Journal of Clinical Virology 51 (2011) 121–125

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Journal of Clinical Virology

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

An automated genotyping tool for enteroviruses and noroviruses夽

A. Kroneman a,∗ , H. Vennema a , K. Deforche b , H.v.d. Avoort a , S. Peñaranda c ,
M.S. Oberste c , J. Vinjé c , M. Koopmans a,d
a
Laboratory of infectious diseases, National Institute for Public Health and the Environment, Antonie van Leeuwenhoeklaan 9, 3720BA Bilthoven, The Netherlands
b
MyBioData, Rotselaar, Belgium
c
Division of Viral Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30333, USA
d
Virology Department, Erasmus MC, Molenwaterplein 10, Rotterdam, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Background: Molecular techniques are established as routine in virological laboratories and virus typing
Received 18 January 2011 through (partial) sequence analysis is increasingly common. Quality assurance for the use of typing data
Accepted 21 March 2011 requires harmonization of genotype nomenclature, and agreement on target genes, depending on the
level of resolution required, and robustness of methods.
Keywords: Objective: To develop and validate web-based open-access typing-tools for enteroviruses and noroviruses.
Phylogeny
Study design: An automated web-based typing algorithm was developed, starting with BLAST analysis
Genotyping
of the query sequence against a reference set of sequences from viruses in the family Picornaviridae or
Norovirus
Enterovirus
Caliciviridae. The second step is phylogenetic analysis of the query sequence and a sub-set of the reference
sequences, to assign the enterovirus type or norovirus genotype and/or variant, with profile alignment,
construction of phylogenetic trees and bootstrap validation. Typing is performed on VP1 sequences of
Human enterovirus A to D, and ORF1 and ORF2 sequences of genogroup I and II noroviruses. For validation,
we used the tools to automatically type sequences in the RIVM and CDC enterovirus databases and the
FBVE norovirus database.
Results: Using the typing-tools, 785(99%) of 795 Enterovirus VP1 sequences, and 8154(98.5%) of 8342
norovirus sequences were typed in accordance with previously used methods. Subtyping into variants
was achieved for 4439(78.4%) of 5838 NoV GII.4 sequences.
Discussion and conclusions: The online typing-tools reliably assign genotypes for enteroviruses and
noroviruses. The use of phylogenetic methods makes these tools robust to ongoing evolution. This should
facilitate standardized genotyping and nomenclature in clinical and public health laboratories, thus
supporting inter-laboratory comparisons.
© 2011 Elsevier B.V. All rights reserved.

1. Background
With the increasing implementation of molecular methods in
routine viral diagnostics, laboratories are increasingly generating
their own genotyping data. Therefore, there is a need for standard-
ization of genotyping methods. The relevance of typing for clinical
practice or hospital epidemiology depends on the pathogen/variant
and on the capabilities of the laboratory.
The routine practice for enterovirus surveillance as part of the
global poliovirus eradication program,1–6 has been the serotyping
of cultured viruses using a neutralization assay with serotype spe-
夽 The findings and conclusions in this article are those of the authors and do not
necessarily represent the views of the funding agency or the Centers for Disease
Control and Prevention (CDC).
∗ Corresponding author. Tel.: +31 302744035.
E-mail address: annelies.kroneman@rivm.nl (A. Kroneman).
cific reference sera.46 Genotyping based on the VP1 gene, which
encodes the capsid protein, is a reliable alternative, and is currently
replacing serotyping.3,7–13
For noroviruses, routine testing is on the rise due to the increas-
ing awareness of the clinical importance of these pathogens.14–19
Since noroviruses cannot be cultured,20 molecular typing tech-
niques have been the method of choice from the beginning. For
clinical virology practice, typing at the level of genotypes may be
relevant, because genotypes differ in their ability to spread, and
food- or water-borne outbreaks are more frequently caused by
strains that are less common in other settings, such as healthcare
institutions.21 This implies that timely genotype information may
help infer routes of transmission and help focus outbreak inves-
tigations. In-depth investigation of a food-borne source is more
likely to be successful if non-GII.4 strains are involved. The cur-
rently circulating GII.4 viruses evolve quite rapidly in a manner
similar to what is known for influenza A viruses.22 This process
of antigenic changes leads to a rapid displacement of subsequent

1386-6532/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jcv.2011.03.006
122 A. Kroneman et al. / Journal of Clinical Virology 51 (2011) 121–125

variants with a turnover of one or a few years. This highest resolu- start
tion of genotyping may be used to unravel sources of transmission
in difficult-to-control healthcare setting outbreaks. Here, sequenc-
ing of the most variable region of the genome, the P2 domain Blast to identify:
Genus, species/genogroup
of the capsid gene, has been used to link cases in outbreaks.23 Sequenced region, length
Because recombinant norovirus strains have been reported, the Reverse complement?
choice of target region (within ORF1, encoding the polymerase, or
ORF2, encoding the capsid protein) for sequence-based typing is Outcome:
•genus, species/genogroup
an important factor in determining the accuracy of the genotype •length and localization
HEV A-D or no
assignment.28 NoV GI-II?
Or: unassigned

2. Objectives yes stop

Overlap
To develop and validate a web-based, open-access molecular (>100nt) no
typing-tool for enteroviruses and noroviruses. Choice of correct reference set
with supported
for phylogenetic analysis on the
region?
basis of the Blast result
yes
3. Study design
Phylogenetic analyses
3.1. Rationale for genotyping to identify type

Genotyping methods using cut-off values for percentage pair-
wise similarity are presently in use for both enteroviruses and
noroviruses.7,11,13,29–32 For enteroviruses amino acid sequences
are often used because of the large nucleotide sequence diver-
sity within serotypes, mainly in the third codon position. Amino
acid-based typing, however, results in a high level of aggregation
of data, and loss of resolution. Also, pairwise similarity comparisons
are difficult to interpret with increasing numbers of sequences,
and prototype strains (the first strains found of a particular geno-
type) are often quite different from more recent enterovirus or
norovirus strains due to accumulation of mutations over time
and geographic bias. Using pairwise similarity cut-offs for type-
assignment, such strains could eventually differ more than the
established threshold for a genotype. Phylogenetic methods per-
mit simultaneous comparison to a large number of reference
sequences resulting in a potentially more robust typing result, if the
reference set is representative. In phylogenetic methods the clus-
tering patterns remain intact, including for older prototype strains,
and in the face of ongoing evolutionary drift within each cluster.
Bootstrapping may also be used as a measure of confidence for
specific clusters. Therefore we chose to develop phylogeny-based
genotyping-tools.
3.2. Development of a phylogeny-based genotyping-tool
We developed two web-based typing-tools with a common
design that is easily adaptable to other viruses or microorgan-
isms. We used building blocks from the open source Rega HIV
subtypingtool33 (Fig. 1). In the first step the query sequence is
analyzed using BLAST against a reference set of whole genome
sequences representing different taxonomic groups.34 The BLAST
algorithm was chosen because an alignment of sufficient qual-
ity for genotyping by phylogenetic methods is not possible for
sequences of different genera. In the BLAST reference set, repre-
sentatives of other genera of the same virus family are also present
in order to be able to name sequences mistakenly submitted as
norovirus or enterovirus sequences. Thus the enterovirus refer-
ence set consists of representatives of all Picornaviridae genera
that contain known human pathogens (Table 1). The initial BLAST
search also groups the query sequence into a species within the
Enterovirus genus (Table 1). The standard enterovirus nomencla-
ture proposed and maintained by the Picornaviridae Study Group
of the ICTV is followed in the tool.11,29 Similarly, the norovirus
(Caliciviridae) BLAST reference set contains strains representing all
human viruses belonging to the family Caliciviridae, with further
Outcome:
Phylogenetic analyses: •sero/genotype, variant
1) Profile alignment (ClustalW) •bootstrap value
•alignment
against reference alignment
2) Phylogenetic analysis, Or: unassigned
NJ (HKY85)
3) Bootstrap analysis
(n = 100, cut off:70)
Fig. 1. Steps in the genotyping algorithm of the typingtools.
segregation into genogroups for the Sapovirus and Norovirus genera
(Table 1) Nomenclature of genotypes and variants follows proposed
standards30 but will be updated based on recommendations of the
NoroNet nomenclature working group (submitted for publication).
The result of the BLAST step is the assignment of a
genus/species/genogroup, given only if the expectation (E)-value
is less than 10−5 .35 During the BLAST analysis, length and
genome localization are determined. Reverse and/or complement
sequences are also recognized and searched.
The second step involves phylogenetic analysis and is only
performed on sequences for which the genus identification is suc-
cessful (or in case of norovirus, a genogroup), which have a specified
minimal length, and are located on a specified region of the genome
(Fig. 1). To maximize flexibility, most genomic regions commonly
used for typing are included in the phylogenetic step (the VP1
region for enteroviruses, and ORF1 and ORF2 for noroviruses.11,31
The correct reference set is chosen according to the BLAST result
(Table 1). For enterovirus, phylogenetic analysis is performed for
enterovirus species A, B, C or D, with ≥ 100 nucleotides of VP1
sequence. For norovirus the phylogenetic analysis proceeds for a
sequence of ≥ 100 nucleotides from genogroup I or II norovirus cap-
sids or a sequence of ≥ 100 nucleotides within 800 nt at the 3 end of
ORF1 (encoding the polymerase). In a third step, only for GII.4 geno-
type sequences, variants are assigned.22 In the typing-tools, profile
alignments are performed using ClustalW against an alignment of
the reference sequences. Phylogenetic trees are constructed using
the neighbor-joining method with a HKY8536 substitution model,
(PAUP*37 ).
The HKY85 model was chosen based on likelihood-based model
selection on the reference alignment. In the PAUP* log file the
bootstrap tree is also presented. A type assignment only takes
place if the bootstrap value exceeds 70%.38 Persons accessing
the tool can download all results, including the complete aligned
reference sets, for their own use. To protect users’ data, query
sequences and results are stored only temporarily on the server,
protected by a randomly generated job ID token. The typing-tools
 A. Kroneman et al. / Journal of Clinical Virology 51 (2011) 121–125 123

Table 1
Taxonomic levels of Picornaviridae and Caliciviridae and representatives in the reference sets of the typing tool. The family Picornaviridae consists of 12 assigned and two
proposed genera29 The human viruses in the genus Enterovirus are sub-divided into seven species, further subdivided into serotypes. Noroviruses are divided into genogroups,
further subdivided into genotypes and variants. The viruses infecting humans are found in genogroups I, II and IV.

BLAST analysis Phylogenetic analysis

Family Genus Species/genogroup Reference sets for NoV Reference sets of NoV GII.4
(species) genotypes and HEV variantsa
serotypes

Picornaviridae Enterovirus HEV-Ab complete VP1 sequences of

17c (of 21) types
HEV-B complete VP1 sequences of
57c (of 59) types
HEV-C complete VP1 sequences of
15c (of 18) types
HEV-D complete VP1 sequences of
3c (of 3) types
HRV-Ad
HRV-B
HRV-C
Parechovirus
Cardiovirus
Aphtoviruse
Hepatovirus
Erboviruse
Kobuvirus
Teschoviruse
Cosavirus
Senecaviruse
Caliciviridae Norovirus GI Complete ORF2 sequences
of 8 genotypes
ORF1 sequencesf of 14 GI
and 31 GII types
GII ORF 1 seqf . of 13 GII.4
variants
Complete ORF2 sequences Complete ORF2 seq. of 13
of 21 genotypes GII.4 variants
GIII
GIV
GV
Sapovirus GI
GII
GIII
GIV
GV
Vesivirus
Lagovirus
Becovirus
Valovirusg
Recovirusg
a
For norovirus GII.4 sequences only.
b
HEV: human enterovirus.
c
serotypes infecting humans as listed at www.picornaviridae.com as of January 2010.
d
HRV: human rhinovirus.
e
Does not contain human species, therefore not included in the typing tool.f 800 nt of the 3’ end of ORF1.
g
Proposed genera.

are available at: http://www.rivm.nl/mpf/enterovirus/typingtool
and http://www.rivm.nl/mpf/norovirus/typingtool.
4. Validation of genotyping-tool
In order to validate the typing-tools, a collection of enterovirus
and norovirus sequences was analyzed using the typing-tools
in comparison with the current typing methods used in our
laboratories. The proportion of untypeable strains and typing mis-
matches were calculated. For enterovirus typing, we used 358 VP1
sequences from 4 different publications,10,39–41 which were previ-
ously typed using the CDC methods,11 and all 437 VP1 sequences
collected as part of the national enterovirus surveillance program
in the Netherlands from 2007 to 2009. This last set was previously
tested using pairwise amino acid similarity scores with a library
of reference sequences using the BioNumerics software (Applied
Maths, Gent, Belgium).
For norovirus we used 8342 sequences >100 nt long, of which
6332 were partial ORF1 sequences and 2010 partial and complete
ORF2 sequences that were collected from 2000 to 2010 by 13
European countries within the FBVE network.42 These sequences
were genotyped using the same library method used for the Dutch
enterovirus set, followed by a manual step. The library used con-
sisted of the reference sequences used in new the typingtool.
Variants were assigned using combined analysis of ORF1 and ORF
2 sequences (when available).
5. Results
The enterovirus and norovirus testsets were typed using the
typing-tools and the results were compared to the previous typing
results (Table 2a and b). For enterovirus 10 (1%) discrepancies were
found, of which three were unfavorable: two sequences were only
typed until genus level (an echovirus 29 (HEV B), and an echovirus
124 A. Kroneman et al. / Journal of Clinical Virology 51 (2011) 121–125

Table 2
Composition of the test sets used in the enterovirus (a) and norovirus (b) typing tools and comparison of the results with previous typing results.

(a) Test sets N Median length Number of Number (%) of Comment

enterovirusa serotypes discrepancies

CDC set 358 333 nt 4 (1%)

HEV A 50 13 serotypes 0
HEV B 263 44 serotypes 3 (1%) 2 only typed until
genus level, 1
mistyped
HEV C 41 11 serotypes, 1 1 (2%) 1 unassigned was
unassigned typed
HEV D 2 0
Rhinovirus 2 0
NL set 437 318 nt 6 (1%)
HEV A 102 6 serotypes 0
HEV B 315 23 serotypes 0
HEV C 10 4 serotypes 0
Rhinovirus 3 0
Unassigned 7 6 (86%) 6 were typed
Total NL + CDC 795 10 (1%)

(b) Test sets norovirusa N Number of Number (%) of Average length

genotypes/variants unassigned assigned/unassigned

ORF1 sequences 6332 126 (2.0%)c

ORF1 GI 502 11 genotypes 13 (2.6%) 212/180
ORF1 GII 5830 20 genotypes 233/160b
ORF1 GII, other than GII.4 1276 50 (3.9%)
ORF1 GII.4 genotype analysis 4554 63 (1.4%)
ORF1 GII.4 variant analysis 4491 10 variants 1173 (26.1%)c 246 / 212b
ORF2 sequences 2010 2 (0.1%)c
ORF2 GI 180 8 genotypes 2 (1.1%) 347
ORF2 GII 1830 17 genotypes 0 351
ORF2 GII.4 variant analysis 1348 12 variants 227 (16.8%)c 370/246b
Total nr of sequences, genotype analysis 8342 128 (1.5%)
Total nr of GII.4 sequences, variant analysis 5839 1400 (21.6%)
a
Type according to previously used typing methods.
b
Average length of assigned sequences is significantly higher, using 1-tailed T test.
c
The difference in % unassigned between ORF1 and ORF2 sequences is significant, using Chi square test.

13 (HEV B) sequence) and one EV69 sequence was mistyped as
E13 (HEV B). One previously untyped enterovirus C was typed as
coxsackievirus A13. Six previously untyped Dutch sequences were
typed as coxsackievirus A16 (HEV A, 4 sequences), coxsackievirus
B5 (HEV B) and echovirus 6 (HEV B).
For norovirus 128 (1.5%) discrepancies were found in the geno-
type assignment and 1400 (21.6%) in the variant assignment. All
discrepancies were due to the inability of the tool to assign a
genotype or variant. Calculation of average lengths of assigned
and unassigned sequences yielded higher lengths for assigned
sequences for all subsets, which were significant for the ORF1 GII
genotype set and the ORF1 and ORF2 GII.4 variant sets. The tool
performed better for ORF2 than for ORF1 sequences, both for geno-
typing and variant typing. These differences were significant.
6. Discussion
The typing-tools performed very well both for the typing of
enterovirus and the genotype assignment of noroviruses, thus
facilitating standardization of nomenclature at this level. For
noroviruses, GII.4 variants are also recognized, and the typing-tool
successfully assigned 78.4% of GII.4 sequences to a variant by the
tool. Even though this is a lower score than at the genotype level,
we believe the variant typing functionality is very useful, because
new variants need to be recognized and shared among laboratories
as they emerge. Taking typing through this tool as a first step nar-
rows down the number of sequences that need to be assigned using
more specialized approaches (e.g. by taking into account signature
sequences and mutation hot spots). This manual typing, compa-
rable to the last step in the previously used typing method, can
be performed using the alignments and dendrograms produced by
the typing-tool, and can be used to type most of the unassigned
sequences, although in this case no bootstrap validation can be
performed.
The use of longer (e.g., full length) sequences will improve the
phylogenetic signal to distinguish GII.4 variant viruses and may
thus lead to fewer unassigned sequences,43 as is indicated by the
significant difference in average lengths of assigned and unassigned
sequences found in this study. Besides the longer average length
of the ORF2 sequences, the greater genetic diversity of the ORF2
region may play a role in the significantly higher percentage of type
assignment for ORF2 sequences.44,43
Similarly, the vast majority of enterovirus sequences were
typed correctly using the current tool. The mistyping of an EV69
enterovirus B sequence as E13 is a consequence of the very low
phylogenetic distance between these two types.39
Comparison of enterovirus and norovirus genotyping results
across laboratories and countries has been hampered by the lack
of standardized primer sets, sequence analysis methods, and typ-
ing nomenclature. Widespread use of the typing-tools is a first step
towards standardization of typing analyses, thus facilitating rapid
exchange of typing information internationally, even if viruses,
samples, or sequences cannot be shared.42,45 For enteroviruses,
new type names are assigned centrally by the Picornaviridae Study
Group of the ICTV.11,29 The tool will need to be updated periodically
with new names and reference strains to stay up to date. Simi-
larly, regular updates with sequences from NoroNet will be needed
to update the norovirus reference set with newly emerged types
or variants. An essential feature of the genotyping-tool is map-
ping of the query sequence to a specific location on the reference
genome(s). The choice of the optimal target regions for genotyping
is a matter of some debate, and it may have a decisive influence
 A. Kroneman et al. / Journal of Clinical Virology 51 (2011) 121–125
on the level of resolution that can be obtained.43,44 Bootstrap val-
ues can be used to indicate robustness of the type attribution
using sequences of different regions. Future plans are to extend the
enterovirus typing-tool with non-human types, and to add other
human picornaviruses that are of clinical importance, including
human parechoviruses, rhinoviruses, and hepatitis A viruses, and
to set up a typing-tool for influenza virus.
Funding
This project was funded through core funding of the RIVM and
CDC.
Competing interests
None declared.
Ethical approval
Not required.
Acknowledgments
We thank the members of the global NoroNet network for test-
ing of the typing-tool, and for providing feed-back on the output
formats desired and all members of the FBVE network for their
contribution to the norovirus sequence database.
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