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The aim of this study was to establish a reliable method of virus detection for the diagnosis of critical enterovirus infections such as acute infective encephalitis, encephalomyelitis and myocarditis. Because histopathological and immunohistochemical analyses of paraffin-embedded tissues play an important role in recognizing infectious agents in tissue samples, six in-house polyclonal antibodies raised against three representative enteroviruses using an indirect immunofluorescence assay and immunohistochemistry were examined. This panel of polyclonal antibodies recognized three serotypes of enterovirus. Two of the polyclonal antibodies were raised against denatured virus particles from enterovirus A71, one was raised against the recombinant VP1 protein of coxsackievirus B3, and the other for poliovirus type 1 were raised against denatured virus particles, the recombinant VP1 protein and peptide 2C. Western blot analysis revealed that each of these antibodies recognized the corresponding viral antigen and none cross-reacted with non-enteroviruses within the family Picornaviridae. However, all cross-reacted to some extent with the antigens derived from other serotypes of enterovirus. Indirect immunofluorescence assay and immunohistochemistry revealed that the virus capsid and non-structural proteins were localized in the cytoplasm of affected culture cells, and skeletal muscles and neurons in neonatal mice experimentally-infected with human enterovirus. The antibodies also recognized antigens derived from recent clinical isolates of enterovirus A71, coxsackievirus B3 and poliovirus. In addition, immunohistochemistry revealed that representative antibodies tested showed the same recognition pattern according to each serotype. Thus, the panel of in-house anti-enterovirus polyclonal antibodies described herein will be an important tool for the screening and pathological diagnosis for enterovirus infections, and may be useful for the classification of different enterovirus serotypes, including coxsackieviruses A and B, echoviruses, enterovirus A71 and poliovirus., (© 2014 Japanese Society of Neuropathology.)

Infectious mononucleosis (IM) is a clinical syndrome most often attributable to Epstein-Barr virus (EBV). Characteristic clinical features of EBV IM include bilateral upper lid edema, exudative or nonexudative pharyngitis, bilateral posterior cervical adenopathy, and splenomegaly ± maculopapular rash. Laboratory features of EBV IM include atypical lymphocytes and elevated levels of serum transaminases. Leukopenia and thrombocytopenia are not uncommon. The syndrome of IM may also be attributable to other infectious diseases, eg, cytomegalovirus (CMV), human herpes virus-6 (HHV-6), or Toxoplasma gondii. Less commonly, viral hepatitis, leptospirosis, brucellosis, or parvovirus B(19) may present as an IM-like infection. To the best of our knowledge, only 2 cases of IM-like infections attributable to Coxsackie B viruses (B(3) and B(4)) have been reported. We present the first reported case of an IM-like syndrome with sore throat, fatigue, atypical lymphocytes, and elevated levels of serum transaminases likely due to Coxsackie A in an immunocompetent adult., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Diagnosis of Coxsackievirus A16 (CA16) infection in China relies mainly on reverse transcription-polymerase chain reaction (RT-PCR) that require expensive equipment and special trained personnel, thus making its wide application in health care settings unlikely. In this study, a novel IgM anti-CA16 assay was developed for the detection of IgM antibodies to CA16 in serum. The responses and diagnostic value of IgM for the CA16 infection were assessed by testing 1970 serum samples. The results showed that sensitivity of IgM test was 84.6% (259/306, 95% CI: 80.1-88.5), and specificity in control subjects and patients with CA16 HFMD was 99.2% (1508/1520, 95% CI: 98.6-99.6) and 90.3% (14/144, 95% CI: 84.2-94.6), respectively. The IgM positive rate reached 56.3% in the sera collected within the first day after onset, increased continuously to 95.3% at day 5 to day 7 after onset, and then reached 100% after more than 8 days. The cross-reaction rate in patients infected with other non-CA16 enteroviruses was 9.7% (14/144). These results suggest that the IgM anti-CA16 assay offers a rapid, convenient, and reliable method to detect acute CA16 infections., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Viral myocarditis may have various clinical presentations, sometimes mimicking acute myocardial infarction or ischaemia. We describe the case of a young man presenting with acute heart failure, who had electrocardiographic changes suggesting myocardial ischaemia and an episode of sustained ventricular tachycardia. The diagnosis of acute myocarditis was confirmed using cardiac magnetic resonance imaging.

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.

The incidence of orofacial cleft (OC) in newborns was compared with the occurrence of virus-neutralizing antibodies to coxsackie viruses in the serum of newborns and their mothers. No significant difference was found when comparing the seropositivity rates between the group of patients and the control group of healthy newborns. If the patients were divided according to the place of residence however, marked differences occurred between the regions. The lowest incidence of both--coxsackie infection and OC was determined in the region of Bratislava and the highest in the region of Zilina. The explanation of these findings recquires a more detailed analysis of genetic background, social and hygienic status, style of life and other factors, known to influence the development of OC as multi-etiological developmental disorder. (Tab. 2, Fig. 4, Ref. 12.).

In 568 cases suffering from upper-respiratory tract infection with sore throat and more than one of following clinical manifestations: arrhythmia, heart failure and chest pain or oppressive sensation over the chest(Group A), and another 108 cases without above clinical manifestations(Group B), myocardial perfusion imaging with 99mTc-MIBI SPECT was studied. The results revealed that the imaging in 404 cases (71.12%) of Group A supported diagnosis of myocarditis, but only 6 cases(5.56%) did so in Group B. Because of lacking the clinical manifestations of myocarditis but positive finding of CVB-IgM antibody was detected in their sera, we considered that the positive SPECT imaging in these six cases of Group B belonged to myocardial reaction due to virus infection. It was possible that some of them were suffering from subclinical type of myocarditis.

Several lines of evidence have shown that viral infections are capable of causing coronary spasm and precipitating or mimicking clinical myocardial infarction. Here we report the case of a 41-year-old woman with recurrent angina who was admitted to our hospital because of ventricular tachycardia. Laboratory examination revealed positive IgM titers against Coxsackie B virus. Coronary angiography showed normal coronary arteries, but following a cold pressure test severe spasm of all coronaries with thrombotic occlusion of the second marginal branch of the circumflex artery occurred. We conclude that coronary spasm should be clinically suspected in patients with chest pain and ventricular arrhythmia in combination with IgM antibodies against Coxsackie B virus. In these patients, a cold pressure test should be avoided, and antithrombotic and antispastic therapy is recommended.

Detection of anti-Coxsackie virus antibodies in sera with suspect enterovirus infection by the Western blotting (WB) method revealed complete agreement of results with virus neutralization tests (VNT) in those patients where in the VNT no antibodies against any of the tested Coxsackie viruses were detected. Comparison of the results of WB and VNT in other patients did not rule out an inter-type cross-reaction. In paired specimens of sera and saliva comparable results were obtained by WB, VNT and the complement fixation method. The rapid and relatively simple character of the WB method makes this method suitable for more detailed elaboration and introduction into routine diagnosis of enterovirus infections.

The introduction of rapid serological diagnosis of enterovirus infections is faced with the problem of inter-type cross-reactions. Therefore the method of choice is still the virus-neutralization test on cell cultures and on suckling mice resp., i.e. methods which are expensive and time consuming and demanding on laboratory equipment. The detection of antibodies against the Coxsackie virus by means of Western blotting revealed a specific reaction of commercial hyperimmune diagnostic rabbit as well as of sera obtained after oral infection of mice. The application of the method in the diagnosis of human enterovirus infections will be the subject of the subsequent part of this study.

By using the indirect immunofluorescence assay and the tissue cells infected with coxackie virus Group B as antigen, the authors detected the antibodies type IgM to coxsackie virus Group B (CVB-IgM) of the sera of 105 children with viral myocarditis (VMC), 59 children with other diseases (COD) and 67 healthy children (HC). The results showed that in VMC, the genometric mean titre (GMT) of CVB-IgM in female (1:16.58) was higher than that in male (1:9.28, P < 0.05); not only in female, but also in male the GMT of VMC was higher than that of HC; also the GMT in female of VMC was higher than the GMT in female of COD, but no considerable difference existed between male of VMC and COD. Without sex distinction the GMT of HC was 0.289, its standard variance was 0.9335, the upper limit titre of normal range was 1:8.6. The authors took 1:10 as the positive criterion, the sensitivity of CVB-IgM in diagnosing VMC was 79.05%, the specialities of differentiating VMC from COD and HC were 42.3% and 91.04%, the consistancy rates of diagnosing VMC from COD and HC were 65.85% and 83.72% respectively.

An immunoglobulin M (IgM) capture enzyme immunoassay was used to detect major overlapping outbreaks of disease in South Australia caused by coxsackieviruses B5 (CBV-5) and B6 (CBV-6). CBV-5-specific IgM was detected in patients presenting in spring 1992 with acute febrile illnesses, rash, severe acute respiratory disease, meningitis, myocarditis and/or pericarditis, while tests for other viruses were negative. CBV-5 was isolated from an early case. In December 1992 it was noted that CBV-6 had replaced CBV-5 as the major cause of disease. The CBV-6 epidemic continued until April 1993. Serum samples from 495 patients (276 inpatients) were submitted for testing. CBV-6 infection was associated with lower respiratory tract infection and persistent cough. This study demonstrated success of the IgM enzyme immunoassay and the need for diagnostic virology laboratories to look for CBV-6 infection in addition to the other five CBVs.

Objectives: This study was conducted to determine the frequency and significance of Coxsackie B virus-specific immunoglobulin-M (IgM) in patients with idiopathic dilated cardiomyopathy and compare them with the frequency in both unmatched and matched control subjects., Background: The principal evidence supporting a pathoetiologic role for Coxsackie B viruses in human dilated cardiomyopathy is derived from retrospective serologic studies. These studies have evaluated patients with end-stage disease and have failed to recognize the importance of assessing both matched and unmatched control subjects., Methods: In this prospective case-control study, we assessed sera for Coxsackie B virus-specific IgM (serotypes B1 to B5) from 114 patients with dilated cardiomyopathy at diagnosis or referral to our center, 94 healthy unmatched control subjects, 41 healthy matched control subjects from the same general practitioner and 32 members of the patients' own households., Results: A higher frequency of positive Coxsackie B virus IgM was observed in patients with dilated cardiomyopathy than in unmatched control subjects (33% vs. 5%; p = 3 x 10(-7)). In patients with dilated cardiomyopathy, the response was monotypic (84%), commonly against serotypes B2 and B5, and was not associated with any clinical or histologic feature. The frequency of positive virus-specific IgM was similar in patients with dilated cardiomyopathy and their 41 matched community control subjects (46% vs. 27%; p = 0.11) and 32 household contacts (37% vs. 28%; p = 0.59). Control subjects who tested positive for virus-specific IgM tended more commonly to be seropositive than did control seronegative subjects (community control subjects 37% vs. 18%, p = 0.32; household contacts 42% vs. 20%; p = 0.36) and had an identical serotypic response in 4 (33%) of 12 cases., Conclusions: The frequency of Coxsackie B virus IgM was higher in patients with dilated cardiomyopathy than in unmatched control subjects but was similar in patients and control subjects who shared the same environment, indicating local spread of infection. The reason for the association between Coxsackie B virus IgM and dilated cardiomyopathy and its relevance to pathogenesis remain to be established.

We report the application of a modified Western blot (WB) micromethod basically relying on a diffusion-blotting technique (Modi-blot) combined with an immunological detection system using monoclonal antibodies and a biotinavidin amplification step for the detection of IgM antibodies against coxsackieviruses B1 (CBV1), CBV2 and CBV4. Fifty-one adult patients with clinical signs of coxsackievirus B infection (e.g. myocarditis and meningitis) were investigated. The test revealed a total of 31 (60%) IgM positives. The majority of IgM antibodies were group reactive (22/31). Type-specific antibodies could be recognized in 9 cases (3 with CBV1, 5 with CBV2 and 1 with CBV4). The highest rate of antibody prevalence was found in sera from patients with acute meningitis (12/14). Controls [healthy adults (n = 13) and individuals with other infections (n = 13)] were all negative for specific IgM against coxsackievirus B1, B2 and B4. Further WB tests of 8 IgM positive specimens with coxsackievirus B4 revealed specific IgA responses in all cases, reinforcing the evidence for a recent infection. In addition, the patterns of IgG antibody subclasses, also investigated in this group, showed a clear predominance of specific IgG1 and IgG3 antibodies.

Objective: To study the association between coxsackie B virus infection and the postviral fatigue syndrome and to assess the immunological abnormalities associated with the syndrome., Design: Case-control study of patients with the postviral fatigue syndrome referred by local general practitioners over one year., Setting: General practitioner referrals in Dunbartonshire, Scotland., Patients: 254 Patients referred with the postviral fatigue syndrome (exhaustion, myalgia, and other symptoms referable to postviral fatigue syndrome of fairly recent onset--that is, several months) and age and sex matched controls obtained from same general practitioner; 11 patients were rejected because of wrong diagnoses, resolution of symptoms, and refusal to participate, leaving 243 patients and matched controls., Main Outcome Measures: Detailed questionnaire (patients and controls) and clinical examination (patients) and blind analysis of blood sample at entry and after six months for determination of coxsackie B virus IgM and IgG antibodies and other variables (including lymphocyte protein synthesis, lymphocyte subsets, and immune complexes)., Results: Percentage positive rates for coxsackie B virus IgM at entry were 24.4% for patients and 22.6% for controls and for coxsackie B virus IgG 56.2% and 55.3% respectively; there were no significant differences between different categories of patients according to clinical likelihood of the syndrome nor any predictive value in a fourfold rise or fall in the coxsackie B virus IgG titre in patients between entry and review at six months. The rates of positive antibody test results in patients and controls showed a strong seasonal variation. Of the numerous immunological tests performed, only a few detected significant abnormalities; in particular the mean value for immune complex concentration was much higher in 35 patients and 35 controls compared with the normal range and mean value for total IgM was also raised in 227 patients and 35 controls compared with the normal range., Conclusions: Serological tests available for detecting coxsackie B virus antibodies do not help diagnose the postviral fatigue syndrome. Percentage positive rates of the antibodies in patients simply reflect the background in the population as probably do the raised concentrations of total IgM and immune complexes.

An antibody capture radioimmunoassay was established for the detection of coxsackievirus B4 and B5-specific IgM. A significant feature of the assay was the use of an unrefined coxsackievirus B (CBV) antigen. The antigen was prepared by freeze thawing, ultrasonication and low speed centrifugation of infected Vero cells with no purification or concentration of the antigen being performed. Results of sera tested were expressed as a serum ratio (SR) by comparison with a low positive control serum. To establish an SR indicating positivity in the assays, 100 antenatal sera collected in late February were tested. The mean SR was calculated and the mean plus three standard deviations was taken as the minimum SR indicating positivity. Although resulting in a relatively insensitive assay, such a value was required to exclude sera giving a low level of reactivity which may be due to residual enterovirus-specific IgM resulting from a remote infection.The homologous CBV-IgM assay was positive in four cases of CBV4 infection and six cases of CBV5 infection. For the CBV4 IgM assay, ten of 20 (50%) sera from infections with CBV other than CBV4 were positive and nine of the 13 (69%) sera from infections with echoviruses or coxsackieviruses A were positive. Five of 18 (27%) sera with an elevated CBV neutralization titre were positive in the CBV4-IgM assay. For the CB5-IgM assay seven of 18 (39%) sera from infections with CBV other than CBV5 were positive and nine of 13 (69%) sera from infections with echoviruses or coxsackieviruses A were positive. The nine sera that were positive from this group in the CBV5-IgM assay were the same nine as were positive in the CBV4-IgM assay. Two of the 18 (11%) sera with an elevated CBV neutralization titre were positive in the CBV5-IgM assay. These two sera were also positive in the CBV4-IgM assay and had an elevated monotypic CBV4 neutralization titre. None of the sera giving positive results gave significant reactivity when tested with control antigen. Twelve rheumatoid factor containing sera and 46 sera from other infections were negative in both assays. Of 24 sera from confirmed CBV infection, seven gave a positive monotypic CBV4 or 5-IgM response, ten were positive in both assays and seven were negative in both assays. The positive results seen with sera from cases of heterologous enterovirus infection may result from an anamnestic IgM response or, more likely, IgM directed against enterovirus cross-reacting antigens. The absence of homologous neutralizing antibody at a dilution of 1:20 in nine of 20 sera that gave a positive CBV-IgM result and the presence of positive results for CBV4 and 5-IgM in a 14 month old infant who had echovirus 7 infection indicates that the IgM need not be directed against neutralizing antigens.Thus the CBV4 and 5-IgM assays developed appeared to be specific for an enterovirus infection but because of the cross-reactivity were not type-specific or group-specific.

IgM antibody capture radioimmunoassays were developed to detect coxsackie virus B1-B5 specific IgM. Specific IgM was detected in sera from all patients with coxsackie B virus infections proved by isolation; however, sera from 13/32 patients with rising neutralising antibody titres were negative in the assay. Frequent heterotypic responses were seen among the positive sera. Thirty seven patients with other enterovirus infections were also studied, and sera from 15 of these patients reacted in the assay, showing that heterotypic coxsackie B IgM responses occur not only in coxsackie B virus infections but also in other enterovirus infections.

We describe a case of meningo-encephalitis caused by Coxsackievirus group B type 2. Diagnosis was suggested by isolation of virus from faeces and high titres of serum antibody in which Coxsackie virus B2 specific IgM antibody was detected. Evidence of intrathecal production of antibody confirmed the diagnosis.

Twenty pairs of acute and convalescent sera were tested for antibody to the coxsackie B viruses types 1-5 by neutralization and fluorescent-antibody techniques. Results by fluorescence were difficult to read and sensitivity was therefore reduced. Rises by neutralization were type specific in the majority of cases, while those by fluorescence were always against more than one viral type.

The role of coxsackie B viruses (CBV) in myo/pericarditis has been well documented; however, interpretation of static high neutralising antibody titres in individual patients has always been difficult. In introducing the mu-antibody capture ELISA test for the detection of CBV-specific IgM, we hoped to overcome this problem. A regimen for the routine serological diagnosis of CBV infections was introduced, using the CBV IgM ELISA as a screening test, followed by neutralisation tests (NT) to confirm the positive results. Seven hundred and sixty patients and 304 healthy adult controls were tested. The percentage CBV IgM positive in each of the clinical categories myo/pericarditis (33%) chest pain (22%), myalgic encephalomyelitis (31%), myalgia/Bornholm (19%) and controls (9%) was similar to those found in previous studies using NT alone. Cross-reactions with other enteroviruses, including hepatitis A (Enterovirus 72), were observed but did not prove to be a problem in the illness studied, since most involved adults. Both homotypic and heterotypic CBV IgM responses were found. Matching IgM and NT indicated a recent CBV infection. Positive IgM with negative NT titres suggested a recent infection with an enterovirus other than a CBV.

The clinical virological survey of 180 cardiological patients aged 16-60 years with clinically diagnosed infectious allergic myocarditis (100), rheumatism (38) and other cardiac diseases (42) was made. A group of 97 practically healthy adults aged 21-60 years was used for control. The survey revealed statistically significant differences in the frequency of occurrence of Coxsackie B virus in healthy persons and in cardiac patients. Thus the criteria of the diagnostic significance of the results of the serologic survey gave grounds for suspicion that diseases in 31.2% of the surveyed patients were connected with the presence of Coxsackie B virus infection. Significantly more frequently Coxsackie B virus infection (predominantly type 4 virus) was found to be associated with infectious allergic myocarditis (42%).

The ELISA technique was shown to be group-specific for the detection of IgM antibodies against coxsackie B viruses, and probably against a wider range of enteroviruses. No evidence was obtained that recent coxsackie B-virus infection predisposes to myocardial infarction.

A solid-phase enzyme immunoassay (EIA) with the capture antibody principle was developed and evaluated for detection of immunoglobulin M (IgM) specific for group B coxsackieviruses (CBV) in human serum. Of 19 patients who were culture positive for CBV type 4 infections, the EIA for CBV type 4 IgM was more sensitive (84%; 16/19 positive) than a standard microneutralization test (16%; 3/19 positive) and a microneutralization assay on the IgM fractions obtained by sucrose density gradient fractionation of human serum (68%; 13/19 positive). The attempt to produce a serotype-specific EIA was not successful, as IgM antibodies were detected to heterotypic CBV. The pooled CBV types 2 to 5 IgM EIA demonstrated an overall sensitivity of 88% (21/24 positive) in comparison to virus isolation for CBV types 4 and 5. The EIA was specific for IgM to CBV, with the exception of some cross-reactions with hepatitis A IgM-positive sera (15%; 3/20). In comparison to the microneutralization test, the EIA for pooled CBV IgM was more rapid, less cumbersome, and more sensitive. Commercially available reagents used in this study enable the CBV IgM EIA to be developed in diagnostic virology laboratories.

A counterimmunoelectrophoresis test was developed for immunoglobulin M (IgM) antibodies to group B coxsackievirus (CB) types 1 through 5. The IgM precipitin line could be identified and differentiated from the IgG line by treating sera with 2-mercaptoethanol. Antigen purity was demonstrated by single precipitin lines occurring only to the homologous antigen when tested with type-specific hyperimmune rabbit sera. Serum pairs from 19 of 22 patients with documented CB type 1,3,4, and 5 infections were positive for IgM antibody to the infecting serotype, whereas 2 of 7 pairs from CB type 2 patients were positive. Heterologous IgM antibodies were present in sera from 14 fo 29 CB patients. Of the 14 patients with heterologous IgM antibodies, 12 also had greater than or equal to 4-fold rises in whole serum neutralizing antibody to heterologous serotypes. Only three control sera from 72 patients with coxsackievirus group A, echovirus, or other viral infections had IgM antibody to CB serotypes.