ABSTRACT Members of the RNA virus order Nidovirales infect hosts ranging from marine invertebrates to terrestrial mammals. As such, understanding the determinants of host range in this group of viruses, as well as their patterns of emergence and disease potential, is of clear importance. The Mesoniviridae are a recently documented family within the Nidovirales . To date, mesoniviruses have only been associated with the infection of arthropod species, particularly mosquitoes, and hence are regarded as insect-specific viruses (ISVs). Herein, we report the first detection of a mesonivirus—Alphamesonivirus-1 —in mammals. Specifically, we utilized genomic and histological techniques to identify Alphamesonivirus-1 in lung and lymph node tissues of two horses (a mare and its foal) from Italy that succumbed to an acute respiratory syndrome. The genome sequences of Alphamesonivirus-1 obtained from the two horses were closely related to each other and to those from a local Culex mosquito pool and an Alphamesonivirus-1 previously identified in Italy, indicative of ongoing local transmission. The discovery of Alphamesonivirus-1 in horse tissues prompts further investigation into the host range of mesoniviruses, the possible role of insect-specific viruses in mammalian disease processes, the determinants of and barriers to cross-species virus transmission, and the potential epizootic threats posed by understudied viral families. IMPORTANCE Alphamesoniviruses, members of the family Mesoniviridaeare, are considered insect-specific RNA viruses with no known association with vertebrate hosts. Herein, we report the identification of Alphamesonivirus-1 in mammals. Using detailed molecular and histological analyses, we identified Alphamesonivirus-1 in lung and lymph node tissues of two horses that presented with an acute respiratory syndrome and that was phylogenetically related to virus sequences found in local Culex mosquitoes. Hence, Alphamesoniviruses may possess a broader host range than previously believed, prompting the investigation of their possible role in mammalian disease. This work highlights the need for increased surveillance of atypical viruses in association with unexplained respiratory illness, including those commonly assumed to be insect-specific, and may have implications for epizootic disease emergence.

Bats are commonly thought to harbour a high diversity and abundance of RNA viruses, some of which are able to jump species boundaries to emerge in new hosts. However, gaps remain in our understanding of the ecological factors that shape the bat virome and influence the diversity, circulation and population persistence of viral infections. Flying foxes (Pteropodidae) are representative of the chiropteran fauna in Australia, holding significant ecological, cultural, and social importance. However, some species have also been linked to the circulation of mammalian pathogens such as Hendra virus and Australian bat lyssavirus. Here, we characterised the RNA virome of the Christmas Island flying fox (Pteropus melanotus natalis), a native and endangered species only found on the remote Australian territory of Christmas Island. Through metatranscriptomic sequencing of 46 samples, including faeces, blood, urine and tissue lesions, we found that these bats exhibit limited RNA virus diversity dominated by dietary viruses. The paucity of RNA viruses likely reflects their small population size (between 1500 and 2600 individuals) and geographic isolation from other bat and mammalian species, except for pests and humans. However, we identified a novel alphacoronavirus in urine, related to viruses circulating in microbats in mainland Australia, and a picorna-like virus related to picornaviruses found in invertebrates. Although this novel picorna-like virus may be of a dietary origin, these flying foxes predominantly eat nectar, pollen and fruit, and viral RNA was also present in blood, urine and wing lesion samples. Overall, these data reveal how ecological factors have a profound impact on RNA virome diversity, highlighting risks to bat conservation, and showing that bats are not always major reservoirs for zoonotic viruses.

Improved RNA virus understanding is critical to studying animal and plant health, and environmental processes. However, the continuous and rapid RNA virus evolution makes their identification and characterization challenging. While recent sequence-based advances have led to extensive RNA virus discovery, there is growing variation in how RNA viruses are identified, analyzed, characterized, and reported. To this end, an RdRp Summit was organized and a hybrid meeting took place in Valencia, Spain in May 2023 to convene leading experts with emphasis on early career researchers (ECRs) across diverse scientific communities. Here we synthesize key insights and recommendations and offer these as a first effort to establish a consensus framework for advancing RNA virus discovery. First, we need interoperability through standardized methodologies, data-sharing protocols, metadata provision and interdisciplinary collaborations and offer specific examples as starting points. Second, as an emergent field, we recognize the need to incorporate cutting-edge technologies and knowledge early and often to improve omic-based viral detection and annotation as novel capabilities reveal new biology. Third, we underscore the significance of ECRs in fostering international partnerships to promote inclusivity and equity in virus discovery efforts. The proposed consensus framework serves as a roadmap for the scientific community to collectively contribute to the tremendous challenge of unveiling the RNA virosphere.

As members of the RNA virus order Nidovirales include those that infect hosts ranging from marine invertebrates to terrestrial mammals, understanding their emergence, host range and disease potential is of clear importance. The Mesoniviridae are a recently documented family of viruses within the Nidovirales. To date, mesoniviruses have only been associated with the infection of arthropods, particularly mosquitoes. Herein, we report the first detection of a mesonivirus – Alphamesonivirus-1 – in mammals. Specifically, we utilized genomic and histological techniques to identify the presence of Alphamesonivirus-1 in lung and lymph node tissues of two horses that succumbed to an acute respiratory syndrome. Notably, no other pathogens typically associated with respiratory disease in horses were detected in these samples. Counter to the previous contention that mesoniviruses only infect insects, our findings suggest a potentially broader host range and cross-species transmission of these viruses. The genome sequences of Alphamesonivirus-1 obtained from the two horses were closely related to those from a local Culex mosquito pool as well as an Alphamesonivirus-1 previously in identified Italy, suggestive of ongoing local transmission. The discovery of Alphamesonivirus-1 in tissues from diseased horses not only challenges current understandings of mesonivirus host range, but prompts further investigation into the role of insect-specific viruses in mammalian disease processes. Our results emphasize the importance of considering atypical pathogens in cases of unexplained animal deaths and suggest a potential zoonotic threat posed by previously overlooked viral families.

More than 70 bat species are found in mainland Australia. While most studies of bat viromes focus on sampling seemingly healthy individuals, little is known about the viruses and bacteria associated with diseased bats. We performed traditional diagnostic techniques and metatranscriptomic sequencing on tissue samples from 43 Australian bats, comprising three flying fox (Pteropodidae) and two microbat species experiencing a range of disease syndromes, including mass mortality, neurological signs, pneumonia and skin lesions. Of note, we identified the recently discovered Hervey pteropid gammaretrovirus in a bat with lymphoid leukemia, with evidence of replication consistent with an exogenous virus. The possible association of Hervey pteropid gammaretrovirus with lymphoid leukemia clearly merits additional investigation. One novel picornavirus and at least three new astroviruses and bat pegiviruses were also identified in a variety of tissue types, as well as a number of likely bacterial pathogens or opportunistic infections, most notably Pseudomonas aeruginosa.

The Andean Cordilleras of Colombia, especially the Cordillera Occidental, are among the areas with the highest diversity of Pristimantis frogs in the world. Within the Cordillera Occidental, the Serrania de los Paraguas is famous for its high diversity of sympatric species of Pristimantis frogs. In this study, we investigated acoustic frequency segregation in five sympatric and syntopic species of the genus Pristimantis inhabiting this Serrania. It is important to point out that three of the five species' calls are described for the first time: Pristimantis brevifrons, P. ptochus, and P. silverstonei. Our results showed that acoustic frequency segregation occurs among these five Pristimantis species. Another notable result was the inverse relationship between dominant frequency and male body size, which was corroborated in all species except in P. alius. Finally, we suggested that for improving our knowledge on species coexistence in Pristimantis, future research should also focus on species history and character evolution, given that the evolutionary history is also fundamental for explaining species coexistence.

Ticks harbour a high diversity of viruses, bacteria and protozoa. The soft tick Carios vespertilionis (Argasidae) is a common ectoparasite of bats in the Palearctic region and is suspected to be vector and reservoir of viruses and other microbial species in bat populations, some of which may act as zoonotic agents for human disease. The Soprano pipistrelle (Pipistrellus pygmaeus, Vespertilionidae) is widely distributed in Europe, where it can be found inside or close to human habitation. We used meta-transcriptomic sequencing to determine the RNA virome and common microbiota in blood-fed C. vespertilionis ticks collected from a Soprano pipistrelle bat roosting site in south-central Sweden. Our analyses identified 16 viruses from 11 virus families, of which 15 viruses were novel. For the first time in Sweden we identified Issuk-Kul virus, a zoonotic arthropod-borne virus previously associated with outbreaks of acute febrile illness in humans. Probable bat-associated and tick-borne viruses were classified within the families Nairoviridae, Caliciviridae and Hepeviridae, while other invertebrate-associated viruses included members of the Dicistroviridae, Iflaviridae, Nodaviridae, Partitiviridae, Permutotetraviridae, Polycipiviridae and Solemoviridae. Similarly, we found abundant bacteria in C. vespertilionis, including genera with known tick-borne bacteria, such as Coxiella spp. and Rickettsia spp. These findings demonstrate the remarkable diversity of RNA viruses and bacteria present in C. vespertilionis and highlight the importance of bat-associated ectoparasite surveillance as an effective and non-invasive means to track viruses and bacteria circulating in bats and ticks.

Arthropods harbor a largely undocumented diversity of RNA viruses. Some arthropods, like mosquitoes, can transmit viruses to vertebrates but are themselves parasitized by other arthropod species, such as mites. Very little is known about the viruses of these ectoparasites and how they move through the host-parasite relationship. To address this, we determined the virome of both mosquitoes and the mites that feed on them. The mosquito Aedes communis is an abundant and widely distributed species in Sweden, in northern Europe. These dipterans are commonly parasitized by water mite larvae (Trombidiformes: Mideopsidae) that are hypothesized to impose negative selection pressures on the mosquito by reducing fitness. In turn, viruses are dual-host agents in the mosquito-mite interaction. We determined the RNA virus diversity of mite-free and mite-detached mosquitoes, as well as their parasitic mites, using meta-transcriptomic sequencing. Our results revealed an extensive RNA virus diversity in both mites and mosquitoes, including thirty-seven putative novel RNA viruses that cover a wide taxonomic range. Notably, a high proportion of viruses (20/37) were shared between mites and mosquitoes, while a limited number of viruses were present in a single host. Comparisons of virus composition and abundance suggest potential virus transfer between mosquitoes and mites during their symbiotic interaction. These findings shed light on virome diversity and ecology in the context of arthropod host-parasite-virus relationships.

Bats are important reservoirs for viruses of public health and veterinary concern. Virus studies in Australian bats usually target the families Paramyxoviridae, Coronaviridae and Rhabdoviridae, with little known about their overall virome composition. We used metatranscriptomic sequencing to characterise the faecal virome of grey-headed flying foxes from three colonies in urban/suburban locations from two Australian states. We identified viruses from three mammalian-infecting (Coronaviridae, Caliciviridae, Retroviridae) and one possible mammalian-infecting (Birnaviridae) family. Of particular interest were a novel bat betacoronavirus (subgenus Nobecovirus) and a novel bat sapovirus (Caliciviridae), the first identified in Australian bats, as well as a potentially exogenous retrovirus. The novel betacoronavirus was detected in two sampling locations 1,375 km apart and falls in a viral lineage likely with a long association with bats. This study highlights the utility of unbiased sequencing of faecal samples for identifying novel viruses and revealing broad-scale patterns of virus ecology and evolution.

Abstract Revealing the determinants of virome composition is central to placing disease emergence in a broader evolutionary context. Fish are the most species-rich group of vertebrates and so provide an ideal model system to study the factors that shape virome compositions and their evolution. We characterized the viromes of nineteen wild-caught species of marine fish using total RNA sequencing (meta-transcriptomics) combined with analyses of sequence and protein structural homology to identify divergent viruses that often evade characterization. From this, we identified twenty-five new vertebrate-associated viruses and a further twenty-two viruses likely associated with fish diet or their microbiomes. The vertebrate-associated viruses identified here included the first fish virus in the Matonaviridae (single-strand, positive-sense RNA virus). Other viruses fell within the Astroviridae, Picornaviridae, Arenaviridae, Reoviridae, Hepadnaviridae, Paramyxoviridae, Rhabdoviridae, Hantaviridae, Filoviridae, and Flaviviridae, and were sometimes phylogenetically distinct from known fish viruses. We also show how key metrics of virome composition—viral richness, abundance, and diversity—can be analysed along with host ecological and biological factors as a means to understand virus ecology. Accordingly, these data suggest that that the vertebrate-associated viromes of the fish sampled here are predominantly shaped by the phylogenetic history (i.e. taxonomic order) of their hosts, along with several biological factors including water temperature, habitat depth, community diversity and swimming behaviour. No such correlations were found for viruses associated with porifera, molluscs, arthropods, fungi, and algae, that are unlikely to replicate in fish hosts. Overall, these data indicate that fish harbour particularly large and complex viromes and the vast majority of fish viromes are undescribed.

The endosymbiont bacteria of the genus Wolbachia are associated with multiple mutualistic effects on insect biology, including nutritional and antiviral properties. Members of the genus Wolbachia naturally occur in fly species of the genus Drosophila, providing an operational model host for studying how virome composition may be affected by its presence. Drosophila simulans populations can carry a variety of strains of members of the genus Wolbachia, with the wAu strain associated with strong antiviral protection under experimental conditions. We used D. simulans sampled from the Perth Hills, Western Australia, to investigate the potential virus protective effect of the wAu strain of Wolbachia on individual wild-caught flies. Our data revealed no appreciable variation in virus composition and abundance between individuals infected or uninfected with Wolbachia associated with the presence or absence of wAu. However, it remains unclear whether wAu might affect viral infection and host survival by increasing tolerance rather than inducing complete resistance. These data also provide new insights into the natural virome diversity of D. simulans. Despite the small number of individuals sampled, we identified a repertoire of RNA viruses, including nora virus, galbut virus, thika virus and La Jolla virus, that have been identified in other species of the genus Drosophila. Chaq virus-like sequences associated with galbut virus were also detected. In addition, we identified five novel viruses from the families Reoviridae, Tombusviridae, Mitoviridae and Bunyaviridae. Overall, this study highlights the complex interaction between Wolbachia and RNA virus infections and provides a baseline description of the natural virome of D. simulans.

The Serrania de los Paraguas is a mountainous region situated in the central part of the Cordillera Occidental of Colombia. It rises some 2500 m above the wet forest and supports a high concentration of species richness and endemism for plants and terrestrial vertebrates (amphibians, birds, and mammals). In the case of amphibians, 61 named species occurring in The Serrania de los Paraguas are recognized in the literature, which are mostly the works of J.D. Lynch and P. Ruiz-Carranza in the 1990s. During a 9-d survey in the wet season (19-27 July 2019) on the Reserva Natural Comunitaria Cerro El Ingles (one of the hills of the Serrania de los Paraguas) on the frontier between Choco and Valle del Cauca departments, we discovered two new species of the genus Pristimantis. Both species share a divided palmar tubercle as well as a distinctive color pattern on the groin, of which both are distinctive within Pristimantis. The first new species is distinguished by its small size and having a distinctive carmine red color pattern on groin, uniform lateral fringes on fingers (no crenulated), palmar tubercle deeply bifid (or divided), advertisement call composed of a short single note distinctly pulsed, and absence of nuptial pads as well as hyperdistal tubercles, inner tarsal tubercle and fold, and partial fusion of Toe IV and V. The second new species is distinguished by having dermal ridges on scapular region, conical tubercle on upper eyelid and heel, palmar tubercle divided, differentiated tympanic membrane, males with vocal slits, orange spots on groin, advertisement call composed of distinctly pulsed 6-8 notes, and absence of dorsolateral folds as well as cranial crest, hyperdistal tubercles, and toe webbing. Considering these new species, the number of amphibians inhabiting the Serrania de los Paraguas has increased to 63 species. It is important to point out that within this outstanding species richness of amphibians, there are 10 endemic species and 17 threatened species according to the International Union for Conservation of Nature Red List (7 Critically Endangered, 5 Endangered, and 5 Vulnerable). Likewise, it is noteworthy that 29 terraranan species can be found in the Reserva Natural Comunitaria Cerro El Ingles. Thus, based on the species richness, endemism, diversity at higher taxonomic levels, and the number of threatened species, it seems reasonable to say that the Serrania de los Paraguas is a priority site for conservation of amphibians in Colombia.

Background Wildlife species carry a remarkable diversity of trypanosomes. The detection of trypanosome infection in native Australian fauna is central to understanding their diversity and host-parasite associations. The implementation of total RNA sequencing (meta-transcriptomics) in trypanosome surveillance and diagnosis provides a powerful methodological approach to better understand the host species distribution of this important group of parasites. Methods We implemented a meta-transcriptomic approach to detect trypanosomes in a variety of tissues (brain, liver, lung, skin, gonads) sampled from native Australian wildlife, comprising four marsupials (koala, Phascolarctos cinereus ; southern brown bandicoot, Isoodon obesulus ; swamp wallaby, Wallabia bicolor ; bare-nosed wombat, Vombatus ursinus ), one bird (regent honeyeater, Anthochaera phrygia ) and one amphibian (eastern dwarf tree frog, Litoria fallax ). Samples corresponded to both clinically healthy and diseased individuals. Sequencing reads were de novo assembled into contigs and annotated. The evolutionary relationships among the trypanosomatid sequences identified were determined through phylogenetic analysis of 18S rRNA sequences. Results We detected trypanosome sequences in all six species of vertebrates sampled, with positive samples in multiple organs and tissues confirmed by PCR. Phylogenetic analysis indicated that the trypanosomes infecting marsupials were related to those previously detected in placental and marsupial mammals, while the trypanosome in the regent honeyeater grouped with avian trypanosomes. In contrast, we provide the first evidence for a trypanosome in the eastern dwarf tree frog that was phylogenetically distinct from those described in other amphibians. Conclusions To our knowledge, this is the first meta-transcriptomic analysis of trypanosomes in native Australian wildlife, expanding the known genetic diversity of these important parasites. We demonstrated that RNA sequencing is sufficiently sensitive to detect low numbers of Trypanosoma transcripts and from diverse hosts and tissues types, thereby representing an effective means to detect trypanosomes that are divergent in genome sequence.

The Red fox (Vulpes vulpes) has established large populations in Australia's urban and rural areas since its introduction following European settlement. The cryptic and highly adaptable nature of foxes allows them to invade cities and live among humans whilst remaining largely unnoticed. Urban living and access to anthropogenic food resources also influence fox ecology. Urban foxes grow larger, live at higher densities, and are more social than their rural counterparts. These ecological changes in urban red foxes are likely to impact the pathogens that they harbour, and foxes could pose a disease risk to humans and other species that share these urban spaces. To investigate this possibility, we used a meta-transcriptomic approach to characterise the virome of urban and rural foxes across the Greater Sydney region in Australia. Urban and rural foxes differed significantly in virome composition, with rural foxes harbouring a greater abundance of viruses compared to their urban counterparts. We identified ten potentially novel vertebrate-associated viruses in both urban and rural foxes, some of which are related to viruses associated with disease in domestic species and humans. These included members of the Astroviridae, Picobirnaviridae, Hepeviridae, and Picornaviridae as well as rabbit haemorrhagic disease virus-2. This study sheds light on the viruses carried by urban and rural foxes and emphasises the need for greater genomic surveillance of foxes and other invasive species at the human-wildlife interface.

The discovery of highly divergent RNA viruses is compromised by their limited sequence similarity to known viruses. Evolutionary information obtained from protein structural modelling offers a powerful approach to detect distantly related viruses based on the conservation of tertiary structures in key proteins such as the RNA-dependent RNA polymerase (RdRp). We utilised a template-based approach for protein structure prediction from amino acid sequences to identify distant evolutionary relationships among viruses detected in meta-transcriptomic sequencing data from Australian wildlife. The best predicted protein structural model was compared with the results of similarity searches against protein databases. Using this combination of meta-transcriptomics and protein structure prediction we identified theRdRp(PB1) gene segment of a divergent negative-sense RNA virus, denotedLauta virus(LTAV), in a native Australian gecko (Gehyra lauta). The presence of this virus was confirmed by PCR and Sanger sequencing. Phylogenetic analysis revealed thatLauta viruslikely represents a newly described genus within the familyAmnoonviridae,orderArticulavirales, that is most closely related to the fish virusTilapia tilapinevirus(TiLV). These findings provide important insights into the evolution of negative-sense RNA viruses and structural conservation of the viral replicase among members of the orderArticulavirales.

The Red fox ( Vulpes vulpes ) has established large populations in Australia’s urban and rural areas since its introduction following European settlement. Foxes’ cryptic and highly adaptable nature allows them to invade cities and live among humans while remaining largely unnoticed. Urban living and access to anthropogenic food resources also influences fox ecology. Urban foxes grow larger, live at higher densities and are more social than their rural counterparts. These ecological changes in urban red foxes are likely to impact the pathogens that they harbour, and foxes could pose a disease risk to humans and other species that share these urban spaces. To assess this possibility, we used a meta-transcriptomic approach to characterise the viromes of urban and rural foxes across the Greater Sydney region in Australia. Urban and rural foxes differed significantly in virome composition, with rural foxes harbouring a greater abundance of viruses compared to their urban counterparts. In contrast, urban fox viromes comprised a greater diversity of viruses compared to rural foxes. We identified nine potentially novel vertebrate-associated viruses in both urban and rural foxes, some of which are related to viruses associated with disease in domestic species and humans. These included members of the Astroviridae, Picobirnaviridae, Hepeviridae and Picornaviridae as well as rabbit haemorrhagic disease virus-2 (RHDV2). This study sheds light on the viruses carried by urban and rural foxes and emphasises the need for greater genomic surveillance of foxes and other invasive species at the human-wildlife interface. Importance Urbanisation of wild environments is increasing as human populations continue to expand. Remnant pockets of natural environments and other green spaces in urban landscapes provide invasive wildlife such as red foxes with refuges within urban areas, where they thrive on the food resources provisioned by humans. Close contact between humans, domestic species and foxes likely increases the risk of novel pathogen emergence. Indeed, the vast majority of emerging infectious diseases in humans originate in wild animals. Here, we explored potential differences in viromes between urban fox invaders and their rural counterparts. Viromes of foxes and their ectoparasites comprise a diversity of viruses including those from the Astroviridae, Picobirnaviridae, Hepeviridae, Caliciviridae and Picornaviridae . Microbial surveillance in foxes and other urban wildlife is vital for monitoring viral emergence and for the prevention of infectious diseases.

Tilapia lake virus (TiLV) has caused mass mortalities in farmed and wild tilapia with serious economic and ecological consequences. Until recently, this virus was the sole member of the Amnoonviridae, a family within the order Articulavirales comprising segmented negative-sense RNA viruses. We sought to identify additional viruses within the Amnoonviridae through total RNA sequencing (meta-transcriptomics) and data mining of published transcriptomes. Accordingly, we sampled marine fish species from both Australia and China and discovered several segments of two new viruses within the Amnoonviridae, tentatively called Flavolineata virus and Piscibus virus, respectively. In addition, by mining vertebrate transcriptome data, we identified nine additional virus transcripts matching to multiple genomic segments of TiLV in both marine and freshwater fish. These new viruses retained sequence conservation with the distantly related Orthomyxoviridae in the RdRp subunit PB1, but formed a distinct and diverse phylogenetic group. These data suggest that the Amnoonviridae have a broad host range within fish and that greater animal sampling will identify additional divergent members of the Articulavirales.

Identifying the components of host ecology that promote virus diversity is crucial for our understanding of the drivers of virus evolution and disease emergence. As the most species rich group of vertebrates that exhibit diverse ecologies, fish provide an ideal model system to study the impacts of host ecology on the composition of their viromes. To better understand the factors that shape virome composition in marine fishes, we characterised the viromes of 23 fish species (19 from this study and four that were sampled previously (Geoghegan et al 2018a)) using unbiased bulk RNA-sequencing (metatranscriptomics) together with both sequence and protein structural homology searches to identify divergent viruses that often evade characterisation. These data revealed that fish virome composition, that is, viral richness, abundance and diversity, were predominantly shaped by the phylogenetic history of their hosts, as reflected in taxonomic order. In addition, preferred mean water temperature, climate, habitat depth, community diversity and whether fish swim in schools or are solitary were identified as important ecological features that shaped virome diversity and abundance in these fish. Our analysis also identified 25 new virus transcripts that could be assigned to 11 different viral families, including the first fish virus in the Matonaviridae. Other viruses identified fell within the Astroviridae, Picornaviridae, Arenaviridae, Reoviridae, Hepadnaviridae, Paramyxoviridae, Rhabdoviridae, Hantaviridae, Filoviridae and Flaviviridae. Our results provide a better understanding of the ecological determinants of virome diversity and support the view that fish harbour a multitude of viruses, of which the vast majority are undescribed.

BACKGROUND Dengue virus type 4 (DENV-4) was first reported in Brazil in 1982 and since then no more cases were detected again in Brazil until 2010, when the virus was reintroduced. Over the following years, the virus spread to several Brazilian states and resulted in about 1,400,000 dengue cases, in 2013. The largest number of cases were documented in the Southeast macro-region. OBJECTIVES To determine the phylogeography of DENV-4 Genotype IIB strains isolated during the epidemics in 2012-2013 in São Paulo, Brazil, we aimed to contextualise the contribution of viruses sampled in different localities across the overall movement of DENV-4 in Brazil. METHODS Based on the envelope gene sequences retrieved from GenBank, we employed a Bayesian phylogeographic approach to assess the spatiotemporal dynamics of DENV-4 Genotype IIB in São Paulo, Brazil. FINDINGS The dispersal dynamics of DENV-4 Genotype IIB in Brazil indicated Rio de Janeiro and Mato Grosso states as the most likely routes toward São Paulo before the 2012-2013 outbreak. Likewise, Guarujá and São José do Rio Preto facilitated viral spread and transmission to other localities in the South and Southeast macro-regions in Brazil. CONCLUSIONS The spread pattern of DENV-4 Genotype IIB strains across the country supports two independent introductions of the virus in São Paulo in a short period of time. Furthermore, São Paulo appears to have played a pivotal role in the dissemination of DENV-4 to other locations in Brazil.