Seroepidemiology of hepatitis A virus infection in Japan: An area of very low endemicity

The incidence of hepatitis A virus (HAV) infection has declined significantly worldwide, including in Japan. A nationwide seroepidemiological study on hepatitis A in Japan has taken place almost every 10 years since 1973, and the last study was performed in 2003. In the present study, we describe the latest seroepidemiological pattern of hepatitis A in Japan using 7867 serum specimens obtained from healthy individuals collected between 2013 and 2017, approximately 10 years after the last study. Among them, 223 were anti-HAV positive. About 68% of individuals aged 60 years and older had anti-HAV antibodies, whereas only 1.1% of those aged below 60 years old had immunity; thus, almost all individuals younger than 60 years of age were HAV susceptible. In comparison with previous investigations, the susceptible population has increased and aged. According to data from the National Epidemiological Surveillance of Infectious Diseases (NESID) program, between 1989 and 2016, the proportion of patients with hepatitis A aged 60 years and older continuously increased with each year. The NESID data also suggested that recently, typical large foodborne outbreaks of hepatitis A have become rare, and cases tend to be reported among at-risk groups; overseas travelers contributed to 25% of hepatitis A cases, and in 2018, the first nationwide hepatitis A outbreak that affected mostly men who have sex with men was reported. The purpose of this study was to determine the current status of HAV infection in Japan, based on both seroepidemiology and the national surveillance data from the NESID.     

A slow spread of adenovirus type 7 infection after its re-emergence in Yamagata, Japan, in 1995

We have continued the epidemiological study on adenovirus type 7 (Ad7), which re-emerged in 1995 in Yamagata, Japan. Between 1999 and 2004, we isolated only four strains from 10,778 throat swab specimens among children with acute respiratory infections. A serological survey of 303 specimens revealed the antibody-positive rate against Ad7 to be 0-7.4% in children under 10 years of age in 2005, although it was 3.3-16.7% in 1997 and 0% in 1993. Our results suggest that a re-emergence does not always provoke a sudden major outbreak, even if the antibody-positive rate against Ad7 is low in the local community.     

黑龙江省乙型肝炎血清流行病学调查分析

为了掌握黑龙江省乙型肝炎(乙肝)病毒(HBV)流行现状和人群免疫水平,评价我省儿童乙肝疫苗预防接种效果,为制订乙肝预防控制策略提供依据。采用横断面调查方法,选择全省7个国家级疾病监测点,在每个监测点随机抽取2个乡级单位,1～59岁作为目标人群共调查3337名人群。黑龙江省1～59岁人群乙肝病毒表面抗原(HBsAg)阳性率、乙肝病毒表面抗体(抗-HBs)阳性率和乙肝病毒核心抗体(抗-HBc)阳性率经标化后为5.65%、55.45%、30.50%,1～3岁人群乙肝表面抗原阳性率明显低于15～59岁人群。1～3岁、4～10岁和11～14岁人群乙肝疫苗全程接种率为99.43%、89.40%和64.81%;首针及时接种率分别为86.64%、75.57%和49.87。城市和农村HbsAg阳性率分别为3.26%和5.04%。医院出生儿童乙肝疫苗首针及时接种率高于在家出生儿童。结果显示接种乙肝疫苗可明显提高抗-HBs阳性率,提高人群对HBV的免疫保护能力,降低HBsAg携带率。     

Enzootic hepatitis A infection in cynomolgus monkeys (Macaca fascicularis)

During a toxicology study in cynomolgus (long-tailed or crab-eating) monkeys (Macaca fascicularis), a randomly distributed incidence of significantly increased hepatic enzyme activity was observed. Premedication hepatic enzyme activity in all monkeys of this study was normal, but increased alanine aminotransferase (ALT) activity was found in 4 of the 24 animals 2 weeks after initiation of the study and in 10 of 24 at 4 weeks. A drug-related effect was considered unlikely initially because the increases were not doserelated, and a 3-year review of 655 cynomolgus monkeys revealed a 15–20% incidence of increased hepatic enzyme activity. Good correlation was subsequently established between increased hepatic enzyme activity, active hepatitis A virus (HAV) infection, and histomorphologic confirmation of hepatitis (chronic periportal inflammation). Follow-up viral serodiagnostic screening of resident macaques revealed an overall incidence of anti-HAV IgG in 80% (155/193) of cynomolgus and in 70% (14/20) of rhesus monkeys. Serial screening demonstrated that several initially negative monkeys became seropositive for anti-HAV IgG, and a few acquired active infection (anti-HAV IgM). Among newly acquired cynomolgus monkeys, 2.5% (2/80) had an acute HAV infection, and 35% (28/80) eventually tested positive for anti-HAV IgG while quarantined in the primate facility. The characterization of an enzootic HAV infection in incoming monkeys posed a significant risk for the primate colony and handlers. Rigorous sanitation, isolation, and quarantine procedures, including personnel training and additional protective clothing for personnel working in the primate colony, reduced tho potential for transmission and arrested the outbreak. Experimenters should be cautious in ascribing toxicity to a test article based solely on increased hepatic enzyme activity associated with chronic periportal inflammation.     

Seroepidemiological study of norovirus infection in Aichi Prefecture, Japan

The serological prevalence of IgG antibody to seven NoV strains (GI.1, GI.4, GII.3, GII.4, GII.10, GII.12 and GII.15) among inhabitants aged 1–62 years of Aichi Prefecture, Japan was studied. Age-related seroprevalence was measured by ELISA using baculovirus-expressed recombinant VLP antigens. Seropositive rates for all seven VLP antigens gradually increased with age. Among the tested antigens, the highest seropositive rate was for the GII.4 strain. This result is consistent with the recent epidemic of NoV infection due to GII.4 strain in Japan.     

Production of monoclonal antibodies with a controlled N‐glycosylation pattern in seeds of Arabidopsis thaliana

Seed‐specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild‐type (wt) plants and glycosylation mutants lacking plant specific N‐glycan residues. We demonstrate that 2G12 is produced with complex N‐glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N‐glycans significant amounts of oligo‐mannosidic structures, which are typical for endoplasmic reticulum (ER)‐retained proteins. A detailed subcellular localization study demonstrated the deposition of both antibodies virtually exclusively in the extracellular space, illustrating their efficient secretion. In addition, although a KDEL‐tagged version of 2G12 exhibited an ER‐typical N‐glycosylation pattern, it was surprisingly detected in protein storage vacuoles. The different antibody variants showed different levels of degradation with hardly any degradation products detectable for HA78 carrying GnGnXF glycans. Finally, we demonstrate functional integrity of the HA78 and 2G12 glycoforms using viral inhibition assays. Our data therefore demonstrate the usability of transgenic seeds for the generation of mAbs with a controlled N‐glycosylation pattern, thus expanding the possibilities for the production of optimally glycosylated proteins with enhanced biological activities for the use as human therapeutics.     

Seroepidemiology of human parechovirus types 1, 3, and 6 in Yamagata,Japan, in 2014

To clarify the seroepidemiology of human parechovirus type 1 (HPeV1), 3 and 6, neutralizing antibodies (NT Abs) were measured in 214 serum specimens collected in 2014 in Yamagata, Japan. The seroprevalence against HPeV1 was 100% in all age groups, while that against HPeV3 and HPeV6 was 79.4% and 66.8%, respectively, overall. The geometric mean titers of NT Abs against HPeV1, 3 and 6 were 755.2, 255.0 and 55.9, respectively, overall. Our findings indicate that HPeV1 is the most prevalent HPeV circulating in Yamagata, followed by HPeV3 and HPeV6.     

The infectivity and pathogenicity of hepatitis A virus live-attenuated vaccine strain H2 in type I interferon receptor-deficient mice

Hepatitis A virus (HAV) live-attenuated vaccine H2 strain has been approved for clinical use for decades with ideal safety profiles in nonhuman primate models and humans. Recently, type I interferon (IFN) receptor-deficient mice were shown to be susceptible to HAV infection. Herein, we sought to determine the infection and replication dynamics of the H2 in Ifnar^-/- mice that lack type I IFN receptor. Following intravenous injection, the H2 failed to cause obvious clinical symptoms in Ifnar^-/- mice, and no significant upregulation in serum alanine aminotransferase (ALT) levels was observed. Notably, the histopathological examination showed that there were significant focal infiltrations of lymphocytes and neutrophils in the portal area, but no focal necrosis was observed in liver tissues. Viral RNAs sustained in the liver, and the infectious virus could be recovered from the liver tissue until 42 days post-infection. More importantly, H2 infection induced obvious viremia and persistent viral shedding in feces. In addition, robust HAV-specific humoral immune responses were induced in Ifnar^-/- mice. Overall, our study revealed the safety profile of H2 in Ifnar^-/- mice, which not only helps understand the attenuation mechanism of H2, but also expands the application of the Ifnar^-/--/- mouse model for HAV studies.     

甲型肝炎病毒Vero细胞适应株的选育研究

将甲肝患者粪便中分离的甲型肝炎病毒在Vero细胞中进行适应性培养 ,选育高滴度适应株应用于甲肝灭活疫苗研究。在Vero细胞上连续传代 ,测抗原滴度和感染性滴度 ,满意后按WHO推荐的甲型肝炎灭活疫苗规程进行灭活疫苗试制研究。经Vero细胞 14次适应性传代后 ,病毒抗原滴度可高达 1∶2 5 6 0 ,感染性滴度为 8.2 3LogC CID50 /ml。试制的灭活疫苗HPSEC检测在 2 80nm时仅有一个高峰 ,SDS PAGE电泳 ,在 2 2kD、2 6kD和 33kD处有三条蛋白带 ,和HAVVP3、VP2和VP1的位置相同。ICR小鼠效力试验表明疫苗剂量 16 0 0EU/ml与Merck疫苗5 0U效果相似。通过研究获得了Vero细胞甲肝病毒适应株YN5株 ,初步证明可作为甲型肝炎灭活疫苗的候选毒株。     

甲、乙型肝炎联合疫苗接种恒河猴的初步观察

为了探索联合接种甲乙肝疫苗、实验性甲乙肝联合疫苗免疫恒河猴的安全性及免疫原性。实验中挑选了甲肝抗体阴性,乙肝两对半阴性,肝功能指标正常的健康恒河猴24只,随机分为10组。混合或分别接种,进行不同毒株的甲肝灭活疫苗与不同厂家的乙肝疫苗的配对效果比较。并接种了实验性甲乙肝联合疫苗;史克甲乙肝联合疫苗试验组。设甲肝单价灭活疫苗L8株、深圳乙肝单价疫苗作为对照。免疫方案0、4、24w。每只恒河猴接种lml。接种3d内,每天观察动物有无不良反应。接种1针和2针后4w内,每2w采集空腹静脉血,以后每4w采血1次检测抗HAV、抗HBs、ALT、AST直至40w。接种疫苗后4、8、24、28w穿刺肝组织做病理学检查。结果显示接种疫苗后3d内,所有恒河猴均无不良反应。ALT、AST无异常升高。4、8、24、28w肝组织无特殊病理改变。注射2针后4w,除3组外,其余各组抗HAV及抗HBs均阳转。3组抗HAV阳转时间迟至12w。全程免疫后12w(即40w),抗HAVGMT为258.75～37489.50mIU/ml;抗HBsGMT为8263.68～60008.064mIU/ml。甲乙肝疫苗联合免疫及实验性的甲乙肝联合疫苗接种恒河猴安全性及免疫原性均良好。     

Causes of vertical transmission of hepatitis B virus under the at‐risk prevention strategy in Japan

The number of hepatitis B virus (HBV) carrier babies has decreased markedly since the introduction in Japan of an “at‐risk” strategy for preventing HBV infection. However, elimination of HBV infection from our country appears difficult. To clarify the limitations of the at‐risk strategy for preventing vertical transmission of HBV, causes of vertical transmission in a single hospital were retrospectively analyzed. The following causes were presumed in 17 carrier pediatric cases: five patients had prenatal HBV infection, HBV infection during/after the immunization program was confirmed in five cases, two patients had prenatal infection or infection during the immunization program and three cases were caused by human error (by the patients' guardians). Because their mothers were HBV‐negative at screening and only developed acute hepatitis B in the perinatal period, another two cases (Cases 3 and 10) did not undergo immunization because they were not subjects of the at‐risk strategy. Sequence analyses in ten patients revealed genotype C (subgenotype, C2/Ce) in nine cases and genotype A (subgenotype, A2/Ae) in one case (Case 3). In Japan, HBV subgenotype Ae has recently been found more frequently among sexually active men with acute hepatitis. There are concerns that horizontal transmission of HBV from these men to their pregnant partners could increase. These data suggest clear limitations to the at‐risk strategy in Japan and the possibility that the increase in genotype A may influence vertical transmission of HBV.     

A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum

Current clinically assays, such as enzyme‐linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high‐throughput analysis. A novel assay based on magnetic beads and time‐resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti‐HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium‐labeled anti‐HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02–700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra‐ and interassay coefficients of variation were 4.7–8.7% and 3.8–7.5%, respectively. The performance of this assay was further assessed against a well‐established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X – 0.017, R = 0.989). In the current study, we demonstrated that this novel time‐resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high‐throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus. Copyright © 2013 John Wiley & Sons, Ltd.     

Complete coding sequence and molecular analysis of hepatitis A virus from a chimpanzee with fulminant hepatitis

Background Hepatitis A virus (HAV) infects both humans and non‐human primates, in experimentally infected chimpanzees is typically milder than in humans. In 1982, Abe and Shikata reported a first case of a chimpanzee with fulminant hepatitis caused by spontaneous HAV infection, and the underlying mechanisms of the disease remain unknown. Methods To characterize denoted CFH‐HAV, we conducted cloning and near full‐length sequence analysis. Results Phylogenetic analyses of VP1‐2A and complete sequence comparison between various genotypes and the sample sequence showed clustering in genotype IB. Based on BLAST analysis, the sequence was most closely related to the wild‐type (HM175/WT) isolate. Amino acid and nucleic acid similarities were 99.8% and 94.41%, respectively. Conclusions The chimpanzee may have been infected with human HAV genotype IB. The substitutions in VP2, VP4, 2B, 2C, and 3D, which may enhance virus proliferation, contributed to disease severity culminating in fulminant hepatic failure.     

Anti-phosphopeptide antibody, P-STM as a novel tool for detecting mitotic phosphoproteins: identification of lamins A and C as two major targets

A polyclonal, phospho-epitope-specific antibody (P-STM) was generated to detect the activated p21-activated kinase 2 (PAK2), based on the regulatory autophosphorylation site Thr(402) of PAK2 [Yu et al., 1998]. In this report, we show that this antibody can also recognize many phosphoproteins in mitotic HeLa and A431 cells. Signal of these phosphoproteins emerged after treating the cells with nocodazole and okadaic acid, and was highly detected in G2-M phase transition of HeLa cells released from double thymidine block. Immunofluorescence analysis revealed that P-STM strongly stained HeLa cells at prometaphase and metaphase, but not at interphase and anaphase. Interestingly, this staining pattern was almost identical to that obtained by staining with MPM2, a monoclonal antibody known to react with phosphoproteins in mitotic HeLa cells. However, the phosphoproteins detected by the two antibodies are quite different. Two-dimensional gel electrophoresis (2DE) and tryptic peptide fingerprint analysis by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry were employed to identify lamins A and C as two of the mitotic cell-specific phosphoproteins recognized by P-STM. Lamins A and C immunoprecipitated from nocodazole-treated cells, but not from untreated cells showed strong reactivity to P-STM, and this reactivity lost completely after protein phosphatase 2A treatment. In summary, our results show that P-STM represents a novel tool for detecting mitotic phosphoproteins, which are different from those recognized by MPM2, and that lamins A and C are the two prominent mitotic phosphoproteins detected by P-STM.     

The decline in antibodies to hepatitis C virus during antiviral therapy

The goal of the study was to analyze B-cell response to hepatitis C virus during antiviral therapy among responders and non-responders. The content of antibodies to individual structural and non-structural HCV proteins was investigated during two years in three groups of patients: initial responders, non-responders and a reference group (without therapy). Treated patients in all groups exhibited the decrease in antibodies to analyzed HCV proteins, but with different patterns. The first statistically significant differences in the decline of the virus-specific antibodies between initial responders and non-responders were observed within the first three months after the beginning of therapy. Some treated patients demonstrated the decrease in antibody levels to HCV proteins after the end of therapy.     

Spontaneous and experimental hepatitis A in Old World monkeys

Virologic, serologic, biochemical, and morphological data characterizing spontaneous hepatitis A (HA) in cynomolgus macaques (Macaca fascicularis) and green monkeys (Cercopithecus aethiops) are reported. Experimental HA was induced in macaques as a result of infection with human hepatitis A virus (HAV-h). Disease similar to human HA was induced in cynomolgus macaques by HAV isolates from spontaneously sick rhesus (M. mulatta) and green monkeys. This experimental model of HA in macaques can be used for vaccine and anti-viral preparation testing.     

细胞培养结合实时荧光RT-PCR法快速检测甲肝病毒滴度的研究

目的建立一种细胞培养与实时荧光RT-PCR相结合的快速检测甲肝病毒滴度的方法。方法根据甲肝病毒(HAV)L-A-1株5'端基因组序列,设计了2条基因特异性引物及一条探针,建立实时荧光RT-PCR法,结合细胞培养检测甲肝病毒滴度,并与ELISA检测法进行比较。结果实验中建立的方法能特异检测甲肝病毒,细胞培养8d检测病毒滴度为lg107.0CCID50/mL。同一样本重复检测3次,批内样本Ct值的变异系数最大为0.89%,批间样本Ct值变异系数最大为1.66%。建立的细胞培养结合实时荧光RT-PCR法(细胞培养8 d)与细胞培养ELISA法(细胞培养28 d)检测甲肝病毒滴度结果差异无统计学意义(P0.05)。结论该方法具有快速、灵敏、特异等优点,应用于疫苗常规检测有良好前景。