Structure guided mutagenesis of Henipavirus Receptor Binding Proteins reveals molecular determinants of receptor usage and antibody binding epitopes.

Nipah virus (NiV) is a highly lethal, zoonotic henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, use EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, identify the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveals regions critical for GhV binding of EFNB2, and describes putative HNV antibody binding epitopes.

Review of critical factors affecting analytical characteristics of serological and molecular assays.

Analytical characteristics of diagnostic tests, encompassing estimates of repeatability, analytical specificity (ASp) and analytical sensitivity (ASe), are determined during Stage 1 of the OIE Assay Validation Pathway. Repeatability (an estimate of assay precision and robustness), ASp (measuring only what an assay is intended to measure) and ASe (synonymous with the lower limit of detection) are fundamental parameters that determine future test performance. Importantly, these parameters provide the basis for deciding whether a prototype assay progresses to the next stage of the OIE Assay Validation Pathway (determination of diagnostic characteristics) or is withdrawn in favour of alternate tests with better analytical performance characteristics. Implicit in the successful development and validation of any assay is a sound understanding of the target pathogen, the disease pathogenesis in susceptible hosts, the fundamental technical principles that underliey each test system, and its intended use. Factors that affect analytical characteristics of diagnostic assays are numerous and may vary according to each assay type. Using, as examples, development of an enzyme-linked immunosorbent assay for detection of antibodies to capripoxviruses, and the comparative assessment of three quantitative real-time polymerase chain reactions for detection of African swine fever virus DNA, the main factors affecting analytical characteristics of serological and molecular assays are considered. As reviewed within, comprehensive and well-designed experiments are required to develop and optimise assays with favourable analytical characteristics. The underlying principles are broadly applicable to all assay types and, when conducted with appropriate rigour, provide the foundations for high-quality diagnostic tests that are fit for their intended purpose(s).

Les caractéristiques de performance analytique des tests diagnostiques, qui recouvrent l'estimation de la répétabilité, de la spécificité analytique (SpA) et de la sensibilité analytique (SeA) d'un test sont déterminées lors de l'étape 1 du processus de l'OIE relatif à la validation des essais. La répétabilité (une estimation de la précision et de la robustesse de l'essai), la SpA (qui mesure uniquement ce que l'essai est destiné à mesurer) et la SeA (synonyme de limite inférieure de détection) sont des paramètres essentiels qui déterminent les futures performances du test. Il est important de noter que ces paramètres apportent les éléments essentiels pour décider si l'essai peut passer à l'étape suivante du processus de validation de l'OIE (détermination des caractéristiques diagnostiques) ou s'il doit céder la place à des tests alternatifs dotés de meilleures caractéristiques de performance analytique. Pour réussir la mise au point et la validation d'un essai, certaines conditions préalables doivent être réunies : bien connaître l'agent pathogène cible et la pathogenèse de la maladie chez les réservoirs sensibles, ainsi que les grands principes techniques sous-jacents à chaque système de test et l'emploi prévu du test. Les facteurs affectant les caractéristiques analytiques d'un essai diagnostique sont nombreux et varient suivant le type d'essai dont il s'agit. À partir d'exemples portant sur une épreuve immuno-enzymatique mise au point pour la détection des anticorps dirigés contre les capripoxvirus et sur l'évaluation comparative de trois techniques d'amplification en chaîne par polymérase quantitative en temps réel pour la détection de l'ADN viral de la peste porcine africaine, les auteurs mettent en exergue les principaux facteurs qui peuvent altérer les caractéristiques analytiques des essais sérologiques et moléculaires. Il ressort de cette évaluation que des expérimentations complètes et bien conçues sont nécessaires pour mettre au point et optimiser des essais possédant les caractéristiques analytiques souhaitées. En général, les principes sous-jacents sont applicables à tous les types d'essai, et s'ils sont appliqués de manière rigoureuse, ils fournissent la garantie de disposer de tests diagnostiques de qualité élevée et aptes à l'emploi ou aux emplois prévus.

La primera etapa del proceso de validación de ensayos de la OIE es aquella en que se determinan las características analíticas de una prueba de diagnóstico, o dicho de otro modo, en que se calculan los valores de repetibilidad (estimación de la precisión y robustez del ensayo), especificidad analítica (es decir, el hecho de que el ensayo mida únicamente lo que está destinado a medir) y sensibilidad analítica (sinónimo referido al límite inferior de detección), que son tres parámetros fundamentales para determinar el futuro rendimiento de una prueba. Un aspecto importante es que estos parámetros sientan las bases a partir de las cuales decidir si un prototipo de ensayo debe pasar a la siguiente etapa del proceso de validación de ensayos de la OIE (determinación de las características de diagnóstico) o si vale más retirarlo en beneficio de otras pruebas que presenten mejores características de rendimiento analítico. Un factor implícito en el éxito de todo proceso de desarrollo y validación de ensayos es un sólido conocimiento del patógeno en cuestión, la patogénesis de la enfermedad en los anfitriones sensibles, los principios técnicos fundamentales en que reposa cada sistema de ensayo y sus usos previstos. Los numerosos factores que influyen en las características analíticas de un ensayo de diagnóstico difieren en función del tipo de ensayo. Utilizando como ejemplo el desarrollo de un ensayo inmunoenzimático de detección de anticuerpos contra capripoxvirus y la evaluación comparativa de tres PCR cuantitativas en tiempo real para detectar ADN del virus de la peste porcina africana, los autores pasan revista a los principales factores que determinan las características analíticas de los ensayos serológicos y moleculares. Como explican, para desarrollar y optimizar ensayos que presenten características analíticas favorables se requieren experimentos completos y bien concebidos. Los principios subyacentes son válidos en general para todo tipo de ensayos y, cuando se aplican con el debido rigor, sientan las bases para obtener pruebas de diagnóstico de gran calidad y adaptadas a la(s) finalidad(es) prevista(s).

Comparative pathogenicity of Vibrio spp., Photobacterium damselae ssp. damselae and five isolates of Aeromonas salmonicida ssp. achromogenes in juvenile Atlantic halibut (Hippoglossus hippoglossus).

Juvenile Atlantic halibut (~100 mg, Hippoglossus hippoglossus) were exposed to Vibrio proteolyticus, a Vibrio spp. isolate, Photobacterium damselae ssp. damselae and five different isolates of Aeromonas salmonicida ssp. achromogenes via an hour-long bath immersion to ascertain their variation in pathogenicity to this fish species. Results were analysed using Kaplan-Meier survival analysis. Analysis of the data from challenges using A. salmonicida ssp. achromogenes revealed three survival values of zero and a spread of values from 0 to 28.43. Challenges using a Vibrio spp isolate, V. proteolyticus and P. damselae resulted in Kaplan-Meier survival estimates of 31.21, 50.41 and 57.21, respectively. As all bacterial species tested could induce juvenile halibut mortalities, they must all be considered as potential pathogens. However, the degree of pathogenicity of A. salmonicida is isolate dependent.

Sutures impregnated with carbazate-activated polyvinyl alcohol reduce intraperitoneal adhesions.

BACKGROUND: Intraperitoneal adhesions cause significant morbidity. They occur after peritoneal trauma, which induces oxidative stress with production of inflammatory cytokines, peroxidized proteins (carbonyls) and lipids (aldehydes). This study aimed to investigate if carbazate-activated polyvinyl alcohol (PVAC), an aldehyde-carbonyl inhibitor, can reduce intraperitoneal adhesions in an experimental model. MATERIAL AND METHODS: Male Sprague-Dawley rats (n=110) underwent laparotomy, cecal abrasion and construction of a small bowel anastomosis. They either were treated with intraperitoneal instillation of PVAC or were sutured with PVAC-impregnated sutures. Thromboelastography analysis was performed using human blood and PVAC. The lipid peroxidation product malondialdehyde (MDA) and inflammatory cytokines IL-1ß and IL-6 were quantified in peritoneal fluid. At day 7, bursting pressure of the anastomosis was measured and adhesions were blindly scored. RESULTS: PVAC in human blood decreased the production of the fibrin-thrombocyte mesh without affecting the coagulation cascade. MDA, IL-1ß and IL-6 were increased after 6h without significant difference between the groups. PVAC-impregnated sutures reduced intraperitoneal adhesions compared to controls (p=0.0406) while intraperitoneal instillation of PVAC had no effect. Anastomotic bursting pressure was unchanged. CONCLUSIONS: Intervention with an aldehyde-carbonyl inhibitor locally in the wound by PVAC-impregnated sutures might be a new strategy to reduce intraperitoneal adhesions.

Review: Capripoxvirus Diseases: Current Status and Opportunities for Control.

Lumpy skin disease, sheeppox and goatpox are high-impact diseases of domestic ruminants with a devastating effect on cattle, sheep and goat farming industries in endemic regions. In this article, we review the current geographical distribution, economic impact of an outbreak, epidemiology, transmission and immunity of capripoxvirus. The special focus of the article is to scrutinize the use of currently available vaccines to investigate the resource needs and challenges that will have to be overcome to improve disease control and eradication, and progress on the development of safer and more effective vaccines. In addition, field evaluation of the efficacy of the vaccines and the genomic database available for poxviruses are discussed.

Growth characteristics of the intracellular pathogen, Piscirickettsia salmonis, in tissue culture and cell-free media.

Piscirickettsia salmonis is an intracellular bacterium that was first isolated and identified in fish cells. Several types of cell lines have been explored for their ability to provide the bacterium with a host cell to replicate in. Tissue culture has been used for growth and cultivation for nearly two decades, until the facultative nature of P. salmonis was confirmed upon the development of blood- and cysteine-based agar. Since then, research has continued to drive the creation of novel agar and broth formulations in order to improve the efficacy of cultivation of P. salmonis. Until now, the techniques and components used for growth have not been thoroughly discussed. In this review, the methods and formulations for growth of P. salmonis in tissue culture and cell-free media will be examined.

The application of protein microarray assays in psychoneuroimmunology.

Protein microarrays are miniaturized multiplex assays that exhibit many advantages over the commonly used enzyme-linked immunosorbent assay (ELISA). This article aims to introduce protein microarrays to readers of Brain, Behavior, and Immunity and demonstrate its utility and validity for use in psychoneuroimmunological research. As part of an ongoing investigation of psychological and behavioral influences on influenza vaccination responses, we optimized a novel protein microarray to quantify influenza-specific antibody levels in human sera. Reproducibility was assessed by calculating intra- and inter-assay coefficients of variance on serially diluted human IgG concentrations. A random selection of samples was analyzed by microarray and ELISA to establish validity of the assay. For IgG concentrations, intra-assay and inter-assay precision profiles demonstrated a mean coefficient of variance of 6.7% and 11.5% respectively. Significant correlations were observed between microarray and ELISA for all antigens, demonstrating the microarray is a valid alternative to ELISA. Protein microarrays are a highly robust, novel assay method that could be of significant benefit for researchers working in psychoneuroimmunology. They offer high throughput, fewer resources per analyte and can examine concurrent neuro-immune-endocrine mechanisms.

The evolutionary dynamics of influenza A virus adaptation to mammalian hosts.

Few questions on infectious disease are more important than understanding how and why avian influenza A viruses successfully emerge in mammalian populations, yet little is known about the rate and nature of the virus' genetic adaptation in new hosts. Here, we measure, for the first time, the genomic rate of adaptive evolution of swine influenza viruses (SwIV) that originated in birds. By using a curated dataset of more than 24 000 human and swine influenza gene sequences, including 41 newly characterized genomes, we reconstructed the adaptive dynamics of three major SwIV lineages (Eurasian, EA; classical swine, CS; triple reassortant, TR). We found that, following the transfer of the EA lineage from birds to swine in the late 1970s, EA virus genes have undergone substantially faster adaptive evolution than those of the CS lineage, which had circulated among swine for decades. Further, the adaptation rates of the EA lineage antigenic haemagglutinin and neuraminidase genes were unexpectedly high and similar to those observed in human influenza A. We show that the successful establishment of avian influenza viruses in swine is associated with raised adaptive evolution across the entire genome for many years after zoonosis, reflecting the contribution of multiple mutations to the coordinated optimization of viral fitness in a new environment. This dynamics is replicated independently in the polymerase genes of the TR lineage, which established in swine following separate transmission from non-swine hosts.

Pathology and viral antigen distribution following experimental infection of sheep and goats with capripoxvirus.

Current understanding of capripoxvirus pathogenesis is limited since there have been no detailed studies examining cell tropism at well-defined intervals following infection. We undertook time-course studies in sheep and goats following inoculation of sheeppox or goatpox viruses in their respective homologous hosts, and examined tissues by light microscopy. A monoclonal antibody generated to a sheeppox virus core protein was used for immunohistochemical detection of viral antigen in tissue sections. Lesions and virus antigen were observed consistently in the skin, lung and lymph nodes. Antigen was detected at 6 and 8 days post inoculation for skin and lung, respectively, within cells which appeared to be of monocyte/macrophage lineage. In sheep skin capripoxvirus immunoreactivity was detected within previously unreported large multinucleated cells. In the lung, double immunolabelling detected the simultaneous expression of capripoxvirus antigen and cytokeratin indicating the presence of virus within pneumocytes. Lung double immunolabelling also detected the expression of capripoxvirus antigen in CD68(+) cells, confirming the presence of viral antigen within macrophages. Based on early detection of infected macrophages, dissemination of virus within the host and localization to tissues likely occurred through cells of the monocyte/macrophage lineage. Histological findings revealed similarities with both monkeypox and smallpox, thus capripoxvirus infection in sheep and goats may represent useful models with which to study strategies for poxvirus-specific virus vaccine concepts and therapeutics.

Experimental study of the susceptibility of Atlantic cod, Gadus morhua (L.), to infection with an IPNV strain pathogenic for Atlantic salmon, Salmo salar L.

Intraperitoneal (IP) injection, cohabitation and immersion routes of infection were used to determine if Atlantic cod, Gadus morhua (L.), of 1 and 3 g are susceptible to infectious pancreatic necrosis (IPN). Mortalities of cod injected IP were significantly higher when challenged with infectious pancreatic necrosis virus (IPNV) than with phosphate buffered saline. This is the first report of Atlantic cod mortalities caused by IPNV. Fish challenged by cohabitation had significantly higher mortalities than the controls, but mortalities of Atlantic cod challenged with IPNV by immersion were not significantly different from controls. Titres of IPNV in the tissues of infected fish were sometimes very high (range 10(2)-10(10) infectious units per gram of tissue) suggesting virus replication and titres of fish that died were generally higher than those of fish which survived. However, the relatively low mortality rates when challenged by cohabitation and immersion (20% and 17%, respectively), compared to the IP injection challenge (100%) suggest that 1 and 3 g cod have low susceptibility to IPN when challenged by more natural routes. These data strongly suggest that the cause of death of experimentally challenged cod was IPNV and further histological evidence for this came from 1 g cod challenged IP with IPNV in which the pancreas showed severe necrosis and heavy immunostaining for IPNV coincidentally with the peak of mortalities.

Detection of antibodies against capripoxviruses using an inactivated sheeppox virus ELISA.

An indirect ELISA was developed to detect antibodies specific for capripoxviruses in goat, sheep and cattle sera. Heat-inactivated Nigerian sheeppox virus was used as the ELISA antigen. Sera obtained from sheep and goats that were experimentally infected with different capripoxvirus isolates were used to develop and evaluate the sensitivity of the ELISA. Virus neutralization indexes were determined for the experimental sera in OA3.Ts cells. The specificity of the ELISA was determined using 231 sera from capripoxvirus naïve sheep and goats from Canada. In addition, the ELISA was tested for cross-reactivity to anti-orf virus antibodies using orf-reactive sera and no cross-reactivity was observed. Using experimentally generated sera obtained from animals infected with virulent sheeppox or goatpox virus isolates, the diagnostic sensitivity of the ELISA was 96% with a diagnostic specificity of 95%, where the diagnostic sensitivity of the virus neutralization assay was 96% with a diagnostic specificity of 100%. Further evaluation of this ELISA, using 276 cattle serum samples that were positive by virus neutralization assays, revealed a diagnostic sensitivity of 88% with a specificity of 97%. These results indicated that the inactivated capripoxvirus ELISA can detect capripoxvirus-specific antibodies in sheep, goats and cattle that have been infected with virulent capripoxvirus isolates. Non-virulent capripoxvirus isolates, in contrast, did not elicit positive (>or=1.5 Log10 neutralization index) antibody responses.

Capripoxviruses: an emerging worldwide threat to sheep, goats and cattle.

Capripoxviruses are the cause of sheeppox, goatpox and lumpy skin disease (LSD) of cattle. These diseases are of great economic significance to farmers in regions in which they are endemic and are a major constraint to international trade in livestock and their products. Although the distribution of capripoxviruses is considerably reduced from what it was even 50 years ago, they are now expanding their territory, with recent outbreaks of sheeppox or goatpox in Vietnam, Mongolia and Greece, and outbreaks of LSD in Ethiopia, Egypt and Israel. Increased legal and illegal trade in live animals provides the potential for further spread, with, for instance, the possibility of LSD becoming firmly established in Asia. This review briefly summarizes what is known about capripoxviruses, including their impact on livestock production, their geographic range, host-specificity, clinical disease, transmission and genomics, and considers current developments in diagnostic tests and vaccines. Capripoxviruses have the potential to become emerging disease threats because of global climate change and changes in patterns of trade in animals and animal products. They also could be used as economic bioterrorism agents.

Quantification of lumpy skin disease virus following experimental infection in cattle.

Lumpy skin disease along with sheep pox and goatpox are the most serious poxvirus diseases of livestock, and are caused by viruses that belong to the genus Capripoxvirus within the subfamily Chordopoxvirinae, family Poxviridae. To facilitate the study of lumpy skin disease pathogenesis, we inoculated eight 4- to 6-month-old Holstein calves intravenously with lumpy skin disease virus (LSDV) and collected samples over a period of 42 days for analysis by virus isolation, real-time PCR and light microscopy. Following inoculation, cattle developed fever and skin nodules, with the extent of infection varying between animals. Skin nodules remained visible until the end of the experiment on day post-inoculation (DPI) 42. Viremia measured by real-time PCR and virus isolation was not observed in all animals but was detectable between 6 and 15 DPI. Low levels of viral shedding were observed in oral and nasal secretions between 12 and 18 DPI. Several tissues were assessed for the presence of virus at DPI 3, 6, 9, 12, 15, 18 and 42 by virus isolation and real-time PCR. Virus was consistently detected by real-time PCR and virus isolation at high levels in skin nodules indicating LSDV has a tropism for skin. In contrast, relatively few lesions were observed systemically. Viral DNA was detected by real-time PCR in skin lesions collected on DPI 42. Cattle developing anti-capripoxvirus antibodies starting at DPI 21 was detected by serum neutralization. The disease in this study varied from mild with few secondary skin nodules to generalized infection of varying severity, and was characterized by morbidity with no mortality.

Enhanced neuronal differentiation in a three-dimensional collagen-hyaluronan matrix.

Efficient 3D cell systems for neuronal induction are needed for future use in tissue regeneration. In this study, we have characterized the ability of neural stem/progenitor cells (NS/PC) to survive, proliferate, and differentiate in a collagen type I-hyaluronan scaffold. Embryonic, postnatal, and adult NS/PC were seeded in the present 3D scaffold and cultured in medium containing epidermal growth factor and fibroblast growth factor-2, a condition that stimulates NS/PC proliferation. Progenitor cells from the embryonic brain had the highest proliferation rate, and adult cells the lowest, indicating a difference in mitogenic responsiveness. NS/PC from postnatal stages down-regulated nestin expression more rapidly than both embryonic and adult NS/PC, indicating a faster differentiation process. After 6 days of differentiation in the 3D scaffold, NS/PC from the postnatal brain had generated up to 70% neurons, compared with 14% in 2D. NS/PC from other ages gave rise to approximately the same proportion of neurons in 3D as in 2D (9-26% depending on the source for NS/PC). In the postnatal NS/PC cultures, the majority of betaIII-tubulin-positive cells expressed glutamate, gamma-aminobutyric acid, and synapsin I after 11 days of differentiation, indicating differentiation to mature neurons. Here we report that postnatal NS/PC survive, proliferate, and efficiently form synapsin I-positive neurons in a biocompatible hydrogel.

Immunostimulation of larvae and juveniles of cod, Gadus morhua L.

Cod larval culture is currently hampered by high mortalities in the first 2-3 weeks after hatching, often due to infectious diseases. The immune system of cod is not fully competent until 2-3 months after hatching. Conventional vaccination is, therefore, not of value before this time, and the larvae are wholly reliant on non-specific parameters for their defence against infection. A range of substances, generally derived from bacterial, fungal or plant origin, can activate these non-specific parameters. During three hatching seasons, 2001-2003, at the Marine Institute's Experimental Station, Stadur, Grindavik, Iceland, the effects of several immunostimulants on survival and disease resistance of cod larvae and juveniles were examined. Both bathing treatments and administration in the feed were used. One of these substances, lipopolysaccharide (LPS), isolated from the bacterium Aeromonas salmonicida (ssp. salmonicida or achromogenes), appeared in some instances to improve survival and have a beneficial effect on disease resistance. Other substances tested had limited effects. The results emphasize the need for further work in this field.

What the patient wore, and why ...

This study set out to compare the wearing habits and attitudes of patients today with those, featured in a study by Cross, fitted prior to 1949. Information was sought in the present time regarding the age, occupation and sex of the wearers in 10 different categories of lens types currently available. The views of the patients were also sought regarding the comfort, distance vision, close vision, convenience and how the present lenses met their expectations. Motivation to wear contact lenses was also compared between the two studies. Results show a considerable change in the age of wearers both at the time of fitting and at the time of the studies, occupations of the wearers, and wearing modalities. Most of all it highlights the huge amount of choice available to the modern wearer, not only in lens types and materials, but also in the location and type of practice fitting lenses now at very much lower costs. Wearing times tend to have dropped since 1949 but through choice rather than necessity.

Development and function of the thymus in teleosts.

The thymus plays a pivotal role in the development of the adaptive immune system, an important factor that separates higher vertebrates from the rest of the animal phyla. The development of functional T-cells from thymocytes is a crucial step in the development of a functional vertebrate immune system and whilst recent advances in molecular and developmental biology have advanced our understanding of T-cell development, they have also provided potential model species across the vertebrate phyla including the zebrafish (Danio rerio). However, this species is one of more than 20,000 species of fish that could assist in elucidating the development of the vertebrate thymus and, consequently, the evolution of the vertebrate immune response. In this paper we review the knowledge of the teleost thymus through the organogenesis and development studies in teleosts together with advances in molecular and functional approaches. Where necessary we will combine this knowledge with that obtained in higher vertebrates.

Completion of the full-length genome sequence of Menangle virus: characterisation of the polymerase gene and genomic 5' trailer region.

Menangle virus (MenV), isolated in 1997 from stillborn piglets during an outbreak of reproductive disease at a large commercial piggery, is the only new paramyxovirus to be identified in Australia since Hendra virus in 1994. Following partial characterisation of the MenV genome, we previously showed that MenV is a novel member of the genus Rubulavirus. Here we report the characterisation of the large (L) polymerase gene and the adjacent 5' trailer region of MenV, which completes the full-length genome sequence of this novel paramyxovirus (15,516 nucleotides), and thereby confirm its taxonomic position within the family Paramyxoviridae.

IPNV carrier Atlantic salmon growers do not express Mx mRNA and poly I:C-induced Mx response does not cure the carrier state.

Injection of infectious pancreatic necrosis virus (IPNV) in post-smolt Atlantic salmon induced a rapid and persistent expression of Mx mRNA from day 1 to at least day 11 when Mx:beta actin ratios were still at peak values of about 1.0. In contrast, an Atlantic salmon grower population, shown to be carriers of IPNV by culture of the virus from plastic adherent kidney leucocytes, showed no evidence of the expression of Mx transcripts. Nevertheless, IPNV-carrier growers showed a typical Mx response following injection of poly I:C, beginning on day 1, peaking on day 3 (mean Mx:beta actin ratio 0.82) and disappearing by day 7. Notwithstanding such treatment, IPNV continued to persist in growers as the virus could still be isolated 14 days after poly I:C injection.

Poly I:C-induced Mx responses in Atlantic salmon parr, post-smolts and growers.

Intraperitoneal injection of 500 microg poly I:C/fish into Atlantic salmon parr in freshwater and post-smolts and growers in seawater (all at 11 degrees C) induced enhanced expression of Mx mRNA in liver tissue 24 h post-injection. The level of Mx transcripts peaked at day 3 (Mx:beta-actin ratio of about 0.8) and the response disappeared by day 7. In post-smolts, mortalities occurred up to day 14 post-injection, which was dose-dependent. Histological examination of tissues revealed severe pathological changes in the liver of poly I:C injected post-smolts resulting from apoptosis and necrosis of hepatocytes. All other organs appeared histologically normal. Levels of Mx mRNA expression on day 3 post-injection were similar for fish with normal and pathological livers. In untreated or control fish injected with PBS, low levels of Mx transcripts (Mx:beta-actin ratio about 0.1) were sometimes detectable in parr but not in growers. Constitutive Mx expression was variable in post-smolts. Some populations had no detectable transcripts while in others moderate ratios (about 0.3) were detectable over a 3-week period of sampling. Poly I:C administered to parr by bath or orally did not induce upregulation of Mx expression.