Genomic characterization of a novel SARS-CoV-2 lineage from Rio de Janeiro, Brazil.

Almost simultaneously, several studies reported the emergence of novel SARS-CoV-2 lineages characterized by their phylogenetic and genetic distinction (1), (2), (3), (4).….

Cryptococcal dissemination to the central nervous system requires the vacuolar calcium transporter Pmc1.

Cryptococcus neoformans is a basidiomycetous yeast and the cause of cryptococcosis in immunocompromised individuals. The most severe form of the disease is meningoencephalitis, which is one of the leading causes of death in HIV/AIDS patients. In order to access the central nervous system, C. neoformans relies on the activity of certain virulence factors such as urease, which allows transmigration through the blood-brain barrier. In this study, we demonstrate that the calcium transporter Pmc1 enables C. neoformans to penetrate the central nervous system, because the pmc1 null mutant failed to infect and to survive within the brain parenchyma in a murine systemic infection model. To investigate potential alterations in transmigration pathways in these mutants, global expression profiling of the pmc1 mutant strain was undertaken, and genes associated with urease, the Ca2+ -calcineurin pathway, and capsule assembly were identified as being differentially expressed. Also, a decrease in urease activity was observed in the calcium transporter null mutants. Finally, we demonstrate that the transcription factor Crz1 regulates urease activity and that the Ca2+ -calcineurin signalling pathway positively controls the transcription of calcium transporter genes and factors related to transmigration.

Comparative genomics of the major fungal agents of human and animal Sporotrichosis: Sporothrix schenckii and Sporothrix brasiliensis.

BACKGROUND: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes. RESULTS: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE's) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style. CONCLUSIONS: Comparative genomic data suggest a unique ecological shift in the Sporothrix lineage from plant-association to mammalian parasitism, which contributes to the understanding of how environmental interactions may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.

Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual.

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein-protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.

Clinical markers of post-Chikungunya chronic inflammatory joint disease: A Brazilian cohort.

BACKGROUND: Chikungunya-fever (CHIKF) remains a public health major issue. It is clinically divided into three phases: acute, post-acute and chronic. Chronic cases correspond to 25-40% individuals and, though most of them are characterized by long-lasting arthralgia alone, many of them exhibit persistent or recurrent inflammatory signs that define post-Chikungunya chronic inflammatory joint disease (pCHIKV-CIJD). We aimed to identify early clinical markers of evolution to pCHIKV-CIJD during acute and post-acute phases. METHODOLOGY/PRINCIPAL FINDINGS: We studied a prospective cohort of CHIKF-confirmed volunteers with longitudinal clinical data collection from symptoms onset up to 90 days, including a 21-day visit (D21). Of 169 patients with CHIKF, 86 (50.9%) completed the follow-up, from whom 39 met clinical criteria for pCHIKV-CIJD (45.3%). The relative risk of chronification was higher in women compared to men (RR = 1.52; 95% CI = 1.15-1.99; FDR = 0.03). None of the symptoms or signs presented at D0 behaved as an early predictor of pCHIKV-CIJD, while being symptomatic at D21 was a risk factor for chronification (RR = 1.31; 95% CI = 1.09-1.55; FDR = 0.03). Significance was also observed for joint pain (RR = 1.35; 95% CI = 1.12-1.61; FDR = 0.02), reported edema (RR = 3.61; 95% CI = 1.44-9.06; FDR = 0.03), reported hand and/or feet small joints edema (RR = 4.22; 95% CI = 1.51-11.78; FDR = 0.02), and peri-articular edema observed during physical examination (RR = 2.89; 95% CI = 1.58-5.28; FDR = 0.002). Furthermore, patients with no findings in physical examination at D21 were at lower risk of chronic evolution (RR = 0.41, 95% CI = 0.24-0.70, FDR = 0.01). Twenty-nine pCHIKV-CIJD patients had abnormal articular ultrasonography (90.6% of the examined). The most common findings were synovitis (65.5%) and joint effusion (58.6%). CONCLUSION: This cohort has provided important insights into the prognostic evaluation of CHIKF. Symptomatic sub-acute disease is a relevant predictor of evolution to chronic arthritis with synovitis, drawing attention to joint pain, edema, multiple articular involvement including small hand and feet joints as risk factors for chronification beyond three months, especially in women. Future studies are needed to accomplish the identification of accurate and early biomarkers of poor clinical prognosis, which would allow better understanding of the disease's evolution and improve patients' management, modifying CHIKF burden on global public health.

The Omicron Lineages BA.1 and BA.2 (Betacoronavirus SARS-CoV-2) Have Repeatedly Entered Brazil through a Single Dispersal Hub.

Brazil currently ranks second in absolute deaths by COVID-19, even though most of its population has completed the vaccination protocol. With the introduction of Omicron in late 2021, the number of COVID-19 cases soared once again in the country. We investigated in this work how lineages BA.1 and BA.2 entered and spread in the country by sequencing 2173 new SARS-CoV-2 genomes collected between October 2021 and April 2022 and analyzing them in addition to more than 18,000 publicly available sequences with phylodynamic methods. We registered that Omicron was present in Brazil as early as 16 November 2021 and by January 2022 was already more than 99% of samples. More importantly, we detected that Omicron has been mostly imported through the state of São Paulo, which in turn dispersed the lineages to other states and regions of Brazil. This knowledge can be used to implement more efficient non-pharmaceutical interventions against the introduction of new SARS-CoV variants focused on surveillance of airports and ground transportation.

Pyrosequencing-based analysis reveals a novel capsular gene cluster in a KPC-producing Klebsiella pneumoniae clinical isolate identified in Brazil.

BACKGROUND: An important virulence factor of Klebsiella pneumoniae is the production of capsular polysaccharide (CPS), a thick mucus layer that allows for evasion of the host's defense and creates a barrier against antibacterial peptides. CPS production is driven mostly by the expression of genes located in a locus called cps, and the resulting structure is used to distinguish between different serotypes (K types). In this study, we report the unique genetic organization of the cps cluster from K. pneumoniae Kp13, a clinical isolate recovered during a large outbreak of nosocomial infections that occurred in a Brazilian teaching hospital. RESULTS: A pyrosequencing-based approach showed that the cps region of Kp13 (cpsKp13) is 26.4 kbp in length and contains genes common, although not universal, to other strains, such as the rmlBADC operon that codes for L-rhamnose synthesis. cpsKp13 also presents some unique features, like the inversion of the wzy gene and a unique repertoire of glycosyltransferases. In silico comparison of cpsKp13 RFLP pattern with 102 previously published cps PCR-RFLP patterns showed that cpsKp13 is distinct from the C patterns of all other K serotypes. Furthermore, in vitro serotyping showed only a weak reaction with capsular types K9 and K34. We confirm that K9 cps shares common genes with cpsKp13 such as the rmlBADC operon, but lacks features like uge and Kp13-specific glycosyltransferases, while K34 capsules contain three of the five sugars that potentially form the Kp13 CPS. CONCLUSIONS: We report the first description of a cps cluster from a Brazilian clinical isolate of a KPC-producing K. pneumoniae. The gathered data including K-serotyping support that Kp13's K-antigen belongs to a novel capsular serotype. The CPS of Kp13 probably includes L-rhamnose and D-galacturonate in its structure, among other residues. Because genes involved in L-rhamnose biosynthesis are absent in humans, this pathway may represent potential targets for the development of antimicrobial agents. Studying the capsular serotypes of clinical isolates is of great importance for further development of vaccines and/or novel therapeutic agents. The distribution of K-types among multidrug-resistant isolates is unknown, but our findings may encourage scientists to perform K-antigen typing of KPC-producing strains worldwide.

Phylodynamic analysis of SARS-CoV-2 spread in Rio de Janeiro, Brazil, highlights how metropolitan areas act as dispersal hubs for new variants.

During the first semester of 2021, all of Brazil has suffered an intense wave of COVID-19 associated with the Gamma variant. In July, the first cases of Delta variant were detected in the state of Rio de Janeiro. In this work, we have employed phylodynamic methods to analyse more than 1 600 genomic sequences of Delta variant collected until September in Rio de Janeiro to reconstruct how this variant has surpassed Gamma and dispersed throughout the state. After the introduction of Delta, it has initially spread mostly in the homonymous city of Rio de Janeiro, the most populous of the state. In a second stage, dispersal occurred to mid- and long-range cities, which acted as new close-range hubs for spread. We observed that the substitution of Gamma by Delta was possibly caused by its higher viral load, a proxy for transmissibility. This variant turnover prompted a new surge in cases, but with lower lethality than was observed during the peak caused by Gamma. We reason that high vaccination rates in the state of Rio de Janeiro were possibly what prevented a higher number of deaths.

Emergence of Within-Host SARS-CoV-2 Recombinant Genome After Coinfection by Gamma and Delta Variants: A Case Report.

In this study, we report the first case of intra-host SARS-CoV-2 recombination during a coinfection by the variants of concern (VOC) AY.33 (Delta) and P.1 (Gamma) supported by sequencing reads harboring a mosaic of lineage-defining mutations. By using next-generation sequencing reads intersecting regions that simultaneously overlap lineage-defining mutations from Gamma and Delta, we were able to identify a total of six recombinant regions across the SARS-CoV-2 genome within a sample. Four of them mapped in the spike gene and two in the nucleocapsid gene. We detected mosaic reads harboring a combination of lineage-defining mutations from each VOC. To our knowledge, this is the first report of intra-host RNA-RNA recombination between two lineages of SARS-CoV-2, which can represent a threat to public health management during the COVID-19 pandemic due to the possibility of the emergence of viruses with recombinant phenotypes.

Pervasive transmission of E484K and emergence of VUI-NP13L with evidence of SARS-CoV-2 co-infection events by two different lineages in Rio Grande do Sul, Brazil.

Emergence of novel SARS-CoV-2 lineages are under the spotlight of the media, scientific community and governments. Recent reports of novel variants in the United Kingdom, South Africa and Brazil (B.1.1.28-E484K) have raised intense interest because of a possible higher transmission rate or resistance to the novel vaccines. Nevertheless, the spread of B.1.1.28 (E484K) and other variants in Brazil is still unknown. In this work, we investigated the population structure and genomic complexity of SARS-CoV-2 in Rio Grande do Sul, the southernmost state in Brazil. Most samples sequenced belonged to the B.1.1.28 (E484K) lineage, demonstrating its widespread dispersion. We were the first to identify two independent events of co-infection caused by the occurrence of B.1.1.28 (E484K) with either B.1.1.248 or B.1.91 lineages. Also, clustering analysis revealed the occurrence of a novel cluster of samples circulating in the state (named VUI-NP13L) characterized by 12 lineage-defining mutations. In light of the evidence for E484K dispersion, co-infection and emergence of VUI-NP13 L in Rio Grande do Sul, we reaffirm the importance of establishing strict and effective social distancing measures to counter the spread of potentially more hazardous SARS-CoV-2 strains.

Intra-host evolution during SARS-CoV-2 prolonged infection.

Long-term infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a challenge to virus dispersion and the control of coronavirus disease 2019 (COVID-19) pandemic. The reason why some people have prolonged infection and how the virus persists for so long are still not fully understood. Recent studies suggested that the accumulation of intra-host single nucleotide variants (iSNVs) over the course of the infection might play an important role in persistence as well as emergence of mutations of concern. For this reason, we aimed to investigate the intra-host evolution of SARS-CoV-2 during prolonged infection. Thirty-three patients who remained reverse transcription polymerase chain reaction (RT-PCR) positive in the nasopharynx for on average 18 days from the symptoms onset were included in this study. Whole-genome sequences were obtained for each patient at two different time points. Phylogenetic, populational, and computational analyses of viral sequences were consistent with prolonged infection without evidence of coinfection in our cohort. We observed an elevated within-host genomic diversity at the second time point samples positively correlated with cycle threshold (Ct) values (lower viral load). Direct transmission was also confirmed in a small cluster of healthcare professionals that shared the same workplace by the presence of common iSNVs. A differential accumulation of missense variants between the time points was detected targeting crucial structural and non-structural proteins such as Spike and helicase. Interestingly, longitudinal acquisition of iSNVs in Spike protein coincided in many cases with SARS-CoV-2 reactive and predicted T cell epitopes. We observed a distinguishing pattern of mutations over the course of the infection mainly driven by increasing A→U and decreasing G→A signatures. G→A mutations may be associated with RNA-editing enzyme activities; therefore, the mutational profiles observed in our analysis were suggestive of innate immune mechanisms of the host cell defense. Therefore, we unveiled a dynamic and complex landscape of host and pathogen interaction during prolonged infection of SARS-CoV-2, suggesting that the host's innate immunity shapes the increase of intra-host diversity. Our findings may also shed light on possible mechanisms underlying the emergence and spread of new variants resistant to the host immune response as recently observed in COVID-19 pandemic.

Genomic surveillance of SARS-CoV-2 tracks early interstate transmission of P.1 lineage and diversification within P.2 clade in Brazil.

The sharp increase of COVID-19 cases in late 2020 has made Brazil the new epicenter of the ongoing SARS-CoV-2 pandemic. The novel viral lineages P.1 (Variant of Concern Gamma) and P.2, respectively identified in the Brazilian states of Amazonas and Rio de Janeiro, have been associated with potentially higher transmission rates and antibody neutralization escape. In this study, we performed the whole-genome sequencing of 185 samples isolated from three out of the five Brazilian regions, including Amazonas (North region), Rio Grande do Norte, Paraíba and Bahia (Northeast region), and Rio de Janeiro (Southeast region) in order to monitor the spread of SARS-CoV-2 lineages in Brazil in the first months of 2021. Here, we showed a widespread dispersal of P.1 and P.2 across Brazilian regions and, except for Amazonas, P.2 was the predominant lineage identified in the sampled states. We estimated the origin of P.2 lineage to have happened in February, 2020 and identified that it has differentiated into new clades. Interstate transmission of P.2 was detected since March, but reached its peak in December, 2020 and January, 2021. Transmission of P.1 was also high in December and its origin was inferred to have happened in August 2020. We also confirmed the presence of lineage P.7, recently described in the southernmost region of Brazil, to have spread across the Northeastern states. P.1, P.2 and P.7 are descended from the ancient B.1.1.28 strain, which co-dominated the first phase of the pandemic in Brazil with the B.1.1.33 strain. We also identified the occurrence of a new lineage descending from B.1.1.33 that convergently carries the E484K mutation, N.9. Indeed, the recurrent report of many novel SARS-CoV-2 genetic variants in Brazil could be due to the absence of effective control measures resulting in high SARS-CoV2 transmission rates. Altogether, our findings provided a landscape of the critical state of SARS-CoV-2 across Brazil and confirm the need to sustain continuous sequencing of the SARS-CoV-2 isolates worldwide in order to identify novel variants of interest and monitor for vaccine effectiveness.

Maternal SARS-CoV-2 Infection Associated to Systemic Inflammatory Response and Pericardial Effusion in the Newborn: A Case Report.

Vertical transmission of SARS-CoV-2 has already been described, while clinical consequences to the fetus are still under investigation. This article reports a case of systemic fetal inflammatory response and pericardial effusion. As far as is known, this is the first case of fetal/neonatal cardiac complications related to SARS-CoV-2 infection.

Turnover of SARS-CoV-2 Lineages Shaped the Pandemic and Enabled the Emergence of New Variants in the State of Rio de Janeiro, Brazil.

In the present study, we provide a retrospective genomic epidemiology analysis of the SARS-CoV-2 pandemic in the state of Rio de Janeiro, Brazil. We gathered publicly available data from GISAID and sequenced 1927 new genomes sampled periodically from March 2021 to June 2021 from 91 out of the 92 cities of the state. Our results showed that the pandemic was characterized by three different phases driven by a successive replacement of lineages. Interestingly, we noticed that viral supercarriers accounted for the overwhelming majority of the circulating virus (>90%) among symptomatic individuals in the state. Moreover, SARS-CoV-2 genomic surveillance also revealed the emergence and spread of two new variants (P.5 and P.1.2), firstly reported in this study. Our findings provided important lessons learned from the different epidemiological aspects of the SARS-CoV-2 dynamic in Rio de Janeiro. Altogether, this might have a strong potential to shape future decisions aiming to improve public health management and understanding mechanisms underlying virus dispersion.

Zika virus targets the human thymic epithelium.

Previous work showed that the thymus can be infected by RNA viruses as HIV and HTLV-1. We thus hypothesized that the thymus might also be infected by the Zika virus (ZIKV). Herein we provide compelling evidence that ZIKV targets human thymic epithelial cells (TEC) in vivo and in vitro. ZIKV-infection enhances keratinization of TEC, with a decrease in proliferation and increase in cell death. Moreover, ZIKV modulates a high amount of coding RNAs with upregulation of genes related to cell adhesion and migration, as well as non-coding genes including miRNAs, circRNAs and lncRNAs. Moreover, we observed enhanced attachment of lymphoblastic T-cells to infected TEC, as well as virus transfer to those cells. Lastly, alterations in thymuses from babies congenitally infected were seen, with the presence of viral envelope protein in TEC. Taken together, our data reveals that the thymus, particularly the thymic epithelium, is a target for the ZIKV with changes in the expression of molecules that are relevant for interactions with developing thymocytes.

Molecular alterations in the extracellular matrix in the brains of newborns with congenital Zika syndrome.

Zika virus (ZIKV) infection during pregnancy can cause a set of severe abnormalities in the fetus known as congenital Zika syndrome (CZS). Experiments with animal models and in vitro systems have substantially contributed to our understanding of the pathophysiology of ZIKV infection. Here, to investigate the molecular basis of CZS in humans, we used a systems biology approach to integrate transcriptomic, proteomic, and genomic data from the postmortem brains of neonates with CZS. We observed that collagens were greatly reduced in expression in CZS brains at both the RNA and protein levels and that neonates with CZS had several single-nucleotide polymorphisms in collagen-encoding genes that are associated with osteogenesis imperfecta and arthrogryposis. These findings were validated by immunohistochemistry and comparative analysis of collagen abundance in ZIKV-infected and uninfected samples. In addition, we showed a ZIKV-dependent increase in the expression of cell adhesion factors that are essential for neurite outgrowth and axon guidance, findings that are consistent with the neuronal migration defects observed in CZS. Together, these findings provide insights into the underlying molecular alterations in the ZIKV-infected brain and reveal host genes associated with CZS susceptibility.

Evolution and epidemic spread of SARS-CoV-2 in Brazil.

Brazil currently has one of the fastest-growing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemics in the world. Because of limited available data, assessments of the impact of nonpharmaceutical interventions (NPIs) on this virus spread remain challenging. Using a mobility-driven transmission model, we show that NPIs reduced the reproduction number from >3 to 1 to 1.6 in São Paulo and Rio de Janeiro. Sequencing of 427 new genomes and analysis of a geographically representative genomic dataset identified >100 international virus introductions in Brazil. We estimate that most (76%) of the Brazilian strains fell in three clades that were introduced from Europe between 22 February and 11 March 2020. During the early epidemic phase, we found that SARS-CoV-2 spread mostly locally and within state borders. After this period, despite sharp decreases in air travel, we estimated multiple exportations from large urban centers that coincided with a 25% increase in average traveled distances in national flights. This study sheds new light on the epidemic transmission and evolutionary trajectories of SARS-CoV-2 lineages in Brazil and provides evidence that current interventions remain insufficient to keep virus transmission under control in this country.

Genome-wide diversity and differentiation in New World populations of the human malaria parasite Plasmodium vivax.

BACKGROUND: The Americas were the last continent colonized by humans carrying malaria parasites. Plasmodium falciparum from the New World shows very little genetic diversity and greater linkage disequilibrium, compared with its African counterparts, and is clearly subdivided into local, highly divergent populations. However, limited available data have revealed extensive genetic diversity in American populations of another major human malaria parasite, P. vivax. METHODS: We used an improved sample preparation strategy and next-generation sequencing to characterize 9 high-quality P. vivax genome sequences from northwestern Brazil. These new data were compared with publicly available sequences from recently sampled clinical P. vivax isolates from Brazil (BRA, total n = 11 sequences), Peru (PER, n = 23), Colombia (COL, n = 31), and Mexico (MEX, n = 19). PRINCIPAL FINDINGS/CONCLUSIONS: We found that New World populations of P. vivax are as diverse (nucleotide diversity π between 5.2 × 10-4 and 6.2 × 10-4) as P. vivax populations from Southeast Asia, where malaria transmission is substantially more intense. They display several non-synonymous nucleotide substitutions (some of them previously undescribed) in genes known or suspected to be involved in antimalarial drug resistance, such as dhfr, dhps, mdr1, mrp1, and mrp-2, but not in the chloroquine resistance transporter ortholog (crt-o) gene. Moreover, P. vivax in the Americas is much less geographically substructured than local P. falciparum populations, with relatively little between-population genome-wide differentiation (pairwise FST values ranging between 0.025 and 0.092). Finally, P. vivax populations show a rapid decline in linkage disequilibrium with increasing distance between pairs of polymorphic sites, consistent with very frequent outcrossing. We hypothesize that the high diversity of present-day P. vivax lineages in the Americas originated from successive migratory waves and subsequent admixture between parasite lineages from geographically diverse sites. Further genome-wide analyses are required to test the demographic scenario suggested by our data.

Isolation of Infective Zika Virus from Urine and Saliva of Patients in Brazil.

BACKGROUND: Zika virus (ZIKV) is an emergent threat provoking a worldwide explosive outbreak. Since January 2015, 41 countries reported autochthonous cases. In Brazil, an increase in Guillain-Barré syndrome and microcephaly cases was linked to ZIKV infections. A recent report describing low experimental transmission efficiency of its main putative vector, Ae. aegypti, in conjunction with apparent sexual transmission notifications, prompted the investigation of other potential sources of viral dissemination. Urine and saliva have been previously established as useful tools in ZIKV diagnosis. Here, we described the presence and isolation of infectious ZIKV particles from saliva and urine of acute phase patients in the Rio de Janeiro state, Brazil. METHODOLOGY/PRINCIPAL FINDINGS: Nine urine and five saliva samples from nine patients from Rio de Janeiro presenting rash and other typical Zika acute phase symptoms were inoculated in Vero cell culture and submitted to specific ZIKV RNA detection and quantification through, respectively, NAT-Zika, RT-PCR and RT-qPCR. Two ZIKV isolates were achieved, one from urine and one from saliva specimens. ZIKV nucleic acid was identified by all methods in four patients. Whenever both urine and saliva samples were available from the same patient, urine viral loads were higher, corroborating the general sense that it is a better source for ZIKV molecular diagnostic. In spite of this, from the two isolated strains, each from one patient, only one derived from urine, suggesting that other factors, like the acidic nature of this fluid, might interfere with virion infectivity. The complete genome of both ZIKV isolates was obtained. Phylogenetic analysis revealed similarity with strains previously isolated during the South America outbreak. CONCLUSIONS/SIGNIFICANCE: The detection of infectious ZIKV particles in urine and saliva of patients during the acute phase may represent a critical factor in the spread of virus. The epidemiological relevance of this finding, regarding the contribution of alternative non-vectorial ZIKV transmission routes, needs further investigation.