Viral encephalitis is a significant cause of human morbidity and mortality in large part due to suboptimal diagnosis and treatment. Murine reovirus infection serves as a classic experimental model of viral encephalitis. Infection of neonatal mice with T3 reoviruses results in lethal encephalitis associated with neuronal infection, apoptosis, and CNS tissue injury. We have developed an ex vivo brain slice culture (BSC) system that recapitulates the basic pathological features and kinetics of viral replication seen in vivo. We utilize the BSC model to identify an innate, brain-tissue specific inflammatory cytokine response to reoviral infection, which is characterized by the release of IL6, CXCL10, RANTES, and murine IL8 analog (KC). Additionally, we demonstrate the potential utility of this system as a pharmaceutical screening platform by inhibiting reovirus-induced apoptosis and CNS tissue injury with the pan-caspase inhibitor, Q-VD-OPh. Cultured brain slices not only serve to model events occurring during viral encephalitis, but can also be utilized to investigate aspects of pathogenesis and therapy that are not experimentally accessible in vivo.

The Zika virus (ZIKV), like other flaviviruses, produces several species of sub-genomic RNAs (sfRNAs) during infection, corresponding to noncoding RNA fragments of different lengths that result from the exonuclease degradation of the viral 3' untranslated region (UTR). Over the course of infection, these sfRNAs accumulate in the cell as a result of an incomplete viral genome degradation of the 3' UTR by the host 5' to 3' exoribonuclease, Xrn1. The halting of Xrn1 in the 3' UTR is due to two RNA pseudoknot structures in the 3' UTR, termed exoribonuclease-resistant RNA1 and 2 (xrRNA1&2). Studies with related flaviviruses have shown that sfRNAs are important for pathogenicity and inhibiting both mosquito and mammalian host defense mechanisms. However, these investigations have not included ZIKV and there is very limited data addressing how sfRNAs impact infection in a whole animal model or specific tissues. In this study, we generate a sfRNA1-deficient ZIKV (X1) by targeted mutation in the xrRNA1 3' UTR structure. We find that the X1 virus lacks the production of the largest ZIKV sfRNA species, sfRNA1. Using the X1 virus to infect adult Ifnar1-/- mice, we find that while the lack of sfRNA1 does not alter ZIKV replication in the spleen, there is a significant reduction of ZIKV genome replication in the brain and placenta compared to wild-type ZIKV infection. Despite the attenuated phenotype of the X1 ZIKV, mice develop a robust neutralizing antibody response. We conclude that the targeted disruption of xrRNA1 results in tissue-specific attenuation while still supporting robust neutralizing antibody responses. Future studies will need to investigate the tissue-specific mechanisms by which ZIKV sfRNAs influence infection and may utilize targeted xrRNA mutations to develop novel attenuated flavivirus vaccine approaches.

West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5' cap with 2'-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

The emergence of SARS-CoV-2, which causes COVID-19, has led to over 400 million reported cases worldwide. COVID-19 disease ranges from asymptomatic infection to severe disease and may be impacted by individual immune differences.

Double negative (DN) B cells (CD27-IgD-) comprise a heterogenous population of DN1, DN2, and the recently described DN3 and DN4 subsets. In autoimmune disease, DN2 cells are reported to be precursors to autoreactive antibody secreting cells and expansion of DN2 cells is linked to elevated interferon levels. Severe SARS-CoV-2 infection is characterized by elevated systemic levels of pro-inflammatory cytokines and serum autoantibodies and expansion of the DN2 subset in severe SARS-CoV-2 infection has been reported. However, the activation status, functional capacity and contribution to virally-induced autoantibody production by DN subsets is not established. Here, we validate the finding that severe SARS-CoV-2 infection is associated with a reduction in the frequency of DN1 cells coinciding with an increase in the frequency of DN2 and DN3 cells. We further demonstrate that with severe viral infection DN subsets are at a heightened level of activation, display changes in immunoglobulin class isotype frequency and have functional BCR signaling. Increases in overall systemic inflammation (CRP), as well as specific pro-inflammatory cytokines (TNFα, IL-6, IFNγ, IL-1β), significantly correlate with the skewing of DN1, DN2 and DN3 subsets during severe SARS-CoV-2 infection. Importantly, the reduction in DN1 cell frequency and expansion of the DN3 population during severe infection significantly correlates with increased levels of serum autoantibodies. Thus, systemic inflammation during SARS-CoV-2 infection drives changes in Double Negative subset frequency, likely impacting their contribution to generation of autoreactive antibodies.

Mosquito-borne flaviviruses (MBFVs) including dengue, West Nile, yellow fever, and Zika viruses have an RNA genome encoding one open reading frame flanked by 5' and 3' untranslated regions (UTRs). The 3' UTRs of MBFVs contain regions of high sequence conservation in structured RNA elements known as dumbbells (DBs). DBs regulate translation and replication of the viral RNA genome, functions proposed to depend on the formation of an RNA pseudoknot. To understand how DB structure provides this function, we solved the x-ray crystal structure of the Donggang virus DB to 2.1Å resolution and used structural modeling to reveal the details of its three-dimensional fold. The structure confirmed the predicted pseudoknot and molecular modeling revealed how conserved sequences form a four-way junction that appears to stabilize the pseudoknot. Single-molecule FRET suggests that the DB pseudoknot is a stable element that can regulate the switch between translation and replication during the viral lifecycle by modulating long-range RNA conformational changes.

All flaviviruses contain conserved RNA structures in the 3' untranslated region (3' UTR) that are important for flavivirus RNA replication, translation, and pathogenesis. Flaviviruses like Zika virus (ZIKV) contain multiple conserved RNA structures in the viral 3' UTR, including the structure known as dumbbell-1 (DB-1). Previous research has shown that the DB-1 structure is important for flavivirus positive-strand genome replication, but the functional role of the flavivirus DB-1 structure and the mechanism by which it contributes to viral pathogenesis are not known. Using the recently solved flavivirus DB RNA structural data, we designed two DB-1 mutant ZIKV infectious clones, termed ZIKV-TL.PK and ZIKV-p.2.5', which disrupt DB-1 tertiary folding. We found that viral positive-strand genome replication of both ZIKV DB-1 mutant clones is similar to wild-type (WT) ZIKV, but ZIKV DB-1 mutants exhibit significantly decreased cytopathic effect due to reduced caspase-3 activation. We next show that ZIKV DB-1 mutants exhibit decreased levels of sfRNA species compared to ZIKV-WT during infection. However, ZIKV DB-1 mutant 3' UTRs exhibit unchanged sfRNA biogenesis following XRN1 degradation in vitro. We also found that ZIKV DB-1 mutant virus (ZIKV-p.2.5') exhibited enhanced sensitivity to type I interferon treatment, and both ZIKV-DB-1 mutants exhibit reduced morbidity and mortality due to tissue-specific attenuated viral replication in brain tissue of interferon type I/II receptor knockout mice. We propose that the flavivirus DB-1 RNA structure maintains sfRNA levels during infection despite maintained sfRNA biogenesis, and these results indicate that ZIKV DB-dependent maintenance of sfRNA levels support caspase-3-dependent, cytopathic effect, type I interferon resistance, and viral pathogenesis in mammalian cells and in a ZIKV murine model of disease. IMPORTANCE The group of viruses termed flaviviruses cause important disease throughout the world and include dengue virus, Zika virus, Japanese encephalitis virus, and many more. All of these flaviviruses have highly conserved RNA structures in the untranslated regions of the virus genome. One of the shared RNA structures, termed the dumbbell region, is not well studied, but mutations in this region are important for vaccine development. In this study, we made structure-informed targeted mutations in the Zika virus dumbbell region and studied the effect on the virus. We found that Zika virus dumbbell mutants are significantly weakened or attenuated due to a decreased ability to produce non-coding RNA that is needed to support infection, support virus-induced cell death, and support escape from the host immune system. These data show that targeted mutations in the flavivirus dumbbell RNA structure may be an important approach to develop future vaccine candidates.

Memory B cells are comprised of unswitched (CD27+IgD+) and switched (CD27+IgD-) subsets. The origin and function of unswitched human memory B cells are debated in the literature, whereas switched memory B cells are primed to respond to recurrent infection. Unswitched memory B cells have been described to be reduced in frequency with severe SARS-CoV2 infection and here we characterize their activation status, BCR functionality, and contribution to virally-induced cytokine production. Analyses of whole blood from healthy individuals, people immunized against SARS-CoV2, and those who have had mild and severe SARS-CoV2 infection, confirm a reduction in the frequency of unswitched memory B cells during severe SARS-CoV2 infection and demonstrate this reduction is associated with increased levels of systemic TNFα. We further document how severe viral infection is associated with an increased frequency of 'IgD+' only memory B cells that correlate with increased IgG autoantibody levels. Unswitched and switched memory B cells from severe SARS-CoV2 infection displayed evidence of heightened activation with a concomitant reduction in the expression of the inhibitory receptor CD72. Functionally, both populations of memory B cells from severe SARS-COV2 infection harbored a signaling-competent BCR that displayed enhanced BCR signaling activity in the unswitched population. Finally, we demonstrate that B cells from mild SARS-CoV2 infection are poised to secrete pro-inflammatory cytokines IL-6 and TNFα. Importantly, unswitched memory B cells were a major producer of IL-6 and switched memory B cells were a major producer of TNFα in response to viral TLR ligands. Together these data indicate that B cells contribute to the inflammatory milieu during viral infection.

Background.  West Nile virus (WNV) infection in humans can result in severe, acute encephalitis typically involving subcortical gray matter brain regions. West Nile virus replication within specific human brain regions from a human case of acute encephalitis has not been studied. Methods.  We describe a fatal case of WNV encephalitis in which we obtained tissue from specific brain regions at autopsy to evaluate viral-host interactions using next-generation sequencing and immunohistochemistry analysis. Results.  We found that WNV populations in the injured subcortical brain regions exhibited increased amino acid variation and increased expression of specific interferon genes compared with cortical tissues despite similar viral burden. Conclusions.  These observational, patient-based data suggest that neuronal injury and the strength of viral selection pressure may be associated with the level of the innate immune responses. Further studies in human and animal models evaluating the role of innate immune responses on injury patterns and viral selection pressure are needed.

Severe SARS-CoV-2 infection is associated with strong inflammation and autoantibody production against diverse self-antigens, suggesting a system-wide defect in B cell tolerance. BND cells are a B cell subset in healthy individuals harboring autoreactive but anergic B lymphocytes. In vitro evidence suggests inflammatory stimuli can breach peripheral B cell tolerance in this subset. We asked whether SARS-CoV-2-associated inflammation impairs BND cell peripheral tolerance. To address this, PBMCs and plasma were collected from healthy controls, individuals immunized against SARS-CoV-2, or subjects with convalescent or severe SARS-CoV-2 infection. We demonstrate that BND cells from severely infected individuals are significantly activated, display reduced inhibitory receptor expression, and restored BCR signaling, indicative of a breach in anergy during viral infection, supported by increased levels of autoreactive antibodies. The phenotypic and functional BND cell alterations significantly correlate with increased inflammation in severe SARS-CoV-2 infection. Thus, autoreactive BND cells are released from peripheral tolerance with SARS-CoV-2 infection, likely as a consequence of robust systemic inflammation.