Daniele Barbosa de Almeida Medeiros

Zika virus in the Americas: Early epidemiological and genetic findings

Zika virus genomes from Brazil The Zika virus outbreak is a major cause for concern in Brazil, where it has been linked with increased reports of otherwise rare birth defects and neuropathology. In a phylogenetic analysis, Faria et al. infer a single introduction of Zika to the Americas and estimated the introduction date to be about May to December 2013—some 12 months earlier than the virus was reported. This timing correlates with major events in the Brazilian cultural calendar associated with increased traveler numbers from areas where Zika virus has been circulating. A correlation was also observed between incidences of microcephaly and week 17 of pregnancy. Science, this issue p. 345 Virus sequencing indicates that Zika arrived in Brazil during the middle of 2013, coincident with a surge in air travelers. Brazil has experienced an unprecedented epidemic of Zika virus (ZIKV), with ~30,000 cases reported to date. ZIKV was first detected in Brazil in May 2015, and cases of microcephaly potentially associated with ZIKV infection were identified in November 2015. We performed next-generation sequencing to generate seven Brazilian ZIKV genomes sampled from four self-limited cases, one blood donor, one fatal adult case, and one newborn with microcephaly and congenital malformations. Results of phylogenetic and molecular clock analyses show a single introduction of ZIKV into the Americas, which we estimated to have occurred between May and December 2013, more than 12 months before the detection of ZIKV in Brazil. The estimated date of origin coincides with an increase in air passengers to Brazil from ZIKV-endemic areas, as well as with reported outbreaks in the Pacific Islands. ZIKV genomes from Brazil are phylogenetically interspersed with those from other South American and Caribbean countries. Mapping mutations onto existing structural models revealed the context of viral amino acid changes present in the outbreak lineage; however, no shared amino acid changes were found among the three currently available virus genomes from microcephaly cases. Municipality-level incidence data indicate that reports of suspected microcephaly in Brazil best correlate with ZIKV incidence around week 17 of pregnancy, although this correlation does not demonstrate causation. Our genetic description and analysis of ZIKV isolates in Brazil provide a baseline for future studies of the evolution and molecular epidemiology of this emerging virus in the Americas.

A live-attenuated Zika virus vaccine candidate induces sterilizing immunity in mouse models

Zika virus (ZIKV) infection of pregnant women can cause a wide range of congenital abnormalities, including microcephaly, in the infant, a condition now collectively known as congenital ZIKV syndrome. A vaccine to prevent or significantly attenuate viremia in pregnant women who are residents of or travelers to epidemic or endemic regions is needed to avert congenital ZIKV syndrome, and might also help to suppress epidemic transmission. Here we report on a live-attenuated vaccine candidate that contains a 10-nucleotide deletion in the 3′ untranslated region of the ZIKV genome (10-del ZIKV). The 10-del ZIKV is highly attenuated, immunogenic, and protective in type 1 interferon receptor–deficient A129 mice. Crucially, a single dose of 10-del ZIKV induced sterilizing immunity with a saturated neutralizing antibody titer, which no longer increased after challenge with an epidemic ZIKV, and completely prevented viremia. The immunized mice also developed a robust T cell response. Intracranial inoculation of 1-d-old immunocompetent CD-1 mice with 1 × 104 infectious focus units (IFU) of 10-del ZIKV caused no mortality, whereas infections with 10 IFU of wild-type ZIKV were lethal. Mechanistically, the attenuated virulence of 10-del ZIKV may be due to decreased viral RNA synthesis and increased sensitivity to type-1-interferon inhibition. The attenuated 10-del ZIKV was incapable of infecting mosquitoes after oral feeding of spiked-blood meals, representing an additional safety feature. Collectively, the safety and efficacy results suggest that further development of this promising, live-attenuated ZIKV vaccine candidate is warranted.

Vaccine Mediated Protection Against Zika Virus-Induced Congenital Disease

The emergence of Zika virus (ZIKV) and its association with congenital malformations has prompted the rapid development of vaccines. Although efficacy with multiple viral vaccine platforms has been established in animals, no study has addressed protection during pregnancy. We tested in mice two vaccine platforms, a lipid nanoparticle-encapsulated modified mRNA vaccine encoding ZIKV prM and E genes and a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation, for their ability to protect against transmission to the fetus. Vaccinated dams challenged with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As modified mRNA and live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their further development in humans to prevent congenital ZIKV syndrome is warranted.

Phylogeography of dengue virus serotype 4, Brazil, 2010-2011.

Multiple origins indicate this serotype was introduced in several episodes.

Diversity and Distribution of Hantaviruses in South America

ABSTRACT Hantaviruses are important contributors to disease burden in the New World, yet many aspects of their distribution and dynamics remain uncharacterized. To examine the patterns and processes that influence the diversity and geographic distribution of hantaviruses in South America, we performed genetic and phylogeographic analyses of all available South American hantavirus sequences. We sequenced multiple novel and previously described viruses (Anajatuba, Laguna Negra-like, two genotypes of Castelo dos Sonhos, and two genotypes of Rio Mamore) from Brazilian Oligoryzomys rodents and hantavirus pulmonary syndrome cases and identified a previously uncharacterized species of Oligoryzomys associated with a new genotype of Rio Mamore virus. Our analysis indicates that the majority of South American hantaviruses fall into three phylogenetic clades, corresponding to Andes and Andes-like viruses, Laguna Negra and Laguna Negra-like viruses, and Rio Mamore and Rio Mamore-like viruses. In addition, the dynamics and distribution of these viruses appear to be shaped by both the geographic proximity and phylogenetic relatedness of their rodent hosts. The current system of nomenclature used in the hantavirus community is a significant impediment to understanding the ecology and evolutionary history of hantaviruses; here, we suggest strict adherence to a modified taxonomic system, with species and strain designations resembling the numerical system of the enterovirus genus.

Air Travel Is Associated with Intracontinental Spread of Dengue Virus Serotypes 1-3 in Brazil

Dengue virus and its four serotypes (DENV-1 to DENV-4) infect 390 million people and are implicated in at least 25,000 deaths annually, with the largest disease burden in tropical and subtropical regions. We investigated the spatial dynamics of DENV-1, DENV-2 and DENV-3 in Brazil by applying a statistical framework to complete genome sequences. For all three serotypes, we estimated that the introduction of new lineages occurred within 7 to 10-year intervals. New lineages were most likely to be imported from the Caribbean region to the North and Northeast regions of Brazil, and then to disperse at a rate of approximately 0.5 km/day. Joint statistical analysis of evolutionary, epidemiological and ecological data indicates that aerial transportation of humans and/or vector mosquitoes, rather than Aedes aegypti infestation rates or geographical distances, determine dengue virus spread in Brazil.

Understanding Zika Virus Stability and Developing a Chimeric Vaccine through Functional Analysis

ABSTRACT Compared with other flaviviruses, Zika virus (ZIKV) is uniquely associated with congenital diseases in pregnant women. One recent study reported that (i) ZIKV has higher thermostability than dengue virus (DENV [a flavivirus closely related to ZIKV]), which might contribute to the disease outcome; (ii) the higher thermostability of ZIKV could arise from an extended loop structure in domain III of the viral envelope (E) protein and an extra hydrogen-bond interaction between E molecules (V. A. Kostyuchenko, E. X. Y. Lim, S. Zhang, G. Fibriansah, T.-S. Ng, J. S. G. Ooi, J. Shi, and S.-M. Lok, Nature 533:425–428, 2016, https://doi.org/10.1038/nature17994 ). Here we report the functional analysis of the structural information in the context of complete ZIKV and DENV-2 virions. Swapping the prM-E genes between ZIKV and DENV-2 switched the thermostability of the chimeric viruses, identifying the prM-E proteins as the major determinants for virion thermostability. Shortening the extended loop of the E protein by 1 amino acid was lethal for ZIKV assembly/release. Mutations (Q350I and T351V) that abolished the extra hydrogen-bond interaction between the E proteins did not reduce ZIKV thermostability, indicating that the extra interaction does not increase the thermostability. Interestingly, mutant T351V was attenuated in A129 mice defective in type I interferon receptors, even though the virus retained the wild-type thermostability. Furthermore, we found that a chimeric ZIKV with the DENV-2 prM-E and a chimeric DENV-2 with the ZIKV prM-E were highly attenuated in A129 mice; these chimeric viruses were highly immunogenic and protective against DENV-2 and ZIKV challenge, respectively. These results indicate the potential of these chimeric viruses for vaccine development. IMPORTANCE Analysis of a recently observed high-resolution structure of ZIKV led to a hypothesis that its unusual stability may contribute to the associated, unique disease outcomes. Here we performed a functional analysis to demonstrate that viral prM-E genes are the main determinants for the high stability of ZIKV. The extra hydrogen-bond interaction (observed in the high-resolution structure) between ZIKV E proteins did not enhance virion stability, whereas the extended loop of E protein (CD loop in domain III) was essential for ZIKV assembly. More importantly, we found that a chimeric ZIKV with DENV-2 prM-E genes and a chimeric DENV-2 with ZIKV prM-E genes were highly attenuated in A129 mice. Mice immunized with these chimeric viruses generated robust neutralizing antibody responses and were fully protected from DENV-2 and ZIKV challenge, respectively, indicating that these chimeric viruses could be further developed as vaccine candidates. IMPORTANCE Analysis of a recently observed high-resolution structure of ZIKV led to a hypothesis that its unusual stability may contribute to the associated, unique disease outcomes. Here we performed a functional analysis to demonstrate that viral prM-E genes are the main determinants for the high stability of ZIKV. The extra hydrogen-bond interaction (observed in the high-resolution structure) between ZIKV E proteins did not enhance virion stability, whereas the extended loop of E protein (CD loop in domain III) was essential for ZIKV assembly. More importantly, we found that a chimeric ZIKV with DENV-2 prM-E genes and a chimeric DENV-2 with ZIKV prM-E genes were highly attenuated in A129 mice. Mice immunized with these chimeric viruses generated robust neutralizing antibody responses and were fully protected from DENV-2 and ZIKV challenge, respectively, indicating that these chimeric viruses could be further developed as vaccine candidates.

Zika virus epidemic in Brazil. I. Fatal disease in adults: Clinical and laboratorial aspects

BACKGROUND Zika virus (ZIKV) was first detected in Brazil in May 2015 and the country experienced an explosive epidemic. However, recent studies indicate that the introduction of ZIKV occurred in late 2013. Cases of microcephaly and deaths associated with ZIKV infection were identified in Brazil in November, 2015. OBJECTIVES To determine the etiology of three fatal adult cases. STUDY DESIGN Here we report three fatal adult cases of ZIKV disease. ZIKV infection in these patients was confirmed by cells culture and/or real-time reverse transcriptase polymerase chain reaction (RT-qPCR) and by antigen detection using immunohistochemical assay. Samples of brain and other selected organs taken at autopsy from three patients were also analyzed by histopathological and immunohistological examination. RESULTS The first patient, a 36-year-old man with lupus and receiving prednisone therapy, developed a fulminant ZIKV infection. At autopsy, RT-qPCR of blood and tissues was positive for ZIKV RNA, and the virus was cultured from an organ homogenate. The second patient, a previously healthy female, 16 years of age, presented classic symptoms of Zika fever, but later developed severe thrombocytopenia, anemia and hemorrhagic manifestations and died. A blood sample taken on the seventh day of her illness was positive RT-PCR for ZIKV RNA and research in the serum was positive for antinuclear factor fine speckled (1/640), suggesting Evans syndrome (hemolytic anemia an autoimmune disorder with immune thrombocytopenic purpura) secondary to ZIKV infection. The third patient was a 20-year-old woman hospitalized with fever, pneumonia and hemorrhages, who died on 13days after admission. Histopathological changes were observed in all viscera examined. ZIKV antigens were detected by immunohistochemistry in viscera specimens of patients 1 and 3. These three cases demonstrate other potential complications of ZIKV infection, in addition to microcephaly and Guillain-Barre syndrome (GBS), and they suggest that individuals with immune suppression and/or autoimmune disorders may be at higher risk of developing severe disease, if infected with ZIKV.

A single-dose live-attenuated vaccine prevents Zika virus pregnancy transmission and testis damage

Zika virus infection during pregnancy can cause congenital abnormities or fetal demise. The persistence of Zika virus in the male reproductive system poses a risk of sexual transmission. Here we demonstrate that live-attenuated Zika virus vaccine candidates containing deletions in the 3′ untranslated region of the Zika virus genome (ZIKV-3′UTR-LAV) prevent viral transmission during pregnancy and testis damage in mice, as well as infection of nonhuman primates. After a single-dose vaccination, pregnant mice challenged with Zika virus at embryonic day 6 and evaluated at embryonic day 13 show markedly diminished levels of viral RNA in maternal, placental, and fetal tissues. Vaccinated male mice challenged with Zika virus were protected against testis infection, injury, and oligospermia. A single immunization of rhesus macaques elicited a rapid and robust antibody response, conferring complete protection upon challenge. Furthermore, the ZIKV-3′UTR-LAV vaccine candidates have a desirable safety profile. These results suggest that further development of ZIKV-3′UTR-LAV is warranted for humans.Zika virus infection can result in congenital disorders and cause disease in adults, and there is currently no approved vaccine. Here Shan et al. show that a single dose of a live-attenuated Zika vaccine prevents infection, testis damage and transmission to the fetus during pregnancy in different animal models.

Immunity and immune response, pathology and pathologic changes: progress and challenges in the immunopathology of yellow fever.

Yellow fever is a viral hemorrhagic fever, which affects people living in Africa and South America and is caused by the yellow fever virus, the prototype species in the Flavivirus genus (Flaviviridae family). Yellow fever virus infection can produce a wide spectrum of symptoms, ranging from asymptomatic infection or oligosymptomatic illness to severe disease with a high fatality rate. In this review, we focus in the mechanisms associated with the physiopathology of yellow fever in humans and animal models. It has been demonstrated that several factors play a role in the pathological outcome of the severe form of the disease including direct viral cytopathic effect, necrosis and apoptosis of hepatocyte cells in the midzone, and a minimal inflammatory response as well as low‐flow hypoxia and cytokine overproduction. New information has filled several gaps in the understanding of yellow fever pathogenesis and helped comprehend the course of illness. Finally, we discuss prospects for an immune therapy in the light of new immunologic, viral, and pathologic tools. Copyright