Molecular epidemiology of enteroviruses with special reference to their potential role in the etiology of insulin-dependent diabetes mellitus (IDDM). A review

Background: Several lines of evidence suggest that enterovirus infections may be involved in the etiology of the insulin-dependent diabetes mellitus (IDDM). Often in the literature, a reference is given to specifically diabetogenic strains of enterovirus but there is no systematic assessment about the generation of such strains in the course of evolution or about their abundance among the 64 enterovirus serotypes pathogenic to man. If enteroviruses truly are involved in the etiology of IDDM, a possibility to prevent the disease with enterovirus vaccines might become feasible. In such a situation it would be important to know which serotypes and strains are the most important ones, and whether there would be differences between the strains as regards the pathogenetic mechanisms involved.Objective: To present a brief summary of the basic biology of enteroviruses, on existing data of genetic variation of enteroviruses, and on molecular epidemiology of human enteroviruses with special reference to the different epidemiological modes of their putative involvement in the pathogenesis of IDDM.Conclusions: Like RNA viruses in general, enteroviruses exist as a quasispecies, a mixture of genetic microvariants with a vast potential to adapt to new environments. This means that specifically beta cell-tropic and potentially diabetogenic variants could, in theory, emerge sporadically during systemic infection of any individual. The patterns of genetic diversification of enteroviruses, cocirculation of separate genetic lineages in the human populations, and the assumed geographical restrictions of endemic transmission of the lineages, allow one to hypothesize that populations with a high persisting IDDM incidence might be endemically infected by some specific strains of enteroviruses. However, so far, there is no systematically collected data supporting this hypothesis.     

Statistical correlation between enterovirus genome copy numbers and infectious viral particles in wastewater samples

Aims: Classic virological tests are time consuming and labour‐intensive; real‐time RT‐PCR has proven to be a fast method to detect and quantify enterovirus genomes in clinical and environmental samples. This method is unable to discriminate between infective and noninfective enterovirus particles; few clinical studies have compared real‐time RT‐PCR and viral culture. We wondered if the enterovirus genome quantification could be correlated to the infectivity. Methods and Results: We used the statistical approach to verify our hypotheses to correlate data, obtained by the standard method (most probable number of cytopathic units—MPNCU) and molecular test (real‐time RT‐PCR), on wastewater treatment plant samples. Chi‐squared test was used, considering several cut‐off values (‘50’‐‘100’‐‘200’ genome copy numbers), to determine statistical significance in comparison of the two methods. Chi‐square value was not significant when cut‐off of 50 (P = 0·103) and 100 (P = 0·178) was assumed but was significant with cut‐off of 200 (P = 0·044). Conclusion: This limit, 200 genome copy, could be used as cut‐off value to indicate enterovirus survival in environmental monitoring. Significant and Impact of the Study: To introduce a fast procedure that is able to compensate for disadvantages of cell culture method for viral environmental analyses.     

Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR

We have developed a quantitative RT-PCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man technology with the ABI Prism 7700 real-time sequence detection system. We optimized a one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the 5' noncoding region of enteroviruses. For accurate quantification of the number of copies, a Mahoney type 1 poliovirus RNA standard was designed and produced using genetic engineering. This fragment, quantified using the Ribogreen method, was used in serial dilutions as an external standard. The method had a 7-log dynamic range (5 to 2 x 10(7)). PCR inhibitors were removed by extracting viral RNA (after virus concentration) using the RNeasy mini kit with added polyvinylpyrrolidone (PVP) and running the amplification reaction with a mixture containing PVP and T4 gene 32 protein. This real-time quantification of enterovirus RNA allows large numbers of samples to be screened. Its sensitivity, simplicity and reproducibility render it suitable as a screening method with which to characterize enteroviruses, the presence of infectious particles being subsequently confirmed by cell culture.     

Coxsackie B virus infection and β cell autoantibodies in newly diagnosed IDDM adult patients

Background: Environmental agents such as viruses have been identified as potentially important determinants of insulin-dependent diabetes mellitus (IDDM). Enterovirus infections, Coxsackievirus B especially, could be linked to the β cell damaging process and to the onset of clinical IDDM.Objectives: Enteroviral (EV) infection and β cell autoimmunity were studied in adult patients at the onset of IDDM.Study design: A total of 14 newly diagnosed-IDDM patients with ketosis or ketoacidosis were compared to, anteriorly diagnosed IDDM patients with metabolic decompensation, non-IDDM patients with metabolic decompensation and healthy adults. EV infection was studied by genomic RNA detection in whole blood using a RT-PCR assay. In order to assess the level of β cell autoantibodies at the time of the initial metabolic decompensation, serum specimens from IDDM patients were tested for GAD65 antibodies and islet cell antibodies (ICAs).Results: Coxsackie B3 or B4 virus genome was detected and genotyped in five of 14 (35.7%) newly diagnosed IDDM patients and in one of 12 (8%) patients in the course of IDDM. By contrast, none of the 12 non-IDDM patients and none of the 15 healthy adults was positive for enterovirus RNA detection in whole blood. Positive GAD65 antibodies and ICAs assays were not significantly correlated to a positive EV-RNA detection.Conclusion: The present study demonstrates that Coxsackie B virus RNA sequences can be detected in the peripheral blood from adult patients at the onset or in the course of IDDM and suggests that a Coxsackie B virus infection could initiate or accelerate β cell autoimmune damaging process.     

Development of Loop-Mediated Isothermal Amplification (LAMP) for Universal Detection of Enteroviruses

Enteroviruses are found in most environments and cause several diseases in humans. Loop-mediated isothermal amplification (LAMP) was adapted and evaluated for the rapid detection of enteroviruses. Based on the highly conserved 5′ untranslated region (5′-UTR) of the human enteroviruses (HEVs), particularly human enterovirus A (HEV-A) and HEV-B, a set of universal primers was designed. The LAMP amplification was carried out under isothermal conditions at 61 °C, depending on the template concentration results were obtained within 45–90 min. The detection limits were found to be 10¹ copies of cloned enterovirus 71 fragments, more sensitive than conventional PCR. Nine water samples collected from drinking water sources during three seasons and 19 stool specimens collected from HFMD patients were analyzed. By using the LAMP assay, the majority of samples was tested positive, 9/9 (100 %) and 18/19 (94.7 %), respectively. LAMP is a practical method for the rapid detection of enteroviruses in environmental and clinical samples.     

Demonstration of persistent enterovirus in the pancreas of diabetic mice by in situ polymerase chain reaction

Background: Although Enterovirus (EV) do not persist in the tissue, which is essential to maintain autoimmunity, they have been associated as the cause of chronic autoimmunity in some cases of insulin dependent diabetes mellitus (IDDM). Convincing reports, demonstrating persistent EV infections in the pancreases, are rare.Objectives: To determine the role of EV in IDDM, a mouse model was tested and in situ polymerase chain reaction (ISPCR) developed. The major problem of ISPCR are the high amounts of non-specific staining. In the current study we developed an ISPCR protocol which minimised non-specific staining and allowed the accurate localisation of the viral RNA in the tissue.Study design: Five mice were infected with coxsackievirus group B4, sacrificed 7 weeks later and the pancreases were harvested. The EV nucleic acid were localised and detected in the pancreases by ISPCR.Results: In the current study non-specific staining of ISPCR, due to DNA repair and diffuse artefacts, were minimised and the EV nucleic acids were localised in the β cells of the endocrine pancreases in all five diabetogenic mice.Conclusion: This study demonstrates an association of viral RNA with the development of diabetes in mice and the usefulness of ISPCR to determine the role of EV in IDDM.     

Clearance of human-pathogenic viruses from sludge: study of four stabilization processes by real-time reverse transcription-PCR and cell culture

Sludges derived from wastewater treatment are foul-smelling, biologically unstable substances. As well as containing numerous pathogenic microorganisms, they also consist of organic matter that can be used as agricultural fertilizer. Legislation nevertheless requires sludges to be virologically tested prior to spreading by the counting of infectious enterovirus particles. This method, based on culture of enterovirus on BGM cells, is lengthy and not very sensitive. The aim of this study was to propose an alternative method of genome quantification for all enteroviruses that is applicable to verifying the elimination of viruses in complex samples such as sludges. Our complete protocol was compared to the official method, consisting of enterovirus enumeration with the most probable number of cythopathic unit (MPNCU) assay through the study of four stabilization procedures: liming, composting, heat treatment, and mesophile anaerobic digestion. Enterovirus quantities at the start of the stabilization procedures were between 37 and 288 MPNCU/g on the one scale and between 4 and 5 log genome copies/g on the other. It was shown that all procedures except mesophile anaerobic digestion were highly effective in the elimination of enterovirus particles and genomes in wastewater sludges. Reduction of viruses by mesophile anaerobic digestion was by only 1 log (infectious particles and genomes). In conclusion, stabilization processes can indeed be checked by virological quality control of sludges with gene amplification. However, the infectivity of genomes needs to be confirmed with cell culture or a correlation model if the virological risk inherent in the agricultural use of such sludges is to be fully addressed.     

2009年云南省2株肠道病毒71型分离株全基因组序列遗传特性分析

对2009年云南省肠道病毒71型分离株KMM09和KM186-09进行全基因组序列测序,并与我国及其它国家流行的EV71基因型进行比较和进化分析。KMM09和KM186-09基因组长为7 409bp,编码2 193个氨基酸,VP1系统进化分析显示2009年云南分离株属于C4基因型的C4a亚型。在结构区,与其它基因型相比较,C基因型之间的核苷酸和氨基酸的同源性高于其它基因型;而在非结构区,C4与B基因型和CA16原型株G10同源性高于其它C基因亚型。通过RDP3重组软件和blast比对分析,发现EV71C4基因型与B3基因型,与CA16原型株G10的基因组在非结构区存在重组。EV71全基因组序列的比较和分析,对了解引起我国手足口病暴发或流行C4基因亚型EV71毒株的遗传特性具有重要意义。     

Sequencing of porcine enterovirus groups II and III reveals unique features of both virus groups

The molecular classification of the porcine enterovirus (PEV) groups II and III was investigated. The sequence of the almost complete PEV-8 (group II) genome reveals that this virus has unique L and 2A gene regions. A reclassification of this group into a new picornavirus genus is suggested. PEV group III viruses are typical enteroviruses. They differ from other enteroviruses by a prolonged stem-loop D of the 5'-cloverleaf structure.     

<Emphasis Type="Italic">hAT</Emphasis> transposable elements and their derivatives: an analysis in the 12 <Emphasis Type="Italic">Drosophila</Emphasis> genomes

Transposable elements (TEs) comprise a significant fraction of the genome, and some models of the TE “life cycle” suggest that, in the last phases of the cycle, TEs should be represented, in the genomes, by inactive and degenerated copies. In this study, we analyzed, using a bioinformatics approach, the autonomous hAT elements and their derivatives (active non-autonomous, MITE relatives and degenerated copies) in 12 Drosophila genomes. We found 28 hAT elements that had derivatives. Most copies had features that suggested that they were active, while only a few degenerated copies were found. Because hAT elements comprise an evolutionarily old superfamily, one should expect to find many degenerated copies within the genome, although this was not observed in our study. These results suggest that primarily active copies of hAT elements are maintained in the euchromatic regions of the genome and that degenerated copies are removed from the genome by natural selection.     

Evaluation of a new assay for HBV DNA quantitation in patients with chronic hepatitis B

Background: The Amplicor™ HBV Monitor Test for quantitative determination of serum hepatitis B virus (HBV) DNA has recently been introduced. This assay is based on PCR and a non-radioactive hybridization and detection system on microwell plates.Objective: The performance of the Amplicor™ HBV Monitor Test was evaluated in a routine diagnostic laboratory. The Amplicor™ HBV Monoitor assay was compared to the Digene Hybrid Capture™ System HBV DNA assay for the quantitation of HBV in patient sera.Study design: Sensitivity and reproducibility were determined with 10-fold dilution series of two Eurohep HBV reference plasma specimens. Furthermore, 196 sera from 14 children with chronic HBV infection and interferon therapy were tested with both assays.Results: The detection limit was found to be 10³ copies/ml with the Amplicor™ PCR assay compared to 10⁶ to 10⁷ copies/ml with the Digene™ hybridization assay. Both assays were quasi-linear over the measurable ranges. The new PCR assay proved to be very reliable. With the Amplicor™ PCR assay, 26.2% of the HBV DNA-positive clinical samples were found between 10³ and 10⁷ copies/ml and all of them tested below the detection limit with the hybridization assay.Conclusion: The Amplicor™ HBV Monitor Test shows excellent sensitivity and provides a valuable tool for the detection of HBV DNA in serum. It can be used for recognizing those patients who might benefit from antiviral therapy, for evaluation of the efficacy of anti-HBV therapy, and for validation of blood products.     

Evidence of recombination among enteroviruses.

Human enteroviruses consist of more than 60 serotypes, reflecting a wide range of evolutionary divergence. They have been genetically classified into four clusters on the basis of sequence homology in the coding region of the single-stranded RNA genome. To explore further the genetic relationships between human enteroviruses and to characterize the evolutionary mechanisms responsible for variation, previously sequenced genomes were subjected to detailed comparison. Bootstrap and genetic similarity analyses were used to systematically scan the alignments of complete genomic sequences. Bootstrap analysis provided evidence from an early recombination event at the junction of the 5' noncoding and coding regions of the progenitors of the current clusters. Analysis within the genetic clusters indicated that enterovirus prototype strains include intraspecies recombinants. Recombination breakpoints were detected in all genomic regions except the capsid protein coding region. Our results suggest that recombination is a significant and relatively frequent mechanism in the evolution of enterovirus genomes.     

Molecular identification and characterization of a new type of bovine enterovirus

Bovine enteroviruses belong to the family Picornaviridae. Little is known about their pathogenic potential; however, they cause asymptomatic infections in cattle and are excreted in feces. In the present study, viruses isolated from environmental samples were sequenced. According to phylogenetic analyses and standard picornavirus nomenclature, these isolates constitute a new type of bovine enterovirus serogroup A.     

Clinical severity of pediatric respiratory illness with enterovirus D68 compared with rhinovirus or other enterovirus genotypes

Background:

Enterovirus D68 (EV-D68) resulted in a reported increase in the number of children needing hospital or critical care admission because of respiratory insufficiency during 2014. It remains unclear, however, whether EV-D68 infections were more severe than rhinovirus or non–EV-D68 enterovirus infections.

Methods:

We evaluated consecutive children presenting to a pediatric hospital between Aug. 1 and Oct. 31, 2014, with positive nasopharyngeal swabs for rhinovirus or enterovirus that were sent automatically for EV-D68 testing. We compared characteristics and outcomes of patients with EV-D68 with those with rhinovirus or non–EV-D68 enterovirus in a matched cohort study.

Results:

A total of 93/297 (31.3%) of rhinovirus or enterovirus samples tested positive for EV-D68, and it was possible to compare 87 matched pairs. Children with EV-D68 infection were more likely to have difficulty breathing (odds ratio [OR] 3.00, 95% confidence interval [CI] 1.47–6.14). There was no significant difference in admission to the critical care unit or death among children with EV-D68 infection compared with those with other rhinovirus or enterovirus infections (adjusted OR 1.47, 95% CI 0.61–3.52). Children with EV-D68 infection were more often admitted to hospital, but not significantly so (adjusted OR 2.29, 95% CI 0.96–5.46).

Interpretation:

Enterovirus D68 seems to be a more virulent pulmonary pathogen than rhinovirus or non–EV-D68 enterovirus, but we did not find a significant difference in death or need for critical care.Rhinoviruses and enteroviruses are closely related, small RNA viruses in the genus Enterovirus. Rhinovirus species are the leading cause of upper respiratory tract disease, whereas enterovirus species generally infect the gastrointestinal tract and may then disseminate systemically. In August of 2014, the first reports of severe respiratory illness caused by enterovirus D68 (EV-D68), described in the media as a “new” pathogen, came from the American Midwest.¹ However, it should be emphasized that EV-D68 has been circulating for decades. It was first detected in 1962 in California,² and has since been detected in several countries, including the Philippines, Japan and the Netherlands, as well as in previous clusters in the United States.^3–5 However, by the end of 2014, a total of more than 1000 cases from 49 states in the US were reported.⁶ Almost 40% of samples submitted to the Centers for Disease Control and Prevention (CDC) for suspected EV-D68 were positive for EV-D68,⁶ and the genotype spread further north into Canada.⁷Subsequent to media reports, numerous clinicians perceived an “increase” in the number of children who required hospital or intensive care admission for respiratory disease, leading to the hypothesis that EV-D68 was more pathogenic than other respiratory viral pathogens. However, this has not yet been determined. The CDC documented a total of 12 deaths under investigation in 1152 confirmed cases for an estimated mortality of 1%,⁶ which does not appear to greatly exceed expected mortality associated with rhinovirus or non–EV-D68 enterovirus infection.⁸ Furthermore, the CDC estimates may have been artificially elevated by spectrum bias, as patients with severe illness would be more likely to have specimens forwarded to reference laboratories for EV-D68 testing.The goal of this study was to compare the severity of illness in children with EV-D68 infection with that of children with rhinovirus or non–EV-D68 enterovirus infection.