Multivalent and Sequential Heterologous Spike Protein Vaccinations Effectively Induce Protective Humoral Immunity against SARS-CoV-2 Variants.

The emergence of new SARS-CoV-2 variants continues to cause challenging problems for the effective control of COVID-19. In this study, we tested the hypothesis of whether a strategy of multivalent and sequential heterologous spike protein vaccinations would induce a broader range and higher levels of neutralizing antibodies against SARS-CoV-2 variants and more effective protection than homologous spike protein vaccination in a mouse model. We determined spike-specific IgG, receptor-binding inhibition titers, and protective efficacy in the groups of mice that were vaccinated with multivalent recombinant spike proteins (Wuhan, Delta, Omicron), sequentially with heterologous spike protein variants, or with homologous spike proteins. Trivalent (Wuhan + Delta + Omicron) and sequential heterologous spike protein vaccinations were more effective in inducing serum inhibition activities of receptor binding to spike variants and virus neutralizing antibody titers than homologous spike protein vaccination. The higher efficacy of protection was observed in mice with trivalent and sequential heterologous spike protein vaccination after a challenge with a mouse-adapted SARS-CoV-2 MA10 strain compared to homologous spike protein vaccination. This study provides evidence that a strategy of multivalent and sequential heterologous variant spike vaccination might provide more effective protection against emerging SARS-CoV-2 variants than homologous spike vaccination and significantly alleviate severe inflammation due to COVID-19.

The critical role of interleukin-6 in protection against neurotropic flavivirus infection.

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are emerging mosquito-borne flaviviruses causing encephalitis globally. No specific drug or therapy exists to treat flavivirus-induced neurological diseases. The lack of specific therapeutics underscores an urgent need to determine the function of important host factors involved in flavivirus replication and disease progression. Interleukin-6 (IL-6) upregulation has been observed during viral infections in both mice and humans, implying that it may influence the disease outcome significantly. Herein, we investigated the function of IL-6 in the pathogenesis of neurotropic flavivirus infections. First, we examined the role of IL-6 in flavivirus-infected human neuroblastoma cells, SK-N-SH, and found that IL-6 neutralization increased the WNV or JEV replication and inhibited the expression of key cytokines. We further evaluated the role of IL-6 by infecting primary mouse cells derived from IL-6 knockout (IL-6-/-) mice and wild-type (WT) mice with WNV or JEV. The results exhibited increased virus yields in the cells lacking the IL-6 gene. Next, our in vivo approach revealed that IL-6-/- mice had significantly higher morbidity and mortality after subcutaneous infection with the pathogenic WNV NY99 or JEV Nakayama strain compared to WT mice. The non-pathogenic WNV Eg101 strain did not cause mortality in WT mice but resulted in 60% mortality in IL-6-/- mice, indicating that IL-6 is required for the survival of mice after the peripheral inoculation of WNV or JEV. We also observed significantly higher viremia and brain viral load in IL-6-/- mice than in WT mice. Subsequently, we explored innate immune responses in WT and IL-6-/- mice after WNV NY99 infection. Our data demonstrated that the IL-6-/- mice had reduced levels of key cytokines in the serum during early infection but elevated levels of proinflammatory cytokines in the brain later, along with suppressed anti-inflammatory cytokines. In addition, mRNA expression of IFN-α and IFN-ß was significantly lower in the infected IL-6-/- mice. In conclusion, these data suggest that the lack of IL-6 exacerbates WNV or JEV infection in vitro and in vivo by causing an increase in virus replication and dysregulating host immune response.

Intrinsic antiviral immunity of barrier cells revealed by an iPSC-derived blood-brain barrier cellular model.

Physiological blood-tissue barriers play a critical role in separating the circulation from immune-privileged sites and denying access to blood-borne viruses. The mechanism of virus restriction by these barriers is poorly understood. We utilize induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (iBMECs) to study virus-blood-brain barrier (BBB) interactions. These iPSC-derived cells faithfully recapitulate a striking difference in in vivo neuroinvasion by two alphavirus isolates and are selectively permissive to neurotropic flaviviruses. A model of cocultured iBMECs and astrocytes exhibits high transendothelial electrical resistance and blocks non-neurotropic flaviviruses from getting across the barrier. We find that iBMECs constitutively express an interferon-induced gene, IFITM1, which preferentially restricts the replication of non-neurotropic flaviviruses. Barrier cells from blood-testis and blood-retinal barriers also constitutively express IFITMs that contribute to the viral resistance. Our application of a renewable human iPSC-based model for studying virus-BBB interactions reveals that intrinsic immunity at the barriers contributes to virus exclusion.

SARS-CoV-2 Infects Primary Neurons from Human ACE2 Expressing Mice and Upregulates Genes Involved in the Inflammatory and Necroptotic Pathways.

Transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) have been extensively used to investigate the pathogenesis and tissue tropism of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Neuroinvasion and the replication of SARS-CoV-2 within the central nervous system (CNS) of K18-hACE2 mice is associated with increased mortality; although, the mechanisms by which this occurs remain unclear. In this study, we generated primary neuronal cultures from K18-hACE2 mice to investigate the effects of a SARS-CoV-2 infection. We also evaluated the immunological response to SARS-CoV-2 infection in the CNS of K18-hACE2 mice and mouse neuronal cultures. Our data show that neuronal cultures obtained from K18-hACE2 mice are permissive to SARS-CoV-2 infection and support productive virus replication. Furthermore, SARS-CoV-2 infection upregulated the expression of genes involved in innate immunity and inflammation, including IFN-α, ISG-15, CXCL10, CCL2, IL-6 and TNF-α, in the neurons and mouse brains. In addition, we found that SARS-CoV-2 infection of neurons and mouse brains activates the ZBP1/pMLKL-regulated necroptosis pathway. Together, our data provide insights into the neuropathogenesis of SARS-CoV-2 infection in K18-hACE2 mice.

Nutritional interventions for improving the endurance performance in athletes.

Endurance refers to the ability of skeletal muscles to perform continuously withstanding the hardships of exercise. Endurance exercises have three phases: pre-, during-, and post-workout phase. The nutritional requirements that drive these phases vary on intensity, type of workout, individual's body composition, training, weather conditions, etc. Generally, the pre-workout phase requires glycogen synthesis and spare glycogen breakdown. While workout phase, requires rapid absorption of exogenous glucose, insulin release to transport glucose into muscle cells, replenish the loss of electrolytes, promote fluid retention, etc. However, post-workout phase requires quick amino acid absorption, muscle protein synthesis, repair of damaged muscle fibres and tendon, ameliorate inflammation, oxidative stress, etc. Therefore, nutritional sources that can help these metabolic requirements is recommended. In this review, various dietary interventions including timing and amount of nutrient consumption that can promote the above metabolic requirements that in turn support in improving the endurance potential in athletes are discussed.HIGHLIGHTSReview article describes nutritional requirements of endurance exercises.It also describes nutritional interventions to enhance the endurance potential in athletes.

DoseCal: a virtual calculator for dosage conversion between human and different animal species.

CONTEXT: The scientists as well as pharmacologists frequently convert the dose of drugs or extracts between human and different animal species. According to the literature, the extrapolation of the dose by simple conversion based only on body weight is not an accurate method. The miscalculation of dosage conversion may result in adverse effects due to overdose or reduced potency due to underdose. AIM: To develop a conversion calculator with factual factor like Km factor. METHOD: The virtual calculator was developed as a web-based application using hypertext pre-processor programming language. My SQL software was used to store the data related to DoseCal. RESULTS: Based on the Km factor, dose conversion calculator called DoseCal was developed. The DoseCal would provide the dosage for both per kg and actual weight of the animal taken for the experiment. CONCLUSION: The DoseCal would assist scientists in the dosage conversion easily and accurately between species.

SARS-CoV-2 variants of concern infect the respiratory tract and induce inflammatory response in wild-type laboratory mice

The emergence of new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern poses a major threat to the public health due to possible enhanced virulence, transmissibility and immune escape. These variants may also adapt to new hosts in part through mutations in the spike protein. In this study, we evaluated the infectivity and pathogenicity of SARS-CoV-2 variants of concern in wild-type C57BL/6 mice. Six-week-old mice were inoculated intranasally with a representative virus from the original B.1 lineage or emerging B.1.1.7 and B.1.351 lineages. We also infected a group of mice with a mouse-adapted SARS-CoV-2 (MA10). Viral load and mRNA levels of multiple cytokines and chemokines were analyzed in the lung tissues on day 3 after infection. Our data show that unlike the B.1 virus, the B.1.1.7 and B.1.351 viruses are capable of infecting C57BL/6 mice and replicating at high concentrations in the lungs. The B.1.351 virus replicated to higher titers in the lungs compared to the B.1.1.7 and MA10 viruses. The levels of cytokines (IL-6, TNF-, IL-1{beta}) and chemokine (CCL2) were upregulated in response to the B.1.1.7 and B.1.351 infection in the lungs. Overall, these data indicate a greater potential for infectivity and adaptation to new hosts by emerging SARS-CoV-2 variants.

Neuroinvasion and Encephalitis Following Intranasal Inoculation of SARS-CoV-2 in K18-hACE2 Mice.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can cause neurological disease in humans, but little is known about the pathogenesis of SARS-CoV-2 infection in the central nervous system (CNS). Herein, using K18-hACE2 mice, we demonstrate that SARS-CoV-2 neuroinvasion and encephalitis is associated with mortality in these mice. Intranasal infection of K18-hACE2 mice with 105 plaque-forming units of SARS-CoV-2 resulted in 100% mortality by day 6 after infection. The highest virus titers in the lungs were observed on day 3 and declined on days 5 and 6 after infection. By contrast, very high levels of infectious virus were uniformly detected in the brains of all the animals on days 5 and 6. Onset of severe disease in infected mice correlated with peak viral levels in the brain. SARS-CoV-2-infected mice exhibited encephalitis hallmarks characterized by production of cytokines and chemokines, leukocyte infiltration, hemorrhage and neuronal cell death. SARS-CoV-2 was also found to productively infect cells within the nasal turbinate, eye and olfactory bulb, suggesting SARS-CoV-2 entry into the brain by this route after intranasal infection. Our data indicate that direct infection of CNS cells together with the induced inflammatory response in the brain resulted in the severe disease observed in SARS-CoV-2-infected K18-hACE2 mice.

Preeclampsia: Pathophysiology and management.

Preeclampsia is a pregnancy-related multisystem disorder, frequently encountered pregnancy-related medical complications next to gestational diabetes mellitus. It is the onset of hypertension during pregnancy. The preeclampsia can be of two types, placental or maternal preeclampsia. Among these two types former, i.e., placental preeclampsia is more severe than the latter. According to the recent survey by National Health Portal of India, the incidence of preeclampsia is about 8-10 % among pregnant women. Though our understanding of preeclampsia has improved in recent years, the development and interpretation of the clinical tests remain difficult for preeclampsia. Hence, we have made an attempt to understand the pathophysiology, associated conditions/consequences, treatment and management/prevention of the condition in this review.

Neuroinvasion and encephalitis following intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can cause neurological disease in humans, but little is known about the pathogenesis of SARS-CoV-2 infection in the central nervous system. Herein, using K18-hACE2 mice, we demonstrate that SARS-CoV-2 neuroinvasion and encephalitis is associated with mortality in these mice. Intranasal infection of K18-hACE2 mice with 105 plaque-forming units of SARS-CoV-2 resulted in 100% mortality by day 6 after infection. The highest virus titers in the lungs were observed at day 3 and declined at days 5 and 6 after infection. In contrast, very high levels of infectious virus were uniformly detected in the brains of all the animals at days 5 and 6. Onset of severe disease in infected mice correlated with peak viral levels in the brain. SARS-CoV-2-infected mice exhibited encephalitis hallmarks characterized by production of cytokines and chemokines, leukocyte infiltration, hemorrhage and neuronal cell death. SARS-CoV-2 was also found to productively infect cells within the nasal turbinate, eye and olfactory bulb, suggesting SARS-CoV-2 entry into the brain by this route after intranasal infection. Our data indicate that direct infection of CNS cells together with the induced inflammatory response in the brain resulted in the severe disease observed in SARS-CoV-2-infected K18-hACE2 mice.

Immune-Boosting, Antioxidant and Anti-inflammatory Food Supplements Targeting Pathogenesis of COVID-19.

The COVID-19 is an acute and contagious disease characterized by pneumonia and ARDS. The disease is caused by SARS-CoV-2, which belongs to the family of Coronaviridae along with MERS-CoV and SARS-CoV-1. The virus has the positive-sense RNA as its genome encoding for ~26 proteins that work together for the virus survival, replication, and spread in the host. The virus gets transmitted through the contact of aerosol droplets from infected persons. The pathogenesis of COVID-19 is highly complex and involves suppression of host antiviral and innate immune response, induction of oxidative stress followed by hyper inflammation described as the "cytokine storm," causing the acute lung injury, tissue fibrosis, and pneumonia. Currently, several vaccines and drugs are being evaluated for their efficacy, safety, and for determination of doses for COVID-19 and this requires considerable time for their validation. Therefore, exploring the repurposing of natural compounds may provide alternatives against COVID-19. Several nutraceuticals have a proven ability of immune-boosting, antiviral, antioxidant, anti-inflammatory effects. These include Zn, vitamin D, vitamin C, curcumin, cinnamaldehyde, probiotics, selenium, lactoferrin, quercetin, etc. Grouping some of these phytonutrients in the right combination in the form of a food supplement may help to boost the immune system, prevent virus spread, preclude the disease progression to severe stage, and further suppress the hyper inflammation providing both prophylactic and therapeutic support against COVID-19.

Cellular microRNA-155 Regulates Virus-Induced Inflammatory Response and Protects against Lethal West Nile Virus Infection.

West Nile virus (WNV) is a flavivirus that has disseminated globally as a significant cause of viral encephalitis in humans. MircoRNA-155 (miR-155) regulates various aspects of innate and adaptive immune responses. We previously reported that WNV infection induces upregulation of miR-155 in mice brains. In the current study, we demonstrate the critical role of miR-155 in restricting the pathogenesis of WNV infection in mice. Compared to wild-type (WT) mice, miR-155 knockout mice exhibited significantly higher morbidity and mortality after infection with either a lethal strain, WNV NY99, or a non-lethal strain, WNV Eg101. Increased mortality in miR-155-/- mice was associated with significantly high WNV burden in the serum and brains. Protein levels of interferon (IFN)-α in the serum and brains were higher in miR-155-/- mice. However, miR-155-/- mice exhibited significantly lower protein levels of anti-viral interleukin (IL)-1ß, IL-12, IL-6, IL-15, and GM-CSF despite the high viral load. Primary mouse cells lacking miR-155 were more susceptible to infection with WNV compared to cells derived from WT mice. Besides, overexpression of miR-155 in human neuronal cells modulated anti-viral cytokine response and resulted in significantly lower WNV replication. These data collectively indicate that miR-155 restricts WNV production in mouse and human cells and protects against lethal WNV infection in mice.

Experimental models of polycystic ovary syndrome: An update.

Polycystic ovary syndrome (PCOS) is one of the major endocrine disorders in women. PCOS is a disorder with alterations in the structure as well as functions of female reproductive organs and is also associated with metabolic disorders. Studies on humans have limitations due to ethical issues, hence animal models are used to understand the different aspects of PCOS. Animal models of PCOS aids in studying various facts beginning from etiology to the treatment, hence, several animal models have been developed. Despite of the establishment of several models and a number of studies on PCOS, lacunae exist. This may be due to the failure in selecting a suitable animal model, as all animal models may not exhibit all the key features of the human PCOS condition or may exhibit traits similar to other diseased conditions in addition to the PCOS which should be excluded. Therefore, in this review, we have discussed the different animal models, features they exhibit, their merits and limitations which may aid in the selection of the relevant animal model of PCOS based upon the investigation's focus. In addition, a few nonmammalian models as an alternative to mammalian models have also been discussed which is to be validated further.

Z-DNA-Binding Protein 1 Is Critical for Controlling Virus Replication and Survival in West Nile Virus Encephalitis.

West Nile virus (WNV), a neurotropic flavivirus, is the leading cause of viral encephalitis in the United States. Recently, Zika virus (ZIKV) infections have caused serious neurological diseases and birth defects, specifically Guillain-Barrè syndrome and microcephaly. Z-DNA binding protein 1 (ZBP1) is a cytoplasmic sensor that that has been shown to play a significant role in initiating a robust immune response. We previously reported that WNV and ZIKV infections induce dramatic up-regulation of ZBP1 in mouse brains as well as in infected primary mouse cells. Herein, we show the critical role of ZBP1 in restricting the pathogenesis of WNV and ZIKV infections. Deletion of ZBP1 resulted in significantly higher morbidity and mortality after infection with a pathogenic WNV NY99 strain in mice. No mortality was observed in wild-type (WT) mice infected with the non-pathogenic WNV strain, Eg101. Interestingly, infection of ZBP1-/- mice with WNV Eg101 was lethal resulting in 100% mortality, suggesting that ZBP1 is required for survival after WNV infection. Viremia and brain viral load were significantly higher in ZBP1-/- mice compared to WT mice. In addition, protein levels of interferon (IFN)-α, and inflammatory cytokines and chemokines were significantly higher in the serum and brains of infected ZBP1-/- mice compared to the WT mice. Primary mouse cortical neurons and mouse embryonic fibroblasts (MEFs) derived from ZBP1-/- mice produced higher virus titers compared to WT cells after infection with WNV NY99 and WNV Eg101. Similarly, neurons and MEFs lacking ZBP1 exhibited significantly enhanced replication of PRVABC59 (Asian) and MR766 (African) ZIKV compared to WT cells. The knockout of ZBP1 function in MEFs inhibited ZBP1-dependent virus-induced cell death. In conclusion, these data reveal that ZBP1 restricts WNV and ZIKV production in mouse cells and is required for survival of a peripheral WNV infection in mice.

Rapid-scan EPR with triangular scans and fourier deconvolution to recover the slow-scan spectrum.

Direct-detected rapid-scan EPR signals were recorded using triangular field scan rates between 1.7 and 150 kG/s for deoxygenated samples of lithium phthalocyanine (LiPc) and Nycomed trityl-CD3. These scan rates are rapid relative to the reciprocals of the electron spin relaxation times and cause characteristic oscillations in the signals. Fourier deconvolution with an analytical function permitted recovery of lineshapes that are in good agreement with experimental slow-scan spectra. Unlike slow-scan EPR, direct detection rapid-scan EPR does not use phase sensitive detection and records the absorption signal directly instead of the first derivative of the absorption signal. The amplitude of the signal decreases approximately linearly with applied magnetic field gradient. Images of phantoms constructed from samples of LiPc and trityl-CD3 were reconstructed by filtered back-projection from data sets with a missing angle. The lineshapes in spectral slices from the image are in good agreement with slow-scan spectra and the spacing between sample tubes matches well with the known sample geometry.

On the analysis of broad Dysonian electron paramagnetic resonance spectra.

We analyze the equation used for simulating the lineshapes of broad electron paramagnetic resonance spectra in conducting samples (i.e., broad Dysonian lineshapes) where it becomes necessary to include the effects of both clockwise and counterclockwise rotating components of the microwave magnetic field. Using symmetry arguments, we propose a modification to the equation. We show that the modified equation fits the experimental results better than the equation used in literature.