EV-D68 Infection Research Articles

Enterovirus D68 Causing Acute Respiratory Infection: Clinical Characteristics and Differences With Acute Respiratory Infections Associated With Enterovirus Non-D68.

Enterovirus (EV) D68 is mainly associated with acute respiratory infection (ARI). Since 2014, when outbreaks in different countries were observed, this emerging virus was considered a potential threat to public health. During 2015-2017, the presence of enterovirus RNA was investigated in all respiratory samples of children younger than 15 years of age with ARI, obtained for virologic studies in the Pediatric Emergency Care Units and wards of 2 hospitals in Gipuzkoa (Spain), using a commercial multiplex real-time polymerase chain reaction. When enterovirus was detected, a polymerase chain reaction to amplify a specific viral polyprotein (VP1) gene region of EV-D68 was performed. In 2016, EV-D68 circulation was associated to ARI, with the highest incidence in the spring months. EV-D68 was detected in 44 children, mean age 30.1 ± 31.7 months old, 23 (52.3%) of them females and 17 (38.6%) with underlying respiratory medical conditions. Thirty-two patients (72%) required hospital admission, receiving the discharge diagnosis of recurrent wheezing (37.5%), asthmatic crisis (37.5%) or bronchiolitis (12.5%). Seven children (15.9%) needed the support of the pediatric intensive care unit. When coinfections were excluded, children with EV-D68 infection presented with increased work of breathing, recurrent wheezing or asthmatic crisis, more frequently than those with ARI associated with EV non-D68. Moreover, clinical outcomes (hospitalization, respiratory support) were more severe. All 44 EV-D68 strains detected belonged to lineage B3. EV-D68 circulated widely in Gipuzkoa during 2016 and was associated with severe ARI. In children with severe ARI of unknown etiology, the presence of EV-D68 should be considered.

Correction: A Novel Neutralizing Antibody Specific to the DE Loop of VP1 Can Inhibit EV-D68 Infection in Mice

Correction: A Novel Neutralizing Antibody Specific to the DE Loop of VP1 Can Inhibit EV-D68 Infection in Mice

Serial MRI findings of acute flaccid myelitis during an outbreak of enterovirus D68 infection in Japan

ObjeciveTo clarify the neuroimaging findings of children with acute flaccid myelitis during an outbreak of EV-D68 infection. MethodsWe performed a detailed review of the spinal and cranial MRI results of 54 children with acute flaccid myelitis. We focused on the range of longitudinal lesions, the localization and appearance of lesions within a horizontal section, Gadolinium-enhancement, and changes over time. ResultsAll children had longitudinal spinal lesions involving central gray matter. Twenty-six children had lesions spanning the entire spine. Six of them had weakness in all limbs, whereas seven had weakness of only one limb. Thirty-eight children had lesions in both gray and white matter and limb weakness tended to be more severe in these children. During the acute period, spinal lesions showed bilateral ill-defined widespread T2 hyperintensity. During the subacute period, lesions were well defined and confined to the anterior horn. The distribution of limb weakness was correlated with the appearance of lesions during the subacute period. Gadolinium enhancement was performed in 37 children, and enhancement was seen in the cauda equina in 29 children. Enhancement was infrequent within 2 days after onset but was seen in almost all children thereafter. Twenty-two children had brainstem lesions continuous with spinal lesions. ConclusionExtensive longitudinal spinal lesions were characteristic in children with acute flaccid myelitis. Lesions were usually bilateral and widespread during the acute period, whereas localization to the anterior horn could become obvious. Although enhancement of the cauda equina was often observed, its appearance was sometimes delayed.

Neurological complications of infections with (novel) enteroviruses

Enterovirus (EV) infections are frequent andpredominantlyaffect children. Neurological manifestations are rare but can induce severe consequences. Since 2014 aglobal rise in EV-D68 and EV-71 infections manifesting with neurological complications, namely rhombencephalitis (EV-71) and acute flaccid myelitis (EV-D68), has been observed with an increased distribution in Europe and isolated occurrences in Germany. Typicallyprodromes withrespiratory infections and gastrointestinal conditionsprecede neurological symptoms. In these cases, an EV infection should be considered as the underlying cause and appropriate diagnostic tests should be performed. Other than the typical inflammatory signs in cerebrospinal fluid (CSF), the changes on magnetic resonance imaging (MRI) in particular are seminal; however, confirmation of the virus is often not possible in the CSF so that other specimens from, e.g. feces or the respiratory tract are required. To date there are no causal therapies for either of these EV infections; therefore, acomprehensive supportive therapy is of major significance. In Taiwan some beneficial effects could be observed by administration of intravenous immunoglobulins; however, areliable study has not yet been published.

A cross-sectional seroepidemiology study of EV-D68 in China

Enterovirus 68 (EV-D68) is associated with respiratory diseases, such as acute upper respiratory tract infections (URTIs), lower respiratory tract infections (LRTIs), pneumonia, neurological diseases, and acute flaccid myelitis (AFM). In recent years, there have been global outbreaks of EV-D68 epidemics. However, there is no effective vaccine against EV-D68, and the understanding of the seroprevalence characteristics of EV-D68 is limited. To evaluate the epidemiological features of this emerging infection in mainland China, serum samples from 20 pairs of pregnant women and their neonates, 405 infants and children (ages 1 month–15 years), and 50 adults were collected to measure EV-D68 neutralizing antibodies (NtAbs). The results showed that the geometric mean titers (GMTs) of pregnant women and their neonates were 168 (95%CI: 93.6–301.7) and 162.3 (95%CI: 89.9–293.1), respectively. The seroprevalence rate of EV-D68 antibodies was negatively correlated with age in 1-month-old to 12-month-old infants (84% for 1-month-old infants vs 10% for 1-year-old infants), whereas it was positively correlated with age for 1-year-old to 15-year-old children (10% for 1-year-old children vs 92% for 15-year-old children). This study evaluated maternal antibodies against EV-D68 in neonates. Our results showed that if mothers had high levels of anti-EV-D68 NtAbs, the NtAbs titers in their neonates were also high. The GMTs and seroprevalence rates of each age group indicated that EV-D68 infection was very common in China. Periodical EV-D68 seroprevalence surveys and vaccination campaigns should be the top priority for preventing EV-D68 infection.

Concurrent Community Transmission of Enterovirus D68 With Human Rhinoviruses and Respiratory Syncytial Virus Among Children in Sendai, Japan.

In the autumn of 2015, we experienced a surge in the number of pediatric cases of wheeze in our hospital, which was suspected to be caused by enterovirus (EV)-D68 transmission in the community. Thus, we implemented an ad hoc retrospective surveillance for EV-D68. Patients <15 years of age with acute respiratory infection were eligible for inclusion in this study. All enrolled patients underwent virus detection test. Additionally, neutralization tests (NTs) were performed using the stored serum samples of the enrolled patients to compare the antigenicity of the virus isolated in this study with that isolated in 2010 and evaluate the anti-EV-D68 antibody prevalence. Respiratory syncytial virus (RSV) was the most commonly detected virus (35%), followed by EV-D68 (19%) and non-EV-D68 enteroviruses/human rhinoviruses (14%). Patients with EV-D68 infection had higher median age than those with RSV infection (P < 0.05). Moreover, patients with EV-D68 infection showed a higher expiratory wheeze prevalence than those with non-EV-D68 enterovirus/rhinovirus and RSV infections. The antigenicity of the isolate from the current study was similar to the virus that circulated in 2010. At the early study phase, children in our community did not have high NT titers, but the median log NT titer increased from 1.5 to 5 over time (P < 0.05). This study showed the concurrent circulation of EV-D68 with non-EV-D68 enteroviruses/rhinoviruses and RSV in infants and children in our community and captured the early stage of EV-D68 transmission.

Monitoring human enteric viruses in wastewater and relevance to infections encountered in the clinical setting: a one-year experiment in central France, 2014 to 2015.

BackgroundHuman enteric viruses are resistant in the environment and transmitted via the faecal-oral route. Viral shedding in wastewater gives the opportunity to track emerging pathogens and study the epidemiology of enteric infectious diseases in the community. Aim: The aim of this study was to monitor the circulation of enteric viruses in the population of the Clermont-Ferrand area (France) by analysis of urban wastewaters. Methods: Raw and treated wastewaters were collected between October 2014 and October 2015 and concentrated by a two-step protocol using tangential flow ultrafiltration and polyethylene glycol precipitation. Processed samples were analysed for molecular detection of adenovirus, norovirus, rotavirus, parechovirus, enterovirus (EV), hepatitis A (HAV) and E (HEV) viruses. Results: All wastewater samples (n = 54) contained viruses. On average, six and four virus species were detected in, respectively, raw and treated wastewater samples. EV-positive samples were tested for EV-D68 to assess its circulation in the community. EV-D68 was detected in seven of 27 raw samples. We collected data from clinical cases of EV-D68 (n = 17), HAV (n = 4) and HEV infection (n = 16) and compared wastewater-derived sequences with clinical sequences. We showed the silent circulation of EV-D68 in September 2015, the wide circulation of HAV despite few notifications of acute disease and the presence in wastewater of the major HEV subtypes involved in clinical local cases. Conclusion: The environmental surveillance overcomes the sampling bias intrinsic to the study of infections associated with hospitalisation and allows the detection in real time of viral sequences genetically close to those reported in clinical specimens.

Crystal Structure and Thermostability Characterization of Enterovirus D68 3Dpol.

Enterovirus D68 (EV-D68) is one of the many nonpolio enteroviruses that cause mild to severe respiratory illness. The nonstructural protein 3Dpol is an RNA-dependent RNA polymerase (RdRP) of EV-D68 which plays a critical role in the replication of the viral genome and represents a promising drug target. Here, we report the first three-dimensional crystal structure of the RdRP from EV-D68 in complex with the substrate GTP to 2.3-Å resolution. The RdRP structure is similar to structures of other viral RdRPs, where the three domains, termed the palm, fingers, and thumb, form a structure resembling a cupped right hand. Particularly, an N-terminal fragment (Gly1 to Phe30) bridges the fingers and the thumb domains, which accounts for the enhanced stability of the full-length enzyme over the truncation mutant, as assessed by our thermal shift assays and the dynamic light scattering studies. Additionally, the GTP molecule bound proximal to the active site interacts with both the palm and fingers domains to stabilize the core structure of 3Dpol Interestingly, using limited proteolysis assays, we found that different nucleoside triphosphates (NTPs) stabilize the polymerase structure by various degrees, with GTP and CTP being the most and least stabilizing nucleosides, respectively. Lastly, we derived a model of the core structure of 3Dpol stabilized by GTP, according to our proteolytic studies. The biochemical and biophysical characterizations conducted in this study help us to understand the stability of EV-D68-3Dpol, which may extend to other RdRPs as well.IMPORTANCE Enterovirus D68 (EV-D68) is an emerging viral pathogen, which caused sporadic infections around the world. In recent years, epidemiology studies have reported an increasing number of patients with respiratory diseases globally due to the EV-D68 infection. Moreover, the infection has been associated with acute flaccid paralysis and cranial nerve dysfunction in children. However, there are no vaccines and antiviral treatments specifically targeting the virus to date. In this study, we solved the crystal structure of the RNA-dependent RNA polymerase of EV-D68 and carried out systematic biophysical and biochemical characterizations on the overall and local structural stability of the wild-type (WT) enzyme and several variants, which yields a clear view on the structure-activity relationship of the EV-D68 RNA polymerase.

Nasal Infection of Enterovirus D68 Leading to Lower Respiratory Tract Pathogenesis in Ferrets (Mustela putorius furo).

Data from EV-D68-infected patients demonstrate that pathological changes in the lower respiratory tract are principally characterized by severe respiratory illness in children and acute flaccid myelitis. However, lack of a suitable animal model for EV-D68 infection has limited the study on the pathogenesis of this critical pathogen, and the development of a vaccine. Ferrets have been widely used to evaluate respiratory virus infections. In the current study, we used EV-D68-infected ferrets as a potential animal to identify impersonal indices, involving clinical features and histopathological changes in the upper and lower respiratory tract (URT and LRT). The research results demonstrate that the EV-D68 virus leads to minimal clinical symptoms in ferrets. According to the viral load detection in the feces, nasal, and respiratory tracts, the infection and shedding of EV-D68 in the ferret model was confirmed, and these results were supported by the EV-D68 VP1 immunofluorescence confocal imaging with α2,6-linked sialic acid (SA) in lung tissues. Furthermore, we detected the inflammatory cytokine/chemokine expression level, which implied high expression levels of interleukin (IL)-1a, IL-8, IL-5, IL-12, IL-13, and IL-17a in the lungs. These data indicate that systemic observation of responses following infection with EV-D68 in ferrets could be used as a model for EV-D68 infection and pathogenesis.

Seroepidemiology of enterovirus D68 infection in China

Human enterovirus 68 (EV-D68) is a rarely reported virus that has been linked to respiratory disease. In recent years, reports about EV-D68 infection have markedly increased worldwide. However, the epidemiological features of this emerging infection are not well understood. To evaluate the emerging EV-D68 epidemic, we isolated the circulating viral strain and investigated the seroprevalence of neutralizing antibodies (NAbs) in Beijing between 2004 and 2011. We found that the titers of EV-D68 NAbs were generally low in all age groups in sampled populations in 2004 but significantly higher in 2009. From 2007 to 2011, the NAbs against EV-D68 significantly increased over time. These findings indicate that EV-D68 has spread widely in the Chinese population in recent years, although only a limited number of cases were reported.Emerging Microbes & Infections (2017) 6, e32; doi:10.1038/emi.2017.14; published online 10 May 2017

Enterovirus (EV)-D68 in Pediatric Patients with Respiratory Tract Infection: the Circulation of new B3 Clade in Italy

BackgroundEV-D68 is an emerging infectious agent that has been found associated with both mild and severe respiratory diseases and neurological clinical manifestations. Four clades with a number of subclades that can circulate or co-circulate during different periods have been identified. However, molecular evolution of EV-D68 is not known as it is the possible association between specific genetic variants and the development of severe cases. To solve these problems, genetics of strains identified during an outbreak of EV-D68 infection that occurred in Italy during the period March-October 2016 were studied.MethodsNasopharyngeal samples obtained from children admitted to the Emergency Room for respiratory infection were tested with a previously validated specific real-time PCR for detection and quantification of EV-D68. Phylogenetic analysis (PhA) of the virus was performed sequencing the major capsid protein (VP1). Moreover, tests for detection of selective pressure were carried out.ResultsRespiratory samples of 390 children were tested. Twenty-two patients (5.6%; median age, 47 months) were infected by EV-D68. All but three had a lower respiratory tract infection but none of these was severe, although in a few cases a transient reduction of SaO2 level was evidenced at admission. All the strains belonged to the EV-D68 subclade B3. No evidence of increased clinical severity associated with specific molecular signatures of VP1 sequence or viral load was shown. B3 strains had 92.3% and 94.7% nucleotide identity with B3 clades identified in other countries where a number of very severe cases was diagnosed. No sign of selective pressure was found.ConclusionEV-D68 subclade B3 was the only cause of the EV-D68 diseases diagnosed in Italy during 2016.The same subclade was found in Northern Europe, China and the USA in the same period. This suggests that all the outbreaks had a common origin and EV-D68 B3 has become the preeminent EV-D68 strain causing disease worldwide. No specific characteristic of EV-D68 VP1 has been found associated with disease characteristics, in contrast with what has been evidenced with other studies. Further studies on full-genome sequencing in larger cohorts of patients are needed.Disclosures All authors: No reported disclosures.

EV-D68 infection in children with asthma exacerbation and pneumonia in Mexico City during 2014 autumn.

BackgroundHuman enterovirus D68 (EV‐D68) recently caused an increase in mild‐to‐severe pediatric respiratory cases in North America and some European countries. Even though few of these children presented with acute paralytic disease, direct causal relationship cannot yet be assumed.ObjectivesThe purposes of this report were to describe the clinical findings of an outbreak of EV‐D68 infection in Mexico City and identify the genetic relationship with previously reported strains.Patients/MethodsBetween September and December 2014, 126 nasopharyngeal samples (NPS) of hospitalized children <15 years of age with ARI were tested for the presence of respiratory viruses using a multiplex RT‐qPCR and EV‐D68‐specific RT‐qPCR. Clinical, epidemiological, and demographic data were collected and associated with symptomatology and viral infections. Phylogenetic analyses were performed using VP1 region.ResultsEnterovirus/rhinovirus infection was detected in 40 patients (31·7%), of which 24 patients were EV‐D68‐positive. EV‐D68 infection prevailed over September and October 2014 and was associated with neutrophilia and lymphopenia, and patients were more likely to develop hypoxemia. Phylogenetic analyses showed that Mexican EV‐D68 belongs to the new B1 clade.ConclusionsThis is the first EV‐D68 outbreak described in Mexico and occurred few weeks after the United States reported similar infections. Although EV‐D68 belongs to new B1 clade, no neurological affection was observed.

Enterovirus 68 Infection–Association with Asthma

A previously sporadic virus called enterovirus 68 (EV-D68) appears to have been associated with asthma-like illness with a predisposition for asthmatics after an outbreak that occurred in North America in 2014. Clinicians should be aware of the clinical associations with EV-D68 particularly its predilection with pre-existing asthma or asthma-like illness as well as the potential association with acute flaccid myelitis. Further elucidation and development of diagnostic and treatments modalities are warranted to better understand and limit the potential public health impact of future outbreaks of EV-D68 infection.

Respiratory Presentation of Pediatric Patients in the 2014 Enterovirus D68 Outbreak.

Background. In the fall of 2014, a North American outbreak of enterovirus D68 resulted in a significant number of pediatric hospital admissions for respiratory illness throughout North America. This study characterized the clinical presentation and risk factors for a severe clinical course in children admitted to British Columbia Children's Hospital during the 2014 outbreak. Methods. Retrospective chart review of patients with confirmed EV-D68 infection admitted to BCCH with respiratory symptoms in the fall of 2014. Past medical history, clinical presentation, management, and course in hospital was collected and analyzed using descriptive statistics. Comparison was made between those that did and did not require ICU admission to identify risk factors. Results. Thirty-four patients were included (median age 7.5 years). Fifty-three percent of children had a prior history of wheeze, 32% had other preexisting medical comorbidities, and 15% were previously healthy. Ten children (29%) were admitted to the pediatric intensive care unit. The presence of complex medical conditions (excluding wheezing) (P = 0.03) and copathogens was associated with PICU admission (P = 0.02). Conclusions. EV-D68 infection resulted in severe, prolonged presentations of asthma-like illness in the hospitalized pediatric population. Patients with a prior history of wheeze and preexisting medical comorbidities appear to be most severely affected, but the virus can also cause wheezing in previously well children.

3C Protease of Enterovirus D68 Inhibits Cellular Defense Mediated by Interferon Regulatory Factor 7.

Human enterovirus 68 (EV-D68) is a member of the EV-D species, which belongs to the EV genus of the Picornaviridae family. Over the past several years, clusters of EV-D68 infections have occurred worldwide. A recent outbreak in the United States is the largest one associated with severe respiratory illness and neurological complication. Although clinical symptoms are recognized, the virus remains poorly understood. Here we report that EV-D68 inhibits innate antiviral immunity by downregulation of interferon regulatory factor 7 (IRF7), an immune factor with a pivotal role in viral pathogenesis. This process depends on 3C(pro), an EV-D68-encoded protease, to mediate IRF7 cleavage. When expressed in host cells, 3C(pro) targets Q167 and Q189 within the constitutive activation domain, resulting in cleavage of IRF7. Accordingly, wild-type IRF7 is fully active. However, IRF7 cleavage abrogated its capacity to activate type I interferon expression and limit replication of EV-D68. Notably, IRF7 cleavage strictly requires the protease activity of 3C(pro). Together, these results suggest that a dynamic interplay between 3C(pro) and IRF7 may determine the outcome of EV-D68 infection. EV-D68 is a globally emerging pathogen, but the molecular basis of EV-D68 pathogenesis is unclear. Here we report that EV-D68 inhibits innate immune responses by targeting an immune factor, IRF7. This involves the 3C protease encoded by EV-D68, which mediates the cleavage of IRF7. These observations suggest that the 3C(pro)-IRF7 interaction may represent an interface that dictates EV-D68 infection.

Prevalence and molecular characterizations of enterovirus D68 among children with acute respiratory infection in China between 2012 and 2014.

EV-D68 is associated with respiratory tract infections (RTIs). Since its first isolation, EV-D68 has been detected sporadically. However, the US and Canada have experienced outbreaks of EV-D68 infections between August and December 2014. This study aimed to investigate the molecular epidemiology and clinical characteristics of EV-D68 in Chongqing, Southwestern China. From January 2012 to November 2014, 1876 nasopharyngeal aspirate specimens (NPAs) were collected from hospitalized children with RTIs. Among the 1876 NPAs, EV-D68 was detected in 19 samples (1.0%, 19/1876). Of these, 13 samples were detected in September and October 2014 (9.8%, 13/132). Phylogenetic analysis showed that all 13 strains detected in the 2014 Chongqing had high homology with the main strains of the 2014 US outbreak. Among the children with EV-D68 infection, 13 (68%) had a history of recurrent wheezing. A total of 13 children had a discharge diagnosis of asthma. Of these, 11 children were diagnosed with acute asthma exacerbation. EV-D68 was the predominant pathogen that evoked asthma exacerbation in September and October 2014. In conclusion, our results found that a history of recurrent wheezing may be a risk factor for the detection of EV-D68 and viral-induced asthma exacerbation may be a clinical feature of EV-D68 infection.

Management of the 2014 Enterovirus 68 Outbreak at a Pediatric Tertiary Care Center

PurposeEnterovirus 68 (EV-D68) is an uncommonly recognized cause of acute respiratory tract infections. During the late summer of 2014, an international EV-D68 outbreak occurred. We review the steps of outbreak recognition and management in the context of 1 hospital’s experience with the EV-D68 outbreak. MethodsWe reviewed the role of Children’s Mercy Hospital as one of the first hospitals to recognize the 2014 EV-D68 outbreak in the United States. The steps of outbreak management were applied to real-life examples as the outbreak unfolded at our hospital. FindingsManagement of the 2014 EV-D68 outbreak was a multifaceted effort requiring close coordination with hospitals, local and state health departments, and the Centers for Disease Control and Prevention. The importance of clear and frequent communication is highlighted both intra- and interinstitutionally. Increased respiratory disease−related pediatric admissions at hospitals nationally were attributed to EV-D68. Outcomes for these children, including the association of EV-D68 with acute flaccid myelitis, remain under investigation. ImplicationsFollowing the steps of outbreak management is critical to providing optimal patient care and ensuring the health of the public. During the 2014 EV-D68 outbreak, close adherence to outbreak principles led to swift recognition of illness, rapid diagnostic measures, institution of appropriate therapies, and dissemination of information to health care providers and the public. Equally important was the subsequent identification of an increase in acute flaccid myelitis cases against the backdrop of an increase in EV-D68 detections nationally. Future prospective studies are needed to determine the true burden of EV-D68 disease, potential vaccines and therapeutics, and outcomes of children with EV-D68 infection.

Enterovirus D68 disease and molecular epidemiology in Australia

BackgroundEnterovirus D68 (EV-D68) has received considerable recent attention as a cause of widespread respiratory illness. Neurological syndromes such as acute flaccid paralysis following EV-D68 infection have also been reported in a small number of cases. ObjectivesTo summarize the clinical and epidemiological characteristics of laboratory confirmed EV-D68 cases in Australia. Study designWe combined EV-D68 data acquired through laboratory surveillance in Western Australia with cases from national enterovirus surveillance and regional acute flaccid paralysis (AFP) surveillance. Clinical data was obtained for EV-D68 cases and capsid protein sequences were used for phylogenetic analysis. ResultsSporadic cases of EV-D68 were recorded in Australia since 2008, with peaks in activity during 2011 and 2013. EV-D68 was primarily associated with respiratory disease, but was also detected in cerebrospinal fluid of one patient and faeces of two patients presenting with AFP. ConclusionsEV-D68 has been circulating in Western Australia and is likely to have also been present in the wider region for a number of years, causing primarily respiratory disease. Detection of EV-D68 in cerebrospinal fluid of one patient and in faeces of two AFP cases reinforces the association between EV-D68 and neurological disease.

A new real-time reverse transcription-PCR assay for detection of human enterovirus 68 in respiratory samples.

A global reemergence of human enterovirus 68 (EV-D68) associated with severe respiratory illness occurred in 2014. We developed and validated an EV-D68-specific real-time reverse transcription-PCR (RT-PCR) for the detection of EV-D68 in respiratory samples. The rapid diagnosis of EV-D68 may contribute to better management of EV-D68 infections.