Evolutionary analysis of the VP1 gene of Echovirus 30 in Yunnan Province, China / 国际生物医学工程杂志

Objective:To study the genetic characteristics and genetic evolution of echovirus 30 (ECHO30) isolates in Yunnan Province, China.Methods:Virus isolation was performed on nucleic acid-positive samples for hand, foot, and mouth disease pathogen surveillance in Yunnan Province, and VP1 gene sequencing was performed. The sequences of eight ECHO30 isolates from Yunnan Province and the gene sequences of the VP1 region of the ECHO30 reference strain downloaded from GenBank were compared and analyzed using MEGA 5.0 software, and then a phylogenetic tree was constructed to measure the homology of nucleotides and amino acids between the isolates.Results:The ECHO30 virus was distributed in Wenshan, Qujing, Chuxiong, and Kunming in Yunnan Province. The ECHO30 virus was relatively common in Wenshan. ECHO30 isolates belonged to the H2 subtype of the H genotype, which was close to the local reference strain LC120939 in Yunnan Province. On the VP1 gene at site 5, the amino acid change ratio was more active, the amino acids were diverse, and mutations also occurred at sites 54, 156, 258, and so on. Nucleotide and amino acid homology were 84.0% - 100.0% and 98.4% - 100.0%, respectively.Conclusions:ECHO30 isolates from Yunnan Province have certain geographical characteristics and belong to H2 of the H genotype. The nucleotide differences in virus sequences among subtypes are small and have a close genetic relationship.

Genetic characteristic analysis of the VP1 gene of echovirus 30 isolated from viral meningitis cases in Yunnan province, 2010-2013 / 中华实验和临床病毒学杂志

Objective@#In this study we analyzed the genetic characteristics of echovirus 30 (E-30) VP1 gene sequences from Yunnan province isolated from viral meningitis (VM) cases in 2010-2013.@*Methods@#RT-PCR and VP1 gene sequencing were done for 9 E-30 strains isolated from VM cases in 2010-2013. VP1 gene sequences of E-30 reference strains were downloaded from the GenBank and their nucleotide (nt) and amino acid (aa) diversities were calculated by MEGA 5.1 software, the phylogenetic tree was constructed and the genetic characteristics and molecular epidemiology were analyzed.@*Results@#In 2010-2013, 9 strains of E-30 viruses were detected from 79 VM cases caused by echoviruses, accounting for 11.39%(9/79), the overall positive rate was 1.63%(9/553). Phylogenetic analysis revealed that E-30 strains can be divided into four genotypes (genotype A, B, C and D), and genotype D can be further divided into seven sub-genotypes. Nine Yunnan VM isolates were distributed in D7 sub-genotype, and can be further clustered into 3 branches: 5 strains isolated in 2010 were clustered in branch 1, it is evident that these viruses were responsible for an aseptic meningitis outbreak in Kunming in that year; one 2011 isolate, together with 2013 isolate and one isolate from healthy children in 2010 were clustered in branch 2, these two branches were Yunnan special branches, and two 2011 isolates had the highest homology with 2003 VM outbreaks′ strains isolated from Shandong, Jiangsu, and Zhejiang, showing that these strains may have the same evolutionary sources.@*Conclusions@#Nine Yunnan VM isolates were distributed in D7 sub-genotype, and these strains have different evolutionary sources, showing that at different times E-30 viruses in the same sub-genotypes branch might prevail in different areas.

Antiviral Activity of Gemcitabine Against Echovirus 30 Infection in Vitro

Echovirus 30 is one of the major causes of meningitis in children and adults. The purpose of our current study was to investigate whether selected antiviral drugs could provide antiviral activity against echovirus 30. Using RD cells, we assessed the cytopathic effect of echovirus 30, including viral RNA levels as indicators of viral replication. The effects of gemcitabine were compared to rupintrivir, a well-known antiviral drug. To understand the activity gemcitabine exerts on the viral life cycle, time course and time-of-addition assays were implemented. The most effective compounds against echovirus 30 were gemcitabine and rupintrivir, as demonstrated by their concentration-dependent activity. Gemcitabine affects the early stages of echovirus 30 infection by disrupting viral replication. However, gemcitabine failed to directly inactivate echovirus 30 particles or impede viral uptake into the RD cells. Gemcitabine can be considered as a lead candidate in the development of echovirus 30 antiviral drugs, specifically in the early stages of echovirus 30 replication.

Complete nucleotide sequence of a human echovirus 30 strain KM/A363/09 isolated in Yunnan, China in 2009 / 中华微生物学和免疫学杂志

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 .

A viral meningitis outbreak associated with Echo30 in drinking water / 中华流行病学杂志

Objective To study the source of infection,the scope of epidemic and control measures in an outbreak involving students having symptoms as fever,dizziness,headache,vomiting and nausea.Methods The suspected-case was defined as fever (armpit temperature ≥37 ℃) and with one or more of the following symptoms:dizziness,headache,vomiting and nausea,among students and teachers at school from Mar 1,2012.Confirmed-case was among suspected case accompanied by both throat and rectal swabs enterovirus positive by RT-PCR.All the cases were collected through checking the medical records from 4 hospitals as well as through the absence records of students and teachers,from Mar 1,2012.We conducted a case-control study with ratio of 1 ∶ 2 and data on the exposures to water among students and teachers was collected prior to the illness.27 cases' throat and rectal swabs were collected and analyzed by RT-PCR and PCR sequence methods.2 warm-water samples were collected for testing the counts on total bacteria and E.coli.Results 103 students' cases were identified in school L,with the attack rate as 4.6％ (103/2255).Students from Grade three had the high attack rate as 18.1％ (72/397) and 77.7％ (80/103) of the cases located in the building with ' multiple-functions'.Epidemic curve of the outbreak showed a pattern with continuous common source of infection.It seemed that the exposure to warm-water appeared to be the major risk factor (OR =18.3,95％CI:2.0-169.5) together with the intake of un-boiled water (OR =15.5,95 ％CI:1.7-141.8).Specimens from 27 students (81.5％,22/27) were identified enterovirus positive by RT-PCR,and 7 of the 9 students were confirmed carrying Echo 30.Bacteria and coli were negative from the 2 warm-water samples.Conclusion This viral meningitis-outbreak was caused by Echo 30,with drinking water as the major risk factor.

Sequencing and Molecular Characterization of the Genome of Echovirus 30 Isolated from a Korean Aseptic Meningitis Patient

Echovirus 30 is one of the distinct serotypes of enteroviruses and commonly isolated agent causing sporadic to large outbreaks with aseptic meningitis in many regions over the world. Recently, an outbreak of echovirus 30 associated with aseptic meningitis occurred in Korea in 2008. In order to analyze echovirus 30 in Korea, the virus was isolated from cerebrospinal fluid samples of a male patient with aseptic meningitis and its genome sequence was determined. The sequence of Korean echovirus 30 isolate was compared with those of reference strains (Bastianni, FDJS03-84, zhejiang-17-03, 14916net87). At the nucleotide level, the P1 region (84.8~89.0%) had the highest identity value; at the amino acid level, the P3 region (97.0~98.5%) showed the highest value. When the cleavage sites were compared, most sites were identical except those between VP1 and 2A; the Bastianni stain had TT/GA, whereas the other four strains contained NT/GA. The China strains (FDJS03-84 and zhejiang-17-03) were grouped together and the other strains were distinct from each branch in the phylogenetic tree based on the complete genome sequences.

RT-PCR for confirmation of echovirus 30 isolated in Belém, Brazil

Echovirus (Echo) 30 or human enterovirus B is the most frequent enterovirus associated with meningitis cases. Epidemics and outbreaks of this disease caused by Echo 30 have occurred in several countries. In Brazil, Echo 30 has been isolated from sporadic cases and outbreaks that occurred mainly in the south and southeast regions. We used RT-PCR to examine Echo 30 isolates from meningitis cases detected from March 2002 to December 2003 in Belém, state of Pará, in northern Brazil. The patients were attended in a Basic Health Unit (State Health Secretary of Pará), where cerebrospinal fluid (CSF) was collected and stored in liquid nitrogen. Weekly visits were made by technicians from Evandro Chagas Institute to the health unit and samples were stored at -70°C in the laboratory until use. HEp-2 and RD cell lines were used for viral isolation and neutralization with specific antisera for viral identification. RNA extraction was made using Trizol reagent. The RT-PCR was made in one step, and the total mixture (50 æL) was composed of: RNA, reaction buffer, dNTP, primers, Rnase inhibitor, reverse transcriptase, Taq polymerase and water. The products were visualized in agarose gel stained with ethidium bromide, visualized under UV light. Among the 279 CSF samples examined, 30 (10.7 percent) were EV positive, 29 being Echo 30 and one was Cox B. Nineteen Echo 30 were examined with RT-PCR; 18 tested positive (762 and 494 base pairs). The use of this technique permitted viral identification in less time than usual, which benefits the patient and is of importance for public-health interventions.