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.

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Rhinovirus (RV), a member of the Picornaviridae family, accounts for many virus-induced asthma exacerbations. RV induces airway cell chemokine expression both in vivo and in vitro. Because of the known interactions of proteases with cellular functions, we hypothesized that RV 3C protease is sufficient for cytokine up-regulation. A cDNA encoding RV16 3C protease was constructed by PCR amplification and transfected into 16HBE14oϪ human bronchial epithelial cells. 3C protease induced expression of both IL-8 and GM-CSF, as well as transcription from both the IL-8 and GM-CSF promoters. 3C expression also induced activator protein 1 and NF-B transcriptional activation. Finally, mutation of IL-8 promoter AP-1 and NF-B promoter sequences significantly reduced 3C-induced responses. Together, these data suggest expression of RV16 3C protease is RVs are the most common upper respiratory pathogens, inducing the majority of common colds worldwide. Viral infections trigger asthma in 80 to 85% of asthma exacerbations in children and 44% in adults (1, 2) RV, a member of the Picornaviridae family of small, positive-stranded RNA viruses, accounts for much of virus-induced asthma exacerbations (1, 2). Cytokines are elaborated in vivo in the nasal mucosa, and most likely also from bronchial epithelium, after RV infection. Neutrophils, eosinophils, and lymphocytes increase in the nasal secretions of RV-infected patients compared with uninfected controls (3, 4).The cytokines IL-8 and GM-CSF have been implicated in the pathogenesis of asthma. IL-8 is a potent activator and chemoattractant of neutrophils (5) and eosinophils (6), and GM-CSF promotes the differentiation and survival of recruited eosinophils (7). Increased levels of IL-8, GM-CSF (8), and ICAM-1 (9), the receptor of 90% of RV serotypes, as well as IL-1 and IL-6, have been found in the nasal secretions of individuals infected with RVs (10 -12). In bronchial mucosal biopsies obtained after experimental infection with RV, Bardin and colleagues (13) noted a significant increase in submucosal lymphocytes and epithelial eosinophils, which, in asthmatics, persisted into convalescence. Although increases in cytokine secretion in the nasal mucosa does not prove increased expression in the epithelium in the lower airways, taken together the above data strongly suggest that cytokine elaboration is increased in the RV-infected lower airway epithelium.RV infection also induces cytokine production and adhesion molecule expression in cell culture. Divergent findings regard-

Mutations which positively affect growth of hepatitis A virus in cell culture may negatively affect growth in vivo. Therefore, development of an attenuated vaccine for hepatitis A may require a careful balancing of mutations to produce a virus that will grow efficiently in cells suitable for vaccine production and still maintain a satisfactory level of attenuation in vivo. Since such a balance could be achieved most directly by genetic engineering, we are analyzing mutations that accumulated during serial passage of the HM-175 strain of hepatitis A virus in MRC-5 cell cultures in order to determine the relative importance of the mutations for growth in MRC-5 cells and for attenuation in susceptible primates. Chimeric viral genomes of the HM-175 strain were constructed from cDNA clones derived from a virulent virus and from two attenuated viruses adapted to growth in African green monkey kidney (AGMK) and MRC-5 cells, respectively. Viruses encoded by these chimeric genomes were recovered by in vitro or in vivo transfection and assessed for their ability to grow in cultured MRC-5 cells or to cause hepatitis in primates (tamarins). The only MRC-5-specific mutations that substantially increased the efficiency of growth in MRC-5 cells were a group of four mutations in the 5 noncoding (NC) region. These 5 NC mutations and a separate group of 5 NC mutations that accumulated during earlier passages of the HM-175 virus in primary AGMK cells appeared, independently and additively, to result in decreased biochemical evidence of hepatitis in tamarins. However, neither group of 5 NC mutations had a demonstrable effect on the extent of virus excretion or liver pathology in these animals.