Here, we report the discovery of two viruses associated with a disease characterized by severe diarrhea on a large-scale goat farm in Jilin province. Electron Microscopy observations revealed two kinds of virus particles with the sizes of 150-210 nm and 20-30 nm, respectively. Detection of 276 fecal specimens from the diseased herds showed the extensive infection of peste des petits ruminants virus (63.77%, 176/276) and caprine enterovirus (76.81%, 212/276), with a co-infection rate of 57.97% (160/276). These results were partially validated with RT-PCR, where all five PPRV-positive and CEV-positive specimens yielded the expected size of fragments, respectively, while no fragments were amplified from PPRV-negative and CEV-negative specimens. Moreover, corresponding PPRV and CEV fragments were amplified in PPRV and CEV double-positive specimens. Histopathological examinations revealed severe microscopic lesions such as degeneration, necrosis, and detachment of epithelial cells in the bronchioles and intestine. An immunohistochemistry assay detected PPRV antigens in bronchioles, cartilage tissue, intestine, and lymph nodes. Simultaneously, caprine enterovirus antigens were detected in lung, kidney, and intestinal tissues from the goats infected by the peste des petits ruminants virus. These results demonstrated the co-infection of peste des petits ruminants virus with caprine enterovirus in goats, revealing the tissue tropism for these two viruses, thus laying a basis for the future diagnosis, prevention, and epidemiological survey for these two virus infections.

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.
OBJECTIVE: 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.
METHODS: 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.

BACKGROUND: Peste des petits ruminants (PPR) is a contagious and fatal disease of sheep and goats. PPR virus (PPRV) infection induces endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR). The activation of UPR signaling pathways and their impact on apoptosis and virus replication remains controversial.
OBJECTIVE: To investigate the role of PPRV-induced ER stress and the IRE1-XBP1 and IRE1-JNK pathways and their impact on apoptosis and virus replication.
METHODS: The cell viability and virus replication were assessed by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, immunofluorescence assay, and Western blot. The expression of ER stress biomarker GRP78, IRE1, and its downstream molecules, PPRV-N protein, and apoptosis-related proteins was detected by Western blot and quantitative reverse transcription-polymerase chain reaction, respectively. 4-Phenylbutyric acid (4-PBA) and STF-083010 were respectively used to inhibit ER stress and IRE1 signaling pathway.
RESULTS: The expression of GRP78, IRE1α, p-IRE1α, XBP1s, JNK, p-JNK, caspase-3, caspase-9, Bax and PPRV-N were significantly up-regulated in PPRV-infected cells, the expression of Bcl-2 was significantly down-regulated. Due to 4-PBA treatment, the expression of GRP78, p-IRE1α, XBP1s, p-JNK, caspase-3, caspase-9, Bax, and PPRV-N were significantly down-regulated, the expression of Bcl-2 was significantly up-regulated. Moreover, in PPRV-infected cells, the expression of p-IRE1α, p-JNK, Bax, and PPRV-N was significantly decreased, and the expression of Bcl-2 was increased in the presence of STF-083010.
CONCLUSIONS: PPRV infection induces ER stress and IRE1 activation, resulting in apoptosis and enhancement of virus replication through IRE1-XBP1s and IRE1-JNK pathways.

The Peste des petits ruminant virus (PPRV) is a member of the Paramyxoviridae family and is classified into the genus Measles virus. PPRV predominantly infects small ruminants, leading to mortality rates of nearly 100%, which have caused significant economic losses in developing countries. Host proteins are important in virus replication, but the PPRV nucleocapsid (N) protein-host interacting partners for regulating PPRV replication remain unclear. The present study confirmed the interaction between PPRV-N and the host protein vimentin by co-immunoprecipitation and co-localization experiments. Overexpression of vimentin suppressed PPRV replication, whereas vimentin knockdown had the opposite effect. Mechanistically, N was subjected to degradation via the ubiquitin/proteasome pathway, where vimentin recruits the E3 ubiquitin ligase NEDD4L to fulfill N-ubiquitination, resulting in the degradation of the N protein. These findings suggest that the host protein vimentin and E3 ubiquitin ligase NEDD4L have an anti-PPRV effect.

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.

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.

A simple, fast, and visual method for detecting antibodies against peste des petits ruminants virus (PPRV) using colloidal gold strips was developed. In this study, the pET-32a-N was transformed into Escherichia coli Rosetta (DE3) for expression. Hybridoma cell lines were generated by fusing SP2/0 myeloma cells with splenocytes from immunized mice with the expressed and purified N protein of PPRV. The PPRV N protein was labeled with colloidal gold particles as the gold-labeled antigen. The N protein served as the gold standard antigen and as the test (T) line-coated antigen, while the monoclonal antibody served as the quality control (C) line-coated antibody to assemble the colloidal gold immunochromatographic test strips for detecting antibodies against the N protein of PPRV. Hybridoma cell line designated as 1F1 was able to stably secrete the monoclonal antibody against the N protein of PPRV. The titer of 1F1 monoclonal antibody in ascites was 1:128 000 determined by indirect enzyme-linked immunosorbent assays (ELISA), and the immunoglobulin subtype of the monoclonal antibody was IgG1, with kappa chain. The obtained monoclonal antibody was able to specifically recognize the N protein of PPRV, as shown by Western blotting and indirect immunofluorescent assay (IFA). The developed colloidal gold test strip method was able to detect PPRV antibodies specifically, and there was no difference between different batches of the test strips. Testing of a total of 122 clinical sera showed that the compliance rate of the test strip with ELISA test was 97.6%.The test strip assay developed in this study has good specificity, reproducibility, and sensitivity, and it can be used for the rapid detection of PPRV antibodies.

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.
Dimethylation-labeling-based quantitative proteomic analysis of the acetylome of PPRV-infected Vero cells was performed.
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.
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.

BACKGROUND: Peste des petits ruminants (PPR), caused by the PPR virus (PPRV), is an acute and fatal contagious disease that mainly infects goats, sheep, and other artiodactyls. Peripheral blood mononuclear cells (PBMCs) are considered the primary innate immune cells.
OBJECTIVE: PBMCs derived from goats were infected with PPRV and analyzed to detect the relationship between PPRV replication and apoptosis or the inflammatory response.
METHODS: Quantitative real-time polymerase chain reaction was used to identify PPRV replication and cytokines expression. Flow cytometry was conducted to detect apoptosis and the differentiation of CD4+ and CD8+ T cells after PPRV infection.
RESULTS: PPRV stimulated the differentiation of CD4+ and CD8+ T cells. In addition, PPRV induced apoptosis in goat PBMCs. Furthermore, apoptosis and the inflammatory response induced by PPRV could be suppressed by Z-VAD-FMK and Z-YVAD-FMK, respectively. Moreover, the virus titer of PPRV was attenuated by inhibiting caspase-1-dependent apoptosis and inflammation.
CONCLUSIONS: This study showed that apoptosis and the inflammatory response play an essential role in PPR viral replication in vitro, providing a new mechanism related to the cell host response.

In this study, we recombinantly expressed the V protein of the peste des petits ruminants virus (PPRV) and evaluated its diagnostic value for PPRV infection using an indirect ELISA (i-ELISA). The optimal concentration of the coated antigen of V protein was 15 ng/well at a serum dilution of 1:400, and the optimal positive threshold value was 0.233. A cross-reactivity assay showed that the V protein-based i-ELISA was specific to PPRV with consistent reproducibility and showed a specificity of 82.6% and a sensitivity of 100% with a virus neutralization test. Using the recombinant V protein as an antigen in ELISA is useful for seroepidemiological studies of PPRV infections.