Bovine Viral Diarrhea Virus Replication Research Articles

Matrine and icariin can inhibit bovine viral diarrhoea virus replication by promoting type I interferon response in vitro

Abstract Introduction Bovine viral diarrhoea virus (BVDV) can cause diarrhoea (BVD) in an animal herd, leading to heavy economic losses. There are limited drugs available for treating and controlling BVD. This research aims to investigate the antiviral and immunoregulatory effects of two traditional Chinese herb extracts against BVDV infection. The extracts are matrine and icariin, which have been proved to have immunostimulant and antiviral effects. Material and Methods A cell counting kit-8 assay was used to analyse the toxicity of matrine and icariin to Madin–Darby bovine kidney (MDBK) cells. The model of MDBK cells infected with BVDV was utilised to uncover the antiviral mechanism of matrine and icariin, which along with their immunoregulatory ability was evaluated by quantitative reverse-transcription PCR and ELISA. Results The results showed that matrine and icariin can significantly inhibit the gene expression level of the BVDV 5′ untranslated region through various pathways. Both matrine and icariin can statistically upregulate the gene expression level of interferon alpha, interferon beta (IFN-β), toll-like receptor 3, retinoic acid–inducible gene I and interferon regulatory factor 3, and raise the concentration of IFN-β after BVDV infection. Conclusion This study proves that both matrine and icariin have inhibitory effects on BVDV replication by activating IFN production and the IFN signalling pathway. The finding is promising and should open up the possibility of larger-scale in vitro research followed by in vivo experiments evaluating matrine and icariin as therapeutic agents in BVD cases.

The activation of liver X receptors in Madin-Darby bovine kidney cells and mice restricts infection by bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) is prevalent worldwide and is an important pathogen that represents a serious threat to the development of the cattle industry by causing significant economic losses. Liver X receptors (LXRs) are members of the nuclear receptor superfamily and have become attractive therapeutic targets for cardiovascular disease. In the present study, we found that LXRs in both Madin-Darby bovine kidney (MDBK) cells and mice were associated with BVDV infection. GW3965, an agonist for LXRs, significantly inhibited BVDV RNA and protein levels in MDBK cells. In vivo studies in a mouse model also confirmed the inhibitory role of GW3965 in BVDV replication and the ameliorating effect of GW3965 on pathological injury to the duodenum. In vitro investigations of the potential mechanisms involved showed that GW3965 significantly inhibited BVDV-induced increases in cholesterol levels and viral internalization. Furthermore, the antiviral activity of GW3965 was significantly reduced following cholesterol replenishment, thus demonstrating that cholesterol was involved in the resistance of GW3965 to BVDV replication. Further studies indicated the role of ATP-binding cassette transporter A1 (ABCA1) and cholesterol-25-hydroxylase (CH25H) in the antiviral activity of GW3965. We also demonstrated the significant antiviral effect of 25hydroxycholesterol (25HC), a product of the catalysis of cholesterol by CH25H. In addition, the anti-BVDV effects of demethoxycurcumin (DMC), cyanidin-3-O-glucoside (C3G), and saikosaponin-A (SSA), three natural agonizts of LXRs, were also confirmed in both MDBK cells and mice. However, the antiviral activities of these agents were weakened by SR9243, a synthetic inhibitor of LXRs. For the first time, our research demonstrated that the activation of LXRs can exert significant anti-BVDV effects in MDBK cells and mice.

The host protein CALCOCO2 interacts with bovine viral diarrhoea virus Npro, inhibiting type I interferon production and thereby promoting viral replication

ABSTRACT Bovine viral diarrhoea-mucosal disease caused by bovine viral diarrhoea virus (BVDV) is a major infectious disease that affects the cattle industry. The nonstructural protein Npro of BVDV antagonizes the type I interferon (IFN-I) pathway, thereby escaping the host immune response. The exact mechanism by which Npro uses host proteins to inhibit IFN-I production is unclear. The host protein CALCOCO2 was identified as a binding partner of Npro using a yeast two-hybrid screen. The interaction between Npro and CALCOCO2 was confirmed by yeast co-transformation, co-immunoprecipitation assays, and GST pull-down assays. The stable overexpression of CALCOCO2 markedly promoted BVDV propagation, while the knockdown of CALCOCO2 significantly inhibited BVDV replication in MDBK cells. Interestingly, CALCOCO2 inhibited IFN-I and IFN-stimulated gene production in BVDV-infected cells. Ectopic expression of CALCOCO2 effectively reduced IRF3 protein levels via the proteasome pathway. CALCOCO2 was found to promote Npro-mediated ubiquitination degradation of IRF3 by interacting with IRF3. Our results demonstrate the molecular mechanism by which Npro recruits the CALCOCO2 protein to regulate IRF3 degradation to inhibit IFN-I production.

Up-regulated lncRNA CYLD as a ceRNA of miR-2383 facilitates bovine viral diarrhea virus replication by promoting CYLD expression to counteract RIG-I-mediated type-I IFN production

Bovine viral diarrhea virus (BVDV) is one of the most important pathogens of cattle, causing numerous economic losses to the cattle industry. To date, many potential mechanisms of BVDV evading or subverting innate immunity are still unknown. In this study, an lnc-CYLD/miR-2383/CYLD axis involved in BVDV-host interactions was screened from RNA-seq-based co-expression networks analysis of long noncoding RNAs, microRNAs and mRNAs in BVDV-infected bovine cells, and underlying mechanisms of lnc-CYLD/miR-2383/CYLD axis regulating BVDV replication were explored. Results showed that BVDV-induced up-regulation of the lnc-CYLD competed for binding to the miR-2383, and then promoted CYLD expression, thereby inhibiting RIG-I-mediated type-I interferon (IFN) production, which was subsequently confirmed by treatment with lnc-CYLD overexpression and miR-2383 inhibitor. However, miR-2383 transfection and small interfering RNA-mediated lnc-CYLD knockdown inhibited CYLD expression and enhanced RIG-I-mediated type-I IFN production, inhibiting BVDV replication. In addition, interaction relationship between lnc-CYLD and miR-2383, and colocalization relationship of lnc-CYLD, miR-2383 and CYLD were confirmed by dual-luciferase assay and in situ hybridization assay. Conclusively, up-regulation of the lnc-CYLD as a competing endogenous RNA binds to the miR-2383 to reduce inhibitory effect of the miR-2383 on the CYLD expression, playing an important role in counteracting type-I IFN-dependent antiviral immunity to facilitate BVDV replication.

Biological activity of recombinant bovine IFN-α and inhibitory effect on BVDV in vitro

Type I interferon has great broad-spectrum antiviral ability and immunomodulatory function, and its receptors are expressed in almost all types of cells. Bovine viral diarrhea virus (BVDV) is an important pathogen causing significant economic losses in cattle. In this study, a recombinant expression plasmid carrying bovine interferon-α(BoIFN-α)gene was constructed and transformed into E. coli BL21 (DE3) competent cells. SDS-PAGE and Westernblotting analysis showed that the recombinant BoIFN-α protein (rBoIFN-α) was successfully expressed. It is about 36KD and exists in the form of inclusion body. When denatured, purified and renatured rBoIFN-α protein stimulated MDBK cells, the expression of interferon stimulating genes (ISGs) such as ISG15, OAS1, IFIT1, Mx1 and IFITM1 were significantly up-regulated, and reached the peak at 12 h (P< 0.001). MDBK cells were infected with BVDV with moi of 0.1 and 1.0, respectively. The virus proliferation was observed after pretreatment with rBoIFN-α protein and post-infection treatment. The results showed that the denatured, purified and renatured BoIFN-α protein had good biological activity and could inhibit the replication of BVDV in MDBK cells in vitro, which provided a basis for BoIFN-α as an antiviral drug, immune enhancer and clinical application of BVDV.

New Insights into the Role and Therapeutic Potential of Heat Shock Protein 70 in Bovine Viral Diarrhea Virus Infection.

Bovine viral diarrhea virus (BVDV), a positive-strand RNA virus of the genus Pestivirus in the Flaviviridae family, is the causative agent of bovine viral diarrhea-mucosal disease (BVD-MD). BVDV's unique virion structure, genome, and replication mechanism in the Flaviviridae family render it a useful alternative model for evaluating the effectiveness of antiviral drugs used against the hepatitis C virus (HCV). As one of the most abundant and typical heat shock proteins, HSP70 plays an important role in viral infection caused by the family Flaviviridae and is considered a logical target of viral regulation in the context of immune escape. However, the mechanism of HSP70 in BVDV infection and the latest insights have not been reported in sufficient detail. In this review, we focus on the role and mechanisms of HSP70 in BVDV-infected animals/cells to further explore the possibility of targeting this protein for antiviral therapy during viral infection.

Ethyl Gallate Inhibits Bovine Viral Diarrhea Virus by Promoting IFITM3 Expression, Lysosomal Acidification and Protease Activity.

Bovine viral diarrhea virus (BVDV) is a highly contagious viral disease which causes economic losses to the cattle industry. Ethyl gallate (EG) is a phenolic acid derivative which has various potentials to modulate the host response to pathogens, such as via antioxidant activity, antibacterial activity, inhibition of the production of cell adhesion factors, and so on. This study aimed to evaluate if EG influences BVDV infection in Madin-Darby Bovine Kidney (MDBK) cells, and to understand the antiviral mechanism. Data indicated that EG effectively inhibited BVDV infection by co-treatment and post-treatment in MDBK cells with noncytotoxic doses. In addition, EG suppressed BVDV infection at an early stage of the viral life cycle by blocking entry and replication steps but not viral attachment and release. Moreover, EG strongly inhibited BVDV infection by promoting interferon-induced transmembrane protein 3 (IFITM3) expression, which localized to the cytoplasm. The protein level of cathepsin B was significantly reduced by BVDV infection, whereas with treatment with EG, it was significantly enhanced. The fluorescence intensities of acridine orange (AO) staining were significantly decreased in BVDV-infected cells but increased in EG-treated cells. Finally, Western blot and immunofluorescence analyses demonstrated that EG treatment significantly enhanced the protein levels of autophagy markers LC3 and p62. Chloroquine (CQ) significantly increased IFITM3 expression, and Rapamycin significantly decreased it. Thus, EG may regulate IFITM3 expression through autophagy. Our results showed that EG could have a solid antiviral activity on BVDV replication in MDBK cells via increased IFITM3 expression, lysosomal acidification, protease activity, and regulated autophagy. EG might have value for further development as an antiviral agent.

Effects of Spatial Expression of Activating Transcription Factor 4 on the Pathogenicity of Two Phenotypes of Bovine Viral Diarrhea Virus by Regulating the Endoplasmic Reticulum-Mediated Autophagy Process.

The endoplasmic reticulum (ER) stress response is a highly conserved stress-defense mechanism and activates the adaptive unfolded protein response (UPR) to mitigate imbalance. The ER stress-activated signaling pathways can also trigger autophagy to facilitate cellular repair. Bovine viral diarrhea virus (BVDV) utilizes the host cellular ER as the primary site of the life cycle. However, the interplay between cellular ER stress and BVDV replication remains unclear. This report reveals that cytopathic (cp) and noncytopathic (ncp) BVDV have distinct strategies to regulate UPR mechanisms and ER stress-mediated autophagy for their own benefit. Immunoblot analysis revealed that cp and ncp BVDV differentially regulated the abundance of ER chaperone GRP78 for viral replication, while the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2 subunit α (eIF2α)-activating transcription factor 4 (ATF4) pathway of the UPR was switched on at different stages of infection. Pretreatment with ER stress inducer promoted virion replication, but RNA interference (RNAi) knockdown of ATF4 in BVDV-infected cells significantly attenuated BVDV infectivity titers. More importantly, the effector ATF4 activated by cp BVDV infection translocated into the nucleus to mediate autophagy, but ATF4 was retained in the cytoplasm during ncp BVDV infection. In addition, we found that cp BVDV core protein was localized in the ER to induce ER stress-mediated autophagy. Overall, the potential therapeutic target ATF4 may contribute to the global eradication campaign of BVDV. IMPORTANCE The ER-tropic viruses hijack the host cellular ER as the replication platform of the life cycle, which can lead to strong ER stress. The UPR and related transcriptional cascades triggered by ER stress play a crucial role in viral replication and pathogenesis, but little is known about these underlying mechanisms. Here, we report that cytopathic and noncytopathic BVDV use different strategies to reprogram the cellular UPR and ER stress-mediated autophagy for their own advantage. The cytopathic BVDV unconventionally downregulated the expression level of GRP78, creating perfect conditions for self-replication via the UPR, and the noncytopathic BVDV retained ATF4 in the cytoplasm to provide an advantage for its persistent infection. Our findings provide new insights into exploring how BVDV and other ER-tropic viruses reprogram the UPR signaling pathway in the host cells for replication and reveal the attractive host target ATF4 for new antiviral agents.

Bta-miR-2904 inhibits bovine viral diarrhea virus replication by targeting viral-infection-induced autophagy via ATG13.

MicroRNAs (miRNAs) are endogenous small and noncoding RNA molecules (18-25 nt) that can regulate expression of their target genes post-transcriptionally. Previously, using high-throughput sequencing data obtained on a Solexa platform, we found that Bos taurus bta-miR-2904 (miR-2904) was significantly upregulated in Madin-Darby bovine kidney (MDBK) cells infected with bovine viral diarrhea virus (BVDV) strain NADL at 2, 6, and 18 h postinfection (hpi) compared to uninfected MDBK cells. Moreover, miR-2904 overexpression significantly reduced BVDV replication. However, the mechanism by which miR-2904 inhibits viral replication remains unclear. In this study, we used electron microscopy, laser confocal microscopy, dual-luciferase reporter analysis, real-time PCR, and Western blot assays to investigate the effect of the miR-2904 expression on BVDV NADL replication and virus-infection-induced autophagy. The results indicate that miR-2904 inhibits autophagy of MDBK cells by targeting autophagy-related gene 13 (ATG13), and overexpression of miR-2904 inhibited the replication of BVDV NADL.

In Vivo and In Vitro Antiviral Activity of Phlorizin Against Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) is one of the most serious pathogens affecting the cattle industry worldwide. Phlorizin, a kind of flavonoids extracted from apple tree roots, leaves, and fruits, has a variety of biological functions and has been widely used as a herbal supplement and food additive. Here, BALB/c mouse and Madin-Darby bovine kidney (MDBK) cells were used to explore the effect and mechanism of phlorizin against BVDV infection. The results showed that phlorizin significantly inhibited CP BVDV replication and improved the histopathological changes of duodenum and spleen in mice. In vitro studies also confirmed the activity of phlorizin against CP BVDV. Exploration on its potential mechanism suggested that phlorizin inhibited CP BVDV-induced beclin-1 level and the conversion rate of LC3B-I to LC3B-II. Interestingly, although phlorizin also showed a protective effect on MDBK cells, which were treated with 3-methyladenine A (3-MA), the effect was significantly weakened. Furthermore, phlorizin suppressed the stage of BVDV replication but showed no effect on stages of attachment and internalization. Our data further indicated that phlorizin promoted IFN-α and IFN-β levels, decreased IL-1β and IL-6 expression, and regulated RIG-I, MDA5, TLR3, and NLRP3 levels. Similar to CP BVDV results, in vivo and in vitro, phlorizin inhibited NCP BVDV (NY-1 and YNJG2020 strains) infection. These results were the first to be discovered that phlorizin might be used as a new dietary strategy for controlling BVDV infection.

Quercetin Inhibits Hsp70 Blocking of Bovine Viral Diarrhea Virus Infection and Replication in the Early Stage of Virus Infection.

Bovine viral diarrhea virus (BVDV), a positive-strand RNA virus of the genus Pestivirus in the Flaviviridae family, is the causative agent of viral diarrheal disease in bovine. BVDV has been used as a surrogate model for the hepatitis C virus (HCV) to evaluate the efficacy of antiviral drugs. The plant flavonol quercetin causes multiple health-promoting effects in humans and animals. It can be made into a variety of additives, and it exerts a variety of immunomodulatory effects with the potential to be used as an antiviral agent. However, quercetin's antiviral effect and mechanism of action on BVDV are still unclear. Therefore, this study was designed to evaluate quercetin's effect on BVDV virus replication in vitro and in vivo and elucidate its mechanism of action. A CCK-8 kit was used to analyze the toxicity of the quercetin to the MDBK cells. Western blot, qRT-PCR, TCID50, and histological analysis were used to determine the mechanism of quercetin's anti-BVDV activity. An oxidative stress kit was used to evaluate the effects of quercetin on ROS, antioxidant enzymes, and MDA indexes. The effect of quercetin on IL-2 and IFN-γ in the serum of mice was determined by using an ELISA kit. The results showed that quercetin inhibits Hsp70, blocks BVDV infection in the early stage of virus infection and inhibits BVDV replication by inhibiting oxidative stress or ERK phosphorylation. In addition, quercetin alleviated the decrease in IFN-γ and IL-2 in the serum of BVDV-infected mice. Quercetin ameliorated BVDV-induced histopathological changes. In summary, this study demonstrated for the first time the role of Hsp70 in BVDV infection and the potential application of quercetin in treating BVDV infection.

Berbamine hydrochloride inhibits bovine viral diarrhea virus replication via interfering in late-stage autophagy

Bovine viral diarrhea virus (BVDV) is a harmful pathogen that easily causes large-scale infections and huge economic losses to the cattle industry. Berbamine hydrochloride (BBH) is a natural product extracted from berberis and has a wide range of pharmacological effects. However, the antiviral effect of BBH against BVDV needs to be further elucidated. This study aimed to evaluate the antiviral activities of BBH against BVDV infection. We mainly used RT-qPCR, Western blotting, immunofluorescence, and TEM assays to assess the inhibitory activity of BBH against BVDV. The results showed that BBH had an inhibitory effect on BVDV and higher inhibitory activity in the viral attachment and release in MDBK cells. This study found that BVDV could induce and use autophagy to replicate itself. Further results showed that BBH inhibited BVDV infection by inhibiting autophagy integrity in BVDV-infected cells, which was proven by the detection of autophagy-related proteins. Our data show that in BBH-treated BVDV-infected cells, the expression of p62 and LC3 increased over time. After the addition of an autophagy inhibitor, chloroquine (CQ), and an autophagy promoter, rapamycin (Rapa), we found that promoting autophagy was beneficial to the replication of BVDV, while inhibiting autophagy could reduce the number of infections by BVDV, which was evidenced by the expression of the BVDV E2 protein. Furthermore, BBH blocked the normal binding of LC3 and LAMP1 in BVDV-infected cells. In conclusion, BBH inhibited BVDV infection by inhibiting BVDV-induced autophagy in cells, and its inhibitory effect was obvious in the viral attachment and release stages. Therefore, our study provides a new idea for exploring novel anti-BVDV drugs.

Downregulation of the Long Noncoding RNA IALNCR Targeting MAPK8/JNK1 Promotes Apoptosis and Antagonizes Bovine Viral Diarrhea Virus Replication in Host Cells.

Bovine viral diarrhea virus (BVDV) is the causative agent of the bovine viral diarrhea-mucosal disease, which is a leading cause of economic losses in the cattle industry worldwide. To date, many underlying mechanisms involved in BVDV-host interactions remain unclear, especially the functions of long noncoding RNAs (lncRNAs). In our previous study, the lncRNA expression profiles of BVDV-infected Madin-Darby bovine kidney (MDBK) cells were obtained by RNA-seq, and a significantly downregulated lncRNA IALNCR targeting MAPK8/JNK1 (a key regulatory factor of apoptosis) was identified through the lncRNA-mRNA coexpression network analysis. In this study, the function of IALNCR in regulating apoptosis to affect BVDV replication was further explored. Our results showed that BVDV infection-induced downregulation of the lncRNA IALNCR in the host cells could suppress the expression of MAPK8/JNK1 at both the mRNA and protein levels, thereby indirectly promoting the activation of caspase-3, leading to cell-autonomous apoptosis to antagonize BVDV replication. This was further confirmed by the small interfering RNA (siRNA)-mediated knockdown of the lncRNA IALNCR. However, the overexpression of the lncRNA IALNCR inhibited apoptosis and promoted BVDV replication. In conclusion, our findings demonstrated that the lncRNA IALNCR plays an important role in regulating host antiviral innate immunity against BVDV infection. IMPORTANCE Bovine viral diarrhea-mucosal disease caused by BVDV is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BVDV-host interactions are complex. To date, most studies focused only on how BVDV escapes host innate immunity. By contrast, how the host cell regulates anti-BVDV innate immune responses is rarely reported. In this study, a significantly downregulated lncRNA, with a potential function of inhibiting apoptosis (inhibiting apoptosis long noncoding RNA, IALNCR), was obtained from the lncRNA expression profiles of BVDV-infected cells and was experimentally evaluated for its function in regulating apoptosis and affecting BVDV replication. We demonstrated that downregulation of BVDV infection-induced lncRNA IALNCR displayed antiviral function by positively regulating the MAPK8/JNK1 pathway to promote cell apoptosis. Our data provided evidence that host lncRNAs regulate the innate immune response to BVDV infection.

Forsythiaside A Improves the Inhibitory Efficiency of Recombinant Protein Vaccines against Bovine Viral Diarrhea Virus Infection.

Bovine viral diarrhea virus (BVDV) is a critical animal pathogen that leads to cattle production losses associated with acute disease, immune dysregulation, reproductive failure, and respiratory disease. Due to the monotonous control technique and neglect of BVDV, increasing prevalence of BVDV has caused significant economic losses in the cattle industry worldwide. Therefore, novel anti-BVDV drugs are essential to prevent and control BVDV. Our previous studies have found that Forsythoside A (FTA) could inhibit the replication of BVDV via TRAF2-dependent CD28-4-1BB signaling in bovine peripheral blood mononuclear cells (PBMCs), but whether they can directly inhibit the BVDV remains unclear. Here, we further investigated the effects of FTA on BVDV and its underlying mechanisms of action. We found that FTA significantly inhibited the replication of BVDV in the MDBK cell directly. The results demonstrated that FTA could reduce the functional activation of Caspase-1 to inhibit the inflammatory response caused by BVDV infection and increase the expression of type I interferon (IFN-I) to clear the virus in vitro. The animal experiment was performed to evaluate the antiviral effect of FTA in vivo. Notably, after challenged with BVDV, mice with FTA + Erns-E2 protein displayed alleviated pathological damage and decreased the viral load in the spleen compared with mice inoculated with Erns-E2 protein. Furthermore, treatment with FTA enhanced body defense and delayed infection by the BVDV. Our results reveal that FTA suppresses BVDV replication both in vitro and in vivo and therefore shows promise as an anti-BVDV agent.

Benzimidazole-2-Phenyl-Carboxamides as Dual-Target Inhibitors of BVDV Entry and Replication.

Bovine viral diarrhea virus (BVDV), also known as Pestivirus A, causes severe infection mostly in cattle, but also in pigs, sheep and goats, causing huge economical losses on agricultural farms every year. The infections are actually controlled by isolation of persistently infected animals and vaccination, but no antivirals are currently available to control the spread of BVDV on farms. BVDV binds the host cell using envelope protein E2, which has only recently been targeted in the research of a potent and efficient antiviral. In contrast, RdRp has been successfully inhibited by several classes of compounds in the last few decades. As a part of an enduring antiviral research agenda, we designed a new series of derivatives that emerged from an isosteric substitution of the main scaffold in previously reported anti-BVDV compounds. Here, the new compounds were characterized and tested, where several turned out to be potent and selectively active against BVDV. The mechanism of action was thoroughly studied using a time-of-drug-addition assay and the results were validated using docking simulations.

Antiviral Effect of Ginsenoside Rb2 and Rb3 Against Bovine Viral Diarrhea Virus and Classical Swine Fever Virus in vitro

Bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) are members of the genus Pestivirus that cause disease in wild and domestic animals and are responsible for extensive economic losses of livestock and biological industry. BVDV is also a significant laboratory contaminant. Currently, no effective antiviral therapeutics are available to control their infection. Ginsenosides, as major pharmacological ingredients in the plants of ginseng, have various biological activities. In the present work, the antiviral activity of 9 ginsenosides and 3 other saponins from Araliaceae plants was investigated against Pestivirus. Ginsenoside Rb2 and Rb3 showed low cytotoxicity and obvious antiviral effect. They were able to inhibit the replication and proliferation of BVDV and CSFV. In addition, our results suggest that the possible antiviral mechanism of Rb2 might be related to its ability to affect the translation of these viruses. Obtained results suggest that ginsenoside Rb2 and Rb3 have a potential for effective treatment against Pestivirus infection.

Gypenoside Inhibits Bovine Viral Diarrhea Virus Replication by Interfering with Viral Attachment and Internalization and Activating Apoptosis of Infected Cells.

Bovine viral diarrhea virus (BVDV) causes a severe threat to the cattle industry due to ineffective control measures. Gypenoside is the primary component of Gynostemma pentaphyllum, which has potential medicinal value and has been widely applied as a food additive and herbal supplement. However, little is known about the antiviral effects of gypenoside. The present study aimed to explore the antiviral activities of gypenoside against BVDV infection. The inhibitory activity of gypenoside against BVDV was assessed by using virus titration and performing Western blotting, quantitative reverse transcription PCR (RT-qPCR), and immunofluorescence assays in MDBK cells. We found that gypenoside exhibited high anti-BVDV activity by interfering with the viral attachment to and internalization in cells. The study showed that BVDV infection inhibits apoptosis of infected cells from escaping the innate defense of host cells. Our data further demonstrated that gypenoside inhibited BVDV infection by electively activating the apoptosis of BVDV-infected cells for execution, as evidenced by the regulation of the expression of the apoptosis-related protein, promotion of caspase-3 activation, and display of positive TUNEL staining; no toxicity was observed in non-infected cells. Collectively, the data identified that gypenoside exerts an anti-BVDV-infection role by inhibiting viral attachment and internalization and selectively purging virally infected cells. Therefore, our study will contribute to the development of a novel prophylactic and therapeutic strategy against BVDV infection.

Quinolinecarboxamides Inhibit the Replication of the Bovine Viral Diarrhea Virus by Targeting a Hot Spot for the Inhibition of Pestivirus Replication in the RNA-Dependent RNA Polymerase.

The bovine viral diarrhea virus (BVDV), a pestivirus from the family of Flaviviridae is ubiquitous and causes a range of clinical manifestations in livestock, mainly cattle. Two quinolinecarboxamide analogues were identified in a CPE-based screening effort, as selective inhibitors of the in vitro bovine viral diarrhea virus (BVDV) replication, i.e., TO505-6180/CSFCI (average EC50 = 0.07 µM, SD = 0.02 µM, CC50 > 100 µM) and TO502-2403/CSFCII (average EC50 = 0.2 µM, SD = 0.06 µM, CC50 > 100 µM). The initial antiviral activity observed for both hits against BVDV was corroborated by measuring the inhibitory effect on viral RNA synthesis and the production of infectious virus. Modification of the substituents on the quinolinecarboxamide scaffold resulted in analogues that proved about 7-fold more potent (average EC50 = 0.03 with a SD = 0.01 µM) and that were devoid of cellular toxicity, for the concentration range tested (SI = 3333). CSFCII resistant BVDV variants were selected and were found to carry the F224P mutation in the viral RNA-dependent RNA polymerase (RdRp), whereas CSFCI resistant BVDV carried two mutations in the same region of the RdRp, i.e., N264D and F224Y. Likewise, molecular modeling revealed that F224P/Y and N264D are located in a small cavity near the fingertip domain of the pestivirus polymerase. CSFC-resistant BVDV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110, LZ37, and BBP) that are known to target the same region of the RdRp. CSFC analogues did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). CSFC analogues likely interact with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110, LZ37, and BBP. This indicates that this region is a “hot spot” for the inhibition of pestivirus replication.

A novel intracellularly expressed NS5B-specific nanobody suppresses bovine viral diarrhea virus replication

Bovine viral diarrhea virus (BVDV) infection causes significant economic losses to the cattle industry worldwide and still represents a huge pressure on agricultural production. Thus, the development of novel anti-BVDV strategies are urgently needed. The nonstructural protein 5 (NS5B) of BVDV is essential for viral replication. Further, the camel single-domain antibody (nanobody) represents a promising antiviral approach with the advantages of small size, stable structure, high specificity and solubility, and the recognition of specific epitopes. However, no NS5B-specific nanobodies against BVDV have been reported. In this study, NS5B-specific nanobodies were isolated from a phage display library of variable domains of Camellidae heavy chain-only antibodies (VHHs). Further, an MDBK cell line stably expressing Nb1 was established to explore antiviral activity. Results showed that Nb1 could markedly suppress BVDV replication and interact with the BVDV NS5B protein. This suggests that nanobodies have potential for the development of novel antiviral drugs against BVDV infection.

Bovine viral diarrhea virus non-structural protein NS4B induces autophagosomes in bovine kidney cells.

Bovine viral diarrhea virus (BVDV) is an important pathogen in cattle that causes economic losses in livestock industries. Autophagy is an essential cell system for the maintenance of homeostasis and is induced by various triggers, including infection by viruses. BVDV infection leads to autophagy in order to enhance its replication in cells. In this study, we investigated the effect of BVDV non-structural proteins on the induction of autophagosomes. We found that NS4B alone could induce autophagosomes, suggesting a novel and important function of NS4B in BVDV replication.