Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus.

Peste des petits ruminants is an acute and highly contagious disease caused by the Peste des petits ruminants virus (PPRV). Host proteins play a crucial role in viral replication. However, the effect of fusion (F) protein-interacting partners on PPRV infection is poorly understood. In this study, we found that the expression of goat plasminogen activator urokinase (PLAU) gradually decreased in a time- and dose-dependent manner in PPRV-infected goat alveolar macrophages (GAMs). Goat PLAU was subsequently identified using co-immunoprecipitation and confocal microscopy as an F protein binding partner. The overexpression of goat PLAU inhibited PPRV growth and replication, whereas silencing goat PLAU promoted viral growth and replication. Additionally, we confirmed that goat PLAU interacted with a virus-induced signaling adapter (VISA) to antagonize F-mediated VISA degradation, increasing the production of type I interferon. We also found that goat PLAU reduced the inhibition of PPRV replication in VISA-knockdown GAMs. Our results show that the host protein PLAU inhibits the growth and replication of PPRV by VISA-triggering RIG-I-like receptors and provides insight into the host protein that antagonizes PPRV immunosuppression.IMPORTANCEThe role of host proteins that interact with Peste des petits ruminants virus (PPRV) fusion (F) protein in PPRV replication is poorly understood. This study confirmed that goat plasminogen activator urokinase (PLAU) interacts with the PPRV F protein. We further discovered that goat PLAU inhibited PPRV replication by enhancing virus-induced signaling adapter (VISA) expression and reducing the ability of the F protein to degrade VISA. These findings offer insights into host resistance to viral invasion and suggest new strategies and directions for developing PPR vaccines.

Altering the competitive environment of B cell epitopes significantly extends the duration of antibody production.

Persistent immunoglobulin G (IgG) production (PIP) provides long-term vaccine protection. While variations in the duration of protection have been observed with vaccines prepared from different pathogens, little is known about the factors that determine PIP. Here, we investigated the impact of three parameters on the duration of anti-peptide IgG production, namely amino acid sequences, protein carriers, and immunization programs. We show that anti-peptide IgG production can be transformed from transient IgG production (TIP) to PIP, by placing short peptides (Pi) containing linear B cell epitopes in different competitive environments using bovine serum albumin (BSA) conjugates instead of the original viral particles. When goats were immunized with the peste des petits ruminants (PPR) live-attenuated vaccine (containing Pi as the constitutive component) and BSA-Pi conjugate, anti-Pi IgG production exhibited TIP (duration < 60 days) and PIP (duration > 368 days), respectively. Further, this PIP was unaffected by subsequent immunization with the PPR live-attenuated vaccine in the same goat. When goats were coimmunized with PPR live-attenuated vaccine and BSA-Pi, the induced anti-Pi IgG production showed a slightly extended TIP (from ~60 days to ~100 days). This discovery provides new perspectives for studying the fate of plasma cells in humoral immune responses and developing peptide vaccines related to linear neutralizing epitopes from various viruses.

SLAM (CD150) receptor homologous peptides block the peste des petits ruminants virus entry into B95a cells.

The fusion of haemagglutinin-neuraminidase (HN) protein of peste des petits ruminant (PPR) virus with signaling lymphocyte activation molecules (SLAM) host cell receptor consequences the virus entry and multiplication inside the host cell. The use of synthetic SLAM homologous peptides (i.e., molecular decoy for HN protein of PPR virus) may check PPR infection at the preliminary stage. Hence, the predicted SLAM homologous peptides using bioinformatics tools were synthesized by solid phase chemistry with standard Merrifield's 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and were purified by reverse phase high performance liquid chromatography. The secondary structures of synthesized peptides were elucidated by circular dichroism spectroscopy. The in vitro interactions of these peptides were studied through indirect Enzyme Linked Immuno Sorbent Assay (ELISA) and visual surface plasmon UV-visible spectroscopy. The SLAM homologous peptides were able to interact with the peste des petits ruminant virus (PPRV) with varying binding efficiency. The interaction of SLAM homologous peptide with the PPR virus was ascertained by the change in the plasmon color from red wine to purple during visual detection and also by bathochromic shift in absorbance spectra under UV-visible spectrophotometry. The cytotoxic and anti-PPRV effect of these peptides were also evaluated in B95a cell line using PPR virus (Sungri/96). The cytotoxic concentration 50 (CC50 ) value of each peptide was greater than 1000 µg mL-1 . The anti-PPRV efficiency of SLAM-22 was relatively high among SLAM homologous peptides, SLAM-22 at 25 µg mL-1 concentration showed a reduction of more than log10 3 virus titer by priming of B95a cell line while the use of SLAM-15 and Muco-17 at the same concentration dropped virus titer from log10 4.8 to log10 2.5 and log10 3.1 respectively. The concentration of SLAM homologous peptide (25 µg mL-1 ) to exert its anti-PPRV effect was much less than its CC50 level (>1000 µg mL-1 ). Therefore, the synthetic SLAM homologous peptides may prove to be better agents to target PPRV.

Peste Des Petits Ruminants Virus Nucleocapsid Protein Interacts with Protein Kinase R-Activating Protein and Induces Stress Granules To Promote Viral Replication.

The pathogenic mechanisms of peste des petits ruminants virus (PPRV) infection remain poorly understood, leaving peste des petits ruminants (PPR) control and eradication especially difficult. Here, we determined that PPRV nucleocapsid (N) protein triggers formation of stress granules (SGs) to benefit viral replication. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N protein interacted with protein kinase R (PKR)-activating protein (PACT), and this interaction was confirmed in the context of PPRV infection. PACT was essential for PPRV replication. Besides, the ectopic expression of N activated the PKR/eIF2α (α subunit of eukaryotic initiation factor 2) pathway through induction of PKR phosphorylation, but it did not induce PKR phosphorylation in PACT-deficient (PACT-/-) cells. PPRV N interacted with PACT, impairing the interaction between PACT and a PKR inhibitor, transactivation response RNA-binding protein (TRBP), which subsequently enhanced the interaction between PACT and PKR and thus promoted the activation of PKR and eIF2α phosphorylation, resulting in formation of stress granules (SGs). Consistently, PPRV infection induced SG formation through activation of the PKR/eIF2α pathway, and knockdown of N impaired PPRV-induced SG formation. PPRV-induced SG formation significantly decreased in PACT-/- cells as well. The role of SG formation in PPRV replication was subsequently investigated, which showed that SG formation plays a positive role in PPRV replication. By using an RNA fluorescence in situ hybridization assay, we found that PPRV-induced SGs hid cellular mRNA rather than viral mRNA. Altogether, our data provide the first evidence that PPRV N protein plays a role in modulating the PKR/eIF2α/SG axis and promotes virus replication through targeting PACT. IMPORTANCE Stress granule (SG) formation is a conserved cellular strategy to reduce stress-related damage regulating cell survival. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N interacted with PACT to regulate the assembly of SGs. N protein inhibited the interaction between PACT and a PKR inhibitor, TRBP, through binding to the M1 domain of PACT, which enhanced the interaction between PACT and PKR and thus promoted PKR activation and subsequent eIF2α phosphorylation as well as SG formation. The regulatory function of N protein was strikingly abrogated in PACT-/- cells. SGs induced by PPRV infection through the PKR/eIF2α pathway are PACT dependent. The loss-of-function assay indicated that PPRV-induced SGs were critical for PPRV replication. We concluded that the PPRV N protein manipulates the host PKR/eIF2α/SG axis to favor virus replication.

Extracellular vesicles derived from PPRV-infected cells enhance signaling lymphocyte activation molecular (SLAM) receptor expression and facilitate virus infection.

Peste des petits ruminants virus (PPRV) is an important pathogen that seriously influences the productivity of small ruminants worldwide. PPRV is lymphotropic in nature and SLAM was identified as the primary receptor for PPRV and other Morbilliviruses. Many viruses have been demonstrated to engage extracellular vesicles (EVs) to facilitate their replication and pathogenesis. Here, we provide evidence that PPRV infection significantly induced the secretion levels of EVs from goat PBMC, and that PPRV-H protein carried in EVs can enhance SLAM receptor expression in the recipient cells via suppressing miR-218, a negative miRNA directly targeting SLAM gene. Importantly, EVs-mediated increased SLAM expression enhances PPRV infectivity as well as the expression of various cytokines related to SLAM signaling pathway in the recipient cells. Moreover, our data reveal that PPRV associate EVs rapidly entry into the recipient cells mainly through macropinocytosis pathway and cooperated with caveolin- and clathrin-mediated endocytosis. Taken together, our findings identify a new strategy by PPRV to enhance virus infection and escape innate immunity by engaging EVs pathway.

Development and characterization of a novel nanobody with SRMV neutralizing activity.

Peste des petits ruminants (PPR) is an acute, contact infectious disease caused by the small ruminant morbillivirus (SRMV), and its morbidity in goats and sheep can be up to 100% with significant mortality. Nanobody generated from camelid animals such as alpaca has attracted wide attention because of its unique advantages compared with conventional antibodies. The main objective of this study was to produce specific nanobodies against SRMV and identify its characteristics. To obtain the coding gene of SRMV-specific nanobodies, we first constructed an immune phage-displayed library from the VHH repertoire of alpaca that was immunized with SRMV-F and -H proteins. By using phage display technology, the target antigen-specific VHHs can be obtained after four consecutive rounds of biopanning. Results showed that the size of this VHH library was 2.26 × 1010 CFU/mL and the SRMV-F and -H specific phage particles were greatly enriched after four rounds of biopanning. The positive phage clones were selected and sequenced, and total of five independent different sequences of SRMV-specific nanobodies were identified. Subsequently, the DNA fragments of the five nanobodies were cloned into E. coli BL21(DE3), respectively, and three of them were successfully expressed and purified. Specificity and affinity towards inactivated SRMV of these purified nanobodies were then evaluated using the ELISA method. Results demonstrated that NbSRMV-1-1, NbSRMV-2-10, and NbSRMV-1-21 showed no cross-reactivity with other antigens, such as inactivated BTV, inactivated FMDV, His-tag labeled protein, and BSA. The ELISA titer of these three nanobodies against inactivated SRMV was up to 1:1000. However, only NbSRMV-1-21 displayed SRMV neutralizing activity at a maximum dilution of 1:4. The results indicate that the nanobodies against SRMV generated in this study could be useful in future applications. This study provided a novel antibody tool and laid a foundation for the treatment and detection of SRMV.

Molecular characterization of Peste des petits ruminants virus (PPRV) in sheep and goats and risk factors associated with it in selected districts of Khyber Pakhtunkhwa-Pakistan.

BACKGROUND: Peste des Petits Ruminants (PPR) is an economically significant transboundary viral disease of sheep and goats caused by the PPRV virus, affecting annual losses of 1.45-2.10 billion US dollars globally. We designed the current study to evaluate the positive cases, molecular characterization, phylogenetic analysis, and risk factors correlated with the disease in various districts of Khyber Pakhtunkhwa, Pakistan, with the aim of contributing to these strategies. METHODS AND RESULTS: A total of 384 samples from three selected districts, i.e., Peshawar, Charsadda and Chitral (n = 128 each), were collected, and the virus was investigated by using the sandwich ELISA, while the N gene of the virus was used as a target for molecular detection via RT-PCR. The confirmed samples were then sequenced, and phylogenetic analysis was performed. According to our findings, the highest positive cases was found in district Peshawar (50.87%), followed by Charsadda and Chitral (24.56%), respectively, while risk factor analysis showed that certain categories, such as species, sex, and age less than two years, have higher risk (P < 0.05) in contrast to their respective categories. Furthermore, sequencing and phylogenetic analysis of representative samples showed that the PPRV strains in the current study clustered in lineage IV, which is circulating in the small ruminant population of Asia, the Middle East, and African countries. Comparative residue analysis highlighted the mutation by representing 242 variable sites out of 371 locations. CONCLUSIONS: PPRV has foremost importance in Pakistan because the virus was detected in a considerable number of samples, and most of which were sourced from subsidiary areas where veterinary services are not prioritized.

Prevalence and associated risk factors of peste des petits ruminants in selected districts of the northern border region of Pakistan.

BACKGROUND: Peste des Petits Ruminants (PPR) is a world organization for animal health (WOAH) notifiable and economically important transboundary, highly communicable viral disease of small ruminants. PPR virus (PPRV) belongs to the genus Morbillivirus of the family Paramyxoviridae. AIM: The present cross-sectional epidemiological investigation was accomplished to estimate the apparent prevalence and identify the risk factors linked with peste des petits ruminants (PPR) in the previously neglected northern border regions of Pakistan. METHOD: A total of 1300 samples (serum = 328; swabs = 972) from 150 flocks/herds were compiled from sheep (n = 324), goats (n = 328), cattle (n = 324), and buffaloes (n = 324) during 2020-2021 and tested using ELISA for detection of viral antibody in sera or antigen in swabs. RESULTS: An overall apparent prevalence of 38.7% (504 samples) and an estimated true prevalence (calculated by the Rogan and Gladen estimator) of 41.0% (95% CI, 38.0-44 were recorded in the target regions. The highest apparent prevalence of 53.4% (85 samples) and the true prevalence of 57.0%, 95% Confidence Interval (CI) were documented in the Gilgit district and the lowest apparent prevalence of 53 (25.1%) and the true prevalence of 26.0%, 95% Confidence Interval (CI), 19.0-33.0) was reported in the Swat district. A questionnaire was designed to collect data about associated risk factors that were put into a univariable logistic regression to decrease the non-essential assumed risk dynamics with a P-value of 0.25. ArcGIS, 10.8.1 was used to design hotspot maps and MedCalc's online statistical software was used to calculate Odds Ratio (OR). Some of the risk factors significantly different (P < 0.05) in the multivariable logistic regression were flock/herd size, farming methods, nomadic animal movement, and outbreaks of PPR. The odds of large-sized flocks/herds were 1.7 (OR = 1.79; 95% Confidence Interval (CI) = 0.034-91.80%) times more likely to be positive than small-sized. The odds of transhumance and nomadic systems were 1.1 (OR = 1.15; 95% Confidence Interval (CI) = 0.022-58.64%) and 1.0 (OR = 1.02; 95% Confidence Interval (CI) = 0.020-51.97%) times more associated to be positive than sedentary and mixed farming systems, respectively. The odds of nomadic animal movement in the area was 0.7 (OR = 0.57; 95% Confidence Interval (CI) = 0.014-38.06%) times more associated to be positive than in areas where no nomadic movement was observed. In addition, the odds of an outbreak of PPR in the area were 1.0 (OR = 1.00; 95% Confidence Interval (CI) = 0.018-46.73%) times more associated to be positive than in areas where no outbreak of PPR was observed. CONCLUSIONS: It was concluded that many northern regions considered endemic for PPR, large and small ruminants are kept and reared together making numerous chances for virus transmission dynamic, so a big threats of disease spread exist in the region. The results of the present study would contribute to the global goal of controlling and eradicating PPR by 2030.

Space-time cluster analysis and maximum entropy modeling of Peste des petits ruminants (PPR) in Türkiye.

Peste des petits ruminants (PPR) is an economically important highly serious transboundary disease that mainly occurs in small ruminants such as sheep and goats. The aim of this study was to identify the probability of risk and and space-time clusters of Peste des Petits Ruminants (PPR) in Türkiye. The occurrence of PPR in Türkiye from 2017 to 2019 was investigated in this study using spatial analysis based on geographic information system (GIS). Between these dates, it was determined that 337 outbreaks and 18,467 cases. The highest number of outbreaks were detected in the Central Anatolia region. It was determined that PPR is seen more intensely in sheep compared to goats in Türkiye. In this study, 34 environmental variables (19 bioclimatic, 12 precipitation, altitude and small livestock density variables) were used to explore the environmental influences on PPR outbreak by maximum entropy modeling (Maxent). The clusters of PPR in Türkiye were identified using the retrospective space-time scan data that were computed using the space-time permutation model. A PPR prediction model was created using data on PPR outbreaks combination with environmental variables. Nineteen significant (p < 0.001) space-time clusters were determined. It was discovered that the variables altitude, sheep density, precipitation in june, and average temperature in the warmest season made important contributions to the model and the PPR outbreak may be strongly related with these variables. In this study, PPR in Türkiye has been characterized significantly spatio-temporal and enviromental factors. In this context, the disease pattern and obtained these findings will contribute to policymakers in the prevention and control of the disease.

Pathogenesis of wild-type- and vaccine-based recombinant peste des petits ruminants virus (PPRV) expressing EGFP in experimentally infected domestic goats.

Peste des petits ruminants virus (PPRV) is a highly contagious morbillivirus related to measles and canine distemper virus, mostly affecting small ruminants. The corresponding PPR disease has a high clinical impact in goats and is characterized by fever, oral and nasal erosions, diarrhoea and pneumonia. In addition, massive infection of lymphoid tissues causes lymphopaenia and immune suppression. This results in increased susceptibility to secondary bacterial infections, explaining the observed high mortality in some outbreaks. We studied the pathogenesis of PPR by experimental inoculation of Dutch domestic goats with a recombinant virulent PPRV strain modified to express EGFP and compared it to an EGFP-expressing vaccine strain of PPRV. After intratracheal inoculation with virulent PPRV, animals developed fever, viraemia and leucopaenia, and shed virus from the respiratory and gastro-intestinal tracts. Macroscopic evaluation of fluorescence at the peak of infection 7 days post-inoculation (dpi) showed prominent PPRV infection of the respiratory tract, lymphoid tissues, gastro-intestinal tract, mucosae and skin. Flow cytometry of PBMCs collected over time demonstrated a cell-associated viraemia mediated by infected lymphocytes. At 14 dpi, pathognomonic zebra stripes were detected in the mucosa of the large intestine. In contrast, vaccine strain-inoculated goats remained largely macroscopically fluorescence negative and did not present clinical signs. A low-level viraemia was detected by flow cytometry, but at necropsy no histological lesions were observed. Animals from both groups seroconverted as early as 7 dpi and sera efficiently neutralized virulent PPRV in vitro. Combined, this work presents a study of the pathogenesis of wild type- and vaccine-based PPRV in its natural host. This study shows the strength of recombinant EGFP-expressing viruses in fluorescence-guided pathogenesis studies.

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.

Peste des Petits Ruminants Virus Upregulates STING to Activate ATF6-Mediated Autophagy.

Peste des petits ruminants virus (PPRV) infection leads to autophagy, and the molecular mechanisms behind this phenomenon are unclear. Here, we demonstrate that PPRV infection results in morphological changes of the endoplasmic reticulum (ER) and activation of activating transcription factor 6 (ATF6) of the ER stress unfolded protein response (UPR). Knockdown of ATF6 blocked the autophagy process, suggesting ATF6 is necessary for PPRV-mediated autophagy induction. Further study showed that PPRV infection upregulates expression of the ER-anchored adaptor protein stimulator of interferon genes (STING), which is well-known for its pivotal roles in restricting DNA viruses. Knockdown of STING suppressed ATF6 activation and autophagy induction, implying that STING functions upstream of ATF6 to induce autophagy. Moreover, the STING-mediated autophagy response originated from the cellular pattern recognition receptor melanoma differentiation-associated gene 5 (MDA5). The absence of MDA5 abolished the upregulation of STING and the activation of autophagy. The deficiency of autophagy-related genes (ATG) repressed the autophagy process and PPRV replication, while it had no effect on MDA5 or STING expression. Overall, our work revealed that MDA5 works upstream of STING to activate ATF6 to induce autophagy. IMPORTANCEPPRV infection induces cellular autophagy; however, the intracellular responses and signaling mechanisms that occur upon PPRV infection are obscure, and whether innate immune responses are linked with autophagy to regulate viral replication is largely unknown. Here, we uncovered that the innate immune sensor MDA5 initiated the signaling cascade by upregulating STING, which is best known for its role in anti-DNA virus infection by inducing interferon expression. We first provide evidence that STING regulates PPRV replication by activating the ATF6 pathway of unfolded protein responses (UPRs) to induce autophagy. Our results revealed that in addition to mediating responses to foreign DNA, STING can cross talk with MDA5 to regulate the cellular stress response and autophagy induced by RNA viruses; thus, STING works as an adaptor protein for cellular stress responses and innate immune responses. Modulation of STING represents a promising approach to control both DNA and RNA viruses.

PPRV-Induced Autophagy Facilitates Infectious Virus Transmission by the Exosomal Pathway.

Peste des petits ruminants virus (PPRV) is an important pathogen that seriously influences the productivity of small ruminants worldwide. We showed previously that PPRV induced sustained autophagy for their replication in host cells. Many studies have shown that exosomes released from virus-infected cells contain a variety of viral and host cellular factors that are able to modulate the recipient's cellular response and result in productive infection of the recipient host. Here, we show that PPRV infection results in packaging of the viral genomic RNA and partial viral proteins into exosomes of Vero cells and upregulates exosome secretion. We provide evidence showing that the exosomal viral cargo can be transferred to and establish productive infection in a new target cell. Importantly, our study reveals that PPRV-induced autophagy enhances exosome secretion and exosome-mediated virus transmission. Additionally, our data show that TSG101 may be involved in the sorting of the infectious PPRV RNA into exosomes to facilitate the release of PPRV through the exosomal pathway. Taken together, our results suggest a novel mechanism involving autophagy and exosome-mediated PPRV intercellular transmission. IMPORTANCE Autophagy plays an important role in PPRV pathogenesis. The role of exosomes in viral infections is beginning to be appreciated. The present study examined the role of autophagy in secretion of infectious PPRV from Vero cells. Our data provided the first direct evidence that ATG7-mediated autophagy enhances exosome secretion and exosome-mediated PPRV transmission. TSG101 may be involved in the sorting of the infectious PPRV RNA genomes into exosomes to facilitate the release of PPRV through the exosomal pathway. Inhibition of PPRV-induced autophagy or TSG101 expression could be used as a strategy to block exosome-mediated virus transmission.

Peste Des Petits Ruminants Virus N Protein Is a Critical Proinflammation Factor That Promotes MyD88 and NLRP3 Complex Assembly.

Inflammatory responses play a central role in host defense against invading pathogens. Peste des petits ruminants virus (PPRV) causes highly contagious acute or subacute disease of small ruminants. However, the precise mechanism by which PPRV regulates inflammatory responses remains unknown. Here, we revealed a novel mechanism by which PPRV induces inflammation. Our study showed that PPRV induced the secretion of interleukin 1ß (IL-1ß) by activating the NF-κB signaling pathway and the NLRP3 inflammasome. Moreover, PPRV replication and protein synthesis were essential for NLRP3 inflammasome activation. Importantly, PPRV N protein promoted NF-κB signaling pathway and NLRP3 inflammasome via direct binding of MyD88 and NLPR3, respectively, and induced caspase-1 cleavage and IL-1ß maturation. Biochemically, N protein interacted with MyD88 to potentiate the assembly of MyD88 complex and interacted with NLPR3 to facilitate NLRP3 inflammasome complex assembly by forming an N-NLRP3-ASC ring-like structure, leading to IL-1ß secretion. These findings demonstrate a new function of PPRV N protein as an important proinflammation factor and identify a novel underlying mechanism modulating inflammasome assembly and function induced by PPRV. IMPORTANCE An important part of the innate immune response is the activation of NF-κB signaling pathway and NLPR3 inflammasome, which is induced upon exposure to pathogens. Peste des petits ruminants virus (PPRV) is a highly contagious virus causing fever, stomatitis, and pneumoenteritis in goats by inducing many proinflammatory cytokines. Although the NF-κB signaling pathway and NLRP3 inflammasome play an important role in regulating host immunity and viral infection, the precise mechanism by which PPRV regulates inflammatory responses remains unknown. This study demonstrates that PPRV induces inflammatory responses. Mechanistically, PPRV N protein facilitates the MyD88 complex assembly by directly binding to MyD88 and promotes the NLRP3 inflammasome complex assembly by directly binding to NLRP3 to form ring-like structures of N-NLRP3-ASC. These findings provide insights into the prevention and treatment of PPRV infection.

A Morbillivirus Infection Shifts DC Maturation Toward a Tolerogenic Phenotype to Suppress T Cell Activation.

Viruses have evolved numerous strategies to impair immunity so that they can replicate more efficiently. Among those, the immunosuppressive effects of morbillivirus infection can be particularly problematic, as they allow secondary infections to take hold in the host, worsening disease prognosis. In the present work, we hypothesized that the highly contagious morbillivirus peste des petits ruminants virus (PPRV) could target monocytes and dendritic cells (DC) to contribute to the immunosuppressive effects produced by the infection. Monocytes isolated from healthy sheep, a natural host of the disease, were able be infected by PPRV and this impaired the differentiation and phagocytic ability of immature monocyte-derived DC (MoDC). We also assessed PPRV capacity to infect differentiated MoDC. Ovine MoDC could be productively infected by PPRV, and this drastically reduced MoDC capacity to activate allogeneic T cell responses. Transcriptomic analysis of infected MoDC indicated that several tolerogenic DC signature genes were upregulated upon PPRV infection. Furthermore, PPRV-infected MoDC could impair the proliferative response of autologous CD4+ and CD8+ T cell to the mitogen concanavalin A (ConA), which indicated that DC targeting by the virus could promote immunosuppression. These results shed new light on the mechanisms employed by morbillivirus to suppress the host immune responses. IMPORTANCE Morbilliviruses pose a threat to global health given their high infectivity. The morbillivirus peste des petits ruminants virus (PPRV) severely affects small-ruminant-productivity and leads to important economic losses in communities that rely on these animals for subsistence. PPRV produces in the infected host a period of severe immunosuppression that opportunistic pathogens exploit, which worsens the course of the infection. The mechanisms of PPRV immunosuppression are not fully understood. In the present work, we demonstrate that PPRV can infect professional antigen-presenting cells called dendritic cells (DC) and disrupt their capacity to elicit an immune response. PPRV infection promoted a DC activation profile that favored the induction of tolerance instead of the activation of an antiviral immune response. These results shed new light on the mechanisms employed by morbilliviruses to suppress the immune responses.

Combining viral genetic and animal mobility network data to unravel peste des petits ruminants transmission dynamics in West Africa.

Peste des petits ruminants (PPR) is a deadly viral disease that mainly affects small domestic ruminants. This disease threaten global food security and rural economy but its control is complicated notably because of extensive, poorly monitored animal movements in infected regions. Here we combined the largest PPR virus genetic and animal mobility network data ever collected in a single region to improve our understanding of PPR endemic transmission dynamics in West African countries. Phylogenetic analyses identified the presence of multiple PPRV genetic clades that may be considered as part of different transmission networks evolving in parallel in West Africa. A strong correlation was found between virus genetic distance and network-related distances. Viruses sampled within the same mobility communities are significantly more likely to belong to the same genetic clade. These results provide evidence for the importance of animal mobility in PPR transmission in the region. Some nodes of the network were associated with PPRV sequences belonging to different clades, representing potential "hotspots" for PPR circulation. Our results suggest that combining genetic and mobility network data could help identifying sites that are key for virus entrance and spread in specific areas. Such information could enhance our capacity to develop locally adapted control and surveillance strategies, using among other risk factors, information on animal mobility.

Quantitative analysis of acetylation in peste des petits ruminants virus-infected Vero cells.

BACKGROUND: Peste des petits ruminants virus (PPRV) is a highly contagious pathogen that strongly influences the productivity of small ruminants worldwide. Acetylation is an important post-translational modification involved in regulation of multiple biological functions. However, the extent and function of acetylation in host cells during PPRV infection remains unknown. METHODS: Dimethylation-labeling-based quantitative proteomic analysis of the acetylome of PPRV-infected Vero cells was performed. RESULTS: In total, 1068 proteins with 2641 modification sites were detected in response to PPRV infection, of which 304 differentially acetylated proteins (DAcPs) with 410 acetylated sites were identified (fold change < 0.83 or > 1.2 and P < 0.05), including 109 up-regulated and 195 down-regulated proteins. Gene Ontology (GO) classification indicated that DAcPs were mostly located in the cytoplasm (43%) and participated in cellular and metabolic processes related to binding and catalytic activity. Functional enrichment indicated that the DAcPs were involved in the minichromosome maintenance complex, unfolded protein binding, helicase activity. Only protein processing in endoplasmic reticulum pathway was enriched. A protein-protein interaction (PPI) network of the identified proteins further indicated that a various chaperone and ribosome processes were modulated by acetylation. CONCLUSIONS: To the best of our knowledge, this is the first study on acetylome in PPRV-infected host cell. Our findings establish an important baseline for future study on the roles of acetylation in the host response to PPRV replication and provide novel insights for understanding the molecular pathological mechanism of PPRV infection.

Dysregulation of the RIG-I-like Receptor Pathway Signaling by Peste des Petits Ruminants Virus Phosphoprotein.

Peste des petits ruminants virus (PPRV) is a Morbillivirus that causes highly contagious and severe disease in various ruminants. PPRV infection leads to a severe inhibition of host antiviral immune response. Our previous study demonstrated that PPRV V protein blocks IFN response by targeting STAT proteins. In the current study, we identified the phosphoprotein (P) as a novel antagonistic factor of PPRV to counteract host antiviral innate immune response. PPRV P protein significantly suppressed RIG-I-like receptor pathway signaling and impaired IFN-ß and ISGs expression by targeting IFN regulatory factor (IRF)3 in both human embryonic kidney 293T cells and primary goat fibroblasts. The 1-102 region of P protein was critical for the antagonistic function of P protein. P protein interacted with IRF association domain (IAD) of IRF3 to block the interaction between TBK1 and IRF3. The interaction between TBK1 and the IAD of IRF3 is responsible for triggering the phosphorylation of IRF3. P protein competed with TBK1 to bind to the IAD of IRF3 that contributed to the decreased phosphorylation of IRF3, which, in turn, interfered with the dimerization of IRF3 and blocked IRF3 nuclear transportation. Besides, we also found that P protein interacted with IRF5 and IRF8. However, the involved mechanism remains unknown. Taken together, our results reveal a novel mechanism by which PPRV P protein antagonizes host antiviral innate immune response by interacting with the transcription factor IRF3, thereby inhibiting the type I IFN production and promoting viral replication.

A recombinant capripoxvirus expressing the F protein of peste des petits ruminants virus and the P12A3C of foot-and-mouth disease virus.

BACKGROUND: Peste des petits ruminants (PPR), foot-and-mouth disease (FMD) and sheep pox and goat pox are three important infectious diseases that infect goats, sheep and other small ruminants. It is well-known that the prevention of three diseases rely mainly on their individual vaccines. However, the vaccines have a variety of different disadvantages, such as short duration of immunity, increasing the number of vaccinations, and poor thermal stability. The purpose of this study is to construct a recombinant goat pox virus (rGPV) capable of expressing the F gene of PPRV and the P12A3C gene of FMDV as a live vector vaccine. RESULTS: The IRES, FMDV P12A3C and PPRV F genes into the multi-cloning site of the universal transfer plasmid pTKfpgigp to construct a recombinant transfer plasmid pTKfpgigpFiP12A3C, and transfected GPV-infected lamb testis (LT) cells with liposomes and produced by homologous recombination Recombinant GPV (rGPV/PPRVF-FMDVP12A3C, rGPV). The rGPV was screened and purified by green florescence protein (GFP) and xanthine-guanine-phosphoribosyltransferase gene (gpt) of Escherichia coli as selective markers, and the expression of rGPV in LT cells was detected by RT-PCR and immunofluorescence techniques. The results showed that the virus strain rGPV/PPRVF-FMDVP12A3C containing FMDV P12A3C and PPRV F genes was obtained. The exogenous genes FMDV P12A3C and PPRV F contained in rGPV were normally transcribed and translated in LT cells, and the expression products could specifically react with PPRV and FMDV antiserum. Then, the rGPV was intradermally inoculated with goats, the animal experiments showed that rGPV/PPRVF-FMDVP12A3C could induce high levels of specific antibodies against GPV, PPRV and FMDV. CONCLUSIONS: The constructed rGPV induced high levels of specific antibodies against GPV, PPRV and FMDV. The study provides a reference for " one vaccine with multiple uses " of GPV live vector vaccine.

Simultaneous detection and identification of Peste des petits ruminants Virus Lineages II and IV by MCA-Based real-time quantitative RT-PCR assay within single reaction.

BACKGROUND: Peste des petits ruminants (PPR) disease is a cross-species infectious disease that severely affects small ruminants and causes great losses to livestock industries in various countries. Distinguishing vaccine-immunized animals from naturally infected animals is an important prerequisite for the eradication of PPR. At present PPRV are classified into lineages I through IV, and only one vaccination strain, Nigeria/75/1, belongs to lineage II, but all of the epidemic strains in China at present are from lineage IV. RESULTS: To achieve this goal, we developed an SYBR Green I real-time qRT-PCR method for rapid detection and identification of PPRV lineages II and IV by analyzing different melting curve analyses. The negative amplification of other commonly circulating viruses such as orf virus, goat poxvirus, and foot-and-mouth disease virus demonstrated that primers targeting the L gene of PPRV were extremely specific. The sensitivity of the assay was assessed based on plasmid DNA and the detection limit achieved was 100 copies of PPRV lineages II and IV. CONCLUSION: Since the method has high sensitivity, specificity, and reproducibility, it will be effectively differentiated PPRV lineages II from PPRV lineages IV in PPRV infected animals.