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Bovine Pestivirus typically involves one or more organ systems, with clinical manifestations ranging from mild to severe fatal systemic illness that lead to significant reproductive, productive, and economic losses. Vaccines face the challenge of addressing the significant variability of pestiviruses, which affects the interaction between viral antigens and the immune system's ability to provide protection. This study aimed to evaluate the serological responses against bovine viral diarrhea virus 1 (Pestivirus A) and Pestivirus B induced by 10 commercial vaccines, including one recombinant (vaccine E), two modified live (MLV multivalent, vaccine I, and MLV monovalent, vaccine J), and seven killed vaccines (KLV, vaccines A to H). Additionally, we evaluated the cross-reactivity between Pestivirus A and B from vaccines and HoBi-like pestivirus (Pestivirus H). In Phase 1, guinea pigs were used to screen for non-MLVs. They were divided into nine groups (n=6 each) and received two doses (⅕ of bovine dose) of eight different non-MLV on Days 0 and 21. Serum samples were collected on Days 0 and 30 for serological analyse. In Phase 2, Holstein × Gir heifers (n= 45) were divided into five groups, comprising 6-9 animals. They were vaccinated either once with MLVs or twice with the top non-MLVs screened in Phase 1. Serum samples were harvested on d0 (vaccination day) and d60 (60 days after the first dose) for MLV and non-MLV. Specific antibody titers were assessed virus neutralization (VN) and transformed in log2 for statistical analysis using PROC-MIXED. Significant effects were observed for vaccine groups, time points, and their interactions concerning neutralizing antibodies against Pestivirus A and B in both Guinea pigs and heifers. The Phase 1 study revealed serological responses against Pestivirus A exclusively in non-MLV D (85.33±13.49) and E (72.00±19.26). In the bovine study, the KLD vaccine D (72.00±15.10), recombinant vaccine E (90.66±25.85), and MLV I (170.66±28.22) resulted in an average of neutralizing antibodies against Pestivirus A that exceeded the protective threshold (≥ 60). However,individual analysis of heifers showed a higher frequency of animals presenting titers of Pestivirus A Ab surpassing 32 following vaccination with MLV I and J. None of the vaccine formulations in either study elicited a protective immune response against Pestivirus B or demonstrated cross-reactivity against Pestivirus H., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Congenital tremor (CT) type A-II in piglets is caused by an emerging atypical porcine pestivirus (APPV), which is prevalent in swine herds and a serious threat to the pig production industry. This study aimed to construct APPV E2 subunit vaccines fused with Fc fragments and evaluate their immunogenicity in piglets. Here, APPV E2Fc and E2ΔFc fusion proteins expressed in Drosophila Schneider 2 (S2) cells were demonstrated to form stable dimers in SDS-PAGE and western blotting assays. Functional analysis revealed that aE2Fc and aE2ΔFc fusion proteins could bind to FcγRI on antigen-presenting cells (APCs), with the affinity of aE2Fc to FcγRI being higher than that of aE2ΔFc. Moreover, subunit vaccines based on aE2, aE2Fc, and aE2ΔFc fusion proteins were prepared, and their immunogenicity was evaluated in piglets. The results showed that the Fc fusion proteins emulsified with the ISA 201VG adjuvant elicited stronger humoral and cellular immune responses than the IMS 1313VG adjuvant. These findings suggest that APPV E2 subunit vaccines fused with Fc fragments may be a promising vaccine candidate against APPV.

Apart from the established pestivirus species Pestivirus A to Pestivirus K novel species emerged. Pigs represent not only hosts for porcine pestiviruses, but are also susceptible to bovine viral diarrhea virus, border disease virus (BDV) and other ruminant pestiviruses. The present study focused on the characterization of the ovine Tunisian sheep-like virus (TSV) as well as Bungowannah virus (BuPV) and BDV strain Frijters, which were isolated from pigs. For this purpose, we performed genetic characterization based on complete coding sequences, studies on virus replication in cell culture and in domestic pigs, and cross-neutralization assays using experimentally derived sera. TSV forms a distinct phylogenetic group more closely related to Pestivirus C (classical swine fever virus, CSFV) than to Pestivirus D (BDV). In contrast to BDV and BuPV, TSV replicates by far more efficiently on ovine than on porcine cells. Nevertheless, pigs were susceptible to TSV. As a consequence of close antigenic relatedness of TSV to CSFV, cross-reactivity was detected in CSFV-specific antibody assays. In conclusion, TSV is genetically closely related to CSFV and can replicate in domestic pigs. Due to close antigenic relatedness, field infections of pigs with TSV and other ruminant pestiviruses can interfere with serological diagnosis of classical swine fever.

Pestiviruses express the unique essential envelope protein E rns , which exhibits RNase activity, is attached to membranes by a long amphipathic helix, and is partially secreted from infected cells. The RNase activity of E rns is directly connected with pestivirus virulence. Formation of homodimers and secretion of the protein are hypothesized to be important for its role as a virulence factor, which impairs the host's innate immune response to pestivirus infection. The unusual membrane anchor of E rns raises questions with regard to proteolytic processing of the viral polyprotein at the E rns carboxy-terminus. Moreover, the membrane anchor is crucial for establishing the critical equilibrium between retention and secretion and ensures intracellular accumulation of the protein at the site of virus budding so that it is available to serve both as structural component of the virion and factor controlling host immune reactions. In the present manuscript, we summarize published as well as new data on the molecular features of E rns including aspects of its interplay with the other two envelope proteins with a special focus on the biochemistry of the E rns membrane anchor.

A novel pestivirus species, termed Lateral-shaking Inducing Neuro-Degenerative Agent virus (LindaV), was discovered in a piglet-producing farm in Austria in 2015 related to severe congenital tremor cases. Since the initial outbreak LindaV has not been found anywhere else. In this study, we determined the seroprevalence of LindaV infections in the domestic pig population of Austria. A fluorophore labeled infectious cDNA clone of LindaV (mCherry-LindaV) was generated and used in serum virus neutralization (SVN) assays for the detection of LindaV specific neutralizing antibodies in porcine serum samples. In total, 637 sera from sows and gilts from five federal states of Austria, collected between the years 2015 and 2020, were analyzed. We identified a single serum showing a high neutralizing antibody titer, that originated from a farm (Farm S2) in the proximity of the initially affected farm. The analysis of 57 additional sera from Farm S2 revealed a wider spread of LindaV in this pig herd. Furthermore, a second LindaV strain originating from this farm could be isolated in cell culture and was further characterized at the genetic level. Possible transmission routes and virus reservoir hosts of this emerging porcine virus need to be addressed in future studies.

Atypical porcine pestivirus (APPV) had been detected in many countries. However, to date, a commercial detection kit is not available because of a lack of specific monoclonal antibodies (mAbs) to APPV. We generated 7 mAbs targeting the NS3 protein of APPV. Isotyping results indicated that all of these mAbs are IgG1 with a kappa light chain. We analyzed the epitopes recognized by mAbs 2B6, 6G11, 8D1, 8D3, and 8F12, which recognized the same linear epitope (GRIKSAYSDE); the 6H3 and 7E10 mAbs recognized 2 different conformational epitopes. Applications of these antibodies were verified by ELISA, western blot, indirect immunofluorescence assay, and flow cytometry. The antibodies were functionally workable for these immunoassays except for 8F12, which could not be used in flow cytometry.

Autonomously replicating subgenomic Bungowannah virus (BuPV) RNAs (BuPV replicons) with deletions of the genome regions encoding the structural proteins C, E RNS , E1, and E2 were constructed on the basis of an infectious cDNA clone of BuPV. Nanoluciferase (Nluc) insertion was used to compare the replication efficiencies of all constructs after electroporation of in vitro -transcribed RNA from the different clones. Deletion of C, E1, E2, or the complete structural protein genome region (C-E RNS -E1-E2) prevented the production of infectious progeny virus, whereas deletion of E RNS still allowed the generation of infectious particles. However, those ΔE RNS viral particles were defective in virus assembly and/or egress and could not be further propagated for more than three additional passages in porcine SK-6 cells. These "defective-in-third-cycle" BuPV ΔE RNS mutants were subsequently used to express the classical swine fever virus envelope protein E2, the N-terminal domain of the Schmallenberg virus Gc protein, and the receptor binding domain of the Middle East respiratory syndrome coronavirus spike protein. The constructs could be efficiently complemented and further passaged in SK-6 cells constitutively expressing the BuPV E RNS protein. Importantly, BuPVs are able to infect a wide variety of target cell lines, allowing expression in a very wide host spectrum. Therefore, we suggest that packaged BuPV ΔE RNS replicon particles have potential as broad-spectrum viral vectors. IMPORTANCE The proteins N PRO and E RNS are unique for the genus Pestivirus , but only N PRO has been demonstrated to be nonessential for in vitro growth. While this was also speculated for E RNS , it has always been previously shown that pestivirus replicons with deletions of the structural proteins E RNS , E1, or E2 did not produce any infectious progeny virus in susceptible host cells. Here, we demonstrated for the first time that BuPV E RNS is dispensable for the generation of infectious virus particles but still important for efficient passaging. The E RNS -defective BuPV particles showed clearly limited growth in cell culture but were capable of several rounds of infection, expression of foreign genes, and highly efficient trans -complementation to rescue virus replicon particles (VRPs). The noncytopathic characteristics and the absence of preexisting immunity to BuPV in human populations and livestock also provide a significant benefit for a possible use, e.g., as a vector vaccine platform., (Copyright © 2020 American Society for Microbiology.)

Members of the Pestivirus genus (family Flaviviridae ) cause severe and economically important diseases in livestock. Serological studies have revealed the presence of pestiviruses in different cervid species, including wild and semi-domesticated Eurasian tundra reindeer. In this retrospective study, serum samples collected between 2006 and 2008 from 3339 semi-domesticated Eurasian reindeer from Finnmark County, Norway, were tested for anti-pestivirus antibodies using an enzyme linked immunosorbent assay (ELISA) and a subset of these by virus neutralization test (VNT). A seroprevalence of 12.5% was found, varying from 0% to 45% among different herding districts, and 20% in western Finnmark, as compared to 1.7% in eastern Finnmark. Seroprevalence increased with age. Pestivirus-specific RNA was not detected in any of the 225 serum samples tested by real-time RT-PCR. Based on VNT results, using a panel of one bovine viral diarrhea virus (BVDV) strain and two border disease virus (BDV) strains, the virus is most likely a reindeer-specific pestivirus closely related to BDV. A characterization of the causative virus and its pathogenic impact on reindeer populations, as well as its potential to infect other domestic and wild ruminants, should be further investigated., Competing Interests: The authors declare no conflict of interest.

The novel pestivirus species known as lateral-shaking inducing neuro-degenerative agent (LINDA) virus emerged in 2015 in a piglet-producing farm in Austria. Affected piglets showed strong congenital tremor as a result of severe lesions in the central nervous system. Here, we report the results of a controlled animal infection experiment. Post-weaning piglets were infected with LINDA to determine the susceptibility of pigs, the clinical consequences of infection and the humoral immune response against LINDA. No clinically overt disease signs were observed in the piglets. Viremia was hardly detectable, but LINDA was present in the spleen and several lymphatic organs until the end of the experiment on day 28 post-infection. Oronasal virus shedding together with the infection of one sentinel animal provided additional evidence for the successful replication and spread of LINDA in the piglets. Starting on day 14 post-infection, all infected animals showed a strong humoral immune response with high titers of neutralizing antibodies against LINDA. No cross-neutralizing activity of these sera with other pestiviral species was observed. According to these data, following postnatal infection, LINDA is a rather benign virus that can be controlled by the pig's immune system. However, further studies are needed to investigate the effects of LINDA on the fetus after intrauterine infection.

Atypical porcine pestivirus (APPV) is a widely distributed pathogen causing congenital tremor (CT) in piglets. So far, no data are available regarding the humoral immune response against APPV. In this study, piglets and their sows from an affected herd were tested longitudinally for viral genome and antibodies. APPV genome was detected in the majority of the piglets (14/15) from CT affected litters. Transient infection of gilts was observed. Kinetics of E rns - and E2-specific antibodies and their neutralizing capacity were determined by recently (E rns ) and newly (E2) developed antibody ELISAs and virus neutralization assays. Putative maternally derived antibodies (MDA) were detected in most piglets, but displayed only low to moderate neutralizing capacity (ND 50 ≤ 112). Horizontal APPV transmission occurred when uninfected and infected piglets were mingled on the flat deck. Horizontally infected piglets were clinically inapparent and showed only transient viremia with subsequently consistently high E2 antibody levels. For piglets from CT affected litters, significantly lower neutralizing antibody titers were observed. Results indicate that E2 represents the main target of neutralizing antibodies. Characterization of the humoral immune response against APPV will help to provide valuable serological diagnosis, to understand the epidemiology of this novel pathogen, and to implement tailored prevention strategies.

Background: Bovine viral diarrhea virus (BVDV) is an economically important viral pathogen of domestic and wild ruminants. Apart from cattle, small ruminants (goats and sheep) are also the susceptible hosts for BVDV. BVDV infection could interfere both of the innate and adaptive immunity of the host, while the genes and mechanisms responsible for these effects have not yet been fully understood. Peripheral blood mononuclear cells (PBMCs) play a pivotal role in the immune responses to viral infection, and these cells were the target of BVDV infection. In the present study, the transcriptome of goat peripheral blood mononuclear cells (PBMCs) infected with BVDV-2 was explored by using RNA-Seq technology., Results: Goat PBMCs were successfully infected by BVDV-2, as determined by RT-PCR and quantitative real-time RT-PCR (qRT-PCR). RNA-Seq analysis results at 12 h post-infection (hpi) revealed 499 differentially expressed genes (DEGs, fold-change ≥ ± 2, p < 0.05) between infected and mock-infected PBMCs. Of these genes, 97 were up-regulated and the remaining 352 genes were down-regulated. The identified DEGs were found to be significantly enriched for locomotion/ localization, immune response, inflammatory response, defense response, regulation of cytokine production, etc., under GO enrichment analysis. Cytokine-cytokine receptor interaction, TNF signaling pathway, chemokine signaling pathway, etc., were found to be significantly enriched in KEGG pathway database. Protein-protein interaction (PPI) network analysis indicated most of the DEGs related to innate or adaptive immune responses, inflammatory response, and cytokine/chemokine-mediated signaling pathway. TNF, IL-6, IL-10, IL-12B, GM-CSF, ICAM1, EDN1, CCL5, CCL20, CXCL10, CCL2, MAPK11, MAPK13, CSF1R and LRRK1 were located in the core of the network and highly connected with other DGEs., Conclusions: BVDV-2 infection of goat PBMCs causes the transcription changes of a series of DEGs related to host immune responses, including inflammation, defense response, cell locomotion, cytokine/chemokine-mediated signaling, etc. The results will be useful for exploring and further understanding the host responses to BVDV-2 infection in goats.

An atypical porcine pestivirus (APPV) causing congenital tremor type A-II in piglets was identified in China in 2016. An increased number of cases of APPV have been reported in various countries all over the world since 2015. This study aimed to develop an effective subunit vaccine against APPV based on the E2 protein, which is the main immunogenicity protein of APPV. In this study, E2 protein was successfully expressed by the baculovirus expression system. E2 protein was confirmed by Western blot assay, which showed that E2 protein possesses N-linked glycosylation sites. The immunogenicity of E2 subunit vaccine was evaluated in mice. The E2 protein emulsified with ISA 201VG adjuvant induced significantly higher levels of APPV-specific antibodies and elicited stronger lymphocyte proliferative responses and higher interleukin-10 secretion than those of the E2 protein emulsified with IMS 1313VG adjuvant. This observation indicates that the E2 subunit vaccine induces a Th2-type immune response. Our results showed that E2 protein can be developed as a safe and effective subunit vaccine for the control of APPV infection.

Hobi-like viruses (HobiPeV) comprise a novel, recently classified species of bovine pestiviruses, originally identified in commercial fetal bovine serum of Brazilian origin and, subsequently, isolated from diseased animals in several countries. Although frequently isolated from clinical cases, most HobiPeV isolates failed to reproduce overt disease in cattle upon experimental inoculation. Herein, we describe the outcome of experimental infection of four to six months-old seronegative calves with two Brazilian HobiPeV isolates. Calves inoculated intranasally with isolate SV478/07 developed viremia between days 2 and 9 post-inoculation (pi) and shed virus in nasal secretions up to day 11pi. These animals presented hyperthermia (day 7 to 10-11 pi) and lymphopenia from days 4 to 8pi. Clinically, all four calves developed varied degrees of apathy, anorexia, mild to moderate respiratory signs (nasal secretion, hyperemia), ocular discharge and pasty diarrhea in the days following virus inoculation. In contrast, calves inoculated with isolate SV757/15 presented only hyperthermia (days 3 to 10-11 pi) and lymphopenia (days 4-8 pi), without other apparent clinical signs. In these animals, viremia was detected up to day 9 pi and virus shedding in nasal secretions lasted up to day 12-14 pi. Both groups seroconverted to the inoculated viruses, developing virus neutralizing (VN) titers from 320 to 5120 at day 28pi. These results extend previous findings that experimental infections of calves with HobiPeV are predominantly mild, yet they also indicate that field isolates may differ in their ability to cause disease in susceptible animals., (Copyright © 2018. Published by Elsevier Ltd.)

Background: Inactivated and subunit bovine viral diarrhea virus (BVDV) vaccines have shown limited protective efficacy. This study aimed to evaluate the effectiveness of a vaccine containing both inactivated BVDV (iBVDV) and baculovirus-expressed recombinant E2 (rE2), an important BVDV antigen with strongly neutralizing epitopes., Results: Four groups of goats were immunized twice with one of four vaccine preparations: 1) iBVDV+rE2, 2) rE2, 3) iBVDV, and 4) saline, and challenged with BVDV. For goats vaccinated with the iBVDV+rE2 vaccine, no viremia was observed after challenge, and clinical signs, pyrexia, and leukopenia were reduced compared to the saline group. In contrast, for goats vaccinated with either iBVDV or rE2 alone, viremia was still detectable., Conclusion: The combination of iBVDV and rE2 elicited stronger protective immune responses against BVDV than iBVDV or rE2 alone.

Pestiviruses including Bovine viral diarrhea virus type 1 (BVDV-1), BVDV-2 and Border disease virus (BDV) have been reported in both sheep and cattle populations, together with the HoBi-like, an emerging group of pestiviruses. Pestivirus control programs in the United States have focused on the control of BVDV-1 and 2. The incidence of pestivirus infection in sheep in the United States and the risk of transmission between cattle and sheep populations are unknown. The aim of this study was to perform serological surveillance for pestivirus exposure in sheep from an important sheep producing state in the Unites States, Wyoming. For this, sera from 500 sheep, collected across the state of Wyoming (US) in 2015-2016, were examined by comparative virus neutralization assay against four species/proposed species of pestiviruses: BVDV-1, BVDV-2, BDV and HoBi-like virus. Rates of exposure varied between geographic regions within the state. The overall pestivirus prevalence of antibodies was 5.6%. Antibodies were most frequently detected against BVDV-1 (4%), and the highest antibody titers were also against BVDV-1. Data from this study highlights understanding of the dynamics of sheep pestivirus exposure, consideration of reference strains used for VN assays, transmission patterns, and potential vaccination history should be taken into account in implementation of control measures against pestiviruses in sheep and for successful BVDV control programs in cattle., (Published by Elsevier B.V.)

Hobi-like viruses comprise an unclassified group of bovine pestiviruses related to bovine viral diarrhea virus 1 (BVDV-1) and 2 (BVDV-2). These viruses were originally identified in fetal bovine serum from Brazilian origin and, subsequently, isolated from diseased animals in several countries. Herein we performed an antigenic characterization of eight Brazilian HoBi-like viruses isolated from persistently infected (PI) animals and from gastroenteric disease (2007-2015). Phylogenetic analysis based on the 5' unstranslated region (UTR) clustered these viruses with other HoBi-like viruses from European and Asiatic origin. Monoclonal antibody (MAb) binding indicated variability in the Hobi-like virus glycoprotein E2 and significant differences from the homologous BVDV-1 and BVDV-2 glycoprotein. Analysis of antigenic relatedness based on virus-neutralizing titers using virus-specific antisera revealed that HoBi-like viruses are antigenically very different from BVDV-1 and, to a lesser extent, from BVDV-2. Cross-neutralizing assays between pairs of HoBi-like viruses and their respective antisera indicated the existence of antigenic variability among these viruses, even for viruses isolated from the same herd in different occasions. Moreover, the identification of a HoBi-like isolate with low antigenic similarity with the other isolates indicates the potential existence of antigenic subgroups among HoBi-like virus isolates. Finally, sera of lambs immunized with commercial BVDV vaccines showed low or undetectable neutralizing activity against HoBi-like isolates. These results indicate significant antigenic differences between BVDV genotypes and Brazilian HoBi-like viruses and the existence of antigenic variability within this atypical group of pestiviruses. These findings extend the knowledge about the antigenic diversity of HoBi-like viruses and reinforce the need for their inclusion in current BVDV vaccines., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Classical swine fever (CSF) is a highly contagious and lethal disease in swine. Serological tests for the diagnosis of CSF need not only to detect antibodies against CSFV, but also need to differentiate these from antibodies against other pestiviruses. To investigate the possibilities of specific peptide-based serology, various synthetic peptides that represent a well-described linear epitope of the CSFV E2 protein (TAVSPTTLR) were used to test the viability of a peptide-based suspension array for the detection of antibodies against pestiviruses in swine. The results show that N-terminally biotinylated peptides can bind to avidin conjugated beads, and function in detection of the corresponding monoclonal antibody WH303. There are indications that the length of the spacer between epitope and biotin affect the efficiency of the peptide-antibody interaction. A protocol was established that enables probing for antibodies in porcine sera, where neutravidin-blocking of serum and the use of empty control beads for normalization was crucial. With a set of porcine sera with antibodies against various pestiviruses, the proof of concept of a peptide-based suspension array for specific detection of antibodies against pestiviruses in porcine sera was demonstrated., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, cellular localization and the distribution pattern of BVDV genome in lymphoid tissues during the course of experimental acute BVDV-1 infection of sheep was investigated. Tonsils, mesenteric lymph nodes (MLN) and spleen were collected on 3, 6, 9, 12 and 15 days post infection (dpi) from twenty 4-month-old lambs, experimentally inoculated intra-nasally with 5 × 10(5) TCID50 of a non-cytopathic (ncp) BVDV-1 isolate, Ind-17555. Tissues collected from ten mock-infected lambs served as controls. In situ hybridization (ISH) was carried out in paraformaldehyde fixed paraffin embedded tissue sections using digoxigenin labelled riboprobe targeting 5'-UTR of BVDV-1. BVDV genome was detected at all the intervals from 3 dpi to 15 dpi in the lymphoid tissues with variations between the intervals and also amongst the infected sheep. During the early phase of acute infection, presence of viral genome was more in tonsils than MLN and spleen, whereas the distribution was higher in MLN during later stages. BVDV-1 genome positive cells included lymphocytes, macrophages, plasma cells, reticular cells and sometimes crypt epithelial cells. Genome distribution was frequently observed in the lymphoid follicles of tonsils, MLN and spleen, besides the crypt epithelium in tonsils, paracortex and medullary sinus and cords of MLN. Most abundant and widespread distribution of BVDV-1 genome was observed on 6 dpi while there was a reduction in number and intensity of positive signals by 15 dpi in most of the infected animals. This is the first attempt made to study the localisation of BVDV-1 in lymphoid tissues of acutely infected sheep by in situ hybridization. The results show that the kinetics of BVDV-1 distribution in lymphoid tissues of experimentally infected non-pregnant sheep follows almost a similar pattern to that demonstrated in BVDV infected cattle.

Pestiviruses including bovine viral diarrhea virus (BVDV), border disease virus (BDV) and classical swine fever virus (CSFV), occur worldwide and are important pathogens of livestock. A large part of their success can be attributed to the induction of central immunotolerance including B- and T-cells upon fetal infection leading to the generation of persistently infected (PI) animals. In the past few years, it became evident that evasion of innate immunity is a central element to induce and maintain persistent infection. Hence, the viral non-structural protease N(pro) heads the transcription factor IRF-3 for proteasomal degradation, whereas an extracellularly secreted, soluble form of the envelope glycoprotein E(rns) degrades immunostimulatory viral single- and double-stranded RNA, which makes this RNase unique among viral endoribonucleases. We propose that these pestiviral interferon (IFN) antagonists maintain a state of innate immunotolerance mainly pertaining its viral nucleic acids, in contrast to the well-established immunotolerance of the adaptive immune system, which is mainly targeted at proteins. In particular, the unique extension of 'self' to include the viral genome by degrading immunostimulatory viral RNA by E(rns) is reminiscent of various host nucleases that are important to prevent inappropriate IFN activation by the host's own nucleic acids in autoimmune diseases such as Aicardi-Goutières syndrome or systemic lupus erythematosus. This mechanism of "innate tolerance" might thus provide a new facet to the role of extracellular RNases in the sustained prevention of the body's own immunostimulatory RNA to act as a danger-associated molecular pattern that is relevant across various species., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Bovine viral diarrhea virus (BVDV) is an economically important pathogen that causes development of mild to severe clinical signs in wild and domesticated ruminants. We previously showed that mice could be infected by BVDV. In the present study, we infected mice intraperitoneally with non-cytopathic (ncp) BVDV1 or ncp BVDV2, harvested the blood and organs of the infected mice at days 4, 7, 10 and 14 postinfection (pi), and performed immunohistochemical analyses to confirm BVDV infection. Viral antigens were detected in the spleens of all infected mice from days 4 through 14 and were also found in the mesenteric lymph nodes, gut-associated lymphoid tissue (GALT), heart, kidney, intestine, and bronchus-associated lymphoid tissue (BALT) of some infected mice. In ncp BVDV2-infected mice, flow cytometric analysis revealed markedly fewer CD4(+) and CD8(+) T lymphocytes and lower expression of costimulatory molecules CD80 (B7-1) and CD86 (B7-2) and major histocompatibility complex (MHC) class II (I-A/I-E) than those in ncp BVDV1-infected mice. Production of the cytokines interleukin (IL)-6 and monocyte chemotactic protein (MCP)-1 was higher in the plasma of ncp BVDV2-infected mice than that in that of ncp BVDV1-infected mice. Our results demonstrate that ncp BVDV1 and ncp BVDV2 interact differently with the host innate immune response in vivo. These findings highlight an important distinction between ncp BVDV1 and ncp BVDV2 and suggest that ncp BVDV2 impairs the host's ability to control the infection and enhances virus dissemination.

HoBi-like viruses are an emerging species of pestiviruses associated with respiratory and reproductive disease in cattle and in water buffaloes. Although cattle appear to be the main natural hosts, little is know about the potential for HoBi-like viruses to be transmitted to other livestock. In this study, seronegative calves, goats and pigs, and sheep harboring pestivirus antibodies (probably due to previous exposure to BVDV) were exposed to HoBi-like viruses either by direct inoculation (GIn) or by contact with calves persistently infected with HoBi-like viruses (GEx). Both GIn and GEx groups were monitored for clinical signs, lymphocyte count, virus in buffy coats and nasal swabs up to day 18 post-inoculation (pi). Evidence of transmission of HoBi-like virus by PI calves was observed in all studied species. No difference in clinical presentation was observed between animals in the GIn or GEx groups. Evidence of infection, depending on the species included lymphocyte depletion, fever, viral RNA detection, and/or seroconversion. Depletion of lymphocytes was observed in calves and goats (35% and 50%, respectively) but not in pigs. Seroconversion was observed in at least one animal of each group and for all exposed species. The rate of seroconversion was higher in animals in the GIn experimental groups. In sheep, pre-existing moderate to high neutralizing titers against BVDV did not prevent viral replication and shed. The study demonstrated that naive cattle, goats and pigs, in addition to antibody positive sheep, can be infected by HoBi-like virus via persistently infected calf and potentially transmit the virus., (Published by Elsevier B.V.)

The aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats.

Development of transplacental infection depends on the ability of the virus to cross the placenta and replicate within the fetus while counteracting maternal and fetal immune responses. Unfortunately, little is known about this complex process. Non-cytopathic (ncp) strains of bovine viral diarrhea virus (BVDV), a pestivirus in the Flaviviridae family, cause persistent infection in early gestational fetuses (<150 days; persistently infected, PI), but are cleared by immunocompetent animals and late gestational fetuses (>150 days; transiently infected, TI). Evasion of innate immune response and development of immunotolerance to ncp BVDV have been suggested as possible mechanisms for the establishment of the persistent infection. Previously we have observed a robust temporal induction of interferon (IFN) type I (innate immune response) and upregulation of IFN stimulated genes (ISGs) in BVDV TI fetuses. Modest chronic upregulation of ISGs in PI fetuses and calves reflects a stimulated innate immune response during persistent BVDV infection. We hypothesized that establishing persistent fetal BVDV infection is also accompanied by the induction of IFN-gamma (IFN-γ). The aims of the present study were to determine IFN-γ concentration in blood and amniotic fluid from control, TI and PI fetuses during BVDV infection and analyze induction of the IFN-γ downstream pathways in fetal lymphoid tissues. Two experiments with in vivo BVDV infections were completed. In Experiment 1, pregnant heifers were infected with ncp BVDV type 2 on day 75 or 175 of gestation or kept naïve to generate PI, TI and control fetuses, respectively. Fetuses were collected by Cesarean section on day 190. In Experiment 2, fetuses were collected on days 82, 89, 97, 192 and 245 following infection of pregnant heifers on day 75 of gestation. The results were consistent with the hypothesis that ncp BVDV infection induces IFN-γ secretion during acute infection in both TI and PI fetuses and that lymphoid tissues such as spleen, liver and thymus, serve both as possible sources of IFN-γ and target organs for its effects. Notably, induction of IFN-γ coincides with a decrease in BVDV RNA concentrations in PI fetal blood and tissues. This is the first report indicating the possible presence of an adaptive immune response in persistent BVDV infections, which may be contributing to the observed reduction of viremia in PI fetuses., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Pestiviruses cause economically important diseases among domestic ruminants and pigs, but they may also infect a wide spectrum of wild species of even-toed ungulates (Artiodactyla). Bovine viral diarrhea virus (BVDV) and Border disease virus of sheep infect their hosts either transiently or persistently. Cellular and humoral immunotolerance to the infecting strain is a unique feature of persistent infection (PI) by ruminant pestiviruses. Persistence, caused by transplacental infection early in fetal development, depends on virally encoded interferon antagonists that inactivate the host's innate immune response to the virus without globally interfering with its function against other viruses. At epidemiological equilibrium, approximately 1-2% of animals are PI. Successful BVDV control programs show that removal of PI animals results in viral extinction in the host population. The nucleotide sequences of ruminant pestiviruses change little during persistent infection. Nevertheless, they display large heterogeneity, pointing to a long history of virus-host coevolution in which avirulent strains are more successful.

Bungowannah virus is the most divergent atypical pestivirus that had been detected up to now, and does not fit into any of the four approved species: Bovine viral diarrhea virus type 1 (BVDV-1) and type 2 (BVDV-2), Classical swine fever virus (CSFV) and Border disease virus (BDV). However, the presence of N(pro) and E(rns) coding regions, which are unique to pestiviruses, provides clear evidence of a pestivirus. Nevertheless, the amino acid identity of Bungowannah virus N(pro) and BVDV-1 N(pro) (strain CP7) is only 51.5%. By using a BVDV-1 backbone, a novel chimeric construct was generated, in which the genomic region encoding the non-structural protein N(pro) was replaced by that of Bungowannah virus (CP7&#95;N(pro)-Bungo). In vitro studies of CP7&#95;N(pro)-Bungo revealed autonomous replication with the same efficacy as the BVDV backbone CP7 and infectious high-titer virus could be collected. In order to compare the ability of interferon (IFN) suppression, two reporter gene assays, specific for type-I IFN, were carried out. In virus-infected cells, no significant difference in blocking of IFN expression between the parental virus CP7, Bungowannah virus and the chimeric construct CP7&#95;N(pro)-Bungo could be detected. In contrast, an N(pro) deletion mutant showed an impaired replication in bovine cells and a marked type-I IFN response. Taken together, our findings reveal the compatibility of non-structural protein N(pro) of atypical Bungowannah virus with a BVDV type 1 backbone and its characteristic feature as an inhibitor of type-I IFN induction with an inhibitor-activity comparable to other pestiviruses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

It was hypothesized that acute postnatal Bovine viral diarrhea virus 1 (BVDV-1) infection leads to leukopenia and lymphoid depletion of gut-associated lymphoid tissues similar to acute disease in calves. The objectives of the current study were to characterize the pathologic effects, viremia, viral shedding, and viral antigen deposition in 6-24-month-old, acutely infected alpacas following experimental infection with noncytopathic BVDV-1 subgenotype 1b (BVDV C0-6). The BVDV-1 isolate was obtained from a cria with naturally occurring persistent infection. Lymphocytopenia occurred 3-7 days postinfection, with a 50% reduction in peripheral lymphocytes in infected alpacas. Depletion of B-cell populations in gut-associated lymphoid tissues was evident microscopically. Populations of T cells in parafollicular zones and in nodular aggregates along the superficial submucosa remained intact. The BVDV antigen was deposited most consistently in submucosal gastrointestinal aggregated lymphoid tissues of ileum, proximal colon, and stomach compartment three. Viral antigen was more variably evident in other lymphoid tissues. Antigen distribution correlated well with histologic lesions in gastrointestinal aggregated lymphoid tissues, confirming the role of virus in lymphoid depletion. Nasal shedding was detected in all challenged alpacas on day 6 and in 4 out of 12 challenged alpacas on day 9. Viremia was present as early as day 3, and present in all challenged alpacas on days 5, 6, 7, and 9 postchallenge. Lymphocytopenia and depletion of gastrointestinal aggregated lymphoid tissues associated with acute BVDV-1 infection likely results in immune compromise and is expected to exacerbate concurrent infections even though uncomplicated BVDV-1 infection was clinically unapparent.

Hobi-like pestivirus, a new tentative species within genus Pestivirus, was firstly detected in foetal bovine serum batches and later associated to respiratory distress and reproductive failures in cattle. In the present study, the cross-antibody response between bovine viral diarrhoea virus 1 (BVDV-1) and the emerging pestivirus was evaluated in the sheep model. Ten sheep were immunised against BVDV-1 or Hobi-like pestivirus using inactivated preparations and the induced antibody responses were evaluated against the homologous and heterologous viruses. The results showed that heterologous antibody titres were significantly lower than the homologous ones, thus suggesting the need to develop specific vaccines against the emerging pestiviral species., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The objective of this study was to compare the mRNA expression of host genes involved in type-I interferon-induced antiviral state (IFN-α, IFN-β, Mx-1, PKR, OAS-1 and ISG-15), and apoptosis (caspase-3, -8, and -9), after experimental infection of beef calves with low or high virulence noncytopathic (ncp) bovine viral diarrhea virus (BVDV) strains. Thirty BVDV-naïve, clinically normal calves were randomly assigned to three groups. Calves were intranasally inoculated with low (LV; n=10, strain SD-1) or high (HV; n=10, strain 1373) virulence ncp BVDV or BVDV-free cell culture medium (Control, n=10). Quantitative RT-PCR was used to determine the target gene expression in tracheo-bronchial lymph nodes and spleen 5 days after infection. Interferon-α and -β mRNA levels were up-regulated in tracheo-bronchial lymph nodes (P<0.05) in the HV group, but not in the LV group, compared with the control group. There was an up-regulation of type I interferon-induced genes in spleen and tracheo-bronchial lymph nodes of HV and LV groups, compared with the control group (P<0.01). mRNA levels of OAS-1 and ISG-15 were significantly higher in LV than HV calves (P<0.05). A significant up-regulation of caspase-8 and -9 was observed in tracheo-bronchial lymph nodes in the LV group (P=0.01), but not in the HV group. In conclusion, experimental infection with either high or low virulence BVDV strains induced a significant expression of the type I interferon-induced genes in beef calves. There was a differential expression of some interferon-induced genes (OAS-1 and ISG-15) and pro-apoptosis markers based on BVDV virulence and genotype., (Published by Elsevier B.V.)

Recently, confirmed occurrences of persistent bovine viral diarrhea virus (BVDV) infection in North American alpacas have raised concerns about the role of persistently infected (PI) alpacas in transmission of virus among herds, yet only limited pathological descriptions of persistent infections in alpacas have been reported. The objective of this study was to characterize BVDV antigen distribution in 10 PI alpacas of varying age and to compare viral antigen distribution and localization in tissues of PI alpacas with 5 PI calves of varying age. Ocular dysplasia was evident in 1 PI alpaca, constituting the first reported congenital ocular lesion in PI alpacas. Viral antigen was widely distributed in alpaca tissues and was prominent in neurons, endothelial cells, and vascular tunica media myocytes but had limited distribution in lymphoid tissues and moderate distribution in epithelium of several organ systems of alpacas. Macrophages in the alpaca gastrointestinal system submucosa and lymph node medullary sinuses often had prominent labeling. In addition, only 1 alpaca had antigen labeling in the bone marrow in contrast to PI cattle. Labeled cells in calf tissues were more widely distributed, occurring prominently in lymphoid and epithelial tissues. Common features of the 2 host species were widespread antigen labeling and absence of lymphoid depletion.

The genus Pestivirus is composed of 4 important pathogens of livestock: Bovine viral diarrhea virus 1 and 2 (BVDV-1 and BVDV-2), Classical swine fever virus (CSFV), and Border disease virus of sheep (BDV). BVDV are major pathogens of cattle, and infection results in significant economic loss worldwide. A new putative pestivirus species, tentatively called "HoBi-like," "BVDV-3," or "atypical pestiviruses," was first identified in Europe in fetal bovine serum (FBS) imported from Brazil. HoBi-like viruses are related to BVDV at the genetic and antigenic levels. Further, the disease caused by these new viruses resembles clinical presentations historically associated with BVDV infection, including growth retardation, reduced milk production, respiratory disease, reduced reproductive performance, and increased mortality among young stock. Current BVDV diagnostic tests may fail to detect HoBi-like viruses or to differentiate between BVDV and HoBi-like viruses. Further, commercial tests for BVDV exposure, based on serological response, do not reliably detect HoBi-like virus exposure, and cross protection against HoBi-like viruses conferred by current BVDV vaccines is likely limited. As many HoBi-like viruses, characterized to date, were isolated from FBS originating from Brazil, it is assumed that the agent is probably widespread in Brazilian herds. Nevertheless, reports of natural infection in Southeast Asia and Europe demonstrate that these viruses are not restricted to South America. Increased demand for FBS has led to widespread distribution of FBS originating in HoBi-like virus endemic regions. The contamination of such FBS with HoBi-like viruses may lead to spread of this virus to other regions.

Transplacental viral infections are dependent upon complex interactions between feto-placental and maternal immune responses and the stage of fetal development at which the infection occurs. Bovine viral diarrhea virus (BVDV) has the ability to cross the placenta and infect the fetus. Infection early in gestation with non-cytopathic (ncp) BVDV leads to persistent infection. Establishment of fetal persistent infection results in life-long viremia, virus-specific immunotolerance, and may have detrimental developmental consequences. We have previously shown that heifers infected experimentally with ncp BVDV type 2 on d. 75 of gestation had transient robust up-regulation of the type I interferon (IFN) stimulated genes (ISGs) 3-15 days after viral inoculation. Blood from persistently infected (PI) fetuses, collected 115 days post maternal infection, demonstrated moderate chronic up-regulation of ISGs. This infection model was used to delineate timing of the development of innate immune responses in the fetus and placenta during establishment of persistent infection. It was hypothesized that: (i) chronic stimulation of innate immune responses occurs following infection of the fetus and (ii) placental production of the type I IFN contributes to up-regulation of ISGs in PI fetuses. PI fetuses, generated by intranasal inoculation of pregnant heifers with ncp BVDV, and control fetuses from uninfected heifers, were collected via Cesarean sections on d. 82, 89, 97, 192, and 245 of gestation. Fetal viremia was confirmed starting on d. 89. Significant up-regulation of mRNA encoding cytosolic dsRNA sensors -RIG-I and MDA5 - was detected on d. 82-192. Detection of viral dsRNA by cytosolic sensors leads to the stimulation of ISGs, which was reflected in significant up-regulation of ISG15 mRNA in fetal blood on d. 89, 97, and 192. No difference in IFN-α and IFN-β mRNA concentration was found in fetal blood or caruncular tissue, while a significant increase in both IFN-α and IFN-β mRNA was seen in cotyledons from PI fetuses on d. 192. It is concluded that fetuses respond to early gestational ncp BVDV infection by induction of the type I IFN pathway, resulting in chronic up-regulation of ISGs. Cotyledonary tissue contributes to up-regulation of ISGs by increased production of IFNs. The innate immune response might partially curtail viral replication in PI fetuses, but is not able to eliminate the virus in the absence of a virus-specific adaptive immune response., (Copyright © 2012 Elsevier B.V. All rights reserved.)

To date, limited information is available on the ability of 'Hobi'-like pestiviruses (putative bovine viral diarrhoea 3) to infect and cause disease in animal species traditionally affected by pestiviruses. In order to obtain new insights into host range and pathogenic potential of this atypical pestivirus, BVDV-seronegative calves (n=5), lambs (n=5) and piglets (n=5) were experimentally infected with the European 'Hobi'-like strain Italy-1/10-1, whereas two animals per species served as uninfected controls. Appearance of clinical signs, leukopenia, viremia, viral shedding and seroconversion were monitored for 28 days post-infection. Calves and lambs were successfully infected, displaying respiratory signs (nasal discharge), moderate hyperthermia and leukopenia, viremia and viral shedding through the nasal and faecal routes. Antibody responses were observed in both animal species by ELISA and virus neutralisation assays. In contrast, inoculated piglets did not display any clinical signs nor leukopenia and viral RNA was not detected in any biological samples. Nevertheless, the presence of detectable antibodies by virus neutralisation accounted for a successful, albeit limited infection of these animals., (Copyright © 2011 Elsevier B.V. All rights reserved.)

All pestiviruses are important veterinary pathogens causing economic losses in cattle, sheep, and pigs. In this study, blood samples randomly collected from 465 sheep were analysed for the presence of antibodies to pestiviruses (bovine viral diarrhea virus, border disease virus) using an enzyme-linked immunosorbent assay in the province of Van and their towns. The seroprevalance were estimated as 75.9% and 60.0-82.5% in the sampled animals and sampled towns, respectively. The results revealed that pestiviruses are important abort pathogens in the province of Van and their towns.

The tripartite motif (TRIM) protein family comprises more than 60 members that have diverse functions in various biological processes. Although a small number of TRIM proteins have been shown to regulate innate immunity, much remains to be learned about the functions of the majority of the TRIM proteins. Here we identify TRIM56 as a cellular protein associated with the N-terminal protease (N(pro)) of bovine viral diarrhea virus (BVDV), a pestiviral interferon antagonist which degrades interferon regulatory factor 3 (IRF3) through the proteasome. We found that TRIM56 was constitutively expressed in most tissues, and its abundance was further upregulated moderately by interferon or virus. The manipulation of TRIM56 abundance did not affect the protein turnover of N(pro) and IRF3. Rather, ectopic expression of TRIM56 substantially impaired, while knockdown of TRIM56 expression greatly enhanced, BVDV replication in cell culture. The antiviral activity of TRIM56 depended on its E3 ubiquitin ligase activity as well as the integrity of its C-terminal region but was not attributed to a general augmentation of the interferon antiviral response. Overexpression of TRIM56 did not inhibit the replication of vesicular stomatitis virus or hepatitis C virus, a virus closely related to BVDV. Together, our data demonstrate that TRIM56 is a novel antiviral host factor that restricts pestivirus infection.

In 2003 an outbreak of sudden deaths occurred in 2-3-week-old pigs on a piggery in New South Wales, Australia. There was a marked increase in the birth of stillborn pigs and preweaning losses associated with a multifocal non-suppurative myocarditis with myonecrosis. The aim of this study was to amplify any infectious agents present in field material to aid the detection and identification of the causative agent of the porcine myocarditis syndrome (PMC). Foetuses were directly inoculated in utero with tissue extracts from field cases of PMC at 56-60, 70-84 or 85-94 days of gestation and euthanased 7-28 days later. The IgG concentration in foetal sera/body fluids was measured, hearts were examined by light microscopy and selected hearts were examined by electron microscopy. An infectious agent was detected in tissues from cases of PMC and its identification as the novel pestivirus Bungowannah virus has recently been reported (Kirkland et al., 2007). Sow sera, foetal tissues and foetal sera/body fluids were tested for Bungowannah virus RNA by qRT-PCR and antibody by peroxidase-linked assay. Bungowannah virus was detected in numerous organs of the porcine foetus. Following direct foetal exposure it is probable that this virus spreads by direct intra-uterine transmission to adjacent foetuses and by trans-uterine transmission to the dam. Data were obtained for both the replication of the virus in the porcine foetus and the humoral immune response in the foetus and sow., (Crown Copyright (c) 2009. Published by Elsevier B.V. All rights reserved.)

Heterogeneity is a hallmark that encompasses all aspects of pestiviruses, including the genetic properties, spectrum of hosts, disease signs and virulence. As an additional feature, pestiviruses are found world-wide, in both domesticated and wild animals, which indicates that these viruses are highly successful. Starting with the strategy by which pestiviruses persist in their host population, we discuss the role virulence and attenuation play in viral transmission and focus on the interaction of these viruses with their hosts' immune systems. The most unusual feature of pestiviruses is their ability to infect their hosts either transiently or persistently. Persistent infection, best known in cattle infected with bovine viral diarrhea virus, is characterized by immunotolerance to the infecting viral strain. This strategy of evading from the adaptive immune response is unique among persistent viral infections. It obviates antigenic variation, as used by lentiviruses, or hiding by latency, as seen in herpesviruses. In addition to establishing and maintaining immunotolerance, bovine viral diarrhea virus also evades innate immunity during persistent infection. Investigations in cultured cells showed that this virus fails to induce interferon type-I. Moreover, while being resistant to the action of interferon during persistent infection in its own host cells, bovine viral diarrhea virus does not interfere with interferon action against unrelated viruses infecting these cells. This novel finding of discrimination between "self" and "non-self" may contribute to the good health status seen in many persistently infected cattle. In evolutionary terms, not globally interfering with a crucial first line of antiviral defence of its persistently infected hosts may improve the chances for bovine viral diarrhea viruses to transmit to new hosts., (Copyright 2009 Elsevier B.V. All rights reserved.)

The aim of the present study was to define efficient immunophysiological parameters in neonatal Holstein calves with an experimentally induced microbial infection. Calves (n = 15) were challenged with classical swine fever virus (LOM strain) and Erysipelothrix insidiosa live vaccine by intravenous injection at 3 wk of age except for control calves (n = 4). The level of total serum IgA was significantly increased at 14 and 19 d post-experimental challenge (DPEC) compared with that in calves at -2 DPEC. At 5 DPEC, relative amounts of bacterial- and viral-specific IgA increased significantly and were sustained until 26 DPEC. In the hematology assay, the neutrophil:lymphocyte ratio (%) in whole blood was significantly decreased at 14 DPEC because of a significant increase in lymphocytes and a coincident decrease in neutrophils. The percentages of CD4+ and CD25+ T cells were significantly decreased at 14 DPEC and returned to initial levels at 19 DPEC. It is intriguing to note that the level of serum lactoferrin was significantly decreased by the microbial challenge within 1 d. The concentration of haptoglobin was increased within 3 d and gradually decreased in calves after microbial challenge. Our results suggest that 1) bovine serum lactoferrin plays an important role in the innate immune response against microbial infection at an early stage and 2) experimentally induced microbial challenge using porcine live bacterial and viral vaccine in calves could be a good experimental model to evaluate the effect of diet or stress induced by environmental change on the immune responses against microbial infection.

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.

In 2003 an outbreak of sudden deaths occurred in 2-3-week-old piglets on a piggery in New South Wales, Australia. There was a marked increase in the birth of stillborn piglets and preweaning losses associated with a multifocal non-suppurative myocarditis with myonecrosis. The aim of this study was to review existing data and to undertake further investigations of specimens from naturally infected pigs to provide evidence to support the hypothesis that Bungowannah virus, a recently recognised pestivirus, causes the porcine myocarditis syndrome (PMC). Sera collected from gilts and sows from affected and unaffected units were tested for Bungowannah virus antibody by a peroxidase-linked assay and Bungowannah virus RNA by qRT-PCR in selected cases. Stillborn piglets from affected and an unaffected unit were also tested for Bungowannah virus antibody and RNA. Body fluid IgG levels and the incidence of myocardial lesions in these stillborn piglets are summarised. Tissue sections from stillborn piglets with myocarditis/myonecrosis were examined for Bungowannah virus RNA by in situ hybridisation. A clear temporal association between the occurrence of PMC on a unit or module and exposure to Bungowannah virus was identified by serological tests in both breeding aged animals and stillborn pigs. In addition, at the individual animal level on affected units, Bungowannah virus RNA was detected in stillborn piglets in large amounts by qRT-PCR and in association with myocardial lesions by in situ hybridisation. The examination of field material from cases of PMC by serology, qRT-PCR and in situ hybridisation provides strong indirect evidence that Bungowannah virus is the causative agent for PMC.

This moleculo-epidemiological and immunological study through cytokine response assessment was done to know the dynamics of cytokines in the initiation, persistence and association to physiological changes of a particular pathogen in water buffaloes. This is important to understand the magnitude and behavior of disease progression. Water buffalo blood samples gathered from different places in the Philippines revealed a 9.4%, 27.6%, 10.3% and 4.4% prevalence of bovine viral diarrhea virus (BVDV), bovine leukemia virus (BLV), Anaplasma marginale and Babesia bigemina infection, respectively. This was the first surveillance study of BVDV and BLV in the country. Furthermore, cytokine expression of these naturally infected animals was also quantified. BVDV-infected animals had up-regulated expressions of TNFalpha, IL-2 and IL-4; and down-regulated expressions of IFNgamma and IL-12p40 while BLV positive animals had an up-regulated IL-4 and IL-6, and highly expressed IL-10 and IL-12p40 with unchanged IFNgamma expression. Meanwhile, animals infected with A. marginale had all interleukins and IFNgamma up-regulated with significant expression of IL-10 and IL-12p40 similar to the BLV positive animals. Since it was also observed that swamp-type buffaloes were more disease tolerant than riverine-type buffaloes based on the gathered infection rate of each examined pathogen, further assessment was done focusing on the two vital cytokines, IFNgamma and TNFalpha. We quantified IFNgamma and TNFalpha expressions in ConA-stimulated PBMC from both swamp and riverine buffaloes by real-time PCR. Cytokine expression from ConA-stimulated PBMC revealed that both IFNgamma and TNFalpha were more highly expressed in swamp than in riverine buffalo. To further examine the probable cause of expression differences, the proximal promoter region of these two cytokines were sequenced for the presence of nucleotide polymorphism followed by luciferase assay to analyze the effect of these polymorphisms in gene transcription. A single nucleotide polymorphism was found in the IFNgamma (-299) while eight polymorphisms in the TNFalpha promoter (-541, -553, -562, -596, -609, -655, -659, -688). Luciferase assay showed that both IFNgamma promoter and TNFalpha promoter in swamp-type water buffalo had higher transcription activity compared to riverine-type water buffalo. These findings confirm that IFNgamma and TNFalpha transcriptions in these animals were highly affected by the disparity in the cytokine promoter region. This suggests that disease tolerance or susceptibility of these buffaloes could be due to the differences in their relative cytokine transcription and may relate to pathogen-host specific pathogenesis.

Bovine viral diarrhea virus (BVDV) is an important pathogen of domestic cattle. Serologic, experimental, and individual case studies explored the presence and pathogenesis of the virus in wild ungulates; however, there remain large gaps in knowledge regarding BVDV infection in nonbovine species. Live twins were born from a white-tailed deer (Odocoileus virginianus) doe infected with noncytopathic BVDV during its first trimester of pregnancy. The twins died at 1 day of age from trauma unrelated to the infection, and tissues were collected for histologic and immunohistochemical examination. The most significant histologic abnormality was diffuse depletion of B-lymphocytes in both fawns. The BVDV antigen was distributed widely throughout many tissues and cell types, most notably epithelium and vascular endothelium, consistent with that reported in cattle. In contrast to cattle, lymphocytes exhibited only very rare positive staining.

Due to antigenic differences between BVDV1 and BVDV2 strains, both pestivirus species are included in U.S. vaccines. The efficacy of these vaccines in preventing acute infections is evaluated based on reduction of clinical disease. While high virulence BVDV2 strains are used in U.S. vaccine efficacy studies, the BVDV1 strain used (NY-1) produces very little in the way of clinical disease. In order to identify a BVDV1 strain that generates a more pronounced clinical presentation, three field strains were compared to NY-1. Infection with two of the field strains resulted in significantly more pronounced clinical disease compared to NY-1. Decreasing the inoculation of a field strain by two logs did not significantly change clinical presentation.

Although commonly associated with infection in cattle, bovine viral diarrhea viruses (BVDV) also replicate in many domestic and wildlife species, including cervids. Bovine viral diarrhea viruses have been isolated from a number of cervids, including mule deer (Odocoileus hemionus), European roe deer (Capreolus capreolus), red deer (Cervus elaphus), white-tailed deer (Odocoileus virginianus), and mouse deer (Tragulus javanicus), but little information is available regarding clinical presentation and progression of infection in these species. In preliminary studies of experimental infection of deer with BVDV, researchers noted seroconversion but no clinical signs. In this study, we infected white-tailed deer fawns that were negative for BVDV and for antibodies against BVDV, with either a type 1 or a type 2 BVDV that had been isolated from white-tailed deer. Fawns were monitored for changes in basal temperature, circulating lymphocytes, and platelets. The clinical progression following inoculation in these fawns was similar to that seen with BVDV infections in cattle and included fever and depletion of circulating lymphocytes. Because free-ranging cervid populations are frequently in contact with domestic cattle in the United States, possible transfer of BVDV between cattle and cervids has significant implications for proposed BVDV control programs.

In this study serological investigations were performed to determine the prevalence of pestiviral infections in sheep in one Federal State of Austria, namely Carinthia. 1527 blood samples from sheep in 147 flocks were collected and tested by Enzyme-linked immunosorbent assay and virus-neutralisation tests for antibodies to ruminant pestiviruses. The estimated flock prevalence was 47.6%, the individual prevalence 16.3%. Significant geographical variations in the flock as well in the individual prevalence were found. The highest prevalence in sheep and in sheep flocks was established in the region Spittal/Drau with 25.9% and 69.7%.The individual and the flock prevalence was significantly higher on farms where cattle or sheep from other farms were present than on farms with no cattle (p < 0.017). All Enzyme-linked immunosorbent assay positive sera were tested for Bovine viral diarrhea virus-1 (strain NADL), Bovine viral diarrhea virus-2 (strain 125) and for Border disease virus (strain MOREDUN) by virus neutralisation tests. Seventy out of 249 positive samples revealed the highest titres (> or = two-fold) to Bovine viral diarrhea virus-1 and 25 to Border disease virus. The remaining positive samples did not show clear results because of cross reactions.

We previously suggested that antenatal pestivirus infection might play a role in the pathogenesis of perinatal brain white matter damage (WMD) in preterm infants. We have now examined 22 brains from stillborns and deceased newborns (both preterm and term) for the presence of bovine virus diarrhoea virus (BVDV) antigen. The brains of five females and five males with WMD (median gestational age 36.5wks), and nine female and three male controls (median gestational age 36.5wks) were used in the study. No BVDV antigen was detected in any of the 22 brains. We conclude that brain infection with BVDV is unlikely to play a role in WMD pathogenesis among preterm or term newborns. Further research is needed to test the hypothesis that intrauterine exposure to pestivirus antigen elicits a fetal inflammatory response which then contributes to WMD.

The aim of the study was to analyse the dynamics of selected lymphocytes subpopulations in peripheral blood of pigs infected with BVDV, using flow cytometric method. The examinations were performed on eighteen healthy, pestivirus-free pigs divided into 3 groups. Pigs in the group 1 were intranasally infected with two virulent reference strains of BVDV: NADL2 and NADL8. Pigs of group 2 were included into experimentally infected ones. Group 3 consisted of control pigs free from infection. The percentage of CD2+ lymphocytes was gradually decreasing during the experiment. Finally a decrease by 20% was noted in group 2 and by 9% in group 1. A slight decrease of CD4+ cells and more significant decrease of CD8+ subpopulation were observed. The CD4:CD8 ratio changed from approximately 3:5 to 1:1 in pigs of group 2, and from 1:2 to 1:3 in pigs of group 1. There were only small fluctuations regarding TcR(gamma)delta+ cells. The level of these lymphocytes increased during first hours post infection and then decreased to the initial value. Obtained results may indicate that infection with species-unspecific pestiviruses causes similar, but less significant, changes within lymphocyte subpopulations than infection with species-specific pestiviruses.