Connectivity preservation control for multiple unmanned aerial vehicles in the presence of bounded actuation.

This paper proposes a novel multi-unmanned aerial vehicle (UAV) connectivity preservation controller, suitable for scenarios with bounded actuation and limited communication range. According to the hierarchical control strategy, controllers are designed separately for the position and attitude subsystems. A distributed position controller is developed, integrating an indirect coupling control mechanism. The innovative mechanism associates each UAV with a virtual proxy, facilitating connections among adjacent UAVs through these proxies. This structuring assists in managing the actuator saturation constraints effectively. The artificial potential function is utilized to preserve network connectivity and fulfill coordination among all virtual proxies. Additionally, an attitude controller designed for finite-time convergence guarantees that the attitude subsystem adheres precisely to the attitude specified by the distributed position controller. Simulation results validate the efficacy of this distributed formation controller with connectivity preservation under bounded actuation conditions. The simulation results confirm the effectiveness of the distributed connectivity preservation controller with bounded actuation.

A fault isolation strategy for industrial processes using outlier-degree-based variable contributions.

In industrial process monitoring, it is always a challenging and practical problem to analyze the causes of the system fault by isolating true fault variables from vast amounts of process data. However, the phenomenon of smearing effect occurs by using the traditional contribution analysis-based isolation methods since the defined isolation indices of different variables affect each other. In this paper, a new fault isolation method is proposed based on local outlier factor and improved k-nearest neighbor rule aiming to improve the isolation accuracy. Firstly, the nearest neighbors of each sample are obtained along the direction of a specific variable. Based on the nearest neighbors, the outlier-degree value of the variable is calculated and regarded as the contribution of the variable. Then, the contribution of the variable in all samples are obtained in the same way, among which the maximum one is selected as the isolation threshold value of this variable. During the online monitoring, the contribution of the variable in the newly collected sample is calculated in real time. Once the contribution is greater than the threshold, the variable is judged to be the dominant factor causing the system fault. Two cases on numerical example and Tennessee Eastman process are conducted to evaluate the effectiveness of the proposed method.

Over-expression of IL-33 enhances rabies virus early antigen presentations and cellular immune responses in mice.

Human inactivated rabies virus (RABV) vaccines have been widely used worldwide over 30 years. The mechanisms of humoral immunity elicited by previously reported rabies candidate vaccines have been fully investigated, but little is known about the cellular immunity profiles. Herein, the recombinant RABV rLBNSE-IL-33 overexpressing the mouse interleukin-33 (IL-33) proliferated well in Neuro-2a cells and had no effects with the parent virus on growth kinetic in vitro and viral pathogenicity in mice. The rLBNSE-IL-33 experienced more antigen presentations by MHC-II on DCs and activated more CD4+ T cells which helped recruit more CD19+CD40+ B cells in blood and promote rapid and robust IgG1 antibodies responses at initial infection stage compared with the parent rLBNSE strain. Simultaneously, the rLBNSE-IL-33 were also presented by MHC-I to CD8+ T cells which contributed to produce high levels of IgG2a. The rLBNSE-IL-33 elicited significantly high levels of RABV-specific IFN-γ secreting memory CD4+ T cells, more RABV-specific IL-4 and IFN-γ secreting memory CD8+ T cells in spleens at early infection stage in mice. Altogether, overexpression of IL-33 in rLBNSE-IL-33 enhanced early antigen presentation, markedly promote CD4+, memory CD4+ and CD8+ T cells-mediated responses and provided a 100 % protection from lethal RABV challenge in mice. These findings provided an alternative novel therapy and vaccine strategy in future.

Persistent and Severe Viral Replication in PBMCs with Moderate Immunosuppression Served an Alternative Novel Pathogenic Mechanism for Canine Morbillivirus.

Measles virus and canine distemper virus (CDV) cause lethal infections in their respective hosts characterized by severe immunosuppression. To furtherly acknowledge the attenuated mechanisms of the regionally ongoing epidemic CDV isolates and provide novel perspectives for designing new vaccines and therapeutic drugs, a recombinant CDV rHBF-vacH was employed with a vaccine hemagglutinin (H) gene replacement by reverse genetics based on an infectious cDNA clone for the CDV wild-type HBF-1 strain. Interestingly, unlike previously published reports that a vaccine H protein completely changed a pathogenic wild-type CDV variant to be avirulent, rHBF-vacH was only partially attenuated by alleviating the degree of viral immunosuppression, and still caused 66.7% lethality in ferrets with a prolonged period of disease. Further comparisons of pathogenic mechanisms proved that the weaker but necessary invasions into peripheral blood mononuclear cells (PBMCs) of rHBF-vacH, and subsequently persistent viral replications in PBMCs and multiple organs, together contributed to its 66.7% mortality. In addition, despite significantly higher titers than the parent viruses, rHBF-vacH would not be a suitable candidate for a live vaccine, with great invasion and infection potentials of PBMCs from 16 tested kinds of host species. Altogether, sustained and severe viral replication in PBMCs with moderate immunosuppression was first proven to be an alternative novel pathogenic mechanism for CDV, which might help us to understand possible reasons for CDV fatal infections among domestic dogs and the highly susceptible wild species during natural transmission. IMPORTANCE Despite widespread vaccine campaigns for domestic dogs, CDV remained an important infectious disease in vaccinated carnivores and wild species. In recent years, the regionally ongoing epidemic CDV isolates have emphasized conservation threats to, and potentially disastrous epidemics in, endangered species worldwide. However, little is known about how to deal with the CDV variants constantly regional epidemic. In this study, we employed a recombinant CDV rHBF-vacH with a vaccine H gene replacement in a CDV wild-type HBF-1 context to attenuate the epidemic CDV variant to design a new vaccine candidate. Interestingly, rHBF-vacH was only partially attenuated by alleviating the degree of viral immunosuppression, and still caused 66.7% lethality in ferrets by weaker but necessary invasions into PBMCs, and subsequently persistent and severe viral replications in PBMCs. Significantly higher virus titers of rHBF-vacH in vitro might indicate the rapid cell-to-cell spreads in vivo that indirectly contribute to fatal infections of rHBF-vacH in ferrets.

Recombinant adeno-associated virus serotype 9 AAV-RABVG expressing a Rabies Virus G protein confers long-lasting immune responses in mice and non-human primates.

Three or four intramuscular doses of the inactivated human rabies virus vaccines are needed for pre- or post-exposure prophylaxis in humans. This procedure has made a great contribution to prevent human rabies deaths, which bring huge economic burdens in developing countries. Herein, a recombinant adeno-associated virus serotype 9, AAV9-RABVG, harbouring a RABV G gene, was generated to serve as a single dose rabies vaccine candidate. The RABV G protein was stably expressed in the 293T cells infected with AAV9-RABVG. A single dose of 2 × 1011 v.p. of AAV9-RABVG induced robust and long-term positive seroconversions in BALB/c mice with a 100% survival from a lethal RABV challenge. In Cynomolgus Macaques vaccinated with a single dose of 1 × 1013 v.p. of AAV9-RABVG, the titres of rabies VNAs increased remarkably from 2 weeks after immunity, and maintained over 31.525 IU/ml at 52 weeks. More DCs were activated significantly for efficient antigen presentations of RABV G protein, and more B cells were activated to be responsible for antibody responses. Significantly more RABV G specific IFN-γ-secreting CD4+ and CD8+ T cells, and IL-4-secreting CD4+ T cells were activated, and significantly higher levels of IL-2, IFN-γ, IL-4, and IL-10 were secreted to aid immune responses. Overall, the AAV9-RABVG was a single dose rabies vaccine candidate with great promising by inducing robust, long-term humoral responses and both Th1 and Th2 cell-mediated immune responses in mice and non-human primates.

A rabies virus vectored severe fever with thrombocytopenia syndrome (SFTS) bivalent candidate vaccine confers protective immune responses in mice.

The Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne hemorrhagic zoonotic disease, which is potentially fatal in human with mortality rates ranging from 16.2%-32%. The rabies virus (RABV) LBNSE vector expressing foreign antigens have shown considerable promise as vaccines against viral diseases, which is effective and safe. In the present study, we generated a recombinant RABV rLBNSE-Gn expressing a SFTSV glycoprotein Gn by reverse genetic technology to control rabies and SFTS in animals. An extra insertion of Gn gene did not impact replication of the recombinant virus rLBNSE-Gn in NA and BHK-21 cells compared to the parent rLBNSE strain. The SFTSV Gn gene together with RABV N and G genes were efficiently expressed in rLBNSE-infected Vero cells by immunostaining and immune blots. A single dose of 107 FFU of the rLBNSE-Gn intramuscularly inoculated in BALB/c mice induced rapid and robust humoral responses against both RABV and SFTSV without any signs of disease or weight loss. Compared to the rLBNSE and DMEM groups, the extra Gn expression contributed to the recruitments and/or activations of the dendritic cells and B cells from inguinal lymph nodes of BALB/c mice vaccinated with rLBNSE-Gn. The protective efficacy of rLBNSE-Gn against SFTSV in C57BL/6 mice was evaluated, and the virus loading in the spleens reduced to 10 TCID50/mg at 7 days post SFTSV infections, which indicated that the rLBNSE-Gn conferred efficacious protective immune responses from SFTSV in C57BL/6 mice. All the mice immunization with rLBNSE-Gn and rLBNSE survived after a lethal RABV challenge, suggesting a 100 % protection from RABV. Therefore, the rLBNSE-Gn would be a promising bivalent candidate vaccine against SFTS and rabies in animals.

[Construction and identification of an infectious clone for CDV-3 strain of canine distemper virus].

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 107·667 TCID50/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 106·667 TCID50/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.

A Bivalent Human Adenovirus Type 5 Vaccine Expressing the Rabies Virus Glycoprotein and Canine Distemper Virus Hemagglutinin Protein Confers Protective Immunity in Mice and Foxes.

The development of a safe and efficient multivalent vaccine has great prospects for application. Both rabies virus (RABV) and canine distemper virus (CDV) are highly infectious antigens, causing lethal diseases in domestic dogs and other carnivores worldwide. In this study, a replication-deficient human adenovirus 5 (Ad5)-vectored vaccine, rAd5-G-H, expressing RABV glycoprotein (G) and CDV hemagglutinin (H) protein was constructed. The RABV G and CDV H protein of rAd5-G-H were expressed and confirmed in infected HEK-293 cells by indirect immunofluorescence assay. The rAd5-G-H retained a homogeneous icosahedral morphology similar to rAd5-GFP under an electron microscope. A single dose of 108 GFU of rAd5-G-H administered to mice by intramuscular injection elicited rapid and robust neutralizing antibodies against RABV and CDV. Flow cytometry assays indicated that the dendritic cells and B cells in inguinal lymph nodes were significantly recruited in rAd5-G-H-immunized mice in comparison with the mock and rAd5-GFP groups. rAd5-G-H also activated the Th1- and Th2-mediated cell immune responses against RABV and CDV in mice, which contributed to 100% survival of a lethal-dose RABV challenge without any clinical signs. In foxes, a single dose of 109 GFU of rAd5-G-H could elicit high levels of neutralizing antibodies against both RABV and CDV in comparison with the mock and rAd5-GFP groups. All foxes in the rAd5-GFP and mock groups died, while the foxes inoculated with rAd5-G-H all survived and showed no clinical signs of disease after being challenged with a lethal wild-type CDV strain. These results suggested that rAd5-G-H has great potential as a bivalent vaccine against rabies and canine distemper in highly susceptible dogs and wildlife animals.

RNA sequencing analyses of gene expressions in a canine macrophages cell line DH82 infected with canine distemper virus.

Virulent morbillivirus infections, including Meals Virus (MeV) and Canine Distemper Virus (CDV), caused severe immune suppression and leukopenia, while attenuated vaccine strains developed protective host immune responses. However, the detailed molecular foundations of host antiviral responses were poorly characterized. In order to better understand the interactions between attenuated vaccine and host antiviral responses, the global gene expression changes in CDV-11-infected DH82 cells, a macrophage-derived cell line from canine, were investigated by transcriptomic analysis, and portions of results were confirmed with quantitative RT-PCR. The results exhibited that 372 genes significantly up-regulated (p < .01) and 119 genes were significantly down-regulated (p < .01) in CDV-infected macrophages DH82 at 48 h p.i.. The enriched functions of the significantly up-regulated (p < .01) genes were closely associated with interferon stimulated genes (ISGs), chemokine genes and pro-inflammatory factor genes. Gene ontology and pathway analysis of differentially expressed genes (DEGs) revealed that the most significantly involved pathways in CDV-infected DH82 cells were NF-κB and TNF signaling pathway, cytokine-cytokine receptor interaction, and pathogen associated molecular patterns (PAMPs), such as Toll-like, RIG-I-like and NOD-like receptor signalings. Thus, the findings indicated that pattern recognition receptors (PRRs) possibly mediated host innate and protective antiviral immune responses in CDV-11 infected DH82 cells.

Antiviral efficacy of favipiravir against canine distemper virus infection in vitro.

BACKGROUND: Canine distemper (CD) is an acute infectious disease with high morbidity rates caused by a highly contagious pathogen (Canine Morbillivirus, also known as canine distemper virus, CDV). CDV can infect a broad range of carnivores resulting in complex clinical signs. Currently, there is no effective method to treat for CDV infections. Favipiravir (T-705), a pyrazine derivative, was shown to be an effective antiviral drug against RNA viruses, acting on RNA-dependent RNA polymerase (RdRp). However, whether the T-705 has antiviral effects following CDV infection is unclear. Here, we investigated the antiviral effect of T-705 against CDV-3 and CDV-11 strains in Vero and DH82 cell lines. RESULTS: Our data demonstrated that T-705 significantly inhibited the replication of CDV-3 and CDV-11 in both Vero and DH82 cells at different concentrations, ranging from 2.441 µg/ml to 1250 µg/ml. Additionally, T-705 exhibited efficacious antiviral effects when administered at different time points after virus infection. Cytotoxicity tests showed a slight decline in viability in Vero cells after T-705 treatment, and no apparent cytotoxicity was detected in T-705 treated DH82 cells. Comparison of anti-CDV polyclonal serum only inhibition of CDV in supernatant, T-705 directly inhibited viral replication in cells, and indirectly reduced the amount of virions in supernatant. The combination application of T-705 and anti-CDV polyclonal serum exhibited a rapid and robust inhibition against virions in supernatant and virus replication in cells. CONCLUSIONS: Our data strongly indicated that T-705 effectively inhibited viral replication following CDV infection in vitro, and could be a potential candidate for treatment for CD.

Development of a TaqMan probe-based quantitative reverse transcription PCR assay for detection of Getah virus RNA.

Getah virus (GETV), a mosquito-borne virus that mainly infects horses and pigs, has emerged and spread in China. We developed a highly specific and reproducible TaqMan probe-based quantitative reverse transcription PCR (RT-qPCR) assay targeting the non-structural protein 1 of GETV, whose detection limit is 25.5 copies/µL, which is 100-fold higher than that of conventional RT-PCR. RT-qPCR was used to detect GETV RNA in mosquito and animal clinical samples, showing that the accuracy of RT-qPCR was higher than that of conventional RT-PCR. The newly developed RT-qPCR assay may be a useful alternative tool for rapid, simple and specific diagnosis of GETV infection.

Outbreak of severe pseudorabies virus infection in pig-offal-fed farmed mink in Liaoning Province, China.

An outbreak of severe pseudorabies virus (PRV) infection in farmed mink occurred in northern China in late 2014, causing significant economic losses in the local fur industry. Here, we report the first case of a PRV outbreak in mink in northeastern China, caused by feeding farmed mink with raw pork or organs contaminated by PRV. Mink infected with virulent PRV exhibited diarrhea, neurologic signs, and higher mortality, which can be misdiagnosed as highly pathogenic mink enteritis virus (MEV), canine distemper virus (CDV), and food poisoning. However, these were excluded as causative agents by PCR or bacteria isolation. The duration of disease was 3-7 days, and the mortality rate was 80-90%. PRV was characterized using indirect immunofluorescence assays (IFA) and electron microscopy (EM). Phylogenetic analysis based on full-length genome sequences and those of individual genes of this novel virus strain showed that it clustered in an independent branch with several other PRV isolates from China.

Development of a Peptide ELISA for the Diagnosis of Aleutian Mink Disease.

Aleutian disease (AD) is a common immunosuppressive disease in mink farms world-wide. Since the 1980s, counterimmunoelectrophoresis (CIEP) has been the main detection method for infection with the Aleutian Mink Disease Virus (AMDV). In this study, six peptides derived from the AMDV structural protein VP2 were designed, synthesized, and used as ELISA antigens to detect anti-AMDV antibodies in the sera of infected minks. Serum samples were collected from 764 minks in farms from five different provinces, and analyzed by both CIEP (a gold standard) and peptide ELISA. A peptide designated P1 (415 aa-433 aa) exhibited good antigenicity. A novel ELISA was developed using ovalbumin-linked peptide P1 to detect anti-AMDV antibodies in mink sera. The sensitivity and specificity of the peptide ELISA was 98.0% and 97.5%, respectively. Moreover, the ELISA also detected 342 early-stage infected samples (negative by CIEP and positive by PCR), of which 43.6% (149/342) were true positives. These results showed that the peptide ELISA had better sensitivity compared with CIEP, and therefore could be preferable over CIEP for detecting anti-AMDV antibodies in serological screening.

Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of Batai Virus in Cattle and Mosquitoes.

Batai virus (BATV) is an arthropod-borne single-stranded RNA virus belonging to the genus Orthobunyavirus of the family Bunyaviridae that is primarily transmitted by mosquitoes. Methods for detecting BATV are currently limited to serological surveillance, virus isolation, and conventional reverse transcription-polymerase chain reaction (RT-PCR) assay. In this study, we sought to develop a BATV detection assay that needs no specialized equipment and is highly specific, sensitive, and simple. We first developed and optimized a reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of BATV that uses two pairs of primers to amplify a conserved region of the BATV M gene. The optimal reaction conditions for this RT-LAMP BATV detection assay were 40 min at 65°C. The amplification products could be visualized directly for color changes. This RT-LAMP method has a detection limit of 2.86 copies/µL and a sensitivity that was approximately 10- and 100-fold greater than real-time and conventional RT-PCR, respectively. RT-LAMP for BATV detection showed no cross-reactivity with other viruses and its sensitivity was validated with cattle blood and mosquito specimens. Our results suggest that this RT-LAMP method was simpler and faster than conventional RT-PCR or real-time RT-PCR. Moreover, RT-LAMP represents a potential tool to test for BATV in clinical and mosquito samples, especially in rural areas of China. This method also shows promise as a diagnostic tool due to its rapid and sensitive detection without the need for sophisticated equipment or complicated protocols.

Isatis indigotica root polysaccharides as adjuvants for an inactivated rabies virus vaccine.

Adjuvants can enhance vaccine immunogenicity and induce long-term enhancement of immune responses. Thus, adjuvants are important for vaccine research. Polysaccharides isolated from select Chinese herbs have been demonstrated to possess various beneficial functions and excellent adjuvant abilities. In the present study, the polysaccharides IIP-A-1 and IIP-2 were isolated from Isatis indigotica root and compared with the common vaccine adjuvant aluminum hydroxide via intramuscular co-administration of inactivated rabies virus rCVS-11-G into mice. Blood was collected to determine virus neutralizing antibody (VNA) titers and B and T lymphocyte activation status. Inguinal lymph node samples were collected and used to measure B lymphocyte proliferation. Splenocytes were isolated, from which antigen-specific cellular immune responses were detected via ELISpot, ELISA and intracellular cytokine staining. The results revealed that both types of polysaccharides induce more rapid changes and higher VNA titers than aluminum hydroxide. Flow cytometry assays revealed that the polysaccharides activated more B lymphocytes in the lymph nodes and more B and T lymphocytes in the blood than aluminum hydroxide. Antigen-specific cellular immune responses showed that IIP-2 strongly induced T lymphocyte proliferation in the spleen and high levels of cytokine secretion from splenocytes, whereas aluminum hydroxide induced proliferation in only a small number of lymphocytes and the secretion of only small quantities of cytokines. Collectively, these data suggest that the polysaccharide IIP-2 exhibits excellent adjuvant activity and can enhance both cellular and humoral immunity.

Production and characterization of a fusion peptide derived from the rabies virus glycoprotein (RVG29).

Gene therapy targeting the brain holds great promise in curing nervous system degenerative diseases in clinical applications. With this in mind, in a previous study a 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) with a nonamer stretch of arginine residues (RVG29-9R) at its carboxy-terminus was exploited as a ligand for brain-targeting gene delivery. Importantly, the report demonstrated that the RVG29-9R vector was able to cross the blood-brain barrier. RVG29-9R is currently synthesized by commercial companies with high associated costs. In this study, in order to reduce the costs of producing RVG29-9R, we have expressed and purified 6mg of a recombinant peptide (RVG29-9R-6His) from 0.4g of cultured Escherichia coli. We assessed the physiochemical properties of RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro 2a cells (which express the acetylcholine receptor). Our results reveal that the RVG29-9R-6His peptide recognized Neuro 2a cells in a dose-dependent manner and it was also able to bind plasmid DNA and deliver it into the Neuro 2a cells effectively. Therefore, our study has demonstrated that the recombinant RVG29-9R-6His peptide retains the functions of RVG29-9R and so may provide an economically viable and alternative production method for the manufacture of RVG29-9R.

Generation of recombinant rabies Virus CVS-11 expressing eGFP applied to the rapid virus neutralization test.

The determination of levels of rabies virus-neutralizing antibody (VNA) provides the foundation for the quantitative evaluation of immunity effects. The traditional fluorescent antibody virus neutralization test (FAVN) using a challenge virus standard (CVS)-11 strain as a detection antigen and staining infected cells with a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody, is expensive and high-quality reagents are often difficult to obtain in developing countries. Indeed, it is essential to establish a rapid, economical, and specific rabies virus neutralization test (VNT). Here, we describe a recombinant virus rCVS-11-eGFP strain that stably expresses enhanced green fluorescent protein (eGFP) based on a reverse genetic system of the CVS-11 strain. Compared to the rCVS-11 strain, the rCVS-11-eGFP strain showed a similar growth property with passaging stability in vitro and pathogenicity in vivo. The rCVS-11-eGFP strain was utilized as a detection antigen to determine the levels of rabies VNAs in 23 human and 29 canine sera; this technique was termed the FAVN-eGFP method. The good reproducibility of FAVN-eGFP was tested with partial serum samples. Neutralization titers obtained from FAVN and FAVN-eGFP were not significantly different. The FAVN-eGFP method allows rapid economical, specific, and high-throughput assessment for the titration of rabies VNAs.

An inactivated recombinant rabies CVS-11 virus expressing two copies of the glycoprotein elicits a higher level of neutralizing antibodies and provides better protection in mice.

The rabies virus (RABV) G protein is the primary contributor to the pathogenicity and protective immunity of RABV. In this study, we generated a recombinant rCVS-11-G strain containing two copies of the G protein derived from the pathogenic wild-type (wt) CVS-11 strain and based on its infectious clone. Compared with the wtCVS-11 strain, the rCVS-11-G strain possessed a larger virion and 1.4-fold more G protein, but it exhibited a similar growth property to the rCVS-11 strain, including passaging stability in vitro. qPCR results showed that the two G genes were over-expressed in BHK-21 cells infected with the rCVS-11-G strain. However, the rCVS-11-G strain presented an 80 % lower LD50 than the wtCVS-11 strain when intracranially (i.c.) inoculated in adult mice. Adult mice that were either intracranially (i.c.) or intramuscularly (i.m.) inoculated with rCVS-11-G strain developed more acute neurological symptoms and greater mortality than those inoculated with the wtCVS-11 strain. Furthermore, the rCVS-11-G strain was more easily and rapidly taken up by neuroblastoma cells. These data indicated that the rCVS-11-G strain might have increased neurotropism because of the over-expression of the pathogenic G protein. The inactivated rCVS-11-G strain induced significantly higher levels of virus neutralization antibodies and provided better protection from street rabies virus challenge in mice. Therefore, the rCVS-11-G strain may be a promising inactivated vaccine strain due to its better immunogenicity.

Selection of an aptamer against rabies virus: a new class of molecules with antiviral activity.

Rabies is a fatal central nervous system (CNS) disease caused by the neurotropic rabies virus (RABV). The therapeutic management of RABV infections is still problematic, and novel antiviral strategies are urgently required. We established the RVG-BHK-21 cell line, which expresses RABV glycoprotein on the cell surface, to select aptamers. Through 28 iterative rounds of selection, single-stranded DNA (ssDNA) aptamers were generated by exponential enrichment (SELEX). A virus titer assay and a real-time quantitative reverse transcription PCR (qRT-PCR) assay revealed that four aptamers could inhibit the replication of RABV in cultured baby hamster kidney (BHK)-21 cells. However, the aptamers did not inhibit the replication of other virus, e.g., canine distemper virus (CDV) and canine parvovirus (CPV). In addition, the GE54 aptamer was found to effectively protect mice against lethal RABV challenge. After inoculation with aptamers for 24h or 48h, followed by inoculation with CVS-11, approximately 25-33% of the mice survived. In summary, we selected aptamers that could significantly protect from a lethal dose of RABV in vitro and in vivo.

CpG/Poly (I:C) mixed adjuvant priming enhances the immunogenicity of a DNA vaccine against eastern equine encephalitis virus in mice.

Eastern equine encephalitis virus (EEEV) poses a serious public health threat in many countries. Therefore, developing efficient vaccine against EEEV remains an important challenge in the field of disease control. To identify immunogenic proteins in EEEV, we constructed an expression vector containing the protein coding genes C, E3, E2, 6k, and E1 (pcDNA3.1-C-E). After verifying the target gene expression in 293 T cells, we immunized BALB/c mice with the pcDNA3.1-C-E vector as a DNA vaccine in conjunction with either CpG or poly (I:C) or a mixture of both adjuvants and monitored various aspects of the immune response. After two immunizations, the mice vaccinated with antigen plus mixed CpG/poly (I:C) adjuvant exhibited significantly stronger IFN-gamma responses and generated high-level CD4(+) cell responses for the cytokines IL-2, IL-4, and IFN-γ and CD8(+) T cell responses for the cytokines IL-2 and IFN-γ compared to the mice vaccinated with the corresponding antigen plus CpG or poly(I:C) alone. In addition, the higher antibody titers against EEEV effectively neutralized the EEEV pseudoviruses in the group immunized with antigen plus mixed CpG/poly (I:C) adjuvant after tertiary immunization. This study demonstrates that the pcDNA3.1-C-E plasmids in conjunction with mixed CpG/poly (I:C) adjuvant priming maximize the cellular immune response and specific antibody generation in mice. Moreover, this mixed adjuvant priming provides a promising strategy for enhancing the immune effectiveness of a DNA vaccine against EEEV.