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Terminal structure of a Densovirus implies a hairpin transfer replication which is similar to the model for AAV.
We cloned the complete sequence of Bombyx DNV (Ina isolate; Bm DNV-1) genome in a bacterial plasmid pUC 119 and determined the nucleotide sequences of both termini, resulting in elucidation of the nucleotide sequence of the complete genomic DNA of DNV. The complete sequence of the DNV DNA (5048 nucleotides) has inverted repeats of 225 nucleotides and the terminal 153 nucleotides are palindromic. The palindromes can fold back on themselves to form a hairpin structure but, unlike AAV, the small internal palindrome which forms a T-shaped conformation was not observed. End-label analysis demonstrated that the palindromic sequences at both termini can exist in either of two orientations (flip or flop) in virion DNA with different frequencies. These data suggest that the hairpin transfer model for AAV replication must be modified to explain the DNV replication. Additionally, a comparison study on the terminal structures of insect, human, and rodent parvoviruses allowed a prediction on the ancestral terminal structure of parvovirus genome.
Restriction maps and sequence homologies of two densovirus genomes.
The genomes of Junonia coenia densonucleosis virus (JcDNV) and Galleria mellonella densonucleosis virus (GmDNV) were analysed by restriction endonuclease analysis and Southern blot hybridization. A total of 37 and 33 restriction sites were mapped on JcDNV and GmDNV DNA, respectively. BglI, HaeII and BstEII were site-specific for JcDNV DNA, and BglII and ClaI for GmDNV DNA. The two genomes had nearly identical maps for several restriction endonucleases and Southern blot hybridization using a total genomic JcDNV probe indicated extensive DNA sequence homologies spanning the entire length of the two genomes. Symmetrical cleavage sites, mapping at the extremities of both genomes, confirmed the presence of inverted terminal repeats of at least 420 to 440 bases in length.
[Features of the organization of the densovirus genome].
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MDV-like endogenous viral elements act as immune rheostats in Aedes cells by modulating defensin A-mediated responses to arboviruses.
Mosquito cell lines are essential tools for arbovirus research. Endogenous viral elements (EVEs) are prevalent in mosquito genomes, yet their functional effects on host immune responses remain unclear, potentially complicating experimental interpretations. In this study, we systematically characterized endogenous mosquito densovirus-like elements (EMLs) within the Aedes aegypti Aag2 cell line and found that these endogenous EMLs are transcriptionally active but translationally defective. The silencing of EML transcripts significantly diminished the replication of Zika virus (ZIKV), Japanese encephalitis virus (JEV), and chikungunya virus (CHIKV), while transiently increasing dengue virus 2 (DENV-2), thereby indicating a virus-dependent regulatory mechanism. Mechanistically, RNA sequencing after EML interference, alongside plasmid-based mimic expression, demonstrated that EML transcripts downregulate defensin A, an antimicrobial peptide produced by mosquitoes. Functional assays using synthetic defensin A showed that this peptide differentially regulates arboviral infection. Binding assays and structural modeling further supported its interaction with viral envelope proteins, while stage-restricted infection assays revealed distinct stages of action: defensin A enhanced adsorption of ZIKV, JEV, and CHIKV, but did not promote DENV-2 adsorption or entry, and instead reduced DENV-2 RNA accumulation at the post-entry replication stage. Our findings highlight a previously unrecognized role of densovirus-derived EVEs in mosquito innate immunity, extending their functional scope from the well-established PIWI-interacting RNA-mediated antiviral defense to the regulation of antimicrobial peptide-associated immune pathways. These findings emphasize the necessity of accounting for EVE activity when analyzing data derived from mosquito cell lines, and suggest that related EVE-mediated immune regulation may contribute to arbovirus dynamics in mosquitoes.IMPORTANCEMosquito-borne viruses such as dengue, Zika, Japanese encephalitis, and chikungunya continue to threaten human health worldwide. Laboratory studies often use Aedes aegypti cell lines to investigate how these viruses interact with their mosquito hosts. Here, we show that the genomes of these cells contain endogenous viral elements derived from mosquito densoviruses. Far from being inert fossils, these sequences are transcriptionally active and regulate mosquito immunity by suppressing the antimicrobial peptide defensin A. This immune modulation influences the replication of different arboviruses in opposite ways, enhancing some while restricting others. Our findings reveal that integrated viral elements can shape the outcome of arbovirus infection, with important implications for interpreting mosquito cell culture experiments and for evaluating endogenous viral element-mediated immune regulation in mosquito-virus interactions.
Structure, restriction map and infectivity of the genomic and replicative forms of AaPV DNA.
We have characterized the genomic and replicative form (RF) DNA of the Aedes albopictus Parvovirus (AaPV), a virus isolated from a chronically infected C6/36 clone of Aedes albopictus cell line [22]. The genome of AaPV virions is a single-stranded linear DNA molecule approximately 4.2 kb in length, essentially (about 90%) encapsidated as minus strand. A restriction map of the RF DNA isolated from infected C6/36 cells was established. Among the 23 restriction enzymes tested, 14 cleaved the AaPV RF DNA and 30 restriction sites were mapped and oriented with respect to the viral genomic DNA. Both viral and RF DNAs were found infectious when transfected to virus-free C6/36 cells. The asymmetrical encapsidation of the viral genome is a property common to most vertebrate autonomous parvoviruses but rather unusual among densoviruses. Both by its small size, the asymmetrical mode of encapsidation and the restriction map, the AaPV genome resembles that of the Aedes Densonucleosis virus [1].
An efficient and easy method of infection of mosquito larvae from virus-contaminated cell cultures.
A new method for efficient infection of Aedes aegypti larvae by the Aedes albopictus densovirus, AaPV is described. It consists of placing first or third instar larvae in culture flasks containing a chronically infected mosquito cell line. After 24 or 48 h of exposure to the contaminated culture, the larvae acquired the virus by feeding on infected cells. Using this technique, up to 95% of first instar Ae. aegypti larvae were found infected by the AaPV.
A parvo-like virus persistently infecting a C6/36 clone of Aedes albopictus mosquito cell line and pathogenic for Aedes aegypti larvae.
We have isolated and partially characterized from an apparently healthy C6/36 subclone of Aedes albopictus cell line a small icosahedral non-enveloped DNA virus, designated AaPV. This virus proved to be highly pathogenic for Aedes aegypti neonate larvae. Viral infection persisted for over 4 years in the cell culture without any cytopathic effect. Attempts to infect suckling mice, Drosophila melanogaster adults and Spodoptera littoralis larvae with AaPV were unsuccessful. Similarly, the AaPV failed to replicate in vertebrate and Drosophila cell lines. Virions, about 22 nm in diameter, had a buoyant density of 1.43 g/cm3 and contained three capsid polypeptides with molecular weights of 53, 41 and 40 kDa. A preliminary study of the viral genome indicated the presence of single-stranded DNA. By its biophysical and biochemical properties, this virus appears to be related to the genus Densovirus within the family Parvoviridae, but lacks serological relationships with the other members of this genus.
Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence.
Recently, a parvo-like virus was newly isolated from silkworm larvae and the two viral DNAs (VD1 and VD2) with different electro-mobilities were identified. We cloned the viral DNAs in a plasmid pUC119 and demonstrated that these two DNAs were not a bimorphic molecules though they shared a common terminal sequence of 53 nucleotides. In addition, the sequence at the 5' terminus of each strand of the viral DNA was located in inverted form at its 3' terminus. On the other hand, the nucleotide sequences of VD1 and VD2 were different from that of the Bombyx densovirus (Ina isolate) DNA.
Expression of densonucleosis virus GmDNV in Galleria mellonella larvae: size analysis and in vitro translation of viral transcription products.
The RNA of densonucleosis virus type 1 (GmDNV), isolated from GmDNV-infected Galleria mellonella larvae, was shown by Northern blotting to contain five polyadenylated, viral-specific RNA species with sizes of 1.8, 2.4, 3.5, 4.0, and 5.0 kb. Poly(A)-containing RNA from whole larvae and hybrid-selected viral RNA were translated in a rabbit reticulocyte lysate system and the translation products were coelectrophoresed with proteins extracted from CsCl-purified virus. All four virion-associated proteins, namely p49, p55, p65, and p94, were present in the in vitro-translated products. However, in the majority of the experiments the most abundant translation product was a 30K polypeptide which is absent from virion extracts. The most abundant viral protein is p49, and the 49K polypeptide was also the most abundant translation product in about 30% of the preparations. The 1.8 kb transcript, which constitutes about half of the total viral RNA, is only slightly larger than the template required for a 30K polypeptide, suggesting that the latter may be a primary translation product of the smallest RNA transcript. The similarities in gene expression between densoviruses and mam malian parvoviruses are discussed.
Characterization of hepatopancreatic parvo-like virus, a second unusual parvovirus pathogenic for penaeid shrimps.
The hepatopancreatic parvo-like virus (HPV) of penaeid shrimp was extracted from infected shrimp tissues, purified and subsequently characterized. The viral particles, icosahedral in shape, are 22 nm in diameter and possess a buoyant density of 1.41 g/ml. They contain ssDNA, of approximately 5 kb in size which encodes a single polypeptide of 54 kDa. On the basis of its general characteristics this pathogenic agent belongs to the Parvoviridae family, but because of two unusual characteristics (capsid protein formed with a single polypeptide and genome structure more closely related to the autonomous parvoviruses rather than the densoviruses), it seems to constitute a novel group in the Parvoviridae family.
Preliminary characterization of virus-like particles in a mosquito (Aedes pseudoscutellaris) cell line (Mos. 61).
Electron microscopic examination of an Aedes pseudoscutellaris mosquito cell line (Mos. 61) revealed the presence of a large number of virus-like particles (VLP) in the cytoplasm of approximately 10% of the cells. These particles have a diameter of 36 nm, do not contain a lipid envelope, and have a buoyant density of 1.40 g/ml in CsCl. VLP contain DNA which appears to be single-stranded by SU nuclease assay. PAGE of the VLP demonstrates the presence of four structural polypeptides with molecular weights of 52K, 70K, 75K and 100K daltons, the smaller one being the majro polypeptide species and accounting for 71% of the the total protein mass. The A. pseudoscutellaris VLP appear to share some, but not all, of the characteristics of the genus Densovirus, family Parvoviridae. Their possible biologic importance and taxonomic location are still unclear.
Future of biotechnology-based control of disease in marine invertebrates.
Infectious disease is the single most devastating problem in mollusc and shrimp aquaculture. Pathogens causing the greatest problems have been identified as viruses, prokaryotes, and protozoans. Two approaches employing methods of biotechnology have been proposed to prevent, manage, and control mollusc and shrimp diseases. The first is development of a diagnostic scheme for detection and identification of pathogens, using molecular probes. This offers the opportunity for prophylactic measures to be taken. Molecular probes have been prepared for the major pathogens of molluscs, but in the case of shrimp pathogens, only a few are available. Monoclonal antibodies have also been prepared and are used in immunodiagnosis, e.g., immunofluorescence detection. Such diagnostic tools are relatively new to aquaculture, but have enormous potential. A second approach to the control of disease in marine invertebrates, notably shrimp, involves use of genetically transformed strains resistant to specific pathogens. Pathogen-resistant transgenic animals have been developed, but such research has only just begun for molluscs and shrimp. Transfection methods applied to mollusc and shrimp embryos have been successful, with preliminary data showing efficiency of heterologous promoters in controlling expression of reporter genes. Other transformation systems also show promise, including transposable elements and densoviruses.
Complete nucleotide sequence and genomic organization of the Aedes albopictus parvovirus (AaPV) pathogenic for Aedes aegypti larvae.
We have cloned the replicative form of the Aedes albopictus parvovirus (AaPV) genome and determined the complete sequence of the viral strand. The sequence is 4176 nucleotides (nt) in length. The first 134 nt at the 3' end and the terminal 182 nt at the 5' end of the viral (minus) strand can both generate by folding and annealing of complementary sequences a typical terminal T-shaped structure although they differ in their sequence. Three large open reading frames (ORFs), each one in a different frame, are present between map units (mu) 8.0 and 87.6 on the complementary (plus) strand. The left, mid (located within the left ORF), and right ORFs have potential coding capacities of 95, 41, and 40 kDa, respectively. Two potential promoters were found upstream from the left and right ORFs, at mu 7.2 and mu 60.0, respectively. Computer search for sequence homologies suggests that the left ORF very likely encodes the nonstructural NS-1 protein since it contains the highly conserved NTP-binding amino acid (aa) domain (GKRN sequence) of all parvoviruses. Comparison with other invertebrate and vertebrate parvoviruses revealed that the AaPV genome shares 77.3% nt sequence homology and between 73 and 78% aa sequence homologies with the Aedes aegypti densonucleosis virus (Aedes DNV). Organization of both genomes was similar except that no potential ORF was found on the minus strand of AaPV. The difference of 167 nt in length between AaPV and Aedes DNV (4009 nt) genomes is due to additional noncoding sequences located between the internal coding region and the terminal palindromes in the AaPV genome. No significant homology was found between AaPV and the two other insect parvoviruses sequenced so far, the Bombyx mori DNV (BmDNV) and the Junonia coenia DNV (JcDNV).
Evidence for two small viruses persistently infecting established cell lines of Phthorimaea operculella, deriving from embryos of the potato tuber moth.
Two small viruses were isolated from established cell lines of P. operculella deriving from embryos. The first one probably related to the Nodaviridae family, is a 30 nm in diameter icosahedral virus, with a bisegmented RNA genome and a single polypeptide of 39 kilodaltons. The second one related to the Parvoviridae family, is a 25 nm in diameter icosahedral virus with a DNA genome and a capsid constituted of 4 polypeptides of respectively, 90,000; 64,000; 56,000 and 43,500 daltons. The two viruses probably chronically infect the cell lines and may be consider latent viruses.