Bovine Viral Diarrhea Virus E2 Research Articles

Exosomes-mediated transmission of standard bovine viral diarrhea strain OregonC24Va in bovine trophoblast cells

Bovine viral diarrhoea virus (BVDV) can infect cows on days 30–110 of gestation and crossing the placental barrier, resulting in persistently infected (PI) and causing significant economic losses to dairy farming. Bovine placental trophoblast cells (BTCs) are the major cells in the early chorionic tissue of the placenta and play important roles in placental resistance to viral transmission. In this study, we have confirmed that BTCs is among a groups of cell types those could be infected by BVDV in vivo, and BVDV infection stimulates the autophagic responses in BTCs and promotes the release of exosomes. Meanwhile, the exosomes derived from BTCs can be used by BVDV to spread between placental trophoblast cells, and this mode of transmission cannot be blocked by antibodies against the BVDV E2 protein, whereas the replication and spread of BVDV in BTCs can be blocked by inhibiting autophagy and exosomogenesis. Our study provides a theoretical and practical basis for scientific prediction and intervention of reproductive disorders caused by BVDV infection in cows of different gestation periods from a novel perspective.

Evaluation of the Mucosal Immunity Effect of Bovine Viral Diarrhea Virus Subunit Vaccine E2Fc and E2Ft

Classified as a class B infectious disease by the World Organization for Animal Health (OIE), bovine viral diarrhea/mucosal disease is an acute, highly contagious disease caused by the bovine viral diarrhea virus (BVDV). Sporadic endemics of BVDV often lead to huge economic losses to the dairy and beef industries. To shed light on the prevention and control of BVDV, we developed two novel subunit vaccines by expressing bovine viral diarrhea virus E2 fusion recombinant proteins (E2Fc and E2Ft) through suspended HEK293 cells. We also evaluated the immune effects of the vaccines. The results showed that both subunit vaccines induced an intense mucosal immune response in calves. Mechanistically, E2Fc bonded to the Fc γ receptor (FcγRI) on antigen-presenting cells (APCs) and promoted IgA secretion, leading to a stronger T-cell immune response (Th1 type). The neutralizing antibody titer stimulated by the mucosal-immunized E2Fc subunit vaccine reached 1:64, which was higher than that of the E2Ft subunit vaccine and that of the intramuscular inactivated vaccine. The two novel subunit vaccines for mucosal immunity developed in this study, E2Fc and E2Ft, can be further used as new strategies to control BVDV by enhancing cellular and humoral immunity.

Novel bovine viral diarrhea virus (BVDV) virus-like particle vaccine candidates presenting the E2 protein using the SpyTag/SpyCatcher system induce a robust neutralizing antibody response in mice.

Bovine viral diarrhea virus (BVDV) is a pathogen of commercial consequence in cattle. Although many modified live and killed vaccines are commercially available, their drawbacks precipitate the need for new effective vaccines. Virus-like particles (VLPs) are a safe and powerful technology used in several human and veterinary vaccines; however, it is difficult to produce large amounts of BVDV VLPs. In this study, we generated red-spotted grouper nervous necrosis virus (RGNNV) VLPs presenting the BVDV E2 protein (domain I to IIIb) of the Nose (BVDV-1) or KZ-91-CP (BVDV-2) strain by exploiting SpyTag/SpyCatcher technology. Mice immunized twice with 30 μg of RGNNV VLPs conjugated with 10 μg of E2 proteins of the Nose or KZ-91-CP strain with a 14-day interval elicited high (1:512,000 to 1:1,024,000) and moderate (1:25,600 to 1:102,400) IgG titers against E2 proteins of homologous and heterologous strains, respectively. In addition, this prime-boost regimen induced strong (1:800 to 1:3,200) and weak (~1:10) neutralization titers against homologous and heterologous BVDV strains, respectively. Our results indicate that conjugation of the E2 protein to RGNNV VLPs strongly enhances the antigenicity of the E2 protein and that RGNNV VLPs presenting the E2 protein are promising BVDV vaccine candidates.

Immunogenicity of a secreted, C-terminally truncated, form of bovine viral diarrhea virus E2 glycoprotein as a potential candidate in subunit vaccine development

Both current live, attenuated, and killed virus vaccines for bovine viral diarrhea virus (BVDV) have their limitations. Here, we report the development of a BVDV subunit vaccine by (i) the expression of a secreted form of a recombinant E2 glycoprotein using BHK21 cells and (ii) determination of the immune responses in mice. The E2 glycoprotein was modified by deletion of the C-terminal transmembrane anchor domain and fusion to a V5 epitope tag. This allowed detection using anti-V5 monoclonal antibodies together with simple purification of the expressed, secreted, form of E2 from the cell media. Furthermore, we genetically fused green fluorescent protein (GFP) linked to E2 via a Thosea asigna virus 2A (T2A) ribosome skipping sequence thereby creating a self-processing polyprotein [GFP-T2A-BVDV-E2trunk-V5], producing discrete [GFP-T2A] and [E2trunk-V5] translation products: GFP fluorescence acts, therefore, as a surrogate marker of E2 expression, BALB/c mice were inoculated with [E2trunk-V5] purified from cell media and both humoral and cellular immune responses were observed. Our antigen expression system provides, therefore, both (i) a simple antigen purification protocol together with (ii) a feasible strategy for further, large-scale, production of vaccines.

Berbamine hydrochloride inhibits bovine viral diarrhea virus replication via interfering in late-stage autophagy

Bovine viral diarrhea virus (BVDV) is a harmful pathogen that easily causes large-scale infections and huge economic losses to the cattle industry. Berbamine hydrochloride (BBH) is a natural product extracted from berberis and has a wide range of pharmacological effects. However, the antiviral effect of BBH against BVDV needs to be further elucidated. This study aimed to evaluate the antiviral activities of BBH against BVDV infection. We mainly used RT-qPCR, Western blotting, immunofluorescence, and TEM assays to assess the inhibitory activity of BBH against BVDV. The results showed that BBH had an inhibitory effect on BVDV and higher inhibitory activity in the viral attachment and release in MDBK cells. This study found that BVDV could induce and use autophagy to replicate itself. Further results showed that BBH inhibited BVDV infection by inhibiting autophagy integrity in BVDV-infected cells, which was proven by the detection of autophagy-related proteins. Our data show that in BBH-treated BVDV-infected cells, the expression of p62 and LC3 increased over time. After the addition of an autophagy inhibitor, chloroquine (CQ), and an autophagy promoter, rapamycin (Rapa), we found that promoting autophagy was beneficial to the replication of BVDV, while inhibiting autophagy could reduce the number of infections by BVDV, which was evidenced by the expression of the BVDV E2 protein. Furthermore, BBH blocked the normal binding of LC3 and LAMP1 in BVDV-infected cells. In conclusion, BBH inhibited BVDV infection by inhibiting BVDV-induced autophagy in cells, and its inhibitory effect was obvious in the viral attachment and release stages. Therefore, our study provides a new idea for exploring novel anti-BVDV drugs.

Co-administration of a plasmid encoding CD40 or CD63 enhances the immune responses to a DNA vaccine against bovine viral diarrhea virus in mice.

Bovine viral diarrhea virus (BVDV) causes substantial economic losses in the livestock industry worldwide. Plasmids encoding the BVDV E2 protein are potential DNA vaccines against BVDV, but their immunogenicity has been insufficient. Here, we investigated the adjuvant effect of CD40 and CD63 plasmids on the immune responses to a BVDV E2 DNA vaccine in mice. We constructed pUMVC4a-based plasmids encoding the BVDV E2 protein (pE2), mouse CD40 (pCD40), or mouse CD63 (pCD63). Protein expression by each plasmid was confirmed through Western blot analysis and immunofluorescence staining of cultured cell lines. BALB/c mice were immunized intradermally twice with pE2 in combination with, or without, pCD40 or pCD63, with 3 weeks between the two doses. pE2 with pCD40 induced significantly higher neutralizing antibody titers against BVDV than pE2 alone. pE2 with pCD63 induced significantly higher anti-E2 IgG2a antibody titers than pE2 alone. Furthermore, pE2 with pCD40 or pCD63 induced significantly increased lymphocyte proliferation and interferon (IFN)-γ production in response to BVDV, compared with E2 alone. These results suggest that a plasmid encoding CD40 or CD63 can be used as an adjuvant to enhance immune responses to DNA vaccines against BVDV.

Establishment and Preliminary Application of Multiplex Fluorescent Quantitative PCR for Simultaneous Detection of BVDV, BRV and BCV

Background: In this study, we aimed to establish a multiplex fluorescence quantitative polymerase chain reaction (PCR) method for the identification and detection of bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV) and bovine coronavirus (BCV). Methods: Based on the highly conserved sequences of BVDV E2 gene, BRV VP6 gene and BCV N gene in GenBank, specific primers were designed to amplify the target gene fragments of each virus and the reaction conditions and system were optimized. Multiple fluorescence quantitative methods were established by fluorescence quantitative PCR. Result: The minimum detection limits of plasmid standards for BVDV, BRV and BCV by multiplex fluorescence quantitative PCR were 1.19×102 copies/μL, 3.89×101 copies/μL and 3.74×101 copies/μL, respectively. The lowest sensitivity of the established method was 100 times higher than that of conventional PCR and had high sensitivity. Furthermore, BVDV, BRV and BCV were amplified specifically, with no cross-reactivity with Escherichia coli (E. coli), Salmonella and infectious bovine rhinotracheitis virus (IBRV). The intra-and inter-group coefficients of variation were less than 1%, showing good assay repeatability. Using the established method and ordinary multiplex PCR to simultaneously detect 150 clinical diarrheal disease material samples, the coincidence rate of samples with mixed infection of the three viruses was 83.3%. The results showed that the multiplex fluorescent quantitative PCR detection method established in this study provides a rapid, sensitive and specific technique for clinical diagnosis and epidemiological monitoring of BVDV, BRV and BCV.

Immunogenicity evaluation of recombinant Lactobacillus casei W56 expressing bovine viral diarrhea virus E2 protein in conjunction with cholera toxin B subunit as an adjuvant

BackgroundBovine viral diarrhea virus (BVDV) is one of the main causes of infectious diseases in cattle and causes large financial losses to the cattle industry worldwide. In this study, Lactobacillus casei strain W56 (Lc W56) was used as antigen deliver carrier to construct a recombinant Lactobacillus vaccine pPG-E2-ctxB/Lc W56 constitutively expressing BVDV E2 protein fused with cholera toxin B subunit (ctxB) as an adjuvant, and its immunogenicity against BVDV infection in mice model by oral route was explored.ResultsOur results suggested that pPG-E2-ctxB/Lc W56 can effectively activate dendritic cells (DCs) in the Peyer’s patches, up-regulate the expression of Bcl-6, and promote T-follicular helper (Tfh) cells differentiation, as well as enhance B lymphocyte proliferation and promote them differentiate into specific IgA-secreting plasma cells, secreting anti-E2 mucosal sIgA antibody with BVDV-neutralizing activity. Moreover, significant levels (p < 0.01) of BVDV-neutralizing antigen-specific serum antibodies were induced in the pPG-E2-ctxB/LC W56 group post-vaccination. The recombinant Lactobacillus vaccine can induce cellular immune responses, and significant levels (p < 0.01) of Th1-associated cytokines (IL-2, IL-12, and IFN-γ), Th2-associated cytokines (IL-4, IL-10) and Th17-associated cytokine (IL-17) were determined in the serum of vaccinated mice. Significantly, the recombinant Lactobacillus vaccine provides immune protection against BVDV infection, which can be cleared effectively by the vaccine post-challenge in orally vaccinated animals.ConclusionsThe genetically engineered Lactobacillus vaccine constructed in this study is immunogenic in mice and can induce mucosal, humoral, and cellular immune responses, providing effective anti-BVDV immune protection. It thus represents a promising strategy for vaccine development against BVDV.

Attenuated porcine-derived type 2 bovine viral diarrhea virus as vector stably expressing viral gene

Infectious bovine viral diarrhea virus (BVDV) cDNA clones have been used for the expression of classical swine fever virus (CSFV) genes for immune prevention and control. However, can it be used for the expression of an allogenetic fragment? To answer this question, a BVDV chimeric virus expressing the spike (S) antigen fragment of porcine epidemic diarrhea virus (PEDV) was constructed. Antigen S499-602 was inserted into pig-derived BVDV-2 infectious cDNA clone pASH28 in tandem by overlapping PCR, located between the seventh and eighth amino acids at the N-terminus of the capsid (C) protein of BVDV. Indirect immunofluorescence assay confirmed that the chimeric virus vASH-dS312 containing double S499-602 sequences was successfully assembled, which could react with the monoclonal antibody (MAb) against BVDV E2 and PEDV S proteins. Further western blot analysis confirmed that the exogenous S499-602 double protein could be stably expressed. Next, the chimeric virus vASH-dS312 was administered to BALB/C mice either orally or by intramuscular injection. The immunized mice were healthy and showed no signs of toxicity. IgG against BVDV and PEDV antibodies could be detected in the mice administered vASH-dS312 by intramuscular injection, which had neutralization activity against BVDV and PEDV. Thus, this study reported a new insertion site in the BVDV infectious cDNA clone that could successfully express an allogenetic antigen.

Bioprocess optimization for purification of chimeric VLP displaying BVDV E2 antigens produced in yeast Hansenula polymorpha

Chimeric virus-like particles (VLP) are known as promising tools in the development of safe and effective subunit vaccines. Recently, a technology platform to produce VLP based on the small surface protein (dS) of the duck hepatitis B virus was established. In this study, chimeric VLP were investigated displaying the 195 N-terminal amino acids derived from the glycoprotein E2 of the bovine viral diarrhea virus (BVDV) on their surface. Isolation of the VLP from methylotrophic yeast Hansenula polymorpha was allowed upon co-expression of wild-type dS and a fusion protein composed of the BVDV-derived antigen N-terminally fused to the dS. It was shown the VLP could be purified by a process adapted from the production of a recombinant hepatitis B VLP vaccine. However, the process essentially depended on costly ultracentrifugation which is critical for low cost production. In novel process variants, this step was avoided after modification of the initial batch capture step, the introduction of a precipitation step and adjusting the ion exchange chromatography. The product yield could be improved by almost factor 8 to 93 ± 12 mg VLP protein per 100 g dry cell weight while keeping similar product purity and antigenicity. This allows scalable and cost efficient VLP production.

Dendritic Cell Targeting of Bovine Viral Diarrhea Virus E2 Protein Expressed by Lactobacillus casei Effectively Induces Antigen-Specific Immune Responses via Oral Vaccination.

Bovine viral diarrhea caused by bovine viral diarrhea virus (BVDV) is an important disease in cattle, resulting in significant economic losses to the cattle industry worldwide. In order to develop an effective vaccine against BVDV infection, we constructed a dendritic cell (DC)-targeting oral probiotic vaccine (pPG-E2-DCpep/LC W56) using Lactobacillus casei as antigen delivery carrier to express BVDV glycoprotein E2 fused with DC-targeting peptide, and the immunogenicity of orally administered probiotic vaccine was evaluated in mice model. Our results showed that after immunization with the probiotic vaccine, significantly levels of antigen-specific sera IgG and mucosal sIgA antibodies (p < 0.05) with BVDV-neutralizing activity were induced in vivo. Challenge experiment showed that pPG-E2-DCpep/LC W56 can provide effective immune protection against BVDV, and BVDV could be effectively cleared from the intestine of immunized mice post-challenge. Moreover, the pPG-E2-DCpep/LC W56 could efficiently activate DCs in the intestinal Peyer’s patches, and significantly levels of lymphoproliferative responses, Th1-associated IFN-γ, and Th2-associated IL-4 were observed in mice immunized with pPG-E2-DCpep/LC W56 (p < 0.01). Our results clearly demonstrate that the probiotic vaccine could efficiently induce anti-BVDV mucosal, humoral, and cellular immune responses via oral immunization, indicating a promising strategy for the development of oral vaccine against BVDV.

Identification of structural glycoprotein E2 domain critical to mediate replication of Classical Swine Fever Virus in SK6 cells

Envelope glycoprotein E2 of Classical Swine Fever Virus (CSFV) is involved in several critical virus functions. To analyze the role of E2 in virus replication, a series of recombinant CSFVs harboring chimeric forms of E2 CSFV and Bovine viral diarrhea virus (BVDV) were created and tested for their ability to infect swine or bovine cell lines. Substitution of native CSFV E2 by BVDV E2 abrogates virus replication in both cell lines. Substitution of individual domains in CSFV Brescia E2 by the homologous from BVDV produces chimeras that efficiently replicate in SK6 cells with the exception of a chimera harboring BVDV E2 residues 93–168. Further mapping revealed a critical area in E2 required for CSFV replication in SK6 cells between protein residues 136–156. This is the first report categorically defining a discrete portion of E2 as essential to pestivirus infection in susceptible cells.

Recombinant E2 protein enhances protective efficacy of inactivated bovine viral diarrhea virus 2 vaccine in a goat model

BackgroundInactivated 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.ResultsFour 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.ConclusionThe combination of iBVDV and rE2 elicited stronger protective immune responses against BVDV than iBVDV or rE2 alone.

Discovery of Novel Bovine Viral Diarrhea Inhibitors Using Structure-Based Virtual Screening on the Envelope Protein E2.

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus within the family Flaviviridae. BVDV causes both acute and persistent infections in cattle, leading to substantial financial losses to the livestock industry each year. The global prevalence of persistent BVDV infection and the lack of a highly effective antiviral therapy have spurred intensive efforts to discover and develop novel anti-BVDV therapies in the pharmaceutical industry. Antiviral targeting of virus envelope proteins is an effective strategy for therapeutic intervention of viral infections. We performed prospective small-molecule high-throughput docking to identify molecules that likely bind to the region delimited by domains I and II of the envelope protein E2 of BVDV. Several structurally different compounds were purchased or synthesized, and assayed for antiviral activity against BVDV. Five of the selected compounds were active displaying IC50 values in the low- to mid-micromolar range. For these compounds, their possible binding determinants were characterized by molecular dynamics simulations. A common pattern of interactions between active molecules and aminoacid residues in the binding site in E2 was observed. These findings could offer a better understanding of the interaction of BVDV E2 with these inhibitors, as well as benefit the discovery of novel and more potent BVDV antivirals.

An improved indirect ELISA for specific detection of antibodies against classical swine fever virus based on structurally designed E2 protein expressed in suspension mammalian cells

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV), a ruminant pestivirus. However, currently available ELISAs based on the full-length E2 protein of CSFV cannot discriminate anti-CSFV from anti-BVDV antibodies. In this study, a truncated CSFV E2 protein (amino acids 690 to 879) covering antigenic domains B/C/D/A (E2B/C/D/A) was designed based on homologous modeling according to the crystal structure of the BVDV E2 protein. The E2B/C/D/A protein was expressed in CHO cells adapted to serum-free suspension culture, and an indirect ELISA (iELISA) was establishedbased on the recombinant protein. No serological cross-reaction was observed for anti-BVDV sera in the iELISA. When testing 282 swine serum samples, the iELISA displayed a high sensitivity (119/127, 93.7%) and specificity (143/155, 92.3%), with an agreement of 92.9% (262/282) and 92.2% (260/282) with virus neutralization test and theIDEXX CSFV blocking ELISA, respectively. Taken together, the newly developed iELISA is highly specific and sensitive and able to differentiate anti-CSFV from anti-BVDV antibodies.

The analyses of relationships among nucleotide, synonymous codon and amino acid usages for E2 gene of bovine viral diarrhea virus

In this study, the systemic analyses of nucleotide, codon and amino acid usages for E2 gene of bovine viral diarrhea virus (BVDV) were carried out for estimating its genetic features. The nucleotide usage pattern at the first codon position was strongly influenced by the overall nucleotide composition, while the nucleotide usage patterns at the second and third codon positions seemed to have little link to the overall nucleotide composition. The result indicated that the mutation pressure from nucleotide composition constraint was not the single evolutionary force for genetic features of BVDV E2 gene. Just 18 out of 59 synonymous codons were similar with synonymous codon usage patterns for E2 gene between BVDV1 and BVDV2, while all synonymous codons which contain CpG dinucleotides were selected at the low level by E2 gene, suggesting that this gene suppressed the usages of codons containing CpG dinucleotides to regulate E2 gene replicate and transcript efficiently and avoid immune response from infected hosts. Amino acid usage patterns of E2 protein were generally different between BVDV1 and BVDV2. The patterns of synonymous codon and amino acid usages for E2 gene might be caused by the equilibrium of evolutionary forces from virus and host. Our work gave new investigations into the role of host origin in the formations of synonymous codon/amino acid usages and the evolutionary trend of BVDV E2 gene. The genetic characteristics that codon/amino acid usages of E2 gene adapted to the internal environment of individual animals might assist in understanding the changes of genetics and antigenicity for newly emerging BVDV.

Replicon Particle Expressing the E2 Glycoprotein of Bovine Viral Diarrhea Virus Immunization and Evaluation of Antibody Response.

The objective of this study was to develop a new antigen delivery system using an alphavirus replicon particle (RP) to induce humoral antibody responses against bovine viral diarrhea virus (BVDV) recombinant antigen produced from envelope glycoprotein E2. An alphavirus RP expressing the E2 glycoprotein of BVDV was used for immunization of pigs. A fluorescent microsphere immunoassay (FMIA) has been applied to detect BVDV E2 antigen-specific antibody isotype in pig immunized with alphavirus RP. Full-length BVDV E2 (aa 1-375) was cleaved into several pieces, eight E2 DNA fragments, including full-length DNA, were cloned into expression vector pHUE, and the recombinant proteins expressed in BL-21 (DE3) Escherichia coli. After successful conjugation of purified proteins with microsphere beads, a multiplex FMIA platform was constructed, and BVDV E2 alphavirus-based RP-immunized animal serum samples were tested in the presence of bead-bound antigen targets. The results were represented as mean fluorescence intensity (MFI); the MFI values were converted to sample value/positive value (S/P) ratios. BVDV E2 (aa 1-183) showed the highest MFI values of eight recombinant E2 fragments when the specific activity of each fragment was tested. In immunized animals, data for BVDV E2-specific IgA, IgG, and IgM in serum and only IgG and IgA in oral fluids were recorded. The MFI values for the positive serum sample showed a 100-fold increase compared with the negative serum sample. Antibody isotype to BVDV E2 antigens showed that IgG > IgM > IgA in serum, whereas IgG > IgA > IgM in oral fluids. The data presented in this study suggested that boosting with the same doses of alphavirus RP in 3-week intervals may potentially enhance antibody response. The experimental results demonstrate that alphavirus RP-expressing BVDV E2 antigen induces antibody response in pig.

Local innate responses and protective immunity after intradermal immunization with bovine viral diarrhea virus E2 protein formulated with a combination adjuvant in cattle

Bovine viral diarrhea virus (BVDV) is one of the most serious pathogens in cattle. Recently, we developed a novel adjuvant platform (TriAdj) that includes a toll-like receptor 3 agonist, poly (I:C); an innate defense regulatory peptide; and water-soluble polymer, poly[di(sodiumcarboxylatoethylphenoxy)]-phosphazene (PCEP). To develop a needle-free intradermal (ID) subunit vaccine, the BVDV type-2 E2 protein was formulated with TriAdj, and immune protection was evaluated in calves against a BVDV-2 strain. Intradermal delivery of E2/TriAdj elicited robust virus neutralizing antibodies and cell-mediated immune responses including CD4+ and CD8+ T-cell responses. The development of CD8+ T-cell responses in vaccinated calves indicates that TriAdj promotes cross-presentation. Upon challenge with virulent BVDV-2, the vaccinated calves showed no weight loss, leukopenia or virus shedding, and almost no temperature increase, in contrast to the control animals, which had severe clinical disease and shed virus for three to six days in nasal fluids and white blood cells. Intradermal vaccination was shown to attract various immune cell populations including dendritic cells, the most important antigen presenting cells. These data demonstrate that ID delivery is suitable as an administration route in cattle and that ID delivered, TriAdj-formulated E2 can protect cattle from BVDV-2.

Morphology and Molecular Composition of Purified Bovine Viral Diarrhea Virus Envelope.

The family Flaviviridae includes viruses that have different virion structures and morphogenesis mechanisms. Most cellular and molecular studies have been so far performed with viruses of the Hepacivirus and Flavivirus genera. Here, we studied bovine viral diarrhea virus (BVDV), a member of the Pestivirus genus. We set up a method to purify BVDV virions and analyzed their morphology by electron microscopy and their protein and lipid composition by mass spectrometry. Cryo-electron microscopy showed near spherical viral particles displaying an electron-dense capsid surrounded by a phospholipid bilayer with no visible spikes. Most particles had a diameter of 50 nm and about 2% were larger with a diameter of up to 65 nm, suggesting some size flexibility during BVDV morphogenesis. Morphological and biochemical data suggested a low envelope glycoprotein content of BVDV particles, E1 and E2 being apparently less abundant than Erns. Lipid content of BVDV particles displayed a ~2.3 to 3.5-fold enrichment in cholesterol, sphingomyelin and hexosyl-ceramide, concomitant with a 1.5 to 5-fold reduction of all glycerophospholipid classes, as compared to lipid content of MDBK cells. Although BVDV buds in the endoplasmic reticulum, its lipid content differs from a typical endoplasmic reticulum membrane composition. This suggests that BVDV morphogenesis includes a mechanism of lipid sorting. Functional analyses confirmed the importance of cholesterol and sphingomyelin for BVDV entry. Surprisingly, despite a high cholesterol and sphingolipid content of BVDV envelope, E2 was not found in detergent-resistant membranes. Our results indicate that there are differences between the structure and molecular composition of viral particles of Flaviviruses, Pestiviruses and Hepaciviruses within the Flaviviridae family.

Expression of Bovine Viral Diarrhea Virus Envelope Glycoprotein E2 in Yeast Pichia pastoris and its Application to an ELISA for Detection of BVDV Neutralizing Antibodies in Cattle

The aim of this article is to express envelope glycoprotein E2 of bovine viral diarrhea virus (BVDV) in yeast Pichia pastoris and its utility as a diagnostic antigen in ELISA. The BVDV E2 gene was cloned into the pPICZαA vector followed by integration into the Pichia pastoris strain X-33 genome for methanol-induced expression. SDS-PAGE and Western blot results showed that the recombinant BVDV E2 protein (72 kDa) was expressed and secreted into the medium at a concentration of 40 mg/L of culture under optimized conditions. An indirect ELISA was then developed by using the yeast-expressed E2 protein. Preliminary testing of 300 field cattle serum samples showed that the E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02% compared to the reference virus neutralization test. The concordance between the E2 ELISA and VNT was 91.67%. This study demonstrates feasibility of BVDV E2 protein expression in yeast Pichia pastoris for the first time and its efficacy as an antigen in ELISA for detecting BVDV neutralizing antibodies in cattle.