Accumulation Of Viral Transcripts Research Articles

MAVS signaling shapes microglia responses to neurotropic virus infection

Viral encephalitis is characterized by a series of immunological reactions that can control virus infection in the brain, but dysregulated responses may cause excessive inflammation and brain damage. Microglia are brain-resident myeloid cells that are specialized in surveilling the local CNS environment and in case of viral brain infection they contribute to the control of the infection and to restriction of viral dissemination. Here, we report that after exposure to neurotropic vesicular stomatitis virus (VSV), murine in vitro microglia cultures showed rapid upregulation of a broad range of pro-inflammatory and antiviral genes, which were stably expressed over the entire 8 h infection period. Additionally, a set of immunomodulatory genes was upregulated between 6 and 8 h post infection. In microglia cultures, the induction of several immune response pathways including cytokine responses was dependent on mitochondrial antiviral-signaling protein (MAVS). Consequently, in Mavs-deficient microglia the control of virus propagation failed as indicated by augmented virus titers and the accumulation of viral transcripts. Thus, in the analyzed in vitro system, MAVS signaling is critically required to achieve full microglia activation and to mediate profound antiviral effects. In Mavs-deficient mice, intranasal VSV instillation caused higher disease severity than in WT mice and virus dissemination was noticed beyond the olfactory bulb. Virus spread to inner regions of the olfactory bulb, i.e., the granular cell layer, correlated with the recruitment of highly inflammatory non-microglia myeloid cells into the olfactory bulb in Mavs−/− mice. Furthermore, increased cytokine levels were detected in the nasal cavity, the olfactory bulb and in other brain regions. Thus, microglial MAVS signaling is critically needed for virus sensing, full microglia activation, and for orchestration of protective immunity in the virus-infected CNS.

Extrachromosomal viral DNA produced by transcriptionally active endogenous viral elements in non-infected banana hybrids impedes quantitative PCR diagnostics of banana streak virus infections in banana hybrids.

The main edible and cultivated banana varieties are intra- and interspecific hybrids of the two main Musa species, Musa acuminata and Musa balbisiana, having diploid genomes denoted A and B, respectively. The B genome naturally hosts sequences of banana streak virus (BSV) named endogenous BSV (eBSV). Upon stress, eBSVs are identified as the origin of BSV infection for at least three BSV species, causing banana streak disease. For each of the three species, BSV and eBSV share >99.9 % sequence identity, complicating PCR-based diagnosis of viral infection in the B genome-containing bananas. Here, we designed a quantitative PCR-based method to only quantify episomal BSV particles produced, overcoming the limitation of eBSV also being detected by qPCR by using it as a 'calibrator'. However, our results revealed unexpected variation of eBSV amplification in calibrator plants composed of a clonal population of 53 replicating virus-free banana hybrids with the same AAB genotype. Our in-depth molecular analyses suggest that this calibrator variation is due to the variable abundance of non-encapsidated extrachromosomal viral DNA, likely produced via the transcription of eBSVs, followed by occasional reverse transcription. We also present evidence that accumulation of viral transcripts in AAB plants is downregulated both at post-transcriptional and transcriptional levels by an RNA interference mechanism that keeps the plants free of virus infection. Finally, we recommend that such eBSV amplification variation be taken into account to establish a quantitative viral diagnostic for banana plants with the B genome.

Zika Virus Infection Activates Proinflammatory Cytokines and Triggers Monocyte Differentiation

In 2015, Zika virus (ZIKV) outbreak in several countries of South America was linked to microcephaly in newborns. Microcephaly, a severe clinical presentation, was first diagnosed in association with ZIKV, which primarily considered self-limiting infection. From infected keratinocytes, virus disseminates crossing blood-tissue barriers and is detected in various tissues including brain, muscles and placenta. One of the mechanisms utilized by viruses to cross tissue barriers involves leukocytes, where monocytes are often identified as the carriers. Recently, circulating monocytes were shown to harbor ZIKV, suggesting that these leukocyte populations could serve as vehicle to transport ZIKV to the fetus. We speculate that ZIKV infection could promote monocyte activation and release of cytokines causing tissue damage. However, little is known about monocyte susceptibility to ZIKV infection. Also, effect of ZIKV infection on transcriptional activation and production remains unclear.Materials and methods. Monocytes were isolated from cord blood buffy coats (University of Colorado Cord Blood Bank, Aurora) using Miltenyi magnetic bead separation kit (CD14 Microbeads; Miltenyi, Auburn, CA). Monocytes were infected with two ZIKV strains, PRVABC59 (South America) and IBH30656 (Nigeria), ATCC (Manassas, VA). Total RNA was extracted and used for Next generation Sequencing (Illustra RNAspin Mini kit (GE Healthcare, Marlborough, MA)). FastQ data generated by the MiSeq (Illumina, San Diego CA) was annotated and the sequence reads were analyzed by CLC workbench 9.0.1 (Qiagen, Germantown, MD) for the detection of viral and cellular genes. Differential expressions of cellular genes based on the Reads Per Kilobase of transcript per Million mapped reads were determined by comparing with mock-infected cells using RNA-seq analysis tool of CLC Workbench. Differentially regulated genes due to ZIKV infection, identified by CLC Workbench, were subjected for determining the deregulated pathways using IPA Ingenuity Pathways analysis tool (Qiagen Germantown, MD). Viral replication was determined by qPCR, Western blot and immunofluorescence. Cytokine activation was analyzed using Bio-Plex Pro Human Cytokine 27-plex Panel (Bio-Rad, Hercules, CA, USA) following manufacturer's instructions.Results. We have shown that South American and Nigerian strains of ZIKV can infect monocytes and actively replicate. Presence of capsid protein and accumulation of viral transcripts was demonstrated in vitro. Infection with PRVABC59 strain changes transcriptional activity of 332 cellular genes, while the Nigerian strain affected the transcription of 521 cellular genes compared to the mock-infected controls. Interestingly, 195 genes were commonly deregulated in cells infected with both strains. Analysis of these commonly affected genes identified multiple pathways including apoptosis, chemotaxis, cell proliferation, inflammation, acute phase response, mitochondria activation. The most amazing observation was upregulation of mitochondrial and inflammasome genes. Mitochondrial genes (MT-ND2, MT-ND5 and MT-ATP8) activated in ZIKV infected monocytes were shown to play a key role in pathogenesis of other neurodegenerative disease.Analysis of cytokine production revealed early activation of proinflammatory cytokines (IL-1β, IL-6 and IL-8) (Table 1). Upregulation of IL-1β by ZIKV infected monocytes is evident for inflammasome activation. Upregulation of IL-6, IL-10 and IL-17α suggested an activation of Th17 pathway in ZIKV infected monocytes. Besides only Nigerian ZIKV strain increased the production of IL-4 in monocytes. IL-4 together with IL-10 could promote Th2 type immune response to ZIKV. Finally, both strains increased production of FGF, while levels of VEGF were increased only in supernatants of PRVABC59 infected monocytes.Conclusions. Human monocytes are susceptible to infection with ZIKV strains, PRVABC59 and IBH30656 and support viral replication. ZIKV infection affects cellular pathways, including mitochondrial and inflammasome ones that could play significant role in pathogenesis. ZIKV infection of monocytes upregulates cytokine production, which can trigger inflammation and leukocyte migration. Analysis of cytokine production by ZIKV infected monocytes suggests activation of Th2 and Th17 leukocytes, supporting the role of this signaling in ZIKV pathogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expression Profiling of Human Hepatoma Cells Reveals Global Repression of Genes Involved in Cell Proliferation, Growth, and Apoptosis upon Infection with Parvovirus H-1

Autonomous parvoviruses are characterized by their stringent dependency on host cell S phase and their cytopathic effects on neoplastic cells. To better understand the interactions between the virus and its host cell, we used oligonucleotide arrays that carry more than 19,000 unique human gene sequences to profile the gene expression of the human hepatocellular carcinoma cell line QGY-7703 at two time points after parvovirus H-1 infection. At the 6-h time point, a single gene was differentially expressed with a >2.5-fold change. At 12 h, 105 distinct genes were differentially expressed in virus-infected cells compared to mock-treated cells, with 93% of these genes being down-regulated. These repressed genes clustered mainly into classes involved in transcriptional regulation, signal transduction, immune and stress response, and apoptosis, as exemplified by genes encoding the transcription factors Myc, Jun, Fos, Ids, and CEBPs. Quantitative real-time reverse transcription-PCR analysis on selected genes validated the array data and allowed the changes in cellular gene expression to be correlated with the accumulation of viral transcripts and NS1 protein. Western blot analysis of several cellular proteins supported the array results and substantiated the evidence given by these and other data to suggest that the H-1 virus kills QGY-7703 cells by a nonapoptotic process. The promoter regions of most of the differentially expressed genes analyzed fail to harbor any motif for sequence-specific binding of NS1, suggesting that direct binding of NS1 to cellular promoters may not participate in the modulation of cellular gene expression in H-1 virus-infected cells.

Accumulation of viral transcripts and DNA during establishment of latency by herpes simplex virus.

Latent infection of mice with wild-type herpes simplex virus is established during an acute phase of ganglionic infection in which there is abundant viral replication and productive-cycle gene expression. Thymidine kinase-negative mutants establish latent infections but are severely impaired for acute ganglionic replication and productive-cycle gene expression. Indeed, by in situ hybridization assays, acute infection by these mutants resembles latency. To assess events during establishment of latency by wild-type and thymidine kinase-negative viruses, we quantified specific viral nucleic acid sequences in mouse trigeminal ganglia during acute ganglionic infection by using sensitive PCR-based assays. Through 32 h postinfection, viral DNA and transcripts representative of the three kinetic classes of productive-cycle genes accumulated to comparable levels in wild-type- and mutant-infected ganglia. At 48 and 72 h, although latency-associated transcripts accumulated to comparable levels in ganglia infected with wild-type or mutant virus, levels of DNA accumulating in wild-type-infected ganglia exceeded those in mutant-infected ganglia by 2 to 3 orders of magnitude. Coincident with this increase in DNA, wild-type-infected ganglia exhibited abundant expression of productive-cycle genes and high titers of infectious progeny. Nevertheless, the levels of productive-cycle RNAs expressed by mutant virus during acute infection greatly exceeded those expressed by wild-type virus during latency. The results thus distinguish acute infection of ganglia by a replication-compromised mutant from latent infection and may have implications for mechanisms of latency.