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Biomedical subjects

K H Eckels

Publications and source records attributed to K H Eckels.

At least 37 records · Page 2Linked to original sources
PubMed1991

Infection of Aedes albopictus and Aedes aegypti mosquitoes with dengue parent and progeny candidate vaccine viruses: a possible marker of human attenuation.

Dengue (DEN-1) and DEN-4 parent (P) and progeny candidate vaccine (CV) viruses were compared in their abilities to infect and to replicate in Aedes aegypti and Aedes albopictus mosquitoes. The DEN CV clones were temperature sensitive (ts) and had small plaque morphology. The DEN-1 and DEN-4 CV viruses differed in their ability to infect, to replicate in, and to be transmitted by mosquitoes. The DEN-1 CV virus was not attenuated for the vector mosquitoes; oral infection rates with the CV virus were as high as or higher than the P virus, and the CV virus replicated efficiently in mosquitoes after oral infection. The DEN-4 CV virus was attenuated; it was less efficient than its P virus in infection and replication in mosquitoes. Thus, the ts phenotype and small plaque morphology are not reliable biological markers for prediction of vector attenuation. Similar results were reported by others for attenuation in man and monkeys. These studies with DEN-1 and DEN-4 viruses, and previously reported studies with DEN-2 virus and with DEN-3 virus suggest that vector and vertebrate host attenuation are genetically linked. Thus, vector attenuation may be a biological marker for human attenuation.

Aedes↗
PubMed1990

Preparation of noninfectious hepatitis A virus hemagglutinin for detecting hemagglutination inhibition antibodies.

Hepatitis A virus (HAV) harvested from infected MRC-5 cells can hemagglutinate various species of erythrocytes at acid pH (Eckels et al., 1989). Further studies revealed that the majority of the hemagglutinin (HA) in MRC-5 and BS-C-1 cells was cell-associated. A simplified procedure for preparing HAV-HA consisted of collecting infected cells in phosphate-buffered saline followed by three cycles of freeze-thawing and sonication. The fluids were clarified and stored at 4 degrees C. The analysis of HA by rate-zonal sucrose gradient centrifugation indicated that the majority of HA co-migrated with infectious virus. Complete inactivation of infectious HAV with 0.03% beta-propiolactone (BPL) did not affect HA activity, while inactivation with 0.05% formalin caused a 16-fold reduction in titer. There was no difference in HAI antibody titers when BPL-treated HA was compared to untreated HA in the hemagglutination inhibition (HAI) test.

Animals↗
PubMed1990

Dengue 3 virus infection of Aedes albopictus and Aedes aegypti: comparison of parent and progeny candidate vaccine viruses.

DEN-3 parent (strain CH53489) and progeny candidate vaccine (clone 24/28) viruses were compared in their abilities to interact with Aedes aegypti and Ae. albopictus. The parent and progeny virus were equivalent in their ability to infect, to replicate in, and to be transmitted by both species of mosquitoes. The candidate vaccine DEN-3 clone was temperature sensitive and had small plaque morphology. These phenotypic markers remained stable during mosquito passage. Thus, temperature sensitivity and small plaque morphology are not reliable biological markers for attenuation.

Aedes↗
PubMed1990

Preparation of an attenuated dengue 4 (341750 Carib) virus vaccine. I. Pre-clinical studies.

Dengue 4 (DEN-4) virus strain 341750 Carib was modified by serial passage in primary canine kidney (PCK) cell cultures. By the 15th PCK passage, this virus was less infectious for monkeys and resulted in a significantly reduced viremia as compared to the parent DEN-4 virus. The 30th PCK passage of DEN-4 341750 Carib was non-infectious for monkeys. A vaccine prepared at the 20th PCK passage in DBS-FRhL-2 cells stimulated the production of both neutralizing and hemagglutination inhibition antibodies in monkeys; these animals were also protected against challenge with the homologous strain as well as a heterologous strain of DEN-4. An ID50 titration in monkeys resulted in a titer of greater than 10(4) plaque-forming units (PFU) for the vaccine virus and 0.5 PFU for the parent virus. Reduced monkey infectivity of this magnitude has been correlated with human attenuation in previous dengue vaccine candidates. The DEN-4 strain 341750 Carib PCK-20/FRhL-4 vaccine has been characterized and sufficiently tested to be considered for safety and immunogenicity trials in humans.

Animals↗
PubMed1990

Preparation of an attenuated dengue 4 (341750 Carib) virus vaccine. II. Safety and immunogenicity in humans.

To determine safety and immunogenicity, a single 0.5 ml dose of a monovalent live-attenuated dengue (DEN) 4 (341750 Carib) vaccine was given sc to 3 groups of flavivirus nonimmune volunteers in increasing concentrations. Two recipients received 10(3) plaque forming units (PFU)/dose (1:100 dilution of stock vaccine). One remained asymptomatic, but became viremic between days 12 and 15, experienced a mild elevation of temperature (37.4 degrees C), and developed DEN-4 specific antibody. Neither recipient of the 10(4) PFU became infected. Eight volunteers then received undiluted vaccine (10(5) PFU). Viremia and antibody (neutralizing, hemagglutination inhibition, and IgM) developed in 5 of the 8 (63%). These 5 volunteers also developed a scarcely noticeable macular, blanching rash and minimal temperature elevations (37.3, 38.1, 37, 37.9, and 37.9 degrees C). Clinically insignificant decreases in total white blood cell, lymphocyte, and polymorphonuclear cell counts and an elevation in mononuclear cell counts occurred in association with viremia. This vaccine is safe, reasonably immunogenic, and suitable for further evaluation.

Adult↗
PubMed1989

Flavivirus entry into cultured mosquito cells and human peripheral blood monocytes.

The 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.

Adsorption↗
PubMed1989

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.

Aedes↗
PubMed1989

Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis.

We have constructed vaccinia virus recombinants expressing dengue virus proteins from cloned DNA for use in experimental immunoprophylaxis. A recombinant virus containing a 4.0-kilobase DNA sequence that codes for three structural proteins, capsid (C), premembrane (pre-M), and envelope (E), and for nonstructural proteins NS1 and NS2a produced authentic pre-M, E, and NS1 in infected CV-1 cells. Mice immunized with this recombinant were protected against an intracerebral injection of 100 50% lethal doses of dengue 4 virus. A recombinant containing only genes C, pre-M, and E also induced solid resistance to challenge. Deletion of the putative C-terminal hydrophobic anchor of the E glycoprotein did not result in secretion of E from recombinant-virus-infected cells. Recombinants expressing only the E protein preceded by its own predicted N-terminal hydrophobic signal or by the signal of influenza A virus hemagglutinin or by the N-terminal 71 amino acids of the G glycoprotein of respiratory syncytial virus produced glycosylated E protein products of expected molecular sizes. These vaccinia virus recombinants also protected mice.

Animals↗
PubMed1989

Monoclonal antibodies for dengue virus prM glycoprotein protect mice against lethal dengue infection.

Five murine monoclonal antibodies (Mabs) reactive against the prM glycoproteins of DEN-3 and -4 were used to passively protect mice in vivo against lethal challenge with homologous and heterologous dengue virus serotypes. Four of the 5 prM-reactive monoclonals cross-protected mice against heterologous challenge, whereas 1 protected against challenge with only the homologous serotype. Although in vitro binding to virions was readily demonstrated, only 2 of the prM Mabs had detectable neutralizing activity. The neutralizing activity could not be enhanced by anti-mouse immunoglobulin or complement. However, 4 of the 5 prM Mabs fixed complement. This is the first report of prM-specific Mabs that are protective in mice.

Antibodies, Monoclonal↗
PubMed1988

Japanese encephalitis virus live-attenuated vaccine, Chinese strain SA14-14-2; adaptation to primary canine kidney cell cultures and preparation of a vaccine for human use.

The Japanese encephalitis (JE) live-attenuated vaccine virus clone SA14-14-2 was adapted to grow in primary canine kidney (PCK) cell culture, and vaccine seeds and a first lot of vaccine were prepared in these cells. Characterization of the PCK-grown virus by various laboratory and animal tests indicated that passage in PCK did not result in detectable phenotypic or genome changes for this virus clone. Markers of attenuation included small plaque size, lack of intracerebral virulence for weanling mice, minimal neurovirulence for rhesus monkeys and a distinct nucleotide pattern compared to the parent SA14 non-attenuated virus. In addition, the seeds and vaccine were free of any detectable adventitious microbial agents that would render these materials unsafe for human immunization. Small-scale clinical trials of the JE SA14-14-2 PCK vaccine can now proceed to test the human safety of this product.

Animals↗
PubMed1988

Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue virus encephalitis.

We have constructed a recombinant baculovirus containing a 4.0-kilobase dengue virus cDNA sequence that codes for the three virus structural proteins, capsid (C) protein, premembrane (PreM) protein, and envelope glycoprotein (E), and nonstructural proteins NS1 and NS2a. Infection of cultured Spodoptera frugiperda cells with this recombinant virus resulted in the production of E and NS1 proteins that were similar in size to the corresponding viral proteins expressed in dengue virus-infected simian cells. Other dengue virus-encoded proteins such as PreM and C were also synthesized. Rabbits immunized with the dengue virus protein products of the recombinant virus developed antibodies to PreM, E, and NS1, although the titers were low, especially to PreM and E. Nevertheless, the dengue virus antigens produced by the recombinant virus induced resistance in mice to fatal dengue encephalitis.

Animals↗
PubMed1987

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

The 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.

Aedes↗
PubMed1987

Maturation process of Japanese encephalitis virus in cultured mosquito cells in vitro and mouse brain cells in vivo.

The 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.

Animals↗
PubMed1987

Monoclonal antibodies against dengue 2 virus E-glycoprotein protect mice against lethal dengue infection.

A panel of 11 murine monoclonal antibodies directed against dengue type 2 was evaluated for antigen specificity by dot immunobinding assay and Western blot analysis and for in vitro and in vivo biological activities. Nine of the 11 monoclonal antibodies reacted with viral E-glycoprotein based on the Western blot analysis; one reacted with a 36 Kd protein present in dengue-infected C6/36 mosquito cells. The nine E-glycoprotein-reactive monoclonal antibodies also neutralized dengue 2 virus in a plaque reduction assay. Of the neutralizing monoclonal antibodies, five passively protected mice in vivo against lethal intracerebral dengue 2 challenge. The protective monoclonal antibodies were directed against viral determinants that fell into at least three spatially separate families of epitopes on E-glycoprotein, the antigenicities of which were preserved after heat/detergent denaturation.

Aedes↗
PubMed1987

Lack of attenuation of a candidate dengue 1 vaccine (45AZ5) in human volunteers.

A dengue type 1, candidate live virus vaccine (45AZ5) was prepared by serial virus passage in fetal rhesus lung cells. Infected cells were treated with a mutagen, 5-azacytidine, to increase the likelihood of producing attenuated variants. The vaccine strain was selected by cloning virus that produced only small plaques in vitro and showed reduced replication at high temperatures (temperature sensitivity). Although other candidate live dengue virus vaccines selected for similar growth characteristics have been attenuated for humans, two recipients of the 45AZ5 virus developed unmodified acute dengue fever. Viremia was observed within 24 hr of inoculation and lasted 12 to 19 days. Virus isolates from the blood produced large plaques in cell culture and showed diminished temperature sensitivity. The 45AZ5 virus is unacceptable as a vaccine candidate. This experience points out the uncertain relationship between in vitro viral growth characteristics and virulence factors for humans.

Adult↗