Development of infectious cDNA clones of Salmonid alphavirus subtype 3

<p>Abstract</p> <p>Background</p> <p>Salmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed.</p> <p>Results</p> <p>Infectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2₁₉₇, nsP3₂₆₃and nsP3₃₂₃severely reduced infectivity, a serine to proline mutation in E2₂₀₆appeared to enhance the virus titer production.</p> <p>Conclusion</p> <p>We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.</p

Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ∼17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production

Enhanced transfection of cell lines from Atlantic salmon through nucoleofection and antibiotic selection

Background Cell lines from Atlantic salmon kidney have made it possible to culture and study infectious salmon anemia virus (ISAV), an aquatic orthomyxovirus affecting farmed Atlantic salmon. However, transfection of these cells using calcium phosphate precipitation or lipid-based reagents shows very low transfection efficiency. The Amaxa Nucleofector technology™ is an electroporation technique that has been shown to be efficient for gene transfer into primary cells and hard to transfect cell lines. Findings Here we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry. Depending on the plasmid promoter, TO cells could be transfected transiently with an efficiency ranging from 11.6% to 90.8% with good viability, using Amaxa's cell line nucleofector solution T and program T-20. A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells. Conclusions The results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin

Apnea of prematurity: from cause to treatment

Apnea of prematurity (AOP) is a common problem affecting premature infants, likely secondary to a “physiologic” immaturity of respiratory control that may be exacerbated by neonatal disease. These include altered ventilatory responses to hypoxia, hypercapnia, and altered sleep states, while the roles of gastroesophageal reflux and anemia remain controversial. Standard clinical management of the obstructive subtype of AOP includes prone positioning and continuous positive or nasal intermittent positive pressure ventilation to prevent pharyngeal collapse and alveolar atelectasis, while methylxanthine therapy is a mainstay of treatment of central apnea by stimulating the central nervous system and respiratory muscle function. Other therapies, including kangaroo care, red blood cell transfusions, and CO2 inhalation, require further study. The physiology and pathophysiology behind AOP are discussed, including the laryngeal chemoreflex and sensitivity to inhibitory neurotransmitters, as are the mechanisms by which different therapies may work and the potential long-term neurodevelopmental consequences of AOP and its treatment

[Very premature births: Dilemmas and management. Second part: Ethical aspects and recommendations].

International audienceIn the first part of this work, the outcome following very premature birth was assessed. This enabled a gray zone to be defined, with inherent major prognostic uncertainty. In France today, the gray zone corresponds to deliveries occurring at 24 and 25 weeks of postmenstrual age. The management of births occurring below and above the gray zone was described. Withholding intensive care at birth for babies born below or within the gray zone does not mean withholding care but rather providing palliative care to prevent pain and suffering during the time period preceding death. Given the high level of uncertainty, making good decisions within the gray zone is problematic. Decisions should be based on the infant's best interests. Decisions should be reached with the parents, who are entitled to receive clear and comprehensive information. Possible decisions to withhold intensive care should be made following the procedures described in the French law of April 2005. Guidelines, based on gestational age and the other prognostic elements, are proposed to the parents before birth. They are applied in an individualized fashion, in order to take into account the individual features of each case. At 25 weeks, resuscitation and/or full intensive care are usually proposed, unless unfavorable factors, such as severe growth restriction, are associated. A senior neonatologist will attend the delivery and will make decisions based on both the baby's condition at birth and the parents' wishes. At 24 weeks, in the absence of unfavorable associated factors, the parents' wishes should be followed in deciding between initiating full intensive care or palliative care. Below 24 weeks, palliative care is the only option to be offered in France at the present time

[Very premature births: Dilemmas and management. Part 1. Outcome of infants born before 28 weeks of postmenstrual age, and definition of a gray zone].

International audienceWith very preterm deliveries, the decision to institute intensive care, or, alternatively, to start palliative care and let the baby die, is extremely difficult, and involves complex ethical issues. The introduction of intensive care may result in long-term survival of many infants without severe disabilities, but it may also result in the survival of severely disabled infants. Conversely, the decision to withhold resuscitation and/or intensive care at birth, which is an option at the margin of viability, implies allowing babies to die, although some of them would have developed normally if they had received resuscitation and/or intensive care. Withholding intensive care at birth does not mean withholding care but rather providing palliative care to prevent pain and suffering during the time period preceding death. The likelihood of survival without significant disabilities decreases as gestational age at birth decreases. In addition to gestational age, other factors greatly influence the prognosis. Indeed, for a given gestational age, higher birth weight, singleton birth, female sex, exposure to prenatal corticosteroids, and birth in a tertiary center are favorable factors. Considering gestational age, there is a gray zone that corresponds to major prognostic uncertainty and therefore to a major problem in making a "good" decision. In France today, the gray zone corresponds to deliveries at 24 and 25 weeks of postmenstrual age. In general, babies born above the gray zone (26 weeks of postmenstrual age and later) should receive resuscitation and/or full intensive care. Below 24 weeks, palliative care is the only option offered in France at the present time. Decisions within the gray zone will be addressed in the 2nd part of this work