RESUMO
Porcine deltacoronavirus (PDCoV) is a recently identified coronavirus that causes intestinal diseases in neonatal piglets with diarrhea, vomiting, dehydration, and post-infection mortality of 50-100%. Currently, there are no effective treatments or vaccines available to control PDCoV. To study the potential of RNA interference (RNAi) as a strategy against PDCoV infection, two short hairpin RNA (shRNA)-expressing plasmids (pGenesil-M and pGenesil-N) that targeted the M and N genes of PDCoV were constructed and transfected separately into swine testicular (ST) cells, which were then infected with PDCoV strain HB-BD. The potential of the plasmids to inhibit PDCoV replication was evaluated by cytopathic effect, virus titers, and real-time quantitative RT-PCR assay. The cytopathogenicity assays demonstrated that pGenesil-M and pGenesil-N protected ST cells against pathological changes with high specificity and efficacy. The 50% tissue culture infective dose showed that the PDCoV titers in ST cells treated with pGenesil-M and pGenesil-N were reduced 13.2- and 32.4-fold, respectively. Real-time quantitative RT-PCR also confirmed that the amount of viral RNA in cell cultures pre-transfected with pGenesil-M and pGenesil-N was reduced by 45.8 and 56.1%, respectively. This is believed to be the first report to show that shRNAs targeting the M and N genes of PDCoV exert antiviral effects in vitro, which suggests that RNAi is a promising new strategy against PDCoV infection.
Assuntos
Infecções por Coronavirus/genética , Coronavirus/genética , Proteínas Virais/genética , Replicação Viral/genética , Animais , Coronavirus/patogenicidade , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Diarreia/genética , Diarreia/patologia , Diarreia/veterinária , Diarreia/virologia , Masculino , Plasmídeos/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Viral/genética , Suínos/virologia , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Testículo/crescimento & desenvolvimento , Testículo/virologiaRESUMO
Porcine deltacoronavirus (PDCoV) is a recently identified coronavirus in the genus Deltacoronavirus that can cause enteric disease including diarrhea, vomiting, dehydration and mortality in neonatal piglets. Serological assays to detect anti-PDCoV antibodies are presently limited to certain laboratories and geographic regions. In this study, a recombinant M protein-based indirect enzyme-linked immunosorbent assay (PDCoV-rM ELISA) was developed and utilized to determine the prevalence of anti-PDCoV IgG in Hebei province. The PDCoV-rM ELISA showed no cross-reaction with antisera against transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PRV), porcine circovirus 2 (PCV2), classical swine fever virus (CSFV) or porcine reproductive and respiratory syndrome virus (PRRSV). The diagnostic sensitivity was 90.6% and the diagnostic specificity was 93.3%. A total of 871 serum samples collected in Hebei from January 2015 to October 2016 were checked for presence of antibodies against PDCoV using the novel PDCoV-rM ELISA. Anti-PDCoV IgG antibodies were detected in 11% (96/871) of the samples and in 25% (10/40) of the investigated farms. The data suggest that PDCoV has a low seroprevalence in pig population in Hebei province, China.
