Peter L. Summers

Flavivirus entry into cultured mosquito cells and human peripheral blood monocytes

SummaryThe entry modes of Japanese encephalitis (JE) and dengue-2 (DEN-2) viruses into C6/36 mosquito cells and of DEN-2 virus into human peripheral blood monocytes in vitro were studied. Inoculation of either JE or DEN-2 virions into C6/36 cells resulted in direct penetration of the virions into the cytoplasm at the cell surface in 3 stages. At stage 1, virions attached to the plasma membrane of host cells by their envelope spikes; at stage 2, the virion envelopes approximated to and eventually overlapped the host plasma membrane, and in the process the plasma membrane at the attachment sites dissolved; and, at stage 3, virions penetrated into the cytoplasm through the plasma-membrane disruptions created at the adsorption sites. Virions themselves apparently disintegrated at or near the penetration sites, for no virions were seen in the deeper cytoplasm. Coated pits did not form at the virion attachment sites, and virion-containing vesicles were not found in the cytoplasm. In the entry of DEN-2 virus into human peripheral blood monocytes, virions were found, adsorbed onto the external surface of the plasma membrane and attached to the luminal surface of macropinocytic vacuolar membranes. The latter apparently occurred as the result of ruffling and macropinocytic activities of the cells. At both sites virions penetrated into the cytoplasm through the plasma or vacuolar membrane in the same manner as they did through the plasma membrane of C6/36 cells. No evidence of viral entry by receptor-mediated endocytosis was observed. Implications of the entry mode of the mosquito cell-generated DEN-2 virus into human peripheral blood monocytes to an early process of natural, mosquito-transmitted infection is discussed.

An electron and immunoelectron microscopic study of dengue-2 virus infection of cultured mosquito cells: Maturation events

SummaryThe maturation process of dengue-2 virus in C6/36 mosquito cells was studied by electron microscopy at 12, 16, 24, 48, and 78 hours postinoculation (p.i.) and by immunoelectron microscopy at 48 and 78 hours p.i. Maturing virions appeared within cytoplasmic vacuoles and on the surface of infected cells from 24 hours p.i. onward in close topographical relationship to the dense particles that occurred concurrently in the cytoplasm. The dense particles measured 25 to 35 nm in diameter; the mature virions measured 50 to 55 nm in diameter, with a dense core measuring 30 to 35 nm in diameter covered by a 10 nm-thick membrane envelope. The morphological observations indicated that the dense particles were dengue nucleocapsids assembled in the cytoplasm and that they apparently budded into the vacuolar lumens and the extracellular space at the vacuolar and plasma membranes, acquiring membrane envelopes and becoming mature virions in the process. The virions that budded into the vacuolar lumens were released extracellularly by exocytosis. In the samples tested with dengue-2 polyclonal antibodies, intense immunostaining occurred at the sites of virus budding on the cell surface; host cell membrane and cytoplasm adjacent to the budding virions stained less intensely. In the samples tested with a dengue-2 monoclonal antibody specific for the envelope glycoprotein, budding virions stained rather exclusively, with no staining occurring in adjacent host membrane or cytoplasm.

Maturation Process of Japanese Encephalitis Virus in Cultured Mosquito Cells in Vitro and Mouse Brain Cells in Vivo

SummaryThe maturation process of Japanese encephalitis (JE) virus in C6/36 cells in vitro and in mouse brain cells in vivo was studied by electron microscopy. In the C6/36 cell infection, 500 to 2250 virions per cell were released into the medium during the period of study; yet, no virus budding process was observed at the host cell membranes. JE virions at various maturation stages appeared within the cisternae of rough endoplasmic reticulum (RER) of infected cells at 24 hours p.i.; and, although C6/36 cells did not show a well-developed Golgi apparatus, the virions appeared to be carried to the cell surface within host-cell secretory vesicles for extracellular release as early as 24 hours p.i. The occurrence of a secretory-type intracellular transport of maturing JE virus particles was well recognizable in brain cells of infected mice, in which JE virus particles were found almost exclusively in the cisternae of RER, in the Golgi apparatus, and in various vesicles, including coated vesicles, in the vicinity of the Golgi apparatus. Our previous study of dengue-2 virus morphogenesis and our present study of JE virus morphogenesis differed substantially at various stages of maturation. Possible mechanisms which explain these differences were discussed.

Flaviviruses can mediate fusion from without in Aedes albopictus mosquito cell cultures

Flavivirus-induced polykaryocytes were detected in monolayers of Aedes albopictus (clone C6/36) mosquito cells as early as 20 min after adsorbing virus to these cells. A high multiplicity of infection with dengue (DEN)-1, 2, 3, 4, Japanese encephalitis, and yellow fever viruses was required to demonstrate fusion from without (FFWO) with these flaviviruses. Optimal conditions for FFWO included exposure of adsorbed virus to pH 6.0 and an incubation temperature of 39 degrees C. DEN-2 monoclonal antibodies to the envelope E glycoprotein inhibited cell fusion, whereas monoclonal antibodies to the prM and NS1 proteins did not inhibit cell fusion. These results indicate that flaviviruses cause FFWO soon after adsorption to C6/36 mosquito cells and the process is most likely mediated by the virion envelope E glycoprotein.

A comparative study of entry modes into C6/36 cells by Semliki Forest and Japanese encephalitis viruses

SummaryThe entry modes of Semliki Forest virus and Japanese encephalitis virus into C6/36 cells were compared by electron microscopic observation. At physiological pH, the two viruses showed characteristically different entry modes. Following attachment to the plasma membrane, many SF virions appeared within plasma membrane invaginations and cytoplasmic vesicles; on the other hand, JE virions remained to be found exclusively at the cell surface, with no virions appearing within cytoplasmic vesicles. Electron microscopic observation, therefore, indicated that SF virus entered C6/36 cells by receptormediated endocytosis, while JE virus penetrated the cells at the surface and disintegrated at or near the adsorption sites. At pH 5.8, SF virus also entered C6/36 cells by direct penetration at the cell surface. On the basis of the present and other findings, the following working hypotheses are presented for future investigations: (a) at physiological pH, the fusion protein of SF virus is in an inactive state and needs to be activated by acidic pH within the endosome in order to act on the host-cell membrane, but that of JE virus is in an active state and is capable of dissolving the host plasma membrane at the cell surface immediately after the attachment; (b) the states of viral fusion proteins (inactive or active) at the time of viral attachment to the cell surface determine which of the two entry modes these viruses follow.

Morphogenesis of flaviviruses.

The flaviviruses consist of about 70 viruses that include some important pathogens that are responsible for a number of serious diseases, such as yellow fever, dengue fever, and various encephalitides (Porterfield, 1980; Shope, 1980). They are transmitted to humans by arthropod vectors, i.e., mosquitoes and ticks (Chamberlain, 1980) and are also called arboviruses. The number of known flaviviruses will undoubtedly increase in the future as more viruses are isolated from various hosts and their vectors. Until recently, the flavivirus genus was included with three others, alphavirus, pestivirus, and rubivirus, as part of the Togaviridae family (Porterfield et al., 1978). With the accumulation of experimental data, it has become increasingly clear that flaviviruses have substantially different genomic organization and mechanisms of replication and gene expression from those of alphaviruses, the other major genus of the Togaviridae family. Therefore, the possibility that the two genera have diverged from the same ancestor is remote. Consequently, flaviviruses are now classified in their own family, Flaviviridae (Westaway et al., 1985).

Comparison of replication rates and pathogenicities between the SA 14 parent and SA 14-14-2 vaccine strains of Japanese encephalitis virus in mouse brain neurons

SummaryThe replication rates and pathogenicities of the SA 14 parent and SA 14-14-2 vaccine strains of Japanese encephalitis (JE) virus in neurons of the mouse brain following intracerebral inoculation were compared. All the mice inoculated with the SA 14 parent strain died within one week postinoculation (p.i.), whereas all the mice inoculated with the SA 14-14-2 vaccine strains survived without showing any signs of central nervous system (CNS) involvement. The virus titers of the mouse brains inoculated with the SA 14 strain reached progressively higher levels until day 5 when the animals died. On the other hand, the virus titers of the mouse brains inoculated with the SA 14-14-2 strain persisted at low levels for several days and could not be detected after 10 days. In the routine electron microscopical study, a majority of neurons in the mouse brains inoculated with the SA 14 strain contained virions and showed characteristic cytopathological changes in connection with viral replication. In the brains inoculated with the SA 14-14-2 strain, however, we failed to find neurons containing virions or showing characteristic cytopathological changes. In the alkaline phosphatase immunostaining of paraffin-embedded sections, a majority of neurons in the brains of mice inoculated with the SA 14 strain stained positively on day 5 p.i., but only a small number of neurons in scattered small foci stained positively in the brains inoculated with the SA 14-14-2 strain. The immunogold staining of Vibratome sections also revealed the identical patterns; moreover, electron microscopical examination of the immunopositive foci of the brain inoculated with the vaccine strain revealed neurons that contained virions in dilated cisternae of rough endoplasmic reticulum (RER), indicating that the SA 14-14-2 strain also replicated, albeit poorly, in neurons. The present results showed that upon intracerebral inoculation into mice the SA 14 parent strain of JE virus grew vigorously in a large number of neurons, killing the animals, while the SA 14-14-2 vaccine strain grew poorly only in a small number of neurons without causing mortality. Possible mechanisms involved in the alteration of pathogenicity between the SA 14 parent virus and the SA 14-14-2 vaccine virus are discussed.

Entry and Replication of Japanese Encephalitis Virus in Cultured Neurogenic Cells

The entry mode and growth pattern of Japanese encephalitis (JE) virus in mouse neuroblastoma N18TG2 cells and mouse neuroblastoma x rat glioma NG108-15 hybrid cells were studied by electron microscopy. At two minutes after inoculation, JE virions adsorbed onto and directly penetrated through the plasma membrane of the hybrid cells, whereas virions did not adsorb nor entered the neuroblastoma cells. Correspondingly, the hybrid cells showed assembling progeny JE virions in the cisternae of rough endoplasmic reticulum (RER) 1 day postinoculation (p.i.) although virions were rarely found on the following days during the experiment. On the other hand, progeny virions did not assemble in the RER cisternae of the neuroblastoma cells throughout the experiment. The morphologic observations, therefore, suggest that (a) the hybrid cells express JE-virus receptors which facilitate the viral attachment onto and entry into the cells, while the neuroblastoma cells do not and (b) JE virus replicates very poorly after the entry into the hybrid cells while it does not replicate at all in the neuroblastoma cells. The virus titrations of the media of the neuroblastoma and hybrid cell cultures showed only titers indicative of residual virus of the inoculum that progressively decreased during the experiment. The present results show therefore that of the two neurogenic cell culture lines studied only the hybrid cell line can be used for the study of viral entry and replication, although it is not suited for virus production. Possible reasons for the poor replication of JE virus in the hybrid cells are discussed.

Expression of the envelope antigen of dengue virus in vaccine strains of Salmonella

The envelope gene of dengue 4 virus (DEN) was cloned in a plasmid under the control of Escherichia coli expression signals. A clone that expressed 93% of the gene was found to be detrimental to the bacterial host. Another clone which carried only 76% of the E gene was found to be quite stable in vitro as well as in vivo. The killed recombinant bacteria induced antibodies in mice which recognized native DEN virus. Attenuated Salmonella typhimurium (SAL) strains carrying the DEN-E plasmid were tested for their efficacy as orally administered live vaccines. Protective immunization was assessed in a mouse model by immunizing three-week old BALB/c mice followed by challenge with DEN virus. It was found that these young mice were highly susceptible to the carrier SAL strains (M206 and aroA SL3261). Moreover, the SAL-infected mice were more susceptible to DEN virus challenge than control mice, suggesting that the SAL infection caused immunosuppression in these young mice.

Cytopathology of PC12 cells infected with Japanese encephalitis virus.

SummaryInfection 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.