Cp Bovine Viral Diarrhea Virus Research Articles

The Involvement of Neutrophil in the Immune Dysfunction Associated with BVDV Infection.

Bovine viral diarrhea virus (BVDV) induces immune dysfunction that often results in a secondary bacterial infection in the infected animals. The underlying mechanism of BVDV-induced immune dysfunction is not well understood. The role of BVDV-infected macrophage-secreted factors was investigated. BVDV-infected monocyte-derived macrophage (MDM) supernatants down-regulated the expression of neutrophil L-selectin and CD18. Regardless of the biotype, phagocytic activity and oxidative burst were downregulated by BVDV-infected MDM supernatants. However, only supernatants from cytopathic (cp) BVDV down-regulated nitric oxide production and neutrophil extracellular traps (NET) induction. Our data suggested that BVDV-induced macrophage-secreted factors caused immune dysfunction in neutrophils. Unlike lymphocyte depletion, the negative impact on neutrophils seems to be specific to cp BVDV biotype. Interestingly the majority of modified live BVDV vaccines are based on cp strain of BVDV.

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.

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.

Isolation and Identification of Type 1d CP Bovine Viral Diarrhea Virus from Inner Mongolia, China

Isolation and Identification of Type 1d CP Bovine Viral Diarrhea Virus from Inner Mongolia, China

Bovine viral diarrhea virus compromises Neutrophil's functions in strain dependent manner

Bovine viral diarrhea virus (BVDV) infection is a major problem that results in economically important diseases of the cattle industry worldwide. The two major consequences of this disease are persistent infection and immune dysfunction. A number of studies have been done to determine the underline mechanisms of BVDV-induced immune dysfunction, in particular targeting antigen-presenting cells, T- and B- cells and cytokine gene expression. However, little research has focused Eon the effect of BVDV on neutrophils. Neutrophils are one of the predominant leukocytes circulating in blood and are considered the first line of defense in the innate immune system along with macrophages. Neutrophils not only eliminate the invading bacteria but also activate innate as well as adaptive immune responses. Therefore, compromised neutrophil function would affect both arms of immune system and caused immune suppression. In the current study, we used virus strains from both BVDV-1 and BVDV-2 species. Including a highly virulent non-cytopathic type 2a BVDV (ncp BVDV2a-1373), moderately virulent non-cytopathic type 2a (ncp BVDV2a 28508-5), and a pair of non-cytopathic type 1b BVDV (ncp BVDV1b TGAN) and cytopathic type 1b BVDV (cp BVDV1b TGAC) strain isolated from a case of mucosal disease. The highly virulent ncp BVDV2a-1373 significantly increased neutrophil apoptosis. However, none of the other BVDV strains affected neutrophil viability. All BVDV strains used significantly reduced CD18 and L-selectin expression on neutrophils as well as their oxidative burst and neutrophil extracellular traps (NET) activity. Cp BVDV significantly reduced neutrophil's phagocytic activity but ncp BVDV did not have any effect on it. On the other hand, ncp BVDV significantly increased neutrophil's CD14 expression and chemotactic activity while cp BVDV did not show any effect either on neutrophil's CD14 expression or on chemotactic activity. In conclusion, BVDV affected neutrophils variability and functional activity in strain dependent manner. Results of the current study will further help in understanding the pathophysiology of different BVDV strains.

Comparative humoral immune response against cytopathic or non-cytopathic bovine viral diarrhea virus infection

Bovine viral diarrhea virus (BVDV) infection causes immune dysfunction. The current study investigated the effect of cytopathic (cp) or noncytopathic (ncp) strains of BVDV on immunomodulation by the levels of total serum immunoglobulin G (IgG), the IgG1, IgG2, BVDV neutralizing antibodies and total white blood cell (WBC) count. Twenty (20) BVDV seronegative dairy calves (5–6 months old) were divided in two groups of ten. The animals were infected with either a cp or ncp virus isolated from the same animal (ncp BVDV1b-TGAN or cp BVDV1b-TGAC). One group of 10 was infected with ncp TGAN while the other group of 10 was infected with cp TGAC. Calves infected with cp BVDV had a significant decrease in total IgG as well as IgG1 concentration at 7 days post infection (DPI) that recovered by 21 DPI (total IgG) and 35 DPI (IgG1), respectively. There was no effect of ncp BVDV infection on total IgG concentration in the first 7 days of infection (DOI); however, IgG1 concentration was significantly reduced and IgG2 concentration was significantly increased at 7 DOI. At 35 DPI, ncp TGAN-infected calves had significantly higher total IgG, IgG1 as well as IgG2 compared to cp TGAC-infected calves. Ncp BVDV induced higher BVDV homologous and heterologous neutralizing antibodies compared to the cp BVDV strain. Calves infected with ncp BVDV had significantly reduced WBC counts at 7 DPI that recovered by 14 DPI. Overall, these findings indicate that humoral immunosuppression occurs early following BVDV infection with the largest effect on IgG1 levels.

CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses.

Pestiviruses like bovine viral diarrhea virus (BVDV) are a threat to livestock. For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections, which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2, the activity of which depends on its cellular cofactor, DNAJC14. Since this chaperone is available in small amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cp BVDV strains arising mostly by RNA recombination show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses has been established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14 dependent. By generating bovine and porcine DNAJC14 knockout cells, we could show that (i) replication of 6 distinct noncp pestivirus species (A to D, F, and G) depends on DNAJC14, (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14, and (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as a pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses.IMPORTANCE Only noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.

The effect of human interferon alpha on replication of different bovine viral diarrhea virus strains.

Bovine viral diarrhea virus (BVDV) exists in two main biotypes: cytopathic (cp) and noncytopathic (ncp). Although some studies were done on the effect of interferon alpha (IFN-α) on BVDV, the effect of exogenous IFN against BVDV biotypes remains unclear. In the present study, we evaluated the comparative effect of exogenous human IFN-α (HuIFN-α) on different BVDV biotypes and genotypes. The results showed that exogenous HuIFN-α greatly inhibited the growth of different BVDV biotypes and genotypes. However, HuINF-α has a significant inhibitory effect on cp biotype compared to ncp one without significant variation between different genotypes. The effect of HuIFN-α on BVDV reached the maximum level at early stages of infection (0-20 h post infection) and increased in a dose-dependent manner (10-500 U/ml). Quantitative real-time RT-PCR was used to evaluate the effect of exogenous HuIFN-α on RNA synthesis of both BVDV biotypes. HuIFN-α reduced RNA production of cp by 4 logs compared to only 2 logs for ncp strains. Additionally, the antiviral effect of IFN-α against both BVDV biotypes seems to be independent of the RNA-dependent protein kinase (PKR) activation as assayed by direct analysis of in vivo phosphorylation of eIF2-α and by 2-aminopurine (2-AP) treatment. Collectively, these results indicated that the exogenous HuIFN-α treatment has an inhibitory effect not only on cp BVDV biotype but also on the ncp BVDV. The antiviral effect of exogenous HuIFN-α was biotype, time, dose but not genotype dependent. PKR has no role in the inhibitory effect suggesting that other IFN-antiviral pathways were involved. Keywords: BVDV biotypes; HuIFN-α; RNA synthesis; PKR-independent.

All is Not Butter that Comes from the Cow: The Bovine Viral Diarrhea. Understanding the Pathogenesis of Cytopathic and Non-Cytopathic Infection

Bovine viral diarrhea infections are seen in all ages and breeds of cattle worldwide and have significant economic impact due to the productive and reproductive losses. The infectious agents that cause this disease, Bovine viral diarrhea viruses (BVDVs) may occur as two biotypes, of which one is non-cytopathic (ncp) and the other cytopathic (cp), classified according to whether or not they produce visible changes in cell culture. Cytopathic biotypes of BVDV can be created through internal deletion or recombination of RNA of ncp biotypes. The mechanisms of this change are not sufficiently understood. In this review, we discuss the work that has been done to date in our laboratory using methods of proteomics, cellular and molecular biology, and immunology. We found that both biotypes cause numerous changes in the expression levels of the proteins in monocytes, including proteins related to professional antigen presentation, enzymes and receptors, and changes in the infected cell functions related to antigen uptake. However, the alterations caused by the cp and not by the ncp biotypes are consistent with the hypothesis that the virus cytotoxicity involves the mitochondrial dysfunction. Overall, our data show that, the three important signals, antigen-specific, co-stimulatory and cytokine, required to promote the effector activation of naive T cells to be delivered by professional antigen presenting cells (APCs) are impaired by both types of BVDV. In addition, cp and ncp BVDVs differentially target mitochondrial proteins and antioxidant enzymes that control the fate of infected cells and determine whether BVDVs produce cytopathic effects or replicate noncytopathically to establish persistent infection. This research is aimed to discover foundational knowledge related to host-pathogen interactions and facilitate the development of innovative disease preventatives for pathogens causing significant animal losses.

Characterization of the cytopathic BVDV strains isolated from 13 mucosal disease cases arising in a cattle herd

Bovine viral diarrhea virus (BVDV) is a positive single stranded RNA virus belonging to the Pestivirus genus of the Flaviviridae family. BVDV has a wide host range that includes most ruminants. Noncytopathic (ncp) BVDV may establish lifelong persistent infections in calves following infection of the fetus between 40 and 120 days of gestation. Cytopathic (cp) BVDV strains arise from ncp strains via mutations. The most common cp mutations are insertions of RNA derived from either host or a duplication of viral sequences into the region of the genome coding for the NS2/3 protein. Superinfection of a persistently infected animal with a cp virus can give rise to mucosal disease, a condition that is invariably fatal. A herd of 136 bred 3-year old cows was studied. These cows gave birth to 41 PI animals of which 23 succumbed to mucosal disease. In this study, we characterized the ncp and cp viruses isolated from 13 of these animals. All viruses belonged to the BVDV type 2a genotype and were highly similar. All the cp viruses contained an insertion in the NS2/3 coding region consisting of the sequences derived from the transcript encoding a DnaJ protein named Jiv90. Comparison of the inserted DnaJ regions along with the flanking viral sequences in the insertion 3′ end of the 13 cp isolates revealed sequence identities ranging from 96% to 99% with common borders. This suggested that one animal likely developed a cp virus that then progressively spread to the other 12 animals. Interestingly, when the inserted mammalian gene replicated within viral genome, it showed conservation of the same conserved motifs between the different species, which may indicate a role for these motifs in the insertion function within the virus genome. This is the first characterization of multiple cp bovine viral diarrhea virus isolates that spread in a herd under natural conditions.

Characterization of the 13 cytopathic BVDV strains from mucosal disease cases from a single herd

Bovine viral diarrhea virus (BVDV) is a positive single-stranded RNA virus belonging to the Pestivirus genus of the Flaviviridae family. BVDV has a wide host range that includes most ruminants. Noncytopathic (ncp) BVDV may establish lifelong persistent infections in calves following infection of the fetus between 40 and 120 days of gestation. Cytopathic (cp) BVDV strains arise from ncp strains via mutations. The most common cp mutations are insertions of RNA derived from either host or a duplication of viral sequences into the region of the genome coding for the NS2/3 protein. Superinfection of a persistently infected animal with a cp virus can give rise to mucosal disease (MD), a condition that is invariably fatal.

The effect of bovine viral diarrhea virus (BVDV) strains on bovine monocyte-derived dendritic cells (Mo-DC) phenotype and capacity to produce BVDV

BackgroundDendritic cells (DC) are important antigen presentation cells that monitor, process, and present antigen to T cells. Viruses that infect DC can have a devastating impact on the immune system. In this study, the ability of bovine viral diarrhea virus (BVDV) to replicate and produce infectious virus in monocyte-derived dendritic cells (Mo-DC) and monocytes was studied. The study also examined the effect of BVDV infection on Mo-DC expression of cell surface markers, including MHCI, MHCII, and CD86, which are critical for DC function in immune response.MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from bovine blood through gradient centrifugation. The adherent monocytes were isolated from PBMCs and differentiated into Mo-DC using bovine recombinant interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GMCSF). To determine the effect of BVDV on Mo-DC, four strains of BVDV were used including the severe acute non-cytopathic (ncp) BVDV2a-1373; moderate acute ncp BVDV2a 28508-5; and a homologous virus pair [i.e., cytopathic (cp) BVDV1b TGAC and ncp BVDV1b TGAN]. The Cooper strain of bovine herpesvirus 1 (BHV1) was used as the control virus. Mo-DC were infected with one of the BVDV strains or BHV-1 and were subsequently examined for virus replication, virus production, and the effect on MHCI, MHCII, and CD86 expression.ResultsThe ability of monocytes to produce infectious virus reduced as monocytes differentiated to Mo-DC, and was completely lost at 120 hours of maturation. Interestingly, viral RNA increased throughout the course of infection in Mo-DC, and the viral non-structural (NS5A) and envelope (E2) proteins were expressed. The ncp strains of BVDV down-regulated while cp strain up-regulated the expression of the MHCI, MHCII, and CD86 on Mo-DC.ConclusionsThe study revealed that the ability of Mo-DC to produce infectious virus was reduced with its differentiation from monocytes to Mo-DC. The inability to produce infectious virus may be due to a hindrance of virus packaging or release mechanisms. Additionally, the study demonstrated that ncp BVDV down-regulated and cp BVDV up-regulated the expression of Mo-DC cell surface markers MHCI, MHCII, and CD86, which are important in the mounting of immune responses.

Intérêt de la stéréolithographie en reconstruction d’oreille

Bovine viral diarrhea virus (BVDV) causes immunosuppression of the adaptive immune response. The level of suppression of the adaptive immune response is strain dependent. The early events of antigen presentation require activation of toll-like receptors that results in the release of pro-inflammatory cytokines. Non-cytopathic (ncp) BVDV infection stimulates cytokines from macrophages in vitro but the effect of BVDV infection in vivo on macrophages or in vitro with monocytes is not clear. Antigen presentation is decreased and co-stimulatory molecules are down regulated. T-lymphocytes numbers are reduced following BVDV infection in a strain dependent manner. There is recruitment of lymphocytes to the bronchial alveolar space following cytopathic (cp) BVDV infection. Depletion of T-lymphocytes occurs in the lymphoid tissue and is strain dependent. BVDV cp T-lymphocyte responses appear to be primarily a T helper 1 response while the response following ncp BVDV induces a T helper 2 response. Cytotoxic T-lymphocytes (CTL), an important BVDV defense mechanism are compromised. The major neutralizing antigens are well characterized but cross-protection between strains is variable. PI animals have normal adaptive immune responses with the exception of the PI strain immunotolerance and mucosal disease may be a function of the level of gamma delta T cells.

The impact of BVDV infection on adaptive immunity

Bovine viral diarrhea virus (BVDV) causes immunosuppression of the adaptive immune response. The level of suppression of the adaptive immune response is strain dependent. The early events of antigen presentation require activation of toll-like receptors that results in the release of pro-inflammatory cytokines. Non-cytopathic (ncp) BVDV infection stimulates cytokines from macrophages in vitro but the effect of BVDV infection in vivo on macrophages or in vitro with monocytes is not clear. Antigen presentation is decreased and co-stimulatory molecules are down regulated. T-lymphocytes numbers are reduced following BVDV infection in a strain dependent manner. There is recruitment of lymphocytes to the bronchial alveolar space following cytopathic (cp) BVDV infection. Depletion of T-lymphocytes occurs in the lymphoid tissue and is strain dependent. BVDV cp T-lymphocyte responses appear to be primarily a T helper 1 response while the response following ncp BVDV induces a T helper 2 response. Cytotoxic T-lymphocytes (CTL), an important BVDV defense mechanism are compromised. The major neutralizing antigens are well characterized but cross-protection between strains is variable. PI animals have normal adaptive immune responses with the exception of the PI strain immunotolerance and mucosal disease may be a function of the level of gamma delta T cells.

Analysis of Bovine Viral Diarrhea Viruses-infected monocytes: identification of cytopathic and non-cytopathic biotype differences

BackgroundBovine Viral Diarrhea Virus (BVDV) infection is widespread in cattle worldwide, causing important economic losses. Pathogenesis of the disease caused by BVDV is complex, as each BVDV strain has two biotypes: non-cytopathic (ncp) and cytopathic (cp). BVDV can cause a persistent latent infection and immune suppression if animals are infected with an ncp biotype during early gestation, followed by a subsequent infection of the cp biotype. The molecular mechanisms that underscore the complex disease etiology leading to immune suppression in cattle caused by BVDV are not well understood.ResultsUsing proteomics, we evaluated the effect of cp and ncp BVDV infection of bovine monocytes to determine their role in viral immune suppression and uncontrolled inflammation. Proteins were isolated by differential detergent fractionation and identified by 2D-LC ESI MS/MS. We identified 137 and 228 significantly altered bovine proteins due to ncp and cp BVDV infection, respectively. Functional analysis of these proteins using the Gene Ontology (GO) showed multiple under- and over- represented GO functions in molecular function, biological process and cellular component between the two BVDV biotypes. Analysis of the top immunological pathways affected by BVDV infection revealed that pathways representing macropinocytosis signalling, virus entry via endocytic pathway, integrin signalling and primary immunodeficiency signalling were identified only in ncp BVDV-infected monocytes. In contrast, pathways like actin cytoskeleton signalling, RhoA signalling, clathrin-mediated endocytosis signalling and interferon signalling were identified only in cp BDVD-infected cells. Of the six common pathways involved in cp and ncp BVDV infection, acute phase response signalling was the most significant for both BVDV biotypes. Although, most shared altered host proteins between both BVDV biotypes showed the same type of change, integrin alpha 2b (ITGA2B) and integrin beta 3 (ITGB3) were down- regulated by ncp BVDV and up- regulated by cp BVDV infection.ConclusionsThis study shows that, as we expected, there are significant functional differences in the host proteins that respond to cp or ncp BVDV infection. The combined use of GO and systems biology network modelling facilitated a better understanding of host-pathogen interactions.

A single point mutation in nonstructural protein NS2 of bovine viral diarrhea virus results in temperature-sensitive attenuation of viral cytopathogenicity.

For Bovine viral diarrhea virus (BVDV), the type species of the genus Pestivirus in the family Flaviviridae, cytopathogenic (cp) and noncytopathogenic (ncp) viruses are distinguished according to their effect on cultured cells. It has been established that cytopathogenicity of BVDV correlates with efficient production of viral nonstructural protein NS3 and with enhanced viral RNA synthesis. Here, we describe generation and characterization of a temperature-sensitive (ts) mutant of cp BVDV strain CP7, termed TS2.7. Infection of bovine cells with TS2.7 and the parent CP7 at 33 degrees C resulted in efficient viral replication and a cytopathic effect. In contrast, the ability of TS2.7 to cause cytopathogenicity at 39.5 degrees C was drastically reduced despite production of high titers of infectious virus. Further experiments, including nucleotide sequencing of the TS2.7 genome and reverse genetics, showed that a Y1338H substitution at residue 193 of NS2 resulted in the temperature-dependent attenuation of cytopathogenicity despite high levels of infectious virus production. Interestingly, TS2.7 and the reconstructed mutant CP7-Y1338H produced NS3 in addition to NS2-3 throughout infection. Compared to the parent CP7, NS2-3 processing was slightly decreased at both temperatures. Quantification of viral RNAs that were accumulated at 10 h postinfection demonstrated that attenuation of the cytopathogenicity of the ts mutants at 39.5 degrees C correlated with reduced amounts of viral RNA, while the efficiency of viral RNA synthesis at 33 degrees C was not affected. Taken together, the results of this study show that a mutation in BVDV NS2 attenuates viral RNA replication and suppresses viral cytopathogenicity at high temperature without altering NS3 expression and infectious virus production in a temperature-dependent manner.

Activation of extracellular signal-regulated kinase in MDBK cells infected with bovine viral diarrhea virus

Our efforts to identify the cellular signaling cascades triggered by bovine viral diarrhea virus (BVDV) infection in MDBK cells revealed marked activation of extracellular signal-regulated kinase 1/2 (ERK). Enhanced phosphorylation of ERK was detected following infection with cytopathogenic (cp) BVDV, but not with noncytopathogenic BVDV. It appears that cp BVDV-induced ERK phosphorylation is caused by oxidative stress, because ERK phosphorylation was inducible by treatment with hydrogen peroxide or serum deprivation and was attenuated by addition of antioxidants. These results indicate that BVDV infection influences the ERK signaling pathway via oxidative stress, depending on the biotype.

Microarray analysis reveals distinct signaling pathways transcriptionally activated by infection with bovine viral diarrhea virus in different cell types

Infection with bovine viral diarrhea virus (BVDV) causes different effects depending on its biotype in vitro; cytopathogenic (cp) strains induce apoptosis, type I interferon (IFN), and various stress-mediated responses, whereas non-cytopathogenic (ncp) strains do not. However, comprehensive transcriptional profiles of the cells infected with BVDV are still unknown. Here we performed microarray analysis of BVDV-infected MDBK epithelial cells and bovine fetal muscle (BFM) fibroblast cells. Infection of both cell types with cp BVDV, but not ncp BVDV, stimulated marked up-regulation of numerous genes belonging to diverse functional classes. However, the pattern of gene expression detected in both cell types was highly distinct from each other. Notably, upon cp BVDV infection, BFM cells exhibited marked induction of IFN-stimulated genes (ISGs), whereas MDBK cells characteristically up-regulated endoplasmic reticulum stress-inducible genes, such as tribbles homolog 3 (TRB3), CHOP and asparagine synthase, and showed much weaker induction of ISGs than BFM cells. This study highlights unexpected diversity in the response of different cell types to BVDV infection.

Bovine Viral Diarrhea Virus infection affects the expression of proteins related to professional antigen presentation in bovine monocytes

The complete annotation of the cattle genome allows reliable protein identification by tandem mass spectrometry (MS 2) and greatly facilitates proteomics. Previously, we reported that differential detergent fractionation (DDF) analysis of bovine monocytes reveals proteins related to antigen pattern recognition, uptake and presentation to immunocompetent lymphocytes. Here we have identified 47 bovine proteins, involved in immune function of professional antigen-presenting cells (APC) that have been significantly altered after cytopathic (cp) Bovine Viral Diarrhea Virus (BVDV) infection. In particular, proteins related to immune responses such as cell adhesion, apoptosis, antigen uptake, processing and presentation, acute phase response proteins, MHC class I- and II-related proteins and other molecules involved in immune function of professional antigen presentation have been significantly altered after BVDV infection. Our data suggest that cp BVDV, while promoting monocyte activation and differentiation, is inhibiting their antigen presentation to immunocompetent T cells, thus resulting in the uncontrolled inflammation mediated by activated macrophages, enhanced viral spread, and impaired anti-viral defense mechanisms in the host.

Bovine viral diarrhea viruses differentially alter the expression of the protein kinases and related proteins affecting the development of infection and anti-viral mechanisms in bovine monocytes

Using a proteomics approach, we evaluated the effect of cytopathic (cp), and non-cytopathic (ncp) bovine viral diarrhea viruses (BVDV) on the expression of protein kinases and related proteins in bovine monocytes. Proteins were isolated from membrane and cytosolic fractions with the differential detergent fractionation (DDF) method and identified with 2D-LC ESI MS 2. Of approximately 10,000 proteins identified, 378 proteins had homology with known protein kinases or related proteins. Eighteen proteins involved in cell differentiation and activation, migration, anti-viral mechanisms (interferon/apoptosis), biosynthesis, sugar metabolism and oncogenic transformation were significantly altered in BVDV-infected monocytes compared to the uninfected controls. Six proteins, mostly related to cell migration, anti-viral mechanisms, sugar metabolism and possibly tumor resistance were differentially expressed between the ncp and cp BVDV-infected monocytes. Particularly, the expression of the receptor of activated C kinase (RACK), of pyridoxal kinase (PK), diacyglycerol kinase (DGK) and Brutons tyrosine kinase (BTK) was decreased in monocytes infected with cp BVDV compared to ncp BVDV, possibly contributing to the cytopathic effect of the virus. This and other findings are discussed in view of the possible role the identified proteins play in the development of viral infection and oncogenic transformation of cells.