Characterization of viral RNA splicing using whole-transcriptome datasets from host species.

RNA alternative splicing (AS) is an important post-transcriptional mechanism enabling single genes to produce multiple proteins. It has been well demonstrated that viruses deploy host AS machinery for viral protein productions. However, knowledge on viral AS is limited to a few disease-causing viruses in model species. Here we report a novel approach to characterizing viral AS using whole transcriptome dataset from host species. Two insect transcriptomes (Acheta domesticus and Planococcus citri) generated in the 1,000 Insect Transcriptome Evolution (1KITE) project were used as a proof of concept using the new pipeline. Two closely related densoviruses (Acheta domesticus densovirus, AdDNV, and Planococcus citri densovirus, PcDNV, Ambidensovirus, Densovirinae, Parvoviridae) were detected and analyzed for AS patterns. The results suggested that although the two viruses shared major AS features, dramatic AS divergences were observed. Detailed analysis of the splicing junctions showed clusters of AS events occurred in two regions of the virus genome, demonstrating that transcriptome analysis could gain valuable insights into viral splicing. When applied to large-scale transcriptomics projects with diverse taxonomic sampling, our new method is expected to rapidly expand our knowledge on RNA splicing mechanisms for a wide range of viruses.

Virus and calcium: an unexpected tandem to optimize insecticide efficacy.

The effective control of insect pests is based on the rational use of the most efficient and safe insecticide treatments. To increase the effects of classical insecticides and to avoid the ability of certain pest insects to develop resistance, it is essential to propose novel strategies. Previous studies have shown that calcium-dependent phosphorylation/dephosphorylation is now considered as a new cellular mechanism for increasing the target sensitivity to insecticides. Because it is known that virus entry is correlated with intracellular calcium concentration rise, this report attempts to present the most important data relevant to the feasibility of combining an insect virus such as baculovirus or densovirus with an insecticide. In this case, the insect virus is not used as a bioinsecticide but acts as a synergistic agent able to trigger calcium rise and to activate calcium-dependent intracellular signalling pathways involved in the increase of the membrane receptors and/or ion channels sensitivity to insecticides. This virus-insecticide mixture represents a promising alternative to optimize the efficacy of insecticides against insect pests while reducing the doses.

Role of the phosphatidylinositol-3-kinase/Akt/target of rapamycin pathway during ambidensovirus infection of insect cells.

The phosphatidylinositol-3-kinase (PI3K)/Akt/target of rapamycin (TOR) signalling pathway controls cell growth and survival, and is targeted by a number of viruses at different phases of their infection cycle to control translation. Whether and how insect viruses interact with this pathway remain poorly addressed. Here, we investigated the role of PI3K/Akt/TOR signalling during lethal infection of insect cells with an insect parvovirus. Using Junonia coenia densovirus (JcDV; lepidopteran ambidensovirus 1) and susceptible insect cells as experimental models, we first described JcDV cytopathology, and showed that viral infection affects cell size, cell proliferation and survival. We deciphered the role of PI3K/Akt/TOR signalling in the course of infection and found that non-structural (NS) protein expression correlates with the inhibition of TOR and the shutdown of cellular synthesis, concomitant with the burst of viral protein expression. Together, these results suggest that NS proteins control the cellular translational machinery to favour the translation of viral mRNAs at the expense of cellular mRNAs. As a consequence of TOR inhibition, cell autophagy is activated. These results highlight new functions for NS proteins in the course of multiplication of an insect parvovirus.

[Rescuing Bombyx mori bidensovirus in BmN cells in vitro].

Bombyx mori bidensovirus (BmBDV) has been identified as causing chronic densonucleosis in Bombyx mori specifically. The replication mechanism of BmBDV remains unknown. Its genome comprises two single stands DNA (VD1 and VD2). In order to rescue infectious virions in vitro, we obtained the total viral DNA extracted from the BmBDV-infected larvae midguts, subsequently cloned the full-length sequence of BmBDV genome fragments by PCR and constructed recombinant plasmids pMD18T-VD1 and pUC-VD2. The linear genome fragments were obtained by digesting recombinant plasmids with corresponding restriction enzymes, and then collectively transfected BmN cells by the method of liposome-embedding. We determined the replication of the virus gene by PCR with the template of demethylated total DNA extracted from the post-transfect BmN cells. Meanwhile, we collected the total proteins from the post-transfect BmN cells and the larvae midgut of feeding the post-transfect BmN cells to perform Western blotting analysis, and detected the expression of viral genes. Here we firstly confirm that infectious virions can be rescued in BmN cells by linear co-transfect method.

Identification of Bombyx mori bidensovirus VD1-ORF4 reveals a novel protein associated with viral structural component.

Bombyx mori bidensovirus (BmBDV) VD1-ORF4 (open reading frame 4, ORF4) consists of 3,318 nucleotides, which codes for a predicted 1,105-amino acid protein containing a conserved DNA polymerase motif. However, its functions in viral propagation remain unknown. In the current study, the transcription of VD1-ORF4 was examined from 6 to 96 h postinfection (p.i.) by RT-PCR, 5'-RACE revealed the transcription initiation site of BmBDV ORF4 to be -16 nucleotides upstream from the start codon, and 3'-RACE revealed the transcription termination site of VD1-ORF4 to be +7 nucleotides downstream from termination codon. Three different proteins were examined in the extracts of BmBDV-infected silkworms midguts by Western blot using raised antibodies against VD1-ORF4 deduced amino acid, and a specific protein band about 53 kDa was further detected in purified virions using the same antibodies. Taken together, BmBDV VD1-ORF4 codes for three or more proteins during the viral life cycle, one of which is a 53 kDa protein and confirmed to be a component of BmBDV virion.

Expression strategy of densonucleosis virus from the German cockroach, Blattella germanica.

Blattella germanica densovirus (BgDNV) is an autonomous parvovirus that infects the German cockroach. BgDNV possesses three mRNAs for NS proteins, two of which are splice variants of the unspliced transcript. The unspliced variant encodes open reading frame 5 (ORF5) (NS3), while NSspl1 encodes ORF3 (NS1) and ORF4 (NS2) and NSspl2 encodes the C-proximal half of NS1. BgDNV possesses three VP transcripts, one of which (VP) is unspliced, while the other two (VPspl1 and VPspl2) are generated by alternative splicing. The unspliced VP transcript contains both ORF1 and ORF2, while in VPspl1, ORF1 and ORF2 are joined in frame. The transcription of NS genes begins at an earlier stage of the virus life cycle than the transcription of VP genes. NS and VP transcripts overlap by 48 nucleotides (nt). BgDNV is characterized by two additional NS transcripts overlapping by more than 1,650 nt with VP-coding transcripts. Four different bands (97, 85, 80, and 57 kDa) corresponding to three BgDNV capsid proteins were detected on SDS-PAGE. Mass spectrometry analysis showed that the amino acid composition of the 85-kDa and 80-kDa proteins is the same. Moreover, both of these proteins are ubiquitinated. The BgDNV PLA(2) domain, which is critical for cellular uptake of the virus, is located in ORF2 and is present only in VP1. In contrast to all of the parvoviruses studied in this respect, VP2 has a unique N terminus that is not contained within VP1 and VP3. In situ recognition with NS1- and VP-specific antibodies revealed an uneven pattern of NS1 expression resembling a halo within the nuclear membrane.

Clearance of Penaeus monodon densovirus in naturally pre-infected shrimp by combined ns1 and vp dsRNAs.

Penaeus monodon densovirus (PmDNV) is one of the major causes of stunted shrimp in Thailand and leads to considerable economic losses in overall shrimp production. Present study shows that the double-stranded RNA corresponding to the non-structural protein gene (ns1) and structural protein gene (vp) of PmDNV effectively inhibit viral propagation in naturally pre-infected shrimp. Multiple application of dsRNA was performed by injection into the haemolymph. The total amount of virus in the hepatopancreas of treated shrimp was measured by semi-quantitative PCR and histological methods. Observations indicated that PmDNV was almost eradicated in comparison to the high viral propagation in the control groups (no dsRNA and non-related dsRNA-gfp). For heavily infected shrimp, simultaneously knock down of ns1 and vp genes exhibited greater potency for viral depletion than dsRNA-ns1 alone. Furthermore, typical hypertrophic nuclei were also reduced in treated shrimp. This study therefore demonstrates the first result of an effective anti-PmDNV therapy in naturally pre-infected shrimp.

Double-stranded RNA confers both preventive and therapeutic effects against Penaeus stylirostris densovirus (PstDNV) in Litopenaeus vannamei.

Penaeus stylirostris densovirus (PstDNV) infection is found widespread in peneaid shrimp, especially in economically important species such as black tiger shrimp Penaeus monodon and Pacific white shrimp Litopenaeus vannamei. Although effective prevention method for viral diseases is not well established in shrimp, the treatment with viral specific double-stranded RNA (dsRNA) or siRNA has given promising results. In present study, dsRNAs corresponding to non-structural (ORF1 and ORF2 overlapping sequence) and structural (ORF3) genes of PstDNV were investigated for their potency to inhibit PstDNV replication in the shrimp. Periodically injection of either ORF1-2 dsRNA or ORF3 dsRNA at three days interval into L. vannamei resulted in substantial inhibition of PstDNV infection. In addition, a possibility for a therapeutic application of dsRNA in PstDNV-infected shrimp was demonstrated by the efficient suppression of PstDNV replication in L. vannamei when the ORF1-2 dsRNA was delivered into the shrimp within 24h post-PstDNV injection. Hence, our results established both the preventive and therapeutic potency of dsRNA to inhibit PstDNV in L. vannamei that could be applied as a potential treatment of PstDNV infection in shrimp.

Transcriptional analysis of Penaeus stylirostris densovirus genes.

Penaeus stylirostris densovirus (PstDNV) genome contains three open reading frames (ORFs), left, middle, and right, which encode a non-structural (NS) protein, an unknown protein, and a capsid protein (CP), respectively. Transcription mapping revealed that P2, P11 and P61 promoters transcribe the left, middle and right ORFs. NS transcript uses the D1/A1 donor/acceptor sites for splicing and has two alternate transcription termination sites (TTS) that were different from the previously predicted TTS. The transcription initiation site (TIS) and the TTS for the middle and the right ORFs conform to predicted sites. PstDNV transcript quantification in infected shrimp revealed that the NS and CP transcripts were expressed at an equivalent level and significantly higher than the middle ORF transcript. In vitro assay showed that P2 had the highest promoter activity followed by P11 and P61. Transcription mapping data provided new insights into PstDNV gene expression strategy.

Production of mosquito densonucleosis viruses by Aedes albopictus C6/36 cells adapted to suspension culture in serum-free protein-free media.

Mosquito densonucleosis viruses (MDVs) have the potential for use as biocontrol agents. To facilitate densovirus production, the Aedes albopictus mosquito cell line C6/36 was adapted to two commercially available serum-free protein-free media (SFPFM), Sf-900 II and Drosophila-SFM. Cells adapted more slowly to growth in Sf-900 II medium, but once adapted, they grew more rapidly and appeared healthier than cells growing in Drosophila-SFM. Cells that were adapted to growth in each of these SFPFM were tested for their ability to be transfected and infected with MDVs. The Sf-900 II-adapted cell line survived transfection and showed infection rates comparable with cells growing in L15 supplemented with 10% fetal bovine serum. Cells adapted to Drosophila-SFM were less infectable and did not survive transfection. Cells adapted to each of these SFPFM were adapted to growth in spinner flasks. Cells in Sf-900 II grew substantially better in spinner flasks than cells in Drosophila-SFM media. Cells grown in Sf-900 II could be frozen and, when thawed, could support the production of densonucleosis viruses in spinner flasks.

Linkage and mapping analysis of a non-susceptibility gene to densovirus (nsd-2) in the silkworm, Bombyx mori.

Nonsusceptibility to Bombyx mori densovirus type 2 (BmDNV-2) is controlled by a recessive non-susceptibility gene, nsd-2 (non-susceptibility to DNV-2) in B. mori. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F1 (BF1) progeny were used for linkage analysis and mapping of nsd-2 using silkworm strains C124 and 902, which are classified as being highly susceptible and non-susceptible to DNV-2, respectively. BF1 larvae were inoculated twice with DNV-2 virus at the first and second instar stages. DNA was extracted from each of the surviving fifth instar larvae and analysed by RFLP inheritance patterns using probes specific to each of the 28 linkage groups of B. mori. Our results indicated that the non-susceptibility gene was linked to linkage group 17, since all surviving larvae showed the homozygous profile of strain 902 in their genotype. The other linkage groups showed mixtures of heterozygous and homozygous genotypes, indicating an independent assortment. A linkage map of 30.6 cM was constructed for linkage group 17 with nsd-2 mapped at 24.5 cM and three closely linked cDNA markers were identified.

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.

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).

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.