Serotyping of Adenoviruses on Conjunctival Scrapings by PCR and Sequence Analysis

Study group and specimens.The Ad prototypes used were obtained from the American Type Culture Collection (ATCC), Rockville, Md. A total of 16 serotypes were studied. They consisted of the seven strains that cause Ad conjunctivitis in Japan: Ad types 3, 4, 7, 8, 11, 19, and 37 (1, 2, 12, 16) and, in addition, Ad types 11, 14, 34, and 35, which belong to subgenus B2; Ad types 1, 2, 5, and 6 in subgenus C; and Ad types 40 and 41 in subgenus F. The study materials consisted of 31 conjunctival scraping specimens collected from patients with Ad conjunctivitis in Japan from 1994 through 1998. The lower palpebral conjunctiva was scraped with two cottonwool swabs, and the virus was extracted by immediately placing each sample in 1.5 ml of Eagle minimal essential medium (MEM) solution (Nissui, Tokyo, Japan). One swab was used for culture isolation, and the other was used for PCR.

Culture isolation-neutralization test (NT).Swabs from the conjunctiva were collected and inoculated onto HEp-2 cells and HEL cells. The viruses were passaged in cell cultures, maintained under Eagle MEM with 2% fetal calf serum, and subpassaged as described previously (13). Infected cells were identified by the immunofluorescent-antibody technique with the mouse monoclonal antibody of Ad (Chemicon International, Inc., Temecula, Calif.). All virus preparations were tested in a preliminary logarithmic serial dilution to determine an optimum working dilution of 100 50% tissue culture infective doses by using four replicate wells per dilution (5). Neutralization tests were performed on HEp-2 cells and HEL cells in 96-well microtiter plates to provide an index of CPE. Antiserum used in this study was obtained from the ATCC.

PCR-RFLP analysis.Serotype identification was performed by PCR-RFLP analysis of all of the specimens prior to carrying out the PCR-sequence method. We described the details of PCR-RFLP analysis previously (30). Briefly, the 1,004-bp conserved region for the hexon was amplified in the first-step PCR and, in the second step, nested PCR was performed to amplify the 956-bp DNA fragment. Differences in the restriction patterns obtained with three restriction enzymes, EcoT14I, HaeIII, and HinfI, were then evaluated, and the serotypes of the clinical specimens were identified by comparisons with the Ad prototypes.

PCR-sequence method. (i) DNA extraction from prototypes and clinical specimens.A 500-μl sample of each of the prototype-infected cell suspensions and conjunctival scrapings extracted with MEM solution was collected in a 1.5-ml microtube and centrifuged at 12,000 × g (High-Speed Microcentrifuge MRX-15; Tomy Seiko Co., Ltd., Tokyo, Japan) at 4°C for 15 min. The pellets were used as samples, and DNA was extracted with SepaGene (Sanko Junyaku Co., Ltd., Tokyo, Japan). After the addition of 50 μl of Tris buffer, 50 μl of guanidine thiocyanate, 350 μl of chloroform containing a protein absorbant, and 200 μl of sodium acetate to the pellet with stirring, the mixture was centrifuged at 12,000 × g at 15°C for 10 min. The supernatant was recovered, 24 μl of acetate buffer and 600 μl of 100% ethanol were added, and the solution was centrifuged at 12,000 × g at 4°C for 10 min. The supernatant was discarded, and 380 μl of 70% ethanol was added to the pellet and, after centrifugation at 12,000 × g at 4°C for 1 min, the supernatant was discarded again. When the DNA pellet had dried, it was dissolved in 25 μl of sterile water.

(ii) PCR primers.Ad is a double-stranded DNA virus, and its full length is approximately 36 kbp (33), with the hexon accounting for about 2.8 kbp (19). The primers to amplify the hexon HVRs were designed for the sites with the highest homology. The first set of primers included HX5-1 (forward primer), 5′-AAGATGGCCACCCCCTCGATGATGCCGCAGT-3′, and HX3-1 (reverse primer), 5′-CACTTATGTGGTGGCGTTGCCGGCCGAGAACGG-3′, which were designed to amplify the region that corresponds to 1 to 2,829 bp in the hexon base sequence of Ad type 3. The second PCR primer set included HX5-3 (forward primer), 5′-CACATCGCCGGACAGGATGCTTCGGAGTA-3′, and HX3-4 (reverse primer), 5′-GTGTTGTGAGCCATGGGGAAGAAGGTGGC-3′. In the second-step PCR, the primers were designed to amplify the region that contains all seven HVRs, which corresponds to bp 40 to 1847 in the Ad type 3 hexon base sequence (Fig. 1).

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Fig. 1.

Ad genome. Gene map of the regions surrounding the Ad hexon protein showing the seven HVRs, the primers, and the regions sequenced.

(iii) Nested PCR.In the first-step PCR, 1 μl of a 100-fold dilution was used when the sample was an Ad prototype whose template DNA was extracted from infected cells, and 2 μl was used when the sample was a clinical specimen extracted from conjunctival scrapings. A 20-μl volume of PCR reaction solution contained Long and Accurate (LA) PCR Buffer II (LA PCR Kit, Ver. 2; Takara Shuzo Biomedicals Co., Ltd., Shiga, Japan), deoxynucleoside triphosphate (dNTP; i.e., dATP, dGTP, dCTP, and dTTP) mixture containing 400 μM concentrations of each, 0.2 μM concentrations of each primer, and 1 U of LA Taq DNA polymerase (Takara Shuzo Biomedicals). Shuttle PCR consisting of a total of 40 cycles of denaturing at 98°C for 10 s, with annealing and extension at 65°C for 6 min, was carried out (Gene Amp PCR System 9600; Perkin-Elmer, Norwalk, Conn.).

In the second-step PCR, the DNA amplified in the first-step PCR (1 μl for the prototypes and 2 μl for the clinical specimens) was placed in 20 μl of PCR solution. The reaction solution contained buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl₂), dNTP mixture containing 200 μM concentrations of each, 0.2 μM concentrations of each primer, and 1 U of Taq DNA polymerase (TaKaRa Taq; Takara Shuzo Biomedicals). The reaction conditions consisted of conventional PCR in a thermal sequencer (Thermal Sequencer TSR-300; Iwaki Glass Co., Ltd., Chiba, Japan) with 40 cycles of denaturing at 94°C for 1 min, annealing at 40°C for 1 min, and extension at 72°C for 2 min.

Each PCR test included a sample of double-distilled water that was treated identically to the virus samples throughout as a negative control. As positive controls, 10 pg of Ad2 or Ad41 DNA was used. Furthermore, to avoid the potential for contamination for DNA amplification, DNA-free handling for reaction mixture preparation and specimen addition for first- and second-step PCRs were performed in separate rooms. Plugged pipette tips, tubes, and other materials used for PCR were all disposable.

(iv) Purification of PCR products.A 1-μl sample of the nested-PCR amplification product was subjected to 1.5% agarose gel electrophoresis and, after being stained with 0.5 mg of ethidium bromide per ml, an approximately 1.8-kbp band of Ad DNA fragment was confirmed under UV light. The confirmed amplified DNA in the PCR products of the specimens was concentrated with Microcon-100 (Amicon, Inc.), and the unreacted primers were removed. After the buffer was completely replaced with deionized water, the purified DNA was electrophoresed, and the DNA concentration was adjusted to approximately 100 ng/ml based on the density of the band.

(v) Sequence primers.The sequences of the 12 serotypes whose base sequences upstream of the hexon had already been elucidated among the 16 standard serotype strains that were the subject of the study were compared, and universal primers were designed in the conserved region present between the individual HVRs (Fig. 1). A total of six primers was used: for sense, S-28 (5′-ACCCACGATGTGACCAC-3′; bp 152 to 172 in the base sequence of Ad type 3), S-29 (5′-GCCAGCACRTWCTTTGACAT-3′; bp 289 to 308), and S-51 (5′-CCCAACAGACCCAAYTACAT-3′; bp 937 to 956); and for antisense, S-52 (5′-CCCATGTTGCCAGTGCTGTTGTARTACA-3′; bp 986 to 1013), S-53 (5′-AAGGGGTTGACGTTGTCCAT-3′; bp 1555 to 1574), and S-54 (5′-CCAGCATTGCGGTGGTGRTT-3′; bp 1576 to 1595). The melting points (T_m s) of all of the primers were calculated from their percent GC content and primer length, and they were designed to have a T_m of ∼60°C.

(vi) Cycle sequence.A total volume of 20 μl, consisting of 8.0 μl of Terminator Ready Reaction Mix (DNA Sequencing Kit; Perkin-Elmer Applied Biosystems, Warrington, Great Britain), 1.0 μl (3.2 μM) of primers for each sequence, 1.0 μl of purified DNA, and 10 μl of sterile water, was placed in a 0.2-ml Gene Amp microtube, and a cycle sequence with a total of 25 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min was carried out (Gene Amp PCR System 9600). The samples were analyzed by means of an autosequencer (ABI Prism 310 Genetic Analyzer; Perkin-Elmer).

Analysis of PCR-sequence method. (i) Homology between prototypes.The sequences of a total of 16 standard serotype strains were compared. The sequences of prototypes previously reported were derived from GenBank. DNASIS (Hitachi Software Engineering Co., Ltd., Tokyo, Japan) was used for sequence alignment and analysis. After referring to the HVR sites described by Crawford-Miksza et al. (7), we compared the HVRs of the 16 serotypes, and the range of each of the new HVRs was reestablished. The rate of homology of each HVR was evaluated.

(ii) Serotype identification of clinical specimens.The predicted amino acid sequences of the clinical specimens were aligned according to the sequences of the 16 serotype prototypes. The homology rate between clinical specimens and Ad prototypes was determined. The serotypes of the clinical specimens were determined by comparing the homology rate. The serotypes identified by the PCR-sequence method were compared with the results of serotype identification by culture isolation-NT and PCR-RFLP analysis.

(iii) Nucleotide sequence accession numbers.Sequence data from this article have been deposited with the GenBank/EMBL/DDBJ data libraries under the following accession numbers: hexon genes Ad11 (AB018424), Ad14 (AB018425), Ad34 (AB018426), and Ad35 (AB018427). The amino acid sequences of these residues were deduced.