Molecular phylogeny and proposed classification of the simian picornaviruses.

The simian picornaviruses were isolated from various primate tissues during the development of general tissue culture methods in the 1950s to 1970s or from specimens derived from primates used in biomedical research. Twenty simian picornavirus serotypes are recognized, and all are presently classified within the Enterovirus genus. To determine the phylogenetic relationships among all of the simian picornaviruses and to evaluate their classification, we have determined complete VP1 sequences for 19 of the 20 serotypes. Phylogenetic analysis showed that A13, SV19, SV26, SV35, SV43, and SV46 are members of human enterovirus species A, a group that contains enterovirus 71 and 11 of the coxsackie A viruses. SA5 is a member of human enterovirus species B, which contains the echoviruses, coxsackie B viruses, coxsackievirus A9, and enterovirus 69. SV6, N125, and N203 are related to one another and, more distantly, to species A human enteroviruses, but could not be definitely assigned to a species. SV4 and SV28 are closely related to one another and to A-2 plaque virus, but distinct from other enteroviruses, suggesting that these simian viruses are members of a new enterovirus species. SV2, SV16, SV18, SV42, SV44, SV45, and SV49 are related to one another but distinct from viruses in all other picornavirus genera, suggesting that they may comprise a previously unknown genus in Picornaviridae. Several simian virus VP1 sequences (N125 and N203; SV4 and SV28; SV19, SV26, and SV35; SV18 and SV44; SV16, SV42, and SV45) are greater than 75% identical to one another (and/or greater than 85% amino acid identity), suggesting that the true number of distinct serotypes among the viruses surveyed is less than 20.     

High rates of detection of respiratory viruses in tonsillar tissues from children with chronic adenotonsillar disease

Chronic tonsillar diseases are an important health problem, leading to large numbers of surgical procedures worldwide. Little is known about pathogenesis of these diseases. In order to investigate the role of respiratory viruses in chronic adenotonsillar diseases, we developed a cross-sectional study to determine the rates of viral detections of common respiratory viruses detected by TaqMan real time PCR (qPCR) in nasopharyngeal secretions, tonsillar tissues and peripheral blood from 121 children with chronic tonsillar diseases, without symptoms of acute respiratory infections. At least one respiratory virus was detected in 97.5% of patients. The viral co-infection rate was 69.5%. The most frequently detected viruses were human adenovirus in 47.1%, human enterovirus in 40.5%, human rhinovirus in 38%, human bocavirus in 29.8%, human metapneumovirus in 17.4% and human respiratory syncytial virus in 15.7%. Results of qPCR varied widely between sample sites: human adenovirus, human bocavirus and human enterovirus were predominantly detected in tissues, while human rhinovirus was more frequently detected in secretions. Rates of virus detection were remarkably high in tonsil tissues: over 85% in adenoids and close to 70% in palatine tonsils. In addition, overall virus detection rates were higher in more hypertrophic than in smaller adenoids (p = 0.05), and in the particular case of human enteroviruses, they were detected more frequently (p = 0.05) in larger palatine tonsils than in smaller ones. While persistence/latency of DNA viruses in tonsillar tissues has been documented, such is not the case of RNA viruses. Respiratory viruses are highly prevalent in adenoids and palatine tonsils of patients with chronic tonsillar diseases, and persistence of these viruses in tonsils may stimulate chronic inflammation and play a role in the pathogenesis of these diseases.     

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.     

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.     

First Detection of an Enterovirus C99 in a Captive Chimpanzee with Acute Flaccid Paralysis,from the Tchimpounga Chimpanzee Rehabilitation Center,Republic of Congo

Enteroviruses, members of the Picornaviridae family, are ubiquitous viruses responsible for mild to severe infections in human populations around the world. In 2010 Pointe-Noire, Republic of Congo recorded an outbreak of acute flaccid paralysis (AFP) in the humans, caused by wild poliovirus type 1 (WPV1). One month later, in the Tchimpounga sanctuary near Pointe-Noire, a chimpanzee developed signs similar to AFP, with paralysis of the lower limbs. In the present work, we sought to identify the pathogen, including viral and bacterial agents, responsible for this illness. In order to identify the causative agent, we evaluated a fecal specimen by PCR and sequencing. A Human enterovirus C, specifically of the EV-C99 type was potentially responsible for the illness in this chimpanzee. To rule out other possible causative agents, we also investigated the bacteriome and the virome using next generation sequencing. The majority of bacterial reads obtained belonged to commensal bacteria (95%), and the mammalian virus reads matched mainly with viruses of the Picornaviridae family (99%), in which enteroviruses were the most abundant (99.6%). This study thus reports the first identification of a chimpanzee presenting AFP most likely caused by an enterovirus and demonstrates once again the cross-species transmission of a human pathogen to an ape.     

Oncolytic enteroviruses

The growing body of knowledge concerning the molecular biology of viruses and virus-cell interactions provides possibilities to use viruses as a tool in an effort to treat malignant tumors. As a rule, tumor cells are highly sensitive to viruses, which can be used in cancer therapy. At the same time, the application of viral oncolysis in cancer treatment requires that the highest possible safety be ensured for both the patient and environment. Human enteroviruses are a convenient source for obtaining oncolytic virus strains, since many of them are nonpathogenic for humans or cause mild disease. The current progress in genetic engineering enables the development of attenuated enterovirus variants characterized with high safety and selectivity. This review focuses on the main members of the Enterovirus genus, such as ECHO, coxsackievirus, and vaccine strains of poliovirus as a promising source for the development of oncolytic agents applicable for cancer therapy. We have summarized the data concerning recently developed and tested oncolytic variants of enteroviruses and discusses the perspectives of their application in cancer therapy, as well as problems associated with their improvement and practical use.     

Development of an enterovirus specific PCR method for the quantification of enterovirus genomes in blood of diabetes patients

Background: Insulin-dependent diabetes mellitus or type 1 diabetes is a disease with a diverse aetiology. Epidemiological studies examining newly diagnosed, recent onset IDDM patients have suggested a role for viruses in the aetiology of IDDM (Yoon, 1995, Diabetes/Metabolism Reviews 11, 83–107). Important candidates are the enteroviruses, in particular coxsackieviruses B3 and B4. The latter can cause diabetes in animals (Clements et al., 1995, Lancet 346, 221–223).Objectives: We have developed a quantitative PCR method for the detection of enterovirus genomes in biological samples. The quantitative PCR will be used to screen for enteroviruses in blood of diabetes patients and their relatives by testing a Blood Diabetes Register.Study design: A substantial amount of data has been collected on enterovirus induced IDDM, our study is original in so far as it will be: (1) a quantitative study, not only the presence of viral genome sequences in blood will be determined, but also their concentrations (viral load); and (2) a longitudinal study, samples are and will be collected as a function of time. Positive PCR samples will be quantified using the standard addition method.Results: The test is specific for enteroviruses, since all enteroviruses were detected with equal sensitivity. Viruses belonging to other picornavirus genera scored negative (even up to 3×10⁶ genome copies). An equal detection limit of 10 genome copies was found for all enteroviruses.Conclusions: The developed method will permit us to generate quantitative and longitudinal data of enterovirus genomes in blood of diabetes patients and their relatives, which might help in the elucidation of the relationship between enteroviruses and IDDM.     

Cell and tissue tropism of enterovirus 71 and other enteroviruses infections

Enterovirus 71 (EV71) is a member of Picornaviridae that causes mild and self-limiting hand, foot, and mouth disease (HFMD). However, EV71 infections can progress to polio-like paralysis, neurogenic pulmonary edema, and fatal encephalitis in infants and young children. Large EV71 outbreaks have been reported in Taiwan, China, Japan, Malaysia, Singapore, and Australia. This virus is considered a critical emerging public health threat. EV71 is an important crucial neurotropic enterovirus for which there is currently no effective antiviral drug or vaccine. The mechanism by which EV71 causes severe central nervous system complications remains unclear. The interaction between the virus and the host is vital for viral replication, virulence, and pathogenicity. SCARB2 or PSGL-1 receptor binding is the first step in the development of viral infections, and viral factors (e.g., 5′ UTR, VP1, 3C, 3D, 3′ UTR), host factors and environments (e.g., ITAFs, type I IFN) are also involved in viral infections. The tissue tropism and pathogenesis of viruses are determined by a combination of several factors. This review article provides a summary of host and virus factors affecting cell and tissue tropism and the pathogenesis of enteroviruses.     

Nucleotide and nucleoside-based drugs: past,present, and future

Nucleotide and nucleoside-based analogue drugs are widely used for the treatment of both acute and chronic viral infections. These drugs inhibit viral replication due to one or more distinct mechanisms. It modifies the virus's genetic structure by reducing viral capacity in every replication cycle. Their clinical success has shown strong effectiveness against several viruses, including ebolavirus, hepatitis C virus, HIV, MERS, SARS-Cov, and the most recent emergent SARS-Cov2. In this review, seven different types of inhibitors have been selected that show broad-spectrum activity against RNA viruses. A detailed overview and mechanism of actionof both analogues are given, and the clinical perspectives are discussed. These inhibitors incorporated the novel SARS-CoV-2 RdRp, further terminating the polymerase activity with variable efficacy. The recent study provides a molecular basis for the inhibitory activity of virus RdRp using nucleotide and nucleoside analogues inhibitors. Furthermore, to identify those drugs that need more research and development to combat novel infections. Consequently, there is a pressing need to focus on present drugs by establishing their cell cultures. If their potencies were evidenced, then they would be explored in the future as potential therapeutics for novel outbreaks.     

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.     

Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

RNA viruses can rapidly mutate and acquire resistance to drugs that directly target viral enzymes, which poses serious problems in a clinical context. Therefore, there is a growing interest in the development of antiviral drugs that target host factors critical for viral replication, since they are unlikely to mutate in response to therapy. We recently demonstrated that phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) and its product phosphatidylinositol-4-phosphate (PI4P) are essential for replication of enteroviruses, a group of medically important RNA viruses including poliovirus (PV), coxsackievirus, rhinovirus, and enterovirus 71. Here, we show that enviroxime and GW5074 decreased PI4P levels at the Golgi complex by directly inhibiting PI4KIIIβ. Coxsackievirus mutants resistant to these inhibitors harbor single point mutations in the non-structural protein 3A. These 3A mutations did not confer compound-resistance by restoring the activity of PI4KIIIβ in the presence of the compounds. Instead, replication of the mutant viruses no longer depended on PI4KIIIβ, since their replication was insensitive to siRNA-mediated depletion of PI4KIIIβ. The mutant viruses also did not rely on other isoforms of PI4K. Consistently, no high level of PI4P could be detected at the replication sites induced by the mutant viruses in the presence of the compounds. Collectively, these findings indicate that through specific single point mutations in 3A, CVB3 can bypass an essential host factor and lipid for its propagation, which is a new example of RNA viruses acquiring resistance against antiviral compounds, even when they directly target host factors.     

肠道病毒调控固有免疫的分子机制研究进展

肠道病毒属于小核糖核酸病毒科,包括脊髓灰质炎病毒等多种重要人类病原体,已成为全球公共卫生安全的重大威胁之一。固有免疫是机体早期抵御病毒感染的重要防线。不同肠道病毒在进化中已经具备了多种途径躲避免疫识别或诱导固有免疫系统失活。本文重点对肠道病毒调控宿主固有免疫的相关分子机制进行综述,系统整理了肠道病毒逃避干扰素依赖与干扰素非依赖的抗病毒固有免疫防御的分子特征与作用规律,为肠道病毒致病机制的探究和抗病毒药物的研发提供参考。     

Viral Etiology of Respiratory Tract Infections in Children at the Pediatric Hospital in Ouagadougou (Burkina Faso)

Background

Acute respiratory infections (ARIs) are a major cause of morbidity and mortality in children in Africa. The circulation of viruses classically implicated in ARIs is poorly known in Burkina Faso. The aim of this study was to identify the respiratory viruses present in children admitted to or consulting at the pediatric hospital in Ouagadougou.

Methods

From July 2010 to July 2011, we tested nasal aspirates of 209 children with upper or lower respiratory infection for main respiratory viruses (respiratory syncytial virus (RSV), metapneumovirus, adenovirus, parainfluenza viruses 1, 2 and 3, influenza A, B and C, rhinovirus/enterovirus), by immunofluorescence locally in Ouagadougou, and by PCR in France. Bacteria have also been investigated in 97 samples.

Results

153 children (73.2%) carried at least one virus and 175 viruses were detected. Rhinoviruses/enteroviruses were most frequently detected (rhinovirus n = 88; enterovirus n = 38) and were found to circulate throughout the year. An epidemic of RSV infections (n = 25) was identified in September/October, followed by an epidemic of influenza virus (n = 13), mostly H1N1pdm09. This epidemic occurred during the period of the year in which nighttime temperatures and humidity were at their lowest. Other viruses tested were detected only sporadically. Twenty-two viral co-infections were observed. Bacteria were detected in 29/97 samples with 22 viral/bacterial co-infections.

Conclusions

This study, the first of its type in Burkina Faso, warrants further investigation to confirm the seasonality of RSV infection and to improve local diagnosis of influenza. The long-term objective is to optimize therapeutic management of infected children.     

New approaches for enhanced detection of enteroviruses from Hawaiian environmental waters

Health risks associated with sewage-contaminated recreational waters are of important public health concern. Reliable water monitoring systems are therefore crucial. Current recreational water quality criteria rely predominantly on the enumeration of bacterial indicators, while potentially dangerous viral pathogens often remain undetected. Human enteric viruses have been proposed as alternative indicators; however, their detection is often hindered by low viral concentrations present in the environment. Reported here are novel and effective laboratory protocols for viral concentration and highly sensitive and optimized RT-PCR for the efficient detection of enteroviruses, an important enteric virus subset, in Hawaiian environmental waters. Eighteen published enterovirus primer pairs were comparatively evaluated for detection sensitivity. The primer set exhibiting the lowest detection limit under optimized conditions, EQ-1/EQ-2, was validated in a field survey of 22 recreational bodies of water located around the island of Oahu, Hawaii. Eleven sites tested positive for enterovirus, indicating fecal contamination at these locations. As an additional means of viral concentration, shellfish were collected from 9 sample sites and subjected to dissection, RNA extraction, and subsequent RT-PCR. Shellfish tissue from 6 of 9 sites tested positive for enterovirus. The techniques implemented here are valuable resources to aid accurate reflection of microbial contamination in Hawaii's environmental waters.     

Evaluation of a Viral Microarray Based on Simultaneous Extraction and Amplification of Viral Nucleotide Acid for Detecting Human Herpesviruses and Enteroviruses

In this study, a viral microarray based assay was developed to detect the human herpesviruses and enteroviruses associated with central nervous system infections, including herpes simplex virus type 1, type 2 (HSV1 and HSV2), Epstein-Barr virus (EBV), cytomegalovirus (CMV), enterovirus 71 (EV71), coxsackievirus A 16 (CA16) and B 5(CB5). The DNA polymerase gene of human herpesviruses and 5’-untranslated region of enteroviruses were selected as the targets to design primers and probes. Human herpesviruses DNA and enteroviruses RNA were extracted simultaneously by using a guanidinium thiocyanate acid buffer, and were subsequently amplified through a biotinylated asymmetry multiplex RT-PCR with the specific primer of enteroviruses. In total, 90 blood samples and 49 cerebrospinal fluids samples with suspected systemic or neurological virus infections were investigated. Out of 139 samples, 66 were identified as positive. The specificities of this multiplex RT-PCR microarray assay were over 96% but the sensitivities were various from 100% for HSV1, HSV2, EV71 and CB5, 95.83% for CMV, 80% for EBV to 71.43% for CA16 in comparison with reference standards of TaqMan qPCR/qRT-PCR. The high Kappa values (>0.90) from HSV1, HSV2, CMV, EV71 and CB5 were obtained, indicating almost perfect agreement in term of the 5 viruses detection. But lower Kappa values for EBV (0.63) and CA16 (0.74) displayed a moderate to substantial agreement. This study provides an innovation of simultaneous extraction, amplification, hybridization and detection of DNA viruses and RNA viruses with simplicity and specificity, and demonstrates a potential clinical utility for a variety of viruses’ detection.     

Prevalence of infection with nonpolio enteroviruses among healthy children from a children's home and in a summer camp

The sera obtained at 6 samplings between June 1981 and May 1982 from 30 children in a children's home were tested for neutralizing antibodies to coxsackieviruses B1 through B5, echovirus serotypes 1, 2, 4, 5, 6, 7, 11, 19, 24, 30 and to enterovirus type 71. Another group of examined children comprised 19 individuals from the children's home and 63 children from families who enrolled as participants of a summer camp recreation. Samples of their sera were obtained at the beginning and the end of camping in July and August of 1981. Virus isolations were attempted in 150 stool specimens collected at various times during the observation period, and in specimens of summer camp swimming pool water collected 2-3 times a week. Overall, 10 strains of enterovirus (CB2, E2, E19, E24) were isolated from stool, but none from swimming pool water. Serologically, fourfold or greater increases in titre of antibody to at least one of the enteroviruses tested were observed 17 children, of whom 4 children showed antibody titre increases simultaneously to two enterovirus serotypes. None of the present viruses showed a tendency to mass spread. Some of the examined children showed the presence of serum antibody to enterovirus before its isolation from stool. Eight of the ten children positive for enterovirus in stool did not react by significant increases in titre of antibodies. Enteroviruses were more frequently isolated in children's home in autumn than in summer camp. Clinically, all virologically or serologically demonstrated enteroviral infections were asymptomatic. Children's home children had a significantly higher prevalence of antibodies to enteroviruses than children from families. Presumably, asymptomatic infections with nonpolio enteroviruses among children appear to be far more frequent than the results of neutralization tests in the study indicated. Moreover, enteroviruses may apparently circulate among humans who are seropositive for the respective enterovirus serotype.     

Sex influences immune responses to viruses,and efficacy of prophylaxis and treatments for viral diseases

The intensity and prevalence of viral infections are typically higher in males, whereas disease outcome can be worse for females. Females mount higher innate and adaptive immune responses than males, which can result in faster clearance of viruses, but also contributes to increased development of immunopathology. In response to viral vaccines, females mount higher antibody responses and experience more adverse reactions than males. The efficacy of antiviral drugs at reducing viral load differs between the sexes, and the adverse reactions to antiviral drugs are typically greater in females than males. Several variables should be considered when evaluating male/female differences in responses to viral infection and treatment: these include hormones, genes, and gender‐specific factors related to access to, and compliance with, treatment. Knowledge that the sexes differ in their responses to viruses and to treatments for viral diseases should influence the recommended course of action differently for males and females. Editor's suggested further reading in BioEssays X‐chromosome‐located microRNAs in immunity: Might they explain male/female differences Abstract     

Human Astrocytic Cells Support Persistent Coxsackievirus B3 Infection

Enteroviruses can frequently target the human central nervous system to induce a variety of neurological diseases. Although enteroviruses are highly cytolytic, emerging evidence has shown that these viruses can establish persistent infections both in vivo and in vitro. Here, we investigated the susceptibility of three human brain cell lines, CCF-STTG1, T98G, and SK-N-SH, to infection with three enterovirus serotypes: coxsackievirus B3 (CVB3), enterovirus 71, and coxsackievirus A9. Persistent infection was observed in CVB3-infected CCF-STTG1 cells, as evidenced by prolonged detection of infectious virions, viral RNA, and viral antigens. Of note, infected CCF-STTG1 cells expressed the nonfunctional canonical viral receptors coxsackievirus-adenovirus receptor and decay-accelerating factor, while removal of cell surface chondroitin sulfate from CCF-STTG1 cells inhibited the replication of CVB3, suggesting that receptor usage was one of the major limiting factors in CVB3 persistence. In addition, CVB3 curtailed the induction of beta interferon in infected CCF-STTG1 cells, which likely contributed to the initiation of persistence. Furthermore, proinflammatory chemokines and cytokines, such as vascular cell adhesion molecule 1, interleukin-8 (IL-8), and IL-6, were upregulated in CVB3-infected CCF-STTG1 cells and human progenitor-derived astrocytes. Our data together demonstrate the potential of CCF-STTG1 cells to be a novel cell model for studying CVB3-central nervous system interactions, providing the basis toward a better understanding of CVB3-induced chronic neuropathogenesis.