Yellow fever disease severity and endothelial dysfunction are associated with elevated serum levels of viral NS1 protein and syndecan-1.

BACKGROUND: Yellow fever virus (YFV) infections are a major global disease concern with high mortality in humans, and as such it is critical to identify clinical correlates of disease severity. While nonstructural protein 1 (NS1) of the related dengue virus is implicated in contributing to vascular leak, little is known about the role of YFV NS1 in severe YF and mechanisms of vascular dysfunction in YFV infections. METHODS: Using serum samples from laboratory-confirmed YF patients with severe (n = 39) or non-severe (n = 18) disease in a well-defined hospital observational cohort in Brazil, plus samples from healthy uninfected controls (n = 11), we investigated factors associated with disease severity and endothelial dysfunction. FINDINGS: We found significantly increased levels of NS1, as well as syndecan-1, a marker of vascular leak, in serum from severe YF as compared to non-severe YF or control groups. We also showed that hyperpermeability of endothelial cell monolayers treated with serum from severe YF patients was significantly higher compared to non-severe YF and control groups, as measured by transendothelial electrical resistance (TEER). Further, we demonstrated that YFV NS1 induces shedding of syndecan-1 from the surface of human endothelial cells. Notably, YFV NS1 serum levels significantly correlated with syndecan-1 serum levels, TEER values, and signs of disease severity. Syndecan-1 levels also significantly correlated with clinical laboratory parameters of disease severity, viral load, hospitalization, and death. INTERPRETATION: This study provides further evidence for endothelial dysfunction as a mechanism of YF pathogenesis in humans and suggests serum quantification of YFV NS1 and syndecan-1 as valuable tools for disease diagnosis and/or prognosis. FUNDING: This work was supported by the US NIH and FAPESP.

Detailed characterization of Redondovirus in saliva of SARS-CoV-2-infected individuals in Sao Paulo, Brazil.

BACKGROUND: Redondovirus (ReDoV) is a DNA virus present in the respiratory tract of many healthy individuals. Since SARS-CoV-2, the virus responsible for COVID-19, also primarily infects the same site, we evaluated whether ReDoV was present at increased frequency in patients with COVID-19 and influenced infection parameters. METHODS: Saliva samples were collected weekly from 59 individuals with COVID-19 and from 132 controls. ReDoV was detected by polymerase chain reaction and the genotypes were identified by metagenomics. Torque Teno Virus (TTV) in these samples were previously reported. RESULTS: ReDoV was detected in saliva more frequently from COVID-19 patients (72.9%) than from controls (50.0%) (p = 0.0015). There were no associations between ReDoV detection and either continuous or intermittent SARS-CoV-2 shedding, the duration of SARS-CoV-2 detection in saliva, patients' sex or if infection was by the B1 or Gamma strain. The two ReDoV strains, Brisavirus and Vientovirus, were present in equivalent frequencies in ReDoV-positive COVID-19 patients and controls. Phylogenetic analysis suggested that the two ReDoV strains in Brazil were similar to strains previously detected on other continents. CONCLUSION: ReDoV expression in saliva is increased in males and females in Brazil with mild COVID-19 but its presence does not appear to influence properties of the SARS-CoV-2 infection.

Expanding the application of graphene vertical devices to dual femtomolar detection of SARS-CoV-2 receptor binding domain in serum and saliva.

The emergence of the graphene-based hybrid electrical-electrochemical vertical device (EEVD) has introduced a promising nanostructured biosensor tailored for point-of-care applications. In this study, we present an innovative EEVD capable of simultaneously detecting the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in both serum and saliva. The foundation of the EEVD lies in a poly-neutral red-graphene heterojunction, which has been enhanced with a bioconjugate of gold nanoparticles and antibodies. The biodevice demonstrates a remarkable limit of detection, registering at the femtomolar scale (2.86 fmol L-1 or 0.1 pg mL-1). Its sensitivity is characterized by a 6.1 mV/decade response, and its operational range spans 10-12 to 10-7 g mL-1 in both serum and saliva samples. With a 20.0 µL of biological samples and a rapid processing time of under 10 min, the EEVD achieves the feat of dual antigen detection. The tests achieved 100.0% specificity, accuracy, and sensitivity in saliva, and 100.0% specificity, 88.9% accuracy, and 80.0% sensitivity in serum. This study highlights the EEVD as a low-cost solution of rapid viral detection during the crucial initial phases of COVID-19 infections.

Performance Evaluation of VIDAS® Diagnostic Assays Detecting Anti-Chikungunya Virus IgM and IgG Antibodies: An International Study.

Chikungunya (CHIK) is a debilitating mosquito-borne disease with an epidemiology and early clinical symptoms similar to those of other arboviruses-triggered diseases such as dengue or Zika. Accurate and rapid diagnosis of CHIK virus (CHIKV) infection is therefore challenging. This international study evaluated the performance of the automated VIDAS® anti-CHIKV IgM and IgG assays compared to that of manual competitor IgM and IgG ELISA for the detection of anti-CHIKV IgM and IgG antibodies in 660 patients with suspected CHIKV infection. Positive and negative agreements of the VIDAS® CHIKV assays with ELISA ranged from 97.5% to 100.0%. The sensitivity of the VIDAS® CHIKV assays evaluated in patients with a proven CHIKV infection confirmed reported kinetics of anti-CHIKV IgM and IgG response, with a positive detection of 88.2-100.0% for IgM ≥ 5 days post symptom onset and of 100.0% for IgG ≥ 11 days post symptom onset. Our study also demonstrated the superiority of ELISA and VIDAS® assays over rapid diagnostic IgM/IgG tests. The analytical performance of VIDAS® anti-CHIKV IgM and IgG assays was excellent, with a high precision (coefficients of variation ≤ 7.4%) and high specificity (cross-reactivity rate ≤ 2.9%). This study demonstrates the suitability of the automated VIDAS® anti-CHIKV IgM and IgG assays to diagnose CHIKV infections and supports its applicability for epidemiological surveillance and differential diagnosis in regions endemic for CHIKV.

Yellow fever disease severity and endothelial dysfunction are associated with elevated serum levels of viral NS1 protein and syndecan-1.

Yellow fever virus (YFV) infections can cause severe disease manifestations, including hepatic injury, endothelial damage, coagulopathy, hemorrhage, systemic organ failure, and shock, and are associated with high mortality in humans. While nonstructural protein 1 (NS1) of the related dengue virus is implicated in contributing to vascular leak, little is known about the role of YFV NS1 in severe YF and mechanisms of vascular dysfunction in YFV infections. Here, using serum samples from qRT-PCR-confirmed YF patients with severe (n=39) or non-severe (n=18) disease in a well-defined hospital cohort in Brazil, plus samples from healthy uninfected controls (n=11), we investigated factors associated with disease severity. We developed a quantitative YFV NS1 capture ELISA and found significantly increased levels of NS1, as well as syndecan-1, a marker of vascular leak, in serum from severe YF as compared to non-severe YF or control groups. We also showed that hyperpermeability of endothelial cell monolayers treated with serum from severe YF patients was significantly higher compared to non-severe YF and control groups as measured by transendothelial electrical resistance (TEER). Further, we demonstrated that YFV NS1 induces shedding of syndecan-1 from the surface of human endothelial cells. Notably, YFV NS1 serum levels significantly correlated with syndecan-1 serum levels and TEER values. Syndecan-1 levels also significantly correlated with clinical laboratory parameters of disease severity, viral load, hospitalization, and death. In summary, this study points to a role for secreted NS1 in YF disease severity and provides evidence for endothelial dysfunction as a mechanism of YF pathogenesis in humans.

SARS-CoV-2 Detection and Culture in Different Biological Specimens from Immunocompetent and Immunosuppressed COVID-19 Patients Infected with Two Different Viral Strains.

Introduction-The dynamics of SARS-CoV-2 shedding and replication in humans remain incompletely understood. Methods-We analyzed SARS-CoV-2 shedding from multiple sites in individuals with an acute COVID-19 infection by weekly sampling for five weeks in 98 immunocompetent and 25 immunosuppressed individuals. Samples and culture supernatants were tested via RT-PCR for SARS-CoV-2 to determine viral clearance rates and in vitro replication. Results-A total of 2447 clinical specimens were evaluated, including 557 nasopharyngeal swabs, 527 saliva samples, 464 urine specimens, 437 anal swabs and 462 blood samples. The SARS-CoV-2 genome sequences at each site were classified as belonging to the B.1.128 (ancestral strain) or Gamma lineage. SARS-CoV-2 detection was highest in nasopharyngeal swabs regardless of the virus strain involved or the immune status of infected individuals. The duration of viral shedding varied between clinical specimens and individual patients. Prolonged shedding of potentially infectious virus varied from 10 days up to 191 days, and primarily occurred in immunosuppressed individuals. Virus was isolated in culture from 18 nasal swab or saliva samples collected 10 or more days after onset of disease. Conclusions-Our findings indicate that persistent SARS-CoV-2 shedding may occur in both competent or immunosuppressed individuals, at multiple clinical sites and in a minority of subjects is capable of in vitro replication.

First case report of monkeypox in Brazil: clinical manifestations and differential diagnosis with sexually transmitted infections.

In 2022, an outbreak of monkeypox is being reported in non-endemic areas, with unusual clinical manifestations. The detailed clinical description of the first patient that received the diagnosis of monkeypox in Brazil is reported here, whose clinical manifestations can easily lead to misdiagnosis of sexually transmitted infections. A 41 years old male presented to an emergency room with a vesicular rash with eight days of evolution. He had traveled to Portugal and Spain and reported non-penetrative sexual involvement with three different male individuals. On the third day of symptoms, he sought medical care and received empirical treatment directed to sexually transmitted infections. As the symptoms did not improve, he sought medical attention at an infectious disease referral center presenting, on admission, an ulcerated penile lesion with central necrotic crusts, a disseminated pleomorphic skin rash and an oropharyngeal ulcer. The monkeypox diagnosis was suspected due to the characteristics of the lesions and the history of intimate contact with casual partners, and it was later confirmed by sequencing the almost complete monkeypox genome. The patient was hospitalized for pain control, which required opiate administration. He developed a secondary bacterial infection on the penile lesions, which were treated with oral antibiotics. He was discharged after 14 days, with lesions in process of re-epithelialization. Given the current outbreak, we must consider the possibility of monkeypox in patients with suggestive lesions, anywhere on the body (including the genitals), added to an epidemiological link or history of intimate contact with strangers or casual partners.

Shotgun metagenomic sequencing of the first case of monkeypox virus in Brazil, 2022.

Monkeypox virus (MPXV), a zoonotic virus endemic to the African continent, has been reported in 33 non-endemic countries since May 2022. We report an almost complete genome of the first confirmed case of MPXV in Brazil. Shotgun metagenomic sequencing was completed in 18 hours, from DNA extraction to consensus sequence generation.

Understanding Sabiá virus infections (Brazilian mammarenavirus).

BACKGROUND: Only two naturally occurring human Sabiá virus (SABV) infections have been reported, and those occurred over 20 years ago. METHODS: We diagnosed two new cases of SABV infection using metagenomics in patients thought to have severe yellow fever and described new features of histopathological findings. RESULTS: We characterized clinical manifestations, histopathology and analyzed possible nosocomial transmission. Patients presented with hepatitis, bleeding, neurological alterations and died. We traced twenty-nine hospital contacts and evaluated them clinically and by RT-PCR and neutralizing antibodies. Autopsies uncovered unique features on electron microscopy, such as hepatocyte "pinewood knot" lesions. Although previous reports with similar New-World arenavirus had nosocomial transmission, our data did not find any case in contact tracing. CONCLUSIONS: Although an apparent by rare, Brazilian mammarenavirus infection is an etiology for acute hemorrhagic fever syndrome. The two fatal cases had peculiar histopathological findings not previously described. The virological diagnosis was possible only by contemporary techniques such as metagenomic assays. We found no subsequent infections when we used serological and molecular tests to evaluate close contacts.

Graphene-based hybrid electrical-electrochemical point-of-care device for serologic COVID-19 diagnosis.

The outbreak of COVID-19 pandemics highlighted the need of sensitive, selective, and easy-to-handle biosensing devices. In the contemporary scenario, point-of-care devices for mass testing and infection mapping within a population have proven themselves as of primordial importance. Here, we introduce a graphene-based Electrical-Electrochemical Vertical Device (EEVD) point-of-care biosensor, strategically engineered for serologic COVID-19 diagnosis. EEVD uses serologic IgG quantifications on SARS-CoV-2 Receptor Binding Domain (RBD) bioconjugate immobilized onto device surface. EEVD combines graphene basal plane with high charge carrier mobility, high conductivity, low intrinsic resistance, and interfacial sensitivity to capacitance alterations. EEVD application was carried out in real human serum samples. Since EEVD is a miniaturized device, it requires just 40 µL of sample for a point-of-care COVID-19 infections detection. When compared to serologic assays such ELISA and other immunochromatographic methods, EEVD presents some advantages such as time of analyses (15 min), sample preparation, and a LOD of 1.0 pg mL-1. We glimpse that EEVD meets the principles of robustness and accuracy, desirable analytic parameters for assays destined to pandemics control strategies.

Shotgun metagenomic sequencing of the first case of monkeypox virus in Brazil, 2022

ABSTRACT Monkeypox virus (MPXV), a zoonotic virus endemic to the African continent, has been reported in 33 non-endemic countries since May 2022. We report an almost complete genome of the first confirmed case of MPXV in Brazil. Shotgun metagenomic sequencing was completed in 18 hours, from DNA extraction to consensus sequence generation.

First case report of monkeypox in Brazil: clinical manifestations and differential diagnosis with sexually transmitted infections

ABSTRACT In 2022, an outbreak of monkeypox is being reported in non-endemic areas, with unusual clinical manifestations. The detailed clinical description of the first patient that received the diagnosis of monkeypox in Brazil is reported here, whose clinical manifestations can easily lead to misdiagnosis of sexually transmitted infections. A 41 years old male presented to an emergency room with a vesicular rash with eight days of evolution. He had traveled to Portugal and Spain and reported non-penetrative sexual involvement with three different male individuals. On the third day of symptoms, he sought medical care and received empirical treatment directed to sexually transmitted infections. As the symptoms did not improve, he sought medical attention at an infectious disease referral center presenting, on admission, an ulcerated penile lesion with central necrotic crusts, a disseminated pleomorphic skin rash and an oropharyngeal ulcer. The monkeypox diagnosis was suspected due to the characteristics of the lesions and the history of intimate contact with casual partners, and it was later confirmed by sequencing the almost complete monkeypox genome. The patient was hospitalized for pain control, which required opiate administration. He developed a secondary bacterial infection on the penile lesions, which were treated with oral antibiotics. He was discharged after 14 days, with lesions in process of re-epithelialization. Given the current outbreak, we must consider the possibility of monkeypox in patients with suggestive lesions, anywhere on the body (including the genitals), added to an epidemiological link or history of intimate contact with strangers or casual partners.

Knowledge about clinical presentation, prevention strategies and sexual transmission of Zika virus infection among women of reproductive age in an endemic area.

BACKGROUND: The recognition of the causal association between Zika virus (ZIKV) infection during pregnancy and congenital abnormalities including microcephaly underlines the importance of preventing this disease in pregnant women (PW) and women of childbearing age (WCA). Although Brazil and other Latin American countries reported a significant reduction in the number of ZIKV infections in recent years, epidemic waves can recur in settings with previous outbreaks as conditions for transmission remain optimal and susceptible populations are continuously replenished. METHODS: In this cross-sectional study, we enrolled 64 PW and 260 non-pregnant WCA attending routine medical appointments in two primary care units in Sao Paulo, Brazil, and assessed knowledge and attitudes about ZIKV infection and prevention. RESULTS: Most women reported knowing that ZIKV is transmitted through the bite of Aedes mosquitos, and most knew that acute symptoms are similar to those seen in Dengue infection. Furthermore, most participants correctly described that ZIKV infection during pregnancy may cause detrimental outcomes for the newborn. However, most ignored that ZIKV infection can be asymptomatic, and only 15% knew about the risk of ZIKV sexual transmission. We found no statistically significant differences between PW and WCA regarding knowledge about ZIKV sexual transmission. Knowledge about ZIKV sexual transmission was significantly associated with education; among participants with ≤12 schooling years, only 9.0% (95%CI 3.4-18.5%) correctly answered that ZIKV can be sexually transmitted, compared to 12.9% (95%CI 8.2-18.8%) among participants with 12-14 schooling years, and to 24.4% (95%CI 15.9-34.9%) of participants with ≥15 schooling years (p = 0.015). Education remained independently associated with knowledge about sexual transmission of ZIKV in a multivariate logistic regression model adjusted for age, race and pregnancy status (p = 0.022). CONCLUSION: Our findings underscore the urgent need of educational and family planning programs that may help prevent detrimental outcomes of ZIKV infection in an endemic area of Brazil.

Neutralisation of SARS-CoV-2 lineage P.1 by antibodies elicited through natural SARS-CoV-2 infection or vaccination with an inactivated SARS-CoV-2 vaccine: an immunological study.

BACKGROUND: Mutations accrued by SARS-CoV-2 lineage P.1-first detected in Brazil in early January, 2021-include amino acid changes in the receptor-binding domain of the viral spike protein that also are reported in other variants of concern, including B.1.1.7 and B.1.351. We aimed to investigate whether isolates of wild-type P.1 lineage SARS-CoV-2 can escape from neutralising antibodies generated by a polyclonal immune response. METHODS: We did an immunological study to assess the neutralising effects of antibodies on lineage P.1 and lineage B isolates of SARS-CoV-2, using plasma samples from patients previously infected with or vaccinated against SARS-CoV-2. Two specimens (P.1/28 and P.1/30) containing SARS-CoV-2 lineage P.1 (as confirmed by viral genome sequencing) were obtained from nasopharyngeal and bronchoalveolar lavage samples collected from patients in Manaus, Brazil, and compared against an isolate of SARS-CoV-2 lineage B (SARS.CoV2/SP02.2020) recovered from a patient in Brazil in February, 2020. Isolates were incubated with plasma samples from 21 blood donors who had previously had COVID-19 and from a total of 53 recipients of the chemically inactivated SARS-CoV-2 vaccine CoronaVac: 18 individuals after receipt of a single dose and an additional 20 individuals (38 in total) after receipt of two doses (collected 17-38 days after the most recent dose); and 15 individuals who received two doses during the phase 3 trial of the vaccine (collected 134-230 days after the second dose). Antibody neutralisation of P.1/28, P.1/30, and B isolates by plasma samples were compared in terms of median virus neutralisation titre (VNT50, defined as the reciprocal value of the sample dilution that showed 50% protection against cytopathic effects). FINDINGS: In terms of VNT50, plasma from individuals previously infected with SARS-CoV-2 had an 8·6 times lower neutralising capacity against the P.1 isolates (median VNT50 30 [IQR <20-45] for P.1/28 and 30 [<20-40] for P.1/30) than against the lineage B isolate (260 [160-400]), with a binominal model showing significant reductions in lineage P.1 isolates compared with the lineage B isolate (p≤0·0001). Efficient neutralisation of P.1 isolates was not seen with plasma samples collected from individuals vaccinated with a first dose of CoronaVac 20-23 days earlier (VNT50s below the limit of detection [<20] for most plasma samples), a second dose 17-38 days earlier (median VNT50 24 [IQR <20-25] for P.1/28 and 28 [<20-25] for P.1/30), or a second dose 134-260 days earlier (all VNT50s below limit of detection). Median VNT50s against the lineage B isolate were 20 (IQR 20-30) after a first dose of CoronaVac 20-23 days earlier, 75 (<20-263) after a second dose 17-38 days earlier, and 20 (<20-30) after a second dose 134-260 days earlier. In plasma collected 17-38 days after a second dose of CoronaVac, neutralising capacity against both P.1 isolates was significantly decreased (p=0·0051 for P.1/28 and p=0·0336 for P.1/30) compared with that against the lineage B isolate. All data were corroborated by results obtained through plaque reduction neutralisation tests. INTERPRETATION: SARS-CoV-2 lineage P.1 might escape neutralisation by antibodies generated in response to polyclonal stimulation against previously circulating variants of SARS-CoV-2. Continuous genomic surveillance of SARS-CoV-2 combined with antibody neutralisation assays could help to guide national immunisation programmes. FUNDING: São Paulo Research Foundation, Brazilian Ministry of Science, Technology and Innovation and Funding Authority for Studies, Medical Research Council, National Council for Scientific and Technological Development, National Institutes of Health. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil.

Cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Manaus, Brazil, resurged in late 2020 despite previously high levels of infection. Genome sequencing of viruses sampled in Manaus between November 2020 and January 2021 revealed the emergence and circulation of a novel SARS-CoV-2 variant of concern. Lineage P.1 acquired 17 mutations, including a trio in the spike protein (K417T, E484K, and N501Y) associated with increased binding to the human ACE2 (angiotensin-converting enzyme 2) receptor. Molecular clock analysis shows that P.1 emergence occurred around mid-November 2020 and was preceded by a period of faster molecular evolution. Using a two-category dynamical model that integrates genomic and mortality data, we estimate that P.1 may be 1.7- to 2.4-fold more transmissible and that previous (non-P.1) infection provides 54 to 79% of the protection against infection with P.1 that it provides against non-P.1 lineages. Enhanced global genomic surveillance of variants of concern, which may exhibit increased transmissibility and/or immune evasion, is critical to accelerate pandemic responsiveness.

Genomics and epidemiology of a novel SARS-CoV-2 lineage in Manaus, Brazil.

Cases of SARS-CoV-2 infection in Manaus, Brazil, resurged in late 2020, despite high levels of previous infection there. Through genome sequencing of viruses sampled in Manaus between November 2020 and January 2021, we identified the emergence and circulation of a novel SARS-CoV-2 variant of concern, lineage P.1, that acquired 17 mutations, including a trio in the spike protein (K417T, E484K and N501Y) associated with increased binding to the human ACE2 receptor. Molecular clock analysis shows that P.1 emergence occurred around early November 2020 and was preceded by a period of faster molecular evolution. Using a two-category dynamical model that integrates genomic and mortality data, we estimate that P.1 may be 1.4-2.2 times more transmissible and 25-61% more likely to evade protective immunity elicited by previous infection with non-P.1 lineages. Enhanced global genomic surveillance of variants of concern, which may exhibit increased transmissibility and/or immune evasion, is critical to accelerate pandemic responsiveness.

Pharmacogenomic Profile and Adverse Drug Reactions in a Prospective Therapeutic Cohort of Chagas Disease Patients Treated with Benznidazole.

Chagas disease remains a major social and public health problem in Latin America. Benznidazole (BZN) is the main drug with activity against Trypanosoma cruzi. Due to the high number of adverse drug reactions (ADRs), BZN is underprescribed. The goal of this study was to evaluate the genetic and transcriptional basis of BZN adverse reactions. METHODS: A prospective cohort with 102 Chagas disease patients who underwent BZN treatment was established to identify ADRs and understand their genetic basis. The patients were classified into two groups: those with at least one ADR (n = 73), and those without ADRs (n = 29). Genomic analyses were performed comparing single nucleotide polymorphisms between groups. Transcriptome data were obtained comparing groups before and after treatment, and signaling pathways related to the main ADRs were evaluated. RESULTS: A total of 73 subjects (71.5%) experienced ADRs. Dermatological symptoms were most frequent (45.1%). One region of chromosome 16, at the gene LOC102724084 (rs1518601, rs11861761, and rs34091595), was associated with ADRs (p = 5.652 × 10-8). Transcriptomic data revealed three significantly enriched signaling pathways related to BZN ADRs. CONCLUSIONS: These data suggest that part of adverse BZN reactions might be genetically determined and may facilitate patient risk stratification prior to starting BZN treatment.

Local Transmission of SARS-CoV-2 Lineage B.1.1.7, Brazil, December 2020.

In December 2020, research surveillance detected the B.1.1.7 lineage of severe acute respiratory syndrome coronavirus 2 in São Paulo, Brazil. Rapid genomic sequencing and phylogenetic analysis revealed 2 distinct introductions of the lineage. One patient reported no international travel. There may be more infections with this lineage in Brazil than reported.

Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing.

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5' end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach 'SMART-9N' and a version compatible rapid adapters  available from Oxford Nanopore Technologies 'Rapid SMART-9N'. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.

Knowledge about clinical presentation, prevention strategies and sexual transmission of Zika virus infection among women of reproductive age in an endemic area

ABSTRACT Background The recognition of the causal association between Zika virus (ZIKV) infection during pregnancy and congenital abnormalities including microcephaly underlines the importance of preventing this disease in pregnant women (PW) and women of childbearing age (WCA). Although Brazil and other Latin American countries reported a significant reduction in the number of ZIKV infections in recent years, epidemic waves can recur in settings with previous outbreaks as conditions for transmission remain optimal and susceptible populations are continuously replenished. Methods: In this cross-sectional study, we enrolled 64 PW and 260 non-pregnant WCA attending routine medical appointments in two primary care units in São Paulo, Brazil, and assessed knowledge and attitudes about ZIKV infection and prevention. Results: Most women reported knowing that ZIKV is transmitted through the bite of Aedes mosquitos, and most knew that acute symptoms are similar to those seen in Dengue infection. Furthermore, most participants correctly described that ZIKV infection during pregnancy may cause detrimental outcomes for the newborn. However, most ignored that ZIKV infection can be asymptomatic, and only 15% knew about the risk of ZIKV sexual transmission. We found no statistically significant differences between PW and WCA regarding knowledge about ZIKV sexual transmission. Knowledge about ZIKV sexual transmission was significantly associated with education; among participants with ≤12 schooling years, only 9.0% (95%CI 3.4-18.5%) correctly answered that ZIKV can be sexually transmitted, compared to 12.9% (95%CI 8.2-18.8%) among participants with 12-14 schooling years, and to 24.4% (95%CI 15.9-34.9%) of participants with ≥15 schooling years (p = 0.015). Education remained independently associated with knowledge about sexual transmission of ZIKV in a multivariate logistic regression model adjusted for age, race and pregnancy status (p = 0.022). Conclusion: Our findings underscore the urgent need of educational and family planning programs that may help prevent detrimental outcomes of ZIKV infection in an endemic area of Brazil.