Densoviruses (DNVs),pathogens for invertebrates belong to the subfamily Densovirinae of the family Porvoviridae (32, 36) and share many characteristics with the defective and autonomous vertebrate parvoviruses (3, 30). Densoviruses have been isolated from several insect species belonging to different orders,mainly from Lepidoptera (butterflies and moths) and a few from other orders,including Diptera,Orthoptera,Dictyoptera,and Odonata. All known DNVs package complementary,linear single strands of the DNA into se-parate virions,as do several parvoviruses of vertebrates (e.g.,B19 and AAV) (8) ,and replicate autonomously in the nucleus where they produce a typical dense nuclear inclusions (24, 35). DNVs have little sequence identity with the vertebrate parvoviruses (7). Further studies indicate that there are at least three distinct DNV groups,one genus (Densovirus),exemplified by Junonia coenia DNV (JcDNV) (9, 15) ,a second genus (Brevidensovirus) by the Aedes (1, 4) and shrimp (28) DNVs,and a third genus (Iteravirus) by the Casphalia DNV (13) and Bombyx mori DNV type 1 (19). Most DNVs remain unclassified,since few are characterized with respect to their molecular biology. DNVs are autonomously replicating small icosahedral non enveloped particles,20 to 23 nm in diameter,with their capsid consisting of 4 structural polypeptides denoted VP1,VP2,VP3 and VP4 ranging from 41 kDa to over 100 kDa (17, 25, 33, 34).
Restriction maps of several densovirus genomes have been determined and many symmetrical sites were found (9, 15).Observation of linear double stranded monomers or concatemers and single stranded circular monomers with "panhandle" structures (6, 16) suggest the presence of inverted terminal repeat (ITRs). This structure was confirmed by nucleotide sequences of cloned densovirus genomes (9, 19, 31, 33). The ITRs were found to play essential roles in the process of DNA replication,DNA excision from plasmid vectors and integration into the host DNA (2, 27, 29).
The densovirus of Diatraea sacchararalis (DsDNV) was isolated by Meynadier et al (23) from the Guadeloupe sugar cane borer,D. saccharalis (Lepidoptera Pyralidae) which is a major sugar cane borer in Brazil and Guadeloupe. The estimation of loses by this borer is about $ 100 M per year (21, 22). High pathogenicity combined with a limited host range gives some densoviruses potential as effective insecticides (11). Several densoviruses have been used successfully in biological control of pests in the world (5, 11, 14). However,safety concerns remain as previous reports (10) suggested that densoviruses may infect and transform L cells (from mouse). El-far et al. (18) recently demonstrated that neither L nor other vertebrate cells support replication or transcription of densovirus,either after infection or after transfection by using molecular biology tools. Therefore,the DsDNV isolated from this insect host repre-sents a potential biological control agent for the D. saccharalis pests (20). In this paper,we report the pathogenicity of the DsDNV in its host larvae,the characte-rization of the genome including visualization of DNA molecules by electron microscopy (EM.),the restriction map of the viral genome and the comparison with two well known densovirus genomes of JcDNV and GmDNV.
Three lots of D. saccharalis third star larvae were used for this experiment. Two lots (Lot Ⅰ and Lot Ⅱ) were infected with DsDNV while the third Group (GIII) was used as a control. Dead larvae were collected and counted every three days. The cumula-tive percentage of dead larvae from the three lots is presented in Fig. 1. The results showed that up to 4 days after inoculation,cumulative mortality curves were similar for the three groups and the infected larvae started to exhibit the infection symptoms from the forth day. Symptoms of infection of larvae started with anorexia and lethargy followed by flaccidity and inhibition of moulting and metamorphosis. Larvae become paralyzed and stop feeding after 7 days. After 5 days of infection,the cumulative mortality of infected larvae in both infected lots increased significantly and reached 60% for Lot Ⅰ and 40% for Lot Ⅱ after 12 days. Finally,100% of mortality was achieved after 21 days of infection for both Lot Ⅰ and Lot Ⅱ,whereas that of the control group was only 10% and 20%,respectively,after same periods of infection,suggesting that the high mortality of infected larvae groups was due to high pathogenicity of DsDNV. This was confirmed by the purification of DsDNV virions from the infected larvae but not from dead larvae of control group.
The dsDNA molecules of DsDNV were extracted and examined under the electronic microscope (EM). They appeared in EM as linear molecules (Fig. 2A). Size was measured and calculated ranging from 1.97 to 2.08 μmol/L from 18 viral DNA molecules. The average size of these molecules was estimated to be 2.03 ±0.03 μmol/L. This value was adjusted according to the size of known length of circular dsDNA (replicative form) of X 174 bacteriophage (Fig. 2B). This DNA marker molecule of 3.4×106 MDa was expected to be 1.77 μm in length and appeared to be 1.80 μm in length when observed by EM according to our method. Thus,it is known that 2.95 kb (1.92 MDa) correspond to 1 μmol/L,the dsDNA of DsDNV was calculated to be 3.9 (±0.05) MDa or 5.9 ±0.08kb.
The restriction fragments were separated in 0.8% to 1.8% agarose gels. An example of this experiment is shown in Fig. 3. For fragments too small to be visualised on agarose gel,8% ~ and 9 % SDS PAGE was used (see materials and methods). 37 restriction endonucleases were used,only 21 of them cut the viral DNA. Some cleaved once the DNA,others generated from 2 to 9 fragments or more. The number of restriction sites and the size of the generated fragments are summari-zed in Table 1. These sizes were corrected by linear regression using DNA MW marker Ⅵ and Ⅶ (Roche). The restriction enzymes Cla Ⅰ and Pst Ⅰ generated 2 fragments each,while enzymes Asp 700,BamH Ⅰ,EcoR Ⅰ,HindⅡ,Hpa Ⅰ,Nco Ⅰ and Nsi Ⅰ generated 3 fragments. The Bcl Ⅰ,Bgl Ⅱ,Xha Ⅰ and Spe Ⅰ generated 4 fragments; Hae Ⅲ and Hha Ⅰ gave 5 fragments,while Sca Ⅰ gave 6 fragments. Alu Ⅰ,Hinf Ⅰ,Dra Ⅰ,Taq Ⅰ and Sau 3A gave 9 or more fragments each. Due to the limitation of our method,we were not able to detect DNA fragments less than 80 bp. Enzymes that did not cut the viral genome were: Ava Ⅰ,Asp 718,Avi Ⅱ,Bgl Ⅰ,BstE Ⅱ,Dpn Ⅰ,EcoR, HinB Ⅲ,Kpn Ⅰ,Ksp Ⅰ,Pvu Ⅱ,Sac Ⅰ,Sal Ⅰ,Sma Ⅰ,Sph Ⅰ and Xho Ⅰ. The average size of the ds DNA calculated by summing the sizes of the fragments gene-rated by each enzyme,was close to 5.95 kb. The gene-rated fragments were listed from the largest to the smallest (A,B,C,D,E,F) (Table 1). The total length of undigested DsDNV genomic dsDNA was also estimated on 1.2 agarose gel along with digested genomic DNA samples isolated from GmDNV and JcDNV and was densoviruses have similar genome sizes. The sizes of the restriction fragments generated by each enzyme were adjusted according to the value of 5.95 kb estimated for the undigested DNA.
Table 1. Restriction fragments of the genomic DNA of DsDNV.
Figure 3. Electrophoretic profile of restricted DsDNV genomic DNA. 1.2% Agarose gel. Lane 1/ 5/ 9,MW marker; Lane 2,Hha Ⅰ; Lane 3,Hha Ⅰ-BamH Ⅰ double digestion; Lane 4,BamH Ⅰ; Lane 6: Eco R Ⅰ; Lane 7,EcoR Ⅰ-Spe Ⅰ double digestion; Lane 8,Spe Ⅰ; Lane 10,Spe Ⅰ-HinC Ⅱ double digestion; Lane 11,Hinc Ⅱ; Lane 12: Nsi Ⅰ-Cla Ⅰ double digestion. Fragments of 80 bp or less were not detectable by this method.
The restriction map of the ds DNA of DsDNV was constructed using combination of total and partial digestion with each of the different enzymes,or simultaneous digestion with pairs of restriction enzymes. Since the orientation of the ssDNA of the DsDNV genomic particle is unknown,we arbitrarily decided that from the two fragments generated by Pst Ⅰ digestion,the largest one of 4 300 bp (A) is positioned on the right side (3'end) and the smaller fragment (B) of about 1650 bp is situated on the left side (5'end) (Fig. 4A). All the restriction fragments obtained using the other enzymes were positioned according to the orientation above. A total of 57 restriction fragments were ordered and allowed the construction of a comprehensive restriction map by the positioning of forty one restriction sites (Fig. 4A). The Sca Ⅰ,BamH Ⅰ and Hha Ⅰ restriction sites were situated symmetrically at both extremities,suggesting that there was an ITR structure at both ends.
Figure 4. Restriction map of DsDNV genomic DNA (A) and its comparison to that of JcDNV and GmDNV (B).
As mentioned above,GmDNV,JcDNV and DsDNV ds DNA have almost similar sizes of about 6 kb. To iden-tify whether there are some similarities of the restriction map among these DNVs,we compared the restriction maps of the genome of the DsDNV genomic DNA to those of JcDNV and GmDNV (Fig. 4B). The results indicated that the Bam HI restriction sites are similar for all three genomes. The three genomic DNA share four identical Hha Ⅰ restriction sites with 2 additional sites on JcDNV and GmDNV genomic DNA. Xba Ⅰ cleaves twice in similar positions the three genomes,but there is one more restriction site positioned differently on each of the genomes. Cla Ⅰ cleaves DsDNV and GmDNV identically. The Asp 700 restriction sites (2) are similarly positioned on JcDNV genome,even one more restriction site of this enzyme were found on JcDNV genomic DNA. The Spe Ⅰ endonuclease cut three times each genome of DsDNV and JcDNV with two similar positions. Nco Ⅰ and Bcl Ⅰ enzymes have only one restriction site similarly positioned on both DsDNV and JcDNV. Restriction enzymes Alu Ⅰ,Dra Ⅰ,Hinf Ⅰ,Sau 3A,and Taq Ⅰ cut both DsDNV and JcDNV many times. These sites were not mapped on the DsDNV genomes.