Molecular Epidemiology of Rabies in Wild Canidae in Tunisia.

Rabies is a viral zoonosis that is transmissible to humans via domestic and wild animals. There are two epidemiological cycles for rabies, the urban and the sylvatic cycles. In an attempt to study the epidemiological role of wild canidae in rabies transmission, the present study aimed to analyze the genetic characteristics of virus isolates and confirm prior suggestions that rabies is maintained through a dog reservoir in Tunisia. Virus strains isolated from wild canidae were subject to viral sequencing, and Bayesian phylogenetic analysis was performed using Beast2 software. Essentially, the virus strains isolated from wild canidae belonged to the Africa-1 clade, which clearly diverges from fox-related strains. Our study also demonstrated that genetic characteristics of the virus isolates were not as distinct as could be expected if a wild reservoir had already existed. On the contrary, the geographic landscape is responsible for the genetic diversity of the virus. The landscape itself could have also acted as a natural barrier to the spread of the virus.

A Novel Terrestrial Rabies Virus Lineage Occurring in South America: Origin, Diversification, and Evidence of Contact between Wild and Domestic Cycles.

The rabies virus (RABV) is characterized by a history dominated by host shifts within and among bats and carnivores. One of the main outcomes of long-term RABV maintenance in dogs was the establishment of variants in a wide variety of mesocarnivores. In this study, we present the most comprehensive phylogenetic and phylogeographic analysis, contributing to a better understanding of the origins, diversification, and the role of different host species in the evolution and diffusion of a dog-related variant endemic of South America. A total of 237 complete Nucleoprotein gene sequences were studied, corresponding to wild and domestic species, performing selection analyses, ancestral states reconstructions, and recombination analyses. This variant originated in Brazil and disseminated through Argentina and Paraguay, where a previously unknown lineage was found. A single host shift was identified in the phylogeny, from dog to the crab-eating fox (Cerdocyon thous) in the Northeast of Brazil. Although this process occurred in a background of purifying selection, there is evidence of adaptive evolution -or selection of sub-consensus sequences- in internal branches after the host shift. The interaction of domestic and wild cycles persisted after host switching, as revealed by spillover and putative recombination events.

Whole Genome Sequencing and Phylogenetic Analysis of Rabies Viruses from Bats in Connecticut, USA, 2018-2019.

We performed whole genome sequencing and genetic characterization of rabies viruses (RABV) detected in bats submitted to the Connecticut Veterinary Medical Diagnostic Laboratory (CVMDL) during 2018-2019. Among 88 bats submitted to CVMDL, six brain samples (6.8%, 95% confidence interval: 1.6% to 12.1%) tested positive by direct fluorescent antibody test. RABVs were detected in big brown bats (Eptesicus fuscus, n = 4), a hoary bat (Lasiurus cinereus, n = 1), and an unidentified bat species (n = 1). Complete coding sequences of four out of six detected RABVs were obtained. In phylogenetic analysis, the RABVs (18-62, 18-4347, and 19-2274) from big brown bats belong to the bats EF-E1 clade, clustering with RABVs detected from the same bat species in Pennsylvania and New Jersey. The bat RABV (19-2898) detected from the migratory hoary bat belongs to the bats LC clade, clustering with the eleven viruses detected from the same species in Arizona, Washington, Idaho, and Tennessee. The approach used in this study generated novel data regarding genetic relationships of RABV variants, including their reservoirs, and their spatial origin and it would be useful as reference data for future investigations on RABV in North America. Continued surveillance and genome sequencing of bat RABV would be needed to monitor virus evolution and transmission, and to assess the emergence of genetic mutations that may be relevant for public health.

Data-Driven Management-A Dynamic Occupancy Approach to Enhanced Rabies Surveillance Prioritization.

Rabies lyssavirus (RABV) is enzootic in raccoons across the eastern United States. Intensive management of RABV by oral rabies vaccination (ORV) has prevented its spread westward and shown evidence of local elimination in raccoon populations of the northeastern US. The USDA, Wildlife Services, National Rabies Management Program (NRMP) collaborates with other agencies to implement broad-scale ORV and conducts extensive monitoring to measure the effectiveness of the management. Enhanced Rabies Surveillance (ERS) was initiated during 2005 and updated in 2016 to direct surveillance efforts toward higher-value specimens by assigning points to different methods of encountering specimens for collection (strange-acting, roadkill, surveillance-trapped, etc.; specimen point values ranged from 1 to 15). We used the 2016-2019 data to re-evaluate the point values using a dynamic occupancy model. Additionally, we used ERS data from 2012-2015 and 2016-2019 to examine the impact that the point system had on surveillance data. Implementation of a point system increased positivity rates among specimens by 64%, indicating a substantial increase in the efficiency of the ERS to detect wildlife rabies. Our re-evaluation found that most points accurately reflect the value of the surveillance specimens. The notable exception was that samples from animals found dead were considerably more valuable for rabies detection than originally considered (original points = 5, new points = 20). This work demonstrates how specimen prioritization strategies can be used to refine and improve ERS in support of wildlife rabies management.

On the Use of Phylogeographic Inference to Infer the Dispersal History of Rabies Virus: A Review Study.

Rabies is a neglected zoonotic disease which is caused by negative strand RNA-viruses belonging to the genus Lyssavirus. Within this genus, rabies viruses circulate in a diverse set of mammalian reservoir hosts, is present worldwide, and is almost always fatal in non-vaccinated humans. Approximately 59,000 people are still estimated to die from rabies each year, leading to a global initiative to work towards the goal of zero human deaths from dog-mediated rabies by 2030, requiring scientific efforts from different research fields. The past decade has seen a much increased use of phylogeographic and phylodynamic analyses to study the evolution and spread of rabies virus. We here review published studies in these research areas, making a distinction between the geographic resolution associated with the available sequence data. We pay special attention to environmental factors that these studies found to be relevant to the spread of rabies virus. Importantly, we highlight a knowledge gap in terms of applying these methods when all required data were available but not fully exploited. We conclude with an overview of recent methodological developments that have yet to be applied in phylogeographic and phylodynamic analyses of rabies virus.

Determination of freedom-from-rabies for small Indian mongoose populations in the United States Virgin Islands, 2019-2020.

Mongooses, a nonnative species, are a known reservoir of rabies virus in the Caribbean region. A cross-sectional study of mongooses at 41 field sites on the US Virgin Islands of St. Croix, St. John, and St. Thomas captured 312 mongooses (32% capture rate). We determined the absence of rabies virus by antigen testing and rabies virus exposure by antibody testing in mongoose populations on all three islands. USVI is the first Caribbean state to determine freedom-from-rabies for its mongoose populations with a scientifically-led robust cross-sectional study. Ongoing surveillance activities will determine if other domestic and wildlife populations in USVI are rabies-free.

Whole-genome sequencing and phylogenetic analysis of rabies viruses from Jordan.

Human fatalities caused by rabies are rarely reported in Jordan; however, domestic animals are more likely to fall victim to rabies compared to wild animals, at least this is the case in Jordan due to the presence of canine rabies. In this study, twelve brain samples from domestic and wild animals suspected of being infected with rabies virus from different regions of Jordan were collected during 2019. Seven of them tested positive using the fluorescent antibody test and real-time SYBR RT-PCR assay. Five specimens were from stray dogs and two from foxes. The whole genome sequences were obtained from the positive samples. Sequence analysis showed that one dog virus from Al Quwaysimah city located in Amman governorate, was closely related to an Israeli strain belonging to a Cosmopolitan ME1a clade. The genomes of the remaining six viruses (four from dogs and two from foxes) collected from different areas of Jordan were genetically-related to each other and clustered together with sequences from Iran and Turkey; all belong to Cosmopolitan ME2 clade. These sequences were analyzed with six other Jordanian rabies virus nucleoprotein (N) gene sequences available in the public database, five of them belong to ME1a clade and one belongs to ME1b clade. Rabies virus whole genome data is scarce across the Middle East. This study provides a better understanding of the molecular epidemiology of rabies virus in the region.

Local Surveillance and Control of Raccoon Rabies Virus in Striped Skunks (Mephitis mephitis) in Southwestern New Brunswick, Canada.

Targeted surveillance for raccoon rabies virus was conducted between February and May 2017, near Waweig, New Brunswick, Canada, in response to detection of a rabid striped skunk (Mephitis mephitis) on 8 February 2017. A total of six skunks, 11 raccoons (Procyon lotor), and two porcupines (Erethizon dorsatum) were live-trapped, euthanized, and tested for rabies virus antigens using the direct rapid immunohistochemical test. Of these, only two skunks tested positive for rabies. All three rabid skunks came from the same location, an abandoned barn used as a denning site. Four of five skunks removed from this barn were males. Feeding, aggression, extreme response to noise and light stimuli, and exposure to porcupine quills were observed in two rabid skunks. No additional cases of rabies in wildlife were detected in the area since 8 March 2017. A targeted surveillance approach that removed potentially infected wildlife followed by localized oral rabies vaccine distribution was implemented in this locality.

Modeling Mongoose Rabies in the Caribbean: A Model-Guided Fieldwork Approach to Identify Research Priorities.

We applied the model-guided fieldwork framework to the Caribbean mongoose rabies system by parametrizing a spatially-explicit, individual-based model, and by performing an uncertainty analysis designed to identify parameters for which additional empirical data are most needed. Our analysis revealed important variation in output variables characterizing rabies dynamics, namely rabies persistence, exposure level, spatiotemporal distribution, and prevalence. Among epidemiological parameters, rabies transmission rate was the most influential, followed by rabies mortality and location, and size of the initial infection. The most influential landscape parameters included habitat-specific carrying capacities, landscape heterogeneity, and the level of resistance to dispersal associated with topography. Movement variables, including juvenile dispersal, adult fine-scale movement distances, and home range size, as well as life history traits such as age of independence, birth seasonality, and age- and sex-specific mortality were other important drivers of rabies dynamics. We discuss results in the context of mongoose ecology and its influence on disease transmission dynamics. Finally, we suggest empirical approaches and study design specificities that would provide optimal contributing data addressing the knowledge gaps identified by our approach, and would increase our potential to use epidemiological models to guide mongoose rabies control and management in the Caribbean.

Development of Monoclonal Antibodies for Detection of Conserved and Variable Epitopes of Large Protein of Rabies Virus.

Rabies virus (RABV) causes fatal neurological encephalitis and results in approximately 6000 human death cases worldwide every year. The large (L) protein of RABV, possessing conserved domains, is considered as the target for detection. In this study, three monoclonal antibodies (mAbs), designated as 3F3, 3A6 and L-C, against L protein were generated by using the recombinant truncated L protein (aa 1431-1754) and the epitopes were also identified using a series of overlapping truncated polypeptides for testing the reactivity of mAbs with different RABV strains. The 1479EIFSIP1484, 1659RALSK1663 and 1724VFNSL1728 were identified as the minimal linear epitopes recognized by mAbs 3F3, 3A6 and L-C, respectively. Amino acid alignment showed epitope 1724VFNSL1728 recognized by mAb L-C is completely conserved among RABV strains, indicating that mAb L-C could be used to detect all of the RABV strains. Epitope 1479EIFSIP1484 is highly conserved among RABV strains except for a P1484S substitution in a China I sub-lineage strain of Asian lineage, which eliminated the reactivity of the epitope with mAb 3F3. However, the epitope 1659RALSK1663 was only completely conserved in the Africa-2 and Indian lineages, and a single A1660T substitution, mainly appeared in strains of the China I belonging to Asian lineage and a Cosmopolitan lineage strain, still retained the reactivity of the epitope with mAb 3A6. While both A1660T and K1663R substitutions in a China I lineage strain, single K1663R/Q substitution in some China II strains of Asian lineage and some Arctic-like lineage strains and R1659Q mutation in a strain of Africa-3 lineage eliminated the reactivity of the epitope with mAb 3A6, suggesting mAb 3A6 could be used for differentiation of variable epitopes of some strains in different lineages. Thus, variability and conservation of the three epitopes of L protein showed the reactive difference of mAbs among RABV strains of different lineages. These results may facilitate future studies in development of detection methods for RABV infection, the structure and function of RABV L protein.

Characterizing and Evaluating the Zoonotic Potential of Novel Viruses Discovered in Vampire Bats.

The contemporary surge in metagenomic sequencing has transformed knowledge of viral diversity in wildlife. However, evaluating which newly discovered viruses pose sufficient risk of infecting humans to merit detailed laboratory characterization and surveillance remains largely speculative. Machine learning algorithms have been developed to address this imbalance by ranking the relative likelihood of human infection based on viral genome sequences, but are not yet routinely applied to viruses at the time of their discovery. Here, we characterized viral genomes detected through metagenomic sequencing of feces and saliva from common vampire bats (Desmodus rotundus) and used these data as a case study in evaluating zoonotic potential using molecular sequencing data. Of 58 detected viral families, including 17 which infect mammals, the only known zoonosis detected was rabies virus; however, additional genomes were detected from the families Hepeviridae, Coronaviridae, Reoviridae, Astroviridae and Picornaviridae, all of which contain human-infecting species. In phylogenetic analyses, novel vampire bat viruses most frequently grouped with other bat viruses that are not currently known to infect humans. In agreement, machine learning models built from only phylogenetic information ranked all novel viruses similarly, yielding little insight into zoonotic potential. In contrast, genome composition-based machine learning models estimated different levels of zoonotic potential, even for closely related viruses, categorizing one out of four detected hepeviruses and two out of three picornaviruses as having high priority for further research. We highlight the value of evaluating zoonotic potential beyond ad hoc consideration of phylogeny and provide surveillance recommendations for novel viruses in a wildlife host which has frequent contact with humans and domestic animals.

Evaluating the Impact of Anthropogenic Factors on the Dissemination of Contemporary Cosmopolitan, Arctic, and Arctic-like Rabies Viruses.

Rabies is a globally prevalent viral zoonosis that causes 59,000 deaths per year and has important economic consequences. Most virus spread is associated with the migration of its primary hosts. Anthropogenic dissemination, mainly via the transportation of rabid dogs, shaped virus ecology a few hundred years ago and is responsible for several current outbreaks. A systematic analysis of aberrant long-distance events in the steppe and Arctic-like groups of rabies virus was performed using statistical (Bayesian) phylogeography and plots of genetic vs. geographic distances. The two approaches produced similar results but had some significant differences and complemented each other. No phylogeographic analysis could be performed for the Arctic group because polar foxes transfer the virus across the whole circumpolar region at high velocity, and there was no correlation between genetic and geographic distances in this virus group. In the Arctic-like group and the steppe subgroup of the cosmopolitan group, a significant number of known sequences (15-20%) was associated with rapid long-distance transfers, which mainly occurred within Eurasia. Some of these events have been described previously, while others have not been documented. Most of the recent long-distance transfers apparently did not result in establishing the introduced virus, but a few had important implications for the phylogeographic history of rabies. Thus, human-mediated long-distance transmission of the rabies virus remains a significant threat that needs to be addressed.

Epidemiologia da raiva humana no estado do Ceará, 1970 a 2019 / Epidemiología de la rabia humana en el estado de Ceará, Brasil, 1970 a 2019 / Epidemiology of human rabies in the state of Ceará, Brazil, 1970 to 2019

Objetivo: Descrever os casos de raiva humana no estado do Ceará, Brasil, no período 1970-2019. Métodos: Estudo descritivo, sobre dados secundários da Secretaria da Saúde e do hospital de referência do Ceará. Resultados: Dos 171 casos, 75,7% ocorreram em homens, 60,0% nas idades até 19 anos e 56,0% em áreas urbanas. O cão foi agente transmissor em 74,0% dos casos; sagui em, 16,7%; e morcego, em 7,3%. Entre 1970 e 1978, houve crescimento do número de casos (pelo Joinpoint Regression Program, percentual da mudança anual [APC] = 13,7 - IC95% 4,6;41,5); e entre 1978 e 2019, redução (APC = -6,7 - IC95% -8,8;-5,9). Houve redução da transmissão por cães (71 casos; último caso em 2010) e aumento relativo por mamíferos silvestres (5 casos a partir de 2005). Conclusão: O estudo evidencia mudança na dinâmica da transmissão da raiva no período observado, com redução da transmissão por cão e incremento de casos por animais silvestres.

Objetivo: Describir los casos de la rabia humana en Ceará, Brasil, 1970-2019. Métodos: Estudio descriptivo con datos secundarios de la Secretaría de Salud y del hospital de referencia de Ceará. Resultados: De los 171 casos, 75,7% ocurrió en hombres, el 60,0% en los 19 años y el 56,0% en áreas urbanas. El perro fue transmisor en 74,0%, el mono tití en 16,7% y el murciélago el 7,3%. Entre 1970 y 1978, hubo un aumento de casos (por el Joinpoint Regression Program, cambio porcentual anual [APC] = 13,7 - IC95% 4,6; 41,5), entre 1978 y 2019 una disminución (APC= -6,7 - IC95% -8,8; -5,9). Hubo una reducción de la transmisión por perros (71 casos, el último en 2010) y un aumento por mamíferos salvajes (5 casos, desde 2005). Conclusión: El estudio muestra un cambio en la dinámica de la transmisión de la rabia en los últimos años, con reducción de la transmisión por perros y aumento de casos por animales salvajes.

Objective: To describe cases of human rabies in Ceará State, Brazil between 1970 and 2019. Methods: This was a descriptive study using secondary data from the Ceará State Department of Health and the state reference hospital. Results: Of 171 cases, 75.7% occurred in males, 60.0% in <19-year-olds, and 56.0% in urban areas. Rabies was transmitted by dogs in 74.0% of cases, marmosets in 16.7% and bats in 7.3%. Between 1970 and 1978, there was an increase of cases (using the Joinpoint Regression Program, annual percentage change [APC] = 13.7 - 95%CI 4.6;41.5), while between 1978 and 2019 there was a decrease (APC = -6.7 - 95%CI -8.8;-5.9). There was a reduction in transmission by dogs (71 cases, last case in 2010) and an increase by sylvatic animals (5 cases since 2005). Conclusion: This study demonstrates changes in rabies transmission dynamics during the period studied, with a reduction in transmission by dogs and an increase of transmission by sylvatic animals.

Evaluation of Two Real-Time, TaqMan Reverse Transcription-PCR Assays for Detection of Rabies Virus in Circulating Variants from Argentina: Influence of Sequence Variation.

In rabies diagnosis, it is essential to count on a rapid test to give a quick response. The combined sensitivity and robustness of the TaqMan RT-PCR assays (qRT-PCR) have made these methods a valuable alternative for rabies virus (RABV) detection. We conducted a study to compare the applicability of two widely used qRT-PCR assays targeting the nucleoprotein gene (LysGT1 assay) and leader sequences (LN34 qRT-PCR assay) of RABV genomes, in all variants circulating in Argentina. A total of 44 samples obtained from bats, dogs, cattle, and horses, that were previously tested for rabies by FAT and conventional RT-PCR, were used in the study. All variants were successfully detected by the pan-lyssavirus LN34 qRT-PCR assay. The LysGT1 assay failed to detect three bat-related variants. We further sequenced the region targeted by LysGT1 and demonstrated that the presence of three or more mismatches with respect to the primers and probe sequences precludes viral detection. We conclude that the LysGT1 assay is prone to yield variant-dependent false-negative test results, and in consequence, the LN34 assay would ensure more effective detection of RABV in Argentina.

Portable Rabies Virus Sequencing in Canine Rabies Endemic Countries Using the Oxford Nanopore MinION.

As countries with endemic canine rabies progress towards elimination by 2030, it will become necessary to employ techniques to help plan, monitor, and confirm canine rabies elimination. Sequencing can provide critical information to inform control and vaccination strategies by identifying genetically distinct virus variants that may have different host reservoir species or geographic distributions. However, many rabies testing laboratories lack the resources or expertise for sequencing, especially in remote or rural areas where human rabies deaths are highest. We developed a low-cost, high throughput rabies virus sequencing method using the Oxford Nanopore MinION portable sequencer. A total of 259 sequences were generated from diverse rabies virus isolates in public health laboratories lacking rabies virus sequencing capacity in Guatemala, India, Kenya, and Vietnam. Phylogenetic analysis provided valuable insight into rabies virus diversity and distribution in these countries and identified a new rabies virus lineage in Kenya, the first published canine rabies virus sequence from Guatemala, evidence of rabies spread across an international border in Vietnam, and importation of a rabid dog into a state working to become rabies-free in India. Taken together, our evaluation highlights the MinION's potential for low-cost, high volume sequencing of pathogens in locations with limited resources.

Quantifying and mapping the burden of human and animal rabies in Iraq.

Rabies was first reported in ancient Iraqi civilizations, yet it remains a poorly quantified and important public health threat in the region. Efforts to control rabies in Iraq including dog population control, and vaccination of livestock and dogs, have increased since 2010. Officially reported data on human rabies, dog bites, and animal rabies cases between 2012 and 2017 are analysed here to assess the effect of existing control efforts, to inform future strategies, and to highlight gaps in surveillance and reporting. The results of molecular characterization of 32 viruses from animal cases from throughout Iraq are presented, to improve the understanding of rabies dynamics in the animal reservoir. Although annual numbers of reported human cases were lower in the period between 2012 and 2017 than prior to 2010, human cases continue. There was a distinct gender and age bias among human cases with nine cases in males for every one female and twice as many cases in children than adults. Spatial clustering analysis and phylogenetic evidence suggests rabies is endemic throughout the country, with no regional variation in risk, but better surveillance and reporting is required to underpin control strategies.

Genetic and Phenotypic Characterization of a Rabies Virus Strain Isolated from a Dog in Tokyo, Japan in the 1940s.

The rabies virus strain Komatsugawa (Koma), which was isolated from a dog in Tokyo in the 1940s before eradication of rabies in Japan in 1957, is known as the only existent Japanese field strain (street strain). Although this strain potentially provides a useful model to study rabies pathogenesis, little is known about its genetic and phenotypic properties. Notably, this strain underwent serial passages in rodents after isolation, indicating the possibility that it may have lost biological characteristics as a street strain. In this study, to evaluate the utility of the Koma strain for studying rabies pathogenesis, we examined the genetic properties and in vitro and in vivo phenotypes. Genome-wide genetic analyses showed that, consistent with previous findings from partial sequence analyses, the Koma strain is closely related to a Russian street strain within the Arctic-related phylogenetic clade. Phenotypic examinations in vitro revealed that the Koma strain and the representative street strains are less neurotropic than the laboratory strains. Examination by using a mouse model demonstrated that the Koma strain and the street strains are more neuroinvasive than the laboratory strains. These findings indicate that the Koma strain retains phenotypes similar to those of street strains, and is therefore useful for studying rabies pathogenesis.

Molecular characterization of rabies viruses from two western provinces of the Democratic Republic of the Congo (2008-2017).

Although rabies is enzootic in the Democratic Republic of the Congo, there is very little molecular epidemiological information about the viruses circulating in animals. In this study, a fragment of the rabies virus (RABV) nucleoprotein gene was amplified and sequenced from 21 animal brain samples collected in two western provinces of the country between 2008 and 2017. The samples tested were from cat (n = 1), dog (n = 17), goat (n = 2), and sheep (n = 1). Phylogenetic analysis revealed that the sequences generated were highly similar to each other and belonged to lineage Africa 1b clustering with a single sample identified in a canine in the Republic of Congo in 2014. This is the first molecular epidemiological study of RABV in the DRC and the data generated will assist authorities in the development of effective control strategies for rabies in the country.

Phylogenetic analysis of the whole genome sequence of a dog lineage rabies virus detected from cattle in eastern China, 2019.

Rabies is an important zoonosis worldwide, and this disease is caused by the rabies virus. Dogs and bat are the major hosts of rabies virus; however, many animals could infect with the rabies virus. By biting or scratching by an infected animal, rabies virus can be transmitted to cattle which is an important domestic animal in animal husbandry. Here, we report a case about a rabies virus (abbreviated JSTZ190314) found in cattle in eastern China in 2019. Our findings suggest that this rabies virus JSTZ190314 was a dog-origin rabies virus and belonged to the Asia clade. Furthermore, we found that this JSTZ190314-like rabies virus has been prevalent in China for more than 13 years and infected six species of animals. Our findings suggested that enhanced surveillance and research of rabies virus infection in bovine populations is needed.

A phylogenetic study of new rabies virus strains in different regions of Iran.

Rabies is the most critical zoonotic disease in Iran, which imposes many extra costs on health care system in each country. The present study aimed to determine the molecular characteristics of the wild circulating strains of the rabies virus (RABV) collected in Iran during 2015-2017. Rabies-suspected samples were collected from different regions of Iran and identified for RABV antigen confirmation using fluorescent antibody tests. Polymerase chain reaction (PCR) was performed on positive samples and gene sequencing was done on rabies nucleoprotein and glycoprotein genes to determine the rabies molecular characteristics. Accordingly, nine street RABVes were isolated. Then, N (802 bp) and G (735 bp) genes were amplified with specific primers using PCR. The sequence of nine strains was determined and compared with another 50 close to them, and the phylogenetic tree was plotted using neighbor-joining method by Mega 7 software. The molecular characteristic results indicated that all new strains belong to RABV wild species. As a result, the most prevalent strains of RABV in northwest, west, center, and south of Iran were identified. The present study may provide a better insight into the identification of all RABV strains, and understanding the evolutionary nature of RABV and how its hosts change in the world over the centuries.