乙型脑炎病毒持续感染株E区序列变异与病毒性状的关系

目的研究乙型脑炎病毒（JaGAr-01株和Nakayama株）持续感染变异株性状与E区基因序列的关系.方法将两种乙脑病毒野生株分别感染人肝癌KN73细胞,建立乙型脑炎病毒持续感染系.采用PFU法进行病毒滴度测定;为探讨持续感染病毒的增殖性,将两种病毒野生株及其持续感染株分别感染KN73细胞;利用E区特异引物以RT-PCR法得到两种病毒E区基因片段,应用基因测序反应进行序列分析,并对两种病毒株E区序列进行比较.结果在KN73细胞中两种持续感染株的增殖性比野生株明显低下.E区基因测序显示：与JaGAr-01野生株比较,其持续感染变异株有4个氨基酸发生置换（第61位酪氨酸→天门冬氨酸,第219位组氨酸→酪氨酸,第384位缬氨酸→谷氨酸,第418位脯氨酸→丙氨酸）;持续感染Nakayama株与其野生株相比有11个氨基酸发生置换（第51位精氨酸→丝氨酸,第61位酪氨酸→天门冬氨酸,第83位赖氨酸→谷氨酸,第123位丝氨酸→精氨酸,第209位精氨酸→赖氨酸,第227位脯氨酸→丝氨酸,第276位天门冬氨酸→丝氨酸,第290位精氨酸→赖氨酸,第387位赖氨酸→精氨酸,第418位亮氨酸→脯氨酸,第454位精氨酸→甘氨酸）.对比还显示基因变异后的持续感染JaGAr-01株和持续感染Nakayama株氨基酸排列的相同性达99.2%.结论乙脑病毒的持续感染变异株增殖性低于野生株;乙脑病毒变异株E区存在基因变异.     

Oligonucleotide fingerprint analysis on Japanese encephalitis virus strains after passage histories

Genetic variation in Japanese encephalitis (JE) virus strains after various passage histories was examined by oligonucleotide fingerprints of the 42S genome RNA. The results indicated that genome changes occurred during passages of new virus isolates through cell lines or mouse brains, especially at earlier passage level in order to adapt quickly to different hosts. However, the changes were not sufficient to explain strain differences observed among fingerprints of various isolates.     

Genetic analysis of Japanese encephalitis virus strains and variants from Russian collection

Genetic analysis of strains and variants of Japanese encephalitis virus from the collection of D. I. Ivanovsky Institute of Virology revealed marked differences in the complex of genetic markers, which makes possible more precise selection of the most interesting clones.     

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.     

Oligonucleotide fingerprint analysis on Japanese encephalitis virus strains isolated in Japan and Thailand

Genetic variation in Japanese encephalitis (JE) virus isolates from Japan and Thailand was examined by oligonucleotide fingerprints of the 42S genome RNA. Japanese isolates were rather similar to each other, as were the recent isolates from Thailand. However, recent Thai isolates were significantly different from recent Japanese isolates. From these results it was presumed that mutations and selections of the JE virus genome would have progressed independently in these geographically distant areas.     

Japanese encephalitis virus glycoproteins

Mature Japanese encephalitis (JE) virus, or N-form virus, contained three structural proteins: V-1, V-2, and V-3. The large membrane protein V-3 was glycosylated, whereas both V-1 (the small membrane protein) and V-2 (the nucleocapsid protein) were not. Intracellular (I-form), immature virions from infected chick embryo cells did not contain V-1 but a larger protein NV-2, which was glycosylated. T-form virions, released by LLC-MK₂ cells incubated with tris(hydroxymethyl)amino-methane (Tris), also contained the glycoprotein NV-2 instead of the nonglycosylated and smaller V-1. We therefore concluded that JE contained two structural membrane glycoproteins, at least one of which is modified during morphogenesis. The NV-2 polypeptide was heterogeneous, and slight differences in electrophoretic mobility were detected among the NV-2 polypeptide peaks from glucosamine-labeled I-form and T-form virions, glucosamine-labeled cell extracts, and amino acid-labeled cell extracts. The significance of these differences is not clear, but they may indicate that NV-2 is composed of several proteins of similar molecular weight. By analyzing extracts of infected cells labeled with glucosamine or amino acids, we tentatively classified the intracellular polypeptide NV-3 as a virus-specified nonstructural glycoprotein; this polypeptide may be a proteolytic fragment of V-3. The virus-specified polypeptides NV-5, NV-4, and NV-1 were classified as nonglycosylated, nonstructural proteins.     

Overview: Japanese encephalitis

Japanese encephalitis (JE) is one of the most important endemic encephalitis in the world especially in Eastern and Southeastern Asia. JE affects over 50,000 patients and results in 15,000 deaths annually. JE virus is a single stranded positive sense RNA virus belonging to family flaviviridae. JE virus is transmitted through a zoonotic cycle between mosquitoes, pigs and water birds. Humans are accidentally infected and are a dead end host because of low level and transient viremia. In the northern region, large epidemics occur during summers whereas in the southern region JE tends to be endemic: cases occur throughout the year with a peak in the rainy season. Occurrence of JE is more closely related to temperature than to humidity. JE is regarded as a disease of children in the endemic areas but in the newly invaded areas, it affects both the adults and children because of the absence of protective antibodies. For every patient of JE, there are large numbers of subclinical cases (25–1000). Symptomatic JEV infection manifests with nonspecific febrile illness, aseptic meningitis or encephalitis. Encephalitis manifests with altered sensorium, seizures and focal neurological deficit. Acute flaccid paralysis may occur due to anterior horn cell involvement. A wide variety of movement disorders especially transient Parkinsonian features and dystonia (limb, axial, orofacial) are reported in 20–60% patients. JE mainly affects thalamus, corpus striatum, brainstem and spinal cord as revealed by MRI and on autopsy studies. Coinfection of JE and cysticercosis occurs because of the important role of pigs in the life cycle of both JEV and cysticercosis.     

Molecular characterization of the full-length genome of the Japanese encephalitis viral strain K87P39

We have determined the complete nucleotide and deduced amino acid sequences of the Japanese encephalitis virus (JEV) strain K87P39, isolated from a pool of circulating Culex tritaeniorhynchus mosquitoes in Korea. In comparison with 27 fully sequenced JEV genomes currently available, we found that the 10968-nucleotide RNA genome of K87P39 has a nine-nucleotide deletion in the 3' nontranslated variable region and that its single open reading frame has a total of eight amino acid substitutions. The K87P39 isolate is highly similar to other JEV isolates, and homology ranges from 97.9 to 89.0% at the nucleotide level, and 99.1 to 96.7% at the deduced amino acid level. Phylogenetic analyses using the full-length sequence of the 27 available JEV genomes showed that the K87P39 strain is most closely related to six Chinese SA14 derivatives and that it is distantly related to the Australian FU, Korean K94P05 and Japanese Ishikawa strains. In addition, we also found that phylogenetic relationships based on the full-length genome are highly similar to those based on the E gene, indicating that phylogenetic analysis of the E gene will be useful for studying the genetic relationships among JEV isolates. We therefore performed a more extensive E gene-based phylogenetic analysis on a selection of 70 JEV isolates available from GenBank, which represent a temporally and geographically wide variety of JEV strains.     

Pathogenic and vaccine strains of Japanese encephalitis virus elicit different levels of human macrophage effector functions

In India, Japanese encephalitis virus (JEV) remains one of the major causative agents of pediatric encephalitis. Macrophages support various neurotropic viruses and influence the immune response. However, the functional status of human macrophages during JEV infection remains unidentified. In this study, we examined the cytokine response and co-stimulatory marker levels in primary human monocyte derived macrophages (MDMs) infected with JE057434 (neurovirulent, primary clinical isolate) or SA14-14-2 (non-neurovirulent, live-attenuated vaccine) JEV strains. We also examined the differential susceptibility of these JEV strains to antiviral effects of interferon and nitric oxide. The results indicate that both JEV strains are capable of inducing various cytokines (type-I IFN, TNFα, IL6 and IL8) and co-stimulatory molecules (CD86 and CD80) in MDMs. However, they varied in replication potential and corresponding interferon sensitivity. SA14-14-2 was highly susceptible to interferon and nitric oxide when compared to JE057434. Thus, reduction in infectious virion production and increased sensitivity of SA14-14-2 towards interferon in MDMs could potentially play a role in limiting viral spread to additional target tissues.     

Molecular phylogenetic and positive selection analysis of Japanese encephalitis virus strains isolated from pigs in China

Japanese encephalitis virus (JEV) is one of the most important virus which causes encephalitis. This disease is most prevalent in the south, southeast and the east region of Asia. In this study, two JEV strains, named JEV/SW/GD/01/2009 and JEV/SW/GZ/09/2004, were isolated from aborted fetuses and seminal fluid of pigs in China. To determine the characteristic of these virus isolates, the virulence of two newly JEV isolates was investigated, the result evidenced that the JEV/SW/GD/01/2009 did not kill mice, while the JEV/SW/GZ/09/2004 displayed neurovirulence with 0.925 log₁₀ p.f.u./LD50. Additionally, the full genome sequences of JEV were determined and compared with other known JEV strains. Results demonstrated that the genome of two JEV isolates was 10,976 nucleotides (nt) in length. As compared to the Chinese vaccine strain SA14-14-2, the JEV/SW/GD/01/2009 and the JEV/SW/GZ/09/2004 showed 99.7% and 97.5% identity at the nucleotide level, 99.6% and 96.7% identity at the amino acid level, respectively. Phylogenetic analysis, based on the full-length genome revealed that two JEV isolates were all clustered into genotype III compared to the reference strains. Furthermore, selection analyses revealed that dominant selective pressure acting on the JEV genome was purifying selection. Four sites under positive selection were identified: codon 521 (amino acid E-227), 2296 (amino acid NS4b-24), 3048 (amino acid NS5-521) and 3055 (amino acid NS5-528). Amino acid E-227 was proved to be related to neurovirulence. Taken together, the molecular epidemiology and functional of positively selected amino acid sites of two newly JEV isolates were fully understood, which might be helpful to predict possible changes in virulence.     

Japanese encephalitis vaccine in travelers

Extensive vaccination against Japanese encephalitis (JE) has been carried out in many Asian countries for the past 20 years and is also increasingly recommended for travelers to endemic areas. Concerns have been raised regarding potential neurological and allergic side effects of the currently available JE vaccine, which is manufactured from mouse brain. A new purified, inactivated JE virus vaccine (IC51) has been developed, which is manufactured in a Vero cell culture substrate. Studies show that the vaccine is both safe and immunogenic and the product will be licensed very soon for use in many industrialized countries. Once a highly immunogenic and safe product is available, wider use of JE vaccine in travelers will be prudent. Currently, vaccination is restricted to travelers with an increased risk of acquiring JE. Individuals at increased risk have been defined quite arbitrarily as travelers with increased behavioral contact to JE-transmitting mosquitoes, in particular, during stays in rural areas and during the transmission season. However, the possibility of an infection with JE virus can never be ruled out when traveling to endemic areas and infection can prove disastrous for the individual concerned. Since a safe product will be available very soon, guidelines and recommendations will have to be reconsidered.     

Japanese encephalitis virus replication: A procedure for the selective isolation and characterization of viral RNA species

Summary The viral RNA species synthesized in a porcine kidney cell line, PS(Y-15), by Japanese encephalitis virus (JEV) are described. Virus titers on these cells ranged between 10⁶ to 10⁷ p.f.u./ml at the end of 2 to 3 days incubation at 35° C. Actinomycin D (AD) could not be used to unmask JEV RNA synthesis since it inhibited virus replication at concentrations necessary to substantially reduce host cell RNA synthesis. Treatment of cells with 1 g AD/ml and removal prior to infection permitted good JEV replication, and at the same time strongly suppressed synthesis of 28 S and 18 S cellular ribosomal RNA. The problem of separating viral RNA from non-ribosomal RNA that was still being synthesized by AD pretreated cells was resolved by the isolation of the cytoplasmic membrane fraction of infected cells. RNA extracted from the membranes of infected AD pretreated cells and analyzed for sedimentation characteristics on sucrose gradients has four RNA species not found in uninfected cells.They are: (1) 45 S single stranded RNA believed to be the infectious RNA found in the virion; (2) a 27 S RNA single stranded RNA; (3) a 20 S ribonuclease resistant RNA believed to be double stranded and (4) an 8 S RNA species. The RNA species found in JEV infected cells, except for the 8 S form, have been found in group A arboviruses. The procedure described utilizing AD pretreatment of host cells and the separation of the cellular cytoplasmic fraction may well have value for the study of the biosynthetic events involved in the replication of other animal viruses that are inhibited by AD.This work was supported by the Bureau of Medicine and Surgery, Department of the Navy, Project MR 041.05.01-0006B3GJ.The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the Naval Service at large.Postdoctoral Research Associate, National Research Council, supported by the Bureau of Medicine and Surgery, Department of the Navy.     

乙型脑炎病毒持续感染株在不同条件下病毒量的变化及分析

目的研究乙脑病毒持续感染株在不同条件下病毒量的变化及探讨影响病毒增殖的因素。方法乙脑病毒野生株JaGAr-01株和Nakayama株分别感染人肝癌细胞株KN73，建立乙脑病毒持续感染模型。采用空泡斑点实验进行病毒滴度测定。用持续感染病毒感染人神经纤维母细胞瘤细胞株IMR．32，在30℃和37℃下检测病毒的温度感受性；分别将两种野生株感染持续感染了病毒的KN73细胞，进行病毒重复感染实验；为探讨持续感染病毒的增殖性，将其感染KN73细胞和IMR．32细胞。结果两种持续感染病毒的增殖量均无温度差异性。重复感染实验结果：JaGAr-01株的病毒量是对照的1．3％和8．8％；Nakayama株的病毒量是对照的80．0％和1．7％。两种野生株及其持续感染株分别感染KN73和IMR．32细胞，前者中两种持续感染株的增殖性比野生株明显低下，而在后者中两株的增殖性与其野生株相近。结论两种持续感染的乙脑病毒均无温度变异株；乙脑病毒的持续感染系中可能存在含有DI粒子的变异病毒；在不同细胞中宿主因子对阻碍持续感染株的增殖存在差异。     

The proteins of Japanese encephalitis virus

Polyacrylamide gel electrophoresis of Japanese encephalitis virus (JEV) grown in both LLC-MK₂ and chick embryo cell culture revealed three principal polypeptides with molecular weights of 8,700, 13,500, and 53,000 (V-1, V-2, and V-3, respectively). Infected chick cells that were treated with actinomycin D and cycloheximide contained seven polypeptides not present in uninfected cells. In addition to V-2 and V-3, polypeptides with molecular weights of 10,500, 19,000, 45,000, 71,000, and 93,000 (NV-1 through NV-5) were found; V-1 was not regularly detected. A similar pattern of polypeptides was obtained by radioimmune precipitation of soluble antigens from cytoplasmic extracts of infected, actinomycin-D treated, chick cells. When virions were treated with NP-40, a dense, RNA-rich structure was detected which contained V-2. An extracellular, slowly sedimenting, RNA-poor, hemagglutinating particle with a density comparable to the virion was present in virus preparations from cell culture and contained V-1, V-3, and NV-2.     

Diagnosis of viral encephalitis by brain biopsy

Prompt and accurate diagnosis of viral encephalitis has acquired increased significance because of the encouraging results obtained in therapeutic trials with antiviral agents such as adenine arabinoside. Currently, brain biopsy remains the only reliable method to confirm the diagnosis of viral encephalitis. This communication, based on the analysis of eighteen confirmed cases of viral encephalitis, illustrates structural abnormalities common to these conditions and suggests biopsy handling procedures that appear most helpful in arriving at a prompt diagnosis. In addition to the traditionally well-known inflammatory changes, such as satellitosis, gliosis, and neuronophagia, a variety of intranuclear inclusion bodies are illustrated, and the judicious application of immunocytochemistry and electronmicroscopy is emphasized.