Molecular epidemiology of adenovirus type 8 (Ad 8) in Taiwan: four subtypes recovered during the period of 1980-1981 from patients with epidemic keratoconjunctivitis

Adenovirus type 8 (Ad 8) has been the major and important causative agent of epidemic keratoconjunctivitis (EKC). By enzymatic cleavage analysis with five endonucleases, PstI, HindIII, BamHI, SalI and SstI, 27 out of 149 Ad 8 isolates recovered from patients with EKC during the period from July, 1980 to July, 1981 in Kaohsiung, Taiwan, were studied. By cleavage patterns, 27 Ad 8 isolates in Kaohsiung were classified into four subtypes which were found to be different from the subtypes (Ad 8A, Ad 8B) prevalent in Sapporo (1).     

Genome type analysis of adenovirus type 4 isolates, recently obtained from clinically different syndromes in some areas in Japan

DNA cleavage analyses with EcoRI, HindIII and BamHI were carried out to investigate genome types of recent Ad 4 isolates obtained from acute respiratory disease (8 strains), and ocular disease (11 strains) in Japan. DNA cleavage patterns of all 19 isolates studied were identical regardless of whether they were recovered from respiratory tract or conjunctiva, but were distinct from that of the prototype strain.     

Enteric adenovirus type 41 isolates: cloning, physical maps and diversity in restriction enzyme cleavage pattern.

Adenovirus (Ad) type 40 and 41 DNAs were directly extracted from stool specimens of children with gastroenteritis. Two new strains of Ad41, Sanekata and Ehime strain, were cloned and their restriction maps were constructed. The left terminal end of the cloned Ad41 genome, EcoRI-E fragment of the Sanekata strain and EcoRI-F fragment of the Ehime strain, had transforming ability in rat 3Y1 cells. Only one of the 35 isolates of Ad40 tested showed a different restriction profile, while three different restriction profiles were found in DNAs from Ad41 isolates.     

A Simple and Practical Method for Typing and Strain Differentiatin of Herpes Simplex Virus Using Infected Cell DNAs

A simple and practical method for typing and strain differentiation of herpes simplex virus (HSV) isolates, based upon analysis of the restriction endonuclease cleavage patterns of viral DNAs, was established by using unlabeled infected cell DNAs. The preparation of infected cell DNA is technically easier than that of purified viral DNA or of radiolabeled viral DNA. The method provides a powerful and practical tool for epidemiological and clinical studies of HSV infection, which can be performed in most diagnostic laboratories. In order to select suitable restriction endonucleases for the study of HSV isolates, the cleavage patterns of viral DNAs (strains MacIntyre, HF, UW-268, and SAV) with 12 enzymes were analyzed. Several enzymes, Bam HI, Kpn I, Pst I, Sal I, Sst I, and Xho I, were found to be useful for both typing and strain differentiation. With this method, HSV isolates from different individuals and from the same individual were analyzed by digestion of their infected cell DNAs with Bam HI. Six isolates from epidemiologically unrelated individuals were readily typed and differentiated from each other. Three isolates from the same individual showed very similar patterns. However, there was a small degree of difference between the first two isolates and the third isolate.     

Genomic comparison of adenovirus type 3 isolates from patients with acute conjunctivitis in Japan, Australia, and the Philippines

A total of 53 Ad3 isolates obtained from patients with acute conjunctivitis in Japan, Australia, and the Philippines during 1973 to 1986 were analyzed for their genome types with 4 restriction endonucleases, BamHI, BglII, HindIII, and SmaI. Two new genome types designated tentatively as Ad3f and Ad3g were identified by combination of BamHI and BglII in the isolates. The changes of restriction sites and sizes of restriction fragments in newly recognized Ad3f and Ad3g were located at the similar regions reported in other Ad3 genome types by O'Donnell et al (1986) on physical maps of the Ad3 prototype strain GB genome. In Japan, 46 Ad3 isolates obtained from 1983 to 1986 were either Ad3f or Ad3g. Yearly alternation of predominance of both genome types were observed in the northern part of Japan during the period. In Australia, two genome types Ad3p and Ad3f were found in 6 isolates, and the former was observed in the 3 isolates obtained before 1981 and the remaining 3 isolates were obtained after 1983. In the Philippines, the only isolate obtained in 1984 was Ad3p.     

Comparison of DNAs of Crypthecodinium cohnii-like Dinoflagellates from Widespread Geographic Locations*

SYNOPSIS. We have examined various properties of DNAs from 7 dinoflagellate isolates of wide geographic distribution; all of the isolates are superficially indistinguishable from a laboratory strain of Crypthecodinium cohnii originally isolated at Woods Hole, Massachusetts (WHd strain). Two isolates, one from Puerto Rico and the other from Honduras, are clearly distinguishable from WHd and the other isolates by their DNA buoyant density values. WHd and the other 5 isolates we have examined are indistinguishable from one another in terms of DNA buoyant densities and melting temperatures. The relationship among the various isolates, including WHd, were evaluated at a finer level through restriction endonuclease cleavage and molecular hybridization to compare ribosomal RNA gene structure in the several DNAs. All the isolates could be further categorized by this method, the patterns of restriction endonuclease cleavage of ribosomal RNA genes in the isolates paralleling exactly their sexual compatibilities established from breeding experiments by Beam & Himes. The DNAs were also treated with a restriction endonuclease sensitive to the presence of the modified base 5-methylcytosine. In all isolates, cytosine residues in both total DNA and DNA specifically containing the ribosomal RNA genes were found to be extensively methylated, as was previously shown for the WHd strain.     

Transforming Region of Group A, B, and C Adenoviruses: DNA Homology Studies with Twenty-Nine Human Adenovirus Serotypes

The 31 human adenovirus (Ad) serotypes form five groups based upon DNA genome homologies: group A (Ad12, 18, 31), group B (Ad3, 7, 11, 14, 16, 21), group C (Ad1, 2, 5, 6), group D (Ad8, 9, 10, 13, 15, 17, 19, 20, 22-30), and group E (Ad4) (M. Green, J. Mackey, W. Wold, and P. Rigden, Virology, in press). Group A Ads are highly oncogenic in newborn hamsters, group B Ads are weakly oncogenic, and other Ads are nononcogenic. However, most or all Ads transform cultured cells. We have studied the homology of Ad5, Ad7, and Ad12 transforming restriction endonuclease DNA fragments with DNAs of 29 Ad types. Ad5 HindIII-G (map position 0-7.3), Ad7 XhoI-C (map position 0-10.8), and Ad12 (strain Huie) EcoRI-C (map position 0-16) and SalI-C (map position 0-10.6) fragments were purified, labeled in vitro (nick translation), and annealed with DNAs of Ad1 to Ad16, Ad18 to Ad24, and Ad26 to Ad31. Hybrids were assayed by using hydroxylapatite. Ad5 HindIII-G hybridized 98 to 100% with DNAs of group C Ads, but only 1 to 15% with DNAs of other types. Ad7 XhoI-C fragment hybridized 85 to 99% with DNAs of group B Ads, but only 6 to 21% with DNAs of other types. Ad12 (Huie) EcoRI-C hybridized 53 to 68% with DNAs of five other Ad12 strains, 53% with Ad18 DNA, 56% with Ad31 DNA, but only 3 to 13% with DNAs of other types. In vitro-labeled Ad12 (Huie) SalI-C hybridized 35 to 71% with DNAs of 6 other Ad12 strains, 44% with Ad18 DNA, 52% with Ad31 DNA, but only 2 to 7% with DNAs Ad7, Ad2, Ad26, or Ad4. When assayed using S-1 nuclease, SalI-C annealed 17 to 44% with DNAs of group A Ads. The melting temperatures of the hybrids of Ad5 HindIII-G with all group C Ad DNAs were 84 degrees C in 0.12 M sodium phosphate (pH 6.8). The melting temperature of the Ad12 (Huie) EcoRI-C hybrid with Ad12 (Huie) DNA was 83 degrees C, but was only 71 to 77 degrees C with DNAs of other group A Ads. Thus, group C and group B Ads both have very homologous transforming regions that are not represented in DNAs of non-group C Ads or non-group B Ads, respectively. Similarily, group A Ads have unique but less homologous transforming regions. These different transforming nucleotide sequences may be reflected in the different oncogenic properties of group A, B, and C Ads.     

Isolation of a non-insecticidal Bacillus thuringiensis strain belonging to serotype H8a8b

Bacillus thuringiensis strains non-toxic to Lepidoptera, Bombyx mori and Diptera, Culex pipiens pallens larvae were isolated from Korean soil samples during an investigation of B. thuringiensis isolates highly toxic to insect pests. One of these isolates, NTB-88, produces parasporal inclusions about 138 kDa in size and is non-toxic to 19 insect species of three orders, Lepidoptera, Diptera and Coleoptera, even though it is highly susceptible to tryptic cleavage. Study of flagellar (H) antibodies of 33 B. thuringiensis strains revealed that NTB-88 has an H antigen identical with that of subsp. morrisoni (serotype 8a8b). Comparison of parasporal inclusion proteins and plasmid DNA patterns of strain NTB-88 with B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. morrisoni PG-14 showed that the isolate is a novel non-insecticidal B. thuringiensis strain belonging to serotype 8a8b.     

Characterization of mitochondrial DNA in various Candida species: isolation, restriction endonuclease analysis, size, and base composition.

A practical and effective method for the extraction of mitochondrial DNA from Candida species was developed. Zymolyase was used to induce yeast protoplasts, and mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations. Restriction endonuclease analyses of mitochondrial DNAs from 19 isolates representing seven species of Candida (C. albicans, C. kefyr, C. lusitaniae, C. maltosa, C. parapsilosis, C. shehatae, and C. tropicalis) and Lodderomyces elongisporus revealed different cleavage patterns that appeared to be specific for the species. Few common restriction fragments were evident. The genome sizes of the mitochondrial DNAs ranged from 26.4 to 51.4 kilobase pairs, and the guanine-plus-cytosine contents ranged from 20.7 to 36.8 mol%. There was no correlation between the base compositions of nuclear and mitochondrial DNAs. Eight isolates of C. parapsilosis, including the type culture, and an ascosporogenous strain of L. elongisporus, which was once proposed as the teleomorph of C. parapsilosis, had similar mitochondrial DNA molecular sizes (30.2 and 28.8 kilobase pairs); however, restriction endonuclease patterns of these organisms were distinct. These data provide additional support for discrimination of these two species. The results of our experiments demonstrate that mitochondrial DNA analyses may provide useful criteria for the differentiation of yeast species.     

Analysis of the DNAs from seven varicella-zoster virus isolates.

The 32P-labeled DNAs from seven different clinical isolates of human varicella-zoster virus (VZV) were independently digested with five site-specific restriction endonucleases, EcoRI, HindIII, SmaI, BamHI, and AvaI. The digestion products were analyzed by electrophoresis on 0.5% agarose gels followed by autoradiography of the dried gels. Evaluation of the restriction enzyme cleavage patterns revealed small variations among the VZV DNAs. The VZV DNAs were also compared based on their buoyant densities in CsCl. No significant buoyant density differences were detected among the VZV DNAs.     

Genetic diversity of Bradyrhizobium japonicum field population revealed by RAPD fingerprinting

RAPD fingerprinting was used for strain identification and the assessment of genetic diversity within a field population of Bradyrhizobium japonicum . Total genomic DNAs from 13 field isolates and two inoculant strains were amplified using six different 10-mer primers. Different and informative band patterns were obtained for all strains analysed. Cluster analysis unexpectedly revealed that none of the field isolates was identical to inoculant strains which were regularly used for soybean inoculation. Among field isolates two highly divergent groups were determined. The results indicate that RAPD is a very discriminative and efficient method for differentiating and studying genetic diversity of B. japonicum strains.     

Kinetic studies on the cleavage of adenovirus DNA by restriction endonuclease Eco RI.

The kinetics of cleavage of DNA from Adenovirus Type 1 (Ad1), Type 5 (Ad5) and Type 6 (Ad6) by restriction endonuclease EcoRI was investigated by quantitative evaluation of the fluorescence from ethidium stained DNA fragments separated on agarose gels. The apparent rate constants of cleavage at different cleavage sites have been determined and large differences in the cleavage rates of the individual sites within one type of DNA were found. From the kinetics of cleavage information on the sequence of the DNA fragments can be obtained. The order of the fragment A, B, C, D of Ad6 DNA obtained after complete cleavage by restriction endonuclease Eco RI was found to be A-D-C-B; the order of the corresponding fragments A, B, C of Ad1 and Ad5 DNA was found to be A-C-B.     

Expression of adenovirus type 12 early region 1 in KB cells transformed by recombinants containing the gene.

The adenovirus type 12 (Ad12) early region 1 (E1) gene was introduced into KB cells by using a dominant selection vector, pSV2-gpt, and over 80 Gpt+ KB cell clones were established. Three types of recombinant DNAs (gAE1A, gARC, and gABA) were constructed. They contained the AccI-H, EcoRI-C, and BamHI-A fragments, respectively, of Ad12 DNA in pSV2-gpt. Five of 50 (10%) gABA-transformed cell clones, 12 of 18 (67%) gAE1A-transformed cell clones, and 10 of 18 (56%) gARC-transformed cell clones complemented the growth of Ad5 dl312 (deletion in E1A) and were designated as Gpt+ Ad+ cell clones. In these cell clones at their early passages, recombinant genome sequences were detected in cellular DNA and were expressed. T antigen g (the E1A gene product) was detected by immunofluorescence. The Gpt+ Ad+ cell clones supported the growth of Ad5 deletion mutants in parallel with the expression of Ad12 E1A or E1A plus E1B genes. After infection of Gpt+ Ad+ cell clones with Ad5 dl312, the early genes of dl312 were efficiently transcribed, indicating the expression of the pre-early function of the Ad12 E1A gene. Two clones each from gAE1A-,gARC-, and gABA-transformed cells were subcultured for a long period to determine the stability of the transfecting DNAs. Subculture in a nonselective medium resulted in cells which lost the transfecting DNAs. Subculture in a selective medium resulted in the selection of cells which maintained the gpt gene expression but lost the Ad12 gene expression. These results indicate that the transfecting DNA is present in an unstable state in KB cells.     

Genome typing of adenovirus strains isolated from conjunctivitis in Japan, Australia, and the Philippines

DNA restriction endonuclease analysis for genome typing of adenovirus (Ad) DNA was carried out on a total of 65 Ad isolates including serotypes Ad4, Ad7, Ad8, Ad11, Ad19, and Ad37 from patients with epidemic keratoconjunctivitis and acute conjunctivitis obtained in Japan from 1982 to 1986, Australia from 1973 to 1986, and the Philippines in 1984. All 4 isolates of Ad7 in Australia were Ad7b. Four of 6 Ad11 isolates obtained in Japan were typed as Ad11 prototype (Ad11p), and the remaining were identified to be new genome types, designated tentatively as Ad11c and Ad11d. An isolate of Ad11 obtained in Australia was typed as Ad11c. Nine Ad8 isolates in Australia and in the Philippines were typed as Ad8p, but 11 Ad8 isolates in Japan were Ad8b. Thirteen Ad19 isolates were identified as Ad19a. All 3 isolates of Ad37 in Japan and three isolates in Australia before 1982 were typed as Ad37p, however, 5 isolates in Australia after 1983 were identified as a new genome type, designated as Ad37d. In Japan, 10 isolates of Ad4 were identified as Ad4a.     

Appearance of new strains associated with group B meningococcal disease and their use for rapid vaccine development

There has been a decrease in the prevalence of disease in the United States due to meningococcal serotypes 2a and 2b containing class 2 proteins with a concomitant increase in nonserotypable strains containing class 3 major outer membrane proteins. A new disease associated strain was identified using monoclonal antibodies as B:4:P1.15. Serotype 4 strains have been heretofore solated almost only from carriers. This B:4:P1.15 strain predominated among group B disease isolates in Cuba from the late 1970s to the present and among Miami, Florida isolates recovered in 1981 and 1982. To determine whether protein vaccines for new strains or serotypes could be prepared using our present methods, a combined vaccine was prepared from a group B strain (B:8:P1.15) recovered during a recent outbreak in Virginia, and a serotype 2b strain, plus group C polysaccharide. The vaccine was prepared with aluminum hydroxide, or with trehalose dimycolate plus monophosphoryl lipid A, or without adjuvant. Four weeks after immunization antibody levels were much higher in mice that received vaccine containing adjuvant.     

Nucleic acid aggregation geometry and the possible evolutionary origin of ribosomes and the genetic code

We probed the structure of mammalian repetitive DNAs with a site-specific mammalian endodeoxyribonuclease, which we recently identified, and which apparently represents a common enzyme activity among the mammals (McKenna et al., 1981). With several of the DNAs (e.g. mouse satellite, guinea pig β-satellite, variable repeated spacer DNA from mouse ribosomal genes and primate alphoid sequences), the endonuclease activity gave highly specific cleavage patterns when the digestion products were analyzed by gel electrophoresis. These patterns were not always identical to those produced by microbial restriction enzymes. However, in other cases (e.g. bovid and caprid satellites and guinea pig α-satellite) the repetitive DNAs appeared to be degraded randomly. Thus, the mammalian enzyme reveals structural features of the repetitive sequences that are not rendered immediately obvious by microbial restriction enzyme analysis. Evidence from mapping data presented here suggests that the mammalian site-specific endonucleases are not sequence specific but have special affinity for imperfect or hyphenated palindromic sequences in repetitive DNAs and in other eukaryotic DNA sequences.     

Pulsed Field Gel Electrophoresis Analysis of Borrelia burgdorferi Sensu Lato Isolated in Japan and Taxonomic Implications with Lyme Disease Spirochetes

Genomic DNAs of Borrelia burgdorferi sensu lato isolates obtained in Japan sharing different rRNA gene ribotypes were digested with rare-cutter restriction endonucleases and the fragments obtained were separated by pulsed field gel electrophoresis (PFGE). The sizes of large restriction cleavage bands with MluI endonuclease were quite similar among isolates in each ribotype group. On the other hand, the PFGE profiles obtained with the other enzymes (NruI, Sal I or SplI) were rather divergent, and Japanese isolates were distinguishable from the United States and European isolates. The Japanese isolates classified as ribotypes group II (Borrelia garinii) and III (B. afzelii) showed different PFGE patterns from that of European isolates. The isolates grouped into ribotype IV revealed distinctively different PFGE profiles. These results indicate that the Japanese isolates may be genetically divergent and distinct from the United States and European isolates.     

Restriction maps of human adenovirus types 2, 5 and 3 for BclI, ClaI, pvul and SphI endonucleases

The sites of cleavage by BclI, ClaI, PvuI and SphI in the DNA from adenovirus (Ad) serotypes 2, 5 and 3 have been located. Certain site coordinates were in accord with nucleotide sequences already published. A small difference in size between the PvuI-E fragments from Ad2 and Ad5 confirmed the existence of a deletion in the N-terminal moiety of Ad5 hexon gene, as previously implied by interserotypic recombinants (Boursnell and Mautner, Virology 112 (1981) 198-209) and more recently by amino acid sequencing (Von Bahr-Lindstr?m et al., Virology 118 (1982) 352-362).     

Cleavage of Epstein-Barr virus DNA by restriction endonucleases EcoRI, HindIII and BamI.

The cleavage of the DNAs of the B95-8 and P3HR-1 virus strains of Epstein-Barr virus by the restriction endonucleases EcoRI, HindIII and BamI was investigated using a new technique for quantitative evaluation of the fluorescence of ethidium stained DNA fragments separated on agarose gels. The results obtained with B95-8 DNA showed that in addition to the limited repetitions of nucleotide sequences observed in the EcoRI and HindIII cleavage patterns, the molecule contained a BamI fragment with a molecular mass of 2.0 megadaltons which was present in a total of about 11 copies and localized to a limited part of the DNA molecule. The same sequences were also present in the P3HR-1 DNA albeit in a lower molar ratio. P3HR-1 DNA yielded restriction enzyme cleavage patterns suggesting DNA sequence heterogeneity of P3HR-1 virus. No fragment was present in more than about 4 copies per molecule of P3HR-1 DNA. Comparison of the restriction enzyme cleavage patterns of P3HR-1 and B95-8 DNA revealed a high degree of structural homology emphasized by nucleic acid hybridization experiments with EBV complementary RNA synthesized in vitro.     

A pair of adjacent genes, cry5Ad and orf2-5Ad, encode the typical N- and C-terminal regions of a Cry5Adelta-endotoxin as two separate proteins in Bacillus thuringiensis strain L366.

A new DNA sequence cry5Ad/orf2-5Ad (GenBank accession number EF219060) was isolated from Bacillus thuringiensis strain L366. This DNA sequence contains two ORFs: cry5Ad (a previously unreported member of the cry5A gene family) and orf2-5Ad. cry5Ad is unique among cry5A genes in that it encodes only the N-terminal region of a typical Cry5Adelta-endotoxin. The cry5Ad sequence includes homology blocks 1-5, which are present in most B. thuringiensisdelta-endotoxins. The usual C-terminal region of a Cry5Adelta-endotoxin (including homology blocks 6-8) is encoded by orf2-5Ad. Both proteins encoded by cry5Ad and orf2-5Ad were found in IPTG-induced Escherichia coli, after a copy of cry5Ad/orf2-5Ad was cloned into the pQE32 expression vector and transformed into pREP4 E. coli cells. Both proteins were also found in parasporal crystal inclusions of B. thuringiensis L366. Sequencing of cDNA derived from transformed E. coli cells showed that the two ORFs are transcribed as a single mRNA. Extracts prepared from the recombinant E. coli expressing Cry5Ad and Orf2-5Ad were not toxic to nematode larvae (Haemonchus contortus), indicating that these two proteins are most likely not responsible for the nematocidal activity seen previously in the B. thuringiensis strain L366.