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  • Research note
  • Open Access

Extending the utility of the WHO recommended assay for direct detection of enteroviruses from clinical specimen for resolving poliovirus co-infection

  • 1, 2,
  • 1,
  • 2,
  • 2,
  • 2,
  • 2,
  • 2,
  • 2 and
  • 1, 3Email author
BMC Research Notes201811:47

https://doi.org/10.1186/s13104-018-3155-6

  • Received: 23 October 2017
  • Accepted: 9 January 2018
  • Published:

Abstract

Objectives

In a polio-free world there might be reduced funding for poliovirus surveillance. There is therefore the need to ensure that enterovirologist globally, especially those outside the global polio laboratory network, can participate in poliovirus surveillance without neglecting their enterovirus type of interest. To accomplish this, assays are needed that allow such active participation.

Results

In this study we describes a sensitive and specific utility extension of the recently recommended WHO RT-snPCR assay that enables independent detection of the three poliovirus types especially in cases of co-infection. More importantly, it piggy-backs on the first round PCR product of the WHO recommended assay and consequently ensures that enterovirologists interested in nonpolio enteroviruses can continue their investigations, and contribute significantly and specifically to poliovirus surveillance, by using the excess of their first round PCR product.

Keywords

  • Enteroviruses
  • Polioviruses
  • WHO
  • Surveillance
  • Nigeria

Introduction

There are 13 species in the genus Enterovirus (EV) (family Picornaviridae, order Picornavirales). The Polioviruses (alongside other nonpolio enteroviruses [NPEVs]) belong to species C and are the prototype members of the genus. In 2015, the WHO included the reverse transcriptase-seminested polymerase chain reaction (RT-snPCR) assay described by Nix and colleagues [1] for direct (cell culture independent) detection of enteroviruses from clinical specimen [2]. The Nix et al. [1] assay is an upgrade (seminested and Consensus Degenerate Hybrid Oligonucleotide Primers [CODEHOP]) version of the Oberste et al. [3] assay. It has been shown that this algorithm is very sensitive for cell culture independent enterovirus detection and identification [46]. However, we [7] have recently shown that the assay lacks the capacity to resolve enterovirus types present in cases of co-infection. Significantly, the prevalence of enteroviruses co-infections in Nigeria is underscored by the independent emergence of 29 lineages of circulating Vaccine Derived Polioviruses (cVDPVs) between 2004 and 2014 [8], most of which are of recombinant origin [9]. This necessitates the need for assays that can detect and resolve enterovirus co-infections.

We had also shown [7] that by including primers 189 and 187 (subsequently referred to in this study as Species Resolution Primers) in the second round PCR of the WHO recommended RT-snPCR assay the resolving power of the assay could be improved. However, in a polio-free world, where there might be reduced funding for enterovirus surveillance, assays are needed that can in one swoop detect and resolve enterovirus co-infections, including different poliovirus types. Therefore, in this study, we extend the utility of the WHO recommended RT-snPCR assay by inclusion of three poliovirus-specific forward primers in the second round assay, thus, making it possible to independently detect the three poliovirus types even in cases of co-infection.

Main text

Sixteen enterovirus isolates (Table 1) previously recovered from sewage contaminated water were used as reference samples in this study and analyzed as depicted in the study algorithm (Fig. 1). Isolation and characterization of samples 1 through 12 have been previously described [10]. Briefly, samples 1 through 8 are enterovirus species B (EV-B), and were isolated on RD cell line [10]. Samples 9 to 12 are enterovirus species C (EV-C) and were isolated on MCF-7 cell line [10]. Samples 13 to 15 are Sabin poliovirus 1–3 respectively. Samples 13 and 15 were isolated and characterized by the WHO Environmental Surveillance (ES) laboratory in Ibadan, Nigeria and provided to us as references for Sabin 1 and 3 polioviruses, respectively. Sample 14 (a poliovirus 2) was isolated as part of a previous study [11] but identified subsequent to the study. Sample 16 is a mixture of six isolates, two species B (samples 1 and 3) and four species C (samples 11, 13, 14 and 15) (Table 1).
Table 1

Identity of isolates determined using the algorithm

S/N

Information prior this study

Results of this study

Previously determined ID

Species

WHO 2015

Species Resolving Modification (SRM)

Poliovirus Resolving Modification (PRM)

Final summary of isolates identified

AN89

189

187

Sab 1

Sab 2

Sab 3

1

E3

EV-B

E3

unx

E3

Neg

Neg

Neg

E3

2

E19

EV-B

E7

unx

E19

Neg

Neg

Neg

E7/E19

3

E7

EV-B

E7

unx

E7

Neg

Neg

Neg

E7

4

E19

EV-B

Neg

E20

Neg

Neg

Neg

Neg

E20/E19

5

E7

EV-B

E7

Neg

E7

Neg

Neg

Neg

E7

6

E19

EV-B

Neg

unx

E19

Neg

Neg

Neg

E19

7

E7

EV-B

E7

unx

E7

Neg

Neg

Neg

E7

8

UNT

EV-B

E7

unx

E7

Neg

Neg

Neg

E7

9

CV-A13

EV-C

CV-A13

unx

CV-A13

Neg

Neg

Neg

CV-A13

10

CV-A13

EV-C

CV-A13

CV-A13

CV-A13

Neg

Neg

Neg

CV-A13

11

CV-A13

EV-C

CV-A13

CV-A13

unx

Neg

Neg

Neg

CV-A13

12

CV-A13

EV-C

CV-A13

CV-A13

Neg

Neg

Neg

Neg

CV-A13

13

PV-1

EV-C

PV-1

PV-1

PV-1

PV-1

PV-2

PV-3

PV-1, PV-2, PV-3

14

PV-2

EV-C

PV-2

PV-2

unx

Neg

PV-2a

unx

PV-2

15

PV-3

EV-C

PV-3

PV-3

unx

Neg

Neg

PV-3b

PV-3

16

PV-1, PV-2, PV-3,

CV-A13 (S/N 11),

E7 (S/N 3), E3 (S/N 1)

EV-B and C

PV-2

unx

PV-1

PV-1

PV-2a

PV-3b

PV-1, PV-2, PV-3

The primers included in the Species and Poliovirus Resolving Modifications (SRMs and PRMs) are referred to in the text as Species and Poliovirus Resolving Primers (SRPs and PRPs), respectively

The PRM did not amplify any of the isolates in samples 1–12

E Echovirus, EV enterovirus, PV Poliovirus, CV Coxsackievirus; unx unexploitable due to bad sequence data, Unt untypable, Neg negative (no amplification)

a, bIsolates are genetically the same

Fig. 1
Fig. 1

The algorithm followed in this study. A—The WHO 2015 recommended assay, B—Species Resolution Modification (SRM) and C—Poliovirus Resolution Modification (PRM)

All 16 samples were subjected to RNA extraction and RT-snPCR as previously described [12] but with slight modifications (Fig. 1). Precisely, instead of three second round PCR assays, there were six as depicted in Figs. 1 and 2. The forward primers included in the remaining three assays are listed in Additional file 1: Table S1. A more detailed description of the molecular detection algorithm is provided in Additional file 2. All amplicons generated from the six second round PCR assays were shipped to Macrogen Inc, Seoul, South Korea, for purification and sequencing. The primers used for each of the second round PCR assays (Fig. 2) were also used for sequencing, respectively. The identities of the sequenced isolates were determined using the enterovirus genotyping tool [13]. Sequences newly generated in this study have been deposited in GenBank. The accession numbers are KX856914-KX856921.
Fig. 2
Fig. 2

A schematic representation of the annealing sites of the different primers used in this study relative to the enterovirus genome and the consequent amplification product (arrows depict primers and their orientation)

The results of this study are detailed in Table 1. For isolates one to eight (all previously known to be EV-Bs [10]), the previously described assays [12] (Table 1; assays WHO 2015 and SRM, respectively) identified the isolates to a large extent in accordance with their previously stated types [10]. It also identified the previously unidentified sample 8 as E7. Furthermore, it showed the presence of E20 in an isolate (sample 4) previously shown to be only E19. As expected, the Poliovirus Resolving Primers (PRPs [Sab 1, 2 and 3]) did not amplify any of the isolates in samples 1 to 8 (Table 1). For isolates nine to twelve which were all previously identified as CV-A13 (EV-C) [10], the assay as previously described [12], identified the isolates in accordance with their previously stated types [10]. The PRPs did not amplify any of the isolates in samples 9 to 12, as expected (Table 1).

For isolates 13 to 15 (previously identified as Sabin PV-1, 2 and 3 respectively), the assay as previously described [12], identified the isolates in accordance with their previously stated types. However, the PRPs resolved the isolates. The Sabin-1 primer only detected sample 13 and confirmed it to be PV-1. The Sabin-2 primer produced the expected amplicon size in both samples 13 and 14. The sequence data subsequently confirmed both to contain two different Sabin 2 viruses that are 99.65% similar (data not shown). In fact, the Sabin 2 in sample 13 has an Isoleusine (I) at position 143 of VP1 while that in sample 14 has an Asparagine (N) at the same position. The Sabin-3 primer also produced the expected amplicon size in both samples 13 and 15. The sequence data also confirmed both to contain two different Sabin-3 viruses that are 99.65% similar (data not shown). Hence, the assay as previously described [12] (i.e. primers AN89 and the SRPs [189 and 187]) did very well in identifying all three polioviruses. The PRPs (Sab1–Sab3) also confirmed the identity of the three samples. The PRPs however, further showed that sample 13, which was previously identified by the GPLN algorithm [14] as PV-1 and confirmed by primers AN89 and the SRPs as such, also contained PV-2 and PV-3 (Table 1).

For sample 16 which contained four EV-Cs (PV-1, PV-2, PV-3 and CV-A13), and two EV-Bs (E3 and E7), all the six primers produced their expected band sizes. Sequence data analysis however showed that while the amplicon from primer 189 produced unexploitable sequence data, primers AN89, 187, Sab-1, Sab-2 and Sab-3 detected PV-2, PV-1, PV-1, PV-2 and PV-3, respectively (Table 1). Otherwise stated, AN89 and the SRPs (primers 189 and 187) only detected PV-2 and PV-1 respectively from the mixture while the PRPs specifically detected all the three poliovirus serotypes. Importantly, the PV-2 and 3 detected in sample 16 were exactly those found in samples 14 and 15 respectively (Table 1).

In this study, by including poliovirus serotype specific forward primers [15] in the second round PCR of the WHO recommended RT-snPCR [2] assay, we were able to selectively and specifically detect and identify the different poliovirus serotypes. More importantly, this was accomplished in a situation where six different enterovirus types were present. The results of this study showed that in such instance, the RT-snPCR assay recommended by the WHO [2] detected only one of the six different enterovirus types present (Table 1; sample 16). It therefore confirmed that the WHO recommended RT-snPCR assay [2] might not be dependable for the resolution of enterovirus mixtures. The results of this study further showed that the WHO recommended RT-snPCR assay [2] can be modified or tailored as described in this study (for detection and identification of the polioviruses) to specifically detect other enterovirus types especially in cases of co-infection. Such serotype-specific modifications would be very valuable for low-income economies as it will broaden the surveillance capacity of enterovirologists in such settings with minimal increase in cost.

It is pertinent to note that this modification appears to be more sensitive for poliovirus detection and identification than both the WHO RT-snPCR algorithm [1, 2] and the current algorithm for poliovirus identification in use by the GPLN [14]. For example, while the GPLN algorithm [14] and the WHO RT-snPCR algorithm [2] identified sample 13 as PV-1, the modification described here showed that sample 13 contained PV-1, 2 and 3. More importantly, sequence comparison showed that the PV-2 and 3 in sample 13, and those in samples 14 and 15 were different (data not shown).

Consequent of this discovery, the initial result of this reference ES sample was retraced in the WHO polio lab at Ibadan Nigeria. It was then discovered that the results of all the inoculated cell culture flasks (five L20B and one RD) [16] showed that PV-1, 2 and 3 were isolated from the parent ES sample, though in different flasks (unpublished data). However, the PV isolate in the L20B flask from which sample 13 was aliquoted was previously identified as PV-1 by the current GPLN poliovirus detection and identification algorithm [14]. Altogether, this suggests that the isolate in sample 13, contained PV-1, 2 and 3 but PV-1 had a titre that is significantly higher than others. Consequently, it was the type detected in sample 13 both by the GPLN algorithm and primers AN89, 189 and 187 (Table 1; sample 13). Further buttressing the influence of PV-1 titre hypothesis is the fact that the PV-2 and 3 in sample 13 could not be detected in the sample 16 mixture. Rather it was the PV-2 and 3 in samples 14 and 15 that were detected in sample 16 (Table 1).

Considering that about 25,000 genomic equivalents are required for the current GPLN algorithm to detect the presence of PV-1 [17], this finding is not unexpected. Rather it suggests that while the genomic equivalents of PV-1 in sample 13 might be up to the required, those of PV-2 and PV-3 are below and account for the inability of the assay to detect both. This has implications for the polio eradication and endgame strategic plan 2013–2018 [18] and particularly the WHO global action plan (GAP III) for poliovirus containment and sequential withdrawal of the Sabin strains [19]. For example, in the course of Sabin PV-2 containment, isolates containing Sabin PV-2 but with titre below the detection limit of the GPLN assay for Sabin PV-2 detection might be missed. It is therefore suggested that for containment, all isolates that contain any of the poliovirus types should be handled as potentially containing the other two types. Furthermore, to reduce the misclassification of mixed isolates with low titre components and consequently enhance the containment programme, effort should be put into increasing the sensitivity of the assays in use by the GPLN and others; like that described in this study. In addition, effort should also be put into mainstreaming serotype-independent NextGen sequencing strategies recently described for the polioviruses [20] and other Species C members [21].

Though in this study; cell culture isolates were used, it is pertinent to note that we have successfully detected PV2 (unpublished data) directly in stool suspension from an AFP case that was missed by the current cell culture based algorithm [16] for poliovirus detection. In a polio-free world there might be reduced funding for poliovirus surveillance. In such potentially resource limited setting, the modification described in this study might be of value because it allows the enlistment of nonessential facilities without the capacity or infrastructure for cell culture to participate in poliovirus surveillance. Particularly, in such settings, enterovirologists interested in nonpolio enteroviruses can continue their investigations, and also contribute significantly and specifically to poliovirus surveillance, by using the excess of their first round PCR product.

Limitations

The limit of detection of the modification described here is currently not known. Hence, effort is ongoing to conduct spiking experiments with plaque purified and titrated reference isolates in a bid to better define the sensitivity of the assay. Furthermore, it is crucial to mention that the sequence data this modification provides do not cover the entire VP1 region. As such, unlike the ECRA assay [17], the sequence data generated might not be sufficient for extensive molecular epidemiology. Consequently, this modification is currently proposed as addenda and not as substitute for either the current GPLN algorithm [6] or the other cell culture independent assays [17, 22] with the capacity to provide sequence data of the entire VP1 region.

Abbreviations

AFP: 

acute flaccid paralysis

PCR: 

polymerase chain reaction

RT-snPCR: 

reverse transcriptase-seminested polymerase chain reaction

RNA: 

ribonucleic acid

cDNA: 

complementary deoxyribonucleic acid

RD: 

human rhabdomyosarcoma

UTR: 

untranslated region

VP1: 

virus protein 1

EV: 

Enterovirus

E: 

Echovirus

CV: 

Coxsackievirus

WHA: 

World Health Assembly

GPEI: 

Global Polio Eradication Initiative

OPV: 

oral polio vaccine

IPV: 

inactivated polio vaccine

ES: 

Environmental Surveillance

GPLN: 

global polio laboratory network

NPEV: 

nonpolio enterovirus

L20B: 

genetically engineered mouse cell line expressing the human cell surface receptor for poliovirus

WHO: 

World Health Organization

CPE: 

cytopathic effect

PE: 

PanEnterovirus

F: 

forward

R: 

reverse

MCF-7: 

breast cancer cell line

Declarations

Authors’ contributions

TOCF, MOA, JAA: study design. TOCF, MOA, NPO, OEO, GA, FWA, OSO, SDO and JAA: acquisition of reagents, laboratory and data analysis. TOCF: wrote the first draft of the manuscript. TOCF, MOA, NPO, OEO, GA, FWA, OSO, SDO and JAA: revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to thank the members of staff of the WHO Environmental Surveillance Laboratory in Ibadan, Nigeria, for providing us with Sabin strains of poliovirus types 1 and 3.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The data set used and/or analysed during the current study are available from the corresponding author on reasonable request.

Consent to publish

Not applicable.

Ethics approval and consent to participate

Not applicable.

Funding

This study was not funded by any organization rather it was funded by contributions from authors.

Publisher’s Note

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Virology, College of Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria
(2)
Department of Microbiology, Faculty of Science, Ekiti State University, Ado Ekiti, Ekiti State, Nigeria
(3)
WHO National Polio Laboratory, University of Ibadan, Ibadan, Oyo State, Nigeria

References

  1. Nix WA, Oberste MS, Pallansch MA. Sensitive, seminested PCR Amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens. J Clin Microbiol. 2006;44(8):2698–704.View ArticlePubMedPubMed CentralGoogle Scholar
  2. World Health Organisation. Enterovirus surveillance guidelines: guidelines for enterovirus surveillance in support of the Polio Eradication Initiative. Geneva: World Health Organisation; 2015.Google Scholar
  3. Oberste MS, Nix WA, Maher K, Pallansch MA. Improved molecular identification of enteroviruses by RT-PCR and amplicon sequencing. J Clin Virol. 2003;26(3):375–7.View ArticlePubMedGoogle Scholar
  4. Faleye TOC, Adewumi MO, Coker BA, Nudamajo FY, Adeniji JA. Direct detection and identification of enteroviruses from faeces of healthy Nigerian children using a cell-culture independent RT-seminested PCR assay. Adv Virol. 2016;2016:12. https://doi.org/10.1155/2016/1412838.View ArticleGoogle Scholar
  5. Rahimi P, Tabatabaie H, Gouya MM, Mahmudi M, Musavi T, Rad KS, Azad TM, Nategh R. Direct identification of non-polio enteroviruses in residual paralysis cases by analysis of VP1 sequences. J Clin Virol. 2009;45(2):139–41.View ArticlePubMedGoogle Scholar
  6. Sadeuh-Mba SA, Bessaud M, Joffret ML, Zanga MCE, Balanant J, Ngole EM, Njouom R, Reynes JM, Delpeyroux F, Rousset D. Characterization of Enteroviruses from non-human primates in cameroon revealed virus types widespread in humans along with candidate new types and species. PLoS Negl Trop Dis. 2014;8(7):e3052. https://doi.org/10.1371/journal.pntd.0003052.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Faleye TOC, Adewumi MO, Kareem SA, Adesuyan YO, Fapohunda FA, Fasanya ST, Jimeto T, Lawrence OE, Obembe AA, Adeniji JA. The impact of a panenterovirus VP1 assay on our perception of the enterovirus diversity landscape of a sample. J Hum Virol Retrovirol. 2016;4(3):00134.Google Scholar
  8. Pons-Salort M, Burns CC, Lyons H, Blake IM, Jafari H, Oberste MS, Kew OM, Grassly NC. preventing vaccine-derived poliovirus emergence during the polio endgame. PLoS Pathog. 2016;12(7):e1005728. https://doi.org/10.1371/journal.ppat.1005728.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Burns CC, Shaw J, Jorba J, Bukbuk D, Adu F, Gumede N, Pate MA, Abanida EA, Gasasira A, Iber J, Chen Q, Vincent A, Chenoweth P, Henderson E, Wannemuehler K, Naeem A, Umami RN, Nishimura Y, Shimizu H, Baba M, Adeniji A, Williams AJ, Kilpatrick DR, Oberste MS, Wassilak SG, Tomori O, Pallansch MA, Kew O. Multiple independent emergences of type 2 vaccine-derived polioviruses during a large outbreak in northern Nigeria. J Virol. 2013;87(9):4907–22. https://doi.org/10.1128/JVI.02954-12.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Faleye TOC, Adeniji JA. Nonpolio enterovirus-C (NPEV-C) strains circulating in South-Western Nigeria and their contribution to the emergence of recombinant cVDPV2 lineages. Br J Virol. 2015;2(5):68–73.View ArticleGoogle Scholar
  11. Adeniji JA, Faleye TOC. Impact of cell lines included in Enterovirus isolation protocol on perception of nonpolio enterovirus species C diversity. J Virol Methods. 2014;2014(207):238–47.View ArticleGoogle Scholar
  12. Adeniji JA, Oragwa AO, George UE, Ibok UI, Faleye TOC, Adewumi MO. Preponderance of Enterovirus Species C in RD-L20B cell culture negative stool samples from children diagnosed with acute flaccid paralysis in Nigeria. Archiv Virol. 2017. https://doi.org/10.1007/s00705-017-3466-2.Google Scholar
  13. Kroneman A, Vennema H, Deforche K, Van der Avoort H, Pe narandac S, Oberste MS, Vinjéc J, Koopmans M. An automated genotyping tool for enteroviruses and noroviruses. J Clin Virol. 2011;51(2):121–5.View ArticlePubMedGoogle Scholar
  14. Kilpatrick DR, Yang CF, Ching K, Vincent A, Iber J, Campagnoli R, Mandelbaum M, De L, Yang SJ, Nix A, Kew OM. Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues. J Clin Microbiol. 2009;47(6):1939–41.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Sadeuh-Mba SA, Bessaud M, Massenet D, Joffret ML, Endegue MC, Njouom R, Reynes JM, Rousset D, Delpeyroux F. High frequency and diversity of species C enteroviruses in Cameroon and neighbouring countries. J Clin Microbiol. 2013;51(3):759–70.View ArticlePubMedPubMed CentralGoogle Scholar
  16. World Health Organisation. Polio laboratory manual. 4th ed. Geneva: World Health Organisation; 2004.Google Scholar
  17. Arita M, Kilpatrick DR, Nakamura T, Burns CC, Bukbuk D, Oderinde SB, Oberste MS, Kew OM, Pallansch MA, Shimizu H. Development of an efficient entire-capsid-coding-region amplification method for direct detection of poliovirus from stool extracts. J Clin Microbiol. 2015;53:73–8.View ArticlePubMedGoogle Scholar
  18. World Health Organisation. Polio eradication & endgame strategic plan 2013–2018. Geneva: World Health Organisation; 2013.Google Scholar
  19. World Health Organisation. Global action plan to minimize poliovirus facility-associated risk. Geneva: World Health Organisation; 2014.Google Scholar
  20. Montmayeur AM, Ng TF, Schmidt A, Zhao K, Magaña L, Iber J, Castro CJ, Chen Q, Henderson E, Ramos E, Shaw J, Tatusov RL, Dybdahl-Sissoko N, Endegue-Zanga MC, Adeniji JA, Oberste MS, Burns CC. High-throughput next generation sequencing of polioviruses. J Clin Microbiol. 2016;52:606–15 (Epub ahead of print).Google Scholar
  21. Bessaud M, Sadeuh-Mba SA, Joffret ML, Razafindratsimandresy R, Polston P, Volle R, Rakoto-Andrianarivelo M, Blondel B, Njouom R, Delpeyroux F. Whole genome sequencing of Enterovirus species C isolates by High-Throughput Sequencing: development of generic primers. Front Microbiol. 2016;26(7):1294. https://doi.org/10.3389/fmicb.2016.01294 (eCollection 2016).Google Scholar
  22. Krasota A, Loginovskih N, Ivanova O, Lipskaya G. Direct identification of Enteroviruses in cerebrospinal fluid of patients with suspected meningitis by nested PCR amplification. Viruses. 2016;8:10.View ArticlePubMed CentralGoogle Scholar

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