Echovirus serotypes circulating in Malaysia from 2002 to 2013

Jeyanthi Suppiah, TS Saraswathy Subramaniam, Amry Khursany Ismail, Apandi Yusof, Zainah Sa’at Virology Unit, Infectious Disease Research Centre, Institute for Medical Research, Kuala Lumpur, Malaysia

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Echovirus serotypes circulating in Malaysia from 2002 to 2013

Jeyanthi Suppiah*, TS Saraswathy Subramaniam, Amry Khursany Ismail, Apandi Yusof, Zainah Sa’at Virology Unit, Infectious Disease Research Centre, Institute for Medical Research, Kuala Lumpur, Malaysia

ABSTRACT

Objective: To identify the circulating serotypes of human echovirus in Malaysia from 2002 to 2013. Methods: A total of 31 retrospective samples from non-polio acute fl acid paralysis, hand-food-and-mouth disease, viral meningitis and enterovirus cases were subjected to amplifi cation of partial VP1 gene by RT-PCR. Results: Sequencing and phylogenetic analysis of the partial sequences identifi ed 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. Conclusions: From the fi ndings, 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 confi dent result to support this evidence.

ARTICLE INFO

Article history:

Received in revised form 20 January 2016

Accepted 15 February 2016

Available online 20 March 2016

Echovirus

VP1

Malaysia

Phylogenetic

Serotype

1. Introduction

The echovirus, being the largest subgroup of Enteroviruses in the Picornaviridae family, consists of 32 serotypes. Echovirus is accounted for causing a huge spectrum of human diseases from asymptomatic or acute febrile illness in infants and young children to fatal encephalitis, aplastic anemia and pulmonary hypertension[1]. It is also reported to be the most common cause of aseptic meningitis[2].

Laboratory diagnosis for echovirus was initially focused on classical method which required viral isolation from cell culture of clinical specimens. However, this method has its limitation whereby not all serotypes were able to grow in cell cultures. To date, molecular assays particularly RT-PCR are being widely used[3-5] as it provided a more sensitive, rapid and accurate platform for characterization and serotyping of echovirus.

The VP1 gene was often targeted for serotyping of non-polio enteroviruses as it codes for the major antigenic sites and most typespecifi c neutralisation determinants[6]. Partial sequence analysis of VP1 gene served as tool of identifi cation of echovirus serotypes, thus discriminating it from other non-polio enteroviruses. Therefore, using this method, we report the molecular epidemiology of echovirus serotypes circulating in Malaysia from 2002 to 2013.

2. Materials and methods

2.1. Samples

A total of 31 patient samples were obtained from the Virology Unit, Institute for Medical Research, Kuala Lumpur, Malaysia over a period of 12 years (2002-2013). Samples were selected from nonpolio acute fl acid paralysis cases (60.0%), hand-food-and-mouth disease cases (3.3%), viral meningitis (26.7%) and enteroviruscases (13.3%) The samples were previously confi rmed as echoviruspositive by culture (86.7%) and pan-enterovirus positive (13.3%) by PCR. The sources of sample procurement include cerebrospinal fl uid, stool, throat swab, rectal swab and lung fl uid.

2.2. RNA isolation

Viral RNA isolation from virus-infected culture supernatant was performed using QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. The isolation procedure was based on spin-column method. The fi nal elution vol?ume of 50 μL containing viral RNA from each sample was stored at -20 ℃for long-term usage.

2.3. VP1 gene partial amplification

Isolated RNA’s were subjected to RT-PCR amplifi cation of partial VP1 gene by using published primers[7]. All amplifi cation reactions were carried out in a 96-well Thermal Cycler (Bio Rad, USA). PCR was undertaken at 50 ℃ for 30 min, 94 ℃ for 3 min followed by thermocycling for 35 cycles at 94 ℃, 30 s; 42 ℃, 30 s and 60 ℃, 30 s and a final incubation at 72 ℃ for 5 min. PCR reaction was composed of 12.5 μL of 2x MyFi RT-PCR Mix (Bioline, USA), 1.0 μL of each oligonucleotides (10 μM), 1.0 μL of Rnase Inhibitor, 0.5 μL of RT enzyme, 5 μL of extracted RNA and 4.0 μL of sterile distilled water.

2.4. Post PCR purification and sequencing

A 15 μL aliquot of each PCR reaction was analyzed on 2% agarose by gel electrophoresis and viewed under UV illumination. The agarose was pre-stained with Red Safe Dye (Intron Biotech, Korea). The corresponding amplicons were extracted from the agarose gel and purified using Gel Extraction Kit (Qiagen, USA) according to the manufacturer’s instruction. Final elution contained 35 μL of purified PCR amplicons from which 5 μL was reanalyzed on 2% agarose gel to confi rm that the purifi cation step was performed precisely. All purifi ed PCR were subjected to sequencing in 3730 genetic analyzer (Applied Biosystem, USA).

2.5. Data analysis

Overlapping sense and antisense sequences derived from sequencing were aligned to produce partial VP1 gene sequence using CLUSTAL Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) bioinformatics tool. Subsequently, the serotype of each isolates was determined by BLAST search of derived VP1 gene sequences against the Genbank NCBI database and further verifi ed by construction of phylogenetic tree by Mega 6.06, neighbor joining method (Tamura-Nei model, bootstrap replication 1000x).

3. Results

3.1. VP1 gene amplification and serotyping

VP1 partial gene amplification was observed in all 31 isolates. Alignment of forward and reverse sequences produced VP1 amplicon of 348 bp. The sequences were deposited in GenBank (Accession numbers: KP204987-KF205039). BLAST search revealed the serotype similarity of Malaysian echovirus isolates with the existing echovirus database in GenBank. It was found that of 31 isolates sequenced, 32.3% (10/31) were echovirus 11 (E11), 19.4% (6/31) were echovirus 6 (E6) and echovirus 7 (E7) respectively 12.9% (4/31) were echovirus 3 (E3) and 3.2% (1/31) were echovirus 1 (E1), echovirus 9 (E9), echovirus 30 (E30), Coxsackie A24 and Coxsackie B3 respectively.

3.2. Phylogenetic analysis

The constructed phylogram revealed a 100% concordance with serotyping result by BLAST search. The phylogram grouped Malaysian isolates into 9 clades comprising of E1, E3, E6, E7, E9, E11, E30, coxsackievirus B3 and coxsackievirus A24 (Figure 1). Majority of the Malaysian echovirus isolates were shown to be closely related to the echovirus isolates from Asian countries such as India, Vietnam, China&Korea.

4. Discussion

Sequencing and phylogenetic analysis of partial VP1 gene of Echovirus revealed E11 as the most common serotype circulating in Malaysia from 2002 to 2013. E11 has been associated with a number of outbreaks in neonatal nurseries[8,9]. In an enterovirus surveillance conducted in United States from 1970 to 2005, E11 was reported to be the second most common serotypes[10]. However, no recent reporting of E11 outbreak was found. Few researches in the Asian region demonstrated E30 as the predominant serotypes[11-13]. Recent outbreaks of E30 were seen in other region of the world such as the United States[14]and Europe[15]. Enteroviruses were known to circulate in variable patterns in diff erent years and regions even inside a country, therefore, inconsistency in circulating serotypes and epidemiological reports are well anticipated[16-18].

Figure 1. Phylogram of partial VP1 sequences of human echovirus and human coxsackievirus isolates.

In the current study, two samples (MY278/05&MY294/12 ST) which were previously identifi ed as echovirus by culture appeared to be coxsackieviruses (Coxsackie A24&B3) as revealed by sequencing and phylogenetic analysis. The misdiagnose could have been due to the fact that some enteroviruses particularly Coxsackie A are not readily detectable in cell culture, therefore a more extensive method such as suckling mice is required for isolation[19]. However, the latter method is no longer commonly used.

The current investigation also found high sequence similarities between diff erent types of samples obtained from the same patient or diff erent passage of the same type of sample (data not shown). Sequence similarities ranged from 97%-100% of which none have induced amino acid changes.

This study finding showed that E11 was the most common echovirus serotype circulating in Malaysia from 2003 to 2013. Detection and analysis by RT-PCR, sequencing and phylogenetic analysis further identifi ed presence of other serotypes such as E6, E7, E3, E9, E30 and E1, coxsackie A24 and coxsackie B3.

Conflict of interest statement

We declare that we have no confl ict of interest.

Acknowledgements

The authors would like to thank the Director General of Health Malaysia for his permission to publish this paper. This study was supported by Institute for Medical Research Operational Grant (Vot 040300/OS27403).

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Document heading 10.1016/j.apjtm.2016.01.037

IF: 1.062

Asian Pacific Journal of Tropical Medicine

journal homepage:www.elsevier.com/locate/apjtm

15 December 2015

*

Jeyanthi Suppiah, Virology Unit, Institute for Medical Research, Jln Pahang, 50588 Kuala Lumpur, Malaysia.

Tel: +60326162674

E-mail: jeyanthi@imr.gov.my

Foundation project: This study was supported by Institute for Medical Research Operational Grant (Vot 040300/OS27403).