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Abstract: Hospital-wide screenings for coronavirus disease (COVID-19) are important to identify healthcare workers at risk of exposure. However, the currently available diagnostic tests are expensive or only identify past infection. Therefore, this single-center observational study aimed to assess the positivity rate of hospital-wide antigen screening tests for COVID-19 and evaluate clinical factors associated with antigen positivity during a COVID-19 institutional outbreak in Sapporo, Japan.We analyzed the data of 1615 employees who underwent salivary or nasal swab antigen tests on November 18, 2020, to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Laboratory confirmation using reverse transcriptase polymerase chain reaction was performed for those with positive viral serology. The demographic characteristics, job titles, and risk of contact with COVID-19 patients were compared between employees with and without COVID-19.A total of 19 employees (1.2%) tested positive for the SARS-CoV-2 antigen. The positivity rate was high among rehabilitation therapists (2.1%) and employees in the low-risk contact group (6.1%). Although there was no association between the job titles and the seropositivity rate, those in the low-risk contact group had an increased risk of testing positive for the viral antigen (odds ratio, 8.67; 95% confidence interval, 3.30-22.8).The antigen positivity rate was low during the hospital outbreak, suggesting that risk assessment of exposure to COVID-19 patients may provide more useful information than using job titles to identify infected health care providers., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Following isolation of pseudorabies virus (PRV) from two hunting dogs in Oita prefecture, Japan, we investigated the PRV antigen distribution in the tissues of the infected animals. At necropsy, PRV-associated lesions included facial oedema, tonsillar and meningeal congestion, blotchy haemorrhages on the pericardium and mitral valves, and incomplete splenic contraction in one dog, with less prominent findings in the other dog. Multiple pulmonary nodules were seen in both cases, caused by the diploid form of Paragonimus westermani lung flukes, as confirmed by PCR-restriction fragment length polymorphism and gene sequencing analyses. Histological examination revealed that the PRV infection was associated with lesions of non-suppurative encephalitis in the brainstem. PRV antigen was detected in the cerebrum, cerebellum, brainstem, submucosal and myenteric plexuses, and mononuclear cells, mainly in the bone marrow, lymph nodes, tonsils and spleen tissues. There was evidence of PRV dissemination to the brain via the trigeminal or olfactory routes, in addition to possible spread to lymphoid organs via infected mononuclear cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Bovine respiratory syncytial virus (BRSV) is an etiologic agent of bovine respiratory disease. The rapid evolutionary rate of BRSV contributes to genetic and antigenic heterogeneity of field strains and causes occasional vaccine failure. We conducted molecular epidemiologic characterization of BRSV circulating in Japan to obtain genetic information for vaccine-based disease control. Phylogenetic analysis of G and F gene sequences revealed that all of the isolated Japanese BRSV strains clustered in the same genetic subgroup, which was distinct from the 9 known groups. We assigned the Japanese group to subgenotype X. The Japanese isolates formed 2 temporal clusters: isolates from 2003 to 2005 clustered in lineage A; isolates from 2017 to 2019 formed lineage B. The alignment of the deduced amino acid sequences of the G gene revealed that the central hydrophobic region responsible for viral antigenicity is conserved in all of the isolates; unique amino acid mutations were found mainly in mucin-like regions. Our results suggest that BRSV has evolved uniquely in Japan to form the new subgenotype X; the antigenic homogeneity of the viruses within this group is inferred.

Respiratory syncytial virus (RSV) antigen-detection kits are used in clinical settings in Japan. Here, we compared the sensitivities of 12 different kits that are commercially available in Japan. Notably, these kits were developed based on the principle of immunochromatography. The comparison was performed using six different test specimens：standard laboratory strains of RSV subgroups A and B, and clinical specimens of nasal aspirates from patients infected with RSV subgroups A or B. The specimens were serially diluted with medium at half log concentration, and the dilutions were applied to the kit tests. The minimum detection limit was determined for each kit and for every specimen by investigating the maximum dilution rate for acquiring positive result. The absolute minimum detection limits were calculated based on the viral RNA copy number of the specimens and the maximum dilution rate ranged from 107 to 109 copies/mL, depending on the specimens and kits. Thus, the sensitivity was revealed to differ among the viral specimens examined using each kit and among the kits used to examine each viral specimen, though the difference was not significant and revealed a maximum of only 10 times. The results were compared with the geometric mean values of the results for all 12 kits which were separately calculated for all 6 specimens. Consequently, some kits with lower or higher sensitivities to all the tested specimens were identified, suggesting that some general differences in sensitivity exist among commercially available RSV antigen-detection kits, although the differences are not significant. Of course, selecting kit with a higher sensitivity would be preferable, but no large differences in sensitivity among the available kits examined in the current study. Therefore, other factors such as cost-effectiveness, convenience, time required for testing, etc., can be taken into account when selecting a kit for use in clinical settings.

Background: Equine influenza (EI) is a highly contagious disease caused by viruses of the H3N8 subtype. The rapid diagnosis of EI is essential to reduce the disease spread. Many rapid antigen detection (RAD) tests for diagnosing human influenza are available, but their ability to diagnose EI has not been systematically evaluated., Objectives: The aim of this study was to compare the performance of 22 RAD tests in the diagnosis of EI., Methods: The 22 RAD tests were performed on fivefold serial dilutions of EI virus to determine their detection limits. The four most sensitive RAD tests (ImmunoAce Flu, BD Flu examan, Quick chaser Flu A, B and ESPLINE Influenza A&B-N) were further evaluated using nasopharyngeal samples collected from experimentally infected and naturally infected horses. The results were compared to those obtained using molecular tests., Results: The detection limits of the 22 RAD tests varied hugely. Even the four RAD tests showing the best sensitivity were 125-fold less sensitive than the molecular techniques. The duration of virus detection in the experimentally infected horses was shorter using the RAD tests than using the molecular techniques. The RAD tests detected between 27% and 73% of real-time RT-PCR-positive samples from naturally infected horses., Conclusions: The study demonstrated the importance of choosing the right RAD tests as only three of 22 were fit for diagnosing EI. It was also indicated that even RAD tests with the highest sensitivity serve only as an adjunct to molecular tests because of the potential for false-negative results., (© 2015 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Migratory water birds are the natural reservoir of influenza A viruses. H5 and H7 influenza viruses are isolated over the world and also circulate among poultry in Asia. In 2010, two H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from fecal samples of water birds on the flyway of migration from Siberia, Russia to the south in Hokkaido, Japan. H7N9 viruses are sporadically isolated from humans and circulate in poultry in China. To monitor whether these viruses have spread in the wild bird population, we conducted virological surveillance of avian influenza in migratory water birds in autumn from 2010 to 2014. A total of 8103 fecal samples from migratory water birds were collected in Japan and Mongolia, and 350 influenza viruses including 13 H5 and 19 H7 influenza viruses were isolated. A phylogenetic analysis revealed that all isolates are genetically closely related to viruses circulating among wild water birds. The results of the antigenic analysis indicated that the antigenicity of viruses in wild water birds is highly stable despite their nucleotide sequence diversity but is distinct from that of HPAIVs recently isolated in Asia. The present results suggest that HPAIVs and Chinese H7N9 viruses were not predominantly circulating in migratory water birds; however, continued monitoring of H5 and H7 influenza viruses both in domestic and wild birds is recommended for the control of avian influenza.

For national regulatory testing in Japan, the Lowry method is used for the determination of total protein content in vaccines. However, many substances are known to interfere with the Lowry method, rendering accurate estimation of protein content difficult. To accurately determine the total protein content in vaccines, it is necessary to identify the major interfering substances and improve the methodology for removing such substances. This study examined the effects of high levels of lactose with low levels of protein in freeze-dried, cell culture-derived Japanese encephalitis vaccine (inactivated). Lactose was selected because it is a reducing sugar that is expected to interfere with the Lowry method. Our results revealed that concentrations of ≥ 0.1 mg/mL lactose interfered with the Lowry assays and resulted in overestimation of the protein content in a lactose concentration-dependent manner. On the other hand, our results demonstrated that it is important for the residual volume to be ≤ 0.05 mL after trichloroacetic acid precipitation in order to avoid the effects of lactose. Thus, the method presented here is useful for accurate protein determination by the Lowry method, even when it is used for determining low levels of protein in vaccines containing interfering substances. In this study, we have reported a methodological adjustment that allows accurate estimation of protein content for national regulatory testing, when the vaccine contains interfering substances.

Green tea is known to contain antiviral components that prevent influenza infection. A limited number of adult clinical studies have been undertaken, but there is a paucity of clinical evidence concerning children. We conducted an observational study to determine the association between green tea consumption and the incidence of influenza infection among schoolchildren. Anonymous questionnaire surveys were undertaken twice during the influenza season from November 2008 to February 2009 (endemic seasonal type A influenza infection); each survey was conducted for 2663 pupils across all elementary schools in Kikugawa City (a tea plantation area), Japan. Each questionnaire was completed and submitted by 2050 pupils (response rate, 77.0%; age range, 6-13 y). The adjusted OR associated with the consumption of green tea for ≥6 d/wk compared with <3 d/wk was 0.60 [(95% CI = 0.39-0.92); P = 0.02] in cases of influenza confirmed by the antigen test. Meanwhile, the adjusted OR inversely associated with the consumption of 1 cup/d to <3 cups/d (1 cup = 200 mL) and 3-5 cups/d compared with <1 cup/d were 0.62 [(95% CI = 0.41-0.95); P = 0.03] and 0.54 [(95% CI = 0.30-0.94); P = 0.03], respectively. However, there was no significant association with the consumption of >5 cups/d. Our findings thus suggest that the consumption of 1-5 cups/d of green tea may prevent influenza infection in children.

We describe an autopsy case of a patient with pandemic influenza (A/H1N1pdm) virus infection in Japan, who developed rapidly progressive viral pneumonia exhibiting diffuse alveolar damage. A 41-year-old female visited our hospital with a fever of 38.7C. She was a public health nurse with no underlying disease and had had contact with a group of elementary school students who had been infected with the influenza (A/H1N1pdm) virus 1 week earlier. She was prescribed oseltamivir and returned to the hotel where she was staying alone. The next day, she was found dead in her hotel room. At autopsy, both lungs were voluminous and microscopic examination revealed acute-stage, severe diffuse alveolar damage with remarkable mononuclear cell infiltration and hyaline membrane formation in the lungs. CD8-positive T lymphocytes were dominantly observed. Immunohistochemically, influenza A viral protein was confirmed in the damaged type II pneumocytes and also in the infiltrated macrophages. Real-time RT-PCR analysis of both pre- and post-mortem pharyngeal swabs confirmed a novel influenza (A/H1N1pdm) virus infection. This is the second autopsy case of influenza (A/H1N1pdm) virus infection in Japan, and the findings indicated that the patient died due to an exceptionally rapid progression of viral pneumonia. This case indicates that patients with influenza (A/H1N1pdm) virus infection should be carefully monitor for acute respiratory distress syndrome.

A total of 90 blood samples were collected from wild boars in the Kyushu region of Japan, and a seroepidemiological survey for 7 arthropod-borne viruses was performed using hemagglutination inhibition tests. The individual seropositive rates for each virus were 52.2% for Japanese encephalitis virus (JEV), 47.8% for Getah virus (GETV), 13.3% for Akabane virus, 10.0% for Aino virus and less than 5% for Bluetongue virus, Chuzan virus and Ibaraki virus. The results indicated that among the 7 viruses, JEV and GETV infections were prevalent among the wild boars and that the animals were involved in the natural transmission cycle of JEV and GETV in Japan. It is necessary to consider the participation of wild animals for the control of arthropod-borne virus infections.

This report describes the clinical characteristics of influenza A(H1N1)v virus infection in Osaka. By the end of May, 171 cases had been reported in Osaka. Most patients were from one school. No patient had a serious underlying medical condition.Clinical symptoms were mild and resembled those of seasonal influenza. The sensitivity of the rapid antigen test was 77%. Antivirals were given to the majority of the cases. Early antiviral treatment may have shortened the duration of fever.

Hokkaido virus (HOKV) is a member of the genus Hantavirus, in the family Bunyaviridae. To investigate HOKV infection in the host Myodes rufocanus, the grey red-backed vole, 199 animals were captured at Tobetsu (October 2004 and July 2005) and Nakagawa (October 2004) in Hokkaido, Japan, for detection of antibody, antigen, and viral RNA. In the surveys in Tobetsu (2004) and Nakagawa (2004), seropositive animals were detected at a frequency of 6.0% (5/84) and 10.4% (5/48), respectively. No seropositive animals were detected in Tobetsu in 2005. Seroprevalence in males in Tobetsu and Nakagawa in 2004 was 25% (1/4) and 45.5% (5/11), respectively, which was higher than in females, at 5.0% (4/80) and 0% (0/37), respectively (P<0.01). These results suggest that male animals play an important role in the maintenance of HOKV in M. rufocanus. Two females were seronegative but viral RNA-positive, indicating that these animals had acute infections before antibody was produced. Another five infected animals in Nakagawa were all male and had high levels of antibodies and viral RNA, suggesting that they had persistent infections. Viral RNA copies in organs of infected animals in Nakagawa were quantified by real-time polymerase chain reaction. Two acutely infected animals had > or = 10 times the number of RNA copies in their lungs compared to those of persistently infected animals. In most cases, lungs or spleen had the highest RNA copy number, regardless of infection status.

Highly pathogenic H5N1 avian influenza viruses were isolated in 9 large-billed crows that died in Kyoto and Osaka prefectures in Japan from March to April in 2004. We studied 3 of the 9 crows using standard histologic methods, immunohistochemistry, and virus isolation. The most prominent lesions were gross patchy areas of reddish discoloration in the pancreas. The consistent histologic lesions included severe multifocal necrotizing pancreatitis, focal degeneration and necrosis of neuron and glial cells in the central nervous system, and focal degeneration of cardiac myocytes. All of these tissues contained immunohistochemically positive influenza viral antigens. The virus was isolated from the brain, lung, heart, liver, spleen, and kidney of the crows examined. Thus we concluded that highly pathogenic avian influenza virus was associated with clinical disease, severe pathologic changes, and death in the 3 crows.

Epidemiology, pathology, and immunohistochemistry were investigated in layer hens affected with H5N1 highly pathogenic avian influenza, which occurred for the first time in 79 years in Japan. The farm, which had a total of 34,640 chickens, experienced up to 43.3% mortality before the chickens were depopulated. Clinically, the affected chickens exhibited mortality without apparent clinical signs. Histologically, hepatocytic necrosis; necrosis of ellipsoids and follicles with fibrin in the spleen; necrosis with glial nodules in the brain stem, cerebrum, and cerebellum; necrosis of acinar cells in the pancreas; and necrosis of lymphoid tissues in intestinal lamina propria were seen. Occasionally, mild bronchiolitis, degeneration of smooth muscle fibers in the cecum, and mild tubulonephrosis were noted. Immunohistochemically, influenza virus antigens were detected often in the liver and spleen, heart, intestine, gizzard, proventriculus, and oviduct. In addition, antigens were seen also in the brain, kidney, pancreas, and ovary, but seldom in the lung and trachea. Virus antigen was mainly detected in the capillary endothelium and parenchymal cells. This suggests that virus excretion from the respiratory tract was not as prevalent as that from the digestive tract in the present cases.

We have previously reported 2 subtypes of hepatitis B virus (HBV) genotype B, one of which has the recombination with genotype C over the precore region plus core gene (Ba) and the other of which does not (Bj). A restriction fragment-length polymorphism method with 2 endonucleases was newly developed for distinguishing between subtypes Ba and Bj and was applied to 313 carriers of HBV genotype B in Japan. Subtype Ba was detected in 38 (12%) and subtype Bj in 275 (88%) of the carriers of HBV genotype B. Hepatitis B e antigen in serum was found more frequently in patients with chronic infection with subtype Ba than in those with chronic infection with subtype Bj (8 [32%] of 25 vs. 25 [9%] of 273; P<.01). The new method for distinguishing between Ba and Bj by restriction fragment-length polymorphism would be useful in examining the distribution of these 2 subtypes in situations in which HBV genotype B is prevalent.

Peripheral blood mononuclear cells (PBMC) obtained from patients with lower respiratory infections were examined for the detection of human respiratory syncytial virus (RSV) sequences in the N region using the reverse transcription polymerase chain reaction (RT-PCR). RSV infection was confirmed by at least one method, i.e., virus isolation, enzyme immunoassay for viral antigen, and RT-PCR of nasopharyngeal secretions (NPS) samples. The detection rate for RSV RNA in PBMC obtained from RSV-infected patients was 40% (38/94), compared to 5% (1/20) in controls (P = 0.002). Between the groups positive (38) and negative (56) for RSV RNA in PBMC, there was no significant difference in clinical parameters. Seven patients had eight episodes of reinfection and RSV RNA was detected in 50% (4/8) during consecutive infections. Sequences of their PBMC samples were distinct from those of prototype strains of subgroup A and B. However, they were not always consistent with those of paired NPS samples. The findings suggested that RSV RNA could be detected in PBMC even during reinfection and as might have the possibility of quasispecies dynamics, reflecting the nature of RNA viruses., (Copyright 2003 Wiley-Liss, Inc.)

Objective: The differences in clinical features between influenza A H1N1, A H3N2, and B in the past three influenza seasons were examined., Methodology: Patients with respiratory symptoms who consulted Kurume University Medical Center, Department of Internal Medicine, Kurume, Fukuoka, Japan, from January to March in 1999, 2000, and 2001 were included. Based on virological and serological findings, the influenza patients were divided into the above three groups for comparison of symptoms and laboratory data., Patients: (n = 196) included 54 with influenza A H1N1, 98 with A H3N2, and 44 with B. Mean ages in the groups were 33 +/- 8.4 years, 41 +/- 15.2 years, and 29 +/- 9.8 years (influenza B patients tended to be younger). Fever was much greater in the A H3N2 group (38.6 +/- 0.46 degrees C) than in the A H1N1 or B groups. This was also true for laboratory indices of viral infection. Gastrointestinal symptoms such as nausea, epigastralgia, and diarrhoea were prominent in influenza B. Myalgia was common in all groups., Conclusions: Influenza A H3N2 infection was more severe than A H1N1 or B in terms of fever, leukopenia, and C-reactive protein. Myalgia and other symptoms such as fever, headache, general malaise and sore throat were equally frequent in influenza A H3N2, A H1N1, and B infections. Gastrointestinal symptoms were more common in influenza B.

Borna disease (BD) was diagnosed in a 3-year-old male Welsh corgi suffering from a severe and acute progressive disorder of the central nervous system. Histopathologically, neuronal lesions were characterized by a non-suppurative encephalomyelitis dominated by large perivascular cuffs consisting of lymphocytes, macrophages and plasma cells; also present were inflammatory cell infiltrates in the neural parenchyma, neuronophagia and focal gliosis. Strong immunolabelling with BD virus (BDV) p40 antibody was diffusely distributed in the cytoplasm of small and large neurons in areas of the brain with and without inflammatory changes, and also in the spinal cord. Positive hybridization signals with BDV p40 sense and antisense riboprobes were seen in the nucleus and cytoplasm of the neurons throughout the whole brain and spinal cord. BDV p24 RNA in formalin-fixed brain tissue was detected by reverse transcriptase (RT)-nested polymerase chain reaction (PCR). BDV p24 RNA-positive signals were detected in the temporal lobe. This is the first report of spontaneous canine BD in Japan., (Copyright Elsevier Science Ltd. All rights reserved.)

Seven new triterpene glycosides, bryoniosides A-G (1-7), have been isolated along with two known triterpene glycosides, cabenoside D (8) and bryoamaride (9), from a methanol extract of the roots of Bryoniadioica. The structures of 1-7 were determined on the basis of spectroscopic and chemical methods. Six compounds, 2, 3, 5, and 7-9, and 11 compounds, 1-9, bryodulcosigenin (10), and bryosigenin (11), respectively, were evaluated for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation (1 microg/ear) in mice and on Epstein-Barr virus early antigen (EBV-EA) activation induced by TPA. All compounds tested showed marked anti-inflammatory effects, with 50% inhibitory doses (ID(50)) of 0.2-0.6 mg per ear. In addition, all of the compounds tested except for compound 5 showed potent inhibitory effects on EBV-EA induction (100% inhibition at 1 x 10(3) mol ratio/TPA).

Primary splenic involvement in lymphoid neoplasms is rare and the clinicopathologic features of splenic lymphoma are not well described compared to nodal non-Hodgkin's lymphoma (NHL). Here we characterized splenic lymphomas in an human T lymphotropic virus type-I (HTLV-I) endemic area of Japan. To assess the pattern of splenic involvement and evaluate prognosis, we reviewed 27 specimens consisting of 26 splenectomies and one necropsy, which were classified using REAL classification. Cases were divided into primary splenic lymphoma in 11 patients and secondary in 16 patients. The incidence of primary splenic lymphoma was 0.3% (11 of approximately 4,000 malignant lymphomas). Primary splenic lymphomas included 7 diffuse large B cell lymphoma (DLBL), 2 follicular lymphomas (FL), and 1 each of splenic marginal zone lymphoma (SMZL) and anaplastic large cell lymphoma (ALCL). Secondary splenic lymphomas included 6 DLBL, 4 mantle cell lymphoma (MCL), 2 FL, 2 Hodgkin's disease (HD), 1 each of hairy cell leukemia and ALCL. Gross examination showed two patterns of splenic involvement; solid type (formation of large nodular mass, n=16) and disseminated type (multiple nodules with diffuse infiltration but no large nodular formation, n=10). The type could not be determined in one case. Most solid types were DLBL or FL, while MCL was of the disseminated type. Immunohistochemistry showed all but each 2 cases of ALCL and HD were of B lineage. Follow-up of 26 patients indicated that all but one patient with primary lymphoma were still alive (range, 1-89 months) and 8 of 15 patients with secondary lymphomas died due to the progression of malignant lymphoma; the survival rate at 2 years was 50% in these patients. No elevation of anti-HTLV-I antibody was found. In situ hybridization for Epstein-Barr virus (EBV) showed no reactivity of lymphoma cells, although a few small lymphocytes were positive for EBV. Hepatitis C virus was observed in 6 of 20 (30%) patients examined and 4 of 11 (36%) cases of primary splenic lymphoma. Our findings indicate that patients with primary splenic lymphoma have a favorable prognosis after splenectomy.

Retrospectively, we demonstrated that seven pigs necropsied in 1989 had characteristic porcine circovirus 2 (PCV-2) lymphoid tissue lesions of severe lymphoid depletion, syncytial giant cell formation, and intracytoplasmic inclusion bodies in macrophages. Immunohistochemically, PCV-2 antigen was detected in these tissues and the distribution of positive staining corresponded to the distribution of inclusion bodies. Electron microscopy demonstrated viral particles compatible with porcine PCV-2. Therefore, the disease occurred in Japan as early as 1989.

A 5-month-old Japanese black bull calf and twenty-seven 1-27-day-old calves exhibiting neurological signs between August and October 1998 were examined. The bull calf exhibited rapid breathing, fever, hypersensitivity, and ataxia and was euthanized 4 days after the onset of symptoms. The 27 calves primarily exhibited ataxia, and 15 had arthrogryposis. Histological examination of the bull calf revealed perivascular infiltraction by mononuclear cells, diffuse to multifocal gliosis, and neuronal necrosis in the brain and spinal cord. Multiple malacic foci were found in the midbrain in 5 cases. In contrast, in the 15 calves necropsied in October, there were fewer inflammatory changes, but there was neuronal cell loss in the ventral horn and a decrease in myelinated axons in the lateral and ventral funiculi. Immunohistochemical examination using a rabbit antiserum against Akabane virus strain OBE-1 revealed a large amount of viral antigen in the degenerating neurons and glial cells of the bull calf, mainly in the spinal gray matter. Small amounts of viral antigen in swollen axons and a few glial cells were found in 5 of 27 calves. Thirteen of the 27 calves had high neutralization antibody titers against the Akabane virus, whereas there was no significant antibody titer in most of the calves necropsied during August. The present study revealed that viral antigen detection was very useful for the diagnosis of Akabane diseases in the 5-month-old bull calf that was suspected to be infected postnatally, while it had limited usefulness in the other young calves.

A birnavirus was recently isolated from cultured ayu Plecoglossus altivelis on Shikoku island, Japan. The diseased fish displayed vertebral or vertical curvature and mild haemorrhage around the brain. Cytopathic effects (CPE) of the virus, including cell roundness, filamentous change and cell lysis, were observed in CHSE-214, RTG-2 and RSBK-2 cells. The virus isolated from ayu, designated the AY-98 strain, was found to be antigenically related to the marine birnavirus (MABV) Y-6 strain that originated from yellowtail Seriola quinqueradiata. AY-98 had a bi-segmented RNA genome and the same nucleotide sequence in the 310 bp VP2/NS junction as MABV Y-6. At the same time that the ayu epizootics occurred, another birnavirus (AM-98) was isolated from amago salmon Oncorhynchus rhodurus which were cultured 66 km away from the ayu farm. AM-98 showed a similar CPE and had the same host cell ranges as AY-98. However, AM-98 was serologically similar to the VR-299 strain of infectious pancreatic necrosis virus (IPNV) and their nucleotide sequences in the VP2/NS junction region showed 98% homology without changes at the amino acid level. In this study, the ayu strain AY-98 was grouped into MABV, whereas the amago salmon strain AM-98 was grouped into IPNV. This indicates that the 2 birnaviruses originated from different sources in spite of the fact that the places where they were isolated are close to one another. The results in this paper show a new aspect of the traditional consensus that the same serogroup of birnavirus distribute in close geographic areas.

Ten strains, eight field and two reference laboratory strains, of canine coronavirus (CCV) were comparatively examined with respect to antigenic relationships and pathogenic potential in dogs. With monoclonal antibodies and hyperimmune antisera to feline coronavirus and CCV, respectively, varying degrees of antigenic diversities were found among the strains by neutralization and immunofluorescence assays, but it was felt that they belong to one serotype. Specific-pathogen-free puppies experimentally inoculated with some CCV strains manifested clinical symptoms, but there was a difference in their virulence. In order to elucidate the prevalence of CCV infections in dogs in Japan, we tested for neutralizing antibodies to CCV in 467 field dogs, and found a prevalence of 44.1%. Moreover, by using nested reverse transcriptase-polymerase chain reaction on rectal swabs of 100 diarrheic dogs recently presented in veterinary clinics, evidence of CCV in 16% of these specimens was found. The results suggested that CCV infection is more widespread than expected in dogs, and that CCV is a significant etiologic factor in canine diarrhea also in Japan.

Mexico virus (MXV) is a genogroup II human calicivirus (HuCV). We conducted an epidemiological study to determine the prevalence of MXV infection in infants and adults in Japan and Southeast Asia by enzyme-linked immunosorbent assays (ELISAs) developed by using baculovirus-expressed recombinant MXV (rMXV) capsids. Of 155 stool specimens obtained from children younger than 10 years old with acute clinical gastroenteritis (diarrhea and vomiting) associated with small, round-structured viruses in Japan from 1987 to 1989, only 2 were positive for MXV antigen. In 42 outbreaks of acute gastroenteritis in Japan from 1986 to 1994, 1 in an infant home and 1 among adults were positive for MXV antigen. The pattern of acquisition of antibody to rMXV was different from that of acquisition of antibody to group A rotavirus, the prototype HuCV Sapporo virus, and Norwalk virus. The prevalence of antibody to rMXV remained low for the first 3 years of life, showed a steep rise during nursery school age, reaching a prevalence of 50%, and another steep rise during adolescence, reaching 80%; and steadily increased thereafter. A high prevalence of antibody (82 to 88%) was observed in adult populations in Japan and Southeast Asia, suggesting that MXV infection is common in these areas. The discrepancy between the high prevalence of antibody to MXV and a low rate of detection of MXV antigen may be explained by a high specificity of the antigen ELISA for the prototype and closely related MXV strains while serological responses can detect responses to a broader group of viruses.

From the winter of 1997 to the spring of 1988, a severe epidemic caused by influenza A (H3N2) developed in Japan. During the epidemic (December 1997 to February 1998), 239 children were admitted to the pediatric ward of Nippon Kokan Hospital: as many as 76 (31.8%) were hospitalized with influenza A (H3N2) infection. Most were young healthy children (mean age, 3.7 years), suggesting the need of influenza immunization for this age group. 110 specimens of hospitalized children were examined by rapid antigen test of influenza A virus (Directigen Flu A, Becton Dickinson, USA). The rapid test showed about 90% sensitivity and specificity, as compared with isolation and serum hemagglutination inhibition test. Directigen Flu A proved to be a reliable, rapid screen for influenza A from symptomatic patients.

Gazelle herpesvirus (GHV-1), correctly designated as equine herpesvirus 9, is a new type of equine herpesvirus immunologically related to equine herpesvirus 1 (EHV-1). As a sequel to a virological study, the neuropathology of encephalitis caused by GHV-1 in Thomson's gazelles (Gazella thomsoni) was examined. Seven gazelles died with or without neurological symptoms between early September and mid-October in 1993. No gross abnormalities were observed at necropsy, but all animals had non-suppurative encephalitis, characterized by necrosis and degeneration of neurons, glial reactions and perivascular cuffing in the cerebrum. Five cases showed intranuclear inclusion bodies, with the appearance of herpesvirus in the degenerating neurons. Immunohistochemically, all seven animals showed a positive reaction to EHV-1 antigen in neurons in the necrotic areas of the cortex. The clinical course and morphological features of GHV-1 encephalitis were distinct from those of EHV-1-induced encephalitis in the horse, which is characterized by vasculitis, thrombosis, ischaemia, and lack of intranuclear inclusions in neurons.

Recombinant baculoviruses were constructed to express the putative proteins VP1, VP2 or VP3 of the chicken anemia virus (CAV). The recombinant VP1, VP2 or VP3 were detected by SDS-PAGE, and their molecular weights were 50, 30/27 and 16 kDa, respectively. The VP2 and VP3 reacted with sera from CAV-infected chickens in Western blot analysis and when used as an enzyme-linked immunosorbent assay (ELISA) antigen, but VP1 did not. Antibodies to CAV were detected, by ELISA using crude insect cell lysates containing VP2 or VP3, from 2 to 20 weeks or 2 to 7 weeks after CAV infection, respectively. These findings indicate that recombinant VP2 and VP3 expressed in the baculovirus vector system can be used as antigens to detect anti-CAV antibodies in ELISA.

New antigenic variants of B/Yamagata/16/88-like lineage which appeared in the season of 1997 as a minor strain tended to predominate in the following season. Also, we could observe for the first time, three peaks of activity caused by H3N2 virus and two variants of B influenza virus. Antigenic and phylogenetic analyses revealed that B/Victoria/2/87-like variants appeared again in Japan in 1997 after a nine-year absence. Influenza B viruses evolved into three major lineages, including the earliest strain (I), B/Yamagata/16/88-like variants (II), which comprised of three sublineages (II-(i), II-(ii), II-(iii)), and B/Victoria/2/87-like variants (III). Evolution of influenza B virus hemagglutinin was apparently distinguishable from that of influenza A virus, showing a systematic mechanism of nucleotide deletion and insertion. This phenomenon was observed to be closely related to evolutionary pathways of I, II-(i), II-(ii), II-(iii) and III lineages. It was noteworthy to reveal that the nucleotide deletion and insertion mechanism of influenza B virus completed one cycle over a fifty-year period, and that a three nucleotide deletion was again observed in 1997 strains belonging to lineage II-(iii). It was evident that amino acid substitutions accompanying nucleotide insertions were highly conserved.

Oral Actinobacillus actinomycetemcomitans strains are serologically classified into 5 distinct groups, a to e. We examined the distribution of A. actinomycetemcomitans serotypes in Japanese patients with periodontitis. A total of 157 A. actinomycetemcomitans clinical isolates from diseased sites of 39 patients with periodontitis were serotyped by using serotype-specific rabbit antisera against A. actinomycetemcomitans serotypes a, b, c, d and e strains. In the immunodiffusion assay, autoclaved extracts of 42, 6, 39, 9 and 41 A. actinomycetemcomitans clinical isolates reacted with serotypes a, b, c, d and e antisera, respectively. Although 37 patients were infected with a serotype strain, 2 patients harbored 2 different serotype strains, b/e and b/untypeable. To establish a correlation between serotype and genotype of A. actinomycetemcomitans clinical isolates from 2 patients who had different serotype strains, we used arbitrarily primed polymerase chain reaction (AP-PCR) to fingerprint clinical isolates of different serotypes. The AP-PCR genotypes among 4 clinical isolates (b/e and b/untypeable) were identical to that of A. actinomycetemcomitans Y4 (serotype b), indicating the presence of multiple A. actinomycetemcomitans serotypes which are genetically homogeneous in the periodontally diseased sites of patients with periodontitis.

The antigenicity and RNA genome structures of five Simbu serogroup bunyaviruses isolated in Japan and Australia were analyzed using monoclonal antibodies (Mabs) raised to Akabane (AKA) virus and oligonucleotide fingerprinting. The virion surface glycoprotein (G1) and the nucleocapsid (N) protein of heterologous viruses showed no reactivity to the Mabs, while the AKA-derived anti-G1 Mab (2F1) reacted with Peaton virus and all three AKA anti-N Mabs reacted with Tinaroo (TIN) virus at almost the same antibody titers as the homologous virus. Oligonucleotide fingerprinting analyses indicated that the three RNA species of all the viruses were unique and distinguishable. However, AKA and TIN viruses exhibited very similar S RNA oligonucleotide fingerprints, while the L and M RNA fingerprints were quite different. The S RNA sequence of TIN virus has been determined and compared with that of AKA and Aino viruses. The results revealed 95.1% S sequence homology between the AKA and TIN viruses. The antigenic and genetic comparisons of AKA and TIN viruses suggest that the two viruses may represent naturally occurring reassortant viruses.

In the winter of 1989 and the spring of 1990, there were large outbreaks of respiratory disease in two swine herds in Nagasaki Prefecture, southern Japan. Serological surveillance indicated that the majority of swine possessed antibodies to swine influenza virus H1 haemagglutinin and neuraminidase of early H3N2 influenza virus strains. Eight viruses were isolated from swine that showed typical clinical symptoms of influenza. The haemagglutinin and neuraminidase of these isolates were closely related to those of swine H1N1 and early human H3N2 viruses, respectively. At least two types of haemagglutinin antigens, distinguished by two monoclonal antibodies, were involved in the outbreaks. Evolutionary analyses indicated that the haemagglutinin gene of the H1N2 reassortants was closely related to those of a recent swine lineage (A/sw/HK/1/74 and A/sw/Ehime/1/80 viruses). However, the neuraminidase genes of the H1N2 reassortants were similar to those of swine N2 viruses which in turn are related to early human H3N2 viruses. A comparison of partial nucleotide sequences revealed that the six other genes of A/sw/Nagasaki/1/89 were derived from those of swine H1N1 virus.

Sixty cases of B-cell nodal non-Hodgkin's malignant lymphoma (B-ML), and 46 cases of T-cell nodal lymphoma (T-ML) were surveyed for Epstein-Barr virus (EBV) genomes, RNA, and associated proteins. We used a Southern blot analysis, polymerase chain reaction (PCR), and EBV-encoded small RNA-1 (EBER-1) in situ hybridization to investigate the presence of EBV. We performed an immunohistochemical study on EBV-related oncoproteins, such as EBV-determined nuclear antigen-2 (EBNA-2), latent membrane protein (LMP), and viral interleukin-10 (vIL-10). In addition, we also analyzed the terminal repetitive sequence of EBV (EBV-TR) to investigate the EBV-infected cell clonality. Non-Hodgkin's lymphomas were grouped into three types by number of EBV-infected cells: I) almost all lymphoma cells showed an EBV presence; II) some scattered lymphoma cells showed an EBV presence; and III) only a few cells showed such a presence, which was probably due to a latent EBV infection. In 25 of 60 B-MLs, EBV-infected cells were found; 7 were type I, 1 was type II, and 17 were type III. In 27 of 46 T-MLs, EBV-infected cells were found; no cases were type I, 5 cases were type II, and 22 cases were type III. Seven B-MLs and 3 T cell lymphomas showed clonal TR bands. Expression of EBNA-2 was found in only three B-MLs, whereas LMP was seen in four B-MLs and six T-MLs. All EBNA-2/LMP-positive cases showed an EBV presence. In B-MLs, expression of EBNA-2 and LMP was detected in almost all lymphoma cells; in T-MLs, however, LMP was found in only a small portion of the lymphoma cells. Expression of IL-10 was closely associated with LMP. In summary, it was thus speculated that EBV infection was associated with the various states of lymphomagenesis.

Based on our previous study using monoclonal antibodies against three Japanese encephalitis (JE) virus strains, Nakayama-RFVL, Beijing 1 and Kamiyama, 25 JE virus strains isolated mainly in Japan and some from Southeast Asia between 1935 and 1979 fell into five antigenic groups, Nakayama, Beijing 1, Kamiyama, Muar and 691004. In the present study, monoclonal antibodies against Muar and 691004 strains were produced to analyze immunological characteristics of JE virus in detail. We obtained the Muar group-specific antibody and three anti-691004 monoclonal antibodies which showed different reactivities. Furthermore, antigenic comparison of the 22 JE virus strains isolated in Asia between 1963 and 1984 was performed by using the 15 monoclonal antibodies characterized by the different reactivities against the above five antigenic groups. Of these 22 strains, the 17 strains reacted with the Kamiyama group-specific monoclonal antibody, but anti-Nakayama, anti-Beijing 1 and anti-Muar group-specific monoclonal antibodies showed no reactivities with any of the strains. This suggested that the currently prevalent JE virus strains in Asia belonged to the Kamiyama group. The other five strains, ThCMP 1982, KE083, KE093, 733913 and Ling, did not react with the above four group-specific monoclonal antibodies, but all except the KE093 strain showed a similar pattern to the Kamiyama strain on the basis of the reactivities against the other 11 antibodies. The KE093 strain isolated from Thailand in 1983 showed immunologically outstanding difference from other strains.

Three strains of influenza A virus (H3N2) were isolated from pigs in Hokkaido, Japan in 1993. The hemagglutinin (HA) antigen of the three isolates was related to that of recent H3N2 viruses of human origin. The reactivity patterns of two of the isolates (A/sw/Obihiro/1/93 and A/sw/Obihiro/2/93) with monoclonal antibodies to the hemagglutinin of A/Bangkok/1/79 strain were similar to that of the human H3N2 strain isolated in Hokkaido in 1988, while that of the other one (A/sw/Obihiro/3/93) was similar to human H3N2 strains of 1993. In the phylogenetic analysis based on the nucleotide sequences of the HA1 regions, the HA genes of the two isolates were shown to be closely related to those of human H3N2 viruses isolated between 1986 and 1988. The remaining one isolate was shown to be closely related to those of current human H3N2 viruses. We have also found serological evidence that the A/sw/Obihiro/1/93 virus is circulating extensively in Obihiro swine. It is clear from these findings that pigs were infected with the recent H3N2 influenza virus during the human epidemic and that the virus has been maintained in pigs for at least five years.

The epidemiology of influenza A in Japan was studied during 1979-91 and viruses isolated from reinfections during 1983-91 were analysed. Of 2963 influenza viruses isolated from reinfections during 1983-91 were analysed. Of 2963 influenza viruses isolated during this period, 922 and 1006 were influenza A(H1N1) and A(H3N2) viruses respectively; the others were influenza B viruses. Influenza A(H1N1) and A(H3N2) caused 5 and 6 epidemics respectively, most accompanied by antigenic drift. Seventeen reinfections with H1N1 and 17 with H3N2 were detected during our study. The primary and reinfection strains isolated from 7 H1N1 and 10 H3N2 cases were studied by haemagglutination-inhibition, and amino acid and nucleotide sequences of the HA1 region of the haemagglutinin. Most of the primary and reinfection strains were antigenically and genetically similar to the epidemic viruses circulating at that time. However, in 4 out of 10 cases of reinfection with influenza H3N2 virus, reinfection strains were genetically different from the epidemic viruses.

The virulence of four herpes simplex virus type 2 (HSV 2) strains (K1-K4) isolated in Okinawa, Japan was investigated, and compared with four strains (C1-C4) from Chiang Mai, Thailand and a standard laboratory strain SAV. Virulence was tested on BALB/c and C57/black mice. After viral inoculation intraperitoneally, the distribution with the passage of time of the virus in the brain and other organs was also studied using the polymerase chain reaction (PCR) method and immunohistochemistry. Generally, Okinawan HSV 2 (K1, K3 and K4) were less virulent than Chiang Mai strains (C1-C4). Among the Okinawan strains, K2 was the most virulent, but slightly less so than C3 and C4. All four Chiang Mai strains (C1-C4) and one Okinawan strain (K2) were more virulent than SAV strain. The virulence of K3 was very weak and no animals died from the intraperitoneal inoculation. In the brain, viral DNA from each strain was demonstrated at 1-9 days after inoculation by the PCR method. However, K3 strain was detected in the brain only between one day and 3 days after virus inoculation, and not after day 5. Immunohistochemically, the virus antigen was first demonstrated around the 3rd ventricle at one day after viral inoculation, then strongly at the ventral hypothalamus and the temporal lobe at 3 days after viral inoculation, and slightly in the frontal lobe, hippocampus, pons and cerebellum (on day 5 after inoculation). Furthermore, in Kupffer cells in the liver and macrophages in the spleen, numerous viral antigens were demonstrated from one to 9 days after viral inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the role of respiratory syncytial virus (RSV) in respiratory tract infections of up-to-3-months old infants. This prospective study was carried out from April 1993 to March 1994. Detection of RSV antigen in nasopharyngeal specimens was done by enzyme immunoassay using TESTPACK RSV at our out patient clinic. During this study period, 65 young infants with respiratory tract infection visited our clinic. Seventeen patients (26%) were diagnosed as having RSV infection. Fifteen out of the 17 infants with RSV infection were observed in spring and winter. Fourteen out of the 17 infants with RSV infection had acute bronchiolitis or pneumonia. In contrast to RSV infection, only three patients out of the 48 infants with non-RSV infection had lower respiratory tract infection. In conclusion, the majority of young infants infected with RSV suffered from bronchiolitis or pneumonia.

The antigenic and genetic relationships among 15 strains of influenza C virus isolated between August, 1988, and May, 1990, in Yamagata City, Japan, were investigated. Based on the results of antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoproteins and oligonucleotide mapping of total vRNA, the isolates were divided into two distinct groups closely related to either C/Yamagata/26/81 or C/Aichi/1/81. Antigenic differences between two groups could be detected clearly with heterogeneous antiviral sera. Comparison of the HE gene sequences between the representative Yamagata isolates and the previous isolates from other countries showed that C/Yamagata/26/81-like and C/Aichi/1/81-like viruses had high degrees of nucleotide sequence homology with C/pig/Beijing/115/81 and C/Johannesburg/1/66, respectively. These observations suggest that two lineages of influenza C virus, markedly different from each other in both antigenic and genetic structures, were simultaneously present in Yamagata City during 1988-1990 and that viruses belonging to each of the two lineages may also be prevalent in other areas of Japan and even in other countries.

Influenza B virus reinfection in Japanese children was studied epidemiologically during 1979-91 and virologically during 1985-91. During this investigation, there were four epidemics caused by influenza B viruses, each of which accompanied antigenic drift. Between the epidemics in 1987/88 and 1989/90, the viruses changed drastically, both genetically and antigenically. The minimum rate of reinfection with influenza B virus during the whole period was 3-25% depending on the influenza seasons. The antigens of primary and reinfection strains of influenza B virus isolated from 18 children during 1985-90, which covered three epidemic periods, were studied by haemagglutination inhibition tests. The results showed that the viruses isolated in the 1984/85 and 1987/88 influenza seasons, which belonged to the same lineage, were antigenically close, and reinfection occurred with these viruses. The results of amino-acid analysis of the HA1 polypeptide of these viruses corresponded with those of antigenic analysis. There were no specific amino-acid changes shared by the primary infection and reinfection influenza B viruses; the patients were infected with the viruses epidemic at that time.

Epizootiological studies of hantavirus infection among urban rats were carried out through the surveys repeated 11 times at the same dumping ground area in 1983 to 1988. A total of 279 rats (Rattus norvegicus) were captured during the surveys. Sero-positive animals to hantavirus strain SR-11 were detected in all the surveys. Overall positive rate of rats 6 months old or more (94/128, 73.4%) was significantly higher than that of younger rats (23/151, 15.2%, x2 = 96.4, P < 0.001). Therefore, age dependent acquisition of hantavirus infection among rats was confirmed. Seven hantavirus strains, KI-83-262 (August, in 1983, designated as strain KI-262 in our previous report (2)), KI-85-1 and 85-2 (July in 1985), KI-88-4, 88-11, 88-15 and 88-24 (October, 1988) were isolated from lung tissues of adult rats which have high titers of neutralizing antibody. Although the serum specimens of virus carrier rats neutralized the infectivity of all the KI isolates, no apparent antigenic change in the isolates was detected by indirect immunofluorescent antibody (IFA) assay using polyclonal and monoclonal antibodies (MAbs) regardless of isolation years. However, neutralization test showed slight difference of antigenicity among KI strains. These results epizootiologically confirmed that hantavirus infected persistently among urban rats in a presence of neutralizing antibody.

Circumstantial evidence from electron microscopic and immunological studies support the view that Paget's disease of bone represents a slow virus infection. However, there is only limited information available regarding its electron microscopic, enzyme and immunocytochemical characteristics. Two cases were studied using electron microscopy with particular emphasis on the inclusions in osteoclasts. Detailed ultrastructural and cytochemical studies including immuno-electron microscopy were performed. Some osteoclasts demonstrated specific virus-like structures composed of aggregations of microtubules in the nucleus and cytoplasm. The structures were easily digested by trypsin or protease, and were sensitive to RNase, which provided substantial evidence of a proteinaceous nature and inclusion of ribonucleic acid. Immunocytochemical examination identified binding of anti-respiratory syncytial virus and anti-measles virus antibodies in the tissue obtained from one of the two cases examined. The presence of viral antigens in structures in the cytoplasm of Pagetic osteoclasts supports the theory of paramyxovirus involvement in this disease.

Osaka, Japan, has one of the highest, primary liver cancer (PLC) incidence-rates in the world, although hepatitis-B virus (HBV) is not endemic. This paper addresses the question of whether the PLC-incidence variation within Osaka Prefecture is due to differences in the prevalence of hepatitis-C virus (HCV) infection. The screening data of antibody to HCV (anti-HCV) and of hepatitis-B virus antigen (HBsAg) in 111,069 male blood-donors, and the incidence data of male PLC obtained from the Osaka Cancer Registry were examined. In a multiple-weighted regression analysis, the age-standardized incidence rate of PLC in the 61 counties within Osaka was correlated significantly with the age-standardized prevalence of anti-HCV with adjustment for that of HBsAg (regression coefficient [RC] = 7.26, P < 0.0001). This finding was consistent with the relationship between the PLC incidence rate and the prevalence of high-titer (> or = 2(12)) anti-HCV (RC = 11.18, P < 0.0001). There was significant association between the prevalence of HBsAg and the PLC incidence rate with adjustment for that of anti-HCV (RC = 7.08, P = 0.018). These findings suggest that the PLC-incidence variation within Osaka is correlated with the geographic pattern of HCV infection as well as that of HBV infection among the residents.

We carried out an epidemiological study on lower respiratory tract infections, concerning the respiratory syncytial virus (RSV) infection, in northern Hokkaido, Japan, during the 2-year-study period from April 1991 to March 1993. There were 467 hospitalized patients with lower respiratory tract infections. We examined the presence of RSV antigen in their nasopharyngeal specimens of 456 out of 467 patients (97.6%) by enzyme immunoassay. The diagnosis of Mycoplasma pneumoniae infection was done by the rise of antibody titer, performed with complement fixation method, in the sera of the acute and convalescent phases. There were 133 patients with RSV infection (28.9%), 45 patients with Mycoplasma pneumoniae infection (9.8%) and 7 patients with measles virus infection (1.5%). RSV played an important role in the lower respiratory tract infections in northern Hokkaido, Japan. The epidemic of RSV infection was observed in spring and early winter in 1991 and only in early winter in 1992. The number of patients with RSV infection from April 1991 to March 1992 was greater than that from April 1992 to March 1993. In contrast with RSV infection, the epidemic of Mycoplasma pneumoniae infection was observed in 1992. We speculated that the RSV and Mycoplasma pneumoniae infections might interfere with each other.