Precision measurement of neutrino oscillation parameters with KamLAND.

The KamLAND experiment has determined a precise value for the neutrino oscillation parameter Deltam21(2) and stringent constraints on theta12. The exposure to nuclear reactor antineutrinos is increased almost fourfold over previous results to 2.44 x 10(32) proton yr due to longer livetime and an enlarged fiducial volume. An undistorted reactor nu[over]e energy spectrum is now rejected at >5sigma. Analysis of the reactor spectrum above the inverse beta decay energy threshold, and including geoneutrinos, gives a best fit at Deltam21(2)=7.58(-0.13)(+0.14)(stat) -0.15+0.15(syst) x 10(-5) eV2 and tan2theta12=0.56(-0.07)+0.10(stat) -0.06+0.10(syst). Local Deltachi2 minima at higher and lower Deltam21(2) are disfavored at >4sigma. Combining with solar neutrino data, we obtain Deltam21(2)=7.59(-0.21)+0.21 x 10(-5) eV2 and tan2theta12=0.47(-0.05)+0.06.

Effect of herpes simplex virus on the DNA of human papillomavirus 18.

Some types of human papillomaviruses (HPV) appear to be associated with carcinoma of the cervix or other tissues, but patients infected with HPV do not necessarily develop carcinoma. Some epidemiological studies of risk factors for cervical carcinoma have indicated the involvement of herpes simplex virus (HSV). To study the effect of HSV on the genome of HPV, total DNAs were extracted and analyzed after HeLa cells, or A431 cells, transiently transfected with HPV18 DNA, were infected with HSV-1 or -2 for 24 hours. In HeLa cells, integrated HPV18 DNA was amplified almost threefold. In A431 cells, HPV 18 DNA fragments, sensitive to the restriction enzyme Mbo I, indicated newly replicated DNA. Replication intermediates were detected when the DNA was resolved by two-dimensional gel electrophoresis. This study showed that HSV caused some amplification of HPV and indicated the possibility of HSV involved in the integration and amplification of HPV in host cells.

Cloning and expression of a putative alcohol dehydrogenase gene of Entamoeba histolytica and its application to immunological examination.

To clone and express the genes encoding major antigens of Entamoeba histolytica, we constructed a lambda gt11 cDNA library for E. histolytica HM1:IMSS and screened it with pooled sera from patients with amoebiasis. A 1,223-bp cDNA was cloned (clone 1223), and its nucleotide sequence was determined. The amino acid sequence predicted to be encoded by the open reading frame of clone 1223 consisted of 396 residues and showed 32.5 and 32.3% homology to the NADH-dependent butanol dehydrogenases I and II (bdhA and bdhB) of Clostridium acetobutylicum, respectively. In addition, 29 of the 34 consensus positions of bdhA and bdhB were also well conserved in clone 1223. The recombinant protein expressed from clone 1223 had an estimated molecular mass of 43.5 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenicity and specificity of the recombinant protein were evaluated by an enzyme-linked immunosorbent assay using sera obtained from two clinical groups of patients with amoebiasis and a group of healthy controls. The recombinant protein had potent and specific antigenicity. In all, 53 serum samples (88.3%) from 60 patients with amoebiasis were positive for immunoglobulin G antibody against the recombinant protein, with a mean optical density value of 0.42. In contrast, 53 of 54 healthy control serum samples were negative, with only 1 positive serum sample showing the lower optical density value. These results suggested that clone 1223 is promising in terms of providing a useful antigen for the accurate serodiagnosis of amoebiasis and that the gene encodes a putative alcohol dehydrogenase of E. histolytica.

Reverse transcription-polymerase chain reaction detection and sequence analysis of small round-structured viruses in Japan.

Between 1985 and 1995, mass outbreaks of acute gastroenteritis caused by small round-structured virus (SRSV), occurred in eight prefectures in Japan. Fecal samples from 59 patients ill during these outbreaks were recently examined in our laboratory by electron microscopy (EM) and by reverse transcription-polymerase chain reaction (RT-PCR). For RT-PCR, we prepared two sets of primers, a set corresponding to the polymerase region of open reading frame 1 (ORF-1) and a set corresponding to the capsid region of ORF-2 of Norwalk virus (NV). The SRSV nucleic acid detection rate with these primers was more than double that achieved with EM. Most samples found by EM to contain virus particles were also positive by PCR. When the two sets of primers were used separately, the virus detection rate differed depending on the primer used, suggesting that the viral strains examined were not genetically not homogeneous. We then selected nine strains of the virus, cloned their PCR products and analyzed their base sequences. The base sequences of these strains were compared with those of reference strains including prototype NV and Snow Mountain agent (SMA). This comparison yielded the following findings: (1) SRSVs that cause mass outbreaks of gastroenteritis in Japan are genetically variable; (2) SRSV strains that are genetically similar to SMA and SRSV-OTH 25/89/J(OTH25) are dominant in Japan, but strains similar to NV are also present in this country; and (3) a strain (MI1/94) which is genetically identical to Southampton virus (SHV) was detected. Detection of SRSV using sensitive RT-PCR and analysis of the sequences of the amplification products seems to provide a useful means of studying the molecular epidemiology of SRSV.

Nucleotide sequence analysis of recent epidemic strains of enterovirus 70.

Nucleotide sequences of the genome of enterovirus 70 (EV70) isolated in Osaka in 1993 were determined, and compared with those of the past epidemic strains. Nucleotide substitution rates in 332 bp, 174 bp, and 178 bp of the genes encoding viral protein (VP)1, VP2, or VP3 were 9.0, 7.5, and 5.6% between Kumoi-2/93 and J670/71, respectively. Likewise, the putative amino acid substitution rates were 1.8, 0, and 0%. It seems that the epidemic strains of EV70 in Japan have been evolving at a constant high nucleotide substitution rate but almost all the substitutions were synonymous.

A large outbreak of acute gastroenteritis associated with astrovirus among students and teachers in Osaka, Japan.

In June 1991, a large outbreak of acute nonbacterial gastroenteritis occurred among students and teachers at 10 primary and 4 junior high schools in Katano City, Osaka, Japan. The outbreak affected > 4700 persons, lasted 5 days, and was believed to have been linked to contaminated food from a common supplier. Astrovirus, identified as the etiologic agent, was detected by direct electron microscopy in 10 of 38 fecal samples obtained from patients with diarrhea. Detection was confirmed by solid-phase immune electron microscopy (IEM), EIA, reverse transcription-polymerase chain reaction, and virus isolation in CaCo-2 cells. Several patients who had astrovirus in their stool also demonstrated a significant antibody response to a reference strain of astrovirus by IEM and EIA and to their own isolate by IEM. Astrovirus can be an important agent of epidemic acute nonbacterial gastroenteritis in school-aged children and adults in Japan.

An occurrence of diarrheal cases associated with group C rotavirus in adults.

Six of the 23 college students who joined a group trip in February of 1991 developed acute nonbacterial gastroenteritis with severe diarrhea. The causal agent was identified as group C rotaviruses by electron microscopy (EM), immune-EM (IEM) and the molecular examinations including polyacrylamide gel electrophoresis (PAGE) and polymerase chain reaction (PCR) on virus particles detected in the extract of watery fecal specimens of the patients. The patients positive for virus isolation showed significant increase in IEM antibody to the isolated virus in their paired sera. These findings suggest that the group C rotavirus is an important etiological agent of diarrhea and may also cause serious food-borne diarrheal disease in adults.

Demonstration of low molecular weight polypeptides associated with small, round-structured viruses by western immunoblot analysis.

Small, round-structured viruses (SRSV) were detected in 14 of 300 fecal specimens obtained from patients with acute gastroenteritis by electron microscopy. These SRSV strains were morphologically indistinguishable from one another. While 11 of these strains had a single usual major structural protein with molecular weight of 63,000 (63K) daltons (p63), interestingly, three strains possessed a single major structural protein with molecular weight of 33K daltons (p33). Treatments of p63-SRSV with proteolytic enzymes or denaturating reagents did not affect the molecular weight of p63, and the p33 was not detectable by Western immunoblot in the ultracentrifugal supernatant of the p63-SRSV suspension. These results suggest that the p33 is neither a definitive subunit of p63 nor disintegrated component derived from the p63-SRSV but a novel polypeptide of SRSV. Immune electron microscopy and Western immunoblot analyses indicated that p63- and p33-SRSVs may share an antigenic determinant(s).

Calculation of efficacy of acellular pertussis vaccine from the number of pertussis patients and vaccinees in three areas.

A study is presented of the efficacy of acellular pertussis vaccine based on the number of pertussis patients and its relationship to vaccination rate in three demographic areas of Osaka Prefecture, Japan. In three separate areas within Osaka Prefecture, the pertussis vaccine is given to children three to six months of age, and in the other two it is given to children two years of age or older. Use of a mathematical method to correct the reported number of pertussis patients per 100,000 population with a constant obtained from analysis of the reported number of exanthem subitum cases allowed us to compare the number of pertussis patients in the three areas with each other. Vaccination rate by age and incidence of pertussis in each city revealed the efficacy of acellular pertussis vaccine to be 94.8%.

Serial observations of chronic rotavirus infection in an immunodeficient child.

Chronic rotavirus infection of an infant with severe combined immunodeficiency (SCID) was studied by virological examinations in association with long-term observation of his symptoms and immune status. During eleven months of hospitalization, the patient was suffering from incurable severe diarrhea with persisting excretion of rotaviruses detected by electron microscopy and the reversed-passive hemagglutination (R-PHA) test and had transient hepatitis symptom despite multiple administrations of human gammaglobulin and high calorie fluids. The detected viruses were morphologically recognized as rotavirus with double capsid structure. Polyacrylamide gel electrophoretic (PAGE) analysis of their genomic RNAs showed the long electropherotype of group A virus with abnormal migration profiles changing considerably from the early to the late phase of illness: (1) The 11th segment became undetectable; (2) the molecular weight of the 6th segment slightly increased; (3) seven to fourteen extra segments appeared; and (4) PAGE patterns of viral genomic RNAs changed every three or four months. These findings suggest that chronic infection with rotavirus accompanied the generation of extra viral genomic segments and their unusual assortments in an immunodeficient host.

Antigenic and genetic analyses of human rotavirus with dual subgroup specificity.

In our previous study (S. Urasawa, T. Urasawa, K. Taniguchi, F. Wakasugi, N. Kobayashi, S. Chiba, N. Sakurada, S. Morita, O. Morita, M. Tokieda, T. Kawamoto, K. Minekawa, and M. Oseto, J. Infect. Dis. 160:44-51, 1989) of antigenic characterization of about 300 human rotavirus (HRV) isolates collected at different localities in Japan, we found 4 HRV isolates having unique antigenic and genetic constructions. The four strains possessed both subgroup I and subgroup II antigens, serotype 3 antigen, and a long RNA electropherotype. The reactivity pattern of these four HRV isolates with three monoclonal antibodies (MAbs) directed to an outer capsid protein, VP4, and with one MAb directed to an inner capsid protein, VP2, was clearly different from those of usual subgroup II HRVs having serotype 1, serotype 3, or serotype 4 specificity and a long RNA pattern, whereas their reactivity pattern was similar to that of strain K8 (subgroup II, serotype 1), which possessed unique VP4 and VP2 proteins. RNA-RNA cross-hybridization analysis indicated that while the four isolates were genetically distinct from the two genetic groups of HRV reported previously, i.e., the Wa family (strains KU, S3, and YO) and the DS-1 family (strain S2), they were closely related to strain K8, a strain having unique antigenic and genetic properties (K. Taniguchi, K. Nishikawa, T. Urasawa, S. Urasawa, K. Midthun, A. Z. Kapikian, and M. Gorziglia, J. Virol. 63:4101-4106, 1989).

Removal of pathogenic human viruses by insoluble pyridinium-type resin.

Cross-linked poly(N-benzyl-4-vinylpyridinium bromide) (BVP resin) was found to be very efficient in removing pathogenic human viruses from aqueous solution. In batch removal experiments using 50 g/l of BVP resin at 35 degrees C, the level of infectivity in suspensions of enterovirus, herpes simplex virus, poliovirus, and human immunodeficiency virus was reduced 1000-100,000 fold during a 2 h period. Those of coxsackievirus and echovirus were reduced 60-600 fold during 1 h contact. The haemagglutination titres of solutions of human rotavirus, influenza virus, human adenovirus, and Japanese encephalitis virus were reduced 16-256 fold during 30 min of contact. In removal experiments by a continuous flow column method for poliovirus, enterovirus, and coxsackievirus with initial infectivities of less than 10(5)/ml, the infectivity of these viruses was no longer detectable in the effluent solution. For poliovirus, coxsackievirus, and echovirus with initial infectivities higher than 10(6), 99.8-99.9998% of the input viruses was removed as indicated by the reduction of infectivity.

Human papillomavirus infection of the uterine cervix analyzed by nonisotopic in situ hybridization.

Some types of human papillomaviruses (HPVs) have been suggested to be strongly related to uterine cervical carcinoma. An attempt to detect these in formalin-fixed, paraffin-embedded sections was made by either immunohistochemical or by in situ hybridization. Anticapsid protein of bovine papillomavirus antibody labeled with peroxidase was used for immunohistochemistry, and biotin was used instead of radioisotopes to label probes for in situ hybridization, which resulted in low background and a rapid procedure. Condylomatous changes were stained immunochemically with this antibody even in invasive carcinoma, whereas the carcinoma itself was not stained. Direct correlation was demonstrated by in situ hybridization between the HPV genome and histopathological structure, which was impossible by Southern or dot hybridization. HPV DNAs were detected in the nuclei of koilocytes and dyskeratinocytes of condylomata and dysplasias. Furthermore, hybridization signals of HPV DNAs in basal and parabasal cells suggested that HPV infection had already begun in the basal cells. In the case of malignant neoplasia accompanied by dysplasia, the same type of HPV was detected both in the malignant neoplasia and accompanying dysplasia. In one case of intraepithelial carcinoma, the very small focus of carcinoma just arisen in the cells of dysplasia was identified, and both were positive for HPV 18. This fact supports the suggestion that the carcinoma arises in dysplasia. Invasive carcinomas were classified further into keratinized, large-cell nonkeratinized, and small-cell nonkeratinized types, and the positive frequency for HPV 16 decreased as the differentiation of the carcinoma decreased. In the case of keratinized type of invasive carcinoma, strong hybridization signals were prominent around the malignant pearl formation.

[A rapid method for serodiagnosis of Japanese encephalitis using persistently infected C6/J-121 cells].

The usefulness of persistently infected C6/36 (C6/J-121) cells with Japanese encephalitis virus (JEV) for a rapid serodiagnostic test was examined with the sera of men, swine and laboratory animals by indirect immunofluorescent antibody (IFA) technique. Detection of specific antibodies was completed within 3 to 5 hours. Nonspecific fluorescence frequently observed in other IFA tests was few. The sensitivity of antibody detection and the serological specificity of IFA (IgG) were similar to those of hemagglutination-inhibition (HI) test. For the detection of IgM antibodies in sera of JE-patients and naturally JEV infected swine, this method was found to be simpler and more sensitive and rapid than the conventional HI test which required 2-ME treatment of the sera.

Survival and disinfection of adenovirus type 19 and enterovirus 70 in ophthalmic practice.

In an experimental model designed to simulate actual clinical situations, the risk period of adenovirus type 19 (AdV-19) and enterovirus 70 (EV-70) transmission by contaminated instruments has been investigated, as well as the effective methods of disinfection against these viruses. No EV-70 could be detected 6 hours after inoculation on applanation tips contaminated with viruses and naturally dried, indicating that drying the instruments and keeping them in a moisture-free environment is a convenient disinfection procedure against EV-70. In contrast, AdV-19 maintained its initial infectious level for 11 days on contaminated and subsequently dried tips. Both viruses when placed in eyedrop solutions at room temperature sustained their infectivity for more than 20 days. It was found that heating at 90 degrees C for 5 seconds is the most simple and effective disinfection against AdV-19 and EV-70. AdV-19 was about 60 times more resistant to ultraviolet irradiation than EV-70.

Three-year survey of the epidemiology of rotavirus, enteric adenovirus, and some small spherical viruses including "Osaka-agent" associated with infantile diarrhea.

Studies were made by electron microscopy (EM) on the viruses associated with diarrhea of outpatients at a pediatric clinic in Osaka Prefecture during the three year period from 1980 through 1982. The viruses detected by EM by negative staining with phosphotungstic acid (PTA) were classified morphologically into 6 groups: rotavirus, adenovirus and four kinds of small spherical viruses, calicivirus, astrovirus, picornavirus/parvovirus (P/P)-like agent and Osaka-agent. Osaka-agent seems to be a newly identified small virus. It is 35-40 nm in diameter with a fringe of spike-like structures on its surface. Viruses were detected in 181 of the 395 cases of diarrhea (45.8%). Rotavirus was detected in 122 (30.9%) of the total cases and in 67.4% of the virus-positive cases, while other viruses were detected in 15% of the total cases; adenovirus in 23 (6%) and small agents in 36 (9%). Rotavirus infection showed a distinctive seasonal variation, being mainly restricted to cooler months, but infections with other viruses did not show any seasonal variation. The age distribution of patients suggested that infants of 0 to 2 years old are very susceptible to all viruses. Attempts to cultivate these viruses in vitro were successful with only two isolates of adenovirus type 5.

Studies on the mechanism of antibody-mediated enhancement of Getah virus infectivity.

Inoculation on BHK-21 cells with Getah virus sensitized with hyperimmune homologous mouse antiserum resulted in higher infective titers than those obtained with non-sensitized control virus. This phenomenon was not observed with Vero cells. Experiments were carried out on the mechanism of this enhancement with the following results. When BHK-21 cells were pretreated with a mixture of UV-irradiated virus and antiserum before inoculation, the enhancement of infectivity of Getah virus was markedly decreased. IgG antibody against Getah virus which had been digested with 1--2% of pepsin did not show any enhancing activity. Sensitized sheep erythrocytes adhered to monolayered cultures of BHK-21 cells. These results indicate that the appearance of enhancing activity of a complex of the virus and antibody is closely related with the existence of a receptor for the Fc part of IgG on ordinary tissue culture cells, BHK-21 cells.

Calicivirus detected in outbreaks of acute gastroenteritis in school children.

Outbreaks of acute gastroenteritis occurred in AK and N Primary Schools of Osaka Prefecture early in March and early in November, respectively, 1977. Epidemiological surveys were carried out in both schools with the following results. The morbidity rates were 21.8% (AK) and 8.5% (N) among all pupils, and 41.7% (AK) and 36.8% (N) in special age groups. The main clinical symptoms were abdominal pain (77.2-91.3%), diarrhea (38.4-41.3%) and vomiting (6.5-41.6%). Electron microscopic observations of negatively stained specimens showed that five of 15 fecal extracts obtained from patients in the two schools contained virus particles of 35-40 nm diameter with some black hollows on the surface of complete particles and ten projection-like structures at the edge of empty shells. These morphological characteristics resemble those of calicivirus particles. Immune electron microscopic examination suggested that outbreak in AK School was associated with this virus. Attempts to cultivate the viruses in various cultured cells and in suckling mice were unsuccessful.

Adenovirus type 10 keratoconjunctivitis with increased intraocular pressure.

An 18-year-old man, with serologically diagnosed adenovirus type 10 infection, had keratoconjunctivouveitis of both eyes after pharyngitis with transient increased intraocular pressure. Increased intraocular pressure, ranging from 46 to 28 mm Hg during medical therapy, lasted for about ten days in the early stage of the disease. Many punctate corneal opacities developed bilaterally two weeks later and disappeared within ten months. Analysis of the clinical findings demonstrated that the increased intraocular pressure may have been secondary glaucoma due to keratoconjunctivitis caused by adenovirus infection.