Ashwin Ashok Raut

The microRNA miR-485 targets host and influenza virus transcripts to regulate antiviral immunity and restrict viral replication

A single miRNA exhibits both an inhibitory effect on the antiviral response and an antiviral effect on viral replication. Targets foreign and domestic The cytosolic protein RIG-I (retinoic acid–inducible gene I) is a sensor of viral RNA, and its activation induces the host’s antiviral response. Ingle et al. found that infection of various human and mouse cells with RNA viruses, including the H5N1 influenza virus, resulted in the increased production of the microRNA miR-485, which targeted RIG-I mRNA for degradation. As a result, antiviral signaling was inhibited and viral replication was enhanced. However, when cells were exposed to increased amounts of virus, mir-485 was expressed, but viral replication was inhibited. Under these conditions, miR-485 targeted PB1 mRNA, which is a viral transcript required for H5N1 replication. Together, these data suggest that miR-485 exhibits bispecificity, with the extent of infection determining its target. MicroRNAs (miRNAs) are small noncoding RNAs that are responsible for dynamic changes in gene expression, and some regulate innate antiviral responses. Retinoic acid–inducible gene I (RIG-I) is a cytosolic sensor of viral RNA; RIG-I activation induces an antiviral immune response. We found that miR-485 of the host was produced in response to viral infection and targeted RIG-I mRNA for degradation, which led to suppression of the antiviral response and enhanced viral replication. Thus, inhibition of the expression of mir-485 markedly reduced the replication of Newcastle disease virus (NDV) and the H5N1 strain of influenza virus in mammalian cells. Unexpectedly, miR-485 also bound to the H5N1 gene PB1 (which encodes an RNA polymerase required for viral replication) in a sequence-specific manner, thereby inhibiting replication of the H5N1 virus. Furthermore, miR-485 exhibited bispecificity, targeting RIG-I in cells with a low abundance of H5N1 virus and targeting PB1 in cells with increased amounts of the H5N1 virus. These findings highlight the dual role of miR-485 in preventing spurious activation of antiviral signaling and restricting influenza virus infection.

IPS-1 differentially induces TRAIL , BCL2 , BIRC3 and PRKCE in type I interferons-dependent and -independent anticancer activity

RIG-I-like receptors are the key cytosolic sensors for RNA viruses and induce the production of type I interferons (IFN) and pro-inflammatory cytokines through a sole adaptor IFN-β promoter stimulator-1 (IPS-1) (also known as Cardif, MAVS and VISA) in antiviral innate immunity. These sensors also have a pivotal role in anticancer activity through induction of apoptosis. However, the mechanism for their anticancer activity is poorly understood. Here, we show that anticancer vaccine adjuvant, PolyIC (primarily sensed by MDA5) and the oncolytic virus, Newcastle disease virus (NDV) (sensed by RIG-I), induce anticancer activity. The ectopic expression of IPS-1 into type I IFN-responsive and non-responsive cancer cells induces anticancer activity. PolyIC transfection and NDV infection upregulate pro-apoptotic gene TRAIL and downregulate the anti-apoptotic genes BCL2, BIRC3 and PRKCE. Furthermore, stable knockdown of IPS-1, IRF3 or IRF7 in IFN-non-responsive cancer cells show reduced anticancer activity by suppressing apoptosis via TRAIL and anti-apoptotic genes. Collectively, our study shows that IPS-1 induces anticancer activity through upregulation of pro-apoptotic gene TRAIL and downregulation of the anti-apoptotic genes BCL2, BIRC3 and PRKCE via IRF3 and IRF7 in type I IFN-dependent and -independent manners.

The role of TLR9 polymorphism in susceptibility to pulmonary tuberculosis.

Mycobacterium tuberculosis (MTB) is the causative agent of pulmonary tuberculosis (PTB), a major health problem that leads to 1.5 million deaths annually. Host genetic factors play a significant role in disease resistance/susceptibility by altering immunity against MTB. Toll-like receptor (TLR) sensors such as TLR2, TLR4, TLR8, and TLR9 are known to play a pivotal role in PTB via modulating sensor expression and/or effector responses. Single-nucleotide polymorphism (SNP) rs187084 (T-1486C) of the TLR9 promoter is associated with various autoimmune disorders and cancers. A recent bioinformatic analysis predicted that the T-1486C SNP is involved in PTB, although its potential role is unclear. To investigate the role of T-1486C in PTB, we stimulated PBMCs with the H37Rv whole cell lysate. We found that the presence of the “C” allele increases the transcriptional activity of the TLR9, which in turn induces high levels of Interferon gamma-induced protein 10 (IP-10), a biomarker for PTB. However, the expression of protective cytokines such as IFNγ and TNFα was observed significantly less with “C” allele in comparison to “T” allele. We further selected three different tribe populations showing differential susceptibility to PTB and performed genotypic analyses for the TLR9 promoter. We found a significantly lower minor allele frequency (MAF) of T-1486C in the Baiga tribe, wherein fewer PTB cases were reported, than that in the Gond and Korku tribes. Collectively, these data suggest that the minor “C” allele at rs187084 locus may be associated with susceptibility to PTB, which may explain the relatively lower PTB rates observed in Baiga tribe members.

Genome Wide Host Gene Expression Analysis in Chicken Lungs Infected with Avian Influenza Viruses.

The molecular pathogenesis of avian influenza infection varies greatly with individual bird species and virus strain. The molecular pathogenesis of the highly pathogenic avian influenza virus (HPAIV) or the low pathogenic avian influenza virus (LPAIV) infection in avian species remains poorly understood. Thus, global immune response of chickens infected with HPAI H5N1 (A/duck/India/02CA10/2011) and LPAI H9N2 (A/duck/India/249800/2010) viruses was studied using microarray to identify crucial host genetic components responsive to these infection. HPAI H5N1 virus induced excessive expression of type I IFNs (IFNA and IFNG), cytokines (IL1B, IL18, IL22, IL13, and IL12B), chemokines (CCL4, CCL19, CCL10, and CX3CL1) and IFN stimulated genes (OASL, MX1, RSAD2, IFITM5, IFIT5, GBP 1, and EIF2AK) in lung tissues. This dysregulation of host innate immune genes may be the critical determinant of the severity and the outcome of the influenza infection in chickens. In contrast, the expression levels of most of these genes was not induced in the lungs of LPAI H9N2 virus infected chickens. This study indicated the relationship between host immune genes and their roles in pathogenesis of HPAIV infection in chickens.

Analysis of the crow lung transcriptome in response to infection with highly pathogenic H5N1 avian influenza virus.

The highly pathogenic avian influenza (HPAI) H5N1 virus, currently circulating in Asia, causes severe disease in domestic poultry as well as wild birds like crow. However, the molecular pathogenesis of HPAIV infection in crows and other wild birds is not well known. Thus, as a step to explore it, a comprehensive global gene expression analysis was performed on crow lungs, infected with HPAI H5N1 crow isolate (A/Crow/India/11TI11/2011) using high throughput next generation sequencing (NGS) (GS FLX Titanium XLR70). The reference genome of crow is not available, so RNA seq analysis was performed on the basis of a de novo assembled transcriptome. The RNA seq result shows, 4052 genes were expressed uniquely in noninfected, 6277 genes were expressed uniquely in HPAIV infected sample and of the 6814 genes expressed in both samples, 2279 genes were significantly differentially expressed. Our transcriptome profile data allows for the ability to understand the molecular mechanism behind the recent lethal HPAIV outbreak in crows which was, until recently, thought to cause lethal infections only in gallinaceous birds such as chickens, but not in wild birds. The pattern of differentially expressed genes suggest that this isolate of H5N1 virus evades the host innate immune response by attenuating interferon (IFN)-inducible signalling possibly by down regulating the signalling from type I IFN (IFNAR1 and IFNAR2) and type II IFN receptors, upregulation of the signalling inhibitors suppressor of cytokine signalling 1 (SOCS1) and SOCS3 and altering the expression of toll-like receptors (TLRs). This may be the reason for disease and mortality in crows.

Identification of novel single nucleotide polymorphisms in the DGAT1 gene of buffaloes by PCR-SSCP

Diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. The DGAT1 gene is a strong functional candidate for determining milk fat content in cattle. In this work, we used PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and DNA sequencing to examine polymorphism in the region spanning exon 7 to exon 9 of the DGAT1 gene in Murrah and Pandharpuri buffaloes. Three alleles (A, B and C) and four novel single-nucleotide polymorphisms were identified in the buffalo DGAT1 gene. The frequencies of the alleles differed between the two buffalo breeds, with allele C being present in Murrah but not in Pandharpuri buffalo. The allele variation detected in this work may influence DGAT1 expression and function. The results described here could be useful in examining the association between the DGAT1 gene and milk traits in buffalo.

Characterization and In Silico Analysis of Pregnancy-Associated Glycoprotein-1 Gene of Buffalo (Bubalus bubalis).

Pregnancy-Associated Glycoproteins (PAGs) are trophoblastic proteins belonging to the Aspartic proteinase family secreted by different placental cells of many mammalian species. They play a pivotal role in placentogenesis, foetomaternal unit remodeling, and implantation. The identification of the genes encoding those proteins will be helpful to unravel the intricate embryogenomic functions during pregnancy establishment. Considering importance of these proteins, the present study was undertaken to characterize the pregnancy associated glycoprotein-1 gene of buffalo. An 1181 base pairs buffalo Pregnancy-Associated Glycoprotein PAG-1 gene was PCR amplified from the RNA obtained from the fetal cotyledons. BLAST analysis of the buffalo PAG-1 sequence retrieved a total of 20 cattle, 5 goat, and 4 sheep PAG sequences, exhibiting more than 80% similarity. Buffalo PAG-1 gene contained an uninterrupted open reading frame of 1140 base pairs encoding 380 amino acids that possess a 15 amino acid signal peptide and mature peptide of 365 amino acids. The phylogenetic study of the buffalo PAG-1 gene revealed buffalo PAG-1 is more related to cattle, goat, and sheep PAG-1 sequences. By this study characterization of buffalo PAG-1 gene and its evolutionary relationship was deduced for the first time.

Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India

Crimean-Congo hemorrhagic fever (CCHF) is an emerging zoonotic disease in India and requires immediate detection of infection both for preventing further transmission and for controlling the infection. The present study describes development, optimization, and evaluation of a novel molecular beacon-based real-time RT-PCR assay for rapid, sensitive, and specific diagnosis of Crimean-Congo hemorrhagic fever virus (CCHFV). The developed assay was found to be a better alternative to the reported TaqMan assay for routine diagnosis of CCHF.

Genome-wide gene expression pattern underlying differential host response to high or low pathogenic H5N1 avian influenza virus in ducks.

The differences in the influenza viral pathogenesis observed between different pathogenic strains are associated with distinct properties of virus strains and the host immune responses. In order to determine the differences in the duck immune response against two different pathogenic strains, we studied genome-wide host immune gene response of ducks infected with A/duck/India/02CA10/2011 and A/duck/Tripura/103597/2008 H5N1 viruses using custom-designed microarray. A/duck/India/02CA10/2011 is highly pathogenic virus (HP) to ducks, whereas A/duck/Tripura/103597/2008 is a low pathogenic (LP) virus strain. Comparative lung tissue transcriptome analysis of differentially expressed genes revealed that 686 genes were commonly expressed, 880 and 1556 genes are expressed uniquely to infection with HP and LP virus, respectively. The up-regulation of chemokines (CCL4 and CXCR4) and IFN-stimulated genes (IFITM2, STAT3, TGFB1 and TGFB3) was observed in the lung tissues of ducks infected with HP virus. The up-regulation of other immune genes (IL17, OAS, SOCS3, MHC I and MHC II) was observed in both infection conditions. The expression of important antiviral immune genes MX, IFIT5, IFITM5, ISG12, β-defensins, RSAD2, EIF2AK2, TRIM23 and SLC16A3 was observed in LP virus infection, but not in HP virus infection. Several immune-related gene ontology terms and pathways activated by both the viruses were qualitatively similar but quantitatively different. Based on these findings, the differences in the host immune response might explain a part of the difference observed in the viral pathogenesis of high and low pathogenic influenza strains in ducks.

Identification of Chicken Pulmonary miRNAs Targeting PB1, PB1-F2, and N40 Genes of Highly Pathogenic Avian Influenza Virus H5N1 In Silico

Highly pathogenic Avian influenza (HPAI) is a notifiable viral disease caused by avian influenza type A viruses of the Orthomyxoviridae family. Type A influenza genome consists of eight segments of negative-sense RNA. RNA segment 2 encodes three proteins, PB1, PB1-F2, and N40, which are translated from the same mRNA by ribosomal leaky scanning and reinitiation. Since these proteins are critical for viral replication and pathogenesis, targeting their expression can be one of the approaches to control and resist HPAI. MicroRNAs are short noncoding RNAs that regulate a variety of biological processes such as cell growth, tissue differentiation, apoptosis, and viral infection. In this study, a set of 300 miRNAs expressed in chicken lungs were screened against the HPAI virus (H5N1) segment 2 with different screening parameter like thermodynamic stability of heteroduplex, seed sequence complementarity, conserved target sequence, and target-site accessibility for identifying miRNAs that can potentially target the transcript of segment 2 of H5N1. Chicken miRNAs gga-mir-133c, gga-mir-1710, and gga-mir-146c* are predicted to target the expression of PB1, PB1-F2, and N40 proteins. This indicates that chicken has genetic potential to resist/tolerate H5N1 infection and these can be suitably exploited in designing strategies for control of avian influenza in chicken.