Genetic variation analyses of porcine epidemic diarrhea virus isolated in mid-eastern China from 2011 to 2013.

Porcine diarrhea outbreaks caused by porcine epidemic diarrhea virus (PEDV) has occurred in China with significant losses of piglets since 2010. In this study, the complete S and ORF3 genes of 15 field PEDV isolates in mid-eastern China from 2011 to 2013 were detected and compared with other reference strains. Based on S gene, all of the PEDV strains could be assigned to 3 genogroups. Only 1 isolate, JS120103, belonged to genogroup 1 and showed a close relationship with previous Chinese strains DX and JS-2004-2, European strain CV777, and Korean strain DR13. The other 14 isolates belonged to genogroup 3 and showed a close relationship with other Chinese strains isolated after 2010. The S genes of those isolates were 9 nucleotides longer in length than JS120103 and the other reference strains in genogroup 1, with 15 bp insertion and 6 bp deletion. Homology analyses revealed that all of the Chinese field isolates, except JS120103, are 97.6% to 100% (95.8% to 100%) identical in nucleotide (deduced amino acid) sequence to each other. Meanwhile, based on the ORF3 gene, all of the PEDV isolates could be separated into 3 genogroups. Eleven of the 15 field isolates in this study belonged to genogroup 3 and were 95.8% to 100% identical in nucleotide sequence or 95.6% to 100% in deduced amino acid sequence to each other. Our results indicate that the variant PEDV strain spread wildly in mid-eastern China. This will be useful to take into consideration in the control and prevention of this disease.

Des épidémies de diarrhée porcine due au virus de la diarrhée épidémique porcine (VDEP) sont présentes en Chine et ont causé des pertes significatives de porcelets depuis 2010. Dans la présente étude, les gènes S et ORF3 de 15 isolats de champs de VDEP provenant de la portion moyen-orientale de la Chine isolés entre 2011 et 2013 furent détectés et comparés à des souches de référence. Sur la base du gène S, toutes les souches de VDEP pouvaient être réparties dans trois génogroupes. Un seul isolat, JS120103, appartenait au génogroupe 1 et montrait une proche parenté avec les souches chinoises précédentes DX et JS-2004-2, la souche européenne CV777, et la souche coréenne DR13. Les 14 autres isolats appartenaient au génogroupe 3, et montraient une proche parenté avec d'autres souches chinoises isolées après 2010. Les gènes S de ces isolats étaient plus longs de 9 nucléotides que JS120103 et les autres souches de référence du génogroupe 1, avec une insertion de 15 paires de base et une délétion de 6 paires de base. Les analyses d'homologie ont révélé que tous les isolats de champs chinois, sauf JS120103, sont 97,6 % à 100 % (95,8 % à 100 %) identiques entre eux en séquence de nucléotides (d'acides aminés déduits). Également, sur la base du gène ORF3, tous les isolats de VDEP pouvaient être séparés en 3 génogroupes. Onze des 15 isolats de champs de la présente étude appartenaient au génogroupe 3 et étaient 95,8 % à 100 % identiques entre eux en séquence de nucléotides ou 95,6 % à 100 % en séquence d'acides aminés déduits. Ces résultats indiquent que les variants des souches de VDEP sont largement dispersés dans la partie moyenne-orientale de la Chine. Ces données seront utiles à prendre en considération pour la prévention et la maitrise de cette maladie.(Traduit par Docteur Serge Messier).

Development of a multiplex TaqMan probe-based real-time PCR for discrimination of variant and classical porcine epidemic diarrhea virus.

Since October 2010, porcine diarrhea outbreaks have occurred widely, resulting in major losses in suckling piglets in China. A variant porcine epidemic diarrhea virus (PEDV), characterized by base deletion and insertion in the S gene, compared to classical PEDV CV777, was shown to be responsible for this outbreak. In this study, a multiplex TaqMan probe-based real-time PCR was developed for detecting PEDV and differentiating the variant from classical PEDV, by using two sets of primers and probes based on the S gene of PEDV. The limits of detection of both variant and classical PEDV were 5×10(2) DNA copies. Specificity was determined using eight other viral pathogens of swine. Reproducibility was evaluated using standard dilutions, with coefficients of variation <1.4%. Standard dilutions included in each test allowed quantification of the amount of PEDV. Among 42 intestinal samples from pigs with severe watery diarrhea, 36 variant PEDV and three classical PEDV samples were detected, with viral loads of 10(2)-10(8) copies/µl and 10(3)-10(5) copies/µl, respectively, which suggested that the variant PEDV was prevalent in China. The multiplex TaqMan probe-based real-time PCR should be a useful tool for quantifying viral load, detecting PEDV, and differentiating variant from classical PEDV.

Subcutaneous and intranasal immunization with Stx2B-Tir-Stx1B-Zot reduces colonization and shedding of Escherichia coli O157:H7 in mice.

The type III secretion system of Escherichia coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E. coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXφ of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B-Tir-Stx1B-Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST·Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs. intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against Stx2B-Tir-Stx1B-Zot, Stx2B-Tir-Stx1B and control agent (GST/PBS) were also monitored. Subcutaneous immunization of mice with Stx2B-Tir-Stx1B-Zot induced significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Mice that were immunized intranasally with Stx2B-Tir-Stx1B-Zot showed dramatically decreased E. coli O157:H7 shedding compared to those of Stx2B-Tir-Stx1B and control agent following experimental infection. Mice immunized subcutaneously with Stx2B-Tir-Stx1B-Zot or Stx2B-Tir-Stx1B both showed reduced shedding in feces, moreover, Stx2B-Tir-Stx1B-Zot did better. These results demonstrate the perspective for the use of Stx2B-Tir-Stx1B-Zot to prevent colonization and shedding of E. coli O157:H7.