Organotypic hippocampal culture model reveals differential responses to highly similar Zika virus isolates.

INTRODUCTION: Zika virus (ZIKV) caused an outbreak in Brazil, in 2015, being associated to microcephaly. ZIKV has a strong neurotropism leading to death of infected cells in different brain regions, including the hippocampus, a major site for neurogenesis. The neuronal populations of the brain are affected differently by ZIKV from Asian and African ancestral lineages. However, it remains to be investigated whether subtle variations in the ZIKV genome can impact hippocampus infection dynamics and host response. OBJECTIVE: This study evaluated how two Brazilian ZIKV isolates, PE243 and SPH2015, that differ in two specific missense amino acid substitutions, one in the NS1 protein and the other in the NS4A protein, affect the hippocampal phenotype and transcriptome. METHODS: Organotypic hippocampal cultures (OHC) from infant Wistar rats were infected with PE243 or SPH2015 and analyzed in time series using immunofluorescence, confocal microscopy, RNA-Seq and RT-qPCR. RESULTS: Unique patterns of infection and changes in neuronal density in the OHC were observed for PE243 and SPH2015 between 8 and 48 h post infection (p.i.). Phenotypic analysis of microglia indicated that SPH2015 has a greater capacity for immune evasion. Transcriptome analysis of OHC at 16 h p.i. disclosed 32 and 113 differentially expressed genes (DEGs) in response to infection with PE243 and SPH2015, respectively. Functional enrichment analysis suggested that infection with SPH2015 activates mostly astrocytes rather than microglia. PE243 downregulated biological process of proliferation of brain cells and upregulated those associated with neuron death, while SPH2015 downregulated processes related to neuronal development. Both isolates downregulated cognitive and behavioral development processes. Ten genes were similarly regulated by both isolates. They are putative biomarkers of early hippocampus response to ZIKV infection. At 5, 7, and 10 days p.i., neuronal density of infected OHC remained below controls, and mature neurons of infected OHC showed an increase in the epigenetic mark H3K4me3, which is associated to a transcriptionally active state. This feature is more prominent in response to SPH2015. CONCLUSION: Subtle genetic diversity of the ZIKV affects the dynamics of viral dissemination in the hippocampus and host response in the early stages of infection, which may lead to different long-term effects in neuronal population.

Mitochondrial DNA as a Possible Ligand for TLR9 in Irinotecan-induced Small Intestinal Mucositis.

Cancer chemotherapy and radiotherapy may result in mucositis characterized by stem cell damage and inflammation in the gastrointestinal tract. The molecular mechanisms underlying this pathology remain unknown. Based on the assumption that mitochondrial CPG-DNA (mtDNA) released and sensed by TLR9 could underlie mucositis pathology, we analyzed the mtDNA levels in sera as well as inflammatory and disease parameters in the small intestine from wild-type (WT) and TLR9-deficient mice (TLR9-/-) in an experimental model of intestinal mucositis induced by irinotecan. Additionally, we verified the ability of WT and TLR9-/- macrophages to respond to CpG-DNA in vitro. WT mice injected with irinotecan presented a progressive increase in mtDNA in the serum along with increased hematocrit, shortening of small intestine length, reduction of intestinal villus:crypt ratio and increased influx of neutrophils, which were followed by higher expression of Nlrp3 and Casp1 mRNA and increased IL-1ß levels in the ileum when compared to vehicle-injected mice. TLR9-deficient mice were protected in all these parameters when compared to WT mice. Furthermore, TLR9 was required for the production of IL-1ß and NO after macrophage stimulation with CpG-DNA. Overall, our findings show that the amount of circulating free CpG-DNA is increased upon chemotherapy and that TLR9 activation is important for NLRP3 inflammasome transcription and further IL-1ß release, playing a central role in the development of irinotecan-induced intestinal mucositis. We suggest that TLR9 antagonism may be a new therapeutic strategy for limiting irinotecan-induced intestinal inflammation.

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.

Protective immunity and safety of a genetically modified influenza virus vaccine.

Recombinant influenza viruses are promising viral platforms to be used as antigen delivery vectors. To this aim, one of the most promising approaches consists of generating recombinant viruses harboring partially truncated neuraminidase (NA) segments. To date, all studies have pointed to safety and usefulness of this viral platform. However, some aspects of the inflammatory and immune responses triggered by those recombinant viruses and their safety to immunocompromised hosts remained to be elucidated. In the present study, we generated a recombinant influenza virus harboring a truncated NA segment (vNA-Δ) and evaluated the innate and inflammatory responses and the safety of this recombinant virus in wild type or knock-out (KO) mice with impaired innate (Myd88 -/-) or acquired (RAG -/-) immune responses. Infection using truncated neuraminidase influenza virus was harmless regarding lung and systemic inflammatory response in wild type mice and was highly attenuated in KO mice. We also demonstrated that vNA-Δ infection does not induce unbalanced cytokine production that strongly contributes to lung damage in infected mice. In addition, the recombinant influenza virus was able to trigger both local and systemic virus-specific humoral and CD8+ T cellular immune responses which protected immunized mice against the challenge with a lethal dose of homologous A/PR8/34 influenza virus. Taken together, our findings suggest and reinforce the safety of using NA deleted influenza viruses as antigen delivery vectors against human or veterinary pathogens.