Genetic characteristics and pathogenicity of the first bluetongue virus serotype 20 strain isolated in China.

Bluetongue virus (BTV), a member of the genus Orbivirus in the family Reoviridae, is transmitted by biting midges and causes severe disease in domestic and wild ruminants. In the present study, a BTV strain, BTV-20/GX015/China/2013 (GX015), was isolated from sentinel cattle in Guangxi, China. Virus neutralization tests and phylogenetic analyses based on genomic segments 2 (S2) and 6 (S6) indicated that GX015 belongs to BTV serotype 20 (BTV-20) and represents a new topotype within BTV-20 strains, which makes GX015 the first BTV-20 strain isolated in China. Genomic analyses suggested that the 10 genomic segments of GX015 originated from a reassortment event, in which S2 and S6 are derived from exotic BTV-20 strains (South Africa or Australia), whereas the remaining eight genomic segments are apparently of Chinese origin and most likely share the same ancestor with a Taiwanese BTV-12 strain. Importantly, we evaluated the infectivity and pathogenicity of the BTV-20 strain in mice lacking the interferon receptor (IFNAR-/- mice, a good animal model for studying the pathogenesis, virulence and transmission of BTVs) and sheep for the first time, and found that GX015 causes severe disease and death in IFNAR-/- mice and clinical signs and viraemia in the natural host sheep. These results improve our understanding of the genetic characteristics, diversity and pathogenicity of BTVs, which is important for developing diagnostic methods and vaccines for the surveillance and prevention of bluetongue disease.

Etx-Y71A as a non-toxic mutant of Clostridium perfringens epsilon toxin induces protective immunity in mice and sheep.

Epsilon toxin (Etx) is an extremely potent toxin produced by Clostridium perfringens toxinotypes B and D, which cause fatal enterotoxemia in many livestock species, mainly sheep and goats. Our previous study demonstrated that the aromatic amino acid (AA) residue at position 71 in domain III of Etx is needed for its cytotoxic activity toward MDCK cells. Here, we first determined that Etx mutants with non-aromatic AA substitutions at Tyr71 lost lethality in mice, indicating that the aromatic AA residue at position 71 is a toxicity determinant of Etx in vivo. After intravenous injection with a high dose of the trypsin-activated Etx-Y71A mutant, mice did not show any histopathological lesions, and confocal microscopy observations further showed that Etx-Y71A lost the ability to cross the blood-brain barrier of the mice. These results suggested that the Etx-Y71A mutant is sufficiently safe in vivo to be a vaccine candidate. Furthermore, the immune efficacy of Etx-Y71A was evaluated in model and host animals. Mice inoculated with this mutant produced high levels of neutralizing antibodies and were completely protected from a 100 LD50 of trypsin-activated Etx challenge. Sheep immunized with Etx-Y71A produced high levels of neutralizing antibodies that provided protection in mice against an activated Etx challenge, and lambs could receive passive immunity through immunization of pregnant ewes. Additionally, homology modeling and circular dichroism analysis showed that Etx-Y71A has structural similarity to Etx, which provides a structural basis for Etx-Y71A retaining the immunogenicity of Etx. Taken together, these results suggest that Etx-Y71A is a potential vaccine candidate against Etx-inducing enterotoxemia.

Identification and complete-genome phylogenetic analysis of an epizootic hemorrhagic disease virus serotype 7 strain isolated in China.

An epizootic hemorrhagic disease virus (EHDV) strain designated YN09-04 was isolated from sentinel cattle in China. The length of its complete genome was 19,344 bp in total, consisting of 10 segments ranging in size from 810 bp (S10) to 3942 bp (S1). Based on phylogenetic analysis of the S2 sequence, YN09-04 clusters with EHDV serotype 7 (EHDV-7) strains form a distinct, well-supported subgroup, indicating that YN09-04 belongs to EHDV-7. However, the origin of the YN09-04 genome is very complex. The S2 and S6 of YN09-04 cluster with those of Japanese EHDV-7 strains, whereas the S1, S3, S4, S5 and S7 of YN09-04 share high nucleotide sequence identity and a close relationship with those of Japanese Ibaraki viruses, and the S8, S9 and S10 nucleotide sequences of YN09-04 are more similar to those of some Australian EHDV strains than to those of other isolates. These results suggest that the genome of YN09-04 likely originated from a reassortment event between EHDV strains that were similar to the current Japanese and Australian strains and that YN09-04 and some EHDVs from Japan and Australia share the same ancestors. This is the first report of the isolation, identification and complete-genome phylogenetic analysis of an EHDV-7 strain from China.

Incorporation of membrane-anchored flagellin or Escherichia coli heat-labile enterotoxin B subunit enhances the immunogenicity of rabies virus-like particles in mice and dogs.

Rabies remains an important worldwide public health threat, so safe, effective, and affordable vaccines are still being sought. Virus-like particle-based vaccines targeting various viral pathogens have been successfully produced, licensed, and commercialized. Here, we designed and constructed two chimeric rabies virus-like particles (cRVLPs) containing rabies virus (RABV) glycoprotein (G), matrix (M) protein, and membrane-anchored flagellin (EVLP-F) or Escherichia coli heat-labile enterotoxin B subunit (EVLP-L) as molecular adjuvants to enhance the immune response against rabies. The immunogenicity and potential of cRVLPs as novel rabies vaccine were evaluated by intramuscular vaccination in mouse and dog models. Mouse studies demonstrated that both EVLP-F and EVLP-L induced faster and larger virus-neutralizing antibodies (VNAs) responses and elicited greater numbers of CD4(+) and CD8(+) T cells secreting IFN-γ or IL-4 compared with a standard rabies VLP (sRVLP) containing only G and M. Moreover, cRVLPs recruited and/or activated more B cells and dendritic cells in inguinal lymph nodes. EVLP-F induced a strong, specific IgG2a response but not an IgG1 response, suggesting the activation of Th1 class immunity; in contrast, Th2 class immunity was observed with EVLP-L. The significantly enhanced humoral and cellular immune responses induced by cRVLPs provided complete protection against lethal challenge with RABV. Most importantly, dogs vaccinated with EVLP-F or EVLP-L exhibited increased VNA titers in sera and enhanced IFN-γ and IL-4 secretion from peripheral blood mononuclear cells. Taken together, these results illustrate that when incorporated into sRVLP, membrane-anchored flagellin, and heat-labile enterotoxin B subunit possess strong adjuvant activity. EVLP-F and EVLP-L induce significantly enhanced RABV-specific humoral and cellular immune responses in both mouse and dog. Therefore, these cRVLPs may be developed as safe and more efficacious rabies vaccine candidate for animals.

Using rabies virus vaccine strain SRV9 as viral vector to express exogenous gene.

Rabies virus (RABV) can cause a fatal neurological disease in human and animals, and vaccines were generally applied for the immunoprophylaxis of rabies. Here, a recombinant viral vector carrying the exogenous gene expression component between phosphoprotein (P) and matrix protein (M) genes of RABV was constructed based on the vaccine strain SRV9 used in China. To develop a reverse genetic system, the full-length cDNA plasmids of SRV9 were constructed using the eukaryotic expression vector pCI or pcDNA3.1(+). However, recovery efficiency based on the pcDNA3.1 vector was significantly higher than that of the pCI vector. The exogenous gene expression component PE-PS-BsiWI-PmeI or PS-BsiWI-PmeI-PE was introduced in different locations between the P and M genes of SRV9. When the enhanced green fluorescent protein (eGFP) was used as a reporter gene, both locations could rescue recombinant RABV (rRABV) expressing eGFP with high efficiency. Characterization of rRABV expressing eGFP in vitro revealed that its growth was similar to that of the parental virus. Animal experiments showed that rRABV expressing eGFP could replicate and express eGFP in the brains of suckling mice. Furthermore, rRABV of SRV9 was nonpathogenic for 3-week-old mice and could be cleared from the central nervous system at 5 days post-inoculation. Our results showed that the recombinant SRV9 virus could be used as a useful viral vector for exogenous gene expression.

Chimeric rabies virus-like particles containing membrane-anchored GM-CSF enhances the immune response against rabies virus.

Rabies remains an important public health threat in most developing countries. To develop a more effective and safe vaccine against rabies, we have constructed a chimeric rabies virus-like particle (VLP), which containing glycoprotein (G) and matrix protein (M) of rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, and membrane-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF), and it was named of EVLP-G. The immunogenicity and protective efficacy of EVLP-G against RABV were evaluated by intramuscular administration in a mouse model. The EVLP-G was successfully produced in insect cells by coinfection with three recombinant baculoviruses expressing G, M, and GM-CSF, respectively. The membrane-anchored GM-CSF possesses a strong adjuvant activity. More B cells and dendritic cells (DCs) were recruited and/or activated in inguinal lymph nodes in mice immunized with EVLP-G. EVLP-G was found to induce a significantly increased RABV-specific virus-neutralizing antibody and elicit a larger and broader antibody subclass responses compared with the standard rabies VLP (sRVLP, consisting of G and M). The EVLP-G also elicited significantly more IFN-γ- or IL-4-secreting CD4+ and CD8+ T cells than the sRVLP. Moreover, the immune responses induced by EVLP-G protect all vaccinated mice from lethal challenge with RABV. These results suggest that EVLP-G has the potential to be developed as a novel vaccine candidate for the prevention and control of animal rabies.

Isolation of ssDNA aptamers that inhibit rabies virus.

Aptamers, functional nucleic acids, capable of binding a variety of molecular targets with high affinity and specificity, have emerged as promising therapeutic agents. In this study, the cell surface-systematic evolution of ligands by exponential enrichment (Cell-SELEX) strategy was used to generate DNA aptamers which targeted to the intact rabies virus-infected live cells. Through 35 iterative rounds of selection, five high-affinity single-stranded DNA (ssDNA) aptamers were generated by cell-SELEX. Virus titer assay and real-time quantitative reverse transcription PCR (qRT-PCR) assay revealed that all five aptamers could inhibit replication of rabies virus (RABV) in cultured baby hamster kidney (BHK)-21 cells; and T14 and F34 aptamers were most effective. The qRT-PCR also showed a dose-dependent inhibitory effect in BHK-21 cells. Collectively, these data show the feasibility of generating functionally effective aptamers against rabies virus-infected cells by the Cell-SELEX iterative procedure. These aptamers may prove clinically useful as therapeutic molecules with specific antiviral potential against RABV infections.

Innate immune response gene expression profiles in central nervous system of mice infected with rabies virus.

The present study was focused on the modulation of innate immune response genes in CNS of mouse in response to rabies virus (RABV) infection. The global gene expression changes in brains of RABV- or mock-infected mice were investigated using DNA microarray analysis and quantitative real-time PCR. Then functional enrichment of the differentially expressed mRNAs was performed. Microarray analysis showed that 390 genes in brain were significantly (P<0.01) regulated in response to RABV infection, with obviously up-regulated genes like interferon (IFN) stimulated genes (ISGs), IFN inducible transcription factors, cytokines and complement, etc. The significant pathways of differentially expressed genes are mainly involved in JAK-STAT signaling pathway, antigen processing and presentation, ubiquitin mediated proteolysis and complement cascades. The results suggest that the modulated genes in infected CNS were possibly involved in pathogenesis of rabies. Conversely, they may have protective effects.