Detection of North American orthopoxviruses by real time-PCR

The prevalence of North American orthopoxviruses in nature is unknown and may be more difficult to ascertain due to wide spread use of vaccinia virus recombinant vaccines in the wild. A real time PCR assay was developed to allow for highly sensitive and specific detection of North American orthopoxvirus DNA in animal tissues and bodily fluids. This method is based on the amplification of a 156 bp sequence within a myristylated protein, highly conserved within the North American orthopoxviruses but distinct from orthologous genes present in other orthopoxviruses. The analytical sensitivity was 1.1 fg for Volepox virus DNA, 1.99 fg for Skunkpox virus DNA, and 6.4 fg for Raccoonpox virus DNA with a 95% confidence interval. Our assay did not cross-react with other orthopoxviruses or ten diverse representatives of the Chordopoxvirinae subfamily. This new assay showed more sensitivity than tissue culture tests, and was capable of differentiating North American orthopoxviruses from other members of Orthopoxvirus. Thus, our assay is a promising tool for highly sensitive and specific detection of North American orthopoxviruses in the United States and abroad

Ectopic Expression of Vaccinia Virus E3 and K3 Cannot Rescue Ectromelia Virus Replication in Rabbit RK13 Cells

Citation: Hand, E. S., Haller, S. L., Peng, C., Rothenburg, S., & Hersperger, A. R. (2015). Ectopic Expression of Vaccinia Virus E3 and K3 Cannot Rescue Ectromelia Virus Replication in Rabbit RK13 Cells. Plos One, 10(3), 15. doi:10.1371/journal.pone.0119189As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV

Soil Moisture and Fungi Affect Seed Survival in California Grassland Annual Plants

Survival of seeds in the seed bank is important for the population dynamics of many plant species, yet the environmental factors that control seed survival at a landscape level remain poorly understood. These factors may include soil moisture, vegetation cover, soil type, and soil pathogens. Because many soil fungi respond to moisture and host species, fungi may mediate environmental drivers of seed survival. Here, I measure patterns of seed survival in California annual grassland plants across 15 species in three experiments. First, I surveyed seed survival for eight species at 18 grasslands and coastal sage scrub sites ranging across coastal and inland Santa Barbara County, California. Species differed in seed survival, and soil moisture and geographic location had the strongest influence on survival. Grasslands had higher survival than coastal sage scrub sites for some species. Second, I used a fungicide addition and exotic grass thatch removal experiment in the field to tease apart the relative impact of fungi, thatch, and their interaction in an invaded grassland. Seed survival was lower in the winter (wet season) than in the summer (dry season), but fungicide improved winter survival. Seed survival varied between species but did not depend on thatch. Third, I manipulated water and fungicide in the laboratory to directly examine the relationship between water, fungi, and survival. Seed survival declined from dry to single watered to continuously watered treatments. Fungicide slightly improved seed survival when seeds were watered once but not continually. Together, these experiments demonstrate an important role of soil moisture, potentially mediated by fungal pathogens, in driving seed survival

Genomic Expression Libraries for the Identification of Cross-Reactive Orthopoxvirus Antigens

Increasing numbers of human cowpox virus infections that are being observed and that particularly affect young non-vaccinated persons have renewed interest in this zoonotic disease. Usually causing a self-limiting local infection, human cowpox can in fact be fatal for immunocompromised individuals. Conventional smallpox vaccination presumably protects an individual from infections with other Orthopoxviruses, including cowpox virus. However, available live vaccines are causing severe adverse reactions especially in individuals with impaired immunity. Because of a decrease in protective immunity against Orthopoxviruses and a coincident increase in the proportion of immunodeficient individuals in today's population, safer vaccines need to be developed. Recombinant subunit vaccines containing cross-reactive antigens are promising candidates, which avoid the application of infectious virus. However, subunit vaccines should contain carefully selected antigens to confer a solid cross-protection against different Orthopoxvirus species. Little is known about the cross-reactivity of antibodies elicited to cowpox virus proteins. Here, we first identified 21 immunogenic proteins of cowpox and vaccinia virus by serological screenings of genomic Orthopoxvirus expression libraries. Screenings were performed using sera from vaccinated humans and animals as well as clinical sera from patients and animals with a naturally acquired cowpox virus infection. We further analyzed the cross-reactivity of the identified immunogenic proteins. Out of 21 identified proteins 16 were found to be cross-reactive between cowpox and vaccinia virus. The presented findings provide important indications for the design of new-generation recombinant subunit vaccines

Papuan mitochondrial genomes and the settlement of Sahul

New Guineans represent one of the oldest locally continuous populations outside Africa, harboring among the greatest linguistic and genetic diversity on the planet. Archeological and genetic evidence suggest that their ancestors reached Sahul (present day New Guinea and Australia) by at least 55,000 years ago (kya). However, little is known about this early settlement phase or subsequent dispersal and population structuring over the subsequent period of time. Here we report 379 complete Papuan mitochondrial genomes from across Papua New Guinea, which allow us to reconstruct the phylogenetic and phylogeographic history of northern Sahul. Our results support the arrival of two groups of settlers in Sahul within the same broad time window (50–65 kya), each carrying a different set of maternal lineages and settling Northern and Southern Sahul separately. Strong geographic structure in northern Sahul remains visible today, indicating limited dispersal over time despite major climatic, cultural, and historical changes. However, following a period of isolation lasting nearly 20 ky after initial settlement, environmental changes postdating the Last Glacial Maximum stimulated diversification of mtDNA lineages and greater interactions within and beyond Northern Sahul, to Southern Sahul, Wallacea and beyond. Later, in the Holocene, populations from New Guinea, in contrast to those of Australia, participated in early interactions with incoming Asian populations from Island Southeast Asia and continuing into Oceania

Y-chromosome descent clusters and male differential reproductive success: young lineage expansions dominate Asian pastoral nomadic populations

International audienceHigh-frequency microsatellite haplotypes of the male-specific Y-chromosome can signal past episodes of high reproductive success of particular men and their patrilineal descendants. Previously, two examples of such successful Y-lineages have been described in Asia, both associated with Altaic-speaking pastoral nomadic societies, and putatively linked to dynasties descending, respectively, from Genghis Khan and Giocangga. Here we surveyed a total of 5321 Y-chromosomes from 127 Asian populations, including novel Y-SNP and microsatellite data on 461 Central Asian males, to ask whether additional lineage expansions could be identified. Based on the most frequent eight-microsatellite haplotypes, we objectively defined 11 descent clusters (DCs), each within a specific haplogroup, that represent likely past instances of high male reproductive success, including the two previously identified cases. Analysis of the geographical patterns and ages of these DCs and their associated cultural characteristics showed that the most successful lineages are found both among sedentary agriculturalists and pastoral nomads, and expanded between 2100 BCE and 1100 CE. However, those with recent origins in the historical period are almost exclusively found in Altaic-speaking pastoral nomadic populations, which may reflect a shift in political organisation in pastoralist economies and a greater ease of transmission of Y-chromosomes through time and space facilitated by the use of horses

Inferring the joint demographic history of multiple populations from multidimensional SNP frequency data

Demographic models built from genetic data play important roles in illuminating prehistorical events and serving as null models in genome scans for selection. We introduce an inference method based on the joint frequency spectrum of genetic variants within and between populations. For candidate models we numerically compute the expected spectrum using a diffusion approximation to the one-locus two-allele Wright-Fisher process, involving up to three simultaneous populations. Our approach is a composite likelihood scheme, since linkage between neutral loci alters the variance but not the expectation of the frequency spectrum. We thus use bootstraps incorporating linkage to estimate uncertainties for parameters and significance values for hypothesis tests. Our method can also incorporate selection on single sites, predicting the joint distribution of selected alleles among populations experiencing a bevy of evolutionary forces, including expansions, contractions, migrations, and admixture. As applications, we model human expansion out of Africa and the settlement of the New World, using 5 Mb of noncoding DNA resequenced in 68 individuals from 4 populations (YRI, CHB, CEU, and MXL) by the Environmental Genome Project. We also combine our demographic model with a previously estimated distribution of selective effects among newly arising amino acid mutations to accurately predict the frequency spectrum of nonsynonymous variants across three continental populations (YRI, CHB, CEU).Comment: 17 pages, 4 figures, supporting information included with sourc

Effects of nutrient addition and soil drainage on germination of N-fixing and non-N-fixing tropical dry forest tree species

To develop generalised predictions regarding the effects of atmospheric nitrogen (N) and phosphorus (P) deposition on vegetation communities, it is necessary to account for the impacts of increased nutrient availability on the early life history stages of plants. Additionally, it is important to determine if these responses (a) differ between plant functional groups and (b) are modulated by soil drainage, which may affect the persistence of added nutrients. We experimentally assessed seed germination responses (germination proportion and germination energy, i.e. time to germination) of commonly occurring N-fixing and non-N-fixing tropical dry forest tree species found in India to simulated N and P deposition in well-drained soils, as well as soils with impeded drainage. When soils were not allowed to drain, germination proportion declined with nutrient addition, while germination energy remained unchanged. Stronger declines in germination proportion were observed for N-fixing species. In free-draining soils, nutrient addition did not affect germination proportion in either functional group. However, we detected a trend of delayed germination with nutrient addition, especially in N-fixers. Our results suggest that nutrient deposition can lead to potential shifts in functional dominance and tree community composition of tropical dry forests in the long term through its effects on early life stages of trees, although the mechanisms underlying the observed germination responses remain unclear. Further, such effects are likely to be spatially variable across the geographic range in which tropical dry forests occur depending on soil drainage properties