Kikuko Miyamura

Nucleotide Sequence Comparison of the Mycobacterial dnaJ Gene and PCR-Restriction Fragment Length Polymorphism Analysis for Identification of Mycobacterial Species

We recently reported a genus-specific PCR for the mycobacterial dnaJ gene. In the present study, we have determined the nucleotide sequences of the dnaJ gene from 19 mycobacterial species (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, M. marinum, M. kansasii, M. gastri, M. simiae, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulare, M. xenopi, M. fortuitum, M. chelonae, M. hemophilum, and M. paratuberculosis). On the basis of the amplified dnaJ gene nucleotide sequences, we constructed a phylogenetic tree of the mycobacterial species by using the neighbor-joining method and unweighted pairwise grouping method of arithmetic average. We found that the phylogenetic relationship inferred within the slowly growing species was in good agreement with the traditional classification, with three major branches corresponding to Runyons groups I, II, and III. An exception was M. simiae, which was phylogenetically closer to the cluster including members of Runyons group III than to that of Runyons group I. On the other hand, the rapid growers, such as M. fortuitum and M. chelonae, did not form a coherent line corresponding to Runyons group IV, indicating that our phylogenetic analysis based on the dnaJ gene reflects the phenotypic characteristics such as pigmentation but not the growth rate. Finally, we revealed the species-specific restriction sites within the amplified dnaJ gene to differentiate most of the mycobacterial DNA by a combination of PCR with restriction fragment length polymorphism analysis.

Sequence analysis of the 3′-end of feline calicivirus genome

Abstract The nucleotide sequence of the 3′-end of the Japanese F4 strain of feline calicivirus (FCV) RNA was determined from a cloned cDNA of 3.5 kbp. We found three open reading frames (ORFs). The largest ORF encoded a 668-amino acid protein of 73,588 Da, which was presumably the capsid precursor protein of FCV and had significant amino acid sequence homology with the VP3 of picornaviruses. A small ORF at the extreme 3′-end was compared with that of the F9 strain of FCV, a vaccine strain originally from the U.S. Highly conserved amino acid sequences were shown, suggesting that this ORF might be functional and encode a putative 106-amino acid protein of 12,153 Da. The other ORF in the 5′-flanking region of the cDNA had consensus amino acid sequences conserved among the RNA-dependent RNA polymerases.

The trend of acquired immunity with live poliovirus vaccine and the effect of revaccination: follow-up of vaccinees for ten years

The persistence of neutralizing antibody (NA) against three types of poliovirus acquired after two doses of trivalent live attenuated poliovirus vaccine (LPV) has been followed up for ten years in individual vaccinees. Sixty-seven children were bled once a year over a five year period following the primary vaccination. More than 80% of them retained NA against all three types of poliovirus. Thirty-two individuals whose NA titres were 1:16 or over for types 1 and 2 and 1:4 or over for type 3 at the fifth year were further followed up for a further five years and it was shown that during this period some of them had a naturally-acquired antibody rise, mostly against type 3 virus. At the sixth to eighth year after the primary vaccination, one further dose of the trivalent vaccine was administered to the children whose NA titres were down to 1:8 or less and the effect of booster vaccination on NA was followed. Other subjects were revaccinated with LPV and their fecal excretion of the vaccine virus was investigated. The results showed that a decrease in serum antibody level could be a good indicator of the local resistance of the alimentary tract and that reinfection could occur if serum NA had decreased to 1:8 or less, which allowed a virus excretion in the stools.

Phylogenetic analysis of a coxsackievirus A24 variant: The most recent worldwide pandemic was caused by progenies of a virus prevalent around 1981

Nucleotide substitutions in the viral-encoded proteinase 3C (3Cpro) region (549 nucleotides) of the RNA genome of a coxsackievirus A24 variant (CA24v), one of the agents causing acute hemorrhagic conjunctivitis (AHC), were studied using 32 isolates collected from the Eastern hemisphere in 1970-1989. Based on regression analysis of nucleotide differences among isolates, the nucleotide substitution rate of CA24v 3Cpro was estimated to be 3.7 x 10(-3)/nucleotide/year. A phylogenetic tree constructed by the modified unweighted pair group method using arithmetic averages (UPGMA) indicated that CA24v had evolved from a common ancestor which appeared in one focal place in November 1963 +/- 21 months, about 7 years before the first isolation of CA24v in Singapore. The tree also revealed that all the recent epidemic isolates in 1985-1989 including Asian and Ghanian strains diverged from each other after 1981. This finding is consistent with the evidence that AHC due to CA24v had been confined to Southeast Asia and the Indian subcontinent until 1985, then suddenly and explosively spread to other areas where no CA24v isolations had been reported.

Evolution of enterovirus 70 in nature: all isolates were recently derived from a common ancestor.

SummaryThe data of large RNase T1-resistant oligonucleotide mapping of enterovirus 70 (EV70) previously reported (Takedaet al., Virology 134, 375–388, 1984) were subjected to further genetical analysis to estimate the evolutionary rate of genome RNA of EV70 and to clarify the phylogenetic relationship among isolates. A proportion of common spots between strains decreased as the year elapsed and eventually, only seven spots were common to all the 16 isolates tested, indicating that the substitution is scattered throughout the genome. On the other hand, some specific sets of spots were conserved among geographically or epidemiologically related strains. Base sequence variation of the isolates was deduced according toAaronsonet al. (Nucleic Acids Res. 10, 237–246, 1982) from pariwise comparison of the common spots and used as a genetic distance between them. The base substitution rate of virus genome was estimated by regression analysis of the genetic distance of the isolates against the sampling time. A fairly constant and rapid rate was obtained; it was 1.83 × 10−3/base/year. Based on the substitution rate, genetic distance and sampling time of the strains, the phylogenetic tree of EV70 was constructed using Unweighted Pair Group Method Using Arithmetic Averages (UPGMA) (Nei, Molecular Population Genetics and Evolution, North Holland, Amsterdam, 1975). The tree supports the previous hypothesis that evolution of EV70 started from a single common ancestor. The time of its emergence was estimated to be 1967±15 months. The virus branched into many strains early during the first pandemic and has evolved in a divergent fashion, yielding genetically polymorphic viruses in the world.

Evolution of enterovirus type 70: Oligonucleotide mapping analysis of RNA genome

Different isolates of enterovirus type 70 (EV70) taken between 1971 and 1981 were studied by molecular biological methods to elucidate their evolutional change. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that only a few isolates had a slight alteration in mobility in some viral proteins. On the contrary, oligonucleotide mapping of virion RNA could clearly delineate the molecular changes among isolates. The number of base changes of isolates became greater as the years elapsed. In addition, the number of base changes among recent isolates from different areas of the world was much greater than those among early isolates. Thus, when the isolates were arranged three-dimensionally according to the number of base changes between each other, the constellation of the strains gave rise to a conical shape. The central axis of the figure was the time of isolation of the strains. The early isolates clustered near the top of the conical figure and the recent isolates tended to disperse divergently at the bottom. The figure indicated that all EV70 strains were derived from a common ancestor, and its top would be the time of emergence of the original strain. It was estimated to be around 1966, 3 years prior to the first epidemic in Accra, Ghana. From the results, it was presumed that EV70 would have emerged at a single focus in Africa as a novel human virus. After having circulated there for a few years, the virus spread to the other parts of the world. Based on the difference in the oligonucleotide spots between the recent isolates and early isolates, the base changes of EV70 that occurred during 10 years was estimated to be 320, about 4% (0.4% a year on the average) of the bases of the total RNA genome.

The nucleotide sequence of 3C proteinase region of the coxsackievirus A24 variant: comparison of the isolates in Taiwan in 1985-1988.

Acute hemorrhagic conjunctivitis caused by coxsackievirus A24 variant (CA24v) first appeared in Taiwan in October 1985, followed by two other sequential epidemics in 1986 and 1988. In order to know the evolutionary relationship of the CA24v strains isolated in Taiwan, we first determined the nucleotide sequence of the 3C proteinase (3Cpro) region of the prototype strain (EH24/70), isolated in Singapore in 1970, by molecular cloning. The nucleotide sequence of the 3Cpro region thus sequenced showed striking homology with polioviruses and coxsackievirus A21.Viral RNA of eight isolates obtained from the three epidemics was reverse transcribed, amplified by the polymerase chain reaction, and cloned into M13 phage for the production of ssDNA for nucleotide sequencing by the dideoxy chain termination method. When the number of nucleotide difference was taken as a genetic distance between isolates, all isolates showed a very similar distance from the EH24/70, the earliest isolate of CA24v, indicating that they evolved at a constant evolutionary rate. Phylogenetic analysis by the unweighted pairwise grouping method of arithmetic average (UPGMA) indicated that the six isolates collected in 1985 and 1986 were closely related, while two 1988 isolates were more distant from them. The branching time between these two groups was estimated to be May 1984, 18 months before the first recognition of the CA24v epidemic in Taiwan.This is the first report of the nucleotide sequence of CA24v genome RNA and of an evolutionary analysis of the virus using the nucleotide sequence.

Evolutionary study on the coxsackievirus A 24 variant causing acute hemorrhagic conjunctivitis by oligonucleotide mapping analysis of RNA genome

SummaryThe evolution of the variant of coxsackievirus A 24 (CA 24 v) which causes acute hemorrhagic conjunctivitis was explored. Using 15 isolates obtained from Southeast Asia during the period 1970–1986, the genetic distance between isolates was estimated from pairwise comparison of nucleotide changes deduced from common spots on oligonucleotide maps of the isolates. From regression analysis of the genetic distance and the time of isolation of the isolates, the evolutionary rate of CA 24 v was estimated to be 3.44×10−4/nucleotide/month. The phylogenetic relationship of these isolates was explored using the neighbor-joining method and the modified unweighted pair group method using arithmetic averages (UPGMA). The phylogenetic tree constructed indicates that CA 24 v appeared from one focal place in July 1968±25 months, very close to the time of the first world epidemic of, then newly recognized, acute hemorrhagic conjunctivitis.

Growth Characteristics of Acute Hemorrhagic Conjunctivitis (AHC) Virus in Monkey Kidney Cells

Acute hemorrhagic conjunctivitis (AHC) virus strains isolated in eight different areas during epidemics of AHC were tested for the reproductive capacity at 33, 37 and 39 degrees. All of the 25 strains tested grew better at 33 degrees but restrictively at 39 degrees. The degree of temperature sensitivity varied slightly from one strain to the other, but generally exceeded that of attenuated poliovirus type 1, strain LSc2ab. Temperature-resistant clones were selected by repeated passages of originally temperature-sensitive prototype strains at supraoptimal temperature. The importance of using a low temperature (32-34 degrees) for isolation of virus from external tissues of the body and for subsequent passages has been emphasized. It was suggested that the relatively low temperature of the conjunctiva has played a role in perpetuating temperature sensitivity of this virus.Monkey kidney cells infected with the J 670/71 strain of acute hemorrhagic conjunctivitis (AHC) virus in the presence of actinomycin D synthesized two species of viral RNA. One species had properties similar to the virion RNA, sedimented in a sucrose gradient with a sedimentation coefficient of 34S, and was sensitive to RNase. The other species sedimented at 18S and was resistant to RNase. The inability of AHC virus to replicate at 39 degrees was attributed to a lack of virus-specific RNA synthesis in infected cells.

Comparison of gelatin particle agglutination and hemagglutination inhibition tests for measles seroepidemiology studies

SummaryThe prevalence of measles antibody in Japan was surveyed with a newly developed gelatin particle agglutination (PA) test, and the results compared with those of the hemagglutination inhibition (HI) test. The two age-distribution curves of the PA antibody-positive rates at ≥1:8 and ≥1:32 were almost the same in all the age groups, except the less-than-1-year-old group for which the rate at ≥1:8 was higher than that at ≥1:32 (p<0.05, χ2 test). In the vaccinated children, all groups older-than-1-year of age had antibody-positive levels of 96% or more. In contrast, in the unvaccinated children, there was a sharp increase in antibody-positive rates between the 1- and 4-year-old groups, indicative that about 80% of the children were infected by wild measles virus at these ages. A significant number of PA antibody-positive specimens were antibody-negative (<1:8) by HI. The percentage of specimens in this category, PA (+) but HI (−), was greatest in infants less than one year old, and least in young children, but it increased with age to 97% of the HI (−) specimens from adults of more than 20 years of age. The PA test therefore detected some measles antibodies that HI could not. This test is simple and useful for making serosurveys in both developed and developing countries.