[Organization of densovirus genome illustrated by the mosquito densovirus].
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Amino Acid Sequence↗
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Insect densoviruses may be widespread in mosquito cell lines.
A diagnostic PCR assay was designed based on conserved regions of previously sequenced densovirus genomic DNA isolated from mosquitoes. Application of this assay to different insect cell lines resulted in a number of cases of consistent positive amplification of the predicted size fragment. Positive PCR results were subsequently confirmed to correlate with densovirus infection by both electron microscopy and indirect fluorescent antibody test. In each case the nucleotide sequence of the amplified PCR fragments showed high identity to previously reported densoviruses isolated from mosquitoes. Phylogenetic analysis based on these sequences showed that two of these isolates were examples of new densoviruses. These viruses could infect and replicate in mosquitoes when administered orally or parenterally and these infections were largely avirulent. In one virus/mosquito combination vertical transmission to progeny was observed. The frequency with which these viruses were detected would suggest that they may be quite common in insect cell lines.
Animals↗
The structure of an insect parvovirus (Galleria mellonella densovirus) at 3.7 A resolution.
BACKGROUND: Parvoviruses infect vertebrates, insects and crustaceans. Many arthropod parvoviruses (densoviruses) are highly pathogenic and kill approximately 90% of the host larvae within days, making them potentially effective as selective pesticides. Improved understanding of densoviral structure and function is therefore desirable. There are four different initiation sites for translation of the densovirus capsid protein mRNA, giving rise to the viral proteins VP1 to VP4. Sixty copies of the common, C-terminal domain make up the ordered part of the icosahedral capsid. RESULTS: The Galleria mellonella densovirus (GMDNV) capsid protein consists of a core beta-barrel motif, similar to that found in many other viral capsid proteins. The structure most closely resembles that of the vertebrate parvoviruses, but it has diverged beyond recognition in many of the long loop regions that constitute the surface features and intersubunit contacts. The N termini of twofold-related subunits have swapped their positions relative to those of the vertebrate parvoviruses. Unlike in the vertebrate parvoviruses, in GmDNV there is no continuous electron density in the channels running along the fivefold axes of the virus. Electron density corresponding to some of the single-stranded DNA genome is visible in the crystal structure, but it is not as well defined as in the vertebrate parvoviruses. CONCLUSIONS: The sequence of the glycine-rich motif, which occupies each of the channels along the fivefold axes in vertebrate viruses, is conserved between mammalian and insect parvoviruses. This motif may serve to externalize the N-terminal region of the single VP1 subunit per particle. The domain swapping of the N termini between insect and vertebrate parvoviruses may have the effect of increasing capsid stability in GmDNV.
Amino Acid Sequence↗
Complete nucleotide sequence of the cloned infectious genome of Junonia coenia densovirus reveals an organization unique among parvoviruses.
We previously constructed a recombinant plasmid, pBRJ, encompassing an infectious Junonia coenia densovirus (JcDNV) genome (M. Jourdan et al. (1990). Virology 179, 403-409). We report here the complete viral sequence of pBRJ. The genome, 5908 nucleotides (nt) long, consists of an internal unique sequence flanked by long (517 nt) inverted terminal repeats. The first 96 bases of one extremity can fold into a typical Y-shaped hairpin structure. The opposite extremity is incomplete, lacking 88 nt. These terminal structures, similar to those of dependoviruses, human parvovirus B19 and Bombyx mori densovirus (BmDNV), strongly suggest a common mechanism of DNA replication for these parvoviruses. JcDNV genomic organization is unique among parvoviruses in that coding sequences are evenly distributed in the 5' half of both strands. On one strand, the major open reading frame (ORF1) encodes the four structural proteins. On the complementary strand, ORF2, ORF3 (included in ORF2), and ORF4 probably encode nonstructural proteins. JcDNV genome has little DNA homology with vertebrate parvoviruses and surprisingly even less with the two densoviruses presently sequenced. ORF1 contains the highly conserved PGY and G-rich regions and ORF2 the NTP-binding domain common to most structural and to all nonstructural vertebrate parvoviral ORFs, respectively. The single homology between JcDNV and BmDNV is unexpectedly located in JcDNV NTP-binding domain and BmDNV ORF2 assumed to encode structural polypeptides. Only a weak homology exists between JcDNV and Aedes DNV in their NTP-binding domain.
Amino Acid Sequence↗
Restriction map of the Casphalia extranea densovirus genome.
A physical map of the Casphalia extranea densovirus genome (CeDNV) was constructed. The size of the intact viral genome was estimated to be 4.9 kilobases or 1.6 MDa (single strand). The double-stranded CeDNV genomic DNA was cleaved with 26 restriction endonucleases and 20 restriction sites were mapped on the genome. The CeDNV DNA restriction map was compared to those of other densoviruses. Southern blotting hybridization experiments failed to reveal any homology between the genomes of CeDNV and Junoniacoenia densovirus (JcDNV).
Blotting, Southern↗
Transduction of Aedes aegypti mosquitoes with vectors derived from Aedes densovirus.
Aedes densovirus (AeDNV)-based constructs that express green fluorescent protein (GFP) from either the P7 or the P61 promoter were made. The construct in which GFP protein was expressed as a fusion protein to the C-terminus of NS1 (NS1-GFP) showed the highest level of GFP expression. This hybrid NS1-GFP protein preserved the biological functions of the parental proteins: it showed GFP fluorescence, it stimulated expression from the virus promoters, and it facilitated rescue and replication of the cloned AeDNV genome. Similar to NS1, the hybrid NS1-GFP localized in the nucleus predominantly in a punctate pattern. Transducing virus particles carrying the NS1-GFP gene infected mosquito larvae. Expression of GFP was detected as early as 48 h postinfection and in larval and pupal stages. Midgut, hindgut, and Malpighian tubule cells expressed GFP soon after transduction. However, the anal papillae were the most commonly infected organ system. The anal papillae are syncytia and regulate ion concentration in the hemolymph of mosquito larvae, and they might be a novel route of mosquito larvae infection with densoviruses.
Aedes↗
Packaging of AeDNV-GFP transducing virus by expression of densovirus structural proteins from a sindbis virus expression system.
Genetic recombination resulting in the production of wild-type infectious virus is an obstacle in the current system for producing densovirus transducing particles. In order to eliminate this problem, a double subgenomic Sindbis virus (TE/3'2J/VP) was engineered that expresses the structural proteins (VPs) of Aedes densonucleosis virus (AeDNV) from the second subgenomic promoter. Expression of AeDNV VPs from TE/3'2J/VP was confirmed by Northern analysis of RNA from infected C6/36 (Aedes albopictus) cells and by indirect immunofluorescence in infected C6/36 cells and BHK-21 cells. TE/3'2J/VP was used to infect C6/36 cells transfected with p7NS1-GFP, a plasmid expressing the nonstructural genes of AeDNV and green fluorescent protein (GFP) as a reporter gene. This infection resulted in the production of AeDNV-GFP transducing virus, which is infectious to C6/36 cells and Aedes aegypti larvae, as determined by GFP expression. The TE/3'2J/VP packaging system produced titers of transducing virus comparable to those produced by the standard two-plasmid method. The possibility of recombination resulting in wild-type infectious virus in transducing densovirus stocks was eliminated by employing an RNA virus expression system to supply AeDNV structural proteins.
Aedes↗
Construction of an infectious clone of Spodoptera frugiperda densovirus and its biological characteristics.
Densoviruses are highly pathogenic to their insect hosts and have great potential for biocontrol. Spodoptera frugiperda densovirus (SfDV) was isolated from diseased larvae of Spodoptera frugiperda, while its biological functions remain unclear. Herein, we successfully constructed an infectious clone of SfDV. The S. frugiperda larvae transfected with the infectious clone exhibited anorexia, stunted growth, and reduced activity. Histopathological analysis further showed that the epidermis, fat body and trachea were infected instead of muscle and midgut tissues. Transmission electron microscopy (TEM) revealed that numerous virions of about 22 nm were distributed within both the nucleoplasm and cytoplasm of epidermal cells. Moreover, many virions were also found contained within vesicles in the cytoplasm. The replication kinetics of the rescued SfDV (rSfDV) was similar to that of the parental SfDV. The median lethal dose (LD50) and median lethal time (LT50) values of rSfDV were 6.63 × 107 viral genome copies (vgc), 5.23 d, respectively, which were also comparable to those of the parental SfDV. Taken together, the infectious clone of SfDV provides an important tool for further exploring the genome function, pathogenesis, and interactions with its hosts.
Animals↗
Organization and nucleotide sequence of a densovirus genome imply a host-dependent evolution of the parvoviruses.
The genome structure of a densovirus from a silkworm was determined by sequencing more than 85% of the complete genome DNA. This is the first report of the genome organization of an insect parvovirus deduced from the DNA sequence. In the viral genome, two large open reading frames designated 1 and 2 and one smaller open reading frame designated 3 were identified. The first two open reading frames shared the same strand, while the third was found in the complementary sequence. Computer analysis suggested that open reading frame 2 may encode all four structural proteins. The genome organization and a part of the nucleotide sequence were conserved among the insect densovirus, rodent parvoviruses, and a human dependovirus. These viruses may have diverged from a common ancestor.
Amino Acid Sequence↗
Densovirus of Aedes aegypti as an expression vector in mosquito cells.
We have constructed an infectious DNA clone containing the genome of Aedes aegypti densovirus (AeDNV) in a bacterial plasmid. When this clone was transfected into Aedes albopictus C6/36 mosquito cells, the AeDNV genome rescued from the plasmid and replicated as the wild-type virus. To investigate the cloned virus as an expression vector, the reporter gene encoding beta-galactosidase (beta-gal) was inserted into four large open reading frames (ORF) observed in the AeDNV genome. When these recombinant constructs were transfected into Aedes albopictus C6/36 cells, the beta-gal was expressed efficiently from the right ORF (encoding capsid proteins, Vps) and the mid ORF (encoding putative nonstructural protein 2). A low level of expression was found from the left ORF (encoding nonstructural protein 1, NS1), and no expression was detected from the ORF observed on the minus strand of the AeDNV genome. The expression from the right, mid, and left ORFs can be trans-activated with NS1. A putative nuclear targeting sequence observed in the N-terminus of the AeDNV Vps is presumed to be responsible for transport of the chimeric beta-gal into nucleus. The recombinant AeDNV genomes (carrying the beta-gal gene) supplied with the AeDNV capsid proteins can be packaged into infectious transducing particles. Our results indicate that the genome of AeDNV can serve as a vector for delivery and expression of foreign genes in mosquito cells with subsequent targeting of the product to the desired cell compartment.
Aedes↗
A densovirus newly isolated from the smoky-brown cockroach Periplaneta fuliginosa.
We purified a causing agent of fetal disease for smoky-brown cockroach Periplaneta fuliginosa, which was designated as "cockroach small spherical virus (CSSV)". Purified virus particles had a diameter of 22 +/- 0.6 nm and contained DNA as a single-stranded form. However, the extraction of DNA under condition of appropriate high salt and elevated temperature yielded a double-stranded DNA with a size of 5,500 nucleotides. These results were quite similar to those of other densoviruses (DNVs). The CSSV had five structural proteins (VP1: 52 KDa, VP2: 56 KDa, VP3: 79 KDa, VP4: 82 KDa, and VP5: 105 KDa). The SDS-PAGE profile of these proteins was quite different from that of the cockroach DNV previously reported and was rather similar to that of Bombyx mori (Bm) DNV-1. An immunochemical study, however, demonstrated that there was no immunological relationship between the CSSV and the Bm DNV-1. These data suggest that the CSSV is a new member of DNV.
Animals↗
A titration procedure of the Junonia coenia densovirus and quantitation of transfection by its cloned genomic DNA in four lepidopteran cell lines.
A sensitive and reproducible tissue culture biossay method was developed based on indirect immunofluorescence to titrate virus suspensions of the Junonia coenia densovirus (JcDNV) and to quantify transfections by its cloned genomic DNA. Four lepidopteran cell lines, the SPC-SL 52 from Spodoptera littoralis, the SPC-PL 40 and the SPC-PL 65 cells derived from Spodoptera litura ovaries and hemocytes, respectively, and the SC-LD 135 from Lymantria dispar were compared for their efficiency to support viral replication. The viral titres expressed as TCID50/ml averaged 10(5) for SPC-SL 52, SPC-PL 40 and SC-LD 135 cells, but were above 10(7) for SPC-PL 65 cells. Even with this most sensitive cell line, the rate of infected cells did not exceed 75% and decreased progressively by serial subcultures. Two transfection protocols were used to compare the sensitivity of the same four cell lines to a recombinant plasmid encompassing an infectious sequence of JcDNV genome. SPC-SL 52 cells were found to be the most sensitive, and the lipofection method resulted in about a 5-fold increase compared to the calcium phosphate precipitation protocol. The rescued virions proved to be infectious and the restriction profiles of their DNA were identical to that of wild type virions.
Animals↗
Molecular cloning and characterization of Bombyx densovirus genomic DNA. Brief report.
A map of the Bombyx densovirus (DNV) genomic DNA has been constructed by cleaving it with several restriction endonucleases. This map shows that this virus and the mammalian parvoviruses are distinct. A restriction fragment containing more than 85 per cent of the complete viral genome has been cloned in a bacterial plasmid.
Chromosome Mapping↗
Cloning of the genome of a densovirus and rescue of infectious virions from recombinant plasmid in the insect host Spodoptera littoralis.
We have cloned an infectious genome of the Junonia coenia densonucleosis virus (JcDNV) into the bacterial plasmid pBR322. The viral genome could be rescued from the recombinant plasmid pBRJ by transfection of pBRJ DNA to sensitive Spodoptera littoralis larvae. pBRJ DNA produced a typical viral infection and a comparable percentage of larvae became infected following inoculation of equivalent amounts of purified virion DNA or cloned viral DNA. Virions extracted from transfected larvae were indistinguishable from wild-type (wt) virions with regard to their biophysical and biological properties. In particular, rescued virions were as infectious as wt virions and showed identical restriction profiles of their genome. In contrast, subcloning of JcDNV DNA deleted at both extremities of a sequence of ca 250 or ca 100 bp resulted in the inability of the recombinant plasmids to initiate a viral infection. These data suggest that, as for vertebrate parvoviruses, the inverted terminal repeats display essential functions in the rescue process and replicative cycle of densoviruses. This is the first report of the molecular cloning of the infectious genome from an insect parvovirus, and more generally from an invertebrate virus. pBRJ should provide an efficient tool to further define the organization of the JcDNV genome and compare it to other parvoviruses.
Animals↗
The densovirus of Junonia coenia (Jc DNV) as an insect cell expression vector.
An infectious genome of the Junonia coenia densovirus (Jc DNV) has been recently cloned and sequenced. We investigated the ability of this cloned genome to be used as expression vector by inserting the lacZ gene of Escherichia coli as fusion gene in the major open reading frame (ORF 1) of the viral sequence. The resulting recombinant plasmid designated pBRJlac Z was transfected into insect SPC-SL 52 cells and the expression of beta-galactosidase (beta-gal) was detected qualitatively or quantitatively by using Xgal or ONPG as chromogenic substrates. Western blot analysis revealed that beta-gal was expressed as chimeric capsid-beta-gal polypeptides. This provided evidence that ORF1 codes for structural polypeptides which share a common C-terminal sequence. Construction of plasmids with alterations or deletions in ORF2, 3 or 4, allowed us to implicate nonstructural (NS) functions in viral DNA replication. Deletions in inverted terminal repeats or in NS functions did not abolish expression of capsid polypeptides but reduced it dramatically. Encapsidation of Jlac Z recombinant genome was achieved by trans-complementation with plasmids bearing intact structural and nonstructural functions. Detection of a beta-gal activity in SPC-SL 52 cells following several subcultures post-transfection suggests that Jlac Z recombinant genome could be maintained in an integrative or episomal state.
Animals↗
[Experimental study of densovirus infection transmission in blood-sucking mosquito populations].
The paper presents the results of laboratory study of the modes of entomopathogenic densonucleasis virus transmission in the population of blood-sucking Culicidae mosquitoes. It was established that densovirus infection was horizontally transmitted through feeding of larvae on the bodies of desonucleasis-infected insects as well as contamination of the environment by infected larvae. Thus, the death rate in populations of various mosquito species attained 25.9-70.8%. Vertical transmission of the virus alters the ratio of sexes at the stage of imago; the survived females and males transmit infection to larvae. Surface sterilization of the ova doesn't prevent larvae infection, death rate among larvae attaining 7.8%.
Animals↗
Autonomous parvovirus and densovirus gene vectors.
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Animals↗