Echovirus Research Articles

Limits for Antibiotic Treatment Set too Narrow

The authors are right to say that purely viral infections do not require antibiotic treatment (1). However, these are rare since secondary bacterial infections frequently occur. During my 45 years as a general practitioner I have noticed that almost all patients with common cold and coughing developed purulent yellow sputum over the course of the infection. Therefore, I believe Holzinger et al. (1) should have mentioned these frequently occurring secondary bacterial infections. The German Pschyrembel Clinical Dictionary (2) mentions as causes of acute bronchitis: „[…] a viral infection with myxoviruses, ECHO viruses, adenoviruses or rhinoviruses; while primary bacterial bronchitis is rather, secondary bacterial infection complicating an existing bronchitis is common (typically caused by Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis)“. Likewise, Herold et al. (3) describe purulent sputum as a sign of secondary bacterial superinfection and then bronchopneumonia as a complication of viral bronchitis. In their article Holzinger et al. state, “Administration of antibiotics can be considered in individual patients with serious chronic diseases or immune deficiencies, because in such cases it is often difficult to rule out pneumonia“. I think that this recommendation is too restrictive. Since I know, also from surgery, that pyogenic pathogens are not viruses but bacteria, such as Staphylococcus aureus, among others, which can be treated with antibiotics (4), the following sentence (1) does not appear to be plausible: “Even in the case of bronchitis with yellow–green sputum or mild fever, the patients can be reassured that the infection is most likely viral, so antibiotic treatment is unnecessary.” The yellow–green sputum mentioned should therefore be regarded as a bacterial superinfection secondary to a viral infection, which I have always treated successfully with antibiotics in adult patients with common cold-associated bronchitis, thereby preventing bronchopneumonia as a complication.

Identification of specific antigenic epitope at N-terminal segment of enterovirus 71 (EV-71) VP1 protein and characterization of its use in recombinant form for early diagnosis of EV-71 infection

Human enterovirus 71 (EV-71) is the main etiologic agent of hand, foot and mouth disease (HFMD). We sought to identify EV-71 specific antigens and develop serologic assays for acute-phase EV-71 infection. A series of truncated proteins within the N-terminal 100 amino acids (aa) of EV-71 VP1 was expressed in Escherichia coli. Western blot (WB) analysis showed that positions around 11–21 aa contain EV-71-specific antigenic sites, whereas positions 1–5 and 51–100 contain epitopes shared with human coxsackievirus A16 (CV-A16) and human echovirus 6 (E-6). The N-terminal truncated protein of VP1, VP16–43, exhibited good stability and was recognized by anti-EV-71 specific rabbit sera. Alignment analysis showed that VP16–43 is highly conserved among EV-71 strains from different genotypes but was heterologous among other enteroviruses. When the GST-VP16–43 fusion protein was incorporated as antibody-capture agent in a WB assay and an ELISA for detecting anti-EV-71 IgM in human sera, sensitivities of 91.7% and 77.8% were achieved, respectively, with 100% specificity for both. The characterized EV-71 VP1 protein truncated to positions 6–43 aa has potential as an antigen for detection of anti-EV-71 IgM for early diagnosis of EV-71 infection in a WB format.

Complete nucleotide sequence of a human echovirus 30 strain KM/A363/09 isolated in Yunnan, China in 2009

Objective To analyze the genetic characteristics of the complete genome of a human echovirus 30 （Echo30） KM/A363/09 strain isolated in Yunnan, China in 2009.Methods Primers specif-ic for Echo30 were designed .The extracted RNA was amplified by using RT-PCR.Seven fragments covering the complete viral genome were sequenced and the complete sequences were aligned with other sequences of enterovirus reference strains downloaded from Genbank . By using Mega5.1, Geneious, RDP3 and SimPlot3.5.1 softwares, the phylogenetic and recombination analysis were carried out .Results The com-plete nucleotide sequence of KM/A363/09 isolate was 7425 bp in length, encoding 2194 amino acids.KM/A363/09 isolate was highly similar with Bastianni prototype strain showing the homology of 81.2%in nucle-otide and 95.8%in amino acid.The eight Echo30 isolates shared 81.2%-88.6%homologies in nucleotide sequences and 95.8%-97.8%in amino acid sequences .Phylogenetic analysis showed that the KM/A363/09 strain belonged to one clade of Echo 30 in China.The genetic recombination of KM/A363/09 isolate was detected in the non-structural region .Conclusion KM/A363/09 isolate belongs to one clade of Echo 30 in China indicating that the evolution of Echo 30 has occurred in China . Key words: Complete genome; Sequence analysis

Environmental surveillance of human enteroviruses in Shandong Province, China, 2008 to 2012: serotypes, temporal fluctuation, and molecular epidemiology.

Environmental surveillance is an effective approach in investigating the circulation of polioviruses (PVs) and other human enteroviruses (EVs) in the population. The present report describes the results of environmental surveillance conducted in Shandong Province, China, from 2008 to 2012. A total of 129 sewage samples were collected, and 168 PVs and 1,007 nonpolio enteroviruses (NPEVs) were isolated. VP1 sequencing and typing were performed on all isolates. All PV strains were Sabin-like, with the numbers of VP1 substitutions ranging from 0 to 7. The NPEVs belonged to 19 serotypes, and echovirus 6 (E6), E11, coxsackievirus B3 (CVB3), E3, E12, and E7 were the six main serotypes, which accounted for 18.3%, 14.8%, 14.5%, 12.9%, 9.0%, and 5.7% of NPEVs isolated, respectively. Typical summer-fall peaks of NPEV were observed in the monthly distribution of isolation, and an epidemic pattern of annual circulation was revealed for the common serotypes. Phylogenetic analysis was performed on environmental CVB3 and E3 strains with global reference strains and local strains from aseptic meningitis patients. Shandong strains formed distinct clusters, and a close relationship was observed between local environmental and clinical strains. As an EV-specific case surveillance system is absent in China and many other countries, continuous environmental surveillance should be encouraged to investigate the temporal circulation and phylogeny of EVs in the population.

Occurrence of B-cell lymphomas in patients with activated phosphoinositide 3-kinase δ syndrome

Occurrence of B-cell lymphomas in patients with activated phosphoinositide 3-kinase δ syndrome

Rubella virus genotype 1G and echovirus 9 as etiologic agents of exanthematous diseases in Brazil: insights from phylogenetic analysis

The aim of the present study was to identify the rubella virus (RV) and enterovirus (EV) genotypes detected during the Epidemiological Surveillance on Exanthematic Febrile Diseases (VIGIFEX) study and to perform phylogenetic analysis. Ten RV- and four EV-positive oropharyngeal samples isolated from cell culture were subjected to RT-PCR and sequencing. Genotype 1G and echovirus 9 (E-9) was identified in RV- and EV-positive samples, respectively. The RV 1G genotype has been persisting in Brazil since 2000-2001. No evidence of E-9 being involved in exanthematic illness in Brazil has been reported previously. Differential laboratory diagnosis is essential for management of rash and fever disease.

POTENTIALS OF SYMPTOMATIC TREATMENT OF ACUTE RESPIRATORY TRACT INFECTIONS IN CHILDREN

Acute infection of upper respiratory tract is one of the most topical medical and social problems: it is respiratory diseases that cause the majority of children’s and adults’ non-attendance of school lessons and working days. Childhood respiratory infections are characterized by prolonged clinical course. The most common causes of upper respiratory tract infections are viruses, such as rhinovirus, respiratory syncytial virus, influenza and parainfluenza viruses, adeno-, corona- and metapneumoviruses as well as Coxsackie virus and ECHO virus. Antiviral agents are efficient only when administered during first 24–48 hours from the onset of disease, and a number of such drugs have only specific activity, therefore the limitation of possibilities of etiotropic therapy of acute respiratory infections can be established. This often leads to excessive inappropriate usage of antibacterial drugs. Such symptoms as nasal stuffiness and cough which accompany acute respiratory tract infections, can significantly affect patients’ and his family’s quality of life. Symptomatic therapy is traditionally used in order to relieve these symptoms. The article contains data on potentials of one of such symptomatic drugs in treatment of upper respiratory tract infections.

Etiological and molecular-epidemiological analysis on enterovirus associated encephalitis in Zhejiang, 2008-2012

In order to investigate etiology and molecular-epidemiological characteristics of enterovirus associated encephalitis (EAE) in Zhejiang, 2008-2012. Method Cerebrospinal fluid and stool specimens were collected from suspected EAE patients, who were admitted to our hospitals. RD and Hep-2 cell lines were used to isolate enterovirus (EV). Serotypes of these EV isolates were identified through neutralization test by using serotype specific anti-sera. VP1 genes of these isolates were sequenced, compared and used for the construction of phylogenetic tree. 127 (20.6%) human enterovirus (HEV) strains were isolated from 616 samples, which were collected from 610 patients. Serotypes of these EV isolates, including 60 coxsackievirus (CV), and 67 Echovirus (E)appeared to be CVA9, CVB1, CVB3-5, E3, E4, E6, E9, E14, E25 and E30, respectively. Predominant EV serotypes on EAE from 2008 to 2012 were seen as CVB3, CVB5, E6, E30 and E30, respectively. The full length of VP1 genes from different EV isolates was between 834 and 918 nucleotides. The VP1 gene similarities between these isolates and the reference strains were from 76.7% to 85.0% (nucleotides level) and 91.1% to 97.9% (amino acids level). The VP1 genes from E6 serotype isolates appeared most diverged, reaching 20.4% (nucleotides level) and 4.8% (amino acids level). Based on the generated phylogenetic tree, all the EV isolates were fallen on the same branch of HEV-B, and the isolates in the same serotype formed one sub-branch, suggesting there existed geographical and temporal effects. E6 isolates diverged into two branchlets. EVs from HEV-B were the etiologic agents for EAE in Zhejiang province from 2008 to 2012. All these EV isolates showed 12 serotypes, with predominant isolates varied every year. E30 was determined as the most dominant serotype while serotype E6 diverged into two sub-gene types.

Prevalence of Nonpolio Enteroviruses in the Sewage of Guangzhou City, China, from 2009 to 2012

The human-pathogenic viruses in urban sewage have been extensively monitored to obtain information on circulating viruses in human communities. Enteroviruses (EVs) excreted by patients who present with diverse clinical syndromes can remain infectious in the environment for several weeks, and limited data on circulating environmental EVs are available. A 4-year (2009 to 2012) surveillance study was conducted to detect nonpolio enteroviruses (NPEVs) in the urban sewage of Guangzhou city, China. After the viruses in the sewage samples were concentrated and isolated, molecular identification was used to detect and type the NPEVs. During the 4-year study, 17 different NPEV serotypes were identified in the sewage of Guangzhou city. The most common serotypes were echovirus 11 (ECHO11), ECHO6, ECHO7, and ECHO12 and coxsackie group B viruses 5 (CVB5) and CVB3. The predominant serotypes were influenced by spatial and temporal factors and differed each year. CVB5 was commonly detected in 2009 and 2010 but was rarely isolated in 2011 and 2012. In contrast, CVB3 was not observed in 2009 and 2010 but was increasingly detected in 2011 and 2012. Our study provides an overview of the serotype distribution and circulation patterns of NPEVs in the sewage of Guangzhou, China. In the absence of a systematic EV disease surveillance system, the detection and characterization of sewage-borne NPEVs will help us better understand the changes in EV disease trends and the epidemic background of circulating EVs, which could help interpret the EV trends and warn of future outbreaks in this area.

Analysis of pathogen spectrum of Encephalitis/Meningitis in northwestern area of China

To learn the characteristics of pathogen spectrum of Encephalitis /Meningitis in northwestern area of China. Between January 1st 2009 and March 31st 2011, a total of 569 patients with clinical symptoms of Encephalitis/Meningitis were selected from the hospitals in Gansu, Qinghai,Inner Mongolia and Xinjiang province. 1514 samples of specimen were collected from the 515 patients, to detect the IgM of Japanese encephalitis virus (JEV), enterovirus (EV, including Coxsackie virus, ECHO virus and enterovirus 71), Mumps virus, Herpes simplex virus (HSV) in blood and cerebrospinal fluid. Meanwhile, Neisseria meningitis (Nm), Haemophilus influenzae Type B (Hib), Staphylococcus, Streptococcus pneumonia, Streptococcus Suis, E. Coli and Cryptococci were also identified. The detection results were analyzed by different region, time and age range. Pathogenic bacteria were identified in the specimen from 16 patients, with the rate at 3.65%, of which the dominant ones were Streptococcus pneumonia (7 patients, 43.75%). Virus were identified in the specimen from 132 patients, with the rate at 27.05%, of which the dominant types were EV and HSV, accounting for 33.33% (44 cases) and 31.82% (42 cases) respectively. The detection rate of virus showed a significant seasonal trend, with the peak appearing between June and November each year. The peak of EV detection was between July and September, with 24 cases detected out; the peak of HSV was between June and August (11 cases detected out); mumps virus was mainly found between July and December (25 cases). There was no significant time-distribution found in the detection of bacteria. The EV and HSV were mainly distributed in Gansu and Qinghai province (70 cases) ;most of mumps virus were found in Gansu province (24 cases);and JEV were only found in Gansu province (20 cases). The viral pathogen spectrum was identified in all ages, and the EV and mumps virus were mainly found in children aged 0-14 years old (42 and 17 cases respectively) ; JEV were identified in people over 15 years old, with 13 detected out of the 20 patients. The main pathogen of acute encephalitis and meningitis in northwestern area of China was virus, and the main pathogens of encephalitis and meningitis in children under 15 years were Herpes simplex virus and Mumps virus.

Molecular diagnosis of enterovirus infections in Chungnam Korea in 2010

The epidemiological data of enteroviruses isolated in Chungnam, Korea since 2005 has been reported, and this current study extends these observations in 2010. During this period, 178 of the total 920 samples (19.3%) were positive for enterovirus, and the types were identified using nucleotide sequence analysis in VP1. The temporal distribution was similar to previously reported data, however, coxsackievirus A6 and coxsackievirus B5 showed different patterns in detail. Children aged < 1 year accounted for 89 (50%) of the total 178 isolates. Most coxsackievirus A6, coxsackievirus A9 and enterovirus 71 isolates were identified in children < 5, < 6 and < 4 years old, respectively. CVB5 was distributed evenly in children < 7 years old. Enterovirus serotypes were identified by sequence determination of the VP1 region, which resolved the isolated enteroviruses into 16 types in 2010; coxsackievirus A4, A5, A6, A9, A10, A12, A16, B2, B3, B4, B5, echovirus 17, 18, 25, 30 and enterovirus 71. The prevalent enterovirus types were coxsackievirus A6 (14.6%), B5 (15.7%), and enterovirus 71 (15.7%). This is the first time E17 has been identified in Korea since surveillance began in 1993. The presentations of enterovirus-infected patients were respiratory illness (30.3%), septicemia (19.7%), central nervous system infections (16.3%), gastrointestinal infections (11.2%), herpangina or hand-foot-mouth disease (10.7%), and genitourinary (5.6%). Key words: Enterovirus, reverse transcriptase-polymerase chain reaction (RT-PCR), epidemiology, molecular type.

Differential Susceptibility and Response of Primary Human Myeloid BDCA1+ Dendritic Cells to Infection with Different Enteroviruses

Coxsackie B viruses (CVBs) and echoviruses (EVs) form the Human Enterovirus-B (HEV-B) species within the family Picornaviridae. HEV-B infections are widespread and generally cause mild disease; however, severe infections occur and HEV-B are associated with various chronic diseases such as cardiomyopathy and type 1 diabetes. Dendritic cells (DCs) are the professional antigen-presenting cells of our immune system and initiate and control immune responses to invading pathogens, yet also maintain tolerance to self-antigens. We previously reported that EVs, but not CVBs, can productively infect in vitro generated monocyte-derived DCs. The interactions between HEV-B and human myeloid DCs (mDCs) freshly isolated from blood, however, remain unknown. Here, we studied the susceptibility and responses of BDCA1+ mDC to HEV-B species and found that these mDC are susceptible to EV, but not CVB infection. Productive EV7 infection resulted in massive, rapid cell death without DC activation. Contrary, EV1 infection, which resulted in lower virus input at the same MOI, resulted in DC activation as observed by production of type I interferon-stimulated genes (ISGs), upregulation of co-stimulatory and co-inhibitory molecules (CD80, CD86, PDL1) and production of IL-6 and TNF-α, with a relative moderate decrease in cell viability. EV1-induced ISG expression depended on virus replication. CVB infection did not affect DC viability and resulted in poor induction of ISGs and CD80 induction in part of the donors. These data show for the first time the interaction between HEV-B species and BDCA1+ mDCs isolated freshly from blood. Our data indicate that different HEV-B species can influence DC homeostasis in various ways, possibly contributing to HEV-B associated pathology.

A single amino acid substitution in viral VP1 protein alters the lytic potential of clone‐derived variants of echovirus 9 DM strain in human pancreatic islets

In vitro studies with primary human pancreatic islets suggest that several enterovirus serotypes are able to infect and replicate in beta cells. Some enterovirus strains are highly cytolytic in vitro whereas others show virus replication with no apparent islet destruction. The capability to induce islet destruction is determined only partially by the virus serotype, since strain specific differences have been detected within some serotypes including echovirus 9 (E-9). In this study, the viral genetic factors determining the outcome of islet infection (i.e., destructive vs. benign) were investigated by constructing parallel infectious clones of lytic E-9-DM strain that was isolated from a small child at the clinical onset of type 1 diabetes. The capabilities of these clone-derived viruses to induce islet destruction were monitored and the lytic potential of clones was modified by site-directed mutagenesis. The lytic capabilities of these clone-derived viruses in human pancreatic islets were modified by a single amino acid substitution (T81A) in the capsid protein VP1. The data presented outline the importance of amino acid point mutations in the pathogenetic process leading to islet necrosis. However, although the amino acid substitution (T81A) modifies the lytic capabilities of E-9-DM strain-derived microvariant strains, it is likely that additional viral genetic determinants of pancreatic islet pathogenicity exist in other E-9 strains.

Molecular evolution and epidemiology of echovirus 6 in Finland

Echovirus 6 (E-6) (family Picornaviridae, genus Enterovirus) is one of the most commonly detected enteroviruses worldwide. The aim of this study was to determine molecular evolutionary and epidemiologic patterns of E-6. A complete genome of one E-6 strain and the partial VP1 coding regions of 169 strains were sequenced and analyzed along with sequences retrieved from the GenBank. The complete genome sequence analysis suggested complex recombination history for the Finnish E-6 strain. In VP1 region, the phylogenetic analysis suggested three major clusters that were further divided to several subclusters. The evolution of VP1 coding region was dominated by negative selection suggesting that the phylogeny of E-6 VP1 gene is predominantly a result of synonymous substitutions (i.e. neutral genetic drift). The partial VP1 sequence analysis suggested wide geographical distribution for some E-6 lineages. In Finland, multiple different E-6 lineages have circulated at the same time.

Evaluation of a Direct Reverse Transcription Loop-Mediated Isothermal Amplification Method without RNA Extraction for the Detection of Human Enterovirus 71 Subgenotype C4 in Nasopharyngeal Swab Specimens

Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD) worldwide and has been associated with neurological complications which resulted in fatalities during recent outbreak in Asia pacific region. A direct reverse transcription loop-mediated isothermal amplification (direct RT-LAMP) assay using heat-treated samples without RNA extraction was developed and evaluated for the detection of EV71 subgenotype C4 in nasopharyngeal swab specimens. The analytical sensitivity and specificity of the direct RT-LAMP assay were examined. The detection limit of the direct RT-LAMP assays was 1.6 of a 50% tissue culture infective dose (TCID50) per reaction and no cross-reaction was observed with control viruses including Cosackievirus A (CVA) viruses (CVA2,4,5,7,9,10,14,16, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3,6,11, and 19). The direct RT-LAMP assay was evaluated and compared to both RT-LAMP and quantitative real-time PCR (qRT-PCR) in detecting EV71 infection with 145 nasopharyngeal swab specimens. The clinical performance demonstrated the sensitivity and specificity of direct RT-LAMP was reported to be 90.3% and 100% respectively, compared to RT-LAMP, and 86.83% and 100% respectively, compared to qRT-PCR. These data demonstrated that the direct RT-LAMP assay can potentially be developed for the point of care screening of EV71 infection in China.

Analysis on epidemiology and the main clinical symptoms of viral encephalitis in Gansu, 2009-2011

To understand the epidemiological characteristics and the main clinical symptoms of viral encephalitis in Gansu. A total of 322 viral encephalitis patients were recruited from province sentinel hospitals in Gansu province from 2009 to 2011, and their basic information were collected as well as their serum samples and cerebrospinal fluid samples. 296 out of the 322 cases were qualified for our study. Based on the patients' epidemiological characteristics and clinical features, we determined the detection of the virus types (at least one kind of virus detection was carried out for each case). ELISA was applied to test the IgM antibody of Japanese encephalitis (JE) virus (JEV), enterovirus (EV: including Coxsackie virus, echovirus, enterovirus 71), mumps virus and herpes simplex virus (HSV) in cerebrospinal fluid and serum specimen. The difference of positive detected rate between types of virus, among patients from different regions, time, or at different ages, as well as the different clinical symptoms between JE patients and other viral encephalitis patients, were analyzed and compared. The positive detected rate of virus in the 296 patients was 27.03% (80/296); the positive rate of JEV, EV, mumps virus, HSV detected was separately 7.53% (22/292), 8.75% (23/263), 13.84% (22/159) and 15.09% (40/265), and the difference was statistically significant (χ(2) = 10.849, P < 0.05). 90.91% (20/22) of the JEV positive cases were distributed in Tianshui, Longnan and Pingliang, and 95.45% (21/22) patients were infected from July to September. All the 23 EV detected positive patients were infected from April to December, while the ages of patients ranged from 1 to 44 years old. Mumps virus, HSV testing positive cases had onset every month. Logistic regression analysis showed that the patients who had the symptoms as disturbance of consciousness (OR = 15.487, 95%CI: 2.266 - 105.852), somnolence (OR = 11.659, 95%CI: 1.783 - 76.242), convulsions (OR = 11.062, 95%CI: 1.687 - 72.530) were more likely to infect JEV. HSV was the principal pathogen of viral encephalitis in Gansu. An obvious central tendency in the regional and time distribution was found in JEV infection; and the clinical symptoms of JE patients were more severe.

Genomic characterization of echovirus 6 causing aseptic meningitis in Hokkaido, Japan: a novel cluster in the nonstructural protein coding region of human enterovirus B

We determined four complete nucleotide sequences of echovirus 6 (E6) isolated from an epidemic of aseptic meningitis (AM) in Hokkaido, Japan, in 2011. Phylogenetic analysis of the genes encoding viral capsid protein 1 revealed that the strains were closely related to E6 strains isolated in China in recent years, but they were distantly related to E6 strains isolated from patients with AM in Osaka Prefecture, Japan, in 2011. The genes encoding the viral protease and RNA-dependent RNA polymerase (3CD) were closely related to those of several non-E6 strains of the species Human enterovirus B isolated in China, South Korea, and Australia from 1999 to 2010, resulting in a novel cluster in the phylogenetic tree. These results suggest that the incidence of AM in Japan in 2011 was caused by at least two lineages of E6 strains, and a lineage of the 3CD gene was interspersed among different serotypic strains isolated in Western Pacific countries.

Molecular identification of enteroviruses associated with aseptic meningitis in children from India

We identified and characterized enteroviruses associated with aseptic meningitis in children between April 2009 and March 2010. Enterovirus RNA was detected in 51 (45.5 %) of 112 CSF samples. Molecular typing by RT-PCR and sequencing of a partial VP1 region revealed the predominance of echovirus (ECV) 32 (n = 20), followed by ECV 11 (n = 10), ECV 13 and ECV 14 (n = 5 each), coxsackievirus (CV) B3 and CV B6 (n = 3 each), CV A2, CV A10 and ECV 30 (n = 1 each). Phylogenetic analysis of ECV 32 showed 0 to 4 % sequence divergence among strains of the present study and 20-23 % from the prototype Puerto Rico strain at the nucleotide level. This is the first report of ECV 32 associated with an aseptic meningitis epidemic and identification of seven different enterovirus serotypes (CV A2, CV A10, CV B3, CV B6, ECV 13, ECV 14 and ECV 32) in meningitis cases from India.

Complete nucleotide sequence of a human echovirus 6 strain KM57-09 isolated in Yunnan, China, in 2009

To analyze the genetic characterization of the complete genome from a human echovirus 6 (Echo6) strain KM57-09 isolated in Yunnan, China, in 2009. Using the RT-PCR, eight fragments containing about 1000 nucleotides which covered the whole viral genome were sequenced. The sequences were aligned with other reference enterovirus sequences downloaded from the GenBank, using Mega 5.05, RDP 3 and SimPlot 3.5.1 softwares. Similar to the other human enterovirus, KM57-09 isolate genome appeared to have 7419 nucleotides in length, encoding for 2191 amino acids. In the complete genome, the rates of homology on nucleotide and amino acid among the seven Echo6 isolates were 79.3% - 80.2% and 93.3% - 94.4%, respectively as well as 79.3% and 93.6% of the rates of homology when compared with that of D' Amor prototype strain. In different segment of genome. The 2C-3A genome region was most similar to the HN-2-E25 strain, the 5' UTR, VP4, 3D and 3' UTR genome region were most similar to the CoxB5-Henan-2010. In the VP1 gene, the rates of homology on nucleotide and amino acid among the China isolates were 80.0% - 96.0% and 95.8% - 99.0%, respectively, and showed 77.6% - 96.0% and 95.2% - 99.0% of the rates on homology when compared to the other Echo6 reference strains isolated from other countries or areas, respectively. from phylogenetic analysis showed that the Echo6 formed five distinct groups, A-E. The KM57-09 strain belonged to clade E. The nucleotide divergence between clades was 15.6% - 23.3%. The putative recombinant event for KM57-09 was detected with RDP 3, SimPlot 3.5.1 and 3D sequence phylogenetic analysis. All the Echo6 isolates could be divided into five clades, the KM57-09 strain belonged to Clade E. The Echo6 strains isolated in China were contributed to several different chains of transmission.

Intercity Spread of Echovirus 6 in Shandong Province, China: Application of Environmental Surveillance in Tracing Circulating Enteroviruses

Environmental surveillance is an effective approach in investigating circulating enteroviruses and had been conducted in the cities of Jinan and Linyi since February 2008 and April 2010, respectively. This study analyzed 46 sewage samples collected in the two cities in 2011 and found that echovirus 6 (E6) was the predominant serotype, with 134 isolates (65 in Jinan and 69 in Linyi) from 23 (50%) samples. This differs from the 2010 data that found 29 E6 isolates in Jinan and only 3 in Linyi. Phylogenetic analysis of the VP1 coding region showed that all environmental E6 samples from 2008 to 2011 (n = 167) segregated into two lineages and revealed an increase in VP1 gene diversity in 2011, suggesting that the increased number of E6 detections reflects a real epidemic in the two cities. Most Linyi isolates (n = 61, or 88%) in 2011 segregated into sublineage 1a, together with 18 Jinan isolates in 2011. Interestingly, the ancestral VP1 sequence of sublineage 1a inferred using the maximum-likelihood method had 100% identity with the sequence of one environmental isolate from Jinan in August 2010, suggesting an intercity spread from Jinan to Linyi. By Bayesian phylodynamic methods, the most recent common ancestor of Linyi isolates in sublineage 1a dated back to 24 December 2010, revealing that this sublineage was likely imported into Linyi from August to December in 2010. This study demonstrates that environmental surveillance is a sensitive method in tracing transmission pathways of circulating enteroviruses among different regions and reveals that E6-associated aseptic meningitis is an emerging concern in China.