Mosquito-borne arboviruses are a major source of human disease. One strategy to reduce arbovirus disease is to reduce the mosquito's ability to transmit virus. Mosquito infection with the bacterial ...endosymbiont
Mel is a novel strategy to reduce
mosquito competency for flavivirus infection. However, experiments investigating cyclic environmental temperatures have shown a reduction in maternal transmission of
Mel, potentially weakening the integration of this strain into a mosquito population relative to that of other
strains. Consequently, it is important to investigate additional
strains. All Zika virus (ZIKV) suppression studies are limited to the
Mel
strain. Here we show ZIKV inhibition by two different
strains:
AlbB (isolated from
mosquitoes) and
Stri (isolated from the planthopper
) in mosquito cells.
strain
Stri inhibited ZIKV most effectively. Single-cycle infection experiments showed that ZIKV RNA replication and nonstructural protein 5 translation were reduced below the limits of detection in
Stri-containing cells, demonstrating early inhibition of virus replication. ZIKV replication was rescued when
was inhibited with a bacteriostatic antibiotic. We observed a partial rescue of ZIKV growth when
-infected cells were supplemented with cholesterol-lipid concentrate, suggesting competition for nutrients as one of the possible mechanisms of
inhibition of ZIKV. Our data show that
AlbB and
Stri infection causes inhibition of ZIKV, making them attractive candidates for further
mechanistic and
studies and future vector-centered approaches to limit ZIKV infection and spread.
Zika virus (ZIKV) has swiftly spread throughout most of the Western Hemisphere. This is due in large part to its replication in and spread by a mosquito vector host. There is an urgent need for approaches that limit ZIKV replication in mosquitoes. One exciting approach for this is to use a bacterial endosymbiont called
that can populate mosquito cells and inhibit ZIKV replication. Here we show that two different strains of
,
AlbB and
Stri, are effective at repressing ZIKV in mosquito cell lines. Repression of virus growth is through the inhibition of an early stage of infection and requires actively replicating
Our findings further the understanding of
viral inhibition and provide novel tools that can be used in an effort to limit ZIKV replication in the mosquito vector, thereby interrupting the transmission and spread of the virus.
Dengue fever (DF), caused by the dengue virus (DENV), is the most burdensome arboviral disease in the world, with an estimated 400 million infections each year. The
mosquito is the main vector of ...DENV and transmits several other human pathogens, including Zika, yellow fever, and chikungunya viruses. Previous studies have shown that the pathogen infection of mosquitoes can alter reproductive fitness, revealing specific vector-pathogen interactions that are key determinants of vector competence. However, only a handful of studies have examined the effect of DENV infection in
, showing a reduction in lifespan and fecundity over multiple blood meals. To provide a more comprehensive analysis of the impact of DENV infection on egg laying and fecundity, we assessed egg laying timing in DENV-2 blood-fed mosquitoes (infected group) compared to mock blood-fed mosquitoes (control group). We confirmed a significant decrease in fecundity during the first gonadotrophic cycle. To further investigate this phenotype and the underlying DENV-2 infection-dependent changes in gene expression, we conducted a transcriptomic analysis for differentially expressed genes in the ovaries of
infected with DENV-2 vs. mock-infected mosquitoes. This analysis reveals several DENV-2-regulated genes; among them, we identified a group of 12 metabolic genes that we validated using reverse transcription-quantitative PCR (RT-qPCR). Interestingly, two genes found to be upregulated in DENV-infected mosquito ovaries exhibited an antiviral role for DENV-2 in an
cell line. Altogether, this study offers useful insights into the virus-vector interface, highlighting the importance of gene expression changes in the mosquito's ovary during DENV-2 infection in the first gonadotrophic cycle, triggering antiviral responses that may possibly interfere with mosquito reproduction. This information is extremely relevant for further investigation of
ability to tolerate viruses since virally infected mosquitoes in nature constitute a powerful source of supporting viruses during intra-epidemic periods, causing a huge burden on the public health system.
Although TLR9 was originally thought to specifically recognize microbial DNA, it is now evident that mammalian DNA can be an effective TLR9 ligand. However, the DNA sequence required for TLR9 ...activation is controversial, as studies have shown conflicting results depending on the nature of the DNA backbone, the route of DNA uptake, and the cell type being studied. In systemic lupus erythematosus, a major route whereby DNA gains access to intracellular TLR9, and thereby activates dendritic cells (DCs), is through uptake as a DNA-containing immune complex. In this report, we used defined dsDNA fragments with a natural (phosphodiester) backbone and show that unmethylated CpG dinucleotides within dsDNA are required for murine DC TLR9 activation induced by a DNA-containing immune complex. The strongest activation is seen with dsDNA fragments containing optimal CpG motifs (purine-purine-CpG-pyrimidine-pyrimidine) that are common in microbial DNA but rare in mammalian DNA. Importantly, however, activation can also be induced by CpG-rich DNA fragments that lack these optimal CpG motifs and that we show are plentiful in CpG islands within mammalian DNA. No activation is induced by DNA fragments lacking CpG dinucleotides, although this CpG-free DNA can induce DC activation if internalized by liposomal transfection instead of as an immune complex. Overall, the data suggest that the release of CpG-rich DNA from mammalian DNA may contribute to the pathogenesis of autoimmune diseases such as systemic lupus erythematosus and psoriasis in which activation of TLR9 in DCs by self DNA has been implicated in disease pathogenesis.
According to WHO, as of July 2, 2021, the pandemic has resulted in 182 101 209 confirmed cases and 3 950 876 deaths, both undoubtedly underestimates of the real toll. Equally essential will be ...ensuring that the field workforce, laboratory facilities, and the health-care community can all work under the safest conditions. ...this pandemic ends, we ask, as we did in February, 2020,1 for solidarity and rigorous scientific data. All of EcoHealth Alliance's work is reviewed and approved by appropriate research ethics committees, the Institutional Animal Care and Use Committee, institutional review boards for biomedical research involving human subjects, P3CO oversight administrators, and biosafety committees, as listed on all relevant publications. JSM is a member of the WHO International Health Regulations Emergency Committee for COVID-19, a member of the One Health High Level Expert Panel that advises the Food and Agriculture Organization of the UN, the World Organisation for Animal Health, the United Nations Environment Programme, and WHO, and a past member of the scientific advisory committee for the Center for Emerging Infectious Diseases of the Wuhan Institute of Virology (2008–11).
Marburg and Ebola viruses cause a severe hemorrhagic disease in humans with high fatality rates. Early target cells of filoviruses are monocytes, macrophages, and dendritic cells. The infection ...spreads to the liver, spleen and later other organs by blood and lymph flow. A hallmark of filovirus infection is the depletion of non-infected lymphocytes; however, the molecular mechanisms leading to the observed bystander lymphocyte apoptosis are poorly understood. Also, there is limited knowledge about the fate of infected cells in filovirus disease. In this review we will explore what is known about the intracellular events leading to virus amplification and cell damage in filovirus infection. Furthermore, we will discuss how cellular dysfunction and cell death may correlate with disease pathogenesis.
The COVID-19 pandemic has drastically impacted work, economy, and way of life. Sensitive measurement of SARS-CoV-2 specific antibodies would provide new insight into pre-existing immunity, virus ...transmission dynamics, and the nuances of SARS-CoV-2 pathogenesis. To date, existing SARS-CoV-2 serology tests have limited utility due to insufficient reliable detection of antibody levels lower than what is typically present after several days of symptoms. To measure lower quantities of SARS-CoV-2 IgM, IgG, and IgA with higher resolution than existing assays, we developed a new ELISA protocol with a distinct plate washing procedure and timed plate development
use of a standard curve. Very low optical densities from samples added to buffer coated wells at as low as a 1:5 dilution are reported using this 'BU ELISA' method. Use of this method revealed circulating SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid protein (N) reactive antibodies (IgG, IgM, and/or IgA) in 44 and 100 percent of pre-pandemic subjects, respectively, and the magnitude of these antibodies tracked with antibody levels of analogous viral proteins from endemic coronavirus (eCoV) strains. The disease status (HIV, SLE) of unexposed subjects was not linked with SARS-CoV-2 reactive antibody levels; however, quantities were significantly lower in subjects over 70 years of age compared with younger counterparts. Also, we measured SARS-CoV-2 RBD- and N- specific IgM, IgG, and IgA antibodies from 29 SARS-CoV-2 infected individuals at varying disease states, including 10 acute COVID-19 hospitalized subjects with negative serology results by the EUA approved Abbott IgG chemiluminescent microparticle immunoassay. Measurements of SARS-CoV-2 RBD- and N- specific IgM, IgG, IgA levels measured by the BU ELISA revealed higher signal from 9 of the 10 Abbott test negative COVID-19 subjects than all pre-pandemic samples for at least one antibody specificity/isotype, implicating improved serologic identification of SARS-CoV-2 infection
multi-parameter, high sensitive antibody detection. We propose that this improved ELISA protocol, which is straightforward to perform, low cost, and uses readily available commercial reagents, is a useful tool to elucidate new information about SARS-CoV-2 infection and immunity and has promising implications for improved detection of all analytes measurable by this platform.
Secreted IgM and complement are important mediators in the optimal initiation of primary T-dependent humoral immune responses. Secreted IgM serves as a natural adjuvant by enhancing the ...immunogenicity of protein antigens, perhaps as a result of IgM's ability to facilitate antigen deposition onto follicular dendritic cells (FDCs) and promote rapid germinal center (GC) formation. To understand how IgM enhances adaptive immune responses, we investigated the mechanism by which IgM-containing immune complexes (IgM-IC) are transported to FDCs as a first step in GC formation. We demonstrate that IgM-IC localize first to the splenic marginal zone (MZ) where the IgM-IC bind MZ B cells in a complement and complement receptor (CR1/2) dependent process. MZ B cells then transport the IgM-IC into the follicle for deposition onto FDCs. Mice with reduced numbers of MZ B cells trap IgM-IC on FDC less efficiently, whereas mice with reduced numbers of follicular B cells trap IgM-IC normally. The functional elimination of MZ B cells abrogates the ability of FDCs to trap IgM-IC. Transfer of B cells with associated IgM-IC into naive mice results in deposition of IgM-IC onto FDC by MZ B cells. The results demonstrate an IgM and complement-dependent role for MZ B cells in the fate of antigen early in the initial phases of T-dependent immune responses. The data also establish an important role for CR1/2 on MZ B cells in the efficient binding and transport of IgM-IC to FDCs, which we suggest is an important first step in initiating adaptive immune responses.
We have investigated the roles of IgM and complement (C) in the enhancement of primary immune responses and the localization of protein antigen (Ag) in the spleen. Pentameric but not monomericIgM ...enhances antibody (Ab) responses in both wild‐type and secretory μ‐deficient (μs–/–) mice, indicating that a single IgM clone is sufficient as long as it activates C. Ag localizes on follicular dendritic cells (FDC) within 16 h after injection of immune complexes (IC) containing pentameric but not monomeric IgM. Surprisingly, pentameric IgM‐containing IC were trapped in spleens of C3‐depleted and Cr2‐deficient mice. However, the IC were found only in the marginal zone (MZ), associated predominantly with MZ macrophages. IC were also detected in the MZ in normal mice within 1 h after injection, but associated with other cells in addition to MZ macrophages. The results demonstrate an important role for pentameric IgM in the initiation of Ag trapping, a step independent of C3 activation and of the interaction of IC with CR1 and CR2. The data also provide direct evidence that C3 activation is required for the next phase of localization, in which Ag moves from the MZ to FDC, with consequent enhancement of specific immune responses.
We have previously shown that rheumatoid factors produced by Fas-deficient autoimmune-prone mice typically bind autologous IgG2a with remarkably low affinity. Nevertheless, B cells representative of ...this rheumatoid factor population proliferate vigorously in response to IgG2a/chromatin immune complexes through a mechanism dependent on the sequential engagement of the BCR and TLR9. To more precisely address the role of both receptors in this response, we analyzed the signaling pathways activated in AM14 B cells stimulated with these complexes. We found that the BCR not only serves to direct the chromatin complex to an internal compartment where it can engage TLR9 but also transmits a suboptimal signal that in combination with the signals emanating from TLR9 leads to NF-kappaB activation and proliferation. Importantly, engagement of both receptors leads to the up-regulation of a group of gene products, not induced by the BCR or TLR9 alone, that include IL-2. These data indicate that autoreactive B cells, stimulated by a combination of BCR and TLR9 ligands, acquire functional properties that may contribute to the activation of additional cells involved in the autoimmune disease process.
PDF
