FLOW CYTOMETRIC ANALYSES OF VIRAL INFECTION IN TWO MARINE PHYTOPLANKTON SPECIES, MICROMONAS PUSILLA (PRASINOPHYCEAE) AND PHAEOCYSTIS POUCHETII (PRYMNESIOPHYCEAE)

Cell characteristics of two axenic marine phytoplankton species, Micromonas pusilla (Butscher) Manton et Parke and Phaeocystis pouchetii (Hariot) Lagerheim, were followed during viral infection using flow cytometry. Distinct differences between noninfected and infected cultures were detected in the forward scatter intensities for both algal species. Changes in side scatter signals on viral infection were found only for P. pouchetii. Chlorophyll red fluorescence intensity per cell decreased gradually over time in the infected cultures. DNA analyses were performed using the nucleic acid–specific fluorescent dye SYBR Green I. Shortly after infection the fraction of algal cells with more than one genome equivalent increased for both species because of the replication of viral DNA in the infected cells. Over time, a population of algal cells with low red autofluorescence and low DNA fluorescence developed, likely representing algal cells just prior to viral lysis. The present study provides insight into basic virus–algal host cell interactions. It shows that flow cytometry can be a useful tool to discriminate between virus infected and noninfected phytoplankton cells.     

Recombination every day: abundant recombination in a virus during a single multi-cellular host infection

Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment—based on data on the timing of coat protein detection—the per base and replication cycle recombination rate was on the order of 2 × 10⁻⁵ to 4 × 10⁻⁵. This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.     

Host factors against plant viruses

Plant virus genome replication and movement is dependent on host resources and factors. However, plants respond to virus infection through several mechanisms, such as autophagy, ubiquitination, mRNA decay and gene silencing, that target viral components. Viral factors work in synchrony with pro-viral host factors during the infection cycle and are targeted by antiviral responses. Accordingly, establishment of virus infection is genetically determined by the availability of the pro-viral factors necessary for genome replication and movement, and by the balance between plant defence and viral suppression of defence responses. Sequential requirement of pro-viral factors and the antagonistic activity of antiviral factors suggest a two-step model to explain plant–virus interactions. At each step of the infection process, host factors with antiviral activity have been identified. Here we review our current understanding of host factors with antiviral activity against plant viruses.     

Viral lysis modifies seasonal phytoplankton dynamics and carbon flow in the Southern Ocean

Phytoplankton form the base of marine food webs and are a primary means for carbon export in the Southern Ocean, a key area for global pCO₂ drawdown. Viral lysis and grazing have very different effects on microbial community dynamics and carbon export, yet, very little is known about the relative magnitude and ecological impact of viral lysis on natural phytoplankton communities, especially in Antarctic waters. Here, we report on the temporal dynamics and relative importance of viral lysis rates, in comparison to grazing, for Antarctic nano- and pico-sized phytoplankton of varied taxonomy and size over a full productive season. Our results show that viral lysis was a major loss factor throughout the season, responsible for roughly half (58%) of seasonal phytoplankton carbon losses. Viral lysis appeared critically important for explaining temporal dynamics and for obtaining a complete seasonal mass balance of Antarctic phytoplankton. Group-specific responses indicated a negative correlation between grazing and viral losses in Phaeocystis and picoeukaryotes, while for other phytoplankton groups losses were more evenly spread throughout the season. Cryptophyte mortality was dominated by viral lysis, whereas small diatoms were mostly grazed. Larger diatoms dominated algal carbon flow and a single ‘lysis event’ directed >100% of daily carbon production away from higher trophic levels. This study highlights the need to consider viral lysis of key Antarctic phytoplankton for a better understanding of microbial community interactions and more accurate predictions of organic matter flux in this climate-sensitive region.Subject terms: Microbial ecology, Virus-host interactions     

Virus ecology of fluvial systems: a blank spot on the map?

The ecology of viruses has been studied only in a limited number of rivers and streams. In light of a recent re‐appraisal of the global fluvial surface area, issues such as abundance and production, host mortality and the influence of suspended particles and biofilms are addressed. Viral life cycles, potential impacts of viruses on water biochemistry and carbon flow, and viral diversity are considered. Variability in trophic levels along with the heterogeneous nature and hydrological dynamics of fluvial environments suggest a prevailingly physical control of virus‐related processes under lotic conditions and more biological control under lentic conditions. Viral lysis likely contributes to a pool of rapidly cycling carbon in environments typically characterized by high proportions of recalcitrant terrestrial carbon. On average, 33.6% (equalling 0.605 Pg C year^?1) of the globally respired carbon from fluvial systems may pass through a viral loop. Virus distribution and the proportion of organic material in horizontal transport versus processes in retention zones remain to be determined in detail. The need for up‐scaling the contribution of virus‐related processes in fluvial systems is of global relevance. Further, the role of climate change and the effect of anthropogenic alterations of fluvial systems on viruses require attention. The identification of these considerable knowledge gaps should foster future research efforts.     

Replication of simian virus 40 DNA in normal human fibroblasts and in fibroblasts from xeroderma pigmentosum.

Simian virus 40 infection of semipermissive human diploid fibroblasts (HF), at early passage in cell culture, was compared with that of permissive established monkey cell lines. Viral DNA can be readily detected at 24 to 48 h postinfection at 37 degrees C with a high multiplicity of infection, approaching 10% of that of monkey cells (TC7). The length of time necessary for replication of an average molecule of viral DNA was found to be indistinguishable in HF and TC7 cells. Strand elongation plus termination were assessed by following the accumulation of DNA I at 40 degrees C from replicative intermediates of tsA30 prelabeled at 33 degrees C, obviating isotope pool problems. Combined initiation and elongation of wild-type viral DNA was measured by density shift experiments involving a 5-bromodeoxyuridine chase of prelabeled [3H]thymidine-labeled viral DNA. Determination of accumulation of viral T and V antigens supports the conclusion that the most likely basis for the reduced virus yield in HF cells results from the inefficiency of an early stage in virus infection, before or during uncoating. Similar results were obtained in fibroblasts derived from patients with xeroderma pigmentosum, suggesting that enzymes of UV repair are not required in unirradiated simian virus 40 DNA synthesis.     

Finding a needle in the virus metagenome haystack--micro-metagenome analysis captures a snapshot of the diversity of a bacteriophage armoire

Viruses are ubiquitous in the oceans and critical components of marine microbial communities, regulating nutrient transfer to higher trophic levels or to the dissolved organic pool through lysis of host cells. Hydrothermal vent systems are oases of biological activity in the deep oceans, for which knowledge of biodiversity and its impact on global ocean biogeochemical cycling is still in its infancy. In order to gain biological insight into viral communities present in hydrothermal vent systems, we developed a method based on deep-sequencing of pulsed field gel electrophoretic bands representing key viral fractions present in seawater within and surrounding a hydrothermal plume derived from Loki's Castle vent field at the Arctic Mid-Ocean Ridge. The reduction in virus community complexity afforded by this novel approach enabled the near-complete reconstruction of a lambda-like phage genome from the virus fraction of the plume. Phylogenetic examination of distinct gene regions in this lambdoid phage genome unveiled diversity at loci encoding superinfection exclusion- and integrase-like proteins. This suggests the importance of fine-tuning lyosgenic conversion as a viral survival strategy, and provides insights into the nature of host-virus and virus-virus interactions, within hydrothermal plumes. By reducing the complexity of the viral community through targeted sequencing of prominent dsDNA viral fractions, this method has selectively mimicked virus dominance approaching that hitherto achieved only through culturing, thus enabling bioinformatic analysis to locate a lambdoid viral "needle" within the greater viral community "haystack". Such targeted analyses have great potential for accelerating the extraction of biological knowledge from diverse and poorly understood environmental viral communities.     

Diverse responses of the bivalve-killing dinoflagellate Heterocapsa circularisquama to infection by a single-stranded RNA virus

Viruses are believed to be significant pathogens for phytoplankton. Usually, they infect a single algal species, and often their infection is highly strain specific. However, the detailed molecular background of the strain specificity and its ecological significance have not been sufficiently understood. Here, we investigated the temporal changes in viral RNA accumulation and virus-induced cell lysis using a bloom-forming dinoflagellate Heterocapsa circularisquama and its single-stranded RNA virus, HcRNAV. We observed at least three host response patterns to virus inoculation: sensitive, resistant, and delayed lysis. In the sensitive response, the host cell culture was permissive for viral RNA replication and apparent cell lysis was observed; in contrast, resistant cell culture was nonpermissive for viral RNA replication and not lysed. In the delayed-lysis response, although viral RNA replication occurred, virus-induced cell lysis was faint and remarkably delayed. In addition, the number of infectious virus particles released to the culture supernatant at 12 days postinoculation was comparable to that of the sensitive strain. By further analysis, a few strains were characterized as variants of the delayed-lysis strain. These observations indicate that the response of H. circularisquama to HcRNAV infection is highly diverse.     

Suppression of PmRab7 by dsRNA Inhibits WSSV or YHV Infection in Shrimp

Viral entry into host cells requires endocytosis machineries of the host for viral replication. PmRab7, a Penaeus monodon small GTPase protein, was investigated for its function in vesicular transport during viral infection. The double-stranded RNA of Rab7 was injected into a juvenile shrimp before challenging with white spot syndrome virus (WSSV) or yellow head virus (YHV). PmRab7 mRNA was specifically decreased at 48 h after dsRNA-Rab7 injection. Silencing of PmRab7 dramatically inhibited WSSV-VP28 mRNA and protein expression. Unexpectedly, the silencing of PmRab7 also inhibited YHV replication in the YHV-infected shrimp. These results suggested that PmRab7 is a common cellular factor required for WSSV or YHV replication in shrimp. Because PmRab7 should function in the endosomal trafficking pathway, its silencing prevents successful viral trafficking necessary for replication. Silencing of PmRab7 could be a novel approach to prevent both DNA virus (WSSV) and RNA virus (YHV) infection of shrimp.     

Early Events in Herpes Simplex Virus Infection: a Radioautographic Study

The early events in herpes simplex virus infection were studied by means of radio-autography. The virus was rapidly taken up by the host cells and uncoated. Viral deoxyribonucleic acid (DNA) reached the nuclear sites of replication in 15 to 30 min after infection. The viral DNA occasionally associated with chromosomes or condensed chromatin but was more frequently found to be randomly distributed. Viral progeny appeared 3 hr after infection. These particles did not show any particular spatial relationship to the parental DNA. The morphological latent period lasted 2.5 hr.     

Interaction between Hantavirus Nucleocapsid Protein (N) and RNA-Dependent RNA Polymerase (RdRp) Mutants Reveals the Requirement of an N-RdRp Interaction for Viral RNA Synthesis

Viral ribonucleocapsids harboring the viral genomic RNA are used as the template for viral mRNA synthesis and replication of the viral genome by viral RNA-dependent RNA polymerase (RdRp). Here we show that hantavirus nucleocapsid protein (N protein) interacts with RdRp in virus-infected cells. We mapped the RdRp binding domain at the N terminus of N protein. Similarly, the N protein binding pocket is located at the C terminus of RdRp. We demonstrate that an N protein-RdRp interaction is required for RdRp function during the course of virus infection in the host cell.     

Assembly and Maturation of the Bacteriophage Lambda Procapsid: gpC Is the Viral Protease

Viral capsids are robust structures designed to protect the genome from environmental insults and deliver it to the host cell. The developmental pathway for complex double-stranded DNA viruses is generally conserved in the prokaryotic and eukaryotic groups and includes a genome packaging step where viral DNA is inserted into a pre-formed procapsid shell. The procapsids self-assemble from monomeric precursors to afford a mature icosahedron that contains a single “portal” structure at a unique vertex; the portal serves as the hole through which DNA enters the procapsid during particle assembly and exits during infection. Bacteriophage λ has served as an ideal model system to study the development of the large double-stranded DNA viruses. Within this context, the λ procapsid assembly pathway has been reported to be uniquely complex involving protein cross-linking and proteolytic maturation events. In this work, we identify and characterize the protease responsible for λ procapsid maturation and present a structural model for a procapsid-bound protease dimer. The procapsid protease possesses autoproteolytic activity, it is required for degradation of the internal “scaffold” protein required for procapsid self-assembly, and it is responsible for proteolysis of the portal complex. Our data demonstrate that these proteolytic maturation events are not required for procapsid assembly or for DNA packaging into the structure, but that proteolysis is essential to late steps in particle assembly and/or in subsequent infection of a host cell. The data suggest that the λ-like proteases and the herpesvirus-like proteases define two distinct viral protease folds that exhibit little sequence or structural homology but that provide identical functions in virus development. The data further indicate that procapsid assembly and maturation are strongly conserved in the prokaryotic and eukaryotic virus groups.     

Protein blotting: principles and applications

Extensive studies on the DNA tumor virus Simian Virus 40 (SV40) have provided a wealth of information regarding the genome organization, regulation of viral gene expression, and the mechanism of DNA replication. SV40 can grow lytically in permissive monkey cells or the viral DNA can integrate into the host genome of nonpermissive rodent cells causing morphological transformation. The viral DNA exists as a minichromosome within the nuclei of lytically infected cells and, as a consequence of DNA replication, there is a significant amplification of the viral genome during infection. These properties suggested that SV40 could be developed as a transducing vector to introduce exogenous DNA into mammalian cells and to express this foreign DNA during the SV40 infectious cycle. In this article the properties of SV40 virus vectors and SV40 hybrid plasmid vectors are described and contrasted.     

Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally infected Cyprinus carpio koi as assessed by real-time TaqMan PCR

The Koi herpesvirus (KHV) is a herpes-like virus now recognized as a worldwide cause of mortality among populations of koi Cyprinus carpio koi and common carp Cyprinus carpio carpio. Temperature is a key factor influencing virus replication both in cell culture and in the tissues of experimentally infected fish. Genomic DNA sequences were used to optimize a rapid real-time TaqMan PCR assay to detect and quantify KHV DNA as found in the tissues of virus-exposed fish. The assay allowed analytical enumeration of target KHV genome copies ranging from 10(1) to 10(7) molecules as present in infected cell lines or fish tissues. The new assay was specific for KHV and did not detect DNA from 3 related herpes-like viruses found in fish, the Cyprinid herpesvirus 1 (CyHV-1), Cyprinid herpesvirus 2 (CyHV-2), Ictalurid herpesvirus 1 (IcHV-1) or the KF-1 cell line used for virus growth. Concentrations of KHV DNA were evaluated in 7 different tissues of replicate groups of virus-exposed koi held at water temperatures of 13, 18, 23 and 28 degrees C. Viral DNA was detected among virus-exposed koi at all 4 water temperatures but mortality was only observed among fish at 18, 23, and 28 degrees C. Time and temperature and the interactions between them affected concentrations of viral DNA detected in tissues of koi exposed to KHV. Although there were no recognized patterns to viral DNA concentrations as found in different tissues over time, KHV genome copies for all tissues increased with time post virus exposure and with water temperature. The remarkably rapid and systemic spread of the virus was demonstrated by the presence of viral DNA in multiple tissues 1 d post virus exposure. The greatest DNA concentrations found were in the gill, kidney and spleen, with virus genome equivalents consistently from 10(8) to 10(9) per 10(6) host cells. High levels of KHV DNA were also found in the mucus, liver, gut, and brain. Koi surviving infection at 62 to 64 d post virus exposure contained lower KHV genome copies (up to 1.99 x 10(2) per 10(6) host cells) as present in gill, kidney or brain tissues.     

Excision of viral DNA from host cell DNA after induction of simian virus 40-transformed hamster cells.

Simian virus 40-transformed hamster cells were induced to produce infectious virus by treatment with mitomycin C or gamma-irradiation. A portion of the simian virus-40 DNA, which is integrated into the host cell genome in uninduced cells, was recovered in a pool of relatively low-molecular-weight DNA early after induction treatment in the absence of DNA replication. The data indicte that excision of the viral genome occurs subsequent to the induction stimulus.     

Chloroplast: the Trojan horse in plant–virus interaction

The chloroplast is one of the most dynamic organelles of a plant cell. It carries out photosynthesis, synthesizes major phytohormones, plays an active part in the defence response and is crucial for interorganelle signalling. Viruses, on the other hand, are extremely strategic in manipulating the internal environment of the host cell. The chloroplast, a prime target for viruses, undergoes enormous structural and functional damage during viral infection. Indeed, large proportions of affected gene products in a virus‐infected plant are closely associated with the chloroplast and the process of photosynthesis. Although the chloroplast is deficient in gene silencing machinery, it elicits the effector‐triggered immune response against viral pathogens. Virus infection induces the organelle to produce an extensive network of stromules which are involved in both viral propagation and antiviral defence. From studies over the last few decades, the involvement of the chloroplast in the regulation of plant–virus interaction has become increasingly evident. This review presents an exhaustive account of these facts, with their implications for pathogenicity. We have attempted to highlight the intricacies of chloroplast–virus interactions and to explain the existing gaps in our current knowledge, which will enable virologists to utilize chloroplast genome‐based antiviral resistance in economically important crops.     

Persistent Hz-1 Virus Infection in Insect Cells: Evidence for Insertion of Viral DNA into Host Chromosomes and Viral Infection in a Latent Status

Persistent/latent viral infections of insect cells are a prominent though poorly understood phenomenon. In this study, the long-term association between the Hz-1 virus and insect host cells, conventionally referred to as persistent viral infection, is described. With the aid of a newly developed fluorescent cell-labeling system, we found that productive viral replication occurs by spontaneous viral reactivation in fewer than 0.2% of persistently infected cell lines over a 5-day period. Once viral reactivation takes place, the host cell dies. The persistently infected cells contain various amounts of viral DNA, and, in an extreme case, up to 16% of the total DNA isolated from infected cells could be of viral origin. Both pulsed-field gel electrophoresis and in situ hybridization experiments showed that some of these viral DNA molecules are inserted into the host chromosomes but that the rest of viral DNA copies are free from host chromosomes. Thus, Hz-1 virus is the first nonretroviral insect virus known to insert its genome into the host chromosome during the infection process. These data also suggest that the previously described persistent infection of Hz-1 virus in insect cells should be more accurately referred to as latent viral infection.