Comparative Susceptibility of Madin-Darby Canine Kidney (MDCK) Derived Cell Lines for Isolation of Swine Origin Influenza A Viruses from Different Clinical Specimens.

Madin-Darby canine kidney (MDCK) cells are commonly used for the isolation of mammalian influenza A viruses. The goal of this study was to compare the sensitivity and suitability of the original MDCK cell line in comparison with MDCK-derived cell lines, MDCK.2, MDCK SIAT-1 and MDCK-London for isolation of swine-origin influenza A viruses (IAV-S) from clinical specimens. One-hundred thirty clinical specimens collected from pigs in the form of nasal swabs, lung tissue and oral fluids that were positive by PCR for the presence of IAV-S RNA were inoculated in the cell cultures listed above. MDCK-SIAT1 cells yielded the highest proportion of positive IAV-S isolations from all specimen types. For nasal swabs, 58.62% of the specimens were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (36.21%), and conventional MDCK and MDCK.2 cells (27.5%). For lung specimens, 59.38% were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (40.63%), and conventional MDCK and MDCK.2 cells (18.75-31.25%). Oral fluids yielded the lowest number of positive virus isolation results, but MDCK-SIAT1 cells were still had the highest rate (35%) of IAV-S isolation, whereas the isolation rate in other cells ranged from 5-7.5%. Samples with lower IAV-S PCR cycle threshold (Ct) values were more suitable for culturing and isolation. The isolated IAV-S represented H1N1-ß, H1N2-α, H1N1pdm and H3N2 cluster IV and cluster IVB viruses. The result of the current study demonstrated the importance of using the most appropriate MDCK cells when isolating IAV-S from clinical samples.

High-throughput sequencing for species authentication and contamination detection of 63 cell lines.

Cell lines are widely used in research and for diagnostic tests and are often shared between laboratories. Lack of cell line authentication can result in the use of contaminated or misidentified cell lines, potentially affecting the results from research and diagnostic activities. Cell line authentication and contamination detection based on metagenomic high-throughput sequencing (HTS) was tested on DNA and RNA from 63 cell lines available at the Canadian Food Inspection Agency's National Centre for Foreign Animal Disease. Through sequence comparison of the cytochrome c oxidase subunit 1 (COX1) gene, the species identity of 53 cell lines was confirmed, and eight cell lines were found to show a greater pairwise nucleotide identity in the COX1 sequence of a different species within the same expected genus. Two cell lines, LFBK-αvß6 and SCP-HS, were determined to be composed of cells from a different species and genus. Mycoplasma contamination was not detected in any cell lines. However, several expected and unexpected viral sequences were detected, including part of the classical swine fever virus genome in the IB-RS-2 Clone D10 cell line. Metagenomics-based HTS is a useful laboratory QA tool for cell line authentication and contamination detection that should be conducted regularly.

Strain-specific monoclonal antibodies to the E2 protein of classical swine fever virus, Paderborn strain.

Monoclonal antibodies (MAbs) against the E2 protein of classical swine fever virus (CSFV) are useful for diagnosis and strain characterization. A purified, baculovirus-expressed CSFV E2 protein from the Paderborn strain was formulated with a saponin adjuvant and successfully used to induce an antigen-specific immune response in mice. After cell fusion a panel, designated F92G, of 12 mouse hybridomas (5-2, 11-1, 14-1, 25-2, 28-2, 31-1, 34-1, 35-2, 37-3, 38-2, 39-1, 41-1) producing CSFV-E2 specific MAbs were selected based on their Ig subclass and secretion level (µg IgG/mL). Nine IgG 1/k, two IgG 2b/k, and one IgG 2a/k MAbs were further characterized using immunoperoxidase reactivity, ELISA, and Western blot analysis. Immunoglobulin concentration-dependent immunoperoxidase and ELISA reactivity was observed for some of the MAbs with certain antigens. In general there were several reactivity patterns exhibited by the MAbs, with CSFV strains representing different genetic subgroups (by immunoperoxidase staining) and recombinant antigens (by ELISA). It was interesting to note that in some cases the strain-specific reactivity of a MAb was dependent on the test, thereby providing a clue regarding the nature of the binding site.

Performance of a foot-and-mouth disease virus reverse transcription-polymerase chain reaction with amplification controls between three real-time instruments.

The foot-and-mouth disease virus (FMDV) is a member of the picornavirus family, possessing an 8-kb single-stranded RNA genome of positive polarity. It is highly contagious among several livestock species and can lead to severe economic consequences, as evidenced by the UK outbreak in 2001. The usage of real-time polymerase chain reaction has facilitated rapid detection of FMDV. Several real-time PCR instruments are available with various capabilities, such as portability and high sample volume analysis. Primers and a dual-labeled TaqMan probe were optimized to detect a single, highly conserved 88-bp segment of the FMDV 3D (RNA polymerase) gene. To increase the confidence of the RT-PCR result, a positive amplification control was synthesized to detect potential false-positive results due to contamination if a wild-type virus is used as positive control. In addition, a preventative measure against false-negative results was developed in which endogenous beta actin mRNA is coamplified by RT-PCR. Assay performance was compared on the LightCycler1.2 (Roche), the SmartCyclerII (Cepheid), and the SDS 7900HT (ABI). These assays successfully identified the FMDV genome and beta actin mRNA from several sources of infected nasal and oral swabs, as well as probang samples.

CHD1 associates with NCoR and histone deacetylase as well as with RNA splicing proteins.

CHD1 is one of a family of nuclear proteins containing two chromodomains, a SWI/SNF-like helicase/ATPase domain and a DNA binding domain. We found that CHD1 co-immunoprecipitates with histone deacetylase (HDAC) activity and that CHD1 also associates with NCoR, a transcriptional corepressor, in yeast two-hybrid and in vitro pull-down assays. NCoR is known to associate with HDACs to effect its repressive activity, suggesting that the predicted chromatin remodeling activity of CHD1 plays a role in this repression. Yeast two-hybrid assays also showed that CHD1 interacts with splicing proteins mKIAA0164, Srp20, and SAF-B. Splicing assays show that CHD1 overexpression can affect alternative splicing. These results suggest that CHD1 may function in both chromatin mediated transcriptional repression and RNA splicing.

Mining single-nucleotide polymorphisms from hexaploid wheat ESTs.

Single-nucleotide polymorphisms (SNPs) represent a new form of functional marker, particularly when they are derived from expressed sequence tags (ESTs). A bioinformatics strategy was developed to discover SNPs within a large wheat EST database and to demonstrate the utility of SNPs in genetic mapping and genetic diversity applications. A collection of > 90000 wheat ESTs was assembled into contiguous sequences (contigs), and 45 random contigs were then visually inspected to identify primer pairs capable of amplifying specific alleles. We estimate that homoeologue sequence variants occurred 1 in 24 bp and the frequency of SNPs between wheat genotypes was 1 SNP/540 bp (theta = 0.0069). Furthermore, we estimate that one diagnostic SNP test can be developed from every contig with 10-60 EST members. Thus, EST databases are an abundant source of SNP markers. Polymorphism information content for SNPs ranged from 0.04 to 0.50 and ESTs could be mapped into a framework of microsatellite markers using segregating populations. The results showed that SNPs in wheat can be discovered in ESTs, validated, and be applied to conventional genetic studies.

The transcriptional repressor Sp3 is associated with CK2-phosphorylated histone deacetylase 2.

Sp1 and Sp3 are ubiquitously expressed mammalian transcription factors that function as activators or repressors. Although both transcription factors share a common domain involved in forming multimers, we demonstrate that Sp1 and Sp3 form separate complexes in estrogen-dependent human breast cancer cells. Sp1 and Sp3 complexes associate with histone deacetylases (HDACs) 1 and 2. Although most HDAC2 is not phosphorylated in the breast cancer cells, HDAC2 bound to Sp1 and Sp3 and cross-linked to chromatin in situ is highly enriched in a phosphorylated form that has a reduced mobility in SDS-polyacrylamide gels. We show that protein kinase CK2 is associated with and phosphorylates HDAC2. Alkaline phosphatase treatment of HDAC2 and Sp1 and Sp3 complexes reduced the associated HDAC activity. Protein kinase CK2 is up-regulated in several cancers including breast cancer, and Sp1 and Sp3 have key roles in estrogen-induced proliferation and gene expression in estrogen-dependent breast cancer cells. CK2 phosphorylation of HDAC2 recruited by Sp1 or Sp3 could regulate HDAC activity and alter the balance of histone deacetylase and histone acetyltransferase activities and dynamic chromatin remodeling of estrogen-regulated genes.

NAPP2, a peroxisomal membrane protein, is also a transcriptional corepressor.

Nuclear factor-erythroid number 2 (NF-E2) is a positive regulatory, DNA binding transcription factor for gene expression in erythroid and megakaryocytic cells. To further understand the mechanisms of NF-E2 function, we used expression cloning to identify coregulators interacting with the erythroid-specific subunit of NF-E2, p45. We have isolated a protein, NAPP2, which contains an aspartic-acid- and glutamic-acid-rich region and a nuclear localization signal. The gene encoding NAPP2, PEX14, is located on chromosome 1p36 and is ubiquitously expressed. The domains of interaction in vitro and in vivo between p45 and NAPP2 were mapped by a yeast two-hybrid system and cotransfection experiments. In mammalian cell culture, ectopically expressed NAPP2 inhibited p45-directed transcriptional activation. Furthermore, NAPP2 functions as a corepressor and interacts specifically with histone deacetylase l (HDAC1), but not HDAC2 or HDAC3. NAPP2 is thus potentially a negative coregulator of NF-E2. NAPP2 is identical to PEX14, an integral membrane protein essential for protein docking onto the peroxisomes. These studies have identified a novel, bifunctional protein capable of acting as a transcriptional corepressor and a polypeptide transport modulator. They also suggest that NF-E2 may function both positively and negatively in the transcription regulation of specific erythroid and megakaryocytic genes.