Noncytopathic Bovine Viral Diarrhea Virus Research Articles

Non-cytopathic bovine viral diarrhea virus (BVDV) inhibits innate immune responses via induction of mitophagy

Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus within the family Flaviviridae. Mitophagy plays important roles in virus-host interactions. Here, we provide evidence that non-cytopathic (NCP) BVDV shifts the balance of mitochondrial dynamics toward fission and induces mitophagy to inhibit innate immune responses. Mechanistically, NCP BVDV triggers the translocation of dynamin-related protein (Drp1) to mitochondria and stimulates its phosphorylation at Ser616, leading to mitochondrial fission. In parallel, NCP BVDV-induced complete mitophagy via Parkin-dependent pathway contributes to eliminating damaged mitochondria to inhibit MAVS- and mtDNA-cGAS-mediated innate immunity responses, mtROS-mediated inflammatory responses and apoptosis initiation. Importantly, we demonstrate that the LIR motif of ERNS is essential for mitophagy induction. In conclusion, this study is the first to show that NCP BVDV-induced mitophagy plays a central role in promoting cell survival and inhibiting innate immune responses in vitro.

Testing and identification of bovine viral diarrhea virus isolates recovered in Russia between 2019 and 2022

Studying the agents of bovine viral diarrhea (BVD) is an important task given the high probability of new isolate introduction into the Russian Federation, as well as the need to take into account the genotype and subgenotype of the virus circulating in a herd when developing vaccines and diagnostic kits for the infection. During the work performed, 6 BVD virus isolates were recovered and identified. The recovery of these isolates in the lamb testicle cell subculture revealed that Bashkiria/2019, Kirov/2020 and Samara/2020 isolates belong to non-cytopathic bovine viral diarrhea virus biotypes, Chelyabinsk/2021 isolate demonstrated the characteristic cytopathic effect in the monolayer and was classified as a cytopathic variant of thevirus, the adaptation of Belgorod/2021 and Udmurtiya/2020 isolates to this cell system was not possible. The study also identified the species of the recovered isolates. Based on the analysis of the nucleotide sequence of genome 5’-untranslated region (5’-UTR) fragment, these isolates were classified as belonging to three genotypes of the virus. The phylogenetic analysis showed that Chelyabinsk/2021 and Udmurtiya/2020 isolates belong togenotype2 and demonstrate, respectively, 98% and99% homology with reference 890 strain of BVD virus. The recovered Bashkiria/2019, Samara/2020, Kirov/2020 isolates were classified as belonging to subtypes 1i, 1f and1b ofgenotype1, and Belgorod/2021 solate represents genotype 3 of the virus. The findings from the study confirm the presence of all three genotypes of bovine viral diarrhea virus in the Russian Federation and reiterate the need for the development of specific prevention and diagnosis tools for the disease.

Sperm and oocyte as carriers for bovine viral diarrhoea virus biotypes during in vitro fertilization.

Bovine viral diarrhoea virus (BVDV) is an important viral agent causing the reproductive failure in cattle. The objectives of the study were to assess the role of male and female gametes, as carriers of cytopathic (CP) and non-cytopathic (NCP) BVDV to embryonic cells during in vitro fertilization. In this respect, sperm and oocytes were separately exposed to concentrations of 104.5 or 105.5 TCID50 /mL CP and NCP BVDV, for 2 h before fertilization. After washing, the intact gametes with the infected gametes were inseminated. Seven days post-fertilization, the virus-exposed embryos were examined for presence of the viral genome by RT-PCR. One-way anova with post-hoc Tukey's HSD test and an independent samples t-test were used to compare within and between groups, respectively. The results presented a significant decrease in the blastocyst rates for CP-infected groups than NCP-infected groups (p ≤ .01). Compared to the controls and the infected oocyte groups, the cleavage rates of the infected sperm groups (NCP and CP BVDV) were significantly reduced both in low (104.5 TCID50 /mL) and high (105.5 TCID50 /mL) titres of the virus (p ≤ .01). The proportion of embryos which was developed to blastocyst stages was significantly lower for CP and NCP-infected groups than the control groups (p ≤ .001). According to the molecular results, all samples of the retarded/degenerated embryos (at least one blastocyst within each one) in CP and NCP groups, one sample (at least one blastocyst in that) within a CP-infected group, and six samples (at least one blastocyst in each one of those) of NCP-infected groups contained the viral nucleic acid. Likewise, the results of viral enrichment showed all reactions in which RT-PCR were positive induced CPEs in MDBK monolayers. In conclusion, it is clear that CP and NCP BVDV were able to traverse zona pellucida during fertilization, and they had also negative effects on embryo development.

NLRP3 Inflammasome Involved with Viral Replication in Cytopathic NADL BVDV Infection and IFI16 Inflammasome Connected with IL-1β Release in Non-Cytopathic NY-1 BVDV Infection in Bovine Macrophages.

Inflammasomes are multiprotein complexes that play a role in the processing of proinflammatory cytokines such as interleukin 1 beta (IL-1β). The secretion of IL-1β in bovine macrophages infected with the bovine viral diarrhea virus (BVDV) cytopathic strain NADL (NADLcp-BVDV) is caspase 1-dependent. In the present study, we found that in macrophages infected with NADL, the NLRP3 inflammasome participated in the maturation of IL-1β as the level decreased from 4629.3 pg/mL to 897.0 pg/mL after treatment with cytokine release inhibitory drug 3 (CRID3). Furthermore, NLRP3 activation has implications regarding viral replication, as there was a decrease in the viral titer until 1 log of a supernatant of macrophages that were inhibited with CRID3 remained. In the case of the non-cytopathic BVDV strain NY-1 (NY-1 ncpBVDV), IL-1β secretion is not affected by NLRP3, but could be related to the IFI16 inflammasome; we found a colocalization of IFI16 with ASC using confocal microscopy in infected macrophages with the NY-1 ncp-BVDV biotype. To relate IFI16 activation to IL-1β release, we used ODN TTAGGG (A151), a competitive inhibitor of IFI16; the results show a decrease in its level from 248 pg/mL to 128.3 pg/mL. Additionally, we evaluated the caspase 1 activation downstream of IFI16 and found a decrease in the IL-1β from 252.9 pg/mL to 63.5 pg/mL when caspase 1 was inhibited with Y-VAD. Our results provide an improved understanding of the mechanisms involved in the viral replication, inflammation and pathogenesis of bovine viral diarrhea.

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.

Characterization of a Highly Virulent Noncytopathic Bovine Viral Diarrhea Virus 2b Isolate Detected in Cattle in Inner Mongolia, China

Bovine viral diarrhea virus (BVDV) infection causes subclinical to severe acute disease in cattle all over the world. Two genotypes of BVDV are recognized, BVDV1 and BVDV2. Currently, the subtypes of BVDV1b and BVDV2a are mainly circulating in China. Here, a newly noncytopathic (ncp) BVDV strain named BVDVNM21 was isolated and identified from cattle. We determined the complete genome sequence of BVDVNM21, phylogenetic analysis of 5′ untranslated region (5′UTR), Npro, and E2 gene, and complete genome showed the virus belongs to BVDV2b subtype and formed an independent clade within BVDV2b. Genome-wide sequence analysis showed that BVDVNM21 had high homology with SD1301 (98.10%) isolated from China. In the experimental infection study of guinea pigs and calves, they all developed similar clinical signs, including a pronounced and prolonged febrile response lasting more than 3 days and pronounced reduction in white blood cells (WBC) and platelets of more than 40%. Moreover, guinea pigs showed more acute infection characteristics after infection, and WBC decreased by more than 50% at 1 day postinfection (dpi), and they recovered in less than 3 days. The results indicated that the BVDVNM21 strain showed high virulence in calves. It was the first confirmed isolation of a highly pathogenic ncpBVDV2b in cattle, as well as the establishment of the BVDV-guinea pig model. This study may provide a basis for further research and control of the prevalence of BVDV2b in China.

Effect of bovine viral diarrhea virus biotypes exposure on bovine gametes in early embryonic development in vitro.

Bovine viral diarrhea virus (BVDV) is an important viral agent causing reproductive failure in cattle. The objectives of the current study were to investigate the interaction between two BVDV biotypes, cytopathic (CP) and Non-cytopathic (NCP) and bovine gametes during in vitro fertilization (IVF) processing, the existence of the virus within embryonic cells and early embryonic development rates. Sperm and ova were exposed separately to CP and NCP BVDV at two concentrations of 104.5 and 105.5 tissue culture infectious dose 50.00% (TCID50) mL-1 prior to IVF, respectively. After five days post-IVF, early embryonic development rates of infected groups were assessed. Several embryos of each group, normal and degenerated, were selected for a viral assay using reverse transcription polymerase chain reaction technique. The result showed that the early embryonic development rates were decreased in treatment groups. The rates in the CP groups were lower than the NCP groups. In the CP groups, the proportions were, respectively, 10.00, 6.00 and 11.00, and 6.00% in the infected sperm and oocyte groups (104.5 and 105.5 TCID50 mL-1) that were higher than 50.00% in the control group. In NCP groups, the rates were, respectively, 25.00, 18.00 and 24.00, and 21.00% in the infected groups compared to 48.00% in the control group. In the CP groups, no BVDV was detected in normal embryos, whereas, all degenerated embryos were completely virus-positive. In the NCP groups, the virus was detected in both normal and degenerated embryos. In conclusion, this study supported detrimental impacts of CP and NCP BVDV on early embryonic development and the role of sperm and the zona pellucida layer as carriers of the virus.

PD-1 Blockade Restores the Proliferation of Peripheral Blood Lymphocyte and Inhibits Lymphocyte Apoptosis in a BALB/c Mouse Model of CP BVDV Acute Infection.

Acute infection of bovine viral diarrhea virus (BVDV) is associated with immune dysfunction and can cause peripheral blood lymphopenia and lymphocyte apoptosis. Our previous study has confirmed that programmed death-1 (PD-1) blockade inhibits peripheral blood lymphocytes (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the situation in vivo remains to be further studied and confirmed. Therefore, in this study, we established a BALB/c mouse model of acute BVDV infection with cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain NY-1). Then, we examined the mRNA and protein levels of PD-1 and programmed death-ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC) from BVDV-infected mice and analyzed the effects of PD-1 blockade on the proportions of CD3+, CD4+, and CD8+ T cell subsets, the apoptosis and proliferation of PBL, and the production of IL-2 and IFN-γ. We found that leukopenia, lymphocytopenia, and thrombocytopenia were developed in both CP and NCP BVDV-infected mice at day 7 of post-infection. The mRNA and protein expression of PD-1 and PD-L1 were significantly upregulated in CP and NCP BVDV-infected mice. Moreover, PD-1/PD-L1 upregulation was accompanied by leukopenia and lymphopenia. Additionally, PD-1 blockade inhibited PBL apoptosis and virus replication, restored the proportions of CD3+, CD4+, and CD8+ T cell subsets, and increased IFN-γ production and p-ERK expression in BVDV-infected mice. However, blocking PD-1 did not significantly affect PBL proliferation and IL-2 production in NCP BVDV-infected mice. Our findings further confirmed the immunomodulatory role of PD-1 in peripheral blood lymphocytopenia in vivo and provided a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.

Elimination of Non-cytopathic Bovine Viral Diarrhea Virus From the LFBK-αvβ6 Cell Line.

The LFBK-αvβ6 cell line is highly sensitive for the isolation of foot-and-mouth disease virus (FMDV) and porcinophilic vesicular viruses. However, LFBK-αvβ6 cells are contaminated with a non-cytopathic bovine viral diarrhea virus (BVDV), which complicates handling procedures in areas where other cell lines are maintained, as well downstream use of viral isolates. In this study, we used an aromatic cationic compound (DB772) to treat LFBK-αvβ6 cells using an approach that has been previously used to eliminate persistent BVDV from fetal fibroblast cell lines. After three cell passages with 4 μM DB772, BVDV could no longer be detected in unclarified cell suspensions using a pan-pestivirus real-time RT-PCR assay, and remained undetectable after treatment was stopped (nine passages) for an additional 28 passages. The analytical sensitivity of the DB772-treated LFBK-αvβ6 cultures (renamed WRL-LFBK-αvβ6) to titrations of FMDV and other vesicular virus isolates was comparable to untreated LFBK-αvβ6 cells. These new BVDV-free cells can be handled without the risk of cross-contaminating other cells lines or reagents, and used for routine diagnostics, in vivo studies and/or preparation of new vaccine strains.

The Effects of Cytopathic and Non-cytopathic Biotypes of Bovine Viral Diarrhea Virus on Sperm Vitality and Viability of Holstein Dairy Bulls in Vitro

BACKGROUND: Bovine viral diarrhea virus (BVDV) is one of the most important pathogens. OBJECTIVES: The aim of this study was to investigate the effects of cytopathic (CP) and non-cytopathic (NCP) biotypes of BVDV on vital status, membrane integrity, and motility of sperm cells in Holstein dairy bulls in vitro. METHODS: BVDV-free frozen semen samples were counted after thawing and centrifuged to separate live sperms. A sample containing 105 spermatozoa/mL was prepared. CP and NCP BVDV with 3 different doses of 105 (high dose), 104 (medium dose), and 103 (low dose) tissue culture infectious dose (TCID) 50/mL were challenged to sperm cells. After 2 hours of incubation at 38.5°C, eosin-nigrosine staining and hypoosmotic swelling (HOS) test were performed to assess the sperm viability and plasma membrane integrity. Computer assisted semen analysis (CASA) was used to evaluate the sperm motility parameters. The obtained data were analyzed using GLM method in SAS software. RESULTS: The percentage of live spermatozoa in the control group was 72±3.60%. However, it decreased significantly with the increase of virus concentration in both groups (p ≤0.05). Sperm integrity in the control group showed that the quality of semen was 65± 3.21. But the effect of virus biotypes resulted in a significant decrease in both high (105) and low (103) concentrations (p ≤0.05). BVDV biotypes are able to reduce different sperm movements as their concentration-increases. CONCLUSIONS: We concluded that CP and NCP biotypes of BVDV had a significant effect (p ≤0.05) on survival, plasma membrane integrity, and motility of sperm cells in vitro.

Bovine viral diarrhoea virus 1b infection associated with congenital tremor and hypomyelination in Holstein calves

Hypomyelination is a rare consequence of in utero bovine viral diarrhoea virus (BVDV) infection. We describe a BVDV outbreak in a naïve Holstein dairy herd in northern Italy, with an unusually high prevalence of calves with neurological signs, generalised tremors and ataxia. Histological analysis showed that hypomyelination was the predominant lesion and that the most typical BVDV neuropathological findings (e.g. cerebellar hypoplasia) were absent. Virological and molecular analyses showed that non-cytopathic BVDV genotype 1b was associated with the calves’ neurological signs and excluded other viruses responsible for congenital infection or neurological disorders. Whole-genome sequencing of BVDVs from the brain of a calf with neurological signs and the whole blood of a persistently infected herd-mate with no such sign showed >99.7 % sequence identity. Analysis of the quasispecies distribution revealed the greatest variation rates in regions coding for the structural proteins E1 and E2. Variation was slightly greater in the brain- than in the blood-derived sequence and occurred at different sites, suggesting the occurrence of distinct evolutionary processes in the two persistently infected calves. Molecular characterisation of BVDV genomes from five other calves with neurological signs from the same farm confirmed that the E1 and E2 regions were the most variable. Several factors, including genetic variability and host factors, appear to have contributed to the observed unique BVDV disease phenotype, characterised by hypomyelination and neurological signs.

Effect of bovine viral diarrhea virus on subsequent infectivity of bovine gammaherpesvirus 4 in endometrial cells in primary culture: An in vitro model of viral co-infection

Bovine viral diarrhea virus (BVDV) and bovine gammaherpesvirus 4 (BoHV-4) infect the uterus of cattle, being responsible for huge economic losses. Most of the pathogenesis of BoHV-4 in the bovine reproductive tract has been elucidated by conducting tests on primary cultures. Thus, it is important to have optimal in vitro conditions, avoiding the presence of other pathogens that can alter the results. BVDV is one of the most frequent viral contaminants of cell cultures. Considering that non-cytopathic (NCP) BVDV biotype can generate persistently infected (PI) cattle, which are the major source for virus transmission in susceptible herds, it is important to check products derived from cattle that are intended to be used in research laboratories. The aim of this work was to evaluate how the natural infection of bovine endometrial cells (BEC) with a NCP BVDV strain (BEC + BVDV) affects BoHV-4 replication. We have demonstrated a delay in BoHV-4 gene expression and a decrease in viral load in the extracellular environment in BEC + BDVD cells compared to BEC (BVDV-free) cells. These results confirm that replication of BoHV-4 in BEC primary cultures is affected by previous infection with BVDV. This finding highlights the importance of ruling out BVDV infection in bovine primary cell cultures to avoid biological interference or misinterpretation of results at the time of performing in vitro studies with BoHV-4.

Alterations in pregnancy-associated glycoprotein concentrations of pregnant sheep experimentally infected with bovine viral diarrhea virus.

To compare pregnancy-associated glycoprotein 1 (PAG1) concentrations in maternal (jugular vein) and fetal (uterine vein) circulations and amniotic fluid samples between pregnant ewes that were and were not experimentally infected with bovine viral diarrhea virus (BVDV). 11 healthy pregnant yearling ewes. Before study initiation, all ewes were naïve to BVDV and confirmed pregnant by transabdominal ultrasonography at approximately 60 days of gestation. At 65 days of gestation, ewes were intranasally inoculated with a noncytopathic BVDV type 1b strain (concentration, 107 TCID50/mL; 2 mL/nostril; n = 6) or an equal volume of BVDV-free viral culture medium (control; 5). A blood sample was collected for measurement of PAG1 concentration before inoculation. At 80 days of gestation, each ewe was anesthetized and underwent an ovariohysterectomy. While sheep were anesthetized, blood samples from the jugular and uterine veins and an amniotic fluid sample were collected for measurement of PAG1 concentration. Fetal tissues underwent real-time PCR analysis for BVDV RNA, and placental specimens underwent histologic evaluation and immunohistochemical staining for BVDV antigen. At 80 days of gestation, BVDV RNA in fetal tissues and mild placentitis were detected in 5 of 6 BVDV-inoculated ewes. Mean PAG1 concentrations in the maternal and fetal circulations of BVDV-inoculated ewes were significantly less than those in control ewes. Mean amniotic fluid PAG1 concentration did not differ significantly between the 2 groups. Concentration of PAG1 in the maternal circulation may be a useful biomarker for determining placental health in sheep after viral infection of the reproductive tract.

PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4+ and CD8+ T Cells During BVDV Infection in vitro

Acute infection of bovine viral diarrhea virus (BVDV) is associated with immune dysfunction and can cause peripheral blood lymphopenia and lymphocyte apoptosis. Our previous study has confirmed that programmed death-1 (PD-1) blockade inhibits peripheral blood lymphocyte (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the immunomodulatory effects of PD-1 pathway on major PBL subsets are unclear and their underlying molecular mechanisms need to be further studied. Therefore, in this study, we examined PD-1 expression in bovine PBL subsets after BVDV infection in vitro and analyzed the effects of PD-1 blockade on the apoptosis and proliferation of CD4+ and CD8+ T cells and expression of PD-1 downstream signaling molecules. The results showed that PD-1 expression was enhanced on CD4+ and CD8+ T cells, but not on CD21+ B cells after cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain KD) infection in vitro and PD-1 blockade significantly reduced the apoptosis of CD4+ and CD8+ T cells after these two strains infection. Remarkably, PD-1 blockade significantly increased the proliferation of CD4+ and CD8+ T cells after CP BVDV infection, but only significantly increased the proliferation of CD4+ T cells after NCP BVDV infection. In addition, we confirmed that PD-1-mediated PI3K/Akt/mTOR, caspase 9/caspase 3 and ERK pathways are involved in regulating the apoptosis and proliferation of CD4+ and CD8+ T cells during BVDV infection in vitro. Notably, ERK is involved in the regulation mechanism PD-1 mediated only when the cells are infected with CP BVDV. Our findings provide a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.

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 Diarrhoea Virus Infection Disrupts Uterine Interferon Stimulated Gene Regulatory Pathways During Pregnancy Recognition in Cows.

In cattle, conceptus-derived interferon tau (IFNT) is the pregnancy recognition (PR) signal. Our previous studies showed that non-cytopathic bovine viral diarrhoea virus (ncpBVDV) infection inhibited IFNT-induced interferon stimulated gene (ISG) expression, potentially causing early embryonic death. This study investigated the effect of bovine viral diarrhoea virus (BVDV) infection on upstream regulatory pathways of ISG production using an established PR model. Uterine endometrial cells from 10 apparently healthy and BVDV free cows were cultured and treated with 0 or 100 ng/mL IFNT for 24 h in the presence or absence of ncpBVDV infection. Microarray and pathway analysis were used to determine the IFNT-induced upstream regulators. Expression of the genes associated with the identified pathways were quantified with qPCR. IFNT challenge activated the signalling pathways associated with IFN receptors, JAK1/TYK2, IRFs and STATs and ncpBVDV infection inhibited the activation of IFNT on this pathway. Inhibition of this upstream signalling pathway may thus reduce ISG production to disrupt maternal PR. In addition, the reduction of uterine immunity by ncpBVDV infection may predispose the animals to uterine infection, which in turn impairs their reproductive performance. This provides a mechanism of how BVDV infection leads to early pregnancy failure in cows.

No effects of noncytopathic bovine viral diarrhea virus type 2 on the reproductive tract of experimentally inoculated boars

Bovine viral diarrhea virus (BVDV) infections in pigs may result in transient leukopenia, chronic gastroenteritis, septicemia, and hemorrhagic lesions. Both classical swine fever virus (CSF) and the atypical porcine pestivirus (APPV) are shed in the semen of infected boars. Because these viruses share conserved regions and present antigenic similarity, they may not be the only species belonging to the genus Pestivirus that can be shed in the semen of infected pigs. The objective of this study was to evaluate the testicular and epididymal changes, seminal parameters, and viral shedding in the reproductive tract of boars experimentally inoculated with noncytopathic BVDV-2. Six males were selected, and samples of blood, semen, and preputial swabs were collected every four days until the 52nd day after inoculation. The samples were tested for the presence of viral RNA by RT-PCR. An aliquot of whole blood was used to perform hematological analyses, which showed a significant reduction in monocyte counts and a significant increase in lymphocyte counts when comparing the pre- and postinoculation periods. The neutralizing antibody titers were determined by the virus neutralization test. None of the animals presented clinical signs or worsening of the seminal parameters that were evaluated. Moreover, BVDV-2 shedding by the reproductive route was not observed.

BVDV permissiveness and lack of expression of co-stimulatory molecules on PBMCs from calves pre-infected with BVDV

Bovine viral diarrhea virus (BVDV) has been detected in peripheral blood mononuclear cells (PBMCs) of immunocompetent animals, not being clear whether the development of a specific humoral immune response can prevent BVDV infection. The aim of this study was to evaluate the ability of non-cytopathic BVDV to replicate and produce infectious virus in PBMCs from calves pre-infected with BVDV and to elucidate the immunomodulatory effect of BVDV on these cells in an in vitro model. Quantification of virus was by quantitative PCR, while its replicative capacity and shedding into the extracellular environment was evaluated by viral titration. Apoptosis was assessed by flow cytometry analysis of annexin V and propidium iodide, and by expression of caspase-3/7. Flow cytometry was used to analyze the expression of CD14/CD11b/CD80, CD4/CD8/CD25, MHC-I/MHC-II and B-B2 markers. Our results showed that PBMCs from cattle naturally infected with BVDV were more susceptible to in vitro BVDV infection and showed a more severe apoptosis response than those from naïve animals. Non-cytopathic BVDV in vitro infection also resulted in a lack of effect in the expression of antigen presentation surface markers. All these findings could be related to the immunosuppressive capacity of BVDV and the susceptibility of cattle to this infection.

Importance of Viral Disease in Dairy Cow Fertility

Many viral diseases are endemic in cattle populations worldwide. The ability of many viruses to cross the placenta and cause abortions and fetal malformations is well understood. There is also significant evidence that viral infections have additional actions in dairy cows, which are reflected in reduced conception rates. These effects are, however, highly dependent on the time at which an individual animal first contracts the disease and are less easy to quantify. This paper reviews the evidence relating to five viruses that can affect fertility, together with their potential mechanisms of action. Acute infection with non-cytopathic bovine viral diarrhea virus (BVDV) in mid-gestation increases abortion rates or causes the birth of persistently infected calves. BVDV infections closer to the time of breeding can have direct effects on the ovaries and uterine endometrium, which cause estrous cycle irregularities and early embryo mortality. Fertility may also be reduced by BVDV-induced immunosuppression, which increases the susceptibility to bacterial infections. Bovine herpesvirus (BHV)-1 is most common in pre-pubertal heifers, and can slow their growth, delay breeding, and increase the age at first calving. Previously infected animals subsequently show reduced fertility. Although this may be associated with lung damage, ovarian lesions have also been reported. Both BHV-1 and BHV-4 remain latent in the host following initial infection and may be reactivated later by stress, for example associated with calving and early lactation. While BHV-4 infection alone may not reduce fertility, it appears to act as a co-factor with established bacterial pathogens such as Escherichia coli and Trueperella pyogenes to promote the development of endometritis and delay uterine repair mechanisms after calving. Both Schmallenberg virus (SBV) and bluetongue virus (BTV) are transmitted by insect vectors and lead to increased abortion rates and congenital malformations. BTV-8 also impairs the development of hatched blastocysts; furthermore, infection around the time of breeding with either virus appears to reduce conception rates. Although the reductions in conception rates are often difficult to quantify, they are nevertheless sufficient to cause economic losses, which help to justify the benefits of vaccination and eradication schemes.

Pathological and virological features of skin lesions caused by BVDV in cattle.

Dermatitis might occur in mucosal disease (MD) caused by bovine viral diarrhea virus (BVDV). This study describes the pathological and virological features of skin lesions associated with BVDV infection in four persistently infected (PI) cattle. Skin samples were reprocessed for histopathology and IHC. BVDV isolates were obtained and were genetically characterized. In addition to upper alimentary system ulcerative lesions, all cattle (one outbreak and three individual cases) presented focal crusty and ulcerative lesions affecting the mucocutaneous and skin-horn junctions, interdigital clefts, pastern, and areas surrounding the dewclaws and diffuse thickened skin within 7-20days of infection. Microscopic analysis revealed parakeratotic hyperkeratosis and single-cell keratinocyte death, accompanied by ballooning degeneration and spongiosis in the epidermis, as well as intraepithelial and subcorneal pustules. IHC showed BVDV antigen in the cytoplasm of keratinocytes undergoing individual cell death. Phylogenetic analysis revealed that the isolates from cattle #1, #2, and #4 belonged to BVDV-1a, whereas that from cattle #3 belonged to BVDV-1d. Cytopathic BVDV was isolated from cattle #2 and #3 (MD), and non-cytopathic BVDV was isolated from cattle #1 and #4. Thus, BVDV infection might cause acute disease, characterized by skin and upper alimentary system ulcerative lesions, in both MD and PI cattle.