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viruses Article Characteristics of the Life Cycle of Porcine Deltacoronavirus PDCoV In Vitro Replication Kinetics Cellular Ultrastructure and Virion Morphology and Evidence of Inducing Autophagy Pan Qin 1 y En Zhong Du 1 2 y Wen Ting Luo 1 Yong Le Yang 1 Yu Qi Zhang 1 Bin Wang 1 and Yao Wei Huang 1 1 Institute of Preventive Veterinary Medicine and Key Laboratory of Animal Virology of Ministry of Agriculture College of Animal Sciences Zhejiang University Hangzhou 310058 China qinpan P Q duenzhong519 E Z D 21617039 W T L yang yl Y L Y 3160100223 Y Q Z wb1990boy B W 2 YEBIO Bioengineering Co Ltd of Qingdao Qingdao 266114 China Correspondence yhuang y These authors contributed equally to this work Received 10 April 2019 Accepted 16 May 2019 Published 18 May 2019 gid00030gid00035gid00032gid00030gid00038gid00001gid00033gid00042gid00045gid00001 gid00048gid00043gid00031gid00028gid00047gid00032gid00046 Abstract Porcine deltacoronavirus PDCoV causes severe diarrhea and vomiting in a ected piglets The aim of this study was to establish the basic in vitro characteristics of the life cycle such as replication kinetics cellular ultrastructure virion morphology and induction of autophagy of PDCoV Time course analysis of viral subgenomic and genomic RNA loads and infectious titers indicated that one replication cycle of PDCoV takes 5 to 6 h Electron microscopy showed that PDCoV infection induced the membrane rearrangements with double membrane vesicles and large virion containing vacuoles The convoluted membranes structures described in alpha and beta coronavirus were not observed PDCoV infection also increased the number of autophagosome like vesicles in the cytoplasm of cells and the autophagy response was detected by LC3 I II and p62 Western blot analysis For the first time this study presents the picture of the PDCoV infection cycle which is crucial to help elucidate the molecular mechanism of deltacoronavirus replication Keywords Porcine deltacoronavirus PDCoV electron microscopy ultrastructure autophagy 1 Introduction Porcine deltacoronavirus PDCoV which belongs to the genus Deltacoronavirus in the subfamily Coronavirinae of the family Coronaviridae order Nidovirales was first reported in Hong Kong in 2012 1 and thereafter isolated from pigs in the United States 2 and many Asian countries including China 3 5 It causes acute diarrhea vomiting dehydration and mortality in nursing pigs 6 7 The PDCoV genome is a positive sense single stranded RNA of approximately 25 4 kb in size The genome organization of PDCoV is similar to those of other reported coronaviruses with the typical gene order 50 ORF1a 1b Spike S Envelope E Membrane M NS6 Nucleocapsid N NS7 30 Ultrastructural characterization of other CoVs in alpha beta or gamma CoV genus such as severe acute respiratory syndrome coronavirus SARS CoV 8 14 human coronavirus NL63 HCoV NL63 15 infectious bronchitis virus IBV 16 transmissible gastroenteritis virus TGEV 17 and porcine epidemic diarrhea virus PEDV 18 has been performed The studies on three CoV genera demonstrated that the architectures of the organelle during CoV infection are distinct between among alpha beta coronaviruses and gammacoronavirus Alpha and beta coronaviruses formed Viruses 2019 11 455 doi 10 3390 v11050455 Viruses 2019 11 455 2 of 14 clusters of the double membrane vesicle DMV which is highly conserved among coronaviruses sometimes linked by a convoluted membrane 11 15 19 21 whereas the gammacoronavirus IBV induced extensive paired membranes and smaller 60 80 nm spherules in addition to the DMVs 16 There is paucity of information on the ultrastructural picture of the newly discovered deltacoronavirus infection The isolation epidemiology and pathogenicity of PDCoV has been recently studied by researchers from di erent countries but a better understanding of the PDCoV life cycle is critically needed to help elucidate the mechanism of virus replication and antiviral activity of the host cells In this study some basic in vitro characteristics of the life cycle for example replication kinetics cellular ultrastructure virion morphology and possible induction of autophagy of PDCoV were studied and elaborated 2 Materials and Methods 2 1 Cell and Virus The PDCoV Chinese Hunan strain was used in this study 22 The virus cultivation was done in a porcine kidney epithelial cell line LLC PK1 ATCC CL 101 at 37 C in 5 CO2 in Dulbecco s modified Eagle s medium DMEM supplemented with 10 fetal bovine serum FBS and 1 w v antibiotics penicillin and streptomycin 2 2 Extracellular and Intracellular Viral RNA Quantitation LLC PK1 cells were seeded at 80 confluence in 6 well culture plates and incubated overnight After the cells were washed with phosphate bu ered saline PBS viruses at a multiplicity of infection MOI of 1 were added to each plate After 1 h of incubation at 37 C the cells were washed four times with PBS and then harvested along with supernatants at an hourly interval until 12 h post infection hpi At least three replicate experiments were performed Viral subgenomic RNA sgRNA load was monitored by one step quantitative reverse transcription polymerase chain reaction qRT PCR targeting the membrane M gene as described previously forward primer 50 ATCGACCACATGGCTCCAA 30 reverse primer 50 CAGCTCTTGCCCATGTAGCTT 30 and probe 50 FAM CACACCAGTCGTTAAGCATGGCAAGCT BHQ 30 23 as the M gene is expressed by the sgRNA and viral genomic RNA gRNA load was detected by qRT PCR targeting the non structural protein nsp 2 encoding region forward primer 50 GAAGGTGAAGATGATAGTG 30 reverse primer 50 GCTCTGGTTTAGGATAGA 30 and probes 50 FAM TACTTCGTCGCTGCTGGTCTT TAMRA 30 as the nsp2 of ORF1a is expressed by the gRNA Standard curves were performed to allow absolute quantitation of PDCoV RNA copy numbers based upon the levels of in vitro transcribed RNAs containing the targeting sequences 24 2 3 Virus Yield Assay LLC PK1 cells were infected with PDCoV at an MOI of 5 and cell supernatants were harvested hourly to determine the onset of progeny virus release from the infected cells The titers of the yield of progeny virus were determined by endpoint dilutions as 50 tissue culture infective dose TCID50 on LLC PK1 cells Viruses 2019 11 455 3 of 14 2 4 Transmission Electron Microscopy TEM LLC PK1 cells were infected with PDCoV at an MOI of 1 The cells were harvested and fixed with 2 5 glutaraldehyde in phosphate bu er 0 1 M pH 7 0 and 1 Osmium tetroxide OsO4 in phosphate at 2 4 6 8 12 16 and 24 hpi Specimens were dehydrated in a series of ethanol dilutions 30 50 70 80 90 95 and 100 for 15 20 min at each step then transferred to absolute acetone for 20 min The specimens were then placed in one of three mixtures of absolute acetone and Spurr resin 1 1 1 3 and pure Spurr resin for 1 h 3 h and overnight respectively Finally ultrathin sections were stained by uranyl acetate and alkaline lead citrate for 5 10 min and observed using a Hitachi Model H 7650 transmission EM 25 2 5 Western Blot Analysis PDCoV infected LLC PK1 cells were lysed in CelLytic M lysis bu er Sigma St Louis MO USA The protein concentration was quantified by the BCA bicinchoninic acid protein assay kit Beyotime Biotechnology Shanghai China Samples were separated by 12 sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE The proteins were transferred onto a polyvinylidene difluoride PVDF membrane that was subsequently blocked with Tris bu ered saline TBS containing 3 bovine serum albumin BSA at room temperature for 2 h and then incubated overnight at 4 C with primary antibodies Rabbit anti LC3 A7198 ABclonal Wuhan China and rabbit anti p62 SQSTM1 P0067 Sigma St Louis MO USA polyclonal antibodies were used in this study The blots were then incubated with corresponding horseradish peroxidase HRP conjugated secondary antibody Thermo Fisher Scientific Waltham MA USA 3 Results and Discussion 3 1 In Vitro Porcine Deltacoronavirus PDCoV Replication Kinetics Viral gRNA was first detected in the supernatants at 5 hpi which increased significantly at 9 hpi p 0 01 and reached a maximum of 1 88 107 copies mL at 12 hpi Figure 1A left panel Intracellular gRNA load remained low until 5 hpi followed by a sharp increase at 6 hpi from 2 92 106 to 2 07 107 copies 106 cells reaching a final titer of 4 47 108 copies 106 cells at 12 hpi Figure 1B left panel Similarly extracellular or intracellular viral sgRNA amount gradually increased from 5 hpi with significant changes at 9 or 8 hpi Figure 1A B right panels Infectious progeny virus could be detected in the culture supernatants at 6 hpi at a low titer 4 5 102 TCID50 mL and then the amounts of progeny virus increased By 12 h post infection infected LLC PK1 cells produced an infectious titer of 1 25 105 TCID50 mL Figure 1C These results indicated that the first generation of progeny was assembled and released from infected cells at about 5 hpi suggesting that one replication cycle of PDCoV takes approximately 5 to 6 h to complete Viruses 2019 11 455 4 of 14Viruses 2019 11 x FOR PEER REVIEW 4 of 14 Figure 1 Determination of the growth kinetics and the time of one life cycle of porcine deltacoronavirus PDCoV infection in porcine kidney epithelial LLC PK1 cells The amounts of extracellular gRNA and sgRNA A as well as intracellular gRNA and sgRNA B were determined by quantitative reverse transcription polymerase chain reaction qRT PCR respectively Samples of supernatants and cells were collected at hourly intervals between 1 to 12 h post infection Viral titers were determined by end point dilutions on fresh LLC PK1 cells and calculated as TCID50 per mL C Data represent mean values standard deviation SD of at least 3 replicates Differences in the RNA copies or virus titer were evaluated by t test Asterisks indicate significant differences between the two groups p 0 05 p 0 01 A B C gRNA sgRNA gRNA sgRNA Extracellular Intracellular Figure 1 Determination of the growth kinetics and the time of one life cycle of porcine deltacoronavirus PDCoV infection in porcine kidney epithelial LLC PK1 cells The amounts of extracellular gRNA and sgRNA A as well as intracellular gRNA and sgRNA B were determined by quantitative reverse transcription polymerase chain reaction qRT PCR respectively Samples of supernatants and cells were collected at hourly intervals between 1 to 12 h post infection Viral titers were determined by end point dilutions on fresh LLC PK1 cells and calculated as TCID50 per mL C Data represent mean values standard deviation SD of at least 3 replicates Di erences in the RNA copies or virus titer were evaluated by t test Asterisks indicate significant di erences between the two groups p 0 05 p 0 01 Viruses 2019 11 455 5 of 14 3 2 PDCoV Infection Induces Cellular Ultrastructural Changes The diameter of known CoV particles ranges from 70 120 nm The size of the purified PDCoV virion by ultracentrifugation the approach described in 25 was first assessed Electron microscopy EM of a negatively stained sample demonstrated that the virus particle was 58 to 70 nm in diameter and had surface projections typical of CoV Supplemental Figure S1 PDCoV infected cells exhibited significant morphologic changes Figure 2A B compared to uninfected controls Figure 2C D at 8 hpi such as the presence of many cytoplasmic vesicles including dilated rough endoplasmic reticulum DMVs and injured mitochondria The cytoplasmic vesicles surrounding the nucleus increased along with infection Figure 2A B The size of PDCoV virions observed with infected cells approximately 50 70 nm in diameter Figure 2E F was in line with the purified viral particle Supplemental Figure S1 CoV infection is initiated by the interaction of the viral S protein with specific host cellular receptors followed by membrane fusion and the cellular receptor for PDCoV entry has just been identified as porcine aminopeptidase N 24 At 4 hpi PDCoV virions were seen attached to the cell surface Figure 2E The process of likely endocytosis of PDCoV was also observed Figure 2F which needs further confirmation Following internalization CoV particles are transported and scattered into the cytoplasm It is well known that viral gRNA is recognized directly by the host cell machinery and translated into non structural proteins which assemble into viral replication transcription complexes RTCs on DMVs or other membrane structures 16 21 26 coordinating the enzymes required for viral RNA transcription proof reading and capping of new viral transcripts Similar events should apply to PDCoV infection as a large number of DMVs were seen at 6 hpi and thereafter After completion of genome replication and RNA transcription the virions of PDCoV start to assemble in the expanded rough endoplasmic reticulum RER with irregular shape as the surface was decorated with ribosomes Figure 2G H Immature PDCoV precursors with an electron dense periphery and clear core were present in the RER Figure 2H or in the lumen of a stack of adjacent cisternae very likely the Golgi apparatus Figure 2I The precursors likely tra cked through the endoplasmic reticulum Golgi intermediate compartment ERGIC completing structural transformation into small infectious virions in the Golgi complex The virions were also observed in large circular organelles Figure 2J These structures named as Large virion containing vacuoles LVCVs have earlier been described for other CoVs infected cells which are thought to originate from Golgi compartments that expand to accommodate numerous virions 27 Mature virions accumulate inside secretory vesicles which tend to merge into bigger vesicles Figure 2J After that vesicles containing mature virions moved to the cell periphery and eventually fuse with the cell membrane In the final phase of infection mature virions must escape the cell PDCoV release was observed to occur via two pathways exocytosis and cytolysis Early virions those generated within the first few replicative cycles may be released by exocytotic fusion of virus containing vesicles with plasma membranes without damage to the cell Figure 2K However as viral infection progressed and the quantity of virus in the cells increased large amounts of virus particles were released by cytolysis resulting in necrosis of the host cell Figure 2L Viruses 2019 11 455 6 of 14 Viruses 2019 11 x FOR PEER REVIEW 6 of 14 Figure 2 Cont Viruses 2019 11 455 7 of 14 Viruses 2019 11 x FOR PEER REVIEW 7 of 14 Figure 2 Electron microscopy EM observation of in vitro PDCoV infection entry assembly and maturation of PDCoV particles LLC PK1 were infected A B with PDCoV or mock infected C D at 8 hr post infection hpi B D Zoomed region highlighted in Figure A and C respectively Compared with mock infected cells large amounts of vesicles including double membrane vesicles DMVs zippered endoplasmic reticulum ER and injured mitochondria are visible in the cytoplasm E Viral particles in the process of attachment on the cell membrane at 4 hpi and one viral particle likely in the process of endocytosis at 4 hpi F One viral particle is likely in the process of endocytosis at 4 hpi G Precursor PDCoV virions assembled predominantly in the rough endoplasmic reticulum RER nearby DMVs white arrow hpi 6 H Precursor virions assembled in the RER or ERGIC Precursor particles were observed in the dilated RER white arrow hpi 8 I Precursor virions likely completed the structural transformation that yielded small size virions Large virion containing vacuoles LVCVs are thought to originate from Golgi compartments that expand to accommodate numerous virions hpi 16 J LVCVs are large circular organelles with a diameter of approximately 400 600 nm that contain numerous virions in their interior hpi 16 K Virion containing vesicles fuse with plasma membranes and then released the virus particles to the extracellular environment without damage to the cell in the first few replicative cycles hpi 12 L Large amounts of viral particles released by cytolysis hpi 24 ER endoplasmic reticulum M mitochondria N nucleus G golgi apparatus CoV replication is closely tied to the formation of membrane bound RTCs that alter cell membrane structures as previously reviewed 19 28 Many such alterations were observed directly in an electron microscope EM of PDCoV infected LLC PK1 cells including DMVs Figure 2G 2H 3A and 3B LVCVs Figure 2I and 2J zippered ER Figure 3C and damaged mitochondria Figure 3D The DMV architecture is a typical and highly conserved feature of CoV infections with a diameter ranging from 150 300 nm with electron dense cores 8 9 11 13 16 26 29 DMVs play a role Figure 2 Electron microscopy EM observation of in vitro PDCoV infection entry assembly and maturation of PDCoV particles LLC PK1 were infected A B with PDCoV or mock infected C D at 8 h post infection hpi B D Zoomed region highlighted in Figure A and C respectively Compared with mock infected cells large amounts of vesicles including double membrane vesicles DMVs zippered endoplasmic reticulum ER and injured mitochondria are visible in the cytoplasm E Viral particles in the process of attachment on the cell membrane at 4 hpi and one viral particle likely in the process of endocytosis at 4 hpi F One viral particle is likely in the process of endocytosis at 4 hpi G Precursor PDCoV virions assembled predominantly in the rough endoplasmic reticulum RER nearby DMVs white arrow hpi 6 H Precursor virions assembled in the RER or ERGIC Precursor particles were observed in the dilated RER white arrow hpi 8 I Precursor virions likely completed the structural transformation that yielded small size virions Large virion containing vacuoles LVCVs are thought to originate from Golgi compartments that expand to accommodate numerous virions hpi 16 J LVCVs are large circular organelles with a diameter of approximately 400 600 nm that contain numerous virions in their interior hpi 16 K Virion containing vesicles fuse with plasma membranes and then released the virus particles to the extracellular environment without damage to the cell in the first few replicative cycles hpi 12 L Large amounts of viral particles released by cytolysis hpi 24 ER endoplasmic reticulum M mitochondria N nucleus G golgi apparatus Viruses 2019 11 455 8 of 14 CoV replication is closely tied to the formation of membrane bound RTCs that alter cell membrane structures as previously reviewed 19 28 Many such alterations were observed directly in an electron microscope EM of PDCoV infected LLC PK1 cells including DMVs Figure 2G H and Figure 3A B LVCVs Figure 2I J zippered ER Figure 3C and damaged mitochondria Figure 3D The DMV architecture is a typical and highly conserved feature of CoV infections with a diameter ranging from 150 300 nm with electron dense cores 8 9 11 13 16 26 29 DMVs play a role not only in the synthesis of viral RNA 21 but also in helping to concentrate on viral proteins and o er protection from cellular antiviral detection and elimination machinery 28 DMVs could be detected at 2 hpi in SARS CoV or mouse hepatitis virus MHV infected cells and then the number of DMVs increased dramatically 11 27 The appearance of DMVs in PDCoV infected cells was seen at 6 hpi Figure 2G The sizes of DMVs ranged from 200 to 400 nm PDCoV induced a low number of DMVs and most of the DMVs were separate entities or in small clusters in the cytoplasm Initially the DMV inner and outer membranes were generally tightly apposed Figure 2G H but occasionally some luminal space between the two lipid bilayers could be discerned Figure 3A Similar observations were previously made for SARS CoV and IBV 11 Most of the DMVs contained few or no fibrous material in the inner vesicles Figure 2G H and Figure 3A Interestingly DMVs containing an electron dense core could also be observed Figure 3B Previous researches confirmed that the detection of this core depended on the protocol used for the preparing of resin embedded specimens In chemically fixed samples the interior of DMV appeared mainly electron translucent However the core can be preserved by