Production of a parvovirus vaccine in plants as viral coat protein fusions

A polynucleotide encoding a fusion protein capable of being expressed in a plant or a plant cell, comprising:

• (a) a coding region that encodes said fusion protein and includes:
• i. a plant viral coat protein from a single-stranded plus-sense RNA virus, and fused thereto,
• ii. a peptide comprising amino acids MGQPDGGQPAVRNERAT (SEQ ID NO:2) or a fragment thereof;
• and
• (b) a promoter functional in plants that is 5′ to the coding region.

A method of making the recombinant plant virus that encodes a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof, comprising the steps of:

• (a) ligating DNA encoding the fusion protein which protein is capable of being expressed in a plant or a plant cell, to a DNA copy of the genome of a single-stranded plussense RNA virus;
• (b) transcribing said ligated DNA to RNA;
• and
• (c) infecting a host plant or plant cell with said transcribed RNA,
• so that said plant or plant cell makes said recombinant plant virus.

A method of making the recombinant plant virus that encodes a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof, comprising the steps of;

• (a) ligating DNA encoding the fusion protein which protein is capable of being expressed in a plant or. a plant cell, to a DNA copy of the genome of a single-stranded plussense RNA virus;
• (b) transcribing said ligated DNA to RNA;
• and
• (c) infecting a host plant or plant cell with said transcribed RNA,
• so that said plant or plant cell makes said recombinant plant virus.

A method of making the recombinant plant virus that encodes a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof, comprising the steps of;

• (a) ligating DNA encoding the fusion protein which protein is capable of being expressed in a plant or a plant cell, to a DNA copy of the genome of a single-stranded plussense RNA virus, under the control of a promoter that is functional in plants;
• and
• (b) transforming or transfecting a host plant or plant cell with said ligated DNA, so that said DNA is expressed in said plant or plant cell,
• whereby said plant or plant cell makes said recombinant plant virus.

A method of isolating a virus, comprising:

• (a) Homogenizing virus-containing plant tissue in Na2S2O5;
• (b) Straining the homogenate to obtain green juice;
• (c) Adjusting the pH of the green juice to 5.0 with acid;
• (d) Heating the green juice to about 47° C. for a period of about 5 minutes followed by cooling to about 5° C.;
• (e) Centrifuging the green juice at about 6000×g for about 3 minutes to obtain a supernatant and pellet;
• (f) Precipitating the supernatant in polyethylene glycol and NaCl to obtain a precipitate;
• (g) Resuspending the precipitate in water at a concentration of about 1 mg per ml;
• (h) Extracting the precipitate in chloroform and butanol and centrifuging the extract
• (i) Recovering and lyophilizing the aqueous phase of the centrifuged material;
• (j) Resuspending the lyophilized material at a concentration of about 5 to about 10 mg per ml water.

A method of isolating a virus, comprising:

• (a) Grinding virus-containing plant material in a buffer to obtain a homogenate;
• (b) Straining the homogenate to obtain green juice, and adding thereto polyethyleneimine to a concentration of about 0.1% (v/v)
• (c) Stirring at about 4° C. for about 30 minutes followed by centrifuging;
• at about 3000×g for about 5 minutes to obtain a supernatant;
• (d) Precipitating the supernatant in polyethylene glycol and NaCl to obtain a precipitate;
• (e) Resuspending the precipitate in water at a concentration of about 1 mg per ml;
• (f) Extracting the resuspended material in chloroform and butanol and centrifuging the extracted material;
• (g) Recovering and lyophilizing the aqueous phase;
• and
• (h) Resuspending the lyophilized material at a concentration of about 5 to about 10 mg per ml water.