Increased interregional virus exchange and nucleotide diversity outline the expansion of chikungunya virus in Brazil.

The emergence and reemergence of mosquito-borne diseases in Brazil such as yellow fever, zika, chikungunya, and dengue have had serious impacts on public health. Concerns have been raised due to the rapid dissemination of the chikungunya virus across the country since its first detection in 2014 in Northeast Brazil. In this work, we carried out on-site training activities in genomic surveillance in partnership with the National Network of Public Health Laboratories that have led to the generation of 422 chikungunya virus genomes from 12 Brazilian states over the past two years (2021-2022), a period that has seen more than 312 thousand chikungunya fever cases reported in the country. These genomes increased the amount of available data and allowed a more comprehensive characterization of the dispersal dynamics of the chikungunya virus East-Central-South-African lineage in Brazil. Tree branching patterns revealed the emergence and expansion of two distinct subclades. Phylogeographic analysis indicated that the northeast region has been the leading hub of virus spread towards other regions. Increased frequency of C > T transitions among the new genomes suggested that host restriction factors from the immune system such as ADAR and AID/APOBEC deaminases might be driving the genetic diversity of the chikungunya virus in Brazil.

Phylogenetic Reconstructions Reveal the Circulation of a Novel Dengue Virus-1V Clade and the Persistence of a Dengue Virus-2 III Genotype in Northeast Brazil.

Dengue fever is among the most significant public health concerns in Brazil. To date, the highest number of Dengue notifications in the Americas has been reported in Brazil, with cases accounting for a total number of 3,418,796 reported cases as of mid-December 2022. Furthermore, the northeastern region of Brazil registered the second-highest incidence of Dengue fever in 2022. Due to the alarming epidemiological scenario, in this study, we used a combination of portable whole-genome sequencing, phylodynamic, and epidemiological analyses to reveal a novel DENV-1 genotype V clade and the persistence of DENV-2 genotype III in the region. We further report the presence of non-synonymous mutations associated with non-structural domains, especially the NS2A (non-structural protein 2A), as well as describe synonymous mutations in envelope and membrane proteins, distributed differently between clades. However, the absence of clinical data at the time of collection and notification, as well as the impossibility of monitoring patients in order to observe worsening or death, restricts our possibility of correlating mutational findings with possible clinical prognoses. Together, these results reinforce the crucial role of genomic surveillance to follow the evolution of circulating DENV strains and understand their spread across the region through inter-regional importation events, likely mediated by human mobility, and also the possible impacts on public health and outbreak management.

Increased interregional virus exchange and nucleotide diversity outline the expansion of the chikungunya virus ECSA lineage in Brazil.

The emergence and reemergence of mosquito-borne diseases in Brazil such as Yellow Fever, Zika, Chikungunya, and Dengue have had serious impacts on public health. Concerns have been raised due to the rapid dissemination of the chikungunya virus (CHIKV) across the country since its first detection in 2014 in Northeast Brazil. Faced with this scenario, on-site training activities in genomic surveillance carried out in partnership with the National Network of Public Health Laboratories have led to the generation of 422 CHIKV genomes from 12 Brazilian states over the past two years (2021-2022), a period that has seen more than 312 thousand chikungunya fever cases reported in the country. These new genomes increased the amount of available data and allowed a more comprehensive characterization of the dispersion dynamics of the CHIKV East-Central-South-African (ECSA) lineage in Brazil. Tree branching patterns revealed the emergence and expansion of two distinct subclades. Phylogeographic analysis indicated that the northeast region has been the leading hub of virus spread towards other regions. Increased frequency of C>T transitions among the new genomes suggested that host restriction factors from the immune system such as ADAR and AID/APOBEC deaminases might be driving CHIKV ECSA lineage genetic diversity in Brazil.

Global SARS-CoV-2 genomic surveillance: What we have learned (so far).

The COVID-19 pandemic has brought significant challenges for genomic surveillance strategies in public health systems worldwide. During the past thirty-four months, many countries faced several epidemic waves of SARS-CoV-2 infections, driven mainly by the emergence and spread of novel variants. In that line, genomic surveillance has been a crucial toolkit to study the real-time SARS-CoV-2 evolution, for the assessment and optimization of novel diagnostic assays, and to improve the efficacy of existing vaccines. During the pandemic, the identification of emerging lineages carrying lineage-specific mutations (particularly those in the Receptor Binding domain) showed how these mutations might significantly impact viral transmissibility, protection from reinfection and vaccination. So far, an unprecedented number of SARS-CoV-2 viral genomes has been released in public databases (i.e., GISAID, and NCBI), achieving 14 million genome sequences available as of early-November 2022. In the present review, we summarise the global landscape of SARS-CoV-2 during the first thirty-four months of viral circulation and evolution. It demonstrates the urgency and importance of sustained investment in genomic surveillance strategies to timely identify the emergence of any potential viral pathogen or associated variants, which in turn is key to epidemic and pandemic preparedness.

Correction: Short Report: Early genomic detection of SARS-CoV-2 P.1 variant in Northeast Brazil.

[This corrects the article DOI: 10.1371/journal.pntd.0009591.].

Machine learning models exploring characteristic single-nucleotide signatures in yellow fever virus.

Yellow fever virus (YFV) is the agent of the most severe mosquito-borne disease in the tropics. Recently, Brazil suffered major YFV outbreaks with a high fatality rate affecting areas where the virus has not been reported for decades, consisting of urban areas where a large number of unvaccinated people live. We developed a machine learning framework combining three different algorithms (XGBoost, random forest and regularized logistic regression) to analyze YFV genomic sequences. This method was applied to 56 YFV sequences from human infections and 27 from non-human primate (NHPs) infections to investigate the presence of genetic signatures possibly related to disease severity (in human related sequences) and differences in PCR cycle threshold (Ct) values (in NHP related sequences). Our analyses reveal four non-synonymous single nucleotide variations (SNVs) on sequences from human infections, in proteins NS3 (E614D), NS4a (I69V), NS5 (R727G, V643A) and six non-synonymous SNVs on NHP sequences, in proteins E (L385F), NS1 (A171V), NS3 (I184V) and NS5 (N11S, I374V, E641D). We performed comparative protein structural analysis on these SNVs, describing possible impacts on protein function. Despite the fact that the dataset is limited in size and that this study does not consider virus-host interactions, our work highlights the use of machine learning as a versatile and fast initial approach to genomic data exploration.

Genomic epidemiology of the SARS-CoV-2 epidemic in Brazil.

The high numbers of COVID-19 cases and deaths in Brazil have made Latin America an epicentre of the pandemic. SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, but important gaps remain in our understanding of virus transmission dynamics at a national scale. We use 17,135 near-complete genomes sampled from 27 Brazilian states and bordering country Paraguay. From March to November 2020, we detected co-circulation of multiple viral lineages that were linked to multiple importations (predominantly from Europe). After November 2020, we detected large, local transmission clusters within the country. In the absence of effective restriction measures, the epidemic progressed, and in January 2021 there was emergence and onward spread, both within and abroad, of variants of concern and variants under monitoring, including Gamma (P.1) and Zeta (P.2). We also characterized a genomic overview of the epidemic in Paraguay and detected evidence of importation of SARS-CoV-2 ancestor lineages and variants of concern from Brazil. Our findings show that genomic surveillance in Brazil enabled assessment of the real-time spread of emerging SARS-CoV-2 variants.

Retrospective Investigation in Horses with Encephalitis Reveals Unnoticed Circulation of West Nile Virus in Brazil.

During these past years, several studies have provided serological evidence regarding the circulation of West Nile virus (WNV) in Brazil. Despite some reports, much is still unknown regarding the genomic diversity and transmission dynamics of this virus in the country. Recently, genomic monitoring activities in horses revealed the circulation of WNV in several Brazilian regions. These findings on the paucity of genomic data reinforce the need for prompt investigation of WNV infection in horses, which may precede human cases of encephalitis in Brazil. Thus, in this study, we retrospectively screened 54 suspicious WNV samples collected between 2017 and 2020 from the spinal cord and brain of horses with encephalitis and generated three new WNV genomes from the Ceará and Bahia states, located in the northeastern region of Brazil. The Bayesian reconstruction revealed that at least two independent introduction events occurred in Brazil. The first introduction event appears to be likely related to the North American outbreak, and was estimated to have occurred in March 2013.The second introduction event appears to have occurred in September 2017 and appears to be likely related to the South American outbreak. Together, our results reinforce the importance of increasing the priority of WNV genomic monitoring in equines with encephalitis in order to track the dispersion of this emerging pathogen through the country.

Dynamics and Determinants of SARS-CoV-2 RT-PCR Testing on Symptomatic Individuals Attending Healthcare Centers during 2020 in Bahia, Brazil.

RT-PCR testing data provides opportunities to explore regional and individual determinants of test positivity and surveillance infrastructure. Using Generalized Additive Models, we explored 222,515 tests of a random sample of individuals with COVID-19 compatible symptoms in the Brazilian state of Bahia during 2020. We found that age and male gender were the most significant determinants of test positivity. There was evidence of an unequal impact among socio-demographic strata, with higher positivity among those living in areas with low education levels during the first epidemic wave, followed by those living in areas with higher education levels in the second wave. Our estimated probability of testing positive after symptom onset corroborates previous reports that the probability decreases with time, more than halving by about two weeks and converging to zero by three weeks. Test positivity rates generally followed state-level reported cases, and while a single laboratory performed ~90% of tests covering ~99% of the state's area, test turn-around time generally remained below four days. This testing effort is a testimony to the Bahian surveillance capacity during public health emergencies, as previously witnessed during the recent Zika and Yellow Fever outbreaks.

Emergence of Dengue Virus Serotype 2 Cosmopolitan Genotype, Brazil.

We used nanopore sequencing and phylogenetic analyses to identify a cosmopolitan genotype of dengue virus serotype 2 that was isolated from a 56-year-old male patient from the state of Goiás in Brazil. The emergence of a cosmopolitan genotype in Brazil will require risk assessment and surveillance to reduce epidemic potential.

SARS-CoV-2 epidemic in Brazil: how the displacement of variants has driven distinct epidemic waves.

Brazil ranks as third in terms of total number of reported SARS-CoV-2 cases globally. The COVID-19 epidemic in Brazil was characterised by the co-circulation of multiple variants as a consequence of multiple independent introduction events occurring through time. Here, we describe the SARS-CoV-2 variants that are currently circulating and co-circulating in the country, with the aim to highlight which variants have driven the different epidemic waves. For this purpose, we retrieved metadata information of Coronavirus sequences collected in Brazil and available at the GISAID database. SARS-CoV-2 lineages have been identified along with eleven variants, labelled as VOCs (Alpha, Gamma, Beta, Delta and Omicron) VOIs (Lambda and Mu) VUMs (B.1.1.318) and FMVs (Zeta, Eta and B.1.1.519). Here we show that, in the Brazilian context, after 24 months of sustained transmission and evolution of SARS-CoV-2, local variants (among them the B.1.1.28 and B.1.1.33) were displaced by recently introduced VOCs firstly with the Gamma, followed by Delta and more recently Omicron. The rapid spread of some of those VOCs (such as Gamma and Omicron) was also mirror by a large increase in the number of cases and deaths in the country. This in turn reinforces that, due to the emergence of variants that appear to induce a substantial evasion against neutralizing antibody response, it is important to strengthen genomic effort within the country and how vaccination still remains a critical process to protect the vulnerable population, still at risk of infection and death.

Genomic epidemiology reveals the impact of national and international restrictions measures on the SARS-CoV-2 epidemic in Brazil.

Brazil has experienced some of the highest numbers of COVID-19 cases and deaths globally and from May 2021 made Latin America a pandemic epicenter. Although SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, important gaps remain in our understanding of virus transmission dynamics at the national scale. Here, we describe the genomic epidemiology of SARS-CoV-2 using near-full genomes sampled from 27 Brazilian states and a bordering country - Paraguay. We show that the early stage of the pandemic in Brazil was characterised by the co-circulation of multiple viral lineages, linked to multiple importations predominantly from Europe, and subsequently characterized by large local transmission clusters. As the epidemic progressed under an absence of effective restriction measures, there was a local emergence and onward international spread of Variants of Concern (VOC) and Variants Under Monitoring (VUM), including Gamma (P.1) and Zeta (P.2). In addition, we provide a preliminary genomic overview of the epidemic in Paraguay, showing evidence of importation from Brazil. These data reinforce the usefulness and need for the implementation of widespread genomic surveillance in South America as a toolkit for pandemic monitoring that provides a means to follow the real-time spread of emerging SARS-CoV-2 variants with possible implications for public health and immunization strategies.

Short Report: Early genomic detection of SARS-CoV-2 P.1 variant in Northeast Brazil.

Tracking the spread of SARS-CoV-2 variants of concern is crucial to inform public health efforts and control the ongoing pandemic. Here, we report genetic evidence for circulation of the P.1 variant in Northeast Brazil. We advocate for increased active surveillance to ensure adequate control of this variant throughout the country.

Betacoronaviruses genome analysis reveals evolution toward specific codons usage: Implications for SARS-CoV-2 mitigation strategies.

Since the start of the coronavirus disease 2019 (COVID-19) pandemic, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly widespread worldwide becoming one of the major global public health issues of the last centuries. Currently, COVID-19 vaccine rollouts are finally upon us carrying the hope of herd immunity once a sufficient proportion of the population has been vaccinated or infected, as a new horizon. However, the emergence of SARS-CoV-2 variants brought concerns since, as the virus is exposed to environmental selection pressures, it can mutate and evolve, generating variants that may possess enhanced virulence. Codon usage analysis is a strategy to elucidate the evolutionary pressure of the viral genome suffered by different hosts, as possible cause of the emergence of new variants. Therefore, to get a better picture of the SARS-CoV-2 codon bias, we first identified the relative codon usage rate of all Betacoronaviruses lineages. Subsequently, we correlated putative cognate transfer ribonucleic acid (tRNAs) to reveal how those viruses adapt to hosts in relation to their preferred codon usage. Our analysis revealed seven preferred codons located in three different open reading frame which appear preferentially used by SARS-CoV-2. In addition, the tRNA adaptation analysis indicates a wide strategy of competition between the virus and mammalian as principal hosts highlighting the importance to reinforce the genomic monitoring to prompt identify any potential adaptation of the virus into new potential hosts which appear to be crucial to prevent and mitigate the pandemic.

Genomic surveillance activities unveil the introduction of the SARS-CoV-2 B.1.525 variant of interest in Brazil: Case report.

The appearance of new variants of SARS-CoV-2 has recently challenged public health authorities with respect to tracking transmission and mitigating the impact in the evolving pandemic across countries. B.1.525 is considered a variant under investigation since it carries specific genetic signatures present in P.1, B.1.1.7, and B.1.351. Here we report genomic evidence of the first likely imported case of the SARS-CoV-2 B.1.525 variant, isolated in a traveler returning from Nigeria.

Field and classroom initiatives for portable sequence-based monitoring of dengue virus in Brazil.

Brazil experienced a large dengue virus (DENV) epidemic in 2019, highlighting a continuous struggle with effective control and public health preparedness. Using Oxford Nanopore sequencing, we led field and classroom initiatives for the monitoring of DENV in Brazil, generating 227 novel genome sequences of DENV1-2 from 85 municipalities (2015-2019). This equated to an over 50% increase in the number of DENV genomes from Brazil available in public databases. Using both phylogenetic and epidemiological models we retrospectively reconstructed the recent transmission history of DENV1-2. Phylogenetic analysis revealed complex patterns of transmission, with both lineage co-circulation and replacement. We identified two lineages within the DENV2 BR-4 clade, for which we estimated the effective reproduction number and pattern of seasonality. Overall, the surveillance outputs and training initiative described here serve as a proof-of-concept for the utility of real-time portable sequencing for research and local capacity building in the genomic surveillance of emerging viruses.

Chikungunya virus ECSA lineage reintroduction in the northeasternmost region of Brazil.

The Northeast region of Brazil registered the second-highest incidence proportion of Chikungunya fever in 2019. In that year, an outbreak consisting of patients presenting with febrile disease associated with joint pain was reported by the public primary health care service in the city of Natal, in the state of Rio Grande do Norte, in March 2019. At first, the aetiological agent of the disease was undetermined. Since much is still unknown about chikungunya virus' (CHIKV) genomic diversity and evolutionary history in this northeasternmost state, we used a combination of portable whole-genome sequencing, molecular clock, and epidemiological analyses that revealed the reintroduction of the CHIKV East-Central-South-African (ECSA) lineage into Rio Grande do Norte. We estimated that the CHIKV ECSA lineage was first introduced into Rio Grande do Norte in early June 2014, while the 2019 outbreak clade diverged around April 2018, during a period of increased Chikungunya incidence in the Southeast region, which might have acted as a source of virus dispersion towards the Northeast region. Together, these results confirm that the ECSA lineage continues to spread across the country through interregional importation events, likely mediated by human mobility.

Multi-epitope based vaccine against yellow fever virus applying immunoinformatics approaches.

Yellow fever disease is considered a re-emerging major health issue which has caused recent outbreaks with a high number of deaths. Tropical countries, mainly African and South American, are the most affected by Yellow fever outbreaks. Despite the availability of an attenuated vaccine, its use is limited for some groups such as pregnant and nursing women, immunocompromised and immunosuppressed patients, elderly people >65 years, infants <6 months and patients with biological disorders like thymus disorders. In order to achieve new preventive measures, we applied immunoinformatics approaches to develop a multi-epitope-based subunit vaccine for Yellow fever virus. Different epitopes, related to humoral and cell-mediated immunity, were predicted for complete polyproteins of two Yellow fever strains (Asibi and 17 D vaccine). Those epitopes common for both strains were mapped into a set of 137 sequences of Yellow fever virus, including 77 sequences from a recent outbreak at the state of Minas Gerais, southeast Brazil. Therefore, the present work uses robust bioinformatics approaches for the identification of a multi-epitope vaccine against the Yellow fever virus. Our results indicate that the identified multi-epitope vaccine might stimulate humoral and cellular immune responses and could be a potential vaccine candidate against Yellow fever virus infection. Hence, it should be subjected to further experimental validations. Communicated by Ramaswamy H. Sarma.

Persistence of chikungunya ECSA genotype and local outbreak in an upper medium class neighborhood in Northeast Brazil.

The chikungunya East/Central/South/Africa virus lineage (CHIKV-ECSA) was first detected in Brazil in the municipality of Feira de Santana (FS) by mid 2014. Following that, a large number of CHIKV cases have been notified in FS, which is the second-most populous city in Bahia state, northeastern Brazil, and plays an important role on the spread to other Brazilian states due to climate conditions and the abundance of competent vectors. To better understand CHIKV dynamics in Bahia state, we generated 5 complete genome sequences from a local outbreak raised in Serraria Brasil, a neighbourhood in FS, by next-generation sequencing using Illumina approach. Phylogenetic reconstructions revealed that the new FS genomes belongs to the ECSA genotype and falls within a single strongly supported monophyletic clade that includes other older CHIKV sequences from the same location, suggesting the persistence of the virus during distinct epidemic seasons. We also performed minor variants analysis and found a small number of SNPs per sample (b_29L and e_45SR = 16 SNPs, c_29SR = 29 and d_45PL and f_45FL = 21 SNPs). Out of the 93 SNPs found, 71 are synonymous, 21 are non-synonymous and one generated a stop codon. Although those mutations are not related to the increase of virus replication and/or infectivity, some SNPs were found in non-structural proteins which may have an effect on viral evasion from the mammal immunological system. These findings reinforce the needing of further studies on those variants and of continued genomic surveillance strategies to track viral adaptations and to monitor CHIKV epidemics for improved public health control.