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[Chronic infection of VERO cell cultures with Japanese encephalitis virus. 2. Certain characteristics of variability of persistent viruses].

Japanese encephalitis virus contained in the medium of chronically infected VERO-K43 cell cultures was shown to be sensitive to the effect of RNA-ase, resistant to treatment with urea, to have no hemagglutinating activity. After 2 passages in suckling mouse brain the virus lost its sensitivity to RNA-ase, was inactivated by urea, and its hemagglutinating activity was restored. The importance of Japanese encephalitis virus variability in the mechanism of chronic infection of cell cultures is discussed.

Animals

[The protective properties in mice of tonate virus and two strains of cabassou virus against neurovirulent everglades Venezuelan encephalitis virus (author's transl)].

Venezuelan encephalitis virus is a constant menace to man and to animals, particularly horses. Two as yet unreported strains have been isolated in French Guyana: Ca An 410d (Tonate) and Ca Ar 508 (Cabassou). They are not neurovirulent after peripheral inoculation into adult mice and guinea pigs. Cabassou is not pathogenic for adult mice after intracerebral inoculation. A third strain, Ca Ar 19007, is antigenically identical by complement-fixation with Cabassou, but appears to cross react with other strains, Tonate, when injected intra-peritoneally or sub-cutaneously, protects adult mice against intra-cerebral or intra-peritoneal challenge with Everglades virus. The Ca Ar 19007 isolate causes total protection against Everglades virus if injected intra-peritoneally, but only partial protection by the sub-cutaneous route. Cabassou, on the other hand, if injected intra-peritoneally will protect against intra-cerebral challenge with Everglades. Tonate and Ca Ar 19007 thus appear to be naturally occurring attenuated variants of the Venezuelan encephalitis virus complex, which can protect mice against intra-cerebral infection with neurovirulent strains.

Animals

Virion and soluble antigens of japanese encephalitis virus.

Japanese encephalitis virions contain a 58 X 10-3-molecular-weight envelope glycoprotein antigen that can be solubilized with sodium lauryl sulfate and separated from other virion structural polypeptides and viral ribonucleic acid by gel filtration chromatography. The 58 X 10-3-molecular-weight envelope protein is the major antigen responsible for cross-reactivity of the virion in complement fixation tests with other closely related arboviruses. A naturally occurring soluble complement-fixing antigen is found in Japanese encephalitis mouse brain preparations after removal of particulate antigens. After partial purification by gel filtration and isoelectric focusing, the 53 X 10-3-molecular weight soluble complement-fixing antigen is more type specific than the Japanese encephalitis envelope antigen in complement fixation tests. Further, the Japanese encephalitis soluble complement-fixing antigen is stable to treatment with sodium lauryl sulfate and 2-mercaptoethanol, whereas virion complement-fixing antigens are unstable after this treatment.

Amino Acids

Intracellular distribution of virus-specific RNA in chick embryo cells infected with Japanese encephalitis virus.

Japanese encephalitis virus (JEV) infected chick embryo (CE) cells were treated with 4 mug actinomycin D/ml and 5 mM-D-glucosamine at 2 or 3 h before harvesting. Production of JEV was not affected by the short-time treatment of these drugs. The radioactivity in virus-specific RNA in the glucosamine-treated cells was a-parently higher than in non-treated cells. Nuclear and cytoplasmic extracts were prepared from the JEV-infected cells pulse-labelled with 3H-uridine at 15 h after infection. Analysis of virus RNA in nuclear extracts on sucrose density gradients showed that most of the radioactivity was in 23S RNA, 26S RNA and 8 to 12S RNA. The radioactivity of virus RNA in cytoplasmic extracts was found in 42S RNA and RNA fragments sedimenting at less than 8S.

Animals

Infections of congenitally athymic (nude) and normal mice with avirulent and virulent strains of Venezuelan encephalitis virus.

Two strains of Venezuelan encephalitis virus that are avirulent for normal BALB/c mice inoculated subcutaneously were also avirulent for infected congenitally athymic (nude) mice of the same strain. Viremias were of similar magnitudes and durations in normal and nude mice. Brain concentrations were higher in nude mice with the one avirulent strain tested, although the periods of detectable virus in brains were similar. No lesions were found in brains, spleens, or lymph nodes by ordinary histopathological examination. Viral neutralizing antibody titers in plasmas at 1 to 3 weeks after infection were lower and more transient in nude than in normal mice, and implantations of thymic tissues into nude mice partially restored their neutralizing antibody responses. Concentrations of spleen cells producing antibodies that lysed sheep erythrocytes 4 days after inoculation of erythrocytes and avirulent virus into nude mice were above the levels of uninfected nude mice. These concentrations were similar in infected and uninfected normal mice. In contrast, two mouse-virulent strains of Venezuelan encephalitis virus killed nude mice faster than normal mice after subcutaneous inoculation. Yet concentrations and durations of virus in bloods and brains were not consistently different between nude and normal mice. There were perivascular monocytes in brains and slight architectural alterations of spleens and lymph nodes. Concentrations of spleen cells producing antibodies hemolytic for sheep erythrocytes 4 days after inoculation with erythrocytes were depressed in nude and normal mice by infection with virulent strains.

Animals

Protease treatment and chemical crosslinking of a flavivirus: tick borne encephalitis virus.

Tick-borne encephalitis virus was treated with pronase or thermolysin. The resulting particles were banded in sucrose gradients and analyzed for polypeptide composition. Both enzymes caused a reduction in particle density from 1.19 to 1.15--1.16 g/cm3. No loss of viral lipid or nucleic acid could be observed. SDS-polyacrylamidegel electrophoresis showed that only the core protein V2 was unchanged whereas the envelope proteins V3 and V1 had disappeared from their original positions in the PAGE profile. Instead a new peptide(s) with molecular weight of 4000--6000 was found in which hydrophobic amino-acids were enriched. Crosslinking by dimethyl-3.3'-dithiobispropionimidate (DTBP) made the virus resistent to solubilization of the envelope proteins by TX-100. This could be interpreted by the formation of a dense envelope protein network around the nucleocapsid preventing its liberation by TX-100. Some data however indicate that direct crosslinking of at least one of the envelope proteins with the core cannot be excluded.

Cross-Linking Reagents

Isolation of Murray Valley encephalitis virus from the brains of three patients with encephalitis.

Murray Valley encephalitis virus was isolated from the brains of three patients who died from encephalitis during the 1974 epidemic. Isolation of the virus from autopsy material was successful when death occurred within two weeks of the onset of illness; however, no isolations were made from specimens collected before death or from autopsy material obtained from patients who died more than two weeks after the onset of symptoms. The virus was recovered most frequently in embryonated eggs, but two strains were isolated in cell culture.

Adolescent

[Effect of the diapause in the Ixodes ricinus (Ixodidae) tick on the multiplication of the tick-borne encephalitis virus in its body].

The tick encephalitis virus was found to multiply more intensively in ticks developing without diapause. Thus, in larvae developing with diapause, in 4 and 8 weeks after they feed on infected animals, the virus titers were 3.0 and 3.5 lg LD50 respectively. At the development without diapause the titers were 4.3 and 5.3, respectively (P less than 0.05). The virus titers in infected nymphs developing without diapause were 2.5 to 3.9 lg LD50 higher than those of the diapausing individuals. In diapausing larvae and nymphs the tick encephalitis virus remained for a long time (8 to 10 weeks) without fall of titers. The fall of virus titers was not recorded either during the moulting of larvae for nymphs and nymphs for imago but in hungry ticks, which were maintained for a long time at 18 to 23 C, the amount of virus gradually diminished.

Animals

Neutralization tests for dengue and Japanese encephalitis viruses by the focus reduction method using peroxidase-anti-peroxidase staining.

Neutralization tests were made on 4 types of dengue (DEN) virus and Japanese encephalitis (JE) virus by incubation of serially diluted antisera and constant amounts of the viruses and then focus assay of surviving virus infectivity with peroxidase-anti-peroxidase (PAP) staining. Neutralization reactions were virtually completed in 2 hr on incubation of serum-virus mixtures at 28 C. A straight regression line was obtained on a probit chart by plotting the focus reduction rates at various dilutions of a given serum against the logarithm of the serum dilution used in the test. The slopes of the probit regression lines for the neutralization for DEN types 1 and 3 were similar, but differed somewhat from those for DEN type 2 and type 4. The slope of the line for JE virus was quite different from those for DEN viruses. Using these relations, the fifty percent focus reduction titer (FR50) of neutralizing antibodies of a given serum could be estimated from the focus reduction rates at several dilutions of the test serum when the latter was between 25-75% of the value of the control.

Animals

Isolation of the structural proteins of western equine encephalitis virus by isoelectric focusing.

Western equine encephalitis virus was disrupted with Triton X-100 and subjected to isoelectric focusing in a sucrose or urea gradient. The two envelope proteins, E1 and E2 were not well separated in a sucrose gradient, while the E1 and E2 proteins were distinguished as two major peaks which focused in a urea gradient at about pH 7.5 and 10, respectively. Isolated E1 protein refocused at pH 6.5 in a sucrose gradient isoelectric focusing column. When Western equine encephalitis virus was treated with Triton X-100 in 0.01 M phosphate buffer (pH6), hemagglutinating E1 protein was solubilized, which isoelectrofocused at pH 6.5. Purified nucleocapsids focused at pH 4 in a sucrose gradient on an isoelectric focusing column. After ribonuclease treatment of the purified nucleocapsid more than 95 per cent of the viral RNA was acid-soluble, and hte nucleocapsid protein isoelectrofocused at about pH 4.

Encephalitis Virus, Western Equine

Experimental double infection of Japanese encephalitis virus and herpes simplex virus in mouse brain.

In our laboratory, 64 autopsy cases of Japanese encephalitis patients were examined by immunofluorescence. Three patients showed some evidences of double infection of Japanese encephalitis virus and herpes simplex virus. Experiments were done to see the mechanisms of double infection. In doubly infected mice, Japanese encephalitis virus antigen was localized in the herpes simplex virus infected areas in the brain. These results suggested that "blood brain barrier" was broken by herpes simplex virus infection and Japanese encephalitis virus gained access to the susceptible cells.

Animals

Proteome Profiling in Cerebrospinal Fluid Reveals Increased Levels of Peroxiredoxin 2 Discriminating Japanese Encephalitis Virus and Scrub Typhus Infection.

Japanese encephalitis virus (JEV) and scrub typhus (ST) are major etiological agents of acute encephalitis syndrome (AES) in India and South Asia. The pathophysiological changes at the molecular level caused by JEV and ST have yet to be studied in detail. The cerebrospinal fluid (CSF) proteomic landscape is a critical indicator of CNS pathology. Here, we conducted label-free quantitative proteomics on CSF from AES patients (n = 15) to identify etiology-specific differentially expressed proteins (DEPs) linked to encephalitis. The key DEPs were validated via ELISA in CSF (n = 49) and serum (n = 33). Our findings revealed 50 proteins exhibited differential expression across JEV and ST groups, with a notable subset of three proteins, PRDX2, KLK6, and TTR. PRDX2 and KLK6 were markedly elevated in JEV CSF (AUC: 0.8933 and 0.9689) but not in ST or non-JEV AES, while TTR was reduced in JEV yet elevated in ST (AUC: 0.5619 vs. 0.8238). Further, PRDX2 upregulation was validated in JEV-infected mouse brains and cortical neurons. Overexpression of PRDX2 in human neuroblastoma cells correlated with enhanced antiviral gene expression, p-STAT1, p-AKT (ser473), and viral replication. Thus, our comprehensive proteomic analysis of CSF identifies PRDX2 as an important circulatory protein, differentially expressed between JEV and ST, with high specificity and enhancing viral propagation, underscoring its role in viral propagation and pathogenesis.

Humans

Persistent infection of cultured mammalian cells by Japanese encephalitis virus.

Persistent infections were established by serial undiluted passage of flavivirus Japanese encephalitis virus in a line of rabbit kidney cells (MA-111). The persistently infected cells resembled uninfected cells in most respects. Low levels of infectious virions were released from a small percentage of cells, and a larger and more variable percentage was shown to possess viral antigen by fluorescent-antibody staining. Released viruses were shown to interfere with replication of wild-type Japanese encephalitis virus. Persistently infected MA-111 cells could not be superinfected with homologous wild-type Japanese encephalitis virus but could be superinfected with two heterologous viruses. Transfer of cell culture medium from persistently infected MA-111 cells to a line of African green monkey kidney cells (Vero) resulted in similar persistent infections in the latter cells. Temperature sensitivity and host-cell interferon production were not involved in establishment or maintenance of persistence. Determination of ratios of physical particles to infectious particles revealed that many defective, noninfectious viruses were present, suggesting that defective interfering particles may be responsible for persistency.

Cell Division

Bergamottin, a bioactive component of bergamot: dual inhibition of Japanese encephalitis virus internalization and genome replication.

Japanese encephalitis virus (JEV) is associated with high mortality and severe neurological sequelae, and existing prevention and control strategies remain insufficient. Therefore, the development of novel antiviral agents is of critical public health importance. This study systematically evaluated the antiviral activity and underlying mechanism of bergamottin, a natural product. Bergamottin exhibited significant dose-dependent inhibitory effects against JEV in multiple cell lines, including BHK-21, HuH-7, and Vero cells, demonstrating potent antiviral efficacy. Mechanistic investigations revealed that bergamottin primarily targeted the internalization and replication stages of the JEV life cycle, thereby effectively suppressing viral proliferation. Additionally, adaptive mutation screening indicated that the D389G mutation in envelope protein E confers drug resistance by potentially changing E protein conformation or reducing endocytic efficiency. In vivo experiment, bergamottin significantly reduced viral loads in mouse brain tissue and effectively improved the survival rate of infected mice. Our findings indicated that bergamottin exerted antiviral activity by dual targeting of key steps in the viral life cycle, making it a highly promising candidate for anti-JEV therapy. Further exploration of the antiviral properties of bergamottin is expected to facilitate its clinical development as a treatment for JEV infection.

Animals

Identification of Saint Louis encephalitis virus mRNA.

Saint Louis encephalitis (SLE) virus-specific RNA was recovered from infected HeLa cells by sodium dodecyl sulfate (SDS)-phenol-chloroform extraction, and the molecular species were resolved by SDS-sucrose gradient centrifugation and agarose gel electrophoresis. Sucrose gradient centrifugation revealed the presence of a 45S species, minor 20 to 30S heterogeneous species, and an 8 to 10 S RNA species in the cytoplasmic extract. Analysis of the same samples by electrophoresis on agarose gels, under both nondenaturing and denaturing conditions, revealed only two virus-specific RNA molecules, the 45S genome-sized RNA and an 8 to 10S species. Varying the gel concentration to facilitate analysis of nucleic acids with molecular weights ranging from 25,000 to 25 X 10(6) failed to reveal additional RNA species, although low levels of a putative double-stranded replicative form could conceivably have escaped detection. From our observations it appears that the heterogeneous RNA species and presumably the 20S RNase-resistant species reported in other investigations of flavivirus RNA are degradation products or conformers of the 45S molecule. Polysomes from SLE virus-infected cells were prepared and separated from contaminating nucleocapsid by centrifugation on discontinuous sucrose gradients. RNA extracted from these polysome preparations was analyzed by sucrose gradient centrifugation and agarose gel electrophoresis. The 45S SLE virus genome-size molecule was found to be the only RNA species associated with the polysomes. This molecule was sensitive to RNase digestion and was released from polysomes by EDTA and puromycin treatment. These findings provide direct evidence that the 45 S SLE virus RNA serves as the messenger during virus replication, in contrast to the 26S RNA species which functions as the predominant messenger during alphavirus replication.

Encephalitis Virus, St. Louis