Establishment and application of a 10-plex liquid bead array for the simultaneous rapid detection of animal species.

BACKGROUND: Meat fraud and adulteration incidents occur frequently in almost all regions of the globe, especially with the increase in the world's population. To ensure the authenticity of meat products, we developed a 10-plex xMAP assay to simultaneously detect ten animal materials: bovine, caprine, poultry, swine, donkey, deer, horse, dog, fox and mink. RESULTS: This method was investigated by analyzing DNA extracts from raw muscle, muscle mixtures, meat products and animal feeds. Our results indicated that the species of interest can be identified, differentiated and detected down to 1 g kg-1 in binary mixtures or 0.01-0.001 ng of genomic DNA from specific species. Testing of 125 commercial samples showed a 97.4% coincidence rate with the method used in routine testing in our lab. CONCLUSION: These results indicated that the method established in this study could detect ten animal materials simultaneously within 3 h, which provides a new, useful tool for animal ingredient analysis in meat products and animal feeds. © 2019 Society of Chemical Industry.

A novel multiplex xMAP assay for generic detection of avian, fish, and ruminant DNA in feed and feedstuffs.

The identification of animal species in feed and feedstuffs is important for detecting contamination and fraudulent replacement of animal components that might cause health and economic problems. A novel multiplex assay, based on xMAP technology and the generic detection of closely related species, was developed for the simultaneous differential detection of avian, fish, and ruminant DNA in products. Universal primers and probes specific to avian, fish, or ruminant species were designed to target a conserved mitochondrial DNA sequence in the 12S ribosomal RNA gene (rRNA). The assay specificity was validated using samples of 27 target and 10 nontarget animal species. The limits of detection of the purified DNA were determined to be 0.2 pg/µL-0.1 ng/µL by testing the meat samples of six species and four feedstuffs. The detection sensitivity of the experimental mixtures was demonstrated to be 0.01% (weight percentage). The assay's suitability for practical application was evaluated by testing feed samples; unlabeled animal ingredients were detected in 32% of the 56 samples. The assay differentially detected the three targeted categories of animal species in less than 2 h, reflecting improvements in speed and efficiency. Based on these results, this novel multiplex xMAP assay provides a reliable and highly efficient technology for the routine detection of animal species in feed and other products for which this information is needed.

Recombinant rabies virus expressing interleukin-6 enhances the immune response in mouse brain.

Rabies, which is caused by the rabies virus (RABV), is an ancient zoonosis that has a high mortality rate. Previous studies have indicated that recombinant RABV expressing canine interleukin-6 (rHEP-CaIL6), induced more virus-neutralizing antibodies than parental RABV in mice following intramuscular immunization. To investigate the immune response induced in the CNS by rHEP-CaIL6 after intranasal or intracranial administration in mice, the permeability of the blood-brain barrier (BBB), the infiltration of CD3 T cells, and innate immune response-related effector molecules in the CNS were examined. It was observed that infection of rHEP-CaIL6 led to enhanced BBB permeability following intranasal infection. More CD3 T cells infiltrated into the central nervous system (CNS) in mice infected with rHEP-CaIL6 than in those infected with the HEP-Flury strain. Furthermore, rHEP-CaIL6 induced an increased expression of innate immune response-related effector molecules, compared with the parental HEP-Flury strain, within the CNS. Taken together, these findings suggest that rHEP-CaIL6 induced stronger immune responses in mice brains, which is more beneficial for virus clearance. These results may also partly illustrate the role of IL6 in RABV infection.

Rescue of a wild-type rabies virus from cloned cDNA and assessment of the proliferative capacity of recombinant viruses.

Reverse genetic systems (RGS) have been widely used for fixed rabies virus (RABV) strains. However, RGS, for wild-type (wt) strains, have been seldom reported despite the value of this approach in defining the biological characteristics of these strains. In this work, we developed a wt RGS using a swine-origin RABV strain (GD-SH-01) for the first time. In order to have a better understanding of the contribution and function of individual gene on viral proliferation for wt RABV isolates, we constructed a full-length cDNA clone of GD-SH-01 and exchanged the single genes encoding RABV protein of a highly attenuated RABV strain HEP-Flury with those of the virulent strain. Analysis of the viral growth kinetics, cell-to-cell spread, and genomic RNA (gRNA) synthesis of the both the rescued and parental virus strains revealed that replacement of the HEP-Flury N or L genes with those from GD-SH-01 resulted in higher proliferative capacity of both chimeric rHEP-shN and rHEP-shL while the former seemed to have a better viral gRNA synthesis ability, the latter spread faster. Replacement of HEP-Flury P gene with GD-SH-01 P gene resulted in reduction of the virus titer in cell culture supernatants with a poor replicative and spreading ability. However, replacement of HEP-Flury M or G genes with those from GD-SH-01 seemed to impact less on viral proliferation. Taken together, we show that we have successfully rescued a wt RABV strain, and assessed the impact of each gene on viral proliferative capacity using a series of single-gene-substituted viruses.

Phosphoprotein Gene of Wild-Type Rabies Virus Plays a Role in Limiting Viral Pathogenicity and Lowering the Enhancement of BBB Permeability.

Enhancement of blood-brain barrier (BBB) permeability is necessary for clearing virus in the central nervous system (CNS). It has been reported that only laboratory-attenuated rabies virus (RABV) induces inflammatory response to lead BBB transient breakdown rather than wild-type (wt) strains. As a component of ribonucleoprotein (RNP), phosphoprotein (P) of RABV plays a key role in viral replication and pathogenicity. To our knowledge, the function of RABV P gene during RABV invasion was unclear so far. In order to determine the role of RABV P gene during RABV infection, we evaluated the BBB permeability in vivo after infection with wt RABV strain (GD-SH-01), a lab-attenuated RABV strain (HEP-Flury), and a chimeric RABV strain (rHEP-SH-P) whose P gene cloned from GD-SH-01 was expressed in the genomic backbone of HEP-Flury. We found that rHEP-SH-P caused less enhancement of BBB permeability and was less pathogenic to adult mice than GD-SH-01 and HEP-Flury. In an effort to investigate the mechanism, we found that the replication of rHEP-SH-P has been limited due to the suppressed P protein expression and induced less response to maintain BBB integrity. Our data indicated that the P gene of wt RABV was a potential determinant in hampering viral replication in vivo, which kept BBB integrity. These findings provided an important foundation for understanding the viral invasion and development of novel vaccine.

The Deoptimization of Rabies Virus Matrix Protein Impacts Viral Transcription and Replication.

Rabies virus (RABV) matrix (M) protein plays several important roles during RABV infection. Although previous studies have assessed the functions of M through gene rearrangements, this interferes with the position of other viral proteins. In this study, we attenuated M expression through deoptimizing its codon usage based on codon pair bias in RABV. This strategy more objectively clarifies the role of M during virus infection. Codon-deoptimized M inhibited RABV replication during the early stages of infection, but enhanced viral titers at later stages. Codon-deoptimized M also inhibited genome synthesis at early stage of infection and increased the RABV transcription rates. Attenuated M through codon deoptimization enhanced RABV glycoprotein expression following RABV infection in neuronal cells, but had no influence on the cell-to-cell spread of RABV. In addition, codon-deoptimized M virus induced higher levels of apoptosis compared to the parental RABV. These results indicate that codon-deoptimized M increases glycoprotein expression, providing a foundation for further investigation of the role of M during RABV infection.

Phenotypic Consequence of Rearranging the N Gene of RABV HEP-Flury.

Nucleoprotein (N) is a key element in rabies virus (RABV) replication. To further investigate the effect of N on RABV, we manipulated an infectious cDNA clone of the RABV HEP-Flury to rearrange the N gene from its wild-type position of 1 (N-P-M-G-L) to 2 (P-N-M-G-L), 3 (P-M-N-G-L), or 4 (P-M-G-N-L), using an approach that left the viral nucleotide sequence unaltered. Subsequently, viable viruses were recovered from each of the rearranged cDNA and examined for their gene expression levels, growth kinetics in cell culture, pathogenicity in suckling mice and protection in mice. The results showed that gene rearrangement decreased N mRNA transcription and vRNA replication. As a result, all viruses with rearranged genomes showed worse replication than that of rHEP-Flury in NA cells at a MOI of 0.01, but equivalent or slightly better replication levels at a MOI of 3. Consequently, the lethality in suckling mice infected with N4 was clearly attenuated compared with rHEP-Flury. However, the protection to mice was not enhanced. This study not only gives us insight into the understanding of the phenotype of RABV N gene rearrangement, but also helps with rabies vaccine candidate construction.

Phosphoprotein Gene Contributes to the Enhanced Apoptosis Induced by Wild-Type Rabies Virus GD-SH-01 In Vitro.

Previous research demonstrated that the matrix protein (M) and glycoprotein (G) of attenuated rabies virus (RABV) strains are involved in the induction of host cell apoptosis. In this work, we show that wild-type (wt) RABV GD-SH-01 induces significantly greater apoptosis than the attenuated strain HEP-Flury. In order to identify the gene(s) accounting for this phenotype, five recombinant RABVs (rRABVs) were constructed by replacing each single gene of HEP-Flury with the corresponding gene of GD-SH-01. By using these rRABVs, we found that not only M and G, but also the phosphoprotein (P) plays an important role in inducing apoptosis. In order to figure out the different role of P gene in inducing apoptosis from the highly divergent background, another rRABV rGDSH-P, which carries the P gene of HEP-Flury in the background of the GD-SH-01 was generated. It was found that infection of NA cells with GD-SH-01 or the recombinant strain rHEP-shP, which carries P gene of GD-SH-01, induced significantly greater apoptosis than HEP-Flury or rGDSH-P in a caspase-dependent pathway that ultimately leads to the activation of the intrinsic apoptotic pathway, which is well characterized with the downregulation of bcl-2, the decrease of mitochondrial membrane potential, the release of mitochondrial cytochrome c, the activation of caspase-9 and caspase-3, and finally the cleavage of poly (ADP-ribose) polymerase. Our results imply that wt P from GD-SH-01 mediates this effect may partly by facilitating viral RNA synthesis but not by viral replication. In sum, we demonstrate a wt RABV strain GD-SH-01 to induce stronger apoptosis than an attenuated RABV HEP-Flury and propose that wt P from GD-SH-01 is involved in this process.

Phenotypic Consequences In vivo and In vitro of Rearranging the P Gene of RABV HEP-Flury.

Phosphoprotein (P) of the Rabies virus (RABV) is critically required for viral replication and pathogenicity. Here we manipulated infectious cDNA clones of the RABV HEP-Flury to translocate the P gene from its wild-type position 2 to 1, 3, or 4 in gene order, using an approach which left the viral nucleotide sequence unaltered. The recovered viruses were evaluated for the levels of gene expression, growth kinetics in cell culture, lethality in suckling mice and protection of mice. The results showed that viral replication was affected by the absolute value of N protein which was regulated by P protein. Viral lethality in suckling mice was consistent with the ratio of P mRNA in one complete transcription. The protection of mice induced by viruses was related to the antibody titer 5 weeks post-infection which might be regulated by G protein. However, the ability to induce cell apoptosis and viral spread were not only related to the viral replication but also to the ratio of related gene which affected by the gene position. These findings might not only improve the understanding of phenotype of RABV and P gene rearrangement, but also help rabies vaccine candidate construction.

Expression of interleukin-6 by a recombinant rabies virus enhances its immunogenicity as a potential vaccine.

Several studies have confirmed that interleukin-6 (IL6) mediates multiple biological effects that enhance immune responses when used as an adjuvant. In the present study, recombinant rabies virus (RABV) expressing canine IL6 (rHEP-CaIL6) was rescued and its pathogenicity and immunogenicity were investigated in mice. We demonstrated that mice received a single intramuscular immunization with rHEP-CaIL6 showed an earlier increase and higher maximum titres of virus-neutralizing antibody (VNA) as well as anti-RABV antibodies compared with mice immunized with the parent strain. Moreover, survival rates of mice immunized with rHEP-CaIL6 were higher compared with mice immunized with parent HEP-Flury according to the challenge assay. Flow cytometry further confirmed that immunization with rHEP-CaIL6 induced the strong recruitment of mature B cells and CD8+ T cells to lymph nodes, which may partially explain the high levels of VNA and enhanced cellular immunity. Quantitative real-time PCR indicated that rHEP-CaIL6 induced stronger inflammatory and immune responses in the central nervous system, which might have allowed virus clearance in the early infection phase. Furthermore, mice infected intranasally with rHEP-CaIL6 developed no clinical symptoms while mice infected with HEP-Flury showed piloerection. In summary, these data indicate that rHEP-CaIL6 induces a strong, protective immune response with a good safety profile. Therefore, a recombinant RABV strain expressing canine IL6 may aid the development of an effective, safe attenuated rabies vaccine.

Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines.

Different rabies virus (RABV) strains have their own biological characteristics, but little is known about their respective impact on autophagy. Therefore, we evaluated whether attenuated RABV HEP-Flury and wild-type RABV GD-SH-01 strains triggered autophagy. We found that GD-SH-01 infection significantly increased the number of autophagy-like vesicles, the accumulation of enhanced green fluorescent protein (EGFP)-LC3 fluorescence puncta and the conversion of LC3-I to LC3-II, while HEP-Flury was not able to induce this phenomenon. When evaluating autophagic flux, we found that GD-SH-01 infection triggers a complete autophagic response in the human neuroblastoma cell line (SK), while autophagosome fusion with lysosomes was inhibited in a mouse neuroblastoma cell line (NA). In these cells, GD-SH-01 led to apoptosis and mitochondrial dysfunction while triggering autophagy, and apoptosis could be decreased by enhancing autophagy. To further identify the virus constituent causing autophagy, 5 chimeric recombinant viruses carrying single genes of HEP-Flury instead of those of GD-SH-01 were rescued. While the HEP-Flury virus carrying the wild-type matrix protein (M) gene of RABV triggered LC3-I to LC3-II conversion in SK and NA cells, replacement of genes of nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) produced only minor autophagy. But no one single structural protein of GD-SH-01 induced autophagy. Moreover, the AMPK signaling pathway was activated by GD-SH-01 in SK. Therefore, our data provide strong evidence that autophagy is induced by GD-SH-01 and can decrease apoptosis in vitro. Furthermore, the M gene of GD-SH-01 may cooperatively induce autophagy.

Ursolic acid inhibits proliferation and reverses drug resistance of ovarian cancer stem cells by downregulating ABCG2 through suppressing the expression of hypoxia-inducible factor-1α in vitro.

Hypoxia in tumors is closely related to drug resistance. It has not been verified whether hypoxia-inducible factor-1α (HIF-1α) or ABCG2 is related to hypoxia-induced resistance. Ursolic acid (UA), when used in combination with cisplatin can significantly increase the sensitivity of ovarian cancer stem cells (CSCs) to cisplatin, but the exact mechanism is unknown. The cell growth inhibitory rate of cisplatin under different conditions was evaluated using Cell Counting Kit-8 (CCK-8) in adherence and sphere cells (SKOV3, A2780, and HEY). The expression of HIF-1α and ABCG2 was tested using quantitative PCR, western blotting, and immuno-fluorescence under different culture conditions and treated with UA. Knockdown of HIF-1α by shRNA and LY294002 was used to inhibit the activity of PI3K/Akt pathway. Ovarian CSCs express stemness-related genes and drug resistance significantly higher than normal adherent cells. Under hypoxic conditions, the ovarian CSCs grew faster and were more drug resistant than under normoxia. UA could inhibit proliferation and reverse the drug resistance of ovarian CSC by suppressing ABCG2 and HIF-1α under different culture conditions. HIF-1α inhibitor YC-1 combined with UA suppressed the stemness genes and ABCG2 under hypoxic condition. The PI3K/Akt signaling pathway activation plays an important functional role in UA-induced downregulation of HIF-1α and reduction of ABCG2. UA inhibits the proliferation and reversal of drug resistance in ovarian CSCs by suppressing the expression of downregulation of HIF-1α and ABCG2.

iTRAQ protein profile analysis of neuroblastoma (NA) cells infected with the rabies viruses rHep-Flury and Hep-dG.

The rabies virus (RABV) glycoprotein (G) is the principal contributor to the pathogenicity and protective immunity of RABV. In a previous work, we reported that recombinant rabies virus Hep-dG, which was generated by reverse genetics to carry two copies of the G-gene, showed lower virulence than the parental virus rHep-Flury in suckling mice with a better immune protection effect. To better understand the mechanisms underlying rabies virus attenuation and the role of glycoprotein G, isobaric tags for relative and absolute quantitation (iTRAQ) was performed to identify and quantify distinct proteins. 10 and 111 differentially expressed proteins were obtained in rHep-Flury and Hep-dG infection groups, respectively. Selected data were validated by western blot and qRT-PCR. Bioinformatics analysis of the distinct protein suggested that glycoprotein over-expression in the attenuated RABV strain can induce activation of the interferon signaling. Furthermore, it may promote the antiviral response, MHC-I mediated antigen-specific T cell immune response, apoptosis and autophagy in an IFN-dependent manner. These findings might not only improve the understanding of the dynamics of RABV and host interaction, but also help understand the mechanisms underlying innate and adaptive immunity during RABV infection.

Novel AAV-based genetic vaccines encoding truncated dengue virus envelope proteins elicit humoral immune responses in mice.

The envelope protein of dengue virus is involved in host cell attachment for entry and induction of protective immunity. Current efforts are focused on producing a tetravalent vaccine by mixing four monovalent vaccine components. In this work, we developed a genetic vaccine based on a novel adeno-associated viral (AAV) vector expressing the carboxy-terminal truncated envelope protein (79E) of dengue virus. The expression of the recombinant 79E protein in HEK 293 cells was confirmed by Western blot. Vectors packaged with novel AAV capsids (AAV2/8 or AAV2/rh32.33) were injected into C57BL/6 mice intramuscularly. Dengue virus antigen was produced in the mice and induced long-lasting antibody responses against the dengue virus still detectable 20 weeks after immunization. AAV2/8 vaccine induced higher anti-dengue virus antibody levels than AAV2/rh32.33 vaccine or AAV plasmid. Furthermore, the anti-dengue antibodies could neutralize homogeneous dengue virus. These results demonstrated that the AAV vaccines possessed appropriate immunogenicity and could be used for the development of an effective dengue vaccine.