1. Results and Discussion Phylogenetic analysis of  determinant L1 and L2 Fig. 4 Phylogenetic trees constructed by different methods were congruent in overall structure, as were trees constructed with deduced amino acid sequences (data not shown). Human adenoviruses clustered in major groups corresponding to their six species, with exception of the only virus of species E, HAdV-E4. (Fig. 4). Fig. 5 Identity of serotypes of the same species was in a range of 61.5% to 97.5% in the L2 region. For comparison, identity between serotypes of different species was lower (51.9% to 67.0%). Distribution of pairwise identity scores of the L2 region between serotypes clearly resulted in two peaks, representing heterologous serotypes of the same species (peak 1) and heterologous serotypes of different species (peak 2) (Fig. 5). Low genetic diversity of HADV-D15 and HAdV-D29 was an exception (0.4% in L2 and 0.0% in L1; * in Fig. 5). Thus, it is doubtful whether HAdV-D15 and HAdV-D29 are distinct serotypes because of extensive cross neutralization. A two step molecular typing scheme for HAdV First step is an established generic PCR frequently used for diagnostic purposes amplifying the conserved 5´end of the hexon gene. Depending on the sequencing results the newly developed L2 PCRs serve as second step. Sequencing of these amplicon permits unequivocal typing as these code partially for the neutralization epitope  (Fig.6). Typing criteria deduced from phylogenetic analysis Values in Fig.7 are nucleic acid sequence diversities to the next prototype sequence in the Genbank databank. Towards a Molecular Typing System of all Human Adenoviruses Derived from Phylogenetic Analysis of Neutralization (  ) and Hemagglutination Inhibition (  ) Immunogenic Determinants Ijad Madisch 1, Daniel Kahlmann 1, Gabi Harste 1, Heidi Pommer 1, Roman Woelfel 2, Albert Heim 1 1 Institute for Virology, Medical School of Hannover 2 Bundeswehr Institute of Microbiology, Munich Introduction Adenoviridae are nonenveloped, double stranded DNA viruses with an icosahedral capsid. The HAdV are classified in 6 species (HAdV-A – HAdV-F) with 51 types, defined mainly by neutralization criteria. Typing is diagnostically important because infections with different HAdV types are often associated with distinct clinical outcomes and epidemiologic features. We focus in this work on epitopes  and  which are relevant for typing and located on the hexon and the fiber capsid protein (Fig.1). Neutralization (SN) with type-specific antisera is the classical reference method. The ε determinant consisting of loop-1 (L1) and loop-2 (L2) (Fig.2) on the hexon protein reacts with type-specific antisera in neutralization tests. The knob region of the fiber protein (γ determinant) has hemagglutinating properties. Hemagglutination inhibition (HI) testing is preferred by many laboratories for typing purposes because it is more rapid than SN. However, virus isolation is required prior to both classical typing methods takes time and may fail in cases of low virus loads, which can be still detected by PCR. Therefore, we developed a two step molecular typing system including two sequential PCRs followed by sequencing. Material &Methods Amplification of hexon L2 comprises two new designed primer pairs in individual PCR reactions, one for the species HAdV-C and -D (CDL and CDR), which produces a 322bp product (Fig.3a) and a primer set for the species B (BL and BR) with a 580bp amplicon (Fig. 3b). For the PCR amplification of the fiber  determinant we designed primer pairs for species B and D. The amplicon size is about 1000bp (data not shown). Sequencing was performed with an ABI Prism 310 automatic sequencer. Phylogenetic trees were constructed with several phylogeny reconstruction algorithms (neighbor-joining, maximum likelihood and maximum parsimony) in order to determine the phylogenetic relationships among HAdV. Direct typing of HAdV in clinical samples Our molecular HAdV typing system was applied to 87 HAdV positive clinical samples without virus isolation. All 87 samples positive with HAdV DNA were typed successfully (Fig. 8). Phylogenetic analysis of the fiber knob (  determinant) Phylogenetic analysis of  determinant sequences proofed to distinguish several prototypes which cross-reacted in hemagglutination inhibition tests but sequence diversity was not sufficient for typing of all HAdV. HAdV-29 was identified as a recombination variant of HAdV-15 (ε- determinant) and a speculative, not yet isolated HAdV prototype (γ determinant, compare fig. 9 and fig. 4). Additionally, γ determinant phylogenetic analysis demonstrated that HAdV-8 did not cluster with -19 and -37 in spite of the same tissue tropism. Identification of intermediate strains The two step molecular typing scheme was designed to substitute only neutralization testing, but not hemagglutination inhibition testing. Intermediate HAdV strains can only be identified, if both methods are applied to a clinical isolate. Intermediate strains are recombinant viruses between two serotypes presenting hexon neutralization epitopes of one serotype and fiber knob hemagglutination epitopes of another serotype. Conclusions Molecular typing by sequencing of immunogenic HAdV determinants is an adequate substitute for classical neutralization and hemagglutination inhibition typing. The proposed two step molecular typing scheme is an easy but sufficient approach to type HAdV in clinical samples even without time consuming virus isolation. As this scheme includes hexon L2 sequencing (  determinant partially) it is a perfect substitute for neutralization typing. L2 sequencing may also help to identify new types more easily. Combination with  determinant sequencing is only required for identification of intermediate strains by specialized laboratories but  determinant sequencing helped to solve several unsettled problems of HAdV typing. Fig.2: Ribbon Diagram of hexon protein main chain, indicating HVRs containing serotype-specific residues. The exterior surface of the capsid is formed by L1, L2 and L4. Six HVRs (HVR1-HVR6) are seen in the L1 loop and the seventh in the L2 loop. Sense Primer CDL Reverse Primer CDR [..]Fig.3a Sense Primer LB Reverse Primer RBFig.3b Hexon 17740-20499 * Fiber 29368-31131 * * refers to HAdV-A12 Fig.4: Phylogenetic analysis of  determinant L1 (A) and L2 (B) with the neigbour joining method based on a Kimura 2 parameter matrix. Bootstrap generated with 1000 pseudoreplicates. 51 Human Adenoviruses PCR (Wadell et al. 1994) or RealTime-PCR (Heim et al. 2002) Final type identification for - HAdV-A (12,18, 31) - HAdV-E (4) - HAdV-F (40,41) HAdV-BHAdV-C and –D First step: Generic PCR Second step Loop2 PCR Final species identification (B,C or D) Sequences submitted to Fasta/Clustal Sequences submitted to Fasta/Clustal Fig.6 Final type identification for * Fig.5 Peak 1: Peak 2 Fig.8:Applying our molecular typing concept on 87 clinical samples positive for HAdV DNA Fig.8: Applying our molecular typing concept on 87 clinical samples positive for HAdV DNA Fig.9: Phylogenetic tree of the  determinant. In many cases the phylogenetic distance is sufficient to differientiate HAdV, which have extensive cross reactions in HI (e.g. HAdV-D15, -D22 and –D42;-D13, -D38 and –D39;-D20 and –D47). Hemagglutination- Inhibition Neutralization Combination of Fiber  -determinant PCR Two step molecular concept Combination of Identification of the intermediate strain Fig.10: classical (A) and molecular (B) identification of intermediate strains AB Applying Step 1 – Generic PCR Species identified if diversity is <9% A, E and F Type identified Applying step 2 – Loop2 PCR B, C and D Type not yet identified <2.5%>2.5% Type identifiedUnknown type? Deduced Amino Acid Loop1 Sequencing Fiber  -determinant sequencing ≤1.5% >1.5% Unknown type? Fig.7 in case of species Deduced Amino Acid Loop1 Sequencing Fiber  -determinant sequencing Schnurr et al. (1995)