A live candidate hepatitis A virus vaccine, developed from the HM-175 strain and adapted to growth in primary African green monkey kidney (AGMK) cells, was adapted to growth in MRC-5 cells. The nucleotide sequence of the MRC-5 cell-adapted virus was determined and compared with the known sequence of the AGMK cell-adapted virus. Thirteen unique mutations, which occurred during passage in MRC-5 cells, were identified. Four of the unique mutations were located in a cluster in the 5' noncoding region (NC), and three of the remaining nine mutations encoded amino acid changes. Infectious chimeric cDNAs were constructed from infectious cDNA clones of the AGMK cell-adapted and wild-type HM-175 viruses and PCR-amplified cDNA segments of the MRC-5 cell-adapted virus. The viruses encoded by these plasmids were recovered after transfection of cultured cells with in vitro transcripts, and their growth phenotypes in fetal rhesus kidney 4 (FRhK-4) and MRC-5 cells were determined. The important growth-enhancing mutations could be divided into three sets. Two of these were located in the 5' NC region, and the third was located in the 2C nonstructural gene. The mutations in the 5' NC region that developed during passage in MRC-5 cells were indispensable for efficient growth in MRC-5 cells, but a combination of the two groups in the 5' NC region and one in the 2C gene were required to increase growth dramatically in MRC-5 cells.

Alzheimer's Disease (AD) is a complex genetic disorder with four loci already identified. Mutations in three of these, the amyloid precursor protein, presenilin I, and presenilin II, cause early-onset AD. The apolipoprotein E (APOE) gene contributes primarily to late-onset AD. The APOE-4 allele acts in a dose-related fashion to increase risk and decrease the age-of-onset distribution in AD. We examined the effect of APOE on AD in a previously unstudied Amish population that has a lower prevalence of dementia compared with other populations. We sampled a large inbred family with 6 late-onset AD members. We also genotyped 53 individuals from the general Amish population as controls for the APOE allele frequency estimates. The frequency of the APOE-4 allele in the Amish controls was 0.037 +/- 0.02. This differed significantly compared with three independent sets of non-Amish white controls (p < 2 x 10(-4), p < 6 x 10(-5), and p < 2 x 10(-6)). In addition, all Amish AD-affected individuals had APOE 3/3 genotypes; no APOE X/4 or 4/4 individuals were observed. We suggest that the lower frequency of dementia in the Amish may be partially explained by the decreased frequency of the APOE-4 allele in this population, and that the inbred nature of this pedigree, with its strong clustering of cases contrasted against the lower frequency of dementia, indicates that additional genetic factors influence late-onset AD.

We have prepared two prototype live hepatitis A virus (HAV) vaccines by serial passage of the HM-175 strain of HAV in African green monkey kidney cells. Passage 21 (P-21) HM-175 virus shows evidence of attenuation for chimpanzees but not for marmosets; passage 32 (P-32) HM-175 virus shows evidence of attenuation for both species. Animals that received P-32 HAV had fewer elevations in levels of liver enzyme activity and evidence of less virus replication in the liver and excretion of virus in stool than did those that received wild-type virus. The P-21 and P-32 viruses were highly immunogenic in both species. The information provided by this study will aid in the development of a live HAV vaccine.