Grapevine fruits produced in China are often infected by grapevine berry inner necrosis virus (GINV). GINV disease is the most economically important viral disease of the grapevine, characterized by discoloration on the fruit surface and necrosis of the flesh. In this study, the transcriptome sequencing technology revealed 1700 differently expressed genes (DEGs), with 846 up-regulated and 854 down-regulated genes in Crimson seedless grapevine during the GINV infection. Both Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to classify the DEGs. The top 10 GO terms were enriched in GINV versus mock, and these enriched GO terms were mainly classified into three categories, namely, 324 cellular components, 2062 biological processes, and 940 molecular functions. Based on the P-values, the KEGG pathway analysis indicated that 96 major pathways were identified in GINV versus mock, and 20 major enrichment pathways were significantly enriched. Using quantitative real-time polymerase chain reaction (qRT-PCR), we validated the differential expression of 12 genes. The expression patterns of 11 genes were consistent with the RNA-sequencing (RNA-seq) results, except for one gene. Thus, our study provides comprehensive transcriptome information on the GINV–grapevine interaction, thereby improving our understanding of virus–host interactions.
Human rhinoviruses are known to cause mild upper respiratory tract infections. They are also now known to cause severe laryngotracheitis and play an important role in triggering asthma attacks. An anti-viral drug against rhinoviruses viruses would be helpful. Acetylsalicylic acid (ASA) might be such a candidate with proven activity against several RNA viruses in vitro and in vivo. ASA was initially mentioned as an anti-inflammatory compound. Viral infections are often accompanied by inflammatory processes. In this study, the anti-viral and anti-inflammatory activities of ASA were examined using the HeLa rhinovirus-infection model. Human rhinovirus (HRV14, major group) infection was quantified by flow cytometry using a monoclonal antibody against the major capsid protein HRV-VP3 in combination with analysis of surface expression of CD54, a key molecule involved in the initiation of an immune response. Our in vitro studies demonstrate on a single cell level HRV14 infection-dependent intra-cellular expression of the HRV-VP3 protein. This expression correlated with HRV14 infection severity and could be dose-dependently blocked with ASA. Further, infection-dependent VP3 expression correlated with a down-regulation of surface antigen expression of CD54. This CD54 down-regulation could also be dose-dependently blocked by ASA supporting the anti-viral efficacy of ASA.
Recombinant herpes simplex virus strains can be constructed by several methods, including homologous recombination, bacterial artificial chromosome manipulation, and yeast genetic methods. Homologous recombination may have the advantage of introducing fewer genetic alterations in the viral genome, but the low level of recombinants can make this method more time consuming if there is no screen or selection. In this study we used complementing cell lines that express Cas9 and guide RNAs targeting the parental virus to rapidly generate recombinant viruses. Analysis of the progeny viruses indicated that CRISPR-Cas9 both promoted recombination to increase recombinant viruses and selected against parental viruses in the transfection progeny viruses. This approach can also be used to enrich for recombinants made by any of the current methods.
Infection with pathogenic viruses is often sensed by innate receptors such as Toll-Like Receptors (TLRs) which stimulate type I and III interferons (IFNs) responses, to generate an antiviral state within many cell types. To counteract these antiviral systems, many viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode non-structural proteins (NSPs) that mediate immune evasion. Using an overexpression system in A549 cells, we demonstrated a significant increase (p ≤ 0.0001) in Vesicular Stomatitis Virus (VSV)-EGFP reporter virus replication in cell lines overexpressing either the SARS-CoV-2 NSP1 or NSP15 when compared to control A549 cells. The increase in VSV-EGFP virus output was associated with a decrease in TLR2, TLR4 and TLR9 protein expression and a lack of antiviral protein production. Truncation of both NSP1 and NSP15 led to an increase in cellular TLR2, TLR4 and TLR9 as well as a decrease in TLR2 expression respectively. This observation can be attributed to the presence of a functional domain in NSP1 and NSP15 between amino acid (aa) 120–180 and aa 230–346, respectively. Both TLR3 and TLR9 ligands but not TLR2 ligand were highly effective at overcoming NSP1 and NSP15 functional interference based on significant decrease (p ≤ 0.0001) in VSV-EGFP virus replication. NSP1 or NSP15 intracellular interactions are likely low affinity interactions that can be easily disrupted by stimulating cells with specific TLR3 and TLR9 ligands. This report provides insights into the role of SARS-CoV-2 NSP1 and NSP15 in limiting specific TLR pathway activation, as an evasive mechanism against host innate responses.
