scholarly journals Genomic Analyses of Acute Flaccid Myelitis Cases among a Cluster in Arizona Provide Further Evidence of Enterovirus D68 Role

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jolene R. Bowers ◽  
Michael Valentine ◽  
Veronica Harrison ◽  
Viacheslav Y. Fofanov ◽  
John Gillece ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Respiratory Illness ◽  
Amplicon Sequencing ◽  
Gastrointestinal Illness ◽  
Metagenomic Sequencing ◽  
Neurologic Disease ◽  
Acute Flaccid Myelitis ◽  
Genomic Analyses ◽  
Enterovirus D68

ABSTRACTEnteroviruses are a common cause of respiratory and gastrointestinal illness, and multiple subtypes, including poliovirus, can cause neurologic disease. In recent years, enterovirus D68 (EV-D68) has been associated with serious neurologic illnesses, including acute flaccid myelitis (AFM), frequently preceded by respiratory disease. A cluster of 11 suspect cases of pediatric AFM was identified in September 2016 in Phoenix, AZ. To determine if these cases were associated with EV-D68, we performed multiple genomic analyses of nasopharyngeal (NP) swabs and cerebrospinal fluid (CSF) material from the patients, including real-time PCR and amplicon sequencing targeting the EV-D68 VP1 gene and unbiased microbiome and metagenomic sequencing. Four of the 11 patients were classified as confirmed cases of AFM, and an additional case was classified as probable AFM. Real-time PCR and amplicon sequencing detected EV-D68 virus RNA in the three AFM patients from which NP swabs were collected, as well as in a fourth patient diagnosed with acute disseminated encephalomyelitis, a disease that commonly follows bacterial or viral infections, including enterovirus. No other obvious etiological causes for AFM were identified by 16S or RNA and DNA metagenomic sequencing in these cases, strengthening the likelihood that EV-D68 is an etiological factor. Herpes simplex viral DNA was detected in the CSF of the fourth case of AFM and in one additional suspect case from the cluster. Multiple genomic techniques, such as those described here, can be used to diagnose patients with suspected EV-D68 respiratory illness, to aid in AFM diagnosis, and for future EV-D68 surveillance and epidemiology.IMPORTANCEEnteroviruses frequently result in respiratory and gastrointestinal illness; however, multiple subtypes, including poliovirus, can cause severe neurologic disease. Recent biennial increases (i.e., 2014, 2016, and 2018) in cases of non-polio acute flaccid paralysis have led to speculations that other enteroviruses, specifically enterovirus D68 (EV-D68), are emerging to fill the niche that was left from poliovirus eradication. A cluster of 11 suspect cases of pediatric acute flaccid myelitis (AFM) was identified in 2016 in Phoenix, AZ. Multiple genomic analyses identified the presence of EV-D68 in the majority of clinical AFM cases. Beyond limited detection of herpesvirus, no other likely etiologies were found in the cluster. These findings strengthen the likelihood that EV-D68 is a cause of AFM and show that the rapid molecular assays developed for this study are useful for investigations of AFM and EV-D68.

Development of Novel PCR Assays for Improved Detection of Enterovirus D68

2021 ◽  
Author(s):  
Tatsuki Ikuse ◽  
Yuta Aizawa ◽  
Hayato Takihara ◽  
Shujiro Okuda ◽  
Kanako Watanabe ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Clinical Manifestations ◽  
Assay Sensitivity ◽  
Acute Flaccid Myelitis ◽  
Specific Pcr ◽  
Enterovirus D68 ◽  
Pcr Assays ◽  
Higher Sensitivity

Enterovirus D68 (EV-D68) causes a range of clinical manifestations, including asthma-like illness, severe respiratory disease, and acute flaccid myelitis. EV-D68 has caused worldwide outbreaks since 2014 and is now recognized as a re-emerging infection in many countries. EV-D68–specific PCR assays are widely used for the diagnosis of EV-D68 infection; however, assay sensitivity is a concern because of genetic changes in recently circulated EV-D68. To address this, we summarized EV-D68 sequences from previously reported world outbreaks from 2014 through 2020 on GenBank, and found several mutations at the primer and probe binding sites of the existing EV-D68–specific PCR assays. Subsequently, we designed two novel assays corresponding to the recently reported EV-D68 sequences: an EV-D68–specific real-time and semi-nested PCR. In an analysis of 22 EV-D68–confirmed cases during a recent EV-D68 outbreak in Japan, the new real-time PCR had higher sensitivity than the existing assay (100% vs. 45%, P < 0.01) and a lower median Ct value (27.8 vs. 32.8, P = 0.005). Sensitivity was higher for the new non-nested PCR (91%) than for the existing semi-nested PCR assay (50%, P < 0.01). The specificity of the new real-time PCR was 100% using samples from non-EV-D68–infected cases (n = 135). In conclusion, our novel assays had higher sensitivity than the existing assay and might lead to more accurate diagnosis of recently circulating EV-D68. To prepare for future EV-D68 outbreaks, EV-D68–specific assays must be continuously monitored and updated.

Genetic analysis of Enterovirus D68 associated with pneumonia in children from South India

2021 ◽  
Vol 70 (5) ◽  
Author(s):  
Ramachandran Erathodi Sanjay ◽  
Sasidharanpillai Sabeena ◽  
Sudandiradas Robin ◽  
John T. Shaji ◽  
M. P. Jayakrishnan ◽  
...  
Keyword(s):  
South India ◽  
Single Case ◽  
Respiratory Illness ◽  
Neurological Complications ◽  
The Novel ◽  
Acute Flaccid Myelitis ◽  
Sporadic Cases ◽  
Enterovirus D68 ◽  
Kerala State ◽  
Unique Amino Acid

EV-D68 is an emerging enterovirus infection associated with severe acute respiratory illness (SARI), acute flaccid myelitis (AFM) and acute flaccid paralysis (AFP). While EV-D68 outbreaks and sporadic cases are reported globally, a single case has been reported from India. The present study aims to investigate the molecular epidemiology and clinical characteristics of EV-D68-associated SARI cases from South India. We screened influenza-negative archived throat swab specimens from Influenza-Like Illness (ILI) and SARI cases (n=959; 2016 to 2018 period) for enteroviruses by pan-enterovirus real-time RT-PCR. Thirteen samples positive for enteroviruses were typed by PCR and sequencing based on VPI, VP2 and/or 5′NCR regions. One EV-D68 RNA sample was subjected to next-generation sequencing for whole genome characterisation. Among 13 enterovirus cases, four were ECHO-11, three EV-D68, two CV-A16 and one each EV-71, CV-B1, CV-B2 and CV-A9. All three cases of EV-D68 infection were reported in children below 2 years of age from Kerala state of South India during June and July 2017. The patients developed pneumonia without any neurological complications. Sequencing based on VPI and 5′NCR regions showed that EV-D68 strains belong to the novel subclade B3. The EV-D68 complete genome identified with two unique amino acid substitutions in VP1 (T-246-I) and 3D (K-344-R) regions. This study reiterates the EV-D68 novel subclade B3 circulation in India and indicates the urgent need for structured EV-D68 surveillance in the country to describe the epidemiology.

scholarly journals 2854. Enterovirus D68 Infections in Pediatric Patients in Central Ohio: Clinical Characteristics of a New Outbreak in 2018

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S74-S74
Author(s):  
Alejandro Diaz ◽  
Huanyu Wang ◽  
Isabel Torrus ◽  
Fatima Ara Montojo ◽  
Maria Mele-Casas ◽  
...  
Keyword(s):  
Clinical Characteristics ◽  
Clinical Presentation ◽  
Gastrointestinal Symptoms ◽  
Respiratory Illness ◽  
Neurologic Manifestations ◽  
Asthmatic Children ◽  
Acute Flaccid Myelitis ◽  
Enterovirus D68 ◽  
Co Detection ◽  
Opsoclonus Myoclonus Syndrome

Abstract Background Many aspects of EV-D68 pathogenesis in children are not fully understood. In 2014, we experienced an outbreak of EV-D68-associated acute respiratory illness affecting mostly asthmatic children with no cases of acute flaccid myelitis identified. Late in 2018, a new outbreak occurred. The objective of this study was to describe the differences in clinical presentation in children diagnosed with EV-D68 infection during the 2018 outbreak. Methods This is a single-center, observational study. Nasopharyngeal (NP) samples from patients <21 years of age that tested positive for rhinovirus/enterovirus (RV/EV) by the FilmArray respiratory panel v1.7 were prospectively collected. EV-D68 was confirmed using a laboratory-developed RT-PCR. Demographic, clinical characteristics, and semiquantitative EV-D68 loads were analyzed according to the clinical presentation. Results From May to October 2018, 1,987/3,633 (55%) samples were RV/EV positive. Of those 399/1,028 (39%) tested positive for EV-D68 (121 outpatients; 278 inpatients). Inpatients were older (3.1 vs. 1.8 year olds; P < 0.01) with no differences in sex or EV-D68 loads (P > 0.05). Within the inpatient cohort, 67 (1.4 year olds) children were previously healthy, 146 (4.1 year olds) had underlying asthma and 65 (2.5 year olds) had chronic medical conditions (24% vs. 53% vs. 23%, respectively). Most patients presented with respiratory symptoms (>95%), followed by fever (51%) or gastrointestinal symptoms (28%). Eleven children (4%) presented with neurologic manifestations including: acute flaccid myelitis in two children, opsoclonus myoclonus syndrome in one child, and seizures in the remaining eight. Rates of viral co-detection were low (8%) and none of the children with neurologic manifestations had another respiratory virus identified. Patients with neurologic findings had lower EV-D68 loads than those who did not (29 vs. 25 Ct values; P = 0.03). Conclusion EV-D68 infection was associated with significant morbidity, affecting children with underlying asthma at greater rates. It was associated with severe neurologic manifestations despite these children having lower EV-D68 loads. Active surveillance for EV-D68 should be routine to allow a better understanding of the epidemiology and severity of disease. Disclosures All Authors: No reported Disclosures.

scholarly journals Evaluation of a Real-Time Reverse Transcription-PCR Assay for Detection of Enterovirus D68 in Clinical Samples from an Outbreak in New York State in 2014

2015 ◽  
Vol 53 (6) ◽  
pp. 1915-1920 ◽  
Author(s):  
Jian Zhuge ◽  
Eric Vail ◽  
Jeffrey L. Bush ◽  
Lauren Singelakis ◽  
Weihua Huang ◽  
...  
Keyword(s):  
New York ◽  
Real Time ◽  
Reverse Transcription ◽  
New York State ◽  
Respiratory Illness ◽  
Clinical Samples ◽  
Sequencing Analysis ◽  
York State ◽  
Reverse Transcription Pcr ◽  
Enterovirus D68

An outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68) infection was reported in mid-August 2014 in the United States. In this study, we evaluated the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (rRT-PCR) that was recently developed by the Centers for Disease Control and Prevention in clinical samples. Nasopharyngeal (NP) swab specimens from patients in a recent outbreak of respiratory illness in the lower Hudson Valley, New York State, were collected and examined for the presence of human rhinovirus or enterovirus using the FilmArray Respiratory Panel (RP) assay. Samples positive by RP were assessed using EV-D68 rRT-PCR, and the data were compared to results from sequencing analysis of partial VP1 and 5′ untranslated region (5′-UTR) sequences of the EV genome. A total of 285 RP-positive NP specimens (260 from the 2014 outbreak and 25 from 2013) were analyzed by rRT-PCR; EV-D68 was detected in 74 of 285 (26.0%) specimens examined. Data for comparisons between rRT-PCR and sequencing analysis were obtained from 194 NP specimens. EV-D68 detection was confirmed by sequencing analysis in 71 of 74 positive and in 1 of 120 randomly selected negative specimens by rRT-PCR. The EV-D68 rRT-PCR showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. Our data suggest that the EV-D68 rRT-PCR is a reliable assay for detection of EV-D68 in clinical samples and has a potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories.

scholarly journals A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Caroline F. Frey ◽  
Jenna R. Oakley ◽  
Vladislav A. Lobanov ◽  
Nelson Marreros ◽  
Janna M. Schurer ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fresh Produce ◽  
Melting Curve ◽  
Amplicon Sequencing ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Analytical Sensitivity ◽  
Leafy Greens ◽  
Food Safety Risk

Abstract Background Zoonotic taeniid cestodes are amongst the most important food-borne parasites affecting human health worldwide. Contamination of fresh produce with the eggs of Echinococcus granulosus (s.l.), Echinococcus multilocularis, and some Taenia species pose a potential food safety risk. However, very few studies have attempted to investigate the potential contamination of fresh produce with taeniid eggs and the available methods are not standardized for this purpose. Established protocols do exist for testing leafy greens and berries for contamination with protozoan parasites and are used in national surveillance programmes. This methodology could be suitable for the detection of taeniids. The objective of this project was to develop and standardize a sensitive and reliable method to detect contamination of leafy greens and berries with eggs of zoonotic taeniids and to differentiate between E. multilocularis, E. granulosus (s.l.) and Taenia spp. Methods We compared the efficacy of different wash solutions to remove Taenia spp. eggs from spiked produce, assessed two DNA extraction kits for their performance on Taenia spp. eggs, and adapted a published conventional multiplex PCR into a real-time PCR with fluorescence melting curve analysis (MCA) that was optimized for use on produce washes. Analytical specificity of this protocol was assessed using non-spiked produce washes as well as a variety of other potentially contaminating parasites. Results The protocol as established in this study had an analytical sensitivity of detecting five eggs per spiked sample for both romaine lettuce and strawberries. Unequivocal identification of E. multilocularis, E. granulosus (s.l.) and Taenia spp. was possible through MCA. Amplicon sequencing allowed identification of Taenia to the species level. The real-time PCR also amplified DNA from Dicrocoelium sp., but with a clearly discernable melting curve profile. Conclusion The new protocol for screening produce for taeniid contamination was highly sensitive. Melting curve analysis and the possibility of amplicon sequencing made this assay very specific. Once further validated, this method could be employed for surveillance of produce for contamination with taeniid parasites to assess potential risks for consumers.

scholarly journals Comparison of the performance of an amplicon sequencing assay based on Oxford Nanopore technology to real-time PCR assays for detecting bacterial biodefense pathogens

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Robert Player ◽  
Kathleen Verratti ◽  
Andrea Staab ◽  
Christopher Bradburne ◽  
Sarah Grady ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Amplicon Sequencing ◽  
Oxford Nanopore ◽  
Pcr Assays

scholarly journals Molecular epidemiological study of enterovirus D68 in hospitalised children in Hong Kong in 2014–2015 and their complete coding sequences

2019 ◽  
Vol 6 (1) ◽  
pp. e000437
Author(s):  
Haichao Wang ◽  
Kinpong Tao ◽  
Cheuk Yin Leung ◽  
Kam Lun Hon ◽  
C M Apple Yeung ◽  
...  
Keyword(s):  
Hong Kong ◽  
Vaccine Development ◽  
Public Awareness ◽  
Respiratory Illness ◽  
Molecular Classification ◽  
Human Enterovirus ◽  
Coding Sequences ◽  
Acute Flaccid Myelitis ◽  
Hospitalised Patients ◽  
Enterovirus D68

BackgroundHuman enterovirus D68 (EV-D68) was first isolated in 1962 and has aroused public concern recently because of a nationwide outbreak among children in 2014–2015 in the USA. The symptoms include fever, runny nose, sneezing, cough and muscle pains. It might be associated with severe respiratory illness in individuals with pre-existing respiratory conditions and its potential association with acute flaccid myelitis is under investigation. In Asia, EV-D68 cases have been reported in several countries.The studyWe aimed to understand the EV-D68 prevalence and their genetic diversity in Hong Kong children.MethodsA total of 10 695 nasopharyngeal aspirate (NPA) samples from hospitalised patients aged <18 years were collected from September 2014 to December 2015 in two regional hospitals. NPAs tested positive for enterovirus/rhinovirus (EV/RV) were selected for genotyping. For those identified as EV-D68, their complete coding sequences (CDSs) were obtained by Sanger sequencing. A maximum-likelihood phylogeny was constructed using all EV-D68 complete coding sequences available in GenBank (n=482).Results2662/10 695 (24.9%) were tested positive with EV/RV and 882/2662 (33.1%) were selected randomly and subjected to molecular classification. EV-D68 was detected in 15 (1.70%) samples from patients with clinical presentations ranging from wheezing to pneumonia and belonged to subclade B3. Eight CDSs were successfully obtained. A total of 10 amino acid residue polymorphisms were detected in the viral capsid proteins, proteases, ATPase and RNA polymerase.ConclusionB3 subclade was the only subclade found locally. Surveillance of EV-D68 raises public awareness and provides the information to determine the most relevant genotypes for vaccine development.

scholarly journals Respiratory and intestinal epithelial cells exhibit differential susceptibility and innate immune responses to EV-D68

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Megan Culler Freeman ◽  
Alexandra I Wells ◽  
Jessica Ciomperlik-Patton ◽  
Michael M Myerburg ◽  
Liheng Yang ◽  
...  
Keyword(s):  
Intestinal Epithelium ◽  
Differential Susceptibility ◽  
Respiratory Illness ◽  
Intestinal Epithelial ◽  
Innate Immune Responses ◽  
Human Airway ◽  
Acute Flaccid Myelitis ◽  
Enterovirus D68 ◽  
Type Iii Interferon ◽  
Severe Respiratory Illness

Enterovirus D68 (EV-D68) has been implicated in outbreaks of severe respiratory illness and is associated with acute flaccid myelitis (AFM). EV-D68 is often detected in patient respiratory samples but has also been detected in stool and wastewater, suggesting the potential for both respiratory and enteric routes of transmission. Here, we used a panel of EV-D68 isolates, including a historical pre-2014 isolate and multiple contemporary isolates from AFM outbreak years, to define the dynamics of viral replication and the host response to infection in primary human airway cells and stem cell-derived enteroids. We show that some recent EV-D68 isolates have decreased sensitivity to acid and temperature compared with earlier isolates and that the respiratory, but not intestinal, epithelium induces a robust type III interferon (IFN) response that restricts infection. Our findings define the differential responses of the respiratory and intestinal epithelium to contemporary EV-D68 isolates and suggest that a subset of isolates have the potential to target both the human airway and gastrointestinal tracts.

scholarly journals Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay

2015 ◽  
Vol 53 (8) ◽  
pp. 2641-2647 ◽  
Author(s):  
Todd N. Wylie ◽  
Kristine M. Wylie ◽  
Richard S. Buller ◽  
Maria Cannella ◽  
Gregory A. Storch
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Limit Of Detection ◽  
Respiratory Illness ◽  
The United States ◽  
Human Enterovirus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcriptase Pcr ◽  
Enterovirus D68

We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses.

scholarly journals Current Understanding of Human Enterovirus D68

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 490 ◽  
Author(s):  
Jing Sun ◽  
Xiao-Yi Hu ◽  
Xiao-Fang Yu
Keyword(s):  
Treatment Options ◽  
Antiviral Agents ◽  
Respiratory Illness ◽  
The United States ◽  
Human Enterovirus ◽  
Genetic Characteristics ◽  
Host Interactions ◽  
Acute Flaccid Myelitis ◽  
Enterovirus D68 ◽  
Neurogenic Disease

Human enterovirus D68 (EV-D68), a member of the species Enterovirus D of the Picornaviridae family, was first isolated in 1962 in the United States. EV-D68 infection was only infrequently reported until an outbreak occurred in 2014 in the US; since then, it has continued to increase worldwide. EV-D68 infection leads to severe respiratory illness and has recently been reported to be linked to the development of the neurogenic disease known as acute flaccid myelitis (AFM), mostly in children, seriously endangering public health. Hitherto, treatment options for EV-D68 infections were limited to supportive care, and as yet there are no approved, specific antiviral drugs or vaccines. Research on EV-D68 has mainly focused on its epidemiology, and its virologic characteristics and pathogenesis still need to be further explored. Here, we provide an overview of current research on EV-D68, including the genotypes and genetic characteristics of recent epidemics, the mechanism of infection and virus–host interactions, and its relationship to acute flaccid myelitis (AFM), in order to broaden our understanding of the biological features of EV-D68 and provide a basis for the development of effective antiviral agents.

Sign in / Sign up

Export Citation Format

Share Document