Phylogenetic Analysis of the Main Neutralization a
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病菌学杂志 2005年第24期
Institut für Virologie, Medizinische Hochschule Hannover, Hannover, Germany
ABSTRACT
Human adenoviruses (HAdV) are responsible for a wide spectrum of diseases. The neutralization determinant (loops 1 and 2) and the hemagglutination determinant are relevant for the taxonomy of HAdV. Precise type identification of HAdV prototypes is crucial for detection of infection chains and epidemiology. and determinant sequences of all 51 HAdV were generated to propose molecular classification criteria. Phylogenetic analysis of determinant sequences demonstrated sufficient genetic divergence for molecular classification, with the exception of HAdV-15 and HAdV-29, which also cannot be differentiated by classical cross-neutralization. Precise sequence divergence criteria for typing (<2.5% from loop 2 prototype sequence and <2.4% from loop 1 sequence) were deduced from phylogenetic analysis. These criteria may also facilitate identification of new HAdV prototypes. Fiber knob ( determinant) phylogeny indicated a two-step model of species evolution and multiple intraspecies recombination events in the origin of HAdV prototypes. HAdV-29 was identified as a recombination variant of HAdV-15 ( determinant) and a speculative, not-yet-isolated HAdV prototype ( determinant). Subanalysis of molecular evolution in hypervariable regions 1 to 6 of the determinant indicated different selective pressures in subclusters of species HAdV-D. Additionally, determinant phylogenetic analysis demonstrated that HAdV-8 did not cluster with -19 and -37 in spite of their having the same tissue tropism. The phylogeny of HAdV-E4 suggested origination by interspecies recombination between HAdV-B (hexon) and HAdV-C (fiber), as in simian adenovirus 25, indicating additional zoonotic transfer. In conclusion, molecular classification by systematic sequence analysis of immunogenic determinants yields new insights into HAdV phylogeny and evolution.
INTRODUCTION
Adenoviridae viruses are nonenveloped, double-stranded DNA viruses with an icosahedral capsid (59). Human adenoviruses (HAdV) are classified into six species (HAdV-A to HAdV-F) which were defined historically as subgenera on the basis of hemagglutination properties (10, 17, 55). Subsequently, oncogenic properties and DNA homology were also used to define subgenera (species) (59). The six HAdV species consist of 51 HAdV types, defined mainly by neutralization criteria (10, 64). Several simian adenoviruses (SAdV), which cannot productively infect humans, are also members of the species HAdV-A (SAdV-2, -4, -6, -9, -10, -11, and -14), HAdV-B (SAdV-21), HAdV-C (SAdV-13), HAdV-E (SAdV-22 to -25), and HAdV-F (SAdV-19).
Precise HAdV typing is essential for epidemiological studies and quality control of diagnostics. Moreover, the greater knowledge about differences in virulence and organ tropism among the adenovirus serotypes has increased the medical value of HAdV classification (59). For example, the more dangerous lower respiratory tract illness is associated primarily with HAdV-B3, -B7, -B21, and -E4 infection (11, 13, 16, 21), whereas the less severe upper respiratory tract illness is frequently caused by HAdV-C1, -C2, -C5, -B3, and -B7 (15, 21). Thus, typing of HAdV isolated from a respiratory tract sample or feces sample may predict the clinical course. Infections of the eye with HAdV-D8, -D19, and -D37 may cause severe epidemic keratoconjunctivitis, whereas mild follicular conjunctivitis and pharyngoconjunctival fever are most frequently caused by HAdV-B3, -B7, and -E4 (6, 37). Disseminated disease in highly immunosuppressed patients, e.g., allogeneic stem cell transplant recipients, is associated with HAdV-A31, -B3, -B7, -C1, -C2, and -C5, most frequently with the last three types (20, 32, 54, 57, 63). Acute hemorrhagic cystitis and acute renal failure in infants, young children, and immunosuppressed patients are associated primarily with the serotypes HAdV-B11, -B21, -B34, and -B35 (3, 12, 20, 23, 38, 57). Due to this background and the wide spectrum of diseases which are caused by HAdV of differing organotropism and virulence, a deeper understanding of the phylogenetic relationships of HAdV types is crucial for the development of molecular classification criteria.
All adenoviruses contain three diagnostically useful antigens, which are part of the three capsid proteins: hexon (polypeptide II), penton (polypeptide III), and fiber (polypeptide IV). The main type-specific epitope, the determinant, consisting of loop 1 (L1) and loop 2 (L2) on the hexon protein, reacts with type-specific antisera in neutralization tests (NT) (24), which are the classical reference method for typing. Cases of failing neutralization with available antisera are an unresolved issue of NT because these require extensive cross-neutralization studies for defining a new HAdV type. The knob region of the fiber protein, which includes the type-specific determinant, has hemagglutinating properties which are used for hemagglutination inhibition (HI) tests. HI is preferred by many laboratories, because it is more convenient and rapid. However, HI cannot differentiate all 51 HAdV because of several cross-reactions (59). As HAdV have a high capability for intraspecies recombination and occasional interspecies recombination, HI results of clinical isolates can be contradictory to NT results ("intermediate strains"). Intermediate strains are recombinant viruses between two serotypes presenting hexon neutralization epitopes of one serotype and fiber knob hemagglutination epitopes of another serotype. Probably, recombination of HAdV may give rise to new types and even species. Retargeting of HAdV types with favorable gene expression and persistence properties (as for example HAdV-C5) with fiber knobs of other HAdV types holds promise for improving HAdV gene therapy and vaccination vector approaches (9, 35). A complete phylogenetic analysis of the fiber knobs of all 51 HAdV should facilitate this approach.
Nowadays, PCRs are frequently used for rapid diagnosis of adenovirus infections (5, 19, 36, 43). Therefore, attempts were made to use PCR also for genotyping of HAdV. Techniques included type-specific PCRs and multiplex PCR protocols using species-specific primers (28, 42, 43, 45, 69) as well as combinations of a generic PCR with restriction digestion of the amplicons (4, 30, 50, 61). These approaches permit rapid molecular typing of a limited number of HAdV types or at least species identification. However, new insights into molecular evolution and epidemiology of HAdV cannot be attained, and new HAdV types cannot be identified.
Based on extensive sequence analysis of the neutralizing epitope (L1 and L2 of the hexon capsid protein) and the hemagglutination epitope , we developed criteria for molecular classification of HAdV and for the identification of new prototype isolates. Sequencing of the short L2 proved to be sufficient for molecular typing whereas additional L1 and determinant sequence data should be generated for the identification of new prototype isolates. L2 sequencing can be recommended as a second step for precise type identification after species identification by diagnostic PCRs.
MATERIALS AND METHODS
HAdV prototype strains and cells. HAdV prototype strains HAdV-B50 (ATCC VR-1501), -C1 (VR-1), -D8 (VR-1368), -D15 (VR-1092), -D20 (VR-255), -D22 (VR-1100), -D23 (VR-258), -D25 (VR-223), -D27 (VR-1105), -D28 (VR-226), -D29 (VR-1107PI/RB), -D30 (VR-273), -D32 (VR-625), -D33 (VR-626), -D36 (VR-913/275), -D37, -D38 (VR-988), -D39 (VR-932), -D42 (VR-1304), -D43 (VR-1305), -D44 (VR-1306), -D49 (VR-1407), and -D51 (VR-1502) were obtained from the American Type Culture Collection (ATCC). HAdV-B14, -D10, -D13, and -D30 prototype strains were obtained from our collection at the German National Reference Laboratory for Adenoviruses, Hannover Medical School, Hannover, Germany, and had been typed previously with cross-neutralization. In a preliminary quality control study using a multiplex PCR protocol (69), the virus delivered by ATCC as HAdV-B50 (VR-1501) turned out to be a member of species D, whereas HAdV-D51 (VR-1502) was a species B virus. In the following correspondence with ATCC, it was stated that ATCC believes that stocks of VR-1501 (HAdV-B50) and VR-1502 (HAdV-D51) have been switched. Subsequently, VR-1501 and VR-1502 were deleted from the ATCC catalogue. For this study, HAdV-B50 (VR-1501) was relabeled as HAdV-D51 and vice versa. All viruses were propagated on A549 cells (ATCC, CCL-185) on 75-cm2 cell culture flasks. When the cytopathogenic effect was above 50%, cells were frozen at –70°C, and DNA was extracted with the QIAGEN blood kit (QIAGEN, Hilden, Germany).
Generic HAdV PCR. HAdV DNA was amplified using either a conventional PCR protocol which was published as a first step of a nested PCR (4) or a real-time PCR protocol (26).
PCR amplification of hypervariable hexon loops. The hexon L1 region was amplified as described previously (43). For the PCR amplification of hexon L2 two primer pairs, one for the species HAdV-B and the other for the species HAdV-C and HAdV-D, were developed (Table 1) with the help of a multiple alignment with GenBank sequence data flanking the L2 region (HAdV-B3, -B7, -B14, -D9, -D10, -D17, -D19, -D23, -D24, -D26, -D45, -D46, -D47, and -D48). Amplification of L2 comprises two primer pairs in individual PCRs, one for the species HAdV-C and -D (CDL and CDR), which produces a 322-bp product, and a primer set for the species B (BL and BR) with a 580-bp amplicon (Table 1). Both primer pairs were designed for an identical annealing temperature of 54°C as calculated with MeltCalc software to permit amplification with a single thermal profile (53). Additionally, L1 and L2 primer pairs were designed for HAdV-A18 to obtain the immunogenically relevant sequences for the phylogenetic analysis (Table 1). These pairs have the same PCR conditions as the corresponding L1 and L2 protocols (Table 1). PCR was performed in a total volume of 100 μl consisting of HotStar Mix (QIAGEN), 1 μM of each primer, and 3 μl of the purified HAdV DNA. The PCR program starts with activation of the "hot start" DNA polymerase for 15 min at 95°C, followed by 40 cycles consisting of denaturation at 94°C for 20 s, primer annealing at 54°C for 20 s, and elongation at 72°C for 40 s, followed by a final extension step of 72°C for 5 min. The sensitivity of the PCR protocols was determined by testing serial dilutions of cell culture-derived HAdV-B3, -C2, and -D8 previously quantified by a generic HAdV real-time PCR protocol (25).
PCR amplification of the fiber knob region. For amplification of HAdV-D, a new sense primer, AdFiDL, and an established antisense primer, AdD2, in the E4 region were combined (69), resulting in a 1,000-bp amplicon (Table 1). The sense primer was designed with the help of a multiple alignment of GenBank sequence data of the 5' end flanking the determinant (sequences of HAdV-B3, -B7, -B11, -B14, -B16, -B21, -B34, -B35, -D8, -D9, -D15, -D17, -D19, -D28, -D30, and -D37). For species B, a new primer set (AdFiBR and AdFiBL) was developed (Table 1), which amplifies the same region as do the species D primers and requires the same thermal cycling profile. PCR was performed in a total volume of 100 μl as described above with a final concentration of 2 μM MgCl2. The thermodynamic profile was equivalent to the previously described hexon PCR program except for the annealing temperature of 56°C and the elongation step for 90 s. New primer pairs were designed for species HAdV-B (FiBL/FiBR) and species HAdV-F (FiFL/FiFR) (Table 1). The PCRs were performed as described above. The sensitivity of the PCR protocols was determined by testing serial dilutions of cell culture-derived HAdV-B3 and -D8 previously quantified by a generic HAdV real-time PCR protocol (26). Furthermore, the fiber knob region of HAdV-A18 was sequenced after amplification with primers located in the fiber (5'-GGCTATGGCTGATGGTG-3') and 3' inverted terminal repeat region (5'-GTTTGGGCGGATGAGG-3').
Gel electrophoresis. PCR products were separated in a 2% agarose gel (1% in the case of fiber amplicons) for 60 min at 120 V. DNA extraction from the agarose gels was performed with the QIAGEN gel extraction kit according to the manufacturer's recommendations.
Sequencing. Cycle sequencing was performed with rhodamine-labeled dideoxynucleotide chain terminator (DNA sequencing kit; ABI, Warrington, England) and analyzed on an ABI Prism 310 automatic sequencer (Applied Biosystems). PCR primers were used for the sequencing reactions.
Phylogenetic analysis. L1, L2, and fiber knob region sequences were compared by sequential pairwise alignment with the Clustal algorithm implemented in the BioEdit software package (version 6.0.5) (62) and adjusted manually to conform to the optimized alignment of deduced amino acid sequences. Phylogenetic relationships were inferred from the aligned nucleic acid sequences by the neighbor-joining method implemented in the programs DNAdist and Neighbor (PHYLIP, version 3.6; University of Washington, Seattle) (22) using the Kimura two-parameter substitution model (31) and a transition/transversion ratio of 10. Support for specific tree topologies was estimated by bootstrap analysis with 1,000 pseudoreplicate data sets. Branch lengths in consensus trees were calculated by the maximum-likelihood quartet-puzzling method, using the nucleotide substitution model of Tamura and Nei (60) as implemented in Tree Puzzle 5.0 (58). The pairwise nucleotide identity matrix was calculated with BioEdit (version 6.0.5). The histogram of the pairwise nucleotide comparisons was plotted with the program PNCH (34). Similarity plots were calculated based on different matrices (PAM250 and EBLOSUM60) with a window of 10 amino acids with the software Plotcon included in the EMBOSS package (47). Bootscans were performed with the software SimPlot (version 3.5.1) (33) with a window of 200 bp (20-bp step) based on a Kimura two-parameter substitution model (31) with a transition/transversion ratio of 2.0.
For making multiple alignments of L1, L2, and fiber knob regions the following GenBank sequences were used: for hexon, HAdV-C1 (X67709), -C2 (AJ293903), -B3 (X76549), -E4 (X84646), -C5 (AF542130), -C6 (X67710), -B7 (Z48571), -D9 (AF161562), -D10 (AB023548), -B11 (AY163756), -A12 (X73487), -B14 (AB018425), -B16 (X74662), -D17 (AF108105), -D19 (AF161565), -B21 (AJ012091), -B34 (AB052911), -D23 (AB023552), -D26 (AB023554), -A31 (X74661), -B35 (AB052912), -D37 (AB023555), -F40 (X51782), -F41 (X51783), -D45 (AB023556), -D46 (AB023557), -D47 (AB023558), -D48 (U20821), and SAdV-E25 (BK000413); for fiber knob ( determinant), HAdV-C1 (AB108423), -C2 (J01917), -B3 (X01998), -C5 (M18369), -C6 (AB108424), -B7 (AF104384), -D8 (AB162768), -D9 (X74659), -B11 (AY163756), -A12 (X73487), -B14 (AB065116), -D15 (X72934), -B16 (U06106), -D17 (Y14241), -D19 (U69131), -D28 (Y14242), -B21 (U06107), -A31 (X76548), -B34 (AB073168), -B35 (U32664), D37 (U69132), -E4 (X76547), -F40 (M28822), -F41 (M60327), and SAdV-E25 (BK000413).
Nucleotide sequence accession numbers. The sequences of L1 of the determinant of HAdV-B14 (AB018425), -B50 (AJ864518), -D13 (AJ749847), -D15 (AJ821891), -D20 (AJ749848), -D25 (AJ749849), -D27 (AJ749850), -D28 (AJ749851), -D29 (AJ749852), -D30 (AJ749853), -D32 (AJ749854), -D33 (AJ749855), -D36 (AJ749856), -D38 (AJ749857), -D39 (AJ749858), -D42 (AJ821893), -D43 (AJ821894), -D44 (AJ749859), -D49 (AJ821895), and -D51 (AJ821896) have been deposited in GenBank, as well as the L2 sequences of HAdV-A18 (AJ821897), -B14 (AJ749860), -B50 (AJ749861), -D13 (AJ745880), -D15 (AJ745881), -D20 (AJ745882), -D22 (AJ745883), -D25 (AJ745884), -D27 (AJ745885), -D28 (AJ745886), -D29 (AJ745887), -D30 (AJ745888), -D32 (AJ745889), -D33 (AJ745890), -D36 (AJ745891), -D37 (AJ745892), -D38 (AJ745893), -D39 (AJ745894), -D42 (AJ745895), -D43 (AJ745896), -D44 (AJ745897), -D49 (AJ745898), and -D51 (AJ745899). Fiber knob ( determinant) sequences of HAdV-A18 (AJ841699), -B50 (AJ811465), -D10 (AJ811442), -D13 (AJ811443), -D20 (AJ811444), -D22 (AJ811445), -D23 (AJ811446), -D24 (AJ811447), -D25 (AJ811448), -D26 (AJ811449), -D27 (AJ811450), -D29 (AJ811451), -D30 (AJ831473), -D32 (AJ811452), -D33 (AJ811453), -D36 (AJ811454), -D38 (AJ811455), -D39 (AJ811456), -D42 (AJ811457), -D43 (AJ811458), -D44 (AJ811459), -D45 (AJ811460), -D46 (AJ811461), -D47 (AJ811462), -D48 (AJ811463), -D49 (AJ811464), and -D51 (AJ811460) have also been submitted to GenBank.
RESULTS
Phylogenetic analysis of determinant L1 and L2. The type-specific neutralization epitope ( determinant) consists of L1, which includes six hypervariable regions (HVR1 to HVR-6), and of L2 with the seventh HVR (14). Sequences of the hypervariable L1 region were generated with the generic primer pair H1.1-H2.1, which has an amplicon size of 800 bp (Table 1). All 45 HAdV prototype viruses of species B, C, D, and A18 were amplified successfully with the new L2 primer sets (Table 1), and all amplicons had the expected length of 322 bp in the case of species C and D, 590 bp in the case of species B, and 656 bp in the case of HAdV-A18. L1 and L2 amplicons of prototype strains were directly sequenced and aligned with previously available GenBank sequence data for human adenovirus prototype strains (for nucleotide sequence accession numbers see Materials and Methods). Phylogenetic trees for L1 based on nucleotide acid positions 19228 to 19828 and for L2 based on positions 19989 to 20272 (both referring to the HAdV-C2 hexon sequence) were constructed with several phylogeny reconstruction algorithms (neighbor joining, maximum likelihood, and maximum parsimony) in order to determine the phylogenetic relationships among HAdV on the species level. The phylogenetic trees constructed by different methods were congruent in overall structure (Fig. 1 depicts neighbor-joining trees), as were trees constructed with deduced amino acid sequences (compare Fig. 1 and 2).
As expected, phylogenetic trees of both L1 and L2 confirmed that human adenoviruses clustered together in major groups corresponding to their six species, with the exception of the only virus of species E, HAdV-E4 (Fig. 1 and 2). HAdV-E4 clustered with the species HAdV-B and had the closest relationship to HAdV-B16 with a high bootstrap value both in nucleic acid and in amino acid phylogenetic trees (100%, Fig. 1 and 2), which supports previously published cross-neutralization data (65). Groups of HAdV-A and HAdV-F were clustering together in one monophyletic group, which correlates well with their common tissue tropism (Fig. 1 and 2) (8). This group clusters together with species HAdV-C.
Identical determinants of HAdV-D15 and HAdV-D29. Genetic divergence of HADV-D15 and HAdV-D29 was as low as 0.0% (nucleic acid sequence) in L1 (Fig. 1A) and 0.4% in L2 (Fig. 1B), resulting in a single amino acid substitution (E422Q), whereas genetic divergence of other HAdV-D serotypes was in the range of 2.5% to 23%. HAdV-D15 and HAdV-D29 also exhibit extensive cross-neutralization (1, 10, 29, 66). Moreover, sequence variability between different strains of HAdV-D15 was in the same range as the difference between HAdV-D15 and HAdV-D29. For example, three different bases resulting in two amino acid substitutions in L2 were found between the HAdV-D15 strain Morrison, available in the GenBank database (X76707), and the HAdV-D15 strain 305 (955; CH. 38; V-215-003-014, ATCC VR-1092) sequenced additionally in this study (AJ745881). Therefore, L1 sequencing, L2 sequencing, and classical neutralization typing concordantly indicate that HAdV-D15 and HAdV-D29 are not distinct serotypes. For comparison, the second highest genetic identity in L2 (97.5%) was observed between HAdV-D39 and -D43, which can be clearly discerned by cross-neutralization. There were eight bases different in the L2 region, resulting in a single amino acid difference between these two serotypes (D435N) (Fig. 1B and 2B). Furthermore, sufficient antigenic divergence between HAdV-D39 and -D43 was plausible because of L1 sequence divergence (7.4% nucleic acid divergence resulting in 16 amino acid exchanges) (Fig. 1A and 2A). These molecular evolutionary data indicated that divergence of HAdV-43 and HAdV-39 into distinct types was the product of a relatively recent evolutionary event.
Evolution of neutralization determinants of species HAdV-D. Species HAdV-D consists of 31 serotypes (counting HAdV-D15 and HAdV-D29 as a single neutralization serotype), and three main subclusters were observed in the L1 amino acid sequence phylogenetic tree (Fig. 2A). Therefore, a subanalysis of the genetic variability related to the HVRs in L1 of the three main subclusters of HAdV-D was performed in order to determine mechanisms of molecular evolution leading to the selection of a multitude of neutralization serotypes. In all three subclusters HVR1 to HVR6 were found (Fig. 3) with the main genetic variability found in HVR4 and HVR5. Similarity plots indicated that HAdV-D prototype viruses of subcluster 1 diverged mainly by additional molecular evolution of HVR3 (Fig. 3A). In contrast to subcluster 1, viruses of subcluster 2 diverged mainly by additional molecular evolution of HVR1 (Fig. 3B), and viruses of subcluster 3 diverged in both HVR1 and HVR2 (Fig. 3C).
Molecular classification criteria derived from determinant phylogeny. The phylogenetic trees of the L1 and L2 regions (Fig. 1 and 2) demonstrated that typing of HAdV is feasible by sequencing either L1 or L2 amplicons because of sufficient genetic distances. The amplicon of the L2 PCR is shorter and easier to sequence than that of the L1 PCR. As the nucleic acid sequence phylogenetic tree of L2 is identical in overall structure to the amino acid sequence tree of L2, we suggest L2 sequencing and comparison of nucleic acid sequence data as routine molecular typing methods. After HAdV-D15 and -D29 were counted as a single neutralization serotype, nucleic acid sequence identity of serotypes of the same species was in the range of 61.5% to 97.5% in the L2 coding region, with the lowest intraspecies nucleic acid identity found between types HAdV-B14 and -B16 and the highest between HAdV-D39 and -D43 (Fig. 1). For comparison, nucleic acid identity between serotypes of different species was lower, between 51.9% and 67.0%, with the lowest interspecies identity found between HAdV-D37 and -F40 and, as an exception, the highest (93.0%) found between HAdV-B16 and -E4 (Fig. 1). Distribution of nucleic acid sequence pairwise identity scores of the L2 region between serotypes clearly resulted in two peaks, representing heterologous serotypes of the same species and heterologous serotypes of different species (Fig. 4). A nucleic acid sequence divergence of <2.5% in the L2 region compared to the next homologous prototype sequence in the data bank is proposed as a criterion for molecular identification of a serotype, because the lowest genetic divergence between two serotypes in the L2 region was 2.5% (HAdV-D39 and -D43) (Fig. 1B). Type identification of a clinical isolate is achieved in the case of a <2.5% genetic divergence of the L2 sequence compared to the most closely related prototype sequence, if the next closely related sequence of another prototype of the same species (in the case of HAdV-E4, HAdV-B16) has a sequence divergence of at least 2.5%. If these criteria are not fulfilled, a deduced amino acid sequence of the clinical isolate has to be aligned with prototype data bank sequences, for example, by using the FASTA Internet server. If the L2 amino acid divergence compared to the next data bank sequence is <1.2%, typing is accomplished (Fig. 2B). However, if the L2 amino acid divergence compared to the next data bank sequence is 1.2%, typing is not feasible and isolation of a new HAdV type may be suspected.
Phylogenetic analysis of the fiber knob determinant. Amplification of the fiber knob determinant of all HAdV species B, D, and HAdV-A18 was achieved with newly developed primer sets (Table 1). New sequence data were generated by direct sequencing and aligned with sequence data previously published in GenBank (nucleic acid positions 31959 to 32766 of HAdV-C2). Phylogenetic trees of all 51 HAdV built by several methods (neighbor joining, maximum likelihood, and maximum parsimony) were identical in overall structure, as were nucleic acid sequence trees (data not shown) and deduced amino acid sequence trees (Fig. 5A depicts a neighbor-joining tree). Trees confirmed several phylogenetic relationships suggested by hemagglutination properties of the HAdV species. Adenovirus fiber knob sequences clustered together in major groups corresponding to the six HAdV species, with the exception of species HAdV-D, which formed three clusters. However, division of species HAdV-D into three clusters was not confirmed by bootstrapping. Surprisingly, pairwise nucleotide comparison of fiber knob sequences resulted in three peaks (Fig. 5B), with peak 1 representing intraspecies relationships, whereas interspecies relationships were divided into two groups (peaks 2 and 3 in Fig. 5B), suggesting a two-step model of HAdV species evolution. Peak 2 is formed by the interspecies relations between species A and F and between species D and C, including HAdV-E4, whereas peak 3 consisted of all other interspecies relationships. Several phylogenetic relationships of species HAdV-D corresponded well to HI cross-reactivity (HAdV-D8 and -D9; HAdV-D10, -D19, and -D37; HAdV-D13, -D38, and -D39; HAdV-D15, -D22, and -D42; HAdV-D20 and -D47; HAdV-D24, -D32, -D33, and -D46) (Fig. 5) (59). As already suggested by cross-neutralization experiments and HI cross-reactions (59), phylogenetic relations of HAdV-D fiber knob sequences did not completely coincide with hexon L1 and L2 sequences (Fig. 2 and 5A). Overall, intraspecies genetic divergence in the fiber knob region was lower than for hexon L1 and L2, with several prototype sequences almost identical (as low as 0.0% difference; Fig. 5A and B).
Phylogenetic criteria for species classification. A determinant species criterion should correlate excellently with the classical species criteria, because the fiber knob region ( determinant) is responsible for the hemagglutination properties of the HAdV. The phylogenetic tree of the fiber knob region demonstrated that a criterion for classification of HAdV species can be easily deduced with as a single exception species HAdV-E clustering with HAdV-C (Fig. 5). Based on an identity matrix of the complete data set of the determinant sequences, we calculated the maximum intraspecies divergence of each species (47%, nucleic acid divergence in species B), and the minimum interspecies divergence between all species was computed (52%, A to F). Therefore, a nucleic acid sequence divergence of 50% from the next data bank prototype sequence can be suggested as a criterion for species definition (Fig. 5B). Based on this species criterion, HAdV-E4 is a member of the HAdV-C species with an average identity of 56% to the other members of the HAdV-C (54.1% to 56.9%). Species identification criteria can also be derived from hexon sequence data sets. For example, many diagnostic laboratories use generic PCR protocols which amplify a conserved region at the 5' end of the hexon gene closely adjacent to the L1 coding region (4, 26). Therefore, we evaluated whether amplicon sequences of these PCRs are appropriate for molecular classification. Figure 6 shows that genetic divergence permits reliable species identification. A generic PCR protocol proposed by Allard et al. (4) allows species identification with a nucleic acid sequence divergence of 9% from the next data bank prototype sequence (Fig. 6A). In the case of a generic real-time PCR protocol (26) with its smaller amplicon size, sequence divergence of <4.8% is required (Fig. 6B). Both protocols even allow a final typing decision, but it is reliable for only six members of the species A, E, and F (criterion, 1.5% nucleic acid sequence divergence from the next homologous prototype sequence) (Fig. 6).
Molecular identification of intermediate strains. Amplification of the immunogenic determinant in combination with the L2 PCR could be a useful alternative for molecular identification of HAdV intermediate strains because the main neutralization epitope and the hemagglutination epitope can be sequenced. Four clinical isolates previously typed as intermediate strains with the classical methods (NT and HI) were analyzed with the newly developed combination of the L2 sequencing and determinant sequencing. In accordance with previous results of classical typing methods, isolates were identified as intermediate strains HAdV-15H9 (two isolates), HAdV-30H44, and HAdV-37H13. The L2 nucleic acid sequences of four analyzed intermediate strains were 100% identical to the prototype strains. Their determinant sequences were 98% to 100% identical to the prototype strains, and the deduced amino acid sequences were 97% to 100% identical. In the case of HAdV-30H44, the fiber determinant of HAdV-D44 was 100% identical to HAdV-D48. This finding correlates well with cross-reactivity in HI between these two viruses (52). HAdV-D30H44 was typed previously with HAdV-D44-specific HI antiserum when HAdV-D48 was not yet isolated, and an HAdV-D48-specific antiserum was not available to us. Therefore, a final differentiation between HAdV-D44 and -D48 was not feasible with both molecular and classical methods.
Molecular evolution of the main antigenic determinants of species HAdV-B and HAdV-E. Species B has been previously divided into two subspecies ("subgenera") because of obvious striking differences in restriction fragment patterns and, in part, tissue tropism. Detailed analysis of the molecular phylogeny of the main immunogenic determinants showed a more complex pattern. In hexon L1 and L2 trees subspecies B1 viruses HAdV-B3 and -B7 were as closely related as anticipated by the subspecies concept, whereas other subspecies B1 viruses (HAdV-B21 and -B50) clustered with subspecies B2 viruses, HAdV-B21 with HAdV-B11 and -35 and HAdV-B50 with HAdV-B14 and -34, supported by high bootstrap values (Fig. 2). Subanalysis of genetic variability related to HVR1 to HVR6 in L1 indicated that HAdV-B3 and -B7 diverged mainly by selection of immunogenic epitopes in HVR5 (Fig. 7A). By contrast, HAdV-B14, -B34, and -B50 diverged mainly by molecular evolution in HVR1 and HVR4 (Fig. 7B), and HAdV-B11, -B21, and -B35 diverged mainly in HVR1 and HVR2 (Fig. 7C). Phylogenetic relations of HAdV-B fiber knob sequences did not coincide with hexon L1 and L2 sequences, indicating intrasubspecies and intersubspecies recombination events in the molecular phylogeny of species B HAdV (compare Fig. 2 and 5A). The close phylogenetic relationship in the species B assumed by HI cross-reactions between HAdV-B7, -B11, and -B14 and between HAdV-B34 and -B35 was confirmed (Fig. 5A) (59).
In the determinant (L1 and L2) HAdV-B16 formed a subcluster with HAdV-E4 and with the more distantly related SAdV-E25 but was not closely related to the other species HAdV-B prototype viruses. However, in the fiber knob region HAdV-B16 clustered with the other species B viruses whereas both HAdV-E4 and SAdV-E25 clustered with species HAdV-C.
DISCUSSION
Previously, HAdV subgenera, now species, were defined by their hemagglutination properties; later oncongenicity, fiber length, and genotyping (restriction fragment analysis) data were also included as subgenus criteria. Phylogenetic analysis of fiber knob ( determinant) sequences of all 51 HAdV confirmed the species concept (Fig. 5A). From our sequence data, DNA homology criteria for species identification were deduced (<50%, Fig. 5). However, phylogenetic relationships between species C and D and between species A and F were closer (peak 2 in Fig. 5B) than between other species, resulting in a unique three-peak pattern of the pairwise nucleotide comparison plot (Fig. 5B).
This finding indicated a more recent divergence in the molecular evolution of the enterotropic species HAdV-A and HAdV-F, as well as in the evolution of HAdV-C and -D, and suggested two main steps of molecular evolution of HAdV species. The species concept of HAdV is also supported by the molecular phylogeny of the determinant L1 and L2 (Fig. 1 and 2), but the two interspecies relationship peaks were not observed in the pairwise nucleotide comparison plot of L2 (Fig. 4). However, the recent divergence of species HAdV-A and -F is also supported by hexon L1 and L2 phylogenetic trees (Fig. 1 and 2), whereas in the case of species HAdV-C and -D a close relationship could not be confirmed by hexon sequence data. Probably, the higher selection pressure resulted in a more rapid evolution of the neutralization determinant than of the determinant. A subanalysis of the genetic variability of HVR1 to HVR6 indicated complex patterns of molecular evolution of the determinant (Fig. 3), leading to the emergence of multiple neutralization types of species HAdV-D. This may have covered up older events of species divergence in the determinant data set. Another hypothesis for the evolution of species HAdV-C and -D viruses would be a recombination event leading to divergence of these species by introducing a more diverse hexon sequence and subsequent evolution in both and determinants, resulting in the multiple types of these species.
Historically, HAdV-E4 was classified as a species of its own mainly because of its difference in GC content from that of species HAdV-B (10), although HAdV-E4 is closely related to species HAdV-B as demonstrated by phylogenetic studies of the hexon and several other genome regions except the fiber gene (8, 25, 44). Recently, the complete genome of HAdV-E4 was sequenced and the close relationship with simian adenoviruses was demonstrated (most closely to SAdV-E25) (46). A zoonotic transmission event was suggested as the possible origin of HAdV-E4 (46). Therefore, we included SAdV-E25 in our phylogenetic analysis of HAdV. Surprisingly, in the neutralization determinant , HAdV-E4 was more closely related to HAdV-B16 (supported by a bootstrap value of 100%) and far more distantly related to SAdV-E25, which also clustered with species HAdV-B (Fig. 1 and 2). By contrast, phylogenetic analysis of the fiber knob region confirmed the close relationship of HAdV-E4 with SAdV-E25 (supported by a bootstrap value of 100%) and both viruses clustered with (but were more distantly related to) species HAdV-C (bootstrap value 100%) (Fig. 5A). Thus, our phylogenetic analysis supported both an interspecies recombination mechanism in the phylogeny of both HAdV-E4 and SAdV-E25, combining a species HAdV-C-like fiber gene with a species HAdV-B genetic backbone, and a zoonotic transmission event as suggested by Purkayastha et al. (46). However, in the determinant (L1 and L2), HAdV-E4 is very closely related to HAdV-B16 as indicated by phylogenetic trees (Fig. 1 and 2) and also by cross-neutralization experiments (27). Therefore, we tried to analyze the phylogenetic relationships of HAdV-B16, HAdV-E4 (AY594253), SAdV-E25 (BK000413), and HAdV-B7 (AY495969). Unfortunately, a complete genomic sequence of HAdV-B16 was not available in GenBank. Phylogenetic analysis of available HAdV-B16 sequences of E1A (AY490821), 13S protein (AY490821), putative pVIII protein and E3 (AB073632), putative 11-kDa protein (AY509991), VA RNA I and II (U10674), and RNA preterminal protein (U52564) sequences in addition to hexon and fiber knob sequences demonstrated that the close relationship of HAdV-B16 and HAdV-E4 is restricted to the hexon region (data not shown). Therefore, a bootscan analysis of the complete HAdV-B16 hexon sequence, compared to HAdV-E4, HAdV-B11, HAdV-B7, HAdV-B3, and SAdV-E25, was performed. Bootscan results indicated an intrahexon recombination event in the origin of HAdV-B16. For nucleotide positions 1 to 1400 of the hexon gene (including the determinant), HAdV-B16 had the highest similarity to HAdV-E4, whereas for positions 1400 to 2823 HAdV-B16 was more closely related to HAdV-B11.
In general, intraspecies recombination is observed much more frequently than interspecies recombination (55, 67). Therefore, most intermediate strains belong either to species B or to species D, both of which include more serotypes (41 of 51) than all other species do. Several cases of strong HI cross-reactions have been described, for example, between HAdV-D10, -D19, and -D37 and between HAdV-D24, -D32, -D33, and -D46 (59), which may suggest that some of these prototype viruses are recombination variants sharing an almost identical fiber knob region. For example, fiber knob sequences of HAdV-D44 and -D48 were 100% identical, as well as HAdV-D19 and -D37 in the determinant, which are closely related to HAdV-D10 (Fig. 5). Therefore, a previous recombination event can indeed be hypothesized in the origin of some HAdV prototypes, e.g., HAdV-D44 and -D48 or HAdV-D19 and -D37. This result is in complete accordance with the organotropism and virulence of HAdV-D19 and -D37, which both cause epidemic keratoconjunctivitis. On the other hand, HAdV-D8 neither clustered with HAdV-D19 and -D37 nor cross-reacted in HI, although it shares similar organotropism and virulence. Recently, sialic acid and CD46 were identified as cellular receptors interacting with the fiber knob of HAdV-D37 (7, 68), whereas the receptor of HAdV-D8 was not yet definitely identified. The phylogenetic relationship of the HAdV-D8 fiber knob to HAdV-D9 suggests CAR as the putative receptor (Fig. 6) (48). This indicates that factors other than cellular receptors are significant for epidemic keratoconjunctivitis.
From a type identification perspective, fiber knob ( determinant) sequencing is clearly secondary to hexon ( determinant) sequencing (compare Fig. 2 and 5A), as is hemagglutination inhibition testing compared to neutralization testing (27, 59, 65). In a diagnostic setting highly sensitive detection of HAdV DNA is currently achieved in many laboratories with extensively validated, generic PCR amplifying the conserved 5' end of the hexon gene (4, 26). Sequencing of these amplicons can be proposed as the first step of a two-step molecular typing scheme. Phylogenetic analysis of these amplicon sequences indicated that species identification is unequivocally feasible (Fig. 6). For unequivocal type identification sequencing of the hypervariable neutralization determinant is required as a second step.
This is the first work including sequences and phylogenetic analysis of both loops of the neutralization determinant of all 51 HAdV. Phylogenetic analysis demonstrated that either L1 or L2 sequencing is sufficient for precise typing (Fig. 1). A <2.5% nucleic acid sequence divergence was deduced as an unequivocal criterion for molecular typing from L2 sequence data. Sequencing of the bigger L1 (compare Fig. 1A and 1B) or use of deduced amino acid data for L2 did not improve type identification (compare Fig. 1B and 2B). Therefore, our results clearly support L2 sequencing as the most simple but sufficient approach for molecular typing of all 51 HAdV. Recently, a sequence-based approach for typing HAdV of species D and E was proposed (56). However, the relevant neutralization determinant was not covered by this approach, nor was an application to HAdV species other than D and E achieved. Another group suggested L2 sequencing for typing all HAdV without defining sequence divergence criteria for type identification or defining the length of the sequence region (51). Furthermore, molecular identification of new prototype HAdV was not possible because of the lack of type identification criteria.
If typing of a wild-type isolate is not feasible with the newly defined L2 molecular typing criterion, isolation of a new HAdV prototype can be suspected. In this case it is important to gain additional L1 sequence data because of the bigger contribution of L1 to the determinant. A 2.4% L1 nucleotide divergence from the next prototype strain (Fig. 1A) in combination with 4.2% amino acid divergence (Fig. 2A) strongly supports identification of a new HAdV neutralization prototype. As a next step, we suggest that the fiber knob region () should be sequenced, in order to predict the immunogenic properties (neutralization and hemagglutination inhibition) of the proposed new prototype sufficiently. However, it should be kept in mind that major cross-reactions in hemagglutination inhibition tests and even identical sequences of the determinant have not been contradictory to identification of a new prototype (Fig. 5A) (10, 59). Other techniques should be used for confirming identification of a new HAdV prototype, e.g., genotyping by restriction fragment analysis (2) and classical cross-neutralization techniques, as taxonomic criteria for type identification have not been modified (10).
Virus typing and subsequent definition of new types on the basis of sequence data of immunogenic epitopes have been applied successfully to human enteroviruses and resulted in the identification of several new types (39-41). Probably, a molecular typing concept as suggested by us for HAdV will also facilitate the identification of new HAdV prototype viruses.
Regarding the suggestions for molecular discovery of new HAdV types, we checked whether the most recently described HAdV types, 50 and 51, would have been indicated as new types (18). The sufficient nucleic acid sequence divergence of the new serotypes HAdV-B50 (16% divergence from the next heterologous serotype HAdV-B34) and -D51 (20% divergence from HAdV-D15 and HAdV-D29) in the L2 region (Fig. 1B) would have clearly indicated the isolation of two new types. This is confirmed by L1 sequences (Fig. 1A) and determinant sequences (Fig. 5A). Thus, our sequencing results and criteria also confirm that these isolates are new HAdV types.
In contrast to HAdV-B50 and -D51, molecular typing is not feasible with the two serotypes HAdV-D15 and -D29. This does not seem to be a drawback of the proposed molecular typing concept because the neutralization method also cannot reliably differentiate these two HAdV (1, 29, 66). Therefore, it was already suggested that the new definition of the serotype HAdV-D29 as a distinct serotype was premature in 1962 (49, 66). Using the proposed molecular typing criteria, HAdV-D29 (AJ811451) is not a separate serotype but a hemagglutination variant (intermediate strain) of HAdV-D15. By contrast, another study comparing the L2 sequences of HAdV-D15 and HAdV-D29 indicated sufficient sequence divergence for molecular typing (51), contradictory to our results and cross-neutralization results (1, 29, 66). This may be explained by the inclusion of hexon sequences adjacent to the immunogenic L2, which do not contribute to the neutralization epitope. Moreover, our L2 typing result is clearly supported by the phylogenetic analysis of L1, which codes for a bigger part of the neutralization epitope (0.0% sequence divergence). As HAdV-D29 differs from HAdV-D15 by its hemagglutination properties as usually intermediate strains do, we tried to identify the phylogenetic origin of the hemagglutination epitope by sequencing of the determinant. Phylogenetic analysis indicated a close relationship of HAdV-D29 (AJ811451) to a previously published data bank sequence of HAdV-D30, strain BP-7 (AF447393). There were only two bases different between the sequences of HAdV-D29 and -D30, resulting in a single amino acid substitution (S104F). This implied that HAdV-D29 and -D30 should have HI cross-reactions. However, HI cross-reactions have never been reported between these two viruses (49, 59, 65). Sequencing of two different strains of HAdV-D30, the BP-7 strain (ATCC VR-273) (AJ831473) and a prototype strain of the German adenovirus reference center, demonstrated that these two had 100% identical sequences which were quite divergent from the HAdV-D30 sequence (AF447393) in the data bank (39% nucleic acid sequence divergence) and from the HAdV-D29 sequence in the data bank (AJ811451) (61.6%). Phylogenetic analysis demonstrated a different clustering of the newly generated HAdV-D30 sequences, which had the closest relationship to HAdV-D49 (Fig. 5A). This relationship is supported by one-way HI cross-reactions between HAdV-D30 and -D49 (52). In summary, our results imply that HAdV-D29 is an intermediate strain originating from a recombination between HAdV-D15 ( determinant) and a so-far-not-isolated, unknown HAdV prototype ( determinant) phylogenetically related to HAdV-D25. The latter result may justify the acknowledgment of HAdV-D29 as a distinct serotype.
In conclusion, molecular typing by sequencing of immunogenic HAdV determinants is an adequate substitute for classical neutralization and hemagglutination inhibition typing. This approach will also help to identify new types more easily. The complete data set of the main immunogenic determinants and may facilitate development of gene therapy vectors and give new insights into the complex molecular evolution of HAdV including multiple recombination events.
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ABSTRACT
Human adenoviruses (HAdV) are responsible for a wide spectrum of diseases. The neutralization determinant (loops 1 and 2) and the hemagglutination determinant are relevant for the taxonomy of HAdV. Precise type identification of HAdV prototypes is crucial for detection of infection chains and epidemiology. and determinant sequences of all 51 HAdV were generated to propose molecular classification criteria. Phylogenetic analysis of determinant sequences demonstrated sufficient genetic divergence for molecular classification, with the exception of HAdV-15 and HAdV-29, which also cannot be differentiated by classical cross-neutralization. Precise sequence divergence criteria for typing (<2.5% from loop 2 prototype sequence and <2.4% from loop 1 sequence) were deduced from phylogenetic analysis. These criteria may also facilitate identification of new HAdV prototypes. Fiber knob ( determinant) phylogeny indicated a two-step model of species evolution and multiple intraspecies recombination events in the origin of HAdV prototypes. HAdV-29 was identified as a recombination variant of HAdV-15 ( determinant) and a speculative, not-yet-isolated HAdV prototype ( determinant). Subanalysis of molecular evolution in hypervariable regions 1 to 6 of the determinant indicated different selective pressures in subclusters of species HAdV-D. Additionally, determinant phylogenetic analysis demonstrated that HAdV-8 did not cluster with -19 and -37 in spite of their having the same tissue tropism. The phylogeny of HAdV-E4 suggested origination by interspecies recombination between HAdV-B (hexon) and HAdV-C (fiber), as in simian adenovirus 25, indicating additional zoonotic transfer. In conclusion, molecular classification by systematic sequence analysis of immunogenic determinants yields new insights into HAdV phylogeny and evolution.
INTRODUCTION
Adenoviridae viruses are nonenveloped, double-stranded DNA viruses with an icosahedral capsid (59). Human adenoviruses (HAdV) are classified into six species (HAdV-A to HAdV-F) which were defined historically as subgenera on the basis of hemagglutination properties (10, 17, 55). Subsequently, oncogenic properties and DNA homology were also used to define subgenera (species) (59). The six HAdV species consist of 51 HAdV types, defined mainly by neutralization criteria (10, 64). Several simian adenoviruses (SAdV), which cannot productively infect humans, are also members of the species HAdV-A (SAdV-2, -4, -6, -9, -10, -11, and -14), HAdV-B (SAdV-21), HAdV-C (SAdV-13), HAdV-E (SAdV-22 to -25), and HAdV-F (SAdV-19).
Precise HAdV typing is essential for epidemiological studies and quality control of diagnostics. Moreover, the greater knowledge about differences in virulence and organ tropism among the adenovirus serotypes has increased the medical value of HAdV classification (59). For example, the more dangerous lower respiratory tract illness is associated primarily with HAdV-B3, -B7, -B21, and -E4 infection (11, 13, 16, 21), whereas the less severe upper respiratory tract illness is frequently caused by HAdV-C1, -C2, -C5, -B3, and -B7 (15, 21). Thus, typing of HAdV isolated from a respiratory tract sample or feces sample may predict the clinical course. Infections of the eye with HAdV-D8, -D19, and -D37 may cause severe epidemic keratoconjunctivitis, whereas mild follicular conjunctivitis and pharyngoconjunctival fever are most frequently caused by HAdV-B3, -B7, and -E4 (6, 37). Disseminated disease in highly immunosuppressed patients, e.g., allogeneic stem cell transplant recipients, is associated with HAdV-A31, -B3, -B7, -C1, -C2, and -C5, most frequently with the last three types (20, 32, 54, 57, 63). Acute hemorrhagic cystitis and acute renal failure in infants, young children, and immunosuppressed patients are associated primarily with the serotypes HAdV-B11, -B21, -B34, and -B35 (3, 12, 20, 23, 38, 57). Due to this background and the wide spectrum of diseases which are caused by HAdV of differing organotropism and virulence, a deeper understanding of the phylogenetic relationships of HAdV types is crucial for the development of molecular classification criteria.
All adenoviruses contain three diagnostically useful antigens, which are part of the three capsid proteins: hexon (polypeptide II), penton (polypeptide III), and fiber (polypeptide IV). The main type-specific epitope, the determinant, consisting of loop 1 (L1) and loop 2 (L2) on the hexon protein, reacts with type-specific antisera in neutralization tests (NT) (24), which are the classical reference method for typing. Cases of failing neutralization with available antisera are an unresolved issue of NT because these require extensive cross-neutralization studies for defining a new HAdV type. The knob region of the fiber protein, which includes the type-specific determinant, has hemagglutinating properties which are used for hemagglutination inhibition (HI) tests. HI is preferred by many laboratories, because it is more convenient and rapid. However, HI cannot differentiate all 51 HAdV because of several cross-reactions (59). As HAdV have a high capability for intraspecies recombination and occasional interspecies recombination, HI results of clinical isolates can be contradictory to NT results ("intermediate strains"). Intermediate strains are recombinant viruses between two serotypes presenting hexon neutralization epitopes of one serotype and fiber knob hemagglutination epitopes of another serotype. Probably, recombination of HAdV may give rise to new types and even species. Retargeting of HAdV types with favorable gene expression and persistence properties (as for example HAdV-C5) with fiber knobs of other HAdV types holds promise for improving HAdV gene therapy and vaccination vector approaches (9, 35). A complete phylogenetic analysis of the fiber knobs of all 51 HAdV should facilitate this approach.
Nowadays, PCRs are frequently used for rapid diagnosis of adenovirus infections (5, 19, 36, 43). Therefore, attempts were made to use PCR also for genotyping of HAdV. Techniques included type-specific PCRs and multiplex PCR protocols using species-specific primers (28, 42, 43, 45, 69) as well as combinations of a generic PCR with restriction digestion of the amplicons (4, 30, 50, 61). These approaches permit rapid molecular typing of a limited number of HAdV types or at least species identification. However, new insights into molecular evolution and epidemiology of HAdV cannot be attained, and new HAdV types cannot be identified.
Based on extensive sequence analysis of the neutralizing epitope (L1 and L2 of the hexon capsid protein) and the hemagglutination epitope , we developed criteria for molecular classification of HAdV and for the identification of new prototype isolates. Sequencing of the short L2 proved to be sufficient for molecular typing whereas additional L1 and determinant sequence data should be generated for the identification of new prototype isolates. L2 sequencing can be recommended as a second step for precise type identification after species identification by diagnostic PCRs.
MATERIALS AND METHODS
HAdV prototype strains and cells. HAdV prototype strains HAdV-B50 (ATCC VR-1501), -C1 (VR-1), -D8 (VR-1368), -D15 (VR-1092), -D20 (VR-255), -D22 (VR-1100), -D23 (VR-258), -D25 (VR-223), -D27 (VR-1105), -D28 (VR-226), -D29 (VR-1107PI/RB), -D30 (VR-273), -D32 (VR-625), -D33 (VR-626), -D36 (VR-913/275), -D37, -D38 (VR-988), -D39 (VR-932), -D42 (VR-1304), -D43 (VR-1305), -D44 (VR-1306), -D49 (VR-1407), and -D51 (VR-1502) were obtained from the American Type Culture Collection (ATCC). HAdV-B14, -D10, -D13, and -D30 prototype strains were obtained from our collection at the German National Reference Laboratory for Adenoviruses, Hannover Medical School, Hannover, Germany, and had been typed previously with cross-neutralization. In a preliminary quality control study using a multiplex PCR protocol (69), the virus delivered by ATCC as HAdV-B50 (VR-1501) turned out to be a member of species D, whereas HAdV-D51 (VR-1502) was a species B virus. In the following correspondence with ATCC, it was stated that ATCC believes that stocks of VR-1501 (HAdV-B50) and VR-1502 (HAdV-D51) have been switched. Subsequently, VR-1501 and VR-1502 were deleted from the ATCC catalogue. For this study, HAdV-B50 (VR-1501) was relabeled as HAdV-D51 and vice versa. All viruses were propagated on A549 cells (ATCC, CCL-185) on 75-cm2 cell culture flasks. When the cytopathogenic effect was above 50%, cells were frozen at –70°C, and DNA was extracted with the QIAGEN blood kit (QIAGEN, Hilden, Germany).
Generic HAdV PCR. HAdV DNA was amplified using either a conventional PCR protocol which was published as a first step of a nested PCR (4) or a real-time PCR protocol (26).
PCR amplification of hypervariable hexon loops. The hexon L1 region was amplified as described previously (43). For the PCR amplification of hexon L2 two primer pairs, one for the species HAdV-B and the other for the species HAdV-C and HAdV-D, were developed (Table 1) with the help of a multiple alignment with GenBank sequence data flanking the L2 region (HAdV-B3, -B7, -B14, -D9, -D10, -D17, -D19, -D23, -D24, -D26, -D45, -D46, -D47, and -D48). Amplification of L2 comprises two primer pairs in individual PCRs, one for the species HAdV-C and -D (CDL and CDR), which produces a 322-bp product, and a primer set for the species B (BL and BR) with a 580-bp amplicon (Table 1). Both primer pairs were designed for an identical annealing temperature of 54°C as calculated with MeltCalc software to permit amplification with a single thermal profile (53). Additionally, L1 and L2 primer pairs were designed for HAdV-A18 to obtain the immunogenically relevant sequences for the phylogenetic analysis (Table 1). These pairs have the same PCR conditions as the corresponding L1 and L2 protocols (Table 1). PCR was performed in a total volume of 100 μl consisting of HotStar Mix (QIAGEN), 1 μM of each primer, and 3 μl of the purified HAdV DNA. The PCR program starts with activation of the "hot start" DNA polymerase for 15 min at 95°C, followed by 40 cycles consisting of denaturation at 94°C for 20 s, primer annealing at 54°C for 20 s, and elongation at 72°C for 40 s, followed by a final extension step of 72°C for 5 min. The sensitivity of the PCR protocols was determined by testing serial dilutions of cell culture-derived HAdV-B3, -C2, and -D8 previously quantified by a generic HAdV real-time PCR protocol (25).
PCR amplification of the fiber knob region. For amplification of HAdV-D, a new sense primer, AdFiDL, and an established antisense primer, AdD2, in the E4 region were combined (69), resulting in a 1,000-bp amplicon (Table 1). The sense primer was designed with the help of a multiple alignment of GenBank sequence data of the 5' end flanking the determinant (sequences of HAdV-B3, -B7, -B11, -B14, -B16, -B21, -B34, -B35, -D8, -D9, -D15, -D17, -D19, -D28, -D30, and -D37). For species B, a new primer set (AdFiBR and AdFiBL) was developed (Table 1), which amplifies the same region as do the species D primers and requires the same thermal cycling profile. PCR was performed in a total volume of 100 μl as described above with a final concentration of 2 μM MgCl2. The thermodynamic profile was equivalent to the previously described hexon PCR program except for the annealing temperature of 56°C and the elongation step for 90 s. New primer pairs were designed for species HAdV-B (FiBL/FiBR) and species HAdV-F (FiFL/FiFR) (Table 1). The PCRs were performed as described above. The sensitivity of the PCR protocols was determined by testing serial dilutions of cell culture-derived HAdV-B3 and -D8 previously quantified by a generic HAdV real-time PCR protocol (26). Furthermore, the fiber knob region of HAdV-A18 was sequenced after amplification with primers located in the fiber (5'-GGCTATGGCTGATGGTG-3') and 3' inverted terminal repeat region (5'-GTTTGGGCGGATGAGG-3').
Gel electrophoresis. PCR products were separated in a 2% agarose gel (1% in the case of fiber amplicons) for 60 min at 120 V. DNA extraction from the agarose gels was performed with the QIAGEN gel extraction kit according to the manufacturer's recommendations.
Sequencing. Cycle sequencing was performed with rhodamine-labeled dideoxynucleotide chain terminator (DNA sequencing kit; ABI, Warrington, England) and analyzed on an ABI Prism 310 automatic sequencer (Applied Biosystems). PCR primers were used for the sequencing reactions.
Phylogenetic analysis. L1, L2, and fiber knob region sequences were compared by sequential pairwise alignment with the Clustal algorithm implemented in the BioEdit software package (version 6.0.5) (62) and adjusted manually to conform to the optimized alignment of deduced amino acid sequences. Phylogenetic relationships were inferred from the aligned nucleic acid sequences by the neighbor-joining method implemented in the programs DNAdist and Neighbor (PHYLIP, version 3.6; University of Washington, Seattle) (22) using the Kimura two-parameter substitution model (31) and a transition/transversion ratio of 10. Support for specific tree topologies was estimated by bootstrap analysis with 1,000 pseudoreplicate data sets. Branch lengths in consensus trees were calculated by the maximum-likelihood quartet-puzzling method, using the nucleotide substitution model of Tamura and Nei (60) as implemented in Tree Puzzle 5.0 (58). The pairwise nucleotide identity matrix was calculated with BioEdit (version 6.0.5). The histogram of the pairwise nucleotide comparisons was plotted with the program PNCH (34). Similarity plots were calculated based on different matrices (PAM250 and EBLOSUM60) with a window of 10 amino acids with the software Plotcon included in the EMBOSS package (47). Bootscans were performed with the software SimPlot (version 3.5.1) (33) with a window of 200 bp (20-bp step) based on a Kimura two-parameter substitution model (31) with a transition/transversion ratio of 2.0.
For making multiple alignments of L1, L2, and fiber knob regions the following GenBank sequences were used: for hexon, HAdV-C1 (X67709), -C2 (AJ293903), -B3 (X76549), -E4 (X84646), -C5 (AF542130), -C6 (X67710), -B7 (Z48571), -D9 (AF161562), -D10 (AB023548), -B11 (AY163756), -A12 (X73487), -B14 (AB018425), -B16 (X74662), -D17 (AF108105), -D19 (AF161565), -B21 (AJ012091), -B34 (AB052911), -D23 (AB023552), -D26 (AB023554), -A31 (X74661), -B35 (AB052912), -D37 (AB023555), -F40 (X51782), -F41 (X51783), -D45 (AB023556), -D46 (AB023557), -D47 (AB023558), -D48 (U20821), and SAdV-E25 (BK000413); for fiber knob ( determinant), HAdV-C1 (AB108423), -C2 (J01917), -B3 (X01998), -C5 (M18369), -C6 (AB108424), -B7 (AF104384), -D8 (AB162768), -D9 (X74659), -B11 (AY163756), -A12 (X73487), -B14 (AB065116), -D15 (X72934), -B16 (U06106), -D17 (Y14241), -D19 (U69131), -D28 (Y14242), -B21 (U06107), -A31 (X76548), -B34 (AB073168), -B35 (U32664), D37 (U69132), -E4 (X76547), -F40 (M28822), -F41 (M60327), and SAdV-E25 (BK000413).
Nucleotide sequence accession numbers. The sequences of L1 of the determinant of HAdV-B14 (AB018425), -B50 (AJ864518), -D13 (AJ749847), -D15 (AJ821891), -D20 (AJ749848), -D25 (AJ749849), -D27 (AJ749850), -D28 (AJ749851), -D29 (AJ749852), -D30 (AJ749853), -D32 (AJ749854), -D33 (AJ749855), -D36 (AJ749856), -D38 (AJ749857), -D39 (AJ749858), -D42 (AJ821893), -D43 (AJ821894), -D44 (AJ749859), -D49 (AJ821895), and -D51 (AJ821896) have been deposited in GenBank, as well as the L2 sequences of HAdV-A18 (AJ821897), -B14 (AJ749860), -B50 (AJ749861), -D13 (AJ745880), -D15 (AJ745881), -D20 (AJ745882), -D22 (AJ745883), -D25 (AJ745884), -D27 (AJ745885), -D28 (AJ745886), -D29 (AJ745887), -D30 (AJ745888), -D32 (AJ745889), -D33 (AJ745890), -D36 (AJ745891), -D37 (AJ745892), -D38 (AJ745893), -D39 (AJ745894), -D42 (AJ745895), -D43 (AJ745896), -D44 (AJ745897), -D49 (AJ745898), and -D51 (AJ745899). Fiber knob ( determinant) sequences of HAdV-A18 (AJ841699), -B50 (AJ811465), -D10 (AJ811442), -D13 (AJ811443), -D20 (AJ811444), -D22 (AJ811445), -D23 (AJ811446), -D24 (AJ811447), -D25 (AJ811448), -D26 (AJ811449), -D27 (AJ811450), -D29 (AJ811451), -D30 (AJ831473), -D32 (AJ811452), -D33 (AJ811453), -D36 (AJ811454), -D38 (AJ811455), -D39 (AJ811456), -D42 (AJ811457), -D43 (AJ811458), -D44 (AJ811459), -D45 (AJ811460), -D46 (AJ811461), -D47 (AJ811462), -D48 (AJ811463), -D49 (AJ811464), and -D51 (AJ811460) have also been submitted to GenBank.
RESULTS
Phylogenetic analysis of determinant L1 and L2. The type-specific neutralization epitope ( determinant) consists of L1, which includes six hypervariable regions (HVR1 to HVR-6), and of L2 with the seventh HVR (14). Sequences of the hypervariable L1 region were generated with the generic primer pair H1.1-H2.1, which has an amplicon size of 800 bp (Table 1). All 45 HAdV prototype viruses of species B, C, D, and A18 were amplified successfully with the new L2 primer sets (Table 1), and all amplicons had the expected length of 322 bp in the case of species C and D, 590 bp in the case of species B, and 656 bp in the case of HAdV-A18. L1 and L2 amplicons of prototype strains were directly sequenced and aligned with previously available GenBank sequence data for human adenovirus prototype strains (for nucleotide sequence accession numbers see Materials and Methods). Phylogenetic trees for L1 based on nucleotide acid positions 19228 to 19828 and for L2 based on positions 19989 to 20272 (both referring to the HAdV-C2 hexon sequence) were constructed with several phylogeny reconstruction algorithms (neighbor joining, maximum likelihood, and maximum parsimony) in order to determine the phylogenetic relationships among HAdV on the species level. The phylogenetic trees constructed by different methods were congruent in overall structure (Fig. 1 depicts neighbor-joining trees), as were trees constructed with deduced amino acid sequences (compare Fig. 1 and 2).
As expected, phylogenetic trees of both L1 and L2 confirmed that human adenoviruses clustered together in major groups corresponding to their six species, with the exception of the only virus of species E, HAdV-E4 (Fig. 1 and 2). HAdV-E4 clustered with the species HAdV-B and had the closest relationship to HAdV-B16 with a high bootstrap value both in nucleic acid and in amino acid phylogenetic trees (100%, Fig. 1 and 2), which supports previously published cross-neutralization data (65). Groups of HAdV-A and HAdV-F were clustering together in one monophyletic group, which correlates well with their common tissue tropism (Fig. 1 and 2) (8). This group clusters together with species HAdV-C.
Identical determinants of HAdV-D15 and HAdV-D29. Genetic divergence of HADV-D15 and HAdV-D29 was as low as 0.0% (nucleic acid sequence) in L1 (Fig. 1A) and 0.4% in L2 (Fig. 1B), resulting in a single amino acid substitution (E422Q), whereas genetic divergence of other HAdV-D serotypes was in the range of 2.5% to 23%. HAdV-D15 and HAdV-D29 also exhibit extensive cross-neutralization (1, 10, 29, 66). Moreover, sequence variability between different strains of HAdV-D15 was in the same range as the difference between HAdV-D15 and HAdV-D29. For example, three different bases resulting in two amino acid substitutions in L2 were found between the HAdV-D15 strain Morrison, available in the GenBank database (X76707), and the HAdV-D15 strain 305 (955; CH. 38; V-215-003-014, ATCC VR-1092) sequenced additionally in this study (AJ745881). Therefore, L1 sequencing, L2 sequencing, and classical neutralization typing concordantly indicate that HAdV-D15 and HAdV-D29 are not distinct serotypes. For comparison, the second highest genetic identity in L2 (97.5%) was observed between HAdV-D39 and -D43, which can be clearly discerned by cross-neutralization. There were eight bases different in the L2 region, resulting in a single amino acid difference between these two serotypes (D435N) (Fig. 1B and 2B). Furthermore, sufficient antigenic divergence between HAdV-D39 and -D43 was plausible because of L1 sequence divergence (7.4% nucleic acid divergence resulting in 16 amino acid exchanges) (Fig. 1A and 2A). These molecular evolutionary data indicated that divergence of HAdV-43 and HAdV-39 into distinct types was the product of a relatively recent evolutionary event.
Evolution of neutralization determinants of species HAdV-D. Species HAdV-D consists of 31 serotypes (counting HAdV-D15 and HAdV-D29 as a single neutralization serotype), and three main subclusters were observed in the L1 amino acid sequence phylogenetic tree (Fig. 2A). Therefore, a subanalysis of the genetic variability related to the HVRs in L1 of the three main subclusters of HAdV-D was performed in order to determine mechanisms of molecular evolution leading to the selection of a multitude of neutralization serotypes. In all three subclusters HVR1 to HVR6 were found (Fig. 3) with the main genetic variability found in HVR4 and HVR5. Similarity plots indicated that HAdV-D prototype viruses of subcluster 1 diverged mainly by additional molecular evolution of HVR3 (Fig. 3A). In contrast to subcluster 1, viruses of subcluster 2 diverged mainly by additional molecular evolution of HVR1 (Fig. 3B), and viruses of subcluster 3 diverged in both HVR1 and HVR2 (Fig. 3C).
Molecular classification criteria derived from determinant phylogeny. The phylogenetic trees of the L1 and L2 regions (Fig. 1 and 2) demonstrated that typing of HAdV is feasible by sequencing either L1 or L2 amplicons because of sufficient genetic distances. The amplicon of the L2 PCR is shorter and easier to sequence than that of the L1 PCR. As the nucleic acid sequence phylogenetic tree of L2 is identical in overall structure to the amino acid sequence tree of L2, we suggest L2 sequencing and comparison of nucleic acid sequence data as routine molecular typing methods. After HAdV-D15 and -D29 were counted as a single neutralization serotype, nucleic acid sequence identity of serotypes of the same species was in the range of 61.5% to 97.5% in the L2 coding region, with the lowest intraspecies nucleic acid identity found between types HAdV-B14 and -B16 and the highest between HAdV-D39 and -D43 (Fig. 1). For comparison, nucleic acid identity between serotypes of different species was lower, between 51.9% and 67.0%, with the lowest interspecies identity found between HAdV-D37 and -F40 and, as an exception, the highest (93.0%) found between HAdV-B16 and -E4 (Fig. 1). Distribution of nucleic acid sequence pairwise identity scores of the L2 region between serotypes clearly resulted in two peaks, representing heterologous serotypes of the same species and heterologous serotypes of different species (Fig. 4). A nucleic acid sequence divergence of <2.5% in the L2 region compared to the next homologous prototype sequence in the data bank is proposed as a criterion for molecular identification of a serotype, because the lowest genetic divergence between two serotypes in the L2 region was 2.5% (HAdV-D39 and -D43) (Fig. 1B). Type identification of a clinical isolate is achieved in the case of a <2.5% genetic divergence of the L2 sequence compared to the most closely related prototype sequence, if the next closely related sequence of another prototype of the same species (in the case of HAdV-E4, HAdV-B16) has a sequence divergence of at least 2.5%. If these criteria are not fulfilled, a deduced amino acid sequence of the clinical isolate has to be aligned with prototype data bank sequences, for example, by using the FASTA Internet server. If the L2 amino acid divergence compared to the next data bank sequence is <1.2%, typing is accomplished (Fig. 2B). However, if the L2 amino acid divergence compared to the next data bank sequence is 1.2%, typing is not feasible and isolation of a new HAdV type may be suspected.
Phylogenetic analysis of the fiber knob determinant. Amplification of the fiber knob determinant of all HAdV species B, D, and HAdV-A18 was achieved with newly developed primer sets (Table 1). New sequence data were generated by direct sequencing and aligned with sequence data previously published in GenBank (nucleic acid positions 31959 to 32766 of HAdV-C2). Phylogenetic trees of all 51 HAdV built by several methods (neighbor joining, maximum likelihood, and maximum parsimony) were identical in overall structure, as were nucleic acid sequence trees (data not shown) and deduced amino acid sequence trees (Fig. 5A depicts a neighbor-joining tree). Trees confirmed several phylogenetic relationships suggested by hemagglutination properties of the HAdV species. Adenovirus fiber knob sequences clustered together in major groups corresponding to the six HAdV species, with the exception of species HAdV-D, which formed three clusters. However, division of species HAdV-D into three clusters was not confirmed by bootstrapping. Surprisingly, pairwise nucleotide comparison of fiber knob sequences resulted in three peaks (Fig. 5B), with peak 1 representing intraspecies relationships, whereas interspecies relationships were divided into two groups (peaks 2 and 3 in Fig. 5B), suggesting a two-step model of HAdV species evolution. Peak 2 is formed by the interspecies relations between species A and F and between species D and C, including HAdV-E4, whereas peak 3 consisted of all other interspecies relationships. Several phylogenetic relationships of species HAdV-D corresponded well to HI cross-reactivity (HAdV-D8 and -D9; HAdV-D10, -D19, and -D37; HAdV-D13, -D38, and -D39; HAdV-D15, -D22, and -D42; HAdV-D20 and -D47; HAdV-D24, -D32, -D33, and -D46) (Fig. 5) (59). As already suggested by cross-neutralization experiments and HI cross-reactions (59), phylogenetic relations of HAdV-D fiber knob sequences did not completely coincide with hexon L1 and L2 sequences (Fig. 2 and 5A). Overall, intraspecies genetic divergence in the fiber knob region was lower than for hexon L1 and L2, with several prototype sequences almost identical (as low as 0.0% difference; Fig. 5A and B).
Phylogenetic criteria for species classification. A determinant species criterion should correlate excellently with the classical species criteria, because the fiber knob region ( determinant) is responsible for the hemagglutination properties of the HAdV. The phylogenetic tree of the fiber knob region demonstrated that a criterion for classification of HAdV species can be easily deduced with as a single exception species HAdV-E clustering with HAdV-C (Fig. 5). Based on an identity matrix of the complete data set of the determinant sequences, we calculated the maximum intraspecies divergence of each species (47%, nucleic acid divergence in species B), and the minimum interspecies divergence between all species was computed (52%, A to F). Therefore, a nucleic acid sequence divergence of 50% from the next data bank prototype sequence can be suggested as a criterion for species definition (Fig. 5B). Based on this species criterion, HAdV-E4 is a member of the HAdV-C species with an average identity of 56% to the other members of the HAdV-C (54.1% to 56.9%). Species identification criteria can also be derived from hexon sequence data sets. For example, many diagnostic laboratories use generic PCR protocols which amplify a conserved region at the 5' end of the hexon gene closely adjacent to the L1 coding region (4, 26). Therefore, we evaluated whether amplicon sequences of these PCRs are appropriate for molecular classification. Figure 6 shows that genetic divergence permits reliable species identification. A generic PCR protocol proposed by Allard et al. (4) allows species identification with a nucleic acid sequence divergence of 9% from the next data bank prototype sequence (Fig. 6A). In the case of a generic real-time PCR protocol (26) with its smaller amplicon size, sequence divergence of <4.8% is required (Fig. 6B). Both protocols even allow a final typing decision, but it is reliable for only six members of the species A, E, and F (criterion, 1.5% nucleic acid sequence divergence from the next homologous prototype sequence) (Fig. 6).
Molecular identification of intermediate strains. Amplification of the immunogenic determinant in combination with the L2 PCR could be a useful alternative for molecular identification of HAdV intermediate strains because the main neutralization epitope and the hemagglutination epitope can be sequenced. Four clinical isolates previously typed as intermediate strains with the classical methods (NT and HI) were analyzed with the newly developed combination of the L2 sequencing and determinant sequencing. In accordance with previous results of classical typing methods, isolates were identified as intermediate strains HAdV-15H9 (two isolates), HAdV-30H44, and HAdV-37H13. The L2 nucleic acid sequences of four analyzed intermediate strains were 100% identical to the prototype strains. Their determinant sequences were 98% to 100% identical to the prototype strains, and the deduced amino acid sequences were 97% to 100% identical. In the case of HAdV-30H44, the fiber determinant of HAdV-D44 was 100% identical to HAdV-D48. This finding correlates well with cross-reactivity in HI between these two viruses (52). HAdV-D30H44 was typed previously with HAdV-D44-specific HI antiserum when HAdV-D48 was not yet isolated, and an HAdV-D48-specific antiserum was not available to us. Therefore, a final differentiation between HAdV-D44 and -D48 was not feasible with both molecular and classical methods.
Molecular evolution of the main antigenic determinants of species HAdV-B and HAdV-E. Species B has been previously divided into two subspecies ("subgenera") because of obvious striking differences in restriction fragment patterns and, in part, tissue tropism. Detailed analysis of the molecular phylogeny of the main immunogenic determinants showed a more complex pattern. In hexon L1 and L2 trees subspecies B1 viruses HAdV-B3 and -B7 were as closely related as anticipated by the subspecies concept, whereas other subspecies B1 viruses (HAdV-B21 and -B50) clustered with subspecies B2 viruses, HAdV-B21 with HAdV-B11 and -35 and HAdV-B50 with HAdV-B14 and -34, supported by high bootstrap values (Fig. 2). Subanalysis of genetic variability related to HVR1 to HVR6 in L1 indicated that HAdV-B3 and -B7 diverged mainly by selection of immunogenic epitopes in HVR5 (Fig. 7A). By contrast, HAdV-B14, -B34, and -B50 diverged mainly by molecular evolution in HVR1 and HVR4 (Fig. 7B), and HAdV-B11, -B21, and -B35 diverged mainly in HVR1 and HVR2 (Fig. 7C). Phylogenetic relations of HAdV-B fiber knob sequences did not coincide with hexon L1 and L2 sequences, indicating intrasubspecies and intersubspecies recombination events in the molecular phylogeny of species B HAdV (compare Fig. 2 and 5A). The close phylogenetic relationship in the species B assumed by HI cross-reactions between HAdV-B7, -B11, and -B14 and between HAdV-B34 and -B35 was confirmed (Fig. 5A) (59).
In the determinant (L1 and L2) HAdV-B16 formed a subcluster with HAdV-E4 and with the more distantly related SAdV-E25 but was not closely related to the other species HAdV-B prototype viruses. However, in the fiber knob region HAdV-B16 clustered with the other species B viruses whereas both HAdV-E4 and SAdV-E25 clustered with species HAdV-C.
DISCUSSION
Previously, HAdV subgenera, now species, were defined by their hemagglutination properties; later oncongenicity, fiber length, and genotyping (restriction fragment analysis) data were also included as subgenus criteria. Phylogenetic analysis of fiber knob ( determinant) sequences of all 51 HAdV confirmed the species concept (Fig. 5A). From our sequence data, DNA homology criteria for species identification were deduced (<50%, Fig. 5). However, phylogenetic relationships between species C and D and between species A and F were closer (peak 2 in Fig. 5B) than between other species, resulting in a unique three-peak pattern of the pairwise nucleotide comparison plot (Fig. 5B).
This finding indicated a more recent divergence in the molecular evolution of the enterotropic species HAdV-A and HAdV-F, as well as in the evolution of HAdV-C and -D, and suggested two main steps of molecular evolution of HAdV species. The species concept of HAdV is also supported by the molecular phylogeny of the determinant L1 and L2 (Fig. 1 and 2), but the two interspecies relationship peaks were not observed in the pairwise nucleotide comparison plot of L2 (Fig. 4). However, the recent divergence of species HAdV-A and -F is also supported by hexon L1 and L2 phylogenetic trees (Fig. 1 and 2), whereas in the case of species HAdV-C and -D a close relationship could not be confirmed by hexon sequence data. Probably, the higher selection pressure resulted in a more rapid evolution of the neutralization determinant than of the determinant. A subanalysis of the genetic variability of HVR1 to HVR6 indicated complex patterns of molecular evolution of the determinant (Fig. 3), leading to the emergence of multiple neutralization types of species HAdV-D. This may have covered up older events of species divergence in the determinant data set. Another hypothesis for the evolution of species HAdV-C and -D viruses would be a recombination event leading to divergence of these species by introducing a more diverse hexon sequence and subsequent evolution in both and determinants, resulting in the multiple types of these species.
Historically, HAdV-E4 was classified as a species of its own mainly because of its difference in GC content from that of species HAdV-B (10), although HAdV-E4 is closely related to species HAdV-B as demonstrated by phylogenetic studies of the hexon and several other genome regions except the fiber gene (8, 25, 44). Recently, the complete genome of HAdV-E4 was sequenced and the close relationship with simian adenoviruses was demonstrated (most closely to SAdV-E25) (46). A zoonotic transmission event was suggested as the possible origin of HAdV-E4 (46). Therefore, we included SAdV-E25 in our phylogenetic analysis of HAdV. Surprisingly, in the neutralization determinant , HAdV-E4 was more closely related to HAdV-B16 (supported by a bootstrap value of 100%) and far more distantly related to SAdV-E25, which also clustered with species HAdV-B (Fig. 1 and 2). By contrast, phylogenetic analysis of the fiber knob region confirmed the close relationship of HAdV-E4 with SAdV-E25 (supported by a bootstrap value of 100%) and both viruses clustered with (but were more distantly related to) species HAdV-C (bootstrap value 100%) (Fig. 5A). Thus, our phylogenetic analysis supported both an interspecies recombination mechanism in the phylogeny of both HAdV-E4 and SAdV-E25, combining a species HAdV-C-like fiber gene with a species HAdV-B genetic backbone, and a zoonotic transmission event as suggested by Purkayastha et al. (46). However, in the determinant (L1 and L2), HAdV-E4 is very closely related to HAdV-B16 as indicated by phylogenetic trees (Fig. 1 and 2) and also by cross-neutralization experiments (27). Therefore, we tried to analyze the phylogenetic relationships of HAdV-B16, HAdV-E4 (AY594253), SAdV-E25 (BK000413), and HAdV-B7 (AY495969). Unfortunately, a complete genomic sequence of HAdV-B16 was not available in GenBank. Phylogenetic analysis of available HAdV-B16 sequences of E1A (AY490821), 13S protein (AY490821), putative pVIII protein and E3 (AB073632), putative 11-kDa protein (AY509991), VA RNA I and II (U10674), and RNA preterminal protein (U52564) sequences in addition to hexon and fiber knob sequences demonstrated that the close relationship of HAdV-B16 and HAdV-E4 is restricted to the hexon region (data not shown). Therefore, a bootscan analysis of the complete HAdV-B16 hexon sequence, compared to HAdV-E4, HAdV-B11, HAdV-B7, HAdV-B3, and SAdV-E25, was performed. Bootscan results indicated an intrahexon recombination event in the origin of HAdV-B16. For nucleotide positions 1 to 1400 of the hexon gene (including the determinant), HAdV-B16 had the highest similarity to HAdV-E4, whereas for positions 1400 to 2823 HAdV-B16 was more closely related to HAdV-B11.
In general, intraspecies recombination is observed much more frequently than interspecies recombination (55, 67). Therefore, most intermediate strains belong either to species B or to species D, both of which include more serotypes (41 of 51) than all other species do. Several cases of strong HI cross-reactions have been described, for example, between HAdV-D10, -D19, and -D37 and between HAdV-D24, -D32, -D33, and -D46 (59), which may suggest that some of these prototype viruses are recombination variants sharing an almost identical fiber knob region. For example, fiber knob sequences of HAdV-D44 and -D48 were 100% identical, as well as HAdV-D19 and -D37 in the determinant, which are closely related to HAdV-D10 (Fig. 5). Therefore, a previous recombination event can indeed be hypothesized in the origin of some HAdV prototypes, e.g., HAdV-D44 and -D48 or HAdV-D19 and -D37. This result is in complete accordance with the organotropism and virulence of HAdV-D19 and -D37, which both cause epidemic keratoconjunctivitis. On the other hand, HAdV-D8 neither clustered with HAdV-D19 and -D37 nor cross-reacted in HI, although it shares similar organotropism and virulence. Recently, sialic acid and CD46 were identified as cellular receptors interacting with the fiber knob of HAdV-D37 (7, 68), whereas the receptor of HAdV-D8 was not yet definitely identified. The phylogenetic relationship of the HAdV-D8 fiber knob to HAdV-D9 suggests CAR as the putative receptor (Fig. 6) (48). This indicates that factors other than cellular receptors are significant for epidemic keratoconjunctivitis.
From a type identification perspective, fiber knob ( determinant) sequencing is clearly secondary to hexon ( determinant) sequencing (compare Fig. 2 and 5A), as is hemagglutination inhibition testing compared to neutralization testing (27, 59, 65). In a diagnostic setting highly sensitive detection of HAdV DNA is currently achieved in many laboratories with extensively validated, generic PCR amplifying the conserved 5' end of the hexon gene (4, 26). Sequencing of these amplicons can be proposed as the first step of a two-step molecular typing scheme. Phylogenetic analysis of these amplicon sequences indicated that species identification is unequivocally feasible (Fig. 6). For unequivocal type identification sequencing of the hypervariable neutralization determinant is required as a second step.
This is the first work including sequences and phylogenetic analysis of both loops of the neutralization determinant of all 51 HAdV. Phylogenetic analysis demonstrated that either L1 or L2 sequencing is sufficient for precise typing (Fig. 1). A <2.5% nucleic acid sequence divergence was deduced as an unequivocal criterion for molecular typing from L2 sequence data. Sequencing of the bigger L1 (compare Fig. 1A and 1B) or use of deduced amino acid data for L2 did not improve type identification (compare Fig. 1B and 2B). Therefore, our results clearly support L2 sequencing as the most simple but sufficient approach for molecular typing of all 51 HAdV. Recently, a sequence-based approach for typing HAdV of species D and E was proposed (56). However, the relevant neutralization determinant was not covered by this approach, nor was an application to HAdV species other than D and E achieved. Another group suggested L2 sequencing for typing all HAdV without defining sequence divergence criteria for type identification or defining the length of the sequence region (51). Furthermore, molecular identification of new prototype HAdV was not possible because of the lack of type identification criteria.
If typing of a wild-type isolate is not feasible with the newly defined L2 molecular typing criterion, isolation of a new HAdV prototype can be suspected. In this case it is important to gain additional L1 sequence data because of the bigger contribution of L1 to the determinant. A 2.4% L1 nucleotide divergence from the next prototype strain (Fig. 1A) in combination with 4.2% amino acid divergence (Fig. 2A) strongly supports identification of a new HAdV neutralization prototype. As a next step, we suggest that the fiber knob region () should be sequenced, in order to predict the immunogenic properties (neutralization and hemagglutination inhibition) of the proposed new prototype sufficiently. However, it should be kept in mind that major cross-reactions in hemagglutination inhibition tests and even identical sequences of the determinant have not been contradictory to identification of a new prototype (Fig. 5A) (10, 59). Other techniques should be used for confirming identification of a new HAdV prototype, e.g., genotyping by restriction fragment analysis (2) and classical cross-neutralization techniques, as taxonomic criteria for type identification have not been modified (10).
Virus typing and subsequent definition of new types on the basis of sequence data of immunogenic epitopes have been applied successfully to human enteroviruses and resulted in the identification of several new types (39-41). Probably, a molecular typing concept as suggested by us for HAdV will also facilitate the identification of new HAdV prototype viruses.
Regarding the suggestions for molecular discovery of new HAdV types, we checked whether the most recently described HAdV types, 50 and 51, would have been indicated as new types (18). The sufficient nucleic acid sequence divergence of the new serotypes HAdV-B50 (16% divergence from the next heterologous serotype HAdV-B34) and -D51 (20% divergence from HAdV-D15 and HAdV-D29) in the L2 region (Fig. 1B) would have clearly indicated the isolation of two new types. This is confirmed by L1 sequences (Fig. 1A) and determinant sequences (Fig. 5A). Thus, our sequencing results and criteria also confirm that these isolates are new HAdV types.
In contrast to HAdV-B50 and -D51, molecular typing is not feasible with the two serotypes HAdV-D15 and -D29. This does not seem to be a drawback of the proposed molecular typing concept because the neutralization method also cannot reliably differentiate these two HAdV (1, 29, 66). Therefore, it was already suggested that the new definition of the serotype HAdV-D29 as a distinct serotype was premature in 1962 (49, 66). Using the proposed molecular typing criteria, HAdV-D29 (AJ811451) is not a separate serotype but a hemagglutination variant (intermediate strain) of HAdV-D15. By contrast, another study comparing the L2 sequences of HAdV-D15 and HAdV-D29 indicated sufficient sequence divergence for molecular typing (51), contradictory to our results and cross-neutralization results (1, 29, 66). This may be explained by the inclusion of hexon sequences adjacent to the immunogenic L2, which do not contribute to the neutralization epitope. Moreover, our L2 typing result is clearly supported by the phylogenetic analysis of L1, which codes for a bigger part of the neutralization epitope (0.0% sequence divergence). As HAdV-D29 differs from HAdV-D15 by its hemagglutination properties as usually intermediate strains do, we tried to identify the phylogenetic origin of the hemagglutination epitope by sequencing of the determinant. Phylogenetic analysis indicated a close relationship of HAdV-D29 (AJ811451) to a previously published data bank sequence of HAdV-D30, strain BP-7 (AF447393). There were only two bases different between the sequences of HAdV-D29 and -D30, resulting in a single amino acid substitution (S104F). This implied that HAdV-D29 and -D30 should have HI cross-reactions. However, HI cross-reactions have never been reported between these two viruses (49, 59, 65). Sequencing of two different strains of HAdV-D30, the BP-7 strain (ATCC VR-273) (AJ831473) and a prototype strain of the German adenovirus reference center, demonstrated that these two had 100% identical sequences which were quite divergent from the HAdV-D30 sequence (AF447393) in the data bank (39% nucleic acid sequence divergence) and from the HAdV-D29 sequence in the data bank (AJ811451) (61.6%). Phylogenetic analysis demonstrated a different clustering of the newly generated HAdV-D30 sequences, which had the closest relationship to HAdV-D49 (Fig. 5A). This relationship is supported by one-way HI cross-reactions between HAdV-D30 and -D49 (52). In summary, our results imply that HAdV-D29 is an intermediate strain originating from a recombination between HAdV-D15 ( determinant) and a so-far-not-isolated, unknown HAdV prototype ( determinant) phylogenetically related to HAdV-D25. The latter result may justify the acknowledgment of HAdV-D29 as a distinct serotype.
In conclusion, molecular typing by sequencing of immunogenic HAdV determinants is an adequate substitute for classical neutralization and hemagglutination inhibition typing. This approach will also help to identify new types more easily. The complete data set of the main immunogenic determinants and may facilitate development of gene therapy vectors and give new insights into the complex molecular evolution of HAdV including multiple recombination events.
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