Viruses may represent the most diverse microorganisms on Earth. Novel viruses and variants continue to emerge. Mosquitoes are the most dangerous animals to humankind. This study aimed at identifying viral RNA diversity in salivary glands of mosquitoes captured in a sylvatic area of Cerrado at the Chapada dos Guimarães National Park, Mato Grosso, Brazil. In total, 66 Culicinae mosquitoes belonging to 16 species comprised 9 pools, subjected to viral RNA extraction, double-strand cDNA synthesis, random amplification and high-throughput sequencing, revealing the presence of seven insect-specific viruses, six of which represent new species of Rhabdoviridae (Lobeira virus), Chuviridae (Cumbaru and Croada viruses), Totiviridae (Murici virus) and Partitiviridae (Araticum and Angico viruses). In addition, two mosquito pools presented Kaiowa virus sequences that had already been reported in South Pantanal, Brazil. These findings amplify the understanding of viral diversity in wild-type Culicinae. Insect-specific viruses may present a broader diversity than previously imagined and future studies may address their possible role in mosquito vector competence.

Viruses are ubiquitous and diverse microorganisms arising as a result of interactions within their vertebrate and invertebrate hosts. Here we report the presence of different viruses in the salivary glands of 1657 mosquitoes classified over 28 culicinae species from the North region of the Brazilian Pantanal wetland through metagenomics, viral isolation, and RT-PCR. In total, 12 viruses were found, eight putative novel viruses with relatively low similarity with pre-existing species of viruses within their families, named Pirizal iflavirus, Furrundu phlebovirus, Pixé phlebovirus, Guampa vesiculovirus, Chacororé flavivirus, Rasqueado orbivirus, Uru chuvirus, and Bororo circovirus. We also found the already described Lobeira dielmorhabdovirus, Sabethes flavivirus, Araticum partitivirus, and Murici totivirus. Therefore, these findings underscore the vast diversity of culicinae and novel viruses yet to be explored in Pantanal, the largest wetland on the planet.

BackgroundHigh throughput sequencing (HTS) boosted the discovery of novel viruses and new variants of known viruses. Here we investigated the presence of viruses in 12 pools of sand flies captured in three climatic periods in RAPELD grids at Rio Claro, Chapada dos Guimarães and at Pirizal, North Pantanal, Mato Grosso State, Midwestern Brazil by HTS, viral isolation of a putative Phlebovirus positive pool in Vero cells, RT-PCR and transmission electron microscopy (TEM).ResultsOne pool containing three Lutzomyia (Lutzomyia) longipalpis sand flies captured in the transitional climatic period in North Pantanal showed a tripartite genomic sequence of a putative novel Phlebovirus belonging to the phlebotomus fever serogroup. Phylogenetic analysis revealed this virus is closely related and share a common ancestor with phleboviruses included in the same clade: Chagres, Urucuri and Uriurana virus. RNA-dependent RNA polymerase (RdRP) presented 60%, 59% and 58% of amino-acid (aa) similarity with these phleboviruses, respectively. Similarity of Nucleoprotein and NSs protein codified by ambissense strategy of segment S was of 49% and 37%, respectively, with the proteins of the closest phlebovirus, Uriurana virus. Glycoproteins (G1, G2) and NSm protein presented 49% and 48% aa similarity with Chagres and Uriurana virus, respectively. Uriurana virus was isolated from sand flies in Brazilian Amazon and Urucuri from rodents in Utinga forest, Pará State. Chagres virus is an arbovirus responsible for outbreaks of febrile illness in Panama. This phlebovirus was isolated in Vero cells, confirmed by TEM and RT-PCR for the L segment of the virus, and named Viola phlebovirus.ConclusionsHTS, viral isolation, RT-PCR and TEM showed the presence of one virus in sand flies from North Pantanal with identity to a putative novel Phlebovirus from phlebotomus fever serogroup, named Viola phlebovirus.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2985-3) contains supplementary material, which is available to authorized users.

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Background: Visceral leishmaniasis (VL) is a zoonotic disease caused by Leishmania chagasi (syn. L. infantum). The dog is the main reservoir for this infectious agent in the urban environment. Among the various systemic signs of viscerotropic infection by L. chagasi, cutaneous lesions, including exfoliative dermatitis, cutaneous ulcers and nodules, alopecia, papular or pustular dermatitis, and onychogryphosis, are the most common in dogs. This study aimed to describe the major cutaneous lesions, evaluate the skin parasite L. chagasi by PCR, and investigate the main dermatoses associated with this zoonosis. Materials, Methods & Results: This study evaluated 50 seropositive dogs of various breeds and sizes for VL by ELISA and IFA and for the dermatological signs associated with VL. Moreover, molecular analysis of skin fragments was performed with primers 150 and 152 for the genus Leishmania, and the species was verified as L. chagasi with RV1 and RV2 primers. Deep skin scraping for mites and fungal culture analysis were performed in all dogs. Of the 50 dogs, 15 (30%) were free of systemic or cutaneous signs; however, changes in skin and annexes were observed in 35 (70%) dogs. Thirty-one dogs (62%) presented infection with dermatophytes, 26 (83.9%) with Microsporum sp., and 5 (16.1%) with Trichophyton sp.; only one dog showed parasitism by Sarcoptes scabiei. A statistically significant association was observed between skin alterations and dermatological infection by dermatophytes (P = 0.61). Of the 29 dogs from which skin fragments were used to perform PCR with specific primers, 19 (65.5%) showed L. chagasi DNA amplification. Discussion: Symptomatic dogs are more common than asymptomatic ones; therefore, sampling was set up in the hospital for reagents dogs with clinical suspicion of this zoonosis. Dermatological signs accounted for 70% of the clinical symptoms presented by the dogs, which were evaluated as described by other authors. Exfoliative dermatitis was the most common skin lesion followed by onychogryphosis and alopecia. This is because of granulomatous or pyogranulomatous inflammation, inflammation in different structures of the skin, or deposition of immune complexes. Only few studies have described the co-existence of VL and dermatophytosis in dogs. We found dermatophyte fungal infection in more than half of the dogs (70%), most frequently Microsporum sp. followed by infection with Trichophyton sp. Regarding the clinical signs, no statistical difference was observed between the dogs with and without dermatophyte infection, which reinforces the lack of specificity in clinical signs that may hinder the diagnosis of both diseases when present as co-morbidities or in isolation. The high frequency of dermatophytosis in dogs with VL may result from a compromised immune system. L. chagasi DNA detected in 65.5% of samples tested by conventional PCR can be related to the host's immune response, as well as to the uneven distribution of the parasite in different tissues. These results support the high freq...