ABSTRACT
Porcine epidemic diarrhea virus (PEDV) inhibits the host typeⅠinterferon and cellular antiviral response, but its inhibition mechanism is unclear, and the roles of PEDV nonstructural proteins in regulating typeⅠinterferon responses have been seldom studied. To study the effect of nsp1 on typeⅠinterferon response, nsp1 gene was cloned into a eukaryotic expression vector pCAGGS. The expression of nsp1 in transfected cells was determined by Western blot and indirect immunofluorescence assay. The effects of nsp1 on the induction of typeⅠinterferon were evaluated by dual luciferase reporter gene assay, ELISA and VSV bioassay. Western blot and indirect immunofluorescence assay showed that nsp1 was highly expressed in transfected cells and PEDV-infected cells. Dual luciferase reporter gene assay results indicated that nsp1 strongly inhibited the IFN-β promoter activity, and the inhibitory effect was nsp1 dose-dependent. ELISA results showed that nsp1 significantly inhibited the expression of IFN-β in protein level. And VSV replication-inhibition bioassay revealed that nsp1 significantly inhibited typeⅠIFN antiviral activities induced by poly(I:C). Our results implied that nsp1 was a highly conserved protein of PEDV and exhibited antagonistic function on interferon promoter activity. The results have laid a foundation for further understanding the immune evasion mechanism of PEDV and for developing new effective vaccine against PEDV.
ABSTRACT
Porcine epidemic diarrhea virus (PEDV) is one of the major etiologies responsible for the acute, highly contagious disease in the digestive tract of pigs, especially neonatal piglets. Since PEDV was first identified in Europe in the late 1970s, it has resulted in significant economic losses in many Asian swine-raising countries, including China. Recently, reverse genetics techniques including targeted RNA recombination, bacteria artificial chromosome system and in vitro ligation have been successfully used to manipulate the genome of PEDV, which providing new strategies for the clear delineation of the functions of the viral proteins, the mechanisms behind PEDV pathogenesis and the design of novel vaccines against PEDV. Here, we review the progresses of different reverse genetics platforms developed for PEDV and their applications, covering the roles of trypsin in PEDV propagation, functions of S and ORF3 protein and the development of next generation PED vaccines, and the perspectives of reverse genetics for PEDV.
ABSTRACT
The porcine epidemic diarrhea virus (PEDV) has recently been shown to cause huge economic losses in the global pork industry. Our results demonstrated that the extract dose-dependently inhibited the replication of PEDV and reduced the visible cytopathic effect (CPE). Treatment with C. heterophylla Fisch extract resulted in marked reduction of PEDVinduced cytokine and chemokine expression. The antiviral activity of C. heterophylla Fisch extract on PEDV replication was found to be primarily exerted at the early stages after infection. Taken together, our data indicate that C. heterophylla Fisch extract may be a good therapeutic agent for use against PEDV and also a potential candidate to be evaluated against other human and animal coronaviruses.
Subject(s)
Animals , Humans , Coronavirus , Corylus , Porcine epidemic diarrhea virus , Red MeatABSTRACT
El artículo busca dar a conocer aspectos generales de los principales coronavirus que afectan los porcinos, su presentación en Colombia y aspectos particulares del virus de diarrea epidémica porcina (PEDV), emergente en diferentes países y que está generando gran impacto en la salud y la economía de la industria porcina. Los coronavirus que afectan la especie porcina son principalmente el virus de gastroenteritis transmisible porcina (TGEV), el coronavirus respiratorio porcino (PRCV), el virus de la encefalomielitis hemaglutinante porcina (PHEV), el PEDV y el deltacoronavirus porcino (PDCoV). Tiempo atrás en Colombia se han tenido registros de TEGV y PRCV asociados a la importación de animales provenientes de Estados Unidos, que se controlaron en las granjas infectadas y en las unidades de cuarentena. Para el caso de PEDV este se detectó por primera vez en Colombia a mediados de marzo de 2014, por lo cual el Instituto Colombiano Agropecuario expidió la alerta sanitaria en Neiva (Huila), Fusagasugá, Silvania (Cundinamarca) y Puerto López (Meta), por la presentación inusual de un cuadro epidémico de vómito y diarrea en animales jóvenes y adultos, aborto en cerdas gestantes, con altas tasas de mortalidad hasta de 100 % en animales de primera semana de edad. En el presente la enfermedad se ha registrado en otros municipios del país lo mismo que en diferentes países con un cuadro clínico similar y mortalidad en cerdos, con altas pérdidas económicas para el sector porcícola.
The article seeks to study general aspects of the main coronaviruses affecting pigs, their presentation in Colombia, and particular aspects of porcine epidemic diarrhea virus (PEDV), emerging in different countries and generating a great impact on the health and economy of the swine industry. The main coronaviruses affecting swine are porcine transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV), PEDV, and porcine deltacoronavirus (PDCoV). Long ago in Colombia there had been reports of TGEV and PRCV associated with the importation of animals from the United States, which was controlled in the infected farms and in quarantine units. PEDV was first detected in Colombia in mid-March 2014; the Colombian Agricultural Institute issued a health alert in Neiva (Huila), Fusagasugá and Silvania (Cundinamarca), and Puerto López (Meta) due to the unusual presentation of epidemic vomiting and diarrhea in young and adult animals, abortion in pregnant sows, with high mortality rates (up to 100%) in animals during the first week of age. At present the disease has been reported in other municipalities of the country as well as in different countries with similar clinical conditions and mortality rates in pigs with high economic losses for the swine sector.
O artigo busca divulgar aspectos gerais dos principais Coronavírus que afetam os suínos, seu aparecimento na Colômbia e aspectos particulares do vírus de diarreia epidêmica suína (PEDV), emergente em diferentes países e que está gerando grande impacto na saúde e a economia da indústria suína. Os Coronavírus que afetam a espécie suína são principalmente o vírus de gastroenterite transmissível suína (TGEV), o Coronavírus respiratório suíno (PRCV), o vírus da encefalomielite hemaglutinante suína (PHEV), o PEDV e o delta Coronavírus suíno (PDCoV). Há um tempo na Colômbia houve relatos de TEGV e PRCV associados à importação de animais provenientes dos Estados Unidos, que se controlaram nas granjas infectadas e nas unidades de quarentena. Para o caso de PEDV, este se detectou pela primeira vez na Colômbia a mediados de março de 2014, razão pela qual o Instituto Colombiano Agropecuário expediu a alerta sanitária em Neiva (Huila), Fusagasugá, Silvania (Cundinamarca) e Puerto López (Meta), pelo aparecimento incomum de um quadro epidêmico de vômito e diarreia em animais jovens e adultos, aborto em porcas gestantes, com altas taxas de mortalidade até de 100 % em animais na primeira semana de idade. Atualmente a doença tem sido reportada em outros municípios do país igualmente que em diferentes países com um quadro clínico similar e mortalidade em porcas, com altas perdas econômicas para o setor da indústria suína.
ABSTRACT
The structure genes spike (S), nucleocapsid (N), membrane (M), small membrane (sM) of a porcine epidemic diarrhea virus (PEDV) strain DX isolated in Gansu province, North-west of China, were cloned, sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S, sM, M and N genes open reading frame (ORF) of DX were 4 152, 231, 681 and 1 326 bases long respectively. There were transcription regulatory sequences (TRSs) upstream of the initiator ATG of the S, N and M genes. The amino acids sequences of S, M and N contained 30, 3 and 7 potential asparagine (N)-linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06, JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China, and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.
ABSTRACT
Porcine epidemic diarrhea virus (PEDV) causes an acute enteritis in pigs of all ages, often fatality for neonates. PEDV occupies an intermediate position between two well characterized members of the coronavirus group I, human coronavirus (HCoV-229E)and transmissible gastroenteritis virus (TGEV) which uses aminopeptidase N (APN), a 150 kDa protein, as their receptors. However, the receptor of the PEDV has not been identified yet. A virus overlay protein binding assay (VOPBA) was used to identify PEDV binding protein in permissive cells. The binding ability of PEDV to porcine APN (pAPN) and the effects of pAPN on infectivity of PEDV in Vero cells were also investigated. VOPBA identified a 150 kDa protein, as a putative PEDV receptor in enterocytes and swine testicle (ST) cells. Further the PEDV binding to pAPN was blocked by anti-pAPN and pAPN enhanced PEDV infectivity in Vero cells. In conclusion, these results suggested that pAPN may act as a receptor of PEDV.