Neurologic Manifestations according to Serotypes of Enterovirus in Pediatric Inpatient in Incheon

PURPOSE: Enterovirus infection in children can manifest various disease and enterovirus have many serotypes. This study was aimed to investigate neurologic manifestations according to serotypes of enterovirus in pediatric inpatients in Incheon. METHODS: We collected the stool samples from the admitted pediatric patients in Inha University Hospital from January 2015 to September 2016. Enterovirus detection and serotypes identification were performed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and semi-nested RT-PCR. RESULTS: A total of 527 samples were collected during study period and 170 patients (32.2%) were diagnosed with enterovirus infections. Genetic sequences of enteroviruses were identified: echovirus 18 (50, 40.5%), enterovirus 71 (12, 9.6%), coxakievirus A10 (10, 8.0%), echovirus 6 (7, 5.6%). Virus in patient with meningitis were identified: echovirus 18 (15, 75%), coxakievirus B5 (2, 10%), enterovirus 71 (2, 10%), and echovirus 6 (1, 5%). Neurologic manifestations of echovirus 18 are headache (15, 30%), vomiting (17, 34%), meningeal irritation sign (10, 20.0%). And enterovirus 71 have headache (3, 25%), vomiting (3, 25%), meningeal irritation sign (2, 16.0%), seizure (1, 8.3%), neurologic sequelae (1, 8.3%). Echovirus 18 and neurologic manifestation have a statistically significant correlation with other serotypes (r=0.701, P < 0.01) CONCLUSION: Echovirus 18 infection was more prominent in neurological symptoms than in other serotypes. The major serotype of meningitis was echovirus 18 but there was no reported neurologic sequelae. Enterovirus infection has different neurological symptoms, depending on the serotypes.

Genetic Characteristics of Echovirus Type 6 Isolated from Hunan Province, China, 2009-2014 / 病毒学报

We wished to understand the genetic characteristics of enteric cytopathic human orphan (ECHO) virus type 6 (ECHO6) circulating in China. First, the partial VP1 coding region of six strains of the ECH-O6 virus isolated from cases of hand, foot and mouth diseases during routine surveillance in Hunan Province (China) from 2009 to 2014 were sequenced. Those sequences were analyzed along with 138 sequences of ECHO viruses covering five provinces of China and countries outside China retrieved from the GenBank database. A phylogenetic tree based on partial VPI was constructed, and it indicated that Chinese strains of the ECHO virus could form two distinct evolutionary branches: branch 1 and branch 2. All isolates of the ECHO virus from Hunan Province belonged to the 2c subranch, which revealed that they may share a common evolutionary origin. ECHO strains in branch 2 may be the predominant strains in China due to their wide geographic distribution and long period of circulation. We used nucleotide differences of >30%o as the basis of cluster division. ECHO, viruses could be divided into four clusters (A-D). Cluster D could be divided further into ten subclusters on the basis of nucleotide differences of 15%-30%. All ECHO6 isolates from Hunan Province belonged to the D7 subcluster. These data showed that the ECHO6 strains that circulated in Hunan Province in 2009-2014 were closely related to each other, and probably shared a common evolutionary origin. In addition, at least four distinct lineages of ECHO viruses have circulated in China.

Analysis of genetic characteristics of ECHO6 virus isolated from an epidemic outbreak of encephalitis in Longyan, China / 病毒学报

This study aimed to analyze the etiology of the encephalitis outbreak in Longyan, Fujian Province, China in 2010, in order to provide valuable information for this prevention and control of this disease. Pathogens were confirmed from cerebrospinal fluid samples with fluorescent RT-PCR, virus isolation (RD cells), and neutralization tests. Then, the VP1 fragments or whole genome nucleotide sequences were determined for four virus strains using PCR. Homology was assessed using the MegAlign software, and a phylogenetic evolutionary tree was drawn using Mega 4.0 software. The results confirmed that the etiology of the outbreak was the ECHO6 intestinal virus, and the nucleotide sequence of the VP1 segment indicated that the C2 subtype was responsible. The genome sequence consisted of 7407 nucleotides, and resembled the genome of other ECHO and CoxB viruses with homology levels of 78.5%-87.3%. The encephalitis outbreak in Longyan in 2010 was caused by the ECHO6 C2 subtype intestinal virus, and its complete genome sequence length is similar to the standard strain (U16283) with a sequence homology of 80.4%.

Genetic characterization of echovirus 6 isolated from meningitis and encephalitis cases in Shandong Province, China / 病毒学报

To analyze the genetic characteristics of echovirus 6 (E6) isolated from meningitis and encephalitis cases in Shandong Province, China, we collected cerebrospinal fluid samples from meningitis and encephalitis cases in Shandong Province from 2007 to 2012 for virus isolation. Viral RNAs were extracted from positive isolates, and complete VP1 coding regions were amplified by RT-PCR and sequenced. Homology comparison and phylogenetic analysis were performed. Six isolates were identified as E6 by microneutralization assay and molecular typing. The homology analysis showed that the six isolates had 78. 6%-99. 8% nucleotide and 95. 5%-100. 0% amino acid identities with each other, as well as 76. 9%-78. 4% nucleotide and 92. 3%-95. 1% amino acid identities with the prototype strain (D' Amori). The phylogenetic analysis based on the integrated VP1 sequences indicated that all Shandong E6 isolates could be separated into four clusters, designated as A, B, C, and D. The six E6 isolates belonged to clusters A, B, and D. Our study reveals high genetic differences between Shandong E6 isolates and suggests different transmission lineages of E6 co-circulated in Shandong Province.

Genetic Diversity of Echovirus 6 Strains Circulating in Korea

Echovirus 6 (ECV6) is the prevalent serotype detected in aseptic meningitis cases in Korea. To analyze the genetic variation of ECV6 isolates recently circulating in Korea, we determined the partial sequence of the VP1 capsid gene from 22 Korean ECV6 isolates and performed pairwise analysis against 42 reference strains from the GenBank database using MegAlign. The 22 Korean ECV6 isolates formed 3 distinct genetic clusters: Kor-lineage I, II, and III. The Korean ECV6 strains showed significant genetic diversity with 14.8~22.8% nucleotide divergence among the 3 different lineages. These ECV6 Kor-lineages were demonstrated to belong to different genetic clusters using VP1 sequence-based phylogenetic analysis, implying that the recently circulating Korean ECV6 strains have potential antigenic variation.

Recently Prevalent Infectious Diseases among Children: Meningitis due to Enteroviral Infection

Enterovirus infections occur throughout the year, but in temperate climates infections are strikingly more prevalent in the summer and autumn. About 50~80% of nonpolio enteroviral infections are completely asymptomatic. The fecal-oral route is thought to be the predominant mode of enterovirus transmission, because viral shedding from the gastrointestinal tract is more prolonged than is shedding from the upper respiratory tract. Even symptomatic infections usually produce undifferentiated febrile illnesses lasting a few days, and often accompanied by symptoms of upper respiratory tract. It has been known that the most common cause of aseptic meningitis is enteroviruses, and this type of meningitis demonstrates benign clinical course and the absence of signs of parenchymal brain involvement. We could see many children with meningitis in the summer of 2008. Among 1,922 children with aseptic meningitis from 1 week to 29 weeks of 2008, 882 (49.9%) was 3~6 years old, and 883 (50.1%) was 7~14 years old. Echovirus 30 (60%) and Echovirus 6 (30%) caused large outbreaks throughout South Korea from May to August 2008, which was analyzed by the Division of Enteric and Hepatitis Viruses, National Institute of Health in Seoul, Korea.

Study on an outbreak of echovirus type 6 of meningitis in Liu'an city, Anhui province / 中华流行病学杂志

<p><b>OBJECTIVE</b>To understand the epidemiological, clinical and etiological characteristics of an Echovirus type 6 meningitis outbreak in Jinzhai county, Liu'an city in Anhui, and to find out the proper way in controlling the aseptic meningitis outbreak.</p><p><b>METHODS</b>A surveillance system for aseptic meningitis was established in Jinzhai to confirm the case definition. Stool or cerebrospinal fluid (CSF) specimens from some cases were collected for entero-viruses isolation and identification. Case-control study was conducted. The case group involved patients while the controls would include: patients' classmate with same gender and the age difference was not over one year. Neutralization antibody in serum specimens were collected and tested in cases and in healthy people.</p><p><b>RESULTS</b>105 cases were distributed in 17 of the 30 towns in Jinzhai county while 41.0% of the cases were in Banzhuyuan town with an incidence rate of 203/10(5). Cases were clustered by school and classroom with age ranging from 3 to 15 years old and the highest as 10.9/10(5) in the 6 to 10 group. The incidence in males was 24.2/10(5) compared to 8.4/10(5) in females. The main clinic characteristics of cases were: fever, headache and vomiting. Echovirus type 6 from 25 of the 72 CSF samples (35%) was isolated. When comparing the cases group with control group, the OR of drinking home-made beverages was 4.1 (95% CI: 1.4-12.0), especially the beverages sacked by plastic bag: 3.3 (95% CI: 1.3-8.8). 6 out of 7 workers engaging in producing home-made beverages were detected to have carried Echovirus type 6 from their stool specimens. The Echovirus type 6 neutralization antibody positive rate in cases (73.5%) was significantly higher than that in 100 healthy people (46.0%) (X2 = 12. 526, P = 0.000).</p><p><b>CONCLUSION</b>This episode of meningitis outbreak was caused by Echovirus type 6. The proportion of drinking home-made beverages, especially the beverages sacked by plastic bag in cases group was higher than in control group.</p>

Updates on Enterovirus Surveillance in Korea

PURPOSE: We identified the causative viruses from patients with aseptic meningitis, acute hemorrhagic conjunctivitis and other enterovirus-related diseases to understand the epidemiological patterns and prevailing strains of enterovirus infections each year. MATERIALS AND METHODS: During 1999-2003, we examined 3,260 specimens from 2,939 patients with aseptic meningitis or other clinical manifestations for the presence of enteroviruses by using both cell culture/ neutralisation test and reverse transcription-polymerse chain reaction-sequencing. To investigate the etiological agents which caused an epidemic of acute haemorrhagic conjunctivitis, conjunctival swab samples from acute haemorrhagic conjunctivitis patients showing cytopathic effects in HEp2 cells were tested by enteroviral specific PCR. RESULTS: We identified 603 isolates of enteroviruses (20.5%) among 2,939 cases and 22 serotypes of human enteroviruses were isolated during this 5 year period. Echovirus 13 and coxsackievirus A24 in 2002 and coxsackievirus A9 in 2003 were the first enterovirus to be indentified in Korea since we began the enterovirus surveillance in 1993. While an epidemic of echovirus 13 infection in Korea began in Gwangju and Jeolla province in 2002 and spread to Seoul, Gyunggi, Busan, Ulsan and other regions, echovirus 6 isolates in 2002 were mainly detected in Busan specimens and some Gwangju samples. From the nucleotide sequencing of enteroviral PCR products of conjunctival swab specimens, we found 85% nucleotide homology to coxsackievirus A24 (D90457). CONCLUSIONS: We isolated 603 enteroviral isolates among 2939 cases during 1999-2003. Echovirus 13 and coxsackievirus A24 were the first enterovirus to be identified in Korea and caused nationwide epidemics in 2002.

Updates on Enterovirus Surveillance in Korea

PURPOSE: We identified the causative viruses from patients with aseptic meningitis, acute hemorrhagic conjunctivitis and other enterovirus-related diseases to understand the epidemiological patterns and prevailing strains of enterovirus infections each year. MATERIALS AND METHODS: During 1999-2003, we examined 3,260 specimens from 2,939 patients with aseptic meningitis or other clinical manifestations for the presence of enteroviruses by using both cell culture/ neutralisation test and reverse transcription-polymerse chain reaction-sequencing. To investigate the etiological agents which caused an epidemic of acute haemorrhagic conjunctivitis, conjunctival swab samples from acute haemorrhagic conjunctivitis patients showing cytopathic effects in HEp2 cells were tested by enteroviral specific PCR. RESULTS: We identified 603 isolates of enteroviruses (20.5%) among 2,939 cases and 22 serotypes of human enteroviruses were isolated during this 5 year period. Echovirus 13 and coxsackievirus A24 in 2002 and coxsackievirus A9 in 2003 were the first enterovirus to be indentified in Korea since we began the enterovirus surveillance in 1993. While an epidemic of echovirus 13 infection in Korea began in Gwangju and Jeolla province in 2002 and spread to Seoul, Gyunggi, Busan, Ulsan and other regions, echovirus 6 isolates in 2002 were mainly detected in Busan specimens and some Gwangju samples. From the nucleotide sequencing of enteroviral PCR products of conjunctival swab specimens, we found 85% nucleotide homology to coxsackievirus A24 (D90457). CONCLUSIONS: We isolated 603 enteroviral isolates among 2939 cases during 1999-2003. Echovirus 13 and coxsackievirus A24 were the first enterovirus to be identified in Korea and caused nationwide epidemics in 2002.

Clinical and Virologic Study of Aseptic Meningitis / 소아과

PURPOSE: The cause of aseptic meningitis remains mostly unknown because viral culture and identification is difficult. Thus, we report a study on 123 children with aseptic meningitis in Gyeongju in 2002 to identify the causing virus and the relationship with the clinical manifestation. METHODS: We prospectively investigated the patients, admitted to Dongguk University Hospital, into two groups between April and October 2002. Group 1 included 123 patients diagnosed as aseptic meningitis. Group 2, the adimssion control, included 120 patients, who suffered from none-enteroviral diseases. Specimens of CSF and stool were collected to perform reverse transcription polymerase chain reaction(RT-PCR), and enteroviral culuture was done in RT-PCR positive patients as well. RESULTS: The male to female sex ratio was 2.2:1 and the mean age was 6.2+/-3.7 years. The clinical manifestations were fever, headache and vomiting. The RT-PCR for enterovirus, performed in 58 cases of CSF in group 1, showed 5.2% positive results and negative result in viral culture. The RT-PCR for enterovirus used in stool specimens showed 89.3% and 41.1% of positive results in group 1 and group 2, respectively. Viral culture of stool specimens showed five cases of echovirus 13 and four cases of echovirus 6 in group 1, whereas three cases of echovirus 6 and one case of coxsackie B4 were detected in group 2. CONCLUSION: The etiologic viruses of the aseptic meningitis outbreak in Gyeongju in 2002 is presumed to be echovirus 13 and echovirus 6. Since echovirus 13 firstly appeared with various age distributions, the outbreak may have emerged due to a lack of acquisition of immunity to this virus.

A Clinical Study of Aseptic Meningitis in the Busan Area in 2002

PURPOSE: There was a outbreak of aseptic meningitis in Busan, 2002. We report the clinical features and causative viruses. METHODS: Two hundred seventy six children with aseptic meningitis who were admitted to the Department of Pediatrics, Maryknoll Hospital between January and December, 2002 were included. CSF, stool and throat swab viral cultures were done in 244 of these children. RESULTS: The male to female ratio was 1.4 : 1. Age of patients varied from five months to fourteen years old. Average age was 5.7+/-6.2 years old and mostly between one and four years.(41.3%) Monthly distribution revealed that the number of patients increased from May to July. The main symptoms were fever, headache and vomiting in this order. Respiratory tract infection symptom was associated from June to July and headache and nausea without fever were characteristically observed in children more than 10 years old from November to December. In peripheral blood examination, leukocytosis(WBC>10,000/mm3) showed in 34.8%, ESR was increased in 56.1%, and CRP was positive value in 61%. Therefore differential diagnosis was difficult through peripheral blood examination. CSF findings revealed mean leukocyte count 86.5+/-180.2/mm3, protein 41.7+/-32.9 mg/dL, glucose 56.4+/-9.9 mg/dL. Median hospitalized period was 4.7+/-7.2 days and compared with non-tapping group, hospitalized period was shorter and subsidance of symptoms was faster, therefore antibiotics injection period was shorter in the spinal tapping group. Virus was isolated in 31 cases of 244. The causative agents were echovirus 6, echovirus 9, echovirus 25, coxsakie virus B3, B4. CONCLUSION: There was an epidemics of aseptic meningitis in Busan, 2002; the causative agent was echovirus 6, 9, 25, coxsakie virus B3, B4.

A Clinical Study of Aseptic Meningitis in Ulsan from May to July, 2002

PURPOSE: There occurred an explosive outbreak of aseptic meningitis in Ulsan, in the year of 2002. In this study, we report clinical manifestations, laboratory data and causative viruses of the outbreak. METHODS: Were assessed 371 children with aseptic meningitis who were admitted to the Department of Pediatrics, Ulsan University Hospital in Ulsan between May and July, 2002. We reviewed the sex, age, clinical manifestations, peripheral blood examinations and CSF examinations of the patients. Virus isolation and serotype identification were performed by cell cultures of cerebrospinal fluid and stool. RESULTS: The ratio of males to females was 1.9:1. The majority of aseptic meningitis occurred in children between 1 and 5 years of age with a mean age of 6.8+/-5.1 years. The clinical manifestations was fever, headache, vomiting, and abdominal pain and 16.2% of the patients developed neck stiffness. The mean duration of fever was 3.2 days. The mean value of peripheral blood leukocytes was 10,355+/-4,800/mm3 and the mean value of CRP was 0.96+/-1.67 mg/dL. Initial CSF findings revealed leukocytes 212+/-385/mm3, protein 37.0+/-18.9 mg/dL and glucose 68.3+/-17.6 mg/dL. Viruses were isolated 67(18.1%) out of 371 patients and the serotypes were echovirus 6 in 10 cases, echovirus 9 in 15 cases, echovirus 11 in 4 cases, echovirus 13 in 18 cases, and unclassified enterovirus in 20 cases. CONCLUSION: Aseptic meningitis was prevalent in Ulsan, in the year of 2002. The clinical manifestations and the laboratory findings of the patients were compatible with those of previous reports. The causative viruses were echovirus 6, echovirus 9, echovirus 11, echovirus 13 and unclassified enterovirus.

A Clinical Study of Aseptic Meningitis in Busan and Kyoungsangnamdo from April to July, 2002

PURPOSE: There was an outbreak of aseptic meningitis in Busan and Kyoungsangnamdo area from April to July, 2002. In this study, we reported the clinical manifestations, laboratory data and causative viruses. METHODS: 184 children with aseptic meningitis who had been admitted to Department of Pediatrics, Inje Paik Hospital in Kaegum and Dongrae between April and July 2002 were assessed. Virus isolation and serotype identification were performed by cell culture and reverse transcription-polymerase chain reaction(RT-PCR) of cerebrospinal fluid. RESULTS: The peak incidence was noted in May. Male-to-female ratio was 2:1. The age ranged from 2 years to 16 years. Mean age was 8.7+/-7.1 years. Clinical manifestations were fever(95.1%), headache(91.8%) and vomiting(89.7%). Mean duration of fever was 2.6 days. The numbers of peripheral blood leukocytes were 1,400-20,000/mm3 and mean value was 9,829/mm3. On cerebrospinal fluid examinations, leukocyte count was in range of 10-2,000(mean 301)/mm3, protein level in range of 10-196(mean 36.7)mg/dL and glucose level from 17 to 155(mean 58.3)mg/dL. Viruses were isolated 13(8.4%) out of 154 patients and serotypes were 6 cases of echovirus 6, 3 cases of echovirus 9, 1 case of echovirus 13, 2 cases of echovirus 25 and 1 case of echovirus 30. CONCLUSION: Aseptic meningitis was prevalent in Busan and Kyoungsangnamdo area from April to July, 2002. We thought that causative viruses were echovirus 6, 9, 13, 25, 30.

Isolation of Causative Viruses from Patients with Aseptic Meningitis in Gwangju Area / 감염

BACKGROUND: Aseptic meningitis is a common illness of children. It seems that viruses are the usual etiologic agents. The distribution of these agents mainly depends on the isolated time and region area. This study was performed to isolate the causative viruses from patients with aseptic meningitis in Gwangju area during recent one year. METHODS:A total of 130 patients with aseptic meningitis were evaluated. Stool and/or cerebrospinal fluid specimens from patients were inoculated into rhabdomyosarcoma (RD), HEp2 and Vero cell lines. The virus propagation was examined by the presence of cytopathic effects. Neutralizing tests using enterovirus serum pool were done on each viral isolates. RESULTS: The isolation rate of enterovirus was 24.6% (32/130). The enterovirus isolates were obtained mostly from stool specimens (29/32). Twenty-two isolates were identified by neutralizing test. Ten isolates disclosed 'untyping' by neutralizing test. The distribution of isolates was coxsackievirus group B2 (11 stains, 34.4%), echovirus 30 (4 strains, 12.5%), echovirus 6 (3 strains, 9.4%), echovirus 9, 11, 25 and coxsackievirus group A16 (1 strain, respectively). These strains were predominantly isolated during summer season (June to July). CONCLUSIONS: The causative viruses from patients with aseptic meningitis in Gwangju area during recent 1 year were coxsackievirus group B2, echovirus 30, 6, 9, 11, 25 and coxsackievirus group A16 which were mostly isolated from stool specimens in summer season.

Serotyping and Phylogenetic analysis of Enteroviruses Isolated from Patients with Aspetic Meningitis / 대한임상미생물학회지

BACKGROUND: The determination of serotype of enteroviruses is useful for the discrimination between sporadic and epidemic infections. The conventional serotyping method is time-consuming and labor-intensive. Recently, molecular method was introduced for the serotyping of enteroviruses. The aim of this study was to establish a method to isolate and analyze enteroviruses from various specimens utilizing molecular biological techniques and to determine which strains were phylogenetically related to clinical samples. METHODS: Clinical samples in this study included 164 cerebrospinal fluid (CSF), 136 stool, 15 sera, 6 throat swab, 5 urine, and 4 sputa, which were obtained from hospitalized patients, primarily infants or children presenting symptoms of aseptic meningitis in 1998. RD cells were used for enterovirus isolation. RT-PCR was performed with RD cell lysate showing CPE. The primers 011 and 012 were used for the VP1 region, and the primers EN1 and EN2 for 5'-UTR. The nucleotide sequences of VP1 region were determined and analyzed with BLAST program. RESULTS: Among 333 samples, only 23 samples produced CPE: 17 samples at first and six samples at the second blind passage. Fifteen isolates were related to coxsackievirus B2 two to echovirus 4, three to echovirus 6, and three to echovirus 18. All 23 viral isolates displayed a nucleotide sequence identity of 80-95%, compared with the reference serotypes. However, the identity was increased up to 93-100% when the VP1 region was translated into amino acids CONCLUSIONS: Since CB2 type was 55% among enteroviral isolates, the CB2 was determined as the major causative serotype of enteroviral meningitis in 1998. CB2 type was emerged between June and July, EC4 and EC6 was limited to July, and EC18 was in August.

Serotyping and Phylogenetic analysis of Enteroviruses Isolated from Patients with Aspetic Meningitis / 대한임상미생물학회지

BACKGROUND: The determination of serotype of enteroviruses is useful for the discrimination between sporadic and epidemic infections. The conventional serotyping method is time-consuming and labor-intensive. Recently, molecular method was introduced for the serotyping of enteroviruses. The aim of this study was to establish a method to isolate and analyze enteroviruses from various specimens utilizing molecular biological techniques and to determine which strains were phylogenetically related to clinical samples. METHODS: Clinical samples in this study included 164 cerebrospinal fluid (CSF), 136 stool, 15 sera, 6 throat swab, 5 urine, and 4 sputa, which were obtained from hospitalized patients, primarily infants or children presenting symptoms of aseptic meningitis in 1998. RD cells were used for enterovirus isolation. RT-PCR was performed with RD cell lysate showing CPE. The primers 011 and 012 were used for the VP1 region, and the primers EN1 and EN2 for 5'-UTR. The nucleotide sequences of VP1 region were determined and analyzed with BLAST program. RESULTS: Among 333 samples, only 23 samples produced CPE: 17 samples at first and six samples at the second blind passage. Fifteen isolates were related to coxsackievirus B2 two to echovirus 4, three to echovirus 6, and three to echovirus 18. All 23 viral isolates displayed a nucleotide sequence identity of 80-95%, compared with the reference serotypes. However, the identity was increased up to 93-100% when the VP1 region was translated into amino acids CONCLUSIONS: Since CB2 type was 55% among enteroviral isolates, the CB2 was determined as the major causative serotype of enteroviral meningitis in 1998. CB2 type was emerged between June and July, EC4 and EC6 was limited to July, and EC18 was in August.