Molecular Characterization of Echovirus 30 Isolated from Environment in North India

Background Echovirus 30 (E30) causes acute aseptic meningitis. Enteroviruses (EVs) are responsible for 30,000 to 50,000 hospitalizations for aseptic meningitis per year in the United States. E30 is one of the most frequently isolated EVs, causing extensive outbreaks in temperate climates in several countries. Methods used this


Abstract
Background Echovirus 30 (E30) causes acute aseptic meningitis. Enteroviruses (EVs) are responsible for 30,000 to 50,000 hospitalizations for aseptic meningitis per year in the United States. E30 is one of the most frequently isolated EVs, causing extensive outbreaks in temperate climates in several countries.
Methods E30 used in this study was isolated from environmental specimens. The virus was con rmed by RT-PCR with speci c primers (1). Virus stock was prepared by infecting RD cells in 25 cm 2 asks (Corning Inc. USA). Virus infected and mock infected cells were incubated at 36.5 ºC with 2% MEM. After 48 h of infection, the culture was aliquoted and kept at-80 ºC for further use.

Results
This present work analyzed the E30 genetic diversity in a fragment of 217 nucleotides of the VP1 region, of environmental strains. The environmental samples of echovirus 30 show mutation in the nonfunctional polyprotein protein (Fig. 1). The result suggested that the information may be obtained by analyzing only a part of the VP1 region.

Conclusion
The present study showed that the E30 environmental sample is more divergent to prototype Bastianni strain.
EVs are responsible for 30,000 to 50,000 hospitalizations for aseptic meningitis per year in the United States. The E30 is one of the most frequently isolated EVs in the United States, comprising 6.8% of all reported EVs isolated from 1970 to 1983 (13) and 9.5% of the EVs isolated from 1993 to 1996 (14). In 1998, E30 accounted for 42% of all the United State.
EV isolations reported to the Centers for Disease Control and Prevention by state and territorial public health laboratories (15). E30 (genus: Enterovirus; family, Picornaviridae) is one of the most frequently isolated EVs, causing extensive outbreaks of E30 in temperate climates in several countries (15,16).
Additionally, this serotype is one of several enteroviruses associated with sporadic cases of the aseptic meningitis (17). In contrast to the highly conserved 5' UTR, the open reading frame encoding capsid protein VP1 is more variable and confers distinct antigenic properties of the virus. Thus, this VP1encoding region of the genome is considered most suitable for sequence analysis, and to determine the Enterovirus genotype and genetic variation (18). In this study, we tried to analyze the E30 (an environmental strain) by RT-PCR and sequencing of the VP1 gene allowed us to understand its genetic diversity.

Materials And Methods
Sewage sample collection Sewage sample (approx one liter) was collected and stored in sterile glass bottle from the collection site by directly dipping into the sewage e uent. The outer surface of the bottle was rinsed with 2% sodium hypo chloride solution, placed in a cool box, and transported to the laboratory. The bottle was frozen at − 20° c, until the process of virus isolation. This procedure is already reported in our previously published article (11).
Sewage sample processing Sewage sample was concentrated by two-phase concentration method described previously (11). In brief, the pH of the sample was adjusted to 7.2 and the sample was centrifuged at 5000 g for 30 min at 4 °C to pellet the solids. 500 ml of clari ed sewage was mixed with de ned amounts of two polymers, dextran and 15% polyethylene glycol (15% PEG6000). The homogenous mixture obtained by vigorous shaking is left to stand overnight at 4° C in a separating funnel. This allows the polymers to separate in two distinct layers (phases) in the funnel. Enterovirus accumulates in the smaller bottom layer and/or at the boundary between the layers (interphase). The bottom layer and interphase were collected drop-wise. The pellet from the initial centrifugation was suspended in this concentration, and treated with chloroform, then centrifuged at 5000g for 30 min at 4°C and assayed for the presence of virus. The concentrated virus suspension was stored at − 20°C until used for virus isolation (19). This method was published in a study which compared three different methods, including direct isolation, centrifugation and two phase separation, and results of their study suggest that the two phase separation method is the best for maximum virus yield (20).

Inoculation of sewage samples
The extracted concentrate was inoculated on fresh monolayer cultures of L20B and RD cells in 50 ml (25 cm2) asks. The cultured asks were incubated at 37 °C and examined at every 24 h for cytopathic effect (CPE). Samples which showed CPE were frozen and thawed two times, then re-passaged on new cells. Samples were observed for CPE up to 7 days before being considered negative. Samples showed CPE were stored for con rmation through serotyping.

RT-PCR
Isolates grown in the RD cells were freeze-thawed three times and centrifuged at 12000 rpm for 10 minutes. The 5µl isolate supernatant was diluted with 10µl of distilled water. Primer E-1 and E-2 were used 50 pmol concentration as described previously (21, 1) RT-PCR product puri cation DNA was eluted from gel and has been puri ed by the QIAGEN gel extraction columns (QIAGEN, Chatsworth, CA, USA). PCR products were examined and randomly ve E30 isolates selected for sequencing.

Sequencing reaction
Sequencing reaction mix was contained, 4 μl of big dye terminator ready reaction mix, 2 μl of each primer (10 pmol λ -1 ), 3 μl Milli-Q water and 1μl of template (100 ng μl -1 ). PCR conditions followed (25 cycles) initial denaturation at 96 °C for 1min, denaturation at 96° C for 10 sec., hybridization at 50 °C for 5 sec. and elongation at 60 °C for 4 min. The ABI 3130 genetic analyzer and chemistry big dye terminator version 3.1 cycle sequencing kit were used for sequencing. Polymer and capillary array used POP_7 polymer 50 cm capillary array and BDTv3-KB-Denovo_v 5.2 protocol were examined, whereas data were analyzed by software Seq Scape_v 5.2 and reaction were examined in the Applied biosystem micro Amp optical 96-well reaction plate.

Construction of phylogenetic trees
Nucleotide BLASTn analysis (http://www.ncbi.nlm.nih.gov/BLAST) was used to identify related genes of the viruses and construct the phylogenetic tree. The reference sequences were obtained from the GenBank. The ClustalX version 2.0.12 (22) was used to perform multiple nucleotide alignment and tree was constructed (Njplot Version 2.3) (23), the neighbor-joining method according to the distances between all pairs of sequences in a multiple alignment. The con dence of sequence clustering was evaluated by bootstrapping (1000 replicates) (24,11) in g.1.

Results
There were 109 sewage samples were collected for this study. 44 out of 109 were detected positive for enterovirus in sewage samples by cell culture and followed by serotyping and RT-PCR. The existence of echovirus was observed throughout the year however the peak season for the maximum detection of other EV was from July to September in each year.

Isolation and typing
The one isolate was isolated from RD cells. The one strain was con rmed to be E30 by the ampli cation of the partial VP1 sequences. The sequence was analysed using an online enterovirus genotyping tool. This was isolated from environmental sample.

VP1 sequence analysis
This present work analyzed the E30 genetic diversity in a fragment of 217 nucleotides of the VP1 region, of environmental strains. The environmental samples of echovirus 30 show mutation in the nonfunctional polyprotein protein (Fig. 1). The result suggested that the information may be obtained by analyzing only a part of the VP1 region. The partial VP1 sequence of the one strain had the highest similarity with other E30 strains in the GenBank database and had a percentage reaching 98%. The isolated strain was identi ed as E30 using an EV serological and molecular typing criteria.

Nucleotide sequence accession number
The accession number of the partial VP1 nucleotide sequence of the E30 strain identi ed in this study is GQ353352.

Discussion
Molecular mechanisms of picornavirus variation and evolution result from point mutations and genomic rearrangement, in particular recombination. Although most attention has been directed towards mutation, recent studies have indicated that recombination might have an important role in enteroviruses. The genetic recombination was involved in a time-correlated manner in their emergence and that drift occurred in all lineages, quasi exclusively by synonymous nucleotide substitutions, indicating strong constraints against amino acid changes in both structural and non-structural genes (27). This pattern of evolution is clearly different from that of other enteroviruses. A single lineage at a time appears to be circulating worldwide. This behavior may be related to the epidemic activity of the E30.
Partial sequencing in the VP1 gene was previously proved to be suitable for serotype identi cation of EVs (28,29,30,15,31), a step which precedes phylogenetic analysis (Fig. 1). This molecular approach for serotyping was proposed to be used in routine diagnosis instead of the laborious and time-consuming seroneutralization assays. For phylogenetic analysis study the VP1 region for several reasons. It is one of these regions of the VP1 that proved to discriminate well between EV serotypes, the use of the same PCR product and sequence data for both serotype identi cation and phylogenetic analysis, being of great interest on the practical level (28). The 3'end of the VP1 has been extensively used in the molecular epidemiology of poliovirus (32) and echovirus 30 (33,34) and proved to be appropriate to discriminate EV isolates in Geno-groups, genotypes and lineages. Many authors have previously used this part of the VP1 for molecular studies of E30; the number of sequences available in the international database, matching the 217 nucleotides considered herein, is higher in comparison to the other parts of the VP1. E30 is among the most commonly isolated EVs in the world. The E30 may cause a full range of EV diseases; some being of particular importance, like severe diseases in neonates and aseptic meningitis with epidemic potential. However, studies on the molecular epidemiology of this infection have been limited to few countries in the world. The present work showed that the genetic characteristics of E30 circulating Page 7/11 strains and the dynamics of genetic evolution may differ from one country to another according to their geographical location and endemicity levels. It also contributes to a better understanding of the epidemiology of the E30.

Conclusion
It could be concluded that the E30 environmental sample is more divergent to prototype Bastianni strain, and the phylogenetic analysis of E30 strain revealed an evolutionary change. This study is also helpful to those who are struggling to eradicate Polio virus in term of better understanding of its presence in community. Phylogenetic tree based on DNA distance inferred from Indian E30 isolate sequence bank accession no. GQ353352 (All HEV isolates sequences available in NCBI sequence database).