Oropouche virus

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N2 - A description of the first outbreaks of Oropouche fever recognized in Amazonas State is presented. AB - A description of the first outbreaks of Oropouche fever recognized in Amazonas State is presented. Microbiology And Immunology.

Overview Fingerprint. Here, motivated by the lack of studies on OROV neurotropism in humans, we have examined whether OROV can directly target human brain cells in a preserved tissue context, using an adult human brain slice culture model. Moreover, this study also opens new possibilities on the use of adult human brain slice cultures to understand viral neuropathogenesis. Oropouche virus strain BeAn was kindly provided by Prof. Conditioned medium from control Vero cells mock were used as negative control.

Cultures were prepared as previously described Mendes et al. Briefly, cortical tissue anterior third of middle temporal gyrus was obtained from patients submitted to an anterior temporal lobectomy with amygdalo-hippocampectomy for the treatment of refractory temporal lobe epilepsy.

No lesion features were detected in both resonance exams and anatomopathological assessments. One third of the culture medium was exchanged for fresh medium every 24 h. The inoculum was removed, the tissue was washed five times with PBS, and once with Neurobasal A medium this medium was stored and referred to as 0 hours post-infection, hpi. In well plates containing 0. H 2 O Merck, 2. After washes, incubation with secondary antibody diluted in TBS-Tx 0.

Finally, slices were placed on frosted cut slides Knittel , dehydrated, and covered with Permount Fisher Scientific and observed under a light microscope Leica. Virus particles in the supernatant from human brain slices were precipitated using polyethylene glycol PEG as described for other Bunyavirus Hover et al. The supernatant was immediately used to viral titration.

The data was normalized by the mass of the slice correspondent to each supernatant. The tissue was imbedded in resin araldite and stained with toluidine blue to select the area of interest.

The tissue was cut with a diamond knife and the contrast were obtained by incubating the tissue with 0. After 24 h, the medium was replaced by differentiation medium containing 0. The differentiation medium was refreshed every 48 h. The results were expressed as picograms of each cytokine analyzed and normalized by the content of total protein in each slice determined by the Bradford assay. Protein concentration was determined using the Bradford assay.

The proteins were transferred to nitrocellulose membranes using a Transblot turbo apparatus Biorad. Primary antibodies were diluted in TBS-T containing 2. Cell viability in the cultured slices was assessed using the MTT assay as in Mendes et al.

Briefly, slices were incubated with 0. The homogenate was centrifuged at 5, rpm for 2 min and the supernatants were collected for quantification of O. The values were normalized by the mass of each slice. On the other hand, no conclusive information about OROV infection in human neural cells is yet available. To address this, we used a novel human brain slice culture model developed by our group Mendes et al. The slices are prepared from tissue collected at the surgical room, from adult patients undergoing surgical resection of epileptic foci.

The tissue fragment used to prepare the slice cultures correspond to the temporal cortex resected to provide access to the hippocampal epileptogenic area. A significant advantage of this model is to preserve the original cellular population and connections found in the adult human brain, as we have previously shown the presence of neurons, microglia and astrocytes by light microscopy in re-sectioned slices submitted to DAB-Nickel immunostaining Fernandes et al.

To evaluate if human neural cells in a preserved tissue architecture are susceptible to OROV infection, we have exposed human brain slices in culture to OROV for either or h, and evaluated the presence of viral antigens in the cells using a polyclonal anti-OROV antibody Rodrigues et al.

We detected viral antigens in human neural cells at both time points tested, with a significant increase in the number of infected cells at 48 hours post infection hpi compared to 24 hpi Figures 1A—C. Sequential images of cells positive for OROV in human brain slices showed virus antigens throughout the cell cytoplasm Figure 1B.

The supernatant collected at 24 hpi was not titrated because only a few cells were positive for OROV antigen by immunostaining at that time point Figure 1. The presence of OROV virus particles in infected human brain slices was also observed by transmission electron microscopy at 48 hpi, which showed structures with typical morphology and diameter expected for family Peribunyaviridae Talmon et al.

These structures were not observed in mock-infected slices data not shown. In conjunction, these data indicate that neural cells in adult human brain tissue are indeed susceptible to OROV. Figure 1. Human neural cells in adult human brain slices are susceptible to ex vivo infection by Oropouche virus OROV.

A Representative confocal images of uninfected control mock and OROV-infected slices at 24 and 48 hours post infection hpi. D Representative cytopathic effect observed in Vero cells inoculated with the 0- or 48 hpi supernatant 10 3 dilution from the one brain slice culture.

F Representative transmission electron microscopy images of a human brain slice 48 hpi. To gain information on the pattern of OROV infection in neocortical areas, we evaluated virus antigens distribution along cortical layers in OROV-infected brain slices.

Although some care must be taken when analyzing cortical tissue obtained from pharmaco-resistant epileptic patients, as it is known that in some cases neurons can present abnormal orientations Al Sufiani and Ang, , in our previous work we have been able to clearly identify all neuronal cortical layers in similar slice cultures Fernandes et al.

Here, we have seen that OROV infection is present in most of the cortical layers identified, being more readily detected in deeper layers Figure 2. In order to determine which neural cell types are infected by OROV in human brain slices in culture, we performed double immunostainings to detect the viral antigens and one of the following neural cell markers: Iba1 for microglia; NeuN for neurons; or GFAP for astrocytes.

We observed that at 48 hpi both microglial cells and neurons were positive for OROV antigen, while no infected astrocytes were detected Figure 3A. In conjunction, these quantifications indicate that microglial cells are preferentially infected by OROV in adult human brain cortex.

Figure 2. Oropouche virus infection in adult human brain slices is distributed along deep cortical layers. Adult human brain slices in culture day in vitro 4 from middle temporal gyrus were labeled with the neuronal marker NeuN, and with anti-OROV antibody C,D. A brightfield image from a control non-infected slice is also shown for reference A.

Cortical brain layers II—VI , assigned according to neuronal morphology and density, are indicated on the left. Figure 3. Oropouche virus targets microglia and neurons in adult human brain slices. We observed intense cytopathic effect at , , and 48 hpi Figure 4A , and infection was confirmed by the detection of strong OROV antigens by immunostaining Figure 4B.

In addition, infectious OROV particle production by infected differentiated SH-SY5Y cells was confirmed by TCID 50 assay in supernatants and remaining cell homogenates, which was time-dependent and reached a plateau at 48 h post infection Figure 4C , roughly in agreement with the time when most cells presented cytopathic effect.

These data reinforced the notion that OROV can infect human neurons, even though the degree of neuronal infection by OROV in the context of preserved human brain tissue seemed to be significantly less abundant than that of microglia.

Figure 4. Differentiated human neuroblastoma cells support OROV replication. The demonstration of OROV infection of both microglia and neurons in human brain slices raised the possibility of augmented release of inflammatory signals by these cells in response to OROV infection. Elevated levels of cytokines have been described as an important initial response to experimental virus infection, including against OROV Rempel et al.

This inflammatory response seems to be not associated to tissue processing prior culturing, since we have observed a clear change in microglial morphology, including increased cell body area and reduced amount and length of branches, known features of activated microglia Tay et al.

Figure 5. Oropouche virus infection induces an inflammatory and toxic response in adult human brain slices. F Cell viability in slices determined by the MTT assay 48 hpi.

Firstly, we have evaluated the expression of iNOS, a functional marker of M1 pro-inflammatory microglia phenotype Lisi et al. Finally, to investigated the impact of OROV infection on neuronal viability, we evaluated Tau phosphorylation Serine levels in OROV-exposed adult human brain slices, as increased pTau Ser has been shown to be a solid marker of neurodegeneration Wang et al.

We observed an increase in pTau ser levels in 2 out of 4 slices tested, indicating neuronal damage in part of the infected slices Figures 5D,E. This reduction in viability was not associated with a reduction in mitochondrial content Supplementary Figure 4 , suggesting that it corresponds to cell death.

Collectively, these data indicated that OROV infection triggers a microglia-mediated inflammatory response and neurotoxicity in adult human brain slices. In the present study, we have used human brain slice cultures, a powerful model in investigations on neural connections and cytoarchitecture typical of the adult human brain, to address the question about human brain susceptibility to OROV infection. We have shown that OROV is capable of infecting human neural cells, namely microglia and neurons, with preponderance of microglia infection.

Source: Emerg Infect Dis. Copy Export. Details: Alternative Title:. Personal Author:. Henry, Ronnie ; Murphy, Frederck A. In September , a virus was isolated from a year-old forest worker from the community of Vega de Oropouche, near the town of Sangre Grande, on the island of Trinidad country: Trinidad and Tobago , who presented with fever, backache, and cough, which resolved spontaneously after 3 days. The urban vector was later identified as the midge Culicoides paraensis, but the sylvatic vector remains unknown.

Virus has been isolated from the three-toed sloth, which is believed to be involved in the sylvatic transmission cycle. The virus was shown to be unique but antigenically related to Simbu virus, which had recently been described from South Africa. It therefore became a member of the large family of bunyaviruses. It derives from an Amerindian word, but the ancient meaning of the word is not clear.

Oropouche virus has since proven to be one of the most common arthropodborne viruses infecting humans in the tropics of the Western Hemisphere. Clinical signs of infection include headache, myalgia, arthralgia, and chills; no deaths have been reported. A newly recognized vesiculovirus, Calchaqui virus, and subtypes of Melao and Maguari viruses from Argentina, with serologic evidence for infections of humans and horses.

A serologic study of California serogroup bunyaviruses in Sri Lanka. Oropouche fever: A review. Viruses ; Zika virus outbreak in Haiti in molecular and clinical data. Mayaro virus in child with acute febrile illness, Haiti, Emerg Infect Dis.

Clinical and epidemiologic patterns of chikungunya virus infection and coincident arboviral disease in a school cohort in Haiti, — Clin Infect Dis. Single-reaction multiplex reverse transcription PCR for detection of zika, chikungunya, and dengue viruses. Chikungunya virus in US travelers returning from India, Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, Bwire G.

Malar J 18, Emergence of Madariaga virus as a cause of acute febrile illness in children, Haiti, — The art of animal cell culture for virus isolation. Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses. J Clin Microbiol. Detection and phylogenetic characterization of arbovirus dual-infections among persons during a chikungunya fever outbreak, Haiti Virol J ; Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription.

Nucleic Acids Res. Consensus amplification and novel multiplex sequencing method for S segment species identification of 47 viruses of the Orthobunyavirus, Phlebovirus and Nairovirus Genera of the Family Bunyaviridae.

Mol Biol Evol ; — Edgar RC. Nucleic Acids Res ; — BMC Bioinformatics ;5: Bioinformatics ; — Huson DH, Bryant D. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol. Improving marginal likelihood estimation for Bayesian phylogenetic model selection. Syst Biol. FastTree 2—approximately maximum-likelihood trees for large alignments.

Bishop DHL. Comprehensive virology, Vol. New York:Plenum Press, 1— Oropouche orthobunyavirus: Genetic characterization of full-length genomes and development of molecular methods to discriminate natural reassortments. Infect Genet Evol. WRBU: Aedes scapularis. Walter Reed Biosystematics Unit. Von B. WRBU: Psorophora ferox. Oropouche fever, an emergent disease from the Americas. Microbes Infect. First outbreak of Oropouche Fever reported in a non-endemic western region of the Peruvian Amazon: Molecular diagnosis and clinical characteristics.

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