Human BST2 inhibits rabies virus release independently of cysteine-linked dimerization and asparagine-linked glycosylation.

The innate immune response is a first-line defense mechanism triggered by rabies virus (RABV). Interferon (IFN) signaling and ISG products have been shown to confer resistance to RABV at various stages of the virus's life cycle. Human tetherin, also known as bone marrow stromal cell antigen 2 (hBST2), is a multifunctional transmembrane glycoprotein induced by IFN that has been shown to effectively counteract many viruses through diverse mechanisms. Here, we demonstrate that hBST2 inhibits RABV budding by tethering new virions to the cell surface. It was observed that release of virus-like particles (VLPs) formed by RABV G (RABV-G VLPs), but not RABV M (RABV-G VLPs), were suppressed by hBST2, indicating that RABV-G has a specific effect on the hBST2-mediated restriction of RABV. The ability of hBST2 to prevent the release of RABV-G VLPs and impede RABV growth kinetics is retained even when hBST2 has mutations at dimerization and/or glycosylation sites, making hBST2 an antagonist to RABV, with multiple mechanisms possibly contributing to the hBST2-mediated suppression of RABV. Our findings expand the knowledge of host antiviral mechanisms that control RABV infection.

Efficiency Comparative Approach of Plant-Produced Monoclonal Antibodies against Rabies Virus Infection.

Rabies encephalitis is a fatal zoonotic viral disease caused by the neurotropic rabies virus. It remains a major public health concern as it causes almost 100% fatality and has no effective medication after the onset of the disease. However, this illness is preventable with the timely administration of effective post-exposure prophylaxis (PEP) consisting of the rabies vaccine and passive immune globulins (HRIG and ERIG). Recently, conventional PEP has been shown to have many limitations, resulting in little support for these expensive and heterologous globulins. Monoclonal antibody (mAb) production via recombinant technology in animal and human cell cultures, as well as a plant-based platform, was introduced to overcome the costly and high-tech constraints of former preparations. We used transient expression technology to produce two mAbs against the rabies virus in Nicotiana benthamiana and compared their viral neutralizing activity in vitro and in vivo. The expression levels of selective mAbs E559 and 62-71-3 in plants were estimated to be 17.3 mg/kg and 28.6 mg/kg in fresh weight, respectively. The plant-produced mAbs effectively neutralized the challenge virus CVS-11 strain in a cell-based RFFIT. In addition, the combination of these two mAbs in a cocktail protected hamsters from rabies virus infection more effectively than standard HRIG and ERIG. This study suggests that the plant-produced rabies antibody cocktail has promising potential as an alternative biological to polyclonal RIG in rabies PEP.

Establishment of Human-Induced Pluripotent Stem Cell-Derived Neurons-A Promising In Vitro Model for a Molecular Study of Rabies Virus and Host Interaction.

Rabies is a deadly viral disease caused by the rabies virus (RABV), transmitted through a bite of an infected host, resulting in irreversible neurological symptoms and a 100% fatality rate in humans. Despite many aspects describing rabies neuropathogenesis, numerous hypotheses remain unanswered and concealed. Observations obtained from infected primary neurons or mouse brain samples are more relevant to human clinical rabies than permissive cell lines; however, limitations regarding the ethical issue and sample accessibility become a hurdle for discovering new insights into virus-host interplays. To better understand RABV pathogenesis in humans, we generated human-induced pluripotent stem cell (hiPSC)-derived neurons to offer the opportunity for an inimitable study of RABV infection at a molecular level in a pathologically relevant cell type. This study describes the characteristics and detailed proteomic changes of hiPSC-derived neurons in response to RABV infection using LC-MS/MS quantitative analysis. Gene ontology (GO) enrichment of differentially expressed proteins (DEPs) reveals temporal changes of proteins related to metabolic process, immune response, neurotransmitter transport/synaptic vesicle cycle, cytoskeleton organization, and cell stress response, demonstrating fundamental underlying mechanisms of neuropathogenesis in a time-course dependence. Lastly, we highlighted plausible functions of heat shock cognate protein 70 (HSC70 or HSPA8) that might play a pivotal role in regulating RABV replication and pathogenesis. Our findings acquired from this hiPSC-derived neuron platform help to define novel cellular mechanisms during RABV infection, which could be applicable to further studies to widen views of RABV-host interaction.

Intracellular Spread of Rabies Virus Is Reduced in the Paralytic Form of Canine Rabies Compared to the Furious Form.

Studies of the furious and paralytic forms of canine rabies at the early stage of disease have shown a more rapid viral colonization of the cerebral hemispheres in the furious form, as measured by viral antigen within neuronal cell bodies and viral RNA levels. Measurement of cellular processes separate from neuronal cell body provides a visual record of the spread of rabies virus which occurs across synapses. In this study, the amount of rabies viral antigen within cell processes was quantitatively assessed by image analysis in a cohort of naturally rabies infected non-vaccinated dogs (5 furious and 5 paralytic) that were sacrificed shortly after developing illness. Measurements were taken at different levels of the spinal cord, brain stem, and cerebrum. Results were compared to the amount of rabies viral antigen in neuronal cell bodies. Generally, the amount of rabies viral antigen in cell processes decreased in a rostral direction, following the pattern for the amount of rabies viral antigen in neuronal cell bodies and the percentage of involved cell bodies. However, there was a delay in cell process involvement following cell body involvement, consistent with replication occurring in the cell body region and subsequent transport out to cell processes. Greater amounts of antigen were seen in cell processes in dogs with the furious compared to paralytic form, at all anatomic levels examined. This difference was even evident when comparing (1) neurons with similar amounts of antigen, (2) similar percentages of involved neurons, and (3) anatomic levels that showed 100% positive neurons. These findings suggest that intracellular transport of the virus may be slower in the paralytic form, resulting in slower viral propagation. Possible mechanisms might involve host-specific differences in intracellular virus transport. The latter could be cytokine-mediated, since previous studies have documented greater inflammation in the paralytic form.

Worldwide rabies deaths prevention--A focus on the current inadequacies in postexposure prophylaxis of animal bite victims.

The World Health Organization reports that over 60,000 humans die of rabies annually, worldwide. Most occur in remote regions of developing countries. Almost all victims received no postexposure rabies prophylaxis (PEP). There are no facilities or health personnel able to provide it in many areas where the disease is prevalent. A first approach to correct this problem would be by extending provision of modern PEP to areas where human rabies is most prevalent.

Alterations in brain cerebral cortex proteome of rabies-infected cat.

Comparative proteome analysis using brain cerebral cortex tissues from cats and dogs infected with/without rabies virus were conducted using both two-dimensional gel-electrophoresis (2-DE) and 2-D fluorescence difference gel- electrophoresis (2D-DIGE) methods. The 2-DE gel images of all samples revealed >1,000 protein spots in each gel. Quantitative intensity analysis revealed the same overall protein pattern in certain regions of the gel, but the rabies-infected brains exhibited more protein spots than the non-infected controls. From approximately 880 protein spots detected by 2D-DIGE, 65 protein spots were increased and 46 were decreased. Eight of these protein spots were randomly selected and annotated by reference to previous known proteome data of rabid dog brains. They were similarly altered in both of the rabies-infected cats and dogs. A more detailed comparison of changes in proteomic profiles of brains between rabid cats and dogs should shed some light on the pathophysiological mechanism of rabies in domestic animals, as most rabies cases have been traceable to or believed to have originated from rabid dogs.

Rabies in Asia: the classical zoonosis.

Rabies remains a constant threat to humans throughout much of Asia. The dog is the main reservoir and vector with wildlife playing a very minor role. No Asian country or region has been declared rabies free by WHO in over two decades and there is evidence of canine rabies spread to new regions during the past 10 years. We now have the knowledge and technology to control canine rabies. The main barrier in managing this costly endemic is lack of motivation by authorities to address this issue along with regional inability of public health and livestock (agriculture) officials to tackle this issue in cooperation and coordination. Rabies is one of the first recognized zoonoses and a model for a true "One Health" management goal where human; veterinary, and government officials must work together in harmony to defeat this disease.

Rabies-related knowledge and practices among persons at risk of bat exposures in Thailand.

BACKGROUND: Rabies is a fatal encephalitis caused by lyssaviruses. Evidence of lyssavirus circulation has recently emerged in Southeast Asian bats. A cross-sectional study was conducted in Thailand to assess rabies-related knowledge and practices among persons regularly exposed to bats and bat habitats. The objectives were to identify deficiencies in rabies awareness, describe the occurrence of bat exposures, and explore factors associated with transdermal bat exposures. METHODS: A survey was administered to a convenience sample of adult guano miners, bat hunters, game wardens, and residents/personnel at Buddhist temples where mass bat roosting occurs. The questionnaire elicited information on demographics, experience with bat exposures, and rabies knowledge. Participants were also asked to describe actions they would take in response to a bat bite as well as actions for a bite from a potentially rabid animal. Bivariate analysis was used to compare responses between groups and multivariable logistic regression was used to explore factors independently associated with being bitten or scratched by a bat. FINDINGS: Of 106 people interviewed, 11 (10%) identified bats as a potential source of rabies. A history of a bat bite or scratch was reported by 29 (27%), and 38 (36%) stated either that they would do nothing or that they did not know what they would do in response to a bat bite. Guano miners were less likely than other groups to indicate animal bites as a mechanism of rabies transmission (68% vs. 90%, p=0.03) and were less likely to say they would respond appropriately to a bat bite or scratch (61% vs. 27%, p=0.003). Guano mining, bat hunting, and being in a bat cave or roost area more than 5 times a year were associated with history of a bat bite or scratch. CONCLUSIONS: These findings indicate the need for educational outreach to raise awareness of bat rabies, promote exposure prevention, and ensure appropriate health-seeking behaviors for bat-inflicted wounds, particularly among at-risk groups in Thailand.

Rabies research in resource-poor countries.

Many cost-benefit/effective rabies research projects need to be carried out in less-developed canine-endemic regions. Among these are educational approaches directed at the public and governments. They would address effective primary wound care, availability, and proper use of vaccines and immunoglobulins, better reporting of rabies, final elimination of dangerous nerve tissue-derived vaccines, and the recognition that rabies is still expanding its geographic range. Such efforts could also reduce deaths in victims who had received no or less than adequate postexposure prophylaxis. There is a need for new technology in canine population control and sustainable vaccination. We have virtually no workable plans on how to control bat rabies, particularly that from hematophagous bats. Preexposure vaccination of villagers in vampire rabies-endemic regions may be one temporary solution. Current efforts to reduce further the time required and vaccine dose required for effective postexposure vaccination need to be encouraged. We still have incomplete understanding of the transport channels from inoculation site to rabies virus antibody generating cells. The minimum antigen dose required to achieve a consistently protective and lasting immune response has been established for intramuscular vaccine administration, but is only estimated for intradermal use. Greater knowledge may have clinical benefits, particularly in the application of intradermal reduced dose vaccination methods. Curing human rabies is still an unattained goal that challenges new innovative researchers.

Human rabies prevention--United States, 2008: recommendations of the Advisory Committee on Immunization Practices.

These recommendations of the Advisory Committee on Immunization Practices (ACIP) update the previous recommendations on human rabies prevention (CDC. Human rabies prevention--United States, 1999: recommendations of the Advisory Committee on Immunization Practices. MMWR 1999;48 [No. RR-1]) and reflect the status of rabies and antirabies biologics in the United States. This statement 1) provides updated information on human and animal rabies epidemiology; 2) summarizes the evidence regarding the effectiveness/efficacy, immunogenicity, and safety of rabies biologics; 3) presents new information on the cost-effectiveness of rabies postexposure prophylaxis; 4) presents recommendations for rabies postexposure and pre-exposure prophylaxis; and 5) presents information regarding treatment considerations for human rabies patients. These recommendations involve no substantial changes to the recommended approach for rabies postexposure or pre-exposure prophylaxis. ACIP recommends that prophylaxis for the prevention of rabies in humans exposed to rabies virus should include prompt and thorough wound cleansing followed by passive rabies immunization with human rabies immune globulin (HRIG) and vaccination with a cell culture rabies vaccine. For persons who have never been vaccinated against rabies, postexposure antirabies vaccination should always include administration of both passive antibody (HRIG) and vaccine (human diploid cell vaccine [HDCV] or purified chick embryo cell vaccine [PCECV]). Persons who have ever previously received complete vaccination regimens (pre-exposure or postexposure) with a cell culture vaccine or persons who have been vaccinated with other types of vaccines and have previously had a documented rabies virus neutralizing antibody titer should receive only 2 doses of vaccine: one on day 0 (as soon as the exposure is recognized and administration of vaccine can be arranged) and the second on day 3. HRIG is administered only once (i.e., at the beginning of antirabies prophylaxis) to previously unvaccinated persons to provide immediate, passive, rabies virus neutralizing antibody coverage until the patient responds to HDCV or PCECV by actively producing antibodies. A regimen of 5 1-mL doses of HDCV or PCECV should be administered intramuscularly to previously unvaccinated persons. The first dose of the 5-dose course should be administered as soon as possible after exposure (day 0). Additional doses should then be administered on days 3, 7, 14, and 28 after the first vaccination. Rabies pre-exposure vaccination should include three 1.0-mL injections of HDCV or PCECV administered intramuscularly (one injection per day on days 0, 7, and 21 or 28). Modifications were made to the language of the guidelines to clarify the recommendations and better specify the situations in which rabies post- and pre-exposure prophylaxis should be administered. No new rabies biologics are presented, and no changes were made to the vaccination schedules. However, rabies vaccine adsorbed (RVA, Bioport Corporation) is no longer available for rabies postexposure or pre-exposure prophylaxis, and intradermal pre-exposure prophylaxis is no longer recommended because it is not available in the United States.

Furious and paralytic rabies of canine origin: neuroimaging with virological and cytokine studies.

Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors studied magnetic resonance imaging (MRI) and cytokine and virus distribution in rabies-infected dogs of both clinical types. MRI examination of the brain and upper spinal cord was performed in two furious and two paralytic dogs during the early clinical stage. Rabies viral nucleoprotein RNA and 18 cytokine mRNAs at 12 different brain regions were studied. Rabies viral RNA was examined in four furious and four paralytic dogs during the early stage, and in one each during the late stage. Cytokine mRNAs were examined in two furious and two paralytic dogs during the early stage and in one each during the late stage. Larger quantities of rabies viral RNA were found in the brains of furious than in paralytic dogs. Interleukin-1beta and interferon-gamma mRNAs were found exclusively in the brains of paralytic dogs during the early stage. Abnormal hypersignal T2 changes were found at hippocampus, hypothalamus, brainstem, and spinal cord of paralytic dogs. More widespread changes of less intensity were seen in furious dog brains. During the late stage of infection, brains from furious and paralytic rabid dogs were similarly infected and there were less detectable cytokine mRNAs. These results suggest that the early stage of furious dog rabies is characterized by a moderate inflammation (as indicated by MRI lesions and brain cytokine detection) and a severe virus neuroinvasiveness. Paralytic rabies is characterized by delayed viral neuroinvasion and a more intense inflammation than furious rabies. Dogs may be a good model for study of the host inflammatory responses that may modulate rabies virus neuroinvasiveness.

Complex genetic structure of the rabies virus in Bangkok and its surrounding provinces, Thailand: implications for canine rabies control.

Dog vaccination and population management have been suggested as priorities in attempts at disease control in canine rabies-endemic countries. Budget limitations and the complexity of social, cultural and religious variables have complicated progress in the developing world. In Bangkok, Thailand, an intensive canine vaccination and sterilization programme has been in place since November 2002. Our objective was to determine if the rabies virus could be mapped according to its genetic variations and geographical location on the small localized scale of Bangkok and its surrounding provinces. Phylogenetic characterization of 69 samples from Bangkok and five neighbouring and two remote provinces, by limited sequence analysis of the rabies virus nucleoprotein gene, distinguished six different clades. Rabies viruses of four clades were intermixed in Bangkok and in the surrounding highly populated regions whereas the other two clades were confined to rural and less populated provinces. Such a complex pattern of gene flow, particularly in Bangkok, may affect the outcome of canine control programmes.

[Bat lyssavirus in Thailand].

A study of bat lyssavirus survey was done in Thailand from 2001 to 2003. A total of 932 bats of 11 species were captured in 8 provinces for blood collection and testing for neutralizing antibodies against rabies virus (RABV), Australian bat lyssavirus (ABLV) and broader panel of other lyssaviruses (Irkut, Aravan and Khujand). All Thai bat samples were negative to RABV Sixteen samples of 394 with sufficient volume of serum had detectable neutralizing antibodies against Irkut, Aravan, Khujand and ABL viruses. Another 13 samples were also found to have antibody to ABLV. However, due to insufficient volume, further analysis to other lyssaviruses could not be performed. Nevertheless, this showed that the prevalence of lyssavirus infection in Thai bats could be as high as 7.3% (29/396). The present study showed that natural occurrence of lyssavirus antibodies found in Thai bats were related to newer putative lyssavirus genotype(s) other than those previously described. These data also suggest that several lyssaviruses are in circulation throughout Thailand as well as other Asian countries, such as in the Philippines, Central Asia, and in certain parts of Russia. The present study and preparation of this article was supported by grants from the Thailand Research Fund and the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand.

Laboratory techniques for rabies diagnosis in animals at QSMI.

The Queen Saovabha Memorial Institute (QSMI), Thai Red Cross Society was founded in 1922 to carry out the production of the nervous tissue vaccines, post-exposure prophylaxis (PEP) treatment, research, and laboratory diagnosis. The diagnostic laboratory had replaced the Seller's staining with direct fluorescent test (DFA) as standard technique in 1987 and the mouse inoculation technique (MIT) had been employed as the confirmatory test to ensure the result of the diagnosis. Other techniques conducted at our facility such as tissue culture infection and polymerase chain reaction techniques are less practical although the sensitivity is competitive with DFA but mainly for research references.

Transmission dynamics of rabies virus in Thailand: implications for disease control.

BACKGROUND: In Thailand, rabies remains a neglected disease with authorities continuing to rely on human death statistics while ignoring the financial burden resulting from an enormous increase in post-exposure prophylaxis. Past attempts to conduct a mass dog vaccination and sterilization program have been limited to Bangkok city and have not been successful. We have used molecular epidemiology to define geographic localization of rabies virus phylogroups and their pattern of spread in Thailand. METHODS: We analyzed 239 nucleoprotein gene sequences from animal and human brain samples collected from all over Thailand between 1998 and 2002. We then reconstructed a phylogenetic tree correlating these data with geographical information. RESULTS: All sequences formed a monophyletic tree of 2 distinct phylogroups, TH1 and TH2. Three subgroups were identified in the TH1 subgroup and were distributed in the middle region of the country. Eight subgroups of TH2 viruses were identified widely distributed throughout the country overlapping the TH1 territory. There was a correlation between human-dependent transportation routes and the distribution of virus. CONCLUSION: Inter-regional migration paths of the viruses might be correlated with translocation of dogs associated with humans. Interconnecting factors between human socioeconomic and population density might determine the transmission dynamics of virus in a rural-to-urban polarity. The presence of 2 or more rabies virus groups in a location might be indicative of a gene flow, reflecting a translocation of dogs within such region and adjacent areas. Different approaches may be required for rabies control based on the homo- or heterogeneity of the virus. Areas containing homogeneous virus populations should be targeted first. Control of dog movement associated with humans is essential.

Survey for bat lyssaviruses, Thailand.

Surveillance for lyssaviruses was conducted among bat populations in 8 provinces in Thailand. In 2002 and 2003, a total of 932 bats of 11 species were captured and released after serum collection. Lyssavirus infection was determined by conducting virus neutralization assays on bat serum samples. Of collected samples, 538 were either hemolysed or insufficient in volume, which left 394 suitable for analysis. These samples included the following: Pteropus lylei (n = 335), Eonycteris spelaea (n = 45), Hipposideros armiger (n = 13), and Rousettus leschennaulti (n = 1). No serum samples had evidence of neutralizing antibodies when tested against rabies virus. However, 16 samples had detectable neutralizing antibodies against Aravan virus, Khujand virus, Irkut virus, or Australian bat lyssavirus; all were specifically associated with fruit bats P. lylei (n = 15) and E. spelaea (n = 1). These results are consistent with the presence of naturally occurring viruses related to new putative lyssavirus genotypes.

Computer based program for rapid canine rabies diagnosis.

Rabies post exposure treatment and rapid diagnosis in an endemic area requires skillful and an experienced medical staff and veterinary specialists. A software program was introduced in 2000 as a tool to diagnose rabies based on clinical symptoms and signs of suspicious dogs where veterinarian service and other laboratory diagnostic options are not immediately available. It is easily accessed from the website: www.soonak.com/rabies.