Development and Evaluation of a TaqMan Real-Time PCR Assay for the Rapid Detection of Cross-Contamination of RD (Human) and L20B (Mouse) Cell Lines Used in Poliovirus Surveillance.

BACKGROUND: The cross-contamination of cell lines in culture is a persistent problem. Genetically modified L20B (Mouse) and RD (Human Rhabdomyosarcoma) cell lines are commonly used in poliovirus research, surveillance, and diagnostics. Cross-contamination between these cell lines leads to unreproducible results and unreliable surveillance data, negatively affecting public health. The gold standard method for cell authentication is Short Tandem Repeats analysis, which is time-consuming and expensive. The disadvantage of STR is limited detection of interspecies contamination. METHODS: This assay targets the mitochondrial cytochrome c oxidase subunit I (MTCO1) gene, a highly conserved and emergent DNA barcode region for detection of cross-contamination in RD and L20B cell lines. The MagNA Pure Compact instrument and ABI 7500 Fast Dx Real-time PCR systems were used for DNA extraction and to perform real-time PCR respectively. RESULTS: The newly developed assay is very sensitive with a limit of detection of 100 RD cells/1 million L20B/mL. The amplification efficiency and R2-value were 102.26% and 0.9969 respectively. We evaluated specificity of the assay with five human and four mouse cell lines, as well as monkey and rat cell lines. The assay showed no cross-reactivity with genomic DNA from human, mouse, rat, or monkey cell lines. The analytical sensitivity was also evaluated by spiking varying amounts of RD cells (0.001% - 10%) into L20B cells. There was no difference in CT values when running single-plex or duplex PCR reactions with similar experimental conditions. CONCLUSIONS: We have developed and validated a TaqMan real-time PCR assay, a sensitive method for the detection of cross-contamination of RD and L20B cell lines.

Improved specificity and reduced subtype cross-reactivity for antibody detection by ELISA using globular head domain recombinant hemagglutinin.

The relative performance of ELISA using globular head domain (GH) and ectodomain hemagglutinins (HAs) as antigens to detect influenza A virus IgG antibody responses was assessed. Assay sensitivity and subtype cross-reactivity were evaluated using sera collected from recipients of monovalent H5N1 vaccine and A(H1N1)pdm09 virus-infected persons. Assay specificity was determined using collections of sera from either individuals unexposed to either H5N1 or A(H1N1)pdm09 viruses or exposed to H5N1 or A(H1N1)pdm09 viruses through vaccination or infection, respectively. ELISA using GH HA showed a similar degree of sensitivity, significantly higher specificity, and significantly lower subtype cross-reactivity compared to ELISA using ectodomain HA.

IgM, IgG, and IgA antibody responses to influenza A(H1N1)pdm09 hemagglutinin in infected persons during the first wave of the 2009 pandemic in the United States.

The novel influenza A(H1N1)pdm09 virus caused an influenza pandemic in 2009. IgM, IgG, and IgA antibody responses to A(H1N1)pdm09 hemagglutinin (HA) following A(H1N1)pdm09 virus infection were analyzed to understand antibody isotype responses. Age-matched control sera collected from U.S. residents in 2007 and 2008 were used to establish baseline levels of cross-reactive antibodies. IgM responses often used as indicators of primary virus infection were mainly detected in young patient groups (≤5 years and 6 to 15 years old), not in older age groups, despite the genetic and antigenic differences between the HA of A(H1N1)pdm09 virus and pre-2009 seasonal H1N1 viruses. IgG and IgA responses to A(H1N1)pdm09 HA were detected in all age groups of infected persons. In persons 17 to 80 years old, paired acute- and convalescent-phase serum samples demonstrated ≥4-fold increases in the IgG and IgA responses to A(H1N1)pdm09 HA in 80% and 67% of A(H1N1)pdm09 virus-infected persons, respectively. The IgG antibody response to A(H1N1)pdm09 HA was cross-reactive with HAs from H1, H3, H5, and H13 subtypes, suggesting that infections with subtypes other than A(H1N1)pdm09 might result in false positives by enzyme-linked immunosorbent assay (ELISA). Lower sensitivity compared to hemagglutination inhibition and microneutralization assays and the detection of cross-reactive antibodies against homologous and heterologous subtype are major drawbacks for the application of ELISA in influenza serologic studies.

Expression of recombinant antigenic proteins from Angiostrongylus cantonensis: a brief report.

Cerebral angiostrongyliasis is an acute inflammation caused by the infection of the nematode Angiostrongylus cantonensis that results in eosinophilic meningitis. The current immunological assay of choice is an immunoblot that detects antibodies to a 31 kDa protein present in crude extracts of the female worm. Recently we have identified diagnostic targets from excretion and secretion products and determined the composition of the 31 kDa antigen after 2-D gel electrophoresis and mass spectrometry. Here we cloned and expressed five proteins in prokaryotic and eukaryotic systems. Recombinant proteins were purified and analysed by Western blot assays and among them 14-3-3, Lec5 and ES7 were recognized by Angiostrongylus-specific serum, although the signal was weak.

Presence of antibodies against genogroup VI norovirus in humans.

BACKGROUND: Noroviruses are important enteric pathogens in humans and animals. Recently, we reported a novel canine norovirus (CaNoV) in dogs with diarrhea belonging to a new genogroup (GVI). No data are available on exposure of humans to this virus. METHODS: Sera from 373 small animal veterinarians and 120 age-matched population controls were tested for IgG antibodies to CaNoV by a recombinant virus like particle based enzyme-linked immunosorbent assay. RESULTS: Antibodies to CaNoV were found in 22.3% of the veterinarians and 5.8% of the control group (p < 0.001). Mean corrected OD450 values for CaNoV antibodies were significantly higher in small animal veterinarians compared to the control group. CONCLUSIONS: These findings suggest that CaNoV may infect humans and small animal veterinarians are at an increased risk for exposure to this virus. Additional studies are needed to assess if this virus is able to cause disease in humans.

Expression and characterization of human group C rotavirus virus-like particles in insect cells.

Group C rotavirus (GpC RV) is a causative agent of acute gastroenteritis in children and adults. We expressed the three major capsid proteins VP2, VP6 and VP7 of human GpC RV in baculovirus and demonstrated the self-assembly of VP2/6/7 or VP6/7 virus-like particles (VLPs) in insect cells. We examined a number of parameters, including the kinetics of protein synthesis in different cell lines and media, to optimize the most favorable conditions for the synthesis of recombinant viral proteins and the production of VLPs in Sf9 cells. Hyperimmune serum to VP2/6/7 and VP6/7 VLPs recognized individual recombinant proteins of human GpC RV by Western blot analysis. This serum also showed specific reactivities with the corresponding GpC VLPs but not GpA RV by using immune electron microscopy (IEM) and enzyme immunoassay (EIA). The ability to produce an unlimited amount of GpC RV antigen and the availability of high quality antibody will allow us to develop sensitive and specific diagnostic assays to better determine the epidemiology and disease burden of GpC RV in humans.

False positive reactivity of recombinant, diagnostic, glycoproteins produced in High Five insect cells: effect of glycosylation.

Baculovirus-mediated expression of recombinant proteins for use in diagnostic assays is commonplace. We expressed a diagnostic antigen for cysticercosis, GP50, caused by the larval stage of Taenia solium, in both High Five and Sf9 insect cells. Upon evaluation of the specificity of recombinant GP50 (rGP50) in a western blot assay, we observed that 12.5% (21/168) of the serum samples from persons with a variety of parasitic infections other than cysticercosis reacted positive when rGP50 was produced in High Five cells. The same samples reacted negative when rGP50 was produced in Sf9 cells. The false positive reactivities of these other parasitic infection sera were abolished when rGP50, expressed in High Five cells, was deglycosylated. In addition, the same sera that reacted with rGP50 from High Five cells also reacted with recombinant human transferrin (rhTf) when expressed in High Five cells, but not Sf9 cells. High Five cells, but not Sf9 cells, modify many glycoproteins with a core alpha(1,3)-fucose. This same modification is found in the glycoproteins of several parasitic worms and is known to be immunogenic. Since the distribution of these worms is widespread and millions of people are infected, the use of recombinant proteins with N-linked glycosylation produced in High Five cells for diagnostic antigens is likely to result in a number of false positive reactions and a decrease in assay specificity.

Identification and authentication of animal cell culture by polymerase chain reaction amplification and DNA sequencing.

Polymerase chain reaction (PCR) amplification and deoxyribonucleic acid (DNA) sequence analysis were used to identify the species origin of cell lines used in a cell culture facility where various cell lines of different species are routinely propagated. The aldolase gene family was selected for PCR amplification because the DNA sequences of this gene are highly conserved over a wide range of animals and humans. A total of 36 cell lines representing 13 different species were selected for this study. The DNA from each cell line was amplified, and PCR products were analyzed by agarose gel electrophoresis. The results showed unique profiles of amplified bands on agarose gels that allowed differentiation among non-closely related species. However, DNA amplification of closely related species, including rat and mouse or human and primate, resulted in similar and indistinguishable banding patterns that could be further differentiated by DNA sequence analysis. These results suggested that aldolase gene amplification coupled with DNA sequence analysis is a useful tool for identification of cell lines and has potential application for use in identification of interspecies cross-contamination.