Coxsackievirus Group B Research Articles

Coxsackievirus Group B Infections during Pregnancy: An Updated Literature Review.

Coxsackievirus group B (CVB), a member of the Picornaviridae family and enterovirus genus, poses risks during pregnancy due to its potential to cause severe fetal and neonatal infections. Transmission primarily occurs through fecal-oral routes, with infections peaking mostly in warmer months. Vertical transmission to the fetus can lead to conditions such as myocarditis, encephalitis, and systemic neonatal disease, presenting clinically as severe myocardial syndromes and neurological deficits. Diagnostic challenges include detecting asymptomatic maternal infections and conducting in utero assessments using advanced techniques like RT-PCR from amniotic fluid samples. Morbidity and mortality associated with congenital CVB infections are notable, linked to preterm delivery, fetal growth restriction, and potential long-term health impacts such as type 1 diabetes mellitus and structural cardiac anomalies. Current treatments are limited to supportive care, with emerging therapies showing promise but requiring further study for efficacy in utero. Preventive measures focus on infection control and hygiene to mitigate transmission risks, which are crucial especially during pregnancy. Future research should aim to fill knowledge gaps in epidemiology, improve diagnostic capabilities, and develop targeted interventions to enhance maternal and fetal outcomes.

Serological characterization of Coxsackievirus B in type 1 diabetes mellitus (T1DM) patients in Basrah city- Iraq

Background: Type 1 diabetes mellitus (T1DM) is a chronic autoimmune illness that causes severe insulin shortage and hyperglycemia. Pancreatic cells may be destroyed by enteroviral (EV) infection either directly or by inducing an inflammatory response in the pancreatic islets that brings autoreactive T lymphocytes to the site of inflammation. Aim of study: investigate whether coxsackievirus group B (CVB) strains are associated with the onset of islet autoimmunity and T1DM. Objective: Serum samples were collected from newly diagnosed patients of type 1 diabetes mellitus who were attending the endocrinology centers at Al-Mawani general hospital and Al-Fayhaa teaching hospital from October 2022 to May 2023. A total of 168 children of both sexes, comprising 86 (51.1%) patients as a patient group. Results and discussion: Showed that there were no significant differences (p≤ 0.871) between patients and control according to age range however, the results showed elevated IgM titers in patients (15.14±2.43) in comparison with the control (8.66±1.56) with highly significant differences (p< 0.000). The age group (11-17) had the highest percentage (57.58%) of positive CVB-IgM patients which was 19 children of the total 33 infected children, while other age groups had 7 patients each (21.21%). Conclusion: Females were significantly more susceptible to T1DM than males. The age group (11-17) significantly more susceptible to T1DM than other age groups. Serological methods were more favorable than molecular methods in diagnosing the Coxsackievirus B. Elevated titers of IgG or IgM in patients in comparison with the control.

Novel Antiviral Activity of Ethyl 3-Hydroxyhexanoate Against Coxsackievirus B Infection.

Coxsackievirus group B (CVB) is a member of the genus Enterovirus in the family Picornaviridae. CVB infection has been implicated as a major etiologic agent of viral myocarditis, dilated cardiomyopathy, meningitis, and pancreatitis among children and young adults. Until date, no antiviral agent has been licensed for the treatment of Coxsackievirus infection. In an effort to identify antiviral agents against diseases caused by the CVB, we found that ethyl 3-hydroxyhexanoate (EHX), a volatile compound present in fruits and food additives, is a potent antiviral compound. In this study, we demonstrated that EHX treatment significantly inhibits CVB replication both in vivo and in vitro. Furthermore, EHX possesses antiviral activity at 50% effective concentration (EC50) of 1.2 μM and 50% cytotoxicity (CC50) of 25.6 μM, yielding a selective index (SI) value as high as 20.8. Insights into the mechanism of antiviral activity of EHX showed that it acts at the step of viral RNA replication. Since EHX has received approval as food additives, treatment of CVB-related infections with EHX might be a safe therapeutic option and may be a promising strategy for the development of semi-synthetic antiviral drugs for viral diseases.

Development of Group B Coxsackievirus as an Oncolytic Virus: Opportunities and Challenges.

Oncolytic viruses have emerged as a promising strategy for cancer therapy due to their dual ability to selectively infect and lyse tumor cells and to induce systemic anti-tumor immunity. Among various candidate viruses, coxsackievirus group B (CVBs) have attracted increasing attention in recent years. CVBs are a group of small, non-enveloped, single-stranded, positive-sense RNA viruses, belonging to species human Enterovirus B in the genus Enterovirus of the family Picornaviridae. Preclinical studies have demonstrated potent anti-tumor activities for CVBs, particularly type 3, against multiple cancer types, including lung, breast, and colorectal cancer. Various approaches have been proposed or applied to enhance the safety and specificity of CVBs towards tumor cells and to further increase their anti-tumor efficacy. This review summarizes current knowledge and strategies for developing CVBs as oncolytic viruses for cancer virotherapy. The challenges arising from these studies and future prospects are also discussed in this review.

Inhibition of Type III Interferon Expression in Intestinal Epithelial Cells-A Strategy Used by Coxsackie B Virus to Evade the Host's Innate Immune Response at the Primary Site of Infection?

Increasing evidence highlights the importance of the antiviral activities of the type III interferons (IFNλs; IL-28A, IL-28B, IL29, and IFNλ4) in the intestine. However, many viruses have developed strategies to counteract these defense mechanisms by preventing the production of IFNs. Here we use infection models, a clinical virus isolate, and several molecular biology techniques to demonstrate that both type I and III IFNs induce an antiviral state and attenuate Coxsackievirus group B (CVB) replication in human intestinal epithelial cells (IECs). While treatment of IECs with a viral mimic (poly (I:C)) induced a robust expression of both type I and III IFNs, no such up-regulation was observed after CVB infection. The blunted IFN response was paralleled by a reduction in the abundance of proteins involved in the induction of interferon gene transcription, including TIR-domain-containing adapter-inducing interferon-β (TRIF), mitochondrial antiviral-signaling protein (MAVS), and the global protein translation initiator eukaryotic translation initiation factor 4G (eIF4G). Taken together, this study highlights a potent anti-Coxsackieviral effect of both type I and III IFNs in cells located at the primary site of infection. Furthermore, we show for the first time that the production of type I and III IFNs in IECs is blocked by CVBs. These findings suggest that CVBs evade the host immune response in order to successfully infect the intestine.

Identification of Immunogenic Epitopes That Permit the Detection of Antigen-Specific T Cell Responses in Multiple Serotypes of Group B Coxsackievirus Infections.

Coxsackievirus group B (CVB) contains six serotypes that can affect various organs. Some of these organ-specific diseases such as myocarditis and pancreatitis can be caused by more than one serotype. Thus, development of immunological tools common to multiple serotypes is desired. This is especially critical for analyzing antigen-specific T cell responses at a single cell level. To this end, we made efforts to identify the immunogenic epitopes of CVB3 leading us to localize three T cell epitopes within the viral protein 1 (VP1) namely, VP1 681–700, VP1 721–740 and VP1 771–790. First, we confirmed their immunogenicity in the immunization settings. Second, we sought to verify the ability of VP1 epitopes to bind major histocompatibility complex (MHC) class II (IAk) molecules. Third, we created MHC class II (IAk) dextramers and tetramers and ascertained the T cell responses to be antigen-specific. Fourth, we analyzed the T cell responses in animals infected with CVB3 and noted the magnitude of antigen-specific T cell responses occurring in the order of VP1 721–740 and VP1 681–700 followed by VP1 771–790 as verified by proliferation assay and IAk tetramer staining. All epitopes induced interferon (IFN)-γ as a major cytokine. Finally, we investigated whether the VP1 tools generated for CVB3 can also be used to verify T cell responses in infections caused by other serotypes. To this end, we established the CVB4 infection model in A/J mice and found that the CVB4 infection led to the induction of IFN-γ-producing T cell responses primarily for VP1 721–740 and VP1 681–700. Thus, the VP1-specific tools, particularly IAk tetramers can be used to monitor anti-viral T cell responses in multiple CVB serotypes.

Aberrant PD-1 ligand expression contributes to the myocardial inflammatory injury caused by Coxsackievirus B infection

Coxsackievirus group B (CVB) is considered as one of the most common pathogens of human viral myocarditis. CVB-induced myocarditis is mainly characterized by the persistence of the virus infection and immune-mediated inflammatory injury. Costimulatory signals are crucial for the activation of adaptive immunity. Our data reveal that the CVB type 3 (CVB3) infection altered the expression profile of costimulatory molecules in host cells. CVB3 infection caused the decrease of PD-1 ligand expression, partially due to the cleavage of AU-rich element binding protein AUF1 by the viral protease 3Cpro, leading to the exacerbated inflammatory injury of the myocardium. Moreover, systemic PD-L1 treatment, which augmented the apoptosis of proliferating lymphocytes, alleviated myocardial inflammatory injury. Our findings suggest that PD1-pathway can be a potential immunologic therapeutic target for CVB-induced myocarditis.

Coxsackievirus B1 infections are associated with the initiation of insulin-driven autoimmunity that progresses to type 1 diabetes.

Islet autoimmunity usually starts with the appearance of autoantibodies against either insulin (IAA) or GAD65 (GADA). This categorises children with preclinical type 1 diabetes into two immune phenotypes, which differ in their genetic background and may have different aetiology. The aim was to study whether Coxsackievirus group B (CVB) infections, which have been linked to the initiation of islet autoimmunity, are associated with either of these two phenotypes in children with HLA-conferred susceptibility to type 1 diabetes. All samples were from children in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study. Individuals are recruited to the DIPP study from the general population of new-born infants who carry defined HLA genotypes associated with susceptibility to type 1 diabetes. Our study cohort included 91 children who developed IAA and 78 children who developed GADA as their first appearing single autoantibody and remained persistently seropositive for islet autoantibodies, along with 181 and 151 individually matched autoantibody negative control children, respectively. Seroconversion to positivity for neutralising antibodies was detected as the surrogate marker of CVB infections in serial follow-up serum samples collected before and at the appearance of islet autoantibodies in each individual. CVB1 infections were associated with the appearance of IAA as the first autoantibody (OR 2.4 [95% CI 1.4, 4.2], corrected p = 0.018). CVB5 infection also tended to be associated with the appearance of IAA, however, this did not reach statistical significance (OR 2.3, [0.7, 7.5], p = 0.163); no other CVB types were associated with increased risk of IAA. Children who had signs of a CVB1 infection either alone or prior to infections by other CVBs were at the highest risk for developing IAA (OR 5.3 [95% CI 2.4, 11.7], p < 0.001). None of the CVBs were associated with the appearance of GADA. CVB1 infections may contribute to the initiation of islet autoimmunity being particularly important in the insulin-driven autoimmune process.

Genotype distribution of human enteroviruses isolated from swage in Shanghai during year 2013-2014

Objective: To explore the time and genotype distribution of human enterovirus (HEV) isolated from sewage in Shanghai in 2013-2014. Methods: One sewage sample each was collected from two local sewage plants located in Minhang District and Jiading District on the same day at the day 24-28 of every month from 2013 to 2014. Each sample weighed 1 L. The specimens were concentrated by anionic membrane absorption, eluted with beef extract solution, and then used to inoculate RD, HEp-2, and L20B cell lines. A total of 249 enterovirus strains were isolated from sewage samples during the study period, including 185 non-polio enterovirus (NPEV) and 64 poliovirus (PV) strains, which were identified as vaccine strains. RT-PCR and Sanger sequencing were performed to identify HEV genotypes. Homologous analysis of VP1 sequences was conducted using BioEdit (version 7.0.0). Phylogenetic analysis was performed using the neighbor-joining method based on the alignment of VP1 gene sequences using MEGA (version 4.0.2). Results: Among 185 NPEV strains, 178 strains were successfully sequenced and classified into 15 genotypes, including coxsackievirus group B (CVB) 2, 3, and 5; enteric cytopathic human orphan (ECHO) virus 1, 3, 6, 7, 11, 13, 19, 20, 24, 25, and 30; and coxsackievirus group A 4. CVB5 and ECHO6 genotypes accounted for 33.5% (56 strains) and 24.9% (43 strains) of NPEV isolates, respectively. During the study period, HEV isolates were mainly isolated in summer and autumn in Minhang District. ECHO6 strains were frequently isolated from June 2013 to July 2014. Thereafter, the number of ECHO6 strains gradually reduced in the second half of 2014. CVB5 strains demonstrated scattered distribution from 2013 to the first half of 2014 and gradually increased in the second half of 2014. The distribution of ECHO6 and CVB5 strains in Jiading District was similar to that in Minhang District. In 2013-2014, CVB5 strains comprised C6 and C8 subgenotypes, which belong to two transmission chains and show large differences compared with foreign strains isolated during the same period. ECHO6 strains comprised C6, C8, and D9 subgenotypes, which belong to three transmission chains. Moreover, ECHO6 subgenotype D9 was a dominant subgenotype in Shanghai, with broad geographical distribution both at home and abroad. Conclusion: Poliovirus was identified as a vaccine strain in environmental surveillance from June 2013 to April 2014 in Shanghai. Several transmission strains of ECHO6 and CVB5 were identified, which were the dominant serotypes.

Coxsackievirus B3 induces the formation of autophagosomes in cardiac fibroblasts both in vitro and in vivo

Coxsackievirus group B (CVB) is one of the common pathogens that cause myocarditis and cardiomyopathy. Evidence has shown that CVB replication in cardiomyocytes is responsible for the damage and loss of cardiac muscle and the dysfunction of the heart. However, it remains largely undefined how CVB would directly impact cardiac fibroblasts, the most abundant cells in human heart. In this study, cardiac fibroblasts were isolated from Balb/c mice and infected with CVB type 3 (CVB3). Increased double-membraned, autophagosome-like vesicles in the CVB3-infected cardiac fibroblasts were observed with electron microscope. Punctate distribution of LC3 and increased level of LC3-II were also detected in the infected cardiac fibroblasts. Furthermore, we observed that the expression of pro-inflammatory cytokines, IL-6 and TNF-α, was increased in the CVB3-infected cardiac fibroblasts, while suppressed autophagy by 3-MA and Atg7-siRNA inhibited cytokine expression. Consistent with the in vitro findings, increased formation of autophagosomes was observed in the cardiac fibroblasts of Balb/c mice infected with CVB3. In conclusion, our data demonstrated that cardiac fibroblasts respond to CVB3 infection with the formation of autophagosomes and the release of the pro-inflammatory cytokines. These results suggest that the autophagic response of cardiac fibroblasts may play a role in the pathogenesis of myocarditis caused by CVB3 infection.

Production of Cross‐Reactive Peptide Antibodies against Viral Capsid Proteins of Human Enterovirus B to Apply Diagnostic Reagent

The coxsackievirus group B (CVB) of the genus Enterovirus and the species human enterovirus B is a nonenveloped virus containing a single-stranded positive-sense RNA genome. Coxsackievirus has icosahedral symmetry and four capsid proteins, VP1, VP2, VP3, and VP4. Specific antibodies against each viral protein are prerequisites for various studies. In this study, we developed seven peptide-derived antibodies directed against coxsackievirus VP1 (NO1-NO5), VP2 (B3), and VP3 (GL3). We developed a type-specific antibody (NO1) and broadly cross-reactive antibodies (NO3 and NO5) to VP1. Anti-VP2 and anti-VP3 antibodies (B3 and GL3, respectively) are also cross-reactive to human enterovirus B such as CVB and echoviruses. Their sensitivities and reactivities are likely to be better than those of the commercial VP1 monoclonal antibody (MAb). The dot-blot analysis also showed that NO5 against VP1 is able to detect less than 1 microg [2x10(6) plaque-forming unit (pfu) of CVB3] of viruses, suggesting that it could be used to develop a diagnostic kit that can directly detect human enterovirus B. The antibodies produced here may allow us to undertake several studies, such as those involving viral trafficking, expression kinetics, and the roles of viral proteins in infection, and the development of diagnostic kits.

Human pancreatic islet endothelial cells express coxsackievirus and adenovirus receptor and are activated by coxsackie B virus infection

Enteroviruses, such as the coxsackievirus (CV) group, have been linked to the induction of inflammatory and autoimmune diseases. Virus tropism and tissue access are modulated by endothelial cells. To examine the susceptibility of microvascular endothelial cells (MECs) derived from pancreatic islets to infection with CV group B (CVB), purified cultured human islet MECs were infected with CVB-4 strain, and the immunological phenotype of the infected cells was analyzed. CVB-4 persistently infected the islet MECs, which expressed the CV receptors human coxsackievirus and adenovirus receptor (HCAR) and decay accelerating factor (DAF) and maintained EC characteristics, without overt cytopathic effects. CVB-4 infection transiently up-regulated expression of the adhesion molecules ICAM-1 and VCAM-1 and increased production of the proinflammatory cytokines IL-1beta and IL-6, and chemokines IL-8 and lymphotactin, as well as IFN-alpha. Mononuclear cell adhesion to CVB infected monolayers was increased, compared to uninfected monolayers. Moreover, infection up-regulated the viral receptors HCAR and DAF and coreceptor alpha(v)beta3 integrin on islet MECs, while down-regulating expression of HCAR on human aortic endothelial cells, indicating potential tissue-specific influence on the pathological outcome of infection. These results provide evidence that islet MECs are natural targets and reservoirs for persistent CVB infection resulting in acute endothelial cell activation by virus, which may contribute to selective recruitment of subsets of leukocytes during inflammatory immune responses, such as insulitis in type 1 diabetes.

The erythrocyte viral trap: Transgenic expression of viral receptor on erythrocytes attenuates coxsackievirus B infection

Viruses rely on attachment to specific cell surface receptors to infect host cells. Selective expression of viral receptors has the potential to attenuate infection of susceptible tissues by redirecting virus to cells that cannot support viral replication. We propose that erythrocytes are an ideal instrument for this strategy, because they are present in vast numbers, permeate every organ, and cannot serve as hosts for viral propagation. To test this hypothesis, we generated a transgenic mouse, termed globin transcription factor 1 (GATA1)-coxsackie and adenovirus receptor (CAR), that expressed the CAR on erythrocytes. Coxsackievirus group B (CVB) adhered to the surface of CAR-expressing erythrocytes and was rendered noninfectious. Upon infection with CVB, GATA1-CAR mice had diminished viremia and reduced viral replication in heart, brain, and liver. Furthermore, when faced with a CVB challenge that was lethal to WT littermates, the survival of GATA1-CAR mice was prolonged, and their ultimate mortality was reduced. The GATA1-CAR mouse model presented here demonstrates that erythrocyte expression of CAR limits CVB pathogenesis. Erythrocytes also may be coated with a variety of receptors by nontransgenic methods, making this a very flexible model for the treatment of infectious diseases in humans.

The Coxsackie-Adenovirus Receptor (CAR) Is Used by Reference Strains and Clinical Isolates Representing All Six Serotypes of Coxsackievirus Group B and by Swine Vesicular Disease Virus

Group B coxsackieviruses are etiologically linked to many human diseases, and cell surface receptors are postulated to play an important role in mediating their pathogenesis. The coxsackievirus adenovirus receptor (CAR) has been shown to function as a receptor for selected strains of coxsackievirus group B (CVB) serotypes 3, 4, and 5 and is postulated to serve as a receptor for all six serotypes. In this study, we demonstrate that CAR can serve as a receptor for laboratory reference strains and clinical isolates of all six CVB serotypes. Infection of CHO cells expressing human CAR results in a 1000-fold increase in CVB progeny virus titer compared to mock transfected cells. CAR was shown to be a functional receptor for swine vesicular disease virus (SVDV), as CHO-CAR cells but not CHO mock transfected controls were susceptible to SVDV infection, produced progeny SVDV, and developed cytopathic effects. Moreover, SVDV infection could be specifically blocked by monoclonal antibody to CAR (RmcB). SVDV infection of HeLa cells was also inhibited by an anti-CD55 MAb, suggesting that this virus, like some CVB, may interact with CD55 (decay accelerating factor) in addition to CAR. Finally, pretreatment of CVB or SVDV with soluble CAR effectively blocks virus infection of HeLa cell monolayers.

Expression of the Adenovirus Receptor and Its Interaction with the Fiber Knob

The coxsackievirus group B (CVB) and adenovirus (Ad) receptor (HCVADR, formerly HCAR) is a cell surface protein with two immunoglobulin-like regions (IG1 and IG2) that serves as a receptor for two structurally unrelated viruses. We have established the tissue distribution of the receptor in the rodent by immunohistochemistry and show that the receptor is broadly expressed during embryonic development in the central and peripheral nervous systems and in several types of epithelial cells. The tissue distribution is more restricted in the adult but remains high mainly in epithelial cells. Using site-directed mutagenesis, based on computer modeling of the IG1 region, Ad5 binding could be inhibited but CVB attachment was unaffected. A double amino acid substitution in a three-stranded anti-parallel β sheet that may form a face of the receptor completely inhibited Ad5 binding. Therefore, we conclude that the molecular interactions critical for Ad5 binding to HCVADR do not overlap with those of CVB3. In fact a specific antibody interfering with only CVB binding recognizes the IG2 domain in the receptor, suggesting that the CVB interacts with this region or an overlap between the IG1 and the IG2 regions.

Roles of the humoral response in coxsackievirus B-induced disease.

Antibodies to a coxsackie virus group B (CVB) may be regarded as a footprint record of trespass in an animal by one of these viruses that resulted in subsequent induction of a humoral immune response toward numerous immunogenic viral protein epitopes. These epitopes will be found in virion polypeptides, virion polypeptides in various stages of completion, nonstructural viral proteins required for replication and the latter proteins in stages of synthesis. Linear, conformational and combinatorial viral epitopes will stimulate production of anti-viral protein antibodies of several immunoglobulin classes and IgG subclasses. Natural challenges to humans by the CVB occur most frequently by ingestion from liquids or foods, contact with fingers or fomites, transplacental passage to the fetus and probably by aerosols during replication in the respiratory tract. The major outcomes of most CVB infections of humans are asymptomatic, but there are numerous transient infections that result in illnesses in the upper respiratory and gastrointestinal tracts and skin. Less frequently, CVB cause serious illnesses involving inflammatory responses in the heart, meninges, brain and pancreas that lead to chronic diseases, some of which terminate in death. To delineate parameters of infection involved in protection or in pathology, murine models of CVB-induced human diseases have been studied for nearly four decades. Reviews of studies on basic mechanisms involved in CVB-induced diabetes mellitus (Barrett-Conner 1985; Toniolo et al. 1988; D’alessio 1992) and in CVB3- and CVB2-induced myocarditis (Lerner and Wilson 1973; Woodruff 1980; Lerner 1985, Gauntt 1987; Gauntt et al. 1993a) show the value of these models in increasing our understanding of the contributions of innate resistance mechanisms and both humoral and cell-mediated immune systems to the resolution or continuation of disease.KeywordsHeart TissueViral MyocarditisCardiac MyosinCVB3 InfectionChronic MyocarditisThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Coxsackie virus infections in rheumatic fever.

The close relationship between Group A beta Hemolytic Streptococci (GABS) and rheumatic fever is a well established one. However, the concept of the streptococcus as the sole etiologic agent of the rheumatic heart disease (RHD) has been challenged over the past years. Since coxsackievirus group B (CVB) has long been proposed as a cause of acquired valvular disease simulating rheumatic fever, we attempted in this study to document infections with this group of viruses in patients with rheumatic fever. We obtained blood samples from 106 patients with old (quiescent) rheumatic fever/rheumatic heart disease [group I], 94 patients with acute rheumatic fever (ARF) [group II], and 74 normal matched controls. We tested for the presence of neutralizing antibodies to the 6 serotypes of CVB by a micro neutralization test. We have found that infection with CVB, especially types B2 and B4, was common in the studied population. Forty-two percent of normal individuals had evidence of infection with any of the 6 serotypes of CVB. Patients of group I had significantly more frequent infections with CVB 2. Patients in group II had significantly more frequent infections with CVB 2 and CVB 6. There was no clear correlation between such infections and the clinical course of rheumatic fever. There was no difference in the incidence of CVB infections between patients with definite ARF, and patients with suspected ARF. We set a low order association between rheumatic fever and infection with CVB types B2 and B6. We emphasize the importance of pursuing the investigation of the role of CVB in relation to RHD.

NON-POLIO ENTEROVIRUS ACTIVITY IN WISCONSIN BASED ON A 20-YEAR EXPERIENCE IN A DIAGNOSTIC VIROLOGY LABORATORY

Laboratory test results, demographic information, and clinical data on 1364 patients who had specimens submitted to the Wisconsin State Laboratory of Hygiene and from whom non-polio enteroviruses were isolated during 1957--1976 were examined. Echoviruses (echo) and Coxsackievirus group B (CB) and group A (CA) were isolated from 719, 389, and 256 of the patients, respectively. Thirty-five different serotypes were identified in the 20-year period. The six most frequently occurring serotypes (echo 9, CB 5, echo 18, CB 2, CB 4, and echo 6) were associated with 57 per cent of all illnesses. The first three serotypes occurred in an epidemic-like manner whereas the next three appeared to be endemic. Eighty-eight per cent of all isolations were from patients whose illnesses began in July through October. The percentage of CA isolates obtained from adults was much smaller than the corresponding percentages seen with CB or echo. A majority of the non-polio enteroviruses were obtained from males. Central nervous system and gastrointestinal tract involvement was greatest with echo and CB 5 tract and lowest with CA and CB 1--4 illnesses. The highest frequency of respiratory tract involvement was with CA and echo 9 illnesses and rash was most frequent with CA illnesses. The highest frequency of hospitalization was with CB 5 patients.