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Peat moss has historically been a key component of soil-free substrates in the greenhouse and nursery industries. However, the increasing expense of peat, negative impacts of peat mining on wetland ecosystems, and growing perception of peat as unsustainable have led to investigation for alternatives. Biochar (BC) is a promising substitute for peat, yet the majority of studies examine additions of BC to peat-based substrates rather than replacing the peat component or employ relatively low substitution rates. Furthermore, at high substitution rates the alkalinity common to many BCs may increase substrate pH and adversely impact plant production. We evaluated BC substitution for peat and pH adjustment of resulting substrates on marigold (Tagetes erecta L.) performance under standard greenhouse conditions. A high pH (10.9) softwood BC (800 °C) was substituted for peat in a standard 70:30 (v/v) peat:perlite mixture at 10% total volume increments. Substrate pH was either not adjusted or adjusted to pH 5.8 using a BC by-product, pyroligneous acid (PLA). Germination was inhibited in pH adjusted substrates with high BC substitution (50–70% total substrate volume) likely due to higher dosages of PLA needed to neutralize pH. At harvest (flowering stage, 9 weeks) the initial pH gradient (4.4–10.4) in substrates that were not pH adjusted had converged to pH 5.6–7.5, and BC substitution for peat did not negatively impact marigold biomass or flowering. At low substitution rates (10–30% total substrate volume), marigold biomass and leaf SPAD values were greater than the control peat-perlite mixture (0% BC). This study demonstrates that softwood BC can be considered as a full replacement for peat in soil-free substrates, and even at high rates (70% total substrate volume) does not require pH adjustment for marigold production. Crop- and BC-specific considerations and economic potential should be investigated for wider application.

Measles remains an important problem in Africa, where human immunodeficiency virus type 1 (HIV-1) infection is prevalent. To identify the consequences of coinfection, Zambian children hospitalized with measles were studied at entry, discharge, and 1 month after discharge. All children had low lymphocyte and eosinophil counts at entry and high leukocyte and monocyte counts during recovery. The death of cultured lymphocytes was more prolonged for HIV-positive children. CD38 and Fas were increased on CD4+ and CD8+ lymphocytes, and CD28 was decreased on CD8+ lymphocytes in all children. Abnormalities in CD4+ and CD8+ lymphocyte percentages and CD28 expression associated with HIV infection were preserved during measles. Therefore, the patterns of changes in leukocyte counts were similar, with little evidence that measles exacerbated HIV-associated lymphocyte abnormalities. Copyright © 2005 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Measles is a highly transmissible systemic viral infection associated with substantial mortality primarily due to secondary infections. Measles induces lifelong immunity to reinfection but loss of immunity to other pathogens. An attenuated live virus vaccine is highly effective, but lapses in delivery have resulted in increasing cases worldwide. Although the primary cause of failure to control measles is failure to vaccinate, waning vaccine-induced immunity and the possible emergence of more virulent virus strains may also contribute., Competing Interests: The authors declare no conflict of interest

Sindbis virus (SINV) infection of mice provides a model system for studying the pathogenesis of alphaviruses that infect the central nervous system (CNS) to cause encephalomyelitis. While studies of human viral infections typically focus on accessible cells from the blood, this compartment is rarely evaluated in mice. To bridge this gap, single-cell RNA sequencing (scRNAseq) was combined with flow cytometry to characterize the transcriptional and phenotypic changes of peripheral blood mononuclear cells (PBMCs) from SINV-infected mice. Twenty-one clusters were identified by scRNAseq at 7 days after infection, with a unique cluster and overall increase in naive B cells for infected mice. Uninfected mice had fewer immature T cells and CCR9 + CD4 T cells and a unique immature T cell cluster. Gene expression was most altered in the Ki67 + CD8 T cell cluster, with chemotaxis and proliferation-related genes upregulated. Global analysis indicated metabolic changes in myeloid cells and increased expression of Ccl5 by NK cells. Phenotypes of PBMCs and cells infiltrating the CNS were analyzed by flow cytometry over 14 days after infection. In PBMCs, CD8 and Th1 CD4 T cells increased in representation, while B cells showed a transient decrease at day 5 in total, Ly6a + , and naive cells, and an increase in activated B cells. In the brain, CD8 T cells increased for the first 7 days, while Th1 CD4 T cells and naive and Ly6a + B cells continued to accumulate for 14 days. Therefore, dynamic immune cell changes can be identified in the blood as well as the CNS during viral encephalomyelitis., Importance: The outcome of viral encephalomyelitis is dependent on the host immune response, with clearance and resolution of infection mediated by the adaptive immune response. These processes are frequently studied in mouse models of infection, where infected tissues are examined to understand the mechanisms of clearance and recovery. However, studies of human infection typically focus on the analysis of cells from the blood, a compartment rarely examined in mice, rather than inaccessible tissue. To close this gap, we used single-cell RNA sequencing and flow cytometry to profile the transcriptomic and phenotypic changes of peripheral blood mononuclear cells (PBMCs) before and after central nervous system (CNS) infection in mice. Changes to T and B cell gene expression and cell composition occurred in PBMC and during entry into the CNS, with CCL5 being a differentially expressed chemokine. Therefore, dynamic changes occur in the blood as well as the CNS during the response of mice to virus infection, which will inform the analysis of human studies., Competing Interests: D.E.G. is on an advisory board for GSK and consulted for Merck.

Importance: Viral encephalomyelitis outcome is dependent on host responses to neuronal infection. Interferon (IFN) is an important component of the innate response, and IFN regulatory factor (IRF) 7 is an inducible transcription factor for the synthesis of IFN-α. IRF7-deficient mice develop fatal paralysis after CNS infection with Sindbis virus, while wild-type mice recover. Irf7 -/- mice produce low levels of IFN-α but high levels of IFN-β with induction of IFN-stimulated genes, so the reason for this difference is not understood. The current study shows that Irf7 -/- mice developed inflammation earlier but failed to clear virus from motor neuron-rich regions of the brainstem and spinal cord. Levels of IFN-γ and virus-specific antibody were comparable, indicating that IRF7 deficiency does not impair expression of these known viral clearance factors. Therefore, IRF7 is either necessary for the neuronal response to currently identified mediators of clearance or enables the production of additional antiviral factor(s) needed for clearance., Competing Interests: DEG is a member of advisory boards for GSK and Takeda Pharmaceuticals.

Millions of people are suffering from Long COVID or post-acute sequelae of COVID-19 (PASC). Several biological factors have emerged as potential drivers of PASC pathology. Some individuals with PASC may not fully clear the coronavirus SARS-CoV-2 after acute infection. Instead, replicating virus and/or viral RNA-potentially capable of being translated to produce viral proteins-persist in tissue as a 'reservoir'. This reservoir could modulate host immune responses or release viral proteins into the circulation. Here we review studies that have identified SARS-CoV-2 RNA/protein or immune responses indicative of a SARS-CoV-2 reservoir in PASC samples. Mechanisms by which a SARS-CoV-2 reservoir may contribute to PASC pathology, including coagulation, microbiome and neuroimmune abnormalities, are delineated. We identify research priorities to guide the further study of a SARS-CoV-2 reservoir in PASC, with the goal that clinical trials of antivirals or other therapeutics with potential to clear a SARS-CoV-2 reservoir are accelerated., (© 2023. Springer Nature America, Inc.)

Measles is a systemic disease initiated in the respiratory tract with widespread measles virus (MeV) infection of lymphoid tissue. Mortality can be substantial, but no licensed antiviral therapy is available. We evaluated both post-exposure prophylaxis and treatment with remdesivir, a broad-spectrum antiviral, using a well-characterized rhesus macaque model of measles. Animals were treated with intravenous remdesivir for 12 days beginning either 3 days after intratracheal infection (post-exposure prophylaxis, PEP) or 11 days after infection at the onset of disease (late treatment, LT). As PEP, remdesivir lowered levels of viral RNA in peripheral blood mononuclear cells, but RNA rebounded at the end of the treatment period and infectious virus was continuously recoverable. MeV RNA was cleared more rapidly from lymphoid tissue, was variably detected in the respiratory tract, and not detected in urine. PEP did not improve clinical disease nor lymphopenia and reduced the antibody response to infection. In contrast, LT had little effect on levels of viral RNA or the antibody response but also did not decrease clinical disease. Therefore, remdesivir transiently suppressed expression of viral RNA and limited dissemination when provided as PEP, but virus was not cleared and resumed replication without improvement in the clinical disease parameters evaluated., (© 2023. The Author(s).)

Viruses depend on host cellular resources to replicate. Interaction between viral and host proteins is essential for the pathogens to ward off immune responses as well as for virus propagation within the infected cells. While different viruses employ unique strategies to interact with diverse sets of host proteins, the multifunctional RNA-binding protein G3BP1 is one of the common targets for many viruses. G3BP1 controls several key cellular processes, including mRNA stability, translation, and immune responses. G3BP1 also serves as the central hub for the protein-protein and protein-RNA interactions within a class of biomolecular condensates called stress granules (SGs) during stress conditions, including viral infection. Increasing evidence suggests that viruses utilize distinct strategies to modulate G3BP1 function-either by degradation, sequestration, or redistribution-and control the viral life cycle positively and negatively. In this review, we summarize the pro-viral and anti-viral roles of G3BP1 during infection among different viral families.

In humans and non-human primates, wild type (WT) measles virus (MeV) replicates extensively in lymphoid tissue and induces an innate response characteristic of NF-κB and inflammasome activation without type I interferon. In contrast, the live attenuated MeV vaccine (LAMV) replicates poorly in lymphoid tissue with little detectable in vivo cytokine production. To characterize the innate responses of macrophages to WT MeV and LAMV infection, we analyzed primary human monocyte-derived macrophages and phorbol myristic acid-matured monocytic THP-1 cells (M0) polarized to inflammatory (M1) and anti-inflammatory (M2) phenotypes 24 h after MeV infection. LAMV infected macrophages more efficiently than WT MeV but produced less virus than WT MeV-infected macrophages. Both strains induced production of NF-κB-responsive cytokines IL-6 and TNFα and inflammasome products IL-1β and IL-18 without evidence of pyroptosis. Analysis of THP-1 cells deficient in inflammasome sensors NOD-like receptor pyrin (NLRP)3, IFN-γ-inducible protein 16 (IFI16) or absent in melanoma (AIM)2; adaptor apoptosis-associated speck-like protein containing a CARD (ASC) or effector caspase 1 showed that IL-18 production was dependent on NLRP3, ASC, and caspase 1. However, M1 cells produced IL-1β in the absence of ASC or caspase 1 indicating alternate pathways for MeV-induced pro-IL-1β processing. Therefore, the innate response to in vitro infection of macrophages with both LAMV and WT MeV includes production of IL-6 and TNFα and activation of the NLRP3 inflammasome to release IL-1β and IL-18. LAMV attenuation impairs production of infectious virus but does not reduce ability to infect macrophages or innate responses to infection.

Chikungunya virus (CHIKV) causes outbreaks of rash, arthritis, and fever associated with neurologic complications, where astrocytes are preferentially infected. A determinant of virulence is the macrodomain (MD) of nonstructural protein 3 (nsP3), which binds and removes ADP-ribose (ADPr) from ADP-ribosylated substrates and regulates stress-granule disruption. We compared the replication of CHIKV 181/25 (WT) and MD mutants with decreased ADPr binding and hydrolase (G32S) or increased ADPr binding and decreased hydrolase (Y114A) activities in C8-D1A astrocytic cells and NSC-34 neuronal cells. WT CHIKV replication was initiated more rapidly with earlier nsP synthesis in C8-D1A than in NSC-34 cells. G32S established infection, amplified replication complexes, and induced host-protein synthesis shut-off less efficiently than WT and produced less infectious virus, while Y114A replication was close to WT. However, G32S mutation effects on structural protein synthesis were cell-type-dependent. In NSC-34 cells, E2 synthesis was decreased compared to WT, while in C8-D1A cells synthesis was increased. Excess E2 produced by G32S-infected C8-D1A cells was assembled into virus particles that were less infectious than those from WT or Y114A-infected cells. Because nsP3 recruits ADP-ribosylated RNA-binding proteins in stress granules away from translation-initiation factors into nsP3 granules where the MD hydrolase can remove ADPr, we postulate that suboptimal translation-factor release decreased structural protein synthesis in NSC-34 cells while failure to de-ADP-ribosylate regulatory RNA-binding proteins increased synthesis in C8-D1A cells.

COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a devastating impact on global public health, emphasizing the importance of understanding innate immune mechanisms and cellular restriction factors that cells can harness to fight viral infections. The multimembrane-spanning zinc metalloprotease ZMPSTE24 is one such restriction factor. ZMPSTE24 has a well-characterized proteolytic role in the maturation of prelamin A, precursor of the nuclear scaffold protein lamin A. An apparently unrelated role for ZMPSTE24 in viral defense involves its interaction with the interferon-inducible membrane proteins (IFITMs), which block virus-host cell fusion by rigidifying cellular membranes and thereby prevent viral infection. ZMPSTE24, like the IFITMs, defends cells against a broad spectrum of enveloped viruses. However, its ability to protect against coronaviruses has never been examined. Here, we show that overexpression of ZMPSTE24 reduces the efficiency of cellular infection by SARS-CoV-2 Spike-pseudotyped lentivirus and that genetic knockout or small interfering RNA-mediated knockdown of endogenous ZMPSTE24 enhances infectivity. We further demonstrate a protective role for ZMPSTE24 in a Spike-ACE2-dependent cell-cell fusion assay. In both assays, a catalytic dead version of ZMPSTE24 is equally as protective as the wild-type protein, indicating that ZMPSTE24's proteolytic activity is not required for defense against SARS-CoV-2. Finally, we demonstrate by plaque assays that Zmpste24 -/- mouse cells show enhanced infection by a genuine coronavirus, mouse hepatitis virus (MHV). This study extends the range of viral protection afforded by ZMPSTE24 to include coronaviruses and suggests that targeting ZMPSTE24's mechanism of viral defense could have therapeutic benefit. IMPORTANCE The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has underscored the importance of understanding intrinsic cellular components that can be harnessed as the cell's first line of defense to fight against viral infection. Our paper focuses on one such protein, the integral membrane protease ZMPSTE24, which interacts with interferon-inducible transmembrane proteins (IFITMs). IFITMs interfere with virus entry by inhibiting fusion between viral and host cell membranes, and ZMPSTE24 appears to contribute to this inhibitory activity. ZMPSTE24 has been shown to defend cells against several, but not all, enveloped viruses. In this study, we extend ZMPSTE24's reach to include coronaviruses, by showing that ZMPSTE24 protects cells from SARS-CoV-2 pseudovirus infection, Spike protein-mediated cell-cell fusion, and infection by the mouse coronavirus MHV. This work lays the groundwork for further studies to decipher the mechanistic role of ZMPSTE24 in blocking the entry of SARS-CoV-2 and other viruses into cells.

Alphaviruses are positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV), the prototype alphavirus, preferentially infects neurons in mice and is a model system for studying mechanisms of viral clearance from the nervous system. Antibody specific to the SINV E2 glycoprotein plays an important role in SINV clearance, and this effect is reproduced in cultures of infected mature neurons. To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons. Differentiated AP-7 rat olfactory neuronal cells, an in vitro model for mature neurons, were infected with SINV and treated with anti-E2 antibody. Whole-cell RNA lysates were collected for sequencing of poly(A)-selected RNA 24, 48, and 72 h after infection. Three primary species of viral RNA were produced: genomic, subgenomic, and defective viral genomes (DVGs) encoding the RNA capping protein nsP1. Antibody treatment resulted in overall lower production of SINV RNA, decreased synthesis of subgenomic RNA relative to genomic RNA, and suppressed production of the nsP1 DVG. The nsP1 DVG was packaged into virus particles and could be translated. Because antibody-treated cells released a higher proportion of virions with noncapped genomes and transient transfection to express the nsP1 DVG improved viral RNA capping in antibody-treated cells, we postulate that one mechanism by which antibody inhibits SINV replication in neurons is to suppress DVG synthesis and thus decrease production of infectious virions containing capped genomes. IMPORTANCE Alphaviruses are important causes of viral encephalomyelitis without approved treatments or vaccines. Antibody to the Sindbis virus (SINV) E2 glycoprotein is required for immune-mediated noncytolytic virus clearance from neurons. We used direct RNA nanopore sequencing to evaluate how anti-E2 antibody affects SINV replication at the RNA level. Antibody altered the viral RNAs produced by decreasing the proportion of subgenomic relative to genomic RNA and suppressing production of a previously unrecognized defective viral genome (DVG) encoding nsP1, the viral RNA capping enzyme. Antibody-treated neurons released a lower proportion of SINV particles with capped genomes necessary for translation and infection. Decreased nsP1 DVG production in antibody-treated neurons led to lower expression of nsP1 protein, decreased genome capping efficiency, and release of fewer infectious virus particles. Capping was increased with exogenous expression of the nsP1 DVG. These studies identify a novel alphavirus DVG function and new mechanism for antibody-mediated control of virus replication.

Multisystem inflammatory syndrome in children (MIS-C) can complicate infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but differences in the immune responses during MIS-C compared to coronavirus disease 2019 (COVID-19) are poorly understood. We longitudinally compared the amounts and avidity of plasma anti-nucleocapsid (N) and spike (S) antibodies, phenotypes of B cells, and numbers of virus-specific antibody-secreting cells in circulation of children hospitalized with COVID-19 (n = 10) and with MIS-C (n = 12). N-specific immunoglobulin G (IgG) was higher early after presentation for MIS-C than COVID-19 patients and avidity of N- and S-specific IgG at presentation did not mature further during follow-up as it did for COVID-19. Both groups had waning proportions of B cells in circulation and decreasing but sustained production of virus-specific antibody-secreting cells for months. Overall, B-cell responses were similar, but those with MIS-C demonstrated a more mature antibody response at presentation compared to COVID-19, suggesting a postinfectious entity., Competing Interests: Potential conflicts of interest. D. G. is on advisory boards for GlaxoSmithKline, Takeda Pharmaceutical, and GreenLight Biosciences. All other authors report no potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Sindbis virus (SINV) causes viral encephalitis in mice with strain-dependent virulence. Fatal encephalomyelitis in C57Bl/6 mice infected with a neuroadapted strain of SINV (NSV) is an immunopathogenic process that involves Th17 cells modulated by the regulatory cytokine IL-10. To further characterize the pathogenic immune response to NSV, we analyzed the regulation of transforming growth factor (TGF)-b in both wild-type (WT) and IL-10-deficient mice. NSV infection upregulated the expression of TGFb1 and TGFb3 in the central nervous system (CNS). In the absence of IL-10, levels of brain Tgfb1 mRNA and brain and spinal cord mature active TGFβ1 and TGFβ3 proteins were higher than in WT mice. Compared to WT mice, IL-10-deficient mice had more TGFβ1-expressing type 3 innate lymphoid cells (ILC3s) and CD4 + T cells infiltrating the CNS, but similar numbers in the cervical lymph nodes. Expression of glycoprotein A repetitions predominant protein (GARP) that binds pro-TGFb on the surface of regulatory T cells was decreased on CNS cells from IL-10-deficient mice. Higher CNS TGFb was accompanied by more expression of TGFbRII receptor, activation of SMAD transcription factors, increased PCKα mRNA, and more RORγt-positive and IL-17A-expressing cells. These results suggest a compensatory role for TGFβ in the absence of IL-10 that fosters Th17-related immunopathology and more rapid death after NSV infection.

DNA viruses often persist in the body of their host, becoming latent and recurring many months or years later. By contrast, most RNA viruses cause acute infections that are cleared from the host as they lack the mechanisms to persist. However, it is becoming clear that viral RNA can persist after clinical recovery and elimination of detectable infectious virus. This persistence can either be asymptomatic or associated with late progressive disease or nonspecific lingering symptoms, such as may be the case following infection with Ebola or Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Why does viral RNA sometimes persist after recovery from an acute infection? Where does the RNA come from? And what are the consequences?, Competing Interests: I have read the journal’s policy and the author of this manuscript has the following competing interests: Membership on advisory boards for Takeda Pharmaceuticals, GreenLight Bioscience, GlaxoSmithKline, Academia Sinica, University of Maryland and University of Vermont; Funding from Gilead, MeVox and the US National Institutes of Health.

Understanding the development and sustainability of the virus-specific protective immune response to infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains incomplete with respect to the appearance and disappearance of virus-specific antibody-secreting cells (ASCs) in circulation. Therefore, we performed cross-sectional and longitudinal analyses of peripheral blood mononuclear cells and plasma collected from 55 hospitalized patients up to 4 months after onset of COVID-19 symptoms. Spike (S)- and nucleocapsid (N)-specific IgM and IgG ASCs appeared within 2 weeks accompanied by flow cytometry increases in double negative plasmablasts consistent with a rapid extrafollicular B cell response. Total and virus-specific IgM and IgG ASCs peaked at 3-4 weeks and were still being produced at 3-4 months accompanied by increasing antibody avidity consistent with a slower germinal center B cell response. N-specific ASCs were produced for longer than S-specific ASCs and avidity maturation was greater for antibody to N than S. Patients with more severe disease produced more S-specific IgM and IgG ASCs than those with mild disease and had higher levels of N- and S-specific antibody. Women had more B cells in circulation than men and produced more S-specific IgA and IgG and N-specific IgG ASCs. Flow cytometry analysis of B cell phenotypes showed an increase in circulating B cells at 4-6 weeks with decreased percentages of switched and unswitched memory B cells. These data indicate ongoing antigen-specific stimulation, maturation, and production of ASCs for several months after onset of symptoms in patients hospitalized with COVID-19.

Emerging and re-emerging viral diseases pose continuous public health threats, and effective control requires a combination of non-pharmacologic interventions, treatment with antivirals, and prevention with vaccines. The COVID-19 pandemic has demonstrated that the world was least prepared to provide effective treatments. This lack of preparedness has been due, in large part, to a lack of investment in developing a diverse portfolio of antiviral agents, particularly those ready to combat viruses of pandemic potential. Here, we focus on a drug target called macrodomain that is critical for the replication and pathogenesis of alphaviruses and coronaviruses. Some mutations in alphavirus and coronaviral macrodomains are not tolerated for virus replication. In addition, the coronavirus macrodomain suppresses host interferon responses. Therefore, macrodomain inhibitors have the potential to block virus replication and restore the host's protective interferon response. Viral macrodomains offer an attractive antiviral target for developing direct acting antivirals because they are highly conserved and have a structurally well-defined (druggable) binding pocket. Given that this target is distinct from the existing RNA polymerase and protease targets, a macrodomain inhibitor may complement current approaches, pre-empt the threat of resistance and offer opportunities to develop combination therapies for combating COVID-19 and future viral threats.

Measles virus (MeV) is highly infectious by the respiratory route and remains an important cause of childhood mortality. However, the process by which MeV infection is efficiently established in the respiratory tract is controversial with suggestions that respiratory epithelial cells are not susceptible to infection from the apical mucosal surface. Therefore, it has been hypothesized that infection is initiated in lung macrophages or dendritic cells and that epithelial infection is subsequently established through the basolateral surface by infected lymphocytes. To better understand the process of respiratory tract initiation of MeV infection, primary differentiated respiratory epithelial cell cultures were established from rhesus macaque tracheal and nasal tissues. Infection of these cultures with MeV from the apical surface was more efficient than from the basolateral surface with shedding of viable MeV-producing multinucleated giant cell (MGC) syncytia from the surface. Despite presence of MGCs and infectious virus in supernatant fluids after apical infection, infected cells were not detected in the adherent epithelial sheet and transepithelial electrical resistance was maintained. After infection from the basolateral surface, epithelial damage and large clusters of MeV-positive cells were observed. Treatment with fusion inhibitory peptides showed that MeV production after apical infection was not dependent on infection of the basolateral surface. These results are consistent with the hypothesis that MeV infection is initiated by apical infection of respiratory epithelial cells with subsequent infection of lymphoid tissue and systemic spread., Competing Interests: The authors declare no competing interest.

While biomolecular condensates have emerged as an important biological phenomenon, mechanisms regulating their composition and the ways that viruses hijack these mechanisms remain unclear. The mosquito-borne alphaviruses cause a range of diseases from rashes and arthritis to encephalitis, and no licensed drugs are available for treatment or vaccines for prevention. The alphavirus virulence factor nonstructural protein 3 (nsP3) suppresses the formation of stress granules (SGs)-a class of cytoplasmic condensates enriched with translation initiation factors and formed during the early stage of infection. nsP3 has a conserved N-terminal macrodomain that hydrolyzes ADP-ribose from ADP-ribosylated proteins and a C-terminal hypervariable domain that binds the essential SG component G3BP1. Here, we show that macrodomain hydrolase activity reduces the ADP-ribosylation of G3BP1, disassembles virus-induced SGs, and suppresses SG formation. Expression of nsP3 results in the formation of a distinct class of condensates that lack translation initiation factors but contain G3BP1 and other SG-associated RNA-binding proteins. Expression of ADP-ribosylhydrolase-deficient nsP3 results in condensates that retain translation initiation factors as well as RNA-binding proteins, similar to SGs. Therefore, our data reveal that ADP-ribosylation controls the composition of biomolecular condensates, specifically the localization of translation initiation factors, during alphavirus infection., Competing Interests: The authors declare no competing interest.

Effects of measles on the immune system are only partially understood. Lymphoid tissue is a primary site of measles virus (MeV) replication where CD150 is the receptor for infection of both B and T cells. Lymphocyte depletion occurs during the acute phase of infection, but initiation of the adaptive immune response leads to extensive lymphocyte proliferation, production of MeV-specific antibody and T cells, the rash and clearance of infectious virus. Viral RNA persists in lymphoid tissue accompanied by ongoing germinal center proliferation, production of antibody-secreting cells, functionally distinct populations of T cells and antibody avidity maturation to establish life-long immunity. However, at the same time diversity of pre-existing antibodies and numbers of memory and naive B cells are reduced and susceptibility to other infections is increased., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The disease spectrum of coronavirus disease 2019 (COVID-19) ranges from no symptoms to multisystem failure and death. Characterization of virus-specific immune responses to severe acute respiratory coronavirus 2 (SARS-CoV-2) is key to understanding disease pathogenesis, but few studies have evaluated T cell immunity. In this issue of the JCI, Sattler and Angermair et al. sampled blood from subjects with COVID-19 and analyzed the activation and function of virus antigen-specific CD4+ T cells. T cells that failed to respond to peptides from the membrane, spike, or nucleocapsid proteins were more common in subjects who died. In those whose T cells had the capacity to respond, older patients with comorbidity had larger numbers of activated T cells compared with patients who had fewer risk factors, but these cells showed impaired IFN-γ production. This cross-sectional study relates activated T cell responses to patient risk factors and outcome. However, T cell response trajectory over the disease course remains an open question.

Alphaviruses are positive-sense, enveloped RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV) is the prototype alphavirus and preferentially infects neurons in rodents to induce an encephalomyelitis similar to the human disease. Using a mouse model of SINV infection of the nervous system, many of the immune processes involved in recovery from viral encephalomyelitis have been identified. Antibody specific to the SINV E2 glycoprotein plays an important role in recovery and is sufficient for noncytolytic suppression of virus replication in vivo and in vitro. To investigate the mechanism of anti-E2 antibody-mediated viral suppression, a reverse-phase protein array was used to broadly survey cellular signaling pathway activation following antibody treatment of SINV-infected differentiated AP-7 neuronal cells. Anti-E2 antibody induced rapid transient NF-κB and later sustained Y705 STAT3 phosphorylation, outlining an intracellular signaling cascade activated by antiviral antibody. Because NF-κB target genes include the STAT3-activating IL-6 family cytokines, expression of these messenger RNAS (mRNAs) was assessed. Expression of leukemia inhibitory factor (LIF) cytokine mRNA, but not other IL-6 family member mRNAs, was up-regulated by anti-E2 antibody. LIF induced STAT3 Y705 phosphorylation in infected differentiated AP-7 cells but did not inhibit virus replication. However, anti-E2 antibody localized the LIF receptor to areas of E2 expression on the infected cell surface, and LIF enhanced the antiviral effects of antibody. These findings identify activation of the NF-κB/LIF/STAT3 signaling cascade as involved in inducing antibody-mediated viral suppression and highlight the importance of nonneutralizing antibody functions in viral clearance from neurons., Competing Interests: The authors declare no competing interest.

My great-grandparents were immigrants from Sweden and settled as farmers in Iowa and Illinois. My father, the oldest of six children, was the first in his family to go to college and had careers as a petroleum geologist and an academic. My mother, the youngest of four children, had older siblings in education, and she focused on early childhood education. My childhood in Oklahoma with two younger sisters was happy and comfortable, and public school prepared me well. My career trajectory into virology did not involve much if any advance planning but was characterized by recognizing the fascinating puzzles of virus diseases, being in good places at the right time, taking advantage of opportunities as they presented themselves, and being surrounded by great mentors, colleagues, trainees, and family. Most of my career was spent studying two diseases caused by RNA viruses, alphavirus encephalomyelitis and measles, and was enriched with several leadership opportunities.

Acute RNA viral encephalomyelitis is a serious complication of numerous virus infections. Antibodies in the cerebral spinal fluid (CSF) are correlated to better outcomes, and there is substantive evidence of antibody secreting cells (ASCs) entering the central nervous system (CNS) and contributing to resolution of infection. Here, we review the RNA viruses known to cause acute viral encephalomyelitis with mechanisms of control that require antibody or ASCs. We compile the cytokines, chemokines, and surface receptors associated with ASC recruitment to the CNS after infection and compare known antibody-mediated mechanisms as well as potential noncytolytic mechanisms for virus control. These non-canonical functions of antibodies may be employed in the CNS to protect precious non-renewable neurons. Understanding the immune-specialized zone of the CNS is essential for the development of effective treatments for acute encephalomyelitis caused by RNA viruses.

Infection with wild-type (WT) measles virus (MeV) is an important cause of childhood mortality that leads to lifelong protective immunity in survivors. WT MeV and the live-attenuated MeV used in the measles vaccine (LAMV) are antigenically similar, but the determinants of attenuation are unknown, and protective immunity induced by LAMV is less robust than that induced by WT MeV. To identify factors that contribute to these differences, we compared virologic and immunologic responses after respiratory infection of rhesus macaques with WT MeV or LAMV. In infected macaques, WT MeV replicated efficiently in B and T lymphocytes with spreading throughout lymphoid tissues resulting in prolonged persistence of viral RNA. In contrast, LAMV replicated efficiently in the respiratory tract but displayed limited spread to lymphoid tissue or peripheral blood mononuclear cells. In vitro, WT MeV and LAMV replicated similarly in macaque primary respiratory epithelial cells and human lymphocytes, but LAMV-infected lymphocytes produced little virus. Plasma concentrations of interleukin-1β (IL-1β), IL-12, interferon-γ (IFN-γ), CCL2, CCL11, CXCL9, and CXCL11 increased in macaques after WT MeV but not LAMV infection. WT MeV infection induced more protective neutralizing, hemagglutinin-specific antibodies and bone marrow plasma cells than did LAMV infection, although numbers of MeV-specific IFN-γ- and IL-4-producing T cells were comparable. Therefore, MeV attenuation may involve altered viral replication in lymphoid tissue that limited spread and decreased the host antibody response, suggesting a link between lifelong protective immunity and the ability of WT MeV, but not LAMV, to spread in lymphocytes., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Clearance of measles virus is complex. Infectious virus is cleared by the adaptive immune response manifested by the characteristic maculopapular rash. CD8 + T cells are major effectors of infectious virus clearance, a process that may fail in individuals with compromised cellular immune responses leading to progressive giant cell pneumonia and/or measles inclusion body encephalitis. In contrast to the usual rapid clearance of infectious virus, clearance of viral RNA is slow with persistence in lymphoid tissue for many months. Persistence of MeV RNA may contribute to the late development of the slowly progressive disease subacute sclerosing panencephalitis in children infected at a young age and to measles-associated immune suppression but also to maturation of the immune response and development of life-long immunity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Sindbis virus (SINV) is an alphavirus that causes age-dependent encephalomyelitis in mice. Within 7-8 days after infection infectious virus is cleared from neurons through the antiviral effects of antibody and interferon-gamma (IFNγ), but RNA persists. To better understand changes in viral RNA associated with immune-mediated clearance we developed recombinant strains of SINV that have genomic and subgenomic viral RNAs tagged with the Broccoli RNA aptamer that binds and activates a conditional fluorophore for live cell imaging of RNA. Treatment of SINV-Broccoli-infected cells with antibody to the SINV E2 glycoprotein had cell type-specific effects. In BHK cells, antibody increased levels of intracellular viral RNA and changed the primary location of genomic RNA from the perinuclear region to the plasma membrane without improving cell viability. In undifferentiated and differentiated AP7 (dAP7) neuronal cells, antibody treatment decreased levels of viral RNA. Occasional dAP7 cells escaped antibody-mediated clearance by not expressing cell surface E2 or binding antibody to the plasma membrane. IFNγ decreased viral RNA levels only in dAP7 cells and synergized with antibody for RNA clearance and improved cell survival. Therefore, analysis of aptamer-tagged SINV RNAs identified cell type- and neuronal maturation-dependent responses to immune mediators of virus clearance.

Sindbis virus (SINV) can infect neurons and cause encephalomyelitis in mice. Nonstructural proteins are translated from genomic RNA and structural proteins from subgenomic RNA. While visualization of viral proteins in living cells is well developed, imaging of viral RNAs has been challenging. RNA aptamers that bind and activate conditional fluorophores provide a tool for RNA visualization. We incorporated cassettes of two F30-scaffolded dimers of the Broccoli aptamer into a SINV cDNA clone using sites in nsP3 (genomic RNA), the 3'UTR (genomic and subgenomic RNAs) and after a second subgenomic promoter resulting in 4-28 Broccoli copies. After addition of the cell-permeable 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI-1T) conditional fluorophore and laser excitation, infected cells emitted green fluorescence that correlated with Broccoli copy numbers. All recombinant viruses replicated well in BHK and undifferentiated neural cells but viruses with 14 or more Broccoli copies were attenuated in differentiated neurons and mice. The signal survived fixation and allowed visualization of viral RNAs in differentiated neurons and mouse brain, as well as BHK cells. Subgenomic RNA was diffusely distributed in the cytoplasm with genomic RNA also in perinuclear vesicle-like structures near envelope glycoproteins or mitochondria. Broccoli aptamer-tagging provides a valuable tool for live cell imaging of viral RNA.

Recovery from measles results in life-long protective immunity. To understand induction of long-term immunity, rhesus macaques were studied for 6 months after infection with wild-type measles virus (MeV). Infection caused viremia and rash, with clearance of infectious virus by day 14. MeV RNA persisted in PBMCs for 30-90 days and in lymphoid tissue for 6 months most often in B cells but was rarely detected in BM. Antibody with neutralizing activity and binding specificity for MeV nucleocapsid (N), hemagglutinin (H), and fusion proteins appeared with the rash and avidity matured over 3-4 months. Lymph nodes had increasing numbers of MeV-specific antibody-secreting cells (ASCs) and germinal centers with late hyalinization. ASCs appeared in circulation with the rash and continued to appear along with peripheral T follicular helper cells for the study duration. ASCs in lymph nodes and PBMCs produced antibody against both H and N, with more H-specific ASCs in BM. During days 14-21, 20- to 100-fold more total ASCs than MeV-specific ASCs appeared in circulation, suggesting mobilization of preexisting ASCs. Therefore, persistence of MeV RNA in lymphoid tissue was accompanied by continued germinal center formation, ASC production, avidity maturation, and accumulation of H-specific ASCs in BM to sustain neutralizing antibody and protective immunity.

Macrodomain (MD), a highly conserved protein fold present in a subset of plus-strand RNA viruses, binds to and hydrolyzes ADP-ribose (ADPr) from ADP-ribosylated proteins. ADPr-binding by the alphavirus nonstructural protein 3 (nsP3) MD is necessary for the initiation of virus replication in neural cells, whereas hydrolase activity facilitates replication complex amplification. To determine the importance of these activities for pathogenesis of alphavirus encephalomyelitis, mutations were introduced into the nsP3 MD of Sindbis virus (SINV), and the effects on ADPr binding and hydrolase activities, virus replication, immune responses, and disease were assessed. Elimination of ADPr-binding and hydrolase activities (G32E) severely impaired in vitro replication of SINV in neural cells and in vivo replication in the central nervous systems of 2-week-old mice with reversion to wild type (WT) (G) or selection of a less compromising change (S) during replication. SINVs with decreased binding and hydrolase activities (G32S and G32A) or with hydrolase deficiency combined with better ADPr-binding (Y114A) were less virulent than WT virus. Compared to the WT, the G32S virus replicated less well in both the brain and spinal cord, induced similar innate responses, and caused less severe disease with full recovery of survivors, whereas the Y114A virus replicated well, induced higher expression of interferon-stimulated and NF-κB-induced genes, and was cleared more slowly from the spinal cord with persistent paralysis in survivors. Therefore, MD function was important for neural cell replication both in vitro and in vivo and determined the outcome from alphavirus encephalomyelitis in mice. IMPORTANCE Viral encephalomyelitis is an important cause of long-term disability, as well as acute fatal disease. Identifying viral determinants of outcome helps in assessing disease severity and developing new treatments. Mosquito-borne alphaviruses infect neurons and cause fatal disease in mice. The highly conserved macrodomain of nonstructural protein 3 binds and can remove ADP-ribose (ADPr) from ADP-ribosylated proteins. To determine the importance of these functions for virulence, recombinant mutant viruses were produced. If macrodomain mutations eliminated ADPr-binding or hydrolase activity, viruses did not grow. If the binding and hydrolase activities were impaired, the viruses grew less well than the wild-type virus, induced similar innate responses, and caused less severe disease, and most of the infected mice recovered. If binding was improved, but hydrolase activity was decreased, the virus replicated well and induced greater innate responses than did the WT, but clearance from the nervous system was impaired, and mice remained paralyzed. Therefore, macrodomain function determined the outcome of alphavirus encephalomyelitis., (Copyright © 2020 Abraham et al.)

Infection of mice with Sindbis virus (SINV) provides a model for examining the role of the immune response to alphavirus infection of the central nervous system (CNS). Interferon-gamma (IFN-γ) is an important component of this response, and we show that SINV-infected differentiated neurons respond to IFN-γ in vitro by induction of antiviral genes and suppression of virus replication. To determine the in vivo effects of IFN-γ on SINV clearance and T cell responses, C57BL/6 mice lacking IFN-γ or IFN-γ receptor-1 were compared to wild-type (WT) mice after intracranial SINV infection. In WT mice, IFN-γ was first produced in the CNS by natural killer cells and then by CD4 + and CD8 + T cells. Mice with impaired IFN-γ signaling initiated clearance of viral RNA earlier than WT mice associated with CNS entry of more granzyme B-producing CD8 + T cells. However, these mice established fewer CD8 + tissue-resident memory T (T RM ) cells and were more likely to experience reactivation of viral RNA synthesis late after infection. Therefore, IFN-γ suppresses the local development of granzyme B-expressing CD8 + T cells and slows viral RNA clearance but promotes CD8 + T RM cell establishment., Competing Interests: The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. The authors declare no conflicts of interest.

Alphaviruses are enveloped, positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV) infects the neurons of rodents and is a model for studying factors that regulate infection of neuronal cells. The outcome of alphavirus infection of the central nervous system is dependent on neuronal maturation status. Differentiated mature neurons survive and control viral replication better than undifferentiated immature neurons. The cellular factors involved in age-dependent susceptibility include higher levels of antiapoptotic and innate immune factors in mature neurons. Because NF-κB pathway activation is required for the initiation of both apoptosis and the host antiviral response, we analyzed the role of NF-κB during SINV infection of differentiated and undifferentiated rat neuronal cells. SINV infection induced canonical NF-κB activation, as evidenced by the degradation of IκBα and the phosphorylation and nuclear translocation of p65. Inhibition or deletion of the upstream IκB kinase substantially reduced SINV replication in differentiated but not in undifferentiated neuronal cells or mouse embryo fibroblasts. NF-κB inhibition did not affect the establishment of infection, replication complex formation, the synthesis of nonstructural proteins, or viral RNA synthesis in differentiated neurons. However, the translation of structural proteins was impaired, phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) was decreased, and host protein synthesis was maintained, suggesting that NF-κB activation was involved in the regulation of translation during infection of mature neurons. Inhibition or deletion of double-stranded RNA-activated protein kinase (PKR) also decreased eIF2α phosphorylation, the translation of viral structural proteins, and virus production. Therefore, canonical NF-κB activation synergizes with PKR to promote SINV replication in differentiated neurons by facilitating viral structural protein translation. IMPORTANCE Mosquito-borne alphaviruses are a significant and growing cause of viral encephalomyelitis worldwide. The outcome of alphaviral neuronal infections is host age dependent and greatly affected by neuronal maturation status, with differentiated, mature neurons being more resistant to infection than undifferentiated, immature neurons. The biological factors that change during neuronal maturation and that influence the outcome of viral infection are currently only partially defined. These studies investigated the role of NF-κB in determining the outcome of alphaviral infection in mature and immature neurons. Inhibition of canonical NF-κB activation decreased alphavirus replication in mature neurons by regulating protein synthesis and limiting the production of the viral structural proteins but had little effect on viral replication in immature neurons or fibroblasts. Therefore, NF-κB is a signaling pathway that influences the maturation-dependent outcome of alphaviral infection in neurons and that highlights the importance of cellular context in determining the effects of signal pathway activation., (Copyright © 2019 American Society for Microbiology.)

Measles virus is directly responsible for more than 100,000 deaths yearly. Epidemiological studies have associated measles with increased morbidity and mortality for years after infection, but the reasons why are poorly understood. Measles virus infects immune cells, causing acute immune suppression. To identify and quantify long-term effects of measles on the immune system, we used VirScan, an assay that tracks antibodies to thousands of pathogen epitopes in blood. We studied 77 unvaccinated children before and 2 months after natural measles virus infection. Measles caused elimination of 11 to 73% of the antibody repertoire across individuals. Recovery of antibodies was detected after natural reexposure to pathogens. Notably, these immune system effects were not observed in infants vaccinated against MMR (measles, mumps, and rubella), but were confirmed in measles-infected macaques. The reduction in humoral immune memory after measles infection generates potential vulnerability to future infections, underscoring the need for widespread vaccination., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Zika virus (ZIKV) is a re-emerged flavivirus transmitted by Aedes spp mosquitoes that has caused outbreaks of fever and rash on islands in the Pacific and in the Americas. These outbreaks have been associated with neurologic complications that include congenital abnormalities and Guillain-Barré syndrome (GBS). The pathogenesis of ZIKV-associated GBS, a potentially life-threatening peripheral nerve disease, remains unclear. Because Schwann cells (SCs) play a central role in peripheral nerve function and can be the target for damage in GBS, we characterized the interactions of ZIKV isolates from Africa, Asia and Brazil with human SCs in comparison with the related mosquito-transmitted flaviviruses yellow fever virus 17D (YFV) and dengue virus type 2 (DENV2). SCs supported sustained replication of ZIKV and YFV, but not DENV. ZIKV infection induced increased SC expression of IL-6, interferon (IFN)β1, IFN-λ, IFIT-1, TNFα and IL-23A mRNAs as well as IFN-λ receptors and negative regulators of IFN signaling. SCs expressed baseline mRNAs for multiple potential flavivirus receptors and levels did not change after ZIKV infection. SCs did not express detectable levels of cell surface Fcγ receptors. This study demonstrates the susceptibility and biological responses of SCs to ZIKV infection of potential importance for the pathogenesis of ZIKV-associated GBS.

The Global Virus Network (GVN) was established in 2011 to strengthen research and responses to emerging viral causes of human disease and to prepare against new viral pandemics. There are now 45 GVN Centers of Excellence and 7 Affiliate laboratories in 29 countries. The 10th International GVN meeting was held from November 28-30, 2018 in Veyrier du Lac, France and was co-hosted by the two GVN Centers of Excellence, the Mérieux Foundation and the University of Veterinary Medicine Hannover (TiHo). The theme of this 10th International GVN meeting was "Eradication and control of (re-) emerging viruses". This report highlights the recent accomplishments of GVN researchers in several important areas of medical virology, including strategies for the eradication of smallpox, measles, polio, SARS and vector-borne or zoonotic infections, emergence and intervention strategies for retroviruses and arboviruses, preparedness for outbreaks of Filo- and other hemophilic viruses, pathogenesis, impact and prevention of respiratory viruses, as well as, viruses affecting the central and peripheral nervous system. Also threats in crisis settings like refugee camps were presented., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Interferon (IFN) regulatory factors (IRFs) are important determinants of the innate response to infection. We evaluated the role(s) of combined and individual IRF deficiencies in the outcome of infection of C57BL/6 mice with Sindbis virus, an alphavirus that infects neurons and causes encephalomyelitis. The brain and spinal cord levels of Irf7, but not Irf3 mRNAs, were increased after infection. IRF3/5/7-/- and IRF3/7-/- mice died within 3-4 days with uncontrolled virus replication, similar to IFNα receptor-deficient mice, while all wild-type (WT) mice recovered. IRF3-/- and IRF7-/- mice had brain levels of IFNα that were lower, but brain and spinal cord levels of IFNβ and IFN-stimulated gene mRNAs that were similar to or higher than WT mice without detectable serum IFN or increases in Ifna or Ifnb mRNAs in the lymph nodes, indicating that the differences in outcome were not due to deficiencies in the central nervous system (CNS) type I IFN response. IRF3-/- mice developed persistent neurological deficits and had more spinal cord inflammation and higher CNS levels of Il1b and Ifnγ mRNAs than WT mice, but all mice survived. IRF7-/- mice died 5-8 days after infection with rapidly progressive paralysis and differed from both WT and IRF3-/- mice in the induction of higher CNS levels of IFNβ, tumour necrosis factor (TNF) α and Cxcl13 mRNA, delayed virus clearance and more extensive cell death. Therefore, fatal disease in IRF7-/- mice is likely due to immune-mediated neurotoxicity associated with failure to regulate the production of inflammatory cytokines such as TNFα in the CNS.

Measles virus (MV) is a highly contagious member of the Morbillivirus genus that remains a major cause of childhood mortality worldwide. Although infection induces a strong MV-specific immune response that clears viral load and confers lifelong immunity, transient immunosuppression can also occur, leaving the host vulnerable to colonization from secondary pathogens. This apparent contradiction of viral clearance in the face of immunosuppression underlies what is often referred to as the 'measles paradox', and remains poorly understood. To explore the mechanistic basis underlying the measles paradox, and identify key factors driving viral clearance, we return to a previously published dataset of MV infection in rhesus macaques. These data include virological and immunological information that enable us to fit a mathematical model describing how the virus interacts with the host immune system. In particular, our model incorporates target cell depletion through infection of host immune cells-a hallmark of MV pathology that has been neglected from previous models. We find the model captures the data well, and that both target cell depletion and immune activation are required to explain the overall dynamics. Furthermore, by simulating conditions of increased target cell availability and suppressed cellular immunity, we show that the latter causes greater increases in viral load and delays to MV clearance. Overall, this signals a more dominant role for cellular immunity in resolving acute MV infection. Interestingly, we find contrasting dynamics dominated by target cell depletion when viral fitness is increased. This may have wider implications for animal morbilliviruses, such as canine distemper virus (CDV), that cause fatal target cell depletion in their natural hosts. To our knowledge this work represents the first fully calibrated within-host model of MV dynamics and, more broadly, provides a new platform from which to explore the complex mechanisms underlying Morbillivirus infection., Competing Interests: The authors have declared that no competing interests exist.