Criteria for a New HAdV Type

The genome provides complete, absolute and readily available information for identifying, characterizing and typing adenoviruses and all viruses.  Databases such as GenBank are resources providing open access adenovirus genome data. The internet provides access to many other databases and bioinformatics tools.

Under the genomics-based paradigm, each candidate type will be evaluated on a “case-be-case” basis, given the complexity and enormity of the genome data.  It should be noted that the serotyping algorithm also required “case-by-case” evaluation, with the criteria modified, over time and on individual basis, to accommodate previously established serotype exceptions (literature citations below).

Previous protocols relied on “interpretations” of the primary sequence, which provided only partial secondary sequence data.  For example, serology uses an interpretation of the antibody-antigen interaction, resulting in a limited amino acid sequence, which may not be contiguously linear, of the 3-D region making up the epitope.  The interaction is not well understood, as not all 3-D structures on the capsid are immunogenic or, if they are, are immunogenic to varying degrees.  In the case of HAdV serum neutralization (SN), the hexon loop 1 epitope represents 2.6% of the genome primary sequence. The only other assay considered for a ‘complete’ serotyping of an HAdV is hemagglutination inhibition (HI) (which is no longer performed on the wet bench).  The epitope resides on the fiber knob and approximately constitutes 1.7% of the genome sequence.  Direct sequencing of these two epitopes only, as “imputed epitopes” and molecular typing, is inadequate and does not constitute a full “genome analysis”.

The genome presents quantitative metrics including the GC%, which may be used to exclude species grouping (e.g., HAdV-G52; PMID: 17360747) or to support species inclusion (diagnostic of species: A (47%), B1 (51%), B2 (49%), C (55%), D (57%), E (57%), F (51%) and G (55%)), and genome percent identity differences between pairs (e.g., HAdV-G52: HAdV-G52 shows a very high whole genome percent nucleotide identity with SAdV-G1, a simian (monkey) AdV, at 95.5%, as opposed to percent identities with the next phylogenetically closest viruses HAdV-F40 and HAdV-F41 at approximately 69%).

As a critical component of the genomics-based paradigm and algorithm, phylogenomics provides important criteria: whole genome, penton base, hexon loop 1, hexon constant and fiber knob (e.g., HAdV-D53 (PMID: 19492050), -B55 (PMID: 20042633) and -D56 (PMID: 21056888)).  The limitations with the enormous amount of data are recognized, as well as the desire of the adenovirus research community to keep the original types as separate unique types.  This is reflected by stating that genome analysis, particularly phylogenetic analysis, does not provide absolute and convenient numerical cut-offs.  Rather, the data provide guidance and add to the important consideration of the individual type’s biology and pathogenicity.  These attributes, of course, are non-predictable and are the basis for scientific inquiry.

Although bootstrap values are not absolute, that is, they may vary slightly with the number of data points, they do provide a relative guide and a reference point.
Note:  Adding more data will not change the bootstrap value significantly.  In the example of HAdV-C57, SN titration of the hexon loop 1 shows it cross-reacts at 8x with HAdV-C6.  The bootstrap value was ~76. Observations with extensive data show bootstrap values within the range 80-70 predict serological cross-reactivity.  This study is on-going.  The equivalent data for the hemagglutination inhibition assay has not been examined.  Bootstrap values above 80 would state the hexon loop 1 are very unrelated and not likely to cross react and below 80 will display cross-reaction.

For guidance and reference, specific examples of the application of genomics-based algorithm and in support of this paradigm shift from using only two antibody epitopes (serology) to whole genome data (genomics) include:

1) HAdV-B55.  This serotypes as HAdV-B11, a renal pathogen, but hemagglutination inhibits as HAdV-B14, a respiratory pathogen.  The restriction enzyme (RE) pattern analysis is highly similar to HAdV-B14 unlike RE patterns for the renal pathogens HAdV-B11b and -B11c.  Originally it was named HAdV-B11a based solely on its serum neutralization.  Genome analysis, including phylogenomics, show this emergent pathogen contains a small portion of the HAdV-B11 genome (2.6%) within a larger chassis of the HAdV-B14 genome (97.4%), and is an example of molecular evolution.  The change in pathogenicity is that it is a “Trojan Horse”, appearing to the host as a renal pathogen and not a respiratory pathogen,  HAdV-B14.  That “B55″ is a very clean example of how recombination can confer new tropism (to “B11, or from the other perspective, to confer immune evation (to “B14″.  It is perhaps a unique case, but that remains to be seen.  In the context of genome-based names (by restriction enzyme mapping data) HAdV-B11, -B11b and -B11c, it is not informative to name this “HAdV-B11a”.

2) HAdV-D53.  HAdV-D53 is imputed to serotype as HAdV-D22 by molecular typing (PMID: 16941356).  HAdV-D22 is not reported as an ocular pathogen whereas HAdV-D53 is a highly contagious EKC agent (ibid).  By the genomics-based algorithm, HAdV-D53 is a new type due to changes in its biology and pathogenicity.  It is insufficient and not productive to refer to this type as a variant of HAdV-D22.  The HAdV-D53 genome contains multiple recombination events and is an example of molecular evolution resulting in a novel HAdV type.

3) HAdV-D56.  HAdV-D56 is imputed to serotype as HAdV-D15 and -D29 by molecular typing (PMID: 19477681); it should be noted HAdV-D15 and -D29 cross-react below the 8x and 16x levels as defined by present ICTV guidelines!  This is an emergent respiratory pathogen causing a respiratory-related fatality and subsequent highly contagious ocular disease amongst the care-givers.  The criteria specified by the genomics-based algorithm define it as being a new HAdV type, with multiple recombination events and an important biology/pathogenic effect (PMID: 21056888). Simultaneously, it was reported in Japan (PMID: 21147954).  Subsequent evaluation of both genomes show they are the same.  It is common sense that the research community refers to this isolate as HAdV-D56, as a unique identifier, rather than as a variant of D15 and -D29, as the serologists desire.  It is unproductive and puzzling to not recognize this is a new type, a product of molecular evolution.

Again, this is an on-going project in developing and refining typing criteria that is open to all in the adenovirus research community for input.  The collective knowledge of the whole is greater than that of an individual; the inclusion of experts in the particular subfields is both important and critically necessary.

It is clear from examining the published and original serology data, and from applying genomics and bioinformatics techniques to the examination of novel HAdV-G52, -D53, -B55 and -D56, that the genome data and its analysis is preferable, morel logical, more informative and more accurate than “serology-only” data for characterizing, defining, understanding, typing and naming human adenoviruses.

Nota bene, despite the widespread misinformed belief that the ICTV has a mandate to provide guidelines below the “species” taxa, it does not– according to the official website and to the current chairman of the appropriate study group.