Non-A, Non-B Hepatitis Virus

169 stove, and required resuscitation. Her parents complained of headache and nausea, and the cat showed generalised convulsions. CO values were 20 and 11%, respectively, after oxygen had been administered by mask and balloon while the babies were in the ambulance. Hence, only a small number of infants with reported severe cot incident were poisoned by carbon monoxide. Emery’s observations and our experience with infants thought to have survived a "near-miss" SIDS both point to CO as a very exceptional cause of SIDS. Sleep Unit, Department of Paediatrics, Free University of Brussels, H&ocirc;pital Saint-Pierre, 1000 Brussels, Belgium ANDR&Eacute; KAHN DANI&Egrave;LE HAESAERTS DENISE BLUM SERUM THIOCYANATE IN PASSIVE SMOKING SIR-Dr Poulton and colleagues (Dec 15, p 1405) suggest that of serum thiocyanate (SCN) concentrations is a useful means of estimating passive smoking in children, and further argue that, because of its longer half-life, SCN has a theoretical advantage over urinary and salivary cotinine levels for this purpose. Mean SCN levels in children exposed at home were, at 97-3mol/1, significantly higher than the value of 54-2mol/1 found in unexposed children. However, their results are seriously at variance with other studies relating SCN to passive smoke exposure and it seems most unlikely that the raised levels they observed were due to passive smoke exposure. Gillies et allhave reported that salivary SCN concentrations were not significantly raised in 10 and 11 year old children from homes with smokers. Among adults, Friedman et a12 found a mean SCN value of 40 mol/1 in unexposed adults rising to 50 mol/1 in those reporting more than 40 h exposure per week. This difference was marginally significant, but the study design did not allow for the possibility that some smokers were falsely claiming to be nonsmokers. In a recent study of stringently defined adult non-smokers we found that measures of cotinine in plasma, saliva, and urine showed a clear dose-response relation with self-reported passive smoke exposure whereas there was no relation with measures of SCN.3Even in those most heavily exposed the smoke dosage appeared to be only about 1% of that seen in active cigarette smokers. In Poulton’s data, by contrast, the average SCN concentration in the exposed group was well into the smoking range, and in several cases the concentrations matched those seen in heavy cigarette smokers. Although deception about true smoking status could be a partial explanation among the older children, this could not account for the values of 175 and 161 mol/1 found in two infants aged less than 18 months. While it would be speculative to advance hypotheses to account for these high levels of SCN, the data do point to a well-known and serious theoretical drawback to SCN as a marker of passive smoking. SCN is derived from a number of dietary sources (eg, brassicas and other leafy vegetables) as well as from smoking, so that raised levels cannot be straightforwardly interpreted. Nicotine, on the other hand, is specific to tobacco, so that any detectable level must be due to either active or passive exposure. Thus SCN possesses neither a theoretical nor a practical advantage as a marker of passive smoking. Cotinine, the major metabolite of nicotine, has been shown to be a sensitive guide to passive exposure in both children4 and adults3,5 and is the marker of choice. measurement Addiction Research Unit, Institute of Psychiatry, London SE5 8AF M. J. JARVIS 1 Gillies PA, Wilcox B, Coates C, Kristmundsdottir F, Reid DJ. Use of objective in the validation of self-reported smoking in children aged 10 and 11 years saliva thiocyanate. J Epidemiol Community Health 1982; 36: 205-08. Friedman GD, Petitti DB, Bawol RD. Prevalence and correlates of passive smoking. Am J Public Health 1983; 73: 401-05. Jarvis MJ, Tunstall-Pedoe H, Feyerabend C, Vesey C, Saloojee Y Biochemical markers of smoke absorption and self-reported exposure to passive smoking. J Epidemiol Community Health 1984; 38: 335-39. Greenberg RA, Haley NJ, Etzel RA, Loda FA. Measuring the exposure of infants to measurement 2 3 4. tobacco smoke. N Engl J Med 1984; 310: 1075-78 5. Wald NJ, Boreham J, Bailey A, Ritchie C, Haddow JE, Knight G. Urinary cotinine asa marker of breathing other people’s tobacco smoke Lancet 1984; i: 230-31. NON-A, NON-B HEPATITIS VIRUS SIR,-Dr Seto and her colleagues (Oct 27, p 941) conclude that retroviruses are the cause of non-A, non-B (NANB) hepatitis. Their conclusion derived mainly from the demonstration of reverse transcriptase activity in sera and plasma-derived products from patients with a record of NANB hepatitis. Seto’s results could be consistent with hepatitis-B-like viruses. Hepatitis-B-like viruses have been related to NANB hepatitis by several investigators, on the basis of clinical and experimental data.l,2 Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of DNA viruses, which contains woodchuck, ground squirrel, and duck hepatitis viruses. Summers and Mason3have demonstrated the replication of the genome of duck HBV by reverse transcription of an RNA intermediate. Synthesis of the viral minus strand DNA required an RNA template, was sensitive to RNase A, and was resistant to actinomycin D. These characteristics are associated with the reverse transcriptase activity of the viral DNA polymerase but duck HBV is not a retrovirus. Replication ofaDNA plant virus (cauliflower mosaic virus) also includes a reverse transcriptase step. These features suggest that reverse transcription is an important mechanism for replication of several DNA viruses. Furthermore, aminoacid sequence homology between retroviral reverse transcriptase and DNA virus polymerases has been demonstratedfirst between the putative polymerase of HBV and cauliflower mosaic virus and retroviral reverse transcriptase (Moloney murine leukaemia virus and Rous sarcoma virus);4and, secondly, between the hepadnavirus DNA polymerases of duck and human HBV and woodchuck hepatitis viruses (especially within a nonapeptide fragment of gene 6 protein and the reverse transcriptases of the Moloney and Rous It would have been useful for hepatitis B sera to have been included in the controls of Seto’s study. To assess the specificity of the reverse transcriptase test, sera from patients with hepatitis related to other viral agents such as hepatitis A virus, cytomegalovirus, and Epstein-Barr virus should have been evaluated. viruses. Nuclear Medicine Service and INSERM Unit 204, H&ocirc;pital Saint-Louis, PHILIPPE POULETTY 75010 Paris, France JEAN KADOUCHE Biology Laboratory, University of South Paris, Orsay PHILIPPE LEBACQ Molecular 1. 2. Dienstag JL. Non-A, non-B hepatitis I: Recognition, epidemiology, and clinical features, II: Experimental transmission, putative virus agents and markers, and prevention. Gastroenterology 1983; 85: 439-62. Feinstone SM, Hoofnagle JH. Non-A, maybe-B hepatitis. N Engl J Med 1984; 311: 185-89. Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 1982; 29: 403-15. 4. Toh H, Hayashida H, Miyata T. Sequence homology between retroviral reverse transcriptase and putative polymerases of hepatitis B virus and cauliflower mosaic virus. Nature 1983; 305: 827-29. 5. Mandart E, Kay A, Galibert F. Nucleotide sequence of a cloned duck hepatitis B virus genome: Comparison with woodchuck and human hepatitis B virus sequences. J Virol 1984; 49: 782-92 3. Summers J, Mason WS. * This letter has been shown to Dr Seto and her colleagues, whose reply follows.-ED. L. SIR,-We are aware that a reverse-transcriptase-requiring step has been proposed for the hepatitis B virus and for other members of the Hepadna family of viruses. We are also aware that an HBV-like nonA, non-B (NANB) hepatitis agent has been proposed as the aetiological agent of this disease. The basis for our conclusion that the agent(s) of NANB hepatitis was either a retrovirus or a retrovirus-like agent and not an HBV-like agent was two-fold. We consistently detected a classical reverse transcriptase in sera obtained at diagnosis, which was associated with particles; the particles and their infectivity banded in sucrose at a density of 1’ 14 g/ml, a density which, in our hands, is identical to that of human T lymphotrophic virus type III. Results from testing sera from patients with hepatitis B, although not reported in our article, were routinely negative under the conditions of our reverse transcriptase assay. Sera from patients , 170 with either hepatitis A or drug-induced hepatitis were also negative. Samples tested containing HBV included sera with more than 107 infectious doses of HBV (in chimpanzee inoculation studies) as well as serum with strong DNA polymerase activity. Preliminary data indicate that HBV and HBV-like viruses are not pelleted under the conditions of our assay, which were designed to pellet retroviruses with S values of up to 1000 (the S value for HBV is 280). Two additional pieces of evidence confirm the non-HBV-like nature of the NANB agent. HBV does not band at 1-14 g/ml in a sucrose gradient. Instead it bands at about 1’ 22 g/ml. In addition, our reverse transcriptase assays are done with exogenous template, something which has never been accomplished for HBV, since HBV is not disrupted by detergents. If the core is disrupted, the conditions necessary to accomplish this will destroy the polymerase enzyme of HBV within the core. In summary, we still feel, on the basis of scientific data as well as virus and disease characteristics, that NANB hepatitis is probably caused by retroviruses or retrovirus-like agents distinct from HBV or HBV-like agents. core Division of Blood and Blood Products, Office of Biologics Research and Review, Food and Drug Administration, Bethesda, Maryland 20205, USA Section on B. SETO R. J. GERETY Pharmacology, The results (table) confirm the high correlation between this antigen/antibody system and post-transfusional NANB hepatitis. 85% of HBsAg-negative PHC patients were positive for NANB antigen or antibody, and this frequency was significantly higher (p<0 - 001) than the frequency found in controls, in HBsAg-positive PHC, or in patients of mixed malignancies with hepatic metastases. Our observations, therefore, suggest a strong association between PHC and serological evidence of past or present NANB infection, as detected by our ELISA. The finding that a retrovirus may be responsible for NANB hepatitis provides additional support for the hypothesis of a causal link between NANB infection and PHC. Although the role of NANB agent(s) in PHC remains to be determined we suggest that similar studies with larger sample size will help in establishing this association. GIOACCHINO ANGARANO GIUSEPPE PASTORE LAURA MONNO TERESA SANTANTONIO ORONZO SCHIRALDI Institute of Infectious Diseases and II Medical Clinic, University of Bari, Bari, Italy possible etiologic role of the hepatitis B virus in hepatocellular evidence from southern Africa In Chisari FV, ed. Advances in hepatitis research. New York Masson, 1984 2. Pastore G, Monno L, Angarano G, Trotta F, Schiraldi O. Development of an ELISA for the detection of an antigen/antibody system in non-A, non-B posttransfusional hepatitis. In: Vyas GN, Dienstag JL, Hoofnagle JH, eds. Viral hepatitis and liver disease. Orlando, Florida: Grune and Stratton, 1984: 624 1. Kew MC. The carcinoma: Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, W. G. National Institutes of Health, Bethesda COLEMAN, JR of Infectious Diseases, Department Ostra Hospital, University of G&oacgr;teborg, Sweden S. IWARSON SIR,-Dr colleagues describe particle-associated reverse transcriptase activity in acute and chronic NANB hepatitis, supporting the hypothesis that a retrovirus or a retrovirus-like agent is responsible for NANB hepatitis. There is extensive evidence for a link between hepatitis B virus (HBV) infection and primary hepatocellular carcinoma (PHC);’ however, in the United States and Europe only 50% of PHC patients are HBsAg positive and 25% have no other HBV marker. Since retroviruses are potentially oncogenic, a natural consequence of Seto and colleagues’ findings is the suggestion that NANB agent(s) have a role in the pathogenesis of the remaining PHC not related to HBV. In the absence of generally accepted serum markers for NANB virus(es) it has been impossible to determine the prevalence and distribution of the chronic carrier state of these virus(es) or to prove an epidemiological association with PHC; however, the preliminary results from our study (presented as a poster at the International Conference on Tumor Viruses, Cremona, April 18-19, 1984), suggest a surprisingly close association between PHC and a NANBrelated antigen/antibody system detected by enzyme-linked immunosorbent assay (ELISA).2 The antigen test was based on the sandwich principle, where unlabelled IgG from convalescent patients was used as capture antibody and, after peroxidase conjugation, as detector antibody. A blocking procedure on a fixed amount of NANB-antigen-positive serum was used in the same test to detect the corresponding antibody. By this method we studied five groups of patients and of healthy controls matched for sex, age, and number of transfusions. Seto and her PREVALENCE OF NANB ANTIGEN AND ANTIBODY SiR.&mdash;Dr Prince and colleagues’ claim (Nov 10, p 1071) that a virus belonging to a hitherto undescribed group of animal viruses has been isolated from chimpanzee liver cell cultures inoculated with non-A, non-B (NANB) hepatitis agent requires comment. The virus illustrated is, by size, morphology, and morphogenesis, identical to foamy virus. 1,2 Foamy viruses have been isolated from a variety of species, including man, and simian foamy virus is often found as a contaminant of primate cell cultures.3These viruses are classified as the subfamily Spumavirinae within the family Retroviridae.4 They are enveloped viruses with a single-stranded RNA genome whose morphology differs from that of other Retroviridae subfamilies, but they do possess a reverse transcriptase within the virion.5,6 Since NANB hepatitis has been shown by Dr Seto and he! colleagues (Oct 27, p 941) to have a particle-associated reverse transcriptase we need to be sure that the virus described is an NANE hepatitis virus and not merely an opportunistic infection of th( culture system used for virus isolation. I thank Dr J. H. Connolly for introduced me to foamy viruses. helpful discussion and Dr J. Department of Microbiology and Immunobiology, Queen’s University of Belfast, K. Clarke wh( EVELYN DERMOTT Belfast BT12 6BN JK, Attridge JT, Gay FW. The morphogenesis of simian foamy agents. J Gen Virol 1969; 4: 183-88. Dermott E, Clarke JK, Samuels J. The morphogenesis and classification of bovine syncytial virus J Gen Virol 1971, 12: 105-09. Hooks JJ, Gibbs CJ. The foamy viruses. Bacteriol Rev 1975; 39: 169-85. 1. Clarke 2. 3. 4 Fenner F. Classification and nomenclature of viruses. Second report of the International Committee on Taxonomy of Viruses Intervirology 1976; 7: 61. 5. Parks W, Todaro G, Scolnick E, Aaronson S. RNA dependent DNA polymerase in primate syncytium-forming (foamy) viruses. Nature 1971; 229: 258-60. 6. Benzair AB, Rhodes-Feuillette A, Emano&iuml;l-Ravicovitch R, P&eacute;ri&egrave;s J. Reverse transcriptase from simian foamy virus serotype 1: Purification and characterization. J Virol 1982; 44: 720-24. ***This letter has whose been shown L. to Dr Prince and his colleagues, reply follows.-ED. SIR,-Dr Dermott notes the close resemblance between the agent reported as replicating in chimpanzee liver cell cultures inoculated with non-A, non-B (NANB) hepatitis materials and the subfamily Spumavirinae (foamy viruses) of the Retroviridiae. The morphological similarity is indeed close, and the fact that similar viruses have been isolated from chimpanzees requires that we we Significantly different * from controls, t between patient groups. consider seriously the for our observations. possibility that such an agent could account 171 The basis for our belief that the agent we identified is related to NANB hepatitis is, first, that we have never seen similar agents in control uninoculated chimpanzee liver cell cultures, or in cultures inoculated with chloroform-treated material derived from human acute-phase NANB inocula despite extensive search. Secondly, the cores of the agent we identified have a characteristic association with the tubules seen within the endoplasmic reticulum in chimpanzees inoculated with NANB. Furthermore, our isolate appears to differ from the foamy viruses in that no intranuclear assembly of core particles has been observed. Nevertheless we recognise the need for caution and will search carefully for foamy viruses in our uninoculated and inoculated cultures using the standard virus isolation techniques for the study of these agents. As stated in our report, conclusive identification of the agent which we observed as a NANB virus will require additional immunological data and reproduction of NANB infection by inoculation of cell culture passaged virus into susceptible chimpanzees. Lindsley F Kimball Research Institute of New York Blood Center, New York, NY 10021, USA 1. Hooks ALFRED M. PRINCE TELLERVO HUIMA BOLANLE A. A. WILLIAMS LUDA BARDINA BETSY BROTMAN JJ, Gibbs CJ, Jr, Cutchins EC, Rogers NG, Lampert P, Gajdusek DC. Characterization and distribution of two new foamy chimpanzees. Arch Gesamte Virusforsch 1971; 38: 38-55. viruses isolated from consist of lipoprotein and pronase-sensitive electron-dense 5 components and do not contain any nucleic acid.5 The "assembly of complete virus-like particles" observed by Prince et al in association with the rough endoplasmic reticulum of their chimpanzee hepatocyte cultures was never observed in our inoculated cultures. The particles described by Prince et al seem to be similar to the intracytoplasmic particles we have demonstrated in patients with NANB hepatitis, 6, These particles were seen adjacent to the rough endoplasmic reticulum in hepatocytes of three patients with transfusion-acquired NANB hepatitis and were observed in about 30% of the human hepatocytes. A dense core-like component was seen in many of these particles, which were 60- 85 nm in diameter. We and colleagues (Oct 27, p 941) reported reverse transcriptase activity in infectious sera from patients with NANB hepatitis and in two infectious plasma-derived products. Reverse transcriptase activity was also found in the serum of the three particle-positive NANB hepatitis patients described above, suggesting that the particles may represent a retrovirus or a retrovirus-like agent. We are puzzled that Prince et al did not suggest that the virus-like particles seen in their chimpanzee cell cultures might be a retrovirus or retrovirus-like agent; their morphology and size seems consistent with our suggestion that a retrovirus or retrovirus-like agent is responsible for most cases of NANB hepatitis. Department of Infectious Diseases, &Ouml;stra Hospital, S-416 85 G&ouml;teborg, Sweden Institute of Pathology and Experimental Cancer Research, Semmelweis Medical University, SIR,-Dr Prince and his colleagues describe membrane-coated virus-like particles (85-90 nm) in cell cultures derived from the liver of chimpanzees inoculated with sera containing a human non-B (NANB) hepatitis agent(s). In similar experiments wel used a continuous line of chimpanzee liver cells (Flow Laboratories, McLean, Virginia; no 03-284) inoculated with a human serum (inoculum I) known to have transmitted NANB hepatitis to a nurse and to chimpanzees. Cells were collected for electron microscopy up to four months after the inoculation. Convoluted membrane’ alterations were observed 4 and 7 days after the inoculation and in about 3% of cells (figure). These results are similar to those of Prince et al, who noted such alterations in their cell cultures 4 and 8 days after inoculation with an infectious NANB hepatitis serum. Convoluted membrane alterations have been observed in association with retrovirus infections. For instance in acquired immunodeficiency syndrome, which is probably caused by the retrovirus human T-lymphotropic type III, tubular alterations in lymphocytes2seem to be identical with the changes found in the hepatocytes of chimpanzees infected with the NANB hepatitis agent. Similar membrane alterations have been found in other retrovirus-infected cells-eg, in MC-29-virus-induced chicken non-A, Budapest, Hungary Office of Biologics, Food and Drug Administration, Bethesda, Maryland, USA ZSUZSA SCHAFF FREDDIE MITCHELL R. J. GERETY S, Gerety RJ. Chronic NANBH carrier state-Transmissible agent documented patient during a six year period. N Engl J Med 1980; 303: 139 2. Schaff ZS, Tabor E, Jackson DR, Gerety RJ. AIDS-associated ultrastructural changes. Lancet 1984; ii: 941-43. 3. Schaff Z, Lapis K, Grimley PM. Undulating membraneous structures associated with the endoplasmic reticulum in tumour cells. Int J Cancer 1976, 18: 697-702. 4. Sebaff Z, Tabor E, Jackson DR, Gerety RJ. Ultrastructural alterations in serial liver biopsy specimens from chimpanzees experimentally infected with human non-A, non-B hepatitis agent. Virchows Arch (Cell Pathol) 1984; 45: 301-12. 5. Schaff Z, Tabor E, Jackson DR, Gerety RJ, Grimley PM. Specificity of ultrastructural 1. Tabor in one alterations in hepatic and lymphoid cells of chimpanzees infected with a human non-A, non-B hepatitis agent. In Vyas GN, Dienstag JL, Hoofnagle JH, eds Viral hepatitis and liver disease. Proceedings of the 1984 International Symposium on Viral Hepatitis (San Francisco). New York: Grune and Stratton. 6 Iwarson S, Schaff Z, Seto B, Norkrans G, Gerety RJ. Retrovirus-like particles in hepatocytes of patients with non-A, non-B hepatitis. European Association for Study of the Liver (EASL) (Bern, Switzerland, Sept 8, 1984): abstr. 7. Iwarson S, Schaff Z, Seto B, Norkrans G, Gerety RJ. Retrovirus-like particles in hepatocytes of patients with transfusion-acquired non-A, non-B hepatitis. J Med Virol (in press). hepatoma.33 What Prince et al describe as "late stage convoluted tubules" resemble the C-III type of cytoplasmic tubules characteristic of NANB hepatitis infected chimpanzee hepatocytes.4These tubules STEN IWARSON RAPID DIAGNOSIS OF CYTOMEGALOVIRUS INFECTION SIR,-A key development in our ability to make a rapid diagnosis of cytomegalovirus (CMV) in the immunosuppressed patient with a radiological diagnosis of pneumonitis has been the development of large volume bronchoalveolar lavage. The paper on CMV infectionI misquoted the method used by R. M. du B.; the volume of instilled sterile isotonic saline should have been 100 ml not 10 ml. We have identified CMV pneumonitis in two further bone marrow transplant recipients, using large volume lavage. Both had CMV detected by the rapid diagnostic technique described. Large volume lavage is especially important in the immunosuppressed patient in whom a vast array of potential pathogens must be excluded, necessitating the distribution of samples to. many different laboratories. now Tubules (arrow) in chimpanzee liver cell culture inoculated with human agent of NANB hepatitis. (xabout 22 000.) Department of Haematology, Royal Free Hospital, London NW3 2QG 1 Griffiths R. M. DU BOIS P. D. GRIFFITHS H. G. PRENTICE PD, Panjwani DD, Stirk PR, et al. Rapid diagnosis of cytomegalovirus infection in immunocompromised patients by detection of early antigen fluorescent foci. Lancet 1984; n: 1242-45.