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Journal of Clinical Microbiology, December 2005, p. 6176-6182, Vol. 43, No. 12
0095-1137/05/$08.00+0 doi:10.1128/JCM.43.12.6176-6182.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Servicio de Virología,1 Servicio de Microbiología Diagnóstica, Centro Nacional de Microbiología, ISCIII, Madrid, Spain,2 The Jerome L. and Dawn Greene Infectious Disease Laboratory, Mailman School of Public Health, Columbia University, New York, New York,3 Cátedra de Virología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina,4 and Barbara Davis Center for Diabetes Research, University of Colorado Health Sciences Center, Denver Colorado5
Received 1 June 2005/ Returned for modification 8 July 2005/ Accepted 7 September 2005
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Molecular typing methods have been established to circumvent practical problems associated with traditional serum neutralization studies (1, 10, 13, 14). Molecular methods also have disadvantages; restriction fragment length polymorphism analysis of adenoviral DNA may fail if mutations are present within the restriction site, and multiplex PCR assays are currently not able to discriminate between serotypes. PCR amplification of the hypervariable portion of the hexon gene followed by DNA sequencing has recently been proposed as a typing method; however, this method was unable to discriminate between species B and E and was validated with only 10 clinical samples (19).
We have previously detected HAdV infection in clinical samples using generic HAdV primers in singleplex (4) and multiplex assays; these assays have been extensively validated and used routinely for clinical diagnosis (6, 8). Here, we report that DNA sequencing and phylogenetic analysis of this moderately conserved region (amino acids 540 to 662) of the hexon gene (9) are sufficient to allow HAdV speciation and, in most cases, serotype identification. We have confirmed and also noted new associations between specific serotypes and clinical presentations.
HAdV infection was detected by two distinct multiplex PCRs (8) in 46 clinical specimens and 25 HAdV culture isolates sent for diagnostic evaluation at Centro Nacional de Microbiologia, ISCIII, Spain. Clinical materials, patient characteristics, and alternate methods to detect HAdV infection, such as cell culture, direct immunofluorescence assay, and latex agglutination, are listed in Table 1.
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Nucleic acids were extracted (7) and the singleplex PCR assay performed as described previously (4). Briefly, 5 µl of the nucleic acid extraction was added to 45 µl of reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 500 µM (each) deoxynucleoside triphosphates, 4 mM MgCl2, 2.5 units of Taq polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk, Conn.), and 20 pmol of the degenerate primers ADHEX1F (5'CAACACCTAYGASTACATGAA3') and ADHEX1R (5'KATGGGGTARAGCATGTT3'). Temperature and time profiles were as follows: 94°C for 1 min, 50°C for 1 min, and 68°C for 1 min for 30 cycles and a final incubation at 68°C for 5 min. Amplification products (475 bp) from this PCR were visualized by agarose gel electrophoresis and sequenced directly. For clinical samples where direct sequencing was not possible due to low DNA yield, two independent nested reactions were performed with 20 pmol of the degenerate primers ADHEX2F (5'CCCITTYAACCACCACCG3') and ADHEX1R or 20 pmol of ADHEX1F and ADHEX2R (5'ACATCCTTBCKGAAGTTCCA3'). Amplified products were purified and sequenced in both directions using an automated ABI PRISM 377 model sequencer.
The consensus sequence was compared and aligned against other sequences from samples or the DNA database using the program CLUSTAL X (version 1.83). The relationships between individual viruses were established using neighbor-joining, unweighted-pair group method using average linkages, and nucleotide substitution methods (Tamura-Nei, Kimura-2p, and Jukes-Cantor). Phylogenetic trees were reconstructed through the neighbor-joining method (MEGA package, version 3) by 1,000 bootstrap resamplings. Pairwise comparisons were also made by global alignment using the Needleman Wunsch algorithm (17), implemented by a program from EMBOSS (18).
The phylogenetic tree showed six different clusters representing species A to F at the nucleotide (Fig. 1) and amino acid levels, with bootstrap values ranging from 59 to 99. Results obtained when the sequences from 46 clinical samples and the 25 HAdV isolates were compared with sequences from the reference strains are presented in Table 2. All clinical samples were speciated, and 42 (91%) of 46 were serotyped. All 25 isolates were speciated, and 22 (88%) of 25 were serotyped. Phylogenetic analysis revealed subclusterings, except in species E, D, and B2; serotype HAdV-7 could be separated into two lineages (14). Serotypes of species D were not clearly discriminated because of high homology. Serotypes 11, 34, and 35 of subgroup B2 were indistinguishable. HAdV-4 is the only member of species E. The Needleman Wunsch pairwise algorithm produced results identical to those obtained via phylogenetic analyses (Table 2) (see Table S2 in the supplemental material). Although both analyses permitted accurate HAdV classification, pairwise similarity analysis has the advantage of speed and simplicity.
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Acute respiratory diseases due to HAdV are attributed primarily to serotypes 3, 4, 7, 14, and 21 (species B and E) (12). We also observed serotypes 1, 2, 5, and 6 (species C) and species D. Pneumonia in children has been associated with serotypes 1 to 3 and 7, whereas pneumonia in adults is predominantly associated with serotypes 4 and 7. We also found serotypes 5 and 6 in young children and subgroup B2 in immunosuppressed patients.
Gastrointestinal manifestations of HAdV infection include diarrhea and hepatitis. In addition to serotypes 40 and 41 (2, 12), we identified cases of diarrhea associated with serotypes 6 (immunocompetent patient); 12, 16, and 31 and a member of species D (bone marrow transplants or human immunodeficiency virus-positive case). The significance of these findings is not clear, since members of species C may be excreted in feces during subclinical infection. HAdV hepatitis has been reported in child recipients of liver transplants associated with serotypes 1, 2, and 5 (species C); in this study we detected the remaining member of species C, serotype 6, in a case of fatal hepatitis.
Epidemic keratoconjunctivitis has been associated with serotypes 8, 19, and 37 (species D) and serotype 11 (species B2). Our sequences revealed not only serotype 8 (species D) but also serotypes 4 and 7 (species E and B1, respectively). Adenovirus 4 (species E) was recently discovered as the cause of either respiratory or mild ocular infections (21, 12) and nosocomial epidemic conjunctivitis in Japan (3).
Cases of acute hemorrhagic cystitis in young children have been associated with species B2 serotypes 11 and 21. In our study, this disease is associated with serotypes 16 and 14 (species B1 and B2), and an indistinguishable member of the cluster 11, 34, and 35 (species B2). Fatal infections due to species B1, serotypes 3 and 7, have been reported (11, 12, 15). The fact that subgroups B1 and B2 use different cellular receptors for viral entry underscores the importance of typing HAdV for epidemiology and pathobiology (20).
Finally, we found serotypes 4 and 5 (species E and C) in throat swabs of four patients with fever, morbilliform rash, Koplik's spots, and cough who had a history of measles, mumps, and rubella vaccination and were negative for measles and rubella. To our knowledge this is the first report of HAdV presenting as a syndrome compatible with measles infection. Case numbers are not sufficient to establish causality between specific serotypes and specific syndromes. Further independent testing is needed to verify these associations.
Using the database and classification system from this study, we have deployed a website (www.greeneidlab.columbia.edu) wherein clinical laboratories can submit hexon sequences to generate an automatic report detailing the serotype, date, and location of the most similar sequence isolate in the database. This system will allow new genotypes to be readily identified, because the classification scheme will fail to relate them to any described serotype. Epidemiological surveillance of HAdV serotypes will improve our understanding of the global burden of HAdV infection. High-throughput systems described here will facilitate HAdV surveillance and enhance understanding of HAdV pathogenesis.
Nucleotide sequence accession numbers. The sequences determined in this work were deposited in the GenBank sequence database under accession numbers AY819809 to AY819926.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the National Institutes of Health (AI51292, AI55466, AI056118, and U54AI057158-Northeast Biodefense Center), the Ellison Medical Foundation, and ISCIII (MPY 1251/00 and MPY 1018/04).
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FOOTNOTES |
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Supplemental material for this article may be found at http://jcm.asm.org/.
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