DDIT3 Targets Innate Immunity via the DDIT3-OTUD1-MAVS Pathway To Promote Bovine Viral Diarrhea Virus Replication.

DNA damage-inducible transcript 3 (DDIT3) plays important roles in endoplasmic reticulum (ER) stress-induced apoptosis and autophagy, but its role in innate immunity is not clear. Here, we report that DDIT3 inhibits the antiviral immune response during bovine viral diarrhea virus (BVDV) infection by targeting mitochondrial antiviral signaling (MAVS) in Madin-Darby bovine kidney (MDBK) cells and in mice. BVDV infection induced high DDIT3 mRNA and protein expression. DDIT3 overexpression inhibited type I interferon (IFN-I) and IFN-stimulated gene production, thereby promoting BVDV replication, while DDIT3 knockdown promoted the antiviral innate immune response to suppress viral replication. DDIT3 promoted NF-κB-dependent ovarian tumor (OTU) deubiquitinase 1 (OTUD1) expression. Furthermore, OTUD1 induced upregulation of the E3 ubiquitin ligase Smurf1 by deubiquitinating Smurf1, and Smurf1 degraded MAVS in MDBK cells in a ubiquitination-dependent manner, ultimately inhibiting IFN-I production. Moreover, knocking out DDIT3 promoted the antiviral innate immune response to reduce BVDV replication and pathological changes in mice. These findings provide direct insights into the molecular mechanisms by which DDIT3 inhibits IFN-I production by regulating MAVS degradation.IMPORTANCE Extensive studies have demonstrated roles of DDIT3 in apoptosis and autophagy during viral infection. However, the role of DDIT3 in innate immunity remains largely unknown. Here, we show that DDIT3 is positively regulated in bovine viral diarrhea virus (BVDV)-infected Madin-Darby bovine kidney (MDBK) cells and could significantly enhance BVDV replication. Importantly, DDIT3 induced OTU deubiquitinase 1 (OTUD1) expression by activating the NF-κB signaling pathway, thus increasing intracellular Smurf1 protein levels to degrade MAVS and inhibit IFN-I production during BVDV infection. Together, these results indicate that DDIT3 plays critical roles in host innate immunity repression and viral infection facilitation.

Cell-to-Cell Transmission Is the Main Mechanism Supporting Bovine Viral Diarrhea Virus Spread in Cell Culture.

After initiation of an infective cycle, spread of virus infection can occur in two fundamentally different ways: (i) viral particles can be released into the external environment and diffuse through the extracellular space until they interact with a new host cell, and (ii) virions can remain associated with infected cells, promoting the direct passage between infected and uninfected cells that is referred to as direct cell-to-cell transmission. Although evidence of cell-associated transmission has accumulated for many different viruses, the ability of members of the genus Pestivirus to use this mode of transmission has not been reported. In the present study, we used a novel recombinant virus expressing the envelope glycoprotein E2 fused to mCherry fluorescent protein to monitor the spreading of bovine viral diarrhea virus (BVDV) (the type member of the pestiviruses) infection. To demonstrate direct cell-to-cell transmission of BVDV, we developed a cell coculture system that allowed us to prove direct transmission from infected to uninfected cells in the presence of neutralizing antibodies. This mode of transmission requires cell-cell contacts and clathrin-mediated receptor-dependent endocytosis. Notably, it overcomes antibody blocking of the BVDV receptor CD46, indicating that cell-to-cell transmission of the virus involves the engagement of coreceptors on the target cell.IMPORTANCE BVDV causes one of the most economically important viral infections for the cattle industry. The virus is able to cross the placenta and infect the fetus, leading to the birth of persistently infected animals, which are reservoirs for the spread of BVDV. The occurrence of persistent infection has hampered the efficacy of vaccination because it requires eliciting levels of protection close to sterilizing immunity to prevent fetal infections. While vaccination prevents disease, BVDV can be detected if animals with neutralizing antibodies are challenged with the virus. Virus cell-to-cell transmission allows the virus to overcome barriers to free virus dissemination, such as antibodies or epithelial barriers. Here we show that BVDV exploits cell-cell contacts to propagate infection in a process that is resistant to antibody neutralization. Our results provide new insights into the mechanisms underlying the pathogenesis of BVDV infection and can aid in the design of effective control strategies.

Variability of E2 protein-coding sequences of bovine viral diarrhea virus in Polish cattle.

Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus of the Flaviviridae family and has worldwide distribution, being one of the main causes of economic losses in cattle raising. The genome of pestiviruses is a single strand of positive-sense RNA with a length of 12.3 kb, which encodes one open reading frame flanked by untranslated regions. E2 glycoprotein is required for binding to cell-surface receptors and it also contains major antigenic determinants. The nucleotide sequence coding E2 is the most variable part of the viral genome. The heterogeneity that exists among circulating strains causes problems in the development of effective vaccines and reliable diagnostics. In this study, and for the first time analysis was made of the E2 glycoprotein coding sequences of 14 Polish BVDV-1 strains which belong to four subtypes: 1b (n = 7), 1f (n = 3), 1s (n = 3), and 1r (n = 1). These sequences showed evidence of strong purifying (negative) selection. However, we also identified positively selected sites. The availability of E2 sequences of Polish BVDV strains for reference, knowledge gained through epitope prediction attempts, and information on protein glycosylation sites can afford a better understanding of host-pathogen interactions.

Experimental infection of Korean native goats (Capra aegagrus hircus) with bovine viral diarrhea virus 1b.

BACKGROUND: Bovine viral diarrhea virus (BVDV) infects various ungulates and causes reproductive failure in infected goats. BVDV has been detected among goats in the Republic of Korea, but the route of transmission remains unclear. Here, we aimed to investigate whether BVDV-1b circulating among Korean cattle can be transmitted to Korean native goats (Capra aegagrus hircus) and characterize the outcomes of BVDV infection in these goats. RESULTS: Four goats were inoculated intranasally with the Korean noncytopathic (ncp) BVDV-1b strain. Two goats exhibited clinical signs of illness, including coughing and nasal discharge. Nasal swabs and blood were collected to screen for viral RNA and BVDV antibodies. Using the 5'-untranslated region (UTR), viral RNA was detected in the nasal swabs of two goats (Goat 1 and 3) on 12 day post-inoculation (dpi) and in the blood sample of one goat (Goat 1) on 7 and 19 dpi. Using the N-terminal protease (Npro) region, viral RNA was detected in the blood sample of Goat 1 on 7 and 12 dpi. Antibodies to BVDV were detected in Goats 1 and 3 on 16-21 dpi using enzyme-linked immunosorbent assay. Sequence analysis of the virus from nasal swabs and blood samples, which was detected via RT-PCR, using the 5'-UTR and Npro regions led to the identification of the strain as ncp BVDV-1b and revealed changes in the nucleotide sequence of these goats. CONCLUSIONS: Our results indicate that changes in the nucleotide sequence are associated with the establishment of BVDV infection in Korean native goats; these changes may be owing to a process required for the establishment of infection in a new host reservoir. Broadly, these findings highlight the importance of BVDV surveillance in ungulates other than cattle.

Evaluation of the serum virome in calves persistently infected with Pestivirus A, presenting or not presenting mucosal disease.

Bovine viral diarrhea virus 1, reclassified as Pestivirus A, causes an economically important cattle disease that is distributed worldwide. Pestivirus A may cause persistent infection in that calves excrete the virus throughout their lives, spreading the infection in the herd. Many persistently infected (PI) calves die in the first 2 years of life from mucosal disease (MD) or secondary infections, probably as a consequence of virus-induced immune depression. Here, high-throughput sequencing (HTS) was applied for evaluation of the total virome in sera of (i) PI calves displaying clinically apparent MD (n = 8); (ii) PI calves with no signs of MD (n = 8); and (iii) control, Pestivirus A-free calves (n = 8). All the groups were collected at the same time and from the same herd. Serum samples from calves in each of the groups were pooled, submitted to viral RNA/DNA enrichment, and sequenced by HTS. Viral genomes of Pestivirus A, Ungulate erythroparvovirus 1, bosavirus (BosV), and hypothetical circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses were identified. Specific real-time PCR assays were developed to determine the frequency of occurrence of such viruses in each of the groups. The absolute number of distinct viral genomes detected in both PI calf groups was higher than in the control group, as revealed by higher number of reads, contigs, and genomes, representing a wider range of taxons. Genomes representing members of the family Parvoviridae, such as U. erythroparvovirus 1 and BosV, were most frequently detected in all the three groups of calves. Only in MD-affected PI calves, we found two previously unreported Hypothetical single-stranded DNA genomes clustered along with CRESS-DNA viruses. These findings reveal that parvoviruses were the most frequently detected viral genomes in cattle serum; its frequency of detection bears no statistical correlation with the status of calves in relation to Pestivirus A infection, since clinically normal or MD-affected/non-affected PI calves were infected with similar U. erythroparvovirus 1 genome loads. Moreover, MD-affected PI calves were shown to support viremia of CRESS-DNA viral genomes; however, the meaning of such correlation remains to be established.

Characterization of thymus-associated lymphoid depletion in bovine calves acutely or persistently infected with bovine viral diarrhea virus 1, bovine viral diarrhea virus 2 or HoBi-like pestivirus.

Naïve pregnant cattle exposed to pestiviruses between 40-125 days of gestation can give birth to persistently infected (PI) calves. Clinical presentation and survivability, in PI cattle, is highly variable even with the same pestivirus strain whereas the clinical presentation in acute infections is more uniform with severity of symptoms being primarily a function of virulence of the infecting virus. The aim of this study was to compare thymic depletion, as measured by comparing the area of the thymic cortex to the medulla (corticomedullary ratio), in acute and persistent infections of the same pestivirus isolate. The same general trends were observed with each pestivirus isolate. Thymic depletion was observed in both acutely and persistently infected calves. The average thymic depletion observed in acutely infected calves was greater than that in age matched PI calves. PI calves, regardless of infecting virus, revealed a greater variability in amount of depletion compared to acutely infected calves. A trend was observed between survivability and depletion of the thymus, with PI calves surviving less than 5 weeks having lower corticomedullary ratios and greater depletion. This is the first study to compare PI and acutely infected calves with the same isolates as well as to evaluate PI calves based on survivability. Further, this study identified a quantifiable phenotype associated with potential survivability.

BVDV alters uterine prostaglandin production during pregnancy recognition in cows.

Embryonic mortality in cows is at least in part caused by failure of pregnancy recognition (PR). Evidence has shown that bovine viral diarrhoea virus (BVDV) infection can disrupt pregnancy. Prostaglandins (PG) play important roles in many reproductive processes, such as implantation. The aim of this study was to investigate the effect of BVDV infection on uterine PG production and PR using an in vitro PR model. Bovine uterine endometrial cells isolated from ten BVDV-free cows were cultured and treated with 0 or 100ng/mL interferon-τ (IFNT) in the absence or presence of non-cytopathic BVDV (ncpBVDV). PGF2α and PGE2 concentrations in the spent medium were measured using radioimmunoassays, and in the treated cells expression of the genes associated with PG production and signalling was quantified using qPCR. The results showed that the IFNT challenge significantly stimulated PTGS1 and PTGER3 mRNA expression and PGE2 production; however, these stimulatory effects were neutralised in the presence of ncpBVDV infection. ncpBVDV infection significantly increased PTGS1 and mPGES1 mRNA expression and decreased AKR1B1 expression, leading to increased PGE2 and decreased PGF2α concentrations and an increased PGE2:PGF2α ratio. The other tested genes, including PGR, ESR1, OXTR, PTGS2, PTGER2 and PTGFR, were not significantly altered by IFNT, ncpBVDV or their combination. Our study suggests that BVDV infection may impair PR by (1) inhibiting the effect of IFNT on uterine PG production and (2) inducing an endocrine switch of PG production from PGF2α to PGE2 to decrease uterine immunity, thereby predisposing the animals to uterine disease.

Experimental infection with non-cytopathic bovine viral diarrhea virus 1 in mice induces inflammatory cell infiltration in the spleen.

Previously, our study showed that oral inoculation of mice with cytopathic (cp) bovine viral diarrhea virus (BVDV) led to lymphocyte depletion and increased numbers of megakaryocytes in the spleen as well as thrombocytopenia and lymphopenia. In the present study, to investigate the possible differences in the detection of viral antigen, histopathological lesions, and hematologic changes between non-cytopathic (ncp) BVDV1 and cp BVDV1, mice were orally administered low and high doses of ncp BVDV1 and were necropsied at days 0, 2, 5, and 9 postinfection (pi). None of the ncp BVDV1-infected mice exhibited clinical signs of illness, unlike those infected with cp BVDV1. Statistically significant thrombocytopenia was observed during ncp BVDV1 infection, and lymphopenia was found only in mice infected with a high dose at day 9 pi. Interestingly, ncp BVDV1 infection increased the numbers of basophils, eosinophils, neutrophils, and monocytes in some infected mice. Viral antigen was detected in the lymphocytes of the spleen, mesenteric lymph nodes, Peyer's patches, and bone marrow by immunohistochemistry. Lymphoid depletion was evident in the mesenteric lymph nodes of mice infected with a high dose and also found in the Peyer's patches of some infected mice. Infiltration of inflammatory cells, including neutrophils and monocytes, and an increased number of megakaryocytes were seen in the spleen. These results suggest that the distribution of viral antigens is not associated with the presence of histopathological lesions. Inflammatory cell infiltration was observed in the spleens as a result of viral replication and may be attributable to the host reaction to ncp BVDV1 infection. Together, these findings support the possibility that mice can be used as an animal model for BVDV infection.

Experimental infection of mice with bovine viral diarrhea virus.

The objective of this study was to test the ability of bovine viral diarrhea virus (BVDV) to infect mice. Two mice each were either mock infected or inoculated with one of three BVDV strains by the intraperitoneal (IP) (n = 8) or intranasal (IN) (n = 8) route. All mice were euthanized at day 7 postinfection (p.i.). None of the infected mice exhibited any clinical signs of illness; however, the tissues harvested after BVDV challenge showed significant histopathological changes. Blood samples from five mice that were injected IP and one mouse that was inoculated IN were positive for BVDV by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemistry (IHC) was used to assess the presence of viral antigen in the organs of mice infected with three BVDV strains. In IP-injected mice, BVDV antigen was detected in the spleen (5/6), mesenteric lymph nodes (4/6), lymphatic tissue of the lung (3/6), lung (1/6), and stomach (1/6) of the infected mice; however, it was not detected in the liver (0/6) or kidney (0/6). In IN-inoculated mice, BVDV antigen was detected in the lung and mesenteric lymph nodes of one BVDV-infected mouse but was not detected in other tissues. The results of this study suggest that the spleen is the most reliable tissue for BVDV antigen detection using IHC in the IP-injected group. Our study demonstrates that mice can be infected by BVDV. This is the first report of BVDV infection in mice.

Role of thymic epithelial cells in lymphoid depletion after experimental infection with the noncytopathogenic BVDV1 strain 7443.

Thymic epithelial cells could play an important role in lymphoid depletion during bovine viral diarrhea virus (BVDV) infection. To evaluate this hypothesis, we examined proliferation of lymphocytes, expression of cytokeratins by thymic epithelial cells, and ultrastructural features at sequential time points after experimental infection of colostrum-deprived calves with the noncytopathogenic BVDV1 strain 7443. Ten clinically healthy Friesian calves were used. Eight were inoculated with the virus, and 2 were used as uninfected controls. Calves were sedated and euthanized in batches between 3 and 14 days postinoculation. At necropsy, thymus samples were collected for structural, immunohistochemical, and ultrastructural study. Thymic lymphoid depletion was accompanied by a decrease in lymphocyte proliferation and immunohistochemical and ultrastructural changes in thymic epithelial cells. Immunohistochemical and ultrastructural results reflect a disturbance of the thymic epithelial cell network, which may explain the decrease in lymphocyte proliferation by defective thymocyte-epithelial cell interactions.

A bovine viral diarrhea virus type 1a strain in China: isolation, identification, and experimental infection in calves.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is one of the most important pathogens in cattle. Previously, BVDV sub-genotypes of 1b, 1c, 1d, and 1 m were detected in China. However, isolation of BVDV type 1a from cattle has not been reported in China. In 2010, twenty nasal swabs and blood samples were collected from the cattle suspected BVDV infection in Henan province, China. A BVDV isolate was isolated using cell culture, and the pathogenesis of the virus isolate was studied. METHODS: Virus isolation was performed on MDBK cells. The virus identification was conducted by RT-PCR, neutralization test and immunofluorescence assay. In order to determine the genotype of the newly isolated virus, the 5' un-translated region (5'UTR) of the virus isolate was cloned, sequenced and phylogenetically analyzed. To evaluate the virulence of the virus isolate, four BVDV sero-negative calves were intranasally inoculated with the virus suspension. Rectal temperatures and clinical signs were recorded daily. Blood samples were analyzed for changes in white blood cell counts, and tissue samples were taken for histopathology analysis. RESULTS: A new isolate of bovine viral diarrhea virus (BVDV), named HN01, was isolated from the nasal swabs using MDBK cell culture. The HN01 strain caused cytopathic effect (CPE) in MDBK cell cultures after two passages. The virus specifically reacted to BVDV1-specific monoclonal antibody in an immunofluorescence assay. A fragment of 288 bp of genome from this isolate was amplified by the RT-PCR. Phylogenetic analysis of 5'UTR indicated that the virus was BVDV 1a. In the pathogenesis study, four calves experimentally infected with the BVDV strain developed depression, cough and other clinical signs. Calves showed high temperature over 40°C, and white blood cell counts dropped more than 40%. CONCLUSIONS: A new subgenotype 1a strain of BVDV was firstly isolated from dairy cattle in China. The experimental infection showed that the virus was moderate pathogenic to cattle and can be used as a BVDV challenge virus to evaluate the efficacy of BVDV vaccines in the target animals.

Identification of differentially expressed gene pathways between cytopathogenic and non-cytopathogenic BVDV-1 strains by analysis of the transcriptome of infected primary bovine cells.

The bovine viral diarrhea virus 1 (BVDV-1), belonging to the Pestivirus genus, is characterized by the presence of two biotypes, cytopathogenic (cp) or non-cytopathogenic (ncp). For a better understanding of the host pathogen interactions, we set out to identify transcriptomic signatures of bovine lung primary cells (BPCs) infected with a cp or a ncp strain. For this, we used both a targeted approach by reverse transcription droplet digital PCR and whole genome approach using RNAseq. Data analysis showed 3571 differentially expressed transcripts over time (Fold Change >2) and revealed that the most deregulated pathways for cp strain are signaling pathways involved in responses to viral infection such as inflammatory response or apoptosis pathways. Interestingly, our data analysis revealed a deregulation of Wnt signaling pathway, a pathway described in embryogenesis, that was specifically seen with the BVDV-1 cp but not the ncp suggesting a role of this pathway in viral replication.

Investigation of a dual fetal infection model with bovine viral diarrhoea viruses (BVDV)-1 and BVDV-2.

Two studies were performed in pregnant heifers to determine whether inoculation with two bovine viral diarrhoea viruses (BVDV), one BVDV-1 and one BVDV-2, inoculated separately into either nostril, results in fetal infection with both viruses. Dual transplacental infection of the fetus with BVDV-1 and BVDV-2 was observed in one case, but not consistently.

The identification of a B-cell epitope in bovine viral diarrhea virus (BVDV) core protein based on a mimotope obtained from a phage-displayed peptide library.

Bovine pestivirus A and B, previously known as bovine viral diarrhea virus (BVDV)-1 and 2, respectively, are important pathogens of cattle worldwide, which causes significant economic losses. B-cell epitopes in BVDV glycoprotein E2 and nonstructural protein NS2/3 have been extensively identified. In this study, we screened a 12-mer phage display peptide library using commercial goat anti-BVDV serum, and identified a mimotope "LTPHKHHKHLHA" referred to as P3. With sequence alignment, a putative B-cell epitope "77ESRKKLEKALLA88" termed as P3-BVDV1/2 residing in BVDV core protein was identified. The synthesized peptides of both P3 and P3-BVDV1/2 show strong reactivity with BVDV serum in immune blot assay. Immunization of mice with these individual peptides leads to the production of antibody that cannot neutralize virus infectivity. Thus for the first time we identified a B-cell epitope, "77ESRKKLEKALLA88", in BVDV core protein. Interestingly, the epitope was highly conserved in Pestivirus A, B, C, D, as well as emerging Pestivirus E and I, but highly variable in Pestiviruses H, G, F, and J, as well as unclassified Pestivirus originated from non-ruminant animals. Whether this putative B-cell epitope is implicated in pestivirus pathogenesis or evolution needs further investigations once large numbers of isolates are available in the future.

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.

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.

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 MS2. 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.

Apoptosis in lymphoid tissues of calves inoculated with non-cytopathic bovine viral diarrhea virus genotype 1: activation of effector caspase-3 and role of macrophages.

The mechanisms responsible for lymphocyte apoptosis in bovine viral diarrhoea have not yet been clarified. Previous work suggests that bovine viral diarrhea virus (BVDV) is only directly responsible for the destruction of a small number of lymphocytes. The aim of this study was to clarify, in vivo, the role of macrophages in lymphocyte destruction through indirect mechanisms linked to the biosynthetic activation of these immunocompetent cells on ileal Peyer's patches, as well as the distribution and quantification of apoptosis. Eight colostrum-deprived calves were inoculated intranasally with a non-cytopathic strain of BVDV genotype 1 and killed in batches of two at 3, 6, 9 and 14 days post-inoculation (p.i.). The progressive depletion of Peyer's patches was found to be due to massive lymphocyte apoptosis, with an increase in cleaved caspase-3 and TUNEL-positive cells. Lymphoid depletion was accompanied, from 3 days p.i., by a significant rise in macrophage numbers both in lymphoid follicles and in interfollicular areas. Some macrophages showed signs of viral infection, together with subcellular changes indicative of phagocyte activation and, in some cases, of secretory activity. However, the number of macrophages that showed positive immunostaining for tumour necrosis factor-alpha and interleukin-1alpha, cytokines with a proven ability to induce apoptosis, remained low throughout the experiment in lymphoid follicles, where most apoptotic cells were found. These results thus appear to rule out a major involvement of macrophages and macrophage-secreted chemical mediators in the apoptosis of follicular B lymphocytes during BVDV infection.

Experimental infection of white-tailed deer fawns (Odocoileus virginianus) with bovine viral diarrhea virus type-1 isolated from free-ranging white-tailed deer.

The objective of the current study was to elucidate the within-host dynamics of bovine viral diarrhea virus (BVDV) type-1 infection to better understand how this virus could be maintained in white-tailed deer (Odocoileus virginianus, WTD) populations. The BVDV type-1 used in this study was originally isolated from a free-ranging WTD in Indiana. Four fawns were intranasally inoculated with 2 ml BVDV type-1 strain 544 WTD at a 10(6) tissue culture infectious dose (TCID(50))/ml. Two fawns were inoculated with sham inoculum (negative controls). Animals were bled on days -7, 0, 1, 7, and 14 postinoculation (PID) for a complete blood count, chemistry panel, buffy coat (BC), real-time RT-PCR, and virus neutralization (VN). On days 7 and 14 PID, nasal and rectal swabs were obtained for RT-PCR and two of the virus-inoculated fawns and one of the negative controls fawns were euthanized. At necropsy, multiple samples were obtained for histopathology and in situ hybridization (ISH). Quantitative RT-PCR was performed on serum, BC, nasal, and rectal swabs. All animals tested negative for BVDV type 1 neutralizing antibodies on day 0 and animals in the control group remained seronegative throughout the study. No gross lesions were observed at necropsy. BVDV was isolated from lung and pooled lymph nodes from all BVDV-inoculated fawns on days 7 and 14 PID. Infected deer had lymphoid depletion, apoptosis, and lymphoid necrosis in the Peyer's patches and mesenteric lymph nodes. BVDV was detected in lymphoid tissues of infected animals by ISH. No lesions or virus were identified in control fawns. On day 7 PID, samples from two virus-inoculated fawns were positive for BVDV by virus isolation and RT-PCR from BC and nasal swab samples. One fawn was also positive on a rectal swab. Nasal and rectal swabs from all animals were negative on day 14. Results indicate that infection of WTD with BVDV is possible, and leads to histologic lesions in variety of tissues. In addition, virus shedding into the environment through feces and other secretions is likely.

Transplacental infection with non-cytopathic bovine viral diarrhoea virus types 1b and 2: viral spread and molecular neuropathology.

Infection with bovine viral diarrhoea virus (BVDV) represents a reproducible natural animal model in which to study mechanisms of transplacental viral infection. In the present study, BVDV-seronegative heifers were challenged intranasally with non-cytopathic BVDV of genotype 1b or 2. Fetuses were retrieved by caesarean section 7-114 days post-challenge of the dam and subjected to virological, histopathological and immunohistochemistry(IHC) studies. Gross and histopathological changes were only seen in fetuses infected at gestational age 75-85 days and retrieved at gestational age 190 days. Viral antigen could be detected in most tissues from 14 days post-infection, but the primary target organs for histopathological changes were brain, liver and spleen. In the brain, microscopical changes included leucomalacia and macrophage infiltration of meninges and neuropil. Viral antigen was detected in neurons, oligodendrocyte precursors and infiltrating macrophages. IHC revealed normal to slightly increased expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in the infected fetuses, with evidence of neuronal apoptosis and induction of inducible nitric oxide synthase (iNOS) and phospho-p38alpha mitogen-activated protein kinase (MAPK). These findings suggest that hypoxia may play only a limited role in the pathogenesis of the neural lesions. By contrast, virus-induced cytokine cascades, as part of the fetal innate immune response, and apoptosis of neurons and glial precursor cells may be central to the development of lesions.