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Epstein-Barr-virus infection and persistence: a B-cell marriage in sickness and in health
Epstein-Barr-virus infection and persistence: a B-cell marriage in sickness and in health After producing the initial troublesome symptoms of acute virus infection, many viruses go on to establish a dormant or latent infection. This chronic relationship with the host is generally harmonious and enables the long-term survival and replication of the virus with no obvious ill-effects to the host. However, persistent infections are increasingly being associated with serious diseases (cancer, AIDS, hepatitis), so understanding of the natural history of these infections and, in particular, how cell types sustain latent infection is important for the development of antiviral strategies for treating or preventing these diseases. One of the commonest persistent infections is that caused by Epstein-Barr virus (EBV), which infects more than 90% of the world’s adults.1 Primary infection with this virus usually occurs in infancy and is symptomless, but delayed infection, a particular feature of affluent countries, can result in infectious mononucleosis. In this disorder, after the initial infection, EBV establishes life-long latency associated with periodic release of infectious virus into saliva, which becomes a source from which virus is transmitted to naïve individuals. Although in most individuals EBV persistence and replication proceed unnoticed, EBV is notorious for its association with cancer. The first such association discovered was with African Burkitt’s lymphoma, but EBV infection is now associated with several malignant tumours of lymphoid or epithelial origins.1 The precise role of EBV in the pathogenesis of these tumours is unknown, but clinical studies using infusions of cytotoxic T lymphocytes (CTL) confirm that the virus makes an essential contribution to the development of B-cell lymphomas in immunosuppressed patients.2 The tumour most consistently associated with EBV is undifferentiated nasopharyngeal carcinoma (NPC), an epithelial tumour prevalent in areas of China and southeast Asia.1 EBV seems to adopt distinct forms of latent infection in the different tumours. What is known about the natural history of EBV infection and persistence, and how does this knowledge influence understanding of EBV-associated tumours? EBV has a strong tropism for B lymphocytes, as shown by the association of the virus with various malignant B-cell tumours and by the ability of EBV to infect and immortalise B cells in vitro.1 The association of EBV with NPC and the demonstration of infectious virus in oropharyngeal secretions (throat washings) suggested that EBV permissively infects epithelial cells.3 This view was endorsed by the discovery that oral “hairy” leucoplakia, a benign lesion of tongue epithelium commonly found in AIDS patients, is a consequence of rampant EBV replication.4 Thus, the conclusion was that EBV persists in basal epithelial cells and is reactivated to enter the virus productive cycle as these cells differentiate.3 A corollary of this concept is that EBV infection of B cells was a secondary event and that, in the absence of an intact immune system or in response to other factors, such as frequent bouts of malaria, this infection can lead to the development of B-cell lymphomas. This view, however, did not sit well with an increasing body of data, the most damning of which was the demonstration that persistent EBV infection could be eradicated from bone-marrow-transplant recipients by obliteration of the patient’s B cells.5 The ready detection of
THE LANCET • Vol 354 • October 2, 1999
EBV-infected B cells in the peripheral blood and lymph nodes of chronic virus carriers and the lack of demonstrable EBV infection in normal epithelial tissues clearly warranted a reappraisal of the EBV-persistence model. Two approaches have served to clarify the issue and have highlighted the usefulness of rare disorders and of new technologies in medical research. Faulkner and colleagues6 searched for EBV infection in patients with X-linked agammaglobulinaemia (XLA), a rare disorder in which there is a lack of mature circulating B cells that is caused by inherited mutations in the gene encoding Bruton tyrosine kinase, an enzyme involved in cell signalling. As assessed by various methods, such as PCR of throat washings and blood and CTL assays, the XLA patients were free of EBV infection. The age-range of the patients and the EBV seropositivity in the patients’ parents and siblings strongly suggest that these individuals were exposed to EBV but could not sustain a primary or persistent infection because of their lack of B cells. In support of this conclusion is the finding by Babcock and colleagues7 who, by using the PCR technique on separated lymphocyte subsets, identified latent EBV infection in resting, memory B cells and virus replication in mucosal lymphoid tissues. These new data support the contention that primary EBV infection and virus persistence are mediated through B cells. EBV seems to have evolved to use the normal mechanisms for the production and maintenance of B-cell memory to persist in a latent state protected from host immune responses. In this concept the EBV-induced proliferation of B cells is necessary for initial colonisation of the B-cell pool but carries with it the danger of malignancy if the infected host is immunosuppressed or if other genetic events perturb B-cell development. Where does this concept leave EBV infection of epithelial cells? Although a role for epithelial cells in EBV persistence and replication cannot be excluded, EBV infection of epithelial cells is probably an accidental event resulting from local reactivation of latently infected B cells.5 In view of the prevalence of EBV infection, not surprisingly an increasing number of carcinomas are EBV positive .R e p o rt s of EBV infection in a proportion of carcinomas of the stomach, liver, and breast are especially intriguing.1,8,9 The contribution of EBV to the oncogenic process in these tumours and in EBV-positive lymphomas is difficult to ascertain and will require a better understanding of latent EBV gene function and of the interaction between virus and host cell. The new evidence firmly implicates B cells as the seat of EBV persistence and highlights the complex strategies that persistent viruses have evolved to integrate their biology with that of their hosts. Furthermore, the findings suggest that therapeutic approaches aimed at preventing the establishment of EBV latency in B cells will thwart the development of virus-associated tumours. Lawrence S Young CRC Institute for Cancer Studies, University of Birmingham Medical School, Birmingham B15 2TA ,U K 1
2
3 4
Rickinson AB, Kieff E. Epstein-Barr virus. In: Fields BN, Knipe DM, Howley PM, eds. Field’s virology, 3rd edn. Philadelphia:LippincottRaven,1996:2397–446. Rooney CM, Smith CA, Ng CYC, et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 1998; 92: 1549–55. Allday MJ, Crawford DH. Role of epithelium in EBV persistence and pathogenesis of B-cell tumours. Lancet 1 9 8 8 ;i :8 5 5 – 5 7 . Greenspan JS, Greenspan D, Lennette ET, et al. Replication of Epstein-
1141
Barr virus within epithelial cells of oral “hairy”leukoplakia, an AIDSassociated lesion. N Engl J Med 1985; 313: 1564–71. 5 Niedobitek G,Young LS. Epstein-Barr virus persistence and virusassociated tumours. Lancet 1994; 343: 333–35. 6 Faulkner GC, Burrows SR, Khanna R, Moss DJ, Bird AG,Crawford DH. X-linked agammaglobulinemia patients are not infected with Epstein-Barr virus:implications for the biology of the virus. JVirol 1999; 73: 155–64. 7 Babcock GJ, Decker LL, Volk M, Thorley-Lawson DA. EBV persistence in memory B cells in vivo. Immunity 1998; 9: 395–404. 8 Sugawara Y, Mizugaki Y, Uchida T, et al. Detection of Epstein-Barr virus in hepatocellular carcinoma tissue: a novel EBV latency characterized by the absence of EBV-encoded small RNA expression. Virology 1999; 256: 196–202. 9 Bonnet M, Guinbretiere J-M, Kremmer E, et al. Detection of EpsteinBarr virus in invasive breast cancers. J Natl Cancer Inst 1999; 91: 1376–81.
Treatment of children with malnutrition and diarrhoea Case-fatality rates for severe protein-energy malnutrition (PEM) have changed little over 50 years.1 Because such rates have been attributed to faulty case-management, Tahmeed Ahmed and colleagues2 examined the value of a standardised protocol for the management of severely malnourished children with diarrhoea at the International Centre for Diarrhoeal Diseases Research (ICDDR) in Dhaka, Bangladesh. The researchers, rightly, thought that random allotment of cases to standardised or unstandardised treatment was unethical, so they followed the standardised protocol for all children admitted in the first 6 months of 1997 and compared the effects with those of non-standardised treatment given to children admitted in the first half of 1996. Both groups were very large (about 300 children), had the same median age (6 months), and were severely stunted as well as wasted. The duration of previous diarrhoea was the same (median 6 days), and so was the prevalence of dehydration (about 40%), pneumonia (about 60%), and septicaemia (about 30%). The only significant difference at baseline was a higher prevalence of oedema among the test group. In both groups the median duration of stay in hospital was only 4 days. So the playing field was level, but there were highly significant differences in outcome.The mortality was 9% in the test group, compared with 17% in the controls. The most important contribution to this difference was probably the better management of dehydration. Only 40% of children in the test group received intravenous infusions, compared with 70% of the controls, and the volume infused and the duration of infusion were lower for the test group. However, whether the mere existence of a standardised protocol imposes more discipline, more attention to detail, and less freedom of choice for the clinician, including freedom to make mistakes, cannot be ruled out. Severe PEM is usually complicated by infection, and in this study the infectious element seemed to predominate. Nevertheless, the relation between mortality and the nature and amount of intravenous therapy suggests that the deaths were due primarily to malnourishment.That malnourished babies differ from normal babies in their response to intravenous infusion has long been recognised.The severely malnourished infant has an excess of water and sodium, even in the absence of clinical oedema, and is deficient in potassium. More than 30 years ago Smith3 recorded pulmonary oedema at necropsy in the majority of babies dying of malnutrition, and attributed it, probably correctly, to over-enthusiatic intravenous therapy. Another point that has not yet been settled is the best composition of oral 1142
rehydration fluid for malnourished children. The group working in Jamaica4 have recommended, for the reasons given above, that the sodium content should be reduced to a half or even a third of that of the standard WHO/ UNICEF oral rehydration solution; on the other hand, extra potassium should be given, as should extra glucose, to prevent hypoglycaemia.5 The ICDDR plans to carry out a comparative trial of the effectiveness of this formulation. The treatment of severe PEM has long been a neglected area. When I presented a paper to the UN Subcommittee on Nutrition some 20 years ago emphasising this deplorable waste of children’s lives, the official response was that resources could not be diverted from prevention to cure. In the past decade the picture has changed, perhaps because of the increasing number of malnourished refugees. WHO has recently produced a comprehensive manual6 on the management of severe malnutrition. This manual covers, in a simple and readable way, all the three conventional phases of treatment—the initial phase, as described in the ICDDR paper, lasting 4–7 days; the maintenance phase, lasting perhaps another week; and the phase of recuperation. In the first phase the priorities are rehydration, treatment of infection, and provision of essential trace elements (magnesium, zinc, and copper). The guidelines are in full agreement with the conclusions of the ICDDR study: to avoid intravenous therapy where possible and to use the low-sodium rehydration solution mentioned above. The manual puts the seal of its approval on the somewhat contentious policy of routinely giving antibiotics to all children, in case there is an undiagnosed infection. For the second phase, the advice on daily requirements is not more than 80–100 kcal/kg of energy and about 1 g protein/kg. The rationale of this regimen, which seemed to be very effective in Jamaica,4 is to avoid overloading the body’s depleted enzyme systems. The final phase begins when appetite has been restored, and highenergy feeds with reasonable amounts of protein can be given without risk. This manual should be widely disseminated in developing countries, perhaps backed up by training for paediatricians and nurses. Feedback is also essential. The next step for WHO is to support schemes to investigate how far the manual’s guidelines can be applied in poor district hospitals, where equipment and staff are in short supply. For the guidelines to be followed in such places requires a definition of the minimum resources needed for the handling of a given number of patients, and also ways of using the resources most effectively. All this may require changes in the traditional attitudes of doctors, nurses and administrators. John Waterlow Department of Public Health and P olicy, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK 1
2
3 4
5 6
Schofield C, Ashworth A .W hy have mortality rates for severe malnutrition remained so high? Bull World Health Organ 1996; 74: 223–29. Ahmed T, Ali M, Ullah MM, et al. Mortality in severely malnourished children with diarrhoea and use of a standardised management protocol. Lancet 1999; 353: 1919–22. Smith R. Hyponatraemia in infantile malnutrition. Lancet 1 9 6 3 ;i : 771–72. Waterlow JC, Golden MHN, Patrick S. Protein-energy malnutrition: treatment.In:Dickerson JWT, Lee HA, eds. Nutrition in the clinical management of disease. London:Edward Arnold,1978:49–71. Waterlow JC. Protein energy malnutrition.London:Edward Arnold, 1992. WHO. Management of severe malnutrition: a manual for physicians and other senior health workers. Geneva:WHO, 1999.
THE LANCET • Vol 354 • October 2, 1999
THE LANCET • Vol 354 • October 2, 1999
EBV-infected B cells in the peripheral blood and lymph nodes of chronic virus carriers and the lack of demonstrable EBV infection in normal epithelial tissues clearly warranted a reappraisal of the EBV-persistence model. Two approaches have served to clarify the issue and have highlighted the usefulness of rare disorders and of new technologies in medical research. Faulkner and colleagues6 searched for EBV infection in patients with X-linked agammaglobulinaemia (XLA), a rare disorder in which there is a lack of mature circulating B cells that is caused by inherited mutations in the gene encoding Bruton tyrosine kinase, an enzyme involved in cell signalling. As assessed by various methods, such as PCR of throat washings and blood and CTL assays, the XLA patients were free of EBV infection. The age-range of the patients and the EBV seropositivity in the patients’ parents and siblings strongly suggest that these individuals were exposed to EBV but could not sustain a primary or persistent infection because of their lack of B cells. In support of this conclusion is the finding by Babcock and colleagues7 who, by using the PCR technique on separated lymphocyte subsets, identified latent EBV infection in resting, memory B cells and virus replication in mucosal lymphoid tissues. These new data support the contention that primary EBV infection and virus persistence are mediated through B cells. EBV seems to have evolved to use the normal mechanisms for the production and maintenance of B-cell memory to persist in a latent state protected from host immune responses. In this concept the EBV-induced proliferation of B cells is necessary for initial colonisation of the B-cell pool but carries with it the danger of malignancy if the infected host is immunosuppressed or if other genetic events perturb B-cell development. Where does this concept leave EBV infection of epithelial cells? Although a role for epithelial cells in EBV persistence and replication cannot be excluded, EBV infection of epithelial cells is probably an accidental event resulting from local reactivation of latently infected B cells.5 In view of the prevalence of EBV infection, not surprisingly an increasing number of carcinomas are EBV positive .R e p o rt s of EBV infection in a proportion of carcinomas of the stomach, liver, and breast are especially intriguing.1,8,9 The contribution of EBV to the oncogenic process in these tumours and in EBV-positive lymphomas is difficult to ascertain and will require a better understanding of latent EBV gene function and of the interaction between virus and host cell. The new evidence firmly implicates B cells as the seat of EBV persistence and highlights the complex strategies that persistent viruses have evolved to integrate their biology with that of their hosts. Furthermore, the findings suggest that therapeutic approaches aimed at preventing the establishment of EBV latency in B cells will thwart the development of virus-associated tumours. Lawrence S Young CRC Institute for Cancer Studies, University of Birmingham Medical School, Birmingham B15 2TA ,U K 1
2
3 4
Rickinson AB, Kieff E. Epstein-Barr virus. In: Fields BN, Knipe DM, Howley PM, eds. Field’s virology, 3rd edn. Philadelphia:LippincottRaven,1996:2397–446. Rooney CM, Smith CA, Ng CYC, et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 1998; 92: 1549–55. Allday MJ, Crawford DH. Role of epithelium in EBV persistence and pathogenesis of B-cell tumours. Lancet 1 9 8 8 ;i :8 5 5 – 5 7 . Greenspan JS, Greenspan D, Lennette ET, et al. Replication of Epstein-
1141
Barr virus within epithelial cells of oral “hairy”leukoplakia, an AIDSassociated lesion. N Engl J Med 1985; 313: 1564–71. 5 Niedobitek G,Young LS. Epstein-Barr virus persistence and virusassociated tumours. Lancet 1994; 343: 333–35. 6 Faulkner GC, Burrows SR, Khanna R, Moss DJ, Bird AG,Crawford DH. X-linked agammaglobulinemia patients are not infected with Epstein-Barr virus:implications for the biology of the virus. JVirol 1999; 73: 155–64. 7 Babcock GJ, Decker LL, Volk M, Thorley-Lawson DA. EBV persistence in memory B cells in vivo. Immunity 1998; 9: 395–404. 8 Sugawara Y, Mizugaki Y, Uchida T, et al. Detection of Epstein-Barr virus in hepatocellular carcinoma tissue: a novel EBV latency characterized by the absence of EBV-encoded small RNA expression. Virology 1999; 256: 196–202. 9 Bonnet M, Guinbretiere J-M, Kremmer E, et al. Detection of EpsteinBarr virus in invasive breast cancers. J Natl Cancer Inst 1999; 91: 1376–81.
Treatment of children with malnutrition and diarrhoea Case-fatality rates for severe protein-energy malnutrition (PEM) have changed little over 50 years.1 Because such rates have been attributed to faulty case-management, Tahmeed Ahmed and colleagues2 examined the value of a standardised protocol for the management of severely malnourished children with diarrhoea at the International Centre for Diarrhoeal Diseases Research (ICDDR) in Dhaka, Bangladesh. The researchers, rightly, thought that random allotment of cases to standardised or unstandardised treatment was unethical, so they followed the standardised protocol for all children admitted in the first 6 months of 1997 and compared the effects with those of non-standardised treatment given to children admitted in the first half of 1996. Both groups were very large (about 300 children), had the same median age (6 months), and were severely stunted as well as wasted. The duration of previous diarrhoea was the same (median 6 days), and so was the prevalence of dehydration (about 40%), pneumonia (about 60%), and septicaemia (about 30%). The only significant difference at baseline was a higher prevalence of oedema among the test group. In both groups the median duration of stay in hospital was only 4 days. So the playing field was level, but there were highly significant differences in outcome.The mortality was 9% in the test group, compared with 17% in the controls. The most important contribution to this difference was probably the better management of dehydration. Only 40% of children in the test group received intravenous infusions, compared with 70% of the controls, and the volume infused and the duration of infusion were lower for the test group. However, whether the mere existence of a standardised protocol imposes more discipline, more attention to detail, and less freedom of choice for the clinician, including freedom to make mistakes, cannot be ruled out. Severe PEM is usually complicated by infection, and in this study the infectious element seemed to predominate. Nevertheless, the relation between mortality and the nature and amount of intravenous therapy suggests that the deaths were due primarily to malnourishment.That malnourished babies differ from normal babies in their response to intravenous infusion has long been recognised.The severely malnourished infant has an excess of water and sodium, even in the absence of clinical oedema, and is deficient in potassium. More than 30 years ago Smith3 recorded pulmonary oedema at necropsy in the majority of babies dying of malnutrition, and attributed it, probably correctly, to over-enthusiatic intravenous therapy. Another point that has not yet been settled is the best composition of oral 1142
rehydration fluid for malnourished children. The group working in Jamaica4 have recommended, for the reasons given above, that the sodium content should be reduced to a half or even a third of that of the standard WHO/ UNICEF oral rehydration solution; on the other hand, extra potassium should be given, as should extra glucose, to prevent hypoglycaemia.5 The ICDDR plans to carry out a comparative trial of the effectiveness of this formulation. The treatment of severe PEM has long been a neglected area. When I presented a paper to the UN Subcommittee on Nutrition some 20 years ago emphasising this deplorable waste of children’s lives, the official response was that resources could not be diverted from prevention to cure. In the past decade the picture has changed, perhaps because of the increasing number of malnourished refugees. WHO has recently produced a comprehensive manual6 on the management of severe malnutrition. This manual covers, in a simple and readable way, all the three conventional phases of treatment—the initial phase, as described in the ICDDR paper, lasting 4–7 days; the maintenance phase, lasting perhaps another week; and the phase of recuperation. In the first phase the priorities are rehydration, treatment of infection, and provision of essential trace elements (magnesium, zinc, and copper). The guidelines are in full agreement with the conclusions of the ICDDR study: to avoid intravenous therapy where possible and to use the low-sodium rehydration solution mentioned above. The manual puts the seal of its approval on the somewhat contentious policy of routinely giving antibiotics to all children, in case there is an undiagnosed infection. For the second phase, the advice on daily requirements is not more than 80–100 kcal/kg of energy and about 1 g protein/kg. The rationale of this regimen, which seemed to be very effective in Jamaica,4 is to avoid overloading the body’s depleted enzyme systems. The final phase begins when appetite has been restored, and highenergy feeds with reasonable amounts of protein can be given without risk. This manual should be widely disseminated in developing countries, perhaps backed up by training for paediatricians and nurses. Feedback is also essential. The next step for WHO is to support schemes to investigate how far the manual’s guidelines can be applied in poor district hospitals, where equipment and staff are in short supply. For the guidelines to be followed in such places requires a definition of the minimum resources needed for the handling of a given number of patients, and also ways of using the resources most effectively. All this may require changes in the traditional attitudes of doctors, nurses and administrators. John Waterlow Department of Public Health and P olicy, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK 1
2
3 4
5 6
Schofield C, Ashworth A .W hy have mortality rates for severe malnutrition remained so high? Bull World Health Organ 1996; 74: 223–29. Ahmed T, Ali M, Ullah MM, et al. Mortality in severely malnourished children with diarrhoea and use of a standardised management protocol. Lancet 1999; 353: 1919–22. Smith R. Hyponatraemia in infantile malnutrition. Lancet 1 9 6 3 ;i : 771–72. Waterlow JC, Golden MHN, Patrick S. Protein-energy malnutrition: treatment.In:Dickerson JWT, Lee HA, eds. Nutrition in the clinical management of disease. London:Edward Arnold,1978:49–71. Waterlow JC. Protein energy malnutrition.London:Edward Arnold, 1992. WHO. Management of severe malnutrition: a manual for physicians and other senior health workers. Geneva:WHO, 1999.
THE LANCET • Vol 354 • October 2, 1999