Evidence of reassortment of avian influenza A (H2) viruses in Brazilian shorebirds.

Influenza A viruses of the H2 subtype represent a zoonotic and pandemic threat to humans due to a lack of widespread specific immunity. Although A(H2) viruses that circulate in wild bird reservoirs are distinct from the 1957 pandemic A(H2N2) viruses, there is concern that they could impact animal and public health. There is limited information on AIVs in Latin America, and next to nothing about H2 subtypes in Brazil. In the present study, we report the occurrence and genomic sequences of two influenza A viruses isolated from wild-caught white-rumped sandpipers (Calidris fuscicollis). One virus, identified as A(H2N1), was isolated from a bird captured in Restinga de Jurubatiba National Park (PNRJ, Rio de Janeiro), while the other, identified as A(H2N2), was isolated from a bird captured in Lagoa do Peixe National Park (PNLP, Rio Grande do Sul). DNA sequencing and phylogenetic analysis of the obtained sequences revealed that each virus belonged to distinct subtypes. Furthermore, the phylogenetic analysis indicated that the genomic sequence of the A(H2N1) virus isolated from PNRJ was most closely related to other A(H2N1) viruses isolated from North American birds. On the other hand, the A(H2N2) virus genome recovered from the PNLP-captured bird exhibited a more diverse origin, with some sequences closely related to viruses from Iceland and North America, and others showing similarity to virus sequences recovered from birds in South America. Viral genes of diverse origins were identified in one of the viruses, indicating local reassortment. This suggests that the extreme South of Brazil may serve as an environment conducive to reassortment between avian influenza virus lineages from North and South America, potentially contributing to an increase in overall viral diversity.

Genetic diversity of adenovirus in neotropical bats from Brazil.

Bats can harbor a diversity of viruses, such as adenovirus. Ten different species of bat adenoviruses (BtAdV A to J) have been previous described worlwide. In Brazil, BtAdV was described in three species of phyllostomid species: Artibeus lituratus, Desmodus rotundus, and Sturnira lilium. There are around 180 bat species in Brazil, with 67% inhabiting the Atlantic Forest, with few information about the circulation of BtAdV in this biome. We aimed to describe the molecular detection and the phylogenetic characterization and suggest a classification of BtAdVs circulating in bats from the Brazilian Atlantic Forest. We collected 382 oral and rectal swabs from 208 bats between 2014-2015 and 2020-2021 from São Paulo, Pernambuco, and Santa Catarina Brazilian states. The adenovirus detection was done by a nested PCR targeting the DNA polymerase gene, and all positive samples were sequenced by the Sanger method. The phylogenetic analyses were based on the amino acid sequences using the MEGA 7 and BEAST software. We obtained 16 positive animals (detection rate 7.7%) belonging to seven bat species: Artibeus lituratus, Carollia perspicillata, Sturnira lilium, Molossus molossus, and the first record of Phyllostomus discolor, Eptesicus diminutus, and Myotis riparius. The phylogenetic analysis based on partial amino acid sequences showed that all obtained AdV sequences belong to the Mastadenovirus genus. We observed a high genetic diversity of BtAdV and identified eleven potential BtAdV species circulating in Brazil (BtAdV K to U). Our results contribute to the epidemiological surveillance of adenovirus, increasing the knowledge about the viral diversity and the distribution of AdV in bats from the Atlantic Forest.

H6N8 avian influenza virus in Antarctic seabirds demonstrates connectivity between South America and Antarctica.

Wild aquatic birds are the natural reservoirs of avian influenza viruses (AIVs). It is estimated that 100 million seabirds live in the Antarctic Peninsula and adjacent islands, regularly encountering migratory birds that use the islands to nest. Between 2010 and 2013, we collected samples from 865 seabirds in Elephant, King George and Livingston islands, around Antarctica Peninsula: chinstrap penguin (n = 143); gentoo penguin (n = 208); Adelie penguin (n = 46); brown skua (n = 90); Cape petrel (n = 115) and southern giant petrel (n = 263). Serum (n = 673) samples were analysed by competitive ELISA and swabs (n = 614) were tested by one step real-time RT-PCR for avian influenza virus (AIV). Sera from 30 chinstrap penguins, 76 brown skuas and a single Adelie penguin were seropositive for AIV. Thirteen swab samples were AIV positive by RT-PCR, and complete genome sequences of H6N8 AIVs isolated from brown skua and chinstrap penguin in 2011 were obtained. Phylogenetic analyses indicated that all gene segments of the H6N8 viruses were closely related to Argentinian and Chilean AIVs. The prevalence with which we identified evidence for AIVs infection in various Antarctic seabirds suggest viral circulation in Antarctic avifauna and interspecies viral transmission in the sub-Antarctic region.

High genetic diversity of alphacoronaviruses in bat species (Mammalia: Chiroptera) from the Atlantic Forest in Brazil.

Bat coronaviruses (Bat-CoVs) represent around 35% of all virus genomes described in bats. Brazil has one of the highest mammal species diversity, with 181 species of bats described so far. However, few Bat-CoV surveillance programmes were carried out in the country. Thus, our aim was to jevaluate the Bat-CoV diversity in the Atlantic Forest, the second biome with the highest number of bat species in Brazil. We analysed 456 oral and rectal swabs and 22 tissue samples from Atlantic Forest bats, detecting Alphacoronavirus in 44 swab samples (9.6%) targeting the RdRp gene from seven different bat species, three of which have never been described as Bat-CoV hosts. Phylogenetic analysis of the amino acid (aa) sequences coding the RdRp gene grouped the sequences obtained in our study with Bat-CoV previously detected in identical or congeneric bat species, belonging to four subgenera, with high aa identity (over 90%). The RdRp gene was also detected in three tissue samples from Diphylla ecaudata and Sturnira lilium, and the partial S gene was successfully sequenced in five tissues and swab samples of D. ecaudata. The phylogenetic analysis based on the partial S gene obtained here grouped the sequence of D. ecaudata with CoV from Desmodus rotundus previously detected in Peru and Brazil, belonging to the Amalacovirus subgenus, with aa identity ranging from 73.6% to 88.8%. Our data reinforce the wide distribution of Coronaviruses in bats from Brazil and the novelty of three bats species as Bat-CoV hosts and the co-circulation of four Alphacoronavirus subgenera in Brazil.

Survey of SARS-CoV-2 genetic diversity in two major Brazilian cities using a fast and affordable Sanger sequencing strategy.

Genetic variants of SARS-CoV-2 have been emerging and circulating in many places across the world. Rapid detection of these variants is essential since their dissemination can impact transmission rates, diagnostic procedures, disease severity, response to vaccines or patient management. Sanger sequencing has been used as the preferred approach for variant detection among circulating human immunodeficiency and measles virus genotypes. Using primers to amplify a fragment of the SARS-CoV-2 genome encoding part of the Spike protein, we showed that Sanger sequencing allowed us to rapidly detect the introduction and spread of three distinct SARS-CoV-2 variants in two major Brazilian cities. In both cities, after the predominance of variants closely related to the virus first identified in China, the emergence of the P.2 variant was quickly followed by the detection of the P1 variant, which became dominant in less than one month after it was first detected.

Discovery of novel astrovirus and calicivirus identified in ruddy turnstones in Brazil.

Birds are the natural reservoir of viruses with zoonotic potential, as well as contributing to the evolution, emergence, and dissemination of novel viruses. In this study, we applied a high-throughput screening approach to identify the diversity of viruses in 118 samples of birds captured between October 2006 to October 2010 in the North and Northeast regions of Brazil. We found nearly complete genomes of novel species of astrovirus and calicivirus in cloacal swabs of ruddy turnstones (Arenaria interpres) collected in Coroa do Avião islet, Pernambuco State. These viruses are positive-sense single-stranded RNA with a genome of ~7 to 8 kb, and were designated as Ruddy turnstone astrovirus (RtAstV) and Ruddy turnstone calicivirus (RTCV), respectively. Phylogenetic analysis showed that RtAstV and RTCV grouped in a monophyletic clade with viruses identified from poultry samples (i.e., chicken, goose, and turkey), including viruses associated with acute nephritis in chickens. Attempts of viral propagation in monkey and chicken cell lines for both viruses were unsuccessful. Also, we found genomes related with viral families that infect invertebrates and plants, suggesting that they might be ingested in the birds' diet. In sum, these findings shed new light on the diversity of viruses in migratory birds with the notable characterization of a novel astrovirus and calicivirus.

Divergent coronaviruses detected in wild birds in Brazil, including a central park in São Paulo.

Coronaviruses are single-stranded positive-sense RNA viruses associated with important avian diseases. Their relatively high rates of mutation and recombination frequencies allow them to adapt to new hosts and ecological niches. Although Brazil has 18% of global avian species diversity, studies regarding the presence of avian viral diseases in wild birds in South America are scarce. In this study, we performed a retrospective analysis of the presence of CoVs in 746 wild birds. Oropharyngeal and cloacal swabs were obtained and placed together in vials containing VTM transport medium collected in different regions of Brazil between 2006 and 2013. Screening for viral nucleic acid was performed using conventional RT-PCR and pancoronavirus nested PCR. Positive samples were characterized by partial sequencing of the RNA-dependent RNA polymerase (RdRp) gene, and ensuing phylogenetic analysis was performed to investigate the association between virus epidemiology and bird migration routes. Coronavirus RNA were detected and sequenced from six samples, in which three were related to gammacoronaviruses group and the other three to deltacoronavirus group. Our study documents the presence of CoVs related to avian gamma- and deltacoronaviruses circulating in both urban- and poultry-farm regions of Brazil, implicating wild birds as potential carriers of CoVs which may represent a risk to poultry farms and public health in Brazil.

Novel Parvoviruses from Wild and Domestic Animals in Brazil Provide New Insights into Parvovirus Distribution and Diversity.

Parvoviruses (family Parvoviridae) are small, single-stranded DNA viruses. Many parvoviral pathogens of medical, veterinary and ecological importance have been identified. In this study, we used high-throughput sequencing (HTS) to investigate the diversity of parvoviruses infecting wild and domestic animals in Brazil. We identified 21 parvovirus sequences (including twelve nearly complete genomes and nine partial genomes) in samples derived from rodents, bats, opossums, birds and cattle in Pernambuco, São Paulo, Paraná and Rio Grande do Sul states. These sequences were investigated using phylogenetic and distance-based approaches and were thereby classified into eight parvovirus species (six of which have not been described previously), representing six distinct genera in the subfamily Parvovirinae. Our findings extend the known biogeographic range of previously characterized parvovirus species and the known host range of three parvovirus genera (Dependovirus, Aveparvovirus and Tetraparvovirus). Moreover, our investigation provides a window into the ecological dynamics of parvovirus infections in vertebrates, revealing that many parvovirus genera contain well-defined sub-lineages that circulate widely throughout the world within particular taxonomic groups of hosts.

Migratory birds in southern Brazil are a source of multiple avian influenza virus subtypes.

BACKGROUND: There is insufficient knowledge about the relation of avian influenza virus (AIV) to migratory birds in South America. Accordingly, we studied samples obtained over a 4-year period (2009-2012) from wild birds at a major wintering site in southern Brazil. METHODS: We obtained 1212 oropharyngeal/cloacal samples from wild birds at Lagoa do Peixe National Park and screened them for influenza A virus by RT-PCR amplification of the matrix gene. Virus isolates were subjected to genomic sequencing and antigenic characterization. RESULTS: Forty-eight samples of 1212 (3.96%) contained detectable influenza virus RNA. Partial viral sequences were obtained from 12 of these samples, showing the presence of H2N2 (1), H6Nx (1), H6N1 (8), H9N2 (1), and H12N5 (1) viruses. As H6 viruses predominated, we generated complete genomes from all 9 H6 viruses. Phylogenetic analyses showed that they were most similar to viruses of South American lineage. The H6N1 viruses caused no disease signs in infected ferrets and, despite genetic differences, were antigenically similar to North American isolates. CONCLUSIONS: Lagoa do Peixe National Park is a source of multiple AIV subtypes, with the levels of influenza virus in birds being highest at the end of their wintering period in this region. H6N1 viruses were the predominant subtype identified. These viruses were more similar to viruses of South American lineage than to those of North American lineage.

Lack of Detection of Avian Influenza, Newcastle Disease, and West Nile Viruses in Wild Birds of Northeastern Brazil.

We tested 529 wild birds captured in northeastern Brazil for infection by avian influenza, Newcastle disease, and West Nile. Viruses were not detected by real-time PCR with the exception of one Tropical Gnatcatcher ( Polioptila plumbea) positive for influenza virus, but this could not be confirmed by viral isolation or gene sequencing.

Survey of pathogens in threatened wild red-tailed Amazon parrot (Amazona brasiliensis) nestlings in Rasa Island, Brazil

ABSTRACT The red-tailed Amazon parrot (Amazona brasiliensis) is a threatened species of psittacine bird that inhabit coastal regions of Brazil. In view of the threat of this species, the aim of this study was to perform a health evaluation in wild nestlings in Rasa Island, determining the prevalence of enterobacteria and infectious agents according to type of nest. Blood samples were collected from 64 birds and evaluated for antibodies of Chlamydia psittaci by commercial dot-blot ELISA. Cloacal and oropharyngeal swabs samples were collected from 23 birds from artificial wooden nests, 15 birds from PVC nests and 2 birds from natural nests for microbiological analysis. Swab samples were collected from 58 parrots for C. psittaci detection by PCR and from 50 nestlings for Avian Influenza, Newcastle Disease and West Nile viruses' detection analysis by real-time RT-PCR. Ten bacterial genera and 17 species were identified, and the most prevalent were Escherichia coli and Klebsiella oxytoca. There was no influence of the type of nest in the nestlings' microbiota. All samples tested by ELISA and PCR were negative. There is currently insufficient information available about the health of A. brasiliensis and data of this study provide a reference point for future evaluations and aid in conservation plans.

Survey of pathogens in threatened wild red-tailed Amazon parrot (Amazona brasiliensis) nestlings in Rasa Island, Brazil.

The red-tailed Amazon parrot (Amazona brasiliensis) is a threatened species of psittacine bird that inhabit coastal regions of Brazil. In view of the threat of this species, the aim of this study was to perform a health evaluation in wild nestlings in Rasa Island, determining the prevalence of enterobacteria and infectious agents according to type of nest. Blood samples were collected from 64 birds and evaluated for antibodies of Chlamydia psittaci by commercial dot-blot ELISA. Cloacal and oropharyngeal swabs samples were collected from 23 birds from artificial wooden nests, 15 birds from PVC nests and 2 birds from natural nests for microbiological analysis. Swab samples were collected from 58 parrots for C. psittaci detection by PCR and from 50 nestlings for Avian Influenza, Newcastle Disease and West Nile viruses' detection analysis by real-time RT-PCR. Ten bacterial genera and 17 species were identified, and the most prevalent were Escherichia coli and Klebsiella oxytoca. There was no influence of the type of nest in the nestlings' microbiota. All samples tested by ELISA and PCR were negative. There is currently insufficient information available about the health of A. brasiliensis and data of this study provide a reference point for future evaluations and aid in conservation plans.

Novel avian paramyxovirus (APMV-15) isolated from a migratory bird in South America.

A novel avian paramyxovirus (APMV) isolated from a migratory bird cloacal swab obtained during active surveillance in April 2012 in the Lagoa do Peixe National Park, Rio Grande do Sul state, South of Brazil was biologically and genetically characterized. The nucleotide sequence of the full viral genome was completed using a next-generation sequencing approach. The genome was 14,952 nucleotides (nt) long, with six genes (3'-NP-P-M-F-HN-L-5') encoding 7 different proteins, typical of APMV. The fusion (F) protein gene of isolate RS-1177 contained 1,707 nucleotides in a single open reading frame encoding a protein of 569 amino acids. The F protein cleavage site contained two basic amino acids (VPKER↓L), typical of avirulent strains. Phylogenetic analysis of the whole genome indicated that the virus is related to APMV-10, -2 and -8, with 60.1% nucleotide sequence identity to the closest APMV-10 virus, 58.7% and 58.5% identity to the closest APMV-8 and APMV-2 genome, respectively, and less than 52% identity to representatives of the other APMVs groups. Such distances are comparable to the distances observed among other previously identified APMVs serotypes. These results suggest that unclassified/calidris_fuscicollis/Brazil/RS-1177/2012 is the prototype strain of a new APMV serotype, APMV-15.

Antibodies Against Henipa-Like Viruses in Brazilian Bats.

Bats are reservoir hosts for many paramyxoviruses, some of which cause human and zoonotic diseases of public health importance. We developed a Nipah virus nucleoprotein enzyme-linked immunosorbent assay to detect cross-reactive antibodies in serum samples from several bat species in Brazil. Our results warrant further investigation of henipa-like virus reservoirs in the Western hemisphere.

Surveillance of Avian Influenza Virus in Aquatic Birds on the Brazilian Amazon Coast.

The occurrence of avian influenza viruses (AIV) has been extensively studied in aquatic birds in the Northern hemisphere; however, much less information is available for the South American region. In 2009-2010, we sampled 1006 wild aquatic birds (90% Charadriiformes, 9% Anseriformes, and 1% other groups) at three locations on the Brazilian Amazon coast, a region that serves as a major stop-over and wintering site along the Atlantic Americas flyway. Real-time RT-PCR identified five samples as positive; however, no AIV isolates could be obtained and Illumina sequencing did not produce gene sequences that would allow further characterization of the virus.

Serological Survey of Hantavirus in Inhabitants from Tropical and Subtropical Areas of Brazil.

Brazil has reported more than 1,600 cases of hantavirus cardiopulmonary syndrome (HPS) since 1993, with a 39% rate of reported fatalities. Using a recombinant nucleocapsid protein of Araraquara virus, we performed ELISA to detect IgG antibodies against hantavirus in human sera. The aim of this study was to analyze hantavirus antibody levels in inhabitants from a tropical area (Amazon region) in Rondônia state and a subtropical (Atlantic Rain Forest) region in São Paulo state, Brazil. A total of 1,310 serum samples were obtained between 2003 and 2008 and tested by IgG-ELISA, and 82 samples (6.2%), of which 62 were from the tropical area (5.8%) and 20 from the subtropical area (8.3%), tested positive. Higher levels of hantavirus antibody were observed in inhabitants of the populous subtropical areas compared with those from the tropical areas in Brazil.

Serological and molecular investigation of viral agents in freeliving jaguars of the Pantanal wetlands, state of Mato Grosso, Brazil / Investigação sorológica e molecular de agentes virais em onças-pintadas de vida livre no Pantanal de Mato Grosso, Brasil

This study investigates the exposure of free-living jaguars from two federal protected areas in the Pantanal of Mato Grosso, Brazil, to a variety viral agents. These viral agents, particularly causing zoonotic diseases, were analyzed using serological and molecular methods. None of the jaguars was positive by RT-PCR for the molecular detection of avian influenza and West Nile Fever (WNF). Only one animal was serologically positive for Eastern Equine Encephalitis (EEE) by virus neutralization test in VERO cell cultures, representing the first reported case of jaguar exposure to EEE virus. However, all the animals were negative for Western Equine Encephalitis (WEE) virus and Venezuelan Equine Encephalitis (VEE) virus. Eleven jaguars were tested by two tests for the detection of antibodies against rabies virus (Simplified Fluorescent Inhibition Microtest ­ SFIMT and Rapid Fluorescent Focus Inhibition Test ­ RFFIT), resulting in five positive animals, two animals in each test and one in both serological tests. Furthermore, three out of 14 samples subjected to the neutralization test were positive for antibodies against canine distemper virus (CDV), and 15 out of 17 samples subjected to the hemagglutination-inhibition test (HI) were positive for antibodies against canine parvovirus (CPV). In view of the findings of this study, it is unlikely that the viruses examined here represent a threat to the jaguar populations in this region.(AU)

Este estudo investigou a exposição de onças-pintadas de vida livre a agentes virais selecionados em duas unidades de conservação federais no Pantanal de Mato Grosso, Brasil. Para a análise desses agentes virais, a maioria de caráter zoonótico, foram utilizados métodos sorológicos e moleculares. Nenhuma das onze onças-pintadas examinadas foi positiva na técnica de real-time RT-PCR para a detecção molecular dos agentes da Influenza aviária e Febre do Nilo Ocidental (WNF). Somente um animal foi positivo sorologicamente para a o vírus da Encefalite Equina do Leste (EEE) pela Microtécnica de vírus neutralização em culturas de células VERO, sendo este o primeiro relato da exposição de onças-pintadas. Todos os animais examinados s foram negativos para o vírus da Encefalite Equina do Oeste (WEE) e Venezuelana (VEE). Amostras de soro colhidas de 11 onças-pintadas foram submetidas a adois testes distintos para a detecção de anticorpos contra o vírus da raiva (Teste Rápido de Inibição de Foco de Fluorescência ­ RFFIT e Microteste Simplificado de Inibição da Fluorescência - SFIMT), resultando em cinco animais positivos, dos quase dois positivos para cada teste e um positivo quando submetido aos dois testes sorológicos. Além disso, três das 14 amostras submetidas a técnica de soroneutralização foram positivas para a pesquisa de anticorpos contra o vírus da cinomose (CDV) e 15 amostras positivas das 17 analisadas para a pesquisa de anticorpos contra o parvovírus canino (CPV) foram identificadas pela técnica de Inibição da Hemaglutinação (HI). De acordo com os resultados deste estudo, é pouco provável que os agentes virais aqui analisados representem ameaça à população de onçaspintadas nesta região.(AU)

Co-circulation in a single biome of the Juquitiba and Araraquara hantavirus detected in human sera in a sub-tropical region of Brazil.

Hantaviruses is an emerging infectious disease. Although HCPS has been reported in several regions of Brazil, more cases of HCPS have recently been reported in Minas Gerais than in any other state. In 2009, we analyzed 27 samples presenting antibodies against hantaviruses. These samples originated from 688 symptomatic patients, as determined based on the Hemorrhagic Fever Protocol. A subsequent SYBR Green-based real-time RT-PCR demonstrated the presence of the virus in 22 of the samples. Among the RT-PCR-positive samples, 17 were analyzed using DNA sequencing; these sequences were compared with others deposited in GenBank and showed similarity with the Araraquara and Juquitiba virus clusters. This work describe the detection of Juquitiba virus, including three fatal cases, in Minas Gerais state, furthermore, showed that it is feasible to characterize the circulating strains using a small fragment of S segment. Finally, the results suggest the co-circulation of Araraquara and Juquitiba virus in a single biome in Minas Gerais state.

Avian influenza virus (H11N9) in migratory shorebirds wintering in the Amazon Region, Brazil.

Aquatic birds are the natural reservoir for avian influenza viruses (AIV). Habitats in Brazil provide stopover and wintering sites for water birds that migrate between North and South America. The current study was conducted to elucidate the possibility of the transport of influenza A viruses by birds that migrate annually between the Northern and Southern Hemispheres. In total, 556 orotracheal/cloacal swab samples were collected for influenza A virus screening using real-time RT-PCR (rRT-PCR). The influenza A virus-positive samples were subjected to viral isolation. Four samples were positive for the influenza A matrix gene by rRT-PCR. From these samples, three viruses were isolated, sequenced and characterized. All positive samples originated from a single bird species, the ruddy turnstone (Arenaria interpres), that was caught in the Amazon region at Caeté Bay, Northeast Pará, at Ilha de Canelas. To our knowledge, this is the first isolation of H11N9 in the ruddy turnstone in South America.

West Nile virus surveillance, Brazil, 2008-2010.

BACKGROUND: West Nile virus (WNV) is an emergent pathogen that is widely distributed in North and Central America. The recent introduction in South America has focused attention on the spread of WNV across Southern American countries. The transmission network involves mosquitoes, birds, horses and humans. METHODS: The serological evaluation of sera from 678 equids and 478 birds was performed using a WNV-specific blocking ELISA, and only the positive results were confirmed by plaque reduction neutralisation tests (PRNTs). Molecular analysis was performed on sera from 992 healthy equids and on 63 macerates of brains from equids that died of encephalitis and had previously tested negative for other pathogens. We also tested swabs from 928 birds. The samples analysed were collected in different biomes of Brazil. RESULTS: We identified WNV antibodies by ELISA in thirteen equids and five birds, and PRNT90 confirmed WNV positivity in four equid samples collected in 2009 in an area between the Amazon and the Pantanal. None of the ELISA positive bird samples were confirmed by PRNT90, and all samples tested by RT-PCR were negative. CONCLUSION: WNV circulation is confirmed by this large scale survey even in the absence of detection of clinical cases.