Persistent Enterovirus Infection Research Articles

Bi-glandular and persistent enterovirus infection and distinct changes of the pancreas in slowly progressive type 1 diabetes mellitus

In slowly progressive type 1 diabetes mellitus (SPIDDM), the pancreas shows sustained islet inflammation, pancreatitis, pancreatic acinar cell metaplasia/dysplasia (ADM), and intraepithelial neoplasia (PanIN), a precancerous lesion. The mechanisms underlying these changes remain unclear. The presence of enterovirus (EV) encoded-capsid protein 1 (VP1) and -2A protease (2Apro) and the innate immune responses of the pancreas were studied using immunohistochemistry and in situ hybridization in 12 SPIDDM and 19 non-diabetic control pancreases. VP1, 2Apro, and EV-RNA were detected in islets and the exocrine pancreas in all SPIDDM pancreases. Innate immune receptor, melanoma differentiation-associated gene 5 (MDA5), and interferon (IFN)-beta1 were intensified in the islets of SPIDDM patients with short disease duration. However, expressions of MDA5 and IFN-beta1were suppressed in those with longer disease duration. CD3+ T cell infiltration was observed in the VP1- and insulin-positive islets (insulitis) and exocrine acinar cells. CD11c+ dendritic cells (DCs) in islets were scarce in long-term SPIDDM. This study showed the consistent presence of EV, suggesting an association with inflammatory changes in the endocrine and exocrine pancreas in SPIDDM. Suppressed expressions of MDA5 and IFN-beta1, as well as decreased numbers of DCs in the host cells, may contribute to persistent EV infection and induction of ADM/PanIN lesions, which may potentially provide a scaffold for pancreatic neoplasms.

Persistent coxsackievirus B infection and pathogenesis of type 1 diabetes mellitus.

Enteroviruses are believed to trigger or accelerate islet autoimmunity in genetically susceptible individuals, thereby resulting in loss of functional insulin-producing β-cells and type 1 diabetes mellitus (T1DM). Although enteroviruses are primarily involved in acute and lytic infections in vitro and in vivo, they can also establish a persistent infection. Prospective epidemiological studies have strongly associated the persistence of enteroviruses, especially coxsackievirus B (CVB), with the appearance of islet autoantibodies and an increased risk of T1DM. CVB can persist in pancreatic ductal and β-cells, which leads to structural or functional alterations of these cells, and to a chronic inflammatory response that promotes recruitment and activation of pre-existing autoreactive T cells and β-cell autoimmune destruction. CVB persistence in other sites, such as the intestine, blood cells and thymus, has been described; these sites could serve as a reservoir for infection or reinfection of the pancreas, and this persistence could have a role in the disturbance of tolerance to β-cells. This Review addresses the involvement of persistent enterovirus infection in triggering islet autoimmunity and T1DM, as well as current strategies to control enterovirus infections for preventing or reducing the risk of T1DM onset.

Persistent Enterovirus Infection: Little Deletions, Long Infections.

Enteroviruses have now been shown to persist in cell cultures and in vivo by a novel mechanism involving the deletion of varying amounts of the 5′ terminal genomic region termed domain I (also known as the cloverleaf). Molecular clones of coxsackievirus B3 (CVB3) genomes with 5′ terminal deletions (TD) of varying length allow the study of these mutant populations, which are able to replicate in the complete absence of wildtype virus genomes. The study of TD enteroviruses has revealed numerous significant differences from canonical enteroviral biology. The deletions appear and become the dominant population when an enterovirus replicates in quiescent cell populations, but can also occur if one of the cis-acting replication elements of the genome (CRE-2C) is artificially mutated in the element’s stem and loop structures. This review discusses how the TD genomes arise, how they interact with the host, and their effects on host biology.

Investigation of the utility of the 1.1B4 cell as a model human beta cell line for study of persistent enteroviral infection

The generation of a human pancreatic beta cell line which reproduces the responses seen in primary beta cells, but is amenable to propagation in culture, has long been an important goal in diabetes research. This is particularly true for studies focussing on the role of enteroviral infection as a potential cause of beta-cell autoimmunity in type 1 diabetes. In the present work we made use of a clonal beta cell line (1.1B4) available from the European Collection of Authenticated Cell Cultures, which had been generated by the fusion of primary human beta-cells with a pancreatic ductal carcinoma cell, PANC-1. Our goal was to study the factors allowing the development and persistence of a chronic enteroviral infection in human beta-cells. Since PANC-1 cells have been reported to support persistent enteroviral infection, the hybrid 1.1B4 cells appeared to offer an ideal vehicle for our studies. In support of this, infection of the cells with a Coxsackie virus isolated originally from the pancreas of a child with type 1 diabetes, CVB4.E2, at a low multiplicity of infection, resulted in the development of a state of persistent infection. Investigation of the molecular mechanisms suggested that this response was facilitated by a number of unexpected outcomes including an apparent failure of the cells to up-regulate certain anti-viral response gene products in response to interferons. However, more detailed exploration revealed that this lack of response was restricted to molecular targets that were either activated by, or detected with, human-selective reagents. By contrast, and to our surprise, the cells were much more responsive to rodent-selective reagents. Using multiple approaches, we then established that populations of 1.1B4 cells are not homogeneous but that they contain a mixture of rodent and human cells. This was true both of our own cell stocks and those held by the European Collection of Authenticated Cell Cultures. In view of this unexpected finding, we developed a strategy to harvest, isolate and expand single cell clones from the heterogeneous population, which allowed us to establish colonies of 1.1B4 cells that were uniquely human (h1.1.B4). However, extensive analysis of the gene expression profiles, immunoreactive insulin content, regulated secretory pathways and the electrophysiological properties of these cells demonstrated that they did not retain the principal characteristics expected of human beta cells. Our data suggest that stocks of 1.1B4 cells should be evaluated carefully prior to their use as a model human beta-cell since they may not retain the phenotype expected of human beta-cells.

Immune Transcriptome of Cells Infected with Enterovirus Strains Obtained from Cases of Autoimmune Thyroid Disease

Background: Hashimoto’s thyroiditis and Graves’ disease are autoimmune thyroid disorders (AITD) of unknown origin. Enterovirus (EV) infection of thyroid cells has been implicated as a possible initiator of cell damage and of organ-specific autoimmunity. We asked whether persistent infection of human epithelial cells with EV strains obtained from thyroid tissue of AITD patients could be associated with transcriptional changes capable of fostering immunopathology. Methods: EV isolates obtained from thyroid tissue of AITD cases were used to infect the AV3 epithelial cell line. AV3 cells incubated with a virus-free medium from thyroid tissue of subjects without evidence of thyroid autoimmunity were used as uninfected controls. Transcripts of immune-related genes were compared in infected vs. uninfected cells. Results: The EV genome and antigens were detected only in the cells exposed to AITD-derived virus isolates, not in control cells. Persistent EV infection, while suppressing transcription of several type I IFN and cytokine determinants, was associated with enhanced transcription of NFKB1/RELA, IFNAR1, JAK1/STAT1, i.e., the determinants that play key immunologic roles. Infection also led to upregulation of the CCL2 chemokine and the IL-18 pro-inflammatory interleukin. Conclusion: As in the case of EV strains obtained from autoimmune diabetes, results show that the EV strains that are present in the thyroid of AITD cases do repress IFN and cytokine pathways. JAK1/STAT1 upregulation supports activation of TLR pathways and aberrant T cell signaling. In the early phases of AITD, our results highlight the potential benefit of interventions aimed at blocking the viral infection and easing the inflammatory response.

Detection of Viral -RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues.

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively.

Extracellular Vesicles Released by Enterovirus-Infected EndoC-βH1 Cells Mediate Non-Lytic Viral Spread.

While human enteroviruses are generally regarded as a lytic virus, and persistent non-cytolytic enterovirus infection in pancreatic beta cells has been suspected of playing a role in type 1 diabetes pathogenesis. However, it is still unclear how enteroviruses could exit the pancreatic beta cell in a non-lytic manner. This study aimed to investigate the role of beta cell-derived extracellular vesicles (EVs) in the non-lytic enteroviral spread and infection. Size-exclusion chromatography and antibody-based immunoaffinity purification were used to isolate EVs from echovirus 16-infected human beta EndoC-βH1 cells. EVs were then characterized using transmission electron microscopy and Multiplex Bead-Based Flow Cytometry Assay. Virus production and release were quantified by 50% cell culture infectious dose (CCID50) assay and qRT-PCR. Our results showed that EVs from echovirus 16-infected EndoC-βH1 cells harbor infectious viruses and promote their spread during the pre-lytic phase of infection. Furthermore, the EVs-mediated infection was not inhibited by virus-specific neutralizing antibodies. In summary, this study demonstrated that enteroviruses could exit beta cells non-lytically within infectious EVs, thereby thwarting the access of neutralizing antibodies to viral particles. These data suggest that enterovirus transmission through EVs may contribute to viral dissemination and immune evasion in persistently infected beta cells.

CORRELATION BETWEEN ENROVIRUS INFECTION AND ACUTE STROKE ON THE GROUND OF VIROLOGICAL, MOLECULAR GENETIC AND SEROLOGICAL METHODS OF DIAGNOSIS

Relevance. Numerous virological studies prove the importance of enteroviruses in human somatic pathology. However, the etiopathogenetic role of enterovirus infection in patients with acute cerebrovascular disorder (GVMK) is not sufficiently highlighted.
 Objective: to establish the value of enterovirus infection as a trigger factor in the pathogenesis of acute stroke.
 Materials and methods. The pear blood serum of 72 patients with acute stroke (main group) and 35 patients with neurological pathology without vascular pathology (group of comparison) were screened for presence of enteroviruses using the virological method, detection of enterovirus genomes using a polymerase chain reaction and the presence of specific Ig M and Ig G to enteroviruses in the enzyme-linked immunosorbent assay (ELISA).
 Results. The enterovirus genomes were isolated from blood serum in 23,6±5,9 % of patients with acute stroke, that is significantly higher than in patients of the comparison group – 2,9±2,8 % (p <0,05). The enteroviruses were isolated in 11 cases of 17 PCR-positive blood serum samples of the main group. These viruses were identified as Coxsackie B viruses (serotypes 2, 3, 4) and ECHO viruses (serotypes 6, 9, 27 (two strains), 29), three strains of viruses could not be identified. The presence of specific Ig M and Ig G in blood serum of 4 patients with HPMC, as well as enterovirus genomes, has been established. It suggest that they have a recent enterovirus infection, or can indicate a recent enterovirus infection or exacerbation of chronic enterovirus infection. Only specific Ig G in the absence of Ig M were detected in blood serum of 4 PCR positive patients, that can indicate chronic enterovirus infection. Only Ig M in the absence of Ig G was detected in blood serum of 6 PCR-positive patients, that can indicate acute enterovirus infection. No Ig M or Ig G in serum from three PCR-positive patients were detected, possibly due to the presence of latent enterovirus infection.
 Conclusions. Acute and chronic persistent enterovirus infection plays a possible trigger role in the development of acute stroke.
 The combination of PCR to detect genomes of enteroviruses, virological for the isolation and identification of viruses, and ELISA for the detection of specific Ig M and Ig G to enteroviruses should be recommended for the diagnosis of persistent enterovirus infection in patients with acute stroke.

No evidence of enteroviruses in the intestine of patients with type 1 diabetes

The purpose of this study was to investigate whether the gut mucosa is a reservoir for enterovirus persistence in patients with type 1 diabetes. Small intestine biopsy samples from 25 individuals at different stages of type 1 diabetes, 21 control individuals and 27 individuals with coeliac disease were analysed for the presence of enterovirus RNA by using both radioactive in-situ hybridisation and real-time RT-PCR and for the presence of enterovirus proteins by immunostaining with antibodies against VP1 and VP4-2-3 capsid proteins and virus polymerase. Lymphocytic enteropathy and serum anti-VP1 antibodies were also evaluated at the time of biopsy. Moreover, high-throughput sequencing was performed to identify viral transcripts or genomes. Enterovirus was not detected by in-situ hybridisation or RT-PCR in any of the individuals tested. Immunohistology revealed a few stained cells in the intestinal epithelium in a low number of individuals, with no difference between diabetic and non-diabetic individuals. Levels of serum IgG against VP1 did not differ between control individuals and those with diabetes or coeliac disease and no evidence of diabetes-related lymphocytic enteropathy was detected. High-throughput sequencing did not reveal specific enterovirus sequences in the gut mucosa of individuals with type 1 diabetes. Prolonged/persistent enterovirus infections in gut mucosa are not common in patients with type 1 diabetes.

Immunopathology of the human pancreas in type-I diabetes

Type 1 diabetes is a chronic autoimmune disease characterised by the selective destruction of pancreatic beta (β) cells. The understanding of the aetiology of this disease has increased dramatically in recent years by the study of tissue recovered from patients, from analysis of the responses of isolated islet and β-cells in tissue culture and via the use of animal models. However, knowledge of the immunopathology of type 1 diabetes in humans is still relatively deficient due largely to the difficulty of accessing appropriate samples. Here we review the state of current knowledge in relation to the histopathological features of the disease in humans. We focus specifically on recent-onset type 1 diabetes cases since in such patients, evidence of the ongoing disease process is still present. We chart the progression of the disease by describing the characteristic features of the pancreas, consider the sequence of immune cell infiltration and discuss the abnormalities of MHC antigen expression. The possibility that these changes might derive from a persistent enteroviral infection of the islet beta cells is examined.

Cerebrospinal fluid detection of enterovirus genome in ALS: A study of 242 patients and 354 controls

Detection of enterovirus (EV) in the spinal cord of patients with amyotrophic lateral sclerosis (ALS) has been reported on post mortem central nervous system tissues. In cases of persistent infection, it is very likely that the EV genome might be detected in the cerebrospinal fluid (CSF). A study was conducted in seven French amyotrophic lateral sclerosis (ALS) centres between 1997 and 2002. A total of 242 ALS patients and 354 age- and sex-matched controls (non-ALS patients) were enrolled. A sensitive RT-PCR method was performed on the CSF to assess the presence of EV RNA; 14.5% of ALS patients were positive compared to 7.6% of controls (chi(2) value, 5.31; p = 0.02). Although EV infection has a seasonal pattern, we observed no seasonality in positive detection of the EV genome among ALS patients. There was no significant relationship among ALS patients between the initial clinical form or survival and the result of the RT-PCR. These findings suggest a relationship between the presence of EV sequences in CSF and ALS. Our study is consistent with the hypothesis that persistent EV infection can be one of the multiple factors involved in the development of ALS.

Detection of enteroviruses in the intestine of type 1 diabetic patients

Enterovirus infections have been diagnosed more frequently in type 1 diabetic patients than in the healthy population, and enteroviruses have also been found in the pancreas of diabetic patients. Primary replication of the virus occurs in the gut, but there are no previous studies evaluating possible presence of virus in the intestine of diabetic patients. The purpose of this study was to investigate if enteroviruses can be found in small intestinal tissue of type 1 diabetic patients. Formalin-fixed, paraffin-embedded upper intestinal biopsy samples were analysed for the presence of enterovirus using in situ hybridization and immunohistochemistry. Enterovirus was detected by in situ hybridization in six (50%) of the type 1 diabetic patients (n = 12) but in none of the control subjects (n = 10, P = 0.015). Immunohistochemistry identified enterovirus in nine (75%) of the patients and one (10%) control subject (P = 0.004). The presence of the virus was confirmed by reverse transcription-polymerase chain reaction in one of the four patients from whom a frozen and unfixed sample was available. Intestinal morphology was normal in all study subjects. The results suggest that a substantial proportion of type 1 diabetic patients have an ongoing enterovirus infection in gut mucosa, possibly reflecting persistent enterovirus infection. This observation opens new avenues for further studies on the possible role of enteroviruses in human type 1 diabetes.

Enteroviruses and postviral fatigue syndrome.

Postviral fatigue syndrome (PFS) occurs both in epidemics and sporadically. Many of the original epidemics were related to poliomyelitis outbreaks which either preceded or followed them. The core clinical symptoms are always the same: severe fatigue made worse by exercise, myalgia, night sweats, atypical depression and excessive sleep. The other common symptoms include dysequilibrium disorders and irritable bowel syndrome. We have detected enteroviral genome sequences in muscle biopsies from cases of PFS, using specific enteroviral oligonucleotide primers in the polymerase chain reaction (PCR). In addition, whole virus particles can be demonstrated in PCR-positive muscle, using solid-phase immuno-electron microscopy. An increase in the number and size of muscle mitochondria was found in 70% of PFS cases, suggesting an abnormality in metabolic function. Evidence of hypothalamic dysfunction was present, particularly involving 5-hydroxytryptamine metabolism. A putative model of PFS, based on persistent enteroviral infection in laboratory mice, revealed resolving inflammatory lesions in muscle with, however, a marked increase in the production of certain cytokines in the brain. This model may help to explain the pathogenesis of PFS.

Sporadic amyotrophic lateral sclerosis: A hypothesis of persistent (non‐lytic) enteroviral infection

Because of recently reported reverse transcriptase polymerase chain reaction evidence of enterovirus in sporadic amyotrophic lateral sclerosis (SALS) and because of newly available anti-enteroviral drugs binding enteroviral capsids, it is reasonable to re-formulate an enteroviral hypothesis of SALS using recent advances in molecular virology. Viral persistence is non-lytic and non-cytopathic infection that evades host's immune surveillance. Enteroviruses are known to cause persistent as well as lytic infection both in vitro and in vivo. Both virion as well as host factors modulate between persistent and lytic infection. Apoptosis, or programmed cell death, is a process of active non-necrotic cell death. It has complex interplay with viruses and may be either promoted or opposed by them. Apoptosis is a major factor in motor neuron death in SALS. Viral tropism is the process by which viruses select and propagate to target cells. It is controlled by capsid conformation and surface receptors on host cells. Enteroviruses have a region on their capsids known as the canyon which docks on such receptors. Docking induces conformational changes of the capsid and genome release. Poliovirus, tropic for motor neurons, docks on the poliovirus receptor, about which much is known. The virus penetrates the motor system focally after crossing either the blood-muscle or the blood-brain barriers. It propagates bidirectionally along axons and synapses to contiguous motor neurons, upper as well as lower, which sequester infection and create avenues for spread over long distances. If chronic and persistent rather than acute and lytic, such viruses trafficking in a finite system of non-dividing cells and inducing apoptosis would cause cell death that summates linearly rather than exponentially. Taken together, these explain signature clinical features of SALS - focal onset weakness, contiguous or regional spread of weakness, confinement to upper and lower motor neurons, and linear rates of progression. The hypothesis predicts the following testable investigations: 1) viral detection may be possible by applying amplification technology to optimally acquired nervous tissue processed by laser microdissection; 2) genetic susceptibility factors such as cell surface receptor polymorphisms may combine with sporadic exposure and chance penetration of the motor system in SALS; 3) a transgenic animal model might be created by inserting such genetic factors into an animal host and inoculating intramuscularly rather than intracerebrally biochemical fractions of SALS motor neurons at vulnerable periods in the developmental life cycle of the transgenic host; and 4) continual long-term administration of anti-enteroviral agents called capsid-binding compounds which stabilize capsids and prevent genome release might be efficacious.

ALS

Polioviruses (and other enteroviruses) have long been considered a possible cause of ALS because these viruses and ALS share the same pathologic target, the motor neuron. 1 In addition, an increased frequency of ALS has been reported to occur in patients with previous poliomyelitis. 2However, these epidemiological reports remain controversial 3 and may relate to earlier confusion with the syndrome of late weakness and atrophy that occurs after poliomyelitis, 4 post-polio syndrome. Since it is now recognized that persistent poliovirus and other enterovirus infections occur in animals, immunodeficient humans, and cell culture systems, it remains important to examine viruses as causative agents of ALS. 1 Over the last 30 years, there have been many attempts to find viruses in ALS tissues. These studies have included antibody assays, standard viral isolation techniques, and electron microscopy for viral particles, and all have been uniformly negative. 5 …

Detection of enterovirus capsid protein VP1 in myocardium from cases of myocarditis or dilated cardiomyopathy by immunohistochemistry: further evidence of enterovirus persistence in myocytes.

The association of enteroviruses with myocardial disease has been investigated extensively by molecular biological techniques to detect viral RNA, but remains controversial. This retrospective study investigated the involvement of enterovirus in myocarditis or dilated cardiomyopathy (DCM) by detection of viral antigens in myocardial samples from a new patient series using an optimized immunohistochemical technique. Formalin-fixed, paraffin-embedded biopsy, autopsy or explanted myocardial tissue samples were obtained from 136 subjects. These comprised histologically proven cases of acute fatal myocarditis (n=10), DCM (n=89, including 10 patients with healing/borderline myocarditis) and a comparison group of samples from 37 unused donor hearts and cases with other conditions. A monoclonal antibody 5-D8/1 directed against a conserved, non-conformational epitope in capsid protein VP1 was employed for broad detection of different enterovirus serotypes. Investigations were performed blindly. Histological sections from 7 of 10 fatal myocarditis cases, 47 of 89 patients (52.8%) with DCM were positive for the viral capsid protein VP1 by immunohistochemical staining. Consecutive sections of positive samples were negative when the antibody was omitted or replaced with subclass- and concentration-matched normal mouse IgG. In contrast, only 3 of 37 samples (8.1%) in the comparison group were positive (Yates corrected chi(2)=19.99, P<0.001: odds ratio =12.68). VP1 staining was distributed in individual or grouped myofibers and localized in the cytoplasm of myocytes. In some cases, VP1 was detected in only a few myofibers within an entire section. These results provide further evidence of enterovirus involvement in a high proportion of DCM cases and demonstrate that VP1 is present in disease stages from acute myocarditis, healing myocarditis to end-stage DCM requiring cardiac transplantation, indicating translation of viral protein during persistent enterovirus infection.

No evidence for persistent enterovirus infection in cattle with end-stage idiopathic dilated cardiomyopathy

No evidence for persistent enterovirus infection in cattle with end-stage idiopathic dilated cardiomyopathy

Quantitative analysis of viral RNA kinetics in coxsackievirus B3-induced murine myocarditis: biphasic pattern of clearance following acute infection, with persistence of residual viral RNA throughout and beyond the inflammatory phase of disease.

Although the association remains controversial, enteroviruses have been implicated in the aetiology of several chronic diseases of humans. To further understand the mechanism of enterovirus persistence and its relationship to organ pathology, virus infectivity and viral RNA kinetics in the heart and other target organs during acute and persistent phases of murine coxsackievirus B3 infection were investigated. These studies revealed a biphasic pattern of virus clearance. Thus, there was a rapid but incomplete clearance of viral RNA from the myocardium following the acute phase of virus replication, which paralleled the elimination of virus infectivity. The mean half-life of viral RNA between days 5 and 14 post-infection (p.i.) was 13.4 h. In contrast, a much slower rate of decline in viral RNA levels was observed during the post-infectious inflammatory phase of myocarditis. The mean half-life of viral RNA between days 14 and 90 p.i. was 14.1 days. Viral RNA persisted in the myocardium beyond the resolution of inflammation and was still detectable in a proportion of animals 90 days after infection. Clearance of viral RNA from other target organs occurred more rapidly, but the rate of clearance was largely independent of the level of viral RNA present during the acute phase of infection. Thus, while antiviral immune responses effectively eliminated infectious virus, clearance of residual viral RNA from the myocardium and other target organs was significantly delayed, despite a prolonged inflammatory response. These findings suggest that clearance of persistent enterovirus infection requires mechanisms different from those responsible for the elimination of virus infectivity.

Enteroviral Capsid Protein VP1 Is Present in Myocardial Tissues From Some Patients With Myocarditis or Dilated Cardiomyopathy

There are still discrepancies in the association of enterovirus and myocardial disease, partially due to lack of data on the detection of virus antigens in tissues. It is desirable to localize enteroviral antigens so as to establish a link between the two and to study mechanisms of virus persistence. Nineteen fixed explanted or postmortem myocardial samples were obtained from patients with myocarditis or dilated cardiomyopathy (DCM). Control samples were collected from 11 subjects who had died accidentally or of noncardiovascular disease. Viral antigen was detected by an improved immunohistochemical technique using an enterovirus group-specific antibody to viral capsid protein VP1. Nine of 11 myocarditis cases (81.8%) and 6 of 8 DCM cases (75%) were positive. Signals were localized in the cytoplasm of myocytes. Intense immunostaining was observed in acute myocarditis, whereas VP1 was detected in scattered myocytes in chronic myocarditis or DCM. Enteroviral RNA was detected in 6 of 11 myocarditis samples (54.5%) and 3 of 8 DCM samples (37.5%) by the reverse transcription-nested polymerase chain reaction, correlating with antigen detection (kappa=0.6+/-0.21). Neither viral antigen nor RNA was detected in any controls. Our findings demonstrate a direct link between enterovirus infection and some myocarditis or DCM cases. The pattern of VP1 detection may correlate with disease stage and severity. The data suggest that viral protein synthesis may be involved in persistent enterovirus infection in the pathogenesis of DCM.

Detection and cellular localization of enterovirus RNA sequences in spinal cord of patients with ALS.

To investigate the possible association of persistent enterovirus (EV) infection with the development of ALS. Although ALS is a clinically well-defined motor neuron disease, little is known about the etiology and pathogenesis of the sporadic cases. Among the different causes that have been hypothesized, conflicting results have been reported about the possible role of persistent enteroviral infection. Reverse transcriptase-PCR (RT-PCR) and direct RT in situ PCR (RT-IS-PCR) were performed in formaldehyde-fixed spinal cord samples of 17 patients with confirmed ALS and 29 control subjects with no history of motor neuron disease. When obtained, PCR products were sequenced subsequently. Using direct RT-IS-PCR, EV nucleic acid sequences were detected in 15 (88.3%) of 17 patients with ALS compared to 1 (3.4%) of 29 control subjects. PCR products were located in neuronal cell bodies of the anterior horns of the spinal cord. The RT-PCR products obtained in 13 of the 17 patients with ALS showed between 94% and 86% homology with echovirus 7 sequences. The 88.3% rate of detection of enterovirus (EV) nucleic acids in the neuronal cell bodies within the gray matter of the spinal cord of patients with ALS strongly suggests association between persistent EV RNA and ALS. Further work is required to confirm that the persisting EV sequences we detected are somehow involved in the development of ALS.