Comparative Study of Influenza Virus Replication in MDCK Cells and in Primary Cells Derived from Adenoids and Airway Epithelium

Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is de- sirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bron- chial epithelium (NHBE cells). Our data showed that despite the fact that all tissue culture models lack a functional adaptive im- mune system, differentiated cultures of human epithelium exhibited the greatest restriction for all H1N1, H3N2, and B vaccine viruses studied among three cell types tested and the best correlation with their levels of attenuation seen in clinical trials with humans. In contrast, the data obtained with MDCK cells were the least predictive of restricted viral replication of live attenuated vaccine viruses in humans. We were able to detect a statistically significant difference between the replication abilities of the U.S. (A/Ann Arbor/6/60) and Russian (A/Leningrad/134/17/57) cold-adapted vaccine donor strains in NHBE cultures. Since live at- tenuated pandemic influenza vaccines may potentially express a hemagglutinin and neuraminidase from a non-human influenza virus, we assessed which of the three cell cultures could be used to optimally evaluate the infectivity and cellular tropism of vi- ruses derived from different hosts. Among the three cell types tested, NHBE cultures most adequately reflected the infectivity and cellular tropism of influenza virus strains with different receptor specificities. NHBE cultures could be considered for use as a screening step for evaluating the restricted replication of influenza vaccine candidates

Cleavage of Influenza A Virus Hemagglutinin in Human Respiratory Epithelium Is Cell Associated and Sensitive to Exogenous Antiproteases

Proteolytic cleavage of the hemagglutinin (HA) of human influenza viruses A/Aichi/2/68 (H3N2) and A/WSN/34 (H1N1) from HA0 to HA1/HA2 was studied in primary human adenoid epithelial cells (HAEC). HAEC contain a mixture of ciliated and nonciliated secretory cells and mimic the epithelium membrane of the human respiratory tract. Pulse-chase labeling with [(35)S]methionine and Western blot analysis with anti-HA antibodies of cellular and virion polypeptides showed that HAEC cleaved newly synthesized HA0 to HA1/HA2 (“cleavage from within”) and significant amounts of cleaved HA accumulated within cells. It was also shown that HAEC was able to cleave HA0 of incoming virions (“cleavage from without”), whereas the HA0 of nonabsorbed virions free in extracellular fluid were not cleaved, supporting the conclusion that HA0 cleavage in HAEC is cell associated. Low-molecular-weight inhibitors of serine proteases, aprotinin and leupeptin, when added to influenza virus-infected HAEC suppressed HA0 cleavage and reduced the amount of cleaved HA1/HA2 both in cells and in progeny virions and thus diminished the infectivity of the virus. In contrast, the addition of fetal bovine serum, containing a number of high-molecular-weight antiproteases that compete for proteases in the extracellular environment, did not inhibit influenza virus growth in HAEC. These data suggest that in human respiratory epithelium the cleavage of influenza virus HA containing a single arginine in the proteolytic site (i) is a cell-associated process accomplished by serine-type protease(s) and (ii) is sensitive to low-molecular-weight exogenous inhibitors of serine proteases

Detection of Viruses in Human Adenoid Tissues by Use of Multiplex PCR▿

By PCR, we detected a high frequency of viruses in adenoids obtained from children without acute respiratory symptoms. Our results suggest that persistent/latent viral infection in the respiratory tract confounds interpretation of the association of pathogen detection by PCR with acute respiratory infection in these sources

Serologic Imprint of Dengue Virus in Urban Haiti: Characterization of Humoral Immunity to Dengue in Infants and Young Children

Dengue is endemic to Haiti but not recognized as an important illness in the autochthonous population. To evaluate the prevalence of antibodies to dengue virus (DENV), serum samples from infants and young children 7–36 months of age (n = 166) were assayed by plaque reduction neutralization assays to each DENV serotype. Dengue virus serotype 1 had infected 40% of this study population, followed by serotype 2 (12%), serotype 3 (11%), and serotype 4 (2%). Fifty-three percent of infants and young children less than 12 months of age had already experienced DENV infection, and the seroprevalence of antibody to DENV increased to 65% by 36 months. Heterotypic antibody responses were an important component of the total dengue immunity profile