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

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

BVDV-1 induces interferon-beta gene expression through a pathway involving IRF1, IRF7, and NF-κB activation.

The bovine viral diarrhea virus (BVDV-1) is a pathogen with the capacity to modulate the interferon type I system. To further investigate the effects of BVDV-1 on the production of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the IFNbeta expression profiles were analyzed. The results showed that cpBVDV-1 was able to induce the production of IFNbeta in a way similar to polyinosinic-polycytidylic acid, but with less intensity. Interestingly, all cpBVDV-1 activities were blocked by pharmacological inhibitors of the IRF-1, IRF-7, and NF-κB signaling pathway, and the level of IFNbeta decreased at the level of transcript and protein. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that cpBVDV-1 regulates IFNbeta expression in bovines through the activation of several key transcription factors. Collectively, the results suggest that during cpBVDV-1 infection, cross talk is evident between various signaling pathways involved in transcriptional activation of IFNbeta in cattle.

Evaluation of protection against bovine viral diarrhea virus type 2 after vaccination of the calves with bovine viral diarrhea virus type 1 combo inactivated vaccine / Avaliação da proteção contra o vírus da diarreia viral bovina tipo 2 após vacinação de bezerros com vacina combinada inativada do vírus da diarreia viral bovina tipo 1

This study was designed to evaluate the extent of the protection for bovine viral diarrhea virus type 2 (BVDV-2) infection, afforded by vaccination with a combo inactivated vaccine, which contains bovine viral diarrhea virus type 1 (BVDV-1) and infectious bovine rhinotracheitis virus (IBRV). Five 3-4-month-old calves were intramuscularly vaccinated with a single dose of the combo vaccine and boosted with same dose three weeks after the first vaccination, with five mock immunized calves serving as a control group. Twenty-one days after the second vaccination, all calves were challenged with BVDV-2 SX08 strain by spray into nostril. The unvaccinated animals developed typical clinical signs of high rectal temperature, diarrhoea with erosions and a dramatic drop in leukocyte counts. These signs occured markedly less in all vaccinated animals, the rectal temperature, leukopenia and virarmia of which, were significantly less than the mock immunized calves. It can be concluded that vaccination with the combo inactivated vaccine affords cross-protection against clinical effects of a challenge-infection with BVDV-2 SX08 strain, although it was part protection.(AU)

Este estudo foi desenvolvido para avaliar a extensão da proteção contra a infecção pelo vírus da diarréia viral bovina tipo 2 (BVDV-2) através da vacinação com uma vacina combinada inativada contendo o vírus da diarréia viral bovina tipo 1 (BVDV-1) e vírus da rinotraqueíte de bovinos infecciosos (IBRV). Cinco bezerros com 3 a 4 meses de idade foram vacinados via intramuscular com uma dose única da vacina combinada e reforçados com a mesma dose três semanas após a primeira vacinação, com cinco bezerros imunizados em simulação servindo como grupo controle. Vinte e um dias após a segunda vacinação, todos os bezerros foram desafiados com a cepa BVDV-2 SX08 por spray na narina. Os animais não vacinados desenvolveram sinais clínicos típicos, como alta temperatura retal, diarréia com erosões e queda drástica na contagem de leucócitos. Estes sinais tiveram ocorrência significativamente menor em todos os animais vacinados, cuja temperatura retal, leucopenia e virarmia eram significativamente menores do que os bezerros simulados. É possível concluir que a vacinação com a vacina combinada inativada proporciona proteção cruzada contra os efeitos clínicos de uma infecção provocada pela cepa BVDV-2 SX08, embora tenha sido parcialmente protegida.(AU)

Serological response against bovine herpesvirus and bovine viral diarrhea virus induced by commercial vaccines in Holstein heifers / Resposta sorológica contra herpesvírus bovino e vírus da diarreia viral bovina induzida por vacinas comerciais em novilhas Holandesas

Vaccination is a strategy to the prevention and control of reproductive diseases caused by bovine viral diarrhea virus (BVDV) and bovine herpesvirus type 1 (BoHV-1), however the various compositions of commercial vaccines should be evaluated for their ability to induce protection mediated by antibodies. The objective of this research was to evaluate the production of specific neutralizing Abs against BVDV-1 and 2, and BoHV-1 induced by commercial vaccines composed by different adjuvants. Holstein heifers were vaccinated and distributed in three experimental groups: Group I (G1) was vaccinated with a commercial vaccine containing inactivated BVDV-1, BVDV-2 and BoHV-1 diluted in alum hydroxide as adjuvant (n=9); Group II (G2) was vaccinated with an product containing inactivated strains of BVDV-1, BVDV-2, BoHV-1 and BoHV-5 diluted in oil emulsion as adjuvant (n=10); Group III (G3) was vaccinated with a commercial vaccine containing inactivated BVDV-1 and BVDV-2, besides live modified thermosensitive BoHV-1, diluted in Quil A, amphigen and cholesterol (n=10); A control, non-vaccinated group (n=6) was mock vaccinated with saline. Heifers received two subcutaneous doses of 5mL of each commercial vaccine on the right side of the neck, with 21 days interval. Humoral immune response was assessed by the virus neutralization test (VN) against BVDV-1 (NADL and Singer strains), BVDV-2 (SV253 strain) and BoHV-1 (Los Angeles strain) in serum samples collected on vaccination days zero (D0), 21 (D21) and 42 (D42; 21 days after boosting). Neutralizing Abs against BVDV-1 NADL was detected only in D42, regardless of the vaccine used. Similar geometric mean titers (GMT) for BVDV-1 NADL were observed between G1 (log2=5.1) and G3 (log2=5.1). The seroconversion rate (%) was higher in G1 (78%) when compared to G2 (10%) and G3 (40%). For BVDV-1 Singer, it was also possible to detect Abs production in G1 (log2=5.8, 100% seroconversion rate) and G3 (log2=3.5, seroconversion rate = 60%), only after the booster dose (D42). Neutralizing Abs to BVDV-2 (SV253) were detected only in G3, observing 90% seroconversion associated with high titers of Abs (log2=6.7) after the 2nd dose of vaccine (D42). Heifers from G1 and G3 responded to BoHV-1 after the first dose (D21): G1 (log2=2.5, seroconversion rate = 67%) and G3 (log2=0.7, seroconversion rate = 80%). In D42, a higher magnitude response was observed in the heifers from G3 (log2=6.1, 100%) compared with G1 (log2=4.3, 100%) and G2 (log2=2.7, 60%). Based on the data obtained, it can be concluded that the commercial vaccine contained aluminum hydroxide (G1) was most effective in the induction of antibodies against BVDV-1. On the other hand, this vaccine did not induce the production of neutralizing Abs against BVDV-2. Only the heifers from G3 (Quil A, amphigen and cholesterol) generated neutralizing Abs against BVDV-2. The animals that received commercial vaccine containing oil emulsion as adjuvant (G2) had a weak/undetectable response against BVDV-1 and BVDV-2. The best protective response against BoHV-1 was observed in heifers vaccinated with the live modified thermosensitive virus.(AU)

A vacinação é utilizada como estratégia para a prevenção e controle das doenças reprodutivas, causadas pelos vírus da diarreia viral bovina (BVDV) e herpesvírus bovino tipo 1 (BoHV-1), entretanto, as diversas composições de vacinas comerciais devem ser avaliadas quanto a sua eficiência protetiva mediada por anticorpos (Acs). O objetivo desta pesquisa foi avaliar a produção Acs neutralizantes específicos para cepas de BVDV-1 e 2, e BoHV-1 induzida por vacinas comerciais contendo diferentes tipos de adjuvantes. Para tal, novilhas Holandesas foram vacinadas e distribuídas em três grupos experimentais: Grupo I (G1) foi vacinado com uma vacina comercial composta por cepas inativadas de BVDV-1, BVDV-2 e BoHV-1 diluídas em hidróxido de alumínio como adjuvante (n=9); Grupo II (G2) foi vacinado com produto contendo as cepas inativadas de BVDV-1, BVDV-2, BoHV-1 e BoHV-5 em uma emulsão oleosa como adjuvante (n=10); O Grupo III (G3) foi vacinado com uma vacina comercial contendo BVDV-1 e BVDV-2 inativado, além do BoHV-1 vivo modificado e termosensivel, diluídos em adjuvante contendo Quil A, Amphigem e colesterol (n=10); O Grupo Controle não vacinado (n=6) foi inoculado com solução salina. As novilhas receberam duas doses das respectivas vacinas ou solução salina (5mL), com intervalo de 21 dias, por via subcutânea, na tábua do pescoço do lado direito. A resposta imune humoral foi avaliada pelo teste de vírus neutralização (VN) contra o BVDV-1 (cepas NADL e Singer), BVDV-2 (cepa SV253) e BoHV-1 (cepa Los Angeles) em amostras de soro coletadas nos dias (D) de vacinação zero (D0), 21 dias após 1ª dose (D21)e 42 (D42; 21 dias após A 2ª dose). Os anticorpos neutralizantes contra o BVDV-1 NADL foram detectados apenas em D42, independentemente da vacina utilizada. Os títulos médios geométricos (GMT) de anticorpos foram semelhantes entre G1 (log2=5,1) e G3 (log2=5,1). A taxa de soroconversão foi maior no G1 (78%) quando comparado ao G2 (10%) e G3 (40%). Para o BVDV-1 Singer, somente após D42 foi observada a produção de Acs no G1 (log2=5,8; taxa de soroconversão de 100%) e G3 (log2=3,5; taxa de soroconversão = 60%). Os anticorpos contra BVDV-2 (SV253) foram detectados apenas nas novilhas do G3, observando-se taxa de soroconversão de 90% com altos títulos de anticorpos neutralizantes (log2=6,7) em D42. Novilhas G1 e G3 responderam ao BoHV-1 após a primeira dose (D21): G1 (log2=2,5; taxa de seroconversão = 67%) e G3 (log2=0,7; taxa de seroconversão = 80%). Em contrapartida, foi observada uma maior magnitude de resposta para as novilhas G3 (log2=6,1; 100%) em D42, em relação aos animais G1 (log2=4,3; 100%) e G2 (log2=2,7; 60%). Com base nos dados obtidos, foi possível concluir que a vacina composta por hidróxido de alumínio (G1) foi mais eficaz na produção de anticorpos contra o BVDV-1, em contrapartida esse produto não induziu anticorpos contra o BVDV-2. Apenas as novilhas do G3 (Quil A, amphigen e colesterol) geraram Acs neutralizantes contra o BVDV-2. Os animais que receberam a vacina em emulsão oleosa (G2) como adjuvante apresentaram uma resposta fraca/indetectável contra o BVDV-1 e BVDV-2. A melhor resposta protetiva contra o BoHV-1 foi observada nas novilhas vacinadas com a vacina viva modificada termosensível.(AU)

Effect of a DNA-based immunostimulant on growth, performance, and expression of inflammatory and immune mediators in beef calves abruptly weaned and introduced to a complete ration.

The effect of a DNA immunostimulant on inflammatory and immune responses, performance, and health in calves following abrupt weaning and introduction to a concentrate diet was tested. Sixty-four single source Angus crossbred steers were weaned on day 1 and assigned to receive a DNA immunostimulant (TRT) or saline (CON) on days 0, 2, 4, and 6. On day 0, steers received clostridial and respiratory vaccines and anthelmintic; they were then transported 2 h, allocated to pens (n = 8 per pen), and introduced to total mixed ration. Daily intake, ADG, and feed efficiency were measured. Serum haptoglobin, tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1ß) were assayed by ELISA or AlphaLISA on days 0, 2, 4, 6, 14, and 28; serum-neutralizing antibodies (SNA) to bovine herpesvirus-1 and bovine viral diarrhea virus-1 (BVDV-1) were quantified on days 0, 28, 68, and 135. In a subset of cattle (n = 6 to 8 per treatment group), the percent macrophages and activated gamma delta (γδ) T cells in blood was determined by flow cytometry on days 2 and 6, and expression of mRNA for TNF-α, interferon-gamma (IFN-γ), IL-4, and IL-10 by stimulated blood mononuclear cells was assessed by real-time reverse transcriptase PCR on day 6. After 70 d, cattle were shipped 1,205 km to a feedlot and performance and health were followed. There was a significant effect of time on serum TNF-α, IL-1ß, haptoglobin, and SNA (P < 0.001); the range in concentration among cattle on each day was large. The ratio of IFN-γ to IL-4 expression was significantly higher (P = 0.03) for TRT cattle, suggesting that treatment activated T-helper type 1 cells. There was a trend toward an improved feed conversion (P = 0.10) for TRT steers over the 70-d backgrounding period. There was no effect of treatment on feedlot performance or carcass merit (P > 0.10). During backgrounding, 1 TRT steer died of enterocolitis. In spite of backgrounding, cattle experienced an outbreak of bovine respiratory disease (BRD) in the feedlot and 1 of 31 TRT cattle and 5 of 32 CON cattle died of BRD. The immunostimulant modified some immune responses during backgrounding. Large variability in inflammatory responses during backgrounding indicated that events around weaning induce systemic inflammation that varies substantially among cattle.

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 established based 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 the IDEXX 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.

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.

Bovine A20 gene overexpression during bovine viral diarrhea virus-1 infection blocks NF-κB pathway in MDBK cells.

Viruses have developed cellular strategies to ensure progeny survival. One of the most interesting is immune camouflage, where the virus triggers a controlled-intensity immune response that prevents total destruction of the infected cell, thus "winning time" for the virus. This study explored the regulatory contexts of the bovine A20 gene during bovine viral diarrhea virus (BVDV)-1 infection, using IL-8 as an immune-response sentinel molecule. Assessments were conducted through RT-qPCR, Western blotting, gene silencing/overexpression, luciferase assays, and the use of pharmacological inhibitors, among other approaches. The results demonstrated that a) BVDV-1 increased A20 levels in Madin-Darby bovine kidney cells, b) increased A20 led to decreased IL-8 expression, and c) the virus affected the NF-κB signaling pathway. Collectively, these data identify bovine A20 as a strong regulator of immune marker expression. In conclusion, this is the first report on BVDV-1 modulating bovine IL-8 activation through the NF-κB/A20 pathway.

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.

Priming Cross-Protective Bovine Viral Diarrhea Virus-Specific Immunity Using Live-Vectored Mosaic Antigens.

Bovine viral diarrhea virus (BVDV) plays a key role in bovine respiratory disease complex, which can lead to pneumonia, diarrhea and death of calves. Current vaccines are not very effective due, in part, to immunosuppressive traits and failure to induce broad protection. There are diverse BVDV strains and thus, current vaccines contain representative genotype 1 and 2 viruses (BVDV-1 & 2) to broaden coverage. BVDV modified live virus (MLV) vaccines are superior to killed virus vaccines, but they are susceptible to neutralization and complement-mediated destruction triggered by passively acquired antibodies, thus limiting their efficacy. We generated three novel mosaic polypeptide chimeras, designated NproE2123; NS231; and NS232, which incorporate protective determinants that are highly conserved among BVDV-1a, 1b, and BVDV-2 genotypes. In addition, strain-specific protective antigens from disparate BVDV strains were included to broaden coverage. We confirmed that adenovirus constructs expressing these antigens were strongly recognized by monoclonal antibodies, polyclonal sera, and IFN-γ-secreting T cells generated against diverse BVDV strains. In a proof-of-concept efficacy study, the multi-antigen proto-type vaccine induced higher, but not significantly different, IFN-γ spot forming cells and T-cell proliferation compared to a commercial MLV vaccine. In regards to the humoral response, the prototype vaccine induced higher BVDV-1 specific neutralizing antibody titers, whereas the MLV vaccine induced higher BVDV-2 specific neutralizing antibody titers. Following BVDV type 2a (1373) challenge, calves immunized with the proto-type or the MLV vaccine had lower clinical scores compared to naïve controls. These results support the hypothesis that a broadly protective subunit vaccine can be generated using mosaic polypeptides that incorporate rationally selected and validated protective determinants from diverse BVDV strains. Furthermore, regarding biosafety of using a live vector in cattle, we showed that recombinant human adenovirus-5 was cleared within one week following intradermal inoculation.

Bovine viral diarrhea virus structural protein E2 as a complement regulatory protein.

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, family Flaviviridae, and is one of the most widely distributed viruses in cattle worldwide. Approximately 60 % of cattle in endemic areas without control measures are infected with BVDV during their lifetime. This wide prevalence of BVDV in cattle populations results in significant economic losses. BVDV is capable of establishing persistent infections in its host due to its ability to infect fetuses, causing immune tolerance. However, this cannot explain how the virus evades the innate immune system. The objective of the present work was to test the potential activity of E2 as a complement regulatory protein. E2 glycoprotein, produced both in soluble and transmembrane forms in stable CHO-K1 cell lines, was able to reduce complement-mediated cell lysis up to 40 % and complement-mediated DNA fragmentation by 50 %, in comparison with cell lines not expressing the glycoprotein. This work provides the first evidence of E2 as a complement regulatory protein and, thus, the finding of a mechanism of immune evasion by BVDV. Furthermore, it is postulated that E2 acts as a self-associated molecular pattern (SAMP), enabling the virus to avoid being targeted by the immune system and to be recognized as self.

Quillaja brasiliensis saponins induce robust humoral and cellular responses in a bovine viral diarrhea virus vaccine in mice.

A saponin fraction extracted from Quillaja brasiliensis leaves (QB-90) and a semi-purified aqueous extract (AE) were evaluated as adjuvants in a bovine viral diarrhea virus (BVDV) vaccine in mice. Animals were immunized on days 0 and 14 with antigen plus either QB-90 or AE or an oil-adjuvanted vaccine. Two-weeks after boosting, antibodies were measured by ELISA; cellular immunity was evaluated by DTH, lymphoproliferation, cytokine release and single cell IFN-γ production. Serum anti-BVDV IgG, IgG1 and IgG2b were significantly increased in QB-90- and AE-adjuvanted vaccines. A robust DTH response, increased splenocyte proliferation, Th1-type cytokines and enhanced production of IFN-γ by CD4(+) and CD8(+) T lymphocytes were detected in mice that received QB-90-adjuvanted vaccine. The AE-adjuvanted preparation stimulated humoral responses but not cellular immune responses. These findings reveal that QB-90 is capable of stimulating both cellular and humoral immune responses when used as adjuvant.

Comparison of reproductive performance of primiparous dairy cattle following revaccination with either modified-live or killed multivalent viral vaccines in early lactation.

The objective of this randomized clinical trial was to compare the effect of revaccination in primiparous dairy cows with modified live viral (MLV) or killed viral (KV) vaccines containing bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BoHV-1) on (1) pregnancy rate following estrus synchronization-timed artificial insemination (TAI), (2) serum progesterone concentrations, and (3) serum neutralizing antibody titers at revaccination and at TAI. Primiparous dairy cows (n=692) that had been previously vaccinated with 4 doses of MLV vaccine as calves or heifers were randomized to receive either an MLV or a KV vaccine between 21 and 28 d in milk and 17 d before initiation of a double-Ovsynch-TAI protocol. Serum was collected within the double-Ovsynch protocol for determination of progesterone concentrations, and at vaccination and TAI for serum neutralizing antibody titers. Ultrasound pregnancy determinations were made at 30 and 60 d after TAI. No differences in pregnancy rates were observed between cows receiving MLV vaccine (44%; n=326) or KV vaccine (43%; n=336). No differences were observed in serum progesterone concentrations during a double-Ovsynch-TAI protocol between cows receiving MLV and KV vaccines. No differences were observed in BVDV 1 or BVDV 2 antibody titers at vaccination and TAI between cows receiving MLV or KV vaccine; however, BoHV-1 antibody titers were greater at TAI in cows receiving KV vaccine. Overall response to vaccination-defined as the percent of all individual cows that had any detectable increase in antibody titer from vaccination to TAI-was 39% for BVDV 1, 45% for BVDV 2, and 61% for BoHV-1. In this research, use of an MLV vaccine did not impede reproduction when revaccination was performed between 21 and 28 DIM and just before enrollment in an estrus synchronization-TAI program in primiparous dairy cows; however, response to vaccination as defined by increases in virus-specific antibody titers could be considered less than ideal for this population of cattle.

Development of an APC-targeted multivalent E2-based vaccine against Bovine Viral Diarrhea Virus types 1 and 2.

The aim of this study was to develop and test a multivalent subunit vaccine against Bovine Viral Diarrhea Virus (BVDV) based on the E2 virus glycoprotein belonging to genotypes 1a, 1b and 2a, immunopotentiated by targeting these antigens to antigen-presenting cells. The E2 antigens were expressed in insect cells by a baculovirus vector as fusion proteins with a single chain antibody, named APCH I, which recognizes the ß-chain of the MHC Class II antigen. The three chimeric proteins were evaluated for their immunogenicity in a guinea pig model as well as in colostrum-deprived calves. Once the immune response in experimentally vaccinated calves was evaluated, immunized animals were challenged with type 1b or type 2b BVDV in order to study the protection conferred by the experimental vaccine. The recombinant APCH I-tE21a-1b-2a vaccine was immunogenic both in guinea pigs and calves, inducing neutralizing antibodies. After BVDV type 1b and type 2 challenge of vaccinated calves in a proof of concept, the type 1b virus could not be isolated in any animal; meanwhile it was detected in all challenged non-vaccinated control animals. However, the type 2 BVDV was isolated to a lesser extent compared to unvaccinated animals challenged with type 2 BVDV. Clinical signs associated to BVDV, hyperthermia and leukopenia were reduced with respect to controls in all vaccinated calves. Given these results, this multivalent vaccine holds promise for a safe and effective tool to control BVDV in herds.

The effects of reference genes in qRT-PCR assays for determining the immune response of bovine cells (MDBK) infected with the Bovine Viral Diarrhea Virus 1 (BVDV-1).

The bovine viral diarrhea virus (BVDV) causes significant economic losses to the dairy industry worldwide, and understanding its infection mechanisms would be extremely useful in designing new and efficient treatments. Due to the limited number of specific antibodies against bovine proteins, differential gene expression analyses are vital for researching host immune responses to viral infection. qRT-PCR provides a sensitive platform to conduct such gene expression analyses, but suitable housekeeping genes are needed for accurate transcript normalization. The present study assessed nine reference genes in bovine kidney cells under conditions of BVDV-1 infection, incubation with pathogen-associated molecular patterns, and co-incubation with BAY117085, a pharmacological inhibitor of the NF-κB signaling pathway. Analyses of Ct values using the BestKeeper and Normfinder programs ranked CD81, RPL4, and GAPDH as the most reliable reference genes. This determination of a stable set of reference genes in this culture system will facilitate analyses of expression levels for genes of interest.

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

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

Preliminary mapping of non-conserved epitopes on envelope glycoprotein E2 of Bovine viral diarrhea virus type 1 and 2.

Bovine viral diarrhea virus (BVDV) belongs together with Classical swine fever virus (CSFV) and Border disease virus (BDV) to the genus Pestivirus in the Flaviviridae family. BVDV has been subdivided into two different species, BVDV1 and BVDV2 based on phylogenetic analysis. Subsequent characterization of both strains revealed major antigenic differences. Because the envelope glycoprotein E2 is the most immunodominant protein for all pestiviruses, the present study focused on epitope mapping by constructing chimeric BVDV type 1 and 2 E2 genes in expression plasmids. These plasmids with chimeric E2-genes were transfected in SK6 cells and transient expression was studied by immunostaining with a panel of MAbs specific for E2 of BVDV1 or BVDV2, resulting in the localization of type-specific antigenic domains at similar regions. These results indicate that E2 glycoproteins of both BVDV types exhibit a comparable antigenic structure, but with type specific epitopes. In addition, the antigenic resemblance with envelope glycoprotein E2 of Classical swine fever virus is discussed.

Efficacy of a BVDV subunit vaccine produced in alfalfa transgenic plants.

Bovine viral diarrhea virus (BVDV) is considered an important cause of economic loss within bovine herds worldwide. In Argentina, only the use of inactivated vaccines is allowed, however, the efficacy of inactivated BVDV vaccines is variable due to its low immunogenicity. The use of recombinant subunit vaccines has been proposed as an alternative to overcome this difficulty. Different studies on protection against BVDV infection have focused the E2 protein, supporting its putative use in subunit vaccines. Utilization of transgenic plants expressing recombinant antigens for the formulation of experimental vaccines represents an innovative and cost effective alternative to the classical fermentation systems. The aim of this work was to develop transgenic alfalfa plants (Medicago sativa, L.) expressing a truncated version of the structural protein E2 from BVDV fused to a molecule named APCH, that target to antigen presenting cells (APCH-tE2). The concentration of recombinant APCH-tE2 in alfalfa leaves was 1 µg/g at fresh weight and its expression remained stable after vegetative propagation. A methodology based an aqueous two phases system was standardized for concentration and partial purification of APCH-tE2 from alfalfa. Guinea pigs parentally immunized with leaf extracts developed high titers of neutralizing antibodies. In bovine, the APCH-tE2 subunit vaccine was able to induce BVDV-specific neutralizing antibodies. After challenge, bovines inoculated with 3 µg of APCH-tE2 produced in alfalfa transgenic plants showed complete virological protection.

Evaluation of envelope glycoprotein E(rns) of an atypical bovine pestivirus as antigen in a microsphere immunoassay for the detection of antibodies against bovine viral diarrhea virus 1 and atypical bovine pestivirus.

Atypical bovine pestiviruses are related antigenically and phylogenetically to bovine viral diarrhea viruses (BVDV-1 and BVDV-2), and may cause the same clinical manifestations in animals. Glycoprotein E(rns) of an atypical bovine pestivirus Th/04_KhonKaen was produced in a baculovirus expression system and was purified by affinity chromatography. The recombinant E(rns) protein was used as an antigen in a microsphere immunoassay for the detection of antibodies against BVDV-1 and atypical bovine pestivirus. The diagnostic performance of the new method was evaluated by testing a total of 596 serum samples, and the assay was compared with enzyme-linked immunosorbent assay (ELISA). Based on the negative/positive cut-off median fluorescence intensity (MFI) value of 2800, the microsphere immunoassay had a sensitivity of 100% and specificity of 100% compared to ELISA. The immunoassay was able to detect antibodies against both BVDV-1 and the atypical pestivirus. This novel microsphere immunoassay has the potential to be multiplexed for simultaneous detection of antibodies against different bovine pathogens in a high-throughput and economical way.

Safety and efficacy of an E2 glycoprotein subunit vaccine produced in mammalian cells to prevent experimental infection with bovine viral diarrhoea virus in cattle.

Bovine viral diarrhea (BVD) infection caused by bovine viral diarrhea virus (BVDV), a Pestivirus of the Flaviviridae family, is an important cause of morbidity, mortality and economical losses in cattle worldwide. E2 protein is the major glycoprotein of BVDV envelope and the main target for neutralising antibodies (NAbs). Different studies on protection against BVDV infection have focused on E2, supporting its putative use in subunit vaccines. A truncated version of type 1a BVDV E2 (tE2) expressed in mammalian cells was used to formulate an experimental oleous monovalent vaccine. Immunogenicity was studied through immunisation of guinea pigs and followed by trials in cattle. Calves of 8-12 months were vaccinated, twice with a 4 week interval, with either a tE2 subunit vaccine (n = 8), a whole virus inactivated vaccine (n = 8) or left untreated as negative control group (n = 8). Four weeks after the last immunisation the animals were experimentally challenged intranasally with a non-cythopathic BVDV strain. Following challenge, BVDV was isolated from all unvaccinated animals, while 6 out of 8 animals vaccinated with tE2 showed complete virological protection indicating that the tE2 vaccine presented a similar performance to a satisfactory whole virus inactivated vaccine.