BackgroundZika 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 FindingsNine 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/SignificanceThe 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.

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Certain major histocompatibility complex class I (MHC-I) alleles (e.g., HLA-B*27) are enriched among human immunodeficiency virus type 1 (HIV-1)-infected individuals who suppress viremia without treatment (termed "elite controllers" [ECs]).Likewise, Mamu-B*08 expression also predisposes rhesus macaques to control simian immunodeficiency virus (SIV) replication. Given the similarities between Mamu-B*08 and HLA-B*27, SIV-infected Mamu-B*08؉ animals provide a model to investigate HLA-B*27-mediated elite control. We have recently shown that vaccination with three immunodominant Mamu-B*08-restricted epitopes (Vif RL8, Vif RL9, and Nef RL10) increased the incidence of elite control in Mamu-B*08 ؉ macaques after challenge with the pathogenic SIVmac239 clone. Furthermore, a correlate analysis revealed that CD8 ؉ T cells targeting Nef RL10 was correlated with improved outcome. Interestingly, this epitope is conserved between SIV and HIV-1 and exhibits a delayed and atypical escape pattern. These features led us to postulate that a monotypic vaccine-induced Nef RL10-specific CD8 ؉ T-cell response would facilitate the development of elite control in Mamu-B*08 ؉ animals following repeated intrarectal challenges with SIVmac239. To test this, we vaccinated Mamu-B*08؉ animals with nef inserts in which Nef RL10 was either left intact (group 1) or disrupted by mutations (group 2). Although monkeys in both groups mounted Nef-specific cellular responses, only those in group 1 developed Nef RL10-specific CD8 ؉ T cells. These vaccine-induced effector memory CD8 ؉ T cells did not prevent infection. Escape variants emerged rapidly in the group 1 vaccinees, and ultimately, the numbers of ECs were similar in groups 1 and 2. High-frequency vaccine-induced CD8 ؉ T cells focused on a single conserved epitope and therefore did not prevent infection or increase the incidence of elite control in Mamu-B*08 ؉ macaques. IMPORTANCESince elite control of chronic-phase viremia is a classic example of an effective immune response against HIV/SIV, elucidating the basis of this phenomenon may provide useful insights into how to elicit such responses by vaccination. We have previously established that vaccine-induced CD8 ؉ T-cell responses against three immunodominant epitopes can increase the incidence of elite control in SIV-infected Mamu-B*08؉ rhesus macaques-a model of HLA-B*27-mediated elite control. Here, we investigated whether a monotypic vaccine-induced CD8 ؉ T-cell response targeting the conserved "late-escaping" Nef RL10 epitope can increase the incidence of elite control in Mamu-B*08 ؉ monkeys. Surprisingly, vaccine-induced Nef RL10-specific CD8 ؉ T cells selected for variants within days after infection and, ultimately, did not facilitate the development of elite control. Elite control is, therefore, likely to involve CD8 ؉ T-cell responses against more than one epitope. Together, these results underscore the complexity and multidimensional nature of virologic control of lentivirus infection.

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