Consiruction Infectious Clone Of Porcine Epidemic Diarrhea Virus And Transcription Analysis Of The Porcine Alveolar Macrophage Response To Porcine Circovirus Type 2

Porcine epidemic diarrhea (PED) is a highly contagious enteric disease which caused by porcine epidemic diarrhea virus (PEDV), it was characterized by acute watery diarrhea, vomiting and dehydration. Variant strains of PEDV associated with large-scale outbreaks of diarrhea with 80%-100% morbidity and 50%-90% mortality in suckling piglets have emerged in China since 2010, afterwards, PED was outbreaks in May 2013 in USA, and caused significant economic loss to the swine industry.Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus associated diseases (PCVAD), which causes significant economic losses to the swine industry. The major target cells of PCV2 are monocytic/marcrophage linage cells (MLCs) and dendritic cells, PCVAD is characterized as lymphoid depletion and histiocytic replacement in lymphoid tissues, which leading to immunosuppression and multisystemic diseases in infected pigs and may render the pigs more susceptible to secondary or concurrent viral and bacterial infections.This study was mainly focused on these two important swine pathogens, and the main results were described as follows:1. The epidemiological study of PEDA total of 455 samples (intestine, stool, and maternal milk) were collected from 57 farms in 12 provinces of China between January 2011 and October 2011. All samples were evaluated by reverse transcription PCR (RT-PCR), using previously described primers. Forty-five (78.95%) of the farms had at least one PEDV-positive sample. Two-hundred-and-seventy-eight (61.10%) of the samples were PEDV-positive, including 253 (of 402; 62.94%) fecal samples,20 (of 31; 64.52%) intestine samples, and 5 (of 22; 22.73%) milk samples, indicates PEDV prevalent in China and with high positive rates, and sow milk might be a possible route for the vertical transmission of PEDV from sow to suckling piglet.2. Isolation and identification of CHGDU strainIsolation of PEDV is extremely difficult, even PEDV was prevalent in the field with high positive rate. In this study, intestinal sample were collected from infected piglets in Guangdong province, then filtered intestinal content suspension were inoculated into monolayer of Vero-CCL81 in the presence of 15μg/mL trypsin, after 3 blind passages, syncytia was observed, then the isolate called CHGDU was further confirmed by RT-PCR and indirect immunofluorescence assay by specific anti-S monoclonal antibody.3. Sequencing and analysis of spike gene of CHGDU strainThe whole spike gene of CHGDU strain was sequenced in this study, strain CHGDU showed 100% identity to another Chinese strain GD-A, and this virus shares the same sequence identity with other identified variant strains, which confirms a variant strain was isolated in this study, the neutralizing epitope region COE and SID were compared to those of vaccine strain CV777, three mutations (T554S, G599S and Q638S) and five mutations (N724S, N728S, P768R, L769S and D771S) were found, respectively. The S1/S2 junction and S2’prediction site were further investigated, CHGDU was acquired the mutation of arginine in these sites, these mutations enable the proprotein concertase (PC) to recognize and cleave the spike protein, which might explain the reason that CHGDU induced bigger syncytia than CV777 did in Vero cell and might induce the virulence increase to piglets. The spike gene of CHGDU was further compared with other 152 PEDV strains available in GenBank, Phylogenetic tree was constructed using MEGA6 software, the results shows all the 153 PEDV strains can be clustered into two genogroups:Genogroup 1 and Genogroup 2, and each of the groups can be further divided into two subgroups:la, lb,2a and 2b. CHGDU strain was clustered into subgroup la, and clustered together with other identified PEDV variants and showed considerable genetic distance from vaccine strain CV777 and DR13.4. Cloning the spike of CHGDU and construction the recombinant virus rPEDV-DR13-SCHGDU-Flag-dORF3/GFPTo analysis the role of spike protein on the pathogensis of PEDV, in this study the spike gene of CHGDU was cloned to construct the plasmid PCAGGS-CHGDU-S-Flag, the plasmid was then transfected into Vero-CCL81 cell, it could induce cell-cell fusion in the presence of 5μg/mL of trypsin. Further, the spike gene was inserted to a transfer vector to construct the plasmid pPEDV-CHGDU-S-Flag-dORF3/GFP, then mRNA was synthesized and then electroporated into mPEDV infected Vero-CCM cell, after which the recombinant virus can be recovered, the recombinant rPEDV-DR13-SCHGDU-Flag-dORF3/GFP was successfully recovered after target recombination, the recombinant virus was further confirmed to need trypsin for propagation, suggests S gene was the key gene which determine the trypsin dependent.5. Construction the stable cell-line of Vero-hTMPRSS2-HA and the role of hTMPRSS2 on the PEDV infectionHuman TMPRSS2 was cloned and fused with a HA tag, the expression of hTMPRSS2-HA was confirmed by western-blot, stable cell-line of Vero-hTMPRSS2-HA was further constructed by MLV system, and the expression of hTMPRSS2-HA on this cell-line was confirmed by IFA and Western-blot. To test the role of hTMPRSS2 on virus entry and releasement, recombinant virus with GFP tag was used to infect Vero-hTMPRSS2-HA, but it seems there have no effect of hTMRPSS2 on the entry and releasement of recombinant virus.6. Transcription analysis of the porcine alveolar macrophage response to porcine circovirus type 2In this study, PCV2-WH strain was used to infect primary alveolar macrophage (PAMs), microarray analysis at 24 and 48 hours post-infection (HPI) revealed 266 and 175 unique genes, respectively, that were differentially expressed (false discovery rate <0.05; fold-change>2). Only six genes were differentially expressed between 24 and 48 HPI.212 and 54 unique genes were significantly μp-regulated and down-regulated at 24 HPI, and 150 and 25 genes were up-regulated and down-regulated at 48 HPI. Differentially expressed genes at 24 HPI were mainly related to cellular movement, hematological system development and function, immune cell trafficking, cell-to-cell signaling and interaction, renal and urological disease et al., according to the corresponding biological process, the majority of these genes related to immune response, inflammatory response, antigen processing and presentation, chemotaxis and anti-apoptosis. Genes differentially expressed at 48 HPI were related to cancer, gastrointestinal disease, tumor morphology, cell death and survival, connective tissue disorders, organismal injury and abnormalities et al., the related biological process were inflammatory response, immune response, chemotaxis, cell-cell signaling and cell adhension et al., a lot of genes related to inflammation were up-regulated in both infection groups, indicating that PCV2 may induce systematic inflammation after infection.