Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus. It was initially reported in 1970s in Europe. However, massive porcine epidemic diarrhea (PED) epidemics did not occur in China until late 2010. PEDV was first introduced into the United States (US) and rapidly spread nationwide in 2013. PEDV infection of suckling piglets causes up to 100% mortality and the US pig industry lost 10% of domestic pig population and $0.9 to $1.8 billion during 2013-2014, posing a great threat to the pork industry sustainability. Live attenuated vaccines (LAVs) given orally to pregnant sows/gilts replicate in the intestines and induce lactogenic immunity in sows/gilts. They are the most effective approach to provide passive immunity to neonatal pigs against PED via colostrum and milk. However, safety concerns associated with potential virulence reversion hinder broad application of LAVs. The goal of this dissertation is to identify promising targets in two viral genes and to develop a recombination-resistant platform for the development of effective and safe PEDV LAVs.
My first objective was to evaluate whether the exonuclease (ExoN) domain within PEDV nsp14 is a good target for LAV development. Based on the infectious cDNA clone of a highly virulent PEDV strain (icPC22A), eight mutants targeting nsp14 ExoN catalytic sites, zinc finger, or Mg2+-binding site were designed. Only one E191A mutant, carrying the mutation in Mg2+-binding site, was rescued that was characterized by poor growth in Vero or IPEC-DQ cells of the early passages no.1-3 (P1-3) with peak titers of 1.80 ± 0.12 and 1.42 ± 0.16 log10 TCID50/mL, respectively. However, the P4 of E191A was characterized by dramatically improved growth characteristics reaching a high infectious titer (5.55 ± 0.35 log10 TCID50/mL) in Vero cells, like icPC22A. Sequence analysis demonstrated that the introduced mutation site has reverted to wildtype in the P4 virus. To evaluate the pathogenesis of the E191A-P1, 4-5-day-old gnotobiotic pigs were orally inoculated with 100 TCID50/pig of the E191A-P1, icPC22A, or mock. All pigs inoculated with icPC22A developed severe diarrhea and died within 6 days post-inoculation (dpi). In comparison, only 2 pigs (2/3, 66.7%) in the E191A-P1 group showed mild diarrhea at 6 dpi. At 22 dpi, surviving pigs were challenged orally with 106 PFU icPC22A. E191A-P1-inoculated pigs were partially protected from severe diarrhea and high level of viral RNA shedding in feces. Sanger sequencing showed that the viral genomes in the fecal samples from some pigs (1/3, 33.3%) in the E191A-P1 group have lost the E191A mutation, suggesting its instability. Collectively, based on the existing literature data and our own experiments, mutations at the essential functional sites within nsp14-ExoN domain of PEDV were either lethal or genetically unstable. So, the nsp14-ExoN domain is not a good attenuating target for PEDV LAV development.
The second objective was to elucidate the role of PEDV nsp1, a potential host interferon (IFN) antagonist, in the development of PEDV LAV candidates. One PEDV nsp1 mutant icPC22A-nsp1-N93/95A (N93/95A) was designed and rescued by targeting the highly conserved residues 93N and 95N of nsp1 of alphacoronaviruses and betacoronaviruses using the reverse genetics system of PEDV strain PC22A. The N93/95A mutant replicated less efficiently (with a peak titer of 5.72 ± 0.83 log10 TCID50/mL in Vero cells and 6.14 ± 0.23 log10 TCID50/mL in LLC-PK cells) than the virulent icPC22A (6.80 ± 0.00 log10 TCID50/mL in Vero cells and 6.63 ± 0.00 log10 TCID50/mL in LLC-PK cells), and it was more sensitive to type I and type III IFNs pre-treatment than icPC22A. Upon viral infection, IPEC-DQ cells infected with N93/95A mutant exhibited increased IFN-β, IFN-λ1, IFN-λ3, and IFN-λ4 mRNA levels than the cells infected with icPC22A. To evaluate the pathogenesis of N93/95A mutant, 5-day-old gnotobiotic piglets were orally inoculated with 100 TCID50/pig of N93/95A mutant, icPC22A, or mock respectively. Pigs in the N93/95A inoculated group showed delayed onset of diarrhea and reduced fecal viral shedding titers (4.80 ± 0.76 log10 TCID50/mL at 2.00 ± 0.00 dpi) compared with the icPC22A-inoculated pigs (5.80 ± 0.33 log10 TCID50/mL at 1.00 ± 0.00 dpi). In addition to the milder clinical signs, decreased mortality rate (25%) was also observed in the N93/95A mutant group than the icPC22A group (100%). At 22 dpi, the surviving pigs were challenged orally with 106 PFU of icPC22A per pig. Pigs in the N93/95A group had no severe diarrhea (0.00 ± 0.00 days) and shorter duration of mild diarrhea (3.33 ± 1.15 days) than the pigs in the mock-challenge group (duration of diarrhea: 7.17 ± 0.98 days and severe diarrhea: 4.50 ± 2.43 days). The N93/95A mutant inoculation also protected pigs from high fecal viral RNA shedding after challenging: pigs in the N93/95A group showed lower peak viral RNA shedding titers (5.57 ± 0.89 log10 copies/mL) than pigs in the mock-challenge group (10.29 ± 0.76 log10 copies/mL). No pigs died in N93/95A group post challenge. However, 33% (2/6) of pigs in the mock-challenge group died. In summary, the N93/95A mutant was attenuated in vivo and induced robust type I and type III IFN responses and protective immunity to virulent PEDV challenging. So, these nsp1 mutation sites N93A and N95A represent feasible targets for the PEDV LAV development.
My third objective was to recode the transcriptional regulatory sequences (TRSs) of PEDV to engineer a recombination-resistant platform for LAV development to increase the safety of PEDV LAVs. First, we generated an icPC22A-derived reporter PEDV dORF3-EGFP, in which the ORF3 of icPC22A was replaced by EGFP. Then, using the dORF3-EGFP as a backbone, we designed and rescued one remodeled TRS mutant (RMT) that carries the recoded leader and body TRSs for all ORFs, except the one for EGFP gene. We found that the RMT mutant formed smaller plaques in Vero cells than the dORF3-EGFP. The sgRNAs for structural proteins spike (S), envelop (E), membrane (M) and nucleocapsid (N) were detected from the RMT- and dORF3-EGFP-infected cells. However, the sgRNA for EGFP was detected exclusively from the dORF3-EGFP but not the RMT infections. Although both viruses showed robust N gene expression by immunofluorescent staining, only the dORF3-EGFP- but not RMT-infected cells exhibited strong green fluorescence signal, indicating the efficient expression of EGFP. These results suggest the incompatibility between the wildtype TRS leading EGFP gene and the recoded leader TRS in the 5’-UTR of the RMT genome, resulting in little EGFP expression in the RMT-infected cells. Multi-step growth kinetics results revealed comparable replication efficiency between the RMT mutant and the dORF3-EGFP. The two viruses reached similar peak infectious titers at similar time points. Experimental infection of neonatal gnotobiotic pigs showed that the RMT mutant had similar attenuation phenotype and replication efficiency to the dORF3-EGFP. Challenge of the pigs with the highly virulent parental strain icPC22A at 19 days post-inoculation showed that both RMT and dORF3-EGFP protected the pigs from severe diarrhea and death, indicating that they retained good immunogenicity. To test whether the RMT decreases recombination rates, we co-infected Vero cells or pigs with RMT (or dORF3-EGFP as a positive control) and a PEDV S-INDEL strain Iowa106, whose S1 sequence can be distinguished from that of PC22A strain due to unique insertions and deletions. We only found potential recombinant viruses from the pigs co-infected with dORF3-EGFP and Iowa106, but not the pigs co-infected with RMT and Iowa106. No recombinant viruses were found in the co-infected Vero cells. These results suggested that the RMT mutant may be more resistant to recombination due to the incompatibility between the recoded and wildtype TRSs and can serve as a platform for LAV development by adding additional desired mutations along its genome.
In summary, we identified promising targets, N93 and N95 within nsp1, for LAV development. The introduced mutations within nsp1 decreased its IFN antagonism and made the N93/95A mutant more sensitivity to IFN responses. Meanwhile, the mutant was attenuated in neonatal pigs and retained good immunogenicity protecting piglets from severe diarrhea and death. However, the ExoN domain within PEDV nsp14 is critical for its life cycle. Engineered viruses carrying mutations within functional sites of ExoN were highly genetically unstable. Although the crippled nsp14-E191A virus was attenuated and provided partial protection against virulent PEDV challenge, it mutated back both in vitro and vivo. Furthermore, we generated one recombinant PEDV genome by recoding the TRS region which is resistant to recombination event mediated by TRS-driven template switching. The remodeled mutant exhibited similar replication as its backbone both in vitro and in vivo. In a recombination assay by experimental co-infection, incompatibility of recoded and wild-type TRSs of the remodeled mutants may contribute to resistance to recombination. The knowledge obtained from this dissertation may aid in the development of efficient and safe LAVs against PEDV infection.