Editorial

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Open AccessEditorial

Special Issue “Advances in Rabies Research”

by Susan A. Nadin-Davis

Viruses 2023, 15(7), 1557; https://doi.org/10.3390/v15071557 - 16 Jul 2023

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Rabies kills approximately 60,000 humans each year, with deaths mostly occurring in developing countries, where rabies lyssavirus (RABV) variants are maintained in dog populations [...] Full article

(This article belongs to the Special Issue Advances in Rabies Research)

Research

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11 pages, 1507 KiB

Open AccessArticle

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay Targeting Rabies-Specific IgM and IgG in Human Sera

by Michelle D. Zajac, Maria Teresa Ortega and Susan M. Moore

Viruses 2023, 15(4), 874; https://doi.org/10.3390/v15040874 - 29 Mar 2023

Cited by 1 | Viewed by 1501

Abstract

Immunity from rabies depends on rabies virus neutralizing antibodies (RVNA) induced after immunization; however, the influence of antibody isotype switching has not been extensively investigated. This has become particularly relevant with changes in World Health Organization (WHO) recommended rabies vaccine regimens that may [...] Read more.

Immunity from rabies depends on rabies virus neutralizing antibodies (RVNA) induced after immunization; however, the influence of antibody isotype switching has not been extensively investigated. This has become particularly relevant with changes in World Health Organization (WHO) recommended rabies vaccine regimens that may influence RVNA isotype kinetics, potentially affecting the peak, and longevity, of RVNA immunoglobulin (IgG) levels. We developed rapid and reliable assays for quantifying the anti-rabies IgM/IgG class switch in human serum based on an indirect ELISA technique. The immune response was tracked in ten individuals naïve to the rabies vaccine by quantifying serum titers weekly, from day seven to day 42 post-immunization, using a serum neutralization assay and the ELISA IgM/IgG assays. The average RVNA IU/mL levels were at D0 ≤ 0.1, D7 0.24, D14 8.36, D21 12.84, D28 25.74 and D42 28.68. Levels of specific IgM antibodies to rabies glycoprotein (EU/mL) were higher, on average, at D7, 1.37, and from D14, 5.49, to D21, 6.59. In contrast, average IgG antibodies (EU/mL) predominated from D28, 10.03, to D42, 14.45. We conclude that levels of anti-rabies IgM/IgG at D28 characterize the isotype class switch. These assays, combined with serum neutralization assays, distinguished the RVNA levels in terms of the IgM/IgG responses and are expected to add to the diagnostic repertoire, provide additional information in establishing rabies vaccine regimens, both post- and pre-exposure prophylaxis, and contribute to research efforts. Full article

(This article belongs to the Special Issue Advances in Rabies Research)

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13 pages, 741 KiB

Open AccessArticle

Comparison of Pan-Lyssavirus RT-PCRs and Development of an Improved Protocol for Surveillance of Non-RABV Lyssaviruses

by Petra Drzewnioková, Sabrina Marciano, Stefania Leopardi, Valentina Panzarin and Paola De Benedictis

Viruses 2023, 15(3), 680; https://doi.org/10.3390/v15030680 - 04 Mar 2023

Cited by 1 | Viewed by 1671

Abstract

Rabies is a zoonotic and fatal encephalitis caused by members of the Lyssavirus genus. Among them, the most relevant species is Lyssavirus rabies, which is estimated to cause 60,000 human and most mammal rabies deaths annually worldwide. Nevertheless, all lyssaviruses can invariably [...] Read more.

Rabies is a zoonotic and fatal encephalitis caused by members of the Lyssavirus genus. Among them, the most relevant species is Lyssavirus rabies, which is estimated to cause 60,000 human and most mammal rabies deaths annually worldwide. Nevertheless, all lyssaviruses can invariably cause rabies, and therefore their impact on animal and public health should not be neglected. For accurate and reliable surveillance, diagnosis should rely on broad-spectrum tests able to detect all known lyssaviruses, including the most divergent ones. In the present study, we evaluated four different pan-lyssavirus protocols widely used at an international level, including two real-time RT-PCR assays (namely LN34 and JW12/N165-146), a hemi-nested RT-PCR and a one-step RT-PCR. Additionally, an improved version of the LN34 assay ((n) LN34) was developed to increase primer–template complementarity with respect to all lyssavirus species. All protocols were evaluated in silico, and their performance was compared in vitro employing 18 lyssavirus RNAs (encompassing 15 species). The (n) LN34 assay showed enhanced sensitivity in detecting most lyssavirus species, with limits of detection ranging from 10 to 100 RNA copies/µL depending on the strain, while retaining high sensitivity against Lyssavirus rabies. The development of this protocol represents a step forward towards improved surveillance of the entire Lyssavirus genus. Full article

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15 pages, 2159 KiB

Open AccessArticle

Comparing Control Intervention Scenarios for Raccoon Rabies in Southern Ontario between 2015 and 2025

by Emily Sohanna Acheson, François Viard, Tore Buchanan, Larissa Nituch and Patrick A. Leighton

Viruses 2023, 15(2), 528; https://doi.org/10.3390/v15020528 - 14 Feb 2023

Cited by 4 | Viewed by 2213

Abstract

The largest outbreak of raccoon rabies in Canada was first reported in Hamilton, Ontario, in 2015 following a probable translocation event from the United States. We used a spatially-explicit agent-based model to evaluate the effectiveness of provincial control programs in an urban-centric outbreak [...] Read more.

The largest outbreak of raccoon rabies in Canada was first reported in Hamilton, Ontario, in 2015 following a probable translocation event from the United States. We used a spatially-explicit agent-based model to evaluate the effectiveness of provincial control programs in an urban-centric outbreak if control interventions were used until 2025, 2020, or never used. Calibration tests suggested that a seroprevalence of protective rabies antibodies 2.1 times higher than that inferred from seroprevalence in program assessments was required in simulations to replicate observed raccoon rabies cases. Our simulation results showed that if control interventions with an adjusted seroprevalence were used until 2025 or 2020, the probability of rabies elimination due to control intervention use was 49.2% and 42.1%, respectively. However, if controls were never used, the probability that initial rabies cases failed to establish a sustained outbreak was only 18.2%. In simulations where rabies was not successfully eliminated, using control interventions until 2025 resulted in 67% fewer new infections compared to only applying controls until 2020 and in 90% fewer new infections compared to no control intervention use. However, the model likely underestimated rabies elimination rates since we did not adjust for adaptive control strategies in response to changes in rabies distributions and case numbers, as well as extending control interventions past 2025. Our agent-based model offers a cost-effective strategy to evaluate approaches to rabies control applications. Full article

(This article belongs to the Special Issue Advances in Rabies Research)

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10 pages, 1445 KiB

Open AccessCommunication

From Field Tests to Molecular Tools—Evaluating Diagnostic Tests to Improve Rabies Surveillance in Namibia

by Conrad M. Freuling, Jolandie van der Westhuizen, Siegfried Khaiseb, Tenzin Tenzin and Thomas Müller

Viruses 2023, 15(2), 371; https://doi.org/10.3390/v15020371 - 28 Jan 2023

Cited by 1 | Viewed by 1978

Abstract

Rabies is endemic in Namibia and is present both in wildlife carnivores and domestic free-roaming dogs. The disease thus represents a challenge for public human and veterinary disease control. Namibia has implemented a national strategic plan to control rabies and the country’s activities [...] Read more.

Rabies is endemic in Namibia and is present both in wildlife carnivores and domestic free-roaming dogs. The disease thus represents a challenge for public human and veterinary disease control. Namibia has implemented a national strategic plan to control rabies and the country’s activities are supported by international organizations. To this end, rabies diagnosis at the Central Veterinary Laboratory (CVL) was improved in the frame of a World Organization for Animal Health (WOAH) laboratory twinning program: from practical sampling techniques and the use of lateral flow devices to a novel universal and discriminatory quantitative real-time Reverse transcription polymerase chain reaction (RT-qPCR), which easily identify dog-associated rabies viruses. The procedures applied and the results can be used as a template to improve rabies laboratory diagnosis. Full article

(This article belongs to the Special Issue Advances in Rabies Research)

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12 pages, 1335 KiB

Open AccessArticle

Factors Associated with Dog Rabies Immunization in Changsha, China: Results of a Cross-Sectional Cluster Survey, 2015–2021

by Chunxiao Ji, Jia Feng, Siying Li, Hui Yang, Hui Wang, Xiangchang Geng, Hongliang Wang, Zengzai Liu, Tao Zhang, Yu He and Wei Liu

Viruses 2023, 15(1), 138; https://doi.org/10.3390/v15010138 - 31 Dec 2022

Cited by 6 | Viewed by 1870

Abstract

The objective of this study was to examine longitudinal trends in the prevalence of dogs that are successfully immunized against rabies virus (as measured by sufficient serum antibodies) in Changsha, an urban center of China. The secondary objective was to investigate the factors [...] Read more.

The objective of this study was to examine longitudinal trends in the prevalence of dogs that are successfully immunized against rabies virus (as measured by sufficient serum antibodies) in Changsha, an urban center of China. The secondary objective was to investigate the factors affecting the seroprevalence of rabies virus antibodies in dogs. In this study, 4515 canine serum samples were collected from 57 pet hospitals (immunization points) during the period of 2015–2021 in five major urban areas of Kaifu, Furong, Tianxin, Yuhua, and Yuelu in Changsha, China. The enzyme-linked immunosorbent assay (ELISA) method was used to analyze the level and trend of rabies virus antibodies in serum and further evaluate the potential factors affecting the immunization effect from five factors: sex, age, time interval after most recent vaccination and sample collection, number of vaccinations, and vaccine manufacturer. The results showed that the seroconversion from the urban dog in Changsha steadily increased from 46.13% to 73.38% during 2015–2017. The seropositivity prevalence remained above the international standard (70%) from 2018 to 2020 and up to 90.99% in 2021. Further analysis showed that the seroconversion of rabies virus among dogs was significantly affected by the age, the number of vaccinations, time interval after the most recent vaccination and sample collection, and vaccine manufacturer, while sex had less influence. The overall rabies vaccination situation in urban areas of Changsha generally meets international standards, with only a few areas showing low levels of antibodies in dogs after vaccination and risk of infectiousness. Therefore, it is recommended that the first vaccination should be given when the dog is about three months old and regularly repeated every year after that. At the same time, antibody concentrations in dogs, especially in newborn puppies and older dogs, need to be tested promptly after vaccination at the required time to ensure that they are at a high level of immune protection, which can strengthen the supervision of rabies. Full article

(This article belongs to the Special Issue Advances in Rabies Research)

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22 pages, 4064 KiB

Open AccessArticle

Susceptibilities of CNS Cells towards Rabies Virus Infection Is Linked to Cellular Innate Immune Responses

by Lena Feige, Tatsuya Kozaki, Guilherme Dias de Melo, Vincent Guillemot, Florence Larrous, Florent Ginhoux and Hervé Bourhy

Viruses 2023, 15(1), 88; https://doi.org/10.3390/v15010088 - 29 Dec 2022

Cited by 5 | Viewed by 2585

Abstract

Rabies is caused by neurotropic rabies virus (RABV), contributing to 60,000 human deaths annually. Even though rabies leads to major public health concerns worldwide, we still do not fully understand factors determining RABV tropism and why glial cells are unable to clear RABV [...] Read more.

Rabies is caused by neurotropic rabies virus (RABV), contributing to 60,000 human deaths annually. Even though rabies leads to major public health concerns worldwide, we still do not fully understand factors determining RABV tropism and why glial cells are unable to clear RABV from the infected brain. Here, we compare susceptibilities and immune responses of CNS cell types to infection with two RABV strains, Tha and its attenuated variant Th2P-4M, mutated on phospho- (P-protein) and matrix protein (M-protein). We demonstrate that RABV replicates in human stem cell-derived neurons and astrocytes but fails to infect human iPSC-derived microglia. Additionally, we observed major differences in transcription profiles and quantification of intracellular protein levels between antiviral immune responses mediated by neurons, astrocytes (IFNB1, CCL5, CXCL10, IL1B, IL6, and LIF), and microglia (CCL5, CXCL10, ISG15, MX1, and IL6) upon Tha infection. We also show that P- and M-proteins of Tha mediate evasion of NF-κB- and JAK-STAT-controlled antiviral host responses in neuronal cell types in contrast to glial cells, potentially explaining the strong neuron-specific tropism of RABV. Further, Tha-infected astrocytes and microglia protect neurons from Tha infection via a filtrable and transferable agent. Overall, our study provides novel insights into RABV tropism, showing the interest in studying the interplay of CNS cell types during RABV infection. Full article

(This article belongs to the Special Issue Advances in Rabies Research)

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13 pages, 1277 KiB

Open AccessArticle

Geographic Range Overlap Rather than Phylogenetic Distance Explains Rabies Virus Transmission among Closely Related Bat Species

by Maude Jacquot, Megan A. Wallace, Daniel G. Streicker and Roman Biek

Viruses 2022, 14(11), 2399; https://doi.org/10.3390/v14112399 - 29 Oct 2022

Cited by 5 | Viewed by 1546

Abstract

The cross-species transmission (CST) of pathogens can have dramatic consequences, as highlighted by recent disease emergence events affecting human, animal and plant health. Understanding the ecological and evolutionary factors that increase the likelihood of disease agents infecting and establishing in a novel host [...] Read more.

The cross-species transmission (CST) of pathogens can have dramatic consequences, as highlighted by recent disease emergence events affecting human, animal and plant health. Understanding the ecological and evolutionary factors that increase the likelihood of disease agents infecting and establishing in a novel host is therefore an important research area. Previous work across different pathogens, including rabies virus (RABV), found that increased evolutionary distance between hosts reduces the frequency of cross-species transmission and of permanent host shifts. However, whether this effect of host relatedness still holds for transmission among recently diverged hosts is not well understood. We aimed to ask if high host relatedness can still increase the probability of a host shift between more recently diverged hosts, and the importance of this effect relative to ecological predictors. We first addressed this question by quantifying the CST frequency of RABV between North American bat species within the genus Myotis, using a multi-decade data set containing 128 nucleoprotein (N) RABV sequences from ten host species. We compared RABV CST frequency within Myotis to the rates of CST between nine genera of North American bat species. We then examined whether host relatedness or host range overlap better explains the frequency of CST seen between Myotis species. We found that at the within genus scale, host range overlap, rather than host relatedness best explains the frequency of CST events. Moreover, we found evidence of CST occurring among a higher proportion of species, and CST more frequently resulting in sustained transmission in the novel host in the Myotis dataset compared to the multi-genus dataset. Our results suggest that among recently diverged species, the ability to infect a novel host is no longer restricted by physiological barriers but instead is limited by physical contact. Our results improve predictions of where future CST events for RABV might occur and clarify the relationship between host divergence and pathogen emergence. Full article

(This article belongs to the Special Issue Advances in Rabies Research)

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20 pages, 2182 KiB

Open AccessArticle

Ampliseq for Illumina Technology Enables Detailed Molecular Epidemiology of Rabies Lyssaviruses from Infected Formalin-Fixed Paraffin-Embedded Tissues

by Susan Angela Nadin-Davis, Allison Hartke and Mingsong Kang

Viruses 2022, 14(10), 2241; https://doi.org/10.3390/v14102241 - 12 Oct 2022

Cited by 1 | Viewed by 1391

Abstract

Whole genome sequencing of rabies lyssaviruses (RABVs) has enabled the generation of highly detailed phylogenies that reveal viral transmission patterns of disease in reservoir species. Such information is highly important for informing best practices with respect to wildlife rabies control. However, specimens available [...] Read more.

Whole genome sequencing of rabies lyssaviruses (RABVs) has enabled the generation of highly detailed phylogenies that reveal viral transmission patterns of disease in reservoir species. Such information is highly important for informing best practices with respect to wildlife rabies control. However, specimens available only as formalin fixed paraffin embedded (FFPE) samples have been recalcitrant to such analyses. Due to the damage inflicted by tissue processing, only relatively short amplicons can be generated by standard RT-PCR methods, making the generation of full-length genome sequences very tedious. While highly parallel shotgun sequencing of total RNA can potentially overcome these challenges, the low percentage of reads representative of the virus may be limiting. Ampliseq technology enables massively multiplex amplification of nucleic acids to produce large numbers of short PCR products. Such a strategy has been applied to the sequencing of entire viral genomes but its use for rabies virus analysis has not been reported previously. This study describes the generation of an Ampliseq for Illumina primer panel, which was designed based on the global sequence diversity of rabies viruses, and which enables efficient viral genome amplification and sequencing of rabies-positive FFPE samples. The subsequent use of such data for detailed phylogenetic analysis of the virus is demonstrated. Full article

(This article belongs to the Special Issue Advances in Rabies Research)

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16 pages, 2620 KiB

Open AccessArticle

Genetic Characterization of Human Rabies Vaccine Strain in Japan and Rabies Viruses Related to Vaccine Development from 1940s to 1980s

by Madoka Horiya, Guillermo Posadas-Herrera, Mutsuyo Takayama-Ito, Yukie Yamaguchi, Itoe Iizuka-Shiota, Hirofumi Kato, Aikou Okamoto, Masayuki Saijo and Chang-Kweng Lim

Viruses 2022, 14(10), 2152; https://doi.org/10.3390/v14102152 - 29 Sep 2022

Cited by 2 | Viewed by 2178

Abstract

The rabies virus is widely distributed and vaccines are an important strategy to prevent its spread. The whole-genome sequences of rabies strains in relation to vaccine development provide essential information to maintain vaccine quality and develop new vaccines. However, the genetic characteristics of [...] Read more.

The rabies virus is widely distributed and vaccines are an important strategy to prevent its spread. The whole-genome sequences of rabies strains in relation to vaccine development provide essential information to maintain vaccine quality and develop new vaccines. However, the genetic characteristics of the purified chick embryo cell culture rabies vaccine, KM Biologics (PCECV-KMB), developed in Japan in the 1970s, have not been explored. In this study, we conducted a genome-wide analysis of the open reading frame regions of rabies strains discovered from the 1940s–1980s and used to develop chick embryo cell-adapted HEP-Flury small plaque-forming (CEF-S) strain, which is a vaccine strain of PCECV-KMB. The genetic characteristic of CEF-S, developed by acclimation of the HEP-Flury-NIID strain to one-day eggs and subsequently to chick embryo cells, were confirmed by comparing the genome identity and revealing the nine amino acid mutations between CEF-S and HEP-Flury-NIID. The efficacy of PCECV-KMB was evaluated using attack strains isolated in Thailand in the 1960s–1970s during vaccine development. Phylogenetic analyses of the attack strains classified them in the same Asian clade as the 2000s imported cases from the Philippines to Japan, suggesting that PCECV-KMB is adequate for preventing the spread of the current rabies virus. Full article

(This article belongs to the Special Issue Advances in Rabies Research)

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15 pages, 2155 KiB

Open AccessArticle

Oral Rabies Vaccine Strain SPBN GASGAS: Genetic Stability after Serial In Vitro and In Vivo Passaging

by Stefan Borutzki, Benjamin Richter, Matthias Proemmel, Izabela Fabianska, Hon Quang Tran, Boris Hundt, Dietmar Mayer, Christian Kaiser, Andreas Neubert and Ad Vos

Viruses 2022, 14(10), 2136; https://doi.org/10.3390/v14102136 - 28 Sep 2022

Cited by 3 | Viewed by 1702

Abstract

Oral vaccination of wildlife has shown to be a very effective management tool in rabies control. Evaluation of the genetic stability of vaccine viruses before distributing vaccine baits in the environment is essential because all available oral rabies vaccines, including the genetically engineered [...] Read more.

Oral vaccination of wildlife has shown to be a very effective management tool in rabies control. Evaluation of the genetic stability of vaccine viruses before distributing vaccine baits in the environment is essential because all available oral rabies vaccines, including the genetically engineered rabies virus vaccine strain SPBN GASGAS (Rabitec), are based on replication-competent viruses. To evaluate the genetic stability of this vaccine strain, five serial passages of the Master Seed Virus (MSV) in the production cell line BHK21 Cl13 were performed. Furthermore, to test possible reversion to virulence, a back-passage study in suckling mouse brain (SMB) was performed. Subsequently, the pooled 5th SMB passage was inoculated intracerebrally (i.c.) in adult and suckling mice. The full genome sequences of the isolated 5th passage, in vivo and in vitro, were compared at both the consensus and the quasispecies level with the MSV. Additionally, the full genome sequence of the 6th SMB passage from the individual animals was determined and compared. Full-length integration of the double glycoprotein and modified base substitutions at amino acid position 194 and 333 of the glycoprotein could be verified in all 5th and 6th passage samples. Overall, 11 single nucleotide polymorphisms (SNPs) were detected in the 5th pooled SMB passage, 4 with frequency between 10 and 20%, and 7 with between 2.5 and 10%. SNPs that resulted in amino acid exchange were found in genes: N (one SNP), G (four SNPs), and L (three SNPs). However, none of these SNPs were associated with reversion to virulence since all adult mice inoculated i.c. with this material survived. In the individual samples of the 6th SMB passage 24 additional SNPs (>2.5%) were found, of which only 1 SNP (L-gene, position 6969) had a prevalence of >50% in 3 of 17 samples. The obtained results confirmed the stable expression of genetic modifications and the genetic stability of the consensus strain after serial in vivo and in vitro passaging. Full article

(This article belongs to the Special Issue Advances in Rabies Research)

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11 pages, 865 KiB

Open AccessArticle

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

by Lisa Dettinger, Crystal M. Gigante, Maria Sellard, Melanie Seiders, Puja Patel, Lillian A. Orciari, Pamela Yager, James Lute, Annette Regec, Yu Li and Dongxiang Xia

Viruses 2022, 14(9), 1845; https://doi.org/10.3390/v14091845 - 23 Aug 2022

Cited by 3 | Viewed by 1901

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Advances in Rabies Research)

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7 pages, 2129 KiB

Open AccessOpinion

Alternative Methods to Current In Vivo Procedures to Address the 3Rs Tenet in Rabies Proficiency Testing

by Maira Zorzan, Morgane Gourlaouen, Stefania Leopardi and Paola De Benedictis

Viruses 2022, 14(8), 1698; https://doi.org/10.3390/v14081698 - 31 Jul 2022

Cited by 1 | Viewed by 1620

Abstract

Canine rabies is responsible for an estimated 59,000 human deaths every year. In an attempt to reach the ZeroBy30 goal, robust disease surveillance coupled with improved diagnostics play a paramount role in ensuring reliable data and gradually attesting rabies control advancements. In this [...] Read more.

Canine rabies is responsible for an estimated 59,000 human deaths every year. In an attempt to reach the ZeroBy30 goal, robust disease surveillance coupled with improved diagnostics play a paramount role in ensuring reliable data and gradually attesting rabies control advancements. In this context, proficiency testing is organized to harmonize rabies diagnostic capacities. In most exercises, rabies-positive samples consist of brains collected from intracerebrally inoculated mice. This procedure causes distress and severe suffering to animals, raising important ethical concerns that can no longer be ignored. In the last decades, the 3Rs tenet (Replace, Reduce, Refine) has been successfully implemented in several scientific areas, and we strongly support its application in the framework of rabies proficiency testing. Here, we discuss cell-based technologies as innovative sustainable in vitro candidate systems to replace in vivo experiments for the production of proficiency testing samples. The application of these alternative methods can allow completely in vitro or ex vivo production of rabies proficiency testing panels, which would represent an important replacement or reduction/refinement for current in vivo procedures. Full article

(This article belongs to the Special Issue Advances in Rabies Research)

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