Clonal analysis of mammalian cell cultures persistently infected with Japanese encephalitis virus.

More than 200 cells were cloned from populations of mammalian cells persistently infected with Japanese encephalitis virus. Only four cloned cultures contained cells that had viral antigen measurable by immunofluorescence and that released infectious virus, yet all clones harbored virus-specific RNA. Superinfection of cloned cells with wild-type Japanese encephalitis virus did not produce cytopathic effects, but resulted in production of viral antigen and infectious virus in formerly nonproducing clones. Cocultivation of nonproducer clone cells with normally permissive cells did not induce virus production, nor did treatment of nonproducer clones with various inhibitors of DNA, RNA, or protein synthesis. It is suggested that the cloning procedure may have selected for a particular subpopulation of cells and that defective virus is also involved in establishment and maintenance of persistent infection.     

Virus-neuron interactions in the mouse brain infected with Japanese encephalitis virus

The virus-host interactions between Japanese encephalitis (JE) virus and mouse brain neurons were analyzed by electron microscopy. JE virus replicated exclusively in the rough endoplasmic reticulum (RER) of neurons. In the early phase of infection, the perikaryon of infected neurons had relatively normal-looking lamellar RER whose cisternae showed focal dilations containing progeny virions and characteristic endoplasmic reticulum (ER) vesicles. The reticular RER, consisted of rows of ribosomes surrounding irregular-shaped, membrane-unbounded cisternae and resembled that observed in JE-virus-infected PC12 cells, were also seen adjacent to the lamellar RER. The appearance of the reticular RER indicated that RER morphogenesis occurred in infected neurons in association with the viral replication. The fine network of Golgi apparatus was extensively obliterated by fragmentation and dissolution of the Golgi membranes and their replacement by the electron-lucent material. As the infection progressed, the lamellar RER was increasingly replaced by the hypertrophic RER which had diffusely dilated cisternae containing multiple progeny virions and ER vesicles. The Golgi apparatus, at this stage, was seen as coarse, localized Golgi complexes near the hypertrophic RER. In the later phase of infection, RER of infected neurons showed a degenerative change, with the cystically dilated cisternae being filled with ER vesicles and virions. Small, localized Golgi complexes frequently showed vesiculation, vacuolation, and dispersion. The present study, therefore, indicated that during the viral replication the normal lamellar RER which synthesized neuronal secretory and membrane proteins was replaced by the hypertrophic RER which synthesized the viral proteins. The hypertrophic RER eventually degenerated into cystic RER whose cisternae were filled with viral products. The constant degenerative change which occurred in the Golgi apparatus during the viral replication suggested that some of the viral proteins transported from RER to the Golgi apparatus were harmful to the Golgi apparatus and that increasing damage to the Golgi apparatus during the viral replication played the principal role in the pathogenesis of JE-virus-infected neurons in the central nervous system.     

Cytogenetic study of continuous pig kidney cell cultures chronically infected with the Kilham virus]

A persistent infection of the continuous embryonal pig kidney cell cultures induced by a rat parvovirus (the Kilhem virus) did not alter morphological or karyological characteristics of the cultures, and caused no transformation of these. The data obtained suggests the resistance of the pig karyotype to the virus under investigation.     

Cytopathology of PC12 cells infected with Japanese encephalitis virus.

Infection of a clonal rat pheochromocytoma cell line, PC12, with Japanese encephalitis (JE) virus produced successively higher titers of virus in the culture fluid during the 72-h experimental period. In electron microscopical observation, JE virus entered PC12 cells by direct penetration through the plasma membrane at 2 min postinoculation (p.i.) and caused marked cellular hypertrophy and extensive proliferation of the cellular secretory system including rough endoplasmic reticulum (RER) and Golgi complexes starting 24 h p.i. The proliferating RER of the virally infected cells contained progeny virions and characteristic endoplasmic reticulum vesicles in its cisternae, and the proliferating Golgi complexes contained virions in their saccules. These findings indicated that the proliferation of the cellular secretory system occurred in association with viral replication and maturation in the system. Seventy-two hours p.i., the cellular secretory system of infected PC12 cells showed degenerative changes with vesiculation, disorganization, and dispersion of the Golgi complexes and fragmentation, focal cystic dilation, and dissolution of the RER in the same manner as those seen in the secretory system of JE-virus-infected neurons in the mouse brain. Thus, JE-virus-infected PC12 cells seem to be a suitable neurogenic cell line for the study of the pathogenic mechanism of JE virus. At the same time, the virally infected cells seem to offer an interesting cell model for the study of the morphogenesis of the cellular secretory system.     

Mechanism of virulence attenuation of glycosaminoglycan-binding variants of Japanese encephalitis virus and Murray Valley encephalitis virus

The in vivo mechanism for virulence attenuation of laboratory-derived variants of two flaviviruses in the Japanese encephalitis virus (JEV) serocomplex is described. Host cell adaptation of JEV and Murray Valley encephalitis virus (MVE) by serial passage in adenocarcinoma cells selected for variants characterized by (i) a small plaque phenotype, (ii) increased affinity to heparin-Sepharose, (iii) enhanced susceptibility to inhibition of infectivity by heparin, and (iv) loss of neuroinvasiveness in a mouse model for flaviviral encephalitis. We previously suggested that virulence attenuation of the host cell-adapted variants of MVE is a consequence of their increased dependence on cell surface glycosaminoglycans (GAGs) for attachment and entry (E. Lee and M. Lobigs, J. Virol. 74:8867-8875, 2000). In support of this proposition, we find that GAG-binding variants of JEV and MVE were rapidly removed from the bloodstream and failed to spread from extraneural sites of replication into the brain. Thus, the enhanced affinity of the attenuated variants for GAGs ubiquitously present on cells and extracellular matrices most likely prevented viremia of sufficient magnitude and/or duration required for virus entry into the brain parenchyma. This mechanism may also account, in part, for the attenuation of the JEV SA14-14-2 vaccine, given the sensitivity of the virus to heparin inhibition. A pronounced loss of the capacity of the GAG-binding variants to produce disease was also noted in mice defective in the alpha/beta interferon response, a mouse strain shown here to be highly susceptible to infection with JEV serocomplex flaviviruses. Despite the close genetic relatedness of JEV and MVE, the variants selected for the two viruses were altered at different residues in the envelope (E) protein, viz., Glu(306) and Asp(390) for JEV and MVE, respectively. In both cases the substitutions gave the protein an increased net positive charge. The close spatial proximity of amino acids 306 and 390 in the predicted E protein structure strongly suggests that the two residues define a receptor-binding domain involved in virus attachment to sulfated proteoglycans.     

Synthesis and localization of Japanese encephalitis virus RNAs in the infected cells

Synthesis and localization of virus-specific RNA in cells infected with Japanese encephalitis virus (JEV) were examined. To prepare specific RNA probes, we constructed four kinds of plasmids which contained DNA fragments corresponding to JEV genomic RNA. Minus probes, JT18V and JT19III, transcribed by T7 RNA polymerase were able to recognize a negative strand of JEV-specific RNA synthesized in cells as early as 6 hr postinfection (p.i.). In the experiments using a plus-strand probe JT19V to hybridize the 3' end of JEV-RNA, not only full-length 42S(+) RNA but also 10S(+) RNA were detected in the infected cells at 24 hr p.i. The positive-strand 42S RNA was found in much greater abundance in the membrane fraction than in the supernatant fraction of the infected cells. In contrast, larger amounts of the negative-strand RNAs existed in the supernatant fraction. It is suggested from the data that the JEV-specific negative- and positive-strand RNAs accumulate at different sites in the infected cells.     

Study on persistent infection of Japanese encephalitis virus Beijing-1 strain in serum-free Sf9 cell cultures

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8 x 10(6) cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0 x 10(5) to 1.5 x 10(6) pfu/ml. The infected Sf9 cells could be sub-cultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.