Pathobiology

A cytopathic stealth virus was cultured from the cerebrospinal fluid (CSF) of a nurse with chronic fatigue syndrome (CFS). Reverse transcriptase - polymerase chain reaction (RT-PCR) performed on the patient's culture yielded positive results with primer sets based on sequences of a previously isolated African green monkey simian cytomegalovirus (SCMV)-derived stealth virus. The same primer sets did not yield PCR products when tested directly on DNA extracted from the cultures. The findings lend support to the possibility of replicative RNA forms of certain stealth viruses and have important implications concerning the choice of therapy in this type of patient.

Atypical cytopathic viruses have been isolated from patients diagnosed as having the chronic fatigue syndrome (CFS) and other neuropsychiatric illnesses (1-6). They have been termed "stealth viruses" primarily because they do not evoke a cellular inflammatory response (2,7). Stealth viral infection can be detected in the blood, cerebrospinal fluid (CSF) and tissues of some patients using low stringency polymerase chain reaction (PCR) assays employing primers cross-reactive with known human herpesviruses (2,5,6,8,9). DNA sequencing studies on PCR products generated from several stealth viruses indicate that the viruses are molecularly heterogeneous (6). The prototype stealth virus, designated stealth virus-1, was isolated from the blood of a CFS patient (2). It has an unstable, fragmented viral genome (10,11) with regions of sequence homology with the Colburn strain of African green monkey simian cytomegalovirus (SCMV). Another SCMV related stealth virus, designated stealth virus-2, was cultured from the cerebrospinal fluid (CSF) of a patient with an acute encephalopathy and prior history of a bi-polar psychotic illness (6). The origins of other stealth viruses on which sequence data are not available is presently uncertain. In several of these cultures, positive PCR results have been achieved by reacting culture-derived DNA with one or more primer sets based on the known sequences of stealth virus-1 (unpublished data). As described in this paper, even if negative results are obtained using DNA with these primers, it may be possible to generate PCR products from RNA sequences by using a reverse transcriptase based PCR (RT-PCR).

This 38 year old patient was working as an emergency room nurse when in September 1992, she had an acute illness characterized by sore throat, cough, fever to 103^oC, episodes of marked diaphoresis, sustained hypotension (60-70/30-40 mm Hg), and bradycardia . The acute respiratory illness lasted approximately 6 weeks. Towards the end of this period, she began to experience frontal headaches and became photophobic. The headaches and photophobia have persisted and accompanied by cognitive deficits including short term memory loss, slowed speech pattern and diminished clarity of through processing. She has had difficulty in initiating and in maintaining nocturnal sleep, yet periods of extreme tiredness during the day. She has developed a heightened sensitivity to spicy foods with altered taste sensations. She has tinnitus and is sensitive to loud noises. She has receive prozac, praxil and zoloft for continuing depression, and additional medications for transient migrating arthralgia and myalgia. The patient has been on disability from the time of her disease onset.

Cerebrospinal fluid (CSF) samples were obtained on several occasions and reported as showing no abnormalities. A SPECT scan performed in July, 1996 showed decreased perfusion to both frontal lobes. A PET scan showed subtle, irregular and patchy uptake throughout the cerebrum but these changes were reported as being of uncertain diagnostic significance. Serological testing repeatedly showed high titers of IgG anti-cytomegalovirus (CMV) antibody often accompanied by an elevated IgM anti-CMV antibody response. In late 1996, the patient was started on intravenous ganciclovir therapy (6 mg/Kg twice a day for 2 weeks, 7 mg/Kg once a day for 2 weeks, 7 mg/kg three times per week thereafter for 4 months). The ganciclovir therapy has lessened the intensity of her headaches and has somewhat improved her cognition. She is also less photophobic but remains quite symptomatic. A CSF sample was submitted for stealth viral testing 3 weeks after the initiation of the ganciclovir therapy.

Viral cultures: The CSF was stored at -70^oC prior to culture. It was thawed and 0.4 ml, diluted into 2 ml of X vivo-15 medium, was added to both MRC-5 human fibroblasts and RhMK primary rhesus monkey kidney cells. The media and cells were obtained from BioWhittaker Inc., MD. The cultures were fed with X-vivo 15 media at 2-3 day intervals and observed for the development of cytopathic effects (CPE). Control cultures were similarly fed and examined. Cells were scrapped from a culture tube in which approximately 50% of the cells showed cellular changes and used for transmission studies and for PCR analysis.

DNA and RNA extraction: Cells were harvested from the patient's positive culture and from uninfected control cultures and used to extract either DNA or RNA. Of several techniques previously used to obtain viral DNA suitable for PCR, the simplest has been to place a tube containing cells suspended in 100 ml of PCR buffer, in a boiling water bath for 10 min. This method was used on the patient's culture and previously used on an SCMV culture to provide a positive PCR control. For RNA extraction, a QIAgen Rneasy kit (QIAgen, Santa Clarita, CA) was used with the cells from a single culture tube reconstituted in 30 ml DEPC-treated water.

PCR assays: Ten ml of the DNA preparations from the patient's culture and from the SCMV positive control, were used in DNA based PCR assays. The assays were run in 100 ml volumes for 34 cycles on a Perkin Elmer Gene Amp 9600 thermocycler. The cycles consisted of: a denaturation step at 95^oC for 30 sec, a primer annealing step at 42^oC for 15 sec, and an extension step at 72^oC for 45 sec. RT-PCR assays were run on the purified RNA preparation using Gene Amp kits supplied by Perkin Elmer (Foster City, CA) and using the same Gene Amp 9600 thermocycler. The assays were run using 2 ml of the RNA preparation, first in a 20 ml volume for one reverse transcription cycle (15 min at 42^oC for reverse transcription, 5 min at 99^oC for denaturation, and a cooling step of 5 min at 5^oC). Upon completion of reverse transcription, 80 ml of reagents were added to bring the total volume to 100 ml for PCR (hold at 95^oC for 105 sec; 35 cycles consisting of the following: denaturation at 95^oC for 15 sec, primer annealing/extension at 60^oC for 30 sec; and a final hold for 7 min at 72^oC). For both PCR and RT-PCR, the primer sets used were based on sequences within three plasmids containing cloned regions of stealth virus-1. The GenBank accession numbers of these plasmids and the sequences of the primers used, are listed in Table 1. Following the PCR, 16 ml of each of the reaction mixtures were electrophoresed in 1 % agarose containing ethidium bromide.

Table I

PCR Primers Based on Sequences of Cloned Regions of Stealth Virus-1

GenBank sequence accession No.	Plasmid designation	Size of PCR product with stealth virus-1	Primer
U27952	C162114	279 bp	5'-AACCATGTCTGCCACATCG-3' 5'-CAATAGGATCTCTCGCGCAC-3'
U27916	3B647	373 bp	5'-CGCTGTCGCTCTCTTCCTT-3' 5'-GAGCACGATACGGTGTTGC-3'
U27888	3B412	374 bp	5'-CCTGTTGTCATCTTGTTCAGG-3' 5'-AATGTTCGACAGTCTGCGC-3'

The RhMK cultures developed numerous foci of rounded, vacuolated cells, with syncytia and cellular destruction. As described elsewhere (2), this type of CPE is readily distinguishable, both in terms of morphology and cell type, from that induced by human cytomegalovirus, adenovirus or enteroviruses. The CPE began after 12 days and slowly spread to involve most of the culture. The CPE was serially passaged using cells scrapped from a positive culture. The initial CPE also developed on MRC-5 cells, but was less prominent than that in RhMK cells. Control cultures showed no CPE.

PCR assays were performed on DNA extracted from the patient's cultures with DNA from an SCMV culture serving as a positive control. Three different primer sets, corresponding to sequences present in stealth virus-1, were used. Each of the primer sets tested gave negative results when tested on DNA extracted from the patient's positive culture (data not shown). When the same primers were used in RT-PCR assay on RNA extracted from patient's culture, clearly defined PCR products were generated (Figure 1). The PCR product generated with the primer set based on plasmid C162114 was similar in size to the 278 base pair (bp) product expected for stealth virus-1. This primer set yielded a similarly sized, but faint product with SCMV. The primer set based on plasmid B614 gives a similar size product with SCMV and stealth virus-1 of 279 bp. A distinctly larger product than seen with SCMV was generated using the patient's culture (480 bp compared to 373 bp). Multiple additional faint bands were also generated. The primer set based on plasmid 3B412, generated two products (550 bp and a less intense band of 425 bp). Both of these products were considerably larger than the product seen with SCMV or predicted for stealth virus-1 (374 bp, see Table 1). Sequencing of the major PCR products has yet to be performed.

Stealth adapted herpesviral infections have been associated with non-inflammatory encephalopathic illnesses in humans (1-6) and in cats (12). Electron microscopy of brain biopsies has provided evidence of incomplete viral particles leading to the suggestion that the viruses may lack components required for evoking an effective anti-viral cellular inflammatory response (4). Agarose gel electrophoresis of undigested viral DNA, together with the multiple bands seen following restriction enzyme digestion of the DNA, suggested that the prototype SCMV-derived stealth virus isolated from a CFS patient consisted of multiple fragments (2). This conclusion was supported by sequencing studies performed on cloned viral DNA (10 ). DNA sequence microheterogeneity, as well as an unpublished observations using RNase digestion, suggested that some of the viral replication was occurring on RNA templates. (10 )

DNA sequences from the stealth virus cultured from the patient described in this paper could not be demonstrated using a DNA based PCR assay. RNA sequences amplified by primer sets based on sequences present in the prototype SCMV-derived stealth virus-1, could be detected using reverse transcriptase to initially generate DNA molecules. Positive results were obtained using all three primer sets used. The formation of more than one PCR product with two of the primer sets, suggests that, like stealth virus-1, this virus may also have sequence microheterogeneity. possibly reflecting impaired fidelity of RNA replication. An understanding of the actual mode of viral replication and the nature of the polymerase being utilized may have important implications with regards to limited response seen in this patient with ganciclovir therapy.

The isolation of a stealth virus from CSF provides an explanation of the neuropsychological symptoms experienced by the patient. Although, the patient was eventually labeled as having CFS, her symptom complex remained undiagnosed for several years. Health care workers are at risk for occupational exposure to stealth viruses, and this possibility should be considered in attending to persons presenting with chronic fatigue and related illnesses.

Acknowledgment

The skilled assistance of Ms. Heidi Torres with the PCR assays is gratefully acknowledged. Dr. Lawrence Cone treated the patient and arranged for a CSF sample to be sent for stealth viral culture. The patient provided an excellent account of her illness. Supported in part by funds from the Theodore and Valda Stanley Foundation and from CAN, Cure Autism Now.

1. Martin W.J. Viral infection in CFS patients. in "The Clinical and Scientific Basis of Myalgic Encephalomyelitis Chronic Fatigue Syndrome." Byron M. Hyde Editor. Nightingdale Research Foundation Press. Ottawa Canada pp 325-327, 1992.

2. Martin WJ, Zeng LC, Ahmed K, Roy M. Cytomegalovirus-related sequences in an atypical cytopathic virus repeatedly isolated from a patient with the chronic fatigue syndrome. Am. J. Path. 145: 441-452, 1994.

3. Martin WJ. Stealth virus isolated from an autistic child. J. Aut. Dev. Dis. 25:223-224,1995

4. Martin WJ. Severe stealth virus encephalopathy following chronic fatigue syndrome-like illness: Clinical and histopathological features. Pathobiology 64:1-8, 1996.

5. Martin WJ. Stealth viral encephalopathy: Report of a fatal case complicated by cerebral vasculitis. Pathobiology 64:59-63, 1996.

6. Martin WJ. Simian cytomegalovirus-related stealth virus isolated from the cerebrospinal fluid of a patient with bipolar psychosis and acute encephalopathy. Pathobiology 64:64-66, 1996.

7. Martin W.J. Stealth viruses as neuropathogens. College of American Pathologist's publication "CAP Today" 8 67-70, 1994

8. Martin W.J. Detection of viral related sequences in CFS patients using the polymerase chain reaction.in "The Clinical and Scientific Basis of Myalgic Encephalomyelitis Chronic Fatigue Syndrome." Byron M. Hyde Editor. Nightingdale Research Foundation Press. Ottawa Canada pp 278-283, 1992.

9. Gollard RP, Mayr A, Rice DA, Martin WJ. Herpesvirus-related sequences in salivary gland tumors. J. Exp. Clin. Can. Res. 15: 1-4, 1996.

10 Martin WJ. Genetic instability and fragmentation of a stealth viral genome. Pathobiology 64:9-17, 1996.

11. Martin WJ, Ahmed KN, Zeng LC, Olsen J-C, Seward JG, Seehrai JS. African green monkey origin of the atypical cytopathic 'stealth virus' isolated from a patient with chronic fatigue syndrome. Clin. Diag. Virol. 4: 93-103, 1995.

12. Martin WJ, Glass RT. Acute encephalopathy induced in cats with a stealth virus isolated from a patient with chronic fatigue syndrome. Pathobiology 63: 115-118, 1995.