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Chapter 1 The discovery of human herpesvirus type 6
Ablashi/Krueger/Salahuddin (eds) Human Herpesvirus-6 8 1992 Elsevier Science Publishers B.V.
3
CHAPTER 1
The discovery of human herpesvirus type 6 SYED ZAKl SALAHUDDIN University of Southern California, School of Medicine, Los Angeles, CA 90033, U.S.A.
1.1. Introduction
This chapter is an effort to summarize the events which led to the discovery of human herpesvirus type 6 (HHV-6). The period of 1984 through 1986 was perhaps one of the most exciting periods in bio-medicine due to the epidemic of acquired immune deficiency syndrome (AIDS). It is important to discuss a segment of this history as it relates to the finding of HHV-6 in the Laboratory of Tumor Cell Biology (LTCB) at the National Cancer Institute (NCI), in Bethesda, MD. HHV-6 was discovered in the latter part of 1984, in 1985 initially reported as human B cell lymphotropic virus (HBLV). This followed the waves of the HIV-1 detection, isolation and other findings. Both 1983 and 1984 were simultaneously exciting and confusing. The initial reports on lymphoadenopathy associated virus (LAV) from the Pasteur Institute in Paris made it impossible not to focus on the new retrovirus, for intellectual as well as for practical reasons. During the latter part of 1983 and early 1984, it began to be clear that this newly discovered retrovirus was present in more patients than only those with lymphoadenopathy. My associates and I, in a subsection of the LTCB, isolated HIV-1 from AIDS patients and from persons who were at high risk for AIDS, e.g., homosexual men, hemophiliacs, etc. (Gallo et al., 1984). We expanded these findings by detection and isolation of HIV-1 from persons at high risk, and from a variety of body fluids and tissues, e.g., peripheral blood, bone marrow, lymph nodes, etc. (Salahuddin et al., 1986). A separate, parallel effort in this area, directed by Drs. Robert C. Gallo and Mikulas Popovic and their associates, focused on the production of HIV-1 and blood testing. This was a very exciting time for LTCB and particularly for Dr. Gallo. Reports from his laboratory in May 1984 established the essential causative relationship to the
4 newly discovered retrovirus, which, partly due to its tropism for T cells, was named human T lymphotropic virus 111 (HTLV-111). These findings were a major leap forward not only in advancing our understanding of AIDS, but also raised hope that perhaps the end of the AIDS epidemic was near.
1.2. Discovery of HHV-6
By the end of 1984, involvement with AIDS had led to my increasing interest in malignancies in AIDS patients which were both devastating and not well understood, viz., AIDS-associated lymphoma and Kaposi’s sarcoma (KS). These diseases were challenging from a virological point of view and because of the diverse biology involved. Kaposi’s sarcoma and lymphoma involve not only a variety of cell types, but were also thought to be mediated by different viruses. For this chapter, I will only discuss our experiences with specimens from AIDS patients with lymphoma because it was these experiments which led to the discovery of human herpesvirus-6 (HHV-6). In case of AIDS-associated lymphoma, the prevailing wisdom was that, since this was a B cell disease, the Epstein-Barr virus (EBV) must play a major role in combination with the immune deficit. It was also thought that the EBV found in this disease might be different from the commonly isolated variety, the P3HR-1 or B95-8 type. In fact, Dr. Ablashi and I looked into this possibility and were surprised to find that AIDS patients generally had high titers of EBV antibodie’s as well as a higher incidence of transforming virus in circulation in the plasma (Ablashi et al., 1987a). When it came to lymphoma in AIDS, the findings were indistinguishable from those of nonlymphoma cases or from normal homosexual males. However, when the leukocytes from the peripheral blood were activated, inducing them to divide, some of the cells ballooned and formed syncytia. These were called ‘juicy cells’ by my associate, Susie Sturzenegger, due to their distinct appearance. To an experienced eye, these cells were distinguishable from those infected by HIV-1. We struggled with these pictures in our minds, and assumed that this might be a cytopathic effect (CPE), perhaps due to a new transmissible agent. I decided to transmit this agent, not knowing whether it was the same in each case or different to normal leukocytes obtained from human cord blood. Transmission to cord blood leukocytes was important to keep them isolated from other adventitious agents, which may be present in cells from adult donors (e.g., EBV, HCMV, etc.), and to separate them from HIV-1. The phenomenon of ‘juicy cells’ was reproducible in the cord blood leukocytes. These transmissions were done by a variety of methods; cell-free culture supernatant, co-culture of donor and target cord blood leukocytes and by cell lysate of donor cells to cord blood leukocytes. The last method was used repeatedly to inactivate any HIV-1 present in the system and transmit only the intracellular virus, if any. We were indeed successful in eliminating HIV-1 from the cell culture systems. The subsequent
5
immunological assays showed that the phenomenon of ‘juicy cells’ was transmissible without positivity for any of the HIV-1 antigens. At this point, I was convinced that I was dealing with a transmissible virus which needed further characterization. This phase took considerable time due to the fact that the work on HIV-1 infection of monocytic cells and the developmental work on Kaposi’s sarcoma had priority. By this time, we had slipped into 1985. Specimen of our transmitted cord blood leukocytes were maintained in culture and the reproducible phenomenon, some cells consistently becoming ‘juicy’and the rapid death of cells in the same culture, continued. The ljuicy cells’ also died within 10-15 days of culture. This helped us in studying the host range of the virus. While this virus was easily transmissible to cord blood leukocytes, it was very difficult to establish infection of established cell lines. Analysis of the primary cells from patients gave indications that the ‘juicy cells’ were, perhaps, immature B cells. This formed the basis for the nomenclature human B cell lymphotropic virus (HBLV). Soon after the initial publications (Josephs et al., 1986; Salahuddin et al., 1986),due to the efforts of Dr. Paulo Lusso, it was established that the cord blood cells were T cells rather than B cells (Lusso et al., 1987).In the meantime, Downing et al. (1987) and Tedder et al. (1987) also wrote in Lancet that the target cells for the new virus were T cells. We agreed and immediately proposed a correction and the renaming of the virus as HHV-6 (Ablashi et al., 1987b).It must be pointed out, however, that in vitro the prototype isolate of HHV-6 infects not only T cells but also B cells (Ablashi et al., 1987b, 1988).At this point, we concluded that we had a transmissible agent, that this was a lytic agent and it might be any of the known or unknown viruses, e.g., EBV, HSV, CMV, or something new. Now that I had eliminated the human T cell lymphotropic viruses I and I1 (HTLV I and 11) and HIV-1 by immunological assay (IFA), it was essential to eliminate the herpesviruses. Specimens were sent to Dr. Bernhardt Kramarsky for electron microscopic analysis. His result showed that the agent I had been working with was a herpesvirus, morphologically indistinguishable from other members of the family. I was, however, very impressed by the following observations: (a) the abundance of particles in the various stages of maturation; (b) the abundance of particles released outside the cells (Salahuddin et al., 1986; Biberfeld et al., 1987); (c) remarkably well-preserved morphology of the released particles; (d) the excellent condition of the cells during the initial stages of virus maturation and release; (e) evidence of mitotic division during the early stages of virus production; (f) unusual release of virus particles from the cell surface; (g) CPE associated only with the late phase. Clearly, this virus deserved a closer look. My initial hope was that this could be the agent responsible for the AIDS-associated lymphoma. Although the virus had unusual features, the virological properties did not fit the disease, their predominant T cell tropism.
6 I had known Dr. Ablashi for a number of years for his work with EBV and other nonhuman primate herpesviruses. He was conveniently located in the same building. When I requested his help in the characterization of this virus he joined the effort enthusiastically. I also approached Dr. Steven Josephs of the LTCB and showed him the data I had developed so far. He was interested and agreed to analyze the virus molecularly. My involvement in virology was limited to retroviruses, an organism with roughly 10 kb genome. Herpesviruses are generally 12-20 times larger, with attending complexity and, therefore, offered a challenge of insurmountable proportions for us. Further disappointment came when I mentioned the data to some colleagues who knew DNA viruses. They could not believe that a member of the herpes family had escaped detection for so long. The last herpesvirus (EBV) was isolated some 20 years ago by Prof. M. Anthony Epstein, from African Burkitt’s lymphoma cells. I discussed the preliminary findings of this virus with Dr. Gallo. He agreed with me that I should invite Dr. Ablashi for collaborative studies on this virus and Dr. Josephs for molecular analysis. The rest is well documented and published. Both these men have contributed enough to justify their being called co-discoverers of HHV-6. It took more than 7 months to prove that this virus was a novel human herpesvirus. Dr. Gary Pearson of Georgetown University, Washington, DC, kindly provided us with some reagents for EBV and HCMV and was a source of information and encouragement throughout this period, and has continued to be since. Dr. Peter Biberfeld, from the Karolinska Institute, Stockholm, Sweden, joined the laboratory in 1985, initially to isolate viruses which might be the causative agent for KS. We had many discussions, and I shared the data on our new herpesvirus with him. He indicated that he would be interested in contributing to the morphological analysis of the virus and immunocytochemistry of the target cells. This contribution resulted in studies that were published as a part of our initial discovery and as an exclusive electron microscopic study (Biberfeld et al., 1987). Dr. Gallo was interested in knowing whether this newly discovered virus had association with any disease. He initiated a major sero-epidemiological study. Dr. Ablashi was given the responsibility of accomplishing this objective. Dr. Paul Levine of the Epidemiology Subdivision of NCI was supposed to coordinate the efforts of all the outside collaborators such as Drs. Mark Kaplan (North Shore Community Hospital, NY), Steven Strauss (NIH, Bethesda, MD) and Tony Komaroff (Brigham and Women’s Hospital, Harvard University, Boston, MA). All the specimens were directed to Drs. Ablashi and Saxinger of the LTCB, who arranged to perform the serological assays under our standardized protocol, developed by Drs. Ablashi, Saxinger, Biberfeld and me. Some assays were done in my laboratory as a measure of system control, but the bulk of them was performed on contract. As it became evident that HHV-6 was a ubiquitous virus and without a clear relationship to any disease, interest in the virus began to diminish. We continued, however, to pursue studies related to the biological aspects as much as we were able to. We were lucky to find Dr. Balachandran (University of Kansas
7 Medical Center, Kansas City, KS) as a collaborator during this period. I have enjoyed knowing him since. Dr. Balachandran is an excellent immunologist and virologist with expertise in immunochemistry. He developed a number of monoclonal antibodies to HHV-6 (Balachandran et al., 1989)which have helped not only in diagnostic work, but also in the analysis of many isolates of HHV-6 into different subgroups (Ablashi et al., 1991). He has been invaluable in this area. During 1986, we initiated collaborative studies with Prof. G.R.F. Krueger of the University of Cologne, Cologne, Germany, to explore the role of HHV-6 in pathogenesis. Professor Krueger contributed a number of scientific publications on the role of HHV-6 in malignant and non-malignant diseases, including EBV and CMV negative infectious mononucleosis (Bertram et al., 1989; Krueger et al., 1990). A lot of effort on the part of many investigators has been published since our original report. I am also most grateful to all of those who helped with these studies and who have remained unnamed. I am referring to the support personnel whose names seldom appear in scientific papers.
References Ablashi, D.V., Fladager, A,, Markham, P.D. and Salahuddin, S.Z. (1987a) Isolation of transforming Epstein-Barr virus plasma of HTLV-III/LAV infected patients. Intervirology 27, 25-31. Ablashi, D.V., Salahuddin, S.Z., Josephs, S.F., Imam, F., Lusso, P., Gallo, R.C., Hung, C.L., Lemp, D. and Markham, P.D. (1987b) HBLV (or HHV-6) in human cells. Nature (London) 329,207. Ablashi, D.V., Lusso, P., Hung, C.L., Salahuddin, S.Z., Llana, T., Kramarsky, B., Biberfeld, P., Markham, P.D. and Gallo, R.C. (1988) The utilization of human haemopoietic cell lines for the propagation, detection and characterization of HBLV (human herpesvirus-6). Int. J. Cancer 42, 787-791. Ablashi, D.V., Balachandran, N., Josephs, S.F., Hung, C.L., Krueger, G.R.F., Kramarsky, B., Salahuddin, S.Z. and Gallo, R.C. (1991) Genomic polymorphism growth properties in human herpesvirus-6 (HHV-6) isolates. Virology, in press. Balachandran, N., Amelese, RYE.,Zhou, W.W. and Chang, C.K. (1989) Identification of proteins specific for human herpesvirus-6 infected T cells. J. Virol. 63, 2835-2840. Bertram, G., Dreiner, N., Krueger, G.R.F., Ablashi, D.V. and Salahuddin, S.Z. (1989) Infectious mononucleosis: clinical features and serologic correlations. In Epstein-Barr Virus and Human Disease 1988, Eds. D.V. Ablashi, A. Faggioni, G.R.F. Krueger, J.S. Pagano and G.R. Pearson, pp. 361-368. Humana Press, Clifton, NJ. Biberfeld, P., Kramarsky, B., Salahuddin, S.Z. and Gallo, R.C. (1987)Ultrastructural characterization of a new human B lymphotropic DNA virus (human herpesvirus-6) isolated from patients with lymphoproliferative disease. J. Natl. Cancer Inst. 79, 933-941. Downing, R.G., Sewankambo, N., Honess, R., Crawford, D., Jarrett, R. and Griffin, B.E. (1987)Isolation of human lymphotropic herpesvirus from Uganda. Lancet ii, 390. Josephs, S.F., Salahuddin, S.Z., Ablashi, D.V., Schachler, F., Wong-Staal, F. and Gallo, R.C. (1986) Genomic analysis of the human B lymphotropic virus. Science 234, 601-603. Krueger, G.R.F. Manak, M., Bourgeois, N., Ablashi, D.V., Salahuddin, S.Z., Josephs, S.F., Buchbinder, A,, Gallo, R.C., Berthold, F. and Tesch, H. (1990) Persistent active herpesvirus infection associated with atypical polyclonal lymphoproliferation (APL) and malignant lymphoma. Cancer Res. 9, 1457-1476. Lusso, P., Josephs, S.F., Ablashi, D.V., Gallo, R.C., Veronese, F.D. and Markham, P.D. (1987) Diverse tropism of HBLV (human herpesvirus-6). Lancet ii, 743-744.
8 Salahuddin, S.Z., Markham, P.D., Popovic, M., Sarngadharan, M.G., Orendorf, S., Fladager, A., Patel, A., Gold, J. and Gallo, R.C. (1985) Isolation of infectious human T cell leukemia/lymphotropic virus type 111(HTLV-111)from patients with acquired immunodeficiency syndrome (AIDS) or AIDSrelated complex (ARC) and from healthy carriers: a study of risk groups and tissue sources. Proc. Natl. Acad. Sci. U.S.A. 82, 5530-5534. Salahuddin, S.Z., Ablashi, D.V., Markham, P.D., Josephs, S.F., Sturzenegger, S., Kaplan, M., Haligan, G., Biberfeld, P., Wong-Staal, F., Kramarsky, B. and Gallo, R.C. (1986) Isolation of a new virus HBLV in patients with lymphoproliferative disorders. Science 234, 596-601. Tedder, R.S., Briggs, M., Cameron, C.H., Honess, R., Robertson, D. and Whittle, H. (1987) A novel lymphotropic herpesvirus. Lancet ii, 390-392.
3
CHAPTER 1
The discovery of human herpesvirus type 6 SYED ZAKl SALAHUDDIN University of Southern California, School of Medicine, Los Angeles, CA 90033, U.S.A.
1.1. Introduction
This chapter is an effort to summarize the events which led to the discovery of human herpesvirus type 6 (HHV-6). The period of 1984 through 1986 was perhaps one of the most exciting periods in bio-medicine due to the epidemic of acquired immune deficiency syndrome (AIDS). It is important to discuss a segment of this history as it relates to the finding of HHV-6 in the Laboratory of Tumor Cell Biology (LTCB) at the National Cancer Institute (NCI), in Bethesda, MD. HHV-6 was discovered in the latter part of 1984, in 1985 initially reported as human B cell lymphotropic virus (HBLV). This followed the waves of the HIV-1 detection, isolation and other findings. Both 1983 and 1984 were simultaneously exciting and confusing. The initial reports on lymphoadenopathy associated virus (LAV) from the Pasteur Institute in Paris made it impossible not to focus on the new retrovirus, for intellectual as well as for practical reasons. During the latter part of 1983 and early 1984, it began to be clear that this newly discovered retrovirus was present in more patients than only those with lymphoadenopathy. My associates and I, in a subsection of the LTCB, isolated HIV-1 from AIDS patients and from persons who were at high risk for AIDS, e.g., homosexual men, hemophiliacs, etc. (Gallo et al., 1984). We expanded these findings by detection and isolation of HIV-1 from persons at high risk, and from a variety of body fluids and tissues, e.g., peripheral blood, bone marrow, lymph nodes, etc. (Salahuddin et al., 1986). A separate, parallel effort in this area, directed by Drs. Robert C. Gallo and Mikulas Popovic and their associates, focused on the production of HIV-1 and blood testing. This was a very exciting time for LTCB and particularly for Dr. Gallo. Reports from his laboratory in May 1984 established the essential causative relationship to the
4 newly discovered retrovirus, which, partly due to its tropism for T cells, was named human T lymphotropic virus 111 (HTLV-111). These findings were a major leap forward not only in advancing our understanding of AIDS, but also raised hope that perhaps the end of the AIDS epidemic was near.
1.2. Discovery of HHV-6
By the end of 1984, involvement with AIDS had led to my increasing interest in malignancies in AIDS patients which were both devastating and not well understood, viz., AIDS-associated lymphoma and Kaposi’s sarcoma (KS). These diseases were challenging from a virological point of view and because of the diverse biology involved. Kaposi’s sarcoma and lymphoma involve not only a variety of cell types, but were also thought to be mediated by different viruses. For this chapter, I will only discuss our experiences with specimens from AIDS patients with lymphoma because it was these experiments which led to the discovery of human herpesvirus-6 (HHV-6). In case of AIDS-associated lymphoma, the prevailing wisdom was that, since this was a B cell disease, the Epstein-Barr virus (EBV) must play a major role in combination with the immune deficit. It was also thought that the EBV found in this disease might be different from the commonly isolated variety, the P3HR-1 or B95-8 type. In fact, Dr. Ablashi and I looked into this possibility and were surprised to find that AIDS patients generally had high titers of EBV antibodie’s as well as a higher incidence of transforming virus in circulation in the plasma (Ablashi et al., 1987a). When it came to lymphoma in AIDS, the findings were indistinguishable from those of nonlymphoma cases or from normal homosexual males. However, when the leukocytes from the peripheral blood were activated, inducing them to divide, some of the cells ballooned and formed syncytia. These were called ‘juicy cells’ by my associate, Susie Sturzenegger, due to their distinct appearance. To an experienced eye, these cells were distinguishable from those infected by HIV-1. We struggled with these pictures in our minds, and assumed that this might be a cytopathic effect (CPE), perhaps due to a new transmissible agent. I decided to transmit this agent, not knowing whether it was the same in each case or different to normal leukocytes obtained from human cord blood. Transmission to cord blood leukocytes was important to keep them isolated from other adventitious agents, which may be present in cells from adult donors (e.g., EBV, HCMV, etc.), and to separate them from HIV-1. The phenomenon of ‘juicy cells’ was reproducible in the cord blood leukocytes. These transmissions were done by a variety of methods; cell-free culture supernatant, co-culture of donor and target cord blood leukocytes and by cell lysate of donor cells to cord blood leukocytes. The last method was used repeatedly to inactivate any HIV-1 present in the system and transmit only the intracellular virus, if any. We were indeed successful in eliminating HIV-1 from the cell culture systems. The subsequent
5
immunological assays showed that the phenomenon of ‘juicy cells’ was transmissible without positivity for any of the HIV-1 antigens. At this point, I was convinced that I was dealing with a transmissible virus which needed further characterization. This phase took considerable time due to the fact that the work on HIV-1 infection of monocytic cells and the developmental work on Kaposi’s sarcoma had priority. By this time, we had slipped into 1985. Specimen of our transmitted cord blood leukocytes were maintained in culture and the reproducible phenomenon, some cells consistently becoming ‘juicy’and the rapid death of cells in the same culture, continued. The ljuicy cells’ also died within 10-15 days of culture. This helped us in studying the host range of the virus. While this virus was easily transmissible to cord blood leukocytes, it was very difficult to establish infection of established cell lines. Analysis of the primary cells from patients gave indications that the ‘juicy cells’ were, perhaps, immature B cells. This formed the basis for the nomenclature human B cell lymphotropic virus (HBLV). Soon after the initial publications (Josephs et al., 1986; Salahuddin et al., 1986),due to the efforts of Dr. Paulo Lusso, it was established that the cord blood cells were T cells rather than B cells (Lusso et al., 1987).In the meantime, Downing et al. (1987) and Tedder et al. (1987) also wrote in Lancet that the target cells for the new virus were T cells. We agreed and immediately proposed a correction and the renaming of the virus as HHV-6 (Ablashi et al., 1987b).It must be pointed out, however, that in vitro the prototype isolate of HHV-6 infects not only T cells but also B cells (Ablashi et al., 1987b, 1988).At this point, we concluded that we had a transmissible agent, that this was a lytic agent and it might be any of the known or unknown viruses, e.g., EBV, HSV, CMV, or something new. Now that I had eliminated the human T cell lymphotropic viruses I and I1 (HTLV I and 11) and HIV-1 by immunological assay (IFA), it was essential to eliminate the herpesviruses. Specimens were sent to Dr. Bernhardt Kramarsky for electron microscopic analysis. His result showed that the agent I had been working with was a herpesvirus, morphologically indistinguishable from other members of the family. I was, however, very impressed by the following observations: (a) the abundance of particles in the various stages of maturation; (b) the abundance of particles released outside the cells (Salahuddin et al., 1986; Biberfeld et al., 1987); (c) remarkably well-preserved morphology of the released particles; (d) the excellent condition of the cells during the initial stages of virus maturation and release; (e) evidence of mitotic division during the early stages of virus production; (f) unusual release of virus particles from the cell surface; (g) CPE associated only with the late phase. Clearly, this virus deserved a closer look. My initial hope was that this could be the agent responsible for the AIDS-associated lymphoma. Although the virus had unusual features, the virological properties did not fit the disease, their predominant T cell tropism.
6 I had known Dr. Ablashi for a number of years for his work with EBV and other nonhuman primate herpesviruses. He was conveniently located in the same building. When I requested his help in the characterization of this virus he joined the effort enthusiastically. I also approached Dr. Steven Josephs of the LTCB and showed him the data I had developed so far. He was interested and agreed to analyze the virus molecularly. My involvement in virology was limited to retroviruses, an organism with roughly 10 kb genome. Herpesviruses are generally 12-20 times larger, with attending complexity and, therefore, offered a challenge of insurmountable proportions for us. Further disappointment came when I mentioned the data to some colleagues who knew DNA viruses. They could not believe that a member of the herpes family had escaped detection for so long. The last herpesvirus (EBV) was isolated some 20 years ago by Prof. M. Anthony Epstein, from African Burkitt’s lymphoma cells. I discussed the preliminary findings of this virus with Dr. Gallo. He agreed with me that I should invite Dr. Ablashi for collaborative studies on this virus and Dr. Josephs for molecular analysis. The rest is well documented and published. Both these men have contributed enough to justify their being called co-discoverers of HHV-6. It took more than 7 months to prove that this virus was a novel human herpesvirus. Dr. Gary Pearson of Georgetown University, Washington, DC, kindly provided us with some reagents for EBV and HCMV and was a source of information and encouragement throughout this period, and has continued to be since. Dr. Peter Biberfeld, from the Karolinska Institute, Stockholm, Sweden, joined the laboratory in 1985, initially to isolate viruses which might be the causative agent for KS. We had many discussions, and I shared the data on our new herpesvirus with him. He indicated that he would be interested in contributing to the morphological analysis of the virus and immunocytochemistry of the target cells. This contribution resulted in studies that were published as a part of our initial discovery and as an exclusive electron microscopic study (Biberfeld et al., 1987). Dr. Gallo was interested in knowing whether this newly discovered virus had association with any disease. He initiated a major sero-epidemiological study. Dr. Ablashi was given the responsibility of accomplishing this objective. Dr. Paul Levine of the Epidemiology Subdivision of NCI was supposed to coordinate the efforts of all the outside collaborators such as Drs. Mark Kaplan (North Shore Community Hospital, NY), Steven Strauss (NIH, Bethesda, MD) and Tony Komaroff (Brigham and Women’s Hospital, Harvard University, Boston, MA). All the specimens were directed to Drs. Ablashi and Saxinger of the LTCB, who arranged to perform the serological assays under our standardized protocol, developed by Drs. Ablashi, Saxinger, Biberfeld and me. Some assays were done in my laboratory as a measure of system control, but the bulk of them was performed on contract. As it became evident that HHV-6 was a ubiquitous virus and without a clear relationship to any disease, interest in the virus began to diminish. We continued, however, to pursue studies related to the biological aspects as much as we were able to. We were lucky to find Dr. Balachandran (University of Kansas
7 Medical Center, Kansas City, KS) as a collaborator during this period. I have enjoyed knowing him since. Dr. Balachandran is an excellent immunologist and virologist with expertise in immunochemistry. He developed a number of monoclonal antibodies to HHV-6 (Balachandran et al., 1989)which have helped not only in diagnostic work, but also in the analysis of many isolates of HHV-6 into different subgroups (Ablashi et al., 1991). He has been invaluable in this area. During 1986, we initiated collaborative studies with Prof. G.R.F. Krueger of the University of Cologne, Cologne, Germany, to explore the role of HHV-6 in pathogenesis. Professor Krueger contributed a number of scientific publications on the role of HHV-6 in malignant and non-malignant diseases, including EBV and CMV negative infectious mononucleosis (Bertram et al., 1989; Krueger et al., 1990). A lot of effort on the part of many investigators has been published since our original report. I am also most grateful to all of those who helped with these studies and who have remained unnamed. I am referring to the support personnel whose names seldom appear in scientific papers.
References Ablashi, D.V., Fladager, A,, Markham, P.D. and Salahuddin, S.Z. (1987a) Isolation of transforming Epstein-Barr virus plasma of HTLV-III/LAV infected patients. Intervirology 27, 25-31. Ablashi, D.V., Salahuddin, S.Z., Josephs, S.F., Imam, F., Lusso, P., Gallo, R.C., Hung, C.L., Lemp, D. and Markham, P.D. (1987b) HBLV (or HHV-6) in human cells. Nature (London) 329,207. Ablashi, D.V., Lusso, P., Hung, C.L., Salahuddin, S.Z., Llana, T., Kramarsky, B., Biberfeld, P., Markham, P.D. and Gallo, R.C. (1988) The utilization of human haemopoietic cell lines for the propagation, detection and characterization of HBLV (human herpesvirus-6). Int. J. Cancer 42, 787-791. Ablashi, D.V., Balachandran, N., Josephs, S.F., Hung, C.L., Krueger, G.R.F., Kramarsky, B., Salahuddin, S.Z. and Gallo, R.C. (1991) Genomic polymorphism growth properties in human herpesvirus-6 (HHV-6) isolates. Virology, in press. Balachandran, N., Amelese, RYE.,Zhou, W.W. and Chang, C.K. (1989) Identification of proteins specific for human herpesvirus-6 infected T cells. J. Virol. 63, 2835-2840. Bertram, G., Dreiner, N., Krueger, G.R.F., Ablashi, D.V. and Salahuddin, S.Z. (1989) Infectious mononucleosis: clinical features and serologic correlations. In Epstein-Barr Virus and Human Disease 1988, Eds. D.V. Ablashi, A. Faggioni, G.R.F. Krueger, J.S. Pagano and G.R. Pearson, pp. 361-368. Humana Press, Clifton, NJ. Biberfeld, P., Kramarsky, B., Salahuddin, S.Z. and Gallo, R.C. (1987)Ultrastructural characterization of a new human B lymphotropic DNA virus (human herpesvirus-6) isolated from patients with lymphoproliferative disease. J. Natl. Cancer Inst. 79, 933-941. Downing, R.G., Sewankambo, N., Honess, R., Crawford, D., Jarrett, R. and Griffin, B.E. (1987)Isolation of human lymphotropic herpesvirus from Uganda. Lancet ii, 390. Josephs, S.F., Salahuddin, S.Z., Ablashi, D.V., Schachler, F., Wong-Staal, F. and Gallo, R.C. (1986) Genomic analysis of the human B lymphotropic virus. Science 234, 601-603. Krueger, G.R.F. Manak, M., Bourgeois, N., Ablashi, D.V., Salahuddin, S.Z., Josephs, S.F., Buchbinder, A,, Gallo, R.C., Berthold, F. and Tesch, H. (1990) Persistent active herpesvirus infection associated with atypical polyclonal lymphoproliferation (APL) and malignant lymphoma. Cancer Res. 9, 1457-1476. Lusso, P., Josephs, S.F., Ablashi, D.V., Gallo, R.C., Veronese, F.D. and Markham, P.D. (1987) Diverse tropism of HBLV (human herpesvirus-6). Lancet ii, 743-744.
8 Salahuddin, S.Z., Markham, P.D., Popovic, M., Sarngadharan, M.G., Orendorf, S., Fladager, A., Patel, A., Gold, J. and Gallo, R.C. (1985) Isolation of infectious human T cell leukemia/lymphotropic virus type 111(HTLV-111)from patients with acquired immunodeficiency syndrome (AIDS) or AIDSrelated complex (ARC) and from healthy carriers: a study of risk groups and tissue sources. Proc. Natl. Acad. Sci. U.S.A. 82, 5530-5534. Salahuddin, S.Z., Ablashi, D.V., Markham, P.D., Josephs, S.F., Sturzenegger, S., Kaplan, M., Haligan, G., Biberfeld, P., Wong-Staal, F., Kramarsky, B. and Gallo, R.C. (1986) Isolation of a new virus HBLV in patients with lymphoproliferative disorders. Science 234, 596-601. Tedder, R.S., Briggs, M., Cameron, C.H., Honess, R., Robertson, D. and Whittle, H. (1987) A novel lymphotropic herpesvirus. Lancet ii, 390-392.