Human outbreaks of a novel reassortant Oropouche virus in the Brazilian Amazon region.

The Brazilian western Amazon is experiencing its largest laboratory-confirmed Oropouche virus (OROV) outbreak, with more than 6,300 reported cases between 2022 and 2024. In this study, we sequenced and analyzed 382 OROV genomes from human samples collected in Amazonas, Acre, Rondônia and Roraima states, between August 2022 and February 2024, to uncover the origin and genetic evolution of OROV in the current outbreak. Genomic analyses revealed that the upsurge of OROV cases in the Brazilian Amazon coincides with spread of a novel reassortant lineage containing the M segment of viruses detected in the eastern Amazon region (2009-2018) and the L and S segments of viruses detected in Peru, Colombia and Ecuador (2008-2021). The novel reassortant likely emerged in the Amazonas state between 2010 and 2014 and spread through long-range dispersion events during the second half of the 2010s. Phylodynamics reconstructions showed that the current OROV spread was driven mainly by short-range (< 2 km) movements consistent with the flight range of vectors. Nevertheless, a substantial proportion (22%) of long-range (>10 km) OROV migrations were also detected, consistent with viral dispersion by humans. Our data provide a view of the unprecedented spread and evolution of OROV in the Brazilian western Amazon region.

Infection of South American coatis (Nasua nasua) with highly pathogenic avian influenza H5N1 virus displaying mammalian adaptive mutations.

Deadly outbreaks among poultry, wild birds, and carnivorous mammals by the highly pathogenic H5N1 virus of the clade 2.3.4.4b have been reported in South America. The increasing virus incidence in various mammal species poses a severe zoonotic and pandemic threat. In Uruguay, the clade 2.3.4.4b viruses were first detected in February 2023, affecting wild birds and backyard poultry. Three months after the first reported case in Uruguay, the disease affected a population of 23 coatis (Nasua) in an ecological park. Most animals became infected, likely directly or indirectly from wild birds in the park, and experienced sudden death. Five animals from the colony survived, and four of them developed antibodies. The genomes of the H5N1 strains infecting coatis belonged to the B3.2 genotype of the clade 2.3.4.4b. Genomes from coatis were closely associated with those infecting backyard poultry, but transmission likely occurred through wild birds. Notable, two genomes have a 627K substitution in the RNA polymerase PB2 subunit, a hallmark amino acid linked to mammalian adaptation. Our findings support the ability of the avian influenza virus of the 2.3.4.4b clade to infect and transmit among terrestrial mammals with high pathogenicity and undergo rapid adaptive changes. It also highlights the coatis' ability to develop immunity and naturally clear the infection.

Molecular Epidemiology of Western Equine Encephalitis Virus, South America, 2023-2024.

Western equine encephalitis virus (WEEV) is a mosquitoborne virus that reemerged in December 2023 in Argentina and Uruguay, causing a major outbreak. We investigated the outbreak using epidemiologic, entomological, and genomic analyses, focusing on WEEV circulation near the Argentina‒Uruguay border in Rio Grande do Sul state, Brazil. During November 2023‒April 2024, the outbreak in Argentina and Uruguay resulted in 217 human cases, 12 of which were fatal, and 2,548 equine cases. We determined cases on the basis of laboratory and clinical epidemiologic criteria. We characterized 3 fatal equine cases caused by a novel WEEV lineage identified through a nearly complete coding sequence analysis, which we propose as lineage C. Our findings highlight the importance of continued surveillance and equine vaccination to control future WEEV outbreaks in South America.

Highly divergent and diverse viral community infecting sylvatic mosquitoes from Northeast Brazil.

Mosquitoes can transmit several pathogenic viruses to humans, but their natural viral community is also composed of a myriad of other viruses such as insect-specific viruses (ISVs) and those that infect symbiotic microorganisms. Besides a growing number of studies investigating the mosquito virome, the majority are focused on few urban species, and relatively little is known about the virome of sylvatic mosquitoes, particularly in high biodiverse biomes such as the Brazilian biomes. Here, we characterized the RNA virome of 10 sylvatic mosquito species from Atlantic forest remains at a sylvatic-urban interface in Northeast Brazil employing a metatranscriptomic approach. A total of 16 viral families were detected. The phylogenetic reconstructions of 14 viral families revealed that the majority of the sequences are putative ISVs. The phylogenetic positioning and, in most cases, the association with a high RNA-dependent RNA polymerase amino acid divergence from other known viruses suggests that the viruses characterized here represent at least 34 new viral species. Therefore, the sylvatic mosquito viral community is predominantly composed of highly divergent viruses highlighting the limited knowledge we still have about the natural virome of mosquitoes in general. Moreover, we found that none of the viruses recovered were shared between the species investigated, and only one showed high identity to a virus detected in a mosquito sampled in Peru, South America. These findings add further in-depth understanding about the interactions and coevolution between mosquitoes and viruses in natural environments. IMPORTANCE: Mosquitoes are medically important insects as they transmit pathogenic viruses to humans and animals during blood feeding. However, their natural microbiota is also composed of a diverse set of viruses that cause no harm to the insect and other hosts, such as insect-specific viruses. In this study, we characterized the RNA virome of sylvatic mosquitoes from Northeast Brazil using unbiased metatranscriptomic sequencing and in-depth bioinformatic approaches. Our analysis revealed that these mosquitoes species harbor a diverse set of highly divergent viruses, and the majority comprises new viral species. Our findings revealed many new virus lineages characterized for the first time broadening our understanding about the natural interaction between mosquitoes and viruses. Finally, it also provided several complete genomes that warrant further assessment for mosquito and vertebrate host pathogenicity and their potential interference with pathogenic arboviruses.

Highly pathogenic avian influenza H5N1 virus infections in pinnipeds and seabirds in Uruguay: Implications for bird-mammal transmission in South America.

The highly pathogenic avian influenza viruses of clade 2.3.4.4b have caused unprecedented deaths in South American wild birds, poultry, and marine mammals. In September 2023, pinnipeds and seabirds appeared dead on the Uruguayan Atlantic coast. Sixteen influenza virus strains were characterized by real-time reverse transcription PCR and genome sequencing in samples from sea lions (Otaria flavescens), fur seals (Arctocephalus australis), and terns (Sterna hirundinacea). Phylogenetic and ancestral reconstruction analysis showed that these strains have pinnipeds most likely as the ancestral host, representing a recent introduction of clade 2.3.4.4b in Uruguay. The Uruguayan and closely related strains from Peru (sea lions) and Chile (sea lions and a human case) carry mammalian adaptative residues 591K and 701N in the viral polymerase basic protein 2 (PB2). Our findings suggest that clade 2.3.4.4b strains in South America may have spread from mammals to mammals and seabirds, revealing a new transmission route.

Molecular epidemiology of Western equine encephalitis virus in Brazil, 2023-2024.

During the ongoing western equine encephalitis virus (WEEV) outbreak in South America, we described three fatal cases in horses from Rio Grande do Sul, Brazil. We sequenced WEEV strains and identified a novel lineage causing these cases. Continued surveillance and horse immunization are needed to mitigate the WEEV burden.

Evidence of Zika Virus Reinfection by Genome Diversity and Antibody Response Analysis, Brazil.

We generated 238 Zika virus (ZIKV) genomes from 135 persons in Brazil who had samples collected over 1 year to evaluate virus persistence. Phylogenetic inference clustered the genomes together with previously reported ZIKV strains from northern Brazil, showing that ZIKV has been remained relatively stable over time. Temporal phylogenetic analysis revealed limited within-host diversity among most ZIKV-persistent infected associated samples. However, we detected unusual virus temporal diversity from >5 persons, uncovering the existence of divergent genomes within the same patient. All those patients showed an increase in neutralizing antibody levels, followed by a decline at the convalescent phase of ZIKV infection. Of interest, in 3 of those patients, titers of neutralizing antibodies increased again after 6 months of ZIKV infection, concomitantly with real-time reverse transcription PCR re-positivity, supporting ZIKV reinfection events. Altogether, our findings provide evidence for the existence of ZIKV reinfection events.

Chikungunya virus infection in the southernmost state of Brazil was characterised by self-limited transmission (2017-2019) and a larger 2021 outbreak.

BACKGROUND: Chikungunya is a mosquito-borne virus that has been causing large outbreaks in the Americas since 2014. In Brazil, Asian-Caribbean (AC) and East-Central-South-African (ECSA) genotypes have been detected and lead to large outbreaks in several Brazilian states. In Rio Grande do Sul (RS), the southernmost state of Brazil, the first cases were reported in 2016. OBJECTIVES AND METHODS: We employed genome sequencing and epidemiological investigation to characterise the Chikungunya fever (CHIKF) burden in RS between 2017-2021. FINDINGS: We detected an increasing CHIKF burden linked to travel associated introductions and communitary transmission of distinct lineages of the ECSA genotype during this period. MAIN CONCLUSIONS: Until 2020, CHIKV introductions were most travel associated and transmission was limited. Then, in 2021, the largest outbreak occurred in the state associated with the introduction of a new ECSA lineage. CHIKV outbreaks are likely to occur in the near future due to abundant competent vectors and a susceptible population, exposing more than 11 million inhabitants to an increasing infection risk.

Reemergence of Dengue Virus Serotype 3, Brazil, 2023.

We characterized 3 autochthonous dengue virus serotype 3 cases and 1 imported case from 2 states in the North and South Regions of Brazil, 15 years after Brazil's last outbreak involving this serotype. We also identified a new Asian lineage recently introduced into the Americas, raising concerns about future outbreaks.

SARS-CoV-2 intra-host diversity, antibody response, and disease severity after reinfection by the variant of concern Gamma in Brazil.

The rapid spread of the SARS-CoV-2 Variant of Concern (VOC) Gamma in Amazonas during early 2021 fueled a second large COVID-19 epidemic wave and raised concern about the potential role of reinfections. Very few cases of reinfection associated with the VOC Gamma have been reported to date, and their potential impact on clinical, immunological, and virological parameters remains largely unexplored. Here we describe 25 cases of SARS-CoV-2 reinfection in Brazil. SARS-CoV-2 genomic analysis confirmed that individuals were primo-infected with distinct viral lineages between March and December 2020 (B.1.1, B.1.1.28, B.1.1.33, B.1.195, and P.2) and reinfected with the VOC Gamma between 3 to 12 months after primo-infection. We found a similar mean cycle threshold (Ct) value and limited intra-host viral diversity in both primo-infection and reinfection samples. Sera of 14 patients tested 10-75 days after reinfection displayed detectable neutralizing antibodies (NAb) titers against SARS-CoV-2 variants that circulated before (B.1.*), during (Gamma), and after (Delta and Omicron) the second epidemic wave in Brazil. All individuals had milder or no symptoms after reinfection, and none required hospitalization. These findings demonstrate that individuals reinfected with the VOC Gamma may display relatively high RNA viral loads at the upper respiratory tract after reinfection, thus contributing to onward viral transmissions. Despite this, our study points to a low overall risk of severe Gamma reinfections, supporting that the abrupt increase in hospital admissions and deaths observed in Amazonas and other Brazilian states during the Gamma wave was mostly driven by primary infections. Our findings also indicate that most individuals analyzed developed a high anti-SARS-CoV-2 NAb response after reinfection that may provide some protection against reinfection or disease by different SARS-CoV-2 variants.

Comparative epidemic expansion of SARS-CoV-2 variants Delta and Omicron in the Brazilian State of Amazonas.

The SARS-CoV-2 variants of concern (VOCs) Delta and Omicron spread globally during mid and late 2021, respectively. In this study, we compare the dissemination dynamics of these VOCs in the Amazonas state, one of Brazil's most heavily affected regions. We sequenced the virus genome from 4128 patients collected in Amazonas between July 1st, 2021, and January 31st, 2022, and investigated the viral dynamics using a phylodynamic approach. The VOCs Delta and Omicron BA.1 displayed similar patterns of phylogeographic spread but different epidemic dynamics. The replacement of Gamma by Delta was gradual and occurred without an upsurge of COVID-19 cases, while the rise of Omicron BA.1 was extremely fast and fueled a sharp increase in cases. Thus, the dissemination dynamics and population-level impact of new SARS-CoV-2 variants introduced in the Amazonian population after mid-2021, a setting with high levels of acquired immunity, greatly vary according to their viral phenotype.

Ancient origin of Jingchuvirales derived glycoproteins integrated in arthropod genomes.

Endogenous virus elements (EVEs) are viral-derived sequences integrated into their host genomes. EVEs of the Jingchuvirales order were detected in a wide range of insect genomes covering several distantly related families. Moreover, Jingchuvirales-derived glycoproteins were recently associated by our group with the origin of a putative new retrovirus based on a glycoprotein captured by a mosquito retrotransposon. But, except for mosquitoes, there is a lack of a more detailed understanding of the endogenization mechanism, timing, and frequency per Jingchuvirales viral lineages. Here we screened Jingchuvirales glycoprotein-derived EVEs (Jg-EVEs) in eukaryotic genomes. We found six distinct endogenization events of Jg-EVEs, that belong to two out of five known Jingchuvirales families (Chuviridae and Natareviridae). For seven arthropod families bearing Jg-EVEs there is no register of bona fide circulating chuvirus infection. Hence, our results show that Jingchuvirales viruses infected or still infect these host families. Although we found abundant evidence of LTR-Gypsy retrotransposons fragments associated with the glycoprotein in Hymenoptera and other insect orders, our results show that the widespread distribution of Jingchuvirales glycoproteins in extant Arhtropods is a result of multiple ancient endogenization events and that these virus fossils are being vertically inherited in Arthropods genomes for millions of years.

The Virome of Bats Inhabiting Brazilian Biomes: Knowledge Gaps and Biases towards Zoonotic Viruses.

Bats host a large variety of viruses, including some that may infect other vertebrates and humans. Research on bat-borne viruses attracted significant attention in recent years mainly due to epizootics caused by viruses having bats as hosts. The characterization of the viral communities of bats was then prioritized, but despite increasing efforts, there are large disparities in the geographical ranges covered and the methodologies employed around the world. As a result, large gaps remain in our current understanding of bat viromes and their role in disease emergence. This is particularly true for megadiverse regions in Latin America. This review aims to summarize the current understanding about bat viruses that inhabit Brazilian biomes, one of the most bat species-rich and diverse regions of the globe. Taking into account all known bat-associated viral families studied in Brazilian biomes, we found that almost half of all bat species (86/181 species) were not investigated for viruses at all. Moreover, only a small fraction of viral lineages or families have been studied more in depth, usually employing targeted methods with limited power to characterize a broad virus diversity. Additionally, these studies relied on limited spatiotemporal sampling and small sample sizes. Therefore, our current understanding of bat viral communities in the Brazilian biomes is limited and biased at different levels, limiting zoonotic risk assessments of bat-borne viruses. Considering these limitations, we propose strategies to bridge the existing gaps in the near future. IMPORTANCE Bat-borne viruses have attracted much attention due to zoonotic outbreaks with large consequences to humans. Because of that, virus characterization in bats has been prioritized in tropical regions of the globe. However, bat-virus research in Latin America and particularly in Brazil, which are among the most bat species-rich regions of the world, are highly biased toward zoonotic viruses and known bat reservoir species. These results have direct implication for virus studies in general but also for new zoonotic virus and spillover events characterization. The limited knowledge we currently have about the virome of Brazilian bats drastically limits any broad assessment of zoonotic viruses they carry and calls for coordinated and large-scale studies to fill this crucial knowledge gap.

Real-life evaluation of a rapid antigen test (DPP SARS-CoV-2 Antigen) for COVID-19 diagnosis of primary healthcare patients, in the context of the Omicron-dominant wave in Brazil.

OBJECTIVES: We aimed to investigate the real-life performance of the rapid antigen test in the context of a primary healthcare setting, including symptomatic and asymptomatic individuals that sought diagnosis during an Omicron infection wave. METHODS: We prospectively accessed the performance of the DPP SARS-CoV-2 Antigen test in the context of an Omicron-dominant real-life setting. We evaluated 347 unselected individuals (all-comers) from a public testing centre in Brazil, performing the rapid antigen test diagnosis at point-of-care with fresh samples. The combinatory result from two distinct real-time quantitative PCR (RT-qPCR) methods was employed as a reference and 13 samples with discordant PCR results were excluded. RESULTS: The assessment of the rapid test in 67 PCR-positive and 265 negative samples revealed an overall sensitivity of 80.5% (CI 95% = 69.1%-89.2%), specificity of 99.2% (CI 95% = 97.3%-99.1%) and positive/negative predictive values higher than 95%. However, we observed that the sensitivity was dependent on the viral load (sensitivity in Ct < 31 = 93.7%, CI = 82.8%-98.7%; Ct > 31 = 47.4%, CI = 24.4%-71.1%). The positive samples evaluated in the study were Omicron (BA.1/BA.1.1) by whole-genome sequencing (n = 40) and multiplex RT-qPCR (n = 17). CONCLUSIONS: Altogether, the data obtained from a real-life prospective cohort supports that the rapid antigen test sensitivity for Omicron remains high and underscores the reliability of the test for COVID-19 diagnosis in settings with high disease prevalence and limited PCR testing capability.

Multiple introductions and country-wide spread of DENV-2 genotype II (Cosmopolitan) in Brazil.

Dengue virus serotype 2, genotype Cosmopolitan (DENV-2-GII), is one of the most widespread DENV strains globally. In the USA, DENV-2 epidemics have been dominated by DENV-2 genotype Asian-American (DENV-2-GIII), and the first cases of DENV-2-GII were only described in 2019, in Peru, and in 2021 in Brazil. To gain new information about the circulation of DENV-2-GII in Brazil, we sequenced 237 DENV-2 confirmed cases sampled between March 2021 and March 2023 and revealed that DENV-2-GII is already present in all geographic regions of Brazil. The phylogeographic analysis inferred that DENV-2-GII was introduced at least four times in Brazil, between May 2020 and August 2022, generating multiple clades that spread throughout the country with different success. Despite multiple introductions of DENV-2-GII, analysis of the country-wide laboratory surveillance data showed that the Brazilian dengue epidemic in 2022 was dominated by DENV-1 in most states. We hypothesize that massive circulation of DENV-2-GIII in previous years in Brazil might have created a population immune barrier against symptomatic homotypic reinfections by DENV-2-GII, leading to sustained cryptic circulation in asymptomatic cases and localized outbreaks of this new genotype. In summary, our study stresses the importance of arboviral genomic surveillance to close monitoring and better understanding the potential impact of DENV-2-GII in the coming years.

Chikungunya virus infection in the southernmost state of Brazil was characterised by self-limited transmission (2017-2019) and a larger 2021 outbreak

BACKGROUND Chikungunya is a mosquito-borne virus that has been causing large outbreaks in the Americas since 2014. In Brazil, Asian-Caribbean (AC) and East-Central-South-African (ECSA) genotypes have been detected and lead to large outbreaks in several Brazilian states. In Rio Grande do Sul (RS), the southernmost state of Brazil, the first cases were reported in 2016. OBJECTIVES AND METHODS We employed genome sequencing and epidemiological investigation to characterise the Chikungunya fever (CHIKF) burden in RS between 2017-2021. FINDINGS We detected an increasing CHIKF burden linked to travel associated introductions and communitary transmission of distinct lineages of the ECSA genotype during this period. MAIN CONCLUSIONS Until 2020, CHIKV introductions were most travel associated and transmission was limited. Then, in 2021, the largest outbreak occurred in the state associated with the introduction of a new ECSA lineage. CHIKV outbreaks are likely to occur in the near future due to abundant competent vectors and a susceptible population, exposing more than 11 million inhabitants to an increasing infection risk.

Molecular characterization of a new SARS-CoV-2 recombinant cluster XAG identified in Brazil.

Recombination events have been described in the Coronaviridae family. Since the beginning of the SARS-CoV-2 pandemic, a variable degree of selection pressure has acted upon the virus, generating new strains with increased fitness in terms of viral transmission and antibody scape. Most of the SC2 variants of concern (VOC) detected so far carry a combination of key amino acid changes and indels. Recombination may also reshuffle existing genetic profiles of distinct strains, potentially giving origin to recombinant strains with altered phenotypes. However, co-infection and recombination events are challenging to detect and require in-depth curation of assembled genomes and sequencing reds. Here, we present the molecular characterization of a new SARS-CoV-2 recombinant between BA.1.1 and BA.2.23 Omicron lineages identified in Brazil. We characterized four mutations that had not been previously described in any of the recombinants already identified worldwide and described the likely breaking points. Moreover, through phylogenetic analysis, we showed that the newly named XAG lineage groups in a highly supported monophyletic clade confirmed its common evolutionary history from parental Omicron lineages and other recombinants already described. These observations were only possible thanks to the joint effort of bioinformatics tools auxiliary in genomic surveillance and the manual curation of experienced personnel, demonstrating the importance of genetic, and bioinformatic knowledge in genomics.

Possible Occupational Infection of Healthcare Workers with Monkeypox Virus, Brazil.

We evaluated epidemiologic and molecular characteristics of monkeypox virus (MPXV) infections sampled from 2 healthcare nurses. Five days after collecting samples from an infected patient, the nurses showed typical MPXV manifestations; quantitative PCR and whole-genome sequencing confirmed MPXV infection, most likely transmitted through contact with fomites.