Natural and experimental infections of Japanese tree sparrows with Japanese encephalitis virus

20 to 37 per cent of wild Japanese tree sparrows had neutralizing antibodies to Japanese encephalitis virus (JEV). Sparrows free of antibodies were inoculated with 10,000 plaque-forming units of JEV. None of the birds became clinically ill. Virus could be detected in blood plasma during the first 3 days after inoculation but not thereafter. Neutralizing antibodies started to appear at 4 days after inoculation and the response was of variable duration, in some cases extending up to 200 days after inoculation.     

Japanese encephalitis and Japanese encephalitis virus in mainland China

Japanese encephalitis (JE), caused by Japanese encephalitis virus (JEV) infection, is the most important viral encephalitis in the world. Approximately 35,000–50,000 people suffer from JE every year, with a mortality rate of 10,000–15,000 people per year. Although the safety and efficacy of JE vaccines (inactivated and attenuated) have been demonstrated, China still accounts for 50% of the reported JE cases worldwide. In this review, we provide information about the burden of JE in mainland China and the corresponding epidemiology from 1949 to 2010, including the morbidity and mortality of JE; the age, gender, and vocational distribution of JE cases; its regional and seasonal distribution; and JE immunization. In addition, we discuss the relationships among vectors, hosts, and JEV isolates from mainland China; the dominant vector species for JEV transmission; the variety of JEV genotypes and the different biological characteristics of the different JEV genotypes; and the molecular evolution of JEV. Copyright © 2012 John Wiley & Sons, Ltd.     

Japanese encephalitis virus infection in fetal mice at different stages of pregnancy. II. Resistance to Japanese encephalitis virus infection

The relationship between the stage of pregnancy of mice at the time of Japanese encephalitis (JE) virus inoculation and the resistance of JE virus infection of their offsprings was investigated. It was found that there was a stronger resistance to JE virus infection in offsprings born of mothers inoculated with JE virus at nine to sixteen days before parturition than in offsprings of mothers inoculated at one to eight days or at seventeen to twenty days before parturition. Resistance of the offsprings to JE virus infection lasted up to the age of 180 days after birth.     

Immunological memory in latent Japanese encephalitis virus infection.

Long term B-cell memory to Japanese encephalitis virus (JEV) in latently infected mice was investigated by adoptive cell transfer. Both IgM and IgG memory were elicited by antigen challenge or cyclophosphamide induced reactivation of virus. A weak antigen-specific IgM response for a brief period and a strong IgG response were detected in Swiss albino mice exposed to secondary infection. A correlation between the secondary IgM antibody and protection against JEV challenge was observed in adoptive transfer experiments. This was abrogated by pretreatment of the serum with 2-mercaptoethanol. Similarly secondary immune splenic T-cells up to day 5 post-reactivation provided protection. These results suggest that a long term antigen-specific IgM and IgG memory was induced by JEV challenge in latently infected mice. Further, the role of IgM antibody and T-cells in the response of mice to secondary JEV infection has been shown.     

福建省新分离3株乙型脑炎病毒的分子特征鉴定

目的　确定福建省新分离乙型脑炎病毒 (JEV)的基因分型及其E区段氨基酸序列特征。方法　用逆转录聚合酶链反应 (RT PCR)扩增并克隆新分离JEV(0 2 4 1、0 2 4 3、0 2 10 2 )的PrM、E区段核苷酸序列 ,测序后应用ClustalX软件做碱基配对和比较分析 ,种系发生采用PHYLIP软件包分析。结果　新分离的 3株JEV属于基因Ⅲ型 ,E区段核苷酸和氨基酸与减毒活疫苗株SA 14 14 2株的同源性均在 96 %以上 ,在关键结构域有部分氨基酸差异。结论　福建省新分离的 3株病毒属于基因Ⅲ型JEV ,E蛋白与疫苗株相比有部分氨基酸差异     

Degradation of Japanese encephalitis virus by neutrophils.

The ability of neutrophils to degrade the phagocytosed Japanese encephalitis (JE) virion, via triggering of the respiratory burst and generation of toxic radicals has been investigated. JEV or JEV-induced macrophage derived factor (MDF) induces increase in intracellular oxidative signals with generation of superoxide anion (O2-), via activation of cytosolic NADPH and subsequent formation of hydrogen peroxide, with maximum activity on day 7 post infection. The response was sensitive to anti-MDF antibody treatment. Further, the study revealed rapid degradation of phagocytosed JE viral protein and nucleic acid. The viral protein degradation was partially dependent on the generation of toxic oxygen species as it could be abrogated by pretreatment of the cells with staurosporine.     

Modeling of persistent HeLa cell infections caused by different Japanese encephalitis virus clones

The authors developed two models of persistent infections of a HeLa cell clone with mildly pathogenic clones of the Nakayama and Peking I strains of Japanese encephalitis virus. The distinguishing features of these models included the noncytocidal nature of the infectious process, predominance of the small-plaque phenotype, further decrease of the infectious properties of the persisting viruses. Altogether during the observation period 84 subpassages of each of the two systems were made without any visible signs of cell degeneration.     

Epitope-blocking enzyme-linked immunosorbent assay to differentiate west nile virus from Japanese encephalitis virus infections in equine sera

West Nile virus (WNV) is now widely distributed worldwide, except in most areas of Asia where Japanese encephalitis virus (JEV) is distributed. Considering the movement and migration of reservoir birds, there is concern that WNV may be introduced in Asian countries. Although manuals and guidelines for serological tests have been created in Japan in preparedness for the introduction of WNV, differential diagnosis between WNV and JEV may be complicated by antigenic cross-reactivities between these flaviviruses. Here, we generated a monoclonal antibody specific for the nonstructural protein 1 (NS1) of WNV and established an epitope-blocking enzyme-linked immunosorbent assay that can differentiate WNV from JEV infections in horse sera. Under conditions well suited for our assay system, samples collected from 95 horses in Japan (regarded as negative for WNV antibodies), including those collected from horses naturally infected with JEV, showed a mean inhibition value of 8.2% and a standard deviation (SD) of 6.5%. However, inhibition values obtained with serum used as a positive control (obtained after 28 days from a horse experimentally infected with WNV) in nine separate experiments showed a mean of 54.4% and an SD of 7.1%. We tentatively determined 27.6% (mean + 3 x SD obtained with 95 negative samples) as the cutoff value to differentiate positive from negative samples. Under this criterion, two horses experimentally infected with WNV were diagnosed as positive at 12 and 14 days, respectively, after infection.     

Congenital infection of mice with Japanese encephalitis virus.

Transplacental transmission of Japanese encephalitis virus (JEV) when given intraperitoneally was demonstrated in pregnant mice as shown by isolation of the virus from placenta and fetal tissues. Furthermore, JEV could be isolated from the brain, liver, and spleen of newborn mice. The effect of JEV at different periods of gestation in pregnant mice was demonstrated for the first time, and the consequences of maternal infection on fetuses and neonates were studied. JEV infection during the 1st week of gestation caused a significantly higher number of fetal and neonatal deaths (66%) than during the 3rd week of gestation (13.8%). The number of abortions, stillbirths, and neonatal deaths was higher in infected mothers than in controls. No congenital abnormalities were found in any of the newborn mice. Sera obtained from 5-week-old health mice delivered by mothers infected during the 3rd week of gestation contained JEV hemagglutination inhibiting and immunoglobulin M antibodies. The results of these preliminary experiments show the usefulness of mice as a model for further elucidation of JEV infection during pregnancy and its effects on the fetus.     

Memory suppressor T cells in latent Japanese encephalitis virus infection.

The generation of secondary suppressor T (Ts) cells has been studied during latent Japanese encephalitis virus (JEV) infection of mice. The mice infected with JEV 27 weeks earlier, on challenge with the homologous virus, showed accelerated generation of secondary Ts cells; these appeared on Day 6, with peak activity on Day 8, and lasted for 27 days. The secondary Ts cells were Thy1.2+, Ly1-2+, antigen-specific, and acted in a dose-dependent manner. The secondary Ts cells could also be generated by reactivation of the JEV in latently infected mice. The findings thus show the presence of memory suppressor T cells in mice latently infected with JEV that can be stimulated to produce secondary Ts cells by exogenous or endogenous virus challenge. This phenomenon could help to persistence of the virus.     

Induction of suppressor cells in Japanese encephalitis virus infected mice.

Adoptive transfer of spleen cells obtained from mice primed with Japanese encephalitis virus (JEV) suppressed IgM antibody plaque forming cells (PFC) against JEV in the spleen. Similar suppression of PFC was also shown in vitro by adding primed spleen cells to JEV-stimulated spleen cell cultures. The suppressor activity appeared sharply in the third week after priming and persisted up to 6 weeks. By using various cell separation procedures it was found that the suppressor activity resided in the T cell enriched fraction and not in B cells or macrophages. Sensitivity of the cells to treatment with anti-Thy 1.2 antiserum and complement confirmed that suppressor cells were T lymphocytes. It was noted that the suppression was effective against dengue virus antigen also. Our findings thus show generation of suppressor T lymphocytes in JEV-infected mice.     

Purification of Japanese encephalitis virus vaccine by zonal centrifugation.

A large-scale procedure for purification and concentration of Japanese encephalitis virus vaccine by a continuous-flow isopycnic banding technique in a sucrose density gradient solution, using a K-III zonal centrifuge rotor, is presented. The quality of zonal-purified vaccine was compared with commercial and Japanese National Institutes of Health reference vaccines for antigenicity, immunodiffusion, and allergic encephalitogenicity tests to show its high purity.     

我国流行性乙型脑炎病毒基因型研究

国外20世纪80年代开始流行性乙型脑炎病毒(乙脑病毒)分子生物学研究,目前结合生物信息学等技术可以进行病毒基因分型、病毒分子流行病学研究等,并用基因分型和进化理论揭示病毒的起源和疾病的预防控制.我国此项工作起步较晚,尤其对乙脑病毒分子特征如毒株基因型别及其在我国的分布等缺乏了解.但是近几年发展很快,有些研究已经达到国际水平.本期杂志收集了一些我国最近几年有关乙脑的研究报告,包括疾病的流行,宿主和媒介以及新分离乙脑病毒病原学研究成果,文章不仅报告了病毒分离与鉴定情况,还包括病毒基因分型研究,将病毒学与分子生物学结合起来,是近年来我国乙脑病毒乃至虫媒病毒研究领域的极大进步.     

Immunological memory in latent Japanese encephalitis virus infection

Long term B-cell memory to Japanese encephalitis virus (JEV) in latently infected mice was investigated by adoptive cell transfer. Both IgM and IgG memory were elicited by antigen challenge or cyclophosphamide induced reactivation of virus. A weak antigen-specific IgM response for a brief period and a strong IgG response were detected in Swiss albino mice exposed to secondary infection. A correlation between the secondary IgM antibody and protection against JEV challenge was observed in adoptive transfer experiments. This was abrogated by pretreatment of the serum with 2-mercaptoethanol. Similarly secondary immune splenic T-cells up to day 5 post-reactivation provided protection. These results suggest that a long term antigen-specific IgM and IgG memory was induced by JEV challenge in latently infected mice. Further, the role of IgM antibody and T-cells in the response of mice to secondary JEV infection has been shown.     

Protection of mice against experimental Japanese encephalitis virus infections by neutralizing anti-glycoprotein E monoclonal antibodies

Neutralizing monoclonal antibodies (MAbs) to glycoprotein E (gpE) of Japanese encephalitis (JE) virus given intraperitoneally (i.p.) (0.1 ml of immune ascitic fluid (AF) diluted 1:10 per mouse) to about 4-week-old Swiss mice 1 day prior or 2 days after the virus challenge (100 LD50 of JE virus administered intracerebrally (i.c.)) resulted in a decreased mortality along with an increased survival of the animals as demonstrated by the HAI-positive virus-specific (Hs) MAbs. The protective effect produced by four Hs MAbs was maximum when given 1 day prior the virus challenge, while other, namely HAI-positive flavivirus cross-reactive (Hx) and HAI-negative virus-specific (NHs) MAbs did not produce any effect. Interestingly, one of the two NHs MAbs, namely NHs-1 showed a reduced survival of mice given the MAb 2 days after the virus challenge. Administration of combinations of two or more Hs MAbs may be recommended due to their possible enhanced protection against JE virus infections in mice.     

Distinct usage of three C-type lectins by Japanese encephalitis virus: DC-SIGN,DC-SIGNR,and LSECtin

Infection with West Nile virus and dengue virus, two mosquito-borne flaviviruses, is enhanced by two calcium-dependent lectins: dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and its related molecule (DC-SIGNR). The present study examined the relationship between Japanese encephalitis virus (JEV) infection and three lectins: DC-SIGN, DC-SIGNR, and liver sinusoidal endothelial cell lectin (LSECtin). Expression of DC-SIGNR resulted in robust JEV proliferation in a lymphoid cell line, Daudi cells, which was otherwise non-permissive to infection. DC-SIGN expression caused moderate JEV proliferation, with effects that varied according to the cells in which JEV was prepared. LSECtin expression had comparatively minor, but consistent, effects, in all cell types used in JEV preparation. While DC-SIGN/DC-SIGNR-mediated JEV infection was inhibited by yeast mannan, LSECtin-mediated infection was inhibited by N-acetylglucosamine β1-2 mannose. Although involvement of DC-SIGN/DC-SIGNR in infection seems to be a common characteristic, this is the first report on usage of LSECtin in mosquito-borne flavivirus infection.     

Immunopathological study of spleen during Japanese encephalitis virus infection in mice.

Following intraperitoneal inoculation, Japanese encephalitis virus replicated in peritoneal macrophages, appeared on day 3 in the splenic macrophages of the perifollicular region and later in cells of the periarteriolar lymphoid sheath (PALS) as shown by indirect immunofluorescence. Productive JEV infection was observed both in macrophages and T-cells. Morphological study of spleen during JEV infection revealed proliferative changes, with increased number of macrophages from day 3 p.i. in the perifollicular region followed by accumulation of polymorphonuclear leucocytes which reached a maximum on day 9 p.i. The T dependent areas were considerably enlarged by day 9 and gradually reduced in size by week 3. At later periods germinal centres appeared in the T independent area and were prominent by day 15. The cells containing virus antigen disappeared with the appearance of germinal centres, thus indicating the role of the latter also in virus clearance.     

St. Louis encephalitis virus: an ultrastructural study of infection in a mosquito vector

The course of St. Louis encephalitis virus infection of Culex pipiens pipiens Linn, mosquitoes was followed sequentially by electron microscopy. At the site of initial viral invasion of mosquito parenchyma in the midgut, epithelial infection involved a rather constant proportion of cells that yielded only moderate numbers of virus particles. Virus was observed in the midgut at locations where spread via the hemolymph could occur. Tissues in intimate contact with hemolymph (abdominal muscles, malpighian tubules, ovarian sheath) became infected, but only modest numbers of virus particles were ever produced. In sharp contrast, an ever increasing number of of virus particles were formed in the epithelial cells of the salivary glands. Virus was primarily yielded into the cisternae of endoplasmic reticulum and then shed from the apical end of cells into the lumen of the glands. Very few particles were associated with lateral or basal margins of salivary gland epithelium, indicating a directional “preference” of virus for shedding through apical plasma membrane. So much virus was shed into the limited space of the glandular lumen and its diverticula that dispersed particles formed into crystalline arrays from day 25 onward; one of the larger of these crystals was estimated to contain more than 50,000 virus particles. Changes in the infected cells of all the mosquito organs examined were interpreted as physiologic variances relative to feeding time and not as specifically due to viral cytopathology.     

Japanese encephalitis virus invasion of cell: allies and alleys

The mosquito‐borne flavivirus, Japanese encephalitis virus (JEV), is the leading cause of virus‐induced encephalitis globally and a major public health concern of several countries in Southeast Asia, with the potential to become a global pathogen. The virus is neurotropic, and the disease ranges from mild fever to severe hemorrhagic and encephalitic manifestations and death. The early steps of the virus life cycle, binding, and entry into the cell are crucial determinants of infection and are potential targets for the development of antiviral therapies. JEV can infect multiple cell types; however, the key receptor molecule(s) still remains elusive. JEV also has the capacity to utilize multiple endocytic pathways for entry into cells of different lineages. This review not only gives a comprehensive update on what is known about the virus attachment and receptor system (allies) and the endocytic pathways (alleys) exploited by the virus to gain entry into the cell and establish infection but also discusses crucial unresolved issues. We also highlight common themes and key differences between JEV and other flaviviruses in these contexts. Copyright © 2015 John Wiley & Sons, Ltd.     

Nuclear immunofluorescence in porcine kidney cells infected with Japanese encephalitis virus.

Acetone-fixed porcine stable kidney (PS) cells infected with Japanese encephalitis (JE) virus were stained in indirect fluorescent antibody (FA) assay with anti-JE virus monoclonal (MoAb) and polyclonal (immune PF) antibodies. First positive immunofluorescence (IF) occurred in the cytoplasm with MoAb Hs-1 (anti-envelope, JE-specific) and immune PF after 7 hr post-infection (p. i.); it became prominent by 15 hr to 48 hr (maximum) when cells reacted strongly also with MoAb Hx-3 (flavivirus crossreactive epitope). In addition, 15 to 20% of the infected cells, which revealed positive cytoplasmic IF, showed intranuclear IF with Hs-1, Hx-3, and immune PF by 20 to 24 hr p.i. By 48 hr, the intranuclear IF was not observed or became diminished. These observations indicate that the JE virus specific epitope Hs-1 appeared first followed by the flavivirus cross-reactive epitope Hx-3. Nuclei of the infected cells seem to play some role in the replication of JE virus.