Japan promotes research related to intractable diseases and financially supports patients with these diseases. Intractable diseases are designated as those that fulfill the following criteria: (1) rarity (affecting less than 0.1% of the population in Japan), (2) unknown etiology, (3) lack of effective treatment, (4) necessity of long-term treatment, and (5) existence of objective diagnostic criteria and not necessarily equal to rare diseases in other countries. The construction of a national database is required to promote research to clarify the pathogenesis of these diseases and to develop pharmaceutical products and medical devices. The Ministry of Health, Labor, and Welfare launched an online registration system in 2001, but many problems associated with gathering and utilizing information on patients with intractable diseases remain. In this paper, we describe the present status of the national registry of designated intractable diseases in Japan and discuss future prospects.

Staphylococcal enterotoxins (SEs) are a common causative agent of food poisoning. Recently, many new SE-like (SEl) toxins have been reported, although the role of SEls in food poisoning remains unclear. In this study, the emetic potentials of SElK, SElL, SElM, SElN, SElO, SElP, and SElQ were assessed using a monkey-feeding assay. All the SEls that were tested induced emetic reactions in monkeys at a dose of 100 mu g/kg, although the numbers of affected monkeys were significantly smaller than the numbers that were affected after consuming SEA or SEB. This result suggests that these new SEs may play some role in staphylococcal food poisoning.

Changing social and environmental factors have been the cause of an increase in the number and variety of animals are being imported into Japan. Moreover, the number of Japanese households are keeping companion animals has also risen. These factors, along with the high density of the Japanese population and the low percentage of registered dogs, have increased the risk of animal-to-human transmission of zoonoses. To control zoonosis outbreaks, the Japanese government has implemented a three-stage approach for the border control of zoonoses and has stipulated the monitoring and reporting of eight companion animal-borne zoonoses under the Rabies Prevention Law and the Infectious Diseases Control Law. The fact that no case of human and animal rabies has been reported over the past 50 years indicates that these measures are highly effective in preventing rabies transmission. Although it is known that the total number of possible companion animal-borne zoonosis outbreaks decreased between 2005 and 2009 when compared with numbers between 2001 and 2004, the number of zoonosis cases that can be attributed to transmission by companion animals remains unclear. Active surveillance should be conducted on a national level to collect the data necessary to determine this number and identify trends in companion-animal transmitted diseases. Using the data collected, regulation systems should be evaluated to determine whether they have met reasonable goals and policy planning conducted for the control of emerging diseases. © 2010 by the authors
licensee MDPI, Basel, Switzerland.

Background: Quantitative survey of research articles, as an application of bibliometrics, is an effective tool for grasping overall trends in various medical research fields. This type of survey has been also applied to infectious disease research; however, previous studies were insufficient as they underestimated articles published in non-English or regional journals.
Methods: Using a combination of Scopus (TM) and PubMed, the databases of scientific literature, and English and non-English keywords directly linked to infectious disease control, we identified international and regional infectious disease journals. In order to ascertain whether the newly selected journals were appropriate to survey a wide range of research articles, we compared the number of original articles and reviews registered in the selected journals to those in the 'Infectious Disease Category' of the Science Citation Index Expanded (TM) (SCI Infectious Disease Category) during 1998-2006. Subsequently, we applied the newly selected journals to survey the number of original articles and reviews originating from 11 Asian countries during the same period.
Results: One hundred journals, written in English or 7 non-English languages, were newly selected as infectious disease journals. The journals published 14,156 original articles and reviews of Asian origin and 118,158 throughout the world, more than those registered in the SCI Infectious Disease Category (4,621 of Asian origin and 66,518 of the world in the category). In Asian trend analysis of the 100 journals, Japan had the highest percentage of original articles and reviews in the area, and no noticeable increase in articles was revealed during the study period. China, India and Taiwan had relatively large numbers and a high increase rate of original articles among Asian countries. When adjusting the publication of original articles according to the country population and the gross domestic product (GDP), Singapore and Taiwan were the most productive.
Conclusion: A survey of 100 selected journals is more sensitive than the SCI Infectious Disease Category from the viewpoint of avoiding underestimating the number of infectious disease research articles of Asian origin. The survey method is applicable to grasp global trends in disease research, although the method may require further development.

Japan is one of the few rabies-free countries. Although 3 imported cases of human rabies were seen in 1970 and 2006, no other cases have been reported for approximate to 50 years. The elimination of rabies in Japan is attributed to not only its geographic isolation but also to effective prevention and control measures, such as registration and vaccination of domestic dogs, required quarantine of susceptible imported animals, and national plans of action based on scientific research. Countermeasures against rabies have been upgraded; an improved management system for domestic dogs under the amended Enforcement Regulations of the Rabies Prevention Law has been in effect since April 2007. The latest regulatory systems for preventing and controlling rabies provide an effective model for elimination of the disease worldwide.

Animal use for scientific purposes, 'animal experimentation', is conducible to scientific development, protecting human health, civilized way of life and so on
however, animal use for human advantage is controversial. Despite of the heated discussion on the rights and wrongs of animal experimentation, animals have been used for research, testing and education in many nations under management systems for the appropriate use of animals from both scientific and ethical viewpoints. Previous efforts to achieve a delicate balance between the scientific rationale and animal protection involve the welfare of animals in scientific procedures. In Japan, each institution where animal experimentation is conducted independently regulates its use of animals under the Ministry's fundamental guidelines, without legal binding force. The management systems are so-called 'self-regulated' or 'self-motivated' management and they are unique, a departure from those in other nations, and fit Japanese religions, traditions and regulatory and administrative frameworks of science and technology. However, several tasks for the management systems for further animal experimentation have become apparent, in response to the new mode of the experimentation developed by the progress of science, technology and animal welfare issues. Here, we describe the current management systems and the future tasks for animal experimentation in Japan. This attempt will provide material for further discussion about animal experimentation and contribute to build an international consensus about animal welfare in scientific procedures. © by São Paulo State University.

We have conducted animal experimentation as a highly effective technique in biological studies. Also in microbiological studies, we have used experimentation to prevent and treat many infectious diseases in humans and animals. In Japan, the 'Law for the Humane Treatment and Management of Animals', which covers the consideration of the three R principles, refinement, replacement and reduction for an international humane approach to animal experimentation came into effect in June 2006. Looking towards the straightforward operation of the law in animal experimentation, three government ministries established new basic guidelines for experimentation performed in their jurisdictional research and testing facilities. For future microbiological studies involving animals in Japan, we need to perform animal experiments according to the basic guidelines in association with overseas management systems. In this report, we discussed essential actions for the management of animal experimentation in microbiological studies in Japan. © 2007 Elsevier Ltd. All rights reserved.

We describe a comprehensive detection system for 18 kinds of classical and newly described staphylococcal superantigenic toxin genes using four sets of multiplex PCR. Superantigenic toxin genotyping of Staphylococcus aureus for 69 food poisoning isolates and 97 healthy human nasal swab isolates revealed 32 superantigenic toxin genotypes and showed that many S. aureus isolates harbored multiple toxin genes. Analysis of the relationship between toxin genotypes and toxin genes encoding profiles of mobile genetic elements suggests its possible role in determining superantigenic toxin genotypes in S. aureus as combinations of toxin gene-encoding mobile genetic elements. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Lactate dehydrogenase-elevating virus (LDV) has a strict species-specificity and can replicate only in a subset of mouse primary macrophages in vitro. Because it is difficult to grow and purify sufficient quantities of LDV virions from the primary macrophages, it has been difficult to further characterize LDV envelope proteins. A few expression systems have been reported for structural analysis of the nonglycosylated envelope protein M/VP-2, however, very few studies of the antigenicity of M/VP-2 have been reported. We cloned and expressed the ORF6 gene, which encodes the M/VP-2, as a fusion protein with a polyhistidine metal-binding tag (6 X His-tag) in Autographa californica nuclear polyhedrosis virus (baculovirus) under the control of the polyhedrin promoter. In Western blotting analysis, the expressed protein was similar in size to the native M/VP-2 plus 6 X His-tag. The usefulness of the baculovirus-expressed LDV ORF6 protein for analysis of the immunogenicity of LDV M/VP-2 was discussed. (C) 2004 Elsevier Ltd. All rights reserved.

We investigated the biological properties of a novel staphylococcal enterotoxin-like putative toxin, staphylococcal enterotoxin-like toxin type R (SEIR). Major histocompatibility complex class 11 molecules were required for T-cell stimulation by SEIR. SEIR stimulated T cells bearing receptors Vbeta 3, 11, 12, 13.2, and 14. These results suggested that SEIR acts as a superantigen.

To develop a diagnostic tool to identify Mycoplasma pulmonis (M. pulmonis) in clinical isolates, we developed a polymerase chain reaction (PCR) assay using primers specific for the 16S-23S rRNA intergenic spacer region (SR) of M. pulmonis. One pair of PCR primers reacted specifically with two reference strains of M. pulmonis tested and seven samples isolated from naturally infected rats. The primer pair did not produce PCR products of the correct size from any other rodent or human mycoplasmas or cellular DNA from rodent lungs. Specificity of the PCR assay was confirmed by Southern blotting with probe specific for the SR of M. pulmonis. The PCR assay for detection of M. pulmonis established in this study is suitable for diagnosis of M. pulmonis infection in clinical cases. (C) 2003 Elsevier Ltd. All rights reserved.

Lactate dehydrogenase-elevating virus (LDV) has a strict species-speciticity. Because only a subset of mouse primary macrophages have been identified that can support LDV replication in vitro, the precise molecular mechanism of viral entry and replication remains unclear. To analyze the LDV envelope proteins, which probably mediate viral attachment to the host cell, we developed a mammalian system for stable co-expression of LDV open reading frame (ORF) 5- and ORF 6-encoded proteins (ORF 5 and ORF 6 proteins), which correspond to envelope VP-3 and M/VP-2, respectively, and compared these expressed proteins to the native ones. Western blotting analysis combined with N-glycanase digestion revealed that ORF 5 and ORF 6 proteins were similar in size to native VP-3 and M/VP-2, and that ORF 5 protein was N-glycosylated, like the native VP-3. Immunofluorescence microscopy revealed that both ORF 5 and ORF 6 proteins were distributed throughout the cytoplasm and were colocalized in most cells. Moreover, ORF 5 protein was localized both in the perinuclear region and the Golgi complex and transported to the cell surface. This mammalian expression system in which the exogenously expressed proteins closely resemble the native proteins will provide the experimental basis for further studies of the interactions between LDV envelope proteins and host cells. (C) 2003 Elsevier Ltd. All rights reserved.

The elucidation of the antigenic structure of the envelope proteins of Arteriviridae which includes lactate dehydrogenase-elevating virus (LDV) will provide further understanding of a mechanism of strict host cell specificity. To analyze the linkage between LDV envelope proteins, M/VP-2 and VP-3, which may play an important role in vital infectivity, We generated specific antibody against M/VP-2 that has not been reported in previous studies. A synthetic polypeptide corresponding to the C-terminal region of LDV strain C (LDV-C) ORF6, which encodes M/VP-2, was chemically synthesized and coupled to keyhole limpet hemocyanin (KLH). The peptide was immunogenic in rabbits and induced antibody specific for viral protein. Western blotting and immunofluorescence analysis of virion M/VP-2 in infected macrophages showed that the antibody was able to react specifically with authentic virion protein. The immunoreactive antibody against LDV M/VP-2 described in this study will be useful for further studies of the specific roles of the envelope proteins in arterivirus assembly and infectivity. (C) 2004 Elsevier Ltd. All rights reserved.

We identified and characterized a novel staphylococcal enterotoxin-like putative toxin, which is named SER. Nucleotide sequencing analysis of the ser gene revealed that ser was most closely related to the seg gene. The ser gene product, SER, was successfully expressed as a recombinant protein in an Escherichia coli expression system, and recombinant SER (rSER) showed significant T-cell stimulation activity. The SER production in ser-harboring Staphylococcus aureus strains was confirmed by Western blot analysis using anti-rSER antibody. Moreover, ser was seen to be encoded by at least two types of plasmids. In particular, one kind of plasmid encoding the ser gene has been known as a sed- and sej-carrying pIB485-related plasmid.

Thirty-four suspected rabid brain samples from 2 humans, 24 dogs, 4 cats, 2 mongooses, 1 jackal and 1 water buffalo were collected in 1995-1996 in Sri Lanka. Total RNA,vas extracted directly from brain suspensions and examined using a one-step reverse transcription-polymerase chain reaction (RT-PCR) for the rabies virus nucleoprotein (N) gene, Twenty-eight samples were found positive for the virus N gene by RT-PCR and also for the virus antigens by fluorescent antibody (FA) test. Rabies virus isolates obtained from different animal species in different regions of Sri Lanka were genetically homogenous. Sequences of 203 nucleotides (nt)-long RT-PCR products obtained from 16 of 27 samples were found identical. Sequences of 1350 nt of N genes of 14 RT-PCR products were determined. The Sri Lanka isolates under study formed a specific cluster that included also an earlier isolate from India but did not include the known isolates from China, Thailand, Malaysia, Israel, Iran, Oman, Saudi Arabia, Russia, Nepal, Philippines, Japan and from several other countries. These results suggest that one type of rabies virus is circulating among human, dog, cat, mongoose, jackal and water buffalo living near Colombo City and in other five remote regions in Sri Lanka.

To investigate whether there is an epidemiological correlation between Borna disease virus (BDV) infection and human neuropsychiatric diseases, we established a reverse-type sandwich enzyme-linked immunosorbent assay (RS-ELISA) for detecting specific antibodies to BDV. In this assay, microplate wells were coated dispersely with BDV p40 antigen, followed by the addition of test samples at a low dilution and then the biotinylated p40. A preformed complex of streptavidin and horseradish peroxidase-conjugated biotin and an enzyme substrate were used to measure the captured biotinylated p40. Theoretically, RS-ELISA should specifically detect anti-BDV antibodies without nonspecific signals; such signals possibly occur in conventional serological assays. Additionally, the RS-ELISA could be applied under the same protocols to test samples from a variety of animals. By using anti-BDV rat and rabbit sera, the assay was standardized so that it had high specificity and sensitivity. When we used the RS-ELISA to determine the presence of anti-BDV antibodies in plasma from 70 patients with chronic schizophrenia as well as 40 healthy individuals in the Tokyo area of Japan, no plasma sample was found to possess specific antibodies to BDV p40, indicating no association between BDV infection and the disease in our testing population. A negative reaction was also shown for the sera that had previously been judged to be seropositive for BDV by an immunofluorescence or immunoblot test. These findings suggested that false-positive cases of infection due to nonspecific reactions may be included in previous seroepidemiological information with regard to BDV.

Wild measles virus isolated in a marmoset lymphoblastoid B95a cell line induced rashes and Koplik's spots when inoculated parenterally in cynomolgus and squirrel monkeys. Marked leukopenia associated with transient decrease in the CD4(+)-to-CD8(+) T-cell ratio also was induced, Virus growth, as well as histologic lesions of necrosis and giant cell formation, was observed in the lymphoid tissues. Thus clinical signs of acute measles were successfully induced in monkeys by inoculation with cell-culture grown measles virus, These nonhuman primate models of measles will be useful for study of the pathogenesis of acute measles virus infection in terms of generalized clinical signs of disease, leukopenia, and changes in the lymphocyte subsets.

We determined the nucleotide sequences of the coding region for the M gene in seven strains of measles virus (MV) that were isolated in Japan between 1984 and 1993. The mutation found among the seven differed from those of laboratory strains, Many of these mutations were the same as those that are characteristic of SSPE viruses, Thus, we suggest that the mutations that have been considered specific to SSPE virus are in fact consensus among prevailing MV.

Measles and rubella skin lesions were immunocytochemically compared by the avidin-biotin-peroxidase complex method for detecting viral antigens. Cryostat sections of biopsied specimens of the skin were stained with mouse monoclonal antibodies to P protein of measles virus and to El protein of rubella virus. The measles virus antigen was concentrated in the corneal layer and the keratinocytes of the epidermis and in the surface part of the dermis in the biopsy secimens taken within 6 days after the onset of rash. On the other hand, the rubella virus antigen was dispersed in all parts of the dermis and the subcutaneous layer but not in the epidermis in the biopsy specimens taken within 2 days after the onset of rash. The differences in the distribution and density of the viral antigen and in the times of its detection suggest distinct patterns of spread of infection with each virus in the skin.

A cloned virus of murine coronavirus JHMV, cl-2, was shown to be highly neurovirulent for rats in comparison with other JHMV variants. We have isolated cl-2-derived Variant viruses resistant to neutralization by monoclonal antibodies (MAbs) specific for the spike (S) protein of cl-2. The variants MM6 and MM13, selected by the MAbs specific for the JHMV S protein, were revealed to have a point mutation located within the N-terminal 100 amino acids (aa) of the S1 protein. The variants MM56, MM85, and MM78, selected by MAbs specific for the larger S protein of JHMV, were shown to have a deletion composed of about 150 aa located in the middle of the S1 subunit (MM56 and MM85) or one amino acid deletion, aspartic acid at number 543 from the N-terminus of the St (MM78). These five MAb-resistant variants were not different from cl-2 in growth pattern on cultured DBT cells. MM6 and MM13 were shown to be highly neurovirulent for Ii-week-old Lewis rats, growing to high titers in the brain and causing as high an incidence of neurological disease and death as the parental cl-2. in contrast, MM56 and MM85 were nonneurovirulent for rats. They did not cause any central nervous system disorders nor did they multiply in the rat brains. MM78 showed intermediate neurovirulence as well as intermediate growth potential in the rat brain. However, there was no apparent difference in neurovirulence between the parental and the MAb-resistant variants for mice; all of these viruses showed high neurovirulence for mice. These results suggest that the domain composed of about 150 aa in the middle of the S1 is critical for high neurovirulence of JHMV for rats. Furthermore, it is suggested that the neurovirulence of cl-2 for mice is controlled by a different viral factor. (C) 1995 Academic Press, Inc.

Seven measles virus (MV) strains were isolated from children who developed clinical signs of fever and rash 3-9 days after measles vaccination. The nucleotide sequence of the H gene, the molecular size of the H protein, the haemadsorption activity on African green monkey red blood cells, and antigenicity as determined by virus neutralization revealed that one strain was of the vaccine type and the remaining six were the wild virus type, Isolation of the virus directly from patients suspected of a vaccine-induced side-reaction and subsequent characterization of such isolated virus may be useful in differentiation between vaccine-induced side-reactions and natural measles.

We have studied the neurovirulence for rats of the MAb-resistant variants isolated from a highly neurovirulent JHMV, c1-2. The variants, MM6 and MM13, with point mutation located within the N terminal 100 amino acids (aa) of the S1 protein showed no alteration in neurovirulence in comparison with c1-2, showing high neurovirulence. The variants, MM65 and MM85, with a deletion composed of about 150 aa located in the middle of the S1 subunit were revealed to be non-neurovirulent. A variant MM78 with one aa deletion, asparagic acid at number 543 from the N terminus of the S1, was shown to be low-virulence. The neurovirulence of these viruses paralleled with the viral growth potential in the rat brain. However, all of these variants as well as parental c1-2 showed high neurovirulence for mice. These results suggest that the domain composed of about 150 aa in the middle of the S1 is critical for high-neurovirulence of JHMV for rats.

Pseudorabies virus (PRV) was inoculated intraocularly into BALB c mice, and the distribution pattern of cells positive for several neurotransmitters and viral antigens in the eyeball, trigeminal nerve ganglia, and superior cervical ganglia was examined immunohistochemically to clarify the neural route of the virus spread. In the eyeball, substance P (SP)- and calcitonin gene-related peptide (CGRP)-positive cells were detected in the ipsilateral iris and ciliary body, neuropeptide tyrosine (NPY)-positive cells were detected in the choloid membrane, and tyrosine hydroxylase (TH)-positive cells were detected in the ipsilateral inner nuclear layer of the retina
all these cells contained viral antigens. In the superior cervical ganglia, viral antigen-positive cells containing TH or NPY were found at bilateral sites. In the trigeminal nerve ganglia, viral antigen-positive cells containing SP or CGRP were found at bilateral sites. These findings indicated that the SP- and CGRP-positive ganglion cells of the trigeminal nerve ganglia innervating the iris or ciliary body, and the NPY-positive ganglion cells of the superior cervical ganglia innervating the iris, ciliary body, and choroid membrane served as the route for the virus spread. These findings also suggested that dopaminergic neurons, such as the TH-positive retinal cells and TH-positive ganglion cells of the superior cervical ganglia, served as the route for virus spread. © 1995.

To investigate the routes of neural spread of pseudorabies (Aujeszky's disease) virus (PRV), the effects of chemical sympathectomy by 6-hydroxydopamine (6-OHDA) on clinical signs and viral spread in mice inoculated intraocularly with PRV were examined. Similar to non-treated mice, the treated mice developed pruritus as a major clinical sign, followed by peracute death, but the time of death tended to be slightly delayed in about half of the treated mice. On immunohistological examination, viral antigens in treated mice were found to be markedly reduced in all the ipsilateral retinae; they were detected diffusely in the forebrain area with a localization in the mammillary area. In the treated mice, viral antigens were also reduced in the ipsilateral trigeminal nerve ganglia as well as in its central nuclei. These findings indicate that both the sympathetic nervous system and the trigeminal nervous system play an important role in the neural spread of PRV. Possible involvement of the dopaminergic nervous system, particularly in the eye, as the main site of viral growth was discussed.

Mechanisms of postinfectious pruritus and peracute death in mice by pseudorabies virus (PRV) were investigated by inoculating the Yamagata-S81 strain of PRV peripherally or intracerebrally into 4-week-old ICR and BALB/c mice. Clinical signs developed most rapidly in mice inoculated intracerebrally, with intermediate speed in mice inoculated intraocularly, and slowly in mice inoculated subcutaneously. Since intraocularly inoculated mice showed an acute reaction and this is considered a peripheral route, the distribution of viral antigens in the nervous system of intraocularly inoculated mice was examined immunohistologically. Viral antigens were mainly detected along the trigeminal and the oculomotor nerves, but neither necrosis nor an inflammatory response was observed in these areas. The infectious virus was efficiently recovered from the viral antigen-positive tissues. In the pruritic skin lesions, viral antigens were not observed. These findings indicate that the main route of viral spread in intraocularly inoculated mice is the trigeminal and oculomotor nerves and that the virus in the trigeminal nerve may trigger pruritus.