Epidemiological and Clinical Insights into the Enterovirus D68 Upsurge in Europe 2021-2022 and Emergence of Novel B3-Derived Lineages, ENPEN Multicentre Study.

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 infections and its clinical impact during the fall-winter season of 2021-2022. From 19 European countries, 58 institutes reported 10 481 (6.8%) EV-positive samples of which 1004 (9.6%) were identified as EV-D68 (including 852 respiratory samples). Clinical data were reported for 969 cases; 78.9% of infections were reported in children (0-5 years); and 37.9% of cases were hospitalized. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases including 6 diagnosed with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of 2 novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale European EV-D68 upsurge with severe clinical impact and the emergence of B3-derived lineages.

Hepatitis B Core-Related Antigen to Indicate High Viral Load: Systematic Review and Meta-Analysis of 10,397 Individual Participants.

BACKGROUND & AIMS: To eliminate hepatitis B virus (HBV) infection, scale-up of testing and treatment in resource-limited countries is crucial. However, access to nucleic acid testing to quantify HBV DNA, an essential test to examine treatment eligibility, remains severely limited. We assessed the performance of a novel immunoassay, HBV core-related antigen (HBcrAg), as a low-cost (less than US $15/assay) alternative to nucleic acid testing to indicate clinically important high viremia in chronic HBV patients infected with different genotypes. METHODS: We searched Medline, Embase, Scopus, and Web of Science databases through June 27, 2018. Three reviewers independently selected studies measuring HBV DNA and HBcrAg in the same blood samples. We contacted authors to provide individual participant data (IPD). We randomly allocated each IPD to a derivation or validation cohort. We applied optimal HBcrAg cut-off values derived from the derivation set to the validation set to estimate sensitivity/specificity. RESULTS: Of 74 eligible studies, IPD were obtained successfully for 60 studies (81%). Meta-analysis included 5591 IPD without antiviral therapy and 4806 treated with antivirals. In untreated patients, the pooled area under the receiver operating characteristic curve and optimal cut-off values were as follows: 0.88 (95% CI, 0.83-0.94) and 3.6 log U/mL to diagnose HBV DNA level of 2000 IU/mL or greater; and 0.96 (95% CI, 0.94-0.98) and 5.3 log U/mL for 200,000 IU/mL or greater, respectively. In the validation set, the sensitivity and specificity were 85.2% and 84.7% to diagnose HBV DNA level of 2000 IU/mL or greater, and 91.8% and 90.5% for 200,000 IU/mL or greater, respectively. The performance did not vary by HBV genotypes. In patients treated with anti-HBV therapy the correlation between HBcrAg and HBV DNA was poor. CONCLUSIONS: HBcrAg might be a useful serologic marker to indicate clinically important high viremia in treatment-naïve, HBV-infected patients.

Enhancing and neutralizing anti-coxsackievirus activities in serum samples from patients prior to development of type 1 diabetes.

BACKGROUND: Studies in prospective cohorts have suggested that enterovirus infections are associated with the appearance of islet autoantibodies that precede later appearance of type 1 diabetes (T1D). It was shown that in addition to an antibody-mediated anti-coxsackievirus (CV)-B neutralizing activity of serum from patients with T1D, there was also enhancing anti-CV-B activity in vitro. In this study, the patterns of enhancing and neutralizing anti-CV activities were analysed from consecutive serum samples collected from children who were followed from birth until they developed T1D in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study and compared to those in non-diabetic control children. METHODS: The titres of serum neutralizing activity were analysed against those CVs which were detected in the stools in these children (CV-B3, CV-B5 or CV-A4) using plaque assay. The enhancing activity of these serum samples was analysed by measuring interferon-alpha (INF-α) production in cultures of peripheral blood mononuclear cells (PBMC) inoculated with a mixture of these viruses and diluted serum. RESULTS: A sustained anti-CV enhancing activity was observed in consecutive serum samples in patients with T1D. The pattern of responses differed between children who developed T1D and control children. In patients, the anti-CV enhancing activity was predominant or even exclusive over the neutralizing activity, whereas in controls the enhancing and neutralising activities were more balanced or the neutralizing activity was largely predominant. CONCLUSIONS: Evaluating the anti-enterovirus neutralizing and enhancing activity of serum samples can be useful to investigate further the relationship between enteroviruses and the development of T1D.

Very slow decline of hepatitis B virus surface antigen and core related antigen in chronic hepatitis B patients successfully treated with nucleos(t)ide analogues.

We investigated the decline of hepatitis B virus surface antigen (HBsAg) and core related antigen (HBcrAg) in chronic hepatitis B patients sussessfully treated with nucleos(t)ide analogues. In patients with plasma viral suppression, the baseline median levels of HBsAg and HBcrAg were 3.1 and 3.0 log U/mL, respectively. The levels in naïve patients were 4.2 and 3.6 log U/mL for HBsAg and HBcrAg, respectively. No significant decline was observed in patients with viral suppression within a year period. A low reduction was observed during the first months after treatment initiation, especially regarding HBcrAg. The dynamics of these antigens after viral suppression should be further investigated.

Persistent coxsackievirus B4 infection induces microRNA dysregulation in human pancreatic cells.

Enterovirus infections are implicated in the development of type 1 diabetes (T1D). MicroRNAs as regulators of gene expression are involved in many physiological and pathological processes. Given that viral infections dysregulate cellular microRNAs, we investigated the impact of persistent coxsackievirus B4 infection on microRNA expression of human pancreatic cells. Next-generation sequencing was used to determine microRNA expression in PANC-1 cells persistently infected (for several weeks) with coxsackievirus B4 and uninfected control cells. Target prediction restricted to T1D risk genes was performed with miRWalk2.0. Functional annotation analysis was performed with DAVID6.7. Expression of selected microRNAs and T1D risk genes was measured by quantitative reverse-transcription polymerase chain reaction. Eighty-one microRNAs were dysregulated in persistently infected PANC-1 cells. Forty-nine of the known fifty-five T1D risk genes were predicted as putative targets of at least one of the dysregulated microRNAs. Most functional annotation terms that were enriched in these 49 putative target genes were related to the immune response or autoimmunity. mRNA levels of AFF3, BACH2, and IL7R differed significantly between persistently infected cells and uninfected cells. This is the first characterization of the microRNA expression profile changes induced by persistent coxsackievirus B4 infection in pancreatic cells. The predicted targeting of genes involved in the immune response and autoimmunity by the dysregulated microRNAs as well as the dysregulated expression of diabetes risk genes shows that persistent coxsackievirus B4 infection profoundly impacts the host cell. These data support the hypothesis of a possible link between persistent coxsackievirus B4 infection and the development of T1D.

Enterovirus D68 detection in respiratory specimens: Association with severe disease.

Molecular techniques increased the number of documented respiratory infections. In a substantial number of cases the causative agent remains undetected. Since August 2014, an increase in Enterovirus(EV)-D68 infections was reported. We aimed to investigate epidemiology and clinical relevance of EV-D68. From June to December 2014 and from September to December 2015, 803 and 847 respiratory specimens, respectively, were tested for respiratory viruses with a multiplex RT-PCR. This multiplex RT-PCR does not detect EV-D68. Therefore, 457 (2014) and 343 (2015) specimens with negative results were submitted to an EV-specific-RT-PCR. EV-positive specimens were tested with an EV-D68-specific-RT-PCR and genotyped. Eleven specimens of 2014 tested positive in the EV-specific-RT-PCR and of these seven were positive in the EV-D68-specific-RT-PCR. Typing confirmed these as EV-D68. Median age of EV-D68-positive patients was 3 years (1 month-91 years). Common symptoms included fever (n = 6, 86%), respiratory distress (n = 5, 71%), and cough (n = 4, 57%). All EV-D68-positive patients were admitted to hospital, 4 (57%) were admitted to intensive care units and 6 (86%) received oxygen. One patient suffered from acute flaccid paralysis. Seven specimens of 2015 were positive in the EV-specific-RT-PCR but negative in the EV-D68-specific-RT-PCR. In conclusion, use of an EV-specific-RT-PCR allowed us to detect EV-D68 circulation in autumn 2014 that was not detected by the multiplex RT-PCR and was associated with severe disease.

Enteroviral pathogenesis of type 1 diabetes: queries and answers.

PURPOSE OF REVIEW: Type 1 diabetes (T1D) results from interplay between genetic predisposition, immune system, and environmental factors. Epidemiological and experimental data strongly suggest a role for enteroviruses in the development of T1D, but a lot of controversies and unanswered questions remained. This review focuses on issues that are fueling debate. RECENT FINDINGS: Beyond HLA genes, which provide genetic susceptibility for T1D, other loci have been identified to be associated with the disease. There is a link between T1D and single-nucleotide polymorphisms (SNPs) in the interferon-induced helicase 1 (IFIH1) gene that encodes melanoma differentiation-associated protein 5 (MDA5). This protein is a cytoplasmic sensor for viruses especially coxsackieviruses B, the most incriminated enteroviruses in T1D pathogenesis. Upon viral infection, MDA5 stimulates the production of mediators of the innate antiviral immune response, which is believed to play a role in a 'bystander activation' scenario. Rare variants of IFIH1 through a lost or reduced expression of the protein are protective against T1D, whereas common IFIH1 SNPs are associated with the disease. However, a clear association has not been yet established between T1D-associated IFIH1 polymorphisms and enterovirus detection. SUMMARY: Literature have accumulated a lot of evidence supporting that enteroviruses can contribute, at least in some patients, to the pathogenesis of T1D through various mechanisms. But it is still a challenge to date to prove a causal relationship between enteroviruses and T1D. Future studies may lead to a better understanding of this relationship and ultimately can help toward disease prevention.

Anti-coxsackievirus B4 (CV-B4) enhancing activity of serum associated with increased viral load and pathology in mice reinfected with CV-B4.

In previous studies it was shown that inoculation of Swiss albino mice with CV-B4 E2 resulted in the production of serum IgG capable of enhancing the CV-B4 E2 infection of murine spleen cells cultures. To investigate whether such an enhancing activity of serum can play a role in vivo, we decided to study the CV-B4 E2 infection in mice exposed to successive inoculations of virus. In Swiss albino mice infected with CV-B4 E2 at the age of 21 days, anti-CV-B4 E2 neutralizing and enhancing activities of their serum peaked after 55 d. In contrast, mice inoculated at the age of 55 d expressed much lower activities. Despite the neutralizing activity of serum, CV-B4 E2 inoculated a second time to 55 day-old animals spread into the host. At the age of 72 and 89 d the levels of viral RNA and infectious particles were higher in organs of animals exposed to 2 successive infections compared with animals infected once at the age of 21 d or 55 d. In animals with 2 successive inoculations of CV-B4 E2 there was a relationship between the anti-CV-B4 E2 enhancing activity of serum and the level of viral RNA in organs and an enhancement of pathology was observed as displayed by histological analysis of pancreas and hyperglycaemia. Altogether our data strongly suggest that an anti-CV-B4 E2 enhancing activity in the host can play a role in the outcome of a secondary infection with this virus.