Variants of hepatitis A virus (pHM175 virus) recovered from persistently infected green monkey kidney (BS-C-1) cells induced a cytopathic effect during serial passage in BS-C-1 or fetal rhesus kidney (FRhK-4) cells. Epitope-specific radioimmunofocus assays showed that this virus comprised two virion populations, one with altered antigenicity including neutralization resistance to monoclonal antibody K24F2, and the other with normal antigenic characteristics. Replication of the antigenic variant was favored over that of virus with the normal antigenic phenotype during persistent infection, while virus with the normal antigenic phenotype was selected during serial passage. Viruses of each type were clonally isolated; both were cytopathic in cell cultures and displayed a rapid replication phenotype when compared with the noncytopathic passage 16 (p16) HM175 virus which was used to establish the original persistent infection. The two cytopathic virus clones contained 31 and 34 nucleotide changes from the sequence of p16 HM175. Both shared a common 5' sequence (bases 30 to 1677), as well as sequence identity in the P2-P3 region (bases 3249 to 5303 and 6462 to 6781) and 3' terminus (bases 7272 to 7478). VP3, VP1, and 3CPr0 contained different mutations in the two virus clones, with amino acid substitutions at residues 70 of VP3 and 197 and 276 of VP1 of the antigenic variant. These capsid mutations did not affect virion thermal stability. A comparison of the nearly complete genomic sequences of three clonally isolated cytopathic variants was suggestive of genetic recombination between these viruses during persistent infection and indicated that mutations in both 5' and 3' nontranslated regions and in the nonstructural proteins 2A, 2B, 2C, 3A, and 3DPo' may be related to the cytopathic phenotype.

The pulmonary collectins surfactant protein (SP)-A and SP-D play important roles in innate lung defense, enhancing opsonization of microbes and limiting lung inflammatory responses. To quantify relationships among collectins, bacteria, and inflammation in early cystic fibrosis (CF) airway secretions, bronchoalveolar lavage fluids (BALFs) were collected from children undergoing clinically indicated bronchoscopy. Quantitative bacteriology, differential cell counts, and ELISA for SP-A and SP-D were assessed. Significantly increased numbers of neutrophils relative to bacteria were noted in BALF from CF compared with non-CF subjects. Although SP-A levels tended to be lower in CF compared with non-CF, this was only significant in the presence of bacterial infection. Among CF patients, SP-A concentrations in BALF were inversely related to inflammation, bacterial colony-forming units per milliliter, and age. SP-D levels were significantly decreased in CF patients, and SP-D was rarely detectable in the presence of infection. Among CF patients, SP-D correlated inversely with inflammation and bacterial colony-forming units per milliliter, and there was decreased immunostaining of BALF cells for SP-D in CF. Immunohistochemistry of CF autopsy lung sections for SP-A and SP-D confirmed their paucity at sites of infection and inflammation. We conclude that relative collectin deficiency occurs early in CF airways and is inversely related to inflammation in CF airways.

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Respiratory synctial virus (RSV) infection of undifferentiated airway epithelial cells has been shown to induce the production of chemokines. The purpose of this study was to investigate the vectorial release of interleukin (IL-8) and Released on Activation, Normal T-cell Expressed and Secreted (RANTES) by polarized, well-differentiated respiratory epithelial cells after RSV infection. Human bronchial epithelial cultures were differentiated under air-liquid interface conditions and infected with RSV by the apical or basolateral route. RSV infection was specific to the apical surface. Supernatants were collected at 6 and 48 hours after RSV inoculation, and IL-8 and RANTES were measured by enzyme-linked immunosorbent assay (ELISA). Both IL-8 and RANTES were significantly released by 48 hours following inoculation with RSV. The secretion of each chemokine was greatest after apical inoculation, and secretion was polarized to the basolateral supernatant. Immunohistochemical staining confirmed that RSV infection was specific to ciliated cells, and immunohistochemical staining for chemokines was localized to RSV-infected ciliated cells. The authors conclude that, in differentiated human airway epithelium in vitro, RSV-induced increases in IL-8 and RANTES release are predominantly in the basolateral direction. In epithelial layers, virus-containing cells are the predominant source of the increased chemokine release. The authors speculate that similar processes in vivo influence recruitment of leukocytes to sites of RSV infection.

Passage of human hepatitis A virus (HAV) in cell culture results in attenuation of the virus as well as progressive increases in the efficiency of virus replication in cell culture. Because the presence of identical mutations within the 5' nontranslated regions (5'NTRs) of several independently isolated cell culture-adapted HAV variants suggests that the 5'NTR may play a role in determining this change in virus host range, we constructed chimeric infectious cDNA clones in which portions of the 5'NTR of cell culture-adapted HM175/p35 virus were replaced with cDNA from either wild-type virus (HM175/wt) or a second independently isolated, but closely related cell culture-adapted virus (HM175/pl6). Substitution of the complete 5'NTR of HM175/p35 with the 5'NTR of HM175/wt resulted in virus with very small replication foci in continuous