. solanivora were immersed for 5 s in a homogenate of virusinfected individuals. Homogenates were prepared at the concentration of 10 larva-equivalents/mL (L-Eq/mL) for AnchV. Ten infected pupae per milliliter were used in the case of Shy-VLP. For both AnchV and Shy-VLP, cross-infections were also carried with larvae of P. operculella and S. tangolias using the same protocol. As an alternate protocol, 4 ll of Shy-VLP homogenate containing 1 mg/ml streptomycin sulfate were injected into 3rd-instar larvae of T. solanivora using a Hamilton 10 ll 701N syringe (Sigma–Aldrich, MO, USA). Controls were immersed in or injected with a homogenate of healthy larvae or pupae. In all cases, the presence of the virus was assessed by electron microscopy or by SDS–PAGE to demonstrate the presence of viral proteins.
To avoid damage to virus particles during gradient centrifugation, AnchV was also purified using either NaCl–polyethylene glycol (PEG) precipitation (Detroy and Still, 1974) or Viraffinity (Biotech Support Group, NJ, USA), a water insoluble elastomeric polyelectrolyte. Briefly, 1=4 volume of Viraffinity was gently mixed with a filtered homogenate (in 0.5 PBS, pH 6.5) of AnchV-infected larvae. After 5 min incubation at room temperature, the mixture was centrifuged 10 min at 1000g. The supernatant was discarded. The pellet was rinsed with 0.5 PBS, pH 6.5 and centrifuged again. This step was repeated three times. Finally, the virus was separated from Viraffinity beads by adding 20 ll of 0.5 PBS, pH 8.5. After a 10 min centrifugation at 1000g, the virus-containing supernatant was stored at 4 °C. TM
TM
TM
2.4. Transmission electron microscopy Virus suspensions were deposited onto 200-mesh grids, stained with 2% phosphotungstic acid (Brenner and Horne, 1959) and then were examined with a Zeiss EM 10 CR transmission electron microscope with an 80 keV acceleration voltage. Virus-infected larvae were fixed for 2 h in glutaraldehyde and then post-fixed in osmium tetroxide, both at concentrations of 2% in cacodylate buffer (pH 7.4, 0.1 M). Tissues were dehydrated in a series of graded acetone solutions (20–100%), embedded in Epon, and cut in 0.1 lm slices with an LKB ultratome. Finally, sections of the insects were double stained in 2% uranyl acetate and lead citrate (Reynolds, 1963) before being examined as described above. 2.5. SDS–polyacrylamide gel electrophoresis (SDS PAGE) of proteins and protein microsequencing Ten microliter virus samples were heat denaturated in 2 loading buffer (80 mM Tris/HCl, pH 6.8; 10% Mercaptoethanol; 2% SDS; 10% Glycerol; 0.01% Bromophenol blue) and loaded onto SDS– PAGE gels (5% stacking gel and 12.5% separating gel) (Laemmli, 1970). Proteins were separated by electrophoresis for 3 h at 75 V and stained with Coomassie Brilliant Blue. Weights were estimated by comparison with protein weight markers using the software PhotoCapt version 10.01 (Vilber Lourmat, France). Viral proteins separated by SDS–PAGE were transferred onto an ABI ProBlot polyvinylidene difluoride (PVDF) membrane (Applied Biosystems Inc., California, USA) using a Z340502 semidry blotter (Sigma, MO, USA). The transfer was performed at 0.8 mA/cm2 of gel using a Tris–Borate buffer (50 mM Tris, 5 mM sodium borate, pH 8.5) following the protocol of the provider. Edman microsequencing of the viral proteins transferred to the PVDF membrane was performed at the Protein Chemistry Laboratory, University of Texas Medical Branch at Galveston, TX, USA.
2.3. Virus purification 2.6. Extraction and electrophoresis of viral nucleic acid Infected larvae were homogenized in a buffer using a PolytronÒ PT 1600E homogenizer (Kinematica, Germany). Buffers tested were 0.5 PBS (1.6 mM Na2HPO4, 0.25 mM KH2PO4, 0.65 mM KCl, 67.5 mM NaCl), TE (10 mM Tris, 1 mM EDTA), TEN (10 mM Tris, 1 mM EDTA, 100 mM NaCl) or RNALater (25 mM sodium citrate, 10 mM EDTA, 5.3 M ammonium sulfate), all at pH 7.0. Insect fragments and debris were eliminated by centrifugation at 10,000g for 20 min at 10 °C. The supernatant was layered onto a 15–45% w/v sucrose (or glycerol) gradient prepared with the same buffer and centrifuged at 100,000g for 2 h at 10 °C. The band corresponding to the virus was collected with a Pasteur pipette, diluted three times in 0.5 PBS and then pelleted at 100,000g for 2.5 h. Finally, the pellet was resuspended in 100 ll of 0.5 PBS. Alternatively, the virus was sometimes directly pelleted (100,000g for 2.5 h) from the supernatant without the gradient step.
Viral nucleic acids were extracted from viral solution using the SV Total RNA Isolation System (Promega, WI, USA). Viral RNAs were heat-denatured and separated on a 1% agarose formaldehyde-containing gel prepared with MOPS buffer (20 mM MOPS, 8 mM sodium acetate, 1 mM EDTA, pH 7.0) as described in Maniatis et al., (1982). After Syber Safe (Promega, USA) staining, the gel was photographed using a UV transilluminator and the sizes of RNA bands were determined by comparison with RNA molecular weight markers using the software Photocapt version 10.01. 2.7. Bioassays Four groups of 60 T. solanivora first-instar larvae from the PUCE virus-free colony were placed on tubers at an infestation ratio of 10
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larvae per 50–70 g tuber. The tubers had been previously immersed in a viral solution at a concentration of 10 L-Eq/mL. Individuals which died within the first 24 h were assumed to have been injured during their manipulation and were eliminated. Mortality was registered daily after the first 24 h. Abbott’s corrected mortality (Abbott, 1925) was calculated using the data from the control (2 60 larvae placed on tubers treated with only 0.5 PBS). Before use, the tubers, of the variety Chaucha, were disinfected with 2% sodium hypochlorite, extensively washed with dH2O and air-dried. Larvae were randomly chosen from the dead and individually checked by SDS–PAGE for the presence of AnchV. Alternatively, two groups of 60 T. solanivora first-instar larvae were directly immersed in the 10 L-Eq/mL viral solution and then placed on non-contaminated tubers. The rest of the bioassay was carried out as above. In the case of P. operculella and S. tangolias, two groups of 50 first-instar larvae were immersed in the 10 L-Eq/mL viral solution and processed as described above for T. solanivora. 3. Results 3.1. Disease symptoms and cross-infection experiments AnchV-infected larvae exhibited slower movements and less feeding activity. They were paralyzed 1 or 2 days after infection. Some dying larvae (and pupae) turned black, mainly in the anterior part of their body, and all were completely black when dead (Fig. 1). The cadavers dehydrated in less than 24 h. Diseased larvae that survived infection had a significant increase in the duration of their larval stages compared to the control population. When almost all individuals from the control had pupated or even had emerged as adults, virus-surviving individuals were still in their larval stage. The disease did not block these individuals at a particular stage of their development but only retarded them. Cross-infections experiments showed AnchV was able to multiply on the two re-
lated potato tuber moth species P. operculella and S. tangolias. All dead individuals analyzed contained large amounts of AnchV proteins and viral particles. Moreover, symptoms produced by the AnchV infection were similar for larvae of the three species tested, T. solanivora, P. operculella and S. plaesiosema. Shy-VLP was found in dead pupae in which no other symptoms were noticed in particular, no change in color was observed. Trials to orally infect larvae with a viral solution were unsuccessful. The same negative result was obtained when larvae were injected with the virus using a micro-injector. Similarly, oral contamination of P. operculella and S. plaesiosema larvae with Shy-VLP was unsuccessful. Because of the impossibility to get an infection using this biological material, the structures observed by TEM were considered as virus-like particles rather than mature virions. 3.2. Transmission electron microscopy For both AnchV and Shy-VLP, large numbers of particles were found in crude extracts of dead or moribund larvae examined by electron microscopy. Negatively stained preparations of AnchV revealed non-occluded, non-enveloped, 32 ± 2 nm in diameter, icosahedral particles (Fig. 2a). Some of the virions exhibited a white core. The large number of presumed capsomers corresponding to particle degradation demonstrated that the conditions for the processing of the virus-infected individuals were not optimal. Similar results (data not shown) were obtained when using other classical buffers, TE, TEN or RNALater. This degradation occurred despite using NaCl-complemented buffers exhibiting characteristics close to the parameters we measured for the hemolymph of potato tuber moth larvae, a neutral pH and an osmolarity of about 300 milliosmoles. Shy-VLP were structurally similar to AnchV virions except that their white core was much less obvious (Fig. 2b). Their diameter was also of approximately 32 ± 2 nm. Because it was impossible to multiply Shy-VLP, no biological material was available for electron microscopy observations of infected cells in larvae. Similarly to what was observed in the case of AnchV preparations, large numbers of free presumed capsomers were noticed in Shy-VLPdead pupae after their homogenization. Electron microscope examination of ultra-thin sections of AnchV-infected cells showed intracytoplasmic aggregates of isometric virus particles located inside vesicles (Fig. 3). These vesicles apparently have a single-layer membrane. Viral particles were also observed free in the cytoplasm but in lower amounts than the vesicle-contained viral particles. This could be corresponding to a later stage of the infection in which the surrounding membrane has disappeared. No paracrystalline arrays were observed, even in heavily infected cells. 3.3. Density gradient centrifugation Even using large groups (>200 individuals) of AnchV-infected larvae, it was impossible to get significant amount of virus by centrifugation on sucrose or glycerol gradients. The viral particles were destroyed during the process, probably due to changes in osmotic pressure. Direct pelleting of the viral particles without the gradient step, allowed recovery of some biological material although more than 90% of the virions were lost as estimated by SDS–PAGE comparison of viral proteins of starting homogenate and final concentrate. NaCl–PEG precipitation allowed the recovery of AnchV virions but other contaminant proteins were also present as seen after SDS–PAGE (data not shown). It was possible to get small quantities of highly purified AnchV using Viraffinity , a water insoluble polyelectrolyte that has been designed for the purification of nonenveloped viruses while preserving infectivity. However, in both TM
Fig. 1. Healthy (left) and Anchilibi virus-dead (right) larvae of Tecia solanivora. (Bar equivalent to 2 mm).
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Fig. 2. Transmission electron microscopy of negatively stained viral particles isolated from Tecia solanivora (crude preparations). (A) Anchilibi virus (Top left corner: Close-up on virions). (B) Shyris virus. EmpVP: empty viral particles; CoVP: core-exhibiting viral particles, Caps: isolated presumed capsomers. Bar equivalent to 100 nm (A and B) or 50 nm (Insert).
Fig. 3. Transmission electron microscopy of an ultra-thin section in an AnchV-infected Tecia solanivora larva infected by Anchilibi virus (VP: viral particles; Mb: unilayer membrane surrounding virus aggregate; Mit: mitochondria; Bar equivalent to 0.3 lm).
cases, SDS–PAGE analysis demonstrated that the loss of virus was very high, in the same order as during gradient purification. Once again, this might be due to sub-optimal conditions of pH or osmolarity encountered by the virions during the purification process.
viral particles also have 3 major proteins (Fig. 4, lane 4) but their molecular weights were different of those of AnchV, 40.4 ± 2.0, 37.7 ± 2.0 and 33.4 ± 2.0 kDa, respectively. The second protein was significantly more abundant than the other two.
3.4. Electrophoresis of virus proteins
3.5. Agarose gel electrophoresis of viral nucleic acids
The AnchV exhibited three main proteins of respectively, 102.0 ± 2.1; 95.8 ± 2.0 and 92.4 ± 1.5 kDa in SDS–PAGE (Fig. 4). Mean sizes were calculated from 14 gel runs. In some cases, the proportional abundance of these three proteins was variable between individual larvae. Several adults emerged from larvae which survived exposure to AnchV were found to be infected and exhibited the same protein pattern as larvae. However, some other AnchV-infected adults collected before they died only had either the two higher weight proteins or the two lower ones (Fig. 4, lane 3). Microsequencing of AnchV proteins did not return an amino acid sequence. Apparently, the three proteins had an N-terminal modification which precluded Edman degradation procedure. Shy-VLP
Three bands (termed L, M and S) were identified by agarose gel electrophoresis of the viral nucleic acids extracted from either AnchV or Shy-VLP (Fig. 5). Treatment of the samples with ribonuclease A in 300 mM NaCl prior to electrophoresis resulted in the disappearance of the bands whereas, the addition of RNAse-free deoxyribonuclease did not eliminate the bands (data not shown). From their electrophoretic mobility in agarose gel, the molecular weights of the three AnchV RNAs were estimated to be 4.1 ± 0.2, 2.8 ± 0.2 and 1.65 ± 0.2 kb, respectively. Surprisingly, those of Shy-VLP RNAs had identical sizes. In the case of AnchV, the relative intensity of the three bands slightly varied between different RNA extractions. This raises the
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3.6. Bioassays Most of the first-instar larvae of T. solanivora exposed to AnchV died within 4 days (Fig. 6). This occurred in both treatments, i.e. larvae fed virus-treated tubers (81.0 ± 2.0% mortality) or larvae directly immersed in the larval homogenate (82.0 ± 8.0% mortality). Despite the mortality rates were almost identical, the limited number of replicates (2) for immersion treatment meant that no statistical comparison could be made between the two treatments. The virulence of the pathogen was obvious considering that, first, only a small amount of the viral solution was deposited on each tuber and, secondly, the period of virus acquisition was reduced as, in general, the mining neonates rapidly entered their galleries following emergence. Using the immersion protocol, AnchV also caused high mortality in P. operculella (54.0 ± 4.0%) and S. plaesiosema (58.5 ± 3.0%) larvae albeit lower than in T. solanivora. Because of the limited numbers of repetitions (2 replicates/species), it was not possible to determine whether the final mortality rates were statistically different between P. operculella and S. plaesiosema larvae contaminated following the immersion method. Identically, more bioassays would be necessary to establish whether T. solanivora is really more susceptible to AnchV than the two latter species. 4. Discussion
Fig. 4. SDS–polyacrylamide gel of viral particles. (A) Lane MW: protein molecular weight marker; Lane AV: Anchilibi virus; (B) Lane MW: protein molecular weight marker; Lane SV: Shyris virus; (C) Lanes Ad1 to Ad3: Anchilibi virus from alive infected adults).
Fig. 5. Agarose gel electrophoresis of formaldehyde-denaturated viral genomes ((A) Lane MW: RNA molecular weight marker with bands of 9.49, 7.46, 4.40, 2.37 and 1.35 kb, respectively; Lane AV: Anchilibi virus; (B) Lane MW: RNA molecular weight marker with bands of 9.49, 7.46, 4.40, 2.37 and 1.35 kb, respectively; Lane SV: Shyris virus).
question of whether the three RNAs were encapsidated together in the same virion or in different viral particles. This phenomenon was not noticed in the case of the RNAs of Shy-VLP.
Insect viruses are presently distributed among 24 taxonomic families, two unassigned genera (Iflavirus and Tenuivirus) and a number of unclassified viruses (Fauquet et al., 2005) which can exhibit almost all types of genome structure: monopartite or pluripartite genomes of single or double-stranded nucleic acids which can be either DNA or RNA. More than half of the groups of invertebrate viruses (13 families and the two genera) exhibit ssRNA genomes. Among them, only the Bunyaviridae are presently known to have a tri-segmented ssRNA viral genome (Fauquet et al., 2005). Characteristics of members of the Bunyaviridae are quite different from those of AnchV and Shy-VLP. Structurally, virions of Bunyaviridae are much larger (80–120 nm in diameter), enveloped and display glycoprotein spikes. The sizes of the virion proteins, which vary among the five genera of the family Bunyaviridae, are quite different from those of AnchV or Shy-VLP. The differences are also obvious when considering the sizes of the three genomic RNAs. In the Bunyaviridae the smaller one (S) ranges between 0.9–3 kb, the medium one (M) between 3–5 kb and the larger one (L) between 6–12 kb. Thus, it is unlikely that AnchV or Shy-VLP could be assigned to Bunyaviridae. Moreover, even more differences exist between AnchV and Shy-VLP and the other groups of insect viruses. Considering that the insects are by far the most numerous group of eukaryotes (several millions of species) and that only about half of the known viruses (around 7000 species) were isolated from insect hosts, it is reasonable to expect to find new types of genomic organization in entomoviruses. The results of the study presented here provide such evidence. AnchV and Shy-VLP were isolated from the same host species and share most of their characteristics as the size and organization of their RNA genome fragments or the size and structure of their viral (or virus-like) particles. The more striking difference was found in their respective protein patterns. While AnchV exhibited high molecular weight capsid proteins (around 90–100 kDa), Shy-VLP only presented proteins in the range 30–40 kDa. Considering this, the most parsimonious hypothesis of the nature of ShyVLP is that they are non-infectious particles of AnchV that have undergone some form of post-mortem degradation in the host that also results in loss of infectivity. These degradations might be due to the fact that viral particles stayed almost two weeks inside their dead hosts in the rearing facility (15–20 °C) before these individu-
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% Cumulative corrected mortality
100 90 80 70 60 A
50
B
40
C
30
D
20 10 0 2
3
4
Days after infection Fig. 6. Time-mortality response of first-instar larvae of the three potato tuber moth species infected with Anchilibi virus (at a concentration of 10 larva-equivalents/L). (A) T. solanivora larvae fed surface-contaminated tubers. (B) T. solanivora larvae contaminated by a 5-s immersion in the virus solution. (C) P. operculella larvae contaminated by a 5s immersion in the virus solution. (D) S. plaesiosema larvae contaminated by a 5-s immersion in the virus solution. (Final mortality rate in controls: A = 7%; B = 7%; C = 6%; D = 8%).
als were diagnosed as dead and then processed. Also, a different processing of capsid proteins could have been occurred in pupae (the source of Shy-VLP) compared to larvae. These alterations would be related to the incapacity for Shy-VLP to multiply in larvae during contamination trials. Indeed, the entry of viruses in host cell generally implies the interactions between cellular receptors and viral proteins located on the capsid surface. Sequence data would be able to definitively establish whether AnchV and Shy-VLP belong to the same virus species or not. There is no known insect virus exhibiting three capsid proteins with sizes between 102 and 92 kDa. Considering the size of the heaviest AnchV capsid protein (more than 100 kDa corresponding to at least 3000 encoding bases), it is obvious that the capsid coding gene can only be located on the L fragment since the others are smaller than this minimum 3 kb size required. Similarly, the presence of the two other proteins of about 95.8 and 92.4 kDa could be explained by the processing of the 102 kDa protein. This assumption is supported by the fact that the proportions of the three proteins were not constant but showed differences in their molarity according to the virus batches analyzed. Also, in some AnchV-infected adults, only two of the three bands were distinguishable on the SDS–PAGE gels. This indicates that the capsid protein processing in adults and larvae may be somewhat different, depending on protease activity and specificity of the insect instar. Probably because of their small sizes, the two small fragments (5–7 kDa) from the processing were not detected on gels. Proteolytic cleavage of a capsid precursor is known from different ssRNA insect virus families: Nodaviridae, Picornaviridae, Tetraviridae, Dicistroviridae (Ball and Johnson, 1998; Fauquet et al., 2005). This strategy can lead to a change in the ratio between proteins (the precursor and its derived products can be more or less abundant according to the efficiency of the proteolytic cleavage). Because of size considerations, it is probable that the replicase gene is located on fragments M or S; the former being the best candidate. Considering the similar sizes of the three RNA fragments in both viruses, it is tempting to assume that, in Shy-VLP, the L fragment might also bear the capsid gene(s). Similarly to AnchV, the capsid proteins could perhaps be generated through the cleavage of a precursor although the cumulative size of the nucleotide sequences necessary for separately coding the three proteins would not exceed the total size of the largest Shy-VLP fragment (L). The molecular weights of Shy-VLP proteins are close to those of insect picorna-like viruses (Christian and Scotti, 1998; Fauquet et al.,
2005). However, these viruses exhibit monopartite ssRNA genomes of 8–10 kb in size, which is quite different from what was found for AnchV and Shy-VLP. Three genera (Bromovirus, Cucumovirus and Ilarvirus) of the plant-infecting and insect-transmitted Begomoviridae family share several characteristics with AnchV and Shy-VLP. First, they exhibit icosahedral virions of 26–35 nm of diameter. Similarly to AnchV and Shy-VLP, they have tri-partite ssRNA genomes whose total length is approximately 8 kb. However, an important difference is that Begomoviridae have a unique capsid protein of 20–24 kDa (Fauquet et al., 2005). As most of the AnchV and Shy-VLP viral particles were damaged when centrifuged on sucrose or glycerol gradients, it was not possible to evidence the presence of distinct virus bands which could be related to the existence of particles exhibiting different densities. So, more investigations will be necessary to establish whether the three RNAs are encapsidated in the same viral particle. However, in the case of AnchV, the molar ratios of the three RNAs were not constant which suggest the existence of virions with different RNA contents. This was not observed for Shy-VLP perhaps because of a reduced number of batches analyzed. It is known that some virus groups encapsidate subgenomic RNAs of viral origin (for example, members of the insect-infecting Betatetravirus) or non viral RNAs as cellular mRNAs, tRNAs or rRNAs (Levin and Seidman, 1979; Adkins and Hunter, 1981; Jiang et al., 1993; Rumenapf et al., 1995; Berkowitz et al., 1996; Rulli et al., 2007). Such a situation could not be excluded in the case of AnchV or/and Shy-VLP but only complete sequencing of the viral genomes will permit to establish whether subgenomic or cellular RNAs are encapsidated in their virions. Genome-fragmented viruses can present a reassortment of genome fragments during replication in the same infected cell (Holland et al., 1982; Lin et al., 2004; Tomohisa et al., 2005; Chare and Holmes, 2006; Hay et al., 2001). This mechanism allows for large-scale evolutionary jumps. Considering this, it is possible that AnchV and Shy-VLP RNA fragments have different phylogenetic origins. Only sequence data will provide unequivocal information on the possible relationships that exist between AnchV or ShyVLP and the known groups of viruses. AnchV appeared as a fast-acting and virulent pathogen against the three species of potato tuber moths. Complementary trials should be carried out in order to establish whether AnchV is more active against T. solanivora. If it were the case, this might be related
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to the fact that AnchV was originally isolated from this species and thus, could be more adapted to its original host than to the two alternate host species tested. However, the three species are sympatric in different areas of Ecuador and are sometimes found in the same potato batches. According to the host range of the virus as determined through laboratory bioassays, it is highly probable that individuals belonging to any of the three potato tuber moth species are infected by AnchV in the wild. Works are underway to confirm this hypothesis. The presence of large amounts of AnchV in apparently healthy adults points to a possible vertical transmission of this pathogen. This would help to a better dissemination of AnchV because flying adults offer a much higher capacity of dispersion than larvae. A particular problem encountered during the study is that AnchV particles presented a marked susceptibility to degradation. The collection of AnchV-infected larvae before their death greatly enhanced the quantity of biologically active particles recovered. Degradation might be caused by proteases and other molecules (and, especifically, polyphenols) released after the lysis of cells (Wilson et al., 2001; Shelby and Popham, 2006). So, early collection before death could limit this phenomenon. On the other hand, important losses which occurred during centrifugation on sucrose gradients were probably due to osmotic pressure changes. Specific experiments should be carried out to solve this problem. This would allow for the preparation of the large amounts of purified virus necessary for further studies, as well as, the analysis of the sedimentation pattern of the virus in a sucrose gradient. This might provide valuable data to determine whether the three RNA segments are all present in the same viral particle. Finally, it is highly probable that both AnchV and Shy-VLP belong to a new taxonomic group of insect viruses as even in prokaryotes, plants and vertebrates, no tri-partite ssRNA virus exhibits all the other characteristics of AnchV and Shy-VLP. Apart of its academic interest, AnchV could offer promising perspectives as a biocontrol agent. Potato is an essential staple food for poor-resource families of the Andean highlands. The high level of damage caused by potato tuber moth larvae threaten the food security of these farmers, as well as, jeopardize their health (Yanggen et al., 2003) due to an increase in chemical insecticide application. All biological control agents with potential to be used in pest management programs should receive attention. In particular, AnchV seems to offer promising opportunities because of its ability to infect the three potato tuber moth species present in the same area. In comparison, PhopGV, which is currently used for the control of P. operculella in Peru, Bolivia and for control of T. solanivora in Venezuela and Colombia (Niño and Notz, 2000; Raman et al., 1987; Das et al., 1992; López and Barreto, 2003) is unable to provide an adequate control of S. tangolias which could limit its use in some areas. Obviously, toxicological tests should first be carried out in order to establish that the entomopathogen does not present a threat to non-target species including vertebrates. Then, trials will determine its real potential for pest control under potato storage conditions. The prevailing environmental conditions (no rain or UV radiation, temperatures generally in the range 5–15 °C) in the storage areas could be favorable to the use of an AnchV-based biopesticide. Acknowledgments The authors gratefully acknowledge two anonymous reviewers. They are also indebted to Dr. Marc Ravallec (Université de Montpellier II, France) and Dr. Yasuji Amano (Instituto Izquieta Perez, Guayaquil, Ecuador) for technical assistance in electron microscopy and Miss. Daniela Chevasco (PUCE) for technical assistance in gel electrophoresis and bioassays. They also acknowledge Dr.
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