Peste des petits ruminants virus non-structural V and C proteins interact with the NF-κB p65 subunit and modulate pro-inflammatory cytokine gene induction.

Peste des petits ruminants virus (PPRV) is known to induce transient immunosuppression in infected small ruminants by modulating several cellular pathways involved in the antiviral immune response. Our study shows that the PPRV-coded non-structural proteins C and V can interact with the cellular NF-κB p65 subunit. The PPRV-C protein interacts with the transactivation domain (TAD) while PPRV-V interacts with the Rel homology domain (RHD) of the NF-κB p65 subunit. Both viral proteins can suppress the NF-κB transcriptional activity and NF-κB-mediated transcription of cellular genes. PPRV-V protein expression can significantly inhibit the nuclear translocation of NF-κB p65 upon TNF-α stimulation, whereas PPRV-C does not affect it. The NF-κB-mediated pro-inflammatory cytokine gene expression is significantly downregulated in cells expressing PPRV-C or PPRV-V protein. Our study provides evidence suggesting a role of PPRV non-structural proteins V and C in the modulation of NF-κB signalling through interaction with the NF-κB p65 subunit.

Changes in m6A RNA methylation of goat lung following PPRV infection.

Peste des petits ruminants (PPR) is an acute, highly contagious viral disease of goats and sheep, caused by the Peste des petits ruminants virus (PPRV). Earlier studies suggest the involvement of diverse regulatory mechanisms in PPRV infection. Methylation at N6 of Adenosine called m6A is a type RNA modification that influences various physiological and pathological phenomena. As the lung tissue represents the primary target organ of PPRV, the present study explored the m6A changes and their functional significance in PPRV disease pathogenesis. m6A-seq analysis revealed 1289 m6A peaks to be significantly altered in PPRV infected lung in comparison to normal lung, out of which 975 m6A peaks were hypomethylated and 314 peaks were hypermethylated. Importantly, hypomethylated genes were enriched in Interleukin-4 and Interleukin-13 signaling and various processes associated with extracellular matrix organization. Further, of the 843 differentially m6A-containing cellular transcripts, 282 transcripts were also found to be differentially expressed. Functional analysis revealed that these 282 transcripts are significantly enriched in signaling by Interleukins, extracellular matrix organization, cytokine signaling in the immune system, signaling by receptor tyrosine kinases, and Toll-like Receptor Cascades. We also found m6A reader HNRNPC and the core component of methyltransferase complex METTL14 to be highly upregulated than the m6A readers - HNRNPA2B1 and YTHDF1 at the transcriptome level. These findings suggest that alteration in the m6A landscape following PPRV is implicated in diverse processes including Interleukin signaling.

Nucleocapsid Protein (N) of Peste des petits ruminants Virus (PPRV) Interacts with Cellular Phosphatidylinositol-3-Kinase (PI3K) Complex-I and Induces Autophagy.

Autophagy is an essential and highly conserved catabolic process in cells, which is important in the battle against intracellular pathogens. Viruses have evolved several ways to alter the host defense mechanisms. PPRV infection is known to modulate the components of a host cell's defense system, resulting in enhanced autophagy. In this study, we demonstrate that the N protein of PPRV interacts with the core components of the class III phosphatidylinositol-3-kinase (PI3K) complex-I and results in the induction of autophagy in the host cell over, thereby expressing this viral protein. Our data shows the interaction between PPRV-N protein and different core components of the autophagy pathway, i.e., VPS34, VPS15, BECN1 and ATG14L. The PPRV-N protein can specifically interact with VPS34 of the PI3K complex-I and colocalize with the proteins of PI3K complex-I in the same sub-cellular compartment, that is, in the cytoplasm. These interactions do not affect the intracellular localization of the different host proteins. The autophagy-related genes were transcriptionally modulated in PPRV-N-expressing cells. The expression of LC3B and SQSTM1/p62 was also modulated in PPRV-N-expressing cells, indicating the induction of autophagic activity. The formation of typical autophagosomes with double membranes was visualized by transmission electron microscopy in PPRV-N-expressing cells. Taken together, our findings provide evidence for the critical role of the N protein of the PPR virus in the induction of autophagy, which is likely to be mediated by PI3K complex-I of the host.

Virus distribution and early pathogenesis of highly pathogenic peste-des-petits-ruminants virus in experimentally infected goats.

INTRODUCTION: Despite causing one of the most dreaded diseases of small ruminants, relatively little is known about the pathogenic events, antigen distribution and the cells responsible for the uptake and transmission of peste-des-petits-ruminants virus (PPRV) during primitive stages of infection. OBJECTIVES: We aimed at deciphering the sequential tissue tropism, pathological events and putative role of M2c macrophages during incubatory, prodromal and invasive stages of PPRV infection. METHODOLOGY: A total of 10 goats were sequentially sacrificed at 1, 2, 3, 4, and 5 days post-infection (dpi, n = 2 per time-point) following intranasal inoculation with a highly virulent strain of PPRV (lineage IV PPRV/Izatnagar/94). Histological evaluation to assess PPRV mediated pathologies, RT-qPCR and immunohistochemistry (IHC) to decipher sequential virus distribution, and dual immunolabelling to determine the role of M2c macrophage in early PPRV uptake and transmission was performed. RESULTS: PPRV/Izatnagar/94 caused major pathologies in the lung tissues. Unprecedentedly, PPRV nucleic acid and antigens were detected in various tissues as early as one dpi. RT-qPCR revealed PPRV in the nasal cavity, trachea, bronchi, tongue and lymph nodes draining these tissues from 1 dpi. IHC affirms cells residing in the lamina propria and submucosa of the respiratory tract and tongue and peribronchiolar areas of lungs as the primary target of PPRV. Following initial replication in the respiratory tract, PPRV is transmitted to the regional lymph nodes where primary viral amplification occurs. After viraemia and secondary replication in generalized lymphoid tissues, PPRV infects and replicates in the epithelial cells. Further, we localized CD163+ M2c macrophages in the goat tissues, but dual IHC elucidated that M2c macrophages do not facilitate uptake and transmission of PPRV during the early stages of infection. CONCLUSION: Our study substantiates the disease establishment process and pathogenesis of PPRV/Izatnagar/94 during the incubatory and prodromal stages of infection. Further, we have also observed M2c macrophage distribution in the goat tissues and demonstrated that they do not pick and transmit PPRV.

A Mouse Model of PPRV Infection for Elucidating Protective and Pathological Roles of Immune Cells.

The study was aimed at developing an accessible laboratory animal model to elucidate protective and pathological roles of immune mediators during Peste des petits ruminants virus (PPRV) infection. It is because of the critical roles of type I IFNs in anti-viral defense, we assessed the susceptibility of IFN receptor knock out (IFNR KO) mice to PPRV infection. IFNR KO mice were exceedingly susceptible to the infection but WT animals efficiently controlled PPRV. Accordingly, the PPRV infected IFNR KO mice gradually reduced their body weights and succumbed to the infection within 10 days irrespective of the dose and route of infection. The lower infecting doses predominantly induced immunopathological lesions. The viral antigens as well as the replicating PPRV were abundantly present in most of the critical organs such as brain, lungs, heart and kidneys of IFNR KO mice infected with high dose of the virus. Neutrophils and macrophages transported the replicating virus to central nervous system (CNS) and contributed to pathology while the elevated NK and T cell responses directly correlated with the resolution of PPRV infection in WT animals. Using an array of fluorescently labeled H-2Kb tetramers, we discovered four immunogenic epitopes of PPRV. The PPRV-peptides interacted well with H-2Kb in acellular and cellular assay as well as expanded the virus-specific CD8+ T cells in immunized or infected mice. Adoptively transferred CD8+ T cells helped control PPRV in infected mice. Our study therefore established and employed a mouse model for investigating the pathogenesis of PPRV. The model could be useful for elucidating the contribution of immune cells in disease progression as well as to test anti-viral agents.