• To the sequence typing tool
• Introduction
• Genotyping process in three steps
• Typing Regions
• Reference set update  

Introduction

By issuing preliminary names for new strains and using a standardized nomenclature, findings in different parts of the world can be compared.

With this tool sequences of any part of the Norovirus (NoV) genome can be assigned to a NoV genogroup, and sequences of all commonly used regions for typing can be assigned to a genotype.

For one specific NoV genotype (II.4) the variant of that genotype is determined.
Sequences of other Caliciviridae are also recognized by the tool and are assigned to their (preliminary) genus. In case of Sapoviruses  a genogroup is also assigned.

A paper describing the norovirus typingtool and the enterovirus typingtool, which was developed using the same platform: 

Kroneman A, Vennema H, Deforche K, Avoort HV, Peňaranda S, Oberste MS, Vinjé J, Koopmans M.
An automated genotyping tool for enteroviruses and noroviruses. J Clin Virol. 2011 Jun;51(2):121-5.

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Genotyping process in three steps

Step 1:

This initial step performs a nucelotide blast scan, using Blast.

The submitted sequence is blasted against a set of whole genome sequences for determination of the genus within the Caliciviridae family and the genogroup (for NoV and for SaV). The start and end positions of the fragment are determined using whole genome sequences.

Step 2:

This second step is performed for sequences which were identified as NoV Genogroups I and II in step 1.

ORF1 sequences and ORF2 sequences are analysed seperately using a different reference set. For each submitted sequence that overlaps sufficiently (= 100nt) with either the ORF1 region or the ORF2 region supported by the tool, an alignment is performed using ClustalW and a phylogenetic tree (NJ) is constructed with the HKY851 method using PAUP* software.

HKY85 is a likelyhood method with 100 bootstrap replicates. The alignment contains the sequence together with the pre-aligned reference sequences for each genotype. In the PAUP* log file  the       bootstrap tree is also presented.

For each genotype two, in some cases more, reference sequences have been selected, representing the level of variance within the cluster. If available (and covering the complete regions) the known prototype strains are used.

Step 3:

When a genotype II.4 is identified in step 2, a second analysis is done to identify a specific variant.

The same phylogenetic method is used as in step 2. Again two representative sequences per subcluster are used in the analysis.

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Typing regions

The submitted sequences are analysed separately for ORF1 and ORF2 type.
The lengths, and positions related to NC_001959 for Genogroup I (GI) sequences and U07611 for Genogroup II (GII) sequences of the supported typing regions for step 2 and 3 are:

                     Starting position      End position             
ORF1 GI         4596                       5357                    
ORF2 GI         5358                       7000
ORF1 GII        4323                       5084
ORF2 GII        5091                       6727 

The length of the individual strains may vary due to gaps in the aligned sequence. 

Figure 1: A representation of the complete norovirus genome with an indication of the regions covered by the different analysis steps in the typing tool.

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Reference sets update

Information on the latest reference sets, used by the tool, can be obtained at noronet@rivm.nl

Latest updates:

• December 2012: Addition of GII.4 Sydney 2012 variant sequences to the reference set
• December 2012: Revision of the GII.4 variant names according to a new international consensus nomenclature (in press, Archives of Virology, 2013)
• April 2013: Addition of VP1 genotype GI.9 and revision of the ORF1 nomenclature according to consensus nomenclature paper

Conversion of GII.4 variant names

1: variant 2003/Asia 2003 is a recombinant with a GII.4 ORF2 and a GII.P12 ORF1
2: Variant Sydney 2012 is a recombinant with a GII.4 ORF2 and a GII.Pe ORF1

Conversion of ORF1 genotype names

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