DESC:FORM 2

THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003

COMPLETE SPECIFICATION
(See section 10 and rule 13)

PROCESS FOR THE OVER EXPRESSION OF dsRNA FOR THE CONTROL OF WHITE SPOT SYNDROME VIRUS

GPS BIOTECH LIMITED
a company incorporated under the companies act, 1956 having address at
House no EWS 86/1, Road No 3, KPHB Colony, Kukatpally, Hyderabad, Telangana - 500072, India.

The following specification particularly describes the invention and the manner in which it is to be performed:
FIELD OF THE INVENTION

The present invention relates to a novel technology for the preparation RNAi / dsRNA for the control of Virus in marine species.
More particularly, the present invention relates to a novel technology for the over expression of Bacteria RNAi / dsRNA technology for the control of Virus in marine species.
More particularly, the present invention relates to novel composition of dsRNA for use a antiviral agent in the control of White Spot Syndrome Virus (WSSV) in shrimps

BACKGROUND OF THE INVENTION
White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. RNA interference (RNAi) is a promising tool against viral infections.
White spot syndrome virus (WSSV) is a major pathogen affecting shrimp farming worldwide. Since its emergence in the mid 90s in various countries in America, it has caused major economic losses to shrimp farmers (Escobedo-Bonilla et al., 2008 and Lightner, 2011). The WSSV genome consists of 531 putative genes (https://www.ncbi.nlm.nih.gov/nuccore/AF440570.1; accessed on 08/20/14), some of which may be essential for WSSV infection and/or replication. These include genes such as vp28, protein kinase, thymidine-thymidylate kinase, DNA polymerase and WSSV449 ( Chen et al., 2002, Liu et al., 2001, Tsai et al., 2000, van Hulten et al., 2001a and Wang et al., 2011). Still, the role of many genes during virus infection is unknown. Silencing genes by specific double-stranded RNA represents a way to determine their putative roles ( Fagutao et al., 2009, Labreuche et al., 2009 and Nakayashiki et al., 2005).
Several studies have used RNA interference (RNAi) to inhibit viral infections in shrimp. Most of the genes used to inhibit WSSV infection encode structural proteins involved in virion architecture (Kim et al., 2007,Robalino et al., 2004, Robalino et al., 2005, Tirasophon et al., 2005 and Yodmuang et al., 2006). WSSV structural proteins such as vp28 and vp26 are involved in virus entry ( Tang et al., 2007, van Hulten et al., 2001a, Youtong et al., 2011 and Zhang et al., 2002). These proteins constitute targets against virus infection by recombinant proteins ( Caipang et al., 2008 and Witteveldt et al., 2006), neutralizing antibodies ( Jiang et al., 2007 and Natividad et al., 2007) and RNAi ( Mejía-Ruíz et al., 2011, Robalino et al., 2004 and Sarathi et al., 2008).
A few studies have assessed the antiviral efficacy of RNAi against non-structural WSSV proteins such as DNA polymerase, protein kinase, ribonucleotide reductase, thymidine-thymidylate kinase or protein WSSV449 (Attasart et al., 2009, Kim et al., 2007, Wang et al., 2011 and Wu et al., 2007).
Since the publication of the complete WSSV genome (Chen et al., 2002, van Hulten et al., 2001b and Yang et al., 2001) many novel WSSV putative open reading frames (ORFs) have been described and some of these have been characterized (Hossain et al., 2004 and Li et al., 2005), but the role of their products in virus infection is largely unknown. One of these genes is orf89, with 4436 nucleotides encoding a peptide 1437 amino acids long and apparent size of 165 kDa. This peptide localizes in the nucleus of transfected Sf9 insect cells. In vitro assays showed that the peptide had a negative regulation function of a WSSV protein kinase (orf61) and a thymidine-thymidylate kinase ( Hossain et al., 2004). Other genes encoding putative enzymes related to nucleotide metabolism include white spot virus (wsv) 067 (thymidylate synthase),wsv112 (dUTP pyrophosphatase), wsv172 (ribonucleotide reductase large subunit), wsv188 (ribonucleotide reductase small subunit [rr]), wsv191 (deoxyribonuclease I) and wsv395 (thymidylate kinase) ( Yang et al., 2001). Of these, gene wsv191 has a sequence of 912 base pairs (bp), which encodes a non-specific nuclease of 304 residues with apparent size of 34.4 kDa. The assumed role of a non-specific nuclease in WSSV infection is degradation of nucleic acids of host cells, viral DNA repair and virus genome synthesis (Li et al., 2005).
Previous works have shown different antiviral efficacies of RNAi molecules depending on the targeted genes (Robalino et al., 2005 and Wu et al., 2007). It is possible that antiviral efficacy depends on the role of the silenced gene in virus infection/replication (Kim et al., 2007). Then, RNAi silencing of WSSV genes with a critical role in virus replication might show stronger virus inhibition and lower shrimp mortality
Journal of King Saud University - Science, Volume 27, Issue 2, April 2015, Pages 182–188 discloses Efficacy of double-stranded RNA against white spot syndrome virus (WSSV) non-structural (orf89, wsv191) and structural (vp28,vp26) genes in the Pacific white shrimp Litopenaeus vannamei.
In view of the importance associated with the use of dsRNA in the treatment of WSSV it is desirable provide a composition for use in the fields specifically Shrimp farms.

OBJECTIVES OF THE PRESENT INVENTION:

It is therefore an objective of the present invention is to provide an optimized large scale in-vivo process for the production of dsRNA for the control of Virus in marine species.
Yet another objective of the present invention is to provide a process for the production of dsRNA on industrial scale.
Still another objective of the present invention is to provide feed formulation with dsRNA or bacteria over expressed with dsRNA specific to viral genes using nanoparticles and study their efficacy to protect the shrimp from WSSV infection.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a novel technology for the preparation RNAi / dsRNA for the control of Virus in marine species.
Yet an embodiment of the present invention relates to a novel technology for the over expression of Bacteria RNAi / dsRNA technology for the control of Virus in marine species.
Yet an embodiment of the present invention relates to a novel technology for the over expression of Bacteria RNAi / dsRNA technology for the control of Virus in marine species which comprises fermenting cultured bacterial cells with a vector in a culture medium containing a peptide and yeast extract in different concentrations at a pH and temperature suitable for the medium.
Yet an embodiment of the present invention relates to a novel technology for the over expression of Bacteria RNAi / dsRNA technology for the control of Virus in marine species which comprises fermenting cultured bacterial cells with a vector in a culture medium containing a peptide and yeast extract in different concentrations at a pH in the range of 5 to 10 and temperature in the range of 25 °C to 50 °C.
Yet an embodiment of the present invention relates to a novel technology for the over expression of Bacteria RNAi / dsRNA technology for the control of Virus in marine species which comprises fermenting cultured bacterial cells with a vector in a culture medium containing a peptide in the concentration range of 0.25 to 4 g/100ml and yeast extract at a concentration in the range of 0.10 to 3.0 g/100ml at a pH in the range of 5 to 10 and temperature in the range of 25 °C to 50 °C.
Yet an embodiment of the present invention relates to a novel technology for the over expression of Bacteria RNAi / dsRNA technology for the control of Virus in marine species wherein the pH is adjusted using an acid or base or a buffering agent.
Yet an embodiment of the present invention relates to provide feed formulation with dsRNA or bacteria over expressed with dsRNA specific to viral genes their efficacy to protect the shrimp from WSSV infection.
Yet an embodiment of the present invention relates to a process for the production of dsRNA on industrial scale.
Yet another embodiment of the present invention relates an optimized large scale in-vivo process for the production of dsRNA specific to VP28 gene and other genes of WSSV.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to make use of efficient gene for large-scale production of dsRNA to protect shrimp from WSSV infection.
The present invention relates to optimizing various parameters for large-scale production of dsRNA specific to selected genes of WSSV and also large-scale production of r-VP28 protein. It also relates to the method of storing the dsRNA and self life period of dsRNA.
Different biocompatible and eco-friendly nanoparticles such as chitosan and its derivative NP are synthesized using chemical methods as described in the literature (Rajeshkuimar et al., 2008; Vimal et al., 2012). The nanoparticles shall be characterized by UV–Vis spectroscopy. Dynamic Light Scattering analysis would be performed to measure the size of the nanoparticles. Transmission electron microscopy (TEM) analysis shall be performed on synthesized nanoparticles. The stability of nanoparticles shall be determined. The efficiency of these nanoparticles to deliver dsRNA will be determined by in vitro using cell lines and in vivo using shrimp.
In one embodiment of present invention nanoparticle compositions herein can be in the form of a tablet, capsule, dry powder, solution or lotion, a paste or cream, an emulsion, or the like.
The methods of present invention to dsRNA nanoparticle compositions are simple, ecofriendly, inexpensive, reproducible, robust and are well adaptable on a commercial scale.
The present invention is further defined by reference to the following methods describing methods of use of the invention. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the invention.

Experimental section:
(a) In vivo production of dsRNA
dsRNA specific to VP28 gene of WSSV is very effective to protect the shrimp from WSSV infection. This viral gene has been selected in the proposed work. In addition other viral genes such as VP15, VP19, and WSSV222 are selected to study their efficacy in different combinations.
The genes of WSSV are amplified using primers already available in collaborator’s lab. For other genes, the primers are designed based on published sequences. All these genes are amplified using standard PCR protocol and the PCR products of target genes of WSSV are cloned in between bi-directional T7 promoter of the LITMUS38i-Vector (New England Biolabs, Ipswich, MA). The cloned plasmid was transformed into E. coli HT115(DE3) strain which lacks the double-strand-specific RNase III and improves the ability to produce dsRNA. The colonies containing transformantsare screened by PCR. The bacterial cells HT115(DE3) are grown over night at 37oC in 3 ml LB medium supplemented with 100 ?g/ml of ampicillin and 12.5 ?g/ml of tetracycline; 250 ?l of the pre culture are transferred to 25 ml LB medium supplemented with ampicillin and tetracycline. After proliferation of the bacteria at 37oC to an OD 595 of 0.4, IPTG (0.5 mM final concentration) is added and incubated for further 4 hours under identical conditions. Bacteria are then pelleted for 15 minutes at 3,000 x g (4oC) and resuspended in 1.25 ml TE (pH 7.5). For large-scale production, various conditions such as temperature, pH and medium conditions are optimized.
(b) Isolation of dsRNA
For nucleic acid isolation, a minimum volume of 0.75 ml of the cell suspension are replenished with an equal volume of phenol - chlorophorm - isoamylalcohol and heated at 65oC for 10 minutes. Then the samples are centrifuged for 10 minutes at 13,000 rpm and the aqueous phase are transferred into a fresh tube. Nucleic acid are then precipitated with 1 volume of isopropanol and 0.2 volume of 4 M LiCl and kept at –20oC for 10 minutes and followed by centrifugation. Supernatant are discarded and the pellet are washed with 70% ethanol. After drying, the pellet are resuspended in 100 ?l Tris-NaCl-MgCl2-DTT buffer; 18 ?l of this solution are supplemented with 1 ?l DNaseI (3 mg/ml) and digested for 1 hour at room temperature to remove DNA. Samples are loaded on a 1.8% agarose gel to confirm the presence of dsRNA.
c. Large-scale production:
Various parameters for large-scale production of dsRNA specific to VP28 gene of WSSV have been determined in fermentation process using standard fermentation protocols with various modifications in temperature, pH, NaCl, and substrate concentrations. The detailed process for large-scale production of dsRNA is summarized below:
1. LB-medium was used and its composition with slight modification to yield maximum amount of dsRNA. The composition consisted of 15 g Tryptone, 1.4 g Yeast extract and 10 g NaCl in 1 L of deionized water and supplemented with antibiotic.
2. The medium was sterilized and allowed to cool at room temperature.
3. After sterilization, optimized the condition of medium with 7.4 pH, 37oC temperature, substrate and NaCl concentration, and DO.
4. Inoculum containing overnight cultured bacterial cells (HT115 {DE3}) with vector (LITMUS – 38i) bearing VP28 gene of WSSV was inoculated into the fermentor.
5. The bacterial cells were allowed to grow to reach the optical density of 0.6, and then IPTG (0.5 mM final concentration) was added into the fermentor under sterile condition to induce the expression of dsRNA specific to VP28 gene.
6. After 5 to 6 hours, bacterial culture was collected and bacterial biomass was harvested by centrifugation.
7. dsRNA was isolated from the bacterial biomass to determine the integrity and quantity of dsRNA produced by agarose gel electrophoresis.
8. The bacterial biomass was inactivated by 0.5% formaldehyde and washed the mass twice using PBS.
9. The inactivated bacterial biomass containing dsRNA was used to prepare the dsRNA formulated feed to test the efficacy of dsRNA-VP28 to protect the shrimp from WSSV infection.
10. The bacterial biomass either stored at -80oC or freeze dried and stored at room temperature prior to use for the experiment.

d. Preparation of formulated feed for oral application:
Formulated feed for oral application will be prepared using the protocol standardized in collaborator’s laboratory. Feed will be coated with bacteria over expressed with dsRNA separately or in combination with other dsRNAs or with r-VP28 protein. Suitable binding agent will be used to avoid leaching of coated materials. This formulated feed will be used for experiments. Dosage and frequency of application will be determined using this formulated feed.
e. Monitoring of dsRNA/r-VP28:
The dsRNA or r-VP28 protein will be monitored at different time intervals in different organs of shrimp fed with dsRNA/r-VP28 protein formulated feed by RT-PCR using specific primers and protocols standardized in collaborator’s laboratory and ELISA using antibody raised against r-VP28 protein. Simultaneously, the clearance of dsRNA/r-VP28 protein will be determined in treated shrimp after ceasing the treatment.
f. Assimilation efficiency:
The assimilation efficiency of gut to absorb the dsRNA from gut will be determined by quantifying the dsRNA in faecal materials collected from shrimp fed with dsRNA formulated feed using the RT-PCR protocol standardized in our laboratory

(g ) Study the efficacy of dsRNA:
(i) In vitro study
Efficacy of dsRNAs specific to genes of WSSV against WSSV for in vitro study are assessed either in fish cell line or crab heart explants established in our lab. The protocol for in vitro study has been already standardized in collaborator’s laboratory. This is done in two methods. In the first method, cells are first transfected with dsRNA (virus specific genes) and then the cells are infected with white spot virus. Viral infectivity and load will be assessed by PCR, Western blot and real time PCR. In the second type, the cells will initially be infected with WSSV and then transfected with dsRNA. Viral infection was assessed by the methods mentioned above.

(ii) In vivo study
For in vivo study, the efficacy of dsRNAs will be studied by directly supplying it to shrimp. As in case of in vitro studies, different dsRNAs with different combinations are tried. As per published works (Kamath et al., 2000; Timmons et al., 2001; Newmark et al., 2003; Turner et al., 2006; Baum et al., 2007; Sarathi et al., 2008b; Whalshe et al., 2009) , dsRNAs are delivered to shrimp by injection or oral through feed coated with dsRNAs or bacteria over expressed with dsRNA or through nanoparticles. The efficacy will be assessed by challenging with WSSV at different time intervals. Viral load and viral clearance will be assessed by PCR, Western blot, real time PCR and bioassay.

Lab trials carried out to study the efficacy of dsRNA-VP28 to protect the shrimp from WSSV infection

Trail Relative percent survival (RPS) of dsRNA treated shrimp challenged with WSSV in each treated group
(n = 12)*
Positive Control

Shrimp fed with WSSV infected meat Shrimp fed with feed coating with bacteria over-expressed dsRNA. (5 µg per g of feed) Shrimp fed with feed coating with bacteria over-expressed dsRNA (10 µg per g of feed) Shrimp fed with feed coating with bacteria over-expressed dsRNA (15 µg per gram of feed)
Trail 1
0 66.68 75.00 83.34
Trail 2
0 58.35 66.68 75.00
Trail 3
0 66.68 75.00 83.34
Trail 4
0 50.00 75.00 83.34
Trail 5
0 66.68 75.00 83.34
Trail 6
0 50.00 66.68 75.00

Healthy shrimp were fed with above mentioned formulated feeds for 10 days. On 11th day, shrimp were fed with WSSV-infected meat for one time (50 mg of tissue per shrimp). After challenging, the shrimp were fed with above respective formulated feeds continuously till the experimental period of 30 days. ,CLAIMS:We Claim,
1. A novel technology for the preparation RNAi / dsRNA for the control of Virus in marine species.
2. The novel technology as claimed in claim 1 for the over expression of Bacteria RNAi / dsRNA technology for the control of Virus in marine species.
3. The novel technology as claimed in claim 1 for the over expression of Bacteria RNAi / dsRNA technology for the control of Virus in marine species which comprises fermenting cultured bacterial cells with a vector in a culture medium containing a peptide and yeast extract in different concentrations at a pH and temperature suitable for the medium.
4. The novel technology as claimed in claim 1 for the over expression of Bacteria RNAi / dsRNA technology for the control of Virus in marine species which comprises fermenting cultured bacterial cells with a vector in a culture medium containing a peptide in the concentration range of 0.25 to 4 g/100ml and yeast extract at a concentration in the range of 0.10 to 3.0 g/100ml at a pH in the range of 5 to 10 and temperature in the range of 25 oC to 50 oC.
5. The novel technology as claimed in claim 4 wherein the pH is adjusted using an acid or base or a buffering agent.
6. Feed formulation with dsRNA or bacteria over expressed with dsRNA specific to viral genes their efficacy to protect the shrimp from WSSV infection.
7. Feed formulation as claimed in claim 6 in the form of nanoparticle compositions wherein the compositions are in the form of a tablet, capsule, dry powder, solution or lotion, a paste or cream, an emulsion.

Dated this Twenty seventh (27th) day of July 2016
_______________________
Dr. S. Padmaja
Agent for the Applicant
IN/PA/883.