Detection of a SARS-CoV-2 variant of concern in South Africa.

Continued uncontrolled transmission of SARS-CoV-2 in many parts of the world is creating conditions for substantial evolutionary changes to the virus1,2. Here we describe a newly arisen lineage of SARS-CoV-2 (designated 501Y.V2; also known as B.1.351 or 20H) that is defined by eight mutations in the spike protein, including three substitutions (K417N, E484K and N501Y) at residues in its receptor-binding domain that may have functional importance3-5. This lineage was identified in South Africa after the first wave of the epidemic in a severely affected metropolitan area (Nelson Mandela Bay) that is located on the coast of the Eastern Cape province. This lineage spread rapidly, and became dominant in Eastern Cape, Western Cape and KwaZulu-Natal provinces within weeks. Although the full import of the mutations is yet to be determined, the genomic data-which show rapid expansion and displacement of other lineages in several regions-suggest that this lineage is associated with a selection advantage that most plausibly results from increased transmissibility or immune escape6-8.

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.

SARS-CoV-2 Spillback to Wild Coatis in Sylvatic-Urban Hotspot, Brazil.

We tested coatis (Nasua nasua) living in an urban park near a densely populated area of Brazil and found natural SARS-CoV-2 Zeta variant infections by using quantitative reverse transcription PCR, genomic sequencing, and serologic surveillance. We recommend a One Health strategy to improve surveillance of and response to COVID-19.

Molecular identification of the emerging Human Gemykibivirus-2 (HuGkV-2) among Brazilian blood donors.

Human gemykibivirus-2 (HuGkV-2) belonging to the Gemykibivirus genus (Genomoviridae family) is an emerging DNA virus which has been described as a component of the virome of a wide variety of samples including clinical ones. So far, the HuGkV-2 DNA prevalence in the human population as well as its clinical impact are completely unknown. The objective of this study was to investigate the HuGkV-2 DNA prevalence among Brazilian healthy blood donors from three different geographic regions. A total of 450 blood samples were screened for HuGkV-2 DNA (150 samples were from the Brazilian Amazon, 150 from Midwest Brazil and 150 from South Brazil). The overall HuGkV-2 DNA prevalence was 7.8 %. Considering the examined regions, the highest prevalence was observed in the Brazilian Amazon (city of Macapa, state of Amapa), 15.3 %, followed by the Midwest Brazil (city of Brasilia, Federal District) (6.0 %) and South Brazil (city of Santa Maria, Rio Grande do Sul State) (2.0 %). This study gives preliminary insights on the molecular prevalence of HuGkV-2 DNA among Brazilian blood donors, highlighting that the highest HuGkV-2 prevalence was recorded in the Brazilian Amazon. However, more studies regarding the prevalence, transmission routes and any possible clinical effects appear to be crucial in order to understand the impact of this emerging viral agent.

A traveling SARS-CoV-2 laboratory as part of a pandemic response among vulnerable Brazilian populations.

BACKGROUND: Brazil has been dramatically hit by the SARS-CoV-2 pandemic and is a world leader in COVID-19 morbidity and mortality. Additionally, the largest country of Latin America has been a continuous source of SARS-CoV-2 variants and shows extraordinary variability of the pandemic strains probably related to the country´s outstanding position as a Latin American economical and transportation hub. Not all regions of the country show sufficient infrastructure for SARS-CoV-2 diagnosis and genotyping which can negatively impact the pandemic response. METHODS: Due to this reason and to disburden the diagnostic system of the inner São Paulo State, the Butantan Institute established the Mobile Laboratory (in Portuguese: LabMovel) for SARS-CoV-2 testing which started a trip of the most important "hotspots" of the most populous Brazilian region. The LabMovel initiated in two important cities of the State: Aparecida do Norte (an important religious center) and the Baixada Santista region which incorporates the port of Santos, the busiest in Latin America. The LabMovel was fully equipped with an automatized system for SARS-CoV-2 diagnosis and sequencing/genotyping. It also integrated the laboratory systems for patient records and results divulgation including in the Federal Brazilian Healthcare System. RESULTS: Currently,16,678 samples were tested, among them 1,217 from Aparecida and 4,564 from Baixada Santista. We tracked the delta introductio in the tested regions with its high diversification. The established mobile SARS-CoV-2 laboratory had a major impact on the Public Health System of the included cities including timely delivery of the results to the healthcare agents and the Federal Healthcare system, evaluation of the vaccination status of the positive individuals in the background of exponential vaccination process in Brazil and scientific and technological divulgation of the fieldwork to the most vulnerable populations. CONCLUSIONS: The SARS-CoV-2 pandemic has demonstrated worldwide the importance of science to fight against this viral agent and the LabMovel shows that it is possible to integrate researchers, clinicians, healthcare workers and patients to take rapid actions that can in fact mitigate this and other epidemiological situations.

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.

Long-term unsustainable patterns of development rather than recent deforestation caused the emergence of Orthocoronavirinae species.

We investigated whether a set of phylogeographical tracked emergent events of Orthocoronavirinae were related to developed, urban and polluted environments worldwide. We explored coronavirus records in response to climate (rainfall parameters), population density, CO2 emission, Human Developmental Index (HDI) and deforestation. We contrasted environmental characteristics from regions with spillovers or encounters of wild Orthocoronavirinae against adjacent areas having best-preserved conditions. We used all complete sequenced CoVs genomes deposited in NCBI and GISAID databases until January 2021. Except for Deltacoronavirus, concentrated in Hong Kong and in birds, the other three genera were scattered all over the planet, beyond the original distribution of the subfamily, and found in humans, mammals, fishes and birds, wild or domestic. Spillovers and presence in wild animals were only reported in developed/densely populated places. We found significantly more occurrences reported in places with higher HDI, CO2 emission, or population density, along with more rainfall and more accentuated seasonality. Orthocoronavirinae occurred in areas with significantly higher human populations, CO2 emissions and deforestation rates than in adjacent locations. Intermediately disturbed ecosystems seemed more vulnerable for Orthocoronavirinae emergence than forested regions in frontiers of deforestation. Sadly, people experiencing poverty in an intensely consumerist society are the most vulnerable.

SARS-COV-2 genomic monitoring in the state of São Paulo unveils two emerging AY.43 sublineages.

Delta VOC is highly diverse with more than 120 sublineages already described as of November 30, 2021. In this study, through active monitoring of circulating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants in the state of São Paulo, southeast Brazil, we identified two emerging sublineages from the ancestral AY.43 strain which were classified as AY.43.1 and AY.43.2. These sublineages were defined by the following characteristic nonsynonymous mutations ORF1ab:A4133V and ORF3a:T14I for the AY.43.1 and ORF1ab:G1155C for the AY.43.2 and our analysis reveals that they might have a likely-Brazilian origin. Much is still unknown regarding their dissemination in the state of São Paulo and Brazil as well as their potential impact on the ongoing vaccination process. However, the results obtained in this study reinforce the importance of genomic surveillance activity for timely identification of emerging SARS-CoV-2 variants which can impact the ongoing SARS-CoV-2 vaccination and public health policies.

Epidemiologic History and Genetic Diversity Origins of Chikungunya and Dengue Viruses, Paraguay.

Paraguay has been severely affected by emergent Zika and chikungunya viruses, and dengue virus is endemic. To learn more about the origins of genetic diversity and epidemiologic history of these viruses in Paraguay, we deployed portable sequencing technologies to strengthen genomic surveillance and determine the evolutionary and epidemic history of arthropod-borne viruses (arboviruses). Samples stored at the Paraguay National Central Laboratory were sequenced and subjected to phylogenetic analysis. Among 33 virus genomes generated, we identified 2 genotypes of chikungunya and 2 serotypes of dengue virus that circulated in Paraguay during 2014-2018; the main source of these virus lineages was estimated to be Brazil. The evolutionary history inferred by our analyses precisely matched the available travel history of the patients. The genomic surveillance approach used was valuable for describing the epidemiologic history of arboviruses and can be used to determine the origins and evolution of future arbovirus outbreaks.

Genome Detective Coronavirus Typing Tool for rapid identification and characterization of novel coronavirus genomes.

SUMMARY: Genome detective is a web-based, user-friendly software application to quickly and accurately assemble all known virus genomes from next-generation sequencing datasets. This application allows the identification of phylogenetic clusters and genotypes from assembled genomes in FASTA format. Since its release in 2019, we have produced a number of typing tools for emergent viruses that have caused large outbreaks, such as Zika and Yellow Fever Virus in Brazil. Here, we present the Genome Detective Coronavirus Typing Tool that can accurately identify the novel severe acute respiratory syndrome (SARS)-related coronavirus (SARS-CoV-2) sequences isolated in China and around the world. The tool can accept up to 2000 sequences per submission and the analysis of a new whole-genome sequence will take approximately 1 min. The tool has been tested and validated with hundreds of whole genomes from 10 coronavirus species, and correctly classified all of the SARS-related coronavirus (SARSr-CoV) and all of the available public data for SARS-CoV-2. The tool also allows tracking of new viral mutations as the outbreak expands globally, which may help to accelerate the development of novel diagnostics, drugs and vaccines to stop the COVID-19 disease. AVAILABILITY AND IMPLEMENTATION: https://www.genomedetective.com/app/typingtool/cov. CONTACT: koen@emweb.be or deoliveira@ukzn.ac.za. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Newborns With Zika Virus-Associated Microcephaly Exhibit Marked Systemic Inflammatory Imbalance.

BACKGROUND: Zika virus (ZIKV) is an emergent flavivirus initially considered a benign and self-limited exanthematic illness. In 2015, a new epidemic emerged in northeastern of Brazil with increased incidence of a previously rare clinical outcome, microcephaly, in newborns from mothers who were infected during pregnancy. Little is known about the immunopathogenesis of ZIKV-associated microcephaly. Understanding the inflammatory profile and degree of inflammation of persons affected with such condition is an important step towards development of innovative therapeutic strategies. METHODS: A case-control study compared plasma levels of several inflammatory biomarkers from newborns with ZIKV microcephaly, asymptomatic ZKV infection, or uninfected controls. Plasma biomarkers were assessed using Luminex. A series of multidimensional analysis was performed to characterize the systemic immune activation profile of the clinical groups. RESULTS: We identified an inflammatory signature associated with ZIKV microcephaly that suggested an increased inflammation. Network analysis suggested that ZIKV microcephaly is associated with imbalanced immune activation and inflammation. The cephalic perimeter was inversely proportional with the degree of inflammatory perturbation. Furthermore, a combination of plasma inflammatory biomarkers could discriminate ZIKV with microcephaly from those with ZIKV without microcephaly or uninfected neonates. CONCLUSIONS: An intense inflammatory imbalance that is proportional to the disease severity hallmarks ZIKV microcephaly.

Yellow Fever Virus Reemergence and Spread in Southeast Brazil, 2016-2019.

The recent reemergence of yellow fever virus (YFV) in Brazil has raised serious concerns due to the rapid dissemination of the virus in the southeastern region. To better understand YFV genetic diversity and dynamics during the recent outbreak in southeastern Brazil, we generated 18 complete and nearly complete genomes from the peak of the epidemic curve from nonhuman primates (NHPs) and human infected cases across the Espírito Santo and Rio de Janeiro states. Genomic sequencing of 18 YFV genomes revealed the estimated timing, source, and likely routes of yellow fever virus transmission and dispersion during one of the largest outbreaks ever registered in Brazil. We showed that during the recent epidemic, YFV was reintroduced from Minas Gerais to the Espírito Santo and Rio de Janeiro states multiple times between 2016 and 2019. The analysis of data from portable sequencing could identify the corridor of spread of YFV. These findings reinforce the idea that continued genomic surveillance strategies can provide information on virus genetic diversity and transmission dynamics that might assist in understanding arbovirus epidemics.IMPORTANCE Arbovirus infections in Brazil, including yellow fever, dengue, zika, and chikungunya, result in considerable morbidity and mortality and are pressing public health concerns. However, our understanding of these outbreaks is hampered by the limited availability of genomic data. In this study, we investigated the genetic diversity and spatial distribution of YFV during the current outbreak by analyzing genomic data from areas in southeastern Brazil not covered by other previous studies. To gain insights into the routes of YFV introduction and dispersion, we tracked the virus by sequencing YFV genomes sampled from nonhuman primates and infected patients from the southeastern region. Our study provides an understanding of how YFV initiates transmission in new Brazilian regions and illustrates that genomics in the field can augment traditional approaches to infectious disease surveillance and control.

Genome Detective: an automated system for virus identification from high-throughput sequencing data.

SUMMARY: Genome Detective is an easy to use web-based software application that assembles the genomes of viruses quickly and accurately. The application uses a novel alignment method that constructs genomes by reference-based linking of de novo contigs by combining amino-acids and nucleotide scores. The software was optimized using synthetic datasets to represent the great diversity of virus genomes. The application was then validated with next generation sequencing data of hundreds of viruses. User time is minimal and it is limited to the time required to upload the data. AVAILABILITY AND IMPLEMENTATION: Available online: http://www.genomedetective.com/app/typingtool/virus/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genomic detection of a virus lineage replacement event of dengue virus serotype 2 in Brazil, 2019.

BACKGROUND Despite efforts to mitigate the impact of dengue virus (DENV) epidemics, the virus remains a public health problem in tropical and subtropical regions around the world. Most DENV cases in the Americas between January and July 2019 were reported in Brazil. São Paulo State in the southeast of Brazil has reported nearly half of all DENV infections in the country. OBJECTIVES To understand the origin and dynamics of the 2019 DENV outbreak. METHODS Here using portable nanopore sequencing we generated20 new DENV genome sequences from viremic patients with suspected dengue infection residing in two of the most-affected municipalities of São Paulo State, Araraquara and São José do Rio Preto. We conducted a comprehensive phylogenetic analysis with 1,630 global DENV strains to better understand the evolutionary history of the DENV lineages that currently circulate in the region. FINDINGS The new outbreak strains were classified as DENV2 genotype III (American/Asian genotype). Our analysis shows that the 2019 outbreak is the result of a novel DENV lineage that was recently introduced to Brazil from the Caribbean region. Dating phylogeographic analysis suggests that DENV2-III BR-4 was introduced to Brazil in or around early 2014, possibly from the Caribbean region. MAIN CONCLUSIONS Our study describes the early detection of a newly introduced and rapidly-expanding DENV2 virus lineage in Brazil.

Co-dependence of HTLV-1 p12 and p8 functions in virus persistence.

HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8. Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26). As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-cell activation and recognition; we compared the CTL response elicited by CD4+ T-cells infected with the different HTLV-1 clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral persistence and spread require the combined functions of p12 and p8.

Palmitoylation and p8-mediated human T-cell leukemia virus type 1 transmission.

The orf-I gene of human T-cell leukemia type 1 (HTLV-1) encodes p8 and p12 and has a conserved cysteine at position 39. p8 and p12 form disulfide-linked dimers, and only the monomeric forms of p8 and p12 are palmitoylated. Mutation of cysteine 39 to alanine (C39A) abrogated dimerization and palmitoylation of both proteins. However, the ability of p8 to localize to the cell surface and to increase cell adhesion and viral transmission was not affected by the C39A mutation.

HTLV-1 in pregnant women from the Southern Bahia, Brazil: a neglected condition despite the high prevalence.

BACKGROUND: As the most frequent pathway of vertical transmission of HTLV-1 is breast-feeding, and considering the higher prevalence in women, it is very important to perform screening examinations for anti-HTLV-1 antibodies as part of routine prenatal care. So far, no studies of HTLV-1 seroprevalence in pregnant women in the Southern region of Bahia, Brazil, have been described. METHODS: Pregnant women were selected at the two regional reference centers for health care from Southern Bahia. A total of 2766 pregnant women attending the antenatal unit between November 2008 and May 2010 have been analyzed. An extra blood sample was drawn during their routine antenatal testing. A standardized questionnaire was applied and all positive plasma samples were tested by ELISA and were confirmed by Western Blot and PCR. Besides that, positive women were contacted and visited. The family members that were present during the visit were asked to be serologically screened to the virus. A prospective study was also carried out and newborns were followed up to two years for evaluation of vertical transmission. RESULTS: HTLV prevalence was 1.05% (CI 95%: 0.70-1.50). There was no association of HTLV-1 infection with age, education, income and ethnic differences. The association with marital status was borderline (OR = 7.99; 95% CI 1.07-59.3; p = 0.042). In addition, 43 family members of the HTLV-1 seropositive women have been analyzed and specific reactivity was observed in 32.56%, including two children from previous pregnancy. CONCLUSION: It is very important to emphasize that the lack of HTLV-1 screening in pregnant women can promote HTLV transmission especially in endemic areas. HTLV screening in this vulnerable population and the promotion of bottle-feeding for children of seropositive mothers could be important cost-effective methods to limit the vertical transmission. Besides that, our data reinforce the need to establish strategies of active surveillance in household and family contacts as important epidemiological surveillance actions for the early detection of virus infection and the prevention of transmission by sexual or and parenteral contact.