ABSTRACT
In this study, we developed a novel, multiplex qPCR assay for simultaneous detection of RIG-1, MDA5, and IFIT-1 at the mRNA level. The assay was validated in A549 cells transfected with in vitro transcribed RNAs. Both exogenous RNA-GFP and self-amplifying (saRNA-GFP) induced significant expression of RIG-1, MDA5, IFIT-1, as well as type I and III interferons. In contrast, native RNA from intact A549 cells did not upregulate expression of these genes. Next, we evaluated RIG-1, MDA5, and IFIT-1 mRNA levels in the white blood cells of patients with influenza A virus (H3N2) or SARS-CoV-2. In acute phase (about 4 days after disease onset) both viruses induced these genes expression. Clinical observations of SARS-CoV-2 typically describe a two-step disease progression, starting with a mild-to-moderate presentation followed by a secondary respiratory worsening 9 to 12 days after the first onset of symptoms. It revealed that the expression of RIG-1, MDA5, and MxA was not increased after 2 and 3 weeks from the onset the disease, while for IFIT-1 it was observed the second peak at 21 day post infection. It is well known that RIG-1, MDA5, and IFIT-1 expression is induced by the action of interferons. Due to the ability of SOCS-1 to inhibit interferon-dependent signaling, and the distinct antagonism of SARS-CoV-2 in relation to interferon-stimulated genes expression, we assessed SOCS-1 mRNA levels in white blood cells. SARS-CoV-2 patients had increased SOCS-1 expression, while the influenza-infected group did not differ from heathy donors. Moreover, SOCS-1 mRNA expression remained stably elevated during the course of the disease. It can be assumed that augmented SOCS-1 expression is one of multiple mechanisms that allow SARS-CoV-2 to escape from the interferon-mediated immune response. Our results implicate SOCS-1 involvement in the pathogenesis of SARS-CoV-2.
Subject(s)
COVID-19 , Interferons , Humans , Interferons/metabolism , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/metabolism , Influenza A Virus, H3N2 Subtype/genetics , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , SARS-CoV-2/genetics , DEAD Box Protein 58/genetics , DEAD Box Protein 58/metabolism , RNA-Binding Proteins , RNA, Messenger/genetics , Antiviral AgentsABSTRACT
Comparative analysis of 16S rRNA gene sequences, DNA-DNA hybridization data and phenotypic properties revealed that 'Sulfobacillus thermosulfidooxidans subsp. thermotolerans' strain K1 is not a member of the genus Sulfobacillus. Phylogenetically, strain K1 is closely related to unclassified strains of the genus Alicyclobacillus: the 16S rRNA gene sequence of strain K1 is similar to that of Alicyclobacillus sp. AGC-2 (99.6 %), Alicyclobacillus sp. 5C (98.9 %) and Alicyclobacillus sp. CLG (98.6 %) and bacterium GSM (99.1 %). The 16S rRNA gene sequence similarity values for strain K1 and species of the genus Alicyclobacillus with validly published names were in the range 92.1-94.6 %, and for S. thermosulfidooxidans VKM B-1269(T) the value was 87.7 %. Sulfobacillus disulfidooxidans SD-11(T) was also phylogenetically related to strain K1 (92.6 % sequence similarity) and thus belonged to the genus Alicyclobacillus. Chemotaxonomic data, such as the major cell-membrane lipid components of strains K1 and SD-11(T) (omega-alicyclic fatty acids) and the major isoprenoid quinone (menaquinone MK-7) of strain K1, supported the affiliation of strains K1 and SD-11(T) to the genus Alicyclobacillus. Physiological and molecular biological tests allowed genotypic and phenotypic differentiation of strains K1 and SD-11(T) from the nine Alicyclobacillus species with validly published names. The G+C content of the DNA of strain K1 was 48.7+/-0.6 mol%; that of strain SD-11(T) was 53+/-1 mol%. DNA-DNA reassociation studies showed low relatedness (22 %) between strains K1 and SD-11(T), and even lower relatedness (3-5 %) between these strains and Alicyclobacillus acidocaldarius subsp. acidocaldarius ATCC 27009(T), DSM 446(T). DNA reassociation of strains K1 and SD-11(T) with Alicyclobacillus cycloheptanicus DSM 4006(T) gave values of 15 and 21, respectively. Based on the phenotypic and phylogenetic characteristics of strains K1 and SD-11(T), Alicyclobacillus tolerans sp. nov. (type strain, K1(T)=VKM B-2304(T)=DSM 16297(T)) and Alicyclobacillus disulfidooxidans comb. nov. (type strain, SD-11(T)=ATCC 51911(T)=DSM 12064(T)) are proposed.