Biomedical subjects
Publications and source records attributed to N J Marchette.
Respiratory syncytial virus was isolated from hospitalized children in Hawaii in each month of the year during the period January 1987 to August 1989. Subgroup A and subgroup B strains cocirculated, with subgroup A predominating. There was an alternating early-season and late-season peak incidence cycle as reported elsewhere.
We tested three dengue type 2 (DEN-2) isolates from children with clinically apparent but mild secondary dengue infections, and 10 isolates from children with moderately severe dengue hemorrhagic fever, and noted significant growth differences in peripheral blood leukocytes, but not in C6/36 cells. We also observed cytopathic effects in C6/36 cells that correlated with disease severity. These preliminary observations suggest the possibility that viral factors, whether surface antigens, attachment sites for entry into leukocytes, or intrinsic replication properties in human mononuclear phagocytes, might contribute to enhanced DEN infection and to the severity of the disease.
Epstein-Barr virus (EBV), a possible cause of Kawasaki syndrome (KS), is not pathenogenically associated with KS in Hawaii. The prevalence of EBV capsid antibody in KS patients was found not to differ significantly from that in controls, and the antibody response in those infected with EBV was the same as that in other children similarly infected. No EBV was isolated from acute-phase patients. All patients with capsid antibody at the onset of KS also had Epstein-Barr nuclear antigen antibody: 36 patients developed antibody within 3 months after onset of KS; in 10, EBV infection could have been coincidental with the disease. Cytomegalovirus (CMV) was isolated from 9 patients with KS and 10 controls. A similar number of controls and patients had antibody to human herpesvirus 6 (HHV6); one patient seroconverted. None of the herpes viruses (EBV, CMV, HHV6, varicella-zoster virus, or herpes simplex virus) plays a unique or dominant role in the etiology or pathogenesis of KS in Hawaii.
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.
Kawasaki syndrome, an acute febrile multisystem illness of young children, is a panvasculitis with prominent rheumatic features. Arthritis and pancarditis are frequent during the acute stage; coronary artery aneurysms occur in 20% of cases and the disease is now the leading cause of acquired heart disease in childhood. A microbial aetiology is suggested by the acute febrile self-limited character of the disease, the regular occurrence of epidemic outbreaks at intervals of 2-3 years, and the virtual restriction to young children, consistent with the early acquisition of immunity. Reports of elevated DNA polymerase activity (assumed to be RNA-dependent reverse transcriptase) in cultured lymphocytes from patients with acute Kawasaki syndrome suggest that a retrovirus might be the causative agent. We have measured supernatant DNA polymerase activity in lymphocyte cultures from 49 Hawaiian patients in acute and convalescent stages of Kawasaki syndrome and have been unable to demonstrate significant reverse transcriptase activity or other evidence of involvement of a retrovirus in the aetiology of the disease.
A dengue 4 (DEN-4, strain 1036-PDK 48) vaccine attenuated by passage in primary dog kidney cells was tested using rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) monkeys to determine its safety, potency, and immunogenicity. 14 rhesus monkeys were divided into 3 groups: group 1, 2 animals given control culture fluid; group 2, 2 animals given DEN-4 parental virus; group 3, 10 animals given DEN-4 vaccine virus. 10 cynomolgus were similarly grouped, but group 3 contained 6 monkeys. No significant neurovirulence was observed with parental or with DEN-4 virus passaged in primary dog kidney (PDK) cells. Both cynomolgus monkeys inoculated with DEN-4 vaccine virus developed minimal (V-1) and mild (V-2) neurovirulence-type lesions in the central nervous system, which were nondestructive in both species. All parental and vaccine viruses produced moderate to high neutralizing antibody titres. Only parental virus produced viraemia, in 2 cynomolgus monkeys. Because of its safety and avirulence in monkeys, PDK 48 is recommended for human trial.
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.
The results of a comparative study of neurovirulence of dengue type 1 virus in two species of Old World monkeys, viz. rhesus monkeys (Macaca mulatta) and cynomolgus monkeys (Macaca fascicularis) are reported. In the present study, parental dengue type 1 (16007) and its vaccine viruses were tested by intrathalamic, intramuscular and intraspinal injections in these two species of monkey. Both species of monkeys inoculated with parental dengue type 1 virus developed neurovirulence-type lesions which were graded as minimal (V-1) and occasionally mild (V-2, in cynomolgus monkeys) in severity. The antibody response to either parental or vaccine virus was slightly less in rhesus monkeys than in cynomolgus inoculated with these strains. This comparative study possibly establishes the cynomolgus monkey as a suitable test model to replace the rhesus monkey for neurovirulence testing of dengue-1 vaccine intended for use in humans.
Antibody-dependent infection enhancement (ADE) was studied with P-388D1 mouse macrophage-like cells, 21 dengue virus type 2 (DEN-2) strains, and 8 monoclonal antibodies reactive with flavivirus group-specific or dengue serotype-specific determinants. Testing a constant number of virions against serial dilutions of antibody for their ability to infect P-388D1 cells, a reproducible 'enhancement profile' was observed. The profile was characterized by (1) appearance, peak, decline, and disappearance of infection enhancement when antibody-containing ascitic fluids were diluted beyond the neutralizing endpoint, and (2) evolution over an approximate 10,000-fold dilutional range. The profiles were similar regardless of whether viruses were complexed with antibody at flavivirus group or serotype determinants, but the antibody dilution at which infection enhancement was maximal varied with the neutralization titer of the antibody. Neutralization and antibody-dependent enhancement of dengue infection appear to be biological outcomes of interactions between antibodies and single viral epitopes at different antibody: virus ratios.
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Cross-infection enhancement of seven African flaviviruses by subneutralising concentrations of antibody in immune ascitic fluids was investigated in P388D1 cell culture. Infection by all the seven flaviviruses tested was enhanced by homologous and at least one of six heterologous immune mouse ascitic fluids (IMAF) tested. Enhancement ratios and enhancing antibody titres were higher in homologous than in heterologous enhancement. Zika, Wesselsbron, Uganda S and West Nile viruses were enhanced in culture by all the IMAF tested. Enhancement of Dakar bat and Yellow fever viruses was produced by five heterologous IMAF, but Potiskum virus was enhanced by one heterologous flavivirus antibody. The antibody to Potiskum virus was the most potent mediator of heterologous infection enhancement; all six heterologous flaviviruses were markedly enhanced by this antibody.
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Vaccines prepared from attenuated virus can cause symptomatic viral infection of the central nervous system. In the present study, dengue-2 parental and its live attenuated viruses were tested by intrathalamic and intraspinal injections in rhesus monkeys. The dengue-2 viruses were found to be only very weakly neurovirulent when injected directly into the brain or spinal cord of rhesus monkeys.
The etiology of Kawasaki syndrome remains unestablished, although a possible role has been suggested for exposure to the application of carpet shampoo, house dust mites, and rickettsial infection. During an outbreak of 20 cases of Kawasaki syndrome that occurred in southeastern Wisconsin from November 1982 through March 1983, a case-control study was done of 15 cases and 30 matched controls. The study included questionnaire administration, dust collection from homes, and serum specimen collection. Only one patient had been exposed to a shampooed carpet within 30 days before onset of illness. No differences were noted between cases and controls in the degree of exposure to house dust mite-associated factors in the home, nor in the occurrence, density and species-specific prevalence of house dust mites in the home. Meadow voles exposed to house dust mites from the homes of patients did not develop serologic or pathologic evidence of infection due to rickettsiae in the spotted fever and typhus groups or Coxiella burnetii. Anti-mite-specific immunoglobulin E was not detected in serum specimens from cases or controls. Results from this study do not support hypotheses suggesting that the development of Kawasaki syndrome is associated with exposure to application of carpet shampoo, house dust mites, or rickettsial infection.
Attempts were made to attenuate prototype dengue (DEN) 4 (H-241) virus. The original viremic human serum was passed once in a susceptible monkey and twice in Aedes albopictus mosquitoes and then serially passed in primary dog kidney (PDK) and African green monkey kidney (GMK) cells. Weekly transfers of undiluted virus were carried to the 50th passage in both primary cell cultures. Biological markers were studied at passages 7, 15, 30 and 50. Parental DEN-4 phenotype characteristics included large plaque formation in LLC-MK2 cells, plaque formation in GMK cells, cytopathic effect in LLC-MK2 cells, growth in human monocyte cultures, growth at 39 degrees C, consistent production of viremia in monkeys and short-incubation neurovirulence in mice. At the seventh passage in both PDK and GMK cell cultures, DEN-4 viruses exhibited reduced plaque-size in LLC-MK2, and failed to plaque in GMK, to produce cytopathic effect in LLC-MK2, or to grow in human monocytes. Serial passage in PDK, as opposed to GMK, resulted in a graduated loss of monkey virulence. Rhesus monkeys inoculated with the PDK 50 strain failed to develop detectable viremia and only 1 of 4 developed an antibody response. Also, replication of PDK 50 was completely shut-off at 39 degrees C. The graduated change in biological properties noted, particularly those in PDK cells, provide a range of potential vaccine candidates for evaluation in human beings.
Uncloned dengue (DEN) 4 (H-241) which had been passaged 15, 30 and 50 times in primary dog kidney (PDK) cells were subjected to two successive terminal dilution procedures. In the first (3Cl), virus was diluted in 10-fold steps in 10 replicate tubes. An infected tube from a dilution row with three or fewer virus-infected tubes was selected for two further passages. In the second (TD3), virus was triple terminal diluted using 2-fold dilution steps and selecting one positive tube out of 10. Both procedures selected virus population which differed from antecedents. Plaque size of PDK 15 was medium, PDK 30, small and PDK 50, pin-point. PDK 19-3Cl were medium and 56-3Cl, 24-TD3, 35-TD3 and 61-TD3 were all small. All cloned virus replication was completely shut-off at 38.5 degrees C; PDK 15 and 30 continued to replicate at this temperature. Uncloned viruses showed a graduated decrease in monkey virulence with PDK passage; cloned viruses were either avirulent for monkeys (19-3Cl, 56-31Cl, 24-TD3 and 35-TD3) or produced revertant large plaque parental-type viremia (35-3Cl and 61-TD3). Those cloned viruses which exhibited temperature sensitivity, reduced monkey virulence and stability after monkey passage may be suitable as vaccine candidates for evaluation in human beings.
Two strains of primary dog kidney-passaged dengue (DEN) 4 (H-241) virus cloned by terminal dilution (PDK 24-TD3 and 35-TD3) were propagated in fetal rhesus lung (FRhL) cells to produce candidate vaccine virus seeds. Both serial passage and prolonged replication of PDK 24-TD3 in FRhL resulted in appearance of medium and large plaques in LLC-MK2 assays. When picked, these plaques proved to contain temperature-resistant, monkey-virulent revertants. Serial passage and prolonged replication of PDK 24-TD3 in LLC-MK2 cells did not result in reversion; but, prolonged replication in PDK cells did. Passage of PDK 35-TD3 in FRhL cells resulted in appearance of medium size plaques which, when picked, yielded temperature sensitive (ts) (38.5 degrees C) viruses of low monkey-virulence. Because of its stability in monkeys and FRhL cells, reduced monkey virulence and ts property. PDK 35-TD3 is a promising candidate for trial in man.