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Since the onset of 2009 H1N1 influenza outbreak, Chiba prefecture had been taking several measures to mitigate its impact, including phone counseling, supporting outpatient clinics, and coordinating hospitals to prevent the overflow of patients. These measures seem to contribute to the very low mortality caused by pandemic (H1N1) 2009 virus infection during the pandemic in Chiba prefecture. At the same time, it gave us several lessons to prepare for the next--possibly more serious--pandemic. Here, we describe those lessons learnt as well as steps to be taken in the future.

Infectivity of adenovirus type 5 (Ad5) to cells depends primarily on its fiber-mediated binding to the coxsackievirus and adenovirus receptor (CAR) on target cells. Down-regulated CAR expression, often found in human tumors, hampered Ad5-mediated gene transfer. Ad 11 and Ad 35, belonging to a subtype B group, use CD46 as their cellular receptors; accordingly, chimeric Ad5 whose fiber structure was substituted with that of the type 11 or 35 (Ad5/11 or Ad5/35) could infect human cells in a different manner from Ad5. We found that Ad5/35 infected human tumors, including pancreatic and breast cancer, and human fibroblasts better than Ad5 and Ad5/11. Infectivity of Ad5/35 to these cells was correlated with that of Ad5/11, but efficacy of Ad5/35- and Ad5/11-mediated transduction was not directly correlated with the expression level of CD46 in the target cells. Infection of human hepatoma cells with measles virus, whose cellular receptor is CD46, down-regulated the CD46 expression and reduced subsequent infectivity of Ad5/35 but not Ad5/11. Infection of Ad5 suppressed subsequent gene transfer by Ad5 but not by Ad5/11 orAd5/35. Likewise infection of Ad5/35 decreased following gene transduction by Ad5/35 and Ad5/11, but to a lesser extent by Ad5. These data collectively showed that combinatory use of Ad5 and the chimeric Ad enables dual gene transfer into target cells and suggest that infectivity of subtype B Ad does not completely depend on CD46 expression and that other receptors possibly influence the infectivity.

Sapovirus (SV), which causes gastroenteritis in humans, is composed of genetically divergent viruses classified into 5 genogroups. In this study, 2.2-kb nucleotide sequences of the 3' terminus of the genome of 15 SV strains detected in Japan were determined. The 15 SV strains could be classified into four genogroups (GI, GII, GIV and GV), and in two of these, GI and GII, 10 genotypes were identified. The amino acid sequences of the central variable region of the capsid protein showed less than 81% identity when strains belonging to different genotypes were compared. It was therefore supposed that antigenic variety exists between different genotypes. These results will be useful for further genetic and antigenic analyses of SV.

Noroviruses (NVs) are common pathogens that consist of genetically divergent viruses that induce gastroenteritis in humans and animals. Between September 1999 and June 2004, 1,898 samples obtained from patients showing sporadic or outbreak gastroenteritis in Chiba Prefecture, Japan, were tested for NVs by reverse transcription-PCR. NVs were detected in 603 samples. Approximately 80% were positive for genogroup GII, 13% were positive for genogroup GI, and the remaining 7% were positive for both genogroups. Phylogenetic analysis showed that the GI and GII genogroups could be further divided into 13 and 16 genotypes (including new genotypes), respectively. The GII-4 genotype, which included five small genetic clusters (subtypes), was the most common in this study and was detected in approximately 40% of positive samples. The P2 regions of 10 strains belonging to each of the five GII-4 subtypes showed 5 to 18% amino acid diversity. The amino acid substitutions accumulated in the protruding (P) region during the 5-year study period. Our data suggest that highly variable NV strains are circulating in Chiba Prefecture, with a high rate of genetic change observed during the 5-year study period.

Two G12 human rotavirus strains, CP727 and CP1030, were isolated from the respective diarrheic stools of an infant and an adult in Japan. VP7 gene sequences of strains CP727 and CP1030 showed high identity with that of the G12 prototype strain L26, and with those of G12 strains reported recently from Thailand, the United States, and India. VP4 gene sequences of strains CP727 and CP1030 showed the highest identity with those of P[9] rotaviruses. In Northern blot hybridization, strains CP727 and CP1030 were found to be closely related to strain AU-1 (G3P[9]); nine RNA segments hybridized to each other. Moreover, all segments each of the two Japanese G12 strains hybridized to those of the Thai G12 strain T152. These results suggest that Japanese G12 strains detected in this study are reassortants between a L26-like strain and a strain in the AU-1 genogroup. A similar reassortant was found in the Thai G12 strain T152., (Copyright 2004 Wiley-Liss, Inc.)

To investigate the prevalence of bovine papillomavirus (BPV) in bovine papilloma and healthy skin, DNA extracted from teat papillomas and healthy teat skin swabs was analysed by PCR using the primer pairs FAP59/FAP64 and MY09/MY11. Papillomavirus (PV) DNA was detected in all 15 papilloma specimens using FAP59/FAP64 and in 8 of the 15 papilloma specimens using MY09/MY11. In swab samples, 21 and 8 of the 122 samples were PV DNA positive using FAP59/FAP64 and MY09/MY11, respectively. Four BPV types (BPV-1, -3, -5 and -6), two previously identified putative BPV types (BAA1 and -5) and 11 putative new PV types (designated BAPV1 to -10 and BAPV11MY) were found in the 39 PV DNA-positive samples. Amino acid sequence alignments of the putative new PV types with reported BPVs and phylogenetic analyses of the putative new PV types with human and animal PV types showed that BAPV1 to -10 and BAPV11MY are putative new BPV types. These results also showed the genomic diversity and extent of subclinical infection of BPV.

Tsutsugamushi disease has been a notifiable disease in Japan since the implementation of the Infectious Diseases Control Law in April 1999. In order to assess the role of public health laboratories in detecting Tsutsugamushi disease, a questionnaire regarding routine testing of suspected cases of Tsutsugamushi disease was sent to 73 regional public health laboratories (47 prefectural laboratories and 26 municipal laboratories) in July 2001. The response rate was 92% (67/73 laboratories). It was found that most prefectural laboratories are well prepared to routinely receive and test specimens of suspected Tsutsugamushi disease cases. Additionally, we found that some regional public health laboratories are using two or more detection methods to improve the accuracy of their routine tests. In southern Japan. Kawasaki and Kuroki strains, strains endemic to the region, are widely used in addition to Kato, Karp, and Gilliam strains, the standard strains used for serum antibody tests in Japan. For the years 2000 and 2001, we found that for some prefectures, the annual number of cases confirmed by regional public health laboratories was nearly equal to the annual number of cases the prefecture reported to the National Notifiable Diseases Surveillance System. In these prefectures, it appears that an effective communication network has been established between physicians, public health laboratories, and local health centers, ensuring laboratory confirmation and proper notification.

A total of 1,335 archived human sera collected in 1985 from an area in Japan where a tick-borne disease is endemic were examined by indirect immunofluorescence antibody test (IFAT) to estimate seroprevalence against three serologically distinct types of Babesia microti-like parasites; namely, Hobetsu, Kobe, and U.S. types. Eighteen sera (1.3%) were found to be IFAT-positive (titer 1:100-1:6,400), of which 14 and three were ascertained by Western blot analysis to be positive against the Hobetsu and Kobe types, respectively. In addition, four sera showed an IFAT titer of 1:100 against the U.S. type, but they appeared to be false-positive because they were cross-reactive against the Hobetsu and Kobe types, and also because a U.S.-type parasite has not been found in Japan. Our results suggest that human babesiosis in Japan occurred prior to the discovery of the index case in 1999 and that the infections were caused mainly by Hobetsu-type parasites.

Surveillance for scrub typhus was conducted in Japan in 1998 using a questionnaire. A total of 462 cases were reported. Scrub typhus occurred in both the fall and spring in the northern part of Honshu (the main island), and in the fall in the central part of Honshu and on the island of Kyushu. The occurrence of the disease varied with age, gender, and activity. Seventy-six percent of the patients were more than 51 years old, and 36% and 16% of the patients were engaged in farm work and forestry, respectively. Fever, rash, and eschar were detected in 98%, 93%, and 97% of the patients, respectively. Elevated levels of C-reactive protein, aspartate transaminase, and alanine transaminase were detected in 96%, 87%, and 77% of the patients, respectively. Disseminated intravascular coagulation developed in 34 cases and had a unique regional distribution. This study shows the status of scrub typhus in Japan in 1998 and provides important information for diagnosis and prevention.

Clinical features of tsutsugamushi disease (scrub typhus) were analyzed, based on 416 cases reported in Japan in 1998. Three major clinical symptoms: eschar, fever and rash were found in 87%, 98% and 92% of the cases, respectively. Elevated levels of CRP, GOT, GPT and LDH were observed in 96%, 85%, 78% and 91%, respectively. These clinical and laboratory findings were observed in the majority of the cases and considered important for diagnosis. Disseminated intravascular coagulation developed in 21 cases, indicating that scrub typhus can be life threatening. Lymphadenopathy was observed in 51% of the cases. Enlarged lymph nodes were limited to the local sites in 75% of these lymphadenopathy cases and most of these sites were adjacent to eschars. Most eschars were scabbed and located in the abdomen and the lower half of the body, especially the feet. This suggests that these parts are frequently exposed to tsutsugamushi mites. Furthermore, the skin is soft in these parts and covered by cloth. These factors may make it possible for mites to keep biting without being noticed for several hours, long enough for rickettsial transmission. Interestingly, eschar and rash were absent in 14% and 8% of the cases, respectively. This result suggests that the cases without the unique symptoms may have been misdiagnosed as common cold or other febrile illnesses. One hundred and fifty-four suspected cases were not scrub typhus cases by the serological tests. The three major clinical symptoms were present in approximately a half of these negative cases, eschar being observed in approximately 70%. This may suggest the presence of new type of scrub typhus can not be diagnosed by the present laboratory tests. Clinical features of scrub typhus in Japan were well revealed, and information obtained in the present study is useful for improving clinical diagnosis. It should, however, be stressed that there were cases that could not be correctly diagnosed only by the clinical symptoms, suggesting that it is important to improve the serological tests.

Epidemiological aspects of tsutsugamushi disease (scrub typhus) in Japan in 1998 were analyzed using questionnaires. Four hundred and sixteen scrub typhus cases were reported in 24 prefectures in 1998. The annual number of the patients in 1998 was similar to those in the preceding three years. There was no sex difference. The patients at the age of 51 or greater accounted for 72% of the total cases. Patients engaged in farming and forestry accounted for 32% and 14% cases, respectively. Fifty-six, 21 and 19% were reported in Kyusyu, Kanto and Tohoku-Hokuriku districts, respectively, 96% of the total cases being reported in these 3 districts. Most cases were reported from April through June with some from October through December in Tohoku-Hokuriku districts, while most cases were reported from October through December in other districts, including Kyusyu and Kanto districts. Thus, there was a difference in epidemic seasons among the districts. Serotypes of scrub typhus rickettsia were analyzed by serum antibody titers in the Kyusyu district. The novel Kawasaki and Kuroki types were major strains; however, no geographical difference was seen within the Kyusyu district. Interestingly, 24 cases were diagnosed only by the new serotypes not by the classical serotypes (Kato, Karp and Gilliam) in serological tests. This result suggests that further investigations are required to determine the prevalent serotypes in each district and to improve the serological tests. This was the first comprehensive report of epidemiology of scrub typus in entire Japan. Information obtained in the present study provides deep insight into prediction, diagnosis, treatment and prevention of scrub typhus in Japan.

We evaluated the incidence of antibodies against canine distemper (CD) virus, canine parvovirus, Toxoplasma gondii (T. gondii), Aujeszky's disease virus, porcine reproductive and respiratory syndrome virus, Rickettsia tsutsugamushi, Rickettsia japonica, and Coxiella burnetii in 30 raccoon dogs and 5 deer captured in suburban areas in Kanagawa Prefecture. Among the raccoon dogs, anti-T. gondii antibody was detected in 1 animal (3.3%) and anti-CD virus antibody in 9 (30.0%). However, antibodies against the other pathogenic microorganisms were not detected in the raccoon dogs. The deer had none of the examined antibodies.

Primer pairs for PCR were designed from the gene encoding the 17,000-molecular-weight genus-common antigen of Rickettsia japonica, Rickettsia rickettsii, Rickettsia conorii, Rickettsia typhi and Rickettsia prowazekii. Primers R1, R2 were designed for amplifying the genomic DNA from spotted fever group (SFG) rickettsiae and epidemic typhus rickettsiae. Primers Rj5, Rj10 were designed for amplifying the genomic DNA from only R. japonica. Using the primers R1, R2, about a 540-bp fragment was observed by amplifying the genomic DNA from R. japonica, R. rickettsii, R. conorii, Thai tick typhus TT-118, Rickettsia sibirica, Rickettsia montana, Rickettsia askari, R. typhi, R. prowazekii and Katayama strain isolated from the patient infected with SFG rickettsiae. Using the primers Rj5, Rj10, the 357-bp fragment was observed by amplifying the genomic DNA from R. japonica and Katayama strain. Therefore, the Katayama strain was identified to belong to R. japonica. With primers R1, R2 and Rj5, Rj10, 537 bp and 357 bp bands were amplified from blood of the patients infected with SFG rickettsiae in Kanagawa prefecture. These findings indicate that the causative agent of SFG rickettsiosis in these two patients was R. japonica. The ticks, Ixodes ovatus and Haemaphysalis flava, were collected by out field research in Kanagawa prefecture. With primers R1, R2 and Rj5, Rj10, 537 bp and 357 bp were amplified from these ticks. This indicates that I. ovatus and H. flava were the vector of R. japonica in Kanagawa prefecture. Also, with the primers R1, R2, about a 540 bp fragment was amplified but with primers Rj5, Rj10, no fragments were amplified from I. ovatus and H. flava. Therefore, these ticks may have SFG rickettsiae other than R. japonica and epidemic typhus rickettsiae.

Polymerase chain reaction (PCR) with nested primer pairs was used to diagnose Tsutsugamushi disease and identify the Rickettsia tsutsugamushi serotype. The primer pairs used for PCR were designed on the basis of the nucleotide sequence of the gene that encodes the 56-kDa antigen. Five serovariants, the Gilliam, Karp, Kato, Kawasaki, and Kuroki strains of Rickettsia tsutsugamushi were detected and identified by nested PCR. The serotypes of patients registered during 1990 to 1992 in Kanagawa Prefecture were identified by nested PCR. Sixty percentage of patients showed Kawasaki types, 20% Karp types, and 20% Kuroki types. This result suggested that the recent Tsutsugamushi disease were mostly caused by Kawasaki types in Kanagawa Prefecture.

In Chiba Prefecture, the first patient of Tsutsugamushi disease was found in the southern part in the 1950's, but after that no patient was detected until 1982. After 1982, patients have been noticed again, the number of patients has been increasing year by year. The number of serologically confirmed cases was 152 and 157 in 1989 and 1990, respectively with indirect immunofluorescence assay. About 90 percent of the patients were found in November and December, prevalently in the southern part of the prefecture. On the other hand, a few patients were found yearly in a harf of towns and villages in this prefecture. The number of isolated strains of Rickettsia tsutsugamushi from patients was 4, i.e., TR6030, TR6310, TR6311, and TR6324 in 1986 and 3, TR1811, TR1827 and TR1829, in 1991. Six isolates except TR6303 reacted with anti-Kawasaki monoclonal antibody but not with other strain specific monoclonal antibodies. Therefore, these 6 isolates were determined as Kawasaki type strain. An isolate. TR6303, reacted with anti-Kuroki monoclonal antibody at a titer of 1:2560 and anti-Karp monoclonal antibody at a titer of 1:320. This result suggested that the recent Tsutsugamushi disease is mostly caused by Kawasaki types in this prefecture.

In Chiba Prefecture, the first patient of infection with spotted fever group Rickettsia was found in 1987, thereafter nine patients were detected serologically by the end of 1990. Patients were found in the villages of Amatsukominato, Katsuura and Ootaki, which were located in southern part of Chiba Prefecture. The illness occurred from June to October. On the other hand, patients with Rickettsia tsutsugamushi were seen from October to September. Difference of prevalent seasons of these two types of rickettsiosis is important to make a clinical diagnosis and serological identification of this spotted fever group rickettsiosis. Antibody of the patients showed the highest titer to YH strain of Rickettsia and showed high cross-reactivity to other spotted fever group rickettsiae. For the diagnosis of the patient serologically, it was confirmed that any strain of spotted fever group Rickettsia were useful.

Acute- and convalescent-phase sera were obtained from a patient with suspected tsutsugamushi disease in July 1987, in Amatsukominato located in the southeastern area of Chiba Prefecture, and showed negative serologic reactions with Rickettsia tsutsugamushi, while the convalescent-phase serum reacted positively with R. montana and a Japanese isolate of spotted fever group rickettsia at IgM and IgG titers of 1:320 and 1:640, respectively, in the indirect immunofluorescence test. These findings showed that a rickettsiosis of the spotted fever group occurred also in Chiba Prefecture like in the southeastern areas of Shikoku and Kyushu islands of Japan.

Five normal hearing adults were examined for power spectral analysis of AER to 1000-Hz pure tone. Major frequency components of responses to stimuli at 50 dB hearing level exist at 1 to 14 c/s. The power spectra of responses to stimuli at 10 dB hearing level are greater than those of the SBA in the frequency components between 3 and 9 c/s; furthermore, the total power of 3 to 9 c/s indicates a clear difference between the 2. Incidentally, at 512-msec analysis time, the total power of 3 to 9 c/s of the AER are significantly greater than that at 1024-msec analysis time, whereas those of the SBA show little discrepancy at the 2; thus at 512-msec analysis time, the total power of 4 to 9 c/s yields a clear difference between the AER and the SBA responses averaged over 50 stimuli even at 10 dB hearing level.