Investigation Of Porcine Enterocoronaviruses,Isolation And Passaging Of The Porcine Deltacoronavirus SCCZ18,and The Pathway Of The Isolate Entering HIEC-6 Cells

Porcine viral diarrhea has always been an intractable problem in the swine industry.Among all porcine diarrhea viruses,porcine enterocoronaviruses are the main pathogens that causing acute diarrhea.At present,four porcine enterocoronaviruses have been identified,including Porcine epidemic diarrhea virus（PEDV）,Swine transmissible gastroenteritis virus（TGEV）,Porcine deltacoronavirus（PDCoV）,and Swine acute diarrhea syndrome coronavirus（SADS-CoV）.In recent years,the prevailing of the PEDV variants and the newly emerged coronaviruses（PDCoV and SADS-CoV）has caused huge economic losses to the swine industry in China.Therefore,it has momentous scientific significance to conduct investigations on the prevalences of enterocoronaviruses and to reveal the genetic characteristics of the circulating strains for the prevention and control of diarrhea.It is worth noting that in addition to causing diarrhea in piglets,PDCoV can also infect humans causing viremia,which has potential risks to public health.As a newly emerged virus,the biological characteristics,infection mechanism,and potential risk of cross-species transmission of PDCoV are still unclear,which deserves great attention.As intracellular parasitic organisms,viruses must enter cells to initiate the life cycle.Receptor binding and entering pathways are the first steps of virus infection.Most viruses can employ the inherent endocytic pathway of cells to enter cells,so it is of great significance to reveal the endocytic pathway of the virus for understanding the mechanism of infection.In this study,the prevalences of four porcine enterocoronaviruses in diarrhea samples from 168 pig farms during 2014-2018 were investigated,and the genetic characteristics of S genes and complete-genomes for some prevailing strains of PEDV and PDCoV were further analyzed.Additionally,a PDCoV field strain was isolated and characterized,and the genome variations and the cell-tropism of the isolates at indicated passages in LLC-PK1 cells were further compared.Finally,the proliferation characteristics of the PDCoV isolate in normal human intestinal epithelial cells（HIEC-6）were evaluated,and the endocytic pathways of the isolate entering HIEC-6 cells were explored.The detailed results are as follows:1.Investigation and analysis of porcine enterocoronaviruses PEDV,TGEV,PDCoV and SADS-CoV.To explore the prevalence and the molecular characteristics of 4enterocoronaviruses（PEDV,TGEV,PDCoV and SADS-CoV）in pig farms with diarrhea in recent years,RT-PCR was used to detect 4 coronaviruses in 698 diarrheal samples of stools and intestinal contents from 168 pig farms during 2014-2018.Furthermore,the genetic characteristics of the S genes and the complete-genomes of the main circulating strains were analyzed.The results showed that PEDV and PDCoV were the main pathogens of diarrhea in pig farms,with an average detection rate（the positive rate for detected farms）of 36.39%（52.98%）and 8.17%（10.12%）respectively.TGEV only occurred in a few pig farms,with a detection rate（the positive rate for farms）of 1.72%（2.98%）.SADS-CoV was not detected in our samples.The co-infection rates of PEDV+PDCoV and PEDV+TGEV were 2.86%（20/698）and 1.86%（13/698）,respectively.No co-infection of PDCoV+TGEV and more than three kinds of coronaviruses were detected.Full-length sequences of the S gene for 52 PEDV field strains from 52 positive pig farms were obtained and analyzed,the result showed that the nucleotide homology of the S gene among 52 strains was 93.11%-99.98%.Phylogenetic analysis showed that these strains can be divided into three subgroups:S-INDEL（G1c）and non-S-INDEL（G2b and G2c）,among which the non-S-INDEL strains（G2b and G2c）were the dominant prevailing strains,with the prevalence rate of 23.08%（12/52）,59.62%（31/52）respectively,while the detection rate of S-INDEL strains was 15.38%（8/52）.Multiple amino acid deletions or insertions,and consecutive amino acid mutations in the S protein were observed between S-INDEL（G1c）strains and non-S-INDEL strains（G2b,G2c）,which lead to unique variation patterns in the N-glycosylation sites and the S10 region of the neutralizing epitope in the S protein.In addition,the complete-genome of nine PEDV field strains（three strains selected from each subgroup,respectively）were further sequenced and analyzed,phylogenetic analysis showed nine strains were all clustered into the GII subgroup（variants）,of which two G2c strains were clustered in GII-b,and the remaining seven strains were clustered into the GII-c subgroup.Recombination analysis showed that six GII-c strains probably derived from recombinations among the GII-c subgroup strains,of which three S-INDEL strains with novel recombination patterns were identified for the first time.The full-length S gene sequences of eight PDCoV field strains were obtained,the homology of the S gene among the eight PDCoV strains was 97.47%-100%,phylogenetic analysis showed that the eight strains were all Chinese lineage strains,of which seven strains were clustering into Chinese lineage Clade 3,and the remaining one was clustered into Chinese lineage Clade 2.Of these eight prevailing PDCoV strains,complete-genome sequences of six strains were successfully obtained,and the genome homology was 98.56%-99.45%.Genome comparison revealed that six strains had variations of nucleotide deletions in the nsp3 of ORF1a and3’UTR region.Phylogenetic analysis based on complete-genome revealed that all six PDCoV strains were Chinese lineage strains,four of which were clustered together into the same branch,while the remaining two strains were clustered into another large branch.In the present part of this study,the prevalences of four porcine enterocoronaviruses in pig farms with diarrhea were investigated,and the genetic characteristics of the prevailing strains for the main diarrhea pathogens（PEDV and PDCoV）were further revealed,which would increase the understanding of the prevalence,genetic characteristics and evolutionary profiles of circulating PEDV and PDCoV strains in China,and provide a scientific reference for the selection of vaccine strains for pig farms.2.Isolation and characterization of porcine deltacoronavirus SCCZ18,and comparative study on the genome variation and cell-tropism of the passaged viruses.To reveal the biological characteristics of PDCoV strains,the isolation and identification of field PDCoV strains were tried,and the variation characteristics in the genome,and differences in cell-tropism of the PDCoV isolate during the passage were further evaluated.The samples from six PDCoV-positive farms were inoculated into LLC-PK1 cells to isolate virus,only one intestinal sample of one farm showed cytopathic effect（CPE）within 36 hours with adding trypsin（10μg/m L）,and can be passaged continuously.Through plaque purification,detection of RT-PCR,indirect immunofluorescence,western blotting and electron microscopy,the PDCoV strain SCCZ18 was successfully isolated.This isolate can proliferate in LLC-PK1cells without trypsin,but on CPE were observed.However,the addition of trypsin can promote the proliferation of SCCZ18 in the infected cells.At present,the SCCZ18 strain has been stably passaged in LLC-PK1 cells for more than 200 passages,and the virus titer can be stabilized above 1×10^13.75 TCID₅₀/m L after the P100 passage.To reveal the variations of PDCoV SCCZ18 during the passage process,the complete-genome of P10,P73,P101,P140,P160,P180 and P200 strains were sequenced and analyzed.The results showed that there was no nucleotide insertion or deletion in the genome during the successive passage in LLC-PK1cells,but multiple nucleotide variations were observed.Compared with the field strain SCCZ18,SCCZ18-P200 has 42 nucleotide mutations in the genome,29 of which were missense mutations,resulting in 28 amino acids changed.These missense mutations were mainly accumulated in the S protein（13 in S1 subunit,9 in S2 subunit）.Additionally,amino acid variations in nsp3（G831W,H1499Y）,nsp5（H2529N）and nsp8（E3203A）in ORF1ab,and 1 serine mutation in each of E protein（R53S）and M protein（R60S）were observed.The cell-tropism comparison of SCCZ18 strain（P10）and its passage strain（P200）in cell lines of different species such as porcine,human,monkey,bovine,canine and avian showed that both SCCZ18-P10 and SCCZ18-P200 can replicate and proliferate in porcine derived intestinal epithelial cells（IEC）and testicular cells（ST）,human derived liver cancer cells（Huh-7）and normal intestinal epithelial cells（HIEC-6）,and african green monkey derived kidney cells（Vero）and embryonic kidney cells（Marc145）,bovine derived kidney cells（MDBK）,avian derived macrophage cells（HD11）and fibroblast cells（DF-1）,except for canine kidney cells（MDCK）and human non-small cell lung cancer cells（A549）.It is worth noting that infections of Vero and MDBK are trypsin-dependent,while other infections don’t depend on trypsin,but the addition of trypsin can promote virus proliferation.The results showed that the gene mutations accumulated in the SCCZ18-P200 strain during the continuous passage of porcine-derived cells（such as mutations at 22 key sites in the S protein）did not substantially affect the cell tropism of the strain.In this part of the study,the PDCoV SCCZ18 was successfully isolated,and its genomic variation characteristics during the passage in LLC-PK1 cells were further revealed.The susceptibility of the SCCZ18 strain and its passaged virus strains in different species of cells were preliminarily evaluated.These results lay the foundation for further development of cell adapted vaccine strains of PDCoV.3.Endocytic pathway of porcine deltacoronavirus SCCZ18 entering HIEC-6 cells.In view of the potential public health significance of the enterotropic virus PDCoV,to reveal the mechanism of PDCoV infecting human intestinal epithelial cells,the proliferative characteristics of PDCoV SCCZ18 in human crypt-like normal intestinal epithelial cells（HIEC-6）were evaluated firstly,and then the endocytic pathway of PDCoV entry into HIEC-6 cells was further studied by using methods of chemical inhibitors,RNAi and confocal microscopy.We found that the HIEC-6 cells supported the complete life cycle of PDCoV,and the virus titer in the cell supernatant can reach 1×10⁶ TCID₅₀/m L at 36 hpi.Study of internalization pathway showed that:the chemical inhibitor of the clathrin-mediated endocytosis（Chlorpromazine）,knockdown of the clathin heavy chain did not affect the infection of PDCoV;the chemical inhibitor of caveola-mediated endocytosis（Genistein）,knockdown of the caveolin-1 did not affect the infection of PDCoV;and the study of confocal laser scanning microscopy also found that the PDCoV virions had no co-localization with clathrin and caveolin-1 during the entering process;these results indicated that the PDCoV entreing HIEC-6 cells are independent of the clathrin-mediated and caveola-mediated endocytosis;the dynamin inhibitors（Dynasore and Mit MAB）,interference with dynamin 2（DNM2）expression with specific si RNA had no significant effects on the infection of PDCoV（P>0.05）;however,pretreated cells with EIPA and Wortmannin,the macropinocytosis pathway inhibitors,had significantly decreased copies of PDCoV and the expression of N protein（P<0.01）;confocal laser scanning microscopy analysis showed that the PDCoV infection could promote the uptake of Dextran（a marker of the macropinocytosis pathway）,and co-localization of virus particles and dextran in cytoplasmic vacuoles were also observed;pretreated HIEC-6 cells with cholesterol blockers MβCD,Nystatin and Filipin could significantly inhibit the infection of PDCoV（P<0.01）,but the infectivity of PDCoV could be recovered to some extent by supplementing exogenous cholesterol during the infection stage,suggesting that PDCoV entering HIEC-6 cells was cholesterol-dependent;moreover,pretreated cells with a tubulin inhibitor Nocodazole and actin stabilizer Jasplakinolide significantly inhibited the infection of PDCoV;actin rearrangement and increasing of filopodia on cell membrane surface were observed at 1,10,30,45,and 60 min after infection,suggesting that PDCoV entering HIEC-6 cells was cytoskeleton-dependent;these above results indicate that PDCoV relies on macropinocytosis for internalization into HIEC-6 cells.The results of the study on the intracellular transport pathway of the virus showed that:endosome acidification inhibitors（Chloroquine,NH₄Cl,and Bafilomycin A1）,and interference with the expression of vacuolar H⁺-ATPase with specific si RNA,could significantly inhibit the infection of PDCoV（P<0.01）;besides,silencing the expression of early endosomal marker protein Rab5a with si RNA had no significant effect on the infection of PDCoV in HIEC-6 cells;but analysis of confocal laser scanning microscopy showed that PDCoV virions could co-locate with another early endosomal marker protein EEA-1 at 5,10,and 30 min after infection,suggesting that the internalized PDCoVs were transported with early endosomes,but did not rely on Rab5a;interference with the late endosomal marker proteins（Rab7a,Rab9a）,the circulating endosomal marker proteins（Rab11a）,and the lysosomal marker（LAMP1）with specific si RNA could significantly inhibit the PDCoV infection in HIEC-6 cells（P<0.01）;analysis of confocal laser scanning microscopy showed that PDCoV virions could co-localize with Rab7a,Rab9a at 30 min after infection,Rab11a and LAMP1 at 45 min after infection,and the co-localization with LAMP1 increased significantly at 90 min after infection;all these evidences indicate that the internalized PDCoV virions were transported into lysosomes depending on early,late,and circulating endosomes.This study for the first time revealed that PDCoV entering HIEC-6 cells rely on macropinocytosis,endosome,and lysosome pathway,which would provide new insights for a better understanding of the mechanism of PDCoV infection,and guiding for the antiviral drug design in the future.