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Bovine respiratory disease complex (BRDC) represents a global acute respiratory condition that imposes substantial economic burdens on the cattle industry due to its high morbidity and mortality rates. Various factors contribute to the development of BRDC, including pathogen infections, environmental stresses, weaning of calves, and herd relocation. Viral pathogens, notably bovine respiratory syncytial virus (BRSV) and bovine viral diarrhea virus (BVDV), play a critical role in the etiology of BRDC, with single or combined viral infections being particularly clinically significant. In this study, we developed a duplex TaqMan-based real-time RT-PCR assay targeting the conserved regions of the F gene of BRSV and the 5′ UTR sequence of BVDV. The limits of detection for BRSV and BVDV were 6.83 copies/μL and 5.24 copies/μL, respectively. Our validation data suggest the assay has excellent sensitivity, specificity and reproducibility. Testing of clinical samples revealed prevalence of BRSV and BVDV in local farms in Jiangsu Province, China. This study provides an efficient diagnostic tool for the epidemiological investigation of BRDC.

Porcine Teschoviruses (PTVs) are associated with polioencephalomyelitis and various diseases, including reproductive and gastrointestinal disorders of pigs and wild boars, but rarely detected in the feces of pigs. In this study, a sample of swine diarrhea that tested positive for PTVs is subjected to high-throughput sequencing. The viral genome was 7221 nucleotides (nt) in length, which was consisted of twelve genes. Phylogenetic analysis showed and it was closely related to the PTV-HNMY(MG755212.1). The nucleotide homology of VP1 gene of PTVs JS2021 with PTV-1AF 296102.1 reached 82.97%, belonging to a branch of PTV-1 serotype. The nucleotide homology of VP1 protein with other serotypes of PTV is quite different from that of other serotypes of PTV. Bioinformatics analysis showed that PTVs have four capsid proteins, namely VP1, VP2, VP3 and VP4. The VP1 encodes a 29 kDa protein, which is the main protective antigen, a theoretical isoelectric point of 6.73, no transmembrane domain, no signal peptide and potential phosphorylation site. The VP1 protein is an unstable hydrophilic intracellular protein, which contains four secondary structures: irregular curl (c), extended chain (e), α-helix (h) and β-folded (t). The tertiary structure is heart-shaped and has multiple B cell epitopes. By analyzing the tertiary structure, we found that the amino acid at position 129 of VP1 mutated and reduction a larger alpha helix. This may lead to the main cause of piglet diarrhea. These findings enriched our knowledge of the viruses in the role of swine diarrhea, and help to develop an effective strategy for disease prevention and control.

Free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), play critical roles in various physiological activities such as cell differentiation, apoptosis, and vascular tension when existing in cells at low levels. However, excessive amounts of free radicals are harmful, causing DNA damage, lipid peroxidation, protein degeneration, and abnormal cell death. Certain viral infections induce cells to produce excessive free radicals, which in multiple ways help the virus to replicate, mature, and exit. Iron is a necessary element for many intracellular enzymes, involved in both cellular activities and viral replication. Ferroptosis, a programmed cell death mode distinct from apoptosis, necrosis, and pyroptosis, is characterized by lipid peroxide accumulation and damage to the antioxidant system, affecting many cellular processes. Viral infection commonly manifests as decreased glutathione (GSH) content and down-regulated glutathione peroxidase 4 (GPX4) activity, similar to ferroptosis. Recent studies have suggested a possible relationship among free radicals, viral infections and ferroptosis. This review aims to elucidate the molecular mechanism linking free radicals and ferroptosis during viral infections and provide a new theoretical basis for studying viral pathogenesis and control.

Genome rearrangement occurs to porcine circovirus type 2 (PCV2) during in vitro and in vivo infections, and a number of rearranged PCV2 genomes have been isolated and characterized. This study was conducted to investigate the role of the rearranged PCV2 (rPCV2) in PCV2 replication and the biological effect of rPCV2 in host cells. Two whole rPCV2 genome sequences (358 nt and 1125 nt in length) were synthesized and recombinant plasmids pBSK(+)-rPCV2 (pBSK(+)-1125 and pBSK(+)-358) were constructed. A novel virus-like agent (rPCV2-1125) was rescued by in vitro transfection of porcine kidney cell line (PK-15) and porcine alveolar macrophage 3D4/21 cells. The data indicate that rPCV2-1125 significantly enhanced PCV2 replication in vitro. Furthermore, rPCV2-1125 led to oxidative stress in host cells, as indicated by decreased intracellular glutathione (GSH) and total superoxide dismutase (SOD) activities, as well as increased malondialdehyde (MDA) levels. These results provide new insights into genome rearrangement of PCV2 and will contribute to future studies of PCV2 replication and associated mechanisms.

Mycoplasma synoviae (MS) is an important avian pathogen causing considerable economic hardship in the poultry industry. A major inflammation caused by MS is synovitis that occurs in the synovial tendon sheath and joint synovium. However, the overall appearance of pathological changes in the tendon sheath and surrounding tissues caused by MS infection at the level of pathological tissue sections was poor. Studies on the role of MS and synovial sheath cells (SSCs) interaction in the development of synovitis have not been carried out. Through histopathological observation, our study found that a major MS-induced pathological change of the tendon sheath synovium was extensive scattered and focal inflammatory cell infiltration of the tendon sheath synovial layer. In vitro research experiments revealed that the CFU numbers of MS adherent and invading SSC, the levels of expression of various pattern recognition receptors, inflammatory cytokines, and chemokines coding genes, such as IL-1β, IL-6, IL-8, CCL-20, RANTES, MIP-1β, TLR7, and TLR15 in SSCs, and chemotaxis of macrophages were significantly increased when the multiplicity of infection (MOI) of MS to SSC were increased tenfold. The expression level of IL-12p40 in SSC was significantly higher when the MOIs of MS to SSC were increased by a factor of 100. The interaction between MS and SSC can activate macrophages, which was manifested by a significant increase in the expression of IL-1β, IL-6, IL-8, CCL-20, RANTES, MIP-1β, and CXCL-13. This study systematically demonstrated that the interaction of MS with chicken SSC contributes to the inflammatory response caused by the robust expression of related cytokines and macrophage chemotaxis. These findings are helpful in elucidating the molecular mechanism of MS-induced synovitis in chickens.

Abstract Background Duck hepatitis A virus type 3 (DHAV-3) is one of the most harmful pathogens in the duck industry. However, the molecular mechanism underlying DHAV-3 infection in ducklings remains poorly understood. To study the genetic regulatory network for miRNA-mRNA and the signaling pathways involved in DHAV-3 infection in ducklings, we conducted global miRNA and mRNA expression profiling of duckling liver tissues infected with lethal DHAV-3 by high-throughput sequencing. Results We found 156 differentially expressed miRNAs (DEMs) and 7717 differentially expressed genes (DEGs) in livers of mock-infected and DHAV-3-infected duckling. A total of 19,606 miRNA-mRNA pairs with negatively correlated expression patterns were identified in miRNA-mRNA networks constructed on the basis of these DEMs and DEGs. Moreover, immune-related pathways, including the cytokine-cytokine receptor interaction, apoptosis, Toll-like receptor, Jak-STAT, and RIG-I-like receptor signaling pathway, were significantly enriched through analyzing functions of mRNAs in the network in response to DHAV-3 infection. Furthermore, apl-miR-32-5p, apl-miR-125-5p, apl-miR-128-3p, apl-miR-460-5p, and novel-m0012-3p were identified as potential regulators in the immune-related signaling pathways during DHAV-3 infection. And some host miRNAs were predicted to target the DHAV-3 genome. Conclusions This is the first integrated analysis of miRNA and mRNA in DHAV-3-infected ducklings. The results indicated the important roles of miRNAs in regulating immune response genes and revealed the immune related miRNA-mRNA regulation network in the DHAV-3-infected duckling liver. These findings increase our knowledge of the roles of miRNAs and their target genes in DHAV-3 replication and pathogenesis. They also aid in the understanding of host-virus interactions.

Abstract Background Porcine circovirus type 2 (PCV2) is the causal agent of postweaning multisystemic wasting syndrome (PMWS), causing large economical losses of the global swine industry. Nitric oxide (NO), as an important signaling molecule, has antiviral activity on some viruses. To date, there is little information on the role of NO during PCV2 infection. Results We used indirect fluorescence assay (IFA), TCID50, real-time RT-qPCR and western blot assay to reveal the role of NO in restricting PCV2 replication. PCV2 replication was inhibited by a form of NO, NO • , whereas PCV2 was not susceptible to another form of NO, NO+. Conclusion Our findings indicate that the form of NO • has a potential role in the fight against PCV2 infection.

Mycoplasma synoviae is an important pathogen of poultry, causing significant economic losses in this industry. Analysis of the unique genes and shared genes among different M. synoviae strains and among related species is helpful for studying the molecular pathogenesis of M. synoviae and provides valuable molecular diagnostic targets to facilitate the identification of M. synoviae species. We selected a total of 46 strains, including six M. synoviae strains, from 25 major animal (including avian) Mycoplasma species/subspecies that had complete genome sequences and annotation information published in GenBank, and used them for comparative genomic analysis. After analysis, 16 common genes were found in the 46 strains. Thirteen single-copy core genes and the 16s rRNA genes were used for genetic evolutionary analysis. M. synoviae was found to have a distant evolutionary relationship not only with other arthritis-causing mycoplasmas, but also with another major avian pathogen, Mycoplasma gallisepticum, that shares the major virulence factor vlhA with M. synoviae. Subsequently, six unique coding genes were identified as shared among these M. synoviae strains that are absent in other species with published genome sequences. Two of the genes were found to be located in the genetically stable regions of the genomes of M. synoviae and were determined to be present in all M. synoviae isolated strains (n = 20) and M. synoviae-positive clinical samples (n = 48) preserved in our laboratory. These two genes were used as molecular diagnostic targets for which SYBR green quantitative PCR detection methods were designed. The two quantitative PCR methods exhibited good reproducibility and high specificity when tested on positive plasmid controls and genomic DNA extracted from different M. synoviae strains, other major avian pathogenic bacteria/mycoplasmas, and low pathogenic Mycoplasma species. The detection limit for the two genes was 10 copies or less per reaction. The clinical sensitivity and specificity of the quantitative PCR methods were both 100% based on testing chicken hock joint samples with positive or negative M. synoviae infection. This research provides a foundation for the study of species-specific differences and molecular diagnosis of M. synoviae.

Caprine parainfluenza virus type 3 (CPIV3) is an emerging respiratory pathogen that affects the sheep and goat industry in China and possibly other countries around the world. Accumulating evidence suggests that microRNAs play important roles in regulating virus-host interactions and can suppress or facilitate viral replication. In this study, we showed that CPIV3 infection induced apoptosis in Madin-Darby bovine kidney (MDBK) cells, as determined by morphological changes and flow cytometry. Caspase activity and the expression of pro-apoptotic genes further indicated that CPIV3 induced apoptosis by activating both the intrinsic and extrinsic pathways. We also demonstrated the involvement of bta-microRNA-98 (bta-miR-98) in regulating CPIV3-induced apoptosis. Bta-miR-98 was downregulated in MDBK cells infected with CPIV3. Overexpression of bta-miR-98 significantly decreased the activities of caspase-3, −8, and −9. Conversely, inhibition of bta-miR-98 had completely opposite effects. Furthermore, our data showed that bta-miR-98 markedly affected CPIV3 replication by regulating apoptosis. Importantly, we found that bta-miR-98 modulated CPIV3-induced apoptosis by targeting caspase-3, an effector of apoptosis. Collectively, our results may suggest that CPIV3 infection induced apoptosis and downregulated the levels of bta-miR-98, and this miRNA regulated viral replication through effected apoptosis. This study contributes to our understanding of the molecular mechanisms underlying CPIV3 pathogenesis.

Mycoplasma synoviae (MS), which causes respiratory disease, eggshell apex abnormalities, infectious synovitis, and arthritis in avian species, has become an economically detrimental poultry pathogen in recent years. In China, the disease is characterized by infectious synovitis and arthritis. However, the mechanism by which MS causes infectious synovitis and arthritis remains unknown. Increasing evidence suggests that synovial fibroblasts (SF) play a key role in the pathogenesis of arthritis. Here, both RNA sequencing and tandem mass tag analyses are utilized to compare the response of primary chicken SF (CSF) following infection with and without MS. The host response between non-infected and infected cells was remarkably different at both the mRNA and protein levels. In total, 2,347 differentially expressed genes (DEGs) (upregulated, n = 1,137; downregulated, n = 1,210) and 221 differentially expressed proteins (DEPs) (upregulated, n = 129; downregulated, n = 92) were detected in the infected group. A correlation analysis indicated a moderate positive correlation between the mRNA and protein level changes in MS-infected CSF. At both the transcriptomic and proteomic levels, 149 DEGs were identified; 88 genes were upregulated and 61 genes were downregulated in CSF. Additionally, part of these regulated genes and their protein products were grouped into seven categories: proliferation-related and apoptosis-related factors, inflammatory mediators, proangiogenic factors, antiangiogenic factors, matrix metalloproteinases, and other arthritis-related proteins. These proteins may be involved in the pathogenesis of MS-induced arthritis in chickens. To our knowledge, this is the first integrated analysis on the mechanism of CSF-MS interactions that combined transcriptomic and proteomic technologies. In this study, many key candidate genes and their protein products related to MS-induced infectious synovitis and arthritis were identified.

Porcine circovirus-like virus P1 is an important pathogen of the current pig industry, the infection mechanism is not entirely clear. Wnt signaling pathway plays an important role in the growth of young animals and infection of some viruses. This study was designed to demonstrate the effects of P1 infection on the Wnt signaling pathway. In vivo experiments, we demonstrated the down-regulatory effects of P1 infection in piglets and mice on the downstream components expression levels of Wnt signaling pathway, and the effects of Wnt signaling pathway activation on the pathogenesis of P1. In vitro studies, we found P1 infection down-regulated protein level of β-catenin and mRNA level of mmp2, prevented the β-catenin from entering into nucleus, abolished the TCF/LEF promoter activity, proved that P1 could inhibit the activation of Wnt signaling pathway in vitro. Finally, we found that VP1 of P1 virus also had the inhibitory effects on Wnt signaling pathway in vitro, elucidated the mechanism of P1’s inhibitory effects on the Wnt signaling pathway and offered the possibility that the suppression of Wnt signaling pathway was involved in the post-weaning multisystemic wasting syndrome (PMWS), laying a foundation for elucidating the pathogenesis of P1.

Background: Nitric oxide (NO), an important signaling molecule with biological functions, has antimicrobial activity against a variety of pathogens including viruses. To our knowledge, little information is available about the regulatory effect of NO on porcine circovirus type 2 (PCV2) infection. This study was conducted to investigate the antiviral activity of NO generated from S-nitrosoglutathione (GSNO), during PCV2 infection of PK-15 cells and BALB/c mice. Results: GSNO released considerable NO in the culture medium of PK-15 cells, and NO was scavenged by its scavenger hemoglobin (Hb) in a dose-dependent manner. NO strongly inhibited PCV2 replication in PK-15 cells, and the antiviral effect was reversed by Hb. An in vivo assay indicated that GSNO treatment reduced the progression of PCV2 infection in mice, evident as reductions in the percentages of PCV2-positive sera and tissue samples and in the viral DNA copies in serum samples. GSNO also improved the growth performance and immune organs (spleens and thymuses) of the PCV2-infected mice to some degree. Conclusions: Our data demonstrate that the NO-generating compound GSNO suppresses PCV2 infection in PK-15 cells and BALB/c mice, indicating that NO and its donor, GSNO, have potential value as antiviral drugs against PCV2 infection. [ABSTRACT FROM AUTHOR]

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhea in piglets and possesses the potential to infect humans. However, there are no commercially available vaccines for PDCoV. In this study, the immune responses to the spike (S) protein and receptor-binding domain (RBD) trimer were examined in mice. Neutralization assays and flow cytometry analysis demonstrated that S protein elicited more robust neutralizing antibodies (NAbs) and cellular immune responses than the RBD trimer. Spike protein and inactivated vaccine were used in the assessment of immunogenicity in piglets and sows. Immunized piglets, sows, and suckling pig showed high NAb titers and S-specific sIgA in colostrum, milk, and serum. The piglets/suckling pig from the immunized group displayed significantly fewer microscopic lesions in the intestinal tissue, with only one or no piglet showing mild diarrhea. However, all piglets/suckling pig showed mild to watery diarrhea and exhibited a high level of viral shedding in the challenged control group. The feces from the piglets/suckling pig in the S protein and inactivated vaccine group exhibited reduced viral load. Of note, vaccine-elicited NAbs last for more than 4 months in immunized piglets. Together, our data demonstrate for the first time the protective efficacy of S-based subunit vaccine, which could be a candidate vaccine against PDCoV. [ABSTRACT FROM AUTHOR]