Antiviral Activity and Adaptive Evolution of Avian Tetherins.

Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.

Empirical evidence of cold stress induced cell mediated and humoral immune response in common myna (Sturnus tristis).

Common myna (Sturnus tristis) is a bird indigenous to the Indian subcontinent that has invaded many parts of the world. At the onset of our investigation, we hypothesized that the immunological profile of myna makes it resistant to harsh/new environmental conditions. In order to test this hypothesis, a number of 40 mynas were caught and divided into two groups, i.e., 7 and 25 °C for 14 days. To determine the effect of cold stress, cell mediated and humoral immune responses were assessed. The macrophage engulfment percentage was significantly (P < 0.05) higher at 25 °C rather than 7 °C either co-incubated with opsonized or unopsonized sheep red blood cells (SRBC). Macrophage engulfment/cell and nitric oxide production behaved in a similar manner. However, splenic cells plaque formation, heterophil to lymphocyte (H/L) ratio, and serum IgM or IgG production remained non-significant. There was a significant increase of IgG antibody production after a second immunization by SRBC. To the best of our knowledge, these findings have never been reported in the progression of this bird's invasion in frosty areas of the world. The results revealed a strengthened humoral immune response of myna and made this bird suitable for invasion in the areas of harsh conditions.

Immune response, oxidative stress and dietary antioxidants in great tit nestlings.

The activation of immune defences counteracts pathogens, but mounting an immune response is costly and can negatively impact life-history traits. Immune activation releases highly reactive species that kill pathogens but can also cause oxidative damage to host tissues, and these negative effects may therefore constrain further investment in immune responses. To offset these toxic effects, animals rely on a complex system of antioxidants. Here, we tested if vitamin E, a dietary antioxidant, can reduce oxidative damage induced by an immune challenge and thus enhance the immune response. In a 2 × 2 experimental design, we supplemented great tit nestlings with either vitamin E or a placebo, and then injected them with either a bacterial lipopolysaccharide (LPS), or a buffer solution (PBS) as a control. LPS-treated nestlings mounted an inflammatory response and increased antioxidant capacity, without any change in ROM (reactive oxygen metabolites), an index of early oxidative damage. These results suggest that the likely transient increase in reactive species of the LPS injection was counteracted by a rise in endogenous antioxidant defences that was independent of supplementary dietary antioxidants. Indeed, vitamin E supplementation neither affected oxidative status nor enhanced the immune response, suggesting that in our experimental condition great tit nestlings were not limited in vitamin E and in antioxidants in general. Overall, our results show that birds can mount an effective antioxidant response to face an immune challenge, and can therefore avoid stress caused by a transient increase in reactive species generated by immune activation.

Dietary antioxidants, lipid peroxidation and plumage colouration in nestling blue tits Cyanistes caeruleus.

Carotenoid pigments are responsible for many of the red, yellow and orange plumage and integument traits seen in birds. One idea suggests that since carotenoids can act as antioxidants, carotenoid-mediated colouration may reveal an individual's ability to resist oxidative damage. In fact, there is currently very little information on the effects of most dietary-acquired antioxidants on oxidative stress in wild birds. Here, we assessed the impacts on oxidative damage, plasma antioxidants, growth and plumage colouration after supplementing nestling blue tits Cyanistes caeruleus with one of three diets; control, carotenoid treatment or α-tocopherol treatment. Oxidative damage was assessed by HPLC analysis of plasma levels of malondialdehyde (MDA), a by-product of lipid peroxidation. Contrary to predictions, we found no differences in oxidative damage, plumage colouration or growth rate between treatment groups. Although plasma lutein concentrations were significantly raised in carotenoid-fed chicks, α-tocopherol treatment had no effect on concentrations of plasma α-tocopherol compared with controls. Interestingly, we found that faster growing chicks had higher levels of oxidative damage than slower growing birds, independent of treatment, body mass and condition at fledging. Moreover, the chromatic signal of the chest plumage of birds was positively correlated with levels of MDA but not plasma antioxidant concentrations: more colourful nestlings had higher oxidative damage than less colourful individuals. Thus, increased carotenoid-mediated plumage does not reveal resistance to oxidative damage for nestling blue tits, but may indicate costs paid, in terms of oxidative damage. Our results indicate that the trade-offs between competing physiological systems for dietary antioxidants are likely to be complex in rapidly developing birds. Moreover, interpreting the biological relevance of different biomarkers of antioxidant status represents a challenge for evolutionary ecology.

Stress and immune responses of nestling tree swallows (Tachycineta bicolor) and eastern bluebirds (Sialia sialis) exposed to nonpersistent pesticides and p,p'-dichlorodiphenyldichloroethylene in apple orchards of southern Ontario, Canada.

To determine the relative effects of pesticides in current use and persistent residues of p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE), we examined endocrine and immune responses in tree swallow (Tachycineta bicolor) and eastern bluebird (Sialia sialis) chicks from pesticide-sprayed apple orchards and reference sites in southern Ontario, Canada, during 2000 to 2001. Nests were exposed to as many as seven individual pesticide applications and up to five mixtures of pesticides during the egg-incubation and chick-rearing stage. Eggs collected from sprayed orchards contained higher p,p'-DDE concentrations than eggs from reference sites. In 16-d-old tree swallows, no significant differences were found in body mass, basal corticosterone concentration, or the corticosterone stress response following a 10-min restraint of chicks sampled from sprayed orchards and reference sites. Challenge with adrenocorticotrophic hormone (ACTH), however, produced a higher level of corticosterone secretion in tree swallow chicks from sprayed orchards relative to chicks from reference sites. Multiple regression analysis revealed no correlation between corticosterone concentrations and exposure to pesticide sprays or p,p'-DDE in tree swallow chicks. In contrast, bluebird chicks from sprayed orchards were less responsive to challenge with ACTH and a significant negative association was found between the response to ACTH challenge and p,p'-DDE concentration in eggs. The phytohemagglutinin (PHA)-induced cutaneous basophil hypersensitivity response was similar between exposure groups in both tree swallow and bluebird nestlings. Examination of immune organs revealed that tree swallow chicks from sprayed orchards had significantly greater thymic lymphocyte density and cortical/ medullary ratios and significant splenic B-cell hyperplasia relative to reference chicks. Our results indicate that modulation in the hypothalamus-pituitary-adrenal axis in songbird chicks tested are most associated with high p,p'-DDE egg concentrations. An alteration in the endocrine or immune system may compromise songbird survival immediately after fledging or during migration.