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Prospective Study Of Human Herpesvirus Six In Multiple Sclerosis
National Multiple Sclerosis Society Grant PP0618

Final progress report
January 30, 2000

Lorri J. Lobeck, M.D.
Department of Neurology
Medical College of Wisconsin
Milwaukee, Wisconsin

Konstance K. Knox, Ph.D. and Donald R. Carrigan, Ph.D.
Institute for Viral Pathogenesis
Milwaukee, Wisconsin
Scientific Summary of Research Progress

The purpose of this study was to validate the use of peripheral blood specimens in detecting active human herpesvirus six (HHV-6.) infections in patients with Multiple Sclerosis (MS). Prior to this study, central nervous system and lymphoid tissues had been assessed and confirmed the presence of active HHV-6. Use of these tissues for routine study has limitations because of the need for invasive procedures to take appointment obtain tissue. We have been successful in determining the presence of active virus in samples of blood from individuals with MS.

Forty patients, with MS, had blood samples drawn at the time of a clinical relapse. A second specimen was to be drawn four to six weeks later. Cross sectional data is available on 37 patients. Paired specimen data is available on 30 patients. These data include patients with two specimens that were either positive or negative for the presence of active virus on both specimens. Seven patients do not have paired sample data, as they either had indeterminate specimens or failed to return for the second blood sample to be obtained. Indeterminate samples were those which were lost due to culture toxicity. The remaining three patients had either two indeterminate specimens or had one indeterminate specimen and failed to return for the second blood sample. The conclusions reviewed below include the cross sectional data and paired specimen data.

Figure 1

The cross sectional study of the incidence of HHV-6 viremia is illustrated in Figure 1. Data from normal control subjects includes 50 healthy blood donors from the Kansas City Blood Bank and 11 healthy laboratory donors. The current study population is indicated as patients seen at the Medical College of Wisconsin Multiple Sclerosis Clinic. Another group of patients has been assessed in St. Luke’s Medical Center in Kansas City, Missouri. The findings confirm the presence of active virus in the peripheral blood of patients with MS.

The clinical features of patients participating in the study are reviewed in Figure 2. This cross-sectional data incorporates the patients viral status at the time of the first blood sample only. Some patients began immunomodulator therapy after the first sample was obtained, but prior to the second. No significant clinical differences were evident between the patients who tested positive or negative for the virus on the first blood sample.

Figure 3 illustrates data from paired specimens. A significant number of patients who were viremic at the time of a clinical relapse demonstrated a lack of viremia when tested 6 to 10 weeks later. Future investigations, to include more frequent samples, would provide insight into the timing and pattern of the HHV-6 viremia.

Figure 2

Figure 3

Figure 4 illustrates the change in clinical status between the time of the first and second blood sample in those patients with paired specimen data. As would be expected, patients with relapsing MS often experience improvement in their symptoms, although not always a return to the pre-relapse condition.
The profile of HHV-6 viremia in paired specimens is further illustrated in Figure 5. The majority of those patients testing positive on the first sample, tested negative on the second, while those negative on the first were most often negative on the second sample.

While the original proposal was to obtain samples 4 to 6 weeks apart, some samples were delayed. This delay led to a fortuitous observation that, as illustrated by Figure 6, more closely spaced samples tended to test alike, and those separated by more time were more likely to be different. This observation raises further questions about the episodic nature of HHV-6 viremia in patients with MS. More frequent and consistent sampling of individuals with MS should define the pattern of viremia.

Figure 4

Figure 5

In conclusion, this project has been successful in demonstrating the presence of active HHV-6 infection in the blood samples of individuals with Multiple Sclerosis at the time of clinical relapse. The virus was not detected in blood samples of normal controls. In addition, several observations are described that suggest viremia is episodic and variable in patients with MS. As a result, we feel additional studies into the pattern of viremia are necessary to gain further understanding of these observations. Further, we feel the use of viral culture for HHV-6 should be incorporated into trials of anti-viral agents for patients with MS as the presence or absence of virus in peripheral blood may provide insight to which patients are most likely to respond to treatment.

Figure 6

Release for the Public

Researchers at the Medical College of Wisconsin and the Institute for Viral Pathogenesis have demonstrated the presence of active human herpesvirus-6 infections, as measured by viral cultures, in patients with Multiple Sclerosis coincident with new clinical relapses. Prior to this study, the active virus infection had been detected in specimens of brain or lymphoid tissue in some patients. The invasive nature of these techniques led to the search to find other ways to identify the active virus infection. These findings will hopefully lead to other studies investigating the role of this virus in Multiple Sclerosis.

Nested PCR

Detection of HHV-6, EBV and HTLV-2 Genomic DNAs by Nested PCR

[For more information about these tests click here.]

Over the past several months, the Institute for Viral Pathogenesis has developed a number of new molecular diagnostic technologies. They are being used in our current studies. These sophisticated new “nested PCR” systems are significantly more sensitive than other currently available PCR technologies.

A schematic of nested PCR is shown in the figure below. (Scroll down to view figure.)

Nested PCR uses two sets of amplification primers. The target DNA sequence of one set of primers (termed “inner” primers) is located within the target sequence of the second set of primers (termed “outer” primers). In practice, a standard PCR reaction is first run with the patient sample using the “outer primers”. Then a second PCR reaction is run with the “inner primers” using the product of the first reaction as the amplification target. This procedure increases the sensitivity of the assay by reamplifying the product of the first reaction in a second reaction. The specificity of the assay is increased because the inner primers amplify only if the first PCR reaction yielded a specific product.

HHV-6 Specific Nested PCR System 
The primers in this PCR system target the HHV-6 U54 gene (Gompels et al, 1995)., Use of the outer primer set, which recognize both the A and B variants of the virus equally in the clinical diagnostic setting has been described previously (Drobysky et al, 1994). The outer PCR primers produce a PCR product that is 383 base pairs in length. Two separate sets of inner primers are used in this system. One set recognizes only the A variant of HHV-6 , while the other set recognizes only the B variant of the virus. Both sets of inner primers produce a product that is 200 base pairs in length.

The PCR product is visualized by agarose gel electrophoresis and ethidium bromide staining. In titration experiments using normal human serum spiked with a plasmid containing the PCR target sequence, it was found that this system could detect less than 10 HHV-6 genomes per milliliter of serum (approximately 2 viral genomes per PCR reaction). We have used this PCR system to detect HHV-6 DNA in the sera of patients with multiple sclerosis, chronic fatigue syndrome and liver transplant recipients.

EBV Specific Nested PCR System
This PCR system targets the EBV gene encoding the viral latent membrane protein one (LMP-1) and detects the two types of EBV equally as assessed using the prototype B95-8 and AG876 strains for type 1 and type 2 viruses, respectively (Kieff and Rickinson, 2001; Sample et al, 1994). The product of the outer primers in this system is 407 base pairs in length while the product of the inner primers is 243 base pairs long. This PCR system has been successfully used to detect EBV DNA in the sera of patients with chronic fatigue syndrome.

HTLV-2 Specific Nested PCR System
Human T Cell Lymphotropic Virus Type 2 (HTLV-2) has been implicated as being involved in some cases of chronic fatigue syndrome (DeFreitas et al, 1991). The Institute has recently been awarded a grant to attempt to confirm this earlier work. In pursuit of this study a nested PCR primer system targeting the viral gag gene has been designed to detect HTLV-2 DNA in patient blood leukocyte samples. The product of the outer primers of this system is 468 base pairs in length while the product of the inner primers is 206 base pairs in length. The figure below shows an ethidium bromide stained agarose gel analyzing a titration of the sensitivity of this PCR system. Serial ten-fold dilutions of a positive control preparation of a plasmid containing the target HTLV-2 DNA were analyzed by PCR with the outer primers alone and with the combined nested PCR primers.

As is evident in the figure, the outer primers were able to detect the viral DNA down to a 10-5 dilution. In contrast, the full nested PCR system detected the viral DNA down to a 10-8 dilution. Thus, the nested system is approximately 1000 times more sensitive than the single primer system.
References Gompels UA, Nicholas J, Lawrence G, Jones M, Thomson BJ, Martin ME, Efstathiou S, Craxton M and Macaulay HA. The DNA sequence of human herpesvirus?6: structure, coding content, and genome evolution. Virology 1995; 209:29?51.

Drobyski WR, Knox KK, Majewski D and Carrigan DR. Fatal encephalitis due to variant B human herpesvirus 6 infection in a bone marrow transplant recipient. N Engl J Med 1994; 330:1356?1360.

Kieff E and Rickinson AB. Epstein-Barr virus and its replication. Fields Virology, Fourth Edition. DM Knipe and PM Howley (eds.) Lippincott Williams and Wilkins. Philadelphia, Pennsylvania. 2001. pp. 2511-2573.

Sample J, Kieff E and Kieff E. Epstein-Barr virus types 1 and 2 have nearly identical LMP-1transforming genes. J Gen Virol 1994; 75:2741-2746.

DeFreitas E, Hilliard B, Cheney PR, Bell DS, Kiggundu E, Sankey D, Wroblewska Z, Palladino M, Woodward JP AND Koprowski H. Retroviral sequences related to human T-lymphotropic virus type II in patients with chronic fatigue immune dysfunction syndrome. Proc Natl Acad Sci 1991; 88:2922?2926.

Chronic Fatigue Syndrome

Persistent Active Human Herpesvirus Six (Hhv-6) Infections In Patients With Chronic Fatigue Syndrome.
Konstance K Knox, Ph.D.¹; Joseph H. Brewer, M.D.² and Donald R. Carrigan, Ph.D.¹ Hospital for Viral Pathogenesis and Wisconsin Viral Research Group; Milwaukee, Wisconsin1 and St. Luke’s Hospital; Kansas City, Missouri².

OBJECTIVE: Data from a number of laboratories have suggested a role for HHV-6 in the pathogenesis of chronic fatigue syndrome (CFS). In the studies described here we sought to explicitly test the hypothesis that a portion of patients with CFS have persistent, active HHV-6 infections.

METHODS: Blood samples from patients with CFS were evaluated by a rapid HHV-6 culture procedure. This technique diagnoses active HHV-6 infections by detecting transfer of the virus from the patient’s blood leukocytes to a target human cell line. CFS patients from two CFS oriented clinics, a large infectious disease practice, and blood samples from CFS patients submitted to our laboratory from physicians and clinics around the United States were studied. Clinical characteristics of and multiple blood samples from a group of CFS patients from the infectious disease practice were evaluated in detail healthpam

RESULTS: The cross-sectional (one blood sample per patient) incidence of active HHV-6 infection was 37% (128/349) in the CFS patients with the incidence being similar at all four sources of samples (range 25% to 47%). This incidence of active HHV-6 infection was significantly higher than the 0% seen in 26 normal controls (p<0.05).

To assess the possibility that HHV-6 infections may be episodic or variable with respect to viral load in patients with CFS, seven patients whose first blood samples were negative for active HHV-6 infection were retested at intervals ranging from 4 to 12 weeks after the initial sample was obtained. Three of the seven patients (43%) were found to be positive for active HHV-6 infection with the second sample. This finding suggested that active HHV-6 infections may be intermittently detectable in patients with CFS. This possibility was examined by testing at least four blood samples from each of four patients with CFS over periods of time ranging from 1 to 5 months. The consecutive blood samples from the four patients were found to be HHV-6 positive 58% (22/38) of the time with the positivity rates for the individual patients ranging from 40% (4/10) to 69% (9/13). These observations suggest that the active HHV-6 infections in patients with CFS are either intermittent or variable with respect to their viral load. Thus, an individual patient’s HHV-6 infection status must be assessed using multiple blood samples obtained over a period of weeks or months. Also, the 37% estimate of the incidence of active HHV-6 infections in patients with CFS should be held as a minimal value since the true incidence may be higher (60% to 70%).

In the course of these studies it was observed that many HHV-6 positive CFS patients had central nervous system (CNS) involvement in their disease. To formally address this, 25 patients with CNS disease (abnormal SPECT or MRI scans, sensory abnormalities, cognitive defects, etc.) seen in the infectious disease practice were evaluated for active HHV-6 infections. Fourteen of the 25 patients (56%) were HHV-6 positive. This incidence of HHV-6 infection was higher (p < 0.08) than that seen in total population of unselected CFS patients (37%) suggesting that the selection for CFS with CNS involvement coselected for active HHV-6 infections. Confirmation of CNS infection with HHV-6 in some patients with CFS was obtained by the detection of HHV-6 DNA in the cerebrospinal fluid (CSF) of 20% (7/35) of the CFS patients studied.

CONCLUSION: These studies demonstrate that a sizable proportion (30% to 70%) of patients with CFS suffer from an active persistent infection with HHV-6 which may account for all or many of the clinical manifestations of their disease. Active HHV-6 infections may be especially prevalent in CFS patients with CNS involvement, consistent with the highly neuroinvasive nature of HHV-6.

Presented at the Fourth International American Association for Chronic Fatigue Syndrome Conference October 12-14, 1998