Marine Snails and Slugs: a Great Place To Look for Antiviral Drugs.

Molluscs, comprising one of the most successful phyla, lack clear evidence of adaptive immunity and yet thrive in the oceans, which are rich in viruses. There are thought to be nearly 120,000 species of Mollusca, most living in marine habitats. Despite the extraordinary abundance of viruses in oceans, molluscs often have very long life spans (10 to 100 years). Thus, their innate immunity must be highly effective at countering viral infections. Antiviral compounds are a crucial component of molluscan defenses against viruses and have diverse mechanisms of action against a wide variety of viruses, including many that are human pathogens. Antiviral compounds found in abalone, oyster, mussels, and other cultured molluscs are available in large supply, providing good opportunities for future research and development. However, most members of the phylum Mollusca have not been examined for the presence of antiviral compounds. The enormous diversity and adaptations of molluscs imply a potential source of novel antiviral compounds for future drug discovery.

Variably lytic infection dynamics of large Bacteroidetes podovirus phi38:1 against two Cellulophaga baltica host strains.

Bacterial viruses (phages) influence global biogeochemical cycles by modulating bacterial mortality, metabolic output and evolution. However, our understanding of phage infections is limited by few methods and environmentally relevant model systems. Prior work showed that Cellulophaga baltica phage ϕ38:1 infects its original host lytically, and an alternative host either delayed lytically or lysogenically. Here we investigate these infections through traditional and marker-based approaches, and introduce geneELISA for high-throughput examination of phage-host interactions. All methods confirmed the lytic, original host infection (70-80 min latent period; approximately eight phages produced per cell), but alternative host assays were more challenging. A 4.5 h experiment detected no phage production by plaque assay, whereas phageFISH and geneELISA revealed phage genome replication and a latent period ≥ 150 min. Longer experiments (26 h) suggested an 11 h latent period and a burst size of 871 by plaque assay, whereas phageFISH identified cell lysis starting at < 5 h and lasting to 11 h, but for only 7% to 21.5% of infected cells, respectively, and with ∼ 39 phages produced per cell. These findings help resolve the nature of the alternative host infection as delayed lytic and offer solutions to methodological challenges for studying inefficient phage-host interactions.

Immunological changes in response to herpesvirus infection in abalone Haliotis laevigata and Haliotis rubra hybrids.

Australian abalone production has been affected by outbreaks of abalone viral ganglioneuritis (AVG) caused by a herpesvirus (AbHV). In this study, we undertook experimental transmission trials by immersion to study the abalone immune response to infection with AbHV. Representative cellular and humoural immune parameters of abalone, including total haemocyte count (THC), superoxide anion (SO) and antiviral activity against herpes simplex virus type 1 (HSV-1), were examined in apparently healthy (sub-clinical) and moribund abalone after challenge. In the early infection, sub-clinical stage (days 1-3), THC was found to increase significantly in infected abalone. TaqMan qPCR confirmed 20.5% higher viral load in moribund abalone compared to apparently healthy abalone, indicating that the abundance of AbHV within abalone is linked to their clinical signs. At the clinical stage of infection, THC was significantly lower in moribund abalone, but increased in AbHV-exposed but apparently healthy abalone, in comparison to non-infected controls. SO was reduced in all abalone that were PCR-positive for AbHV. THC and SO level were found to be negatively correlated with the presence of AbHV in abalone, but no effect of AbVH exposure was observed on the haemolymph antiviral activity. These results suggest that abalone mount an initial cellular immune response to AbHV infection, but this response cannot be sustained under high viral loads, leading to mortality.

A simple methodology for conversion of mouse monoclonal antibody to human-mouse chimeric form.

Passive immunotherapy has mainly been used as a therapy against cancer and inflammatory conditions. Recent studies have shown that monoclonal antibody-(mAb-) based passive immunotherapy is a promising approach to combat virus infection. Specific mouse mAbs can be routinely generated in large amounts with the use of hybridoma technology but these cannot be used for therapy in human beings due to their immunogenicity. Therefore, the development of chimeric and humanized mAbs is important for therapeutic purpose. This is facilitated by a variety of molecular techniques like recombinant DNA technology and the better understanding of the structure and function of antibody. The human-mouse chimeric forms allow detailed analysis of the mechanism of inhibition and the potential for therapeutic applications. Here, a step-by-step description of the conversion process will be described. The commercial availability of the reagents required in each step means that this experimentation can be easily set up in research laboratories.

Influence of elevated temperatures on the immune response of abalone, Haliotis rubra.

Elevated water temperature can act as a stressor impacting the immune responses of molluscs, potentially increasing their susceptibility to microbial infections. Abalone are commercially important marine molluscs that have recently experienced disease outbreaks caused by a herpesvirus and Vibrio bacteria. Sampling of wild-caught Haliotis rubra showed a significant correlation between water temperature and both antiviral and antibacterial activity, with higher activity in summer than in winter months. However, antibacterial activity was compromised in favour of antiviral activity as the water temperatures peaked in summer. A controlled laboratory experiment was then used to investigate several immune responses of H. rubra, including total haemocyte count (THC), stimulated superoxide anion production (SO), antiviral activity against a model herpesvirus, herpes simplex virus type 1 and antibacterial activity against a representative pathogenic bacterium, Vibrio anguillarum, over one week after raising water temperature from 18 to 21 or 24 °C. THC and SO increased at day 1 and then dropped back to control levels by days 3 and 7. By comparison, the humoural immune parameters showed a delayed response with antibacterial and antiviral activity significantly increasing on days 3 and 7, respectively. Consistent with the field study, antibacterial activity became significantly depressed after prolonged exposure to elevated temperatures. A principal components analysis on the combined immune parameters showed a negative correlation between antiviral and antibacterial activity. SO was positively correlated to THC and neither of these cellular parameters were correlated to the humoural antimicrobial activity. Overall, this study indicates that abalone may have more resilience to viruses than bacterial pathogens under conditions of elevated temperature, such as those predicted under future climate change scenarios.

In vitro antiviral activity against herpes simplex virus in the abalone Haliotis laevigata.

As viruses are extremely abundant in oceans, marine organisms may have evolved novel metabolites to protect themselves from viral infection. This research examined a well-known commercial gastropod, abalone (Haliotidae), which in Australia have recently experienced disease due to a neurotropic infection, abalone viral ganglioneuritis, caused by an abalone herpesvirus (AbHV). Due to the lack of molluscan cell lines for culturing AbHV, the antiviral activity of the abalone Haliotis laevigata was assessed against another neurotropic herpesvirus, herpes simplex virus type 1 (HSV-1), using a plaque assay. The concentration range at which abalone extract was used for antiviral testing caused minimal (<10 %) mortality in Vero cells. Haemolymph (20 %, v/v) and lipophilic extract of the digestive gland (3000 µg ml(-1)) both substantially decreased the number and size of plaques. By adding haemolymph or lipophilic extract at different times during the plaque assay, it was shown that haemolymph inhibited viral infection at an early stage. In contrast, the antiviral effect of the lipophilic extract was greatest when added 1 h after infection, suggesting that it may act at an intracellular stage of infection. These results suggest that abalone have at least two antiviral compounds with different modes of action against viral infection, and provide a novel lead for marine antiviral drug discovery.

Identification of human haploinsufficient genes and their genomic proximity to segmental duplications.

Despite the significance of haploinsufficiency in human disease, no systematic study has been reported into the types of genes that are haploinsufficient in human, or into the mechanisms that commonly lead to their deletion and to the expression of the haploinsufficient phenotype. We have applied a rigorous text-searching and database-mining strategy to extract, as comprehensively as possible, from PubMed and OMIM an annotated list of currently known human haploinsufficient genes, including their functions and associated diseases. Gene-set enrichment analysis shows that genes-encoding transcription factors, and genes that function in development, the cell cycle, and nucleic acid metabolism are overrepresented among haploinsufficient genes in human. Many of the phenotypes associated with loss-of-function or deletion of one copy of a haploinsufficient gene describe mental retardation, developmental or metabolic disorders, or tumourigenesis. We also found that haploinsufficient genes are less likely than the complete set of human genes to be situated between pairs of segmental duplications (SDs) that are in close proximity to each other on the same chromosome. Given that SDs can initiate non-allelic homologous recombination (NAHR) and the deletion of adjacent genomic regions, this suggests that the location of haploinsufficient genes between SD pairs, from whence they may suffer intra-genomic rearrangement and loss, is selectively disadvantageous. We describe a custom-made Java visualization tool, HaploGeneMapper, to aid in visualizing the proximity of human haploinsufficient genes to SDs and to enable identification of haploinsufficient genes that are vulnerable to NAHR-mediated deletion.

Towards quantitative viromics for both double-stranded and single-stranded DNA viruses.

BACKGROUND: Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation). METHODS: Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. RESULTS: Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. DISCUSSION: Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Gluconeogenesis and fasting in cerebral malaria.

BACKGROUND: In healthy subjects after an overnight fast, glucose production is for approximately 50% derived from glycogenolysis. If the fast is prolonged, glucose production decreases due to a decline in glycogenolysis, while gluconeogenesis remains stable. In cerebral malaria, glucose production is completely derived from gluconeogenesis after an overnight fast. It is not known if glucose production also decreases during fasting when its only source is gluconeogenesis. DESIGN: Glucose production was measured by infusion of [6,6-2H2]glucose in seven patients with cerebral malaria after prolonging a fast from 20.30 to 00.30 hours. RESULTS: Glucose production decreased by approximately 10% (27.4 +/- 2.1 to 24.7 +/- 1.6 micromol/kg/min, p = 0.05), without changes in the plasma concentrations of glucoregulatory hormones, FFA or precursors. CONCLUSIONS: In the patients with cerebral malaria, glucose production decreases during fasting due to a decrease in the rate of gluconeogenesis. These data suggest that the decrease in the rate of glucose production during short-term fasting is actively regulated and not simply due to shrinkage of glycogen content, as in the absence of glycogenolysis, glucose production decreases at the same rate as normally seen in healthy subjects whose glucose production is for approximately 50% derived from glycogen and in whom gluconeogenesis is stable.

Emerging methods to study bacteriophage infection at the single-cell level.

Bacteria and their viruses (phages) are abundant across diverse ecosystems and their interactions influence global biogeochemical cycles and incidence of disease. Problematically, both classical and metagenomic methods insufficiently assess the host specificity of phages and phage-host infection dynamics in nature. Here we review emerging methods to study phage-host interaction and infection dynamics with a focus on those that offer resolution at the single-cell level. These methods leverage ever-increasing sequence data to identify virus signals from single-cell amplified genome datasets or to produce primers/probes to target particular phage-bacteria pairs (digital PCR and phageFISH), even in complex communities. All three methods enable study of phage infection of uncultured bacteria from environmental samples, while the latter also discriminates between phage-host interaction outcomes (e.g., lytic, chronic, lysogenic) in model systems. Together these techniques enable quantitative, spatiotemporal studies of phage-bacteria interactions from environmental samples of any ecosystem, which will help elucidate and predict the ecological and evolutionary impacts of specific phage-host pairings in nature.

Evolution of gene function and regulatory control after whole-genome duplication: comparative analyses in vertebrates.

The significance of whole-genome duplications (WGD) for vertebrate evolution remains controversial, in part because the mechanisms by which WGD contributed to functional evolution or speciation are still incompletely characterized. Fish genomes provide an ideal context in which to examine the consequences of WGD, because the teleost lineage experienced an additional WGD soon after divergence from tetrapods and because five teleost genomes are available for comparative analysis. Here we present an integrated approach to characterize these post-duplication genomes based on genome-scale synteny, phylogenetic, temporal, and spatial gene expression and on protein sequence data. A minimum of 3%-4% of protein-coding loci have been retained in two copies in each of the five fish genomes, and many of these duplicates are key developmental genes that function as transcription factors or signaling molecules. Almost all duplicate gene pairs we examined have diverged in spatial and/or temporal expression during embryogenesis. A quarter of duplicate pairs have diverged in function via the acquisition of novel protein domains or via changes in the subcellular localization of their encoded proteins. We compared the spatial expression and protein domain architecture of zebrafish WGD-duplicates to those of their single mouse ortholog and found many examples supporting a model of neofunctionalization. WGD-duplicates have acquired novel protein domains more often than have single-copy genes. Post-WGD changes at the gene regulatory level were more common than changes at the protein level. We conclude that the most significant consequence of WGD for vertebrate evolution has been to enable more-specialized regulatory control of development via the acquisition of novel spatiotemporal expression domains. We find limited evidence that reciprocal gene loss led to reproductive isolation and speciation in this lineage.