Dendritic cells, macrophages, NK and CD8+ T lymphocytes play pivotal roles in controlling HSV-1 in the trigeminal ganglia by producing IL1-beta, iNOS and granzyme B.

BACKGROUND: Herpes simplex virus type 1 (HSV-1) cause not only mild symptoms but also blindness and encephalitis. It was previously shown that the immune response against HSV-1 occurs mainly in the trigeminal ganglia (TG) and that Toll-like receptors 2 and 9 (TLR2/9) are important in mediating this response. It was also demonstrated that iNOS (nitric oxide synthase) and interleukin 1 beta (IL-1ß) play an essential role in the defense against HSV-1 infection. Importantly, the present work aimed to identify the primary cells responsible for iNOS and IL-1ß production and search for other important molecules and cells that might or might not depend on TLR2/9 receptors to mediate the immune response against HSV-1. METHODS: C57BL/6 (wild type, WT) and TLR2/9-/- mice were infected by the intranasal route with HSV-1 (1 × 106 p.f.u.). Cells were obtained from the TG and spleen tissues and the profile of immune cells was determined by flow cytometry in infected and mock infected WT and knockout mice. The percentage of cells producing iNOS, IL-1ß, granzyme B and perforin was also determined by flow cytometry. Chemokine monocyte chemoattractant protein-1 (MCP1) was measured by Cytometric Bead Array (CBA) in the TG, spleen and lung. Expression of type I interferons (IFNs), interleukins (IL) 5 and 10, IL-1ß and granzyme B were quantified by real time PCR. RESULTS: The results indicate that dendritic cells (DCs) and monocytes/macrophages (Mo/Mϕ) were the main sources of IL-1ß and iNOS, respectively, which, together with type I IFNs, were essential for the immune response against HSV-1. Additionally, we showed that granzyme B produced by CD8+ T and NK lymphocytes and MCP-1 were also important for this immune response. Moreover, our data indicate that the robust production of MCP-1 and granzyme B is either TLR-independent or down regulated by TLRs and occurs in the TG of TLR2/9-/- infected mice. CONCLUSION: Taken together, our data provide strong evidence that the responses mediated by DCs, Mo/Mϕ, NK and CD8+ T lymphocytes through IL-1ß, iNOS and granzyme B production, respectively, together with the production of type I IFN early in the infection, are crucial to host defense against HSV-1.

Retrospective Investigation in Horses with Encephalitis Reveals Unnoticed Circulation of West Nile Virus in Brazil.

During these past years, several studies have provided serological evidence regarding the circulation of West Nile virus (WNV) in Brazil. Despite some reports, much is still unknown regarding the genomic diversity and transmission dynamics of this virus in the country. Recently, genomic monitoring activities in horses revealed the circulation of WNV in several Brazilian regions. These findings on the paucity of genomic data reinforce the need for prompt investigation of WNV infection in horses, which may precede human cases of encephalitis in Brazil. Thus, in this study, we retrospectively screened 54 suspicious WNV samples collected between 2017 and 2020 from the spinal cord and brain of horses with encephalitis and generated three new WNV genomes from the Ceará and Bahia states, located in the northeastern region of Brazil. The Bayesian reconstruction revealed that at least two independent introduction events occurred in Brazil. The first introduction event appears to be likely related to the North American outbreak, and was estimated to have occurred in March 2013.The second introduction event appears to have occurred in September 2017 and appears to be likely related to the South American outbreak. Together, our results reinforce the importance of increasing the priority of WNV genomic monitoring in equines with encephalitis in order to track the dispersion of this emerging pathogen through the country.

Viability of SARS-CoV-2 in river water and wastewater at different temperatures and solids content.

COVID-19 patients can excrete viable SARS-CoV-2 virus via urine and faeces, which has raised concerns over the possibility of COVID-19 transmission via aerosolized contaminated water or via the faecal-oral route. These concerns are especially exacerbated in many low- and middle-income countries, where untreated sewage is frequently discharged to surface waters. SARS-CoV-2 RNA has been detected in river water (RW) and raw wastewater (WW) samples. However, little is known about SARS-CoV-2 viability in these environmental matrices. Determining the persistence of SARS-CoV-2 in water under different environmental conditions is of great importance for basic assumptions in quantitative microbial risk assessment (QMRA). In this study, the persistence of SARS-CoV-2 was assessed using plaque assays following spiking of RW and WW samples with infectious SARS-CoV-2 that was previously isolated from a COVID-19 patient. These assays were carried out on autoclaved RW and WW samples, filtered (0.22 µm) and unfiltered, at 4 °C and 24 °C. Linear and nonlinear regression models were adjusted to the data. The Weibull regression model achieved the lowest root mean square error (RMSE) and was hence chosen to estimate T90 and T99 (time required for 1 log and 2 log reductions, respectively). SARS-CoV-2 remained viable longer in filtered compared with unfiltered samples. RW and WW showed T90 values of 1.9 and 1.2 day and T99 values of 6.4 and 4.0 days, respectively. When samples were filtered through 0.22 µm pore size membranes, T90 values increased to 3.3 and 1.5 days, and T99 increased to 8.5 and 4.5 days, for RW and WW samples, respectively. Remarkable increases in SARS-CoV-2 persistence were observed in assays at 4 °C, which showed T90 values of 7.7 and 5.5 days, and T99 values of 18.7 and 17.5 days for RW and WW, respectively. These results highlight the variability of SARS-CoV-2 persistence in water and wastewater matrices and can be highly relevant to efforts aimed at quantifying water-related risks, which could be valuable for understanding and controlling the pandemic.

Swine influenza A virus subtypes circulating in Brazilian commercial pig herds from 2012 to 2019.

The swine influenza A virus (SIAV) subtypes/lineages H1N1pdm09, H3N2, H1N2, and H1N1 of seasonal human origin are widespread in Brazilian swine herds. A monovalent inactivated H1N1pdm09 vaccine was licensed in Brazil in 2014. However, there are concerns about its efficacy due to the limited vaccine cross-protection against heterologous viruses and the potential for exacerbated reactions against vaccine strains. Thus, monitoring SIAVs subtypes/lineages that are circulating in the Brazilian swine population is important, by applying a fast and efficient diagnostic test in herd field samples. A RT-PCR assay was developed, using primers specific for HA subtyping of Brazilian SIAV, and was used to evaluate the occurrence of subtypes from samples collected between 2012 and 2019. From 167 field samples positive for influenza A, 117 were subtyped by nested RT-PCR assay. A higher occurrence of H1N1pdm was observed from 2012 to 2015, H3N2 in 2017, and H1hu in 2017 to 2019. A hemagglutination inhibition test was performed in serum samples received from 2017 to 2019, confirming these data. The molecular data highlights the importance of H1hu and H3N2 detection since there are no vaccines available for the subtypes/lineages and raises an alert of H1hu for its potential to infect humans. Serological data suggest a cyclical profile of occurrence between the H3N2 and H1N1pdm over time. Monitoring SIAVs circulating in Brazilian swine herds is necessary, which provides the relevant information for field veterinarians to apply effective control measures on the properties.

Avaliação de modelos murinos utilizando camundongos imunocompetentes e vírus não adaptados para o estudo da Dengue

RESUMO: Existem vários modelos murinos para estudar dengue. Alguns deles utilizam camundongos imunologicamente deficientes ou humanizados, enquanto outros grupos utilizam vírus modificados. Nosso grupo relatou anteriormente um inoculo intracraniano de DENV altamente virulento em camundongos imunologicamente competentes (C57BL/6), causando a morte dos camundongos. A infecção intracraniana realizada no presente trabalho teve o intuito de comparar a perda de peso ocorrida com este tipo de infecção previamente descrita com a perda de peso obtida após a inoculação subcutânea, utilizando a mesma amostra de camundongos e o mesmo vírus altamente virulento. Neste trabalho, apresentamos um modelo com os mesmos camundongos C57BL/6 usados anteriormente, infectados por via subcutânea com DENV altamente virulento.Neste modelo murino de inoculo subcutâneo, os níveis de citocinas, o hemograma e a produção de óxido nítrico, juntamente com a medida da perda de peso e de mortalidade, foram utilizados para a comparação dos animais infectados em relação aos não infectados, permitindo uma melhor compreensão da doença. Os camundongos foram inoculados pela via intracraniana ou subcutânea e avaliados pelos sinais clínicos da dengue. O hemograma foi realizado pela coloração panótica nos leucócitos para a contagem diferencial e contagem total de leucócitos, hemácias e plaquetas foram realizadas em câmara de Neubauer. As itocina s foram medidas por citometria de fluxo e PCR quantitativa (qPCR). O óxido nítrico foi medido pela reação de Griess após exposição de macrófagos murinos ao virus. Os camundongos infectados com DENV subcutaneamente apresentaram perda de peso, mas não apresentaram nenhum outro sinal clínico. Embora o número de hemácias não tenha sido diferente entre camundongos infectados e não infectados nos camundongos por via subcutanea, o número de leucócitos e de plaquetas diminuiu após a infecção. As citocinas TNF alfa e IFN gama aumentaram após a infecção subcutanea. A infecção subcutânea protegeu os camundongos de uma segunda infecção com o vírus por via intracraniana. A titulação viral no cérebro após a infecção intracraniana ou a soro-neutralização após a infecção subcutânea foram realizadas para confirmar a infecção dos camundongos. Também estudamos um modelo de infecção utilizando a picada do mosquito (previamente infectado com DENV) em camudongos. O resultado neste modelo foi um aumento da expressão de nos2 no cérebro dos camundongos infectados através desta picada de Aedes aegypti infectados. Foi observada também uma maior produção de óxido nítrico ex vivo nos macrófagos de C57BL/6, quando estimulados com DENV, em comparação com a produção em macrófagos não estimulados. Concluindo, o modelo subcutâneo apresentado neste trabalho tem suas limitações, como todos os outros modelos já mencionados na literatura, mas é um novo modelo, que usa camundongos imunologicamente competentes, sendo infectados pela via subcutânea com um DENV altamente infeccioso, resultando em alterações no peso, produção de IFN gama e TNF alfa e diminuição do número das plaquetas. Este modelo subcutâneo apresentado é um modelo adicional interessante para o estudo da resposta imune contra dengue.