Multiply-primed rolling-circle amplification (MPRCA) of PCV2 genomes: Applications on detection, sequencing and virus isolation

Multiply-primed rolling-circle amplification (MPRCA) was used to amplify porcine circovirus type 2 (PCV2) genomes isolated from tissues of pigs with signs of post-weaning multisystemic wasting syndrome (PMWS). Two of the amplified PCV2 genomes were cloned in prokaryotic plasmids and sequenced. Both were nearly identical (1767 nt) except for one silent substitution in the region coding for the capsid protein (ORF2). In addition, they showed high nucleotide sequence similarity with PCV2 isolates from others countries (93–99%). To investigate whether the MPRCA amplified PCV2 genomes could be used to produce infectious virus, the cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 10^5.55 TCID₅₀/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes aiming at sequencing and virus isolation strategies, where particularly useful is the fact that it allows straightforward construction of PCV2 infectious clones from amplified genomes. However, it was less sensitive than PCR for diagnostic purposes.     

Local and regional environmental variation influences the growth of tropical trees in selection trials in the Republic of Panama

Native Neotropical trees are being increasingly planted for restoration purposes and timber production, but we lack species-specific data on growth responses to different regional climates and local environmental variation. We used regression trees and variance components to quantify the effects of within- and among-site environmental variation on the basal area (BA) of 21 Neotropical and two exotic tree species at three selection trials in the Republic of Panama. Sites represented distinct regional climates in which annual rainfall varied from 1100 to 2226 mm, with dry seasons of 4.1–6.7 months. Local environmental variables included measures of slope steepness and position, soil texture, soil color, and indicators of soil condition, such as subsoil rockiness.     

Early growth and survival of 49 tropical tree species across sites differing in soil fertility and rainfall in Panama

Reforestation in the tropics takes place across a wide variety of edaphic and climatic conditions. Reforestation trials have demonstrated that edaphic conditions may have a strong effect on species growth and survival. However it is unclear how the relative importance of soil conditions influences species survival and growth under varying amounts of rainfall and lengths of dry season.Two-year growth and mortality of 49 tree species were evaluated in four sites across Panama, representing a soil fertility-rainfall matrix. Despite strong contrasts in environmental conditions, 65% of individual species did not show consistent differences in growth between high- and low-fertility sites or between wet and dry sites. However, early growth and survival were more strongly affected by soil fertility than by rainfall patterns for the second-largest group; 30% of the species grew significantly better in both high-fertility sites than in both low-fertility sites, compared to 6% in both wet sites vs. both dry sites. In the two high-fertility sites, growth of 47% and 69% of the species was better than their across-site means. On the other hand, 55% and 73% of the species grew significantly slower than their across-site averages in the two low-fertility sites.Survival did not appear to be associated to either soil fertility or rainfall. In each site, only a few species had a significantly higher or lower within-site survival than across-site survival.Diversifying the choice of tree species increases the options for reforestation strategies that match species characteristics to local site conditions and to the objectives and management possibilities of landholders. Testing the performance of potential species under different site conditions in screening trials is paramount, both to inform selection from among the vast diversity of tree species in the tropics that show good growth and survival under different local site conditions and to filter out unsuitable species and avoid early failure of the reforestation effort.     

Efficacy of an inactivated,recombinant bovine herpesvirus type 5 (BoHV-5) vaccine

Bovine herpesvirus type 5 (BoHV-5) is the causative agent of bovine herpetic encephalitis. In countries where BoHV-5 is prevalent, attempts to vaccinate cattle to prevent clinical signs from BoHV-5-induced disease have relied essentially on vaccination with BoHV-1 vaccines. However, such practice has been shown not to confer full protection to BoHV-5 challenge. In the present study, an inactivated, oil adjuvanted vaccine prepared with a recombinant BoHV-5 from which the genes coding for glycoprotein I (gI), glycoprotein E (gE) and membrane protein US9 were deleted (BoHV-5 gI/gE/US9⁻), was evaluated in cattle in a vaccination/challenge experiment. The vaccine was prepared from a virus suspension containing a pre-inactivation antigenic mass equivalent to 10^7.69 TCID₅₀/dose. Three mL of the inactivated vaccine were administered subcutaneously to eight calves serologically negative for BoHV-5 (vaccinated group). Four other calves were mock-vaccinated with an equivalent preparation without viral antigens (control group). Both groups were boostered 28 days later. Neither clinical signs of disease nor adverse effects were observed during or after vaccination. A specific serological response, revealed by the development of neutralizing antibodies, was detected in all vaccinated animals after the first dose of vaccine, whereas control animals remained seronegative. Calves were subsequently challenged on day 77 post-vaccination (pv) with 10^9.25 TCID₅₀ of the wild-type BoHV-5 (parental strain EVI 88/95). After challenge, vaccinated cattle displayed mild signs of respiratory disease, whereas the control group developed respiratory disease and severe encephalitis, which led to culling of 2/4 calves. Searches for viral DNA in the central nervous system (CNS) of vaccinated calves indicated that wild-type BoHV-5 did not replicate, whereas in CNS tissues of calves on the control group, viral DNA was widely distributed. BoHV-5 shedding in nasal secretions was significantly lower in vaccinated calves than in the control group on days 2, 3, 4 and 6 post-challenge (pc). In addition, the duration of virus shedding was significantly shorter in the vaccinated (7 days) than in controls (12 days). Attempts to reactivate latent infection by administration of dexamethasone at 147 days pv led to recrudescence of mild signs of respiratory disease in both vaccinated and control groups. Infectious virus shedding in nasal secretions was detected at reactivation and was significantly lower in vaccinated cattle than in controls on days 11–13 post-reactivation (pr). It is concluded that the inactivated vaccine prepared with the BoHV-5 gI/gE/US9⁻ recombinant was capable of conferring protection to encephalitis when vaccinated cattle were challenged with a large infectious dose of the parental wild type BoHV-5. However, it did not avoid the establishment of latency nor impeded dexamethasone-induced reactivation of the virus, despite a significant reduction in virus shedding after challenge and at reactivation on vaccinated calves.     

The function of the cranial crest and jaws of a unique pterosaur from the Early Cretaceous of Brazil

The discovery of a previously undescribed pterosaur, Thalassodromeus sethi, yields information on the function of cranial crests and the feeding strategy developed by these extinct flying reptiles. The material consists of a large skull (length: 1420 millimeters, including the crest) with a huge bony crest that was well irrigated by blood vessels and may have been used for regulation of its body temperature. The rostrum consists of two bladelike laminae, the arrangement of which is analogous to the condition found in the bird Rynchops, which skims over the water to catch food, indicating that T. sethi also may have been a skimmer.