Potential infectivity of blood from HBsAG asymptomatic carriers due to the presence of HBV-DNA and comparison with other markers of hbv infection

Tedeschi, M. Valeria; Padilla, Cláudia Figueiredo; Camargo, Iná Ferraz De; Yoshida, Clara F. Tachibana

doi:10.1590/S0036-46651989000600003

Abstracts

Serum samples from 356 HBsAg positive asymptomatic carriers, which were titrated by reverse passive hemagglutination, were analysed for the presence of HBV-DNA, HBsAg and IgM anti-HBc. The samples were divided in three classes, according to the titers of HBsAg and IgM anti-HBc and the distribution of HBV-DNA and HBsAg among these classes was studied. In the high titer class of HBsAg, 65% of samples have one or both markers against only 19% in the low titer class. From the total of 356 samples, 121 gave positive results for IgM anti-HBc (33.9%). From these, 38.9% of HBV-DNA and 47.9% of HBeAg were observed, whereas in samples with absence of IgM anti-HBc, 18.3% and 16.6% were respectively found. A higher frequency of agreement between all these markers was found in the class of high titers of HBsAg; however, HBV-DNA was detected in the low titer class of HBsAg and little or no IgM anti-HBc, showing potential blood infectivity even in HBsAg positive borderline samples.

HBV-DNA; HBsAg; IgM anti-HBc Asymptomatic carriers

Em 356 soros HBsAg positivos de portadores assintomáticos, titulados por hemaglutinação passiva reversa, foi analisada a presença de HBV DNA, HBeAg e IgM anti-HBc, da seguinte forma: as amostras foram divididas em três classes de acordo com o título de HBsAg e IgM anti-HBc e foi verificada a distribuição de HBV DNA e HBsAg nestas classes. Em amostras com títulos elevados de HBsAg, 65% das amostras tiveram um ou ambos os marcadores HBV-DNA e HBsAg e as mesmas foram encontradas em apenas 19% das amostras com baixo título de HBsAg. Do total de 356 amostras, 121 foram positivas para IgM anti-HBc (33.9%). Destas, 38.9% de HBV-DNA e 47.9% de HBeAg foram observadas, ao passo que em amostras com ausência de IgM anti-HBc, 18.3% e 16.6% foram respectivamente encontrados. A concordância entre os três marcadores foi encontrada em. amostras com alto título de HBsAg, entretanto HBV-DNA foi também detectado em grupo de baixo título de HBsAg na ausência de IgM anti-HBc ou em níveis baixos, mostrando o potencial de infectividade mesmo em amostras de HBsAg com valores próximos ao limite da positividade.

ORIGINAL ARTICLES

Potential infectivity of blood from HBsAG asymptomatic carriers due to the presence of HBV-DNA and comparison with other markers of hbv infection

Potencial de infectividade em sangue de portadores assintomáticos de HBsAg caracterizado pela presença de HBV-DNA e comparação com outros marcadores da Hepatite B.

M. Valeria Tedeschi^I; Cláudia Figueiredo Padilla^I; Iná Ferraz De Camargo^II; Clara F. Tachibana Yoshida^I

^IDepartamento de Virologia, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil

^IIBio-Manguinhos, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil

^{Address for correspondence} Address for correspondence: Dra. M. Valeria Tedeschi Fundação Oswaldo Cruz Departamento de Virologia Av. Brasil 4365 CEP 21040 Rio de Janeiro, RJ, Brazil.

SUMMARY

Serum samples from 356 HBsAg positive asymptomatic carriers, which were titrated by reverse passive hemagglutination, were analysed for the presence of HBV-DNA, HBsAg and IgM anti-HBc. The samples were divided in three classes, according to the titers of HBsAg and IgM anti-HBc and the distribution of HBV-DNA and HBsAg among these classes was studied. In the high titer class of HBsAg, 65% of samples have one or both markers against only 19% in the low titer class. From the total of 356 samples, 121 gave positive results for IgM anti-HBc (33.9%). From these, 38.9% of HBV-DNA and 47.9% of HBeAg were observed, whereas in samples with absence of IgM anti-HBc, 18.3% and 16.6% were respectively found.

A higher frequency of agreement between all these markers was found in the class of high titers of HBsAg; however, HBV-DNA was detected in the low titer class of HBsAg and little or no IgM anti-HBc, showing potential blood infectivity even in HBsAg positive borderline samples.

Key words: HBV-DNA; HBsAg; IgM anti-HBc Asymptomatic carriers.

RESUMO

Em 356 soros HBsAg positivos de portadores assintomáticos, titulados por hemaglutinação passiva reversa, foi analisada a presença de HBV DNA, HBeAg e IgM anti-HBc, da seguinte forma: as amostras foram divididas em três classes de acordo com o título de HBsAg e IgM anti-HBc e foi verificada a distribuição de HBV DNA e HBsAg nestas classes.

Em amostras com títulos elevados de HBsAg, 65% das amostras tiveram um ou ambos os marcadores HBV-DNA e HBsAg e as mesmas foram encontradas em apenas 19% das amostras com baixo título de HBsAg. Do total de 356 amostras, 121 foram positivas para IgM anti-HBc (33.9%). Destas, 38.9% de HBV-DNA e 47.9% de HBeAg foram observadas, ao passo que em amostras com ausência de IgM anti-HBc, 18.3% e 16.6% foram respectivamente encontrados.

A concordância entre os três marcadores foi encontrada em. amostras com alto título de HBsAg, entretanto HBV-DNA foi também detectado em grupo de baixo título de HBsAg na ausência de IgM anti-HBc ou em níveis baixos, mostrando o potencial de infectividade mesmo em amostras de HBsAg com valores próximos ao limite da positividade.

Full text available only in PDF format.

Texto completo disponível apenas em PDF.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge Messias da Silva, Luiz Antonio C. Mercadante and Sergio da Silva e Mouta Jr. for technical assistance. Claudia F. Padilla was a trainee in our Department with a fellowship from CNPq.

Part of this work was supported by CNPq, grant number 40.0081.89.0.

Recebido para publicação em 16/5/1989.

1. ALMEIDA, J. D.; RUBINSTEIN, D. & STOTT, E. J. - New antigen antibody system in Australia antigen positive hepatitis. Lancet, 2: 1225-1227, 1971.
2. BENSABATH, G.; HADLER, S. C; PEREIRA SOARES, M. C; FIELDS, H. & MAYNARD, J. E. - Epidemiologic and serologic studies of acute viral hepatitis in Brazil's Amazon Basin. Bull. Pan Amer. Hlth. Org., 21: 16-27, 1987
3. BENSABATH, G.; HADLER, S. C; PEREIRA SOARES, M. C; FIELDS, H.; DIAS, L. B.; POPPER, H. & MAY-NARD, J. - Hepatitis Delta virus infection and Labrea Hepatitis prevalence and role in fulminant Hepatitis in the Amazon Basin. J. Amer. med. Ass., 258: 479-483, 1987.
4. BERNINGER, M.; HAMMER. M.; HOYER, B. & GERIN, J. - An assay for the detection of the DNA genome of Hepatitis B virus in serum. J. med. Virol, 9: 57-68, 1982.
5. BRECHOT, C. & HADCHOUEL, M.; SCOTTO, J.; DE GOS, F.; CHARNAY, P.; TREPO, C. & TIOLLAIS, P. - Detection of Hepatitis B virus DNA in liver and serum: A direct appraisal of the chronic carrier state. Lancet, 2: 765-768, 1981.
6. BONINO, F.; HOYER, B.; NELSON, J.; ENGLE, R.; VER ME, G. & GERIN, J. - Hepatitis B virus DNA in the sera of the HBsAg carriers: a marker of active hepatitis B virus replication in the liver. Hepatology, 1: 386-391, 1981.
7. CAMARGO, I. F.; GASPAR, A. M. C. & YOSHIDA, C. F. T. - Comparative ELISA reagents for detection of He patitis B surface antigen (HBsAg). Mem. Inst. Oswaldo Cruz, 82: 181-187, 1987.
8. DANE, D. S.; CAMEROM, C. H. & BRIGGS, M. - Virus like particles in serum of patients with Australia antigen associated hepatitis. Lancet, 1: 695-698, 1970.
9. DEINHARDT, F. - Predictive value of markers of Hepa titis virus infection. J. infect. Dis., 141: 299-305, 1980.
10. DIEGUTIS, P. S.; BURNETT, L.; NIGHTINGALE, B. N.; LOWE, S. B.; GIUNEY, R. C; COSSART, Y. E.; KEIR NAN, E.; WILLIANS, G.; PARSONS, C; McCAUGHAN, G.; FREIMAN, J.; BRITTON, W. J. & GALLAGER, N. D. - Relationship between hepatitis B virus DNA in blood and serological markers of hepatitis B infection. Med. J. Aust., 144: 351-355, 1986.
11. DORMEYER, H. H.; ARNOLD, W.; KRYGER, P.; NIELSEN, J. O. & MEYER ZUM BUSCHENFELDE, K. H. - IgM antibody to hepatitis B core antigen (anti HBc IgM) in "healthy" HBsAg carriers: A longitudinal study of 75 cares. Klin. Wschr., 59: 675-678, 1981.
12. FAY, O. H.; HADLER, S. C; MAYNARD, J. E. & PINHEI-RO, F. - Hepatitis in the Americas. Bol. epidem. (OPS), 6(5): 1-7, 1985.
13. FIGUEIREDO MENDES, F.; SIMONETTI, J. P.; FONSE CA, J. C. F.; SIMONETTI, S. R. R.; PITELLA, A. M.; SCHATZMAYR, H. G.; LIMA FERREIRA, L. C; MIRANDA SANTOS, I. K.; MEXAS, P. P. F. & HERBERT, B. A. - O impacto da hepatite Delta. Mod. Hepat., 9: 16-22, 1984.
14. GERLICH, W. H.; LUER, W.; THOMSSEN, R. & the study group for viral hepatitis of the Deutsche Forschungsge-meinchaft. - Diagnosis of acute and inapparent hepatitis B virus infections by measurement of IgM antibody to hepatitis B core antigen. J. infect. Dis., 142: 95-101, 1980.
15. KAPLAN, P. M., FORD, E. C; PURCELL, R. H. & GERIN, J. L. - Demonstration of subpopulation of Dane particles. J. Virol., 17: 885-893, 1976.
16. KAPLAN, P. M.; GREENMAN, R. L.; GERIN, J. L.; PUR CELL, R. H. & ROBINSON, W. S. - DNA polymerase associated with human hepatitis B antigen. J. Virol., 12: 995-1005, 1973.
17. KRYGER, P.; MATHIESEN, L. R.; ALDERSHVILE, J. & NIELSEN, J. O. - Presence and meaning of anti HBc IgM as determined by ELISA in patients with acute type B hepatitis and healthy HBsAg carriers. Hepatology, 12: 50-53, 1982.
18. MATSUYAMA, Y.; OMATA, M.; YOKOSUKA, O.; IMAZEKI, F.; ITO, Y. & OKUDA, K. - Discordance of Hepatitis B e Antigen/Antibody and Hepatitis B Virus Deoxyribonucleic Acid in serum. Gastroenterology, 89: 1104-1108, 1985.
19. MERCADANTE, L. A. C; ENNES, I. C; GASPAR, A. M. C. & YOSHIDA, C. F. T. - An enzyme immunoassay for the detection of hepatitis e antigen and its antibody. In: ENCONTRO NACIONAL DE VIROLOGIA, 3 , São Lourenço, MG, 1986. Resumos p. 36.
20. ROBINSON, W. S. & GREENMAN, R. L. - DNA polymerase in the core of the hepatitis B virus candidate. J. Virol., 13: 1231-1236, 1974.
21. ROGGENDORF, M.; DEINHARDI, F.; FROSNER, G. G.; SCHEID, R.; BAYERL, B. & ZACHOUVAL, R. - Immunoglobulin M antibodies to hepatitis B core antigen: evolution of enzyme immunoassay for diagnosis of hepatitis B virus infection. J. clin. Microbiol., 13: 618-626, 1981.
22. RIGBY, P. W. I.; DIECKMANN, M.; RHODES, C. & BERG, P. - Labelling deoxynucleic acid to high specific activity in vitro by nick translation with DNA polymerase. J. molec. Biol., 113: 237-251, 1977.
23. SCOTTO, J.; HADCHOVEL, M.; HERY, C; YUART, J.; TIOLLAIS, P. & BRECHOT, C. - Detection of hepatitis B virus DNA in serum by a simple spot hybridization technique: comparison with results of other viral markers. He-patology, 3: 279-284, 1983.
24. TAKAHASHI, K.; AKAHANE, Y.; GOTANDA, T.; MI SHIRO, T.; IMAI, M.; MIYAKAWA, Y. & MAYUMI, M. - Demonstration of hepatitis B e antigen in the core of Dane particles. J. Immunol., 122: 275-279, 1979.
25. TAKAHASHI, K.; IMAI, M.; TSUDA, F.; TAKAHASHI, T.; MIYAKAWA, Y. & MAYUMI, M. - Association of Dane particles with e antigen in the serum of asymptomatic carriers of hepatitis surface antigen. J. Immunol, 117: 102-105, 1976.
26. TAKAHASHI, K.; MIYAKAWA, Y.; GOTANDA, T.; MISHIRO, T.; IMAI, M. & MAYUMI, M. - Shift from "small" Hepatitis B e Antigen to IgG-bound "large" form in the circulation of human beings and chimpanzee acutely in fected with Hepatitis B Virus. Gastroenterology, 77: 1193-1199, 1979.
27. THIERS, V.; BOUCHARDEAU, F.; COUROUCÉ, A. M.; TIOLLAIS, P. & BRECHOT, C. - L'ADN du virus de l'hépatite B comme marqueur de multiplication virale: comparison avec l'antigène HBe et l'anticorps anti HBe. Presse méd., 15: 1219-1222, 1986.
28. WELLER, I. V. D.; FOWLER, M. J. F.; MONJARDINO, J. & THOMAS, H. C. - The detection of HBV-DNA in serum by molecular hybridization: A more sensitive me thod for the detection of complete HBV particles. J. med. Virol, 9: 273-280, 1982.
29. YOSHIDA, C. F. T.; MERCADANTE, L. A. C; GASPAR, A. M. C; GOMES, D. F. D. L. & SCHATZMAYR, H. G. - Identification of an endemic area to HBV (hepatitis B virus) infection in a rural population of the south east of Brazil. Hepatitis Scientific Memoranda: 32-34, March 1986.
30. YOSHIDA, C. F. T.; MERCADANTE, L. A. C; GASPAR, A. M. C; MAIA, M. M. S.; BERR, O. J.; ALMEIDA, E. C. C. & SCHATZMAYR, H. G. - Anticorpos anti HBs obtidos em coelhos e sua utilização como reagentes para testes sorológicos no diagnóstico da Hepatite B. Mem. Inst. Oswaldo Cruz, 79: 329-336, 1984.
31. YOSHIZAWA, H.; ITOH, Y.; SIMONETTI, J. P.; TAKA-HASHI, T.; MACHIDA, A.; MIYAKAWA, Y. & MAYUMI, M. - Demonstration of hepatitis B e antigen in hepatitis B core particles obtained from the nucleus of hepatocytes infected with hepatitis B virus. J. gen. Virol., 42: 513-519, 1979.

Address for correspondence:

Dra. M. Valeria Tedeschi

Fundação Oswaldo Cruz

Departamento de Virologia

Av. Brasil 4365

CEP 21040 Rio de Janeiro, RJ, Brazil.

Publication Dates

Publication in this collection
01 Dec 2010
Date of issue
Dec 1989

History

Received
16 May 1989

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. ALMEIDA, J. D.; RUBINSTEIN, D. & STOTT, E. J. - New antigen antibody system in Australia antigen positive hepatitis. Lancet, 2: 1225-1227, 1971.

[2] 2. BENSABATH, G.; HADLER, S. C; PEREIRA SOARES, M. C; FIELDS, H. & MAYNARD, J. E. - Epidemiologic and serologic studies of acute viral hepatitis in Brazil's Amazon Basin. Bull. Pan Amer. Hlth. Org., 21: 16-27, 1987

[3] 3. BENSABATH, G.; HADLER, S. C; PEREIRA SOARES, M. C; FIELDS, H.; DIAS, L. B.; POPPER, H. & MAY-NARD, J. - Hepatitis Delta virus infection and Labrea Hepatitis prevalence and role in fulminant Hepatitis in the Amazon Basin. J. Amer. med. Ass., 258: 479-483, 1987.

[4] 4. BERNINGER, M.; HAMMER. M.; HOYER, B. & GERIN, J. - An assay for the detection of the DNA genome of Hepatitis B virus in serum. J. med. Virol, 9: 57-68, 1982.

[5] 5. BRECHOT, C. & HADCHOUEL, M.; SCOTTO, J.; DE GOS, F.; CHARNAY, P.; TREPO, C. & TIOLLAIS, P. - Detection of Hepatitis B virus DNA in liver and serum: A direct appraisal of the chronic carrier state. Lancet, 2: 765-768, 1981.

[6] 6. BONINO, F.; HOYER, B.; NELSON, J.; ENGLE, R.; VER ME, G. & GERIN, J. - Hepatitis B virus DNA in the sera of the HBsAg carriers: a marker of active hepatitis B virus replication in the liver. Hepatology, 1: 386-391, 1981.

[7] 7. CAMARGO, I. F.; GASPAR, A. M. C. & YOSHIDA, C. F. T. - Comparative ELISA reagents for detection of He patitis B surface antigen (HBsAg). Mem. Inst. Oswaldo Cruz, 82: 181-187, 1987.

[8] 8. DANE, D. S.; CAMEROM, C. H. & BRIGGS, M. - Virus like particles in serum of patients with Australia antigen associated hepatitis. Lancet, 1: 695-698, 1970.

[9] 9. DEINHARDT, F. - Predictive value of markers of Hepa titis virus infection. J. infect. Dis., 141: 299-305, 1980.

[10] 10. DIEGUTIS, P. S.; BURNETT, L.; NIGHTINGALE, B. N.; LOWE, S. B.; GIUNEY, R. C; COSSART, Y. E.; KEIR NAN, E.; WILLIANS, G.; PARSONS, C; McCAUGHAN, G.; FREIMAN, J.; BRITTON, W. J. & GALLAGER, N. D. - Relationship between hepatitis B virus DNA in blood and serological markers of hepatitis B infection. Med. J. Aust., 144: 351-355, 1986.

[11] 11. DORMEYER, H. H.; ARNOLD, W.; KRYGER, P.; NIELSEN, J. O. & MEYER ZUM BUSCHENFELDE, K. H. - IgM antibody to hepatitis B core antigen (anti HBc IgM) in "healthy" HBsAg carriers: A longitudinal study of 75 cares. Klin. Wschr., 59: 675-678, 1981.

[12] 12. FAY, O. H.; HADLER, S. C; MAYNARD, J. E. & PINHEI-RO, F. - Hepatitis in the Americas. Bol. epidem. (OPS), 6(5): 1-7, 1985.

[13] 13. FIGUEIREDO MENDES, F.; SIMONETTI, J. P.; FONSE CA, J. C. F.; SIMONETTI, S. R. R.; PITELLA, A. M.; SCHATZMAYR, H. G.; LIMA FERREIRA, L. C; MIRANDA SANTOS, I. K.; MEXAS, P. P. F. & HERBERT, B. A. - O impacto da hepatite Delta. Mod. Hepat., 9: 16-22, 1984.

[14] 14. GERLICH, W. H.; LUER, W.; THOMSSEN, R. & the study group for viral hepatitis of the Deutsche Forschungsge-meinchaft. - Diagnosis of acute and inapparent hepatitis B virus infections by measurement of IgM antibody to hepatitis B core antigen. J. infect. Dis., 142: 95-101, 1980.

[15] 15. KAPLAN, P. M., FORD, E. C; PURCELL, R. H. & GERIN, J. L. - Demonstration of subpopulation of Dane particles. J. Virol., 17: 885-893, 1976.

[16] 16. KAPLAN, P. M.; GREENMAN, R. L.; GERIN, J. L.; PUR CELL, R. H. & ROBINSON, W. S. - DNA polymerase associated with human hepatitis B antigen. J. Virol., 12: 995-1005, 1973.

[17] 17. KRYGER, P.; MATHIESEN, L. R.; ALDERSHVILE, J. & NIELSEN, J. O. - Presence and meaning of anti HBc IgM as determined by ELISA in patients with acute type B hepatitis and healthy HBsAg carriers. Hepatology, 12: 50-53, 1982.

[18] 18. MATSUYAMA, Y.; OMATA, M.; YOKOSUKA, O.; IMAZEKI, F.; ITO, Y. & OKUDA, K. - Discordance of Hepatitis B e Antigen/Antibody and Hepatitis B Virus Deoxyribonucleic Acid in serum. Gastroenterology, 89: 1104-1108, 1985.

[19] 19. MERCADANTE, L. A. C; ENNES, I. C; GASPAR, A. M. C. & YOSHIDA, C. F. T. - An enzyme immunoassay for the detection of hepatitis e antigen and its antibody. In: ENCONTRO NACIONAL DE VIROLOGIA, 3 , São Lourenço, MG, 1986. Resumos p. 36.

[20] 20. ROBINSON, W. S. & GREENMAN, R. L. - DNA polymerase in the core of the hepatitis B virus candidate. J. Virol., 13: 1231-1236, 1974.

[21] 21. ROGGENDORF, M.; DEINHARDI, F.; FROSNER, G. G.; SCHEID, R.; BAYERL, B. & ZACHOUVAL, R. - Immunoglobulin M antibodies to hepatitis B core antigen: evolution of enzyme immunoassay for diagnosis of hepatitis B virus infection. J. clin. Microbiol., 13: 618-626, 1981.

[22] 22. RIGBY, P. W. I.; DIECKMANN, M.; RHODES, C. & BERG, P. - Labelling deoxynucleic acid to high specific activity in vitro by nick translation with DNA polymerase. J. molec. Biol., 113: 237-251, 1977.

[23] 23. SCOTTO, J.; HADCHOVEL, M.; HERY, C; YUART, J.; TIOLLAIS, P. & BRECHOT, C. - Detection of hepatitis B virus DNA in serum by a simple spot hybridization technique: comparison with results of other viral markers. He-patology, 3: 279-284, 1983.

[24] 24. TAKAHASHI, K.; AKAHANE, Y.; GOTANDA, T.; MI SHIRO, T.; IMAI, M.; MIYAKAWA, Y. & MAYUMI, M. - Demonstration of hepatitis B e antigen in the core of Dane particles. J. Immunol., 122: 275-279, 1979.

[25] 25. TAKAHASHI, K.; IMAI, M.; TSUDA, F.; TAKAHASHI, T.; MIYAKAWA, Y. & MAYUMI, M. - Association of Dane particles with e antigen in the serum of asymptomatic carriers of hepatitis surface antigen. J. Immunol, 117: 102-105, 1976.

[26] 26. TAKAHASHI, K.; MIYAKAWA, Y.; GOTANDA, T.; MISHIRO, T.; IMAI, M. & MAYUMI, M. - Shift from "small" Hepatitis B e Antigen to IgG-bound "large" form in the circulation of human beings and chimpanzee acutely in fected with Hepatitis B Virus. Gastroenterology, 77: 1193-1199, 1979.

[27] 27. THIERS, V.; BOUCHARDEAU, F.; COUROUCÉ, A. M.; TIOLLAIS, P. & BRECHOT, C. - L'ADN du virus de l'hépatite B comme marqueur de multiplication virale: comparison avec l'antigène HBe et l'anticorps anti HBe. Presse méd., 15: 1219-1222, 1986.

[28] 28. WELLER, I. V. D.; FOWLER, M. J. F.; MONJARDINO, J. & THOMAS, H. C. - The detection of HBV-DNA in serum by molecular hybridization: A more sensitive me thod for the detection of complete HBV particles. J. med. Virol, 9: 273-280, 1982.

[29] 29. YOSHIDA, C. F. T.; MERCADANTE, L. A. C; GASPAR, A. M. C; GOMES, D. F. D. L. & SCHATZMAYR, H. G. - Identification of an endemic area to HBV (hepatitis B virus) infection in a rural population of the south east of Brazil. Hepatitis Scientific Memoranda: 32-34, March 1986.

[30] 30. YOSHIDA, C. F. T.; MERCADANTE, L. A. C; GASPAR, A. M. C; MAIA, M. M. S.; BERR, O. J.; ALMEIDA, E. C. C. & SCHATZMAYR, H. G. - Anticorpos anti HBs obtidos em coelhos e sua utilização como reagentes para testes sorológicos no diagnóstico da Hepatite B. Mem. Inst. Oswaldo Cruz, 79: 329-336, 1984.

[31] 31. YOSHIZAWA, H.; ITOH, Y.; SIMONETTI, J. P.; TAKA-HASHI, T.; MACHIDA, A.; MIYAKAWA, Y. & MAYUMI, M. - Demonstration of hepatitis B e antigen in hepatitis B core particles obtained from the nucleus of hepatocytes infected with hepatitis B virus. J. gen. Virol., 42: 513-519, 1979.