Lyssavirus matrix protein inhibits NLRP3 inflammasome assembly by binding to NLRP3.

Lyssavirus is a kind of neurotropic pathogen that needs to evade peripheral host immunity to enter the central nervous system to accomplish infection. NLRP3 inflammasome activation is essential for the host to defend against pathogen invasion. This study demonstrates that the matrix protein (M) of lyssavirus can inhibit both the priming step and the activation step of NLRP3 inflammasome activation. Specifically, M of lyssavirus can compete with NEK7 for binding to NLRP3, which restricts downstream apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. The serine amino acid at the 158th site of M among lyssavirus is critical for restricting ASC oligomerization. Moreover, recombinant lab-attenuated lyssavirus rabies (rabies lyssavirus [RABV]) with G158S mutation at M decreases interleukin-1ß (IL-1ß) production in bone-marrow-derived dendritic cells (BMDCs) to facilitate lyssavirus invasion into the brain thereby elevating pathogenicity in mice. Taken together, this study reveals a common mechanism by which lyssavirus inhibits NLRP3 inflammasome activation to evade host defenses.

RTP4 restricts lyssavirus rabies infection by binding to viral genomic RNA.

Rabies, caused by lyssavirus rabies (Rabies lyssavirus, RABV), is a fatal disease among humans and almost all warm-blooded animals. In this study, we found that RABV infection induces the up-regulation of receptor transporter protein 4 (RTP4) in mouse brains and different cells of nervous tissue. Over-expression of RTP4 reduces the viral titer of RABV in different neuronal cells. Furthermore, a recombinant RABV expressing RTP4, named rRABV-RTP4, was constructed and displayed a lower viral titer in different neuronal cells due to the expression of RTP4. Moreover, the survival rates of mice infected with rRABV-RTP4 were significantly higher than those of mice infected with parent virus rRABV or control virus rRABV-RTP4(-). In terms of mechanism, RTP4 could bind viral genomic RNA (vRNA) of RABV, and suppress the whole viral genome amplification. In addition, we found that the zinc finger domain (ZFD) of RTP4 exerts the antiviral function by truncation analysis, and an important amino acids site (C95) in the RTP4 3CxxC motif which is essential for its antiviral function was identified by mutation analysis. This study contributes to our understanding of how RTP4 or other RTP proteins play a role in defense against the invasion of RABV or other viruses.

The first confirmed human case of rabies, Timor-Leste, 2024.

In March 2024, the first ever human case of rabies, following a dog bite, was detected in Timor-Leste. This paper briefly discusses the circumstances of transmission, clinical presentation, palliative care of the case and public health measures taken. Timor-Leste was previously considered rabies-free. Any person who is bitten or scratched by an animal that could potentially transmit rabies virus (especially dogs, bats, monkeys or cats) in Timor-Leste should be assessed for consideration of provision of rabies post-exposure prophylaxis.

Evaluation of LN34 Pan-Lyssavirus RT-qPCR assay for rabies diagnosis in Brazil.

Rabies, a fatal zoonotic viral disease affecting mammals, including humans, remains a significant global health concern, particularly in low-income countries. The disease, primarily transmitted through infected animal saliva, prompts urgent diagnosis for timely post-exposure prophylaxis (PEP). The gold standard diagnostic test, direct fluorescent antibody test (dFAT), while sensitive, suffers from limitations such as subjective interpretation and high costs. As a confirmatory technique, the LN34 Pan-Lyssavirus RT-qPCR assay has emerged as a promising tool for universal Lyssavirus detection. This study evaluated its performance using 130 rabies virus isolates representing eleven Brazilian variants and 303 clinical samples from surveillance operations. The LN34 assay demonstrated 100% sensitivity and 98% specificity compared to dFAT. Additionally, it detected all samples, including those missed by dFAT, indicating superior sensitivity. The assay's specificity was confirmed through Sanger nucleotide sequencing, with only a minimal false-positive rate. Comparative analysis revealed higher accuracy and concordance with dFAT than traditional rabies tissue culture infection tests (RTCIT). False-negative RTCIT results were attributed to low viral load or suboptimal sampling. These findings underscore the LN34 assay's utility as a confirmatory technique, enhancing rabies surveillance and control in Brazil. Its widespread adoption could significantly improve diagnostic sensitivity, crucial for effective PEP and public health interventions.

Reemergence of a Big Brown Bat Lyssavirus rabies Variant in Striped Skunks in Flagstaff, Arizona, USA, 2021-2023.

Background: Throughout the Americas, Lyssavirus rabies (RV) perpetuates as multiple variants among bat and mesocarnivore species. Interspecific RV spillover occurs on occasion, but clusters and viral host shifts are rare. The spillover and host shift of a big brown bat (Eptesicus fuscus) RV variant Ef-W1 into mesocarnivores was reported previously on several occasions during 2001-2009 in Flagstaff, Arizona, USA, and controlled through rabies vaccination of target wildlife. During autumn 2021, a new cluster of Ef-W1 RV cases infecting striped skunks (Mephitis mephitis) was detected from United States Department of Agriculture enhanced rabies surveillance in Flagstaff. The number of Ef-W1 RV spillover cases within a short timeframe suggested the potential for transmission between skunks and an emerging host shift. Materials and Methods: Whole and partial RV genomic sequencing was performed to evaluate the phylogenetic relationships of the 2021-2023 Ef-W1 cases infecting striped skunks with earlier outbreaks. Additionally, real-time reverse-transcriptase PCR (rtRT-PCR) was used to opportunistically compare viral RNA loads in brain and salivary gland tissues of naturally infected skunks. Results: Genomic RV sequencing revealed that the origin of the 2021-2023 epizootic of Ef-W1 RV was distinct from the multiple outbreaks detected from 2001-2009. Naturally infected skunks with the Ef-W1 RV showed greater viral RNA loads in the brain, but equivalent viral RNA loads in the mandibular salivary glands, compared to an opportunistic sample of skunks naturally infected with a South-Central skunk RV from northern Colorado, USA. Conclusion: Considering a high risk for onward transmission and spread of the Ef-W1 RV in Flagstaff, public outreach, enhanced rabies surveillance, and control efforts, focused on education, sample characterization, and vaccination, have been ongoing since 2021 to mitigate and prevent the spread and establishment of Ef-W1 RV in mesocarnivores.

The matrix protein of lyssavirus hijacks autophagosome for efficient egress by recruiting NEDD4 through its PPxY motif.

Lyssaviruses are well-known worldwide and often cause fatal encephalitis. Previous studies have shown that autophagy is beneficial for the replication of rabies virus (RABV), the representative lyssavirus, but the detailed mechanism remains obscure. In this study, we showed that the rabies virus matrix protein (RABV-M) used its PPxY motif to interact with the E3 ubiquitin-protein ligase NEDD4. NEDD4 then recruited MAP1LC3/LC3 via its LC3-interacting region (LIR). Interestingly, after binding to the ubiquitinated RABV-M, NEDD4 could bind more LC3 and enhance autophagosome accumulation, while NEDD4 knockdown significantly reduced M-induced autophagosome accumulation. Further study revealed that RABV-M prevented autophagosome-lysosome fusion and facilitated viral budding. Inhibition of RABV-M-induced autophagosome accumulation reduced the production of extracellular virus-like particles. We also found that M proteins of most lyssaviruses share the same mechanism to accumulate autophagosome by hijacking NEDD4. Collectively, this study revealed a novel strategy for lyssaviruses to achieve efficient viral replication by exploiting the host autophagy system.Abbreviations: ABLV: Australian bat lyssavirus; ATG5: autophagy related 5; Baf A1:bafilomycin A1;co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI:4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EBLV:European bat lyssavirus; GFP: green fluorescent protein; GST:glutathione S-transferase; hpi: hours post-infection; hpt: hourspost-transfection; LIR: LC3-interactingregion;MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry:red fluorescent protein; MOI: multiplicity of infection; NC: negativecontrol; MVB: multivesicular body; NEDD4: neural precursorcell-expressed developmentally down-regulated 4; RABV: rabies virus;SQSTM1/p62: sequestosome 1; VLP: virus-like particle; VPS4B: vacuolarprotein sorting 4B; TEM: transmission electron microscopy; WB:western blotting; WT: wild-type; µm: micrometer; µM: micromole.

Deforestation and Bovine Rabies Outbreaks in Costa Rica, 1985-2020.

In Latin America, rabies virus has persisted in a cycle between Desmodus rotundus vampire bats and cattle, potentially enhanced by deforestation. We modeled bovine rabies virus outbreaks in Costa Rica relative to land-use indicators and found spatial-temporal relationships among rabies virus outbreaks with deforestation as a predictor.

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.

Lyssavirus M protein degrades neuronal microtubules by reprogramming mitochondrial metabolism.

Infection with neurotropic viruses may result in changes in host behavior, which are closely associated with degenerative changes in neurons. The lyssavirus genus comprises highly neurotropic viruses, including the rabies virus (RABV), which has been shown to induce degenerative changes in neurons, marked by the self-destruction of axons. The underlying mechanism by which the RABV degrades neuronal cytoskeletal proteins remains incomplete. In this study, we show that infection with RABV or overexpression of its M protein can disrupt mitochondrial metabolism by binding to Slc25a4. This leads to a reduction in NAD+ production and a subsequent influx of Ca2+ from the endoplasmic reticulum and mitochondria into the cytoplasm of neuronal cell lines, activating Ca2+-dependent proteinase calpains that degrade α-tubulin. We further screened the M proteins of different lyssaviruses and discovered that the M protein of the dog-derived RABV strain (DRV) does not degrade α-tubulin. Sequence analysis of the DRV M protein and that of the lab-attenuated RABV strain CVS revealed that the 57th amino acid is vital for M-induced microtubule degradation. We generated a recombinant RABV with a mutation at the 57th amino acid position in its M protein and showed that this mutation reduces α-tubulin degradation in vitro and axonal degeneration in vivo. This study elucidates the mechanism by which lyssavirus induces neuron degeneration.IMPORTANCEPrevious studies have suggested that RABV (rabies virus, the representative of lyssavirus) infection induces structural abnormalities in neurons. But there are few articles on the mechanism of lyssavirus' effect on neurons, and the mechanism of how RABV infection induces neurological dysfunction remains incomplete. The M protein of lyssavirus can downregulate cellular ATP levels by interacting with Slc25a4, and this decrease in ATP leads to a decrease in the level of NAD+ in the cytosol, which results in the release of Ca2+ from the intracellular calcium pool, the endoplasmic reticulum, and mitochondria. The presence of large amounts of Ca2+ in the cytoplasm activates Ca2+-dependent proteases and degrades microtubule proteins. The amino acid 57 of M protein is the key site determining its disruption of mitochondrial metabolism and subsequent neuron degeneration.

Cross-Neutralization Activities of Antibodies against 18 Lyssavirus Glycoproteins.

Some lyssaviruses, including the rabies virus (RABV), cause lethal neurological symptoms in humans. However, the efficacy of commercial vaccines has only been evaluated against RABV. To assess cross-reactivity among lyssaviruses, including RABV, sera from rabbits inoculated with human and animal RABV vaccines and polyclonal antibodies from rabbits immunized with expression plasmids of the glycoproteins of all 18 lyssaviruses were prepared, and cross-reactivity was evaluated via virus-neutralization tests using Duvenhage lyssavirus (DUVV), European bat lyssavirus-1 (EBLV-1), Mokola lyssavirus (MOKV), Lagos bat lyssavirus (LBV), and RABV. The sera from rabbits inoculated with RABV vaccines showed cross-reactivity with EBLV-1 and DUVV, both belonging to phylogroup I. However, reactivity with MOKV and LBV in phylogroup II was notably limited or below the detection level. Next, we compared the cross-reactivity of the polyclonal antibodies against all lyssavirus glycoproteins. Polyclonal antibodies had high virus-neutralization titers against the same phylogroup but not different phylogroups. Our findings indicate that a new vaccine should be developed for pre- and post-exposure prophylaxis against lyssaviral infections.

Human Rabies Treatment-From Palliation to Promise.

Rabies encephalitis has plagued humankind for thousands of years. In developed countries, access to preventive care, both pre-exposure and post-exposure, has significantly reduced the burden of suffering and disease. However, around the world, rabies remains a neglected tropical disease, largely due to uncontrolled dog rabies, and tens of thousands perish each year. Currently, the standard of care for management of rabies encephalitis is palliation. Heroic attempts to treat human rabies patients over the last few decades have yielded glimpses into our understanding of pathophysiology, opening the door to the development of new antiviral therapies and modalities of treatment. Researchers continue to investigate new compounds and approaches to therapy, yet there remain real challenges given the complexity of the disease. We explore and review some of the promising therapies on the horizon in pursuit of a salvage treatment for rabies.

Field testing Australian bat lyssavirus risk communication resources.

ISSUE ADDRESSED: Australian bat lyssavirus (ABLV) is a fatal zoonosis, which can be transmitted to humans through scratches or bites from infected bats. Currently, there is a lack of research evaluating risk communication resources about ABLV or the dangers from handling bats. The purpose of this study was to field test resources aimed at educating the public about risks to humans and bats from human-bat interaction, then update these resources based upon feedback to ensure they were relevant and appropriately targeted to the public. METHODS: Thirteen semi-structured interviews with a purposive sample of participants chosen for maximum variation of age and sex were conducted. Two investigators analysed the data independently using a deductive approach and then came to consensus by discussion. RESULTS: The main themes were a wide-ranging level of knowledge and opinions about bats, the resources having an effect on people, and messaging in relation to children and pets being particularly important. CONCLUSION: This study highlighted the complexities of risk communication to a broad audience with varied experience and knowledge about bats, and the importance of evaluation prior to implementation to ensure risk communication is relevant and appealing to the intended audience. SO WHAT?: Field testing of health education material prior to implementation is an effective way to ensure key messages are understood, and is important when communicating about fatal but preventable zoonoses such as ABLV.

Establishment of serological neutralizing tests using pseudotyped viruses for comprehensive detection of antibodies against all 18 lyssaviruses.

Rabies is a fatal zoonotic, neurological disease caused by rabies lyssavirus (RABV) and other lyssaviruses. In this study, we established novel serological neutralizing tests (NT) based on vesicular stomatitis virus pseudotypes possessing all 18 known lyssavirus glycoproteins. Applying this system to comparative NT against rabbit sera immunized with current RABV vaccines, we showed that the current RABV vaccines fail to elicit sufficient neutralizing antibodies against lyssaviruses other than to those in phylogroup I. Furthermore, comparative NT against rabbit antisera for 18 lyssavirus glycoproteins showed glycoproteins of some lyssaviruses elicited neutralizing antibodies against a broad range of lyssaviruses. This novel testing system will be useful to comprehensively detect antibodies against lyssaviruses and evaluate their cross-reactivities for developing a future broad-protective vaccine.

[Postexposure prophylaxis after bite of a broad-winged bat with evidence of European bat lyssavirus 1 (EBLV-1)]. / Postexpositionsprophylaxe nach Biss einer Breitflügelfledermaus mit Nachweis des Europäischen Fledermaus-Lyssavirus 1 (EBLV-1).

Germany has been considered free of terrestrial rabies since 2008 as a result of intensive vaccination and surveillance efforts but reservoirs of the lyssaviruses EBLV­1 and EBLV­2 persist in bat colonies and thus pose a potential risk of infection. We report on a patient who suffered a bat bite in an urban setting in which European bat lyssavirus 1 (EBLV-1) was detected in the euthanized bat. We performed active and passive postexposure prophylaxis (PEP). This case study illustrates the ongoing risk of rabies infection due to close bat contacts in Germany and is intended to sensitize primary care physicians to take such exposure events seriously and to perform a regular PEP including administration of rabies immunoglobulin.

Evaluating the benefit of serology during potential Australian bat lyssavirus and rabies post-exposure prophylaxis.

Post-exposure prophylaxis (PEP) for potential lyssavirus exposures consists of wound management, rabies vaccination and may include rabies immunoglobulin (RIG). Rabies serology is sometimes indicated if there is risk of PEP failure. OBJECTIVES: Evaluate the benefit of serology by indication. METHODS: Chart review of potential lyssavirus exposures managed at a Public Health Unit (June 2015 - December 2022) where serology was requested was conducted. The proportion of non-therapeutic titres was compared by sex, age, Indigenous status, serology indication, and whether RIG was given. RESULTS: 46 notifications with serology were included. Males (5/19) and people over 40 (3/16) were more likely to demonstrate a non-therapeutic response. 2/3 of cases where vaccine doses were not given in the deltoid were non-therapeutic. The rate of non-therapeutic titres was similar for RIG given into the ipsilateral arm (2/11) and given excess RIG for weight (1/4). Although this small sample was inconclusive in isolation, it was also noted that all cases who did not receive RIG had therapeutic serology, whereas 6/35 of those receiving RIG had non-therapeutic serology. CONCLUSIONS: This study supports broader literature questioning the utility of systemic RIG administration as likely limited and potentially detrimental considering the increased risk of immune interference. IMPLICATIONS FOR PUBLIC HEALTH: Highlights a need to review Australian national guidelines to align with World Health Organization advice recommending local RIG administration only.

Naturally Acquired Rabies in White-Eared Opossum, Brazil.

Opossums are considered resistant to rabies. Nonhematophagous bats are reservoirs of rabies in urban areas of South America. We analyzed bats and opossums tested for rabies during 2021 in a highly urbanized city in Brazil to understand spillover in an urban setting. Wildlife surveillance is necessary to prevent rabies in humans and domestic animals.

Molecular Characterization of Lyssaviruses Originating from Domestic and Wild Cats Provides an Insight on the Diversity of Lyssaviruses and a Risk of Rabies Transmission to Other Susceptible Mammals and Humans in South Africa.

Rabies is one of the most significant public and veterinary health problems, causing approximately 59,000 human deaths annually in the developing countries of Asia and Africa. The aetiologic agent, a viral species of the Lyssavirus genus, is highly neurotropic and has a wide host range, including terrestrial mammals and several Chiropteran species. The Lyssavirus mokola (MOKV) was first isolated in the late 1960s from organ pools of shrews (Crocidura flavescens manni) in the Mokola forest (Nigeria). To date, at least 30 MOKV isolations have been confirmed, all exclusively from Africa, with 73% from southern Africa. There is limited knowledge about the epidemiology of MOKV, and the reservoir host species is unknown. Here, we report on the molecular characterization of rabies viruses originating from both domestic and African wild cats. A partial region of the lyssavirus genome, encoding the nucleoprotein, was amplified and sequenced. Nucleotide sequence analysis demonstrated that 98% of cats were infected with both the canid and mongoose rabies virus variants, as well as a rare lyssavirus, Lyssavirus mokola, from a domestic cat from Eswatini. Furthermore, the nucleotide sequence divergence between the recently identified MOKV isolate and the historical Lyssavirus mokola isolates ranged from 6.8% to 8.3%. This study further highlights the association between the potential host species of Lyssavirus mokola and the domestic cat as an incidental host, and the important role cats may play in rabies transmission dynamics in the country. Therefore, continuous vaccination of domestic cats against rabies is crucial, even after the elimination of dog-mediated rabies, as spillover related to sylvatic rabies cycles is likely to occur.

Dispersion and diversification of Lyssavirus rabies transmitted from haematophagous bats Desmodus rotundus: a phylogeographical study.

Rabies is worldwide zoonosis caused by Lyssavirus rabies (RABV) a RNA negative sense virus with low level of fidelity during replication cycle. Nucleoprotein of RABV is the most conserved between all five proteins of the virus and is the most used gene for phylogenetic and phylogeographic studies. Despite of rabies been very important in Public Health concern, it demands continuous prophylactic care for herbivores with economic interest, such as cattle and horses. The main transmitter of RABV for these animals in Brazil is the hematophagous bats Desmodus rotundus. The aim of this study was to determine the dispersion over time and space of RABV transmitted by D. rotundus. Samples of RABV from the State of São Paulo (SP), Southeast Brazil isolated from the central nervous system (CNS) of cattle, were submitted to RNA extraction, RT-PCR, sequencing and phylogeographic analyzes with BEAST (Bayesian Evolutionary Analysis Sampling Trees) v 2.5 software. Was possible to identify high rate of diversification in starts sublineages of RABV what are correlated with a behavior of D. rotundus, the main transmitter of rabies to cattle. This study also highlights the importance of continuous monitoring of genetic lineages of RABV in Brazil.

Human rabies encephalomyelitis in the background of rabies outbreak in animals in Gelephu, Bhutan, 2023: a case report.

BACKGROUND: Rabies continues to pose significant public health challenges in many developing countries including Bhutan. A probable case of rabies was admitted to our hospital and its reporting led to the uncovering of an outbreak in domestic and wild animals. We discuss the challenges in the diagnosis and management of rabies in a resource-limited setting. CASE PRESENTATION: A 35-year-old male presented with intermittent fever, bilateral lower limb weakness that was rapidly progressive, urinary incontinence with episodes of palpitations and sweating. He had sustained a Category III bite on the right lower thigh with four bite marks, inflicted by a stray dog. He had received post-exposure prophylaxis with intra-dermal anti-rabies vaccine. On initial examination, the patient was in distress but cooperative for the interview. He had pulse rate ranging from 60 to 100/min with episodes of diaphoresis and palpitations, but with normal capillary blood glucose. In the lower limb, the muscle power was zero with absent tendon reflexes in the lower limb and impaired abdominal reflex below T10 level. He had hyperaesthesia below T8, hydrophobia, aerophobia and photophobia. He had multiple spontaneous fasciculations in both the thighs and right deltoid and these later involved the intercostal muscles, neck and face muscles. He had altered sensorium and desaturation for which he required mechanical ventilation. Polymerase chain reaction for rabies virus was negative in cerebrospinal fluid and saliva. Rabies virus neutralizing antibody was negative in cerebrospinal fluid but had high titres in the serum. He received Human Rabies Immunoglobulin after admission. He was managed in the intensive care unit and died 23 days later. After this case was notified, a rapid response team was deployed in the field, and uncovered rabies outbreak in animals in the locality. CONCLUSIONS: This case called for a serious evaluation of the country's efforts in achieving zero rabies deaths by 2030. The management of this case identified several critical areas of context-specific interventions in Bhutan. There is also an urgent need to improve diagnostic capabilities at the national reference laboratory and enhance the technical competencies of healthcare workers in the management of dog bite cases.

Phylogenetic analysis of rabies surveillance samples from north and northeast Brazil.

Viruses of the Lyssavirus genus are classified into several genotypes (GT1 to GT7), of which only GT1 (classic rabies virus-RABV) has a cosmopolitan distribution and circulates in Brazil. GT1 is subdivided into several antigenic variants (AgV) maintained in independent cycles with a narrow host range and distinct geographic distributions, namely, AgV1 and AgV2 found in dogs, AgV3 in the vampire bats Desmodus rotundus, and AgV4 and AgV6 in bats non-hematophagous Tadarida brasiliensis and Lasiurus cinereus, a common variant of marmoset (Callithrix jacchus), and crab-eating fox (Cerdocyon thous). In this study, we performed phylogenetic analysis to identify at the antigenic variant level; six RABV genomes derived from the Rabies Surveillance in the north and northeast regions of Brazil. The analysis resulted in the formation of 11 monophyletic clusters, each corresponding to a particular variant, with high bootstrap support values. The samples were positioned inside the AgV3, AgV6, and Callithrix variant clades. This is the first report of the AgV6 variant found in northern Brazil, which provides valuable information for rabies surveillance in the country. The possibility of viral spillover has been much debated, as it deals with the risk of shifting transmission from a primary to a secondary host. However, more genomic surveillance studies should be performed, with a greater number and diversity of samples to better understand the transmission dynamics of each variant to detect changes in its geographic distribution and spillover events.