Rabies Virus Antigen Research Articles

A Global Perspective on Oral Vaccination of Wildlife against Rabies.

The long-term mitigation of human-domestic animal-wildlife conflicts is complex and difficult. Over the last 50 yr, the primary biomedical concepts and actualized collaborative global field applications of oral rabies vaccination to wildlife serve as one dramatic example that revolutionized the field of infectious disease management of free-ranging animals. Oral vaccination of wildlife occurred in diverse locales within Africa, Eurasia, the Middle East, and North America. Although rabies is not a candidate for eradication, over a billion doses of vaccine-laden baits distributed strategically by hand, at baiting stations, or via aircraft, resulted in widespread disease prevention, control, or local disease elimination among mesocarnivores. Pure, potent, safe, and efficacious vaccines consisted of either modified-live, highly attenuated, or recombinant viruses contained within attractive, edible baits. Since the late 1970s, major free-ranging target species have included coyotes (Canis latrans), foxes (Urocyon cinereoargenteus; Vulpes vulpes), jackals (Canis aureus; Lupulella mesomelas), raccoons (Procyon lotor), raccoon dogs (Nyctereutes procyonoides), and skunks (Mephitis mephitis). Operational progress has occurred in all but the latter species. Programmatic evaluations of oral rabies vaccination success have included: demonstration of biomarkers incorporated within vaccine-laden baits in target species as representative of bait contact; serological measurement of the induction of specific rabies virus neutralizing antibodies, indicative of an immune response to vaccine; and most importantly, the decreasing detection of rabies virus antigens in the brains of collected animals via enhanced laboratory-based surveillance, as evidence of management impact. Although often conceived mistakenly as a panacea, such cost-effective technology applied to free-ranging wildlife represents a real-world, One Health application benefiting agriculture, conservation biology, and public health. Based upon lessons learned with oral rabies vaccination of mesocarnivores, opportunities for future extension to other taxa and additional diseases will have far-reaching, transdisciplinary benefits.

Histopathological and immunohistochemical examination of the brains of rabid dogs in the Philippines.

Dogs are the primary transmitters of the rabies virus in the Philippines; however, to the best of our knowledge, no published studies have examined its detailed neuropathology. The present study analyzed the neuropathology in the cerebrum, hippocampus, thalamus, and brainstem of 70 rabid dogs with confirmed rabies infection in the Philippines. Histopathologically, inclusion bodies (Negri bodies) were detected in the hippocampus (87.14%), cerebrum (70%), and thalamus (2.86%) of the dogs. The inclusion bodies in the cytoplasm of the hippocampal and cerebral cortical pyramidal cells were large and round to oval in shape. Whereas the inclusion bodies in the neurons of the thalamus and brainstem were small, fine, and granular. In contrast to the high prevalence of inclusion bodies in the hippocampus and cerebrum, perivascular cuffing and glial nodules were more prominent in the thalamus and brainstem. Immunohistochemically using the anti-phosphoprotein (anti-P), the sensitivity of viral antigen detection was 100% in the hippocampus, thalamus, and brainstem and 97.14% in the cerebrum. Our findings confirmed that observing the inclusion bodies in the hippocampus and cerebral cortex by histopathology could facilitate rabies diagnosis in the dogs in the Philippines, and furthermore, using immunohistochemistry on the brainstem could also be useful to detect rabies virus antigens with high sensitivity.

Prevalence and public health significance of rabies virus in bats in the North Region of Cameroon.

Rabies is a zoonotic disease of all warm-blooded animals including humans. There is a paucity of data on the status of rabies in wild animals in Cameroon and the disease is endemic in the country with dogs being the main source of transmission. Bat habitats are widespread in Cameroon, but there is limited information on the prevalence of rabies in bats, and their role of as potential reservoirs of rabies virus. A cross sectional study was carried out to estimate the prevalence and to assess risk factors of rabies virus in bats in the North Region of Cameroon. A total of 212 bats belonging to three families (Pteropodidae, Vespertilionidae and Molossidae) and 5 species were sampled in 7 localities in the North Region of Cameroon and were tested for rabies virus antigen using direct Immunofluorescence Test (IFA). Overall, 26.9% (57/212) of the bats collected showed an IFA positive reaction. The prevalence was significantly higher (P<0.05) in adult bats (33.3% (36/108)) compared to young individuals (20.2%; 21/104). The main risk factors identified in the study for human exposure to bats were gender (Male), religion (Christianity), localities (Babla and Lagdo), the practice of bat hunting, bat consumption, unawareness of bat rabies and cohabitation with bats in close proximity. The study revealed the first evidence of Lyssavirus in bats in Cameroon. This finding showed that bat rabies are real and represents a potential public health concern in communities with bat habitats in the North Region of Cameroon. Enhancing the level of public awareness and health education on the potential of bats as reservoirs of Lyssavirus in Cameroon as well as the integration of the "One Health" approach for effective management of animal and human rabies should be emphasized.

Immunochromatographic test-system for express diagnostics of rabies

Relevance. Rabies is included in the group of transmissible viral zooanthroponoses that globally affect public health and the public economy. In Russian Federation, according to the State Standard 26075-2013, the method of virus isolation is a priority for the diagnosis of rabies, followed by detection by the method of fluorescent antibodies (MFA), however, the call of the World Health Organization to abandon research using animals makes it relevant to further develop and improve diagnostic methods in vitro.The purpose of the study. Designing a test system based on immunochromatographic analysis (IHA) for rapid diagnosis of rabies.Methods. Colloidal gold nanoparticles (CGN) with an optimal diameter (25–30 nm) were synthesized by citrate reduction of chloroauric acid and morphometrically characterized by the results of transmission electron microscopy. The conjugation of CGN with anti-rabies antibodies was carried out by non-covalent cross-linking, as a result of which stable conjugates were obtained with an immobilization coefficient of 91.8 ± 1.7%.Results. The classical scheme of direct (sandwich) was used IHA, in which the liquid phase (a sample containing the desired antigen) binds to labeled specific antibodies and, during migration through the solid phase, forms a colored complex with applied specific antibodies in the test zone («sandwich» NCHKZ-antibody — antigen — antibody). ICA was performed with positive, negative and heterologous antigens. The results of the study of samples by ICA were in direct correlation with the results of enzyme-linked immunosorbent assay (ELISA) and MFA and demonstrated 100% specificity. The presented data confirm the functionality of ICA for the detection of rabies virus antigen in pathological material.

A simplified method for measuring neutralising antibodies against rabies virus

Rabies virus (RABV) causes a fatal encephalitis that can be prevented through timely vaccination. The levels of virus neutralising antibodies against rabies virus induced by vaccination can be measured using the fluorescent antibody virus neutralisation (FAVN) test. Following incubation of live virus with sera, this method involves the fixation of cell monolayers and staining of rabies virus-specific antigen using fluorescein isothiocyanate (FITC) -conjugated antibody to enable visualisation of rabies virus antigen using a fluorescence microscope. To simplify this procedure, a fluorescent recombinant rabies virus was constructed using reverse genetics by inserting the gene for the mCherry fluorescent protein in front of the ribonucleoprotein gene of the SAD B-19 genome and replacing its glycoprotein with that of the Challenge Virus Standard (CVS)-11 RABV strain to ensure antigenic authenticity with the FAVN. This new recombinant virus (termed mCCCG) expressed the mCherry protein to high levels enabling direct observation of infected cells. In vitro growth kinetics of mCCCG were indistinguishable from that of CVS-11. The stability of the recombinant virus was assessed by sequencing several passages of the rescued virus and only minor changes were detected. Comparative assessment of the virus neutralisation test using mCherry producing virus (NTmCV) against the FAVN demonstrated that test results were equivalent to each other; therefore, mCCCG can be used as an alternative to CVS-11 for measuring antibody titres against the rabies virus. The use of NTmCV removes the need for expensive antibody conjugates and significantly reduces assay time. This would be particularly beneficial for RABV serological assessment in resource limited settings. Moreover, the reading of the plates can be automatically using a cell imaging reader.

Molecular characterization of rabies virus in trade dogs from Plateau state, Nigeria

Rabies is one of the most dreaded diseases known to mankind and it is endemic in Nigeria. This study was aimed at the detection and molecular characterisation of the rabies virus antigen in trade dogs in Plateau State. A cross-sectional study was performed to determine the prevalence of rabies antigen in the brain tissues of slaughtered dogs, from February to June 2018 using a direct fluorescent antibody test and mouse inoculation technique. Direct Fluorescent Antibody Test, Polymerase Chain Reaction (PCR), sequencing and phylogenetic analysis of the identified rabies virus were also undertaken. Phylogenetic analysis was done using molecular evolutionary genetic analysis (MEGA) 10 to determine their evolutionary relationship with other rabies virus sequences deposited in Genbank. Results indicated, out of 450 dog brain samples tested 14(3.1%) were positive for rabies virus antigen. Zoographic data obtained showed 66.3% of dogs were female, and the dogs were kept mainly for security (n=54) and breeding (n=28). Fourteen positive samples were used to inoculate 70 adult mice, only 8 samples were positive and 2 samples were also positive by RT PCR. Phylogenetic analysis of the nucleotide sequences showed that the two rabies virus sequences in this study, sequence MZ148311 had very close identity with EU038106 (99.8% homology) and sequence MZ148312 had perfect identity with EU038087 (100% homology) was most closely related to lyssavirus that has been reported to be circulating previously in Plateau and Nigeria, belonging to the Africa 2 lineage.

Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen.

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately burdensome to those living in low- and middle-income countries (LMICs), where the majority of deadly exposures to rabies occur. Different drug delivery strategies have been explored to condense vaccine regimens to a single injection by encapsulating antigens into polymeric particles. However, harsh stressors during the encapsulation process can cause denaturation of the encapsulated antigen. This article describes a method for encapsulating the rabies virus (RABV) antigen into polymeric microparticles that exhibit tunable pulsatile release. This method, termed Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED), generates microparticles using soft lithography to create inverse polydimethylsiloxane (PDMS) molds from a multi-photon, 3D-printed master mold. Poly(lactic-co-glycolic acid) (PLGA) films are then compression-molded into the PDMS molds to generate open-faced cylinders that are filled with concentrated RABV using a piezoelectric dispensing robot. These microstructures are then sealed by heating the top of the particles, allowing the material to flow and form a continuous, nonporous polymeric barrier. Post-fabrication, an enzyme-linked immunosorbent assay (ELISA) specific to the detection of intact trimeric rabies virus glycoprotein is used to confirm the high recovery of immunogenic antigen from the microparticles.

Rabies virus infection is associated with variations in calbindin D-28K and calretinin mRNA expression levels in mouse brain tissue.

Rabies virus (RABV) infection leads to a fatal neurological outcome in humans and animals and is associated with major alterations in cellular gene expression. In this study, we describe the effects of RABV infection on the mRNA expression levels of two genes, encoding the Ca2+-binding proteins (Ca-BPs) calbindin D-28K (Calb1) and calretinin (Calb2), in the brains of BALB/c mice. Sixty 4-week-old mice were divided into two test groups and one control group. Mice were inoculated intramuscularly with either a street rabies virus (SRV) strain or a challenge virus standard (CVS-11) strain and sacrificed at 3-day intervals up to day 18 postinfection. A direct fluorescent antibody test (DFAT) was used to verify the presence of RABV antigen in brain tissues, and real-time quantitative PCR (RT-PCR) was used to assess gene expression. Infection with both RABV strains resulted in significant (p < 0.05) increases in Calb1 and Calb2 expression in the test animals when compared with the controls at various time points in the study. Correlation analysis indicated very weak insignificant (p > 0.05) negative and positive relationships, respectively, between Calb1 expression (r = -0.04) and Calb2 expression (r = 0.08) with viral load (CVS-11 strain). Insignificant (p > 0.05) relationships were also observed Calb1 expression (r = -0.28) and Calb2 expression (r = 0.06) and viral load for the SRV strain.The observed alterations in Calb1 and Calb2 expression in this study indicate possible impairments in neuronal Ca2+ buffering and Ca2+ homeostasis as a result of RABV infection and, consequently, possible involvement of calbindin-D28K and calretinin in the neuropathogenesis of rabies.

Adaptation of the rabies virus of the production strain "sheep" GNKI to the transplanted cultures of NGUK-1 and ВНК-21/13 CELLS

The selection of the optimal line of the transplanted cell culture and the adaptation of the rabies virus to it makes it possible to exclude the use of laboratory animals, fully control the process of obtaining viruscontaining material with high infectious activity in large quantities and with a faster and shorter production cycle. The adaptive potential of various strains of rabies virus varies significantly, in this regard, the aim of the study was to study the sensitivity of the production strain of the rabies virus "Sheep" GNKI to the transplanted cultures of NGUK-1 and ВНК-21/13 cells. The adaptation of the rabies virus to the transplanted cultures of NGUK-1 and VNK-21/13 cells was carried out by sequential passivation. The titer of the virus was calculated by the number of fluorescent foci, the concentration of rabies virus antigen was determined by the ELISA method, the pathogenicity of the virus at the level of 11 passages on different cell lines – on white mice.It was found that the transplanted ВНК-21/13 cell line provided a faster adaptation of the virus and the achievement of maximum titers within 36-48 hours, whereas the NGUK-1 line maintained relatively slow replication and ensured the achievement of maximum titers after 96-120 hours. The optimal multiplicity of infection with NGUK-1 and ВНК-21/13 was 0.1 KKID50/cell, while the titers of the "Sheep" virus strains were 4.11±0.26 and 6.17±0.49 lg KKID50/cm3, respectively. Virus replication in ВНК-21/13 cells was characterized by greater intensity: the antigenic titer of the virus at all passage levels was 1.5-2 times (p&lt;0.05) higher than that in NGUK-1 cells. The positive dynamics of the accumulation of the viral titer persisted until passage 8-9, after which this indicator remained stable until passage 11 inclusive. The results of the assessment of the pathogenicity of the rabies virus at the level of passage 11 on different cell lines showed that the virus adapted to both NGUK-1 and ВНК-21/13 has not lost pathogenicity for white mice. Thus, it was found that the transplanted ВНК-21/13 cell line significantly exceeds the NGUK-1 cell line in terms of replicative capabilities and can be used to develop viral raw materials for the production of diagnostic tools and specific prevention.

Immunogenicity and Antigenicity of the Ectodomain of Rabies Virus Glycoprotein Stably Expressed in HEK293T Cells.

Rabies continues to be a huge threat to public health. The rabies virus envelope glycoprotein (RABV G) is a major rabies virus antigen and contains neutralizing epitopes, which are primary candidates for subunit vaccines and diagnostic antigens. However, the production and purification of rRABV G while retaining its antigenic and immunogenic remains to be a challenge. Here, we aimed to establish a platform for rRABV G production and purification, and determine the immunogenicity and antigenicity of rRABV G. The cDNA fragment encoding the soluble form of RABV G was synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-RABV G-eGFP was packaged, titered, and then transduced into HEK 293T cells. The cell culture supernatant was purified using nickel affinity chromatography and subsequently confirmed through Western Blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). The ELISA utilized human sera obtained from individuals who had been vaccinated with the human commercial Purified Vero Cells Rabies Vaccine (PVRV). Notably, we observed a neutralizing antibody response in immunized pigs rather than in mice. This discrepancy could potentially be attributed to factors such as the instability of the rRABV G protein, variations in host responses, and variances in the adjuvant used. Taking all these findings into account, the rRABV G protein generated in this study exhibits promise as a potential vaccine candidate for the prevention of rabies.

Rabies Virus Seroprevalence among Dogs in Limpopo National Park and the Phylogenetic Analyses of Rabies Viruses in Mozambique.

Rabies is considered a neglected disease among many developing Asian and African countries, including Mozambique, where its re-emergence is often attributed to low dog parenteral vaccination coverage. The objectives of this study were two-fold: (1) to assess the level of antibodies against rabies virus in dogs (n = 418) in Limpopo National Park (LNP), and (2) to genetically characterise selected rabies viruses from brain tissue samples collected in 2017 and 2018. To meet the first objective, we used the BioProTM Rabies blocking ELISA antibody kit, and the results were expressed as the percentage of blocking (%PB). Dog sera with PB ≥ 40% were considered positive for antibodies to rabies virus, whereas sera with PB < 40% were negative. Just under ninety percent (89.2%; n = 373) of dogs were seronegative, and the rest (10.8%; n = 45) had detectable levels of rabies virus-specific antibodies. All eight brain tissue samples were positive for rabies virus antigen using a direct fluorescent antibody test and amplified in a quantitative real-time PCR, but only five (n = 4 from dogs and n = 1 from a cat) were amplified in a conventional reverse-transcription PCR targeting partial regions of the nucleoprotein (N) and the glycoprotein (G) genes. All samples were successfully sequenced. Phylogenetically, the rabies viruses were all of dog origin and were very closely related to each other (Africa 1b rabies virus lineage). Furthermore, the sequences had a common progenitor with other rabies viruses from southern Africa, confirming the transboundary nature of rabies and the pivotal role of dogs in maintaining rabies cycles. The study demonstrates the principal application of the BioProTM rabies ELISA antibody for the detection of anti-lyssavirus-specific antibodies in the serum samples of dogs, and most importantly, it highlights the low levels of antibodies against rabies virus in this dog population.

Detection of Apparent Early Rabies Infection by LN34 Pan-Lyssavirus Real-Time RT-PCR Assay in Pennsylvania.

The Pennsylvania Department of Health Bureau of Laboratories (PABOL) tested 6855 animal samples for rabies using both the direct fluorescent antibody test (DFA) and LN34 pan-lyssavirus reverse transcriptase quantitative PCR (RT-qPCR) during 2017–2019. Only two samples (0.03%) were initially DFA negative but positive by LN34 RT-qPCR. Both cases were confirmed positive upon re-testing at PABOL and confirmatory testing at the Centers for Disease Control and Prevention by LN34 RT-qPCR and DFA. Rabies virus sequences from one sample were distinct from all positive samples processed at PABOL within two weeks, ruling out cross-contamination. Levels of rabies virus antigen and RNA were low in all brain structures tested, but were higher in brain stem and rostral spinal cord than in cerebellum, hippocampus or cortex. Taken together, the low level of rabies virus combined with higher abundance in more caudal brain structures suggest early infection. These cases highlight the increased sensitivity and ease of interpretation of LN34 RT-qPCR for low positive cases.

Application of conjugate based on colloidal gold for express diagnostics of rabies in dot immunoanalysis

The aim of the research was to develop a direct dot-immunoassay on a nitrocellulose membrane (NCM) using a conjugate based on colloidal gold for the qualitative determination of the presence of rabies virus antigen in pathological material. As the test samples, we used brain samples of various animal species, which were positive during the initial study by methods of fluorescent antibodies: foxes and mice. IFA and indirect ELISA carried out using diagnostic kits manufactured at the Federal Center for Toxicological, Radiation and Biological Safety. The materials present the results of primary laboratory tests of the test system for the indication of rabies antigen based on direct dotimmunoassay (DIA) 81 samples of pathological material, the brain of various animal species. It was shown that DIA has 100% specificity, and its optical signals correlate with the results of indirect ELISA. The proposed DIA method, in addition to independent application, can also serve as a basis for the design of test systems based on immunochemical analysis.

Use of a Direct, Rapid Immunohistochemical Test for Diagnosis of Rabies Virus in Bats

Rabies, a zoonotic encephalitis due to transmission of a lyssavirus, such as rabies virus (RABV), has the highest case fatality of any infectious disease. A global program for the elimination of human rabies caused by dogs is proposed for realization by 2030. Sensitive, specific, and inexpensive diagnostic tests are necessary for enhanced surveillance to detect infection, inform public health and veterinary professionals during risk assessments of exposure, and support overall programmatic goals. Multiple laboratory techniques are used to confirm a suspect case of rabies. One method for the detection of lyssavirus antigens within the brain is the direct rapid immunohistochemical test (dRIT), using light microscopy, and suitable for use under field conditions. Besides dogs, other major RABV reservoirs reside among mammalian mesocarnivores and bats. To date, use of the dRIT has been applied primarily for the diagnosis of RABV in suspect mesocarnivores. The purpose of this study was to assess the usefulness of the dRIT to the diagnosis of rabies in bats, compared to the gold-standard, the direct fluorescent antibody test (DFAT). Brains of 264 suspect bats, consisting of 21 species from Arizona and Texas, were used in the evaluation of the dRIT. The overall sensitivity of the dRIT was 100% (0.969–1.0, 95% CI) and the specificity was 94.6% (0.896–0.976, 95% CI), comparable to the DFAT. This preliminary study demonstrated the utility of the dRIT in the confirmation of RABV infection in bats. Future studies should include additional geographic, lyssavirus, and mammalian species representations for broader application during enhanced rabies surveillance, with incorporation of any potential adjustments to standard protocols, as needed.

Comparative effectiveness of liquid and freeze-dried vaccines for oral vaccination of dogs against rabies

Currently, vaccination is the main measure to combat rabies in domestic and wild animals. Inactivated and live vaccines are used for this purpose. Oral vaccines for wild carnivores contain live attenuated rabies viruses in liquid or freeze-dried form, which are packaged inside edible baits. Since there are no consistent data showing advantages of liquid and freeze-dried oral products for vaccine-induced immunity against rabies in animals, we compared effectiveness of these rabies vaccines produced from rabies virus strain VRC-RZ2. Immunogenicity was tested in mongrel dogs aged 3 months and older that are seronegative for rabies virus antigens. The animals were randomly divided into three groups: two experimental and one control group. Group One was fed a block-type bait containing a blister with liquid virus-containing suspension, Group Two was given a block-type bait containing a gelatin capsule with freeze-dried virus suspension. On Day 21 post vaccination, blood samples were taken from all the animals and the obtained sera were examined in virus neutralization test to measure virus neutralizing antibodies titers. The level of the immune response against rabies in the vaccinated dogs was assessed by intracerebral infection of animals with virulent rabies virus strain CVS. The carried out research demonstrated that both groups of the vaccinated dogs had approximately the same titers of virus neutralizing antibodies that ranged from 3.25 to 4.33 log 2 . The virus neutralizing antibodies observed in the immunized dogs ensured good protection from virulent CVS strain. All animals of the control group died after infection demonstrating clinical signs of paralytic rabies. The results obtained show that both forms of the oral rabies vaccines are effective.

Direct fluorescent antibody test in laboratory diagnosis of animal rabies in Ukraine

The aim of the study was to analyze the role of the direct fluorescent antibody (DFA) test in the system of laboratory diagnosis of animal rabies in Ukraine. For the analysis, materials of official veterinary reporting were used according to Form No. 2-VET “Report on the work of the state laboratories of veterinary medicine” regarding the results of laboratory studies of pathological material suspicious of rabies, the State Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise (SRILDVSE) and virology departments of the State Regional Laboratories of the State Food and Consumer Service of Ukraine, and analytical materials: Report “On the assessment of the risk of spread of rabies among domestic and farm animals under the current animal rabies control system in Ukraine” and the Report “On the assessment of the risk of spread of rabies among wild animals in Ukraine”. It has been determined that, over the past 15 years (2006–2020), 194,079 tests of the pathological material were carried out in state laboratories. The direct fluorescent antibody test is the main technique for the diagnosis of animal rabies in Ukraine, when used in 94.5% of cases, the final diagnosis of rabies is made. We have used standardization of approaches, including the use of the reference rabies virus CVS-11 (ATCC VR 959), to the organization and conducting of interlaboratory rounds of professional testing VET-TEST to identify of rabies virus antigen within the requirements ISO 17043:2017 “Conformity assessment. General requirements for testing professional level”. DFA test is the main reaction for the diagnosis of rabies in animals in Ukraine. Standardized approaches were introduced and interlaboratory rounds of professional testing BET-TEST have been organized in 2020 for the detection of rabies virus antigen in accordance with the quality standard ISO 17043:2017. The necessity of introducing new methods of laboratory diagnostics of rabies, such as viral isolations in tissue culture and polymerase chain reaction, has been established

Risk Factors and Protective Immunity Against Rabies in Unvaccinated Butchers Working at Dog Slaughterhouses in Northern Vietnam

Vietnam is a rabies-endemic country where eating dog meat is customary. However, the risks of rabies transmission to dog slaughtering and processing workers have not been identified. This study aimed to determine the rabies neutralizing antibody (NTA) and risk factors in dog slaughterers to propose appropriate intervention methods for this occupational group. In 2016, a cross-sectional study on NTA against rabies virus and related factors was conducted among 406 professional dog slaughterers in Vietnam. The participants were interviewed using a structured questionnaire, and their sera were tested for rabies NTA by a rapid focus fluorescence inhibition test. Statistical algorithms were used to analyze the data. The results showed that most of the professional dog butchers (344/406 subjects, 84.7%) had no rabies NTA. Interestingly, 7.8% (29/373) had NTA without a rabies vaccination history. Over 5 years of experience as a dog butcher was positively associated with the presence of NTA in unvaccinated individuals (OR = 6.16, P = 0.001). The NTA in vaccinated butchers was present in higher titer and for longer persistence to those of other previously reported professionals, which is possibly as a result of multiple exposures to low levels of rabies virus antigens during dog slaughtering. Our study demonstrated that professional dog butchers in Vietnam are at a high risk of rabies virus infection, apart from those with common bite experiences. In countries where dog meat consumption is customary, rabies control and prevention activities should focus on safety during dog trading and slaughtering.

Rabies epidemiology, prevention and control in Nigeria: Scoping progress towards elimination.

BackgroundHuman rabies remains a significant public health problem in Africa with outbreaks reported in most countries. In Nigeria–the most populous country in Africa–rabies causes a significant public health burden partly due to perennial obstacles to implementing a national prevention and control program.MethodsWe conducted a scoping review using standard Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to identify and select published articles from Nigeria during 1978–2020 reporting on rabies virus infections (human, canine, livestock, and wildlife), canine bites, knowledge, attitudes and practices (KAP) surveys on rabies and canine ecology studies. We extracted information on study location, year and additional details of each study such as rabies prevalence, general characteristics of offending dogs, dog vaccination status and health-seeking behaviours.FindingsBetween 1978 and 2020, 90 published articles met our inclusion criteria. The prevalence of rabies virus antigen detection varied between 3% and 28%, with more studies in the north. Most bites were unprovoked from dog bite studies (36.4%-97%), by dogs with low vaccination rates (12–38%). A more significant proportion of biting dogs were owned (31–90%). Laboratory confirmation for biting was available for only a small proportion of studies (6%; n = 2/32). Of the dogs surveyed during ecology studies, indigenous dogs accounted for the majority (62–98%), used mostly for security purposes (52–98%), with the vaccination rate between 15% and 38% in most states. Studies conducted in areas distant from rabies diagnostic facilities accounted for more human rabies cases and fewer dog rabies cases.ConclusionSignificant improvements are necessary to achieve the elimination of human rabies mediated via dogs by 2030.

Formulation optimization of chitosan nanoparticles incorporated rabies viral antigen and its influence on the release kinetics, immune potency and biosafety potential

In the present study, optimization of parameters for the preparation of nano formulated whole attenuated rabies vaccine with high immune potency and biocompatibility was synthesised. Response Surface Methodology (RSM) was adapted to study the influence of factors such as the concentration of chitosan (mg/mL), PEG (mg), acetic acid (%), sodium tripolyphosphate (M) and antigen (I.U.) on the structural, functional properties of chitosan PEG nanoparticles loaded rabies whole attenuated viral antigen (CS-PEG-NP-RVAg). Mathematical models were used to study the release kinetics of the nanoformulation adopting various plots such as Zero-order kinetics, First-order kinetics, Huguchi, and Huguchi-Crowell models. The evaluation provides insight into the controlled release of the rabies vaccine. The use of acetic acid (1%), sodium tripolyphosphate (0.1 M), antigen 1.5 (I.U.), PEG (60 µL), chitosan (75 mg) yields the desirable size of nanoformulations, immune potency, notable control release pattern Biocompatibility of the prepared nanovaccine was tested on Vero cell line, peripheral blood cells and probiotic bacterial strains. Nanoformulation, which obtained at the optimum condition was not inducing any toxic sign-on tested model system. Our study provides the basis of developing as effective CS-PEG-OV-RVAg as an effective and biocompatible vaccine formulation.

Quantitation of Rabies Virus in Various Bovine Brain Structures.

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major zoonotic risk. Here, our objective was to utilize a laboratory protocol to determine the relative copy number of the rabies virus (RABV) genome in different bovine brain anatomical structures using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR quantifies the specific number of gene copies present in a sample based on fluorescence emitted after amplification that is directly proportional to the amount of target nucleic acid present in the sample. This method is advantageous owing to its short duration, reduced risk of contamination, and potential to detect viral nucleic acids in different samples more easily compared to other techniques. The brains of six rabid animals were divided into six anatomical structures, namely the Ammon's horn, cerebellum, cortex, medulla, pons, and thalamus. All brains were identified as positive for RABV antigens based on a direct immunofluorescence test. The same anatomical structures from the brains of four RABV-negative bovines were also assessed. RNA was extracted from each structure and used for qRT-PCR. An assay was performed to determine the copy numbers of RABV genes using an in vitro transcribed nucleoprotein gene. The standard curve used to quantify viral RNA exhibited an efficiency of 100% and linearity of 0.99. Analysis revealed that the cortex, medulla, and thalamus were the ideal CNS portions for use in RABV detection, based on the observation that these structures possessed the highest levels of RABV. The test specificity was 100%. All samples were positive, no false positives were detected. This method can be used to detect RABV in samples that contain low levels of RABV during diagnosis of BPR.