Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly.

The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle.

Assessment of neutralizing and non-neutralizing antibody responses against Porcine circovirus 2 in vaccinated and non-vaccinated farmed pigs.

Vaccination is the most efficacious procedure to curtail Porcine circovirus 2 (PCV2)-associated diseases (PCVAD). Experimental studies indicate that PCV2 vaccine-induced virus-neutralizing antibodies play a major role in protection from PCVAD. However, the immune response to PCV2 vaccination of pigs on farms is less clear. Analysing groups of age-matched vaccinated and non-vaccinated farmed pigs, we found significantly increased levels of virus-neutralizing antibodies only in vaccinated pigs belonging to the age group with the highest risk for developing PCVAD. Serum levels of PCV2 genomes were not different between corresponding age groups. Levels of antibodies directed against a linear peptide from the PCV2 capsid protein correlated with those of virus-neutralizing antibodies and reached the highest levels in older, non-vaccinated animals, pointing towards an intense interaction between PCV2-infected cells and the immune system. In conclusion, current PCV2 vaccines are in need of improvement to induce stronger and more rapid immunity to prevent PCV2 infection.

Acute BVDV-2 infection in beef calves delays humoral responses to a non-infectious antigen challenge.

Immunosuppressive effects of an intranasal challenge with non-cytopathic bovine viral diarrhea virus (BVDV) 2a (strain 1373) were assessed through acquired and innate immune system responses to ovalbumin (OVA). Concurrent BVDV infection was hypothesized to delay and reduce the humoral response to ovalbumin (administered on days 3 and 15 post-inoculation). Infected animals followed the expected clinical course. BVDV titers, and anti-BVDV antibodies confirmed the course of infection and were not affected by the administration of OVA. Both the T-helper (CD4(+)) and B-cell (CD20(+)) compartments were significantly (P < 0.05) reduced in infected animals, while the gamma-delta T-cell population (Workshop cluster 1+, WC1(+)) decreased slightly in numbers. Infection with BVDV delayed the increase in OVA IgG by approximately 3 d from day 12 through day 21 post-inoculation. Between days 25 and 37 post-inoculation following BVDV infection the IgM concentration in the BVDV- group decreased while the OVA IgM titer still was rising in the BVDV+ animals. Thus, active BVDV infection delays IgM and IgG responses to a novel, non-infectious antigen.

Une infection aiguë par le BVDV-2 chez les veaux retarde les réponses humorales face à un test à l'aide d'un antigène non infectieux. Les effets immunosuppressifs d'une inoculation défin intranasale à l'aide du virus non cytopathogène de la diarrhée virale bovine (VBVD) 2a (souche 1373) ont été évalués par les réactions acquises et innées du système immunitaire à l'ovalbumine (OVA). On a émis l'hypothèse que l'infection concomitante par le VBVD retardait et réduisait la réaction humorale à l'ovalbumine (administrée aux jours 3 et 15 après l'inoculation). Les animaux infectés ont suivi le cheminement clinique prévu. Les titres de BVDV et les anticorps anti-BVDV ont confirmé le déroulement de l'infection et ils n'ont pas été affectés par l'administration d'OVA. Les compartiments des lymphocytes T auxiliaires (CD4+) et des cellules B (CD20+) étaient significativement réduits (P < 0,05) chez les animaux infectés, tandis que la numération de la population de cellules T gamma-delta (WC1+) a diminué légèrement. L'infection par le VBVD a retardé l'augmentation de l'OVA IgG d'environ 3 jours, à compter du jour 12 jusqu'au jour 21 après l'inoculation. Entre les jours 25 et 37 après l'inoculation suivant l'infection par le BVDV, la concentration d'IgM dans le groupe VBVD a diminué tandis que le titre d'OVA IgM augmentait toujours chez les animaux positifs pour le VBVD. Par conséquent, l'infection active par le VBVD retarde les réactions IgM et IgG face à un antigène non infectieux nouveau.(Traduit par Isabelle Vallières).

Cytotoxicity of ORF3 proteins from a nonpathogenic and a pathogenic porcine circovirus.

Porcine circovirus type 2 (PCV2) infection is associated with significant and serious swine diseases worldwide, while PCV1 appears to be a nonpathogenic virus. Previous studies demonstrated that the ORF3 protein of PCV2 (PCV2ORF3) was involved in PCV2 pathogenesis via its proapoptotic capability (J. Liu, I. Chen, Q. Du, H. Chua, and J. Kwang, J. Virol. 80:5065-5073, 2006). If PCV2ORF3-induced apoptosis is a determinant of virulence, PCV1ORF3 is hypothesized to lack this ability. The properties of PCV1 and PCV2 ORF3, expressed as fusion proteins to an enhanced green fluorescent protein (eGFP), were characterized with regard to their ability to cause cellular morphological changes, detachment, death, and apoptosis. PCV1ORF3 significantly induced more apoptotic cell death and was toxic to more different cell types than PCV2ORF3 was. PCV1ORF3-associated cell death was caspase dependent. PCV1ORF3 also induced poly(ADP-ribose) polymerase 1 (PARP) cleavage; however, whether PARP was involved in cell death requires further studies. Truncation of PCV1 and elongation of PCV2 ORF3 proteins revealed that the first 104 amino acids contain a domain capable of inducing cell death, whereas the C terminus of PCV1ORF3 contains a domain possibly responsible for enhancing cell death. These results suggest that the pathogenicity of PCV2 for pigs is either not determined or not solely determined by the ORF3 protein.

Immune Responses in Laying Hens after an Infectious Bronchitis Vaccination of Pullets: A Comparison of Two Vaccination Strategies.

For decades, vaccinations have been used to limit infectious bronchitis (IB) in both the broiler and layer industries. Depending on the geographical area, live attenuated vaccines are used either alone or in combination with inactivated vaccines to control infectious bronchitis virus (IBV) infections. It has been shown that administering inactivated vaccines preceded by priming with live attenuated vaccines in pullets protects laying hens against IB. However, the immunological basis of this protective response has not been adequately investigated. The objective of the study was to compare two vaccination strategies adapted by the Canadian poultry industry in terms of their ability to systemically induce an adequate immune response in IBV-impacted tissues in laying hens. The first vaccination strategy (only live attenuated IB vaccines) and second vaccination strategy (live attenuated and inactivated IB vaccines) were applied. Serum anti-IBV antibodies were measured at two time points, i.e., 3 weeks and 10 weeks post last vaccination. The recruitment of T cell subsets (i.e., CD4+ and CD8+ T cells), and the interferon (IFN)-γ mRNA expression were measured at 10 weeks post last vaccination. We observed that vaccination strategy 2 induced significantly higher serum anti-IBV antibody responses that were capable of neutralizing an IBV Mass variant associated with a flock history of shell-less egg production better than a Delmarva (DMV)1639 variant, as well as a significantly higher IFN-γ mRNA expression in the lungs, kidneys, and oviduct. We also observed that both vaccination strategies recruited CD4+ T cells as well as CD8+ T cells to the examined tissues at various extents. Our findings indicate that vaccination strategy 2 induces better systemic and local host responses in laying hens.

In Ovo Delivered Toll-Like Receptor 7 Ligand, Resiquimod Enhances Host Responses against Infectious Bronchitis Corona Virus (IBV) Infection.

Toll-like receptor (TLR) 7 ligand, resiquimod, has been studied as an adjuvant and antiviral agent against several pathogens in chicken. Yet, the effectiveness of resiquimod against infectious bronchitis virus (IBV) infection has not been evaluated. In this study, we investigated the effectiveness of resiquimod delivered pre-hatch (in ovo) against IBV infection post-hatch identifying key mechanisms involved in resiquimod driven immune activation. First, we found an upregulation of interleukin (IL)-1ß and interferon (IFN)-γ mRNA levels and considerable expansions of macrophage and cluster of differentiation (CD) 8α+ T cell populations in lungs of chicken as early as day one post-hatch, following pre-hatch delivery of resiquimod. Second, we observed that resiquimod was able to act as an adjuvant when resiquimod was delivered pre-hatch along with an inactivated IBV vaccine. Finally, when the resiquimod pretreated one-day-old chickens were infected with IBV, reduction in viral shedding via oral and fecal routes was observed at 3 days post- infection. Overall, this study shows that the pre-hatch delivered resiquimod increases cell-mediated immune responses in lungs with an advantage of reduction in IBV shedding.

Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2.

A novel reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2 has been developed. The LAMP assay achieves a comparable limit of detection (25-50 copies per reaction) to commonly used RT-PCR protocols using clinical samples quantified by digital droplet PCR. Precision, cross-reactivity, inclusivity, and limit of detection studies were performed according to regulatory standards. Clinical validation of dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 98.48 % (95 % CI 91.84%-99.96%) and NPA 100.00 % (95 % CI 93.84%-100.00%) based on the E gene and N2 gene reference RT-PCR methods. The method has implications for development of point of care technology using isothermal amplification.

Animal models of henipavirus infection: a review.

Hendra virus (HeV) and Nipah virus (NiV) form a separate genus Henipavirus within the family Paramyxoviridae, and are classified as biosafety level four pathogens due to their high case fatality rate following human infection and because of the lack of effective vaccines or therapy. Both viruses emerged from their natural reservoir during the last decade of the 20th century, causing severe disease in humans, horses and swine, and infecting a number of other mammalian species. The current review summarises current published data relating to experimental infection of small and large animals, including the natural reservoir species, the Pteropus bat, with HeV or NiV. Susceptibility to infection and virus distribution in the individual species is discussed, along with the pathogenesis, pathological changes, and potential routes of transmission.

Cloning, expression and immunoassay detection of ferret IFN-gamma.

Ferrets (Mustela putorius furo) develop symptoms upon influenza infection that resemble those of humans, including sneezing, body temperature variation and weight loss. Highly pathogenic strains of influenza A, such as H5N1, have the capacity to cause severe illness or death in ferrets. The use of ferrets as a model of influenza infection is currently limited by a lack of species-specific immunological reagents. Interferon gamma (IFN-gamma) plays a key role in the development of innate and adaptive immunity and the regulation of Th1-type immune responses. Here we describe the cloning of the full-length cDNA for ferret IFN-gamma. Multiple sequence alignment of the predicted amino acid sequence with those of other species indicates that the predicted ferret protein shares the highest identity with Eurasian badger IFN-gamma. We raised two hybridoma clones expressing monoclonal antibodies against recombinant ferret IFN-gamma capable of detecting IFN-gamma protein derived from mitogen-stimulated ferret PBMCs by immunoblotting, ELISA and ELISPOT assay. Finally, an ELISA utilizing the ferret-specific antibodies detected elevated levels of IFN-gamma in serum samples from H3N2 influenza A-infected ferrets.

The In Ovo Delivery of CpG Oligonucleotides Protects against Infectious Bronchitis with the Recruitment of Immune Cells into the Respiratory Tract of Chickens.

The in ovo delivery of cytosine-guanosine (CpG) oligodeoxynucleotides (ODNs) protects chickens against many bacterial and viral infections, by activating the toll-like receptor (TLR)21 signaling pathway. Although the delivery of CpG ODNs in ovo at embryo day (ED) 18 has been shown to reduce infectious bronchitis virus (IBV) loads in embryonic chicken lungs pre-hatch, whether in ovo delivered CpG ODNs are capable of protecting chickens against a post-hatch challenge is unknown. Thus, our objectives were to determine the protective effect of the in ovo delivery of CpG ODNs at ED 18 against IBV infection encountered post-hatch and, then, to investigate the mechanisms of protection. We found significantly higher survival rates and reduced IBV infection in the chickens following the pre-treatment of the ED 18 eggs with CpG ODNs. At 3 days post infection (dpi), we found an increased recruitment of macrophages, cluster of differentiation (CD)8α+ and CD4+ T lymphocytes, and an up-regulation of interferon (IFN)-γ mRNA in the respiratory tract of the chickens. Overall, it may be inferred that CpG ODNs, when delivered in ovo, provide protection against IBV infection induced morbidity and mortality with an enhanced immune response.

The interaction between the Nipah virus nucleocapsid protein and phosphoprotein regulates virus replication.

Continued outbreaks of Henipaviruses in South Asia and Australia cause severe and lethal disease in both humans and animals. Together, with evidence of human to human transmission for Nipah virus and the lack of preventative or therapeutic measures, its threat to cause a widespread outbreak and its potential for weaponization has increased. In this study we demonstrate how overexpression of the Nipah virus nucleocapsid protein regulates viral polymerase activity and viral RNA production. By overexpressing the Nipah virus nucleocapsid protein in trans viral transcription was inhibited; however, an increase in viral genome synthesis was observed. Together, the bias of polymerase activity towards genome production led to the severe inhibition of viral progeny. We identified two domains within the nucleocapsid protein, which were each independently capable of binding the viral phosphoprotein. Evident by our data, we propose that the nucleocapsid protein's ability to interact with the phosphoprotein of the polymerase complex causes a change in polymerase activity and subsequent deficiency in viral replication. This study not only provides insights into the dynamics of Henipavirus RNA synthesis and replication, but also provides insight into potential targets for antiviral drug development.

Induction of humoral immune response in piglets after perinatal or post-weaning immunization against porcine circovirus type-2 or keyhole limpet hemocyanin.

The objective of this study was to test the hypothesis that porcine circovirus type-2 (PCV2) vaccination is efficacious when administered in the first week of life. Three groups of pigs were vaccinated with Circumvent either early (at the end of week 1), late (at the end of week 4), or not at all. All 3 groups were later challenged intranasally with PCV2 (at the end of week 5). Two other groups were immunized with keyhole limpet hemocyanin (KLH) as a novel antigen at the end of either week 1 or week 4. Weight, PCV2 genome copy number in serum and saliva, anti-KLH antibody titer, and serum PCV2-neutralizing antibodies were measured weekly. Early PCV2 vaccination or KLH antigen exposure resulted in earlier humoral responses that were slower to develop than in older piglets, yet converged with the responses to later vaccination within 5 wk. Both groups of vaccinated piglets had periods of higher PCV2-neutralizing antibody titers and lower viral levels shortly after weaning and PCV2 challenge, thus supporting the recent labelling of 1 Canadian PCV2 vaccine for use in week 1 and suggesting that early PCV2 vaccination can reduce piglet handling without compromising vaccine efficacy.

L'objectif de la présente étude était de vérifier l'hypothèse que la vaccination contre le circovirus porcin de type 2 (CVP2) est efficace lorsqu'administrée durant la première semaine de vie. Trois groupes de porcs ont été vaccinés avec Circumvent soit hâtivement (à la fin de la semaine 1), tardivement (à la fin de la semaine 4), ou pas du tout. Les trois groupes ont plus tard été inoculés par voie intranasale avec CVP2 (à la fin de la semaine 5). Deux autres groupes ont été immunisés avec de l'hémocyanine de patelle (KLH) à titre de nouvel antigène à la fin de soit la semaine 1 ou la semaine 4. Le poids, le nombre de copies du génome de CVP2 dans le sérum et la salive, le titre d'anticorps anti-KLH, et le titre d'anticorps sériques neutralisants CVP2 ont été mesurés à chaque semaine. La vaccination tôt contre CVP2 ou l'exposition à l'antigène KLH a donné des réponses humorales plus hâtives qui étaient plus lentes à se développer que chez les porcs plus vieux, mais qui convergeaient vers les réponses de la vaccination tardive à l'intérieur d'un délai de 5 sem. Les deux groupes de porcelets vaccinés avaient des périodes de titres d'anticorps neutralisants contre CVP2 plus élevés et des charges virales plus basses peu de temps après le sevrage et le challenge avec CVP2, soutenant ainsi l'étiquetage récent d'un vaccin canadien contre CVP2 pour utilisation dans la semaine 1 et suggérant qu'une vaccination tôt contre CVP2 peut réduire la manipulation des porcelets sans compromettre l'efficacité du vaccin.(Traduit par Docteur Serge Messier).

Production and characterization of monoclonal antibodies against binary ethylenimine inactivated Nipah virus.

Nipah virus, a zoonotic paramyxovirus which emerged recently was chemically inactivated using binary ethylenimine (BEI). The inactivated virus was concentrated and purified by sucrose gradient centrifugation. The gradient fractions were examined by electron microscopy and Western immunoblot, and gradient fraction containing mainly Nipah matrix (M) and nucleocapsid (N) proteins was used for immunizing BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones identified five strongly positive clones producing IgG monoclonal antibodies (mAbs) reactive to the Nipah virus antigen. The protein specificity of these mAbs was determined by Western immunoblot using Nipah virus and recombinant Nipah virus proteins expressed in mammalian cells. Four mAbs reacted with Nipah N protein and one reacted with Nipah M protein. None of the mAbs neutralized Nipah virus infectivity in vitro. However, all mAbs recognized Nipah virus in ELISA and immunofluorescence assay. F45G2 mAb was most suitable for immunohistochemistry on long term formalin-fixed Nipah virus infected swine tissues. Three of the anti-nucleocapsid mAbs (F45G2, F45G3 and F45G6) showed cross-reactivity with closely related Hendra virus N protein in both immunofluorescence and Western Immunoblot assays. Two of the mAbs were specific for the Nipah virus only, F45G4 (anti-N) and F45G5 (anti-M), and could be used in the primary identification of Nipah virus. The use of these immunoreagents to develop new diagnostic assays is discussed.

Rapid, differential diagnosis of orthopox- and herpesviruses based upon real-time PCR product melting temperature and restriction enzyme analysis of amplicons.

Orthopoxviruses tend to have non-specific early symptoms that cannot be differentiated readily from other infectious exanthemas, such as varicella-zoster virus (VZV) or disseminated herpes simplex virus (HSV) infections. A rapid assay was developed for the differential diagnosis of orthopoxviruses and herpesviruses based upon the melting temperatures of real-time PCR amplicons. A mean melting temperature difference of 8.7 degrees C was observed between the products amplified from the two virus families. Further identification of individual pathogens was made using restriction enzyme analysis. The assay was able to identify correctly viruses from quality control panels of herpes and orthopoxviruses.

Probe-free real-time reverse transcription polymerase chain reaction assays for the detection and typing of porcine reproductive and respiratory syndrome virus in Canada.

Porcine reproductive and respiratory syndrome (PRRS) has tremendous impact on the pork industry in North America. The molecular diagnosis of infection with PRRS virus (PRRSV) is hampered by its considerable strain diversity. In this study, 43 previously published or newly developed primers for probe-free real-time reverse transcription polymerase chain reaction (RT-PCR) were evaluated on their sensitivity, specificity, reproducibility, and repeatability, using a diverse panel of 36 PRRSV strains as well as other arteriviruses and unrelated porcine viruses. Three primer pairs had excellent diagnostic and analytical sensitivity on par with a probe-based reference assay, absolute specificity to virus genotype and species, as well as over 95% reproducibility and repeatability across a wide dynamic range.

Le syndrome dysgénésique et respiratoire du porc (SDRP) a un impact énorme sur l'industrie du porc en l'Amérique du Nord. Le diagnostic moléculaire de l'infection avec le virus du SDRP est entravée par sa diversité considérable. Dans cette étude, 43 amorces publiées antérieurement ou nouvellement développés pour RT-PCR en temps réel sans d'une sonde spécifique ont été évalués sur leur sensibilité, la spécificité, la reproductibilité et la répétabilité. Un groupe varié de 36 souches de virus du SDRP, d'autres virus appartenant au genre des arterivirus et les virus porcins indépendants a été utilisé. Trois paires d'amorces ont une excellente sensibilité de diagnostic and d'analyse qui équivaut à un test de référence basé sur des sondes. Ils avaient une spécificité absolue de génotype et des espèces du virus, et la reproductibilité et la répétabilité a été plus de 95 % sur une large plage dynamique.(Traduit par les auteurs).

In ovo delivery of CpG DNA reduces avian infectious laryngotracheitis virus induced mortality and morbidity.

Endosomal toll-like receptor-21 and -9 sense CpG DNA activating production of pro-inflammatory mediators with antimicrobial effects. Here, we investigated the induction of antiviral response of in ovo delivered CpG DNA against infectious laryngotracheitis virus (ILTV) infection. We found that in ovo delivered CpG DNA significantly reduces ILTV infection pre-hatch correlating with the expression of IL-1ß and increase of macrophages in lungs. As assessed in vitro, CpG DNA stimulated avian macrophages could be a potential source of IL-1ß and other pro-inflammatory mediators. Since we also found that in ovo CpG DNA delivery maintains increased macrophages in the lungs post-hatch, we infected the chickens on the day of hatch with ILTV. We found that in ovo delivered CpG DNA significantly reduces mortality and morbidity resulting from ILTV infection encountered post-hatch. Thus, CpG DNA can be a candidate innate immune stimulant worthy of further investigation for the control of ILTV infection in chickens.

Microglial cell activation in demyelinating canine distemper lesions.

Microglia cells are the principal immune effector elements of the brain responding to any pathological event. To elucidate the possible role of microglia in initial non-inflammatory demyelination in canine distemper virus (CDV) infection, microglia from experimentally CDV infected dogs were isolated ex vivo by density gradient centrifugation and characterized immunophenotypically and functionally using flow cytometry. Results from dogs with demyelinating lesions were compared to results from recovered dogs and two healthy controls. CDV antigen could be detected in microglia of dogs with histopathologically confirmed demyelination. Microglia of these dogs showed marked upregulation of the surface molecules CD18, CD11b, CD11c, CD1c, MHC class I and MHC class II and a tendency for increased expression intensity of ICAM-1 (CD54), B7-1 (CD80), B7-2 (CD86), whereas no increased expression was found for CD44 and CD45. Functionally, microglia exhibited distinctly enhanced phagocytosis and generation of reactive oxygen species (ROS). It was concluded that in CDV infection, there is a clear association between microglial activation and demyelination. This strongly suggests that microglia contribute to acute myelin destruction in distemper.

Characterization of canine microglial cells isolated ex vivo.

Microglia are the principal immune effector elements of the brain sharing immunophenotypic and functional characteristics of macrophages as well as of antigen presenting cells (APCs). The purpose of this study was to isolate canine microglial cells and make them available for ex vivo characterizations of their functions and immunophenotype. After isolation, carried out by density gradient centrifugation, microglial cells accumulated on distinct interfaces of 1.077 and 1.066 g/ml of a Percoll gradient. Identification of microglial cells in other species is realized by their specific immunophenotype of CD11b/c+ and CD45low. Our results indicate, that expression of CD45 is very low or even absent in canine microglial cells. In addition, they expressed CD18 and CD11b/c+, as determined by flow cytometry and immunohistochemistry. Fourteen additional monoclonal antibodies (mAbs) were used to characterize and compare canine microglial cells with monocytes. Microglia and monocytes can be clearly distinguished by their differential expression intensity of surface antigens (CD45, CD44, CD14). Functional characterization was assessed by a reactive oxygen species (ROS)-generation test and phagocytosis assay using flow cytometry. In conclusion, ex vivo examination of microglia is possible in dogs and most probably reflects the conditions in vivo. The measurement of tissue culture artifacts can be largely avoided using this method.

Porcine circovirus-2 capsid protein induces cell death in PK15 cells.

Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis.

Rapid detection of neutralizing antibodies against bovine viral diarrhoea virus using quantitative high-content screening.

Bovine viral diarrhoea virus (BVDV) is an important cause of morbidity, mortality and economic losses in cattle worldwide. Humoral immunity to BVDV plays a major role in the protection against infection and disease. In vitro serum neutralization tests can quantify humoral responses, but standard protocols are time-consuming and labour-intensive. The objective of this study was to develop a highly sensitive assay based on high-content cell-by-cell screening that is faster and less subjective than the conventional protocols. It can detect a neutralizing antibody response within the first week after infection of an animal, takes less than 24h to complete and excludes operator bias by automated data acquisition and analysis.