The rapid and disorderly urbanization in the Amazon has resulted in the insertion of forest fragments into cities, causing the circulation of arboviruses, which can involve hematophagous arthropods and free-ranging birds in the transmission cycles in urban environments. This study aimed to evaluate the circulation of arboviruses in free-ranging birds and hematophagous arthropods captured in an Environmental Protection Area in the Belem metropolitan area, Brazil. Birds were captured using mist nets, and hematophagous arthropods were collected using a human protected attraction technique and light traps. The birds’ sera were subjected to a hemagglutination inhibition test to detect antibodies against 29 arbovirus antigens. Arthropod macerates were inoculated into C6/36 and VERO cell cultures to attempt viral isolation and were tested using indirect immunofluorescence, subsequent genetic sequencing and submitted for phylogenetic analysis. Four bird sera were positive for arbovirus, and one batch of Psorophora ferox was positive for Flavivirus on viral isolation and indirect immunofluorescence. In addition, the Ilheus virus was detected in the sequencing and phylogenetic analysis. The presence of antibodies in sera from free-ranging birds and the isolation of Ilheus virus in Psorophora ferox indicate the circulation of arboviruses in forest remnants in the urban center of Belem.

Key words: arbovirus; domestic pigeons; clinical; serological tests

Advances in diagnostic techniques coupled with ongoing environmental changes have resulted in intensified surveillance and monitoring of arbovirus circulation in the Amazon. This increased effort has resulted in increased detection of insect-specific viruses among hematophagous arthropods collected in the field. This study aimed to document the first isolation of Agua Salud alphavirus in mosquitoes collected within the Brazilian Amazon. Arthropods belonging to the family Culicidae were collected within a forest fragment located in the Environmental Protection Area of the metropolitan region of Belem. Subsequently, these specimens were meticulously identified to the species level. Afterward, the collected batches were macerated, and the resulting supernatant was then inoculated into C6/36 and Vero cell cultures to facilitate viral isolation. The presence of arboviruses within the inoculated cell cultures was determined through indirect immunofluorescence analysis. Furthermore, positive supernatant samples underwent nucleotide sequencing to precisely identify the viral strains present. Notably, a batch containing Culex (Melanoconion) mosquitoes was identified to be positive for the genus Alphavirus via indirect immunofluorescence. This study is the first report on insect-specific alphavirus isolation in Brazil and the first-ever description of Agua Salud alphavirus isolation within Amazon Forest remnants.

Mosquitoes are recognized as the arthropod group with the greatest vectorial capacity, and the viruses they transmit constitute a significant concern in the context of global One Health. In addition, these insects act as hosts for a wide diversity of insect-specific viruses (ISVs), which exclusively infect arthropods. Expanding knowledge of ISVs is particularly relevant, given their potential influence on arbovirus replication and their role in elucidating the evolutionary processes that shape virus–vector interactions. In this study, we report the isolation and molecular analysis of three negeviruses associated with different mosquito species of the genera Culex, Coquillettidia, Mansonia, and Ochlerotatus, collected in Belém, Pará State, in the Brazilian Amazon: Loreto virus, Wallerfield virus, and a putative new species, designated Terra firme virus. Eleven pools exhibited cellular alterations consistent with cytopathic effects in invertebrate C6/36 cells but showed no evidence of replication in vertebrate Vero cells. Notably, simultaneous infections by two or three negeviruses were detected in some mosquito pools, indicating the occurrence of multiple viral infections within individual samples. Genomic analyses revealed that the isolated strains share conserved domains with previously described isolates from other countries. Phylogenetic inferences demonstrated that the investigated strains are classified within the clades Nelorpivirus and Sandewavirus. Taken together, these findings expand the currently known diversity of the negevirus group and contribute to a more comprehensive understanding of its host range and geographic distribution.

Wild birds have great prominence on transmission of diseases to humans, mainly due to their ease of access to human population, raising concerns about the potential impact of that proximity in context of the One Health. Studies referring to circulation of avian reovirus (ARV) and picobirnavirus (PBV) in wild birds are limited, in addition to reinforcing the development of researches that describe the prevalence, characterize the variants and evaluate the potential impact of these infections on the wild ecosystem and public health. The present study reports the occurrence of ARV and PBV in wild birds collected from an environmental protection area in the Amazon biome. RT-PCR analysis showed ARV infection prevalence in 0.6% (1/155) and PBV infection in 1.29% (2/155) on the samples. ARV strain isolated in this study demonstrated more phylogenetically related to other ARVs previously circulating in poultry in the same region. The two PBV strains obtained belong to genogroup I, and showed phylogenetically related to other PBV isolated from different animal species in different geographic regions. This study is a pioneer in the detection of ARV in wild birds in Brazil and presents a report of the first occurrence of PBV in wild birds of Guira guira specie. Additional studies in wild birds are required to increase the epidemiology, origin, evolution and emergence of new viruses that may provoke problems in the context of One Health.

Free-living Columbidae are considered the second largest reservoir of Chlamydia psittaci, transmitting the agent to humans and other animals. The present study aimed to identify the presence of Chlamydia spp. in samples of lung and stool collected from free-living domestic pigeons (Columba livia domestica) captured at the Mangal das Garças Naturalistic Park in Belém, Pará, Brazil, using semi-nested polymerase chain reaction, and also correlate the clinical and post-mortem findings of animals positive for Chlamydia spp. Among the 45 animals analyzed, 10 (22.2%) were positive for Chlamydia spp.; the positive findings originated from 5 (50%) lung samples and 5 (50%) stool samples, with no overlap between animals and samples. None of the animals evaluated in this study showed clinical signs of chlamydiosis; rather, these were only found during necropsy of positive animals, mainly through pulmonary, hepatic, splenic, and intestinal changes. These findings demonstrate that free-living pigeons can be reservoirs of Chlamydia spp. and transmit the agent silently to humans and animals, which is concerning for public and animal health, since these birds are easily found in urban areas cohabitating with humans, other species of birds, and other animals.