Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to
DISSECTING THE ROLE OF THE ORF3 GENE OF PORCINE EPIDEMIC DIARRHEA VIRUS IN VIRAL PATHOGENICITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1008096
Grant No.
2016-67012-24716
Project No.
VA-Rogers
Proposal No.
2015-03682
Multistate No.
(N/A)
Program Code
A7201
Project Start Date
Dec 15, 2015
Project End Date
Dec 14, 2018
Grant Year
2016
Project Director
Rogers, A. J.
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
(N/A)
Non Technical Summary
Porcine Epidemic Diarrhea Virus (PEDV) is a disease which has circulated in Europe and Asia since the late 70s and was firstintrouduced to the United States in 2013. The virus induces severe diarrhea in infected pigs which is oftenfatal to newborn piglets. In 2014, an estimated 8 million piglets were killed by this disease in the United States. While some vaccines have been in use in parts of the world where PEDV is endemic for decades, the virus has thus far escaped vaccine control, suggesting the need for better vaccine candidates to control the US outbreak.Previous vaccines generated by passaging the virus in tissue culture have a characteristic deletion of all or part of open reading frame 3 (ORF3) along with other mutations. Outside of the host, ORF3 is not required, but loss of the gene seems to reducethe virus's ability to infect and replicate in the host. We will, therefore, use a strain of PEDV our laboratory has isolated from Colorado and mutate ORF3 on its own, leaving the rest of the virus as close to the circulating US strains as possible, with the idea of evaluating it as a vaccine candidate. Once the mutants are generated, we will look at changes in the viral life cycle in cell culture, as well as performing animal experiments with pigs to look for any reduction in disease severity for infected piglets.Ultimately, the goal of this project is to determine if ORF3 deleted PEDV is a viable vaccine candidate. If pathogenecity is reduced or removed in piglets, the virus can then be evaluated for its ability to stimulate an immune response in nursing sows which can be passed on to newborns to protect them from this disease. If succesful, a vaccine against PEDV would greatly curb the spread of PEDV through american hog farms.
Animal Health Component
100%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31135101101100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1101 - Virology;
Goals / Objectives
The major goal of this project is to investigate the role of Open Reading Frame 3 (ORF3) in the viral pathogenesis of Porcine Epidemic Diarrhea Virus (PEDV) as a means of exploring ORF3 deletion as a vaccine generation candidate. The objectives of the project are:1) Generate an ORF3 deletion mutant from a Colorado strain of PEDV from our laboratory.2) Utilize tissue culture techniques to observe the effect of ORF3 deletion on the viral life cyclein vitro.3) Determine the effect of ORF3 deletion on virulence and pathogenesis in piglets4) Generate ORF3 mutants with deletion of domains predicted to be important including: Amino acids 1-40, 81-100, 152-170, and 182-224.5) Observe effects of ORF3 targeted deletion in tissue culture compared to deletion of the full ORF6) Observe reduced pathogenesis and virulence of PEDV ORF3 targeted mutants as compared to full length deletion mutant
Project Methods
EffortsI will utilize targeted mutagenesisto rapidly introduce a series of mutations to the ORF3 gene of a U.S. PEDV-Colorado strain. Recombinant sequences of PEDV encompassing 7,832 nt from ORF1b to the 3' terminus will be synthetically generated by Genescript Inc. This synthetic sequence, containing the 1,263 nt ectodomain of the MHV spike protein along with the transmembrane domain and cytoplasmic tail of PEDV S (61 aa), will be cloned into an expression vector with a T7 promoter. This transfer vector will then be transfected into Vero cells infected with U.S. PEDV-Colorado strain, which will then be overlaid onto a Murine L cell monolayer. Recombinant mPEDV will be recovered by two subsequent rounds of plaque selection and purification on L cells and confirmed after recovery by RT-PCR and sequencing at the Virginia Bioinformatic Institute. This mPEDV will then be used to generate all subsequent PEDVΔORF3 mutants. An oligonucleotide containing PEDV S protein and deleted ORF3 (nt 24,789 to 25,463) will be cloned into a T7 expression vector. L cells infected with mPEDV will be transfected with this expression vector and overlaid onto a Vero cell monolayer supplemented with trypsin. The resulting recombinant virus will be recovered by two rounds of plaque selection. After deleting PEDV ORF3 completely, smaller deletion mutants of ORF3 will remove domains which may play a role in protein function. Other mutagenic studies have shown that Y170 and H152 are important for potassium ion activity and a pair of protein-protein interaction sites were predicted at aa 157 and 158, so another mutant will delete these (PEDV ΔORF3 152-170,PEDVΔORF3 1-40,PEDVΔORF3 152-170, PEDVΔORF3 182-224, andthe characteristic attenuation mutant of PEDV, PEDVΔORF3 81-100.Overexpression of ORF3 and deletion mutants in vitro to identify ORF3 colocalization and protein-protein interaction partners: First, I propose to examine the ORF3 protein for its subcellular localization and interaction with host protein(s) outside the context of virus infection. I will identify differences in cell death with trypan blue staining for cells with or without wt ORF3 expression and look for changes in vesicle formation and/or Golgi fragmentation (tracked by co-transfection with Golgi marker Gal-CFP) in the cells, as described for SARS-CoV 3A. I will also determine the colocalization of ORF3 with markers of the trans-Golgi network as a starting point to identify potential protein-protein interaction partners which can then be confirmed in vitro by co-immunoprecipitation. Lyso tracker red DND-99, a fluorophoric basic amine that selectively accumulates in acidic compartments of the lysosome or endosome, will allow for observation if ORF3 localizes within these structures. Additionally, we will examine infected cells for interaction between ORF3 mutants and the autophagosome.Infection of Vero cells with ORF3 Mutant PEDV Viruses: In parallel to the work proposed above, I will also examine the effects of ORF3 mutations in the context of PEDV infection in Vero cells. Wild-type and mutant viruses generated by targeted mutagenesiswill be used to infect Vero cells. Morphological changes in the Golgi, ER, and double membrane vesicle structures in the mutant viruses-infected cells different than those infected with wild-type PEDV-Colorado strain will be studied. Antibodies specific for double-stranded RNA will be used to determine the differences in PEDV replication efficiency in the absence of wt ORF3, which will be confirmed with qRT-PCR. Virion formation and release will be monitored by qRT-PCR in the cell culture media. Additionally, isolation of infectious virus will be achieved through gradient ultracentrifugation of infected cells. Virions will then be analyzed by western blot to determine if ORF3 is included within and, if so, if any of the ORF3 mutants are no longer incorporated.Determination of the role of ORF3 in PEDV pathogenicity: Using the ORF3-deletion PEDV mutant viruses as well as the ORF3 domain mutants that have been demonstrated to express mutant PEDVs (as opposed to those with rapid turnover or degradation of ORF3, which would presumably be indistinguishable from the ORF3 deletion mutant), I will perform comparative pathogenicity studies in piglets to determine the role of ORF3 in PEDV pathogenicity. A total of 42 piglets will be used to determine the effect of PEDV ORF3 deletion on disease manifestation in piglets. Newborn piglets that are free of PEDV infection will be obtained from the Virginia Tech Swine Center. Pigs will be randomly assorted into 3 groups of 14 pigs each and housed within a BSL-2 facility at the Virginia Tech BSL-2 Swine Research Facility (Building 450). Piglets at 3-day-old will be inoculated orally with media (as negative control), wild-type PEDV-Colorado strain (104.0 TCID50 per pig), and mutant PEDV with ORF3 deleted (PEDV ΔORF3) (104.0 TCID50 per pig). The PEDV-Colorado strain and the mutant PEDV ΔORF3 virus stocks will be propagated in Vero cells. The piglets will be observed over a 5-day period for severity of clinical symptoms. Diarrhea scores will be assessed visually and recorded daily by two separate evaluators with a score from 1 to 5 (1=normal, 2=moist feces, 3=mild diarrhea, 4=severe diarrhea, 5=watery diarrhea).Fecal and blood samples will be collected daily, and tested for PEDV viral RNA loads by a quantitative rt-PCR assay. Seven piglets from each group will be necropsied at 3 days post-inoculation, and the remaining 7 piglets will be necropsied at 5 days post-inoculation (Table 1). By performing a 2nd necropsy at 5 dpi, we will increase the likelihood of identifying the pathology when delayed disease progression may be most evident in the piglets infected with the PEDV ΔORF3. Macroscopic lesions in the intestines, lung, lymph nodes and a panel of other tissues will be recorded and scored. Microscopic lesions will be evaluated in a blind fashion by an experienced veterinary pathologist. Sections of small intestines and colon will be scored for the presence of lesions and inflammation. Sections of lymphoid node and lungs will be evaluated for the presence of lymphohistiocytic inflammation and scored from 0 (none) to 3 (severe). A PEDV immunohistochemistry assay (available at ISU VDL) will be utilized to quantify the amount of PEDV antigen in tissues for this study. Summary statistics for all quantitative assays will be calculated to assess the overall quality of the data.Table 1. Comparative pathogenicity study in newborn piglets between wild-type PEDV and its ORF3-deletion mutant PEDVGroupNumber of PigsDay 0Day 3Day 5Oral inoculationNecropsyNecropsy114Culture Medium77214PEDV Colorado strain77314PEDV ΔORF377Determination of the role of specific ORF3 domains in PEDV pathogenicity: Once we have determined that the PEDV ΔORF3 is attenuated in piglets, a further animal experiment will be performed to determine if any of the ORF3-specific domain mutants, PEDV ORF3 partial deletion mutants [PEDV ΔORF3 1-40, ΔORF3 81-100, ΔORF3 157-158, or ΔORF3 182-224)], possess a similar effect on virus pathogenicity compared to the full ORF3-deletion mutant.Sample collection and analyses will be identical to those performed in the above comparative pathogenicity study.Evaluation-Mutants generated from ORF3 will be evaluated by sequencing to ensure complete and accurate deletion of the entire ORF3 or partial deletion for later generated mutants.-Results from tissue culture/in vitro/animal infection studies will be evaluated by the lab group and the mentor, Dr. XJ Meng, with presentation at monthly laboratory meetings.-Results obtained from the study will be evaluated by peer reviewers during the publication submission process and by peers when presented at annual meetings.

Progress 12/15/15 to 12/14/18

Outputs
Target Audience: Nothing Reported Changes/Problems:The first step of this process required the generation of a chimeric PEDV virus with a large portion of the spike gene replaced with the analogous portion of the spike protein from Murine Hepatitisvirus, another coronavirus. As has been previously demonstrated in literature, this method can be used as a selection tool to generate viral mutants downstream. We confirmed by sequencing that, several times, we were able to successfully generate these murine PEDV (mPEDV) mutants. However, when we attempted to move them into Murine L Cells (which should support mPEDV replication) the virus was rapidly lost after 1 passage. This, unfortunately, prevented completion of the objective of creating viral knock-out mutants for ORF3, as we could not continue with the mutation process. Some independent research completed during the process cast doubt on the validity of continuing with this line of work. For one thing, sequencing results of our laboratory strains of PEDV demonstrated that, in all cases, the viruses had rapidly mutated ORF3 after a few passages to eliminate expression of the gene in tissue culture. As such, most of our lab strains already have mutated ORF3 and are fully capable of replicating in both tissue culture and in animals. Additionally, a personal communication with a collaborator who was following a similar line of inquiry with Chinese strains of PEDV had demonstrated the virus was fully capable of replicating and completing the full course of disease in animals with similar virulence and pathogenesis with a knocked out ORF3. As such, it was determined that this was no longer a fruitful line of inquiry, and it was decided to investigate other avenues. To continue our studies of ORF3 protein molecular function, we instead determined that the best move was to focus on understanding ORF3's function in cells.Towards this end, we attempted to create a lentiviral expression vector for ORF3 to create a stable expression vector in cells. This was initially hindered by difficulties in finding lab strains which had not already mutated during the course of adaptation to replication in cell culture. As one example, a strain of PEDV isolated from Minnesota by our laboratory developed mutations to ORF3 by 5 passages in cell culture. As such, the effect of ORF3 deletion on viral life cycle in vitro appeared to be beneficial or, perhaps, even necessary for viral success. This is supported by observation from literature that a number of attenuated PEDV strains used for vaccines also feature a deleted ORF3. To create our lentiviral expression vector, we instead created a synthetic full-length ORF3 cloned into the lentiviral vector plix402. We are currently in progress with efforts to transduce Vero cells with this lentiviral vector. The transduction has been completed and the cells are growing under Puromycin selection prior to clonal selection. Additionally, we cloned an expression vector for the viral membrane (M) protein and envelope protein (E), the two components required for construction of VLPs in cells. Based on our understanding of analogous coronavirus proteins, we predicted that ORF3 would rearrange cellular components and, in doing so, alter the localization and/or secretion of these proteins from cells. To begin working with this lentiviral vector prior to my resignation from Virginia Tech, I used transient transfection of this vector with Doxycycline to activate ORF3 expression. By immunofluorescence assay, I was able to show that T7-tagged ORF3 expression was occurring in the Vero cells. Additionally, by transiently transfecting the Vero cells, inducing ORF3-T7 expression by Doxycycline, and infecting the cells with an ORF3 deficient strain of PEDV isolated from Colorado, I was able to demonstrate that ORF3 expressed in trans interfered with virus production, reducing the pfu/mL of produced virus by one log compared to mock transfected cells. I am confident these results will be even more pronounced with the lentivirus transduced cells, once the experiment is repeated. What opportunities for training and professional development has the project provided?During the course of this work, I participated in the Network Learning Initiative, a program designed around training future educators in educational technology and tools for teaching and conducting research. I additionally made use of the Virginia Polytechnic Institute and State University Career Services Office to improve my application and interview skills for job hunting. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?After my resignation from the laboratory, a collaborator from Dr. Meng's laboratory will continue the experiments. The objective of this is to determine if ORF3 is secreted from cells when expressed, if its expression alters localization/expression/ and/or secretion of the envelope or membrane proteins from vero cells, and confirming that ORF3 disrupts virus success in tissue culture. If these results merit it, they will be present as a poster at the annual CRWAD meeting in Chicago.

Impacts
What was accomplished under these goals? Generate an ORF3 deletion mutant from a Colorado strain of PEDV from our laboratory. A great deal of effort was expended during the early portion of the project to utilize a targeted recombination method to generate an ORF3 deletion mutant of PEDV. The first step of this process required the generation of a chimeric PEDV virus with a large portion of the spike gene replaced with the analogous portion of the spike protein from Murine Hepatitisvirus, another coronavirus. As has been previously demonstrated in literature, this method can be used as a selection tool to generate viral mutants downstream. We confirmed by sequencing that, several times, we were able to successfully generate these murine PEDV (mPEDV) mutants. However, when we attempted to move them into Murine L Cells (which should support mPEDV replication) the virus was rapidly lost after 1 passage. Exhaustive efforts were used to optimize replication conditions to attempt to make the next stages of the study possible, but we were unsuccessful. This, unfortunately, prevented completion of the objective of creating viral knock-out mutants for ORF3, as we could not continue with the mutation process. Some independent research completed during the process cast doubt on the validity of continuing with this line of work. For one, sequencing results of our laboratory strains of PEDV demonstrated that, in all cases, the viruses had rapidly mutated ORF3 after a few passages to eliminate expression of the gene in tissue culture. As such, most of our lab strains already have mutated ORF3 and are fully capable of replicating in both tissue culture and in animals. Additionally, a personal communication with a collaborator who was following a similar line of inquiry with Chinese strains of PEDV had demonstrated the virus was fully capable of replicating and completing the full course of disease in animals with similar virulence and pathogenesis with a knocked out ORF3. As such, it was determined that this was no longer a fruitful line of inquiry, and it was decided to investigate other avenues. Utilize tissue culture techniques to observe the effect of ORF3 deletion on the viral life cycle in vitro. Towards our new focus of investigating the molecular mechanisms of ORF3 in tissue, we attempted to create a lentiviral expression vector for ORF3 to create a stable expression vector in cells. This was initially hindered by difficulties in finding lab strains which had not already mutated during the course of adaptation to replication in cell culture. As one example, a strain of PEDV isolated from Minnesota by our laboratory developed mutations to ORF3 by as early as 5 passages in cell culture. Any lab strains with intact ORF3 propagated to a much lower level, making amplifying cDNA of the ORF3 gene more difficult.As such, the effect of ORF3 deletion on viral life cycle in vitro appeared to be beneficial or, perhaps, even necessary for viral success. This is supported by observation from literature that a number of attenuated PEDV strains used for vaccines also feature a deleted ORF3. To create our lentiviral expression vector, we instead created a synthetic full-length ORF3 cloned into the lentiviral vector plix402. We are currently in progress with efforts to transduce Vero cells with this lentiviral vector. The transduction has been completed and the cells are growing under Puromycin selection prior to clonal selection. Additionally, we cloned an expression vector for the viral membrane (M) protein and envelope protein (E), the two components required for construction of VLPs in cells. Based on our understanding of analogous coronavirus proteins, we predicted that ORF3 would rearrange cellular components and, in doing so, alter the localization and/or secretion of these proteins from cells. To begin working with this lentiviral vector prior to my resignation from Virginia Tech, I used transient transfection of this vector with Doxycycline to activate ORF3 expression. By immunofluorescence assay, I was able to show that T7-tagged ORF3 expression was occurring in the Vero cells. Additionally, by transiently transfecting the Vero cells, inducing ORF3-T7 expression by Doxycycline, and infecting the cells with an ORF3 deficient strain of PEDV isolated from Colorado, I was able to demonstrate that ORF3 expressed in trans interfered with virus production, reducing the pfu/mL of produced virus by one log compared to mock transfected cells. I am confident these results will be even more pronounced with the lentivirus transduced cells, once the experiment is repeated. Determine the effect of ORF3 deletion on virulence and pathogenesis in piglets As stated, personal communication with a colleague in China demonstrated that ORF3 had no effect on virulence or pathogenesis in piglets. 4) Generate ORF3 mutants with deletion of domains predicted to be important including: Amino acids 1-40, 81-100, 152-170, and 182-224. 5) Observe effects of ORF3 targeted deletion in tissue culture compared to deletion of the full ORF 6) Observe reduced pathogenesis and virulence of PEDV ORF3 targeted mutants as compared to full length deletion mutant Since we have only just begun working with full length ORF3, we have not attempted performing site-directed mutagenesis of our ORF3 expression vectors to identify critical domains for protein function. If results of future experiments indicate this is warranted, we will investigate it as a possibility.

Publications


    Progress 12/15/16 to 12/14/17

    Outputs
    Target Audience: Nothing Reported Changes/Problems:As stated, our initial method of inducing mutation into PEDV virions through targetted recombination has met with significant difficulty. Though our rt-PCR assays suggest that we have generated recombinant virus during multiple attempts to create the chimeric PEDV capable of infecting murine L cells, this recombinant virus does not seem to efficiently infect these cells or propogate to a large enough degree to allow for the next step of the process, reverting the virus to PEDV's tissue tropism while introducing Orf3 deletion. The reason for this is unclear, other than the likelihood that the resultant chimeras are simply ineficient/deficient at replicating in L cells. Additionally, as stated, one of our collaborators has communicated results with us from his own efforts to mutate Orf3 in Chinese strains of the virus, determining Orf3 knock-out PEDV to still be virulent and capble of inducing full disease in animals. As our initial intent was to explore the possible use of Orf3 knock out as a vaccine candidate, this suggested to us that we focus our efforts on better characterizing the functional role of this protein by focusing on the in vitro methods and determining the role of Orf3 in the viral lifecycle. If this can be determined, it may offer insights into potential therapeutic or preventative methods to protect swine herds from PEDV infection. Additionally, the Orf3 accessory protein is present in a number of Cornaviruses that cause human disease, including SARS. Better understanding of this protein's function could also yield to insights in the molecular mechanisms of these viruses as well. What opportunities for training and professional development has the project provided?During the previous year, I participated in the Network Learning Initiative program at Virginia Tech, as well as training offered from the Office of the Vice President of Research. The courses are listed below. OVPRI Data Management Planning for Research Grants 1/12th Budgeting for Grants and Contracts 11/29th Successful Grant Writing 11/29th NLI EFAR: Introduction to Virginia tech's Electronic Faculty Activity Reporting System Managing References for Endnote, January 30th 10AM Project Sites: What Tool is Right for Me?, January 31st 1:30PM Before you sign your next publication agreement: An author rights discussion, February 1, 12:20PM Research Data Management Planning and Practices: February 2, 12PM Making Visible the Invisible: Data Visualization and Poster Design. 9 a.m Feb. 21 Mindfulness in Academia. 10:30 a.m Feb. 23 Google Apps as a Project Site, Feb 23rd, 2:30PM Introducing CollabVT, a Research Profile system for Virginia Tech. 10 a.m. March 02 Google Sites as a Project Site, March 28th 12PM NLI Canvas Self-Paced Workshops How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?PEDV Orf3 is an ion channel protein and regulates viral production. Porcine epidemic diarrhea virus Orf3 prolongs S-phase and facilitates formation of vesicles. Expression of Orf3 interferes with recovery of PEDV virus in cell culture through reverse genetic systems, while truncating it in a manner similar to that observed in attenuated strands reverses this effect. More specifically, point mutations to disrupt one of these trafficking motifs at amino acid position 81 has been shown as being critical to restoration of virus rescue in reverse genetic systems. Tellingly, all of our laboratory strains of tissue-culture adapted PEDV have mutated to introduce stop codons which truncate the protein at or around amino acid 80, just before this key tracking motif. This includes our lab isolated Minnesota strain, which we've found to possess truncated Orf3 after only 5 passages in Vero cells. One possible explanation of this is the fact that, in animals, PEDV spreads primarily by basolateral spread through intestinal cells, while in tissue culture this is replaced with a mechanism primarily reliant on lysis and spread through trypsin-treated media. Mutation of Orf3 may be a critical step in this adaptation. For Human Cytomegalovirus, the tegument protein pUL-71 contains a number of Yxx? trafficking motifs that are required for the protein to perform its function of efficient envelope formation. Mutation of the tyrosine or the entire motif results in accumulation of the protein at the plasma membrane and through the cytoplasm and results in reduced viral yields in supernatants and clusters of maturing capsids accumulating around viral assembly complexes. It thus appears to be a critical factor in efficient secondary endocytosis for this virus. Yxx? motifs have previously been shown to be involved in normal cellular processes such as receptor internalization from the plasma membrane, protein targeting to lysosomes, endosomal compartments, the basolateral surface of polarized cells, and the trans-Golgi network.SARS Orf3a protein, a homolog of PEDV Orf3,similarly contains Yxx? motifs and is trafficked to the cell membrane before undergoing endocytosis. PEDV Orf3 has a number of putative Yxx? motifs, including one that begins at AA 81, which we previously stated is important for inhibiting viral replication in tissue culture. We propose that these trafficking motifs, particularly the one at amino acid 80, play a role in secondary envelopment and basolateral release of the virus. Truncation of the protein changes this mechanism, potentially altering the packaging mechanisms and/or viral release. We have begun investigating this by cloning truncated and full-length synthetic Orf3 sequence into pLix402, a retroviral expression vector with doxycycline induced expression. Using this, we can induce constitutive, temporary Orf3 expression in cells transfected with our lab strains of virus or the two factors required for virus-like particle formation, PEDV proteins Envelope (E) and Membrane (M), tagged with flag epitopes. We can then use budding assays and immunofluorescence assays to track packaging, transport, and budding of the virus-like particles within infected/transfected cells. Further, we can test the effect expression of full length Orf3 in trans has on virus production of our laboratory strains. Once a system is completed to examine the effect or absence of functional, full length Orf3 on VLP formation and envelopment, we can go on to generate mutants to explore the mechanisms in closer detail. We have already cloned and generated cell-lines expressing the truncated Orf3 from Colorado PEDV. The full-length synthetic PEDV Orf3 can then be used to perform site-directed mutagenesis to try and identify the specific residues responsible for Orf3 protein function. The Tyrosine residue at amino acid 80 is one obvious target, to disrupt what appears to be an important trafficking motif. Additionaly, there are several other Yxx? motifs in the 3' end of the protein that can be investigated as well. Also, SARS Orf3a protein interacts with caveolin(20), an important factor in receptor independent endocytosis. By searching for similar motifs, a pair of putative caveolin motifs is present in PEDV Orf3 at AA 55-60 and 185-190. These also make interesting targets for investigation.

    Impacts
    What was accomplished under these goals? Over the course of this year's work, we have successfully performed targeted recombination to create chimeric virus to which contains the PEDV structural proteins and the MHV spike protein, the first steps to creating a recombinant for use in rapid and efficient mutagenesis of the PEDV virus. This was to be used for deletion of the ORF3 to investigate its use as a vaccine candidate. However, the subsequent chimeric virus, despite being positive by PCR tests, does not propagate well, spreads slowly, and cannot be recovered after a passage in tissue culture. Further, we have been made aware by a collaborator that PEDV with deleted ORF3 is still virulent and initiates the full virulent disease in pigs. Therefore, we have determined to use the rest of the allocated time on this project to focus on Specific Aim 2 of the grant: Determine the Effort of ORF3 mutations/deletions on virus life cycle in vitro. Towards this end, we have clones a truncated Orf3 from our lab tissue-culture adapted strains of PEDV into a retroviral vector, pLix402, and have used it to create a stable cell-line with tetracycline controlled expression of truncated Orf3. We have also had a full-length, non-truncated Orf3 synthesized by Genscript and have cloned it into pLix402 as well. We have developed a simple budding assay to track viral release, and have optimized assays to determine virus production by measuring TCID50 of supernatant collected from infected plates.

    Publications


      Progress 12/15/15 to 12/14/16

      Outputs
      Target Audience: Nothing Reported Changes/Problems: Recombinant viruses formed thus far from this process, if present, do not appear to replicate efficiently. While these virions, if used to infect L cells after passaging, have some weak expression of MHV spike visible by IFA, no obvious CPE or halting of cell growth is visible in infected plates. Results of mPEDV RNA by rt-PCR assay have also been inconsistent. We will attempt to address these difficulties using the methods mentioned above. In addition, we have obtained an infectious clone based on Chinese strains of PEDV inside a Bacterial Artificial Chromosome (BAC) vector. If the targeted recombination method continues to fail, we can introduce the desired mutations into this vector directly using methods such as CRISPR based Targeted Mutagenesis and site-directed mutagenesis. We believe this should at least be sufficient to knock out ORF3 expression in this clone within the next year and test for any reduced virulence in the resultant virus. In tandem with these efforts, we are working to generate a PEDV virus line which is adapted to cell culture rather than infecting the host. In order to replicate efficiently in VERO cells, virus must be initially incubated with Trypsin to induce proteolytic cleavage of the Spike protein, and a concentration of Trypsin must be maintained in the cell's media. However, this method is not practical for the Murine L cells required for mPEDV replication, as these cells do not tolerate Trypsin in their media. The initial experiments demonstrating the efficacy of the initially proposed targeted recombination method used a line of PEDV adapted to no longer need this Trypsin. We are, therefore, passaging the Colorado strain of PEDV with gradually reduced amounts of Trypsin in order to select for adaptation away from this Trypsin requirement. This method has been used previously with Asian strains of PEDV to generate commercially available PEDV vaccines. When successful, we can use this resultant strain for the targeted recombination and, potentially, increase recombination efficiency and improve the growth of mPEDV virus in L cells. Additionally, this work could potentially result in a publication and/or viable vaccine candidate in and of itself by attenuating the virulence of the Colorado PEDV strain. Once passaged a sufficient number of times, we intend to sequence the viral genome to identify mutations acquired during the attenuation process. What opportunities for training and professional development has the project provided? Dr. Rogers has taken advantage of the Office of the Vice President of Research's Professional Development program, attending seminars on Grant Budget and Contract Preparation, Successful Grant Writing, and is enrolled in courses for Data Management, Building the NIH Grant, Finding Funding, and using Grants.gov. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?In order to progress to the next steps, mutagenesis of the virus itself, an efficiently replicating mPEDV virus will be required. Whether this can be achieved by passing the already generated mPEDV lines in L cells to allow them to adapt (which carries its own risks, as further mutagenesis could result in inefficient recombination with mutated PEDV for downstream applications) or further attempts to improve the efficiency of mutagenesis and obtain more efficient replicators, further efforts will be required before we can move on to the phase wherein mutations will be introduced directly into the PEDV genome for downstream testing. We intend to continue with the targeted recombination method by repeating the initial recombination with larger amounts of mPEDV RNA and continuing the experiment until an efficiently replicating mPEDV clone is obtained. From there, we will continue with the 2nd round of targeted recombination to rescue the PEDV spike and introduce deletion of ORF3. We anticipate this will be a quicker process, as we will have optimized the Targetted Recombination method in the first round of mutation. In addition, during the next year, Dr. Rogers will participate in the CIDER program during the Spring semester of Virginia Tech. This will offer additional professional development on grant management, instruction of students, and operating a laboratory. If possible, it is still Dr. Meng's intention to allow Dr. Rogers to mentor an undergraduate student in the laboratory as well.

      Impacts
      What was accomplished under these goals? The efforts to construct a hybrid coronavirus with murine hepatitis virus (MHV) spike protein inserted in place of the Porcine Epidemic Diarrhea Virus spike are progressing. We have ordered and received the T7 based expression vector described in the initial grant application from Genscript Incorporated. Using the Ribomax Large Scale RNA Production System-T7 we have produced capped and poly-adenylated murine PEDV RNA for recombination (from here on referred to as mPEDV RNA) synthetically and transfected them into PEDV infected VERO cells, allowing for recombination to occur. These are then overlaid onto murine L cells and allowed to grow for four days. We are in the process of verifying whether these recombinants contain the correct sequences and performing plaque purification to have a clonal line of PEDV for the next steps of the experiment.

      Publications