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In China, cats cause about 5% of human rabies cases. Rabies control in cats plays a role in achieving the ultimate goal of elimination of dog rabies-mediated human deaths. However, there is no cat-specific rabies vaccine in China yet. In this study, we constructed a recombinant rabies vaccine by using a felid herpesvirus 1 (FHV-1) isolate, and deleted the gI/E in the FHV-1 and replaced the region with a glycoprotein (G) of rabies virus (RABV) strain BD06 through homologous recombination. The recombinant virus FHV-RVG was recovered and purified, and the expression of RABV glycoprotein was verified by indirect immunofluorescent assay. For potency in cats, each animal was inoculated intranasally with 1 ml FHV-RVG at 106.5 TCID50. Blood samples were collected at defined intervals for antibody titration. The animals were challenged by herpes and rabies after completion of vaccination on day 180 and day 194, respectively. Our results demonstrated all vaccinated cats generated antibodies against both FHV-1 and RABV, and reached an arbitrary protective titer > 0.5 IU/ml for rabies viral neutralizing antibody (VNA) by day 14 post inoculation (dpi) and titer peaked on 30 dpi with VNA at 24.5 ± 10.23 IU/ml. All vaccinated cats presented no clinical signs of FHV-1 infection and survived rabies challenge, while the control cats had severe rhinotracheitis and died from rabies after challenge. All this demonstrated that the FHV-based recombinant vaccine is effective in protection against both FHV-1 and RABV infections.

A painless skin delivery of vaccine for disease prevention is of great advantage in improving compliance in patients. To test this idea as a proof of concept, we utilized a pDNA vaccine construct, pDNAg333-2GnRH that has a dual function of controlling rabies and inducing immunocontraception in animals. The pDNA was administered to mice in a nanoparticulate form delivered through the skin using the P.L.E.A.S.E.® (Precise Laser Epidermal System) microporation laser device. Laser application was well tolerated, and mild skin reaction was healed completely in 8 days. We demonstrated that adjuvanted nanoparticulate pDNA vaccine significantly upregulated the expression of co-stimulatory molecules in dendritic cells. After topical administration of the adjuvanted nano-vaccine in mice, the high avidity serum for GnRH antibodies were induced and maintained up to 9 weeks. The induced immune response was of a mixed Th1/Th2 profile as measured by IgG subclasses (IgG2a and IgG1) and cytokine levels (IFN-γ and IL-4). Using flow cytometry, we revealed an increase of CD8+ T-cells and CD45R B cells upon the administration of the adjuvanted vaccine. Our previous study used the same pDNA nanoparticulate vaccine through an IM route, and a comparable immune response was induced using P.L.E.A.S.E. However, the vaccine dose in the current study was four-fold less than what was applied through the IM route.We concluded that laser-assisted skin vaccination has a potential of becoming a safe and reliable vaccination tool for rabies vaccination in animals or even in humans for pre- or post-exposure prophylaxis.

The NIH potency test for human rabies vaccines has disadvantages for use, especially in developing countries where rabies is endemic and prophylaxis needs ample, rapid, and reliable vaccine supplies. In China, 60–75 million doses of human rabies vaccines are administered each year. Vaccine quality control is of paramount importance, as is the release of potency-validated vaccines. We intended to design an alternative to the NIH in vivo method, and developed a relative potency test using an ELISA. Using Pearson's correlation analysis, we found a close relationship between the rabies vaccine glycoprotein content in vitro and the potency values in vivo. We suggest the relative potency test developed here as a simplified method for human rabies vaccine quality control in China and a possible alternative to the NIH method.

Plasmid DNA (pDNA) vaccines have the potential for protection against a wide range of diseases including rabies but are rapid in degradation and poor in uptake by antigen-presenting cells. To overcome the limitations, we fabricated a pDNA nanoparticulate vaccine. The negatively charged pDNA was adsorbed onto the surface of cationic PLGA (poly ( d , l -lactide-co-glycolide))-chitosan nanoparticles and were used as a delivery vehicle. To create a hydrogel for sustainable vaccine release, we dispersed the pDNA nanoparticles in poloxamer 407 gel which is liquid at 4 °C and turns into soft gels at 37 °C, providing ease of administration and preventing burst release of pDNA. Complete immobilization of pDNA to cationic nanoparticles was achieved at a pDNA to nanoparticles ratio (P/N) of 1/50. Cellular uptake of nanoparticles was both time and concentration dependent and followed a saturation kinetics with Vmax of 11.389 µg/mL h and Km of 139.48 µg/mL. The in vitro release studies showed the nanoparticulate vaccine has a sustained release for up to 24 days. In summary, pDNA PLGA-chitosan nanoparticles were non-cytotoxic, their buffering capacity and cell uptake were enhanced, and sustained the release of pDNA. We expect our pDNA vaccine’s potency will be greatly improved in the animal studies.

On September 18, 2014, the Missouri Department of Health and Senior Services (MDHSS) was notified of a suspected rabies case in a Missouri resident. The patient, a man aged 52 years, lived in a rural, deeply wooded area, and bat sightings in and around his home were anecdotally reported. Exposure to bats poses a risk for rabies. After two emergency department visits for severe neck pain, paresthesia in the left arm, upper body tremors, and anxiety, he was hospitalized on September 13 for encephalitis of unknown etiology. On September 24, he received a diagnosis of rabies and on September 26, he died. Genetic sequencing tests confirmed infection with a rabies virus variant associated with tricolored bats. Health care providers need to maintain a high index of clinical suspicion for rabies in patients who have unexplained, rapidly progressive encephalitis, and adhere to recommended infection control practices when examining and treating patients with suspected infectious diseases.

Attenuated strains of rabies virus (RABV) have been used for oral vaccination of wild carnivores in Europe and North America. However, some RABV vaccines caused clinical rabies in target a...

Human rabies is an encephalitic disease transmitted by animals infected with lyssaviruses. The most common lyssavirus that causes human infection is rabies virus (RABV), the prototypic member of the genus. The incubation period of RABV in humans varies from few weeks to several months in some instances. During this prodromal period, neither antibodies nor virus is detected. Antibodies, antigen and nucleic acids are detectable only after the onset of encephalitic symptoms, at which point the outcome of the disease is nearly 100% fatal. Hence, the primary intervention for human RABV exposure and subsequent post-exposure prophylaxis relies on testing animals suspected of having rabies. The most widely used diagnostic tests in animals focus on antigen detection, RABV-encoded nucleoprotein (N protein) in brain tissues. N protein accumulates in the cytoplasm of infected cells as large and granular inclusions, which are visualized in infected brain tissues by immuno-microscopy using anti-N protein antibodies. In this study, we explored a mass spectrometry (MS) based method for N protein detection without the need for any specific antibody reagents or microscopy. The MS-based method described here is unbiased, label-free, requires no amplification and determines any previously sequenced N protein available in the database. The results demonstrate the ability of MS/MS based method for N protein detection and amino acid sequence determination in animal diagnostic samples to obtain RABV variant information. This study demonstrates a potential for future developments of rabies diagnostic tests based on MS platforms., Author summary Although rabies is almost always fatal after the symptom onset phase, it can be prevented by timely administration of post-exposure prophylaxis (PEP), which involves passive antibody transfer and vaccination. One of the primary laboratory confirmatory tests for RABV infection is antigen detection, directed against the RABV encoded N protein using anti-N protein specific antibodies, in central nervous system (CNS) tissue samples of animals. This immuno-microscopy based detection utilizes either fluorescent tags (direct detection) or chromogenic substrates (indirect) in brain impressions from animals in which rabies is suspected. In this study, we explored the detection of N protein by a novel mass spectrometry (MS) based method that is label-free and does not require target amplification. The MS method specifically detected N protein in brain tissue and identified RABV variants based on amino acid sequence information. To our knowledge, this is the first report of an N protein detection method that does not utilize either antibodies or microscopy. This method provides an alternative platform for the development of future rabies diagnostic tests.

The rabies virus (RABV) glycoprotein (G) is responsible for inducing neutralizing antibodies against rabies virus. Development of recombinant vaccines using the G genes from attenuated strains rather than street viruses is a regular practice. In contrast to this scenario, we generated three human adenovirus type 5 recombinants using the G genes from the vaccine strains SRV9 and Flury-LEP, and the street RABV strain BD06 (nrAd5-SRV9-G, nrAd5-Flury-LEP-G, and nrAd5-BD06-G). These recombinants were non-replicative, but could grow up to ~10(8) TCID50/ml in helper HEK293AD cells. Expression of the G protein was verified by immunostaining, quantitative PCR and cytometry. Animal experiments revealed that immunization with nrAd5-BD06-G can induce a higher seroconversion rate, a higher neutralizing antibody level, and a longer survival time after rabies virus challenge in mice when compared with the other two recombinants. Moreover, the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly higher in mice immunized with nrAd5-BD06-G, which might also contribute to the increased protection. These results show that the use of street RABV G for non-replicative systems may be an alternative for developing effective recombinant rabies vaccines.

OBJECTIVE To assess the immunogenicity of thermostable live-attenuated rabies virus (RABV) preserved by vaporization (PBV) and delivered to the duodenal mucosa of a wildlife species targeted for an oral vaccination program. ANIMALS 8 gray foxes (Urocyon cinereoargenteus). PROCEDURES Endoscopy was used to place RABV PBV (n = 3 foxes), alginate-encapsulated RABV PBV (3 foxes), or nonpreserved RABV (2 foxes) vaccine into the duodenum of foxes. Blood samples were collected weekly to monitor the immune response. Saliva samples were collected weekly and tested for virus shedding by use of a conventional reverse-transcriptase PCR assay. Foxes were euthanized 28 days after vaccine administration, and relevant tissues were collected and tested for presence of RABV. RESULTS 2 of 3 foxes that received RABV PBV and 1 of 2 foxes that received nonpreserved RABV seroconverted by day 28. None of the 3 foxes receiving alginate-encapsulated RABV PBV seroconverted. No RABV RNA was detected in saliva at any of the time points, and RABV antigen or RNA was not detected in any of the tissues obtained on day 28. None of the foxes displayed any clinical signs of rabies. CONCLUSIONS AND CLINICAL RELEVANCE Results for this study indicated that a live-attenuated RABV vaccine delivered to the duodenal mucosa can induce an immune response in gray foxes. A safe, potent, thermostable RABV vaccine that could be delivered orally to wildlife or domestic animals would enhance current rabies control and prevention efforts.

Immunocontraceptive vaccines may be an alternative to surgical sterilization. Dual rabies vaccination and dog population management is a helpful tool for rabies prevention. A synthetic gonadotropin-releasing hormone (GnRH) peptide coupled to a carrier protein or T cell epitope is efficacious in inducing immunocontraception in a variety of mammals. However, virus-vectored GnRH recombinant vaccines have advantages over the conjugation method. In a previous in vitro study, we were able to insert a GnRH-coding sequence into the rabies virus (RABV) glycoprotein (G) gene, and the recombinant viruses grew to high titers in cells. Here, we further focused on the RABV G in accepting various copy numbers of GnRH. We demonstrated although RABV G protein with up to 4 copies of GnRH was well expressed, the recombinant virus was recovered only when 2 copies of GnRH (20 amino acids) were incorporated into the G, indicating a possible insertion limit in making a full infectious clone. The investigation provides insight into the utility of RABV G as a carrier for small peptides and its suitability for vaccine studies. Following our previous study, we selected ERAg3p/2GnRH and tested the construct in mice. The vaccine induced ⩾80% infertility after three doses without any adjuvant, in live (8 of 10 mice infertility) or inactivated (13 of 14 mice infertility) formulations; while the pregnancy rate was 100% (10 of 10 mice) in the controls. This initial success of immunocontraception in mice is promising, and we are now optimizing the vaccine formulation by using adjuvants and exploring novel delivery methods to minimize the dosage.

A major challenge for global rabies prevention and control is the lack of sufficient and affordable high quality vaccines. Such candidates should be pure, potent, safe, effective and economical to produce, with broad cross-reactivity against viral variants of public health and veterinary importance. The history of licensed human vaccines reviewed herein demonstrates clearly how the field has evolved to the current state of more passive development and postexposure management. Modern cell culture techniques provide adequate viral substrates for production of representative verified virus seeds. In contrast to outdated nervous tissue-based rabies vaccines, once a suitable substrate is identified, production of high titer virus results in a major qualitative and quantitative difference. Given the current scenario of only inactivated vaccines for humans, highly cell-adapted and stable, attenuated rabies viruses are ideal candidates for consideration to meet the need for seed viruses in the future.

While current rabies post-exposure prophylaxis (PEP) is highly effective, it is costly and the vaccination regimen is complicated, requiring both inactivated vaccines and immunoglobulins. A one-dose rabies vaccine for human PEP remains a long-term goal. Here, we describe development of a highly attenuated rabies virus ERAg3m, with a mutation in the glycoprotein (G) gene and a switch of the G gene with the matrix protein gene in the viral genome. After a one-dose intramuscular vaccination, the ERAg3m virus protected 100% of mice and hamsters from lethal challenge. In co-infections, using a lethal dose of street rabies virus mixed with ERAg3m, 100% of hamsters and 90% of mice survived and were protected against subsequent infection. A mock co-infection, using inactivated commercial human rabies vaccine and a lethal dose of street rabies virus, protected 100% and 40% of hamsters and mice, respectively. In co-infections, when vaccine was administrated in the left leg and challenge virus in the right leg, the ERAg3m virus protected 40% of mice, while the inactivated vaccine showed no protection. Therefore, live attenuated rabies virus when given pre-exposure or co-infected with street rabies virus, is capable of preventing rabies in two different animal models. Overall, this highly attenuated live rabies virus offered better protection than the inactivated vaccine.

Rabies in Asia is emerging as a serious public health issue. To explore the possible origin, phylogenetic relationships, and evolutionary dynamics of Asian Rabies viruses (RABV), we examined 200 complete nucleoprotein (N) gene sequences from RABV isolates in the region. Phylogeny supported the classification of Asian RABVs into five distinct clusters in lyssavirus genotype 1. Our geospatial and temporal analyses demonstrated that China appears to be the prime source of Asian RABVs. Understanding of rabies transmission and associated human activities, such as dog translocation, can help rabies control and elimination in Asia through collaborative efforts or programs.

The effectiveness of rabies vaccination in both humans and animals is determined by the presence of virus neutralizing antibodies (VNAs). The Rapid Fluorescent Focus Inhibition Test (RFFIT) is the method traditionally used for detection and quantification of VNAs. It is a functional in vitro test for assessing the ability of antibodies in serum to bind and prevent infection of cultured cells with rabies virus (RABV). The RFFIT is a labor intensive, low throughput and semi-quantitative assay performed by trained laboratorians. It requires staining of RABV-infected cells by rabies specific fluorescent antibodies and manual quantification of fluorescent fields for titer determination. Although the quantification of fluorescent fields observed in each sample is recorded, the corresponding images are not stored or captured to be used for future analysis. To circumvent several of these disadvantages, we have developed an alternative, automated high throughput neutralization test (HTNT) for determination of rabies VNAs based on green fluorescent protein (GFP) expression by a recombinant RABV and compared with the RFFIT. The HTNT assay utilizes the recombinant RABV ERA variant expressing GFP with a nuclear localization signal (NLS) for efficient quantification. The HTNT is a quantitative method where the number of RABV-infected cells are determined and the images are stored for future analysis. Both RFFIT and HTNT results correlated 100% for a panel of human and animal positive and negative rabies serum samples. Although, the VNA titer values are generally agreeable, HTNT titers tend to be lower than that of RFFIT, probably due to the differences in quantification methods. Our data demonstrates the potential for HTNT assays in determination of rabies VNA titers., Author summary The potency of rabies vaccine is demonstrated by the presence of virus neutralizing antibodies (VNAs) in serum. It is critical to evaluate immunologic status of individuals who work directly with rabies virus (RABV) (laboratorians) or at high risk of infection due to interaction with animals (veterinarians and animal control workers). In addition, rabies vaccination records and demonstration of VNAs in animals are mandatory before initiating pet travel to rabies-free counties or regions. Rabies VNAs are currently determined by the rapid fluorescent focus inhibition test (RFFIT) and the fluorescent antibody virus neutralization (FAVN) test, which measure the ability of antibodies to bind and prevent infection of RABV in vitro. Both assays require staining of infected cells using anti-rabies antibodies and manual observation of infected cells by a fluorescent microscope to determine VNA titers. In this study, we have developed a GFP reporter-based high throughput neutralization test (HTNT) for automated quantification of infected cells. This method has the advantages of allowing investigators to analyze and store the results, and can accommodate large sample sizes. Overall, the results from HTNT exhibited 100% correlation with that of RFFIT, albeit with differences in rabies VNA titer values due to quantification methods.

Background Rabies is a serious reemerging zoonosis in China. The molecular evolution and transmission patterns of rabies virus inferred from historical data can provide guidelines for better disease control and prevention in the future. Objectives To investigate the epidemiology and evolutionary dynamics of the rabies virus in China. Study design The molecular evolution of 132 viral glycoprotein gene sequences of Chinese rabies viruses collected in 17 provinces and 3 municipalities between 1969 and 2009 was analyzed. Results Phylogenetic analysis revealed that Chinese rabies viruses are subdivided into 6 lineages (A–F) within Lyssavirus genotype 1. Lineage A represents the widely dispersed cosmopolitan lineage while lineage B is closely related to Arctic-like rabies viruses. The remaining lineages (C–F) are typical of those circulating across much of Southeast Asia. The evolutionary rate for Chinese rabies virus was 1.532×10 −4 substitutions per site per year, and the corresponding common ancestor was in about 1115. Conclusions The phylogeographic structure demonstrated Chinese rabies viruses have been transmitted intra-provincially and extra-provincially due to human-related activities.

Enhancing DNA vaccine effectiveness remains a challenge, especially if the desired goal is immunization efficacy after a single dose. The glycoprotein gene from the rabies virus Evelyn-Rokitnicki-Abelseth (ERA) strain was modified by mutation at amino acid residue 333 from arginine to glutamine. The modified and original unmodified glycoprotein genes were cloned separately and developed as DNA vaccines for immunization in mice. The intramuscular (IM) route using a single dose (100 microg) of a modified DNA vaccine showed virus neutralizing antibody induction by d30, and 80% of the mice survived a challenge in which 100% of unvaccinated controls succumbed. Similar results were obtained using a single dose (10 microg) by the intradermal (ID) route with one-tenth amount of the DNA administered. Administration of single dose of DNA vaccine with unmodified G did not result in the production of detectable levels of virus neutralizing antibody by d30. The results of the IM and the ID routes of administration were statistically significant (P0.01). Based on these preliminary results, a modified glycoprotein gene from the ERA rabies virus strain may be an ideal candidate for DNA vaccine efficacy enhancement.

Rabies prevention and appropriate population management of free-ranging animals have an important role to play in the eventual elimination of rabies in dogs. An effective sterilant based on rabies vaccines has the potential to create a supportive measure of public acceptability and to reduce associated clinic visit costs. We inserted the coding sequence of gonadotropin-releasing hormone (GnRH) into different locations within the rabies virus ERA glycoprotein (G) gene, and demonstrated that the amino terminus (N), antigenic site IIa, and the junction between the ecto- and cytoplasmic domains (C) of the G were suitable sites for GnRH insertion. The rescued recombinant rabies viruses ERA-N-GnRH and ERA-C-GnRH grew as well as the parental ERA virus, reaching 1x10(9)ffu/ml in cell culture. Insertion and expression of the GnRH were stable in the viruses after multiple passages in vitro. To increase immunogenicity of the GnRH peptide, two copies of GnRH, aligned in tandem, were fused to the N terminus of the G. The recombinant rabies virus ERA-N-2GnRH was recovered and grown to high titers in cell culture. All GnRH-carrying rabies viruses induced antibodies against GnRH in immunized mice and protected 100% of the animals after rabies virus challenge. The recombinant viruses reacted strongly with the serum from a GonaCon-immunized animal. The GnRH-carrying rabies viruses have significant potential in rabies and animal population control.

Despite progress in vaccine development in the past century the mechanisms behind immune responses elicited by rabies biologics or via natural infection remain largely unknown. In this study, we compared protection elicited by standard, early, or delayed prophylaxis with a reduced number of vaccine doses using inactivated and live-attenuated vaccines. Two-month-old Syrian hamsters, 4-week-old ICR mice or adult rhesus macaques were inoculated with canine rabies virus variants. Thereafter, prophylaxis was initiated 6 h, 1, 2, 3, 4, 5, 6 or 7 days post-exposure (p.e.). One or several doses of inactivated (HDCV), or reverse genetically attenuated (live), or gamma-irradiated (inactivated)-ERAG333 vaccines were administered intramuscularly. The dynamics of virus spread were measured over time in the rodent models. Rabies virus reached the spinal cord at day 4 and brain at day 6 p.e. All hamsters succumbed in groups in which live ERAG333 was delayed until days 5 and 6 p.e. However, 78%, 44%, 56% and 22% of hamsters survived when one dose of live ERAG333 was administered 6 h, 1, 2, 3, and 4 days p.e., respectively. Similarly, 67% survived when inactivated ERAG333 was administered at 24 h p.e. All hamsters succumbed when standard prophylaxis (the Essen regimen) was delayed until days 3–6, but 67% and 33% of hamsters survived when PEP began 1 or 2 days p.e., respectively. Macaques were protected by one dose of attenuated ERAG333 at 24 h p.e. The highly attenuated (live) and inactivated ERAG333 vaccines elicited potent protective immune responses, even when prophylaxis initiation was delayed. When 2–5 doses of commercial vaccine and HRIG were administered according to the Essen scheme, 89–100% of the animals survived. Reduced vaccine schedules provided efficacious intervention, regardless of the total number of vaccine doses administered.

Standardized protocols and diagnostic-based surveillance are imperative for detection and elimination., Rabies is a reemerging disease in China. The high incidence of rabies leads to numerous concerns: a potential carrier-dog phenomenon, undocumented transmission of rabies virus from wildlife to dogs, counterfeit vaccines, vaccine mismatching, and seroconversion testing in patients after their completion of postexposure prophylaxis (PEP). These concerns are all scientifically arguable given a modern understanding of rabies. Rabies reemerges periodically in China because of high dog population density and low vaccination coverage in dogs. Mass vaccination campaigns rather than depopulation of dogs should be a long-term goal for rabies control. Seroconversion testing after vaccination is not necessary in either humans or animals. Human PEP should be initiated on the basis of diagnosis of biting animals. Reliable national systemic surveillance of rabies-related human deaths and of animal rabies prevalence is urgently needed. A laboratory diagnosis–based epidemiologic surveillance system can provide substantial information about disease transmission and effective prevention strategies.

Individual lyssavirus genes were evaluated for phylogenetic studies from available full genome sequences. The full genome of the ERA rabies virus was sequenced and its accuracy was confirmed through virus recovery by reverse genetics. The full length of the ERA is 11,931 nucleotides (nt), with a leader sequence of 58 nt, the nucleoprotein (N) gene of 1350 nt, phosphoprotein (P) gene of 891 nt, matrix protein (M) gene of 606 nt, glycoprotein (G) gene of 1572 nt, RNA-dependent RNA polymerase (L) gene of 6384 nt, Psi-region (or G–L intergenic region) of 400 nt, and a trailer region of 70 nt. The five mono-cistrons are separated by intergenic regions of 2, 5, 5 and 24 nt, respectively. One obvious difference between the ERA and SAD-B19 rabies virus strains was the putative stop/polyadenylation signal of the G gene, with a poly(A 8 ) tract for ERA, and a poly(A 5 ) for SAD-B19. The TGpoly(A 8 ) sequence tract was identified to be a leaky termination signal in the ERA strain. Through analyses of nt diversity, protein co-variations, structural and functional constraints, and reconstruction of phylogenetic trees from comprehensive datasets, we propose lyssavirus genes probably are of similar value for phylogenetic analyses.

It has been reported that phosphorylation of rabies virus N plays an important role in the process of viral transcription and replication. Rabies virus N is phosphorylated when expressed alone, indicating that cellular kinase phosphorylates rabies virus N. To identify what cellular kinase phosphorylates rabies virus N, the N was expressed in Escherichia coli and purified by metal affinity chromatography. The recombinant N was phosphorylated by BHK cellular extracts and by purified CK-II. In addition, the phosphorylation of the recombinant N in vitro can be blocked by a CK-II inhibitor, heparin. Furthermore, N phosphorylation in the virus-infected cells can be inhibited by a CK-II specific inhibitor, 5,6-dichloro-beta-D-ribofuranosyl benzimidazole. However, PKC did not phosphorylate the recombinant N in vitro; nor did staurosporine, a PKC and other kinase inhibitor, prevent rabies virus N from phosphorylation. Thus, our data demonstrate that cellular CK-II phosphorylates rabies virus N.

A rabies vaccine that is thermostable over a range of ambient environmental temperatures would be highly advantageous, especially for tropical regions with challenging cold-chain storage where canine rabies remains enzootic resulting in preventable human mortality. Live attenuated rabies virus (RABV) strain ERAg333 (R333E) was preserved by vaporization (PBV) in a dry, stable foam. RABV stabilized using this process remains viable for at least 23 months at 22°C, 15 months at 37°C, and 3 hours at 80°C. An antigen capture assay revealed RABV PBV inactivated by irradiation contained similar levels of antigen as a commercial vaccine. Viability and antigen capture testing confirmed that the PBV process stabilized RABV with no significant loss in titer or antigen content. Live attenuated and inactivated RABV PBV both effectively induced RABV neutralizing antibodies and protected mice from peripheral rabies virus challenge. These results demonstrate that PBV is an efficient method for RABV stabilization.

Even though rabies is almost uniformly fatal, it is readily preventable with currently available tools. Vaccination is highly efficacious for the pre-exposure prophylaxis (PrEP) of rabies in humans and animals, and prompt postexposure prophylaxis (PEP) with vaccine and rabies immune globulin (RIG) can reliably prevent disease in humans. However, access to these tools and knowledge of their proper use are often limited, especially in impoverished, rabies-enzootic countries with the highest disease burden. In the absence of reliable diagnostic capacity and risk assessments, vaccines and RIG are often administered inappropriately, leading to chronic supply shortages and otherwise preventable deaths. Rather than focusing solely on human prophylaxis, it is more cost-effective over the long term to eliminate canine rabies in its natural terrestrial reservoirs. Because more than 99% of human rabies deaths result from dog bites, prevention efforts should focus on dogs. A versatile “One Health” strategy for canine rabies elimination should aim to create sustainable herd immunity in dogs, using proven vaccination strategies at the local level, coupled with community education and humane population management. Such strategies have succeeded in both developed and developing countries, and can be adapted to any locality. Numerous examples in Africa, Asia, and Latin America have shown that community-based, locally guided vaccination and education programs, based on a shared vision and long-term commitment, can eliminate canine rabies. Such programs should have specific goals and measurable outcomes, and should be conducted under the guidance of supportive governments, in collaboration with international partners and nongovernmental organizations. In addition to currently available tools, rabies prevention can be augmented by new dose-sparing human vaccine schedules, alternative routes of vaccine administration, monoclonal antibodies as an alternative to RIG, sensitive and specific point-of-care diagnostics and the development of canine immunocontraceptive methods. Accurate risk assessments of potential human exposures and support for decentralized laboratory capacity will be essential to ensure the most effective utilization of vaccines and RIG until canine rabies has been eliminated.

We present an unconventional approach to antiviral drug discovery, which is used to identify potent small molecules against rabies virus. First, we conceptualized viral capsid assembly as occurring via a host-catalyzed biochemical pathway, in contrast to the classical view of capsid formation by self-assembly. This suggested opportunities for antiviral intervention by targeting previously unappreciated catalytic host proteins, which were pursued. Second, we hypothesized these host proteins to be components of heterogeneous, labile, and dynamic multi-subunit assembly machines, not easily isolated by specific target protein-focused methods. This suggested the need to identify active compounds before knowing the precise protein target. A cell-free translation-based small molecule screen was established to recreate the hypothesized interactions involving newly synthesized capsid proteins as host assembly machine substrates. Hits from the screen were validated by efficacy against infectious rabies virus in mammalian cell culture. Used as affinity ligands, advanced analogs were shown to bind a set of proteins that effectively reconstituted drug sensitivity in the cell-free screen and included a small but discrete subfraction of cellular ATP-binding cassette family E1 (ABCE1), a host protein previously found essential for HIV capsid formation. Taken together, these studies advance an alternate view of capsid formation (as a host-catalyzed biochemical pathway), a different paradigm for drug discovery (whole pathway screening without knowledge of the target), and suggest the existence of labile assembly machines that can be rendered accessible as next-generation drug targets by the means described.

Purpose: Pakistan is one of the few countries where Rabies is endemic and a great threat to humans as well as live stocks. A large number of individuals died of rabies exposure due to either the limited access to rabies vaccines or the side-effects of sheep brain -originated Sample vaccine. Cell culture-derived rabies vaccines are mostly unaffordable to the needed population under rabies threat. Development of a safer and more affordable vaccine is necessary in Pakistan. Here, we developed two novel recombinant rabies virus vaccines using a local Pakistan rabies virus glycoprotein gene, and tested the efficacy of vaccines in mice. Methods: The glycoprotein gene (RVG) of vector ERAg3p and ERAg3m was substituted, respectively, with a modified Pakistani RVG. The resulting recombinant vectors were applied for reverse genetics to recover two vaccine viruses, PK-SG and PK-DG. The efficacy of PK-SG and PK-DG were tested in mice by intramuscular injection and oral delivery. Results: All mice survived the challenge after intramuscular vaccination using PK-SG, or PK-DG. In the oral vaccination groups, 80% mice with PK-SG and 90% mice with PK-DG survived the challenge. Meanwhile, 80% of the unvaccinated control mice succumbed after challenge. The mean rabies virus neutralizing antibody titers was ≥0.5 IU/ml in all vaccinated groups. Conclusion: Our results demonstrated the efficacy of PK-SG and PK-DG in rabies vaccination in mice. The two recombinant virus strains may be good vaccine candidates for the target animals and humans in Pakistan. Detailed investigations are necessary in the future.

We characterized the proteomes of murine N2a cells following infection with three rabies virus (RV) strains, characterized by distinct virulence phenotypes (i.e., virulent BD06, fixed CVS-11, and attenuated SRV9 strains), and identified 35 changes to protein expression using two-dimensional gel electrophoresis in whole-cell lysates. The annotated functions of these proteins are involved in various cytoskeletal, signal transduction, stress response, and metabolic processes. Specifically, a-enolase, prx-4, vimentin, cytokine-induced apoptosis inhibitor 1 (CIAPIN1) and prx-6 were significantly up-regulated, whereas Trx like-1 and galectin-1 were down-regulated following infection of N2a cells with all three rabies virus strains. However, comparing expressions of all 35 proteins affected between BD06-, CVS-11-, and SRV9-infected cells, specific changes in expression were also observed. The up-regulation of vimentin, CIAPIN1, prx-4, and 14-3-3 theta/delta, and downregulation of NDPK-B and HSP-1 with CVS and SRV9 infection were ≥ 2 times greater than with BD06. Meanwhile, Zfp12 protein, splicing factor, and arginine/serine-rich 1 were unaltered in the cells infected with BD06 and CVS- 11, but were up-regulated in the group infected with SRV9. The proteomic alterations described here may suggest that these changes to protein expression correlate with the rabies virus' adaptability and virulence in N2a cells, and hence provides new clues as to the response of N2a host cells to rabies virus infections, and may also aid in uncovering new pathways in these cells that are involved in rabies infections. Further characterization of the functions of the affected proteins may contribute to our understanding of the mechanisms of RV infection and pathogenesis.

In recent years, no major paradigm shifts have occurred in the utilization of new products for the prevention and control of rabies. Development of new cost-effective rabies biologics and antiviral drugs is critical in continuing to prevent and reduce disease. Current rabies vaccines are highly effective but have developed largely based on technical improvements in the vaccine industry. In the future, alternative approaches for improved vaccines, including novel avirulent rabies virus (RABV) vectors, should be pursued. Any rabies vaccine that is effective without the need for rabies immune globulin (RIG) will contribute fundamentally to disease prevention by reducing the cost and complexity of postexposure prophylaxis (PEP). The lack of high quality, affordable RIG is a continuing problem. Virus-specific monoclonal antibodies (mAbs) will soon fulfill the PEP requirement for passive immunity, currently met with RIG. Several relevant strategies for mAb production, including use of transgenic mice, humanization of mouse mAbs, and generation of human immune libraries, are underway. As a result of successful PEP and pre-exposure prophylaxis in developed countries, until recently, no significant focused efforts have been devoted to RABV-specific antiviral agents. To date, combination therapy including broad spectrum antiviral agents has been successful in only one case, and reports of antiviral activity are often conflicting. Current antiviral strategies target either the nucleoprotein or phosphoprotein, but drugs targeting the viral polymerase should be considered. Considering the lag from creation of new concepts to experimental development and clinical trials, many years will likely elapse between today's ideas and tomorrow's practices.

Background The frequent occurrence of ferret badger-associated human rabies cases in southeast China highlights the lack of laboratory-based surveillance and urges revisiting the potential importance of this animal in rabies transmission. To determine if the ferret badgers actually contribute to human and dog rabies cases, and the possible origin of the ferret badger-associated rabies in the region, an active rabies survey was conducted to determine the frequency of rabies infection and seroprevalence in dogs and ferret badgers. Methods A retrospective survey on rabies epidemics was performed in Zhejiang, Jiangxi and Anhui provinces in southeast China. The brain tissues from ferret badgers and dogs were assayed by fluorescent antibody test. Rabies virus was isolated and sequenced for phylogenetic analysis. The sera from ferret badgers and dogs were titrated using rabies virus neutralizing antibodies (VNA) test. Results The ferret badgers presented a higher percentage of rabies seroconversion than dogs did in the endemic region, reaching a maximum of 95% in the collected samples. Nine ferret badger-associated rabies viruses were isolated, sequenced, and were phylogenetically clustered as a separate group. Nucleotide sequence revealed 99.4-99.8% homology within the ferret badger isolates, and 83-89% homology to the dog isolates in the nucleoprotein and glycoprotein genes in the same rabies endemic regions. Conclusions Our data suggest ferret badger-associated rabies has likely formed as an independent enzootic originating from dogs during the long-term rabies infestation in southeast China. The eventual role of FB rabies in public health remains unclear. However, management of ferret badger bites, rabies awareness and control in the related regions should be an immediate need.

Ferret badger–associated human rabies cases emerged in China in 1994. We used a retrospective epidemiologic survey, virus isolation, laboratory diagnosis, and nucleotide sequencing to document its reemergence in 2002–2008. Whether the cause is spillover from infected dogs or recent host shift and new reservoir establishment requires further investigation.

Unlike vesicular stomatitis virus, rabies virus glycoprotein gene has not been successfully relocated closer to promoter-proximal regions by reverse genetics. Here we describe an efficient system for the Evelyn-Rokitnicki-Abelseth (ERA) rabies virus with the glycoprotein gene switched with the matrix protein gene, creating a reshuffled virus ERAgm (gene order N-P-G-M-L). With the aid of an autogene plasmid, the T7 RNA polymerase containing a nuclear location signal from the SV40 large T antigen facilitated virus recovery. The rearranged ERAgm rabies virus replicated as well as the parental ERA (gene order N-P-M-G-L) virus, reaching 10(9) ffu/ml in infected BSR cells. The altered glycoprotein gene position in viral genome presented an alternative way to study the pathogenicity of rabies virus. This also provides a potential novel method for rabies vaccine development.

The objective of this study was to determine if a replication defective recombinant adenovirus expressing rabies virus glycoprotein (Adrab.gp) given through a non-invasive vaccination route (by topical application) onto the skin (NIVS) could elicit an immune response and/or protection against rabies. Groups of mice were immunized by NIVS with various doses of Adrab.gp. For comparison, groups of mice were immunized intramuscularly, subcutaneously, or intradermally with Adrab.gp. Mice received two booster immunizations at 1 and 2 months after the first immunization. Virus neutralizing antibody (VNA) titers were measured at day 21 after the first and second immunizations and at day 14 after the third immunization. Fifty percent of the mice immunized by NIVS with 2 x 10(7) and 2 x 10(8)pfu Adrab.gp vaccine developed VNA, whereas none of the control mice or the mice immunized by NIVS with the lowest dose (2 x 10(6)pfu) of Adrab.gp virus developed VNA. However, this low dose induced high titers of VNA in mice immunized by parenteral routes. Two weeks after the last immunization, all the mice were challenged with a lethal dose of rabies virus. More than 70% of the animals immunized by NIVS with > or = 2 x 10(7)pfu Adrab.gp virus survived the challenge, whereas all the mice in the negative control group and the group immunized by NIVS with the lowest dose of Adrab.gp succumbed to rabies. Taken together, the results suggest that NIVS with Adrab.gp can induce VNA production and protection against lethal challenge with rabies virus in mice.