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HHV-6

Human herpesvirus 6 or HHV-6 was serendipitously discovered in Robert Gallo's lab in 1986 when enlarged balloon-like cells were seen in the PBMC’s of AIDS and cancer patients. Its two variants, HHV-6A and HHV-6B, were uncovered in 1991 by Ablashi and others (Ablashi et. al .1991). HHV-6 is now the focus of a great deal of research mostly with regard to complications from organ transplantation but also with reference to a wide array of diseases including multiple sclerosis, cardiomyopathy, hepatitis, optic neuropathy, etc. PubMed citations for HHV-6 in 2005 number around 150.

HHV-6A is also able to readily induce T-cells to form giant ‘syncytia’ while HHV-6B cannot (Mori et. al. 2002). Syncytia are cells that have fused together to form giant polynucleate cells. Giant multinucleate cells are believed to signal HHV-6 infection in some diseases.

As research into the two ‘variants’ proceeds evidence continues to grow of their distinctiveness. Both Campadelli-Fiume and Mirandola in the late 1990’s asserted HHV-6A and 6B were different enough to warrant identifying them as separate species yet most studies still collapse the distinction between the two and simply study ‘HHV-6’ (Mirandola et. al 1998, Campadelli-Fiume 1999).The co-discoverer of the AIDS virus, Robert Gallo, called for the International Taxonomy Committee to recognize HHV-6A as a distinct virus and to be renamed, possibly as HHV-9.

Prevalence - HHV-6b is almost ubiquitous but it is not clear how common HHV-6a is. Tests discriminating between the two invariably find that HHV-6B is the predominant strain in healthy people (Caserta et. al. 2003). While HHV-6B is almost ubiquitous in children past a certain age, HHV-6A has rarely been detected in children. A recent paper stating HHV-6A ‘is rarely identified in patients’ (Boutolleau et al. 2005) found, using real time PCR that HHV-6A occurred in only 5% of the plasma and saliva samples positive for HHV-6 from AIDS and transplant patients and healthy controls. The two variants are, in fact, rarely found found together; in one study both viruses were found in only one of 186 samples (Boutolleau et. al. 2005).

Location - Both HHV-6A and B are found throughout the brain but it appears HHV-6A has a greater affinity for the CNS (is more neurotropic) and is more associated with CNS disease. HHV-6A, for instance, appears to be more commonly found in the cerebrospinal fluid (CSF) of AIDS patients and in multiple sclerosis while HHV-6B is more frequently found in the cerebrospinal fluid (CSF) of healthy controls (Ahlqvist et. al. 2005). Some studies have found increased levels of HHV-6A in infections of the CNS. HHV-6B is increased, however, in virally derived encephalitic conditions (Dewhurst 2004). Ablashi asserts that HHV-6A is the predominant strain in CFS, multiple sclerosis (MS) and AIDS patients. HHV-6A is more affinity for skin cells than HHV-6B does.

It has become clear that HHV-6B is the strain most commonly reactivated during organ and stem cell transplantation. Several lab studies, on the other hand, indicate HHV-6A is more effective at producing productive infections in immune and central nervous system cells. An in vitro study found that while HHV-6B was unable to effectively establish itself in premature oligodendrocytes, HHV-6A was able to infect, kill and establish a latent infection in them (Ahlqvist et. al. 2005). An ex vivo study found HHV-6A is also more effective in mounting productive infections in cytotoxic T-cells (Grivel et. al. 2003). HHV-6A is also reported to more easily infect NK cells than HHV-6B.

Although HHV-6 is able to infect many cells in only a few is it able to progress to the ‘lytic phase’. The lytic phase occurs when a virus enters the cells and uses the machinery to produce virions which usually end up killing the cell, allowing them to escape en masse. HHV-6 can replicate in B cells and NK cells but productive HHV-6 infection is essentially confined to T lymphocytes, particularly in T-helper (CD4+) cells. and monocytes/macrophages and perhaps oligodendrocytes (Kakimoto et. al. 2002). Ex vivo tests found productive HHV-6 infection in both T helper and cytotoxic T cells (CD8+) (Grivel et. al. 2003). Interestingly even in the most productive infections viral yields are low compared to other viruses (Campadelli-Fiume et. al. 1999).

A recent study employing real-time PCR found HHV-6B was found in most healthy controls, transplant and AIDS patients. HHV-6A was more commonly found in patients with central nervous system (CNS) dysfunctions (Boutolleau et.al. 2005).

While reactivation during organ transplantation is common HHV-6’s effects - usually consisting of nothing more than a fever and rash and often not even visible - are usually benign (Caserta et. al. 2003, Ward 2005). Bone marrow suppression, transplant rejection, pneumonitis and encephalitis may, however, be associated with HHV-6 reactivation in a small percentage of transplant patients. CNS complications were significantly more likely in liver transplant patients with evidence of HHV-6 reactivation than in those without it.

In vitro studies indicate that HHV-6 infection of B-cells triggers the production of a nuclear protein (called Zebra) that ‘transactivates’ genes active early in the life cycle of the another herpesvirus, Epstein-Barr virus (EBV). One gene it turns on plays a crucial role in switching EBV from latency to activity (Flammand et. al. 1993). Glaser intriguingly posits the chronic production of early EBV enzymes with or without viral replication could cause many of the symptoms of CFS. HHV-6 reactivation can also lead to a subsequent reactivation of another herpesvirus, the cytomegalovirus (HCMV). As with AIDS, HCMV patients often exhibit HHV-6 reactivation but it does not appear to negatively effect the progression of their disease (Caserta et. al. 2003). Nor does HHV-6 infection appear to increase the risk of mycoplasma or chlamydiae infection in CFS patients (Nicholson et. al. 2003).

Laboratory evidence suggests HHV-6 infection of CNS cells can have pathologic effects. Both oligodendrocytes and astrocytes originate from glial precursor cells (GPC’s). The responsibility of these cells for re-myelination makes them of special interest in demyelinating diseases such as MS where they appear to have been turned off. 

A recent in vitro study found HHV-6 is able to infect, replicate in and profoundly alter the morphology of GPC’s and may reduce the rate of re-myelination in MS patients (Dietrich et. al. 2004). HHV-6 also appears to tilt the production of oligodendrocytes towards a type which is particularly vulnerable to a pro-inflammatory cytokine, TNF-a, commonly found in MS lesions (Dietrich et. al. 2004). Despite these alterations HHV-6 infection did not result in increased GPC mortality. The authors noted, however, that several neurotropic viruses are able to disrupt cell functioning without altering cell survival. Could this process be responsible for the increased choline peaks but no sign of increased cell death or inflammation in the basal ganglia of CFS patients seen by Chaudhuri and Behan and others (See Choline on the Brain?).

Another way HHV-6 might cause CNS problems is by causing a vasculitis. Vasculitis, inflammation of a blood or lymph vessel, can result in reduced blood flows. Brain scans and other evidence suggest to Dr. Hyde that vasculitis of the CNS and other parts of the body plays a major role in CFS (Click here). In vitro studies that indicate HHV-6 can infect the endothelial cells which line the blood vessels suggest it could induce vasculitis. Interestingly, given Chaudhuri’s and Behan’s theory basal ganglia dysfunction in CFS a PET scan of one case of HHV-6 encephalopathy found hypoperfusion of the basal ganglia that researchers suggested was due to HHV-6 caused vasculitis.

While HHV-6 reactivation (or activation) is associated with several neurological diseases there is as yet no conclusive evidence that it causes them. Even with regard to febrile seizures – a complication always associated with primary HHV-6B infection in review articles - the causal role of HHV-6B is unclear. The incidence of HHV-6 infection in both febrile seizure patients and healthy controls is the same and some febrile seizure patients show no evidence of HHV-6 CNS infection at all (Dewhurst 2004).

For other diseases the evidence is even murkier. While HHV-6 reactivation can often be found in CNS diseases it is by no means always found in them and in some diseases only has been documented in small subsets of patients. As was noted earlier most cases of HHV-6 reactivation are benign; even though the virus is present and replicating in most cases the patients remains asymptomatic. A PCR finding of HHV-6 DNA in the cerebrospinal fluid (CSF) does not, therefore, necessarily indicate a significant pathological process is occurring. HHV-6 DNA has been found in the CSF of immunocompromised patients who did not display neurological symptoms (Dewhurst 2004).

It is clear that HHV-6 reactivation does occur in at least a subset of patients with several CNS diseases. Whether it causes or contributes to them or is simply a bystander exploiting an already damaged and vulnerable environment is unclear.

HHV-6 enters cells through a ubiquitous protein called CD 46 that spans the outer membranes on almost all cells. This transmembrane protein protects them from being attacked during complement activation and plays a role in T-cell stimulation and nitric oxide (NO) production by macrophages. HHV-6 appears to be able to down regulate the expression of this protein simply binding to it. Since the CD 46 protein plays a protective role during complement activation HHV-6 binding could result in increased complement activation and tissue damage. One study found exercise in CFS patients increased the activity of one part of the complement system. A parallel down regulation of another receptor (CD3) by HHV-6 that is involved in T-cell stimulation could result in immune suppression. HHV-6 infection also markedly enhances the secretion of a RANTES chemokine by T-cells. Chemokines facilitate leukocyte movement and activation.

Dendritic cells (DC’s) are the main antigen presenting cells (APC’s) in the body. APC’s present evidence of infection to T and B cells. Not only do they help activate T and B cells they also help determine the direction (Th1 vs. Th2) of the immune response. They do this by altering the makeup of the surface molecules (CD markers) they display and cytokines they produce. IL-12 production by DC cells, for instance, drives T-cells to produce the Th1 oriented cytokines (IFN-y, IL-2) that are most effective in battling intracellular invaders.

A recent study found that HHV-6 was able, by significantly inhibiting IL-12 production, to inhibit the Th1 response (Smith et. al. 2005). HHV-6 again appeared able to do this simply by binding to the CD 46 protein.

As mentioned earlier HHV-6A but not HHV-6B has been found to induce T-cells to fuse together forming giant polynucleate cells (Mori et. al. 2002). This fusion process was believed to be induced by proteins formed during HHV-6A’s replicative process but a recent study found that HHV-6A is able to induce cellular fusion when proteins already present on the viral envelope interact with the CD 46 protein. Since epithelial and endothelial cells appear most at risk from this process it is possible HHV-6 can alter blood vessel functioning as it travels through the blood stream.

HHV-6 like some other herpesviruses appears able to drive the immune system toward a state of Th2 dominance. HHV-6 ‘infected’ DC cells demonstrated a marked reduction in their ability to stimulate T cells and were associated with a ‘trend’ towards reduced TNF-a and increased IL-10 production (Smith et. al. 2005). Natelson has found increased IL-10 production in the CSF of a subset of CFS patients.

At least six methods are used by researchers to detect HHV-6:

The HHV-6 Foundation states that while both IgM and IgG tests can detect HHV-6 infection infection, only IgM can detect an active HHV-6 infection. There is a proviso to this; according to the Foundation very high IgG titers (4x’s normal) also suggest an active infection. Wallace states that at least with regard to CFS patients high antibody titers are insufficient to determine if an active infection is present, and that culture studies are needed to verify them (Wallace et. al. 1999). The IgG antibody test is routinely positive for about 90% of adults. While the IgM test can provide evidence of active infection since its levels are high only early in the disease it does not provide a suitable test for low-level chronic infections.

Polymerase Chain Reaction (PCR) - PCR tests for specific amino acid sequences known to occur in viral DNA. Unfortunately the lack of a standard primer for HHV-6 makes comparing results across studies more difficult (Opsahl and Kennedy 2005). Three different kinds of PCR (nested, standard, RT-PCR) are used to detect the presence of HHV-6 DNA.

According to an Immunesupport.com article by Tamara Schmidt, the researchers Knox and Carrigan state positive PCR tests don’t necessarily indicate an active infection is present. They also believe inhibitors in the blood may cause high false negative rates in these tests. Like Wallace they believe that culture tests are the only truly accurate diagnostic tools. (Schmidt ImmuneSupport.com 2000).

Culture Tests – In culture tests a sample is cultured to see if HHV-6 is present. At the completion of the culture period samples are tested for the presence of specific proteins produced by actively replicating HHV-6.

Testing for Active HHV-6 Infection – According to the HHV-6 Foundation the following tests have been used to detect active HHV-6 infection; PCR on serum and plasma, IgM early antibody tests, viral isolation, CPE/IFA positive, cell cultures. Not all of these are commercially available. PCR tests on serum or plasma presumably are able to pick up evidence of active infection because they searching the medium through which active viral particles pass as they attempt to infect other cells. PCR tests on peripheral blood mononuclear cells (PBMC’s) and IgG antibody tests usually pick up indications of latent (or past) infection. Since HHV-6 is often present in its latent form in peripheral blood mononuclear cells (PBMC’s) the HHV-6 Foundation believes these tests are essentially useless.

While many commercial labs in the U.S. are proficient at picking up acute HHV-6 infections, the HHV-6 Foundation asserts none are capable at identifying the kind of chronic, low-level infection they believe is characteristically found in CFS patients. According to the Foundation "the most sensitive test for active infection was available briefly to researchers in the late 90’s and has never made it into commercial production; it was based on antibodies to the early antigen (EA) or one of the first proteins produced by the virus in the early stages of infection."

The HHV-6 Foundation provides a list of testing facilities, and costs and recommendations for CFS and other patients with possible HHV-6 infection. They state the development of a sensitive test for HHV-6 infection is the Foundations top priority.

Researchers at the HHV-6 Foundation are not the only ones dissatisfied with the state of HHV-6 diagnosis and testing. It has been difficult to determine the accurate levels of the variants when they are found in the same sample. Two papers presented in 2005 utilized new PCR technology that they assert enabled them to adequately identify both variants (Boutolleau et. al 2005, Reddy and Manna 2005).

Both Wallace and Reeves assert most studies on HHV-6 in CFS have been flawed; they have either relied on serological studies, have been too small, lacked adequately matched controls or may have mischaracterized HHV-7 as HHV-6 (Wallace et. al. 1999, Reeves et. al. 2000). HHV-7 mischaracterization can occur when researchers unknowingly use primers for PCR tests that contain genetic sequences found in both HHV-6 and HHV-7. Ablashi has noted that "many of the early studies were done on whole blood, a technique that picks up latent virus, leaving the results muddled’ This would seem to suggest these studies exaggerated the amount of active infection in CFS patients. Indeed most of the early studies of HHV-6 serology in CFS used ‘extremely insensitive assays for HHV-6 antibodies’. As more sensitive antibody tests were developed several studies found rates of seroprevalence in CFS patient did not differ from healthy controls (Soto and Strauss 2001). Ablashi would, however, argue that continued refinement of the testing process, i. e. the creation of tests able to pick up evidence of active infection, has resulted in the opposite.

The HHV-6 Foundation has produced a chart which breaks up the studies into those which differentiated between an active and latent virus and those which did not. This chart indicates that the rate of HHV-6 infection is much more likely to be significantly increased in CFS patients relative to controls (83-53%) when researchers look for the active virus. This suggests CFS patients are more likely to harbor the active virus than healthy controls.

Ablashi et. al. found that a far greater percentage of CFS patients (57-16%) carried antibodies for an early IgM antigen (p41/38) in their serum than controls, a finding that indicated viral reactivation commonly occurred in CFS. Culture studies found HHV-6 infection was present in about half of CFS patients (n=22) and that most of the HHV-6 found was variant A (70%) (Ablashi et. al. 2000). It is rare that one comes across a scientific paper in which a statistical analysis was not done but this was one. The authors stated the results may not have been statistically significant because of the wide amount of variability in the data.

In a large study (n=145) Dr. Peterson found active HHV-6A (but not HHV-6B) was present in the cerebral spinal fluid (CSF) of a subset of CFS patients (20%) with CNS abnormalities (abnormal MRI, severe cognitive problems, headaches, paresthesias (burning, pricking, tickling, tingling sensations). How to reconcile the lower percentage of actively infected CFS patients in this CSF study compared to many of those testing for it in the serum? One would think that if HHV-6 activity in the CNS was the problem it would show up in far higher amounts in CSF than in the blood. This doesn’t appear to be true; levels of HHV-6 are also lower in CSF than in the serum or blood of MS patients as well (Fotheringham and Jacobsen 2005). In another recent study PCR evidence of active HHV-6 infection in the blood was found in 30% of CFS patients vs. 9% of controls (Nicholson et. al. 2003). In a report from the 2006 HHV-6/7 Conference in Barcelona Knox found that 22-25% of CFS patients (n=75) tested positive for HHV-6 infection using rapid culture, antigenemia and nested PCR.  Another report from the conference indicated Murovska and Chapenko found 40% of CFS patients (n=17) had active HHV-6 and HHV-7 infections compared with 0% of the controls (n=30). Peterson also reported laboratory findings suggesting a significant number of CFS patients with active HHV-6 infections are at risk from lymphoma.

Ablashi has suggested HHV-6A is a co-factor that enhanced the disease process in CFS (Ablashi et. al. 2000). No one has yet suggested HHV-6A is found in every CFS patient; Ablashi, the foremost proponent of HHV-6A in CFS, estimates it’s present in a significant subset of CFS patients. Given the controversy over the testing procedures it appears to be impossible to state with real certainty in what percentage of CFS patients HHV-6A is found or how active it is or where it occurs.

Probably the best evidence that HHV-6 or other herpesvirus infection may play a major role in some CFS patients appears to be the success that antiviral therapy has had in well defined subsets of CFS patients. Anecdotal reports from Dr. Ablashi and Dr. Peterson indicate that significant symptom reduction can occur when CFS patients with HHV-6A infection in the cerebrospinal fluid undergo antiviral therapy (Ampligen, cidofovir). Positive experiences from some CFS patients taking Ampligen indicate that viral infection is important in at least a subset of CFS patients. Antiviral treatments have also been effective in several small studies of CFS patients who demonstrate high titers of another herpesvirus EBV (Lerner et. al. 2002). Dr. Montoya reported from the 2007 International HHV-6/7 Conference that antiviral treatment (Valcyte 900 mg/day - 6 months) was highly effective in treating 9/12 patients elevated HHV-6 and EBV titers who had demonstrated long-term fatigue.

So why are researchers still interested in HHV-6 infection in CFS? One rather flippant answer is that most of them are not. Research into HHV-6 peaked in the early to mid 1990’s. Between 1989 and 1995 18 studies examined HHV-6 in CFS. In the ten years since then seven studies have been published and only one in the last two years. Of the last six papers published on HHV-6 and CFS (PubMed) only two have found evidence of increased HHV-6 infection in CFS. Three papers published around 2000 by established researchers with well characterized study groups that did not find increased HHV-6 prevalence in CFS patients relative to controls may have settled the question for many researchers (Wallace et. al. 1999, Reeves et. al. 2000, Koelle et. al. 2002). (Wallace used PCR, Reeves used three types of PCR, including one looking for an early gene, Koelle used PCR to examine plasma and PBMC’s for HHV-6. None used IgM antibody tests or culture studies. Whether these tests were sufficient to detect a low level HHV-6 infection depends on who you talk to.)

HHV-6, however, appears to be too intriguing a virus, despite all the conflicting evidence, for it to be ignored. It is able to affect two systems, the immune and central nervous system (CNS), that are of great interest in CFS. Several factors in CFS suggest immune dysfunction possibly tied to viral activity is present. These include decreased NK cell activity and numbers, increased T-cell activation, a tendency towards Th2 cytokine production and increased rates of RNase L fragmentation and activation. It is also clear that viral infection is a risk factor for CFS; several pathogens (EBV - infectious mononucleosis only, Ross-River VIrus, Coxiella burnetii) are known to induce CFS in a subset (@10%) of the infected patients.

The evidence of CNS dysfunction in CFS also continues to mount. Several small studies have found increased levels of choline in the basal ganglia of CFS patients (See Choline on the Brain?). Other studies have found reduced motor cortex excitation, reduced levels of gray matter, and altered serotonin receptor activity (See The Fatigue in CFS – Is it Central?). Natelson recently found increased protein and cytokine levels in the cerebral spinal fluid of a subset of CFS patients. A recent proteomics study found  unusual protein production in the CSF of CFS patients (Baraniuk et. al. 2005) (click here). HHV-6A activation or reactivation presents a plausible scenario for CFS patients suffering from both immune and CNS related problems.

Advocates of HHV-6’s role in CFS assert that too much regarding HHV-6 in CFS - its prevalence, activity, the type found, etc. - is unclear for the door to be closed on HHV-6’s role in CFS. They assert that until blood tests able to track chronic low-level HHV-6A infections in the CNS are developed it will be impossible to determine how significant a role it plays in CFS. Indeed, as HHV-6 testing has become more and more sophisticated evidence of active infection in CFS has risen.

They argue that HHV-6A’s location in the brain makes it inherently difficult to study. Its ability to effect cells simply by binding to their receptors suggests tests based on antigens produced late in the virus’s life cycle can miss evidence of considerable pathology. Ablashi states "there is good reason that it has taken so long to build a case for this virus playing a role in chronic fatigue syndrome – its very difficult to find…..Its quite possible to have a significant infection in brain tissues but no virus in the serum by DNA testing’. Issues like this have left the door for HHV-6a's role in CFS, if not wide open, at least still ajar.

Some basic questions need to be answered with regard to CFS and HHV-6A.

(1) The argument for HHV-6 in CFS rests largely on HHV-6A, not HHV-6B. A standard test needs to be devised (and become commercially available) that accurately measures the rate of low-level HHV-6A infection in CFS and other diseases. It seems astonishing that 14 years after HHV-6A was first described most papers still do not differentiate between HHV-6A and HHV-6B – they simply measure 'HHV-6'.

(2) The pathogenic role of HHV-6A badly needs to be clarified. Is it present but benign, present and mildly bothersome or present and dangerous or can it take on all these guises? Recent in vitro tests suggest it has the potential to be highly pathogenic.

(3) If HHV-6A is present in CFS where is it located? Since no consistent pattern can be seen in the locations of the lesions occurring in the brains of CFS one wonders where in the brains of CFS patients it might appear? One should note, however, a recent study found that normal appearing white matter (NAWM) in MS patients was anything but normal and and that these tissues exhibited greatly increased rates of HHV-6 infection relative to controls. (Opsahl and Kennedy 2005). This suggests our scanning technology is unable to detect all relevant signs of pathology and that the absence of obvious lesions in the brains of CFS patients is not necessarily synonymous with the absence of infectious activity.

(4) If HHV-6A is present and active what role does it play in CFS? Is it possible for it to be active and largely benign or does it tend to play, as several reports of successful antiviral therapy suggest, a major role in a subset of CFS patients.

Research into HHV-6 appears to be increasing. PubMed citations for 2005 (@150) will probably be about 40% higher than in 2004. Laboratory tests continue to elucidate HHV-6A’s effects on immune, endothelial and nervous system cells. These tests usually indicate that HHV-6’s effects, at least in the laboratory, are anything but benign. Given their sometimes dramatic findings  it appears the scientific community will continue to closely examine the role HHV-6 plays in the body. New PCR tests will hopefully enable researchers to better differentiate HHV-6A and B infections.

I am unaware of any current studies on HHV-6 and CFS. This certainly doesn’t mean one or several are not in progress now. (Please e-mail me at phoenixcfs@yahoo.com to update me.) The HHV-6 Foundation’s association with the new CFS research center being developed in Reno, Nevada will hopefully spark new research. Please note the HHV-6 Foundation will soon (???) begin publishing a free online newsletter and that it funds research and accepts donations.

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Ablashi, D., Eastman, H., Owen, C., Roman, M., Friedman, J., Zabriskie, J., Peterson, D., Pearson, G. and J. Whitman. 2000. Frequent HHV-6 reactivation in multiple sclerosis (MS) and chronic fatigue syndrome (CFS) patients. Journal of Clinical Virology 36, 179-191.