Originalia W. Siegert, J. Grunst, W. WiImanns, G. G. FrOsner, F. Deinhardt
Quantitative Correlation between the Dane Particle-Associated DNA Polymerase and the Hepatitis B e Antigen Summary: In order to evaluate the potential infectivity of blood of hepatitis B patients, the Dane particle associated DNA polymerase was determined, which is a reliable marker for the presence of complete viral particles. Enzyme activities were compared with hepatitis B e antigen (HBeAg) titers determined by radioimmunoassay. Detectable DNA polymerase activity was only present in HBeAg positive blood, preferentially in samples with high antigen titers (1 : 1000 and above). These samples therefore have to be considered as highly infectious. However, blood with low HBeAg levels and free of detectable polymerase activity can still be infectious, since the polymerase reaction is rather insensitive compared to the radioimmunological HBeAg determination. Zusammenfassung: Quantitative Korrelation zwischen der Dane Partikel-assoziierten DNS-Polymerase und dem Hepatitis B e Antigen. Zur Beurteilung der m6glichen Infektiosit~it yon Blut yon an Hepatitis B erkrankten Patienten wurde die Dane Partikel-assoziierte DNS-Polymerase bestimmt. Diese ist ein zuverliissiger Marker ftir die Anwesenheit kompletter Viruspartikel. Die Enzymaktivit~iten wurden mit den im Radioimmunassay bestimmten Hepatitis B e Antigen (HBeAg)-Titern verglichen. MeBbare DNS-Polymeraseaktivitiit war nut in HBeAg-positivem Blut vorhanden, vorzugsweise in Proben mit hohen Antigentitern (1:t000 und dariiber). Aus diesem Grund miissen solche Proben als h6chst infekti6s angesehen werden. Jedoch kann Blur mit niedrigen HBeAg-Spiegeln und ohne nachweisbare DNS-Polymerase trotzdem infekti6s sein, da die Polymerasereaktion relativ unempfindlich ist, verglichen mit der radioimmunologischen HBeAg-Bestimmung.
Introduction As a rule, the potential infectivity of patients suffering from hepatitis B can be judged only indirectly, since the demonstration of viral particles or the performance of biological tests for infectivity (1) are not routine procedures. The close correlation between the presence of hepatitis B e antigen (HBeAg) or Dane particles in the blood (2, 3, 4, 5) and its infectivity has been demonstrated in several clinical studies (6, 7, 8, 9). Newborns of HBeAg positive mothers frequently become infected, in contrast to children of women who are hepatitis B surface antigen (HBsAg) carriers but HBeAg negative (8). Accidental inoculation with HBeAg positive blood frequently results in overt or inapparent infection (9), thus making HBeAg a valuable marker of infectivity. Recently, the development of a highly sensitive solid phase radioimmunoassay for HBeAg has been reported (10). HBeAg can be detected regularly in the serum during the acute stage of hepatitis B and there seems to be a good correlation between the presence of Dane particles and HBeAg. Nevertheless, it has not been established with
220
Infection 7 (1979) Nr. 5
certaimy whether HBeAg is a structural viral protein, a virus-coded non-structural antigen or a virus induced host cell antigen (11, 12, and for review see 13). Most recent data seem to indicate, however, that HBeAg is indeed a structural component of the Dane particle, located between the envelope and the core (Zuckerman, personal communication). To obtain more direct evidence for the presence of infectious viral particles in HBeAg positive plasma, we have determined the activity of the Dane particle associated D N A polymerase and compared it with the HBeAg titers determined by radioimmunoassay in the same blood sample.
Materials and Methods Plasma specimens: Samples of heparinized plasma were drawn from 44 HBsAg positive patients with acute and histologically confirmed chronic hepatitis B. Forty-seven HBsAg negative healthy blood donors served as controls. All samples were assayed for the presence of HBsAg, HBeAg, and the particle-associated DNA polymerase. Serological determinations: HBeAg was determined by solid phase radioimmunoassay as already described (10). For titration plasma specimens were assayed undiluted and diluted 1 : 10, 1 : 100 and 1 : 1000 in 0.9% NaC1. HBsAg determinations were performed by solid phase radioimmunoassay (Ausria II). DNA polymerase assay: The enzyme assay was performed essentially according to the method of Kaplan et al. (14). The radioactivity values given in Figures I and 2 were obtained by trichloracetic acid precipitation of standard incubation mixtures composed of 100 ~1 Dane particle suspension, corresponding to 2.0 ml plasma, and 50 ,ul salt mixture after an incubation for four hours at 37°.
Results and Discussion The plasma samples were grouped into three categories according to their HBAg pattern -- 1) HBsAg negative/ HBeAg negative, 2) HBsAg positive/HBeAg negative and 3) HBsAg positive/HBeAg positive -- and tested for their D N A polymerase content. Figure 1 shows the incorpora-
Received: 18 May 1979 Dr. W. Siegert, PD Dr. J. Grunst, Prof. Dr. W. Wilmanns, III. und II. Medizinische Klinik im Klinikum Groghadern, Marchioninistrage 15, D-8000 Munich 70, West Germany; PD Dr. G. G. Fr&sner, Prof. Dr. F. Deinhardt, Max von Pettenkofer Institut ftir Hygiene und medizinische Mikrobiologie, PettenkoferstraBe 9a, D-8000 Munich 2, West Germany.
W. Siegert et aI.: Hepatitis B e Antigen
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Figure 1: Dane particle-associated DATA polymerase activity was determined in HBsAg and HBeAg negative (Group 1), in HBsAg positive/HBeAg negative (Group 2) and in HBsAg positivejHBeAg positive (Group 3) plasma. The incorporation o/tritiated triphosphates into DNA was determined by TCA precipitation and liquid scintillation counting after performing the standard assay with/our hour incubation at 37° .
150
'7, o x
100
E
50
20
!
10
o
I 1110
1:100
o
if- ,y
I:1000
>1:1000
HBeAg
present. Due to the lack of sufficient material this test could not be repeated to exclude an artifact. Moreover, within the HBeAg positive group (Group 3) there was a considerable proportion of samples with low and some with no measurable enzyme activity. If a correlation between HBeAg and infectivity exists, one m a y also expect a correlation between HBeAg and the activity of the Dane particle-associated D N A polymerase. To test this assumption, all HBeAg positive samples were titrated for their HBeAg content, and the individual enzyme activities were plotted against the corresponding HBeAg titer (Figure 2). Most plasmas with high polymerase activity also had high HBeAg titers ( ~ 1 : 1000). Samples with medium to low HBeAg titers generally had low or undetectable enzyme activity. However, this correlation m a y not be very strict, since in a few cases samples with medium HBeAg titers exhibited high enzyme activity, and a few samples with high HBeAg titers had low or undetectable polymerase activities. In conclusion, these experiments indicate that blood with high HBeAg titers usually also exhibits moderate to high polymerase activity and therefore must be considered as highly infectious. The situation is less clear in samples which do not contain detectable polymerase activity but low titers of HBeAg. One likely explanation would be that the HBeAg determination is a more sensitive marker for infectivity than the D N A polymerase assay. HBeAg was determined in this series with a very sensitive radioimmunoassay, whereas the polymerase reaction is less sensitive because it requires rather high particle concentrations. F o r this reason it is conceivable that samples with low HBeAg titers also contain infectious particles, but in concentrations too low for the detection by the polymerase reaction. However, if HBeAg is not a structural component of the Dane particle, or if HBeAg is produced in excess, it could be present in the absence of Dane particles and therefore not be a definite marker of infectivity. Further studies of the correlation between Dane particle counts, polymerase activity and HBeAg titers may define more accurately the potential infectivity of blood or blood products. But until such information is available, biological materials positive for HBeAg must be considered to be infectious.
-titer
Figure 2: DNA polymerase activity measured by the incorporation of tritiated triphosphates into DNA in HBeAg positive plasma specimens plotted versus the corresponding HBeAg titer determined by radioimmunoassay. tion of TCA-precipitable radioactivity during a standard incubation. It is obvious and in agreement with previous findings that there is detectable polymerase activity only in the HBeAg positive group (Group 3). In a single case we unexpectedly detected enzyme activity in a HBsAg positive/HBeAg negative blood sample from a patient with chronic hepatitis B. This fact cannot be explained at
Acknowledgement Part of this study was supported by a grant from the Deutsche Forschungsgemeinschaft (Fr 400/6). Literature 1. Deinhardt, F.: Hepatitis in primates. Adv. Virus Res. 20 (1976) 113-157. 2. Nordenfelt, E., Andren-Sandberg, M.: Dane particle-associated DNA polymerase and e antigen: Relation to chronic hepatitis among carriers of hepatitis B surface antigen. J. infect. Dis. 134 (1976) 85-89. 3. Hess, G., Arnold, W., Shih, W.-K., Kaptan, P. M., Purcell, R. H., Gerin, 1. L., Meyer zum Bueschenfelde, K. H.: Expression of hepatitis B virus-specific markers in asymptomatic
Infection 7 (1979) Nr. 5
221
W. Siegert et al.: Hepatitis B e Antigen hepatitis B surface antigen carriers. Infect. Immun. 17 (1977) 550-554.
4. Nordenfelt, E., K]ell~n, L,: Dane particles, D N A polymerase, and e antigen in two different categories of hepatitis B antigen carriers. Intervirology 5 (1975) 225-232.
5. Magnius, L. 0., Espmark, ]. A.: New specificities in Australia antigen positive sera distinct from the Le Bouvier determinants. J. Immunol. 109 (1972) 1017-1021. 6. Shikata, T., Karasawa, T., Abe, K., Uzawa, T., Suzuki, H., Oda, T., Imai, M., Mayumi, M., Moritsugu, Y.: Hepatitis B e antigen and infectivity of hepatitis B virus. J. infect. Dis. 136 (1977) 571-576.
7. Maynard, J. E., Barrett, D. H., Murphy, B. L., Bradley, D. W., Berquist, K. R., Bender, T. R.: Relation of e antigen to hepatitis B virus infection in an area of hyperendemicity. J. infect. Dis. 133 (1976) 339-342.
8. Okada, K., Kamiyama, I., lnomata, M., Irnai, M., Miyakawa, Y., Mayumi, M.: E antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of positive and negative transmission of hepatitis B virus to their infants. New Engl. J. Med. 294 (1976) 746-749.
9. Alter, J. H., See//, L. B., Kaplan, P. M., McAuli]/e, V. J., Wright, E. C., Gerin, 1. L., Purcell, R. H. Holland P. V.,
222
Infection 7 (1979) Nr. 5
Zimmermann, H. l.: Type B hepatitis: The infectivity of blood positive for e antigen and D N A polymerase after accidental needlestick exposure. New Engl. J. Med. 295 (1976) 909-913. 10. Fr6sner, G. G., Brodersen, M., Papaevangelou, G., Sugg,
U., Haas, H., Mushahwar, L K., Ling, C.-M., Overby, L. R., Deinhardt, F.: Detection of HBeAg and anti-HBe in acute hepatitis B by a sensitive radioimmunoassay. J. med. Virol. 3 (1978) 67-76. 11. Takahashi, K., Yamashita, S., Imai, M., Miyakawa, Y., Mayumi, M.: Failure of antibody to e antigen to precipitate Dane particles containing D N A polymerase activity and hepatitis B core antigen. J. Gen. Virol. 38 (1978) 431-436. 12. Neurath, A. R., Trepo, C., Chen, M., Prince, A. M.: Identification of additional antigenic sites on Dane particles and the tubular forms of hepatitis B surface antigen. J. Gen. Virol. 30 (1976) 277-285. 13. Millman, I., Magnius, L. 0.: HBeAg and anti-HBe. In: Vyas, G. N., Cohen, S. N., Sehmid, R. (ed.): Viral hepatitis. A contemporary assessment of etiology, epidemiology, pathogenesis and prevention, p. 159-216. Franklin Institute Press, Philadelphia 1978. 14. KapIan, P. M., Greenman, R. L., Gerin, J. L., Purcell, R. H., Robinson, W. S.: D N A polymerase associated with human hepatitis B antigen. J. Virol. 12 (1973) 995-1005.
Quantitative Correlation between the Dane Particle-Associated DNA Polymerase and the Hepatitis B e Antigen Summary: In order to evaluate the potential infectivity of blood of hepatitis B patients, the Dane particle associated DNA polymerase was determined, which is a reliable marker for the presence of complete viral particles. Enzyme activities were compared with hepatitis B e antigen (HBeAg) titers determined by radioimmunoassay. Detectable DNA polymerase activity was only present in HBeAg positive blood, preferentially in samples with high antigen titers (1 : 1000 and above). These samples therefore have to be considered as highly infectious. However, blood with low HBeAg levels and free of detectable polymerase activity can still be infectious, since the polymerase reaction is rather insensitive compared to the radioimmunological HBeAg determination. Zusammenfassung: Quantitative Korrelation zwischen der Dane Partikel-assoziierten DNS-Polymerase und dem Hepatitis B e Antigen. Zur Beurteilung der m6glichen Infektiosit~it yon Blut yon an Hepatitis B erkrankten Patienten wurde die Dane Partikel-assoziierte DNS-Polymerase bestimmt. Diese ist ein zuverliissiger Marker ftir die Anwesenheit kompletter Viruspartikel. Die Enzymaktivit~iten wurden mit den im Radioimmunassay bestimmten Hepatitis B e Antigen (HBeAg)-Titern verglichen. MeBbare DNS-Polymeraseaktivitiit war nut in HBeAg-positivem Blut vorhanden, vorzugsweise in Proben mit hohen Antigentitern (1:t000 und dariiber). Aus diesem Grund miissen solche Proben als h6chst infekti6s angesehen werden. Jedoch kann Blur mit niedrigen HBeAg-Spiegeln und ohne nachweisbare DNS-Polymerase trotzdem infekti6s sein, da die Polymerasereaktion relativ unempfindlich ist, verglichen mit der radioimmunologischen HBeAg-Bestimmung.
Introduction As a rule, the potential infectivity of patients suffering from hepatitis B can be judged only indirectly, since the demonstration of viral particles or the performance of biological tests for infectivity (1) are not routine procedures. The close correlation between the presence of hepatitis B e antigen (HBeAg) or Dane particles in the blood (2, 3, 4, 5) and its infectivity has been demonstrated in several clinical studies (6, 7, 8, 9). Newborns of HBeAg positive mothers frequently become infected, in contrast to children of women who are hepatitis B surface antigen (HBsAg) carriers but HBeAg negative (8). Accidental inoculation with HBeAg positive blood frequently results in overt or inapparent infection (9), thus making HBeAg a valuable marker of infectivity. Recently, the development of a highly sensitive solid phase radioimmunoassay for HBeAg has been reported (10). HBeAg can be detected regularly in the serum during the acute stage of hepatitis B and there seems to be a good correlation between the presence of Dane particles and HBeAg. Nevertheless, it has not been established with
220
Infection 7 (1979) Nr. 5
certaimy whether HBeAg is a structural viral protein, a virus-coded non-structural antigen or a virus induced host cell antigen (11, 12, and for review see 13). Most recent data seem to indicate, however, that HBeAg is indeed a structural component of the Dane particle, located between the envelope and the core (Zuckerman, personal communication). To obtain more direct evidence for the presence of infectious viral particles in HBeAg positive plasma, we have determined the activity of the Dane particle associated D N A polymerase and compared it with the HBeAg titers determined by radioimmunoassay in the same blood sample.
Materials and Methods Plasma specimens: Samples of heparinized plasma were drawn from 44 HBsAg positive patients with acute and histologically confirmed chronic hepatitis B. Forty-seven HBsAg negative healthy blood donors served as controls. All samples were assayed for the presence of HBsAg, HBeAg, and the particle-associated DNA polymerase. Serological determinations: HBeAg was determined by solid phase radioimmunoassay as already described (10). For titration plasma specimens were assayed undiluted and diluted 1 : 10, 1 : 100 and 1 : 1000 in 0.9% NaC1. HBsAg determinations were performed by solid phase radioimmunoassay (Ausria II). DNA polymerase assay: The enzyme assay was performed essentially according to the method of Kaplan et al. (14). The radioactivity values given in Figures I and 2 were obtained by trichloracetic acid precipitation of standard incubation mixtures composed of 100 ~1 Dane particle suspension, corresponding to 2.0 ml plasma, and 50 ,ul salt mixture after an incubation for four hours at 37°.
Results and Discussion The plasma samples were grouped into three categories according to their HBAg pattern -- 1) HBsAg negative/ HBeAg negative, 2) HBsAg positive/HBeAg negative and 3) HBsAg positive/HBeAg positive -- and tested for their D N A polymerase content. Figure 1 shows the incorpora-
Received: 18 May 1979 Dr. W. Siegert, PD Dr. J. Grunst, Prof. Dr. W. Wilmanns, III. und II. Medizinische Klinik im Klinikum Groghadern, Marchioninistrage 15, D-8000 Munich 70, West Germany; PD Dr. G. G. Fr&sner, Prof. Dr. F. Deinhardt, Max von Pettenkofer Institut ftir Hygiene und medizinische Mikrobiologie, PettenkoferstraBe 9a, D-8000 Munich 2, West Germany.
W. Siegert et aI.: Hepatitis B e Antigen
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®
®
160
120
100
o
E
80
m 60
20
i
8 5
2
Figure 1: Dane particle-associated DATA polymerase activity was determined in HBsAg and HBeAg negative (Group 1), in HBsAg positive/HBeAg negative (Group 2) and in HBsAg positivejHBeAg positive (Group 3) plasma. The incorporation o/tritiated triphosphates into DNA was determined by TCA precipitation and liquid scintillation counting after performing the standard assay with/our hour incubation at 37° .
150
'7, o x
100
E
50
20
!
10
o
I 1110
1:100
o
if- ,y
I:1000
>1:1000
HBeAg
present. Due to the lack of sufficient material this test could not be repeated to exclude an artifact. Moreover, within the HBeAg positive group (Group 3) there was a considerable proportion of samples with low and some with no measurable enzyme activity. If a correlation between HBeAg and infectivity exists, one m a y also expect a correlation between HBeAg and the activity of the Dane particle-associated D N A polymerase. To test this assumption, all HBeAg positive samples were titrated for their HBeAg content, and the individual enzyme activities were plotted against the corresponding HBeAg titer (Figure 2). Most plasmas with high polymerase activity also had high HBeAg titers ( ~ 1 : 1000). Samples with medium to low HBeAg titers generally had low or undetectable enzyme activity. However, this correlation m a y not be very strict, since in a few cases samples with medium HBeAg titers exhibited high enzyme activity, and a few samples with high HBeAg titers had low or undetectable polymerase activities. In conclusion, these experiments indicate that blood with high HBeAg titers usually also exhibits moderate to high polymerase activity and therefore must be considered as highly infectious. The situation is less clear in samples which do not contain detectable polymerase activity but low titers of HBeAg. One likely explanation would be that the HBeAg determination is a more sensitive marker for infectivity than the D N A polymerase assay. HBeAg was determined in this series with a very sensitive radioimmunoassay, whereas the polymerase reaction is less sensitive because it requires rather high particle concentrations. F o r this reason it is conceivable that samples with low HBeAg titers also contain infectious particles, but in concentrations too low for the detection by the polymerase reaction. However, if HBeAg is not a structural component of the Dane particle, or if HBeAg is produced in excess, it could be present in the absence of Dane particles and therefore not be a definite marker of infectivity. Further studies of the correlation between Dane particle counts, polymerase activity and HBeAg titers may define more accurately the potential infectivity of blood or blood products. But until such information is available, biological materials positive for HBeAg must be considered to be infectious.
-titer
Figure 2: DNA polymerase activity measured by the incorporation of tritiated triphosphates into DNA in HBeAg positive plasma specimens plotted versus the corresponding HBeAg titer determined by radioimmunoassay. tion of TCA-precipitable radioactivity during a standard incubation. It is obvious and in agreement with previous findings that there is detectable polymerase activity only in the HBeAg positive group (Group 3). In a single case we unexpectedly detected enzyme activity in a HBsAg positive/HBeAg negative blood sample from a patient with chronic hepatitis B. This fact cannot be explained at
Acknowledgement Part of this study was supported by a grant from the Deutsche Forschungsgemeinschaft (Fr 400/6). Literature 1. Deinhardt, F.: Hepatitis in primates. Adv. Virus Res. 20 (1976) 113-157. 2. Nordenfelt, E., Andren-Sandberg, M.: Dane particle-associated DNA polymerase and e antigen: Relation to chronic hepatitis among carriers of hepatitis B surface antigen. J. infect. Dis. 134 (1976) 85-89. 3. Hess, G., Arnold, W., Shih, W.-K., Kaptan, P. M., Purcell, R. H., Gerin, 1. L., Meyer zum Bueschenfelde, K. H.: Expression of hepatitis B virus-specific markers in asymptomatic
Infection 7 (1979) Nr. 5
221
W. Siegert et al.: Hepatitis B e Antigen hepatitis B surface antigen carriers. Infect. Immun. 17 (1977) 550-554.
4. Nordenfelt, E., K]ell~n, L,: Dane particles, D N A polymerase, and e antigen in two different categories of hepatitis B antigen carriers. Intervirology 5 (1975) 225-232.
5. Magnius, L. 0., Espmark, ]. A.: New specificities in Australia antigen positive sera distinct from the Le Bouvier determinants. J. Immunol. 109 (1972) 1017-1021. 6. Shikata, T., Karasawa, T., Abe, K., Uzawa, T., Suzuki, H., Oda, T., Imai, M., Mayumi, M., Moritsugu, Y.: Hepatitis B e antigen and infectivity of hepatitis B virus. J. infect. Dis. 136 (1977) 571-576.
7. Maynard, J. E., Barrett, D. H., Murphy, B. L., Bradley, D. W., Berquist, K. R., Bender, T. R.: Relation of e antigen to hepatitis B virus infection in an area of hyperendemicity. J. infect. Dis. 133 (1976) 339-342.
8. Okada, K., Kamiyama, I., lnomata, M., Irnai, M., Miyakawa, Y., Mayumi, M.: E antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of positive and negative transmission of hepatitis B virus to their infants. New Engl. J. Med. 294 (1976) 746-749.
9. Alter, J. H., See//, L. B., Kaplan, P. M., McAuli]/e, V. J., Wright, E. C., Gerin, 1. L., Purcell, R. H. Holland P. V.,
222
Infection 7 (1979) Nr. 5
Zimmermann, H. l.: Type B hepatitis: The infectivity of blood positive for e antigen and D N A polymerase after accidental needlestick exposure. New Engl. J. Med. 295 (1976) 909-913. 10. Fr6sner, G. G., Brodersen, M., Papaevangelou, G., Sugg,
U., Haas, H., Mushahwar, L K., Ling, C.-M., Overby, L. R., Deinhardt, F.: Detection of HBeAg and anti-HBe in acute hepatitis B by a sensitive radioimmunoassay. J. med. Virol. 3 (1978) 67-76. 11. Takahashi, K., Yamashita, S., Imai, M., Miyakawa, Y., Mayumi, M.: Failure of antibody to e antigen to precipitate Dane particles containing D N A polymerase activity and hepatitis B core antigen. J. Gen. Virol. 38 (1978) 431-436. 12. Neurath, A. R., Trepo, C., Chen, M., Prince, A. M.: Identification of additional antigenic sites on Dane particles and the tubular forms of hepatitis B surface antigen. J. Gen. Virol. 30 (1976) 277-285. 13. Millman, I., Magnius, L. 0.: HBeAg and anti-HBe. In: Vyas, G. N., Cohen, S. N., Sehmid, R. (ed.): Viral hepatitis. A contemporary assessment of etiology, epidemiology, pathogenesis and prevention, p. 159-216. Franklin Institute Press, Philadelphia 1978. 14. KapIan, P. M., Greenman, R. L., Gerin, J. L., Purcell, R. H., Robinson, W. S.: D N A polymerase associated with human hepatitis B antigen. J. Virol. 12 (1973) 995-1005.
