Adeno-associated virus type 2 in US children with acute severe hepatitis.

As of August 2022, clusters of acute severe hepatitis of unknown aetiology in children have been reported from 35 countries, including the USA1,2. Previous studies have found human adenoviruses (HAdVs) in the blood from patients in Europe and the USA3-7, although it is unclear whether this virus is causative. Here we used PCR testing, viral enrichment-based sequencing and agnostic metagenomic sequencing to analyse samples from 16 HAdV-positive cases from 1 October 2021 to 22 May 2022, in parallel with 113 controls. In blood from 14 cases, adeno-associated virus type 2 (AAV2) sequences were detected in 93% (13 of 14), compared to 4 (3.5%) of 113 controls (P < 0.001) and to 0 of 30 patients with hepatitis of defined aetiology (P < 0.001). In controls, HAdV type 41 was detected in blood from 9 (39.1%) of the 23 patients with acute gastroenteritis (without hepatitis), including 8 of 9 patients with positive stool HAdV testing, but co-infection with AAV2 was observed in only 3 (13.0%) of these 23 patients versus 93% of cases (P < 0.001). Co-infections by Epstein-Barr virus, human herpesvirus 6 and/or enterovirus A71 were also detected in 12 (85.7%) of 14 cases, with higher herpesvirus detection in cases versus controls (P < 0.001). Our findings suggest that the severity of the disease is related to co-infections involving AAV2 and one or more helper viruses.

Evaluation of 3D Human Intestinal Organoids as a Platform for EV-A71 Antiviral Drug Discovery.

Enteroviruses are a leading cause of upper respiratory tract, gastrointestinal, and neurological infections. Management of enterovirus-related diseases has been hindered by the lack of specific antiviral treatment. The pre-clinical and clinical development of such antivirals has been challenging, calling for novel model systems and strategies to identify suitable pre-clinical candidates. Organoids represent a new and outstanding opportunity to test antiviral agents in a more physiologically relevant system. However, dedicated studies addressing the validation and direct comparison of organoids versus commonly used cell lines are lacking. Here, we described the use of human small intestinal organoids (HIOs) as a model to study antiviral treatment against human enterovirus 71 (EV-A71) infection and compared this model to EV-A71-infected RD cells. We used reference antiviral compounds such as enviroxime, rupintrivir, and 2'-C-methylcytidine (2'CMC) to assess their effects on cell viability, virus-induced cytopathic effect, and viral RNA yield in EV-A71-infected HIOs and cell line. The results indicated a difference in the activity of the tested compounds between the two models, with HIOs being more sensitive to infection and drug treatment. In conclusion, the outcome reveals the value added by using the organoid model in virus and antiviral studies.

Current status of hand-foot-and-mouth disease.

Hand-foot-and-mouth disease (HFMD) is a viral illness commonly seen in young children under 5 years of age, characterized by typical manifestations such as oral herpes and rashes on the hands and feet. These symptoms typically resolve spontaneously within a few days without complications. Over the past two decades, our understanding of HFMD has greatly improved and it has received significant attention. A variety of research studies, including epidemiological, animal, and in vitro studies, suggest that the disease may be associated with potentially fatal neurological complications. These findings reveal clinical, epidemiological, pathological, and etiological characteristics that are quite different from initial understandings of the illness. It is important to note that HFMD has been linked to severe cardiopulmonary complications, as well as severe neurological sequelae that can be observed during follow-up. At present, there is no specific pharmaceutical intervention for HFMD. An inactivated Enterovirus A71 (EV-A71) vaccine that has been approved by the China Food and Drug Administration (CFDA) has been shown to provide a high level of protection against EV-A71-related HFMD. However, the simultaneous circulation of multiple pathogens and the evolution of the molecular epidemiology of infectious agents make interventions based solely on a single agent comparatively inadequate. Enteroviruses are highly contagious and have a predilection for the nervous system, particularly in child populations, which contributes to the ongoing outbreak. Given the substantial impact of HFMD around the world, this Review synthesizes the current knowledge of the virology, epidemiology, pathogenesis, therapy, sequelae, and vaccine development of HFMD to improve clinical practices and public health efforts.

Trend of hand, foot, and mouth disease from 2010 to 2021 and estimation of the reduction in enterovirus 71 infection after vaccine use in Zhejiang Province, China.

BACKGROUND: Zhejiang, ranked in the top three in HFMD (hand, foot, and mouth disease) incidence, is located in the Yangtze River Delta region of southeast China. Since 2016, the EV71 vaccine has been promoted in Zhejiang Province. This study aimed to investigate the trend and seasonal variation characteristics of HFMD from 2010 to 2021 and estimate the reduction in enterovirus 71 infection after vaccine use. METHODS: The data on HFMD cases in Zhejiang Province from January 2010 to December 2021 were obtained from this network system. Individual information on cases and deaths was imported, and surveillance information, including demographic characteristics and temporal distributions, was computed by the system. The Joinpoint regression model was used to describe continuous changes in the incidence trend. The BSTS (Bayesian structural time-series models) model was used to estimate the monthly number of cases from 2017 to 2021 based on the observed monthly incidence during 2010-2016 by accounting for seasonality and long-term trends. The seasonal variation characteristics of HFMD pathogens were detected by wavelet analysis. RESULTS: From 2010 to 2021, the annual incidence rate fluctuated between 98.81 cases per 100,000 in 2020 and 435.63 cases per 100,000 in 2018, and 1711 severe HFMD cases and 106 fatal cases were reported in Zhejiang Province, China. The annual percent change (APC) in EV71 cases was -30.72% (95% CI: -45.10 to -12.50) from 2016 to 2021. The wavelet transform of total incidence and number of cases of the three pathogens all showed significant periodicity on the 1-year scale. The average 2-year scale periodicity was significant for the total incidence, EV71 cases and Cox A16 cases, but the other enterovirus cases showed significant periodicity on the 30-month scale. The 6-month scale periodicity was significant for the total incidence, EV71 case and Cox A16 case but not for the other enteroviruses case. The relative error percentage of the performance of the BSTS model was 0.3%. The estimated number of cases from 2017 to 2021 after the EV-A71 vaccines were used was 9422, and the reduction in the number of cases infected with the EV71 virus was 73.43% compared to 70.80% when the impact of the COVID-19 epidemic in 2020 was excluded. CONCLUSIONS: Since 2010, the incidence of EV71 infections has shown an obvious downward trend. All types of viruses showed significant periodicity on the 1-year scale. The periodicity of the biennial peak is mainly related to EV71 and Cox A16 before 2017 and other enteroviruses since 2018. The half-year peak cycle of HFMD was mainly caused by EV71 and Cox A6 infection. The expected incidence will be 2.76 times(include the cases of 2020) and 2.43 times(exclude the cases of 2020) higher than the actual value assuming that the measures of vaccination are not taken. EV71 vaccines are very effective and should be administered in the age window between 5 months and 5 years.

Comprehensive profiling and characterization of cellular microRNAs in response to coxsackievirus A10 infection in bronchial epithelial cells.

Coxsackievirus A10 (CV-A10), the causative agent of hand, foot, and mouth disease (HFMD), caused a series of outbreaks in recent years and often leads to neurological impairment, but a clear understanding of the disease pathogenesis and host response remains elusive. Cellular microRNAs (miRNAs), a large family of non-coding RNA molecules, have been reported to be key regulators in viral pathogenesis and virus-host interactions. However, the role of host cellular miRNAs defensing against CV-A10 infection is still obscure. To address this issue, we systematically analyzed miRNA expression profiles in CV-A10-infected 16HBE cells by high-throughput sequencing methods in this study. It allowed us to successfully identify 312 and 278 miRNAs with differential expression at 12 h and 24 h post-CV-A10 infection, respectively. Among these, 4 miRNAs and their target genes were analyzed by RT-qPCR, which confirmed the sequencing data. Gene target prediction and enrichment analysis revealed that the predicted targets of these miRNAs were significantly enriched in numerous cellular processes, especially in regulation of basic physical process, host immune response and neurological impairment. And the integrated network was built to further indicate the regulatory roles of miRNAs in host-CV-A10 interactions. Consequently, our findings could provide a beneficial basis for further studies on the regulatory roles of miRNAs relevant to the host immune responses and neuropathogenesis caused by CV-A10 infection.

Comparison of Neutralizing Antibody Response Kinetics in Patients with Hand, Foot, and Mouth Disease Caused by Coxsackievirus A16 or Enterovirus A71: A Longitudinal Cohort Study of Chinese Children, 2017-2019.

Hand, foot, and mouth disease (HFMD), which is mainly caused by coxsackievirus A16 (CVA16) or enterovirus A71 (EV-A71), poses a serious threat to children's health. However, the long-term dynamics of the neutralizing Ab (NAb) response and ideal paired-serum sampling time for serological diagnosis of CVA16-infected HFMD patients were unclear. In this study, 336 CVA16 and 253 EV-A71 PCR-positive HFMD inpatients were enrolled and provided 452 and 495 sera, respectively, for NAb detection. Random-intercept modeling with B-spline was conducted to characterize NAb response kinetics. The NAb titer of CVA16 infection patients was estimated to increase from negative (2.1, 95% confidence interval [CI]: 1.4-3.3) on the day of onset to a peak of 304.8 (95% CI: 233.4-398.3) on day 21 and then remained >64 until 26 mo after onset. However, the NAb response level of EV-A71-infected HFMD patients was much higher than that of CVA16-infected HFMD patients throughout. The geometric mean titer was significantly higher in severe EV-A71-infected patients than in mild patients, with a 2.0-fold (95% CI: 1.4-3.2) increase. When a 4-fold rise in titer was used as the criterion for serological diagnosis of CVA16 and EV-A71 infection, acute-phase serum needs to be collected at 0-5 d, and the corresponding convalescent serum should be respectively collected at 17.4 (95% CI: 9.6-27.4) and 24.4 d (95% CI: 15.3-38.3) after onset, respectively. In conclusion, both CVA16 and EV-A71 infection induce a persistent humoral immune response but have different NAb response levels and paired-serum sampling times for serological diagnosis. Clinical severity can affect the anti-EV-A71 NAb response.

Molnupiravir and Its Active Form, EIDD-1931, Show Potent Antiviral Activity against Enterovirus Infections In Vitro and In Vivo.

Enterovirus infections can cause hand, foot, and mouth disease (HFDM), aseptic meningitis, encephalitis, myocarditis, and acute flaccid myelitis, leading to death of infants and young children. However, no specific antiviral drug is currently available for the treatment of this type of infection. The Unites States and United Kingdom health authorities recently approved a new antiviral drug, molnupiravir, for the treatment of COVID-19. In this study, we reported that molnupiravir (EIDD-2801) and its active form, EIDD-1931, have broad-spectrum anti-enterovirus potential. Our data showed that EIDD-1931 could significantly reduce the production of EV-A71 progeny virus and the expression of EV-A71 viral protein at non-cytotoxic concentrations. The results of the time-of-addition assay suggest that EIDD-1931 acts at the post-entry step, which is in accordance with its antiviral mechanism. The intraperitoneal administration of EIDD-1931 and EIDD-2801 protected 1-day-old ICR suckling mice from lethal EV-A71 challenge by reducing the viral load in various tissues of the infected mice. The pharmacokinetics analysis indicated that the plasma drug concentration overwhelmed the EC50 for enteroviruses, suggesting the clinical potential of molnupiravir against enteroviruses. Thus, molnupiravir along with its active form, EIDD-1931, may be a promising drug candidate against enterovirus infections.

Pan-Enterovirus Amplicon-Based High-Throughput Sequencing Detects the Complete Capsid of a EVA71 Genotype C1 Variant via Wastewater-Based Epidemiology in Arizona.

We describe the complete capsid of a genotype C1-like Enterovirus A71 variant recovered from wastewater in a neighborhood in the greater Tempe, Arizona area (Southwest United States) in May 2020 using a pan-enterovirus amplicon-based high-throughput sequencing strategy. The variant seems to have been circulating for over two years, but its sequence has not been documented in that period. As the SARS-CoV-2 pandemic has resulted in changes in health-seeking behavior and overwhelmed pathogen diagnostics, our findings highlight the importance of wastewater-based epidemiology (WBE ) as an early warning system for virus surveillance.

Human Intestinal Organoids Recapitulate Enteric Infections of Enterovirus and Coronavirus.

Enteroviruses, such as EV-A71 and CVA16, mainly infect the human gastrointestinal tract. Human coronaviruses, including SARS-CoV and SARS-CoV-2, have been variably associated with gastrointestinal symptoms. We aimed to optimize the human intestinal organoids and hypothesize that these optimized intestinal organoids can recapitulate enteric infections of enterovirus and coronavirus. We demonstrate that the optimized human intestinal organoids enable better simulation of the native human intestinal epithelium, and that they are significantly more susceptible to EV-A71 than CVA16. Higher replication of EV-A71 than CVA16 in the intestinal organoids triggers a more vigorous cellular response. However, SARS-CoV and SARS-CoV-2 exhibit distinct dynamics of virus-host interaction; more robust propagation of SARS-CoV triggers minimal cellular response, whereas, SARS-CoV-2 exhibits lower replication capacity but elicits a moderate cellular response. Taken together, the disparate profile of the virus-host interaction of enteroviruses and coronaviruses in human intestinal organoids may unravel the cellular basis of the distinct pathogenicity of these viral pathogens.

Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2.

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.