Method to Produce Virus in Cultured Cells

Abstract

A method for improving virus production in a host cell infected with the virus is provided.

1 . A method for producing a virus comprising the step of culturing a host cell infected with a virus under conditions appropriate for producing the virus, wherein the conditions include a fatty acid in an amount and for a time effective to permit virus production, optionally wherein the conditions include the presence of cholesterol, and optionally wherein the conditions further include cholesterol and scavenging compound. 
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1  wherein the conditions include the presence of a fatty acid and cholesterol. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1  wherein the conditions further include a scavenging compound. 
     
     
         6 . The method of  claim 5  wherein the virus is produced in an amount greater in the presence of the scavenger compound compared to virus produced in the method performed without the scavenger compound. 
     
     
         7 . The method of  claim 1  wherein the conditions include no more than one, two, three or four fatty acids. 
     
     
         8 - 10 . (canceled) 
     
     
         11 . The method of  claim 1  wherein the conditions include at least two, at least three, at least four or four or more different fatty acids. 
     
     
         12 - 21 . (canceled) 
     
     
         18 . The method of  claim 1  further comprising the step of infecting the host cells with the virus. 
     
     
         19 - 23 . (canceled) 
     
     
         24 . The method of  claim 18  further comprising the step of incubating the host cells with an infecting virus for an adsorption period. 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 18  further comprising the step of i) introducing the fatty acid, cholesterol and/or scavenging compound prior to infecting the host cell with the virus or with virus infected cells; or
 ii) introducing the fatty acid, cholesterol and/or scavenging compound after infecting the host cell with the virus. 
 
     
     
         27 . (canceled) 
     
     
         28 . The method of  claim 1  further comprising the step of introducing the fatty acid, cholesterol and/or scavenging compound at more than one time during the step of culturing the cells. 
     
     
         29 - 33 . (canceled) 
     
     
         34 . The method of  claim 1  wherein the host cell is mammalian. 
     
     
         35 - 37 . (canceled) 
     
     
         38 . The method of  claim 1  wherein the virus is an enveloped virus. 
     
     
         39 - 45 . (canceled) 
     
     
         46 . The method of  claim 1  wherein the virus is an RNA virus, a nonenveloped RNA virus, an enveloped RNA virus, a DNA virus, a nonenveloped DNA virus, and enveloped DNA virus, a pox virus, a picorna virus, poliovirus, rhinovirus, hepatitis A virus, foot and mouth disease virus, influenza virus, herpes simplex virus, Epstein Barr virus, hepatitis C virus, Dengue virus, HIV, mumps virus, measles virus, rotavirus and/or parainfluenza virus. 
     
     
         47 - 48 . (canceled) 
     
     
         49 . The method of  claim 1  wherein the fatty acid is a long chain fatty acid or a very long chain fatty acid. 
     
     
         50 . The method of  claim 1  wherein the fatty acid is selected from the group consisting of an omega-3 fatty acid, an omega-6 fatty acid, a naturally-occurring fatty acid, a derivative of a naturally-occurring fatty acid, a non-naturally-occurring fatty acid, a free fatty acid, a fatty acid ester and a fatty acid derivative. 
     
     
         51 - 61 . (canceled) 
     
     
         62 . The method of any one of  claims 1 - 61  wherein the fatty acid has at least 18 carbons, at least 20 carbons, at least 22 carbons, at least 24 carbons, at least 26 carbons, at least 28 carbons, at least 30 carbons, at least 32 carbons, at least 34 carbons, at least 36 carbons, at least 38 carbons or at least 40 carbons. 
     
     
         63 - 73 . (canceled) 
     
     
         74 . The method of  claim 1  wherein the fatty acid is saturated, is unsaturated or is polyunstaruated. 
     
     
         75 - 76 . (canceled) 
     
     
         77 . The method of  claim 1  wherein the fatty acid has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more double bonds. 
     
     
         78 - 88 . (canceled) 
     
     
         89 . The method of  claim 1  wherein the fatty acid is selected from the group consisting of: linoleic acid (LA), α-linolenic acid (LLA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (AA), hexacosanoic acid (HSA) and octacosanoic acid, OSA) 
     
     
         90 - 93 . (canceled) 
     
     
         94 . The method of  claim 1  wherein the scavenging compound is a carbonyl scavenging compound or a free radical scavenging compound. 
     
     
         95 . The method of  claim 1  further comprising a carbonyl scavenging compound and a free radical scavenging compound. 
     
     
         96 . (canceled) 
     
     
         97 . The method of  claim 1  wherein the fatty acid is present at a concentration of i) at least 5 μM, at least 10 μM, at least 15 μM, at least 20 μM, at least 25 μM, at least 30 μM, at least 35 μM, at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, at least 65 μM, at least 70 μM, at least 75 μM, at least 80 μM, at least 85 μM, at least 90 μM, at least 95 μM, at least 100 μM, at least 110 μM, at least 120 μM, at least 130 μM, at least 140 μM, at least 150 μM or more, and wherein the fatty acid is present at a concentration of 500 μM or less, or at a concentration that is not toxic to the host cell; or
 ii) no more than 5 μM, no more than 10 μM, no more than 15 μM, no more than 20 μM, no more than 25 μM, no more than 30 μM, no more than 35 μM, no more than 40 μM, no more than 45 μM, no more than 50 μM, no more than 55 μM, no more than 60 μM, no more than 65 μM, no more than 70 μM, no more than 75 μM, no more than 80 μM, no more than 85 μM, no more than 90 μM, no more than 95 μM, no more than 100 μM, no more than 110 μM, no more than 120 μM, no more than 130 μM, no more than 140 μM, or no more than 150 μM. 
 
     
     
         98 . The method of  claim 1  wherein cholesterol is present at a concentration of i) at least 5 μM, at least 10 μM, at least 15 μM, at least 20 μM, at least 25 μM, at least 30 μM, at least 35 μM, at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 at least 65 μM, at least 70 μM, at least 75 μM, at least 80 μM, at least 85 μM, at least 90 μM, at least 95 μM, at least 100 μM, at least 110 μM, at least 120 μM, at least 130 μM, at least 140 μM, at least 150 μM or more and wherein cholesterol is present at a concentration of 500 μM or less, or at a concentration that is not toxic to the host cell; or
 ii) no more than 5 μM, no more than 10 μM, no more than 15 μM, no more than 20 μM, no more than 25 μM, no more than 30 μM, no more than 35 μM, no more than 40 μM, no more than 45 μM, no more than 50 μM, no more than 55 μM, no more than 60 μM, no more than 65 μM, no more than 70 μM, no more than 75 μM, no more than 80 μM, no more than 85 μM, no more than 90 μM, no more than 95 μM, no more than 100 μM, no more than 110 μM, no more than 120 μM, no more than 130 μM, no more than 140 μM, or no more than 150 μM. 
 
     
     
         99 . The method of  claim 1  wherein the scavenging compound is present at a concentration of i) at least 1 μm, at least 2 μM, at least 3 μM, at least 4 μM, at least 5 μM, at least 6 μM, at least 7 μM, at least 8 μM, at least 9 μM, at least 10 μM, at least 15 μM, at least 20 μM, at least 25 μM, at least 30 μM, at least 35 μM, at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, at least 65 μM, at least 70 μM, at least 75 μM, at least 80 μM, at least 85 μM, at least 90 μM, at least 95 μM, at least 100 μM, at least 110 μM, at least 120 μM, at least 130 μM, at least 140 μM, at least 150 μM or more, and wherein the scavenging compound is present at a concentration of 500 μM, or less, or at a concentration that is not toxic to the host cell; or
 ii) no more than 5 μM, no more than 10 μM, no more than 15 μM, no more than 20 μM, no more than 25 μM, no more than 30 μM, no more than 35 μM, no more than 40 μM, no more than 45 μM, no more than 50 μM, no more than 55 μM, no more than 60 μM, no more than 65 μM, no more than 70 μM, no more than 75 μM, no more than 80 μM, no more than 85 μM, no more than 90 μM, no more than 95 μM, no more than 100 μM, no more than 110 μM, no more than 120 μM, no more than 130 μM, no more than 140 μM, or no more than 150 μM. 
 
     
     
         100 - 102 . (canceled)