The best way to culture HHV-6 from the PBMCs is to separate the lymphocytes, PHA stimulate for 48 hours and then co-cultivate with cord blood mononuclear cells that are already PHA stimulated. Add 10% IL-2 to the culture. This is a very difficult virus to isolate; therefore, the success rate to get an isolate is very remote. HHV-6B is much easier to isolate from children with primary infection and exanthem subitum or reactivation with febrile illness. Isolation from bone marrow is easier than from peripheral blood. Also, the virus has been isolated frequently from he saliva. HHV-7 can be easily isolated from the saliva again by infecting with PHA stimulated cord blood mononuclear cells.
Once HHV-6A or B is growing in cord blood mononuclear cells (CBMC) then it can be co-cultivated with continuous T-cell lines. HHV-6A infected CBMCs can be co-cultivated with immature T-cell line, i.e. HSB2 as well as SupT-1 cells and J-Jhan HHV-6B infected CBMC can be co-cultivated with MOLT-3 (WCI strain). Since the virus is highly cell associated, there is cell to cell infection and very little virus is released in the cell culture supernatant. Therefore, co-cultivating infected cells with uninfected cells (a ratio of 1 infected cell to 5 uninfected cells) has much greater success.
The best cell line to grow HHV-6B is from the cord blood infected cells in MOLT-3 (NCI strain.) MOLT-3 is a mature CD4+ T-cell. One can also use SuptT-1 (an immature T-cell.)
The various primers people use which are either extracted from the early genes or late genes. See paper by Flamand et al 2008 J Clinical Microbiology 46:2700-2706. Here, participating labs had success with different primer sequences they used.
The best cells to propagate fresh HHV-6A or HHV-6B isolates is to use PHA stimulated cord blood mononuclear cells or use human CD4+ T-cells which are the PCR negative PBMCs. One could also try the T-cell lines, i.e. HSB2, Supt., MOLT-3, J-Jhan, but success will be limited.
If you want to isolate HHV-6A/B from blood, you should use PBMC. It is best to stimulate the PBMC with PHA (1-5 micrograms/ml) for 48 hours and then add 10-50 units of human IL-2. Because the virus resides in the T-cells and monocytes, if the T-cells and monocytes are separated, you will have better success. If PHA-stimulated cord blood cells are available, then use them to co-cultivate with PBMC. The appearance of large, multinucleated giant cells is a good indication that these are infected celMonitor them with HHV-6 specific monoclonal antibodies (MABs).
It is best to stimulate the PBMCs with PHA (5 mg/ml) for 48 hours and then add 10% human IL-2. Because the virus resides in the T-cells and monocytes, if the T-cells and monocytes are separated, you will have better success. If PHA stimulated cord blood cells are available, then use them to co-cultivate with PBMCs. The appearance of large, multinucleated giant cells is a good indication that these are infected cells. Monitor them with HHV-6 specific monoclonal antibodies (MABs).
If you want to grow primary HHV-6A/B isolates, you should use cord blood mononuclear cells (CBMC as these cells typically do not contain any HHV-6A/B. It is best to stimulate the CBMC with PHA (1-5 micrograms/ml) for 48 hours and then add 10-50 units of human IL-2. When the cells proliferate actively (3-4 days after PHA), spin and count the cells and infect the cell pellet for several hours (with mixing every hour) with HHV-6A/B at a MOI of approximately 0.1. Resuspend the cells at 500,000 cells/ml in culture medium with IL-2 (10-20 U/mL) and observe the cells on a daily basis. If the medium gets yellow, add some culture medium. After a few days, the appearance of large, multinucleated giant cells is a good indication that these are infected cel When infection is progressing nicely (>70% infected cells), expand by adding uninfected PHA-stimulated CBMC to the infected culture. Add 10 times more uninfected PHA-stimulated CBMC relative to the number of infected cells. When most cells are large and refractile (and infected), spin the cells at 4˚C. Collect supernatant and freeze-thaw the cell pellet 3 times using dry ice/methanol and 37˚C water bath. Spin at maximum speed and collect supernatant and mix with culture supernatant. At this point you can aliquot or centrifuge the culture supernatant to concentrate the virus if you desire.
Since cord blood, PBMCs, HSB2, MOLT-3, Supt., and J-Jhan cells are lymphocytes, the best medium to use is RPMI-1640 with 10%-20% fetal bovine serum (heat inactivated) and the usual amounts of antibiotics.
In the life cycle of HHV-6 replication, IE antigen appears between 4-8 hours post infection followed by early antigen (P41) which appears between 12-18 hours. After this viral DNA synthesis starts, late proteins, such as gp116/64/54, appear which tells you that virus replication is taking place. Therefore, use IE-2 (HHV-6A specific), P41 and gp116/64/54 to monitor the infection.
There will be cell clumping, i.e. infected medium and large cells surrounded by uninfected small cells. The appearance of large cells is a good sign. The large giant cells are multinucleated and they will lyse very quickly and release the virus.
The best time to freeze occurs when cells show that 50%-60% infection. One can tell by the appearance of large cells and also by using the late viral protein MABs, i.e. gp116/64/54. Freeze the infected cells using the following mixture. RPMI-1640 + 20% fetal bovine serum + 10% DMSO and the usual amounts of antibiotics. Remember, when you freeze the cells, you start lowering the temperature slowly starting from 4° C. If you do not have access to a cell-freezing machine then use a Styrofoam box. Put the cell vial in the box wrapped in tissue paper. Seal the box and leave it overnight in a -80°C freezer. Next day, transfer the vial into a liquid nitrogen tank.
Marmosets. Common marmosets, cynomolgus and African green monkeys are susceptible to HHV-6 infection. Claude Genain at CPMG has reported CNS manifestations from HHV-6 infection in common marmosets. Gessain, et al, identified a homologue for HHV-6 in a chimpanzee. Claude Genain at CPMG has reported success with infecting mice with a CD46 transgenic line. He warns, however, that it is not an easy process. David Mock at the University of Rochester uses murine oligodendroglial precursor cells and infects them with HHV-6 for a murine in vitro model. Margolis and Lusso at the NIH have studied HHV-6 in human lymphoid tissue ex vivo.
A group in Madrid found a genetic subset of MS patients with HHV-6A active replication and MHC2TA rs4,774C or another polymorphism in tight linkage disequilibrium with it. There is an association between a polymorphism in the IL-12p40 gene and cytomegalovirus reactivation after kidney transplantation. HLA class I polymorphisms may predispose patients to development of IM upon primary EBV infection.