Increased PDT Efficacy When Associated with Nitroglycerin: A Study on Retinoblastoma Xenografted on Mice.

PURPOSES: The aim of the study was to assess the efficacy of a treatment protocol that combines photodynamic therapy (PDT) and nitroglycerin (NG) on human retinoblastoma tumors xenografted on mice. We aimed to increase the PDT efficiency (in our least treatment-responsive retinoblastoma line) with better PS delivery to the tumor generated by NG, which is known to dilate vessels and enhance the permeability and retention of macromolecules in solid tumors. METHODS: In vivo follow-up of the therapeutic effects was performed by sodium MRI, which directly monitors variations in sodium concentrations non-invasively and can be used to track the tumor response to therapy. NG ointment was applied one hour before PDT. The PDT protocol involves double-tumor targeting, i.e., cellular and vascular. The first PS dose was injected followed by a second one, separated by a 3 h interval. The timelapse allowed the PS molecules to penetrate tumor cells. Ten minutes after the second dose, the PS was red-light-activated. RESULTS: In this study, we observed that the PDT effect was enhanced by applying nitroglycerin ointment to the tumor-bearing animal's skin. PDT initiates the bystander effect on retinoblastomas, and NG increases this effect by increasing the intratumoral concentration of PS, which induces a higher production of ROS in the illuminated region and thus increases the propagation of the cell death signal deeper into the tumor (bystander effect).

Comparison of the Ability of High and Low Virulence Strains of Non-cytopathic Bovine Viral Diarrhea Virus-1 to Modulate Expression of Interferon Tau Stimulated Genes in Bovine Endometrium.

Bovine Viral Diarrhea virus (BVDV) is a pestivirus with a single-stranded, positive sense RNA genome. It is endemic in many cattle populations, causing major economic losses in part due to reduced fertility. BVDV exhibits great genetic diversity and is classified as type 1 or 2 (BVDV-1, BVDV-2) with either non-cytopathogenic (ncp) or cytopathogenic (cp) biotypes. Differing strains of ncpBVDV differ in virulence, affecting clinical outcome. BVDV replicates in the reproductive tract, affecting host immunity and embryo survival. This study used an in vitro model of primary bovine endometrial cell cultures to compare the effects of two BVDV ncp type 1a strains of differing virulence (termed HO and KY) on endometrial transcription of candidate interferon stimulated genes (ISG) using qPCR. Half the cultures were stimulated with interferon tau (IFNT, the conceptus produced pregnancy recognition factor) in the presence or absence of viral infection. Cultures were replicated on cells from 10 BVDV-free cows. IFNT treatment stimulated transcription of 10 candidate ISGs, whereas both ncpBVDV-1 strains alone inhibited transcription of 8/10 ISGs. In combined BVDV-1+IFNT cultures, the stimulatory effect of IFNT on expression of GBP4, ISG15, HERC5, RSAD2, IFIH1, IFIT3, and MX1 was significantly inhibited by HO, but only ISG15, RSAD2, IFI27, and IFIT3 were decreased by KY. Inhibition by HO was generally greater. The IFNT-induced expression of TRIM56 was, however, increased by HO. These data show that HO, the more virulent ncpBVDV-1 strain, has a greater capacity to inhibit key antiviral pathways. These differences need confirmation at the protein level but may influence immune tolerance of the host. They could also reduce fertility by increasing uterine susceptibility to bacterial infection and disrupting IFNT-mediated pregnancy recognition.

Importance of Viral Disease in Dairy Cow Fertility.

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

Serological survey of wild cervids in England and Wales for bovine viral diarrhoea virus.

BACKGROUND: Bovine viral diarrhoea (BVD) is a production disease commonly found in British cattle herds. Species other than cattle have been shown to be infected with the virus, thereby providing a potential source of infection for livestock. This study surveyed serum samples taken from 596 culled wild deer from England and Wales, between 2009 and 2010, for the presence of BVD antibodies. METHODS: 596 samples were tested with the SVANOVIR BVDV p80-Ab ELISA and a subset of 64 were tested with the IDEXX BVDV p80-Ab ELISA. ELISA results were confirmed using serum neutralisation tests. RESULTS: 2/596 samples (0.35 per cent) tested positive for BVD antibodies using the Svanova test and one of these tested positive and the other inconclusive using the IDEXX test; both were confirmed positive with serum neutralisation tests. These were both red deer stags, one from Devon and the other from East Anglia. CONCLUSIONS: The results indicate that it is unlikely that BVD virus is widely circulating within the wild deer population and particularly unlikely that persistently infected deer are present in the populations surveyed. These results suggest that wild deer are unlikely to be a significant reservoir of BVD infection in cattle.

Low retinal toxicity of intravitreal carboplatin associated with good retinal tumour control in transgenic murine retinoblastoma.

BACKGROUND: Retinoblastoma is a rare intraocular malignancy in children. Current treatments have many adverse effects. New therapeutic approaches like intravitreal injections of chemotherapies are currently being developed but their toxicities need to be evaluated on animal models. This study compares the efficacy and toxicity of intravitreal melphalan, topotecan and carboplatin, alone or in combination (sequential administration), in the LHBetaTag retinoblastoma mice. METHODS: Mice were divided into nine groups: control, carboplatin 1.5 and 4 µg, melphalan 0.1 and 1 µg, topotecan 0.1 and 1 µg, carboplatin 4 µg/topotecan 0.1 µg and melphalan 1 µg/topotecan 0.1 µg. The follow-up was performed using fundus imaging and optical coherence tomography combined with histopathological analysis. Absence of tumour and presence of calcified tumours were the criteria for therapeutic response assessment. Ocular complications were assessed after four weekly injections. Retinal toxicity was defined by the decrease of retinal thickness and of the number of retinal layers. RESULTS: Topotecan was inactive on retinal tumours. Melphalan (1 µg) led to a complete tumour control in 91.7% of eyes. Carboplatin strongly decreased the tumour burden (85.7-93.8% of eyes without retinal tumour). The intravitreal injection itself led to ocular complications (25% of media opacities and 45.7% of retinal detachment). Only melphalan at 1 µg showed a strong retinal toxicity. The two combinations showed a good efficacy in reducing the number of eyes with retinal tumours with a reduced retinal toxicity. CONCLUSIONS: This preclinical study suggests that intravitreal injection of carboplatin has a low toxicity and could be evaluated in clinical practice to treat patients suffering from retinoblastoma.

Mechanisms linking bovine viral diarrhea virus (BVDV) infection with infertility in cattle.

Bovine viral diarrhea virus (BVDV) is an important infectious disease agent that causes significant reproductive and economic losses in the cattle industry worldwide. Although BVDV infection is known to cause poor fertility in cattle, a greater part of the underlying mechanisms particularly associated with early reproductive losses are not clearly understood. Previous studies reported viral compromise of reproductive function in infected bulls. In females, BVDV infection is thought to be capable of killing the oocyte, embryo or fetus directly, or to induce lesions that result in fetal abortion or malformation. BVDV infections may also induce immune dysfunction, and predispose cattle to other diseases that cause poor health and fertility. Other reports also suggested BVDV-induced disruption of the reproductive endocrine system, and a disruption of leukocyte and cytokine functions in the reproductive organs. More recent studies have provided evidence of viral-induced suppression of endometrial innate immunity that may predispose to uterine disease. Furthermore, there is new evidence that BVDV may potentially disrupt the maternal recognition of pregnancy or the immune protection of the conceptus. This review brings together the previous reports with the more recent findings, and attempts to explain some of the mechanisms linking this important virus to infertility in cattle.

Iterative Variance Stabilizing Transformation Denoising of Spectral Domain Optical Coherence Tomography Images Applied to Retinoblastoma.

BACKGROUND: Due to the presence of speckle Poisson noise, the interpretation of spectral domain-optical coherence tomography (SD-OCT) images frequently requires the use of data averaging to improve the signal-to-noise ratio. This implies long acquisition times and requires patient sedation in some cases. Iterative variance stabilizing transformation (VST) is a possible approach by which to remove speckle Poisson noise on single images. METHODS: We used SD-OCT images of human and murine (LH Beta-Tag mouse model) retinas with and without retinoblastoma acquired with 2 different imaging devices (Bioptigen and Micron IV). These images were processed using a denoising workflow implemented in Matlab. RESULTS: We demonstrated the presence of speckle Poisson noise, which can be removed by a VST-based approach. This approach is robust as it works in all used imaging devices and in both human and mouse retinas, independently of the tumor status. The implemented algorithm is freely available from the authors on demand. CONCLUSIONS: On a single denoised image, the proposed method provides results similar to those expected from the SD-OCT averaging. Because of the friendly user interface, it can be easily used by clinicians and researchers in ophthalmology.

Looking for the Most Suitable Orthotopic Retinoblastoma Mouse Model in Order to Characterize the Tumoral Development.

Purpose: Because retinoblastoma therapies have many adverse effects, new approaches must be developed and evaluated on animal models. We describe orthotopic xenograft models of retinoblastoma using different strains of mice, suitable for this purpose. Methods: Human retinoblastoma tumors were established on immunodeficient mice by subcutaneous engraftment of tumors from enucleated eyes. The orthotopic model was obtained by subretinal injections of suspension cells into the right eye of immunodeficient (Swiss-nude, severe combined immunodeficiency [SCID]) and immunocompetent mice (C57BL/6N, B6Albino). In vivo tumor growth was monitored by fundus and spectral-domain optical coherence tomography (SD-OCT) imaging and compared with histology. Results: Retinal and vitreal tumor growth was achieved both in immunocompetent and immunodeficient strains after the subretinal injection of tumor cells. The best tumor engraftment rate was obtained in the SCID mice (68.8%). No tumor growth was observed in the C57BL/6N strain. Chronic retinal detachment may occur in most strains after the subretinal injection, in particular the Swiss-nude strain, which exhibits retinal degeneration. Conclusions: The setting up of an orthotopic mouse model depends mainly on the choice of the engrafted cells (cell lines or patient-derived xenografts) but it can also depend on the xenografted mouse strain. Severe combined immunodeficiency mice (an immunodeficient strain) achieved the best tumor engraftment rate (68.8%). However, intraocular tumor growth was also satisfactory (50%) in the immunocompetent strain B6Albino, and this strain will allow to exploit the immune response after a tumor treatment. Both of these strains may therefore be recommended when setting up orthotopic retinoblastoma xenografts.

Enhanced neutralising antibody response to bovine viral diarrhoea virus (BVDV) induced by DNA vaccination in calves.

DNA vaccination is effective in inducing potent immunity in mice; however it appears to be less so in large animals. Increasing the dose of DNA plasmid to activate innate immunity has been shown to improve DNA vaccine adaptive immunity. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA pattern receptor required for innate immune activation in response to viral infection. RIG-I recognise viral RNA and trigger antiviral response, resulting in type I interferon (IFN) and inflammatory cytokine production. In an attempt to enhance the antibody response induced by BVDV DNA in cattle, we expressed BVDV truncated E2 (E2t) and NS3 codon optimised antigens from antibiotic free-plasmid vectors expressing a RIG-I agonist and designated either NTC E2t(co) and NTC NS3(co). To evaluate vaccine efficacy, groups of five BVDV-free calves were intramuscularly injected three times with NTC E2t(co) and NTC NS3(co) vaccine plasmids individually or in combination. Animals vaccinated with our (previously published) conventional DNA vaccines pSecTag/E2 and pTriExNS3 and plasmids expressing RIG-I agonist only presented both the positive and mock-vaccine groups. Our results showed that vaccines coexpressing E2t with a RIG-I agonist induced significantly higher E2 antigen specific antibody response (p<0.05). Additionally, E2t augmented the immune response to NS3 when the two vaccines were delivered in combination. Despite the lack of complete protection, on challenge day 4/5 calves vaccinated with NTC E2t(co) alone or NTC E2t(co) plus NTC NS3(co) had neutralising antibody titres exceeding 1/240 compared to 1/5 in the mock vaccine control group. Based on our results we conclude that co-expression of a RIG-I agonist with viral antigen could enhance DNA vaccine potency in cattle.

Cellular density, a major factor involved in PDT cytotoxic responses: Study on three different lines of human retinoblastoma grafted on nude mice.

BACKGROUND: PDT represents a very localized and non-mutagen antitumoral treatment using a photosensitive molecule (porphyrin family) light activated. The first way of cell damage is a direct one, active on the very site where ROSs have been produced. The second one is indirect by activating and transmitting the processes of cellular death signaling. In order to seek for a better characterization of the photo-biology involved in in vivo PDT and to better understand the differences on the treatment outcome, we have used three different human retinoblastomas xenografted on mice. METHODS: Mice were treated according to the double targeting protocol exposed in a previous paper. One i.v. dose (0.6 mg/kg) of PS was followed by a second dose, separated by a 3 h interval (double targeting PDT). As a consequence both cancer cells and blood vessels were targeted. The treatment was repeated two times, at 4 days interval. RESULTS: First of all, sodium MRI revealed qualitative differences in the sodium average content of the three retinoblastoma lines before treatment. After the PDT treatments the tumor responses were different between the lines as revealed by sodium MRI and later on by histology. CONCLUSIONS: We have put into evidence that PDT is accompanied by a bystander effect that may propagate the cellular death triggered by the initial photoreaction. This effect is highly dependent on the cellular density of the tissue; therefore this factor is to be taken into account in clinical PDT protocols.

A phylogenetic analysis of Bovine Viral Diarrhoea Virus (BVDV) isolates from six different regions of the UK and links to animal movement data.

Bovine Viral Diarrhoea Virus (BVDV) is a pestivirus which infects cattle populations worldwide and is recognised as a significant source of economic loss through its impact on health and productivity. Studies investigating the molecular epidemiology of BVDV can give invaluable information about the diversity of viral strains present in a population and this, in turn, can inform control programs, drive vaccine development and determine likely infection sources. The current study investigated 104 viral isolates from forty farms across the UK. Through phylogenetic and nucleotide sequence analysis of the 5'UTR and Npro regions of the isolates investigated, it was determined that BVDV 1a was the predominant sub-genotype. However, BVDV 1b, 1e and 1i were also identified and, for the first time in the UK, BVDV 1d. Through analysis of animal movement data alongside the phylogenetic analysis of these BVD isolates, it was possible to link animal movements to the viral isolates present on several premises and, for the first time, begin to elucidate the routes of viral transmission. With further work, this type of analysis would enable accurate determination and quantification of the true biosecurity risk factors associated with BVDV transmission.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus.

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

PDT induced bystander effect on human xenografted colorectal tumors as evidenced by sodium MRI.

BACKGROUND: Previous in vivo studies on photodynamic therapy (PDT)-treated, high cellular density tumors showed evidences of a bystander effect accompanying the therapy, cellular death continuing beyond the limits of the photochemical reactions in time and space. This process is generated by the initially damaged cells on the light pathway. The aim of this study was to determine if the bystander effect may be induced as well in colorectal xenografted tumors (less compact structure) and if the cellular signaling depends primarily on cellular proximity or not. METHODS: The photosensitizer was a glycoconjugated, meso substituted porphyrin derivative synthesized at Institut Curie. The longitudinal follow-up of the tumors was carried out by (23)Na/(1)H MRI, ideal imaging modality for mapping the extracellular compartment. Two regimens were followed in order to target either blood vessels alone or blood vessels and cancer cells simultaneously. RESULTS: The antivascular PDT did not succeed to arrest the tumors growth at the end of the follow-up. For double targeting PDT, we managed to stop the tumoral evolution. Sodium MRI evidenced a bystander effect. CONCLUSION: The results obtained showed that the bystander effect is more difficult to induce for the type of colorectal tumors used in this work. It needs a double treatment, 4 days apart, in order to be promoted.

Potential evidence for biotype-specific chemokine profile following BVDV infection of bovine macrophages.

Chemokines play a key role in initiating the innate and subsequently adaptive immune response by recruiting immune cells to the site of an infection. Monocytes/macrophages (MØ) are part of the first line of defence against invading pathogens, and have been shown to release a variety of chemokines in response to infection. Here, we reveal the early transcriptional response of MØ to infection with cytopathogenic (cp) and non-cytopathogenic (ncp) bovine viral diarrhoea strains (BVDV). We demonstrate up-regulation of several key chemokines of the CCL and CXCL families in MØ exposed to cpBVDV, but not ncpBVDV. In contrast, infection of MØ with ncpBVDV led to down-regulation of chemokine mRNA expression compared to uninfected cells. Data suggest that ncpBVDV can shut down production of several key chemokines that play crucial roles in the immune response to infection. This study helps to further our understanding of the pathogenesis of BVDV infection, highlighting biotype-specific cellular responses.

A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins.

Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

Validation of endogenous reference genes for RT-qPCR normalisation in bovine lymphoid cells (BL-3) infected with Bovine Viral Diarrhoea Virus (BVDV).

Reverse transcription quantitative PCR (RT-qPCR) is a highly sensitive tool that can be used for accurate and reliable gene expression analysis; however, careful normalisation to a set of stably expressed endogenous reference genes is essential. Expression levels of many reference genes in RT-qPCR analyses can be extremely variable under different experimental conditions, producing potentially erroneous results (Bustin, 2002). This limitation can be overcome with a systematic evaluation of candidate reference genes to determine the most stable. In the present study eight candidate reference genes were evaluated in a bovine lymphoid (BL-3) cell culture system over seven different time points in response to three different Bovine Viral Diarrhoea Virus (BVDV) strains. Data were analysed using BestKeeper (Pfaffl et al., 2004), geNorm (Vandesompele et al., 2002), and NormFinder (Andersen et al., 2004) validation programs and results enable the candidate reference genes to be ranked from most to least stable. Quantification cycle (C(q)) variability was determined between samples, i.e. between treatment groups and time points, and variability was also observed between the three validation programs. The reference gene combination of beta-actin and hypoxanthine-guanine phosphoribosyl transferase (HPRT) was found to be the most stable in Norm Finder. BestKeeper and geNorm both demonstrated beta-microglobulin and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase (YWHAZ) as the most stable. The determination of a stable set of reference genes in the BL-3 cell culture system facilitates analysis of expression levels for appropriate genes of interest. This study further emphasises the need to accurately validate candidate reference genes before use in gene expression RT-qPCR studies.

23Na MRI longitudinal follow-up of PDT in a xenograft model of human retinoblastoma.

BACKGROUND: Photodynamic therapy is an established cancer treatment in which a photosensitizing agent is activated by exposure to light thus generating cytotoxic reactive oxygen species that cause cellular damage. METHODS: A new photosensitizer synthesized at Curie Institute was used to treat retinoblastoma xenografts in mice, a glycoconjugated meso substituted porphyrin derivative, that showed some retinoblastoma cell affinity. The longitudinal follow-up of the tumors was carried out by (23)Na MRI (without adding exogenous contrast agents) to map the extracellular compartment and to characterize cell packing. Two regimens were followed to target either blood vessels alone or blood vessels and cancer cells simultaneously. RESULTS AND CONCLUSIONS: Only the protocol targeting both cancer cells and blood vessels effectively induces cellular death, confirmed by histology at the end of the experiment. Sodium MRI evidences a huge change in the cellular density of tumors only 24h after a double targeting (vascular and cellular) PDT treatment. We suggest that this change was possibly due to a bystander effect that can be promoted by the intercellular signaling favored by the high cellular density of retinoblastoma. These results indicate that non-invasive (23)Na imaging (which detects the tumor response to treatment from very early stages) in association with non-mutagenic therapies represents an effective option for tailored and individualized clinical treatments.

Spontaneous regression of human cancer cells in vitro: potential role of disruption of Cdk1/Cdk4 co-expression.

BACKGROUND: Although well-acknowledged in vivo, spontaneous death of cancer cells in vitro is less widely appreciated. MATERIALS AND METHODS: Colony formation was studied in untreated control plates of standard clonogenic assays and measurements of actual and potential doubling times performed in asynchronous cultures of human cancer cells lines. Western blotting of lung large cell carcinoma, COR-L23 cells actively undergoing spontaneous cell death was also carried out. RESULTS: Catastrophic disintegration of mature colonies could be seen in the untreated plates of lung large cell carcinoma, H460 and colon adenocarcinoma, SW620 human cancer cell lines and a significant cell loss factor was present in the cell lines growing as adherent cells in continuous culture. Western blotting demonstrated alterations of relative cyclin dependent kinase (Cdk)1 to Cdk4 protein expression in dying COR-L23 cells. CONCLUSION: The phenomenon of spontaneous cell death should be considered a hallmark of cancer and may be the result of failure to stabilise unstable, fully developed cancer cells due to the disruption of Cdk1/Cdk4 co-expression in those cells.

Infectivity of pestivirus following persistence of acute infection.

Bovine viral diarrhoea virus (BVDV) is an endemic pathogen worldwide and eradication strategies focus on the identification and removal of persistently infected (PI) animals arising after in utero infection. Despite this, acute infections with BVDV can persist for months or years after the removal of the PI source despite repeated screening for PIs and tight biosecurity measures. Recent evidence for a prolonged duration of viraemia in the testicles of bulls following acute BVDV infection suggests the possibility of a form of chronic persistence that may more closely resemble the persistence strategies of hepatitis C virus (HCV). To investigate the potential for virus transmission from infected and recovered cattle to virus naïve hosts we established an acute infection of 5 BVDV-naïve calves and monitored animals over 129 days. Infectious BVDV was detected in white blood cells between days 3 and 7 post-challenge. The animals seroconverted by day 21 post-infection and subsequently were apparently immune and free from infectious virus and viral antigen. Animals were further monitored and purified white blood cells were stimulated in vitro with phytohaemagglutinin A (PHA) during which time BVDV RNA was detected intermittently. Ninety-eight days following challenge, blood was transferred from these apparently virus-free and actively immune animals to a further group of 5 BVDV-naïve calves and transmission of infection was achieved. This indicates that BVDV-infected, recovered and immune animals have the potential to remain infectious for BVDV-naïve cohorts for longer than previously demonstrated.

Evaluation of efficacy of mammalian and baculovirus expressed E2 subunit vaccine candidates to bovine viral diarrhoea virus.

Bovine viral diarrhoea virus (BVDV) is a worldwide pathogen of cattle causing a wide spectrum of clinical disease. The major envelope glycoprotein of BVDV, E2, induces the production of neutralising antibodies. In this study we compared the protection afforded to cattle after BVDV challenge by two separate E2 vaccine candidates produced by different heterologous protein expression systems. E2 antigen was expressed using the baculovirus expression system (brE2) and a mammalian cell expression system (mrE2). In the first vaccination study the quantity of recombinant protein expressed by the two systems differed. Vaccination of cattle with a higher dose of brE2 or low dose mrE2 gave comparable protection from viral challenge. Immunised animals showed no pyrexia and reduced leucopaenia which contrasted to the unvaccinated controls. In addition virus shedding from the nasal mucosa was decreased in the vaccinated groups and strong humoral responses were evident post-challenge. However, the efficacy of the brE2 vaccine was greatly diminished when a reduced dose was tested, indicating the importance of assessing the type of expression system used in antigen production.