Retroviruses in autoimmune liver disease: genetic or environmental agents?

Retroviruses have been implicated in the pathogenesis of several human autoimmune conditions including Sj?gren's syndrome, primary biliary cirrhosis, immune mediated diabetes, and multiple sclerosis. The human intracisternal A type particle derived from Sj?gren's syndrome patients' salivary glands was the first retrovirus to be isolated from a human autoimmune disorder but the agent has yet to be cloned. In primary biliary cirrhosis patients, virus like particles have been observed by electron microscopy in biliary epithelium, endogenous retroviral sequences have been cloned from liver samples, and antibody reactivity to the human intracisternal A type particle has been observed in the majority of patients tested. However, there is no evidence to link the endogenous retroviral sequences in primary biliary cirrhosis patients to the retroviral antibody reactivity or virus like particles. In other patients with liver disease, reactivity to the human intracisternal A type particle was observed in a small but significant proportion of patients with hepatitis C virus infection. If the intracisternal A type particle is an endogenous retrovirus, it is interesting to speculate that hepatitis C virus infection may modulate the endogenous retroviral expression, as chronic hepatitis C has been linked with the development of Sj?gren's syndrome. Furthermore, many patients with chronic hepatitis C virus infection have reactivity to an autoantigen of unknown significance known as GOR that has protein sequence homology with both hepatitis C virus nucleocapsid protein as well as HTLV-1 gag. This may be an another example of an endogenous retroviral protein acting as an autoantigen in liver disease patients. At this time, there is little evidence to suggest that endogenous retroviruses are infectious agents that cause autoimmune disease but they may be implicated as either genetic elements or antigens. Further studies will be required to characterize the role that both exogenous and endogenous retroviruses play in the pathogenesis of autoimmune liver diseases.     

Human foamy virus antigens in thyroid tissue of Graves' disease patients.

Human foamy virus (HFV) is a recently characterized member of the retrovirus subfamily Spumaretrovirinae. HFV has a complex structure: it encodes the classical three retroviral structural genes gag, pol and env, but also possesses additional regulatory, so-called bel sequences. Foamy viruses have been discussed occasionally as being possibly involved in the pathogenesis of autoimmune thyroiditis. By indirect immunofluorescence we were able to demonstrate the expression of HFV gag proteins on the epithelial cells of 7/7 thyroid glands of patients with Graves' disease, but not in those of 9 patients with struma parenchymatosa, 3 with follicular carcinoma and 2 normal thyroids. The thyroids of 5 patients with Hashimoto's disease were also negative with the exception of a single small focus in 1 case. These observations may have importance for our understanding of the development of Graves' disease.     

Human herpesviruses in chronic fatigue syndrome

We have conducted a double-blind study to assess the possible involvement of the human herpesviruses (HHVs) HHV6, HHV7, Epstein-Barr virus (EBV), and cytomegalovirus in chronic fatigue syndrome (CFS) patients compared to age-, race-, and gender-matched controls. The CFS patient population was composed of rigorously screened civilian and Persian Gulf War veterans meeting the Centers for Disease Control and Prevention's CFS case definition criteria. Healthy control civilian and veteran populations had no evidence of CFS or any other exclusionary medical or psychiatric condition. Patient peripheral blood mononuclear cells were analyzed by PCR for the presence of these HHVs. Using two-tailed Fisher's exact test analyses, we were unable to ascertain any statistically significant differences between the CFS patient and control populations in terms of the detection of one or more of these viruses. This observation was upheld when the CFS populations were further stratified with regard to the presence or absence of major axis I psychopathology and patient self-reported gradual versus acute onset of disease. In tandem, we performed serological analyses of serum anti-EBV and anti-HHV6 antibody titers and found no significant differences between the CFS and control patients.     

HTLV-Associated Diseases: Human Retroviral Infection and Cutaneous T-Cell Lymphomas

An array of neurologic, oncologic, and autoimmune disorders are associated with infection with the human pathogenic retroviruses human T-cell leukemia virus types I and II (HTLV-I, II), as well as the human immunodeficiency viruses (HIV). The cutaneous T-cell lymphomas, mycosis fungoides (MF) and its hematogenous variant Sezary Syndrome (SS), share similar clinical and pathological features to HTLV-I-associated adult T-cell leukemia (ATL) and speculation of a retroviral link to MF and SS, especially in areas non-endemic for ATL, has lead to an intensified search for HTLV- and HIV-like agents in these diseases. To further explore a potential role for human retroviruses in MF and SS, skin biopsy-derived or peripheral blood mononuclear cell-derived DNA from 17 patients (MF, n=7; erythrodermic MF (EMF), n=5; SS, n=5) from the North Eastern United States were screened using gene amplification by PCR and a liquid hybridization detection assay. Previously published primers and probes for HTLV-I (LTR, gag, pol, env, and pX), and our own primers and probes for HTLV-I (gag, pol, and env), HTLV-II (pol and env) and HIV-I (gag and pol) were employed. Serum antibodies to HTLV-I were negative in all but one EMF patient. The single HTLV-I seropositive patient carrying a diagnosis of EMF generated positive amplified signals for all of the eight HTLV-I regions tested. Ultimately, this individual evolved to exhibit clinical manifestations indistinguishable from ATL. The other 16 patients were negative for all 12 HTLV and HIV retroviral regions. Our findings suggest that none of the known prototypic human retroviruses are associated with seronegative MF and SS. The uniformly positive results for HTLV-I in the seropositive patient suggests that this patient initially presented with a smoldering form of ATL and illustrates the difficulty that sometimes may be encountered in the differential diagnosis of MF, SS, and ATL based solely on clinical and histopathological criteria.     

Lack of association between HLA genotype and chronic fatigue syndrome.

Although the aetiology of chronic fatigue syndrome is controversial, evidence that infective agents including viruses may have a role in the development of the condition has led to studies seeking an association with the immunomodulatory HLA genes. In the present study, we sought to extend previous work using a well-characterized patient group and modern HLA genotyping techniques. Fifty-eight patients were phenotyped for HLA A and B by microcytotoxicity and genotyped for HLA DRB, DQB and DPB by PCR oligoprobing, and the frequencies of antigens so assigned were compared with those from a control group of 134. No significant differences in HLA frequencies were found between patient and control groups. Thus, this study does not confirm previous findings of an HLA association with chronic fatigue syndrome, suggesting that neither presentation of viral antigen by HLA class I nor antigen processing genes in the HLA region is a major contributory factor in the development of the disease.     

Cytomegalovirus-related sequence in an atypical cytopathic virus repeatedly isolated from a patient with chronic fatigue syndrome.

An atypical virus, cytopathic for human and animal fibroblasts, was repeatedly cultured from a patient with chronic fatigue syndrome. Viral particles, suggestive of cytomegalovirus (CMV) were seen by electron microscopy. Infected cells did not, however, stain with antisera specific for CMV, herpes, simplex virus, or human herpes-virus-6. Polymerase chain reaction (PCR) assays for these viruses were also negative. Two distinct products of approximately 1.5 kilobase pairs were amplified from virally infected cells using the human T lymphotropic virus-II tax gene reactive primer, SK44, in low stringency PCR. Sequencing of one of the amplified products showed a region of highly significant partial homology with the UL34 gene of CMV. The sequence of the other PCR product did not correspond with CMV or any other virus. DNA was extracted from the material pelleted by ultracentrifugation of filtered culture supernatants. It migrated in agarose gels as a single band of approximately 20 kpb. The banded DNA was digested with EcoRI and cloned. A 2.2 kbp plasmid containing the CMV-related sequence identified within the PCR product was recovered. Sequencing of this plasmid extended the region of partial sequence homology with CMV to include a portion of the UL35 gene of CMV. Initial sequencing of additional plasmids has confirmed the partial relatedness to CMV. The data indicate a novel type of CMV-related "stealth" virus that is able to establish a clinically persistent human infection.     

Human foamy virus genome in the thymus of myasthenia gravis patients

The etiological relationship of human foamy virus (HFV), which is a spumaretrovirus, with human diseases is not clear. We analyzed thymus specimens from four patients with myasthenia gravis for the presence of HFV proviral genome by polymerase chain reaction (PCR). The results showed the presence of both 257 base pair (bp) and 299 bp DNA fragments representing a part of gag and bel-2 sequences, respectively, in all four thymuses. Their specificity was confirmed by Southern blot hybridization with the corresponding probes. This was also confirmed by sequence analysis, although there were some point mutations. We confirmed the presence of gag related sequence, a 1353 bp Xba I-cleaved DNA fragment in all four thymus samples, a 693 bp fragment in two (#3 and #4) and a 4300 bp Hind III-cleaved DNA fragment in another two (#1 and #4), indicating possible chromosomal integration of the HFV partial genome. To our knowledge, this is the first report on the presence of HFV genome in thymus tissues of myasthenia gravis patients. Our efforts to isolate the infectious HFV by cultivation of the tissues were not successful. Low titers of neutralizing antibody were detected in all four patients' serum samples. The possible role of the HFV in this autoimmune disease needs further investigation.     

Human Herpesviruses in Chronic Fatigue Syndrome

We have conducted a double-blind study to assess the possible involvement of the human herpesviruses (HHVs) HHV6, HHV7, Epstein-Barr virus (EBV), and cytomegalovirus in chronic fatigue syndrome (CFS) patients compared to age-, race-, and gender-matched controls. The CFS patient population was composed of rigorously screened civilian and Persian Gulf War veterans meeting the Centers for Disease Control and Prevention’s CFS case definition criteria. Healthy control civilian and veteran populations had no evidence of CFS or any other exclusionary medical or psychiatric condition. Patient peripheral blood mononuclear cells were analyzed by PCR for the presence of these HHVs. Using two-tailed Fisher’s exact test analyses, we were unable to ascertain any statistically significant differences between the CFS patient and control populations in terms of the detection of one or more of these viruses. This observation was upheld when the CFS populations were further stratified with regard to the presence or absence of major axis I psychopathology and patient self-reported gradual versus acute onset of disease. In tandem, we performed serological analyses of serum anti-EBV and anti-HHV6 antibody titers and found no significant differences between the CFS and control patients.     

Detection of enterovirus-specific RNA in serum: The relationship to chronic fatigue

The serum of 88 chronic fatigue patients was screened for enteroviral specific sequences by polymerase chain reaction (PCR) assay. The PCR method used was “nested” PCR targetting the 5′ nontranslated region of the enteroviral genome which yielded a final fragment length of 264 base pairs. Samples were obtained from patients during 1990–1991. In addition, buffy coat specimens and stool specimens were examined in some patients. Samples from two cohorts of comparison individuals were also obtained. The comparison groups were firstly, acutely ill individuals with symptoms consistent with a presumed enteroviral infection (matched by age, sex, and date of receipt of specimen) and secondly, healthy individuals (matched by age and date of receipt of specimen). Enteroviral specific sequences were detected in 36 of 88 serum samples from chronic fatigue patients, 22 of 82 acutely ill individuals, and 3 of 126 healthy individuals. The enteroviral PCR positivity did not correlate with any one particular feature of chronic fatigue nor did it reflect any history of illness at onset of fatigue, duration of fatigue, or age of patient. These results provide new evidence for the presence of enteroviral specific sequences in serum, buffy coat, and stool samples in many patients with chronic fatigue. This may reflect a persistent enterovirus infection in a proportion of chronic fatigue patients. © 1995 Wiley-Liss, Inc.     

Comparison of Coxsackie B neutralization and enteroviral PCR in chronic fatigue patients

Coxsackie B enteroviruses have been implicated repeatedly as agents associated with chronic fatigue syndrome (CFS). The objective of this study was to compare the serological evidence for the presence of Coxsackie B virus neutralising antibody, with the polymerase chain reaction (PCR) detecting a portion of the 5′ nontranslated region (NTR) of the enterovirus genome. Serum samples from 100 chronic fatigue patients and from 100 healthy comparison patients were used in this study. In the CFS study group, 42% patients were positive for enteroviral sequences by PCR, compared to only 9% of the comparison group. Using the neutralisation assay, 34% of study patients were positive, compared to 41% of comparison patients. In the study group, 66/100 patient results correlated, i.e., they were either positive/positive or negative/negative for both tests. Of those that did not correlate, the majority were PCR-positive/Coxsackie B antibody-negative (21/34). In the comparison group, 58/100 patient results correlated. Of those that did not, the majority were PCR-negative/Coxsackie B antibody-positive (37/42). The Coxsackie B antibody neutralisation assay was not able to differentiate the CFS study group from the healthy comparison group, and thus the clinical relevance of this assay may be questioned. The PCR assay did differentiate the two groups with significantly more CFS patients having evidence of enterovirus than the comparison group. © 1995 Wiley-Liss, Inc.     

Lack of association between HLA genotype and chronic fatigue syndrome

Although the aetiology of chronic fatigue syndrome is controversial, evidence that infective agents including viruses may have a role in the development of the condition has led to studies seeking an association with the immunomodulatory HLA genes. In the present study, we sought to extend previous work using a well‐characterized patient group and modern HLA genotyping techniques. Fifty‐eight patients were phenotyped for HLA A and B by microcytotoxicity and genotyped for HLA DRB, DQB and DPB by PCR oligoprobing, and the frequencies of antigens so assigned were compared with those from a control group of 134. No significant differences in HLA frequencies were found between patient and control groups. Thus, this study does not confirm previous findings of an HLA association with chronic fatigue syndrome, suggesting that neither presentation of viral antigen by HLA class I nor antigen processing genes in the HLA region is a major contributory factor in the development of the disease.     

Expression of antigen reactive with a monoclonal antibody to HTLV-1 P19 in salivary glands in Sjögren''s syndrome.

To examine the possible involvement of retroviruses in Sjögren's syndrome (SS), labial salivary gland sections from 99 individuals were probed with three MoAbs to core (gag) proteins of human T cell leukaemia virus-1 (HTLV-1) and two MoAbs to HIV-1. Sections from 31% of 39 patients with primary SS (pSS) contained an epithelial cytoplasmic protein reactive with a MoAb(197) to the p19 group specific antigen (gag) of HTLV-1. The antigen was also detected in samples from 24% of 17 patients with rheumatoid arthritis (RA) and SS. 21% of 14 patients with sicca symptoms and 12.5% of 16 patients with other connective tissue diseases. It was not found in the salivary glands of 13 normal controls. A second MoAb to p19 gag, a MoAb to the p24 gag of HTLV-1 and MoAbs to HIV-1 p17and p24 gags gave negative reactions. Serum antibodies to HTLV-1 were negative, confirming that the antigen was not part of HTLV-1. The antigen showed properties consistent with an endogenous retrovirus in that it was absent in healthy tissues or resting cells but inducible by stimulation with phytohaemagglutinin (PHA) or interferon-gamma (IFN-γ) It appeared to be distinct from the endogenous retroviral sequence HRES-1. These data suggest the presence of an endogenous retrovirus in salivary gland epithelium which could contribute to the chronic inflammation of SS.     

Comparative humoral responses to human immunodeficiency virus type 1-p24gag linear B-cell epitopes among individuals showing atypical western immunoblotting reactions and implications for diagnosis.

Serum specimens from 25 individuals with an isolated human immunodeficiency virus type 1 (HIV-1) core antigen reactivity in a Western immunoblot test were examined for their reactivities with HIV-1 virions, control cellular antigens, HIV-1-Bru p24gag recombinant protein (p24gag), and a panel of 22 p24gag-derived peptides. The results were as follows: (i) serum specimens from eight HIV-1-uninfected subjects did bind to virions but failed to bind to p24gag; (ii) sera from 13 HIV-1-uninfected subjects and from one HIV-2-infected patient reacted with HIV-1 virions and p24gag but failed to bind to any of the peptides expressing major p24gag epitopes, and (iii) 3 serum specimens obtained from one neonate carrying anti-HIV-1 maternal antibody and from two HIV-1-infected subjects who had seroconverted during the study reacted with HIV-1 virions, p24gag, and one or more peptides containing the major p24gag epitopes. Our data suggest that the combination of p24gag and appropriate peptides could be useful for resolution when atypical Western immunoblot results are encountered.     

Neurasthenia in the 1980s: chronic mononucleosis, chronic fatigue syndrome, and anxiety and depressive disorders

In the 1980s, patients suffering from unexplained fatigue and what seemed like a prolonged attack of acute mononucleosis were given the diagnosis of chronic mononucleosis or chronic infection with the Epstein-Barr virus. Although the diagnosis has great appeal, the Epstein-Barr virus does not cause the syndrome (CFS) of chronic fatigue, which has been renamed and redefined chronic fatigue syndrome to remove the inference that the virus is its cause. From a historical perspective, both syndromes represent the 1980s equivalent of neurasthenia, a disease of fatigue that influenced the development of psychiatric nosology. Because patients with depression and anxiety also have chronic fatigue and because most patients with CFS have an affective disorder, the assessment of organic causes of this syndrome requires careful psychiatric diagnosis and treatment. Defining chronic fatigue syndrome as a medical disorder may deprive patients of competent treatment of their affective disorder.     

Human foamy virus proteins accumulate in neurons and induce multinucleated giant cells in the brain of transgenic mice.

Human foamy virus (HFV) is a retrovirus encoding structural genes and, like human immunodeficiency virus and human T cell leukemia virus I, several ancillary reading frames collectively termed the be1 genes. We have previously shown that HFV transgenic mice develop an encephalopathy with neuronal loss in hippocampus and cerebral cortex. We have now raised and characterized rabbit antisera to various recombinant portions of gag, pol, env, and bel-1, the viral trans-activator. Immunoreactivity for gag and bel-1 was observed in nuclei and processes of hippocampal and cortical neurons before the onset of morphological lesions and correlated with the appearance of HFV mRNA. Astrocyte-derived multinucleated giant cells containing HFV proteins were present in the brain of transgenic mice coexpressing full-length HFV genes but not in mice expressing truncated gag and env, suggesting that these genes contain a fusogenic domain. Expression of full-length structural genes decreased the life expectancy of transgenic mice, implying an adjuvant role for these proteins in HFV-induced brain damage.