Human Echovirus Research Articles

Genetic characterizations and molecular epidemiology of human echovirus 30 isolated from Ningxia, China

In October 2017, a small outbreak of echovirus 30 (E30) associated with aseptic meningitis in nine cases occurred at a primary school in the Ningxia Hui Autonomous Region. That year, we observed a significant increase in E30 levels in an acute flaccid paralysis (AFP) case surveillance system. To investigate their phylogenetic relationships, we determined the whole genomic sequences of 12 strains isolated from aseptic meningitis cases, AFP cases, and healthy children. We found that the E30 strains circulating in Ningxia belong to two lineages (H and J). The strains isolated in 2010, 2012, and 2016 belonged to the H lineage. In 2017, a new lineage, J, emerged as the dominant lineage. Phylogenetic trees were constructed based on the whole genome and P1, P2, and P3 regions; clustering with other types of enterovirus species B was found, suggesting that recombination events had occurred. The recombination sites were mainly in regions 2B, 2C, and 3D. This study confirmed that the E30 strains in Ningxia in 2010, 2012, and 2016 had different recombination patterns and were recombined with different enteroviruses. The 2017 epidemic E30 originated from another new lineage with a complex recombination pattern and formed an independent transmission chain in Ningxia.

Metagenomic search of viral coinfections in herpes simplex encephalitis patients

Little is known about concomitant central nervous system (CNS) infections by more than one virus. Current diagnostics are based on molecular tests for particular pathogens making it difficult to identify multi-viral infections. In the present study, we applied DNA- and RNA-based next-generation sequencing metagenomics (mNGS) to detect viruses in cerebrospinal fluids from 20 patients with herpes simplex encephalitis. Coinfection was detected in one patient: sequences in cerebrospinal fluids matched enterovirus A (2.660 reads; 4% of recovered genome) and enterovirus B (1.571 reads; 13% of recovered genome). Subsequent PCR combined with serotyping allowed to identify human echovirus 6, a representative of enterovirus B. Several other mNGS hits (human pegivirus, Merkel cell polyomavirus, human papillomavirus type 5) were not considered to represent a genuine signal as they could not be confirmed by specific RT-PCR/PCR. HSV DNA, while being detectable by PCR in every patient, was detected by mNGS in only one. In conclusion, contaminations and false signals may complicate mNGS interpretation; however, the method can be useful in diagnostics of viral coinfections in CNS, particularly in the case of rare pathogens.

Molecular detection and phylogenetic tree of echovirus in children at Thi-Qar province Iraq

The current study was conducted in Thi-Qar province, southern Iraq, A number of 100 blood samples collected from sick Children in Mohammed Al-Mousawi Hospital for Children (ages between 2 days - 3 years), males and females, during the period from November 2021 to February 2022.This study aimed to detects the presence of Echovirus RNA in patients serum by RT-PCR and compare sequences results of current study isolates with previously described isolates from other region of the world and then draw its phylogenetic tree. Based on the results recorded among 100 child have different symptoms only 9% of patients infected with Echovirus, the results also noted a significant difference in viral prevalence in babies patients. The results from the sequencing analysis indicated that three viral isolates belong to the human echovirus, out of nine positive samples for PCR analysis. The results of the viral sequencing showed that the Iraqi strain is identical to the American isolate, China's isolate and Russia's isolate ,all of them have 100% sequencing identical.

Cucurbit[7]uril as a Broad-Spectrum Antiviral Agent against Diverse RNA Viruses.

The emergence and re-emergence of RNA virus outbreaks highlight the urgent need for the development of broad-spectrum antivirals. Polyamines are positively-charged small molecules required for the infectivity of a wide range of RNA viruses, therefore may become good antiviral targets. Cucurbit[7]uril (CB[7]), a synthetic macrocyclic molecule, which can bind with amine-based organic compounds with high affinity, has been shown to regulate bioactive molecules through competitive binding. In this study, we tested the antiviral activity of CB[7] against diverse RNA viruses, including a panel of enteroviruses (i.e. human enterovirus A71, coxsackievirus A16, coxsackievirus B3, and echovirus 11), some flaviviruses (i.e. dengue virus and Zika virus), and an alphavirus representative Semliki forest virus. CB[7] can inhibit virus replications in a variety of cell lines, and its mechanism of action is through the competitive binding with polyamines. Our findings not only for the first time provide evidence that CB[7] can be a promising broad-spectrum antiviral agent, but more importantly, offer a novel therapeutic strategy to fight against RNA viruses by supramolecular sequestration of polyamines.

Modeling the complete kinetics of coxsackievirus B3 reveals human determinants of host-cell feedback.

Complete kinetic models are pervasive in chemistry but lacking in biological systems. We encoded the complete kinetics of infection for coxsackievirus B3 (CVB3), a compact and fast-acting RNA virus. The model consists of separable, detailed modules describing viral binding-delivery, translation-replication, and encapsidation. Specific module activities are dampened by the type I interferon response to viral double-stranded RNAs (dsRNAs), which is itself disrupted by viral proteinases. The experimentally validated kinetics uncovered that cleavability of the dsRNA transducer mitochondrial antiviral signaling protein (MAVS) becomes a stronger determinant of viral outcomes when cells receive supplemental interferon after infection. Cleavability is naturally altered in humans by a common MAVS polymorphism, which removes a proteinase-targeted site but paradoxically elevates CVB3 infectivity. These observations are reconciled with a simple nonlinear model of MAVS regulation. Modeling complete kinetics is an attainable goal for small, rapidly infecting viruses and perhaps viral pathogens more broadly. A record of this paper's transparent peer review process is included in the Supplemental information.

Determining sensitivity of novel animal-derived cell cultures to clinical isolates of human enterovirus Echovirus 11 and Coxsackievirus B5

The cell culture laboratory of Ekaterinburg Research Institute of Viral Infections established and characterized diploid cell cultures of fetal porcine larynx, lungs, kidneys and muscles for the first time. Sensitivity of the novel animal-derived cell cultures to the priority clinical isolates of human enterovirus, Coxsackievirus B5 and Echovirus 11, was evaluated. Experimental studies with a Coxsackievirus B5 (CB5–8100) strain detected high sensitivity to the virus in the diploid laryngeal and renal cells and absence of such sensitivity in the cells of the lung and muscle tissues. We also documented the total absence of sensitivity to Echovirus 11 (111/RD) in all the cell cultures under study. The results will be used for further studies of animal-derived cell cultures. Certification of the novel fetal porcine renal and laryngeal cells will open up new opportunities for a wide variety of their uses in virology for studying the etiology of enteroviral infections caused by Coxsackievirus B5.

Advances on Antiviral Activity of Morus spp. Plant Extracts: Human Coronavirus and Virus-Related Respiratory Tract Infections in the Spotlight.

(1) Background: Viral respiratory infections cause life-threatening diseases in millions of people worldwide every year. Human coronavirus and several picornaviruses are responsible for worldwide epidemic outbreaks, thus representing a heavy burden to their hosts. In the absence of specific treatments for human viral infections, natural products offer an alternative in terms of innovative drug therapies. (2) Methods: We analyzed the antiviral properties of the leaves and stem bark of the mulberry tree (Morus spp.). We compared the antiviral activity of Morus spp. on enveloped and nonenveloped viral pathogens, such as human coronavirus (HCoV 229E) and different members of the Picornaviridae family—human poliovirus 1, human parechovirus 1 and 3, and human echovirus 11. The antiviral activity of 12 water and water–alcohol plant extracts of the leaves and stem bark of three different species of mulberry—Morus alba var. alba, Morus alba var. rosa, and Morus rubra—were evaluated. We also evaluated the antiviral activities of kuwanon G against HCoV-229E. (3) Results: Our results showed that several extracts reduced the viral titer and cytopathogenic effects (CPE). Leaves’ water-alcohol extracts exhibited maximum antiviral activity on human coronavirus, while stem bark and leaves’ water and water-alcohol extracts were the most effective on picornaviruses. (4) Conclusions: The analysis of the antiviral activities of Morus spp. offer promising applications in antiviral strategies.

Relationship Between Inactivation and Genome Damage of Human Enteroviruses Upon Treatment by UV254, Free Chlorine, and Ozone.

Quantitative PCR (qPCR) is a convenient tool for monitoring virus concentrations in water and wastewater treatment trains, though it only informs about virus presence, but not infectivity. This limitation can be overcome if the relationship between infectivity loss and genome decay induced by a given disinfectant is known. Here, we performed inactivation experiments using two human enteroviruses, Coxsackievirus B5 and Echovirus 11, with three disinfection methods: low-pressure ultraviolet light (UV254), free chlorine (FC), and ozone. We compared the inactivation rates as measured by culturing to the decay rates of the whole genome, to evaluate the extent of qPCR-measurable genome damage as a function of inactivation. To determine genome damage, we used an approach that estimates damage across the full viral genome from the measured decay of multiple amplicons distributed across the viral genome. Correlations between inactivation and genome decay were observed for all viruses and all disinfection treatments, but results showed that even among closely related viruses, disinfection methods can damage the viral genome to different extents and that genome damage does not necessarily translate to inactivation. For both viruses, UV254 treatment had the closest relationship between inactivation and genome decay and with ozone, the rate of genome decay exceeded the inactivation rate. Finally, for FC, the ratios between methods were vastly different between viruses. This work provides the basis to relate qPCR measurements to infectivity loss and enables the establishment of molecular monitoring tools for assessing enterovirus inactivation during disinfection treatments of water and wastewater.

Sequential electrocoagulation-electrooxidation for virus mitigation in drinking water

Electrochemical water treatment is a promising alternative for small-scale and remote water systems that lack operational capacity or convenient access to reagents for chemical coagulation and disinfection. In this study, the mitigation of viruses was investigated using electrocoagulation as a pretreatment prior to electrooxidation treatment using boron-doped diamond electrodes. This research is the first to investigate a sequential electrocoagulation-electrooxidation treatment system for virus removal. Bench-scale, batch reactors were used to evaluate mitigation of viruses in variable water quality via: a) electrooxidation, and b) a sequential electrocoagulation-electrooxidation treatment train. Electrooxidation of two bacteriophages, MS2 and ΦX174, was inhibited by natural organic matter and turbidity, indicating the probable need for pretreatment. However, the electrocoagulation-electrooxidation treatment train was beneficial only in the model surface waters employed. In model groundwaters, electrocoagulation alone was as good or better than the combined electrocoagulation-electrooxidation treatment train. Reduction of human echovirus was significantly lower than one or both bacteriophages in all model waters, though bacteriophage ΦX174 was a more representative surrogate than MS2 in the presence of natural organic matter and turbidity. Compared to conventional treatment by ferric salt coagulant and free chlorine disinfection, the electrocoagulation-electrooxidation system was less effective in model surface waters but more effective in model groundwaters. Sequential electrocoagulation-electrooxidation was beneficial for some applications, though practical considerations may currently outweigh the benefits.

Enterovirus particles expel capsid pentamers to enable genome release

Viruses from the genus Enterovirus are important human pathogens. Receptor binding or exposure to acidic pH in endosomes converts enterovirus particles to an activated state that is required for genome release. However, the mechanism of enterovirus uncoating is not well understood. Here, we use cryo-electron microscopy to visualize virions of human echovirus 18 in the process of genome release. We discover that the exit of the RNA from the particle of echovirus 18 results in a loss of one, two, or three adjacent capsid-protein pentamers. The opening in the capsid, which is more than 120 Å in diameter, enables the release of the genome without the need to unwind its putative double-stranded RNA segments. We also detect capsids lacking pentamers during genome release from echovirus 30. Thus, our findings uncover a mechanism of enterovirus genome release that could become target for antiviral drugs.

Long-term monitoring of virus antibody titers in human intravenous immunoglobulin lots derived from donors in Japan.

Intravenous immunoglobulin (IVIG) contains immunoglobulin G against various viruses, except those that have been screened, such as human immunodeficiency and hepatitis C viruses. Antivirus titers reflect the serostatus of the blood donor population in the collection region and are of clinical interest. During the past 10 years, measles, mumps, rubella, varicella-zoster, hepatitis A and B, Epstein-Barr, and human respiratory syncytial viruses; human parainfluenza viruses 1, 2, and 3; human herpes simplex viruses 1 and 2; human herpesvirus 6; cytomegalovirus (CMV); human adenoviruses (HAdVs) 1, 2, 3, 7, and 11; human parvovirus B19; and human echovirus 9 and 11 titers in IVIG lots have been measured by a commercial testing facility. A viral neutralizing assay for CMV has been used at our facility. Herein, we summarize the measurements and results of a regression analysis of the trends in virus antibody titers. IVIG lots contained significant titers against all of the above viruses, except for HAdV 7. Three patterns-stable, increasing, and decreasing-were observed, without any drastic changes. Although these trends reflect the seroprevalence in Japan, the titers were not obviously affected by the cycle of epidemics. On the other hand, the prevalence data suggest that titers against hepatitis A virus and other viruses will decrease in the near future, although they are currently stable. Monitoring the titer of IVIG lots and seroprevalence of donor populations is important for anticipating future changes in virus antibody titers of IVIG lots and can provide useful information of clinical interest.

Molecular characterization of echovirus 12 strains isolated from healthy children in China

Human echovirus 12 (E-12) belongs to the enterovirus B species. To date, only one full-length genome sequence of E-12 (prototype strain Travis) is available in the GenBank database. This study determined the complete sequence of three E-12 strains, which were isolated from the stools of three healthy children in Yunnan, China, in 2013. We revealed that the three Yunnan E-12 strains had only 80.8–80.9% nucleotide identity and 96.4–96.8% amino acid identity with the Travis strain based on pairwise comparisons of the complete genome nucleotide and amino acid sequences. The three Yunnan strains shared 99.7% nucleotide identity and 99.1–99.5% amino acid similarity. Phylogenetic and similarity plot analyses showed that intertypic recombination occurred in the non-structural regions of the three Yunnan E-12 strains. This is the first report of the complete genome sequence of E-12 in China and it enriches the complete genome sequences of E-12 in the GenBank database.

Molecular characterization of two novel echovirus 18 recombinants associated with hand-foot-mouth disease

Human echovirus 18 (E-18) is a member of the enterovirus B species. To date, sixteen full-length genome sequences of E-18 are available in the GenBank database. In this study, we describe the complete genomic characterization of two E-18 strains isolated in Yunnan, China. Pairwise comparisons of the nucleotide sequences and the deduced amino acid sequences revealed that the two Yunnan E-18 strains had 87.5% nucleotide identity and 96.3–96.5% amino acid identity with the Chinese strain. Phylogenetic and bootscanning analyses revealed the two E-18 strains had the highest identity with other several EV-B serotypes than the other E-18 strains in the P3 coding region, especially, 3B region of the Swine Vesicular disease virus (SVDV) strain HK70, indicated that frequent intertypic recombination might have occurred in the two Yunnan strains. This study contributes the complete genome sequences of E-18 to the GenBank database and provides valuable information on the molecular epidemiology of E-18 in China.

Experimental adaptation of human echovirus 11 to ultraviolet radiation leads to resistance to disinfection and ribavirin.

Ultraviolet light in the UVC range is a commonly used disinfectant to control viruses in clinical settings and water treatment. However, it is currently unknown whether human viral pathogens may develop resistance to such stressor. Here, we investigate the adaptation of an enteric pathogen, human echovirus 11, to disinfection by UVC, and characterized the underlying phenotypic and genotypic changes. Repeated exposure to UVC lead to a reduction in the UVC inactivation rate of approximately 15 per cent compared to that of the wild-type and the control populations. Time-series next-generation sequencing data revealed that this adaptation to UVC was accompanied by a decrease in the virus mutation rate. The inactivation efficiency of UVC was additionally compromised by a shift from first-order to biphasic inactivation kinetics, a form of ‘viral persistence’ present in the UVC resistant and control populations. Importantly, populations with biphasic inactivation kinetics also exhibited resistance to ribavirin, an antiviral drug that, as UVC, interferes with the viral replication. Overall, the ability of echovirus 11 to adapt to UVC is limited, but it may have relevant consequences for disinfection in clinical settings and water treatment plants.

Integrin \u03b12\u03b21 in nonactivated conformation can induce focal adhesion kinase signaling

Conformational activation of integrins is generally required for ligand binding and cellular signalling. However, we have previously reported that the nonactivated conformation of α2β1 integrin can also bind to large ligands, such as human echovirus 1. In this study, we show that the interaction between the nonactivated integrin and a ligand resulted in the activation of focal adhesion kinase (FAK) in a protein kinase C dependent manner. A loss-of-function mutation, α2E336A, in the α2-integrin did not prevent the activation of FAK, nor did EDTA-mediated inactivation of the integrin. Full FAK activation was observed, since phosphorylation was not only confirmed in residue Y397, but also in residues Y576/7. Furthermore, initiation of downstream signaling by paxillin phosphorylation in residue Y118 was evident, even though this activation was transient by nature, probably due to the lack of talin involvement in FAK activation and the absence of vinculin in the adhesion complexes formed by the nonactivated integrins. Altogether these results indicate that the nonactivated integrins can induce cellular signaling, but the outcome of the signaling differs from conventional integrin signaling.

Cellular Metabolomics of Rhabdomyosarcoma Cell during Echovirus 30 Infection

The Human echovirus 30 causes acute aseptic meningitis. Viral replication requires energy and macromolecular precursors derived from the metabolic network of the host cell. The effect of viral infection within a host cell metabolic activity remains unclear. To study an insight of cell-virus interaction during echovirus 30 infection we used human rhabdomyosarcoma cell line. The new approach of metabolomics the 1H NMR was used to measure the level of various cellular metabolites at different times of infections and morphological examination of the cells. The 1H NMR metabolite spectrum signals were observed between uninfected and infected cells. Both uninfected and infected cells utilized the glucose through metabolic pathways and released the metabolic end products. After infection the concentration of Alanine, Lactate, Acetate, Glutamate, Tyrosine, Histidine, Phenylalanine, Creatine, Choline and Formate, were increased and all these augmented metabolites were decreased at the end of the infection. The cells showed wide-ranging lipid signals at the end of the infections, which correlates with the morphological changes as apoptosis of cells was observed. Progressive breakdown and utilization of all cellular components were observed as the infections were increased. This study is useful for monitoring the cellular metabolic changes during virus infection.

Echovirus 1 internalization negatively regulates epidermal growth factor receptor downregulation

We have demonstrated previously that the human picornavirus Echovirus 1 (EV1) triggers an infectious internalization pathway that follows closely, but seems to stay separate, from the epidermal growth factor receptor (EGFR) pathway triggered by epidermal growth factor (EGF). Here, we confirmed by using live and confocal microscopy that EGFR and EV1 vesicles are following intimately each other but are distinct entities with different degradation kinetics. We show here that despite being sorted to different pathways and located in distinct endosomes, EV1 inhibits EGFR downregulation. Simultaneous treatment with EV1 and EGF led to an accumulation of EGFR in cytoplasmic endosomes, which was evident already 15min p.i. and more pronounced after 2hr p.i. EV1 treatment led to reduced downregulation, which was proven by increased total cellular amount of EGFR. Confocal microscopy studies revealed that EGFR accumulated in large endosomes, presumably macropinosomes, which were not positive for markers of the early, recycling, or late endosomes/lysosomes. Interestingly, EV1 did not have a similar blocking effect on bulk endosomal trafficking or transferrin recycling along the clathrin pathway suggesting that EV1 did not have a general effect on cellular trafficking pathways. Importantly, EGF treatment increased EV1 infection and increased cell viability during infection. Simultaneous EV1 and EGF treatment seemed to moderately enhance phosphorylation of protein kinase C α. Furthermore, similar phenotype of EGFR trafficking could be produced by phorbol 12-myristate 13-acetate treatment, further suggesting that activated protein kinase C α could be contributing to EGFR phenotype. These results altogether demonstrate that EV1 specifically affects EGFR trafficking, leading to EGFR downregulation, which is beneficial to EV1 infection.

Surveillance and genetic characteristics of echovirus 6 strains isolated from environmental sewage and aseptic encephalitis cases

Objective To analyze the genetic characteristics of echovirus 6 (E-6) strains isolated from patients with acute meningitis/encephalitis syndrome (AMES) in 2014 and sewage samples in 2013—2014 in Shandong province and to investigate their correlations. Methods Enterovirus strains were isolated from cerebrospinal fluid, stool and throat swab samples collected from 940 cases of AMES and 96 sewage samples used for environmental surveillance. The positive isolates were identified by molecular typing method. Homologous and phylogenetic analyses based on the VP1 sequences of E-6 isolates were performed. Results Altogether 47 E-6 strains were isolated from patients with AMES in 2014, accounting for 29.56% of all isolated enteroviruses (EVs) strains. No E-6 strains were isolated from AMES cases in 2013. Data of the environmental surveillance showed that E-6 virus strains had been frequently detected in sewage samples since the summer of 2013 to the end of 2014. In total, 40 E-6 virus strains were isolated (7.87% of total isolated EVs strains) in 2013 and 139 E-6 virus strains (26.18%) in 2014. Phylogenetic analysis indicated that the E-6 isolates recruited in this study belonged to clusters A and C with high intracluster sequence identities between AMES and environmental isolates. The nucleotide identities were 98.3%-100% among cluster A E-6 virus strains isolated from AMES cases in 2014 and 96.6%-100% among cluster A E-6 virus environmental isolates during the surveillance year 2013—2014. The cluster A E-6 virus strains shared 97.1%-100% nucleotide identities between the AMES and environmental isolates. For cluster C E-6 virus strains, the nucleotide identities were 100%, 98.7%-100% and 99.1%-100%, respectively. Conclusion The epidemic of viral encephalitis in Shandong province in 2014 was associated the transmission of two lineages of E-6 virus. Environmental surveillance revealed the potential epidemic of E-6 virus even before the epidemic of viral encephalitis in Shandong province, indicating the possibility of using environmental surveillance for early warning of related diseases. Key words: Viral meningitis; Viral encephalitis; Environmental surveillance; Human echovirus 6; Sequence homology; Phylogeny

Echovirus serotypes circulating in Malaysia from 2002 to 2013.

To identify the circulating serotypes of human echovirus in Malaysia from 2002 to 2013. A total of 31 retrospective samples from non-polio acute flacid paralysis, hand-food-and-mouth disease, viral meningitis and enterovirus cases were subjected to amplification of partial VP1 gene by RT-PCR. Sequencing and phylogenetic analysis of the partial sequences identified presence of human echovirus and human coxsackie viruses. It was found that echovirus 11 was the commonly circulating serotype followed by echovirus 6, echovirus 7, echovirus 3, echovirus 9, echovirus 30 and echovirus 1 in decreasing order. Additionally two types of human coxsackie virus isolates were detected which were coxsackie A24 and B3. From the findings, there is a possibility that echovirus 11 is the predominant serotype among Malaysian patients with echovirus infection. However, a larger sample size will yield a more confident result to support this evidence.

Molecular characterization of a new human echovirus 11 isolate associated with severe hand, foot and mouth disease in Yunnan, China, in 2010.

Human echovirus 11 (E-11), a member of the species Enterovirus B, frequently causes aseptic meningitis and hand, foot and mouth disease (HFMD). We determined the complete genome sequence of strain 520K/YN/CHN/2010, isolated from a subject with HFMD and aseptic meningitis in Yunnan Province, China, in 2010. The strain shared 78.8% and 81.1% nucleotide sequence similarity with prototype strain Gregory in the complete VP1 gene and the complete genome, respectively. Only the VP2-VP3-VP1 genome region of 520K/YN/CHN/2010 was similar to that of the E-11 strain; the other genome regions were most similar to those of other members of the species Enterovirus B. Using phylogenetic analysis and sequence comparisons of the complete VP1 gene, E-11 strains could be divided into five genogroups, and the 520K/YN/CHN/2010 strain was found to belong to genogroup A. Recombination analysis showed evidence of recombination with other member of the species Enterovirus B, especially the E-9 strain MSH/KM812/2010. Persistent surveillance of HFMD pathogens might help predict potential emerging viruses and related disease outbreaks.