Construction of pseudorabies virus variant attenuated vaccine: codon deoptimization of US3 and UL56 genes based on PRV gE/TK deletion strain.

Since 2011, pseudorabies based on the pseudorabies virus (PRV) variant has emerged as a serious health issue in pig farms in China. The PRV gE/TK or gE/gI/TK deletion strains protect against emerging PRV variants. However, these variants may cause lethal infections in newborn piglets without PRV antibodies. Previous studies have shown that codon deoptimization of a virulence gene causes virus attenuation. Accordingly, we deoptimized US3-S (US3 gene encoding a short isoform that represents approximately 95% of the total US3 transcription) and UL56 genes (first 10 or all codons) of PRV gE/TK deletion strain (PRVΔTK&gE-AH02) to generate six recombinant PRVs through bacterial artificial chromosome technology. In swine testicular cells, recombinant PRVs with all codon deoptimization of US3-S or UL56 genes were grown to lower titers than the parental virus. Notably, US3-S or UL56 with all codon deoptimization reduced mRNA and protein expressions. Subsequently, the safety and immunogenicity of recombinant PRVs with codon deoptimization of US3-S or UL56 are evaluated as vaccine candidates in mice and piglets. The mice inoculated with recombinant PRVs with codon deoptimization of US3-S or UL56 showed exceptional survival ability without severe clinical signs. All codons deoptimized (US3-S and UL56) significantly decreased virus load and attenuated pathological changes in the brains of the mice. Moreover, the protection efficiency offered by recombinant PRVs with codon deoptimization of US3-S or UL56 showed similar effects to PRVΔTK&gE-AH02. Remarkably, the 1-day-old PRV antibody-negative piglets inoculated with PRVΔTK&gE-US3-ST-CD (a recombinant PRV with all codon deoptimization of US3-S) presented no abnormal clinical symptoms, including fever. The piglets inoculated with PRVΔTK&gE-US3-ST-CD showed a high serum neutralization index against the PRV variant. In conclusion, these results suggest using codon deoptimization to generate innovative live attenuated PRV vaccine candidates.

Porcine epidemic diarrhea virus 3CLpro causes apoptosis and collapse of mitochondrial membrane potential requiring its protease activity and signaling through MAVS.

Porcine epidemic diarrhea (PED) is a highly contagious and virulent intestinal infectious disease characterized by diarrhea, vomiting and dehydration. Although PEDV-induced apoptosis has been characterized in vitro and vivo, the functional proteins related to this event and the mechanism still need further research. Here, we firstly demonstrated that PEDV epidemic strain JS2013 could trigger apoptosis in a dose- and time-dependent manner. Then, PEDV 3CLpro was further identified as a crucial inducer of PEDV-triggered apoptosis. In addition, using site-directed mutagenesis to disrupt the protease activity of 3CLpro by His41 and Cys144 mutations, we found that 3CLpro-induced apoptosis and mitochondrial damage significantly reduced, suggesting that the protease activity of 3CLpro was essential for apoptosis and mitochondrial damage. Furthermore, PEDV 3CLpro could synergistically promote MAVS-mediated apoptosis and MAVS was involved in the signaling pathway of 3CLpro-induced apoptosis, but no direct interaction between PEDV 3CLpro and MAVS was detected by immunoprecipitation assays. Our findings provide important insights into the role of 3CLpro in the pathogenicity of PEDV.

NM23 Is a CP-Binding Protein Involved in Infectious Hypodermal and Hematopoietic Necrosis Virus Infection in Shrimp.

The aim of this study was to identify the putative host cell receptor for Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) CP in the gill membrane of L. vannamei. Putative CP binding partners were screened first using a 2-dimensional Virus Overlay Protein Blot Assay (VOPBA) to probe isolated gill membrane proteins using recombinant CP. Putative binding partners were identified using mass spectrometry. A Phage Display Random Dodecapeptide Library was used to screen for dodecapeptides and motifs that bound to CP. Finally, putative binding pairs were confirmed using GST(glutathione-S-transferase) pulldown assays. 2-Dimensional VOPBA identified NM23 as a putative binding partner for IHHNV CP. GST pulldown experiments confirmed the direct interaction of NM23 and IHHNV CP. The phage display library was used to identify six groups of dodecapeptides that bound to CP. From these peptides, three characteristic binding motifs were identified, SW*Y, SKWV, and PQR. Interestingly, the SW*Y motif was also found in NM23. We are the first to implicate NM23 in IHHNV infection and postulate that it may bind to the CP using the SW*Y motif, although this remains to be confirmed.

Evaluation of the transcriptional regulatory efficacy of transcription regulatory sequences of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteropathogenic coronavirus causing severe watery diarrhea and high mortality in piglets. In order to investigate the role of the transcription regulatory sequences (TRSs) in regulation of gene expression and replication of PEDV, the enhanced green fluorescent protein (EGFP) gene, under control of different TRSs of PEDV, were inserted between the N gene and 3' UTR of the PEDV genome using a reverse genetic system. The EGFP expression from different chimeric PEDVs was analyzed for each TRS. TRSs of all the structural and accessory protein genes of PEDV positively regulate EGFP expression at different levels, and the TRS of M protein gene produced the highest level of EGFP. Moreover, this is the first study to show that exogenous gene could be inserted between N gene and 3' UTR of PEDV, and the EGFP insertion had no effect on PEDV replication. Taken together, our study enriched the information of PEDV TRSs on gene expression and replication of PEDV.

Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum.

The widespread avian pathogen Mycoplasma gallisepticum is a causative agent of respiratory disease. The wall-less prokaryotes lack some tricarboxylic acid cycle enzymes, therefore, the glycolysis metabolic pathway is of great importance to these organisms. Pyruvate kinase (PK) is one of the key enzymes of the glycolytic pathway, and its immunological characteristics in Mycoplasma are not well known. In this study, the M. gallisepticum pyruvate kinase fusion protein (PykF) was expressed in a pET system. The full-length of the gene was subcloned into the expression vector pET28a(+) to construct the pET28a-rMGPykF plasmid, which was then transformed into Escherichia coli strain BL21 (DE3) cells. The expression of the 62 kDa recombinant protein of rMGPykF in E. coli strain BL21 (DE3) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGPykF exhibited PK catalytic activity, which could reflect the conversion of NADH to NAD(+). Mouse anti-PykF antibodies were generated by immunization of mice with rMGPykF. Immunoblot and immunoelectron microscopy assays identified PykF as an immunogenic protein expressed on the surface of M. gallisepticum cells. Bactericidal assay showed that anti-rMGPykF antiserum killed 70.55% of M. gallisepticum cells, suggesting the protective potential of PykF. Adherence inhibition assay on immortalized chicken fibroblasts (DF-1) cells revealed more than 39.31% inhibition of adhesion in the presence of anti-rMGPykF antiserum, suggesting that PykF of M. gallisepticum participates in bacterial adhesion to DF-1 cells.

Development of a loop-mediated isothermal amplification targeting a gene within the pyruvate dehydrogenase complex, the pdhA gene, for rapid detection of Mycoplasma gallisepticum.

Mycoplasma gallisepticum infections impose a significant economic burden on the poultry industry. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed and optimized to detect M. gallisepticum based on a gene within the pyruvate dehydrogenase complex, the pdhA gene, which codes for the major subunit (E1α) in the complex. The reaction conditions were optimized, and the specificity was confirmed by successful amplification of several M. gallisepticum strains, while no amplification was detected with 20 other major bacterial and viral pathogens of poultry. Additionally, the LAMP assay achieved 10-fold higher sensitivity than an existing polymerase chain reaction (PCR) method. The LAMP assay was applied to swab samples collected from poultry farms and compared with PCR. The positive detection rate was 20.2% (37/183) by LAMP and 13.1% (24/183) by PCR. The LAMP assay could provide a cost-effective, quick, and sensitive method for the detection of M. gallisepticum.

Toxinotyping of Clostridium perfringens fecal isolates of reintroduced Père David's deer (Elaphurus davidianus) in China.

Clostridium perfringens is an important pathogen causing sudden death syndrome, necrotic enteritis, and gas gangrene in ruminants, especially some deer species. Père David's deer (Elaphurus davidianus) is one of the world's rare species and is an endangered and protected species in China. Some Père David's deer in the Chinese Shishou Père David's Deer Preserve died due to C. perfringens infection. We investigated the toxin types and C. perfringens enterotoxin-positive (cpe(+)) strains of isolated C. perfringens in Père David's deer in China. We collected 155 fecal samples from the Beijing Nanhaizi Père David's Deer Park and the Jiangsu Dafeng Père David's Deer National Nature Reserve between July 2010 and July 2011. Bacteria isolated using blood agar and mannitol agar plates were identified by Gram staining and nested PCR for 16S rRNA. We isolated C. perfringens from 41 fecal samples and used PCR amplification of five toxin genes to identify the toxinotypes and the cpe(+) strains of C. perfringens. Twenty-one isolates were type A, 15 were type E, and five were type D. Fifteen isolates were cpe(+) strains, including eight that were type A and seven that were type E.

The genome sequence of a novel simian adenovirus in a chimpanzee reveals a close relationship to human adenoviruses.

Recently, several reports have revealed that some simian adenoviruses (AdVs) strains show a close relationship to human AdVs. In the present study, a simian AdV strain named SAdV-ch1 was detected in chimpanzees in China, and its complete genome was determined. Phylogenetic analysis revealed SAdV-ch1 clustering in a clade that was separate from all of the other simian AdVs but genetically close to a human AdV strain, HAdV-18 (GenBank no. GU191019), sharing 92.5 % sequence identity with it. Recombination analysis provided evidence that a recombination event had occurred between SAdV-ch1 and HAdV-61 (JF964962), where SAdV-ch1 exchanged partial of its hexon gene segment with HAdV-61, leading to the recombinant HAdV-31 (AM749299).

Analysis of variable genomic loci in white spot syndrome virus to predict its origins in Procambarus clarkii crayfish farmed in China.

Variable genomic loci were examined in 4 white spot syndrome virus (WSSV) isolates (08HB, 09HB, 08JS and 09JS) from Procambarus clarkii crayfish collected from Jiangsu and Hubei Provinces in China in 2008 and 2009. In ORF75, sequence variation detected in the 4 isolates, as well as in isolates sequenced previously, suggested that WSSV might have segregated into 2 lineages since first emerging as a serious pathogen of farmed shrimp in East Asia in the early-mid 1990s, with one lineage remaining in East Asia and the other separating to South Asia. In ORF23/24, deletions of 9.31, 10.97, or 11.09 kb were evident compared to a reference isolate from Taiwan (WSSV-TW), and, in ORF14/15, deletions of 5.14 or 5.95 kb were evident compared to a reference isolate from Thailand with the largest genome size (TH-96-II). With respect to these genome characteristics, the crayfish isolates 08HB, 09HB and 08JS were similar to WSSV-TW and the isolate 09JS was similar to a reference isolate from China (WSSV-CN). In addition to these loci, sequence variation was evident in ORF94 and ORF125 that might be useful for differentiating isolates and in epidemiological tracing of WSSV spread in crayfish farmed in China. However, as all 4 crayfish isolates possessed a Homologous Region 9 sequence identical to isolate WSSV-TW and another Thailand isolate (WSSV-TH), and as their transposase sequence was identical to isolates WSSV-CN and WSSV-TH, these 2 loci were not useful in predicting their origins.