Simultaneous Production of a Virus-Like Particle Linked to dsRNA to Enhance dsRNA Delivery for Yellow Head Virus Inhibition.

A co-expressed Penaeus stylirostris densovirus (PstDNV) capsid and dsRNA specific to the yellow head virus (YHV) protease (CoEx cpPstDNV/dspro) has been shown to suppress YHV replication in the Pacific white-legged shrimp (Litopenaeus vannamei). However, maintaining two plasmids in a single bacterial cell is not desirable; therefore, a single plasmid harboring both the PstDNV capsid and the dsRNA-YHV-pro gene was constructed under the regulation of a single T7 promoter, designated pET28a-Linked cpPstDNV-dspro. Following induction, this novel construct expressed an approximately 37-kDa recombinant protein associated with a roughly 400-bp dsRNA (Linked cpPstDNV-dspro). Under a transmission electron microscope, the virus-like particles (VLP; Linked PstDNV VLPs-dspro) obtained were seen to be monodispersed, similar to the native PstDNV virion. A nuclease digestion assay indicated dsRNA molecules were both encapsulated and present outside the Linked PstDNV VLPs-dspro. In addition, the amount of dsRNA produced from this strategy was higher than that obtained with a co-expression strategy. In a YHV infection challenge, the Linked PstDNV VLPs-dspro was more effective in delaying and reducing mortality than other constructs tested. Lastly, the linked construct provides protection for the dsRNA cargo from nucleolytic enzymes present in the shrimp hemolymph. This is the first report of a VLP carrying virus-inhibiting dsRNA that could be produced without disassembly and reassembly to control virus infection in shrimp.

A multi-target dsRNA for simultaneous inhibition of yellow head virus and white spot syndrome virus in shrimp.

Outbreaks of diseases caused by yellow head virus (YHV) and white spot syndrome virus (WSSV) infection in shrimp have resulted in economic losses worldwide. DsRNA-mediated RNAi has been used to control these viruses, and the best target genes for efficient inhibition of YHV and WSSV are the protease and ribonuleotide reductase small subunit (rr2), respectively. However, one dsRNA can suppress only one virus, and therefore the production of multi-target dsRNA to effectively inhibit both YHV and WSSV is needed. In this study, plasmids pETpro-rr2_one stem and pETpro-rr2_two stems were constructed to produce two different forms of multi-target dsRNA in E. coli, which were designed specifically to both YHV protease and WSSV rr2 genes. The potency of each dsRNA in inhibiting YHV and WSSV and reducing shrimp death were investigated in L. vannamei. Shrimp were injected with the dsRNAs into the hemolymph before challenge with YHV or WSSV. The results showed that both dsRNAs could inhibit the viruses, however the one stem construct was more effective than the two stems construct when shrimp were infected with WSSV. This study establishes a potential strategy for dual inhibition of YHV and WSSV for further application in shrimp aquaculture.

Presence of Penaeus monodon densovirus in the ovary of chronically infected P. monodon subadults.

Shrimp infected with Penaeus monodon densovirus (PmoDNV) usually display no specific gross signs, but heavy infections can kill postlarvae and retard juvenile growth. In the present study, samples of hepatopancreas, feces, gonads and hemolymph were isolated from male and female P. monodon subadults chronically infected by PmoDNV. Each sample of hepatopancreas and gonad was divided into 2 parts: one for PmoDNV detection by polymerase chain reaction (PCR), and the other for routine histology and immunohistochemistry. The frequency of positive findings via PCR assays was 92% in the hepatopancreas, 57% in feces, 50% in ovary, 35% in hemolymph and 0% in the testis. Using the densitometric value (DV) of the specific band for PmoDNV relative to that of the ß-actin gene as an index of the viral load in the samples, no significant differences were observed among sample types and sexes. Hematoxylin-eosin staining of infected hepatopancreas revealed typical PmoDNV inclusions in the nuclei of infected cells. The ovaries with high DV (>1) contained various types of inclusions along the row of the follicular cells or possibly in the connective tissue cells surrounding the oocytes. Using immunohistochemistry with specific probes to detect PmoDNV proteins, a positive reaction was observed in viral inclusions found in infected hepatopancreas and in ovaries with high DV, specifically in the ovarian capsule, hemolymph, oocytes and nuclear inclusions. These results suggest that the localization of PmoDNV in P. monodon is not confined to the hepatopancreas, but rather that the virus can also occur in the ovary; hence, trans-ovarian, vertical transmission of the virus is highly possible.

Administration of co-expressed Penaeus stylirostris densovirus-like particles and dsRNA-YHV-Pro provide protection against yellow head virus in shrimp.

The activation of the innate RNA interference pathway through double-stranded RNAs (dsRNAs) is one of the approaches to protecting shrimp from viruses. Previous studies have shown that injection of specific dsRNAs can successfully inhibit viral infection in shrimp. However, inhibition requires high levels of dsRNA and dsRNA stability in shrimp is limited. Virus-like particles (VLPs) have been applied to deliver nucleic acids into host cells because of the protection of dsRNAs from host endonucleases as well as the target specificity provided by VLPs. Therefore, this study aimed to develop Penaeus stylirostris densovirus (PstDNV) VLPs for dsRNA deliver to shrimp. The PstDNV capsid protein was expressed and can be self-assembled to form PstDNV VLPs. Co-expression of dsRNA-YHV-Pro and PstDNV capsid protein was achieved in the same bacterial cells, whose structure was displayed as the aggregation of VLPs by TEM. Tested for their inhibiting yellow head virus (YHV) from infecting shrimp, the dsRNA-YHV-Pro-PstDNV VLPs gave higher levels of YHV suppression and a greater reduction in shrimp mortality than the delivery of naked dsRNA-YHV-Pro. Therefore, PstDNV-VLPs are a promising vehicle for dsRNA delivery that maintains the anti-virus activity of dsRNA in shrimp over a longer period of time as compared to native dsRNAs.

Inhibition of Plasmodium falciparum proliferation in vitro by double-stranded RNA nanoparticle against malaria topoisomerase II.

The need to develop new effective antimalarial agents is urgent due to the rapid emergence of drug resistance to all current drugs by the most virulent human malaria parasite, Plasmodium falciparum. A promising avenue is in the development of antimalarials based on RNA interference targeting expression of malaria parasite vital genes, viz. DNA topoisomerase II gene (PfTOP2). Biodegradable chitosan nanoparticle system has proven to be effective in delivering DNA and small double-stranded interfering RNA to target cells. We have employed a long double-stranded (dsRNA) targeting the coding region of PfTOP2 that is complexed with chitosan nanoparticles in order to interfere with the cognate mRNA expression and examined its effect on P. falciparum growth in culture. Exposure of ring stage-infected erythrocytes to 10 µg/ml PfTOP2 chitosan/dsRNA nanoparticles for 48 h resulted in 71% growth inhibition as determined by [(3)H] hypoxanthine incorporation and microscopic assays, compared with 41% inhibition using an equivalent amount of free PfTOP2 dsRNA or 12% with unrelated chitosan/dsRNA nanoparticles. This inhibition was shown to occur during maturation of trophozoite to schizont stages. RT-PCR analysis indicated 56% and 38% decrease in PfTOP2 transcript levels in P. falciparum trophozoites treated with PfTOP2 dsRNA nanoparticles and free PfTOP2 dsRNA respectively. These results suggest that chitosan-based nanoparticles might be a useful tool for delivering dsRNA into malaria parasites.

A formulated double-stranded RNA diet for reducing Penaeus monodon densovirus infection in black tiger shrimp.

Penaeus monodon densovirus (PmDNV) is one of the major causes of stunted shrimp in the aquaculture industry in Thailand. Significant reductions in levels of PmDNV as assessed by PCR analysis of shrimp hepatopancreas were seen in both prophylactic and curative experiments after feeding shrimp with a formulated diet containing mixed inactivated bacteria harboring dsRNAs corresponding to the PmDNV ns1 and vp genes. Significant reductions of approximately 88% (prophylactic) and 64% (curative) of PmDNV were observed, suggesting that this diet has a high potential for application in commercial aquaculture for reducing PmDNV associated stunted growth of shrimp.

Protection of yellow head virus infection in shrimp by feeding of bacteria expressing dsRNAs.

Although prevention of shrimp mortality from yellow head virus (YHV) infection via dsRNA injection has been well demonstrated for many years, it has not yet been applied in a farm culture because of its impracticality. Hence, oral administration of dsRNA becomes an alternative and desirable approach. This study is the first to demonstrate that oral feeding of Escherichia coli expressing shrimp Rab7 gene (dsRab7) or YHV protease gene (dsYHV) could inhibit YHV replication and lowered shrimp mortality. E. coli HT115 expressing dsRab7 or dsYHV or a combination of these dsRNAs were embedded in agar and used to feed vannamei shrimp at early juvenile stage before YHV challenge. After 4 days of continuous feeding of dsRNAs, strong inhibitory effect on shrimp mortality was observed in which dsRab7 gave the highest effect (70% reduction from the control) whereas dsYHV showed a 40% reduction. Our results reveal the potential of anti-YHV strategy via orally delivered dsRNA for application in the shrimp farm industry.

Ingestion of bacteria expressing dsRNA triggers specific RNA silencing in shrimp.

RNAi activation in shrimp through dsRNA injection has been well demonstrated but oral delivery of dsRNA remains controversial. Therefore, this study was conducted to determine whether RNAi was induced in shrimp by ingestion of bacteria expressing dsRNA. We fed shrimp, Penaeus monodon and Litopenaeus vannamei, with inactivated bacteria expressing dsRNA specific to the shrimp genes (Rab7 and STAT). Forty-eight hours after 6 day-continuous feeding, the level of the targeted gene transcript was measured by semi-quantitative RT-PCR. Significant reduction of Rab7 as well as STAT transcript was observed when compared to that of control shrimp fed with bacteria containing the empty vector or bacteria expressing non-related dsRNA (GFP). Moreover, the suppression was detected not only in the hepatopancreas but also in the gills indicating the successful systemic induction of RNAi via oral delivery of dsRNA. Our results suggested that RNAi in shrimp could be triggered by ingestion of dsRNA expressing bacteria. Therefore, oral feeding is a practical approach which can be used to deliver dsRNA for further viral inhibition in farmed shrimp.

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.

Inhibition of Penaeus monodon densovirus replication in shrimp by double-stranded RNA.

Stunted shrimp caused by Penaeus monodon densovirus (PmDNV) infection is one of the main problems leading to a significant economic loss in Thailand. To control this pandemic disease, a double-stranded-RNA-mediated virus-specific gene silencing approach was applied to inhibit viral replication. In this study, two dsRNAs corresponding to the non-structural protein (ns1) and the structural protein (vp) genes of PmDNV were synthesized and introduced into shrimp haemolymph prior to viral challenge. After allowing viral replication for two weeks, the suppression effect by each dsRNA was evaluated by semi-quantitative PCR and compared with the control. A reduction of PmDNV in shrimp treated with each dsRNA was observed. In contrast, a high level of viral infection was detected in the control group (NaCl). Based on a limited sample number, we reached the tentative conclusion that virus-specific dsRNA can inhibit PmDNV replication, in which the dsRNA-ns1 was more effective than the dsRNA-vp.

Inhibition of white spot syndrome virus replication in Penaeus monodon by combined silencing of viral rr2 and shrimp PmRab7.

Although a significant progress has been achieved on dsRNA mediated anti-virus strategy development, there is still no effective means to control the virulent white spot syndrome virus (WSSV). Six double-stranded RNAs specific to different essential genes of WSSV (ie1, ie3, pol (DNA polymerase), rr2 (ribonucleotide reductase small subunit), vp26, and vp28) were employed to suppress viral replication in shrimp. At the condition that non-specific inhibitory effect was overwhelmed, the relative protective degree of these dsRNAs against WSSV infection (rr2>ie3>vp26, vp28>ie1>pol) was observed by semi-quantitative PCR. Besides, more than one injection of dsRNA was needed for an efficient viral inhibition. To improve viral protection in Penaeus monodon, synchronized blocking of viral cellular transport (by dsRNA-PmRab7) and viral essential gene synthesis (by dsRNA-rr2) was first performed in this study. The suppression effects of shrimp mortality by either combined dsRNAs of rr2 and PmRab7 or dsRNA-rr2 alone was monitored for 8 days after viral challenge. Approximately 95% of shrimp survivals were detected from both combined dsRNAs and dsRNA-rr2 alone whereas all shrimp without dsRNA were dead. It revealed that there was no additive inhibitory effect of the combined dsRNAs over dsRNA-rr2 alone.

Complete nucleotide sequence and genomic organization of hepatopancreatic parvovirus (HPV) of Penaeus monodon.

We have determined the genome of hepatopancreatic parvovirus (HPV), a minus, single-stranded DNA virus isolated from infected Penaeus monodon in Thailand. Its genome consisted of 6321 nucleotides, representing three large open reading frames (ORFs) and two non-coding termini. The left (ORF1), mid (ORF2), and right (ORF3) ORFs on the complementary (plus) strand may code for 428, 579, and 818 amino acids, equivalent to 50, 68, and 92 kDa, respectively. The 5' and 3' ends of viral genome contained hairpin-like structure length of approximately 222 and 215 bp, respectively. No inverted terminal repeat (ITR) was detected. The ORF2 contained conserved replication initiator motif, NTP-binding and helicase domain similar to NS-1 of other parvoviruses. Therefore, it most likely encoded the major nonstructural protein (NS-1). The ORF1 encoded putative nonstructural protein-2 (NS-2) with unknown function. The ORF3 of the HPV genome encoded a capsid protein (VP) of approximately 92 kDa. This may be later cleaved after arginine residue to produce a 57-kDa structural protein. A phylogenetic tree based on conserved amino acid sequences (119 aa) revealed that it is closely related to Brevidensoviruses, which are shrimp parvovirus (IHHNV) and mosquito densoviruses (AaeDNV and AalDNV). However, the overall genomic organization and genome size of HPV were different from these parvoviruses, for instance, the non-overlapping of NS1 and NS2, the larger VP gene, and the bigger genome size. This suggested that this HPV virus is a new type in Parvoviridae family. We therefore propose to rename this virus P. monodon densovirus (PmDNV).