ATP-P2X7 signaling mediates brain pathology while contributing to viral control in perinatal Zika virus infection.

Zika virus (ZIKV), the causative agent of Zika fever, is a flavivirus transmitted by mosquitoes of the Aedes genus. Zika virus infection has become an international concern due to its association with severe neurological complications such as fetal microcephaly. Viral infection can induce the release of ATP in the extracellular environment, activating receptors sensitized by extracellular nucleotides, such as the P2X7 receptor. This receptor is the primary purinergic receptor involved in neuroinflammation, neurodegeneration, and immunity. In this work, we investigated the role of ATP-P2X7 receptor signaling in Zika-related brain abnormalities. Wild-type mice (WT) and P2X7 receptor-deficient (P2X7-/-) C57BL/6 newborn mice were subcutaneously inoculated with 5 × 106plaque-forming units of ZIKV or mock solution. P2X7 receptor expression increased in the brain of Zika virus-infected mice compared to the mock group. Comparative analyses of the hippocampi from WT and P2X7-/-mice revealed that the P2X7 receptor increased hippocampal damage in CA1/CA2 and CA3 regions. Doublecortin expression decreased significantly in the brains of ZIKV-infected mice. WT ZIKV-infected mice showed impaired motor performance compared to P2X7-/- infected mice. WT ZIKV-infected animals showed increased expression of glial markers GFAP (astrocytes) and IBA-1 (microglia) compared to P2X7-/- infected mice. Although the P2X7 receptor contributes to neuronal loss and neuroinflammation, WT mice were more efficient in controlling the viral load in the brain than P2X7 receptor-deficient mice. This result was associated with higher induction of TNF-α, IFN-ß, and increased interferon-stimulated gene expression in WT mice than P2X7-/-ZIKV-infected. Finally, we found that the P2X7 receptor contributes to inhibiting the neuroprotective signaling pathway AKT/mTOR while stimulating the caspase-3 activation, possibly two distinct pathways contributing to neurodegeneration. These findings suggest that ATP-P2X7 receptor signaling contributes to the antiviral response in the brain of ZIKV-infected mice while increasing neuronal loss, neuroinflammation, and related brain abnormalities.

Effect of Demographics and Time to Sample Processing on the qPCR Detection of Pathogenic Leptospira spp. from Human Samples in the National Reference Laboratory for Leptospirosis, Brazil.

Leptospirosis diagnosis by MAT requires antibody levels that are typically present only after the first week of symptoms, many days after infection. To improve testing capacity and to develop a fast and reliable solution for the diagnosis of this disease in the first few days after clinical manifestations, the National Reference Laboratory for Leptospirosis/WHO Collaborating Center in Brazil implemented a duplex molecular method by qPCR for human samples for the detection of the gene lipL32, conserved in pathogenic Leptospira spp. In this paper, we describe the overall performance of this protocol in the first 3 months as a standard routine. Detection of pathogenic Leptospira spp. DNA was similar between blood, plasma, and tissue samples, with a limit of detection as low as one cell per sample, and among 391 samples from suspected cases, 174 (44.6%) were positive. The average RNASEP1 control gene detection cycle thresholds (Ct) were 28.4 and 29.8 for positive and negative samples, respectively. The median sample collection interval from the beginning of symptoms was 3 days for positive and 4 days for negative samples, respectively. Neither age, sex, nor the time intervals between sample collection and DNA extraction significantly influenced the results. Surprisingly, positivity was related to the time between DNA extraction and the qPCR reaction. These data support the use of this routine as a diagnostic approach to strengthen the molecular detection of leptospirosis and to develop new strategies.

Skeletal Muscle Is an Early Site of Zika Virus Replication and Injury, Which Impairs Myogenesis.

Zika virus (ZIKV) infection became a worldwide concern due to its correlation with the development of microcephaly and other neurological disorders. ZIKV neurotropism is well characterized, but the role of peripheral viral amplification to brain infection remains unknown. Here, we found that ZIKV replicates in human primary skeletal muscle myoblasts, impairing its differentiation into myotubes but not interfering with the integrity of the already-formed muscle fibers. Using mouse models, we showed ZIKV tropism to muscle tissue either during embryogenesis after maternal transmission or when infection occurred after birth. Interestingly, ZIKV replication in the mouse skeletal muscle started immediately after ZIKV inoculation, preceding viral RNA detection in the brain and causing no disruption to the integrity of the blood brain barrier, and remained active for more than 2 weeks, whereas replication in the spleen and liver were not sustained over time. In addition, ZIKV infection of the skeletal muscle induces necrotic lesions, inflammation, and fiber atrophy. We also found a reduction in the expression of regulatory myogenic factors that are essential for muscle repair after injury. Taken together, our results indicate that the skeletal muscle is an early site of viral amplification and lesion that may result in late consequences in muscle development after ZIKV infection. IMPORTANCE Zika Virus (ZIKV) neurotropism and its deleterious effects on central nervous system have been well characterized. However, investigations of the initial replication sites for the establishment of infection and viral spread to neural tissues remain underexplored. A complete description of the range of ZIKV-induced lesions and others factors that can influence the severity of the disease is necessary to prevent ZIKV's deleterious effects. ZIKV has been shown to access the central nervous system without significantly affecting blood-brain barrier permeability. Here, we demonstrated that skeletal muscle is an earlier site of ZIKV replication, contributing to the increase of peripheral ZIKV load. ZIKV replication in muscle promotes necrotic lesions and inflammation and also impairs myogenesis. Overall, our findings showed that skeletal muscle is involved in pathogenesis and opens new fields in the investigation of the long-term consequences of early infection.

Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Advantages of Stain-Free Technology.

Western blotting is one of the most used techniques in research laboratories. It is popular because it is an easy way of semiquantifying protein amounts in different samples. In Western blotting, the most commonly used method for controlling the differences in the amount of protein loaded is to independently quantify housekeeping proteins (typically actin, GAPDH or tubulin). Another less commonly used method is total protein normalization using stains, such as Ponceau S or Coomassie Brilliant Blue, which stains all the proteins on the blots. A less commonly used but powerful total protein staining technique is stain-free normalization. The stain-free technology is able to detect total protein in a large linear dynamic range and has the advantage of allowing protein detection on the gel before transblotting. This chapter discusses the theory, advantages, and method used to do total protein quantification using stain-free gels for normalization of Western blots.

Development of standard methods for Zika virus propagation, titration, and purification.

The emergence of Zika virus (ZIKV) infection has stimulated several research groups to study and collaborate to understand virus biology and pathogenesis. These efforts may assist with the development of antiviral drugs, vaccines and diagnostic tests, as well as to promote advancements in public health policies. Here, we aim to develop standard protocols for propagation, titration, and purification of ZIKV strains, by systematically testing different cell types, kinetics, multiplicity of infection and centrifugation protocols. ZIKV produces a productive infection in human, non-human primate, and rodents-derived cell lines, with different efficacies. The highest yield of ZIKV-AFR and ZIKV-BR infectious progeny was obtained at 7days post infection in C6/36 cells (7×107 and 2×108 PFU/ml, respectively). However, high titers of ZIKV-AFR could be obtained at earlier time points in Vero cells (2.5×107PFU/ml at 72hpi), whereas ZIKV-BR titers reached 108 PFU/ml at 4dpi in C6/36 cells. High yield of purified virus was obtained by purification through a discontinuous sucrose gradient. This optimized procedure will certainly contribute to future studies of virus structure and vaccine development. Beyond the achievement of efficient virus propagation, the normalization of these protocols will also allow different laboratories around the world to better compare and discuss data regarding different features of ZIKV biology and disease, contributing to more efficient collaborations and progression in ZIKV research.