A real-time PCR to detect and analyze virulent EMCV loads in sows and piglets

Abstract

A real-time polymerase chain reaction with SYBR Green was developed for the detection and quantification of encephalomyocarditis virus (EMCV) in porcine tissues; the method uses two primers specific for the 3D gene. The detection limit of this assay was 22 gene copies/reaction, equivalent to 0.001 TCID(50)/ml. The assay was linear over a 10(7) dilution range of template concentrations and was specific for EMCV; it did not amplify other porcine pathogens (porcine circovirus 2, porcine reproductive and respiratory virus, classical swine fever virus, pseudorabies virus, or porcine teschovirus). This assay detected EMCV titers at least 10(4) smaller than the routine PCR assay. To increase our understand of EMCV pathogenesis, the new method was used to quantify levels of EMCV genome in various tissues of artificially challenged sows and piglets. The virus was found mainly in the heart, lung, spleen, kidney, and endometrium of sows, and mainly in the heart, spleen, lung, and testis of fetuses. The real-time PCR method described here should be useful for the study of EMCV infection and distribution in pigs.

Isolation and Characterization of Encephalomyocarditis Virus from Dogs in China

“…The EMCV-specific primer set described by Wang et al . 16 was used as follows: forward primer (position 1116–1140) 5′-GACGCTTGAAGACGTTGTCTTCTTA-3′; reverse primer (position 1302–1326) 5′-CCCTACCTCACGGAATGGGGCAAAG-3′. The primers were designed based on a 3D sequence and have been confirmed to be highly specific for EMCV.…”

Encephalomyocarditis Virus 3C Protease Relieves TRAF Family Member-associated NF-κB Activator (TANK) Inhibitory Effect on TRAF6-mediated NF-κB Signaling through Cleavage of TANK

“…Cell Lines and Viruses-Human embryonic kidney 293T (HEK293T) and baby hamster kidney (BHK)-21 cells were purchased from the ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories, Inc., Logan, UT), 100 units/ml penicillin, and 100 g/ml streptomycin at 37°C in a 5% CO 2 -humidified atmosphere. EMCV HB10 strain and FMDV (Asia 1/YS/CHA/05) were propagated in BHK-21 cells as described previously (21,22). In brief, cellular supernatants of EMCV-infected BHK-21 cells were diluted 10-fold serially.…”

Human SNORA31 variations impair cortical neuron-intrinsic immunity to HSV-1 and underlie herpes simplex encephalitis

“…Cells were collected at various time points and frozen. DNA was extracted from fibroblasts and EMCV genome copy number was measured by qPCR as previously described 51 .…”