C-Terminal Part of Glutamate-Ammonia-Ligase Adenyltransferase Gene Identified by RAPD-HRM with 3H Primer for E. Coli Screening.

A single random oligonucleotide 3H primer has been previously applied in random-amplified- polymorphic-DNA (RAPD)-PCR to distinguish stocked bacteria E. coli within a cocktail mixture also containing Enterococcus faecalis, Bifidobacterium longum and Ruminococcus gnavus. In this study, we demonstrate that a 702 base pair (bp) gene fragment can be amplified as a unique pattern by RAPD-PCR using a 3H primer in human faeces containing E. coli. This unique 702 bp amplicon contained a 687 bp gene fragment identified as the C-terminal region of the glutamate-ammonia-ligase adenyltransferase (glnE) gene of E. coli. By high-resolution melt (HRM) analysis, a mean melt-curve temperature of this 702 bp amplicon was determined to be approximately 88.1 ± 0.22 degrees Celsius (°C). A combination of RAPD with HRM in one single reaction based on this amplicon can achieve semi-quantitative detection of up to 102 CFU/ml of E. coli. To increase the signal intensity of HRM, a primer pair capable of screening E. coli directly from fresh human faeces was re-designed from the 687 bp gene segment, giving a mean peak melt-curve temperature at 88.35 ± 0.11 °C. Finally, single-nucleotide polymorphisms of this 687 bp gene segment were analysed for pathogenic E. coli strains, including UMN026, O83:H1, O104:H4, O157:H7 and O169:H41. We conclude that this 687 bp segment of the glnE gene has a high potential for screening of human faecal E. coli, including pathogenic strains, in contaminated food and water.

Nonstructural proteins nsp2TF and nsp2N of porcine reproductive and respiratory syndrome virus (PRRSV) play important roles in suppressing host innate immune responses.

Recently, we identified a unique -2/-1 ribosomal frameshift mechanism in PRRSV, which yields two truncated forms of nonstructural protein (nsp) 2 variants, nsp2TF and nsp2N. Here, in vitro expression of individual PRRSV nsp2TF and nsp2N demonstrated their ability to suppress cellular innate immune responses in transfected cells. Two recombinant viruses were further analyzed, in which either nsp2TF was C-terminally truncated (vKO1) or expression of both nsp2TF and nsp2N was knocked out (vKO2). Host cellular mRNA profiling showed that a panel of cellular immune genes, in particular those involved in innate immunity, was upregulated in cells infected with vKO1 and vKO2. Compared to the wild-type virus, vKO1 and vKO2 expedited the IFN-α response and increased NK cell cytotoxicity, and subsequently enhanced T cell immune responses in infected pigs. Our data strongly implicate nsp2TF/nsp2N in arteriviral immune evasion and demonstrate that nsp2TF/nsp2N-deficient PRRSV is less capable of counteracting host innate immune responses.