Discovery and Classification of Human Herpesvirus6 HHV6

Dharam V. Ablashia,b aHHV-6 Foundation, 285 San Ysidro Road, Santa Barbara, CA 93108, USA bDepartment of Microbiology & Immunology, Georgetown University School of Medicine, Washington, DC, USA

Discovery

The discovery of herpesvirus-6 (HHV-6) dates back to early 1985 when Zaki Sal-ahuddin, in Dr. Robert Gallo's Laboratory of Tumor Cell Biology, was establishing long-term cultures from peripheral blood and splenic tissue of AIDS patients. He frequently found large syncytia that were distinct from HIV-1-induced syncytia. What he really saw in the peripheral blood mononuclear cells (PBMCs) of at least 6-8 patients with B-cell lymphoma were large, refractile cells (Fig. 1), always either single or, occasionally, two or more together. These cells began to disappear after a few days in culture, even in the presence of IL-2. The individuals with these cells were all AIDS patients with or without lymphoma. When these cells were stained with Giemsa, they were often multinucleated, or two large nuclei basically covered the entire cell (Fig. 2). The PBMCs of one particular lymphocytic leukemia patient, a 17-year-old boy, received in March 1985 from Dr. Gregory Halligan of Philadelphia, showed these bizarre-looking cells following mitogen stimulation. These PBMCs were sent to Dr. Matthew Gonda at the Frederick Cancer Research Center, Frederick, MD. Herpesvirus-like particles were observed in large numbers (Fig. 3), and a great majority of these particles were extra cellular, with an enveloped virion diameter of 160-200 nm. A repeat sample of PBMCs from this patient was obtained on April 17, 1985, and similar cells reappeared in the culture.

Fig. 1 Peripheral blood mononuclear cells (PHA stimulated), cultured from AIDS patient with B-cell lymphoma, showing large refractile cells. (for colour version: see colour section on page 345).
Fig. 2 Giemsa-stained peripheral blood mononuclear cells, containing refractile cells, showing multinucleated giant cells. (for colour version: see colour section on page 345).

After careful analysis for the presence of HIV-1, HTLV-I, and HTLV-II, the only virus particles evident were the herpesvirus-like particles. Since it was a herpesvirus and found in a PBMCs culture, not much was done except storing the virus in a -70 °C freezer, and cells were stored in liquid nitrogen.

Because of my interest in the role of EBV in AIDS B-cell lymphomas, I was invited to join Dr. Gallo's group in June 1985 and I began to look at the infection of EBV with HIV-1 (IIIB) (these data were published). During these investigations, it was evident that B-cells lacking EBV could not be infected with HIV-1. When,

however, B-cells were converted to EBV, positivity or cell lines carrying the EBV genome expressing CD4 + , receptor could be infected with HIV-1. One day, while this line of research was progressing, Zaki Salahuddin and Dr. Gallo asked me to look at another herpesvirus isolate, which they had frozen. They felt that since it was found in the PBMCs of their AIDS patient with B-cell lymphoma, it might be a variant of EBV that might be immortalizing cells. Zaki and I were able to transmit the cell-free supernatant obtained from cell cultures infected with this virus to fetal cord blood mononuclear cells. It was very clear that PHA must stimulate such cells; otherwise, the infection would not be effective. More than 50% of the inoculated cord blood mononuclear cells showed bizarre-looking, extremely large cells, which appeared between three and seven days, post infection. We called these cells as "juicy cells''. We consulted Dr. Bernard Kramarsky (then from Electronucleonics, Inc., of Silver Spring, MD), who had knowledge and experience with the ultrastructure of viruses. He believed that these herpesvirus particles could not be EBV since this virus shows very few extracellular particles. He also noted that the tegument was much more pronounced than that of EBV and similar to CMV (Salahuddin et al., 1986; Biberfeld et al., 1987).

The task assigned to me by Dr. Gallo was to characterize the herpesvirus isolate and to rule out any possibility of contamination by other viruses. I spent about six months studying its biologic and immunologic characteristics. At this time, Dr. Gary Pearson of the Georgetown University School of Medicine was brought into the picture. He provided me with all the EBV and CMV monoclonal antibodies as well as some good suggestions. I tested all the available monoclonal antibodies of human herpesviruses and herpesvirus Saimiri, a primate herpesvirus that can grow in human cells. I also used sera from Rhesus monkeys, African green monkeys, chimpanzees, and baboons to see whether there was any cross-reactivity. The results were clearly negative. Dr. Steve Josephs, a molecular virologist in Dr. Gallo's laboratory, trained in retrovirology by Dr. Flossy Wong-Staal, worked with me to analyze the isolate by generating probes and performing hybridization with human and simian herpesviruses. Our first dot-blot hybridization showed slight reactivity to CMV, but not to EBV, HSV, or VZV (Josephs et al., 1986).

Since Dr. Pearson and I were experienced with EBV immunofluorescence, I used the indirect immunofluorescence assay (IFA) to test the patient's sera for IgG antibody. The patient's serum and infected cord blood mononuclear cells provided us with the way to screen sera from other patients (Fig. 4). Dr. Peter Biberfeld of the Karolinska Institute, Stockholm, Sweden, performed the immune electron microscopy using patients' serum. His results, which were published in the Journal of the National Cancer Institute (Biberfeld et al., 1987), showed very strong positivity. We also performed the adsorption studies using the patients' plasma and showed the specificity of the serum to this virus after adsorption with other herpesviruses (Buchbinder et al., 1989). We were then convinced that we had a new herpes agent, which we called GS isolate. Since we had found this agent in AIDS patients more frequently, we thought it might be the causative agent of AIDS-associated lymphoma. Since our original patients were either AIDS or other lymphoproliferative disorder or lymphoma patients, we decided to call this virus HBLV. When we presented our data to Dr. Gallo, he was of the opinion that we should be certain that this was not a contamination by another herpesvirus. He also said that since he was not a herpes virologist, we should seek the opinion of an established herpes virologist. Dr. Gallo, at my suggestion, called Dr. Bernard Roizman of the University of Chicago, to discuss our HBLV data with him. Dr. Roizman suggested that since Dr. Elliot Kieff, his former student and an expert on EBV, was coming to Washington, we should show our data to him, which we did. After looking through

Fig. 4 Immunofluorescent staining of HHV-6-infected human cordblood mononuclear cells with patient GS serum. (for colour version: see colour section on page 346).

our data books, Dr. Kieff asked whether we could give him some viral DNA so that he could check it in his own laboratory. After about two weeks, Dr. Kieff called us to say that we could publish our data on this virus, as a new herpesvirus. Dr. Roizman was also comfortable with these data. The last human herpesvirus reported prior to this was EBV in 1966 by Sir M.A. Epstein. Because of such a long gap in the discovery of a new herpesvirus, most people we talked to were unwilling to believe that we had found a new herpesvirus. In fact, those who reviewed our manuscript for Science were very critical and believed that we had found a CMV variant. Before our two papers on HBLV appeared in Science (Josephs et al., 1986; Salahuddin et al., 1986), Dr. Debra Barrens from Science visited our lab and spent two hours with us looking through the HBLV cultures, IFA slides, and other data. She was very excited when she realized that this was something new and could be associated with AIDS. After the papers on these studies were published in Science, people called and wrote to us saying that they had also seen these strange-looking cells in the culture of PBMCs from AIDS patients.

The name HBLV—was it a mistake? I do not say that it was. At present, all lymphomas found with HHV-6 DNA are of B-cell origin (Josephs et al., 1988), with the exception of one disseminated T-cell lymphoma (Jarrett et al., 1988). Later, I was unable to infect B-cells with HHV-6 in vitro unless EBV DNA was present. In fact, the laboratories of Dr. Jose; Menezes, University of Montreal, Canada, and Dr. Jonas Luka, Eastern Virginia Medical School in Norfolk, VA, showed that not only are the EBV genome-positive B-cells infectable with HHV-6, but that HHV-6 can also activate EBV antigens such as EA, VCA, and Zebra protein (Flamand et al., 1993). After Dr. Paolo Lusso, then a post-doc in Dr. Gallo's laboratory, characterized the infected cells as T-cells (Lusso et al., 1987, 1988), we were the first group to change the name from HBLV to HHV-6 in a brief report (Ablashi et al., 1987). Later Lusso et al. (1988) conducted more detailed studies of the T-cells infectable with HHV-6. So far, it is evident that HHV-6 has a somewhat wide host range (Ablashi et al., 1988).

To summarize, was the discovery of HHV-6 a lucky chance, or was it keen observation on the part of Zaki Salahuddin and Dharam Ablashi? Once this occurred, Zaki and I, along with Drs. Joseph, Kramarsky, and Lusso, helped to characterize HBLV. We would have never found this virus if our clinical collaborators, Drs. Halligan and Mark Kaplan of the North Shore University Hospital, Long Island, NY, had not provided us with the specimens. Drs. Robert Gallo and Flossy Wong-Staal not only gave us moral support and encouragement, but also made necessary resources available to us for the discovery of HHV-6. We also acknowledge the critical help of Drs. Roizman and Kieff. The rest is history, because we can now look back and say that we made a valuable contribution to the advancement of science.

Nomenclature and classification

HHV-6 is a double-stranded DNA virus belonging to the human herpesviridae family (Braun et al., 1997). Herpesviruses are generally highly disseminated in nature. To date, herpesviruses examined from animals and humans are able to remain latent in their natural host. The cell harboring latent virus genomes take the form of closed circular molecules and only a small subset of viral genes are expressed. Latent genes retain the capacity to replicate and cause disease upon reactivation from the latent state, with HHV-6 not being an exception to this process. This may differ from one virus to another. The International Committee on the Taxonomy of Viruses (ICTV) endorsed nomenclature consists of the designation of herpesviruses by serial Arabic number and the family or sub-family of the natural host of the virus (e.g. HHV-6, HHV-7, etc.). The ICTV classified human herpesviruses into three sub-families, i.e. alpha, beta, and gamma, and eight human herpesviruses, i.e. HSV-1, HSV-2, VZV, CMV, HHV-6, HHV-7, EBV, and HHV-8 were put into these sub-families (Fig. 5) on the basis of their biological properties, before DNA sequences of the individual members of the family were known. ICTV also classified a small number of herpesviruses as to genera, based on DNA sequence homology and similarities in genomic sequence demonstrated by immuno-logic methods.

HHV-6 is ubiquitous, with 90% seropositivity in adults. HHV-6 variants HHV-6A and HHV-6B are classified as members of the beta-herpesvirus sub-family (Braun et al., 1997; Campadelli-Fiume et al., 1999; Krueger and Ablashi, 2003; DeBolle et al., 2005). The other two members are human cytomegalovirus (HCMV) and HHV-7. A non-exclusive characteristic of the members of beta-herpesvirinae is a restricted host range. The reproductive cycle is long and the infection progresses

HUMAN HERPESVIRUSES (HHV)

HHV-1 HHV-2 HHV-3

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