RESUMO
BACKGROUND: Zika fever has been a global health security threat, especially in the tropical and subtropical regions where most of the cases occur. The disease is caused by Zika virus (ZIKV), which belongs to the family Flaviviridae, genus Flavivirus. The virus is transmitted by Aedes mosquitoes, mostly by Aedes aegypti, during its blood meal. In this study we present a descriptive analysis, by transmission electron microscopy (TEM), of ZIKV infection in A. aegypti elected tissues at the 3rd day of infection. ZIKV vertical transmission experiments by oral infection were conducted to explore an offspring of natural infection. RESULTS: Gut and ovary tissues harbored a higher number of viral particles. The ZIKV genome was also detected, by RT-qPCR technique, in the organism of orally infected female mosquitoes and in their eggs laid. CONCLUSIONS: The data obtained suggest that the ovary is an organ susceptible to be infected with ZIKV and that virus can be transmitted from mother to a fraction of the progeny.
Assuntos
Aedes/virologia , Mosquitos Vetores/virologia , Zika virus/fisiologia , Animais , Feminino , Intestinos/virologia , Microscopia Eletrônica de Transmissão , Ovário/virologia , Óvulo/virologia , RNA Viral/genética , Vírion/ultraestrutura , Zika virus/ultraestrutura , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologiaRESUMO
During flavivirus infection, some viral proteins move to the nucleus and cellular components are relocated from the nucleus to the cytoplasm. Thus, the integrity of the main regulator of the nuclear-cytoplasmic transport, the nuclear pore complex (NPC), was evaluated during infection with dengue virus (DENV) and Zika virus (ZIKV). We found that while during DENV infection the integrity and distribution of at least three nucleoporins (Nup), Nup153, Nup98, and Nup62 were altered, during ZIKV infection, the integrity of TPR, Nup153, and Nup98 were modified. In this work, several lines of evidence indicate that the viral serine protease NS2B3 is involved in Nups cleavage. First, the serine protease inhibitors, TLCK and Leupeptin, prevented Nup98 and Nup62 cleavage. Second, the transfection of DENV and ZIKV NS2B3 protease was sufficient to inhibit the nuclear ring recognition detected in mock-infected cells with the Mab414 antibody. Third, the mutant but not the active (WT) protease was unable to cleave Nups in transfected cells. Thus, here we describe for the first time that the NS3 protein from flavivirus plays novel functions hijacking the nuclear pore complex, the main controller of the nuclear-cytoplasmic transport.
Assuntos
Vírus da Dengue/metabolismo , Poro Nuclear/metabolismo , Serina Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Zika virus/metabolismo , Transporte Ativo do Núcleo Celular , Dengue/metabolismo , Dengue/virologia , Vírus da Dengue/ultraestrutura , Immunoblotting , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Zika virus/ultraestrutura , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologiaRESUMO
In Brazil and in other tropical areas Zika virus infection was directly associated with clinical complications as microcephaly in newborn children whose mothers were infected during pregnancy and the Guillain-Barré syndrome in adults. Recently, research has been focused on developing new vaccines and drug candidates against Zika virus infection since none of those are available. In order to contribute to vaccine and drug development efforts, it becomes important the understanding of the molecular basis of the Zika virus recognition, infection and blockade. To this purpose, it is essential the structural determination of the Zika virus proteins. The genome sequencing of the Zika virus identified ten proteins, being three structural (protein E, protein C and protein prM) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Together, these proteins are the main targets for drugs and antibody recognition. Here we examine new discoveries on high-resolution structural biology of Zika virus, observing the interactions and functions of its proteins identified via state-of-art structural methodologies as X-ray crystallography, nuclear magnetic resonance spectroscopy and cryogenic electronic microscopy. The aim of the present study is to contribute to the understanding of the structural basis of Zika virus infection at an atomic level and to point out similarities and differences to others flaviviruses.(AU)
Assuntos
Zika virus/ultraestrutura , Proteínas Virais/ultraestrutura , Elementos Estruturais de Proteínas , Genoma Viral , Espectroscopia de Ressonância MagnéticaRESUMO
BACKGROUND: During pregnancy, the Zika virus (ZIKV) replicates in the placenta and central nervous system (CNS) of infected fetuses; nevertheless, the ability of ZIKV to replicate in other fetal tissues has not been extensively characterized. METHODS: We researched whether dissemination of congenitally-acquired ZIKV outside the CNS exists by searching for the accumulation of the viral envelope protein, ZIKV ribonucleic acid (RNA), and infectious viral particles in different organs of a deceased newborn with Congenital Zika Syndrome. A real-time qualitative polymerase chain reaction (qPCR) was used to detect ZIKV RNA in the brain, thymus, lungs, kidneys, adrenal glands, spleen, liver, and small intestine. The same tissues were analyzed by indirect immunofluorescence and immunoperoxidase assays using the monoclonal antibody 4G2 to detect ZIKV envelope antigens. Isolation of infectious ZIKV in a cell culture was carried out using brain and kidney samples. RESULTS: A postmortem, virological analysis of multiple organs, such as the kidneys (epithelial cells in the renal tubules), lungs (bronchial epithelia), thymus (epithelial cells inside the Hassall's corpuscles), and brain (neurons, ependymal cells, and macrophages) revealed the presence of ZIKV RNA and envelope antigens. Other tissues of the deceased newborn tested positive by qPCR for Epstein-Barr virus and human herpesvirus 6, including the brain cortex (Epstein-Barr) and the thymus, kidneys, and adrenal glands (human herpesvirus 6). The kidneys were identified as a significant niche for viral replication, given that infectious particles were successfully isolated from renal tissues. CONCLUSIONS: Our findings demonstrate the ability of congenitally-acquired ZIKV to produce disseminated infections and the viral tropism towards epithelial cells.
Assuntos
Complicações Infecciosas na Gravidez/diagnóstico , Complicações Infecciosas na Gravidez/virologia , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologia , Zika virus/genética , Antígenos Virais , Autopsia , Biópsia , Coinfecção , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Transmissão Vertical de Doenças Infecciosas , Nefropatias/patologia , Nefropatias/virologia , México/epidemiologia , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Vigilância em Saúde Pública , RNA Viral , Adulto Jovem , Zika virus/imunologia , Zika virus/ultraestrutura , Infecção por Zika virus/epidemiologia , Infecção por Zika virus/transmissãoRESUMO
We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.
Assuntos
Evolução Molecular , Interações Hospedeiro-Patógeno , Infecção por Zika virus/virologia , Zika virus/fisiologia , Brasil/epidemiologia , Citocinas/metabolismo , Feminino , Genitália Masculina/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Masculino , Sêmen/metabolismo , Sêmen/virologia , Zika virus/classificação , Zika virus/ultraestrutura , Infecção por Zika virus/epidemiologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/transmissãoRESUMO
Zika virus (ZIKV) infection has been documented within Central and South America, Asia, and Africa. Here we report the isolation of virus from a patient infected with ZIKV returning to Japan from the Dominican Republic. The ZIKV strain was imaged by electron microscopy and its complete genome sequence was analyzed. Phylogenetic analysis and molecular characterization revealed that the strain was of Asian lineage, and carried 2 unique mutations in its NS5 region. These mutations are characteristic of strains that originated in the Dominican Republic and the USA in 2016.
Assuntos
Genoma Viral/genética , Infecção por Zika virus/epidemiologia , Zika virus/genética , Adulto , República Dominicana/epidemiologia , Feminino , Humanos , Japão/epidemiologia , Microscopia Eletrônica , Mutação/genética , Filogenia , Viagem , Zika virus/isolamento & purificação , Zika virus/ultraestrutura , Infecção por Zika virus/virologiaRESUMO
The recent outbreaks of Zika virus (ZIKV) disease have caused worldwide concerns. Guangdong province is one of the commercial centers in China and communicates frequently with the epidemic areas. To date, 65.2% of the ZIKV infection cases in China were imported via port of entry in Guangdong. The continuous surveillance of imported cases is crucial for the prevention and control of potential ZIKV infection outbreak in China. In this study, a strain of ZIKV was isolated from the serum of a 6-year-old child returning from Venezuela. The morphology of the ZIKV was analyzed in vivo and in vitro by electron microscopy, and clusters of virus particles were found in the loose cytoplasmic membrane structures. The genomic sequence of the isolated ZIKV was determined, and the alignment and phylogenetic analysis identified one unique amino acid substitution occurring in the non-structural protein 4B (NS4B), and the isolated virus belonged to the Asian lineage.
Assuntos
Infecção por Zika virus/diagnóstico , Infecção por Zika virus/transmissão , Zika virus/isolamento & purificação , Zika virus/ultraestrutura , Sequência de Aminoácidos , Animais , Criança , Genoma Viral , Humanos , Camundongos , Microscopia Eletrônica , Filogenia , RNA Viral/genética , Saliva/virologia , Homologia de Sequência de Aminoácidos , Urina/virologia , Venezuela/epidemiologia , Carga Viral , Zika virus/genética , Infecção por Zika virus/epidemiologiaRESUMO
Zika virus (ZKV) infection is a huge public health problem in Brazil because of the increased incidence of microcephaly in neonates from infected mothers. Detection of specific IgG antibodies in maternal serum samples constitutes an important approach for diagnosing ZKV infection and evaluating its relationship with neonatal microcephaly. However, as there is no serological test produced in Brazil to detect IgM and IgG antibodies against ZKV, we sought to examine specific IgG in serum samples from patients or suspected mothers to detect previous infection and to test for specificity with regard to flaviviral infections occurring in the same area. Brazilian Zika virus native antigens were obtained from infected Vero cell layers or free virions in the culture medium and then used in ELISA. We tested sera from eight ZKV RNA-diagnosed infected patients (ZKVR), seven neonates with microcephaly and their mothers after delivery (MM), 140 dengue virus IgM-positive (DM) and IgG (DG)-positive patients, and 100 yellow fever (YF)-vaccinated patients. According to the ELISA, ZKVR samples were mostly positive (7/8), and all the MM serum samples were positive for ZKV IgG (7/7). In contrast, cross-reactions for dengue or yellow fever-vaccinated patients were observed, including DM (48/95), DG (10/45) or YF (3/100) serum samples; however, these cross-reactions exhibited low antigen avidity so that 6 M urea largely removed this cross-reactivity, with only a few cross-reacting samples remaining (8/140). ELISA based on extracted virions was much more specific, with all ZKVR (8/8) and MM sera being positive for ZKV IgG (7/7) and only borderline cross-reactivity found for DM (6/95), DG (3/45) or YF (4/100)-vaccinated serum samples. This technique (ELISA) can identify specific IgG in ZKV-infected patients and may be helpful in diagnosing congenital infetions after maternal RNA virus clearance or in epidemiological studies.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Imunoglobulina G/sangue , Infecção por Zika virus/diagnóstico , Zika virus/imunologia , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Epitopos , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Células Vero , Zika virus/ultraestruturaRESUMO
Zika virus (ZIKV) has infected thousands of Brazilian people and spread to other American countries since 2015. The introduction of ZIKV brought a strong impact to public health in Brazil. It is of utmost importance to identify a susceptible cell line that will enable the isolation and identification of the virus from patient samples, viral mass production, and testing of drug and vaccine candidates. Besides real-time reverse transcriptase polymerase chain reaction diagnosis for detecting the viral genome, virus isolation in cell lines was useful in order to study the structure of the viral particle and its behaviour inside cells. Analysis of ZIKV infected cell lines was achieved using transmission electron microscopy (TEM). Blood was obtained from a Brazilian patient during the first days after presenting with signs of the disease, and ZIKV from the patient's blood was isolated in the C6/36 mosquito cell line. Afterwards, Vero cells were inoculated with the viral suspension, fixed six days after inoculation, embedded in polymers, and ultra-thin cut. Like dengue viruses, this flavivirus showed numerous virus particles present inside cellular vesicles thereby confirming the susceptibility of the Vero cell line to ZIKV replication. TEM is a unique technique available to make the virus visible.
Assuntos
Vírion/ultraestrutura , Zika virus/ultraestrutura , Animais , Técnicas de Cultura de Células , Chlorocebus aethiops , Genoma Viral , Humanos , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real , Células Vero , Replicação ViralRESUMO
Zika virus (ZIKV) has infected thousands of Brazilian people and spread to other American countries since 2015. The introduction of ZIKV brought a strong impact to public health in Brazil. It is of utmost importance to identify a susceptible cell line that will enable the isolation and identification of the virus from patient samples, viral mass production, and testing of drug and vaccine candidates. Besides real-time reverse transcriptase polymerase chain reaction diagnosis for detecting the viral genome, virus isolation in cell lines was useful in order to study the structure of the viral particle and its behaviour inside cells. Analysis of ZIKV infected cell lines was achieved using transmission electron microscopy (TEM). Blood was obtained from a Brazilian patient during the first days after presenting with signs of the disease, and ZIKV from the patient’s blood was isolated in the C6/36 mosquito cell line. Afterwards, Vero cells were inoculated with the viral suspension, fixed six days after inoculation, embedded in polymers, and ultra-thin cut. Like dengue viruses, this flavivirus showed numerous virus particles present inside cellular vesicles thereby confirming the susceptibility of the Vero cell line to ZIKV replication. TEM is a unique technique available to make the virus visible.