Biologicals (1992) 20, 83-85
SHORT NOTE Inactivation of Viruses Related to Hepatitis C Virus by Pasteurization in Human Plasma Derivatives Thomas Nowak, Ulrich Klockman and Joachim Hilfenhaus Research Laboratories of Behringwerke AG, Emil-von-Behring-StraBe 76, PO Box 11 40, D-3550 Marburg, Germany
Q. L. Choo et al.1 were the first to clone the genome of hepatitis C virus (HCV). Based on their findings it was possible to clone and sequence the genomes of various HCV isolates. 2 From the analysis of the genome of this RNA virus it was concluded t h a t HCV belongs to the family of the flaviviridae. 3According to the current nomenclature the flavivirus family is divided in three genera, the flaviviruses, the pestiviruses and the hepatitis G virus group. From the genome analyses it is further concluded t h a t HCV is more related to the pestiviruses than to the flaviviruses. This conclusion has recently been supported by a paper published by Bradley et al. 4 who studied the buoyant density of hepatitis C virus in a sucrose gradient. HCV is one of the h u m a n pathogenic viruses t h a t can be transmitted from blood or plasma donors to the recipients of whole blood transfusions or h u m a n plasma derivatives. ~ Since HCV cannot be propagated in tissue cultures or animals, apart from chimpanzees, there is no routine method available to assay infectious HCV. H u m a n blood and plasma protein preparations cannot therefore be investigated for a contamination with infectious HCV and it is not possible to study the elimination/inactivation of HCV by the manufacturing procedures used for the production of h u m a n plasma derivatives, as requested by the CPMP guidelines. 6 For the time being, experimental studies with another member of the flaviviridae which can be grown in tissue cultures will be the only possibility. We therefore did a series of experiments with three members of the flaviviridae family to find out whether they were equally sensitive to heat treatment in an aqueous solution at 60°C for 10 h (i.e. pasteurization). Pasteurization is the virus inactivation method introduced into the manufacturing procedures of our plasma derivatives. 7The viruses used in these experiments were two flaviviruses, yellow 1045-1056/92/010083 + 03 $03.00/0
fever virus (YFV) and tick-borne encephalitis virus (TBEV), and one pestivirus bovine diarrhoea virus (BVDV). s For virus growth and titration of infectious v i r u s t h e following virus/cell s y s t e m s were used: BVDV/bovine epithelial foetal lung cells, TBEV/human carcinoma cell line (A549) and YFV strain 17 D/vero cells. Virus titres were determined in flat bottomed wells containing cell monolayers, which were incubated with 0.1 ml of tenfold serial dilutions in complete medium of the virus samples to be assayed. Eight wells per dilution were used. Microtitre plates were incubated at 37°C for 14 days and evaluated microscopically for cytopathic effects. The infectivity titres (CCIDso m1-1) were calculated according to the REED and MUENCH method. If no infectious virus was detected by microtitration, four 25 cm 2 or 100 cm 2 cell culture vessels were incubated with 1 ml each of the original sample diluted in cell culture medium. These cell cultures were also incubated at 37°C for 14 days and studied microscopically for cytopatic effects. If all four cultures remained negative, i.e. no infectious virus was detectable in four 1 ml aliquots of the original sample, the virus titre of this sample was given as less than 10°CCID~o ml-L The capability of pasteurization to inactivate viruses was tested by spiking stabilized plasma protein samples taken from a production lot with each of the three viruses. For this purpose one volume of the virus preparation was added to nine volumes of the product sample, and the resulting virus-spiked protein preparation was subjected to heat treatment at 60°C for 10 h. Aliquots were taken before heat treatment and at various times during heat treatment and assayed for infectious virus. For these studies a preparation of a highly purified factor VIII: G concentrate (tradename: Beriate P), an antithrombin III (tradename: Kybernin P), an im 7s immunoglobulin © 1992 The International Association of Biological Standardization
84
T. Nowak e t al.
(tradename: Beriglobin P) and an iv 5s immunoglobulin (tradename: Gamma-Venin P) were used. The plasma proteins were stabilized by high concentrations of sucrose and glycine which were added to the
aqueous protein preparation before heat t r e a t m e n t # The kinetics of the inactivation of the th re e flaviviridae tested in the above-mentioned protein solutions are shown in Fig. 1. The infectivity titres of
7 6 5 4 5 2 T 0 rm
I
0
No virus detectable
(.9 0 Cn 0
0
I
2
3
4
5
7
6
8
9
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>
5 4
2I-
O-
i
~ No virus detectable
I
0
,
I I
I
I 2
,
I 3
l
I 4
i
I 5
I
I 6
i
I
7
i
I
8
i
1
9
i 0
Heat treatment ( h ) Figure 1. Kinetics of the inactivation of tick-borne encephalitis virus (TBEV), yellow fever virus (YFV) and bovine viral diarrhoea virus (BVDV) by heat treatment at 60°C in aqueous solutions of various stabilized plasma proteins. The following preparations spiked with TBEV or YFV were tested: ( a ) - - T - - , TBEV/7s immunoglobulin; --~7--, TBEV/5s iv immunoglobulin; --O--, YFV/antithrombin II; III--[:]--, YFV/factor VIII:G concentrate of high purity; - - i - - , YFV/7s im immunoglobulin; --O--, YFV/5s iv immunoglobulin. The inactivation of BVDV was analysed in the following protein preparations: (b) --O--, antithrombin III; --[2--, factor VIII concentrate of high purity; - - I - - , YFV/7s im immunoglobulin; --O--, YFV/5s iv. immunoglobulin.
Inactivation of viruses by pasteurization
the 'spiked' plasma samples before heat t r e a t m e n t varied between 105 and more t h a n 107 CCID~o m1-1. On average, the infectivity titres of the virus harvests of YFV and BVDV, used for spiking, were one order of magnitude lower t h a n t h a t of TBEV. Therefore, the infectivity titres of the plasma protein samples spiked with BVDV or YFV were also one order of magnitude lower t h a n those spiked with TBEV. All three viruses tested were completely inactivated after a 6 h heat t r e a t m e n t at maximum. As expected, slight variations of the virus inactivation were observed, depending on the protein sample in which the virus inactivation occurred. When comparing the inactivation kinetics in Fig. l(a) and l(b) it is obvious, however, t h a t u n d e r these conditions, BVDV is more stable than the two flaviviruses TBEV and YFV. Our data demonstrate t h a t all three members of the flaviviridae are efficiently inactivated by pasteurization and t h a t the pestivirus BVDV is the most stable of all the viruses tested. Since, according to genome analysis and first studies on the physicochemical properties ofHCV 3.4 (including the heat stability described here), this virus is more closely related to the pestivirus group t h a n to the flavivirus group, we choose BVDV as a candidate virus (being closely related to HCV) for routine in-vitro virus safety studies.
Acknowledgements We are grateful to Mrs R. Volk, Mrs E. Weber and Mr K. Weber for their skilful technical assistance and Dr D. B e r n h a r d t for providing us with BVDV and the well-established assay system for this virus.
85
References
1. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A/non-B viral hepatitis C genome. Science 1989; 244: 359-362. 2. Kato N, Hijikata M, Ootsuyama Y, Nakagawa M, Ohkoshi S, Sugimura T, Shimotohno K. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, and non-B hepatitis. Proc Natl Acad Sci USA 1990; 87: 9524-9528. 3. Choo QL, Richman KH, Han JH, Berger K, Lee C, Dong C, Gallegos C, Coit D, Meddina-Selby A, Barr PJ, Weiner AJ, Bradley DW, Kuo G, Houghton M. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci USA 1991; 88: 2451-2455. 4. Bradley D, McCaustland K, Krawczynski K, Spelbring J, Humphrey C, Cook EH. Hepatitis C virus: buoyant density of the factor VIII-derived isolate in sucrose. J Med Virol 1991; 34: 206-208. 5. Prince AM, Horowitz B, Horowitz MS, Zang E. The development of virus-free labile blood derivatives-a review. Eur J Epidemiol 1987; 3: 103-118. 6. Commission of the European Communities: note for guidance of the CPMP: Validation of virus removal and inactivation procedures'. Biologicals 1991; 19: 247-251. 7. Hilfenhaus J, He~:l:mann A, Mauler R, Prince AM. Inactivation of the AIDS causing retrovirus and other human viruses in antihemophilic plasma protein preparations by pasteurization. Vox Sang 1986; 50: 208-211. 8. Collett MS, Anderson DK, Retzel E. Comparisons of the pestivirus bovine viral diarrhea virus with members of the flaviviridae. J Gen Virol 1988; 69: 2637-2643.
Received for publication 7 October 1991; accepted 6 November 1991.
SHORT NOTE Inactivation of Viruses Related to Hepatitis C Virus by Pasteurization in Human Plasma Derivatives Thomas Nowak, Ulrich Klockman and Joachim Hilfenhaus Research Laboratories of Behringwerke AG, Emil-von-Behring-StraBe 76, PO Box 11 40, D-3550 Marburg, Germany
Q. L. Choo et al.1 were the first to clone the genome of hepatitis C virus (HCV). Based on their findings it was possible to clone and sequence the genomes of various HCV isolates. 2 From the analysis of the genome of this RNA virus it was concluded t h a t HCV belongs to the family of the flaviviridae. 3According to the current nomenclature the flavivirus family is divided in three genera, the flaviviruses, the pestiviruses and the hepatitis G virus group. From the genome analyses it is further concluded t h a t HCV is more related to the pestiviruses than to the flaviviruses. This conclusion has recently been supported by a paper published by Bradley et al. 4 who studied the buoyant density of hepatitis C virus in a sucrose gradient. HCV is one of the h u m a n pathogenic viruses t h a t can be transmitted from blood or plasma donors to the recipients of whole blood transfusions or h u m a n plasma derivatives. ~ Since HCV cannot be propagated in tissue cultures or animals, apart from chimpanzees, there is no routine method available to assay infectious HCV. H u m a n blood and plasma protein preparations cannot therefore be investigated for a contamination with infectious HCV and it is not possible to study the elimination/inactivation of HCV by the manufacturing procedures used for the production of h u m a n plasma derivatives, as requested by the CPMP guidelines. 6 For the time being, experimental studies with another member of the flaviviridae which can be grown in tissue cultures will be the only possibility. We therefore did a series of experiments with three members of the flaviviridae family to find out whether they were equally sensitive to heat treatment in an aqueous solution at 60°C for 10 h (i.e. pasteurization). Pasteurization is the virus inactivation method introduced into the manufacturing procedures of our plasma derivatives. 7The viruses used in these experiments were two flaviviruses, yellow 1045-1056/92/010083 + 03 $03.00/0
fever virus (YFV) and tick-borne encephalitis virus (TBEV), and one pestivirus bovine diarrhoea virus (BVDV). s For virus growth and titration of infectious v i r u s t h e following virus/cell s y s t e m s were used: BVDV/bovine epithelial foetal lung cells, TBEV/human carcinoma cell line (A549) and YFV strain 17 D/vero cells. Virus titres were determined in flat bottomed wells containing cell monolayers, which were incubated with 0.1 ml of tenfold serial dilutions in complete medium of the virus samples to be assayed. Eight wells per dilution were used. Microtitre plates were incubated at 37°C for 14 days and evaluated microscopically for cytopathic effects. The infectivity titres (CCIDso m1-1) were calculated according to the REED and MUENCH method. If no infectious virus was detected by microtitration, four 25 cm 2 or 100 cm 2 cell culture vessels were incubated with 1 ml each of the original sample diluted in cell culture medium. These cell cultures were also incubated at 37°C for 14 days and studied microscopically for cytopatic effects. If all four cultures remained negative, i.e. no infectious virus was detectable in four 1 ml aliquots of the original sample, the virus titre of this sample was given as less than 10°CCID~o ml-L The capability of pasteurization to inactivate viruses was tested by spiking stabilized plasma protein samples taken from a production lot with each of the three viruses. For this purpose one volume of the virus preparation was added to nine volumes of the product sample, and the resulting virus-spiked protein preparation was subjected to heat treatment at 60°C for 10 h. Aliquots were taken before heat treatment and at various times during heat treatment and assayed for infectious virus. For these studies a preparation of a highly purified factor VIII: G concentrate (tradename: Beriate P), an antithrombin III (tradename: Kybernin P), an im 7s immunoglobulin © 1992 The International Association of Biological Standardization
84
T. Nowak e t al.
(tradename: Beriglobin P) and an iv 5s immunoglobulin (tradename: Gamma-Venin P) were used. The plasma proteins were stabilized by high concentrations of sucrose and glycine which were added to the
aqueous protein preparation before heat t r e a t m e n t # The kinetics of the inactivation of the th re e flaviviridae tested in the above-mentioned protein solutions are shown in Fig. 1. The infectivity titres of
7 6 5 4 5 2 T 0 rm
I
0
No virus detectable
(.9 0 Cn 0
0
I
2
3
4
5
7
6
8
9
I0
{,-
-I.-(D I,.-
>
5 4
2I-
O-
i
~ No virus detectable
I
0
,
I I
I
I 2
,
I 3
l
I 4
i
I 5
I
I 6
i
I
7
i
I
8
i
1
9
i 0
Heat treatment ( h ) Figure 1. Kinetics of the inactivation of tick-borne encephalitis virus (TBEV), yellow fever virus (YFV) and bovine viral diarrhoea virus (BVDV) by heat treatment at 60°C in aqueous solutions of various stabilized plasma proteins. The following preparations spiked with TBEV or YFV were tested: ( a ) - - T - - , TBEV/7s immunoglobulin; --~7--, TBEV/5s iv immunoglobulin; --O--, YFV/antithrombin II; III--[:]--, YFV/factor VIII:G concentrate of high purity; - - i - - , YFV/7s im immunoglobulin; --O--, YFV/5s iv immunoglobulin. The inactivation of BVDV was analysed in the following protein preparations: (b) --O--, antithrombin III; --[2--, factor VIII concentrate of high purity; - - I - - , YFV/7s im immunoglobulin; --O--, YFV/5s iv. immunoglobulin.
Inactivation of viruses by pasteurization
the 'spiked' plasma samples before heat t r e a t m e n t varied between 105 and more t h a n 107 CCID~o m1-1. On average, the infectivity titres of the virus harvests of YFV and BVDV, used for spiking, were one order of magnitude lower t h a n t h a t of TBEV. Therefore, the infectivity titres of the plasma protein samples spiked with BVDV or YFV were also one order of magnitude lower t h a n those spiked with TBEV. All three viruses tested were completely inactivated after a 6 h heat t r e a t m e n t at maximum. As expected, slight variations of the virus inactivation were observed, depending on the protein sample in which the virus inactivation occurred. When comparing the inactivation kinetics in Fig. l(a) and l(b) it is obvious, however, t h a t u n d e r these conditions, BVDV is more stable than the two flaviviruses TBEV and YFV. Our data demonstrate t h a t all three members of the flaviviridae are efficiently inactivated by pasteurization and t h a t the pestivirus BVDV is the most stable of all the viruses tested. Since, according to genome analysis and first studies on the physicochemical properties ofHCV 3.4 (including the heat stability described here), this virus is more closely related to the pestivirus group t h a n to the flavivirus group, we choose BVDV as a candidate virus (being closely related to HCV) for routine in-vitro virus safety studies.
Acknowledgements We are grateful to Mrs R. Volk, Mrs E. Weber and Mr K. Weber for their skilful technical assistance and Dr D. B e r n h a r d t for providing us with BVDV and the well-established assay system for this virus.
85
References
1. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A/non-B viral hepatitis C genome. Science 1989; 244: 359-362. 2. Kato N, Hijikata M, Ootsuyama Y, Nakagawa M, Ohkoshi S, Sugimura T, Shimotohno K. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, and non-B hepatitis. Proc Natl Acad Sci USA 1990; 87: 9524-9528. 3. Choo QL, Richman KH, Han JH, Berger K, Lee C, Dong C, Gallegos C, Coit D, Meddina-Selby A, Barr PJ, Weiner AJ, Bradley DW, Kuo G, Houghton M. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci USA 1991; 88: 2451-2455. 4. Bradley D, McCaustland K, Krawczynski K, Spelbring J, Humphrey C, Cook EH. Hepatitis C virus: buoyant density of the factor VIII-derived isolate in sucrose. J Med Virol 1991; 34: 206-208. 5. Prince AM, Horowitz B, Horowitz MS, Zang E. The development of virus-free labile blood derivatives-a review. Eur J Epidemiol 1987; 3: 103-118. 6. Commission of the European Communities: note for guidance of the CPMP: Validation of virus removal and inactivation procedures'. Biologicals 1991; 19: 247-251. 7. Hilfenhaus J, He~:l:mann A, Mauler R, Prince AM. Inactivation of the AIDS causing retrovirus and other human viruses in antihemophilic plasma protein preparations by pasteurization. Vox Sang 1986; 50: 208-211. 8. Collett MS, Anderson DK, Retzel E. Comparisons of the pestivirus bovine viral diarrhea virus with members of the flaviviridae. J Gen Virol 1988; 69: 2637-2643.
Received for publication 7 October 1991; accepted 6 November 1991.
