Replication of Rocio virus in primary cultures of mouse neural cells.

OBJECTIVE: This study verified the replication efficiency of the Rocio virus in a primary culture of mouse neural cells. METHODS: Mixed primary cultures (neurons/glia) obtained from the brains of newborn isogenic BALB/c mice were inoculated with Rocio virus on the 7 th day of culture, and the development of cytopathogenic effects was monitored. The infection was confirmed via immunocytochemistry (anti-ROCV), while viral replication was quantified in infected primary cultures. The titration method used depended on the infection period. RESULTS: Rocio virus efficiently infected primary cultured neural cells, with the highest viral titer causing cytopathic changes was observed at 2 days post infection. The virus-infected primary culture survived for up to 7 days post infection, and viral load quantitation showed viral replication kinetics compatible with the cell death kinetics of cultures. CONCLUSION: The findings of this study suggest that mouse neural cell primary cultures support Rocio virus replication and could be used as an alternative system for studying Flavivirus infection in the central nervous system.

Alterações histopatológicas testiculares associadas a infecção experimental por vírus Zika em macacos-de-cheiro / Testicular histopathological changes associated with experimental infection by zika virus in squirrel monkeys

The Zika virus belongs to the family Flaviviridae, genus Flavivirus. It is an arbovirus whose main vectors are mosquitoes of the genus Aedes. Studies in rodents have shown that the persistence of the virus in the testicles causes damage to the reproductive tissue. This work aimed to study the effect of experimental infection by the Zika virus on fertility in non-human primates of the species Saimiri collinsi. Five pre-pubertal males (≤2 years old) of the species Saimiri collinsi were used. Three animals were infected (infected group) with the strain of Zika virus BE H815744. Two other uninfected males were used as a negative control (uninfected group). Twenty-one days after infection, infected and uninfected males were euthanized. After euthanasia, they were referred for necroscopic examination for macroscopic and microscopic evaluation. During the necropsy, the testicles were collected and fixed in 10% formaldehyde. After fixation, the tissues were processed routinely and embedded in paraffin. The slides were stained with hematoxylin and eosin for histopathological evaluation. Histopathological changes were observed in the testis of three of the five animals. Different degrees of inflammation were identified, in addition to degeneration and/or necrosis. The three animals presented a reduced number of sperm cells, with no sperm and severe necrosis. The results obtained conclude that the Zika virus can cause pathological changes in the reproductive system of males of the species Saimiri collinsi.

Alterações histopatológicas testiculares associadas a infecção experimental por vírus Zika em macacos-de-cheiro / Testicular histopathological changes associated with experimental infection by zika virus in squirrel monkeys

The Zika virus belongs to the family Flaviviridae, genus Flavivirus. It is an arbovirus whose main vectors are mosquitoes of the genus Aedes. Studies in rodents have shown that the persistence of the virus in the testicles causes damage to the reproductive tissue. This work aimed to study the effect of experimental infection by the Zika virus on fertility in non-human primates of the species Saimiri collinsi. Five pre-pubertal males (≤2 years old) of the species Saimiri collinsi were used. Three animals were infected (infected group) with the strain of Zika virus BE H815744. Two other uninfected males were used as a negative control (uninfected group). Twenty-one days after infection, infected and uninfected males were euthanized. After euthanasia, they were referred for necroscopic examination for macroscopic and microscopic evaluation. During the necropsy, the testicles were collected and fixed in 10% formaldehyde. After fixation, the tissues were processed routinely and embedded in paraffin. The slides were stained with hematoxylin and eosin for histopathological evaluation. Histopathological changes were observed in the testis of three of the five animals. Different degrees of inflammation were identified, in addition to degeneration and/or necrosis. The three animals presented a reduced number of sperm cells, with no sperm and severe necrosis. The results obtained conclude that the Zika virus can cause pathological changes in the reproductive system of males of the species Saimiri collinsi.(AU)

Favipiravir inhibits in vitro Usutu virus replication and delays disease progression in an infection model in mice.

Usutu virus (USUV) is an emerging flavivirus that causes Usutu disease mainly in birds, but infection of mammals such as rodents, bats and horses has also been demonstrated. In addition, human cases (both in immunocompromised and -competent individuals) were also reported. Large outbreaks with other flaviviruses, such as West Nile virus and Zika virus, indicate that one should be vigilant for yet other outbreaks. To allow the identification of inhibitors of USUV replication, we established in vitro antiviral assays, which were validated using a small selection of known flavivirus inhibitors, including the broad-spectrum viral RNA polymerase inhibitor favipiravir (T-705). Next, an USUV infection model in AG129 (IFN-α/ß and IFN-γ receptor knockout) mice was established. AG129 mice proved highly susceptible to USUV; an inoculum as low as 102 PFU (1.3 × 105 TCID50) resulted in the development of symptoms as early as 3 days post infection with viral RNA being detectable in various tissues. Treatment of mice with favipiravir (150 mg/kg/dose, BID, oral gavage) significantly reduced viral load in blood and tissues and significantly delayed virus-induced disease. This USUV mouse model is thus amenable for assessing the potential in vivo efficacy of (novel) USUV/flavivirus inhibitors.

The Role of Flaviviral Proteins in the Induction of Innate Immunity.

Flaviviruses are positive, single-stranded, enveloped cytoplasmic sense RNA viruses that cause a variety of important diseases worldwide. Among them, Zika virus, West Nile virus, Japanese encephalitis virus, and Dengue virus have the potential to cause severe disease. Extensive studies have been performed to elucidate the structure and replication strategies of flaviviruses, and current studies are aiming to unravel the complex molecular interactions between the virus and host during the very early stages of infection. The outcomes of viral infection and rapid establishment of the antiviral state, depends on viral detection by pathogen recognition receptors and rapid initiation of signalling cascades to induce an effective innate immune response. Extracellular and intracellular pathogen recognition receptors play a crucial role in detecting flavivirus infection and inducing a robust antiviral response. One of the main hallmarks of flaviviral nonstructural proteins is their multiple strategies to antagonise the interferon system. In this chapter, we summarize the molecular characteristics of flaviviral proteins and discuss how viral proteins target different components of the interferon signalling pathway by blocking phosphorylation, enhancing degradation, and downregulating the expression of major components of the Janus kinase/signal transducer and activator of transcription pathway. We also discuss how the interactions of viral proteins with host proteins facilitate viral pathogenesis. Due to the lack of antivirals or prophylactic treatments for many flaviviral infections, it is necessary to fully elucidate how these viruses disrupt cellular processes to influence pathogenesis and disease outcomes.

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus).

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

Cytopathological changes induced by selected Brazilian flaviviruses in mouse macrophages.

Scanning and transmission electron microscopy were used to analyse the ultrastructure of peritoneal mouse macrophage cells infected with Brazilian flavivirus (yellow fever, Rocio, Bussuquara and Saint Louis encephalitis viruses). Macrophage cells collected 3 days after viral infection had a flattened shape, with an increased number of large spikes of cytoplasm prolongations, giving an appearance of hairy cells. Cytopathological changes to the macrophage cells were similar regardless of the infecting flavivirus. Rough and smooth endoplasmic reticulum of the macrophage cells infected with flavivirus were abundant, hypertrophic and enlarged. A large number of free ribosomes were seen in the cytoplasm of these infected cells. Spherical particles approximately 50-70 nm in diameter, some of which were empty, were observed in the cytoplasm, generally inside vesicles. These particles probably correspond to viral particles.

Arboviroses da Amazônia: estudo do mecanismo de morte celular e da resposta fenotípica do hospedeiro na febre amarela / Arboviruses of the Amazonia: study of the cell death mechanism of cell death and the fenotipic host response in the yellow fever[

Os eventos histológicos no fígado foram quantificados, usando técnica de imunomarcação para avaliar 53 amostras hepáticas provenientes de viscerotomia de pacientes vitimados por febre amarela silvestre. A análise quantitativa dos eventos demonstrou amplo predomínio do componente apoptótico sobre a necrose. O infiltrado inflamatório é desproporcional em intensidade à morte dos hepatócitos. Houve predomínio de linfócitos TCD4+, ocorrendo em menor proporção linfócitos TCD8+, linfócitos B, células NK e apresentadoras de antígenos (S100). A expressão citocínica foi de perfil Th1, com expressão de TNF-a, IFN-g e acompanhada de intensa imunomarcação para TGF-b. Frente aos achados acreditamos que a predileção pela localização médio zonal das lesões do fígado poderia decorrer de fenômenos de hipóxia por hipofluxo secundária à vasculopatia sistêmica e associada ao efeito citopático viral.In this work, the histological events in the liver were quantified, using a immunomarking technique to evaluate 53 hepatic samples from the viscerotomy of patients with the sylvatic form of yellow fever. Quantitative analysis of the events demonstrated an ample prevalence of an apoptotic component in the necrosis. The intensity of the inflammatory infiltration is disproportionate to the death of the hepatocytes. There is a prevalence of TCD4+ lymphocytes, with a smaller proportion of TCD8+ lymphocytes, B lymphocytes, NK cells and antigen-presenting cells (S100). The cytokine expression presented a profile of Th1, with expression of TNF-a, IFN-g and accompanied by intense immunomarking by TGF-b. Faced with these findings, we consider that the predilection for the midzonal location of the lesions in the liver could arise from the phenomena of hypoxia secondary to systemic vasculopathy associated to the viral cytopathic effect...