Reconstruction of the Evolutionary Dynamics of A(H3N2) Influenza Viruses Circulating in Italy from 2004 to 2012

Background

Influenza A viruses are characterised by their rapid evolution, and the appearance of point mutations in the viral hemagglutinin (HA) domain causes seasonal epidemics. The A(H3N2) virus has higher mutation rate than the A(H1N1) virus. The aim of this study was to reconstruct the evolutionary dynamics of the A(H3N2) viruses circulating in Italy between 2004 and 2012 in the light of the forces driving viral evolution.

Methods

Phylodinamic analyses were made using a Bayesian method, and codon-specific positive selection acting on the HA coding sequence was evaluated.

Results

Global and local phylogenetic analyses showed that the Italian strains collected between 2004 and 2012 grouped into five significant Italian clades that included viral sequences circulating in different epidemic seasons. The time of the most recent common ancestor (tMRCA) of the tree root was between May and December 2003. The tMRCA estimates of the major clades suggest that the origin of a new viral strain precedes the effective circulation of the strain in the Italian population by 6–31 months, thus supporting a central role of global migration in seeding the epidemics in Italy. The study of selection pressure showed that four codons were under positive selection, three of which were located in antigenic sites. Analysis of population dynamics showed the alternation of periods of exponential growth followed by a decrease in the effective number of infections corresponding to epidemic and inter-epidemic seasons.

Conclusions

Our analyses suggest that a complex interaction between the immune status of the population, migrations, and a few selective sweeps drive the influenza A(H3N2) virus evolution. Our findings suggest the possibility of the year-round survival of local strains even in temperate zones, a hypothesis that warrants further investigation.     

Sex chromosome composition revealed in Characidium fishes (Characiformes: Crenuchidae) by molecular cytogenetic methods

The W chromosome of the fishes Characidium cf. fasciatum, Characidium sp. and Characidium cf. gomesi is heterochromatic, as is usually seen in most Characidium species. Samples of W-chromatin were collected by mechanical microdissection and amplified by DOP-PCR (degenerate oligonucleotide-primed polymerase chain reaction), to be used as painting probes (DCg and CgW) and for sequence analysis. FISH (fluorescence in situ hybridization) with DCg probe painted the whole W chromosome, the pericentromeric region of Z chromosomes and the terminal region of B chromosomes. DOP-PCR-generated fragments were cloned, sequenced and tested by in situ hybridization, but only CgW4 produced positive hybridization signals. Clone sequence analysis recovered seven distinct sequences, of which six did not reveal any similarity to other known sequences in the GenBank or GIRI databases. Only CgW9 clone sequence was recognized as probably derived from a Helitron-transposon similar to that found in the genome of the zebrafish Danio rerio. Our results show that the composition of Characidium’s W chromosome does seem rich in repetitive sequences as well as other W chromosomes found in several species with a ZW sex-determining mechanism.     

The anticancer drug perillyl alcohol is a Na/K-ATPase inhibitor

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that suppresses adaptive T-cell immunity by catabolizing tryptophan from the cellular microenvironment. Inhibition of IDO pathway might enhance the efficacy of immunotherapeutic strategies for cancer. We synthesized 1-alkyl-tryptophan targeted IDO inhibitors and compared their effects on IDO expression and activity in dendritic cells (DCs) with the common IDO inhibitor 1-methyl-dl-tryptophan (1-MT). The IDO gene expression was examined by RT-PCR and realtime PCR. The toxicity of these analogs on the proliferation of DCs was detected by MTT assay. All of these analogs inhibited IDO expression and activity induced by IFN-γ and showed no cytotoxicity to DCs at 100 μM. 1-MT intensively suppressed IDO1 expression and activity in DCs, and 1-propyl-tryptophan (1-PT) and 1-isopropyl-tryptophan (1-isoPT) moderately inhibited them. 1-Butyl-tryptophan (1-BT) and 1-ethyl-tryptophan (1-ET) mainly inhibited IDO2 expression. Our results suggest that those analogs differed in their inhibitory activity on IDO expression may give us a clue for developing active IDO inhibitors.     

Effects of anti-TNF therapy on glucose metabolism in patients with ankylosing spondylitis,psoriatic arthritis or juvenile idiopathic arthritis

The objective of this study was to evaluate the influence of anti-tumor necrosis factor (anti-TNF) in juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS) or psoriatic arthritis (PsA). Sixty-two patients were investigated: 7 JIA; 37 AS; and 18 PsA. Caucasian race accounted for 79% and 29% were female. Mean age was 40.4 ± 12.6years. None of the patients had a history of diabetes, and none had used oral hypoglycemic agents or insulin. Treatment was with adalimumab, infliximab and etanercept. Glucose, inflammatory markers and prednisone dose were assessed at baseline, as well as after three and six months of treatment. The mean erythrocyte sedimentation rate was significantly lower at three months and six months than at baseline (13.7 ± 18.0 and 18 ± 22.5 vs. 27.9 ± 23.4 mm; p = 0.001). At baseline, three months and six months, we found the following: mean C-reactive protein levels were comparable (22.1 ± 22.7, 14.5 ± 30.7 and 16.0 ± 23.8 mg/L, respectively; p = 0.26); mean glucose levels remained unchanged (90.8 ± 22.2 mg/dl, 89.5 ± 14.6 mg/dl and 89.8 ± 13.6 mg/dl, respectively; p = 0.91); and mean prednisone doses were low and stable (3.9 ± 4.9 mg/day, 3.7 ± 4.8 mg/day and 2.6 ± 4.0 mg/day, respectively; p = 0.23). During the first six months of treatment, anti-TNF therapy does not seem to influence glucose metabolism in JIA, AS or PsA.     

Population structure in the South American tern Sterna hirundinacea in the South Atlantic: two populations with distinct breeding phenologies

The South American tern Sterna hirundinacea is a migratory species for which dispersal, site fidelity and migratory routes are largely unknown. Here, we used five microsatellite loci and 799 bp partial mitochondrial DNA sequences (Cytochrome b and ND2) to investigate the genetic structure of South American terns from the South Atlantic Ocean (Brazilian and Patagonian colonies). Brazilian and Patagonian colonies have two distinct breeding phenologies (austral winter and austral summer, respectively) and are under the influence of different oceanographic features (e.g. Brazil and Falklands/Malvinas ocean currents, respectively), that may promote genetic isolation between populations. Results show that the Atlantic populations are not completely panmictic, nevertheless, contrary to our expectations, low levels of genetic structure were detected between Brazilian and Patagonian colonies. Such low differentiation (despite temporal isolation of the colonies) could be explained by demographic history of these populations coupled with ongoing levels of gene flow. Interestingly, estimations of gene flow through Maximum likelihood and Bayesian approaches has indicated asymmetrical long term and contemporary gene flow from Brazilian to Patagonian colonies, approaching a source–sink metapopulation dynamic. Genetic analysis of other South American tern populations (especially those from the Pacific coast and Falklands–Malvinas Islands) and other seabird species showing similar geographical distribution (e.g. royal tern Thalasseus maximus), are fundamental in gaining a better understanding of the main processes involved in the diversification of seabirds in the southern hemisphere.     

Nitrogen fluxes from treefrogs to tank epiphytic bromeliads: an isotopic and physiological approach

Diverse invertebrate and vertebrate species live in association with plants of the large Neotropical family Bromeliaceae. Although previous studies have assumed that debris of associated organisms improves plant nutrition, so far little evidence supports this assumption. In this study we used isotopic (¹⁵N) and physiological methods to investigate if the treefrog Scinax hayii, which uses the tank epiphytic bromeliad Vriesea bituminosa as a diurnal shelter, contributes to host plant nutrition. In the field, bromeliads with frogs had higher stable N isotopic composition (δ¹⁵N) values than those without frogs. Similar results were obtained from a controlled greenhouse experiment. Linear mixing models showed that frog feces and dead termites used to simulate insects that eventually fall inside the bromeliad tank contributed, respectively, 27.7% (±0.07 SE) and 49.6% (±0.50 SE) of the total N of V. bituminosa. Net photosynthetic rate was higher in plants that received feces and termites than in controls; however, this effect was only detected in the rainy, but not in the dry season. These results demonstrate for the first time that vertebrates contribute to bromeliad nutrition, and that this benefit is seasonally restricted. Since amphibian–bromeliad associations occur in diverse habitats in South and Central America, this mechanism for deriving nutrients may be important in bromeliad systems throughout the Neotropics.     

Unidirectional Na(+) and Ca (2+) fluxes in two euryhaline teleost fishes, Fundulus heteroclitus and Oncorhynchus mykiss, acutely submitted to a progressive salinity increase

Na⁺ and Ca²⁺ regulation were compared in two euryhaline species, killifish (normally estuarine-resident) and rainbow trout (normally freshwater-resident) during an incremental salinity increase. Whole-body unidirectional fluxes of Na⁺ and Ca²⁺, whole body Na⁺ and Ca²⁺, and plasma concentrations (trout only), were measured over 1-h periods throughout a total 6-h protocol of increasing salinity meant to simulate a natural tidal flow. Killifish exhibited significant increases in both Na⁺ influx and efflux rates, with efflux slightly lagging behind efflux up to 60% SW, but net Na⁺ balance was restored by the time killifish reached 100% SW. Whole body Na⁺ did not change, in agreement with the capacity of this species to tolerate daily salinity fluctuations in its natural habitat. In contrast, rainbow trout experienced a dramatic increase in Na⁺ influx (50-fold relative to FW values), but not Na⁺ efflux between 40 and 60% SW, resulting in a large net loading of Na⁺ at higher salinities (60–100% SW), and increases in plasma Na⁺ and whole body Na⁺ at 100% SW. Killifish were in negative Ca²⁺ balance at all salinities, whereas trout were in positive Ca²⁺ balance throughout. Ca²⁺ influx rate increased two- to threefold in killifish at 80 and 100% SW, but there were no concomitant changes in Ca²⁺ efflux. Ca²⁺ flux rates were affected to a larger degree in trout, with twofold increases in Ca²⁺ influx at 40% SW and sevenfold increases at 100% SW. Again, there was no change in Ca²⁺ efflux with salinity, so plasma Ca²⁺ concentration increased in 100% SW. As the killifish is regularly submitted to increased salinity in its natural environment, it is able to rapidly activate changes in unidirectional fluxes in order to ensure ionic homeostasis, in contrast to the trout.     

Hierarchical status and colour preference in Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

We studied the colour preference of isolated Nile tilapia (Oreochromis niloticus) and whether previous residence or body size can affect environmental colour choice. In the first phase, a cylindrical tank was divided into five differently coloured compartments (yellow, blue, green, white and red), a single fish was introduced into the tank and the frequency at which this fish visited each compartment was recorded over a 2-day study period. An increasingly larger fish (approx +2 cm in length each time) was then added into the tank on each of days 3, 5 and 7 (=four fish in the tank by day 7), and the frequency at which each fish visited the different compartments of the tank was observed twice a day to obtain visit frequency data on the differently sized fishes. This experiment was replicated six times. In the first phase, the solitary fish established residence inside the yellow compartment on the first and second days. Following the introduction of a larger fish, the smaller fish was displaced from the occupied compartment. Nile tilapia possibly shows this preference for yellow as a function of its visual spectral sensitivity and/or the spectral characteristics of its natural environment. Moreover, body size is an important factor in determining hierarchical dominance and territorial defence, and dominant fish chose the preferred environmental colour compartment as their territory.     

Paracoccidioidomycosis in wild monkeys from Paraná State, Brazil

The aim of this study was to evaluate the seroprevalence of Paracoccidioides brasiliensis infection in wild New World monkeys (Cebus sp. and Alouatta caraya). A total of 93 animals (Cebus sp., n = 68 and Alouatta caraya, n = 25) were captured in the Paraná River basin, Paraná State, Brazil and the serum samples were analyzed by ELISA and immunodiffusion using P. brasiliensis gp43 and exoantigen as antigens, respectively. The seropositivity observed by ELISA was 44.1% and 60% for Cebus sp. and A. caraya, respectively, while by immunodiffusion test Cebus sp. showed positivity of 2.9% only. No significant difference was observed in relation to age and sex. This is the first report of paracoccidioidomycosis in wild capuchin monkeys and in wild-black and golden-howler monkeys. The high positivity to P. brasiliensis infection in both species evaluated in our study and the positivity by immunodiffusion test in Cebus sp. suggest that natural disease may be occurring in wild monkeys living in paracoccidioidomycosis endemic areas.     

Antimicrobial Activity of Aroma Compounds against Saccharomyces cerevisiae and Improvement of Microbiological Stability of Soft Drinks as Assessed by Logistic Regression

The combined effects of a mild heat treatment (55°C) and the presence of three aroma compounds [citron essential oil, citral, and (E)-2-hexenal] on the spoilage of noncarbonated beverages inoculated with different amounts of a Saccharomyces cerevisiae strain were evaluated. The results, expressed as growth/no growth, were elaborated using a logistic regression in order to assess the probability of beverage spoilage as a function of thermal treatment length, concentration of flavoring agents, and yeast inoculum. The logit models obtained for the three substances were extremely precise. The thermal treatment alone, even if prolonged for 20 min, was not able to prevent yeast growth. However, the presence of increasing concentrations of aroma compounds improved the stability of the products. The inhibiting effect of the compounds was enhanced by a prolonged thermal treatment. In fact, it influenced the vapor pressure of the molecules, which can easily interact within microbial membranes when they are in gaseous form. (E)-2-Hexenal showed a threshold level, related to initial inoculum and thermal treatment length, over which yeast growth was rapidly inhibited. Concentrations over 100 ppm of citral and thermal treatment longer than 16 min allowed a 90% probability of stability for bottles inoculated with 10⁵ CFU/bottle. Citron gave the most interesting responses: beverages with 500 ppm of essential oil needed only 3 min of treatment to prevent yeast growth. In this framework, the logistic regression proved to be an important tool to study alternative hurdle strategies for the stabilization of noncarbonated beverages.