Comparative study of influenza virus replication in Vero and MDCK cell lines.

The choice of a cell line for the production of influenza vaccines is determined by how well the virus is able to replicate and how easily the cell line can be maintained. Madin-Darby canine kidney (MDCK) cells have long been known to successfully support influenza growth. Vero cells are also another well studied candidate cell line. In this work, we have compared these two cell lines for their ability to propagate type A and type B cold-adapted and wild type influenza viruses. The growth of these viruses has been measured as plaque forming units (via plaque assay) as well as viral particle formation (via a novel quantitative RT-PCR assay) to assess the suitability of these cell lines to support the development of live attenuated influenza vaccines. The novel qRT-PCR assay outlined in this work was demonstrated to be an efficient, sensitive and reproducible method for measuring wild type (wt) and cold-adapted (ca) influenza strains. Replicates of six per sample consistently showed an average variation around +/-10%. In this study we have also found qRT-PCR to be a useful method for differentiating between wt and ca influenza strains based on their differing growth characteristics at varying temperatures. This can subsequently be used to assess reassortants prepared from ca donor strains for the purposes of live viral vaccine development. For type A and B influenza viruses studied in this work, MDCK cells supported a more rapid viral growth (measured in terms of genome copies) compared with Vero cells. For the type A viruses studied here, the genome copies: infectious unit (genome copy, gc:infectious unit, iu) ratio was found to be more favorable for Vero cells compared with MDCK cells. For the type B viruses studied in this work, the gc:iu was equivalent in both cell lines tested. Ultimately, however, the use of any new cell line would need to be approved by regulatory agencies prior to its commercial application.

The I1307K APC mutation does not predispose to colorectal cancer in Jewish Ashkenazi breast and breast-ovarian cancer kindreds.

An increased incidence of colorectal cancer has been observed in breast and breast-ovarian cancer syndrome families, including those of Ashkenazi origin. Recently, a germ-line missense mutation in the APC gene, I1307K, was identified that may indirectly cause colorectal cancer in Ashkenazi Jews. To determine whether the excess of colon cancer in some breast-ovarian cancer families is related to the I1307K mutation, we evaluated 264 Ashkenazi Jews from 158 families. Most of these individuals had either a personal or a family history of breast and/or ovarian cancer, and 19.3% (51 of 264) carried one of the recurrent BRCA1 (185delAG or 5382 insC) or BRCA2 (6174delT) mutations. We detected the APC I1307K mutation in 7% (11 of 158) of the Ashkenazi Jewish families and in 4.5% (12 of 264) of the individuals participating in these studies. Of the families studied, 26.6% (42 of 158) had at least one case of colorectal cancer in a first-, second-, or third-degree relative of the proband. Significantly, of the 12 individuals who possessed the I1307K mutation, none was diagnosed with colorectal cancer and none had a known first-, second-, or third-degree relative diagnosed with colon cancer. The results suggest that factors other than the I1307K mutation contribute to the increased incidence of colon cancer in Ashkenazi breast-ovarian cancer families. Our results emphasize that only a subset of Ashkenazi Jewish individuals with a family history of colorectal cancer should be viewed as candidates for genetic susceptibility testing for the I1307K APC mutation.