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.

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.

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.

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).

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.

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).

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).

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.

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.

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.

Induction of Toll-like receptor 4 signaling in avian macrophages inhibits infectious laryngotracheitis virus replication in a nitric oxide dependent way.

LPS is one of the pathogen associated molecular patterns that activates Toll-like receptor 4 (TLR4) signaling pathway eliciting antiviral host responses in mammals although information on such responses in avian species is scarce. Our objectives were to characterize the LPS induced innate responses particularly the expression of LPS receptors (TLR4, CD14) in avian macrophages and observe whether TLR4 mediated induction of NO can elicit antiviral response against infectious laryngotracheitis virus (ILTV) replication. We found that LPS was capable of inducing the expression of TLR4, CD14 and NO production but not the type 1 interferons in an avian macrophage cell line, MQ-NCSU. We also showed that TLR4 mediated NO production can lead to antiviral response against ILTV replication when MQ-NCSU cells were treated with LPS and the resultant supernatant was then transferred to ILTV replicating cells to assess antiviral activity. Antiviral activity of NO was blocked by a selective inhibitor, S-methylisothiourea sulfate that inhibits inducible NO synthase. This observation confirms that the antiviral activity is positively correlated with NO production. The data show that LPS can be a potential innate immune stimulant that can be used against ILTV infection in chickens that require further evaluation in vivo.

Development and evaluation of one-step rRT-PCR and immunohistochemical methods for detection of Rift Valley fever virus in biosafety level 2 diagnostic laboratories.

Rift Valley fever virus (RVFV) is a zoonotic insect transmitted virus endemic to Africa and the Arabian Peninsula. Infection causes abortions and high mortality in newborn ruminants. The overall human infection rate is <1%; however, fatality rates in those with severe clinical disease have been reported as high as 29%. The potential of RVFV as a bioterrorism agent and/or being accidentally introduced into North America is widely recognized. Currently, regional veterinary biosafety level 2 (BSL-2) diagnostic laboratories lack safe, modern, validated diagnostic tests to detect RVFV. An existing one-step real-time RT-PCR (rRT-PCR) assay was modified for quick virus inactivation for use in BSL-2 laboratories, evaluated on serum and tissue samples from experimentally infected lambs and calves, and compared to virus isolation. Viremia was detected in all inoculated sheep with titers reaching 10(6.5) plaque forming units/ml, or up to 10(10) viral RNA copies/ml. Viremia in calves was lower and not detected in all inoculated animals; however, all animals became transiently febrile and were infected as determined by rRT-PCR of tissues. Virus was isolated from rRT-PCR-positive liver and/or spleen in 33% of lamb and 41% of calf samples between 2 and 7 days post inoculation. For RVFV antigen detection, reagents are typically produced at BSL-3Ag or BSL-4 conditions and require inactivation and safety testing for use outside of containment. In this study, antiserum against recombinant RVFV-nucleocapsid (N) was produced to develop an immunohistochemical (IHC) assay which was subsequently evaluated on formalin fixed lamb and calf tissues at BSL-2 laboratory conditions. Antigen was detected by IHC in 79% of rRT-PCR-positive sheep and 70% of rRT-PCR-positive calf tissues tested. Once validated and approved by national regulatory agencies, these assays can be safely produced and distributed to regional diagnostic laboratories, providing capacity for early detection of RVFV in suspected ruminant samples.

Anti-tumor necrosis factor therapy and influenza: keeping it in perspective.

The association of opportunistic infections in the context of anti-tumor necrosis factor (TNF) antibody therapies have attracted widespread attention. The recent H1N1 influenza pandemic brought this into sharp focus with numerous patient queries and physician anxieties. The following short review gives a scientific perspective to this issue including the role of vaccination.

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.

Genomic analysis of porcine circovirus type-2 isolates in Alberta pigs demonstrating clinical porcine circovirus associated disease (PCVAD).

Nineteen pigs with clinical signs of porcine circovirus associated diseases (PCVAD) on 5 Alberta pig farms were examined pathologically, including gross pathology, histopathology, and immunohistochemistry. Polymerase chain reaction (PCR) for porcine circovirus type-2 (PCV-2) and sequence analysis was performed on tissue samples of 12 animals. Results showed that new strains of porcine circovirus type-2 genogroup b were present in most pigs that were positive for PCV-2. Furthermore, a mixed infection with PCV-2a and PCV-2b occurred in the liver and lungs of 1 pig. Comparison of whole genome sequences representing known viruses and the newly discovered Alberta viruses revealed mutations distributed over the entire genome of PCV-2; however, sequences encoding for immunodominant epitopes of PCV-2 appear to be unaffected by these mutations.

Molecular, biochemical and genetic characteristics of BSE in Canada.

The epidemiology and possibly the etiology of bovine spongiform encephalopathy (BSE) have recently been recognized to be heterogeneous. In particular, three types [classical (C) and two atypical (H, L)] have been identified, largely on the basis of characteristics of the proteinase K (PK)-resistant core of the misfolded prion protein associated with the disease (PrP(res)). The present study was conducted to characterize the 17 Canadian BSE cases which occurred prior to November 2009 based on the molecular and biochemical properties of their PrP(res), including immunoreactivity, molecular weight, glycoform profile and relative PK sensitivity. Two cases exhibited molecular weight and glycoform profiles similar to those of previously reported atypical cases, one corresponding to H-type BSE (case 6) and the other to L-type BSE (case 11). All other cases were classified as C-type. PK digestion under mild and stringent conditions revealed a reduced protease resistance in both of these cases compared to the C-type cases. With Western immunoblotting, N-terminal-specific antibodies bound to PrP(res) from case 6 but not to that from case 11 or C-type cases. C-terminal-specific antibodies revealed a shift in the glycoform profile and detected a fourth protein fragment in case 6, indicative of two PrP(res) subpopulations in H-type BSE. No mutations suggesting a genetic etiology were found in any of the 17 animals by sequencing the full PrP-coding sequence in exon 3 of the PRNP gene. Thus, each of the three known BSE types have been confirmed in Canadian cattle and show molecular characteristics highly similar to those of classical and atypical BSE cases described from Europe, Japan and the USA. The occurrence of atypical cases of BSE in countries such as Canada with low BSE prevalence and transmission risk argues for the occurrence of sporadic forms of BSE worldwide.

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.