Abstract
Echovirus 9 (E9) has been detected in an increased number of symptomatic patient samples received by the National Enterovirus Reference Laboratory in Hungary during 2018 compared to previously reported years.
Formerly identified E9 viruses from different specimen types detected from patients of various ages and showing differing clinical signs were chosen for the detailed analysis of genetic relationships and potential variations within the viral populations. We used next generation sequencing (NGS) analysis of 3,900 nucleotide long amplicons covering the entire capsid coding region of the viral genome without isolation, directly from clinical samples.
Compared to the E9 reference strain, the viruses showed about 79% nucleotide and around 93% amino acid sequence similarity. The four new viral genome sequences had 1-20 nucleotide differences between them also resulting in 6 amino acid variances in the coding region, including 3 in the structural VP1 capsid protein. One virus from a patient with hand, foot, and mouth disease had two amino acid changes in the VP1 capsid protein. An amino acid difference was also detected in the non-structural 2C gene of one virus sequenced from a throat swab sample from a patient with meningitis, compared to the faecal specimen taken two days later. Two amino acid changes, one in the capsid protein, were found between faecal samples of meningitis patients of different ages.
Sequencing the whole capsid genome revealed several nucleotide and amino acid differences between E9 virus strains detected in Hungary in 2018.
Introduction
Surveillance efforts initiated by the Global Polio Laboratory Network and regular, detailed reporting of non-polio enterovirus (EV) epidemics increased scientific awareness for the importance of enterovirus genetic characterisation. With molecular typing mostly based on the VP1 gene encoding the viral capsid protein increasingly replacing traditional serotyping of virus isolates, we have a greater resolution of enteroviruses detected in patient samples.
The name echovirus (for enteric, cytopathogenic, human, orphan virus) was originally chosen for a group of related viruses whose association with human disease was unknown at the time of their discovery (Committee on the ECHO viruses, 1955). It subsequently became obvious that infections by individual echovirus serotypes are indeed associated with a wide variety of clinical manifestations ranging from non-specific febrile illness, mild skin or mucosal lesions and meningitis to overwhelming diseases [1]. Echoviruses are also generally recognised as important viral agents of neurological infections [2]. Echovirus 9 is classified as an Enterovirus B, which species was identified as the most common cause of aseptic meningitis, an important reason for hospitalisation affecting mainly children [3].
E9 along with echoviruses 30 and 6 were the three most abundant enteroviruses found in a Belgian study from 2007–2018 [4]. In the United States from 1970 to 2005, E9 was the most prevalent enterovirus serotype and was usually associated with aseptic meningitis outbreak [3]. A Chinese study found E9 as the sole pathogen in 17.17% of 367 detected enterovirus infections of HFMD patients with confirmed encephalitis and meningitis [5]. E9 was among the ten most prevalent serotypes in a European study aggregating reported EV cases between 2015 and 2017 [6], with a 4% incidence and neurological symptoms being recorded in 69% of 177 patients with E9 infection. in In our earlier Hungarian study E9 was detected in 3.5% of 284 EV cases reported between 2010 and 2018 [7].
EVs have a ∼7.4 kb long genome of positive-sense single-stranded RNA that contains 5′ and 3′ untranslated regions (UTRs) flanking a single open reading frame encoding 4 structural proteins (VP1-4) and 7 non-structural proteins (2A, 2B, 2C, 3A, 3B (VPg), 3C and 3D polymerase) [8]. While VP1-3 interact with each other to construct the outer architecture of the viral capsid, their N-termini, together with VP4, line the capsid interior.
High mutation rates and recombination drive great genetic variability resulting in diverse and dynamic viral populations. Tracking molecular changes and their association with virus pathogenicity and transmission is crucial for monitoring outbreaks and understanding the underlying causes of severe disease.
Routinely generated nucleotide sequencing information about EVs from clinical samples is usually limited to short, partial genomic sequences of VP1 that enable identifying the genotype but have limited use for detailed genetic characterisation. Several approaches to EV NGS have been described previously for sequencing viruses directly from clinical samples and from cell culture isolates with metagenomics or target-specific techniques, still, available E9 virus sequences covering the whole capsid are scarce [9–13].
In this study we aimed to generate E9 whole capsid sequences to identify nucleotide and amino acid sequence differences and virus variants. We also investigated their genetic relationships to other reported E9 viruses and the possible significance of sequence divergence.
Materials and methods
Samples, EV detection
The Hungarian National Enterovirus Reference Laboratory carries out diagnostic testing for EV from clinical specimens received from across the country. Received samples are processed for routine EV detection and genotyping as described previously [7].
The viral load of identified E9 samples was checked with an in-house panEV real-time one-step RT-PCR assay targeting the conserved 5′ untranslated region of the genome with qScript XLT 1-Step RT-qPCR ToughMix (Quantabio, Beverly, MA, USA) using the following primers and probe:
PanEV_F_qPCR: 5′- CTCCGGCCCCTGAATG
PanEV_R_qPCR: 5′- TTGTCACCATAAGCAGYCA
PanEV_probe: 5′-[FAM]-CGGAACCGACTACTTTGGGTG-[BHQ1]
Nine E9 cases were detected in 2018 [7] and connected clinical specimens were chosen for further characterisation based on availability, sample type, reported symptoms and age of patient (Table 1, Fig. 1A).
Details of Echovirus 9 cases detected in Hungary in 2018
| Sample ID | Date of sampling | Sample type | Reported symptoms | Age (years) | Location | Ct value |
| Sz2519 | 11 May 2018 | Faeces | cardiac failure | 44 | Monaj | NA |
| Li2548 | 19 May 2018 | Liquor | aseptic meningitis | 8 | Örkény | 29.5 |
| Sz2549 | 19 May 2018 | Faeces | 26.1 | |||
| Li2638 | 24 May 2018 | Liquor | meningitis | 29 | Örkény | 25.7 |
| Gm2642 | 24 May 2018 | Throat swab | 26.6 | |||
| Sz2650 | 26 May 2018 | Faeces | 27.9 | |||
| Li3370 | 25 June 2018 | Liquor | encephalitis | 6 | Monor | NA |
| Li4467 | 09 August 2018 | Liquor | aseptic meningitis | 5 | Kecskemét | NA |
| Li5340 | 30 August 2018 | Liquor | meningitis serosa | 5 | Ráckeve | 32.7 |
| Sz5380 | 07 September 2018 | Faeces | 26.2 | |||
| Sz5346 | 07 September 2018 | Faeces | hand, foot and mouth disease | 1.5 | Tiszaszőlős | 30.0 |
| Sz5378 | 10 September 2018 | Faeces | meningitis serosa | 5 | Ráckeve | 27.1 |
| Gm5573 | 12 September 2018 | Throat swab | fever, headache, herpangina | 11 | Ráckeve | 29.5 |
Notes: Whole-capsid nucelotide sequences for samples shown in bold were generated in this study. Samples derived from the same patient are grouped together.
A: Enterovirus genome schematic with PCR strategies, B: Genome coverage of E9 entire capsid regions following reverse-transcription polymerase chain reaction (RT-PCR) and NGS. Filtered reads were mapped to respective consensus sequences and the number of reads at each nucleotide is shown
Citation: Acta Microbiologica et Immunologica Hungarica 2022; 10.1556/030.2022.01788
A: Enterovirus genome schematic with PCR strategies, B: Genome coverage of E9 entire capsid regions following reverse-transcription polymerase chain reaction (RT-PCR) and NGS. Filtered reads were mapped to respective consensus sequences and the number of reads at each nucleotide is shown
Citation: Acta Microbiologica et Immunologica Hungarica 2022; 10.1556/030.2022.01788
A: Enterovirus genome schematic with PCR strategies, B: Genome coverage of E9 entire capsid regions following reverse-transcription polymerase chain reaction (RT-PCR) and NGS. Filtered reads were mapped to respective consensus sequences and the number of reads at each nucleotide is shown
Citation: Acta Microbiologica et Immunologica Hungarica 2022; 10.1556/030.2022.01788
Entire capsid-coding region amplification by RT-PCR
We used an echovirus adapted version of the entire capsid-coding region amplification (ECRA) method originally designed for poliovirus by Arita et al. [14] and modified for enteroviruses [13, 15, 16] (Fig. 1A). Where available, viral RNA was freshly extracted from the clinical samples with Zymo Quick RNA kit (Zymo Research, Irvine, CA, USA) or Qiagen Viral RNA kit (Qiagen, Dusseldorf, Germany) following the manufacturers' instructions with added on-column DNase digestion.
The approximately 3,900 nucleotide (nucleotides 531–4456, numbering as in Echovirus 9 Barty strain, GenBank accession number: X92886) long amplicons were produced with SuperScript III reverse transcriptase (Invitrogen, Waltham, MA, USA) in a two-step RT-PCR amplification with Q5 High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) using NIBSC_EV_F4 and NIBSC_EV_R3 primers:
NIBSC_EV_F4: 5′-YRGCGGAACCGACTACTTT-3′
NIBSC_EV_R3: 5′-TCAATRCGRCATTTGGACTTRAAYTG-3′
PCR products were purified with AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA) and quantified using Qubit Fluorometers with dsDNA High Sensitivity assay (Invitrogen).
Whole-capsid nucleotide sequencing, quality trimming of sequencing reads
Sequencing libraries were prepared with Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) and sequenced on a MiSeq instrument using MiSeq Reagent Kit Micro v2 (Illumina) following manufacturer's protocols. NGS data were quality trimmed and analysed using the Geneious R10 software package (Biomatters, Auckland, New Zealand). Raw sequence data were then imported into Geneious and paired end reads combined. Data were filtered and trimmed to have no bases with a quality <Q30 no ambiguities, and no sequencing primer and adapter sequences. Following this, reads <50 nt in length were discarded and duplicate reads removed.
Generation of E9 whole capsid sequence by reference-guided assembly
The filtered reads were mapped to a set of Echovirus9 sequences by using a sequence database obtained from GenBank and contig sequences were generated. Consensus sequences from the contig with most mapped reads producing the longest single contig was used for iterative reassembly with a minimum 50 base overlap, minimum overlap identity of 95%, maximum 5% mismatches per read and only using paired hits during assembly. To build whole-capsid contig sequences re-assembly was repeated until no further filtered reads could be mapped. Final consensus sequences were obtained by assigning the most common nucleotide sequence at each nucleotide position.
SNP and phylogenetic analysis of E9 strains
Single nucleotide polymorphisms (SNPs) were identified using Geneious 10.2.3 default settings. Variants with coverage <100, average quality <30, variant frequency <10%, variant P-value >10−6 and the number of total variant reads <100 were excluded.
Echovirus 9 full capsid sequences obtained in this study were compared to those of other E9 sequences available in the GenBank database. Genome sequences were aligned using the program Clustal Omega 1.2.2 (within Geneious 10.2.3) software and used for phylogenetic analyses performed in MEGA X software [17]. The evolutionary history of aligned sequences was calculated using the Maximum Likelihood tree building method with genetic distances computed using the Jukes-Cantor model [18]. Bootstrap test with 1,000 replicates was applied.
Results
Thirteen E9 viruses previously identified from clinical specimens linked to nine separate E9 infections were considered for suitability of whole capsid genome amplification. Where enough sample was available, fresh viral RNA was extracted, otherwise previously purified RNA was first used to set up real-time RT-PCR with panEV primers and probe that target the well conserved 5′ UTR. Cycle threshold values of 25.7–32.7 representing the accumulating fluorescent signal crossing the threshold of background indicated moderate to reasonable viral load in the 10 samples available for testing (Table 1, Fig. 1A).
Long amplicon RT-PCRs were prepared, and PCR products checked for size and amplification specificity before magnetic bead purification followed by quantification. Six samples produced appropriate amplicons for sequencing library preparation and viral genomes of 4 selected E9 strains were characterised by high resolution sequencing. Final libraries were selected based on a few criteria. We wanted to compare at least two samples from different specimens derived from the same patient. Comparison of two samples from patients with different clinical symptoms and samples from patients exhibiting similar clinical signs, but of different ages was also pursued.
All four libraries produced high numbers of good quality filtered sequencing reads (between 98.000 and 198.000) allowing assembly of full-size capsid sequences with 3700–7500× average coverage per nucleotide (Fig. 1B, Table 2). Consensus sequences were deposited in the GenBank database with accession numbers OM743921-4 and molecular typing by comparative nucleotide alignment with sequences available in GenBank confirmed the E9 genotypes.
Genetic properties of E9 strains
| Sample ID | GM2642 | Sz2650 | Sz5346 | Sz5378 | E9 Barty | |
| Accession No.1 | OM743923 | OM743924 | OM743922 | OM743921 | X92886 | |
| Number of reads2 | 1,28,250 | 1,97,512 | 1,38,452 | 98,232 | - | |
| Mean coverage3 | 4,896 | 7,517 | 5138 | 3,712 | - | |
| Protein | Nucleotide4 | |||||
| 5′ UTR | ||||||
| 704 | A | A | A | C | C | |
| VP4 | ||||||
| 794 | C | C | T | C | C | |
| VP2 | ||||||
| 1082 | C | C | C | T | C | |
| 1421 | T | T | T | C | T | |
| 1493 | T | T | T | C | C | |
| 1628 | T | T | T | C | T | |
| 1697 | C | C | T | C | C | |
| VP3 | ||||||
| 1874 | G | G | G | A | G | |
| 2016 | T | T | T | C | C | |
| 2177 | T | T | C | T | C | |
| 2372 | T | T | T | C | C | |
| VP1 | ||||||
| 3081 | A | A | G (Thr>Ala)5 | A | A | |
| 3182 | C | C | T | C | C | |
| 3221 | C | C | T | T | T | |
| 3250 | C | C | T (Ala>Val)5 | C | C | |
| 3274 | A | A | A | G (Asn>Ser)5 | A | |
| 2A | ||||||
| 3410 | T | T | T | C | C | |
| 3662 | T | T | C | T | A | |
| 2B | ||||||
| 2C | ||||||
| 4192 | A | A | A | G (Lys>Arg)5 | A | |
| 4246 | A | A | G (Lys>Arg)5 | A | A | |
| 4266 | G (Ser>Gly)5 | A | A | A | A | |
| 4371 | T | T | C | T | C | |
Notes: 1GenBank database accession number. 2Number of filtered sequencing reads mapping to consensus sequence. 3Mean number of sequence reads at each nucleotide position. 4Nucleotide position of X92886 strain. 5Amino acid change as a result of nucleotide variation compared to consensus.
Comparison of full capsid consensus sequences of the four virus strains revealed up to 20 nucleotide differences between them (Table 2). These nucleotide changes also resulted in 6 amino acid variances in the coding region, including 3 in the structural VP1 capsid protein.
Gm2642 and Sz2650 were two viruses from the same 29-year-old patient with meningitis, a throat swab, and a faecal sample, respectively, taken 2 days apart. The two viral sequences showed only a single nucleotide divergence between them in the non-structural 2C protein gene, resulting a Serine to Glycine amino acid change at 2C-57 amino acid position.
In GM2642 variant analysis revealed a SNP in VP3 at nucleotide 2055 (20.7% C and 79.3% T, no amino acid change effect), one transition in VP1 at nucleotide 2502 (26.2% G and 73.7% A, ATA->ATG, Ile->Met amino acid substitution) and an additional polymorphism at nucleotide 4267 (28.1% A and 71.9% G, GGT->AGT transition, Gly->Ser amino acid substitution) in the non-structural 2C protein. In Sz2650 two nucleotide transitions were detected during SNP analysis. One at position 2502 in VP1 (18.2% G and 81.8% A), and another at nucleotide 4267 in 2C 48.9% G and 51.1% A. Sz2650 also had a minor variant in VP1 with only 8.6% frequency.
Sz5346, a virus from an 18 months old patient with hand, foot and mouth disease symptoms had two amino acid changes (VP1-213 Thy>Ala, VP1-269 Ala>Val) in VP1 capsid protein and a third (2C-50 Lys>Arg) in protein 2C in comparison to the other three E9 viruses from 2018, all of which derived from patients with meningitis. Sz5346 had no SNPs detected.
Two amino acid differences, including one in the capsid protein, were found between faecal samples of meningitis patients of different ages. Sz5378 had an Asparagine to Serine change at VP1-277 and a Lysine to Arginine change at 2C-32. Sz5378 had two minor variants below 10% frequency, one in 5′UTR and one in VP1.
Phylogenetic analysis was carried out on E9 sequences from NCBI GenBank database available on eighth May 2022, that shared at least 80% nucleotide sequence and were of appropriate length (Fig. 2). Evolutionary analysis also included E9 serotype strain Hill and an echovirus 15 strain (MT641434, isolated in Great Britain in 2018). Year and country of collection was researched for each included sequence and this information is shown where it was available.
Phylogenetic tree based on 45 VP1 sequences (982bp) of E9 isolates from around the world. The evolutionary history was inferred by using the Maximum Likelihood method and Jukes-Cantor model [18] in MEGA X [17]. The tree with the highest log likelihood (−11292.75) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Sequences produced in this study are displayed in bold text
Citation: Acta Microbiologica et Immunologica Hungarica 2022; 10.1556/030.2022.01788
Phylogenetic tree based on 45 VP1 sequences (982bp) of E9 isolates from around the world. The evolutionary history was inferred by using the Maximum Likelihood method and Jukes-Cantor model [18] in MEGA X [17]. The tree with the highest log likelihood (−11292.75) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Sequences produced in this study are displayed in bold text
Citation: Acta Microbiologica et Immunologica Hungarica 2022; 10.1556/030.2022.01788
Phylogenetic tree based on 45 VP1 sequences (982bp) of E9 isolates from around the world. The evolutionary history was inferred by using the Maximum Likelihood method and Jukes-Cantor model [18] in MEGA X [17]. The tree with the highest log likelihood (−11292.75) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Sequences produced in this study are displayed in bold text
Citation: Acta Microbiologica et Immunologica Hungarica 2022; 10.1556/030.2022.01788
Discussion
BLAST searches for the complete sequences returned an E9 virus isolated in 2008 from a meningitis case in Taiwan (GenBank accession number: MF422581) with 82.5–82.7% nucleotide identity and 96.91% amino acid identity as the closest match to the new Hungarian sequences. Another E9 strain MH752986, from the USA, isolated in 2004 also showed 96.91% amino acid sequence identity and 81.7–81.8% nucleotide sequence similarity. Comparisons restricted to VP1 sequences found a virus isolated in India between 2007–2009 from an AFP case (JN203737) as the closest relative. JN203737 shares 92.2% and 92.4% nucleotide sequence similarity with Sz5643 and GM2642, Sz2650, Sz5378 respectively, with VP1 amino acid sequence similarities ranging 96.7%–97.3%, equal to 8-10 amino acid differences.
Due to their error-prone polymerase, EVs change rapidly and show great genetic diversity within genotypes. E9 sequences available in GenBank based on their capsid have evolved into eight clusters (A-H) since 1953, with the main epidemic strains belonging to D, F and H clusters [19]. Following this classification, the four Hungarian strains from 2018 belong to cluster F along with the two Indian viruses from AFP cases from 2007–2009, strains isolated from suspected meningitis cases in Russia in 2012 and viruses detected in 2006 in France. The VP1 phylogenetic tree infers that several strains have been co-circulating, since viruses detected within the same year cluster separately from each other and many main clusters contain strains isolated in different decades.
VP1 is the most variable of the capsid proteins, the N-terminus of VP1, which contains the BC loop, is highly conserved within individual enteroviral serotypes [20]. The exposed BC loop has been shown to be important for the reactivity of type-specific antibodies [21]. Antigenic differences between enterovirus types are assumed to be the results of amino acid variations in the exposed surfaces of the virion, like the BC loop of the VP1 protein [22]. In polioviruses some of the conformational changes required for infectivity and involved in the control of capsid stability and neurovirulence in mice occur in the surface exposed loops of VP1 [23]. The sequence of the E9 BC loop, GDPESTDRFDA (amino acids 83–93 of the VP1 protein) diverged to GNPESTDRFDA in our four strains as the Aspartic acid residue changed to an Asparagine compared to the Barty strain. 18 out of 40 additional inspected E9 sequences containing the BC loop shared the same amino acid change. Several additional third base nucleotide differences appear in the BC loop amongst the compared viral sequences, along with the occasional alternative single amino acid difference seen in a few isolates. Alanine at amino acid position 81, which is exposed at the surface near the BC loop, is shared by most E9 strains, including the viruses sequenced in this study. An E9 strain (AF524867), that was isolated from a child at the clinical onset of type 1 diabetes, carried the T81A substitution in VP1 with lytic capability toward human pancreatic islets [24]. Apart from that reported strain, only one additional virus sequence (HF948097) out of 44 analysed, contained Threonine at this site.
At the carboxy terminus of VP1 capsid protein and RGD motif is present in E9 strain Barty, isolated form the cerebrospinal fluid of a child with aseptic meningitis in 1957 is highly virulent in new-born mice. Hill, the E9 prototype strain isolated from a healthy child in 1953 lacks this extension and is non-pathogenic in new-born mice [25–27]. Apart from Hill, all E9 virus full capsid sequences downloaded from GenBank possess this motif, just as all four Hungarian strains characterised in this study, including Sz5346, the virus detected in a patient with HFMD. This result agrees with previous findings of E9 viruses isolated from meningitis cases containing the RGD peptide motif [28, 29] Presence of the RGD motif is important for pathogenicity both in mice and humans, the virus-host cell interaction occurs via contact between the RGD motif and the integrin αvβ3 (vitronectin receptor, VNR) [30, 31]. Researching human E9 isolates with RGD motif showed a spectrum of virulence in new-born mice from apathogenic to highly lethal, suggesting that the site itself is not the only determinant of pathogenicity [32].
Partial nucleotide sequences of the VP1 region routinely used for genotyping and often the basis of phylogenetic analysis would have only detected one of the 20 nucleotide differences without any amino acid sequence variation within the characterised four viral sequences. The method used in our study produces much longer nucleotide sequences for each E9 strain (approximately 3900bp) covering the entire capsid region and part of the region coding for non-structural proteins, hence allowing more in-depth genetic analyses. The straightforward approach used in this study generated whole capsid enterovirus PCR amplicons straight from the sample that could be used for high resolution sequencing directly, is arguably a valuable tool in any enterovirus laboratory. Further studies need to be carried out to generate and compare E9 viral genomes from patient samples, ideally from a broad spectrum of diseases with full records of samples including clinical signs. Our whole capsid genome sequences will provide a useful resource for any such future studies investigating enteroviral phenotypic determinants and monitoring the emergence of altered pathogenicity and tropism.
Conflict of interest
The authors declare that they have no conflict of interest.
Authors contributions
Erika Bujaki and Mária Takács contributed to study conception and design. Material preparation, data collection and analysis were performed by Erika Bujaki and Ágnes Farkas. First draft of the manuscript was written by Erika Bujaki and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Acknowledgement
The authors would like to thank Javier Martin and Manasi Majumdar at NIBSC, UK for their advice and training, Anna Nagy, Éva Áy, Nóra Magyar, Bernadett Pályi and Ákos Tóth at the National Public Health Center, Hungary for helpful discussions and practical help.
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