automated genotyping tool for
   Group A rotaviruses.  Read More

Classification process


  • Classification strategy

    Complete ORF analysis: Once the complete ORF of a rotavirus gene under investigation has been determined, it will be compared to other complete ORFs of  cognate genes available in the GenBank database through a BLAST (Basic Local Alignment Search Tool) –search. If pairwise nucleotide identities between the gene of the novel strain under investigation (strain X) and strains belonging to an established genotype A are above the cut off value of that gene segment (Table 1), strain X can be assigned to genotype A. The exact relationship between the gene of strain X and cognate genes of all established genotypes, has to be obtained phylogenetically. When all the pairwise nucleotide identities between a gene of the new strain Y, and the cognate genes of all the established genotypes are below the cut-off value for that gene segment (Table 1), strain Y may be the prototype of a new genotype.
    In order to avoid the appearance of duplications in the literature and identical numbering of different new genes, a Rotavirus Classification Working Group (RCWG) including molecular virologists, infectious disease physicians, epidemiologists and public health specialists has been formed, who would be prepared and responsible to help validate the new sequence and to assign an appropriate successive genotype number, in close consultation with the Reoviridae Study Group of the ICTV.
    Partial ORF analysis: If only the partial ORF sequence of a rotavirus genome segment is available, assigning it to a certain genotype is less certain because the genotypic diversity across the ORF is not a constant value. Some regions of the ORF may be highly variable, while others may be more conserved. Since the cut-off percentage values for each of the 11 genome segments has been calculated based on entire ORFs, applying these cut off percentages to only a part of the ORF, might lead to erroneous conclusions. Only under certain circumstances when all three of the following restrictions are obeyed, a partial gene sequence might be used to assign a rotavirus gene to an established genotype:

    1) At least 50% of the ORF sequence should be determined.
    2) At least 500 nt of the ORF should be determined.
    3) Identity between strain X and a strain belonging to an established genotype A should be at least 2% above the appropriate cut-off sequence (Table 1), before strain X can be assigned to genotype A.

    Remarks: To assign a genotype to a new ORF sequence, whether complete or partial, the comparison should only be done to strains for which the genotype has been established based on the entire ORF analysis, and not with other partial ORF sequences. Due to intra-segmental recombination [30-32, 40] or to different rates of diversification throughout a genome segment, classification assignments based on partial ORFs may yield misleading or incorrect results.
     

  • Practical Recommendations

    Any sequence of a rotavirus RNA segment that had been analyzed as described above, and found to clearly belong to one of the established genotypes, can be assigned to that genotype. When a potential new genotype is found, the complete ORF should be determined and can be sent (in confidence and out of competition) to a member of the RCWG. The sequence will be analyzed and, if appropriate, a new successive genotype number will be assigned. The new number can be used for publication. Most reputable journals will request to provide the GenBank accession number of the new sequence, and this should be obtained as soon as possible and the correct new genotype be assigned to it. The whole process of the RCWG: (i) receiving the sequence, (b) appropriate phylogenetic analysis, (c) presentation to the RCWG members, and (d) approval by the RCWG, should not take more than 6 weeks. An annual report of the RCWG will be published summarizing all newly assigned genotypes.


  • Table 1. Nucleotide percentage identity cut-off values defining genotypes for 11 rotavirus gene segments.

    Gene product

    Percentage identity cut-off values

    Genotypes

    Name of genotypes

    VP7

    80%

    27 G types

    Glycosylated

    VP4

    80%

    34 P types

    Protease sensitive

    VP6

    85%

    16 I types

    Inner capsid

    VP1

    83%

    9 R types

    RNA-dependent RNA polymerase

    VP2

    84%

    9 C types

    Core protein

    VP3

    81%

    8 M types

    Methyltransferase

    NSP1

    79%

    16 A types

    Interferon Antagonist

    NSP2

    85%

    9 N types

    NTPase

    NSP3

    85%

    12 T types

    Translation enhancer

    NSP4

    85%

    14 E types

    Enterotoxin

    NSP5

    91%

    11 H types

    pHosphoprotein