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Mucosal immunity plays a crucial role in controlling upper respiratory infections, including influenza. We established a quantitative ELISA to measure the amount of influenza virus-specific salivery IgA (sIgA) and salivary IgG (sIgG) antibodies using a standard antibody broadly reactive to the influenza A virus. We then analyzed saliva and serum samples from seven individuals infected with the A(H1N1)pdm09 influenza virus during the 2019-2020 flu seasons. We detected an early (6-10 days post-infection) increase of sIgA in five of the seven samples and a later (3-5 weeks) increase of sIgG in six of the seven saliva samples. Although the conventional parenteral influenza vaccine did not induce IgA production in saliva, vaccinated individuals with a history of influenza infection had higher basal levels of sIgA than those without a history. Interestingly, we observed sIgA and sIgG in an asymptomatic individual who had close contact with two influenza cases. Both early mucosal sIgA secretion and late systemically induced sIgG in the mucosal surface may protect against virus infection. Despite the small sample size, our results indicate that the saliva test system can be useful for analyzing upper mucosal immunity in influenza., Competing Interests: The authors have declared that no competing interests exist.

We conducted a seroprevalence investigation of the healthy population of animals in Kagoshima Prefecture, an area in which severe fever with thrombocytopenia syndrome (SFTS) is endemic. Of 104 domestic cat and 114 dog samples, 2 (1.9%) and 11 (9.6%) were positive for anti-SFTS virus (SFTSV) IgG by indirect ELISA, respectively. Viral RNA was detected in one dog (0.9%) by RT-PCR. Of the 102 wild boar (Sus scrofa) and 107 deer (Cervus nippon) samples tested, 55 (53.9%) and 37 (34.7%) were positive for anti-SFTSV IgG, respectively. Only one wild boar (1.0%) was positive for viral RNA. Although symptomatic SFTSV infections in domestic cats have increased in this area, the seroprevalence of the healthy population of domestic cats tends to be lower than those of other animals. We developed a Gaussia luciferase immunoprecipitation system (GLIPS) using mammalian cells expressing a recombinant SFTSV nucleoprotein (SFTSV-rNP) for the detection of SFTSV-specific antibodies in samples from various animal species. The sensitivity of the assay was highly consistent with that of indirect ELISA, indicating that it could serve as a useful tool for a large-scale surveillance of SFTSV across multiple species of animals., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The objective of this study was to provide a screening scheme of persistently infected (PI) cattle on dairy herds by combining reverse-transcription polymerase chain reaction (RT-PCR) to detect bovine viral diarrhea virus (BVDV) in milk tanker samples and commercial enzyme-linked immunosorbent assay to detect BVDV antibodies in bulk tank milk. We conducted a pilot survey and regional survey targeting all dairy farms in Ibaraki Prefecture by using milk tanker and bulk tank milk samples to screen PI cattle. Farms with positive samples underwent a follow-up test to identify PI cattle. In the pilot study, all virus-positive samples in bulk tank milk were included in the positive milk tanker samples. The RT-PCR assay successfully detected BVDV at dilutions of 1:1,600 by using two PI cows' milk. In the regional survey, 5 of 79 milk tanker samples were virus-positive. The virus was detected in three PI lactating cows and one PI calf on three farms. Antibody screening using bulk tank milk samples revealed 15 of 363 samples were positive, and 12 of 348 farms were BVDV antibody-positive. Follow-up tests on one farm identified three PI calves. Thus, eight PI cattle on five farms were identified in this study. In conclusion, combining BVDV detection using milk tanker samples and antibody detection using bulk tank milk is a feasible and economical method to efficiently screen PI cattle and confirm the PI-free status among dairy herds.

The prevalence of bovine foamy virus (BFV) infections in cattle on farms in the Kanto region of Japan was determined using agar gel immunodiffusion (AGID) test and polymerase chain reaction (PCR). Six out of 20 farms contained BFV-positive cattle. Furthermore, 16.7% (91/545) of all cattle tested positive for BFV. This suggested that BFV-infected cattle are widely prevalent in Japan. Positive results for BFV infection were consistent between AGID and PCR tests. Additionally, we tested for bovine leukemia virus (BLV) infections at nine farms, primarily those containing BFV-infected cows. At each farm, the infection rate of BFV was lower than that of BLV. Further, cattle that were PCR-positive but antibody-negative, indicating immune tolerance to BFV, were not detected.

Purpose: Acute retinal necrosis (ARN) is a severe necrotizing retinitis caused by varicella-zoster virus or herpes simplex virus (HSV) that often results in blindness. Occasionally, HSV-caused ARN develops after herpes simplex encephalitis (HSE). It remains unknown, however, when and how often ARN develops after HSE. To investigate the actual conditions of patients with ARN following HSE and the interval period between the prior HSE and the onset of ARN, a retrospective nationwide survey of the Japanese population was performed., Study Design: Retrospective., Methods: Questionnaires were sent out to the neurology and ophthalmology departments of teaching hospitals in Japan. They inquired about HSE patients in neurology departments and ARN patients diagnosed with HSV in ophthalmology departments. The proportion of the HSV-ARN patients with a history of HSE and their interval periods were obtained from the questionnaires returned by the ophthalmology departments., Results: Neurology departments of 324 hospitals responded to the questionnaires (response proportion: 40.9%), and 53 HSE cases were reported. Ophthalmology departments of 535 hospitals responded the questionnaires (response proportion: 54.3%), and 67 HSV-ARN cases were reported. Among the 67 HSV-ARN cases, 16 (23.9%) had histories of prior HSE. Although the interval periods from the prior HSE to the onset of HSV-ARN varied among cases, nearly half developed HSV-ARN within 2 years., Conclusions: This nationwide survey of the Japanese population showed that HSV-ARN develops after HSE in higher frequency within 2 years. Neurologists and ophthalmologists should be aware that HSE survivors have a risk of contracting HSV-ARN.

Background: Hepatitis E virus (HEV) is prevalent in pigs and may serve as a reservoir for human infection. However, data on HEV infections in pigs in Ibaraki Prefecture, Japan, are limited. Here, we clarified the process and course of HEV in naturally infected pigs. Serum (n = 160) and liver (n = 110) samples were collected from pigs at the slaughterhouse. Furthermore, serum samples were collected from 45 breeding sows and serum and feces samples were collected from 7 piglets once a week (raised until 166 days of age). HEV antigen and antibodies were evaluated, and the genotype was identified based on molecular phylogenetic tree analysis., Results: The samples collected from the slaughterhouse revealed that few pigs were HEV carriers but most possessed anti-HEV antibodies. Most breeding sows possessed antibodies, and the piglets excreted HEV on the farm at approximately 10 weeks of age. One pig was initially infected, and in a few weeks, the other pigs living in the same sty became infected., Conclusions: Most pigs in Ibaraki Prefecture were with HEV. On the farm, most piglets were infected with HEV by the time they reached slaughter age. We confirmed that HEV infection is successively transmitted among piglets living in the same sty.

In October 2014, the varicella vaccination policy in Japan was changed from a single voluntary inoculation to two routine inoculations. This paper reports the results of booster vaccination in children who did not show seroconversion after initial vaccination (i.e., primary vaccine failure : PVF) over a 7-year period prior to the introduction of routine varicella vaccination. Between November 2007 and May 2014, 273 healthy children aged between 1.1 and 14.5 years (median : 1.7 years) underwent varicella vaccination. Before and 4 to 6 weeks after vaccination, the antibody titers were measured using an immune adherence hemagglutination (IAHA) assay and a glycoprotein-based enzyme-linked immunosorbent assay (gpELISA). In addition, side reactions were examined during the four-week period after vaccination. Children who did not show IAHA seroconversion (PVF) were recommended to receive a booster vaccination, and the measurement of antibody titers and an assessment of side reactions were performed after the booster dose. In May 2015, a questionnaire was mailed to each of the 273 participants to investigate whether they had developed varicella and/or herpes zoster after vaccination. After initial vaccination, the IAHA seroconversion rate was 75% and the mean antibody titer (Log2) with seroconversion was 4.7, while the gpELISA seroconversion rate was 84% and the mean antibody titer (Log10) with seroconversion was 2.4. Among children with PVF, 54 received booster vaccination within 81 to 714 days (median : 139 days) after the initial vaccination. After booster vaccination, the IAHA seroconversion rate was 98% and the mean antibody titer (Log2) with seroconversion was 5.8. Both the seroconversion rate and the antibody titer were higher compared with the values after the initial vaccination (p < 0.01). After booster vaccination, the gpELISA seropositive rate was 100% and the mean positive antibody titer (Log 10) was 3.6 ; similar results were obtained for the IAHA assay, with a significantly higher, antibody response than that after the initial vaccination (p < 0.01). Side reactions were generally minor, including fever (≥ 37.5 degrees C), rash at the injection site, and rash at other sites. There were no significant differences in the incidences of side reactions between the initial and booster vaccinations. A total of 185 participants responded to the questionnaire (response rate : 68%), and the period between receiving the initial vaccination and their response to the questionnaire ranged from 1.0 to 7.5 years (median : 4.0 years). The prevalence of breakthrough varicella after the initial vaccination was 17% among seroconverters who did not receive booster vaccination and 14% among non-seroconverters who received booster vaccination, showing no significant difference between the two groups. In conclusion, there are no safety issues regarding the administration of a booster vaccination to children with PVF after an initial varicella vaccination, and,a good antibody response can be expected.

Background: For effective non-routine vaccination in women of childbearing age as a countermeasure against congenital rubella syndrome (CRS), it is important to know the specific age group in which fewer members have rubella immunity., Methods: We analyzed rubella hemagglutination-inhibition (HI) antibody data (about 890,000) accumulated from 2008 through 2013 at a commercial diagnostic laboratory, which originated from the serum specimens sent mainly from obstetrics & gynecology clinics and hospital departments in Japan. Changes during the above period in the pattern of the annual curve for the prevalence of rubella antibodies (HI antibody titer > or = 8) by age, were observed., Results: The antibody prevalence among women in their twenties decreased gradually from 2008 to 2013. However, the prevalence at < or = 22 years of age in the 2013 specimens was found to have risen, which we believe is the effect of the five-year interim vaccination program for high-school students that began in 2008. The lowest antibody prevalence at 23 years of age in 2013 corresponded to the highest numbers of female patients at the same age in the 2013 rubella epidemic among the adult population., Conclusions: The analysis of extensive data accumulated for rubella antibodies assayed at the commercial diagnostic laboratory was useful for monitoring the susceptibility of the women to rubella infection in Japan, and that the Oct. 1987 to Mar. 1990 birth cohort (age 23 - 25 in 2013) is a high-risk group for CRS.

Aim: Measles during pregnancy has deleterious effects on both perinatal and maternal outcomes. In Japan, local epidemics of measles and cases of measles during pregnancy are still being reported; therefore, the seroprevalence of antibodies to measles is suspected to be still not sufficient. The aim of this study was to analyze the seroprevalence of antibodies to measles in Japanese pregnant women and estimate the percentage of these women who require vaccination or revaccination against measles., Material and Methods: We analyzed the seroprevalence of immunity to measles by the neutralization test in 10 349 pregnant women in the first trimester managed at the National Center for Child Health and Development between February 2004 and December 2010. The neutralization test titers were interpreted as follows: ≧1:8, seropositive; =4, low-positive; ≦4, seronegative., Results: Of the total number of pregnant women tested, 7408 (71.6%) were seropositive, 1864 (18.0%) were low-positive, and 1079 (10.4%) were seronegative for measles antibodies, respectively., Conclusion: Our results revealed that 28% of our pregnant population was seronegative or low-positive for measles antibodies, and thought to require revaccination or vaccination. Screening for measles immunity might be advisable for women of childbearing age., (© 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.)

Background: Respiratory syncytial virus (RSV) infection is a major cause of hospitalization during the winter among infants and young children. In 2002 palivizumab was introduced to high-risk infants for RSV hospitalization in Japan. It is important to characterize the hospitalized children due to RSV infection after the introduction of palivizumab., Methods: A survey was conducted to collect the data from the hospitalized children at 12 participating hospitals during the winter of 2007., Results: From October 2007 through April 2008, 8163 children were admitted to participating hospitals, with RSV infection accounting for 811 of those hospitalizations. Mean age in children with RSV infection at hospitalization was 12.4 ± 12.7 months, and children under 24 months of age accounted for 86.4%. The mean gestational age of those at birth was 38.0 ± 2.6 weeks, with 82.4% of the children born at term. Palivizumab was administered in 24 cases of RSV infection, while there were 28 patients who were not treated with palivizumab, even though they met the indication for palivizumab. Death, in a total of five cases, occurred in children who were not treated with palivizumab., Conclusions: Palivizumab has been widely used in high-risk infants who were covered by health insurance, and most of the hospitalized children with RSV infection in the study hospitals were not treated with palivizumab., (© 2011 The Authors. Pediatrics International © 2011 Japan Pediatric Society.)

Breast milk contains immunological factors, such as IgA antibody, which help to prevent infectious diseases. A total of 197 paired samples of colostrum and breast milk was collected from postpartum mothers in Gunma City, Japan, and examined for anti-rubella IgA antibody by enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB). The anti-rubella virus IgA ranged from 0.5 to 78.5 U/ml with a mean of 6.05 U/ml and a median of 3.6 U/ml in colostrum, and from 0.5 to 32.7 U/ml with a mean of 2.74 U/ml and a median of 2 U/ml in milk. The differences between the means of titers of total IgA and anti-rubella virus IgA in colostrum and in milk were significant statistically. The levels of anti-rubella virus IgA in both colostrum and breast milk correlated positively with the anti-rubella virus hemagglutination inhibition (HI) titers in the sera of mother, indicating that the levels of these different classes of antibodies correlated. Based on WB, anti-rubella virus IgA in both colostrum and breast milk reacted with the rubella viral protein E1 and C, but not with the E2 protein., ((c) 2010 Wiley-Liss, Inc.)

This study aimed to assess the outcome of cardiovascular diseases for patients with chronic active Epstein-Barr virus infection (CAEBV). The study enrolled 15 patients (7 boys and 8 girls) who fulfilled the diagnostic criteria for CAEBV, including 10 patients with T-cell type and 3 patients with natural killer (NK)-cell type. The median age at the CAEBV onset was 6.3 years (range, 1.2-17.8 years). Regular cardiologic studies were performed during the median follow-up period of 8 years (range, 2-20 years). Nine patients (60%) had cardiac diseases including coronary artery lesion (CAL) (n = 4, 44%), decreased left ventricular ejection fraction and pericardial effusion in (n = 3, 33%), complete atrioventricular block (n = 1), and sudden arrest (n = 1). The frequency of fever (78%, p = 0.04) or cytopenias (100%, p = 0.01), as the major symptom among patients with cardiac complications, was higher than among those without complications. The median time from disease onset to detection of CAL was 3.4 years (range, 1.8-8.6 years). The mean z-score increased to 3.98. Seven patients (78%) with cardiac complications died of disease progression, hematopoietic stem cell transplantation-related events, or both. In two patients, CAL regressed after allogeneic cord blood transplantation. Among CAEBV patients, CAL was the most common cardiac complication and could not be controlled without the eradication of EBV-infected T- and NK-cells.

Background: H5N2 avian influenza virus infection of humans has not been reported thus far. The first H5N2 avian influenza infection of poultry in Japan occurred in Ibaraki., Methods: The subjects were workers at 35 chicken farms in Ibaraki Prefecture, where the H5N2 virus or antibody was isolated from chickens. None of the subjects exhibited influenza symptoms. The H5N2-neutralizing antibody titers of the first and second paired sera samples were compared. To investigate the possible factors for this increase, the H5N2-neutralizing antibody titer (1:40 or more) was calculated for the second samples. A logistic regression analysis was performed to examine the association of these factors with H5N2-neutralizing antibody positivity., Results: We performed Wilcoxon matched-pairs signed-ranked test on data collected from 257 subjects, and determined that the H5N2 antibody titers of the second paired sera samples were significantly higher than those of the first samples (P < 0.001). The H5N2 antibody titers of paired sera of 13 subjects without a history of seasonal influenza vaccination within the previous 12 months increased 4-fold or more. The percentage of antibody positivity was 32% for subjects with a history of seasonal influenza vaccination (28% of all subjects) and 13% for those without a history of the same. The adjusted odds ratio of H5N2-neutralizing antibody positivity was 4.6 (95% confidence interval: 1.6-13.7) for those aged over 40 and 3.1 (95% confidence interval: 1.6-6.1) for those with a history of seasonal influenza vaccination within the previous 12 months., Conclusion: The results suggest that this may have been the first avian influenza H5N2 infection of poultry to affect humans. A history of seasonal influenza vaccination might be associated with H5N2-neutralizing antibody positivity.

To analyze human herpesvirus 6 (HHV-6) infection in adult living related liver transplantation, we performed a virological analysis, including viral isolation, serological assay, and real-time polymerase chain reaction, of serially collected blood samples from 67 recipients. In addition, cytokine levels were measured to determine their role in viral reactivation. HHV-6 was isolated from only 4 recipients (6.0%), and viral DNA was detected in 15 (22.4%) of the 67 recipients. A significant increase in HHV-6 immunoglobulin G antibody titers was observed in 19 (28.4%) of the 67 recipients. Finally, 26 recipients (38.8%) had HHV-6 reactivation 2-6 weeks after transplantation. HHV-6 associated clinical features were analyzed in the 17 recipients presenting with either viremia or DNAemia. Two recipients with viremia and 3 recipients with DNAemia had unexplained fever at the time of viral infection. An increase in aminotransferase levels was observed in 2 recipients with viremia and 3 recipients with DNAemia. Recipients with liver cirrhosis caused by hepatitis B virus or hepatitis C virus infection as the underlying disease were more likely to have HHV-6 infection (P = 0.025). Mortality at the last follow-up in recipients with HHV-6 reactivation was significantly higher than in those without viral reactivation (P = 0.0118). Plasma interleukin-6 levels were significantly higher in the recipients with HHV-6 viremia than in the recipients without viremia at 4 weeks post-transplant (P = 0.0411). Moreover, tumor necrosis factor alpha levels were also higher in recipients with HHV-6 viremia (P < 0.0001) or reactivation (P = 0.0011) than in recipients without viremia or reactivation 4 weeks post-transplant., ((c) 2007 AASLD.)

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.

Background: Transmission routes of hepatitis A virus (HAV) in Japan have changed. The present study investigated changes of transmission routes in relation to genetic drift., Methods: All 60 patients who were admitted between 1993 and 2003 with a diagnosis of hepatitis A were retrospectively analyzed. Nucleotide sequences of the VP1/2A region of the HAV recovered from their sera were determined., Results: The suspected transmission routes were household contact, 19 (31%); food or waterborne, 16 (27%); homosexual activity, 11 (18%); international travel, 4 (7%); and unknown 10. (17%). The 11 patients presumably infected through homosexual activity were found exclusively in 1998 and 1999. The proportion of patients exposed through homosexual behavior and household contact was higher in those 2 years than in other years. Nucleotide sequences could be determined for 58 patients. Fifty-seven of the 58 sequences belonged to genotype IA HAV, with less than 10% nucleotide diversity. Of the 27 sequences isolated during 1998 and 1999, 25 had an identical nucleotide sequence regardless of the suspected transmission route. In contrast, sequences obtained in the other years differed from one another. A phylogenetic tree constructed from sequences recovered from patients without a history of travel abroad showed several clusters., Conclusions: Our results suggest that (1) HAV acquired through homosexual activity may be transmitted to nonhomosexual individuals; (2) hepatitis A in metropolitan areas in Japan is caused mainly by sporadic infection with genotype IA HAV; and (3) several subtypes of genotype IA HAV are endemic in Japan.

Recent reductions in numbers of human and equine Japanese encephalitis (JE) cases in Japan have seen calls to end JE vaccination. Here, we analyzed yearly variations of natural JE virus activity, using sera collected serially in 1998-2003 from racehorses residing in Ibaraki and Shiga prefectures, both located in central Japan. A total of 208 sera from 24 individuals in Ibaraki and 259 from 27 in Shiga were examined for antibodies to JE virus nonstructural 1 (NS1) protein, a marker of natural infection. The natural infection rate in epizootic seasons, which was determined by a significant increase in NS1 antibody level, was 4.2-26.7% in Ibaraki and 0-41.7% in Shiga, indicating that high levels of JE virus activity still existed in central Japan.

Following gelatin-containing varicella vaccine (1994-1999: 1,410,000 distributed doses), 28 serious anaphylactic reactions and 139 non-serious allergic reactions were reported, with no serious and only five non-serious reactions following gelatin-free vaccine (1999-2000: 1,300,000 distributed doses). All nine sera available from children with serious reactions tested positive for gelatin-specific IgE, whereas 55 of the 70 available from those with non-serious reactions were positive, with one false positive. There was no correlation between gelatin-specific IgE antibody titers and severity of allergic reaction. Post-immunization anti-varicella antibody titers were comparable for both gelatin-free and gelatin-containing vaccine groups. The new gelatin-free varicella vaccine is thought to be safe, with similar immunogenicity to the earlier gelatin-containing vaccine.

Twenty-two and 37 infants and young children received two doses of influenza HA vaccine before the 2001-2002 influenza season and before the 2002-2003 season, respectively. Two or three serial specimens were obtained, before and 1 month after the first vaccination as well as 1 month after the second vaccination. Infants showed a significantly poor HI antibody rise and lymphocyte response compared with young children aged > or =12 months. Time kinetics of the lymphoproliferative responses to influenza antigen among young children varied whereas their activities in infants were typically negative before immunization and increased after vaccination. Infants responded poorly to HA influenza vaccine compared with young children.

In most cases of hepatitis E in Japan, patients acquire the viral infection abroad in countries where hepatitis E is endemic. However, in Nagano Prefecture, Japan, we encountered a patient with hepatitis E who had never been abroad. The diagnosis was made on finding hepatitis E viral RNA and antibodies against the virus in the serum. Prompt normalization of liver function test occurred without medication. The nucleotide sequence of the virus isolated from this patient was closely related to the sequence of previously isolated viruses of genotype III. The source of infection could not be identified.

Japanese encephalitis virus (JEV) causes fatal diseases in equines as well as humans. In Japan, racehorses are vaccinated with inactivated JE vaccine every year and no equine JE cases have been reported since 1986. However, the current reduction in JEV activity in nature has raised an argument against the requirement of continuous vaccination. Here, we studied natural infection rates in racehorses to address the issue. To identify naturally-infected individuals from vaccinated populations, we used an immunostaining method for detecting antibodies to JEV nonstructural 1 (NS1) protein. A total of 779 horses in eight nationwide locations showed NS1 antibody prevalences ranging from 15 to 73%. NS1 antibody prevalences among 2-year-old individuals that had spent one epizootic season in a particular location, therefore representing annual infection rates, were 15-67%. Individuals aged >/=3 years showed higher NS1 antibody titers than the 2-year-olds, suggesting that NS1 antibody levels were boosted by repeated exposures to JEV antigen over >/=2 epizootic seasons. These results indicate that horses in Japan are exposed to natural JEV infections, confirming the need for continuous vaccination for protecting horses from JE.

The ELISA we developed was able to determine the antigen content and was suitable for a potency test, and we described a relative potency assay method which determines the potency of test vaccines by comparing the ELISA value of a test vaccine to that of a reference vaccine. In the present study, we standardized the reference vaccine used for determining the potencies of test vaccines, and established a potency test by ELISA. We evaluated the proposed reference vaccine by the neutralizing antibody responses in dogs after vaccination, by the challenge protection test in guinea pigs (GP potency test), which is the earlier official potency test used in Japan, and by the NIH potency test, which is widely used throughout the world. The results showed that a 4-fold dilution of the proposed reference vaccine induced sufficient immunity in dogs. A 3-fold dilution of the proposed reference vaccine passed the GP potency test. The international units (IU) calibrated by the NIH potency test were 3.7 IU/dose. From the results and the WHO recommendation that veterinary rabies vaccines should have a potency of at least 1.0 IU/dose, we determined to dilute the proposed reference vaccine by 3 fold and regarded it as the reference vaccine. Finally, we confirmed that there is a good agreement between the results of the potency test by ELISA and the results of the GP potency test. The establishment of the potency test by ELISA has made it possible to monitor the potency in the production process and has contributed to the stable production of the vaccine.

Bovine viral diarrhea virus (BVDV) has been segregated into two genotypes, type 1 and type 2. To determine the efficacy of the commercially available bovine viral diarrhea type 1 vaccine used in Japan against BVDV type 2, calves were infected with BVDV type 2 strain 890 4 weeks after administration of the vaccine. The vaccinated calves did not develop any clinical signs and hematological changes such as observed in unvaccinated calves after the challenge. Furthermore, the challenge virus was not recovered from the vaccinated calves throughout the duration of the experiment, whereas it was recovered from all unvaccinated calves. The bovine viral diarrhea vaccine used in Japan is efficacious against infection with BVDV type 2 strain 890.

Three cattle were experimentally infected with bovine herpesvirus type 4 (BoHV-4), strain B11-41, isolated from the spinal cord of a cow, and monitored for clinical symptoms. None of them showed any clinical signs except increases of leukocyte numbers in two of them, and the body temperature remained normal throughout the experiment. Antibody titers against BoHV-4 continuously increased for one month and were maintained at a high level for more than 1 year by enzyme-linked immunosorbent assay (ELISA). The virus was isolated only from serum and peripheral blood leukocytes (PBL) of one cow in the early stage of infection, but the viral genome was detected in PBL continuously by PCR. When they were euthanized, the viral genome was detected in the lymph nodes and nervous tissues such as medulla, spinal cord, and trigeminal ganglion. These results indicate that cattle are infected with the virus latently and persistently, and the latency site would be in the tissues of the central nervous system as well as lymphoid tissues. When a seroepidemiological survey was performed on antibodies to BoHV-4 among cattle in Japan by ELISA, the rate of antibody-positive cattle was 8.9% and they were found irregularly on certain farms.

Borna disease virus (BDV)-specific antibodies were monitored in Misaki feral horses annually for 4 years using an electrochemiluminescence immunoassay (ECLIA). Among 130 horses examined, 35 (26.9%) with an ECLIA count above 1000 once or more were judged as BDV seropositive. Throughout the study period, p24 antibodies were more frequent than p40 antibodies in almost all positive animals. Among the 35 seropositive horses, the ECLIA count was consistently high in 12 cases. Eleven horses seroconverted from negative to positive and 7 underwent reversal. The count in the remaining 95 horses (73.1%) remained low for 4 years and these animals were judged as seronegative.

A rare subset of HIV lymphoma, known as primary effusion lymphoma (PEL), is a high-grade tumour carrying human herpes virus 8 (HHV-8). Very little is known about genomic aberration in PEL, and only a few HIV-negative PEL have been reported. Here we report the results of chromosomal analysis and comparative genomic hybridisation (CGH) conducted to detect regions of gain and loss, in five HIV-negative Japanese cases of HHV-8-negative PEL. All patients except one (35-year-old female) were elderly men and the morphologic examination showed large cell type. PEL expressed B-cell-associated and activation-associated antigens, and exhibited clonal immunoglobulin genes. No HHV-8 was detected in all four examined cases, but Epstein-Barr virus (EBV) was detected in one case. Genomic abnormalities and aberrations were identified in all HHV-8/HIV-negative PEL. CGH studies showed gain in 19 of 24 chromosomes. Gains of 3q13-27, 8q24, 10q21-23 and Yq were detected in two of the five cases, but other gains were noted in each case. Chromosomal analysis revealed complex abnormalities both in numbers and structures. Burkitt lymphoma-associated t(8;22) was detected in one case, but +8 chromosome and c-myc amplification were detected in the other three cases by Southern blot and/or fluorecence in situ hybridization (FISH). Abnormality of chromosome 8, which associates with c-myc, was detected in four of the five HHV-8/HIV-negative PEL. However, the other common genomic abnormalities of HHV-8/HIV-negative PEL were not detected in our study, but the complex abnormalities seemed to be true rather than the usual large B-cell lymphoma. Our results suggest that multi-step genomic abnormalities might be associated in HHV-8/HIV-negative PEL tumorigenesis.

We analyzed anti-Cytomegalovirus (CMV) IgG and IgM antibody (EIA) and anti-Epstein-Barr virus (EBV) viral capsid antigen (VCA) IgG and IgM antibody (FA) in adults during 1994-1999. We examined these IgM sero-positive patient's medical records, and diagnosed CMV mononucleosis and EBV mononucleosis. Anti-CMV antibody positive rates decreased from 87.6% in 1994 to 77.8% in 1999. Especially in twenties, anti-CMV antibody positive rates decreased from 65.2% in 1994 to 53.3% in 1999. On the other hand, anti-EBV VCA antibody positive rates were not changed (91-94%). Number of cases of CMV mononucleosis increased from 2 cases in 1994 to 16 cases in 1999, but EBV mononucleosis was not changed. These results suggested that increasing cases of CMV mononucleosis was influenced by decreasing anti-CMV antibody positive rate.

Clinical investigations and hematological, serum biochemical, and serological surveys were carried out on 11 male and 6 female Iriomote cats (Felis iriomotensis) in Japan. Examined Iriomote cats were considered clinically healthy by the inspection for the general physical conditions. However, urinalysis suggested the inflammation of the urinary tract in all the cats. Antibody for feline panleukopenia virus was positive in one of the examined Iriomote cats, which suggested a previous infection.

Purpose: A seroepidemiological study of hepatitis E virus (HEV) infection was conducted in Japan, where HEV infection is not considered endemic., Methods: IgG and IgM class antibodies to HEV were measured with a newly developed enzyme-linked immunosorbent assay in which recombinant virus-like particles were used as an antigen. A total of 1253 individuals (401 males and 852 females; age range, 6-89 years) were enrolled from two different areas: area 1 (n = 478), in which hepatitis C was endemic; and area 2 (n = 775), in which it was not endemic., Results: The HEV antibody (IgG class) positive rate was 6.7% in area 1 and 4.6% in area 2. Similarly, the HAV antibody (IgG class) positive rates were 65.3% and 72.3%. The age- and sex-specific prevalence of both HAV and HEV antibodies was quite similar in the two areas, and the HAV antibody positive rate clearly increased with age in both males and females. On the other hand, the HEV antibody positive rate showed a slight tendency to increase with age in males, but not in females. None of the 32 individuals with the HEV antibody who were interviewed had a history of visiting countries in which hepatitis E was endemic. In both areas, the mean age, percentage of males, and HAV antibody positive rate were significantly higher in the group of individuals with the HEV antibody than in the group of those without it, according to conventional statistical analyses. Of the three factors age, male sex, presence of HAV antibody, and the area factor, only male sex was statistically significant (P < 0.001) on multivariate logistic regression analysis. Two (0.2%) of the total of 1253 individuals were positive for the IgM class antibody to HEV., Conclusions: Our results suggest the possibility that HEV infection is circulating in Japan at a low level. HEV infection was associated with male sex, but not with HAV infection.

Study Objectives: The etiology of sarcoidosis remains unknown, but recently it was reported that human herpesvirus 8 (HHV-8) may be detected in sarcoid tissue in a high proportion of patients. This study was performed to determine whether HHV-8 is implicated in sarcoidosis in Japan., Materials and Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 100 patients with sarcoidosis and 100 healthy donors living in central Japan. Additionally, 19 samples of sarcoid tissue, 10 of tuberculous tissue, and 10 of lung cancers were examined. DNA was extracted from PBMC or tissue samples, and a hemi-nested polymerase chain reaction assay was performed for HHV-8 detection., Results: In the PBMC study, the detection rates for HHV-8 in patients with sarcoidosis and in normal donors were 2% and 1%, respectively, the difference not being significant (p>0.99). In the tissue study, HHV-8 was detected in 10.5% of sarcoid and in 15% of nonsarcoid tissues, again not a significant difference (p>0.99)., Conclusions: From these results, we conclude that HHV-8 is not implicated in sarcoidosis in Japanese patients.

Purpose: To determine the type of herpes simplex virus in acute retinal necrosis syndrome associated with herpes simplex virus., Methods: Herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, Epstein-Barr virus, and cytomegalovirus were examined by polymerase chain reaction in intraocular specimens from 16 patients with acute retinal necrosis syndrome. Anti-herpes simplex virus type 1 and anti-herpes simplex virus type 2 type-specific antibodies in serum from the patients were detected by enzyme immunoassay., Results: Of 16 patients with acute retinal necrosis syndrome, seven were polymerase chain reaction positive for herpes simplex virus type 2 and nine were positive for varicella-zoster virus. Anti-herpes simplex virus type 2 antibody was positive and anti-herpes simplex virus type 1 antibody was negative in the sera of the seven patients with herpes simplex virus type 2 DNA-positive acute retinal necrosis syndrome. In contrast, anti-herpes simplex virus type 2 antibody was absent in all nine varicella-zoster virus DNA-positive acute retinal necrosis syndrome patients., Conclusion: Herpes simplex virus type 2 has been demonstrated to be the major causative agent in acute retinal necrosis syndrome associated with herpes simplex virus by molecular biological and serological methods. Negative preexisting anti-herpes simplex virus type 1 antibody may play an important role in acute retinal necrosis syndrome associated with herpes simplex virus type 2.

It is well known that Epstein-Barr virus (EBV) is excreted from oral regions in the patients with infectious mononucleosis. We analyzed the prevalence of EBV in saliva and throat washings from healthy people in Japan by the polymerase chain reaction assay. EBV DNA was detected in 43 (90%) of the 48 throat washings from healthy adults (21 to 57 years old) and in 35 (38%) of the 93 salivas from healthy children (0 to 6 years old). The percentages of the EBV DNA-positive ratio in salivas increased in proportion relative to the increase of the children's ages. EBV type 1 was predominant and was detected in 86 and 94% of adults and children, respectively. Umbilical cord lymphocytes were transformed by some throat washings from EBV seropositive donors. EBV DNA was detected in throat washings from two healthy adults whose EBV antibody was not detected. In both cases, higher amounts of EBV DNA were detected in their peripheral blood mononuclear cells than in those of other, EBV antibody-positive donors. These results demonstrated the incidence of EBV excretion in oral regions of healthy individuals in Japan and defined a novel type of EBV infection in healthy adults.

A total of 15 (T-1-T-15) domestic cats with neurological disorders in Tokyo area were examined for association with Borna disease virus (BDV). None had detectable antibodies to feline immunodeficiency virus (FIV), feline leukemia virus, feline infectious peritonitis virus and Toxoplasma gondii, and only cat T-8 had detectable antibody to FIV. Serological and molecular epidemiological studies revealed a significantly high prevalence of BDV infection in these cats: antibodies against BDV p24 and/or p40 proteins in 10/15 (66.7%) and p24 and/or p40 RNA in peripheral blood mononuclear cells in 8/15 (53.3%). Further, in situ hybridization and immunohistochemistry analyses of the autopsied brain samples derived from one of the cats (T-15) revealed BDV RNA predominantly in neuronal cells in restricted regions, such as olfactory bulb and medulla of cerebrum. Thus, BDV is present in Japanese domestic cats with neurological disorders at a high prevalence.

The rubella vaccination rate remarkably decreased in Japan after a revised vaccination law was passed in 1994, as a result of which individual vaccination replaced mass vaccination at each school. We investigated the rubella antibody of 887 students and the accumulative rubella vaccination rate in an area of Okayama. The vaccination rate in Ibara, where children were inoculated individually, was 22.8%. In contrast, in Yoshii, where the vaccination was done as mass at each school, the rate was 89.8%. After 2003, when the transit period of the law ends, children 12-90 months of age will be the subjects for rubella vaccine. We presume that the vaccination rate in the future will reach 40-50% through accumulative vaccination. Such a rate of vaccination cannot prevent rubella epidemics and congenital rubella syndrome. In addition, an individual's rubella vaccination is generally done once during lifetime. We doubt whether the immune response induced by a vaccine can be maintained for more than 20 years without boosting. We think that a second vaccination is necessary for at least young girls, and this should be performed as mass at each school.

A sero-epizootiological survey was conducted for Japanese encephalitis virus (JEV) and Getah virus (GeV) at 10 to 20 regional horse race tracks from 1991 to 1997 in Japan. It was observed that geometrical mean (GM) antibody titer to JEV and GeV was 10 to 50 times higher than others at several race courses (RCs) almost every year. Of them, several race horses showing high antibody titer, which were suggested to be infected with the virus, were also observed in this survey. These data suggested that the viruses have spread among race horses almost every year in Japan, although, fortunately, no horse showing clinical illness due to these viruses was observed. The calendar years and the names of the race courses in which the spread of JEV was suggested were Sonoda and Nakatsu RCs in 1991, Nakatsu RC in 1992, Arao RC in 1993, Nagoya RC in 1994, Kaminoyama, Urawa, Saga and Arao RCs in 1995 and Ooi and Saga RCs in 1997. Spread of JEV was not observed in 1996. The calendar year and name of the race courses at which the spread of GeV was suggested were at Ooi, Sonoda and Nakatsu RCs in 1991, Arao RC in 1992, Nakatsu RC in 1994 and 1995, Funabashi RC in 1996, Saga RC in 1997. It was suggested that surveillance of JEV and GeV should be continued in the future in at least the southern to middle parts of Japan. It has also been suggested that it is necessary to promote the wider use of vaccines to persons related to horse racing.

Objective: An immunoassay for antibodies against an hepatitis G virus (HGV) protein (anti-E2) was recently developed that might serve as a useful marker for diagnosing recovery from HGV infection., Methods: We investigated the intraspousal transmission of GB virus C/hepatitis G virus (GBV-C/HGV) using both reverse transcription hemipolymerase chain reaction (RT-hemi-PCR for the 5' untranslated region) and a recently developed anti-E2., Results: Thirty-two GBV-C/HGV-infected index subjects were selected from an hepatitis C virus hyperendemic area in Japan. Of the 32 subjects, seven (6.4%) were GBV-C/HGV RNA-positive, 24 (21.8%) were anti-E2-positive, and one (0.9%) was both GBV-C/HGV RNA- and anti-E2-positive. Among the 32 spouses of these subjects, GBV-C/HGV RNA, anti-E2, and both GBV-C/HGV RNA and anti-E2 positivity were detected in 0, 6, (18.8%), and one (3.1%) spouses, respectively (the total prevalence of GBV-C/HGV was 7 spouses [21.9%]). Thus, the intraspousal transmission of GBV-C/HGV was undeniable in these seven couples. The respective positive rates of 175 sex- and age-matched controls were 7 (4.0%), 26 (14.9%), and 0 (the total prevalence of GBV-C/HGV was 34 [19.4%]). No significant difference in positive rates was observed between the subjects/spouses and the controls. Five spouses among the seven couples who were positive for any of GBV-C/HGV markers had parenteral risk factors such as blood transfusion, acupuncture, and major surgery., Conclusion: Based on these observations, we cannot draw a definitive conclusion that intraspousal transmission of GBV-C/HGV had occurred among these seven couples.

On March 22, 1998, a mature, male, hyposthenic Pacific striped dolphin (Lagenorhynchus obliquidens) was stranded at Aoshima Beach in Miyazaki prefecture, Japan. A necropsy performed 14 hr after death revealed mild diffuse congestion and edema of the leptomeninges and mild pulmonary atelectasis. Histopathologically, non-purulent inflammatory were observed throughout the cerebrum, thalamus, midbrain, pons, medulla oblongata, and spinal cord. Hematoxylin and eosin stain revealed no viral inclusion bodies. Immunohistochemistry using a monoclonal antibody against nucleoprotein of canine distemper virus (CDV-NP) revealed a number of CDV-NP-positive granular deposits in the cytoplasm and cell processes of the degenerating or intact neurons. The present paper is a first report of spontaneously occurred morbillivirus infection in a dolphin at the Pacific Ocean around Japan.

Serum samples collected from 739 free-living wild birds of 44 species from Gifu, Mie and Hyogo Prefectures in Japan during the period 1989 to 1997 were tested for antibodies to infectious bursal disease virus (IBDV) serotypes 1 and 2 by a virus neutralization test. Serological evidence of infection with serotypes 1 and 2 was found in 15 (2%) of the sera of 6 species and 36 (4.9%) of the sera of 11 species, respectively. Antibodies to IBDV were detected from both sedentary and migratory species. These findings suggest that free-living wild birds have an important role in the natural history of IBDV. These findings raise the possibility that the IBDV prevalent in the breeding grounds of these birds in other countries could be imported by the migratory species. This is the first report of an extensive serological survey of IBDV in wild birds.

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.

Seroprevalence of Bartonella henselae and Toxoplasma gondii was investigated among 471 pet cats obtained from seven private animal hospitals in Kanagawa and Saitama Prefectures during the period from May 1994 to June 1995. 'Furthermore, 67 randomly selected from the 471 serum samples were examined for the feline immunodeficiency virus (FIV) antibody and feline leukemia virus (FeLV) antigen. The antibody to B. henselae was examined by an indirect immunofluorescent antibody test. T. gondii, FIV and FeLV infections in cats were detected with respective commercial kits. Of the cat serum samples tested, 43 (9.1%) were found to be seropositive for B. henselae and 41 (8.7%) for T. gondii. The B. henselae-positive rate (12.9%) of male cats was significantly higher than that (5.2%) of female cats. On the other hand, T. gondii-positive rate was 9.1% in male and 8.7% in female cats and there was no significant difference in the positivity between sexes. The positive rate in each hospital varied from 0 to 19.5% for B. henselae and 4.9 to 18.8% for T. gondii. The ages of B. henselae- and T. gondii-positive cats were distributed from < 1-year-old to 14-year-old and the seropositivity increased with age of cats. Of the 67 cat serum samples, 16 and 6 cases were positive for FIV and FeLV, respectively. There was no relationship between these viral and B. henselae infections in cats.

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.

We examined seroprevalence of cytomegalovirus (CMV) of pregnant women for 18 years since 1980 with complement-fixing antibody (CF) and specific IgG antibody. CF seropositive rate decreased gradually from 93.2% to 66.7%. CMV-IgG Seropositive rates were 87.4% in 1985, and 75.2% in 1996 to 1997. Age and parity of pregnant women associated with the immune status; 35.6% of recent young primipara were susceptible.

In order to know the rate of occurrence of varicella among vaccinees (breakthrough varicella: BV), questionnaire postcards were sent to 593 healthy children who had received varicella vaccine (Oka strain) from March 1987 to December 1989. The questionnaire survey was repeated once a year until January 1996. The annual attack rate from the 1st to 3rd questionnaire was approximately 12%: however, from the 5th to 8th one it was 1-4%. To February 1996, the cumulative attack rate was 157/459 (34.2%). This rate was comparable to that among vaccinees who had confirmed seroconversion; namely, 51/132 (38.6%). These rates are much higher than those reported by other authors. All BV cases were clinically mild; even subjects who had received the vaccine 7 years prior to the disease showed mild symptoms. The high incidence may be partly explained by the regional epidemiology of varicella. The decrease in annual incidence with time after vaccination may be due to the following reasons: some vaccinees remained free from BV owing to reinforcement of their immunity from subclinical infection of varicella-zoster virus (VZV) and others from diminution of opportunity for exposure to VZV with increasing age. Varicella vaccine seems to be effective in modifying the symptoms of varicella, but not potent enough in protecting from VZV infection.