Shudo Yamazaki

Quantitative Detection and Rapid Identification of Human Adenoviruses

ABSTRACT We have established a method of quantitative detection and rapid identification of human adenoviruses (hAdVs). Using LightCycler PCR with a primer set, we were able to amplify 554 bp of the hexon gene from each of 51 prototype strains of hAdVs. The sensitivity of LightCycler PCR was 10 copies of hAdV DNA/reaction. When LightCycler PCR was performed using a set of primers, hAdV was positive for 74.4% (99 of 133) of conjunctivitis patients and for 27.3% (81 of 297) of respiratory infection patients. We also attempted to measure hAdV in the potentially contaminated eye drops used by patients, detecting 5.4 × 102 to 1.6 × 106 copies/ml of hAdV. We determined the 350-bp nucleotide sequence of the amplified hexon gene and compared it with the sequences of the 51 prototype strains. Phylogenetic analysis based on 350 bp of the hexon gene identified 99 positive conjunctival swabs as 24 cases of AdV type 3 (AdV-3), 14 cases of AdV-4, 1 case of AdV-8, 19 cases of AdV-19a, and 41 cases of AdV-37. The 81 sequences from pharyngeal or nasal mucus swabs were identified as 29 cases of AdV-2, 18 cases of AdV-1, 18 cases of AdV-5, 12 cases of AdV-4, 2 cases of AdV-37, 1 case of AdV-3, and 1 case of AdV-6. LightCycler PCR followed by phylogenetic analysis provides an effective tool for the rapid identification of hAdVs and for studying molecular epidemiology.

Human rhinovirus 87 identified as human enterovirus 68 by VP4-based molecular diagnosis

Human rhinoviruses (HRVs) are the major cause of respiratory infections. We developed a diagnostic method for HRVs based on the reverse-transcription polymerase chain reaction (RT-PCR) and VP4-based phylogenetic analysis. A set of primers used in the RT-PCR of human enteroviruses (EVs) appeared to be capable of amplifying all prototype strains of HRVs, each of which generated a 530-bp fragment. The single exception was HRV-87, which generated a 650-bp fragment, as observed in human EVs. The VP4 nucleotide sequence of HRV-87 showed more than 97% nucleotide identity with human EV-68, and formed a monophyletic cluster along with the prototype strain of EV-68 in the human EV-D cluster. HRV-87 showed the second highest homology (76.8%) with EV-70, another member of the human EV-D, in a sample of 66 human EVs and 12 HRVs. Therefore, HRV-87 should be reclassified into the cluster containing human EV-68.

Five New Genome Types of Adenovirus Type 37 Caused Epidemic Keratoconjunctivitis in Sapporo, Japan, for More Than 10 Years

ABSTRACT Human adenovirus type 37 (HAdV-37) is a major cause of epidemic keratoconjunctivitis and has recently been the largest causative agent of keratoconjunctivitis in Japan. To investigate the genetic characteristics of HAdV-37 strains isolated in Sapporo, we analyzed the genome types and genetic relationships of 51 strains isolated there from 1990 through 2001. By using DNA restriction analysis, eight genome types (HAdV-37/D1, HAdV-37/D3, and HAdV-37/D6 to HAdV-37/D11) were identified, including five new ones. The restriction fragments of these genome types shared more than 95% identity with those of the prototype strain. By DNA sequence analysis, five and three single nucleotide substitutions, respectively, were found in partial sequences of the hexon and fiber genes. The combinations of mutations resulted in four hexon and fiber types (hx1 to hx4 and f1 to f4) and six hexon/fiber pairs (hx1/f1, hx2/f1, hx1/f2, hx1/f3, hx3/f4, and hx4/f4). The six pairs correlated well with certain genome types. In all three epidemics of keratoconjunctivitis to strike Sapporo in the past 12 years, specific genome types and fiber types were usually isolated: in the first epidemic, HAdV-37/D1 (f1) and HAdV-37/D3 (f1); in the second, HAdV-37/D6 (f2) and HAdV-37/D8 (f3); and in the third, HAdV-37/D10 (f4) and HAdV-37/D11 (f4). We conclude that mutations in the adenovirus genome occurred chronologically and that certain mutations were correlated with the epidemics of adenoviral keratoconjunctivitis.

Monoclonal Antibodies to Herpes Simplex Viruses Antigens-Specificity and Utility in Rapid Serotyping of Clinical Isolates

Sixteen hybridomas secreting antibodies to HSV-1 and 22 hybridomas secreting antibodies to HSV-2 were derived from fusion of SP2/0 myeloma cells with spleen cells from BALB/c mice immunized with each respective virus. Four of the former 16 hybridomas and seven of the latter 22 hybridomas were subcloned and injected into pristane-primed mice to obtain high titers of monoclonal antibodies. Antigen specificity of these monoclonal antibodies were determined by the Western blotting (WB) assay. Two out of four monoclonal antibodies that showed selective reactivity for HSV-1 in IFA, reacted with HSV-1 specific proteins; #1 reacted with 100 KD and 70 KD proteins and #4 with a 150 KD protein, respectively, while the remaining two antibodies reacted only with a 50 KD protein that is type-common antigen. On the other hand, two out of seven antibodies which showed selective reactivity for HSV-2 in IFA, reacted with HSV-2 specific proteins: #5 with a 100 KD protein and #10 with three proteins of 30, 25, and 20 KD, and the other two antibodies reacted with a 50 KD protein that is a type-common antigen. The remaining three antibodies, two of which were found to be immunoglobulin type IgM, reacted with neither HSV-1 nor HSV-2 antigens in WB assay. In order to determine their utility in serotyping, 11 monoclonal antibodies were examined by IFA test for reactivity to cells that were infected with 20 HSV-1 or 16 HSV-2 isolates which had been typed by neutralization test.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on uptake of macrolide antibiotics by Chlamydia host cell

Minimum inhibitory concentration (MIC) of Macrolide antibiotic (ML) against C. trachomatis was found to greatly vary with the cell culture system used for the assay. We then investigated the ability of various cell cultures to uptake MLs in the relation to MIC determination. Penetration of the 14C-labeled MLs, Erythromycin, Jasamycin and Rokitamycin into cells was quantitatively studied by measuring the radioactivity incorporated into McCoy, HeLa229W and HeLa229F cells. It was found that HeLa229W cell showed the lowest MIC for the drugs, followed by HeLa229F cell and then by McCoy cell. Reversely, McCoy cell showed the lowest intracellular concentration of an ML, followed by HeLa229F cell and then by HeLa229W cell. These results indicate that MIC of an ML significantly varies depending on the ability of the test cell to uptake the drug.