An attenuated EMCV-HB10 strain acts as a live viral vector delivering a foreign gene

Abstract

We successfully constructed a full-length cDNA infectious clone of the encephalomyocarditis virus (EMCV) HB10 strain and obtained a partially attenuated rEMCV-C 9 virus with a shorter poly(C) tract. Our results showed that the length of the EMCV-HB10 poly(C) tract was related to the pathogenicity of the EMCV-HB10 strain in vivo. Using pEMCV-C 9 as the backbone, we constructed the novel viral vector pC 9 -MCS-D2A by inserting a cDNA fragment containing a 127 amino acid deletion in the 2A protein, a primary cleavage cassette, a FLAG tag and a multiple cloning site (MCS) at the junction of VP1 and D2A. Additionally, the enhanced green fluorescent protein (egfp) gene was cloned into the MCS of pC 9 -MCS-D2A to test its capacity to express foreign proteins. Insertion of the egfp gene did not affect viral replication, and a decrease in EGFP expression was observed within five serial passages. Furthermore, we found that rC 9 -EGFP-D2A was avirulent in vivo, induced neutralizing antibody production and conferred protective immune responses against lethal challenge with EMCV in mice. Taken together, our results demonstrated that we had constructed an attenuated live vector based on an EMCV-HB10 strain with two modified critical virulence factors (the poly(C) tract and 2A protein) that could be used as a candidate live vaccine and a potential live viral vector for foreign antigen delivery.

Isolation and Characterization of Encephalomyocarditis Virus from Dogs in China

“…1, negatively stained samples contained a small virus with a diameter of 27–30 nm 18 . Virus growth was confirmed by IFA using an anti-EMCV VP1 specific monoclonal antibody 19 . The EMCV VP1 protein stained by the monoclonal antibody was distributed in the cytoplasm but not in the nucleus (Fig.…”

“…At 48 h post-infection, the infected BHK-21 cells were fixed with 4% formaldehyde for 30 min at room temperature. The cells were washed three times with PBS, blocked with 10% FBS for 1 h at room temperature, washed three more times with PBS and incubated with mouse anti-VP1 mAb 19 (1:200 dilution) for 1 h. The cells were washed three times with PBS and incubated with FITC-conjugated goat anti-mouse IgG or TRITC-conjugated goat anti-mouse IgG (1:100 dilution) for 1 h at 37 °C. After three more washes in PBS, the cells were imaged under an inverted fluorescence microscope (EVOS f1, AMG, USA).…”

“…An equal amount of each sample was loaded onto a 12% SDS-PAGE gel, followed by protein transfer to a PVDF membrane (Millipore). Each membrane was blocked for 1 h with blocking buffer (5% skimmed milk and 0.1% Tween-20 in PBS) and incubated with anti-VP1 primary antibody (1:1,000 dilution) 19 or with mouse anti-GAPDH antibodies (Cell Signaling Technology, Inc.) to detect endogenous GAPDH, which served as a protein-loading control. Each membrane was washed three times with washing buffer (0.1% Tween-20 in PBS) and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution) for 1 h, after which the membranes were washed and exposed to the detection agent 3,3′-N-diaminobenzidine tertrahydrochloride (DAB, CWBIO, China).…”

The long-lasting enigma of polycytidine (polyC) tract

“…An infectious clone obtained from the pathogenic strain EMCV-2887A carrying a short polyC tract of 27 nucleotides total (C 10 UCUC 3 UC 10 ) was 100 times more attenuated in mice than its wild type counterpart [ 44 ]. Similarly, a recombinant virus differing from its parental EMCV-HB10 strain only for the length of the polyC tract (C 9 ) showed a 200-fold attenuation [ 58 ]. Infectious clones derived from the extremely pathogenic EMCV-PV21 strain and designed to carry a long polyC tract of approximately 150 nucleotides—close to the natural isolate 158 nucleotide–long polyC tract C 141 UCUC 3 UC 10 —showed the same levels of pathogenicity, virulence, and biodistribution of their parent strain in NMRI mice [ 59 ].…”

Investigation of Antibody Levels During Three Doses of Sinopharm/BBIBP Vaccine Inoculation

“…Vaccination is widely acknowledged as the most effective strategy for preventing and controlling infectious diseases. Inactivated vaccinations, mRNA vaccines, and viral vaccines have all been widely utilized ( 3 , 4 ). Furthermore, the inactivated vaccine has been widely used to prevent diseases caused by the influenza virus and poliovirus ( 5 , 6 ), which has a high level of safety and immunogenicity.…”