Identification of four insertion sites for foreign genes in a pseudorabies virus vector

Abstract Background Pseudorabies virus (PRV) is a preferred vector for recombinant vaccine construction. Previously, we generated a TK&gE-deleted PRV (PRVΔTK&gE−AH02) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4 ~ 5 week-old PRV antibody negative piglets and provide rapid and 100 % protection in weaned pigs against lethal challenge with the PRV variant strain. It suggests that PRVTK&gE−AH02 may be a promising live vaccine vector for construction of recombinant vaccine in pigs. However, insertion site, as a main factor, may affect foreign gene expression. Results In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same spike (S) expression cassette of a variant porcine epidemic diarrhea virus strain in different noncoding regions (UL11-10, UL35-36, UL46-27 or US2-1) from AH02LA BAC with TK, gE and gI deletion. The successful expression of S gene (UL11-10, UL35-36 and UL46-27) in recombinant viruses was confirmed by virus rescue, PCR, real-time PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in swine testicular cells infected with PRV-S(UL11-10)ΔTK/gE and PRV-S(UL35-36)ΔTK/gE compared to that of PRV-S(UL46-27)ΔTK/gE at 6 h post infection (P < 0.05). Moreover, at 12 h post infection, cells infected with PRV-S(UL11-10)ΔTK/gE exhibited higher S gene mRNA expression than those infected with PRV-S(UL35-36)ΔTK/gE (P = 0.097) and PRV-S(UL46-27)ΔTK/gE (P < 0.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRVΔTK&gE−AH02). Conclusions This study focuses on identification of suitable sites for insertion of foreign genes in PRV genome, which laids a foundation for future development of recombinant PRV vaccines.

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Identification of the Suitable Insertion Site for Expression of Spike Gene of A Porcine Epidemic Diarrhea Virus Variant in A Pseudorabies Virus Vector

10.21203/rs.3.rs-124699/v1 ◽

2020 ◽

Author(s):

Chuanjian Zhang ◽

Shiqi Guo ◽

Rongli Guo ◽

Saisai Chen ◽

Yating Zheng ◽

...

Keyword(s):

Mrna Expression ◽

Post Infection ◽

Porcine Epidemic Diarrhea Virus ◽

Pseudorabies Virus ◽

Insertion Site ◽

Diarrhea Virus ◽

Artificial Chromosomes ◽

S Gene ◽

Porcine Epidemic Diarrhea ◽

Gene Mrna

Abstract Background: The emergence of variant porcine epidemic diarrhea virus (PEDV) strain and pseudorabies virus (PRV) in China in recent years has decreased the effectiveness of CV777 and Bartha K61 vaccines, causing significant loss to the swine industry. Previously, we generated a TK&gE-deleted PRV (PRVTK&gE-AH02) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4～5 week-old PRV antibody negative piglets and provide rapid and complete protection in weaned pigs against lethal challenge with the PRV variant strain. PRVTK&gE-AH02 may be a promising live vaccine vector for construction of recombinant vaccine in pigs. PEDV spike (S) protein is mainly used in the development of PEDV vaccines. Therefore, the gene-deleted PRV (from PRV variants) vectored vaccine expressing variant PEDV S gene may be viable PEDV and PRV vaccine candidates. However, insertion site is an important factor affecting foreign gene expression and vaccine efficacy. Results: In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same S expression cassette in different noncoding regions (UL11-10, UL35-36, UL46-27 or US2-1) from AH02LA BAC with TK, gE and gI deletion. The successful expression of S gene (UL11-10, UL35-36 and UL46-27) in recombinant viruses was confirmed by virus rescue, PCR, RT-PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in Swine testicular cells infected with PRV-S(UL11-10)ΔTK/gE and PRV-S (UL35-36)ΔTK/gE compared to that of PRV-S(UL46-27)ΔTK/gE at 6 hour post infection (P < 0.05). Moreover, at 12 hour post infection, cells infected with PRV-S (UL11-10)ΔTK/gE exhibited higher S gene mRNA expression than those infected with PRV-S (UL35-36)ΔTK/gE (P = 0.097) and PRV-S (UL46-27)ΔTK/gE (P < 0.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRVTK&gE-AH02).Conclusions: The identification and comparison of the insertion sites in PRV genome laids a foundation for future development of recombinant PRV vaccines.

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Natural Bovine Coronavirus Infection in a Calf Persistently Infected with Bovine Viral Diarrhea Virus: Viral Shedding, Immunological Features and S Gene Variations

Animals ◽

10.3390/ani11123350 ◽

2021 ◽

Vol 11 (12) ◽

pp. 3350

Author(s):

Annamaria Pratelli ◽

Maria Stella Lucente ◽

Marco Cordisco ◽

Stefano Ciccarelli ◽

Roberta Di Fonte ◽

...

Keyword(s):

Bovine Viral Diarrhea Virus ◽

Post Infection ◽

Fecal Excretion ◽

Bovine Viral Diarrhea ◽

Bovine Coronavirus ◽

Persistently Infected ◽

Diarrhea Virus ◽

Viral Diarrhea ◽

S Gene ◽

Viral Diarrhea Virus

The evolution of a bovine coronavirus (BCoV) natural infection in a calf persistently infected with bovine viral diarrhea virus (BVDV) was described. The infected calf developed intermittent nasal discharge, diarrhea and hyperthermia. The total number of leukocytes/mL and the absolute differential number of neutrophils and lymphocytes resulted within the normal range, but monocytes increased at T28 (time 28 post-infection). Flow-cytometry analysis evidenced that the CD8+ subpopulation increased at T7 and between T28 and T35. BCoV shedding in nasal discharges and feces was detected up to three weeks post infection and high antibody titers persisted up to T56. The RNA BCoV load increased until T14, contrary to what was observed in a previous study where the fecal excretion of BCoV was significantly lower in the co-infected (BCoV/BVDV) calves than in the calves infected with BCoV only. We can suppose that BVDV may have modulated the BCoV infection exacerbating the long viral excretion, as well as favoring the onset of mutations in the genome of BCoV detected in fecal samples at T21. An extensive study was performed to verify if the selective pressure in the S gene could be a natural mode of variation of BCoV, providing data for the identification of new epidemic strains, genotypes or recombinant betacoronaviruses.

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Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

Microorganisms ◽

10.3390/microorganisms9051005 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1005

Author(s):

Olga Chervyakova ◽

Elmira Tailakova ◽

Nurlan Kozhabergenov ◽

Sandugash Sadikaliyeva ◽

Kulyaisan Sultankulova ◽

...

Keyword(s):

Fluorescent Protein ◽

Viral Vector ◽

Foot And Mouth Disease ◽

Open Reading Frames ◽

Foreign Gene ◽

Mouth Disease Virus ◽

Foreign Genes ◽

Green Fluorescent ◽

Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

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Epidemiological investigation of porcine circovirus type 2 and its coinfection rate in Shandong province in China from 2015 to 2018

BMC Veterinary Research ◽

10.1186/s12917-020-02718-4 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Zicheng Ma ◽

Mengda Liu ◽

Zhaohu Liu ◽

Fanliang Meng ◽

Hongyu Wang ◽

...

Keyword(s):

Pseudorabies Virus ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Shandong Province ◽

Porcine Circovirus ◽

Respiratory Syndrome Virus ◽

Limited Information ◽

Serum Samples ◽

Diarrhea Virus

Abstract Background Porcine circovirus type 2 (PCV2) is one of the crucial swine viral pathogens, caused porcine circovirus associated diseases (PCVAD). Shandong province is one of the most important pork producing areas and bears a considerable economic loss due to PCVAD. However, there is limited information on epidemiology and coinfection rate of PCV2 with other critical swine diseases in this area, such as porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Pseudorabies virus (PRV), and porcine epidemic diarrhea virus (PEDV). Results Overall, 89.59% serum samples and 36.98% tissue samples were positive for PCV2 specified ELISA and PCR positive for PCV2, respectively. The coinfection rates of PCV2 with PRRSV, PRV, CSFV, and PEDV were 26.73%, 18.37%, 13.06%, and 3.47%, respectively. Moreover, genetic characteristic of PCV2 were analyzed based on the cap genes showing that PCV2d is the dominant sub-genotype circulating in the province. Conclusions Our findings reveal that PCV2d, as the dominant strain, is prevailing in pig farms in Shandong province at high levels. There was a high frequency of coinfection of PCV2 and PRRSV.

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TACROLIMUS DECREASES, CYCLOSPORINE INCREASES, AND RAPAMYCIN HAS NO EFFECT ON PROFIBROTIC GENE MRNA EXPRESSION IN RENAL TISSUE

Transplantation Journal ◽

10.1097/00007890-200407271-01684 ◽

2004 ◽

Vol 78 ◽

pp. 625

Author(s):

G R Bicknell ◽

N R Brook ◽

J R Waller ◽

M L Nicholson

Keyword(s):

Mrna Expression ◽

Renal Tissue ◽

Gene Mrna

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Novel Porcine Epidemic Diarrhea Virus (PEDV) Variants with Large Deletions in the Spike (S) Gene Coexist with PEDV Strains Possessing an Intact S Gene in Domestic Pigs in Japan: A New Disease Situation

PLoS ONE ◽

10.1371/journal.pone.0170126 ◽

2017 ◽

Vol 12 (1) ◽

pp. e0170126 ◽

Cited By ~ 31

Author(s):

Nguyen Van Diep ◽

Junzo Norimine ◽

Masuo Sueyoshi ◽

Nguyen Thi Lan ◽

Ryoji Yamaguchi

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

New Disease ◽

Domestic Pigs ◽

Diarrhea Virus ◽

S Gene ◽

Large Deletions ◽

Porcine Epidemic Diarrhea

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Coronaviruses as Vectors: Stability of Foreign Gene Expression

Journal of Virology ◽

10.1128/jvi.79.20.12742-12751.2005 ◽

2005 ◽

Vol 79 (20) ◽

pp. 12742-12751 ◽

Cited By ~ 28

Author(s):

Cornelis A. M. de Haan ◽

Bert Jan Haijema ◽

David Boss ◽

Frank W. H. Heuts ◽

Peter J. M. Rottier

Keyword(s):

Genetic Stability ◽

Vaccine Development ◽

Hepatitis Virus ◽

Firefly Luciferase ◽

Foreign Gene ◽

Rna Recombination ◽

Large Deletions ◽

Group 2 ◽

Attenuated Viruses ◽

Foreign Genes

ABSTRACT Coronaviruses are enveloped, positive-stranded RNA viruses considered to be promising vectors for vaccine development, as (i) genes can be deleted, resulting in attenuated viruses; (ii) their tropism can be modified by manipulation of their spike protein; and (iii) heterologous genes can be expressed by simply inserting them with appropriate coronaviral transcription signals into the genome. For any live vector, genetic stability is an essential requirement. However, little is known about the genetic stability of recombinant coronaviruses expressing foreign genes. In this study, the Renilla and the firefly luciferase genes were systematically analyzed for their stability after insertion at various genomic positions in the group 1 coronavirus feline infectious peritonitis virus and in the group 2 coronavirus mouse hepatitis virus. It appeared that the two genes exhibit intrinsic differences, the Renilla gene consistently being maintained more stably than the firefly gene. This difference was not caused by genome size restrictions, by different effects of the encoded proteins, or by different consequences of the synthesis of the additional subgenomic mRNAs. The loss of expression of the firefly luciferase was found to result from various, often large deletions of the gene, probably due to RNA recombination. The extent of this process appeared to depend strongly on the coronaviral genomic background, the luciferase gene being much more stable in the feline than in the mouse coronavirus genome. It also depended significantly on the particular genomic location at which the gene was inserted. The data indicate that foreign sequences are more stably maintained when replacing nonessential coronaviral genes.

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Bacterial Lipopolysaccharide Induced Alterations of Genome-Wide DNA Methylation and Promoter Methylation of Lactation-Related Genes in Bovine Mammary Epithelial Cells

Toxins ◽

10.3390/toxins11050298 ◽

2019 ◽

Vol 11 (5) ◽

pp. 298 ◽

Cited By ~ 9

Author(s):

Jingbo Chen ◽

Yongjiang Wu ◽

Yawang Sun ◽

Xianwen Dong ◽

Zili Wang ◽

...

Keyword(s):

Dna Methylation ◽

Immune Response ◽

Mrna Expression ◽

Promoter Methylation ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Bovine Mammary Epithelial Cells ◽

Genome Wide ◽

Immune Response Pathway ◽

Gene Mrna

Bacterial lipopolysaccharide (LPS) could result in poor lactation performance in dairy cows. High methylation of DNA is associated with gene repression. However, it is unclear whether LPS could suppress the expression of lactation-related genes by inducing DNA methylation. Therefore, the objective of this study was to investigate the impact of LPS on genome-wide DNA methylation, using methylated DNA immunoprecipitation with high-throughput sequencing (MeDIP-seq) and on the promoter methylation of lactation-related genes using MassArray analysis in bovine mammary epithelial cells. The bovine mammary epithelial cell line MAC-T cells were treated for 48 h with LPS at different doses of 0, 1, 10, 100, and 1000 endotoxin units (EU)/mL (1 EU = 0.1 ng). The results showed that the genomic methylation levels and the number of methylated genes in the genome as well as the promoter methylation levels of milk genes increased when the LPS dose was raised from 0 to 10 EU/mL, but decreased after further increasing the LPS dose. The milk gene mRNA expression levels of the 10 EU/mL LPS treatment were significantly lower than these of untreated cells. The results also showed that the number of hypermethylated genes was greater than that of hypomethylated genes in lipid and amino acid metabolic pathways following 1 and 10 EU/mL LPS treatments as compared with control. By contrast, in the immune response pathway the number of hypomethylated genes increased with increasing LPS doses. The results indicate LPS at lower doses induced hypermethylation of the genome and promoters of lactation-related genes, affecting milk gene mRNA expression. However, LPS at higher doses induced hypomethylation of genes involved in the immune response pathway probably in favor of immune responses.

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In Vivo Dynamics of Reporter Flaviviridae Viruses

Journal of Virology ◽

10.1128/jvi.01191-19 ◽

2019 ◽

Vol 93 (22) ◽

Cited By ~ 8

Author(s):

Tomokazu Tamura ◽

Manabu Igarashi ◽

Bazarragchaa Enkhbold ◽

Tatsuya Suzuki ◽

Masatoshi Okamatsu ◽

...

Keyword(s):

Luciferase Activity ◽

Viral Proteins ◽

Solvent Accessible Surface Area ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Recombinant Viruses ◽

Diarrhea Virus ◽

Foreign Genes

ABSTRACT Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo. Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro. The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals. IMPORTANCE In vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo. Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.

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