UNASSIGNED: It remains unclear whether BPIV3 infection leads to stress granules formation and whether G3BP1 plays a role in this process and in viral replication. This study aims to clarify the association between BPIV3 and stress granules, explore the effect of G3BP1 on BPIV3 replication, and provide significant insights into the mechanisms by which BPIV3 evades the host\'s antiviral immunity to support its own survival.
UNASSIGNED: Here, we use Immunofluorescence staining to observe the effect of BPIV3 infection on the assembly of stress granules. Meanwhile, the expression changes of eIF2α and G3BP1 were determined. Overexpression or siRNA silencing of intracellular G3BP1 levels was examined for its regulatory control of BPIV3 replication.
UNASSIGNED: We identify that the BPIV3 infection elicited phosphorylation of the eIF2α protein. However, it did not induce the assembly of stress granules; rather, it inhibited the formation of stress granules and downregulated the expression of G3BP1. G3BP1 overexpression facilitated the formation of stress granules within cells and hindered viral replication, while G3BP1 knockdown enhanced BPIV3 expression.
UNASSIGNED: This study suggest that G3BP1 plays a crucial role in BPIV3 suppressing stress granule formation and viral replication.

Bovine parainfluenza virus 3 (BPIV3) is an important respiratory pathogen of both young and adult cattle. The pathways of cell entry are highly related to viral transmission and pathogenicity. In previous studies, we demonstrated that macropinocytosis and clathrin-dependent endocytosis play critical roles in the entry of BPIV3 into MDBK cells. Macropinocytosis is special endocytic process which need to activate signaling pathways that remodle the actin cytoskeleton. Parainfluenza viruses (PIVs) initiate infection by binding to sialic acid receptors on cell surfaces. Nevertheless, sialic acids are not able to transmit signals across the plasma membrane, indicating the necessity for additional signaling receptors. Here, we have demonstrated that specific inhibitors and siRNAs targeting EGFR inhibit the entry of BPIV3 into MDBK cells. BPIV3 productive infection in MDBK cells led to activation of EGFR. Inactivation of EGFR suppressed BPIV3-induced rearrangement of the F-actin cytoskeleton. In addition, PI3K-Akt and ERK1/2 were activated in an EGFR-dependent manner during BPIV3 infection. Specific inhibitors targeting these canonical downstream effectors of EGFR could significantly reduce viral entry efficacy. Moreover, we also demonstrated that the important regulators of macropinocytosis, Rac1 and Pak1, are downstream mediators of EGFR during BPIV3 internalization. These results indicated that EGFR is a host-entry cofactor used by BPIV3 to enter MDBK cells.

Bovine respiratory disease complex (BRDC) occurs widely in cattle farms. The main viral pathogens include bovine viral diarrhea virus (BVDV), Bovine herpesvirus 1 (BoHV-1), bovine parainfluenza virus type 3 (BPIV3), and bovine respiratory syncytial virus (BRSV), and the newly emerged influenza D virus (IDV). In this study, we have developed a one-step multiplex real-time Polymerase Chain Reaction (PCR) capable of simultaneously detecting these five viral pathogens causing BRDC. The established assay could specifically detect targeted viruses without cross-reaction with others. The detection limit was ~10 copies/reaction for single real-time PCR and 100 copies/ reaction for multiplex real-time PCR assay. A total of 213 nasal samples from cattle with signs of respiratory tract disease were then collected for performance evaluation of the established platform, proving that the method has good specificity and sensitivity. The surveillance data suggested that BVDV and BoHV-1 infections are the dominant cause of BRDC in the herd, whereas the detection rate of IDV, BIPV3, and BRSV is relatively lower. In summary, the established assay provides technical support for rapid clinical detection of BRDC associated viral pathogens to guide the formulation of BRDC prevention and control measures.

Bovine Respiratory Syncytial virus (BRSV) and Bovine Parainfluenza 3 virus (BPIV3) are closely related viruses involved in and both important pathogens within bovine respiratory disease (BRD), a major cause of morbidity with economic losses in cattle populations around the world. The two viruses share characteristics such as morphology and replication strategy with each other and with their counterparts in humans, HRSV and HPIV3. Therefore, BRSV and BPIV3 infections in cattle are considered useful animal models for HRSV and HPIV3 infections in humans.The interaction between the viruses and the different branches of the host\'s immune system is rather complex. Neutralizing antibodies seem to be a correlate of protection against severe disease, and cell-mediated immunity is thought to be essential for virus clearance following acute infection. On the other hand, the host\'s immune response considerably contributes to the tissue damage in the upper respiratory tract.BRSV and BPIV3 also have similar pathobiological and epidemiological features. Therefore, combination vaccines against both viruses are very common and a variety of traditional live attenuated and inactivated BRSV and BPIV3 vaccines are commercially available.

Bovine parainfluenza virus 3 (BPIV3) is an important respiratory pathogen of both young and adult cattle. No specific therapies are available for BPIV3. Understanding the viral internalization pathway of BPIV3 will provide new strategies for the development of antiviral treatments. Here, the entry mechanism of BPIV3 into MDBK cells was analyzed using chemical inhibitors and RNA silencing. Our data demonstrated that treatment with an inhibitor targeting the clathrin-mediated pathway or clathrin heavy chain (CHC) knockdown suppressed the entry of BPIV3 into MDBK cells. In contrast, sequestration of cellular cholesterol by nystatin or silencing of caveolin-1 had no effect on viral entry. Moreover, inhibition of critical modulators of macropinocytosis significantly reduced BPIV3 uptake. In addition, fluid-phase uptake was significantly increased in cells infected with BPIV3, which is indicative of virus-induced facilitation of macropinocytosis. These results suggest that BPIV3 enters MDBK cells via macropinocytosis and clathrin- but not caveolar-dependent endocytosis. Furthermore, inhibition of endosomal acidification and activation of cathepsin blocked BPIV3 entry, demonstrating that BPIV3 entered MDBK cells in a acid-dependent manner and required cathepsin L. Finally, we demonstrated that macropinocytosis but not clathrin-mediated endocytosis is dependent on actin dynamics during BPIV3 infection.

Bovine parainfluenza virus 3 (BPIV3) is a crucial causative agent of respiratory disease in young and adult cattle. No specific therapies are available for BPIV3 infection. Understanding the internalization pathway of the virus will provide a new strategy for the development of antiviral therapy. Here, the mechanism of BPIV3 entry into HeLa cells was analyzed using RNA silencing and pharmacological inhibitors. Treatment of HeLa cells with hypertonic medium prevented BPIV3 internalization. These results indicated that BPIV3 entered HeLa cells via receptor-mediated endocytosis. Moreover, removing cell membrane cholesterol through MβCD treatment hampered viral penetration but not viral replication. In addition, BPIV3 infection was inhibited by pretreatment with dynasore or chlorpromazine (CPZ) or knockdown of dynamin II or clathrin heavy chain. However, virus entry was unaffected by nystatin, EIPA, wortmannin, or cytochalasin D treatment or caveolin-1 knockdown. These data demonstrated that the entry of BPIV3 into HeLa cells was dependent on clathrin-mediated endocytosis but not on caveolae-mediated endocytosis or the macropinocytosis pathway. Many viruses are transported to endosomes, which provide an acidic environment and release their genome upon separation from primary endocytic vesicles. However, we found that BPIV3 infection required endosomal cathepsins, but not a low pH. In summary, we show, for the first time, that BPIV3 enters HeLa cells through the clathrin-mediated endocytosis pathway, presenting novel insights into the invasion mechanism of Paramyxoviridae.

Two defective bovine parainfluenza virus type 3 (BPIV3) strains were generated, one lacking the membrane (M) protein gene and expressing EGFP (ΔM-EGFP) and the other lacking the fusion (F) protein gene and expressing mStrawberry (ΔF-mSB), by supplying deficient proteins in trans. When Madin-Darby bovine kidney (MDBK) cells were co-infected with ΔM-EGFP and ΔF-mSB at a multiplicity of infection (MOI) of 0.1, complemented viruses were easily obtained. Complemented viruses grew as efficiently as wild-type BPIV3 and could be passaged in MDBK cell cultures even at an MOI of 0.01, possibly due to multiploid virus particles containing genomes of both ΔM-EGFP and ΔF-mSB. This reciprocal complementation method using two defective viruses would be useful to express large or multiple proteins in cell cultures using paramyxovirus vectors.

Lipid rafts are specialized lipid domains enriched in cholesterol and sphingolipid, which can be utilized in the lifecycle of numerous enveloped viruses. Bovine parainfluenza virustype3 (BPIV3) entry to cell is mediated by receptor binding and membrane fusion, but how lipid rafts in host cell membrane and BPIV3 envelope affect virus infection remains unclear. In this study, we investigated the role of lipid rafts in the different stages of BPIV3 infection. The MDBK cells were treated by methyl-β-cyclodextrin (MβCD) to disrupt cellular lipid raft, and the virus infection was determined. The results showed that MβCD significantly inhibited BPIV3 infection in a dose-dependent manner, but didn\'t block the binding of virus to the cell membrane. Whereas, the MDBK cells treated by MβCD after virus-entry had no effects on the virus infection, to suggest that BPIV3 infection was associated with lipid rafts in cell membrane during viral entry stage. To further confirm lipid rafts in viral envelope also affected BPIV3 infection, we treated BPIV3 with MβCD to determine the virus titer. We found that disruption of the viral lipid raft caused a significant reduction of viral yield. Cholesterol reconstitution experiment showed that BPIV3 infection was successfully restored by cholesterol supplementation both in cellular membrane and viral envelope, which demonstrated that cholesterol-rich lipid rafts played a critical role in BPIV3 infection. These findings provide insights on our understanding of the mechanism of BPIV3 infection and imply that lipid raft might be a good potential therapeutic target to prevent virus infection.

Paramyxovirus C protein targets the host interferon (IFN) system for virus immune evasion. To identify its unknown anti-IFN activity, we examined the effect of Sendai virus C protein on activation of the IFN-α promoter via various signaling pathways. This study uncovers a novel ability of C protein to block Toll-like receptor (TLR) 7- and TLR9-dependent IFN-α induction, which is specific to plasmacytoid dendritic cells. C protein interacts with a serine/threonine kinase IKKα and inhibits phosphorylation of IRF7. This anti-IFN activity of C protein is shared across genera of the Paramyxovirinae, and thus appears to play an important role in paramyxovirus immune evasion.