Deep sequencing of PRRSV isolates: rapid and large-scale characterization of viral genomes

Porcine reproductive and respiratory syndrome virus (PRRSV) is a single stranded, positive sense RNA virus with a genome size of approximately 15 kb. Much of the genetic characterization or viral genotyping of PRRSV isolates is limited to one or two viral genes only (ORF5 and/or ORF7) for a number of reasons, for example: (1) characterizing one or two ORFs is sufficient for diagnostics; (2) genome characterization is laborious because traditional (Sanger) sequencing yields only a single sequence of 800-1000 bases per reaction; (3) large-scale genome characterization is time-consuming and costly. Collectively, this hinders the study of PRRSV genomic evolution at different levels (host, regional, and global). We demonstrate here the use of 454 technology to rapidly sequence PRRSV genomic nucleic acid from different sources (cell culture and swine tissue), genotypes (type 1 and type 2), and genome structure (non-deletion vs. deletion variants). Samples (n=16) were multiplexed to bring down cost per genome sequence. Assembly of sample specific reads resulted in a single contig in almost all instances (15 out of 16). Average genome coverage was 96.7% with reference to prototype isolates (Lelystad virus for type 1 and ATCC VR2332 for type 2). Average sequence depth was 405 reads per nucleotide position. This high sequence depth allowed characterization of variants from quasispecies that occurred at frequencies even lower than 1%. In summary, next generation sequencing technology offers unparalleled opportunity to quickly and efficiently characterize near complete length PRRSV genomes in an economical manner. This allows experiments to be designed with considerations to viral genomic evolution rather than those with limited insights from select viral genes only.postprin

The Critical period for weed control in spring maize in North-West India

Spring maize is becoming popular in Punjab state owing to its higher yield potential and is a new introduction. A field experiment was conducted at Punjab Agricultural University, Ludhiana with 12 treatments viz. crop kept weedy for 20, 30, 40, 50, 60 DAS, crop kept weed free for 20, 30, 40, 50, 60 DAS, weed free throughout and weedy check. The weed density showed a linear progression as the weedy interval was increased from 30 to 40 days, 40 to 50 days and 50 to 60 days. The results showed that yield attributes and grain yield declined with the increased duration of crop- weed interference period and increased with long weed free durations. Significant reduction in grain yield was recorded with increased weed infestation from 30 to 60 DAS, however, the differences observed between 20 and 30 days weedy were statistically at par. This suggested the tolerance of weed interference up to 30 days in spring maize. The 60 days weed free treatment resulted in grain yield statistically same with that of weed free throughout but significantly better from all other weed free duration treatments. This marked the end of critical period of crop-weed competition in spring maize. A significant negative linear correlation was observed for weed biomass and grain yield. The critical period of crop-weed competition in spring maize started at 30 DAS and ended at 60DAS. This period needs immediate attention for the adoption of weed control measure

Tumor Grafting Induces Changes of Gut Microbiota in Athymic Nude Mice in the Presence and Absence of Medicinal Gynostemma Saponins

Recent findings have revealed that gut microbiota plays a substantial role in modulating diseases such as autism, rheumatoid arthritis, allergies, and cancer that occur at sites distant to the gut. Athymic nude mice have been employed for tumorigenic research for decades; however, the relationships between the gut microbiome and host’s response in drug treatment to the grafted tumors have not been explored. In this study, we analyzed the fecal microbiome of nonxenograft and xenograft nude mice treated with phytosaponins from a popular medicinal plant, Gynostemma pentaphyllum (Gp). Analysis of enterobacterial repetitive intergenic consensus (ERIC)-PCR data showed that the microbiota profile of xenograft mice departed from that of the nonxenograft mice. After ten days of treatment with Gp saponins (GpS), the microbiota of the treated mice was closer to the microbiota at Day 0 before the implantation of the tumor. Data obtained from 16S pyrosequencing of fecal samples reiterates the differences in microbiome between the nonxenograft and xenograft mice. GpS markedly increased the relative abundance of Clostridium cocleatum and Bacteroides acidifaciens, for which the beneficial effects on the host have been well documented. This study, for the first time, characterizes the properties of gut microbiome in nude mice responding to tumor implant and drug treatment. We also demonstrate that dietary saponins such as GpS can potentially regulate the gut microbial ecosystem by increasing the number of symbionts. Interestingly, this regulation of the gut ecosystem might, at least in part, be responsible for or contribute to the anticancer effect of GpS.published_or_final_versio

The diversity of Porcine Reproductive and Respiratory Syndrome virus type 1 and 2 in Denmark

Session - Viral Heterogeneity and EvolutionBoth Type 1 and Type 2 PRRS viruses are circulating among Danish pigs. The first appearance of Type 1 PRRSV in Denmark was in 1992 whereas the Type 2 PRRSV was introduced in 1996 after the use of a live attenuated vaccine that reverted to virulence. Since then, vaccination to control the disease for both PRRSV genotypes has been widely used in Denmark and it is therefore highly relevant to monitor the diversity of currently circulating PRRSV strains. Only subtype 1 of the Type 1 PRRSV strains and vaccine-like Type 2 PRRSV strains were previously detected in Denmark, however, only few Danish PRRSV strains were sequenced. Denmark exports more than 50.000 living pigs each month. A portion of these pigs inevitably harbor PRRSV. Thus, the diversity of PRRSV in Denmark is of interest to other countries besides Denmark. The main objective of the present study was to close the gap in knowledge on the genetic diversity of currently circulating PRRSV stains in Danish pigs by sequencing ORF5 and ORF7 of approximately 41 Type 1 and 50 Type 2 strains isolated between 2003 and 2013. Furthermore, full genome analysis was performed on nine Type 1 and nine Type 2 selected strains. The preliminary assessment of the results showed that the Type 1 strains all belonged to subtype 1. Based on the ORF5 sequences, the Danish Type 1 viruses clustered into two groups. These two groups shared 84 % to 92 % and 94 % to 99 % nucleotide identity to the Lelystad virus, respectively. The sequenced Type 2 viruses showed a significant higher level of identity in that the ORF5 sequences were 94 - >99 % identical at the nucleotide level. Most of the Type 2 viruses, shared high level of identity to the VR2332 vaccine strain (Ingelvac MLV), but a few more diverse isolates were also identified, including strains with interesting deletions in NSP2 and other genes. The full genome sequences of Danish strains showed an overall nucleotide identity of 88-98 % (Type 1) and 94 % to >99 % (Type 2). The impact of these results will be discussed.postprin

Evaluation of Oryza sativa x O. glaberrima derived progenies for resistance to rootknot nematode and identification of introgressed alien chromosome segments using SSR markers

The genus Oryza has two cultivated species, Asian rice (Oryza sativa L.) and African rice (Oryza glaberrima Steud.) and 22 wild species. O. glaberrima is low yielding but has useful genes for resistance to biotic and abiotic stresses. Introgression lines derived from backcrossing of O. sativa x O. glaberrima, using O. sativa as recurrent parent, were evaluated for tolerance to root-knot nematode (Meloidogyne graminicola). Testing in sick plots infested with nematodes showed reduction in plant height, shoot and root biomass and leaf area index compared to the control. Based on gall rating and the ratio of the final population to the initial population of nematodes (Pf/Pi ratio), three introgression lines were found to be resistant to nematodes (IR80311-9-B-B-1-2 and IR80311-2-B-B-1-2 under screenhouse and IR80311-48-BB- 1 under phytotron conditions). Gall rating and the Pf/Pi ratio showed positive correlation (r = 0.61). Analysis of 122 introgression lines using simple sequence repeat (SSR) markers detected introgression of O. glaberrima segments into O. sativa

Towards a metagenomic understanding on enhanced biomethane production from waste activated sludge after pH 10 pretreatment

BACKGROUND: Understanding the effects of pretreatment on anaerobic digestion of sludge waste from wastewater treatment plants is becoming increasingly important, as impetus moves towards the utilization of sludge for renewable energy production. Although the field of sludge pretreatment has progressed significantly over the past decade, critical questions concerning the underlying microbial interactions remain unanswered. In this study, a metagenomic approach was adopted to investigate the microbial composition and gene content contributing to enhanced biogas production from sludge subjected to a novel pretreatment method (maintaining pH at 10 for 8 days) compared to other documented methods (ultrasonic, thermal and thermal-alkaline). RESULTS: Our results showed that pretreated sludge attained a maximum methane yield approximately 4-fold higher than that of the blank un-pretreated sludge set-up at day 17. Both the microbial and metabolic consortium shifted extensively towards enhanced biodegradation subsequent to pretreatment, providing insight for the enhanced methane yield. The prevalence of Methanosaeta thermophila and Methanothermobacter thermautotrophicus, together with the functional affiliation of enzymes-encoding genes suggested an acetoclastic and hydrogenotrophic methanogenesis pathway. Additionally, an alternative enzymology in Methanosaeta was observed. CONCLUSIONS: This study is the first to provide a microbiological understanding of improved biogas production subsequent to a novel waste sludge pretreatment method. The knowledge garnered will assist the design of more efficient pretreatment methods for biogas production in the future.published_or_final_versio

The Ecm11-Gmc2 complex promotes synaptonemal complex formation through assembly of transverse filaments in budding yeast

During meiosis, homologous chromosomes pair at close proximity to form the synaptonemal complex (SC). This association is mediated by transverse filament proteins that hold the axes of homologous chromosomes together along their entire length. Transverse filament proteins are highly aggregative and can form an aberrant aggregate called the polycomplex that is unassociated with chromosomes. Here, we show that the Ecm11-Gmc2 complex is a novel SC component, functioning to facilitate assembly of the yeast transverse filament protein, Zip1. Ecm11 and Gmc2 initially localize to the synapsis initiation sites, then throughout the synapsed regions of paired homologous chromosomes. The absence of either Ecm11 or Gmc2 substantially compromises the chromosomal assembly of Zip1 as well as polycomplex formation, indicating that the complex is required for extensive Zip1 polymerization. We also show that Ecm11 is SUMOylated in a Gmc2-dependent manner. Remarkably, in the unSUMOylatable ecm11 mutant, assembly of chromosomal Zip1 remained compromised while polycomplex formation became frequent. We propose that the Ecm11-Gmc2 complex facilitates the assembly of Zip1 and that SUMOylation of Ecm11 is critical for ensuring chromosomal assembly of Zip1, thus suppressing polycomplex formation

Characterizing genomic alterations in cancer by complementary functional associations.

Systematic efforts to sequence the cancer genome have identified large numbers of mutations and copy number alterations in human cancers. However, elucidating the functional consequences of these variants, and their interactions to drive or maintain oncogenic states, remains a challenge in cancer research. We developed REVEALER, a computational method that identifies combinations of mutually exclusive genomic alterations correlated with functional phenotypes, such as the activation or gene dependency of oncogenic pathways or sensitivity to a drug treatment. We used REVEALER to uncover complementary genomic alterations associated with the transcriptional activation of β-catenin and NRF2, MEK-inhibitor sensitivity, and KRAS dependency. REVEALER successfully identified both known and new associations, demonstrating the power of combining functional profiles with extensive characterization of genomic alterations in cancer genomes

Aberrant Epigenetic Silencing Is Triggered by a Transient Reduction in Gene Expression

Aberrant epigenetic silencing plays a major role in cancer formation by inactivating tumor suppressor genes. While the endpoints of aberrant silencing are known, i.e., promoter region DNA methylation and altered histone modifications, the triggers of silencing are not known. We used the tet-off system to test the hypothesis that a transient reduction in gene expression will sensitize a promoter to undergo epigenetic silencing.The tet responsive promoter (P(TRE)) was used to drive expression of the selectable human HPRT cDNA in independent transfectants of an Hprt deficient mouse cell line. In this system, high basal HPRT expression is greatly reduced when doxycycline (Dox) is added to the culture medium. Exposure of the P(TRE)-HPRT transfectants to Dox induced HPRT deficient clones in a time dependent manner. A molecular analysis demonstrated promoter region DNA methylation, loss of histone modifications associated with expression (i.e., H3 lysine 9 and 14 acetylation and lysine 4 methylation), and acquisition of the repressive histone modification H3 lysine 9 methylation. These changes, which are consistent with aberrant epigenetic silencing, were not present in the Dox-treated cultures, with the exception of reduced H3 lysine 14 acetylation. Silenced alleles readily reactivated spontaneously or after treatment of cells with inhibitors of histone deacetylation and/or DNA methylation, but re-silencing of reactivated alleles did not require a new round of Dox exposure. Inhibition of histone deacetylation inhibited both the induction of silencing and re-silencing, whereas inhibition of DNA methylation had no such effect.This study demonstrates that a transient reduction in gene expression triggers a pathway for aberrant silencing in mammalian cells and identifies histone deacetylation as a critical early step in this process. DNA methylation, in contrast, is a secondary step in the silencing pathway under study. A model to explain these observations is offered

Genetic diversity of PRRSV 1 in Central Eastern Europe in 1994-2014: origin and evolution of the virus in the region

More than 20 years after the first outbreaks, the phylogenetic picture of PRRSV is still incomplete and full of gaps, especially in regards of PRRSV 1. Due to the exceptional diversity observed at the eastern borders of Europe and the low number of available sequences from Central Eastern European countries, the authors collected and analyzed both recent as well as already submitted sequences comparing them to a large backbone set of available ORF5 sequences representing the full spectrum of PRRSV 1 Subtype 1 diversity to conduct a systematic phylogenetic analysis and reclassification elucidating the diversity of the virus in these countries. Moreover, further analyses of the EUROSTAT data regarding the live pig movement trends revealed their influence of virus diversity and evolution. The results indicate that besides the effect of local, isolated divergent evolution and the use of modified live vaccines, the most important factor influencing a given country’s virus diversity is the transboundary movement of live, infected animals