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Retinoic acid-inducible gene I (RIG-I), a crucial member of cytoplasmic pattern recognition receptors (PRRs), is initially characterized as a dsRNA-binding protein triggering interferon (IFN) induction in response to virus invasion. While the antiviral regulatory mechanism of RIG-I remains largely unclear. In this study, the mechanism of CiRIG-I ( Ctenopharyngodon idella RIG-I ) against grass carp reovirus (GCRV) would be revealed from the perspective of epigenetics. By prediction, three CpG islands (CGIs) were located in 5′-flanking region and the first exon. The first CGI and the second one, both located in the 5′-flanking region, were 109 base pairs (bp) and 134 bp in length, involving five CpG sites and four loci, respectively. The third CGI was of 386 bp spanning the 5′-flanking region and the first exon, densely possessing 24 CpG sites. DNA methylation statuses of CpG sites were identified by virtue of the bisulfite sequencing PCR (BSP) in spleen of all susceptible and resistant individuals post the challenge experiment. The resistance association analysis was performed with Chi-square test. And the relationship between DNA methylation and gene expression in CiRIG-I was investigated by quantitative real-time RT-PCR (qRT-PCR). Results indicated only the methylation of -534 CpG site in the second CGI possessing tight association with the resistance against GCRV, which was significantly higher in the susceptible individuals than that in the resistant individuals. In addition, the average expression of CiRIG-I was down-regulated in the susceptible group compared with the resistant one demonstrating gene transcription may be negatively-regulated by DNA methylation in CiRIG-I . Collectively, the methylation statuses of CiRIG-I were extremely related to the resistance against GCRV and maybe serve as a negative modulator on antiviral transcription of CiRIG-I . This study revealed the underlying antiviral regulatory mechanism of CiRIG-I and laid a theoretical foundation for the nosogenesis of hemorragic diseases in C. idella . [ABSTRACT FROM AUTHOR]

Melanoma differentiation-associated gene 5 (MDA5) plays a crucial role in recognizing intracellular viral infection, activating the interferon regulatory factor pathways as well as inducing antiviral response. While the antiviral regulatory mechanism of MDA5 remains unclear. In the present study, CiMDA5 ( Ctenopharyngodon idella MDA5 ) against grass carp reovirus (GCRV) would be initially revealed from the perspective of DNA methylation, a pivotal epigenetic modification. Two CpG islands (CGIs) were predicted located in the first exon of CiMDA5 , of which the first CpG island was 427 bp in length possessed 29 candidate CpG loci and 34 CpA loci, and the second one was 130 bp in length involving 7 CpG loci as well as 10 CpA loci. By bisulfite sequencing PCR (BSP), the methylation statuses were detected in spleen of 70 individuals divided into resistant/susceptible groups post challenge experiment, and the resistance-association analysis was performed with Chi-square test. Quantitative real-time RT-PCR (qRT-PCR) was carried out to explore the relationship between DNA methylation and gene expression in CiMDA5 . Results indicated that the methylation levels of CpA/CpG sites at +200, +202, +204, +207 nt, which consisted of a putative densely methylated element (DME), were significantly higher in the susceptible group than those in the resistant group. Meanwhile, the average transcription of CiMDA5 was down-regulated in the susceptible individuals compared with the resistant individuals. Evidently, the DNA methylation may be the negative modulator of CiMDA5 antiviral expression. Collectively, the methylation levels of CiMDA5 demonstrated the tight association with the resistance against GCRV and the negative-regulated roles in mRNA expression. This study first discovered the resistance-associated gene modulated by DNA methylation in teleost, preliminary revealed the underlying regulatory mechanism of CiMDA5 transcription against GCRV as well as laid a theoretical foundation on molecular nosogenesis of hemorrhagic diseases in C. idella . [ABSTRACT FROM AUTHOR]

As an intracellular pattern recognition receptor (PRR), laboratory of genetics and physiology 2 (LGP2) plays a pivotal role in detecting nucleic acids of invading pathogens and simultaneously modulating signaling by retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) in type I interferon (IFN–I) pathway. Nevertheless, the underlying antiviral transcription mechanism of LGP2 remains obscure. The present study attempted to reveal the methylation levels of CiLGP2 ( Ctenopharyngodon idella LGP2 ) in muscle and spleen of grass carp and their association with the resistance against grass carp reovirus (GCRV). By prediction, the CpG island was 133 bp in length in 5′-flanking region, containing six candidate CpG loci, whose methylation statuses were investigated by virtue of the bisulfite sequencing PCR (BSP) among muscle and spleen tissues in 120 individuals that were divided into resistant/susceptible groups after a challenge experiment, and the association analysis was performed with Chi-square test. Quantitative real-time RT-PCR (qRT-PCR) was employed to ascertain the interrelation between methylation status and transcription of CiLGP2 . The CpG sites at −1394, −1366, −1331 and −1314 nt were identified as hypermethylated, inversely unmethylated at −1350 CpG site. The −1411 CpG site presented six methylation patterns as well as one mentionable type of mutation triggered by spontaneous deamination. Although there was no statistically significant difference on DNA methylation with resistance against GCRV at −1411 CpG site, the methylation levels were significantly lower in spleen than those in muscle, accompanied by higher mRNA expression of CiLGP2 in spleen. Notably, DNA methylation may be conceivably serve as an essential regulatory factor for CiLGP2 antiviral transcription in spleen. This research first demonstrated the relationship between DNA methylation and LGP2 gene expression, preliminary revealed the underlying transcription mechanism of CiLGP2 against GCRV as well as provided potential references and laid a theoretical foundation for viral recognition and regulation research of LGP2 in vertebrates. [ABSTRACT FROM AUTHOR]

Toll-like receptor 8 (TLR8), a prototypical intracellular member of TLR family, is generally linked closely to antiviral innate immune through recognizing viral nucleic acid. In this study, 5′-flanking region of Ctenopharyngodon idella TLR8 ( CiTLR8 ), 671 bp in length, was amplified and eight SNPs containing one SNP in the intron, three SNPs in the coding region (CDS) and four SNPs in the 3′-untranslated region (UTR) were identified and characterized. Of which 4062 A/T was significantly associated with the susceptibility/resistance to GCRV both in genotype and allele ( P < 0.05), while 4168 C/T was extremely significantly associated with that ( P < 0.01) according to the case (susceptibility)–control (resistance) analysis. Following the verification experiment, further analyses of mRNA expression, linkage disequilibrium (LD), haplotype and microRNA (miRNA) target site indicated that 4062 A/T and 4168 C/T in 3′-UTR might affect the miRNA regulation, while the exertion of antiviral effects of 4062 A/T might rely on its interaction with other SNPs. Additionally, the high-density of SNPs in 3′-UTR might reflect the specific biological functions of 3′-UTR. And also, the mutation of 747 A/G in intron changing the potential transcriptional factor-binding sites (TFBS) nearby might affect the expression of CiTLR8 transcriptionally or post-transcriptionally. Moreover, as predicted, the A/G transition of the only non-synonymous SNP (3846 A/G) in CDS causing threonine/alanine variation, could shorten the length of the α-helix and ultimately affect the integrity of the Toll-IL-1 receptor (TIR) domain. The functional mechanism of 3846 A/G might also involve a threonine phosphorylation signaling. This study may broaden the knowledge of TLR polymorphisms, lay the foundation for further functional research of CiTLR8 and provide potential markers as well as theoretical basis for resistance molecular breeding of grass carp against GCRV. [ABSTRACT FROM AUTHOR]