The genomes of coronaviruses carry accessory genes known to be associated with viral virulence. The single accessory gene of porcine epidemic diarrhea virus (PEDV), ORF3, is dispensable for virus replication in vitro, while viral mutants carrying ORF3 truncations exhibit an attenuated phenotype of which the underlying mechanism is unknown. Here, we studied the effect of ORF3 deletion on the proliferation of PEDV in Vero cells. To this end, four recombinant porcine epidemic diarrhea viruses (PEDVs) were rescued using targeted RNA recombination, three carrying the full-length ORF3 gene from different PEDV strains, and one from which the ORF3 gene had been deleted entirely. Our results showed that PEDVs with intact or naturally truncated ORF3 replicated to significantly higher titers than PEDV without an ORF3. Further characterization revealed that the extent of apoptosis induced by PEDV infection was significantly lower with the viruses carrying an intact or C-terminally truncated ORF3 than with the virus lacking ORF3, indicating that the ORF3 protein as well as its truncated form interfered with the apoptosis process. Collectively, we conclude that PEDV ORF3 protein promotes virus proliferation by inhibiting cell apoptosis caused by virus infection. Our findings provide important insight into the role of ORF3 protein in the pathogenicity of PEDV.

In the present study, a polymerase chain reaction-linked single-strand conformation polymorphism (PCR-SSCP) approach combined with DNA sequencing was used to characterise samples of Fasciola spp. from different host species and geographical locations in mainland China. The first internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was amplified by PCR from individual Fasciola and analysed by SSCP. SSCP analyses displayed three different banding profiles that allowed the identification of all Fasciola samples examined into three groups: Fasciola hepatica, F. gigantica and the "intermediate" Fasciola. Then, the ITS-1 rDNA was sequenced from representative Fasciola samples, and analysis of the complete ITS-1 sequences supported the identification of all Fasciola samples by SSCP approach. The length of the ITS-1 sequences was 422 bp for all Fasciola samples sequenced. Although there was no variation in length or composition of the ITS-1 sequences among multiple specimens within each of the taxa, F. hepatica and F. gigantica differed by 1.2% in their ITS-1 sequences, whereas the "intermediate" Fasciola was unique, in which two different ITS-1 sequences exist in the rDNA array within a single Fasciola worm. One of the sequences is identical to that of F. hepatica, and the other is identical to that of F. gigantica. This study demonstrated that PCR-SSCP analysis of the ITS-1 rDNA followed by selective sequencing provides a reliable approach for the accurate identification of Fasciola spp., and also supports the existence of the "intermediate" Fasciola between F. hepatica and F. gigantica in mainland China.

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Among the helminths infecting ruminants in China are three taxa belonging to the genus Fasciola: F. hepatica, F. gigantica and the so-called 'intermediate form' that appears to lie between these two species. Based on the sequences of the second internal-transcribed spacers (ITS-2) within the parasites' nuclear ribosomal DNA (rDNA), a pair of primers (DSJf/DSJ3) specific for F. hepatica and a pair (DSJf/DSJ4) specific for F. gigantica were designed and used to develop PCR-based assays. These assays allowed the identification and differentiation of F. hepatica, F. gigantica and the 'intermediate' Fasciola, with no amplicons produced from heterologous DNA samples. The results of sequencing confirmed the species-specific identity of the amplified products. The assays showed good sensitivity, giving positive results with as little as 0.11 ng of F. hepatica DNA and 0.35 ng of F. gigantica DNA. This meant that the DNA from a single Fasciola egg or a single infected snail was sufficient for identification of the Fasciola taxon. The developed PCR assays could provide useful tools for the detection, identification and epidemiological investigation of Fasciola infection in humans, other mammals and snails.