The Perils of Pathogen Discovery: Origin of a Novel Parvovirus-Like Hybrid Genome Traced to Nucleic Acid Extraction Spin Columns

Next-generation sequencing was used for discovery and de novo assembly of a novel, highly divergent DNA virus at the interface between the Parvoviridae and Circoviridae. The virus, provisionally named parvovirus-like hybrid virus (PHV), is nearly identical by sequence to another DNA virus, NIH-CQV, previously detected in Chinese patients with seronegative (non-A-E) hepatitis. Although we initially detected PHV in a wide range of clinical samples, with all strains sharing ∼99% nucleotide and amino acid identity with each other and with NIH-CQV, the exact origin of the virus was eventually traced to contaminated silica-binding spin columns used for nucleic acid extraction. Definitive confirmation of the origin of PHV, and presumably NIH-CQV, was obtained by in-depth analyses of water eluted through contaminated spin columns. Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture. The confirmation of PHV/NIH-CQV as laboratory reagent contaminants and not bona fide infectious agents of humans underscores the rigorous approach needed to establish the validity of new viral genomes discovered by next-generation sequencing.     

Reolysin is a novel reovirus-based agent that induces endoplasmic reticular stress-mediated apoptosis in pancreatic cancer

Activating mutation of KRas is a genetic alteration that occurs in the majority of pancreatic tumors and is therefore an ideal therapeutic target. The ability of reoviruses to preferentially replicate and induce cell death in transformed cells that express activated Ras prompted the development of a reovirus-based formulation for cancer therapy called Reolysin. We hypothesized that Reolysin exposure would trigger heavy production of viral products leading to endoplasmic reticular (ER) stress-mediated apoptosis. Here, we report that Reolysin treatment stimulated selective reovirus replication and decreased cell viability in KRas-transformed immortalized human pancreatic duct epithelial cells and pancreatic cancer cell lines. These effects were associated with increased expression of ER stress-related genes, ER swelling, cleavage of caspase-4, and splicing of XBP-1. Treatment with ER stress stimuli including tunicamycin, brefeldin A, and bortezomib (BZ) augmented the anticancer activity of Reolysin. Cotreatment with BZ and Reolysin induced the simultaneous accumulation of ubiquitinated and viral proteins, resulting in enhanced levels of ER stress and apoptosis in both in vitro and in vivo models of pancreatic cancer. Our collective results demonstrate that the abnormal protein accumulation induced by the combination of Reolysin and BZ promotes heightened ER stress and apoptosis in pancreatic cancer cells and provides the rationale for a phase I clinical trial further investigating the safety and efficacy of this novel strategy.     

Development of novel real‐time TaqMan® PCR assays for the species and sex identification of otter (Lutra lutra) and their application to noninvasive genetic monitoring

Developing strategies to maintain biodiversity requires baseline information on the current status of each individual species. The development of genetic techniques and their application to noninvasively collected samples have the potential to yield information on the structure of elusive animal populations and so are important tools in conservation management. Using DNA isolated from faecal samples can be challenging owing to low quantity and quality. This study, however, presents the development of novel real‐time polymerase chain reaction assays using fluorescently labelled TaqMan^® MGB probes enabling species and sex identification of Eurasian otter (Lutra lutra) spraints (faeces). These assays can also be used in determining an optimum microsatellite panel and can be employed as cost‐saving screening tools for downstream genetic testing including microsatellite genotyping and haplotype analysis. The techniques are shown to work efficiently with L. lutra DNA isolated from tissue, hair, spraint, blood and anal jelly samples.     

Identification of Immune Traits Correlated with Dairy Cow Health,Reproduction and Productivity

Detailed biological analyses (e.g. epidemiological, genetic) of animal health and fitness in the field are limited by the lack of large-scale recording of individual animals. An alternative approach is to identify immune traits that are associated with these important functions and can be subsequently used in more detailed studies. We have used an experimental dairy herd with uniquely dense phenotypic data to identify a range of potentially useful immune traits correlated with enhanced (or depressed) health and fitness. Blood samples from 248 dairy cows were collected at two-monthly intervals over a 10-month period and analysed for a number of immune traits, including levels of serum proteins associated with the innate immune response and circulating leukocyte populations. Immune measures were matched to individual cow records related to productivity, fertility and disease. Correlations between traits were calculated using bivariate analyses based on animal repeatability and random regression models with a Bonferroni correction to account for multiple testing. A number of significant correlations were found between immune traits and other recorded traits including: CD4⁺:CD8⁺ T lymphocyte ratio and subclinical mastitis; % CD8⁺ lymphocytes and fertility; % CD335⁺ natural killer cells and lameness episodes; and serum haptoglobin levels and clinical mastitis. Importantly these traits were not associated with reduced productivity and, in the case of cellular immune traits, were highly repeatable. Moreover these immune traits displayed significant between-animal variation suggesting that they may be altered by genetic selection. This study represents the largest simultaneous analysis of multiple immune traits in dairy cattle to-date and demonstrates that a number of immune traits are associated with health events. These traits represent useful selection markers for future programmes aimed at improving animal health and fitness.     

Depth-Dependent Differences in Community Structure of the Human Colonic Microbiota in Health

Objective

The aims of this study were to develop techniques for spatial microbial assessment in humans and to establish colonic luminal and mucosal spatial ecology, encompassing longitudinal and cross-sectional axes.

Design

A microbiological protected specimen brush was used in conjunction with a biopsy forceps to sample the colon in nine healthy volunteers undergoing colonoscopy. Terminal Restriction Fragment Length Polymorphism analysis was used to determine the major variables in the spatial organization of the colonic microbiota.

Results

Protected Specimen Brush sampling retrieved region-specific, uncontaminated samples that were enriched for bacterial DNA and depleted in human DNA when compared to biopsy samples. Terminal Restriction Fragment Length Polymorphism analysis revealed a segmentation of bacterial communities between the luminal brush and biopsy-associated ecological niches with little variability across the longitudinal axis of the colon and reduced diversity in brush samples.

Conclusion

These results support the concept of a microbiota with little longitudinal variability but with some degree of segregation between luminal and mucosal communities.     

Prevalence of systemic diseases in patients undergoing minor oral surgeries

It is of interest to evaluate the prevalence of systemic disorders in patients undergoing minor oral surgeries at a dental hospital. This will help to take necessary precautions prior to oral surgeries. We used the digital case records of 1288 patients who underwent minor oral surgeries in a hospital. Demographic details and systemic diseases of the patients were recorded from digital case records. Data shows that 103 patients (7.9%) of the total number of patients undergoing minor oral surgeries had systemic diseases with 3.8% of patients diagnosed with diabetes. Statistically significant associations were found between type of minor oral surgery and the type of systemic disease (p<0.001); age of patients and type of minor oral surgery (p<0.001); age and type of systemic diseases (p<0.001) and gender of patient and type of minor oral surgery (p = 0.005). Thus, data shows the prevalence of systemic diseases in patients undergoing minor oral surgeries was 7.9%.     

An assessment of pathogen removal during composting of the separated solid fraction of pig manure

The aim was to investigate pathogen survival during composting of pig manure solids with and without bulking agents in two trials of 56 days duration, each with four treatments. Salmonella was detected in the sawdust and straw bulking agents but was undetectable in the compost, except in one treatment at day 0. Enteric indicator organisms were reduced by day 7 (P<0.001) and were undetectable in the final compost, except for coliform which were present at 3.66-4.43 log?? CFU/g. Yeasts and moulds were reduced and aerobic spore-formers remained stable in one trial but both increased in the other (P<0.001). Bacillus licheniformis and Clostridium sporogenes were the predominant culturable spore-forming bacteria recovered. Microbial counts were influenced by the bulking agent but only at particular time points (P<0.05). Overall, the pig manure-derived compost complied with EU regulations for processed manure products, as E. coli and Enterococcus were below limits and it was Salmonella-free.     

Host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures

The mechanisms by which RNA arboviruses, including chikungunya virus (CHIKV), evolve and maintain the ability to infect vertebrate and invertebrate hosts are poorly understood. To understand how host specificity shapes arbovirus populations, we studied CHIKV populations passaged alternately between invertebrate and vertebrate cells (invertebrate ↔ vertebrate) to simulate natural alternation and contrasted the results with those for populations that were artificially released from cycling by passage in single cell types. These CHIKV populations were characterized by measuring genetic diversity, changes in fitness, and adaptability to novel selective pressures. The greatest fitness increases were observed in alternately passaged CHIKV, without drastic changes in population diversity. The greatest increases in genetic diversity were observed after serial passage and correlated with greater adaptability. These results suggest an evolutionary trade-off between maintaining fitness for invertebrate ↔ vertebrate cell cycling, where maximum adaptability is possible only via enhanced population diversity and extensive exploration of sequence space.Emergence of pathogenic RNA viruses is associated with their genomic variability and environmental changes that lead to novel host contacts. Many new and reemerging RNA viruses have been introduced into humans (10). Arthropod-borne viruses (arboviruses), including dengue virus (DENV), have caused recent epidemics by changing their host ranges to increase infection of humans (54). Adaptation to the urban mosquito Aedes albopictus may have expanded a 2005-2006 outbreak of Chikungunya virus (CHIKV) in Reunion Island, France (46), that subsequently circulated among humans in the absence of other amplifying hosts.Despite these emergence events, the evolutionary processes that mediate arbovirus host range changes are poorly understood, partly since arbovirus evolution is understudied. Arboviruses are transmitted horizontally between arthropod vectors and vertebrate reservoir hosts. They replicate rapidly and achieve large population sizes. Polymerases of RNA viruses lack proofreading to repair errors, leading to one substitution per ∼10⁻⁴ nucleotides (nt) copied (11, 36), corresponding to one error per 10-kb genome. This polymerase infidelity leads to diversification that produces closely related but nonidentical RNAs that together form a spectrum of mutants. Although arbovirus mutant spectra have been observed in nature (1, 8, 20, 55, 57), the diversity and divergence within the spectrum are not well described, and the phenotypic roles of minority RNAs are unknown. Understanding the mutation distribution in a heterogeneous arbovirus population is important, given that any variant can be favored by selection and ultimately affect fitness (12, 13). However, the relationships between fitness and RNA virus population diversity are poorly understood.Studies with other RNA viruses, including human immunodeficiency virus (HIV) (3, 58, 60), hepatitis C virus (14, 15, 25), and poliovirus (30, 52), indicate that intrahost population diversity is important for virus evolution, fitness, and pathogenesis. Unlike these vertebrate-only RNA viruses, arboviruses obligately cycle between vertebrates and arthropods, a process that imposes additional selective constraints on evolution and adaptation. Sequence comparisons of RNA arbovirus isolates show that they are relatively stable (18, 19), and genetic studies reveal that evolution is dominated by purifying selection (20-22, 57). This constancy of consensus sequence may derive from the need to infect disparate hosts that present conflicting demands for adaptation where sequence changes that improve fitness in one host may not be maintained in the alternate organism.Experimental evolution studies have been performed to study fitness trade-offs and the unique ability of arboviruses to simultaneously evolve to vertebrate and invertebrate hosts. In vitro evolution studies reveal three general patterns of arbovirus evolution: (i) fitness gains after serial passage in vertebrate or invertebrate cells (except in certain cases [7]) and losses in bypassed host cell types (DENV, Eastern equine encephalitis virus [EEEV], Sindbis virus [SINV], and vesicular stomatitis virus [VSV]) (17, 28, 51, 56); (ii) reduced fitness in new cells (VSV) (28), and (iii) fitness increases after alternating (invertebrate ↔ vertebrate) passage (DENV, EEEV, SINV, VSV) (17, 28, 51, 56). Together, these studies suggest that constraints on fitness differ in insect and vertebrate cells and can be virus specific but that arbovirus fitness is not limited by alternating between vertebrate and invertebrate hosts.An in vivo study revealed a similar pattern of arbovirus evolution: vertebrate-passaged Venezuelan equine encephalitis virus (VEEV) was five times more fit than its unpassaged parent, and mosquito-passaged VEEV was more infectious for vectors (6). Consensus sequences revealed that, despite becoming more fit, mutations in passaged populations were slight or absent. This suggests that fitness increases were mediated by minority genomes in the mutant spectrum. However, a major limitation of the in vivo experiments and other arbovirus evolution studies is that sequencing of individual RNAs from the mutant spectrum in passaged intrahost populations was not performed (although notable exceptions for flaviviruses exist [5, 22]). The identity of minority variants within intrahost arbovirus populations and their influences on phenotype have not been extensively examined.The goal of this study, therefore, is to understand how obligate host cycling shapes an intrahost arbovirus population. RNA viruses can tolerate increased niche breadth when they evolve in heterogeneous environments (48), a trait which may promote easier emergence and therefore help explain why arboviruses frequently emerge or reemerge to cause human and veterinary disease. One limitation of increasing breadth may be restricted genetic diversity, where only genomes accepted in both hosts can be maintained. To explore this possibility, we described arbovirus populations after invertebrate ↔ vertebrate cell cycling and compared them to populations that were artificially released from alternating passage via serial vertebrate or invertebrate cell transfer. Since previous studies indicate that intrahost RNA virus population diversity can determine phenotype (3, 37, 52) and given that increases in arbovirus fitness are not always associated with consensus genome changes (6, 27), we determined how arbovirus population diversity (mutation frequency) and distance (number of mutations by which each RNA differs from the consensus) relate to fitness. We predicted that genetic diversity and distance between RNAs in the mutant spectrum and the corresponding consensus are correlated with fitness and that diverse populations better escape varied selective pressures than genetically homogeneous populations since, by chance, they are more likely to contain an advantageous mutation(s) when the pressure is applied.Chikungunya virus (family Togaviridae, genus Alphavirus) was selected primarily because of its medical relevance. CHIKV has caused outbreaks of human disease characterized by arthralgia and myalgia since the 18th century and since 2004 in Africa, Indian Ocean islands, Southeast Asia, and Italy (32). Furthermore, no adaptation studies have been performed for CHIKV, and no intrahost population studies have been conducted for an alphavirus. An infectious clone was generated from the strain used for passages and was genetically marked to differentiate its RNAs from passaged CHIKV, so that after direct competition, the fitnesses of passaged populations could be compared to those for progenitors.