Phylogeography, genetic diversity, and intraspecific genetic structure of the black-horned capuchin (Sapajus nigritus).

Recent molecular studies have clarified the overarching taxonomy of capuchin monkeys, but intraspecific genetic diversity remains unexplored for most capuchin species. One example is Sapajus nigritus, the southernmost capuchin monkey, found in Brazil and Argentina; its phenotypic diversity has been recognized as two geographic subspecies, but the intraspecific genetic structure of this taxon is poorly known. Here, we sampled across most of this species' geographic distribution, producing a newly sequenced data set for genetic analyses that included 78 individuals from 14 populations. We investigated the intraspecific diversity, genetic structure, and evolutionary history using three mitochondrial markers. Our results indicated that S. nigritus populations exhibited high levels of genetic structure. We found strong support for two monophyletic clades within this species with a deep phylogenetic split, and clear separation from other related taxa. Vicariance events seem to have played a prevalent role in shaping S. nigritus genetic differentiation. The Paraíba do Sul River may have driven the deep divergence between southern and northern clades, whereas the Tietê River may have had a weaker, more recent effect on the divergence of populations within the southern clade.

Untargeted Metabolomics Insights into Newborns with Congenital Zika Infection.

Zika virus (ZIKV), an emerging virus belonging to the Flaviviridae family, causes severe neurological clinical complications and has been associated with Guillain-Barré syndrome, fetal abnormalities known collectively as congenital Zika syndrome, and microcephaly. Studies have shown that ZIKV infection can alter cellular metabolism, directly affecting neural development. Brain growth requires controlled cellular metabolism, which is essential for cell proliferation and maturation. However, little is known regarding the metabolic profile of ZIKV-infected newborns and possible associations related to microcephaly. Furthering the understanding surrounding underlying mechanisms is essential to developing personalized treatments for affected individuals. Thus, metabolomics, the study of the metabolites produced by or modified in an organism, constitutes a valuable approach in the study of complex diseases. Here, 26 serum samples from ZIKV-positive newborns with or without microcephaly, as well as controls, were analyzed using an untargeted metabolomics approach involving gas chromatography-mass spectrometry (GC-MS). Significant alterations in essential and non-essential amino acids, as well as carbohydrates (including aldohexoses, such as glucose or mannose) and their derivatives (urea and pyruvic acid), were observed in the metabolic profiles analyzed. Our results provide insight into relevant metabolic processes in patients with ZIKV and microcephaly.

Epidemiology and evolution of Zika virus in Minas Gerais, Southeast Brazil.

Autochthonous Zika virus (ZIKV) transmission in Brazil was first identified in April 2015 in Brazil, with the first ZIKV-associated microcephaly cases detected in October 2015. Despite efforts on understanding ZIKV transmission in Brazil, little is known about the virus epidemiology and genetic diversity in Minas Gerais (MG), the second most populous state in the country. We report molecular and genomic findings from the main public health laboratory in MG. Until January 2020, 26,817 ZIKV suspected infections and 86 congenital syndrome cases were reported in MG state. We tested 8552 ZIKV and microcephaly suspected cases. Ten genomes were generated on-site directly from clinical samples. A total of 1723 confirmed cases were detected in Minas Gerais, with two main epidemic waves; the first and larger epidemic wave peaked in March 2016, with the second smaller wave that peaked in March 2017. Dated molecular clock analysis revealed that multiple introductions occurred in Minas Gerais between 2014 and 2015, suggesting that the virus was circulating unnoticed for at least 16 months before the first confirmed laboratory case that we retrospectively identified in December 2015. Our findings highlight the importance of continued genomic surveillance strategies combined with traditional epidemiology to assist public health laboratories in monitoring and understanding the diversity of circulating arboviruses, which might help attenuate the public health impact of infectious diseases.

Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing.

Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas.

An observational clinical case of Zika virus-associated neurological disease is associated with primary IgG response and enhanced TNF levels.

Descriptive clinical data help to reveal factors that may provoke Zika virus (ZIKV) neuropathology. The case of a 24-year-old female with a ZIKV-associated severe acute neurological disorder was studied. The levels of ZIKV in the cerebrospinal fluid (CSF) were 50 times higher than the levels in other compartments. An acute anti-flavivirus IgG, together with enhanced TNF-alpha levels, may have contributed to ZIKV invasion in the CSF, whereas the unbiased genome sequencing [obtained by next-generation sequencing (NGS)] of the CSF revealed that no virus mutations were associated with the anatomic compartments (CSF, serum, saliva and urine).

Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum.

The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1-4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-µl serum sample, the sensitivity and specificity values of the DENV1-4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-µl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction-positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses.

The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication.

Zika virus (ZIKV) is a member of the Flaviviridae family, along with other agents of clinical significance such as dengue (DENV) and hepatitis C (HCV) viruses. Since ZIKV causes neurological disorders during fetal development and in adulthood, antiviral drugs are necessary. Sofosbuvir is clinically approved for use against HCV and targets the protein that is most conserved among the members of the Flaviviridae family, the viral RNA polymerase. Indeed, we found that sofosbuvir inhibits ZIKV RNA polymerase, targeting conserved amino acid residues. Sofosbuvir inhibited ZIKV replication in different cellular systems, such as hepatoma (Huh-7) cells, neuroblastoma (SH-Sy5y) cells, neural stem cells (NSC) and brain organoids. In addition to the direct inhibition of the viral RNA polymerase, we observed that sofosbuvir also induced an increase in A-to-G mutations in the viral genome. Together, our data highlight a potential secondary use of sofosbuvir, an anti-HCV drug, against ZIKV.

Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study.

BACKGROUND: The incidence of microcephaly in Brazil in 2015 was 20 times higher than in previous years. Congenital microcephaly is associated with genetic factors and several causative agents. Epidemiological data suggest that microcephaly cases in Brazil might be associated with the introduction of Zika virus. We aimed to detect and sequence the Zika virus genome in amniotic fluid samples of two pregnant women in Brazil whose fetuses were diagnosed with microcephaly. METHODS: In this case study, amniotic fluid samples from two pregnant women from the state of Paraíba in Brazil whose fetuses had been diagnosed with microcephaly were obtained, on the recommendation of the Brazilian health authorities, by ultrasound-guided transabdominal amniocentesis at 28 weeks' gestation. The women had presented at 18 weeks' and 10 weeks' gestation, respectively, with clinical manifestations that could have been symptoms of Zika virus infection, including fever, myalgia, and rash. After the amniotic fluid samples were centrifuged, DNA and RNA were extracted from the purified virus particles before the viral genome was identified by quantitative reverse transcription PCR and viral metagenomic next-generation sequencing. Phylogenetic reconstruction and investigation of recombination events were done by comparing the Brazilian Zika virus genome with sequences from other Zika strains and from flaviviruses that occur in similar regions in Brazil. FINDINGS: We detected the Zika virus genome in the amniotic fluid of both pregnant women. The virus was not detected in their urine or serum. Tests for dengue virus, chikungunya virus, Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus, HIV, Treponema pallidum, and parvovirus B19 were all negative. After sequencing of the complete genome of the Brazilian Zika virus isolated from patient 1, phylogenetic analyses showed that the virus shares 97-100% of its genomic identity with lineages isolated during an outbreak in French Polynesia in 2013, and that in both envelope and NS5 genomic regions, it clustered with sequences from North and South America, southeast Asia, and the Pacific. After assessing the possibility of recombination events between the Zika virus and other flaviviruses, we ruled out the hypothesis that the Brazilian Zika virus genome is a recombinant strain with other mosquito-borne flaviviruses. INTERPRETATION: These findings strengthen the putative association between Zika virus and cases of microcephaly in neonates in Brazil. Moreover, our results suggest that the virus can cross the placental barrier. As a result, Zika virus should be considered as a potential infectious agent for human fetuses. Pathogenesis studies that confirm the tropism of Zika virus for neuronal cells are warranted. FUNDING: Consellho Nacional de Desenvolvimento e Pesquisa (CNPq), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Molecular characterization of a rare, human-porcine reassortant rotavirus strain, G11P[6], from Ecuador.

The Pan-American Health Organization established a rotavirus pre-vaccination disease burden and strain surveillance network in Latin America and the Caribbean in 2004. During strain surveillance in Ecuador in 2005-2006, a rare rotavirus genotype, G11P[6], was detected among common strains. Sequencing and phylogenetic analysis of this strain identified a novel lineage of the G11 VP7 gene, most closely related to A253 (91.8% nt identity), a porcine rotavirus strain identified in Venezuela. Most genes of this strain clustered with porcine, human-porcine or bovine-porcine reassortant strains; only VP6 and perhaps NSP2 genes were more closely related to cognate genes of human rotaviruses. Thus, this strain was likely generated by gene reassortment between porcine and human parental strains. Our study provides further evidence that animal rotaviruses play an important role in genetic and antigenic diversity of rotaviruses pathogenic for humans.

Quantification of dengue virus type 3 RNA in fatal and non-fatal cases in Brazil, 2002.

We examined levels of dengue virus type 3 (DENV-3) RNA in association with the type of infection (primary or secondary) in 42 patients with fatal and non-fatal outcomes in Rio de Janeiro, Brazil, 2002. Subjects with fatal outcomes had mean virus titers significantly higher than those who survived (12.5 vs. 7.9 log(10) RNA copies/ml). Because primary infections were confirmed among the fatal cases (52.1%), antibody-dependent enhancement alone did not explain all the cases of severe disease in this study population. These findings suggest that high levels of DENV-3 may have contributed to the severe form of dengue in Rio de Janeiro, 2002.

Vector competence of Brazilian Aedes aegypti and Ae. albopictus for a Brazilian yellow fever virus isolate.

Because the potential urban yellow fever (YF) mosquito vectors Aedes aegypti and Ae. albopictus are at historical highs in Brazil, both in terms of density and geographical range, we assessed the risk of an urban YF epidemic in Brazil. We evaluated and confirmed in a laboratory setting the vector competence of Brazilian Ae. aegypti for a currently circulating strain of YF virus, and investigated the potential for Brazilian Ae. albopictus to transmit YF.