Human Rabies during the COVID-19 Pandemic: Insights into Rabies Worldwide and Brazil

ABSTRACT Human Rabies (HR) is a fatal zoonotic disease caused by lyssaviruses, with the rabies virus (RABV) identified as the causative agent. While the incidence of HR transmitted by dogs has decreased in Latin America, there has been a corresponding rise in transmission via wild animals. Given the lack of effective treatments and specific therapies, the management of HR relies on the availability of post-exposure prophylaxis and animal control measures. This review examines the dynamics and spread of HR during the global pandemic.

Lesiones ocasionadas por mordeduras de perros en niños atendidos en un hospital de tercer nivel de Bolivia / Dog bite injuries in children seen at a tertiary care hospital in Bolivia

Introducción. Este estudio pretende caracterizar las lesiones provocadas por perros en niños de un hospital pediátrico de Bolivia. Población y métodos. Se realizó un estudio observacional, retrospectivo, en pacientes atendidos del 2017 al 2021. Resultados. Se estudiaron 769 pacientes. Las lesiones representaron el 5,6 % de las emergencias y el 0,8 % de las internaciones. Fueron más frecuentes en niños de hasta 5 años (55,1 %), en quienes se observó mayor gravedad de las lesiones (p = 0,008), antecedente de provocación al animal (p = 0,048), un animal agresor conocido (p <0,036), el contexto doméstico del accidente (p = 0,021), mayor frecuencia de profilaxis con suero luego de la exposición (p = 0,005) y regiones afectadas principalmente maxilofaciales (p <0,001). Observamos 3 casos de mortalidad por rabia humana y 1 por shock hipovolémico. Conclusión. Las lesiones producidas por perros son causas frecuentes de visita a emergencia y hospitalización en pediatría, y tienen características particulares en niños de hasta 5 años de edad.

Introduction. The objective of this study is to describe the characteristics of dog bite injuries in children seen at a children's hospital in Bolivia. Population and methods. This was an observational, retrospective study in patients seen between 2017 and 2021. Results. A total of 769 patients were studied. Dog bite injuries accounted for 5.6% of emergency visits and 0.8% of hospitalizations. They were more frequent in children younger than 5 years (55.1%), in whom the following were observed: greater injury severity (p = 0.008), history of animal provocation (p = 0.048), known attacking animal (p < 0.036), domestic accident (p = 0.021), greater frequency of post-exposure prophylaxis with anti-rabies serum (p = 0.005), and maxillofacial area as the main region involved (p < 0.001). There were 3 deaths due to human rabies and 1 due to hypovolemic shock. Conclusion. Dog bite injuries are a frequent cause of visit to the emergency department and hospitalization in pediatrics and have specific characteristics in children younger than 5 years.

Historia de la rabia en Chile: tres tesis médicas

En la antigüedad ya se describía la rabia como una enfermedad zoonótica fatal cuyo pronóstico inexorable superaba todas las alter-nativas terapéuticas de los más célebres médicos. La realidad chilena sobre esta enfermedad a fines del siglo XIX fue descrita certeramente por el médico mártir Pedro Videla Órdenes en su tesis "La rabia" de 1879, destacando la descripción clínica de la rabia, su pronóstico fatal y la ausencia de tratamientos eficaces. Tan sólo seis años después, en 1885, el aclamado químico y microbiólogo Louis Pasteur desarrolló la vacuna antirrábica, logrando por primera vez en la historia de la humanidad prevenir esta terrible enfermedad. En Chile, se inició rápidamente la implementación de la vacuna Pasteur, vacunando al primer chileno el 7 de julio de 1896. Los doctores Milcíades Espinosa y Arturo Atria, en sus tesis "Generalidades sobre la rabia" (1898) y "Sobre la rabia y su profilaxia en Chile" (1905), respectivamente, abordaron esta primera etapa del desarrollo de la vacuna antirrábica en el país.

In antiquity, rabies was already described as a fatal zoonotic disease whose inexorable prognosis exceeded all the therapeutic alternatives of the most famous doctors. The Chilean reality about this disease at the end of the 19th century was accurately described by the martyred doctor Pedro Videla Ordenes in his thesis "La rabia" of 1879, highlighting in it his description about the unknown etiological agent, the fatal prognosis of the disease and the absence of effective treatments. Just six years later, in 1885, the acclaimed chemist and microbiologist Louis Pasteur developed the rabies vaccine, managing to prevent this terrible disease for the first time in human history. In Chile, the implementation of the Pasteur vaccine began rapidly, vaccinating the first Chilean on July 7, 1896. Doctors Milcíades Espinosa and Arturo Atria, in their theses "Generalidades sobre la rabia" (1898) and "Sobre la rabia y su profilaxia en Chile" (1905), respectively, addressed this first stage of the development of the rabies vaccine in the country.

Virología molecular de la rabia: un enfoque clínico / Rabies molecular virology: a clinical approach

Introducción: La rabia es una enfermedad zoonótica asociada al virus RABV, el cual tiene características neurotrópicas. El virus se transmite por el contacto con saliva de animales infectados; la mordedura de un perro es la causa más común. Es un virus que causa la muerte de miles de personas cada año. Objetivo: Describir a profundidad los principios moleculares de la infección por rabia, así como su patogenia, diagnóstico y tratamiento. Métodos: Se realizó una búsqueda de bibliografía en PubMed, SciELO, Scopus, Researchgate; se consultaron 163 referencias y se seleccionaron 51 fuentes que contenían la información más relevante para cumplir con el objetivo del trabajo. Conclusión: Actualmente es posible entender de mejor manera los mecanismos de transmisión y propagación del virus en el organismo; existe nuevo conocimiento sobre los receptores involucrados, así como la función de estos en la replicación viral. Sin embargo, el objetivo de la erradicación de la rabia a corto plazo es complejo. La invasión de territorios selváticos vuelve a la rabia un posible patógeno reemergente; la vacunación de especies transmisoras es el medio ideal para conseguir el control de la enfermedad.

Introduction: Rabies is a zoonotic disease associated with the RABV virus, which has neurotropic characteristics. The virus is transmitted by contact with saliva from infected animals; a dog's bite is the most common cause. This virus causes the death of thousands of people every year. Objective: To describe in depth the molecular principles of rabies infection, as well as its pathogenesis, diagnosis and treatment. Methods: A literature search was conducted in PubMed, SciELO, Scopus, and Researchgate. A total of 163 references were consulted, and 51 sources containing the most relevant information were selected to fulfill the objective of the work. Conclusions: It is currently possible to better understand the mechanisms of transmission and spread of the virus in the organism; there is new knowledge about the receptors involved, as well as their function in viral replication. However, the goal of eradicating rabies in the short term is complex. The invasion of wild territories makes rabies a possible re-emerging pathogen; vaccination of transmitting species is the ideal means to achieve disease control.

A Study of Clinico-social Profile of Animal Bite Patients Attending the Antirabies Clinic of BRD Medical College, Gorakhpur

Background: Rabies is one of zoonotic viral disease, estimated to cause 59000 human deaths annually in over 150 countries, of which 20,000 are from India alone; about 40% of which are in children under the age of 15. Rabies though 100% fatal is preventable with post-exposure prophylaxis which includes wound washing, anti-rabies vaccination and rabies immunoglobulin. Objective: To describe the clinico-social profile of animal bite patients attending the anti-rabies clinic of BRD Medical College, Gorakhpur. Methodology: A cross-sectional study was conducted in the anti-rabies clinic of Nehru hospital, BRD Medical College, Gorakhpur from January 2022 to May 2022. Study participants were interviewed by using a pre-phrased, pre-designed and pre-tested questionnaire. Data regarding socio-demographic and clinical profile of the study participants following animal bite exposure was collected. Results: The total number of animal bite victims were 250, in which majority of them were males (76.77%) and highest percentage was of adult population (20-59 years). Maximum number of victims were from rural area (78.70%). 19.35% were working and 39.35% were students. 77.43% were category III bites and in 50.96% cases lower limb was the site of bite and dogs were responsible for 89.67% of the bites. 60.64% victims did not wash the wound properly before reaching the anti-rabies clinic. Conclusion: This study concludes that as majority of the animal bite victims were students and majority of victims were unaware about the importance of wound care, therefore a step can be taken to create awareness in various schools.

Inovação e ética: a adoção de técnicas substitutivas ao uso de animais no diagnóstico laboratorial da raiva / Innovation and ethics; the adoption of alternative techniques to the use of animals in the laboratory diagnosis of rabies

Abordagens interdisciplinares vêm ganhando maior reconhecimento e destaque nas comunidades de saúde humana e animal, principalmente pela (re)emergência de diversas doenças infecciosas que emanam da interface humano-animal-ambiente. A raiva, zoonose grave, considerada endêmica no Brasil e globalmente negligenciada, é um exemplo. Tanto a vigilância epidemiológica quanto a confirmação dessa doença dependem do diagnóstico laboratorial, que é realizado, frequentemente, por meio dos testes de Imunofluorescência Direta (IFD) e de Isolamento Viral em Camundongo (IVC), via inoculação intracerebral da amostra suspeita em camundongos lactentes ou desmamados. Entretanto, recentemente, a Organização Mundial da Saúde reconheceu a Transcrição Reversa seguida da Reação em Cadeia da Polimerase (RT-PCR) como uma técnica primária válida para esse diagnóstico, podendo ser empregada como alternativa ao uso de animais, evitando sofrimento e morte. Esta dissertação apresenta uma discussão sobre as implicações técnicas e éticas da (não) adoção desse método substitutivo, considerando que todos os animais devem ser respeitados e entendidos como sujeitos singulares em suas percepções do mundo, não como objetos de pesquisa. Esse fato corrobora a necessidade de novas perspectivas que ressignifiquem nossas relações com os animais não humanos, o que é primordial para o estabelecimento de mudanças sistêmicas, de caráter ético-político, que visem o fim da instrumentalização animal e de seu uso no âmbito científico, bem como de qualquer forma de opressão.

Interdisciplinary approaches have been gaining greater recognition and prominence in the human and animal health communities, mainly due to the (re)emergence of several infectious diseases that emanate from the human-animal-environment interface. Rabies is an example, considered a serious zoonosis endemic in Brazil and globally neglected. Both epidemiological surveillance and confirmation of this disease depend on laboratory diagnosis, which is usually performed by the direct fluorescent antibody test (DFAT) and the mouse inoculation test (MIT) via intracranial inoculation of the suspected sample into suckling or weanling mice. However, the World Health Organization recently recognized the reverse transcription polymerase chain reaction (RT-PCR) as a valid primary technique for this diagnosis, which can replace the use of animals, avoiding suffering and death. This study presents a discussion about the technical and ethical implications of (not) adopting this alternative method, considering that all animals must be respected and understood as unique beings with their perceptions of the world, not as objects of research. It also further corroborates the need for new perspectives that reframe our relationships with non-human animals, which is fundamental for the implementation of systemic ethical-political changes, aiming at the end of animal instrumentalization and use in scientific research, as well as all forms of oppression.

Associação entre fatores socioeconômicos e demográficos e vacinação antirrábica de cães e gatos domésticos / Association between socioeconomic and demographic factors and rabies vaccination of dogs and cats

Resumo Introdução A raiva é uma doença endêmica no Brasil, cuja forma de combate em humanos é a vacinação de animais. Objetivo Buscou-se analisar associação entre fatores sociodemográficos e econômicos de adultos com vacinação antirrábica de animais domésticos com amostra da Pesquisa Nacional de Saúde realizada em 2013. Método Estudo transversal tendo como desfecho vacinação antirrábica de cães e gatos e exposições: sexo, idade, escolaridade, renda per capita e cor da pele autodeclarada do chefe de domicílio. Foram realizadas análises bi e multivariadas por meio de regressão logística. Resultados No Brasil, 59,3% (IC95%59,1-59,5) dos domicílios tinham animal de estimação. Em relação à vacinação antirrábica, a região Sudeste possui a maior prevalência, em que 84,8% (IC95%83,3-86,2) dos domicílios vacinaram todos os cães e gatos, contra 63,7% (IC95%60,9-66,3) na região Sul. Indivíduos com ≥50 anos têm 64% mais chances de vacinar cães e gatos, comparados àqueles na faixa etária mais jovem (18-29 anos). Quanto à escolaridade, indivíduos com ≥12 anos de estudo têm 2,4 vezes mais chances de vacinar todos os animais do domicílio, em relação àqueles com 0-8 anos de estudo. Conclusão Os resultados deste estudo apontam para a necessidade de políticas públicas que orientem a população sobre guarda responsável e que ofertem serviços de imunização.

Abstract Background In Brazil, rabies is an endemic disease and the vaccination of animals is a form of combat for humans. Objective The study aimed to analyze the association between sociodemographic and economic factors of adults and rabies vaccination of domestic animals, using samples from the National Health Survey conducted in 2013. Method This is a cross-sectional study with the outcome of rabies vaccination of dogs and cats and exposures: sex, age, education, per capita income, and skin color of the head of household. Bi and multivariate analyses were performed by logistic regression. Results In Brazil, 59.3% (95% CI 59.1-59.5) of households have pets. Regarding rabies vaccination, the southeast region has the highest prevalence, with 84.8% (95% CI 83.3-86.2) of households vaccinating all dogs and cats against 63.7% (95% CI 60.9-66.3) in the south region. Individuals in the age group ≥50 years are 64% more likely to vaccinate dogs and cats compared to those in the younger age group (18-29 years old). Regarding education, individuals with ≥12 school years are 2.4 times more likely to vaccinate all animals in the household compared to those with 0-8 school years. Conclusion The results of this study point to the fact that there's a demand for public policies to guide the population on responsible custody and to provide immunization services.

Survey of euthanasia methods used in neotropical chiropterans in São Paulo State, Brazil: critical analyses and proposal for standardization of bat euthanasia methods / evantamento dos métodos de eutanásia utilizados em quirópteros neotropicais no estado de São Paulo, Brasil: análise crítica e proposta de padronização de métodos de eutanásia em morcegos

Zoonotic Surveillance Divisions (ZSDs) rescue and euthanize bats in contact with humans. The euthanasia procedure should be conducted safely for the animal and caretaker and minimal stress for the animal is important. The objectives of this study were: i. evaluate the methods of euthanasia used by ZSDs in the State of São Paulo; ii. compare the methods with national and international guidelines for animal euthanasia practices; iii. assess the methodologies considering taxonomy and eating habits of the main bat species, and iv. propose standardization of euthanasia procedures. Sixty-five ZSDs locations received an online questionnaire or were contacted by telephone and 33 ZSDs (50.8%) responded and are distributed in 11 mesoregions in the state to remain anonymous. The euthanasia methods were divided into chemical (injectable or inhalation), physical, or mixed methods. Bat specimens (n = 550) were identified and classified to evaluate the main genera found in the state. The location of the ZSD, species, eating habits, and the method of euthanasia used were analyzed. The specimens by bat families were Molossidae (n = 340), Phyllostomidae (n = 171), and Vespertilionidae (n = 39). Chemical methods were used in 25 ZSDs (75.75%), physical in 5 (15.15%), and mixed in 3 (9.09%). There is no uniformity or standardization in bat euthanasia methods used by ZSDs, although most are based on acceptable chemical methods. It was proposed an algorithm to assist the veterinarian in choosing the method of euthanasia for bats that will allow standardizing euthanasia procedures for this species, considering physiological differences, and respecting technical, bioethical, and animal welfare guidelines.(AU)

As Divisões de Vigilância de Zoonoses (DVZs) coletam e eutanasiam morcegos que tiveram contato com humanos. O procedimento de eutanásia precisa ocorrer de modo seguro para o animal e para o manipulador, sendo importante garantir o mínimo de estresse ao animal. Os objetivos deste estudo foram: i. avaliar os métodos de eutanásia praticados nas DVZ do estado de São Paulo; ii. comparar esses métodos com os guias nacionais e internacionais de práticas de eutanásia animal; iii. avaliar as metodologias aplicadas considerando a taxonomia e os hábitos alimentares das principais espécies de ocorrência de morcegos; e iv. propor a padronização dos procedimentos de eutanásia. Sessenta e cinco DVZs receberam o questionário online ou foram contatadas por telefone, dessas, trinta e três DVZs (50,8%) que responderam ao questionário foram incluídas no estudo e distribuídas em 11 mesorregiões para garantir anonimato. Os métodos de eutanásia reportados foram divididos em métodos químicos (injetáveis; inalatórios), físicos ou mistos. Os espécimes de morcegos (n = 550) foram identificados e classificados para avaliar os principais gêneros encontrados no estado. A localização da DVZ, as espécies, os hábitos alimentares e o método de eutanásia utilizado foram analisados. Os espécimes por família de espécies de morcegos foram Molossidae (n = 340), Phyllostomidae (n = 171) e Vespertilionidae (n = 39). Métodos químicos foram utilizados em 25 DVZs (75,75%), físicos em 5 (15,15%) e mistos em 3 (9,09%). Não há uniformidade ou padronização nos métodos de eutanásia de morcegos, embora a maioria seja baseada em métodos químicos aceitáveis. É proposto um algoritmo para auxiliar o veterinário na escolha do método de eutanásia, que permite a padronização desses procedimentos para os morcegos, considerando as diferenças fisiológicas e respeitando as diretrizes técnicas, bioéticas e de bem-estar animal.(AU)

Profile of human anti-rabies care and post-exposure prophylaxis in the state of São Paulo

ABSTRACT Background: Rabies is an anthropozoonosis that greatly impacts public health and is transmitted by infected mammals. Aggression by animals is notifiable and may result in anti-rabies post-exposure prophylaxis (PEP). This study aimed to characterize anti-rabies PEP notifications in São Paulo state, Brazil. Methods: A descriptive study was conducted using data provided by the SINAN between 2013 and 2017. Results: A total of 572,889 aggressions were recorded during the study period, characterized mostly by dogs (83.5%), single wounds (56.9%), superficial wounds (58.6%), and hands/feet (34.6%). Conclusions: Animal observation was the most frequent recommendation, even in cases of attacks from non-domestic animals.

Factors associated with delayed rabies post-exposure prophylaxis among dog bite victims in Nyagatare District, Rwanda, 2017 to 2019

Background Rabies is an incurable zoonotic disease of public health concern. After exposure the only effective intervention is Post-Exposure Prophylaxis (PEP). Surveillance data in Rwanda shows that a high number of dog bites do not get PEP in time. Objectives Determine factors associated with delays in post-exposure prophylaxis among victims of dog bites. Methods The retrospective cross-sectional study was conducted in Nyagatare District. It targeted people bitten by dogs and who attended Nyagatare district from January 2017 to December 2019. Review of registers and interview with victims were used to collected data using excel and analyze them using STATA version 15. Odds ratio (OR) and confidence interval (CI) were reported. Results Among 412 dog bite victims, 161 (39%) delayed getting PEP. The more likely to delay in getting PEP, were people living in rural areas, AOR = 3.54, 95%CI [2.12, 5.90], and those without medical insurance, AOR = 4.40, 95%CI [1.82-10.62]. Conclusion Seeking PEP among dog bites victims is subject to delay in Nyagatare district. Local leaders should establish initiatives to support people to get medical insurance while public health officers need to prioritize sensitization for PEP for people living in rural areas.

Epidemiological characteristics of rabies and post-exposure immunization failure cases in Hubei Province in 2015-2021 / 公共卫生与预防医学

Objective To analyze the epidemic characteristics and causes of post-exposure immunization failure of rabies in Hubei Province from 2015 to 2021, and to provide evidence for the prevention and control of rabies in Hubei Province. Methods The investigation data of rabies cases in Hubei Province from 2015 to 2021 were collected, and descriptive epidemiological methods were used for data analysis. Results A total of 127 cases of rabies were reported in Hubei Province from 2015 to 2021, with an average annual incidence of 0.31/million, showing a downward trend. The male to female ratio was 1.70:1. Farmers accounted for 82.67% of the total cases, and the 50-79 years old group accounted for 75.59%. The incidence was mainly concentrated in Xiangyang, Shiyan, Yichang and Jingmen, accounting for 77.17%. Most of the cases were concentrated in summer and autumn. Exposure of grade Ⅱand Ⅲ accounted for 24.79% and 75.21%, respectively. Hands, lower limbs below knee, head, arms and lower limbs above knee accounted for 46.15%, 25.21%, 9.40%, 8.55% and 7.69% of the exposed parts, respectively. Dogs, cats and wild animals accounted for 95.73%, 3.42% and 0.85% of the exposed animals, respectively. Stray animals, domesticated animals, neighbors' animals and wild animals accounted for 41.88%, 37.61%, 19.66% and 0.85% of animal sources, respectively. Neither the neighbors’ animals nor domesticated animals were vaccinated against veterinary rabies virus. After exposure, 8.55% of patients went to medical institutions for standard treatment of wounds, 9.40% were vaccinated with human rabies vaccine, and 4.55% of patients with grade III exposure were injected with rabies virus immunoglobulin. The incubation period within 6 months, from 6 months to 1 year, and over 1 year accounted for 72.22%, 14.74%, and 12.04%, respectively. The exposure degree (Z=-1.98, P 2=10.91, P 2=15.73, P < 0.05) had statistically significant effects on the incubation period. Among the 11 cases of post-exposure immunization failure, all were grade Ⅲ exposure, 63.63% were exposed to the head and face, 81.81% were not fully vaccinated with human rabies virus vaccine, 63.63% were not immunized with immunoglobulin, and 27.27% were inappropriate wound treatment. Conclusion The key to rabies prevention and control is to standardize dog management, strengthen rabies education, standardize post-exposure wound treatment, timely vaccinate against rabies virus, and inject rabies virus immunoglobulin when necessary.

Development and validation of size exclusion column-high performance liquid chromatography for purity determination of bulk material of freeze-dried rabies vaccine for human use / 中国生物制品学杂志

@#Objective To develop and validate size exclusion column-high performance liquid chromatography(SEC-HPLC)for determination of the purity of bulk material of freeze-dried rabies vaccine for human use.Methods Chromatography column TSK-gel G6000PW_(XL)(7.8 mm × 30 cm,13 μm)was used for the determination(column temperature 30 ℃)with mobile phase of 0.1 mol/L PB buffer(pH 7.8)at a flow rate of 0.5 mL/min.The detection wavelength was 280 nm and injection volume was 20 μL.The method was validated for system suitability,specificity,precision and durability and determined for detection limit and quantitation limit,which was applied to analyze bulk material purity of freeze-dried rabies vaccine for human use of 3 batches of Vero cells and 1 batch of human diploid cells.Results The resolution of target protein spectrum peak of bulk material of reference sample and freeze-dried rabies vaccine for human use prepared with two substrates was more than 1.5 with a tailing factor less than 1.5;The blank solvent showed no absorption peak at the position of target protein peak with no interference in the determination;The RSDs of retention time and peak area in precision verification were both less than 2.0%;The quantitative limit was 10 μg/mL,and the detection limit was 4 μg/mL;The reference sample was injected three times continuously at three different detection wavelengths of 278,280 and 282 nm,and the RSDs of retention time and peak area were also less than 2.0%.The purity of 4 batches of freeze-dried rabies vaccine bulk material for human use was all more than 97%.Conclusion The developed SEC-HPLC for determination of the purity of freeze-dried rabies vaccine bulk material for human use showed good specificity,precision and durability,which provided a reliable method for the quality control of human rabies vaccine.

Establishment and preliminary application of dual fluorescent quantitative PCR for detection of RV and CAV2 / 公共卫生与预防医学

Objective To investigate the shedding of CAV2-ΔE3-CGS after immunization and the background of canine adenovirus (CAV) infection, and to establish a dual fluorescent quantitative PCR detection method for rabies virus (RV) and canine adenovirus type 2 (CAV2). Methods A dual fluorescent quantitative PCR detection method was established by designing specific primers and probes for E1 gene of CAV and G gene of RV for the detection of CAV2-ΔE3-CGS. Oral swabs, anal swabs and environmental samples of stray dogs from experimental animal farm and dog detention center were tested. Results The standard curves generated by this method were Y=-3.351 × logX + 44.895, R2 = .999 and Y=-3.413 × logX + 45.192, R2=0.996, respectively. The linear relationships were good, and the minimum detection limits were both 102 copies/μL. CAV2-ΔE3-CGS was not detected in experimental animal farm. CAV was detected in dog detention center, and the positive rates were 5.88% (5/85) in oral swabs, 8.24% (7/85) in anal swabs, and 4% (1/25) in environmental samples. Conclusion The dual fluorescent quantitative PCR method can be used for the detection of CAV2-ΔE3-CGS after immunization and the investigation of CAV infection. The present study has shown that no CAV2-ΔE3-CGS has been detected after immunization and CAV infection rate of stay dogs is low in Shanghai. CAV2-ΔE3-CGS oral immunization meets requirement and is applicable.

Construction and verification of recombinant chimeric vesicular stomatitis virus expressing rabies virus G protein / 中国生物制品学杂志

@#Objective To construct recombinant chimeric vesicular stomatitis virus(VSV)expressing G protein of rabies virus(RV)using VSV as vector.MethodsTo rescue the recombinant virus,G gene of VSV antigenome was replaced with G gene of RV vaccine CTN-1 strain,and co-transfected into 293T cells with helper plasmids coding T7 RNA polymerase and proteins N,P and L of VSV. The expression of RV G gene and G protein was detected by RT-PCR,immunofluorescence assay and Western blot. The recombinant virus was subcultured in Vero cells,the virus titer of different generations was detected and the virus growth curve was drawn.ResultsThe recombinant virus VSV-RVG was successfully rescued. RTPCR results demonstrated that the RV G gene was successfully inserted into the genome of the recombinant virus,and the expression of RVG protein was detected by immunofluorescence assay and Western blot. The recombinant virus was continuously passaged for 5 generations,and the virus titer was stable within 7. 5 ～ 8. 5 lgTCID50/mL.ConclusionThe recombinant chimeric VSV expressing RV G protein was successfully constructed with good genetic stability,which lays a foundation of the construction of reverse genetics technology platform based on VSV vector.

Safety and immunogenicity of pre-exposure prophylaxis of approved freeze-dried rabies vaccine(Vero cell) for human use in adults ≥18 years of age / 中国生物制品学杂志

@#Objective To evaluate the safety and immunogenicity of pre-exposure prophylaxis of the approved freeze-dried rabies vaccine(Vero cell)(rabies vaccine in brief)for human use in adults ≥18 years of age.Methods Participants aged ≥18 years in Guizhou and Shanxi provinces from June 2022 to September 2022 were enrolled,vaccinated with 1 dose of rabies vaccine at 0,7,28 d respectively,and collected for 5. 0 mL of venous blood before the first dose and 14 d after the third dose. Immunofluorescence foci assay was used to detect rabies virus neutralizing antibodies,and adverse events were collected 0～7 d after each dose of vaccine.Results A total of 120 participants were enrolled,120 participants received the first dose of rabies vaccine,119 participants received the second dose and 118 participants received the third dose.Before the first dose of rabies vaccine,the seropositive rate of antibody was 2. 54% and the geometric mean concentration(GMC)was 0. 40 IU/mL. After the third dose of rabies vaccine,the seropositive rate and seroconversion rate were 100%,the GMC was 9. 68 IU/mL,and the geometric mean increase(GMI)was 23. 99. The incidence of adverse events 0～7 d after the first,second and third doses of rabies vaccine were 22. 50%,13. 45% and 4. 24%,respectively. The local adverse events were mainly pain,and the systemic adverse events were mainly fever,fatigue/weakness,dizziness,joint pain,and diarrhea.Conclusion Approved rabies vaccine showed good safety and immunogenicity in pre-exposure prophylaxis of people ≥ 18 years old.

Collaborative calibration of the 9th national standard for human rabies vaccine / 中国生物制品学杂志

@#Objective To collaboratively calibrate and finally assign the potency of the 9th national standard for human rabies vaccine for candidate.Methods Qualified laboratories for the production,research and development of human rabies vaccines were organized to determine the potency of candidate national standard for rabies vaccines by using NIH method,with the 7th international standard for rabies vaccines(NIBSC code:16/204)as the reference standard. The detection results were statistically analyzed,and the geometric mean of effective detection values was taken as the final potency value of the candidate standard. According to the requirements of the preparation of national drug reference materials,the candidate standard was destroyed by heat acceleration,and then detected for the glycoprotein antigen to investigate the stability.Results A total of 20 laboratories participated in the collaborative calibration,of which the two laboratories that did not strictly follow the collaborative calibration SOP were excluded,and the data of the remaining collaborative laboratories were valid. After statistical analysis,the final potency of the 9th national standard for rabies vaccine was 11. 4 IU/mL,the 95% confidence limit was 10. 9～11. 9 IU/mL,and the 95% reference range of ED_(50) was 2. 10～2. 75. There was no significant difference in the results of glycoprotein antigen detection under different time conditions(2,4,8 and 16 weeks)at 37 ℃.Conclusion The collaborative calibration research of the 9th national standard for human rabies vaccine(batch number:201906001)has been completed,the potency assignment is scientific and rigorous,the data was reliable,and the thermal stability meets the requirements. At present,this standard has been approved by the National Drug Reference Material Committee,which is of great significance to the quality control of human rabies vaccine,especially the quality control of effectiveness.

Development and verification of qPCR method for detection of residual host DNA in human rabies vaccine(Vero cells) / 中国生物制品学杂志

@#Objective To develop and verify a qPCR method for the qualitative and quantitative analysis of residual host DNA in human rabies vaccine(Vero cells) stock solution.Methods The qPCR standard curve was established by using the Vero cell DNA quantitative national standard,and the residual host DNA was extracted using magnetic beads.The specificity,repeatability,intermediate precision,accuracy and durability of the method were verified,and the linear range and limit of quantification were determined.The residual DNA of three batches of human rabies vaccine(Vero cells) stock solution was quantitatively analyzed and the fragment size was qualitatively analyzed by using this method.Results The correlation coefficients(R~2) of Vero cell DNA quantitative national standard amplification standard curve were all more than 0.99 by qPCR,and the quantitative range was 0.3 pg/mL～30 ng/mL.The method showed good specificity and repeatability.In the verification of intermediate precision,accuracy and durability,the relative standard deviations(RSD)of detection results of the samples were all less than 10%.The residual DNA content of Vero cells in three batches of stock solution was 0.20～0.77 ng/dose,which met the relevant standard of Chinese Pharmacopoeia(Volume Ⅲ,2020edition).The residual DNA fragments greater than 154 bp accounted for 52%～63%.Conclusion The developed qPCR method for the detection of residual DNA in human rabies vaccine(Vero cells) stock solution had good specificity,repeatability,intermediate precision and durability,and qualitatively and quantitatively analyzed the residual DNA rapidly and accurately,which was of great significance for improving the detection and control of residual DNA content in the production process and final product of human rabies vaccine(Vero cells).

Rabies virus viability in vitro / 中华微生物学和免疫学杂志

Objective:To investigate the in vitro viability of rabies virus in tissues and body fluid samples. Methods:The viability of rabies virus in tissues and suspensions was analyzed by virus titer determination method, direct immunofluorescence, RT-PCR and laboratory techniques for virus isolation.Results:With the increase of temperature, the viability of rabies virus in brain tissues and suspensions decreased gradually. Rabies virus lost infectivity after 30 min at 56℃, but remained viable in tissues for 7 d at 37℃. The virus showed no viability after 1 h at pH9.6. The rabies virus in suspensions could be completely inactivated after the stimulation with ethanol at a final concentration above 30%, sodium hypochlorite above 500 mg/L or benzalkonium bromide above 100 mg/L for 3 min. It was found that 80% acetone had the strongest inactivation effect on rabies virus in tissues, and no virus could be isolated after soaking for 4 h.Conclusions:Rabies virus was not tolerant to high temperature and relatively stable in the environment with pH6.8-7.4. Common disinfectants could kill the virus. This study provided detailed data about the viability of rabies virus in vitro, which would be conducive to the prevention and control of rabies.

Comparison of immunization strategies for human rabies pre-exposure prophylaxis in laboratory animals / 中华微生物学和免疫学杂志

Objective:To compare the differences in the safety, efficacy and protective effects of rabies vaccine using the current pre-exposure prophylaxis schedule in China (0-7-21 or 28) and the newly recommended immunization program of WHO (0-7), aiming to provide data support for modifying the related content of Technical Guideline for Human Rabies Prevention and Control. Methods:The mice were randomly divided into five groups, namely 0-7-21 group (3-injection regimen), 0-7 group (2-injection regimen), 0-14 group, 0-21 group and control group, according to the current 3-injection regimen (0-7-21) in China and the 2-injection regimen (0-7) recommended by WHO. The survival status of the mice was observed. The mice were weighed every five days to compare the safety of different immunization procedures. Rabies virus neutralizing antibodies (RVNA) were detected 7, 14, 21, 28 and 35 d after the initial immunization. On day 35, the mice in each group were challenged with lethal dose of CVS-11 rabies virus to evaluate the protective effects of different pre-exposure immunization procedures.Results:There was no significant difference in weight gain of mice after vaccination. The positive rate of RVNA was 100% in all immunized groups from day 14, which could provide complete protection to mice. There was a significant difference in RVNA levels between 0-7-21 and 0-7 groups at 35 d( P<0.05), but there was no statistical difference at other time points ( P>0.05). RVNA level had a significant difference between 0-7 and 0-21 groups at 21 d and 35 d ( P<0.05). There was no statistical difference in RVNA level between other groups at each time point ( P>0.05). In the protective test, the survival rates of mice in all immune groups were 100%. Conclusions:The current 3-injection pre-exposure immunization procedure for rabies vaccine (0-7-21) and the newly recommended 2-injection immunization procedure (0-7) had similar efficacy and protective effects in animal tests. In view of the cost saving and better compliance of the 2-injection immunization procedure, it was recommended that relevant departments should conduct clinical trials as soon as possible to promote the implementation of this program.

Meta-analysis of safety of human purified Vero cell rabies vaccine after exposure / 中国生物制品学杂志

@#Objective To evaluate the safety of human purified Vero cell rabies vaccine(PVRV)after exposure in China by Meta-analysis.Methods With rabies,vaccine and safety as key words,a systematic search was performed in PubMed,EMBASE,Cochrane and China National Knowledge Infrastructure(CNKI),supplemented by manual retrieval.A Meta-analysis was performed to analyze the incidence of adverse events of two immunization regimens Zagreb and Essen using Review Manager 5.4 software after literature screening and data extraction according to the inclusion and exclusion criteria.Results A total of 12 studies were included,of which 7 were prospective studies and 5 were retrospective studies.Most included in the studies showed a low risk of bias.The incidence of adverse events in Zagreb regimen was significantly higher than that in Essen regimen[relative risk(RR)= 1.01,95% CI = 0.90 ～ 1.14;I2= 73.00%,P<0.05],but there was a high degree of heterogeneity.The incidence of fever,pain and induration in Zagreb regimen was significantly higher than that in Essen regimen(RR = 1.14,0.92 and 0.86,95% CI = 0.82 ～ 1.60,0.73 ～ 1.14 and 0.29 ～ 2.51;I2= 73.00%,P<0.05],but there was a high degree of heterogeneity.The incidence of fever,pain and induration in Zagreb regimen was significantly higher than that in Essen regimen(RR = 1.14,0.92 and 0.86,95% CI = 0.82 ～ 1.60,0.73 ～ 1.14 and 0.29 ～ 2.51;I2= 81%,65% and 92%,respectively,P<0.01).Conclusion Two regimens of PVRV vaccination after exposure showed good safety.However,when adopting Zagreb regimen,attention should be paid to the physical conditions of children and the elderly with relatively poor immunity to avoid adverse events.