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.     

Incorporation of3H-Uridine into bovine cells doubly infected with the virus of bovine viral diarrhea and a bovine enterovirus

Summary Tritiated uridine was selectively incorporated into the cytoplasm but not the nucleus of BEK cells infected with bovine enterovirus. This was accompanied by complete shutdown of cellular RNA synthesis. Bovine embryonic spleen cells infected with the BVD virus incorporated³H-uridine into the nucleus and cytoplasm, while uninfected cells showed only nuclear and nucleolar incorporation. Bovine embryonic spleen cells that were preinfected with BVD virus for 20 hours and then superinfected with bovine enterovirus showed complete absence of nuclear and nucleolar labelling in the early stages of superinfection with the bovine enterovirus, but nuclear labelling returned in reduced amounts 8 to 10 hours after superinfection and persisted throughout the remainder of the double infection ending in cell death.This research was supported by funds of Regional Research Project W-88 and W-112 through Colorado Agricultural Experiment Station Project 252 and 220 and is published as paper number 1630.     

Mucosal disease induced in cattle persistently infected with bovine viral diarrhea virus by antigenically different cytopathic virus

Summary.  Four cattle persistently infected with non-cytopathic (NCP) bovine viral diarrhea (BVD) virus were challenged with cytopathic (CP) BVD virus that was antigenically different from the persistent virus. Two of the animals were injected with dexamethasone (DM) and then challenged. They developed mucosal disease on days 21 and 33 post-challenge. CP-BVD viruses were isolated from their lymph nodes but not from the sera. The isolates were antigenically different from the persistent virus and the nucleotide sequence of a 787 base region in the E2 gene was markedly different. One of the isolates was indistinguishable from the challenge virus by virus neutralization tests and the nucleotide sequence showed high homology with that of the challenge CP-BVD virus. The other two cattle, challenged with the CP-BVD virus without DM treatment, developed mucosal disease at 30 and 264 days post-inoculation. CP-BVD virus was isolated from the sera as well as the lymph nodes of the cattle and was antigenically and genetically similar to the persistent virus and different from the challenge CP-BVD virus. The present results indicate that cattle persistently infected with NCP-BVD virus can develop mucosal disease induced by antigenically different CP-BVD viruses when their cellular immunity is suppressed, although they are not immunotolerant to the virus. Accepted November 1, 2000 Received July 25, 2000     

Alteration of leukocyte populations in calves concurrently infected with bovine respiratory syncytial virus and bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) infection altered leukocyte populations in calves that were reflected by depression of T, BoCD4+, and BoCD8+ lymphocytes in the thymus and depression of B lymphocytes in Peyer's patches (PP). The present study was based on mononuclear leukocyte preparations from eighteen 9- to 12-month-old crossbred calves that were each exposed to either bovine respiratory syncytial virus (BRSV), BVDV, or BRSV and BVDV concurrently, or served as mock-infected controls. Peripheral blood leukocytes were collected on postinfection days (PID) 0, 2, 4, 6, and 8, and cell populations from thymus, spleen, mesenteric lymph node, and PP were collected at necropsy on PID 9. The leukocytes were analyzed using flow cytometry for lymphocyte subpopulations expressing antigens specific for BoCD2, BoCD4, BoCD8, BoWC1, lambda light chain of bovine immunoglobulin, BoCD11b and major histocompatibility complex (MHC) class II. Concurrent BRSV and BVDV infections caused exaggerated alterations in leukocyte populations with a greater percentage of T-lymphocytes harvested from the PP. Alterations in the leukocyte populations in lymphatic tissues and in peripheral circulation due to BVDV infection may be an important mechanism for causation of clinically severe diseases of the respiratory and digestive tracts during concurrent BRSV and BVDV infections.     

Soluble antigen of bovine viral diarrhea virus

Summary Complement-fixing (CF) soluble antigen (SA) was detectable intra-cellularly prior to the appearance of infectious NADL-MD bovine viral diarrhea (BVD) virus during synthesis in roller flask cultures of bovine embryonic kidney cells. The release of infective virus into the extracellular fluid was concomitant with the release of SA.The SA was separated from the infective virus by sedimentation in a sucrose density gradient and by DEAE-cellulose chromatography.The SA was heat labile at 56° C, but stable in buffers at pH 5, 7, and 9 at 37° C. The SA was irreversibly precipitated in buffers at pH of 3 or below.Trypsin and -chymotrypsin completely inactivated the SA, whereas ribonuclease and deoxyribonuclease had no detectable effect on the CF activity.There was no apparent CF or neutralizing relationship between the SA and infectious NADL-MD BVD virus and arbovirus group B and lymphocytic choriomeningitis virus antisera.     

Depression of bovine monocyte chemotactic responses by bovine viral diarrhea virus.

Incubation of bovine peripheral blood monocytes with bovine viral diarrhea virus (either Singer or NY-1 strain) caused a consistent, statistically significant decrease in their random locomotion (no chemoattractant) and chemotaxis towards a chemotactic lymphokine. Chemotaxis was determined by a modification of the Boyden method. Incubation of bovine viral diarrhea virus with mononuclear cells depressed chemotaxis by a mean of 56% (P less than 0.0005). Heat-killed virus had no effect on monocyte motility. Data suggest that bovine viral diarrhea virus can rapidly suppress monocyte functions in vitro, but by unknown mechanisms, not by killing cells.     

Comparison of viral replication and IFN response in alpaca and bovine cells following bovine viral diarrhea virus infection

Alpacas develop diminished disease following bovine viral diarrhea virus (BVDV) infection compared to cattle. We hypothesized that alpaca and bovine cells have differential permissiveness and responses to BVDV infection. To characterize alpaca testicular (AT) and bovine turbinate (BT) cells BVDV infection permissiveness, viral replication and interferon (IFN) synthesis was evaluated. BVDV replicated 3-4 logs lower in AT cells with diminished antigen deposition compared to BT cells. BVDV infection inhibited IFN response in both AT and BT cells. Compared to BT cells, BVDV-infected AT cells had a 2-5 fold increase in IFN synthesis following dsRNA stimulation. The greater IFN response of AT cells compared to BT cells following poly I:C stimulation with or without ncp BVDV infection, may be the basis for the decreased BVDV permissiveness of AT cells and may contribute to the clinical differences following BVDV infection of alpacas and cattle.     

Immunobiologic heterotypic activity associated with viral and soluble components of bovine virus diarrhea virus

Summary The V and S antigens of bovine virus diarrhea (BVD) virus were studied by pig inoculation experiments to determine the basis for the bovine virus diarrheahog cholera heterotypic relationship. BVD virus infected tissue cultures were harvested and separated by ultracentrifugation and ultrafiltration. V antigen was prepared by Tween-ether-urea inactivation of virus. S antigen was quantitated in filtration samples and in density gradients by specific complement fixation. Pigs inoculated with crude, concentrated V antigen survived virulent hog cholera (HC) virus exposure. However, V antigen partially purified by ultracentrifugation failed to protect pigs. Neutralizing antibody to BVD, but not to HC, was synthesized in inoculated pigs following HC exposure. S antigen separated by 10 m, ultrafiltration and purified by potassium tartrate density gradient ultracentrifugation protected pigs from virulent HC. No BVD or HC antibody was detected at the time of HC virus exposure but HC antibody was produced at an accelerated rate following exposure. It thus appeared that an S antigen less than 10 m in size having a density between 1.05 and 1.10 g/ml which was produced during BVD virus replication in cells initiated the potential for a protective response.     

Analysis of codon usage in bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) is a widespread virus in beef and dairy herds. BVDV has been grouped into two genotypes, genotype 1 and genotype 2. In this study, the relative synonymous codon usage (RSCU) values, effective number of codon (ENC) values and nucleotide content were investigated, and a comparative analysis of codon usage patterns for open reading frames (ORFs) of 22 BVDV genomes, including 14 of genotype 1 and 8 of genotype 2, was carried out. A high A+U content and low codon bias were found in BVDV genomes. Depending on the RSCU data, it was found that there was a significant variation in bias of codon usage between the two genotypes, and a geographic factor exists only in genotype-1 of BVDV. The RSCU data have a negative correlation with general average hydrophobicity (GRAVY), aromaticity and nucleotide content. Furthermore, the overall abundance of C and U has no effect on the synonymous codon usage patterns. In contrast, the A and G content showed a significant correlation with the nucleotide content at the third position. In addition, the codon usage patterns of BVDV are similar to those of 22 conserved genes of Bos taurus. Taken together, the genetic characteristics of BVDV possibly result from interactions between natural seclection and mutation pressure.     

Comparison of low- and high-passage bovine turbinate cells for assay of bovine viral diarrhea virus

Summary The preparation and maintenance of a culture of bovine turbinate cells is described. The cell line is free of bovine viral diarrhea, parainfluenza-3, and infectious bovine rhinotracheitis viruses; and although the modal chromosome number changed significantly between the 9th passage and the 81st passage, no definite morphological changes were observed. Low- and high-passage bovine turbinate cells were equally susceptible to bovine viral diarrhea virus.Mention of commercial products does not constitute endorsement.     

Elimination of contaminating bovine viral diarrhea virus from bovine respiratory syncytial virus stock

During experiments to investigate the replication of certain bovine respiratory viruses, it was observed that Vero cells supported replication of bovine respiratory syncytial virus (BRSV) but not bovine viral diarrhea virus (BVDV). The selective replication characteristics of these viruses were used to free a BRSV stock from BVDV contamination.     

Roles of bovine viral diarrhea virus envelope glycoproteins in inducing autophagy in MDBK cells

Macroautophagy (autophagy) is an evolutionarily conserved control process that maintains cellular homeostasis in eukaryotic cells. Autophagy principally serves an adaptive role to degrade dysfunctional proteins and to clean damaged organelles in response to pathogenic, viral, or microbial infection, nutrient deprivation and endoplasmic reticulum (ER) stress. In previous study, we showed bovine viral diarrhea virus (BVDV) NADL infection induced autophagy and significantly elevated the expression levels of autophagy-related genes, Beclin1 and ATG14, at 12 h post-infection in MDBK cells. However, the specific mechanisms involved in controlling autophagic activity remain unclear. Here, we investigate the effects of BVDV NADL envelope glycoproteins overexpression on inducing autophagy. The results show that viral envelope glycoproteins E^rns and E2 overexpression mediated by lentivirus increase the formation of autophagosome, the percentage of GFP-LC3 puncta-positive cells and the expression levels of Beclin1 and ATG14. Whereas E1 overexpression doesn't affect autophagic activity. Collectively, these findings suggest that the viral envelope glycoproteins E^rns and E2 are involved in inducing autophagy, and provide a mechanistic insight into the regulation of autophagy in viral infected cells.     

Monoclonal antibodies with neutralizing activity segregate isolates of bovine viral diarrhea virus into groups

Summary Isolates of bovine viral diarrhea (BVD) virus were differentiated by monoclonal antibodies (MoAbs) reactive with the 56kD viral polypeptide. Patterns of neutralizing activity of the MoAbs indicate that multiple epitopes are involved in virus neutralization.