Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/µl while the LOD for the RT-qPCR was 0.2 PFU/µl. Good agreement (69.4-100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

Determination of the complete genome sequence of infectious hematopoietic necrosis virus (IHNV) Ch20101008 and viral molecular evolution in China.

This study determined the complete genomic sequence of the infectious hematopoietic necrosis virus (IHNV) strain Ch20101008 isolated from farmed brook trout (Salvelinus fontinalis) that died from a disease caused by the virus in northeast China. The sequence was determined from 10 overlapping clones obtained through RT-PCR amplification. The whole genome length of Ch20101008 comprised 11,129 nucleotides (nt), and the overall organization was typical of that observed for all other IHNV strains. The phylogenetic analysis results of the 65 IHNV glycoprotein genes and 47 IHNV partial nucleoprotein genes presented five major genogroups (J, U, L, E and M). The J genogroup included the J Nagano and J Shizuoka subgroups. The IHNV Ch20101008 strain belonged to the J Nagano subgroup of the J genogroup and was significantly related to other Chinese IHNV strains. All Chinese IHNV isolates are monophyletic, with a recent common ancestor, except for the BjLL strain. The N, P, M, G, NV and L genes of Ch20101008 were compared with the available IHNV sequences in GenBank. The results indicated that 198 nt were substituted, 53 of which exhibited amino acid change in the Ch20101008 genome. An adenine nucleotide deletion was found at position 4959 of the 5' UTR of the L gene. In the G gene, specific nucleotides and amino acid variations of the Chinese IHNV strains were observed when compared with 23 isolates from other countries. Of the 15 nucleotide sites that changed, seven resulted in amino acid substitution. The data further demonstrated that the J genogroup IHNV was introduced to and evolved in salmon farm environments in China.

Generation of an attenuated Tiantan vaccinia virus by deletion of the ribonucleotide reductase large subunit.

Attenuation of the virulence of vaccinia Tiantan virus (VTT) underlies the strategy adopted for mass vaccination campaigns. This strategy provides advantages of safety and efficacy over traditional vaccines and is aimed at minimization of adverse health effects. In this study, a mutant form of the virus, MVTT was derived from VTT by deletion of the ribonucleotide reductase large subunit (R1) (TI4L). Compared to wild-type parental (VTT) and revertant (VTT-rev) viruses, virulence of the mutant MVTT was reduced by 100-fold based on body weight reduction and by 3,200-fold based on determination of the intracranial 50% lethal infectious dose. However, the immunogenicity of MVTT was equivalent to that of the parental VTT. We also demonstrated that the TI4L gene is not required for efficient replication. These data support the conclusion that MVTT can be used as a replicating virus vector or as a platform for the development of vaccines against infectious diseases and for cancer therapy.

[Detection of Angiostrongylus cantonensis larvae with a high resolution melt PCR assay].

OBJECTIVE: To develop a detection method of the third-stage larvae of Angiostrongylus cantonensis by real-time PCR and high-resolution melt curve analysis. METHODS: A pair of specific primers was designed based on the internal transcribed spacer 1 (ITS1) region of the nuclear ribosomal DNA of A. cantonensis. The third-stage larvae of A. cantonensis were detected by real-time PCR and high-resolution melt curve analysis. The specificity of the method was analyzed by testing DNAs of A. cantonensis, Clonorchis sinensis, and Gnathostoma spinigerum. The genomic DNA were extracted from 1 to 10 third-stage larvae of A. cantonensis, respectively, and used to identify the sensitivity of the method. RESULTS: This method could specifically detect A. cantonensis and the detection limit reached to one larva. No amplification curve and melt curve were found in C. sinensis and G. spinigerum. CONCLUSION: Real-time PCR and high-resolution melt curve analysis show good specificity and sensitivity for detecting the third-stage larvae of A. cantonensis.

Characterization of an attenuated TE3L-deficient vaccinia virus Tian Tan strain.

An attenuated vaccinia virus (VACV), TE3L(-)VTT, was evaluated for virulence and safety to determine its potential use as a vaccine or as a recombinant virus vector to express foreign genes. The virulence of TE3L(-)VTT was compared with that of the wild-type VTT both in vivo and in vitro. The humoral and cellular immune responses were detected in a mouse model to test the vaccine efficacy of the TE3L mutant. The results suggested that deletion of the TE3L gene decreased the virulence and neurovirulence significantly in mice and rabbit models, yet retained the immunogenicity. Thus, the deletion of TE3L improved the safety of the VTT vector; this approach may yield a valuable resource for studies of recombinant VACV-vectored vaccines.

Attenuation of vaccinia Tian Tan strain by removal of viral TC7L-TK2L and TA35R genes.

Vaccinia Tian Tan (VTT) was attenuated by deletion of the TC7L-TK2L and TA35R genes to generate MVTT3. The mutant was generated by replacing the open reading frames by a gene encoding enhanced green fluorescent protein (EGFP) flanked by loxP sites. Viruses expressing EGFP were then screened for and purified by serial plaque formation. In a second step the marker EGFP gene was removed by transfecting cells with a plasmid encoding cre recombinase and selecting for viruses that had lost the EGFP phenotype. The MVTT3 mutant was shown to be avirulent and immunogenic. These results support the conclusion that TC7L-TK2L and TA35R deletion mutants can be used as safe viral vectors or as platform for vaccines.

Immune responses to the oral administration of recombinant Bacillus subtilis expressing multi-epitopes of foot-and-mouth disease virus and a cholera toxin B subunit.

Bacillus subtilis has been engineered successfully to express heterologous antigens for use as a vaccine vehicle that can elicit mucosal and systemic immunity response. In this study, a recombinant B. subtilis expressing the B subunit of cholera toxin (CT-B) and an epitope box constituted with antigen sites from foot-and-mouth disease virus (FMDV) type Asia 1 was constructed and named 1A751/CTB-TEpiAs. Its capability to induce mucosal, humoral, and cellular responses in mice and guinea pigs was evaluated after oral administration with vegetative cells of 1A751/CTB-TEpiAs. In addition, its capability to protect guinea pigs against homologous virus challenge was examined. All animals were given booster vaccination at day 21 after initial inoculation and guinea pigs were challenged 3 weeks after booster vaccination. The control groups were inoculated with a commercial vaccine or administered orally with 1A751/pBC38C or an oral buffer. All animals vaccinated with 1A751/CTB-TEpiAs developed specific anti-FMDV IgA in lung and gut lavage fluid, serum ELISA antibody, neutralizing antibody as well as T lymphocyte proliferation, and IFN-γ secretory responses. Three of the five guinea pigs vaccinated with 1A751/CTB-TEpiAs were protected completely from the viral challenge. The results demonstrate the potential viability of a B. subtilis-based recombinant vaccine for the control and prevention of FMDV infections.

Construction, expression and characterization of a chimeric multi-domain protein mediating specific DNA transfer.

The delivery of plasmid DNA to target cells using a simple, defined, non-viral system is an area of intense research in gene therapy. Here, we describe a novel DNA carrier protein termed TG, consisting of the DNA-binding domain of the yeast transcriptional activator GAL4 and human immunodeficiency virus type 1 Tat protein, which can transfer modified naked plasmid DNA into target cells to express foreign genes of interest. The TG protein was expressed in Escherichia coli (E. coli), refolded and purified on an immobilized Ni(2+) affinity chromatography column. SDS-PAGE and Western blotting revealed that the fusion protein was highly expressed with a yield of approximately 275 mg/L. We also constructed the pIRES-UAS-EGFP DNA vector, consisting of upstream activating sequences (UASs) for the specific binding of the DNA-binding protein and the enhanced green fluorescent protein (EGFP) gene. The TG protein could bind specifically to pIRES-UAS-EGFP, forming a complex which could efficiently transfect target cells and result in detectable EGFP protein expression. Thus, these results provide a basis for development of efficient non-viral DNA transfer vectors for further improvements of gene therapy strategies.

[Establishment of a fluorescent real-time quantitative RT-PCR assay for detection of genotype 4 hepatitis E virus in swine stools].

The primers and probes for the Real-time RT-PCR were designed based on the multiple sequence (swine and humans HEV strains) alignments of the ORF3 region of genotype 4 HEV. A rapid, sensitive and stable TaqMan Real-time RT-PCR assay was established, and its specificity and sensitivity were assessed, and comparison of the Real-time RT-PCR with conventional and nested RT-PCR was performed. The results found that the crossing points showed linearly proportional to the logarithm of the input copy number. The correlation coefficient (R2) and the slope value of the standard curves with plasmid DNA were 0.994 and -3.312, respectively. The efficiency (E) of the PCR was 100%. Coefficients of variation values of the different diluted plasmid DNA were low in the same or different repeated experimental group. In addition, the assay was able to correctly detect genotype 4 HEV RNA from swine fecal samples. The sensitivity of established assay was 100-fold higher than that of conventional RT-PCR and 10-fold higher than nested RT-PCR.

Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo.

BACKGROUND: Oncolytic virotherapy is an attractive drug platform of cancer gene therapy, but efficacy and specificity are important prerequisites for success of such strategies. Previous studies determined that Apoptin is a p53 independent, bcl-2 insensitive apoptotic protein with the ability to specifically induce apoptosis in tumor cells. Here, we generated a conditional replication-competent adenovirus (CRCA), designated Ad-hTERT-E1a-Apoptin, and investigated the effectiveness of the CRCA a gene therapy agent for further clinical trials. RESULTS: The observation that infection with Ad-hTERT-E1a-Apoptin significantly inhibited growth of the melanoma cells, protecting normal human epidermal melanocytes from growth inhibition confirmed cancer cell selective adenoviral replication, growth inhibition, and apoptosis induction of this therapeutic approach. The in vivo assays performed by using C57BL/6 mice containing established primary or metastatic tumors expanded the in vitro studies. When treated with Ad-hTERT-E1a-Apoptin, the subcutaneous primary tumor volume reduction was not only observed in intratumoral injection group but in systemic delivery mice. In the lung metastasis model, Ad-hTERT-E1a-Apoptin effectively suppressed pulmonary metastatic lesions. Furthermore, treatment of primary and metastatic models with Ad-hTERT-E1a-Apoptin increased mice survival. CONCLUSIONS: These data further reinforce the previously research showing that an adenovirus expressing Apoptin is more effective and advocate the potential applications of Ad-hTERT-E1a-Apoptin in the treatment of neoplastic diseases in future clinical trials.