Current Understanding of West Nile Virus Clinical Manifestations, Immune Responses, Neuroinvasion, and Immunotherapeutic Implications

West Nile virus (WNV) is the most common mosquito-borne virus in North America. WNV-associated neuroinvasive disease affects all ages, although elderly and immunocompromised individuals are particularly at risk. WNV neuroinvasive disease has killed over 2300 Americans since WNV entered into the United States in the New York City outbreak of 1999. Despite 20 years of intensive laboratory and clinical research, there are still no approved vaccines or antivirals available for human use. However, rapid progress has been made in both understanding the pathogenesis of WNV and treatment in clinical practices. This review summarizes our current understanding of WNV infection in terms of human clinical manifestations, host immune responses, neuroinvasion, and therapeutic interventions

Tumor Necrosis Factor-Alpha Signaling May Contribute to Chronic West Nile Virus Post-Infectious Proinflammatory State

Background: West Nile virus (WNV) causes a spectrum of human disease ranging from a febrile illness (WNV fever) to severe neuroinvasive disease (meningitis, encephalitis, acute flaccid paralysis). Since WNV gained entry into North America in 1999, clinicians caring for WNV survivors have observed persistent neurological symptoms occurring long-after the production of neutralizing antibodies and clearance of the virus. Accordingly, alternative pathogeneses other than direct viral invasion have been hypothesized to explain these post-infectious symptoms. The dominant hypothesis is that antiviral inflammatory responses triggered initially to clear WNV may persist to promote a post-infectious proinflammatory state. Methods: In 4 serologically-confirmed WNV patients with persistent post-infectious symptoms (3 WNV fever, 1 neuroinvasive disease), we ordered a comprehensive cytokine panel at weeks 8, 10, 12, and 36 months post-onset of illness, respectively, to better understand the pathophysiology of the protracted symptoms. Results: All patients had abnormally elevated tumor necrosis factor alpha (TNF-α), a major molecule triggering antiviral cytokines and chronic inflammation in many human autoimmune diseases, but heretofore not reported to be upregulated in human WNV infection. Three patients also had elevations of other proinflammatory proteins. Major symptoms included fatigue, arthralgias, myalgias, generalized or multifocal pain or weakness, imbalance, headaches, cognitive problems, and symptoms of dysautonomia. Conclusion: The findings provide support for an extended post-infectious proinflammatory state that may contribute to chronic inflammation and long-term morbidity in some WNV survivors and further suggest that TNF-α may play a pathogenic role in initiating this inflammatory environment. Clinical trials may be warranted to determine if TNF-α inhibitors or other immunosuppressive agents can improve patient outcomes

Differential Expression of Genes Related to Innate Immune Responses in Ex Vivo Spinal Cord and Cerebellar Slice Cultures Infected With West Nile Virus

West Nile virus (WNV) infection results in a spectrum of neurological symptoms, ranging from a benign fever to severe WNV neuroinvasive disease with high mortality. Many who recover from WNV neuroinvasive infection present with long-term deficits, including weakness, fatigue, and cognitive problems. While neurons are a main target of WNV, other cell types, especially astrocytes, play an important role in promoting WNV-mediated central nervous system (CNS) damage. Conversely, it has been shown that cultured primary astrocytes secrete high levels of interferons (IFNs) immediately after WNV exposure to protect neighboring astrocytes, as well as neurons. However, how intrinsic responses to WNV in specific cell types and different regions of the brain modify immune protection is not fully understood. Here, we used a mouse ex vivo spinal cord slice culture (SCSC) and cerebellar slice culture (CSC) models to determine the innate immune responses specific to the CNS during WNV infection. Slices were prepared from the spinal cord and cerebellar tissue of 7⁻9-day-old mouse pups. Four-day-old SCSC or CSC were infected with 1 × 10³ or 1 × 10⁵ PFU of WNV, respectively. After 12 h exposure to WNV and 3 days post-infection in normal growth media, the pooled slice cultures were processed for total RNA extraction and for gene expression patterns using mouse Affymetrix arrays. The expression patterns of a number of genes were significantly altered between the mock- and WNV-treated groups, both in the CSCs and SCSCs. However, distinct differences were observed when CSC data were compared with SCSC. CSCs showed robust induction of interferons (IFNs), IFN-stimulated genes (ISGs), and regulatory factors. Some of the antiviral genes related to IFN were upregulated more than 25-fold in CSCs as compared to mock or SCSC. Though SCSCs had twice the number of dysregulated genes, as compared CSCs, they exhibited a much subdued IFN response. In addition, SCSCs showed astrogliosis and upregulation of astrocytic marker genes. In sum, our results suggest that early anti-inflammatory response to WNV infection in CSCs may be due to large population of distinct astrocytic cell types, and lack of those specialized astrocytes in SCSC may make spinal cord cells more susceptible to WNV damage. Further, the understanding of early intrinsic immune response events in WNV-infected ex vivo culture models could help develop potential therapies against WNV

Congenital Zika Virus Infection in Immunocompetent Mice Causes Postnatal Growth Impediment and Neurobehavioral Deficits

A small percentage of babies born to Zika virus (ZIKV)-infected mothers manifest severe defects at birth, including microcephaly. Among those who appeared healthy at birth, there are increasing reports of postnatal growth or developmental defects. However, the impact of congenital ZIKV infection in postnatal development is poorly understood. Here, we report that a mild congenital ZIKV-infection in pups born to immunocompetent pregnant mice did not display apparent defects at birth, but manifested postnatal growth impediments and neurobehavioral deficits, which include reduced locomotor and cognitive deficits that persisted into adulthood. We found that the brains of these pups were smaller, had a thinner cortical layer 1, displayed increased astrogliosis, decreased expression of microcephaly- and neuron development- related genes, and increased pathology as compared to mock-infected controls. In summary, our results showed that even a mild congenital ZIKV infection in immunocompetent mice could lead to postnatal deficits, providing definitive experimental evidence for a necessity to closely monitor postnatal growth and development of presumably healthy human infants, whose mothers were exposed to ZIKV infection during pregnancy

Corticosteroids for Severe Coronavirus Disease 2019: High Time for High Dose Clinical Trials?

Over 3 million deaths into the COVID-19 pandemic, low dose corticosteroids are still the chief therapeutic intervention to decrease mortality in critically ill COVID-19 patients. However, the persistently high mortality rate (approximately 30%) and the global surge in cases in 2021 associated with new more contagious variants of SARS-CoV-2 underscore the urgent need to evaluate whether the current corticosteroid regimens achieve the best possible outcomes. Our review of clinical, virological, and immunological data suggest that corticosteroids reduce COVID-19 mortality more when initiated in the second or third weeks after symptom onset, after viral infectivity has waned and SARS-CoV-2 immunopathology is maximal. At this time, clinical deterioration is driven by aberrant hyperinflammation. To counteract the hyperinflammation, we present credible arguments that high dose corticosteroids, defined in this Perspective as intravenous (i.v.) methylprednisolone 500-1000 mg/day for 3-7 days or the equivalent doses of other corticosteroids, can be administered with little or no risk of worsening viral replication and with expected benefits that greatly outweigh potential harm. Since the 1970s, the strategy of administering supraphysiologic doses of i.v. methylprednisolone (termed ‘pulse’ therapy) over a short period to enhance therapeutic benefit while decreasing harmful effects has become an accepted standard of care to effectively suppress inflammation in many autoimmune and immune-mediated disorders. It is particularly useful in conditions requiring rapid immunosuppression and anti-inflammatory effect and has recently been used to control inflammation in SARS-CoV-2 related complications, including multisystem inflammatory syndrome in children (MIS-C) and organizing pneumonia. In aggregate, awareness of the need for more aggressive immunosuppression and lessons learned over a half-century from the successful treatment of various inflammatory disorders, provide a sound scientific and ethics rationale for expeditiously conducting high dose corticosteroid randomized clinical trials (RCTs) in critically ill patients with COVID-19

Microsomal Ca²⁺ flux modulation as an indicator of heavy metal toxicity

737-743Inositol 1,4,5-trisphosphatee (IP3), an intracellular messenger, releases Ca2+ from microsomes. Ca2+ plays a major role in regulating various cellular events like neural transmission and regulation of hormones and growth factors. Aluminum (Al), lead (Pb) and mercury (Hg) were reported to alter Ca2+-regulated events thereby causing neurotoxicity. Hence, an attempt was made characterize IP3 mediated Ca2+ release from rat brain microsomes under the influence of Al, Pb and Hg. Different concentrations of metals were tested over a designated time scale and their effects on IP3 mediated Ca2+ release from microsomes were monitored using Fura-2 technique. All the three metals inhibited IP3 mediated Ca2+ release, Pb being more potent. The order of potency of these three metals was Pb>Hg>Al. Except for Al, both Hg and Pb independently released Ca2+ from microsomes. Re-uptake of Ca2+ into microsomes was inhibited by all the three metals, Pb being more potent. Microsomal Ca2+-ATPase activity was also inhibited by all the three metals. These results suggest that neurotoxicity exerted by Al, Pb and Hg may be due to the interference of these metals with IP3 mediated calcium release and also interfering with the microsomal Ca2+ sequestration mechanism. Differential effects of heavy metal induced changes in Ca2+ flux can be used as an index of relative toxicity

Effects of UV Inactivated West Nile Particles on Astrocytic Morphology and Expression of Marker Proteins

West Nile virus (WNV) infection results in a spectrum of neurological symptoms ranging from a bening fever to severe West Nile neuroinvasive disease (WNND). The paralytic form of WNND has a poor prognosis, yet potential causes of neuronal death are not well understood. Significantly high levels of reactive astroglial marker proteins have been reported in the serum and cerebrospinal fluid of WNV patients. The spinal cord postmortem tissue shows areas of swollen axons, hypertrophied neuronal cell bodies, and a marked increase in astroglial reactivity. Thus, we hypothesized that factors secreted by astrocytes during or after acute WNV infection trigger neurodegeneration that contributes to WNND. To test this, astrocytic cell lines and dissociated astrocytic primary cultures from mouse brain were treated with UV inactivated WNV particles. We found significantly increased astrocyte specific factors in WNV treated astroglial cells compared with the mock controls. Real-Time PCR showed upregulated mRNA expression of some of those factors. The results suggest that higher levels of astrocytic proteins within the brain or circulating in the CSF or blood may stimulate secondary pathways leading to neurodegeneration. More studies are needed to detertmine whether these changes are associated with severe and prolonged WNV neuroinvasive disease