• Foscarnet prophylaxis improved engraftment and survival in cord blood transplant patients

    Six-month overall survival was 96% in the treated group compared to 72% in the untreated group.

  • Latent HHV-6B infection of astrocytes may increase the risk of depression by activating the HPA axis

    Japanese investigators show that a protein expressed by latent HHV-6B in astrocytes, results in an influx of extra-cellular calcium, triggering the onset of depression.

  • Mothers with iciHHV6 have an increased risk of spontaneous abortion

    27.6% of Japanese mothers with iciHHV6 had a spontaneous abortion compared to 14.8% of controls. iciHHV6 mothers were also 6.4X more likely to have two or more spontaneous abortions.

  • Significantly worse outcomes in critically ill hematology patients associated with HHV-6 infection.

    Two reports associate detectable HHV-6 DNA in body fluids with increased risk of mortality in patients being treated for hematologic malignancies.

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Researcher FAQ

What is the best way to culture HHV-6 virus?
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.
What is the best cell line to grow HHV-6A?
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.
What is the best cell line to grow HHV-6B?
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.)
What is the best primer to use?

A review of 46 recent papers found 17 different primer sets at multiple locations throughout the genome, but only U31, U65 and U66 used more than once.  See Flamand 2008 and de Pagter 2010 for a comparison of assays.

What is the best way to propagate fresh isolates?
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.
What is the best way to isolate primary HHV-6A/B isolates
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).
What is the best way to get fresh isolates from PBMC's?
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).
What is the best way to grow primary HHV-6A/B isolates?
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.
What cell culture medium is needed to grow the infected and uninfected cells?
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.
What mab(s) is important to initially monitor infected cells?
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.
What morphological changes should you look for when the cells are infected?
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.
When do you freeze the infected cells?
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.
Is there an animal model for HHV-6?

Marmosets. Common marmosets, cynomolgus and African green monkeys are susceptible to HHV-6 infection. CD46 transgenic mice are susceptible to HHV-6A but not HHV-6B (Reynaud 2014). Porcine cytomegalovirus or PCMV is closely related to HHV-6 and HHV-7 has recently been designated a roseolovirus closely related to HHV-6A/B.  MTV or murine thymus virus is now known to be a roseolovirus and was renamed murine roseolovirus (MRV). David Mock at the University of Rochester has used 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.

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