Bacteriophage (phage) are viruses that specifically infect bacteria. Phage come in a large variety of sizes and shapes. They are globally classified in terms of morphology and genome type. A phage particle is composed of a single type of nucleic acid (either DNA or RNA) and a protein capsid that protects the genetic material. The vast majority of phage also possess a tail (made of proteins) that enables the specific recognition of a receptor at the surface of the host bacterium. Phage are now recognized as the most abundant biological entities on our planet and they play major roles in the ecological balance of the microbial life. transition:
Bacteriophage can be found in any environment occupied by a suitable host; even in hosts that have been starved for long periods of time. Phage have been isolated from soil using specific enrichment with a variety of soil bacteria, including Arthrobacter, Bacillus, Nocardia, Pseudomonas, Rhodococcus, and Streptomyces, suggesting that phage are common inhabitants of soil. Early measurements of phage abundance in natural environments were quite low; however, these measurements were made using plaque formation in specific host bacteria and were not representative of the total bacteriophage populations. More recently, microscopic methods have been used to enumerate bacteriophage in environmental samples and the counts have increased by several orders of magnitude from previous determinations.
Major breakthroughs in our understanding of microbial ecology have come through technical improvements in direct enumeration of microorganisms. In the 1970s, application of epifluorescent light microscopy (EFM) to enumeration of bacteria revealed that natural abundance of this group in water and soil samples exceeds that obtained through cultivation-based techniques by 100- to 1000-fold. This realization has been pivotal in justifying the search for cultivation-independent approaches to the study of bacterial diversity such as the array of molecular genetic techniques based on small-subunit ribosomal ribonucleic acid (SSU rRNA). In addition, accurate estimation of bacterial abundance has led to dramatic improvements in understanding carbon and energy flow through natural ecosystems. The concept of the ‘microbial loop,’ in which a significant proportion of primary production, which would otherwise be lost to higher-order consumers, is incorporated into the bacterioplankton is supported by more recent knowledge of the high abundance of bacteria in many marine systems.
In comparison with the evolution of bacterial direct-counting approaches, the discovery of abundant viral populations within water samples was a dramatic, serendipitous event. Francisco Torrella and Richard Morita were the first directly to observe virus particles using transmission electron microscopy (TEM). In 0.2-μm filtered water samples, c. 104 virus-like particles (VLPs) per milliliter of seawater were observed; however, they felt natural abundance was probably higher, as prefiltration may have removed VLPs. A decade later, in a project originally designed to quantify the elemental composition of marine bacteria using TEM, extraordinarily high abundances of VLPs in water samples (c. 107–108 ml−1) from a variety of marine environments were observed. In general, this and other early studies have demonstrated that viral abundance typically exceeds co-occurring host abundance by 10-fold; thus viruses are now widely acknowledged to be the most abundant members of marine microbial communities. The role of viruses and viral infection in influencing the composition, diversity, and productivity of marine microbial communities continues to be an open area of investigation.
TEM provided the earliest evidence of high viral abundance in water samples and continues to be the standard through which all succeeding EFM-based enumeration techniques are judged. The principal advantage of TEM-based enumeration is the provision of morphological data (e.g., capsid size and head–tail morphology) that confirms positive identification of a virus particle and enables additional characterization of a viral community. However, in comparison with epifluorescence techniques, TEM is difficult with samples that contain even a small proportion of particulate material and requires more time for sample preparation and observation. Several nucleic acid-binding fluorescent stains have been employed for direct EFM enumeration of viruses within water samples, including 4′-6′-diamidino-2-phenylindole (DAPI), YoPro, and the SYBR stains. Of these, the SYBR stains appear to be the most widely accepted due to ease of staining, exceptional brightness, and low nonspecific binding and fluorescence. Because EFM provides no morphological detail, counts using these techniques are reported as VLPs. Numerous comparative studies have shown that TEM virus counts are similar, but generally lower than counts of VLPs using EFM. The possible interference of particulates in TEM counting and nonviral, stain-positive particles in EFM are hypothesized to result in under- and overestimates of these two approaches, respectively. Nevertheless, the ease and relative precision of EFM techniques have resulted in widespread application of these approaches to viral enumeration.
In comparison with aquatic environments, there are only two reports of cultivation-independent observation or enumeration of indigenous viruses within soils. TEM has been used to show that there is an abundance of virus particles within British agricultural soils (1.5 × 107 g−1). Corresponding bacterial direct counts, estimated by EFM and acridine orange staining, are c. 200-fold higher. However, control experiments indicate a 40-fold loss of bacteriophage particles to binding with the soil matrix. Adjusting for extraction efficiency, actual viral abundance is estimated to be 10-fold higher, yielding a of 0.04. This low VBR estimate for soil samples is among the lowest reported by direct counting.
EFM using SYBR Gold staining has been a robust means of estimating VLP abundance in 0.2-μm filtered extracts of Delaware agricultural soils. In an investigation of suitable methods for extraction of autochthonous viruses from soils, a grand mean of 4.3 × 108 VLP g−1 dry soil has been observed using EFM. Brightly staining particles are almost entirely encapsulated double-stranded DNA (dsDNA) because treatment with heat followed by DNase digestion completely eliminates VLPs (Figure 1). Treatment with DNase alone removes a statistically insignificant number of particles. While the DNase experiment demonstrates that SYBR Gold-positive particles contain dsDNA, conclusive evidence that VLPs seen using EFM are actually viruses requires TEM examination of soil extracts. Initial attempts to examine directly 0.2-μm filtered soil extracts using TEM have failed owing to excessive interference from particulate matter. Subsequently, viral extracts of soil are purified by CsCl gradient centrifugation prior to TEM. With this combination of techniques (extraction, 0.2-μm filtration, and CsCl gradient centrifugation), a morphologically diverse range of virus particles have been detected in soil samples (Figure 2). TEM examinations of both Matapeake and Evesboro soil samples indicate a viral abundance of 1.5 × 108 g−1 dry soil (grand mean). Both EFM and TEM direct counts of VLPs in Delaware soil samples exceed those reported from British soils by 10 and 28 times; however, the significantly greater abundance of viruses in Delaware soils is probably due to methodological differences in extraction of virus particles
Figure 1. These potatoes have been infected by the potato spindle tuber viroid (PSTV), which is typically spread when infected knives are used to cut healthy potatoes, which are then planted. (credit: Pamela Roberts, University of Florida Institute of Food and Agricultural Sciences, USDA ARS)
In 1971, Theodor Diener, a pathologist working at the Agriculture Research Service, discovered an acellular particle that he named a viroid, meaning “virus-like.” Viroids consist only of a short strand of circular RNA capable of self-replication. The first viroid discovered was found to cause potato tuber spindle disease, which causes slower sprouting and various deformities in potato plants (see Figure 1). Like viruses, potato spindle tuber viroids (PSTVs) take control of the host machinery to replicate their RNA genome. Unlike viruses, viroids do not have a protein coat to protect their genetic information.
Viroids can result in devastating losses of commercially important agricultural food crops grown in fields and orchards. Since the discovery of PSTV, other viroids have been discovered that cause diseases in plants. Tomato planta macho viroid (TPMVd) infects tomato plants, which causes loss of chlorophyll, disfigured and brittle leaves, and very small tomatoes, resulting in loss of productivity in this field crop. Avocado sunblotch viroid (ASBVd) results in lower yields and poorer-quality fruit. ASBVd is the smallest viroid discovered thus far that infects plants. Peach latent mosaic viroid (PLMVd) can cause necrosis of flower buds and branches, and wounding of ripened fruit, which leads to fungal and bacterial growth in the fruit. PLMVd can also cause similar pathological changes in plums, nectarines, apricots, and cherries, resulting in decreased productivity in these orchards, as well. Viroids, in general, can be dispersed mechanically during crop maintenance or harvesting, vegetative reproduction, and possibly via seeds and insects, resulting in a severe drop in food availability and devastating economic consequences.
A second type of pathogenic RNA that can infect commercially important agricultural crops are the virusoids, which are subviral particles best described as non–self-replicating ssRNAs. RNA replication of virusoids is similar to that of viroids but, unlike viroids, virusoids require that the cell also be infected with a specific “helper” virus. There are currently only five described types of virusoids and their associated helper viruses. The helper viruses are all from the family of Sobemoviruses. An example of a helper virus is the subterranean clover mottle virus, which has an associated virusoid packaged inside the viral capsid. Once the helper virus enters the host cell, the virusoids are released and can be found free in plant cell cytoplasm, where they possess ribozyme activity. The helper virus undergoes typical viral replication independent of the activity of the virusoid. The virusoid genomes are small, only 220 to 388 nucleotides long. A virusoid genome does not code for any proteins, but instead serves only to replicate virusoid RNA.
Virusoids belong to a larger group of infectious agents called satellite RNAs, which are similar pathogenic RNAs found in animals. Unlike the plant virusoids, satellite RNAs may encode for proteins; however, like plant virusoids, satellite RNAs must coinfect with a helper virus to replicate. One satellite RNA that infects humans and that has been described by some scientists as a virusoid is the hepatitis delta virus (HDV), which, by some reports, is also called hepatitis delta virusoid. Much larger than a plant virusoid, HDV has a circular, ssRNA genome of 1,700 nucleotides and can direct the biosynthesis of HDV-associated proteins. The HDV helper virus is the hepatitis B virus (HBV). Coinfection with HBV and HDV results in more severe pathological changes in the liver during infection, which is how HDV was first discovered.
At one time, scientists believed that any infectious particle must contain DNA or RNA. Then, in 1982, Stanley Prusiner, a medical doctor studying scrapie (a fatal, degenerative disease in sheep) discovered that the disease was caused by proteinaceous infectious particles, or prions. Because proteins are acellular and do not contain DNA or RNA, Prusiner’s findings were originally met with resistance and skepticism; however, his research was eventually validated, and he received the Nobel Prize in Physiology or Medicine in 1997.
A prion is a misfolded rogue form of a normal protein (PrPc) found in the cell. This rogue prion protein (PrPsc), which may be caused by a genetic mutation or occur spontaneously, can be infectious, stimulating other endogenous normal proteins to become misfolded, forming plaques (see Figure 2). Today, prions are known to cause various forms of transmissible spongiform encephalopathy (TSE) in human and animals.
Figure 2. Endogenous normal prion protein (PrPc) is converted into the disease-causing form (PrPsc) when it encounters this variant form of the protein. PrPsc may arise spontaneously in brain tissue, especially if a mutant form of the protein is present, or it may originate from misfolded prions consumed in food that eventually find their way into brain tissue. (credit b: modification of work by USDA)
TSE is a rare degenerative disorder that affects the brain and nervous system. The accumulation of rogue proteins causes the brain tissue to become sponge-like, killing brain cells and forming holes in the tissue, leading to brain damage, loss of motor coordination, and dementia Infected individuals are mentally impaired and become unable to move or speak. There is no cure, and the disease progresses rapidly, eventually leading to death within a few months or years.
5)SATELLITES: Satellites are subviral agents which lack genes that could encode functions needed for replication. Thus for their multiplication they depend on the co-infection of a host cell with a helper virus. Satellite genomes have a substantial portion or all of their nucleotide sequences distinct from those of the genomes of their helper virus.
According to this definition, two major classes of satellites may be distinguished. Satellite viruses encode a structural protein that encapsidates their genome and so have nucleoprotein components distinct from those of their helper viruses. Satellite nucleic acids encode either non-structural proteins, or no proteins at all, and are encapsidated by the CP of helper viruses.
In addition to the true satellites, this chapter also describes subviral agents (nucleic acids) that depend upon viruses in a variety of ways. Satellite-like nucleic acids resemble satellites because they do not encode a replicase but differ because they encode a function necessary for the biological success of the associated virus. They can therefore be considered as components that remedy a deficiency in a defective virus. They have sometimes been classified as part of the genome of the virus they assist but they can also be dispensable because they are in not always found in association with their helper virus. Examples include RNAs associated with groundnut rosette virus (genus Umbravirus) or with beet necrotic yellow vein virus (genus Benyvirus), that contribute to vector transmissibility and DNAs associated with begomoviruses (betasatellites) that encode a pathogenicity determinant.
A final group of agents described are nucleic acids capable of autonomous replication and which therefore are not strictly satellites although the term has sometimes been loosely applied to them. These agents are dependent on their helper viruses for various functions such as encapsidation, cell-to-cell and long-distance movement and vector transmission. Examples are the alphasatellites (DNAs that encode a replication initiator protein) or the RNAs associated with some poleroviruses that appear to encode a carmovirus-like RdRp.
The distinction between satellite nucleic acids, satellite-like nucleic acids and virus genomic components can be subtle and these agents are not always easy to categorize.
Satellites are genetically distinct from their helper virus with a nucleotide sequence that is substantially different from that of their helper virus. However, the genomes of most satellites have short sequences, often at the termini, that are identical to the genome of the helper virus. This is presumably linked to the dependence of nucleic acids of both satellite and helper virus on the same viral polymerase and host-encoded proteins for replication. Satellites are distinct from defective interfering (DI) RNAs or defective RNAs because such RNAs are derived from their “helper” virus genomes. Nevertheless, satellite viruses may form their own DI RNAs that specifically interfere with the satellite virus genomic RNA, as has been shown for satellite panicum mosaic virus. Recombination can occur between satellites and their helper viruses. For example, chimeric molecules can be formed from a satellite RNA associated with turnip crinkle virus (genus Carmovirus) and parts of the helper virus genome.
Satellites do not constitute a homogeneous taxonomic group and are not formally classified into species and higher taxa by ICTV. The descriptions in this section are meant only to provide a classification framework and a nomenclature to assist in the description and identification of satellites and other virus-dependent nucleic acids. The arrangement adopted is based largely on features of the genetic material of the satellites. The physicochemical and biological features of the helper virus and of the helper virus host are important secondary characters.
There appears to be no taxonomic correlation between the viruses that are associated with satellites. Satellites would appear to have arisen independently a number of times during virus evolution. A further complication is that some viruses are associated with more than one satellite and some satellites are supported by more than one species of helper virus. Satellites can even depend on both a second satellite and a helper virus for multiplication.
The first satellites characterized were mostly ssRNA satellites that use ssRNA plant viruses as helpers. It can be very difficult to distinguish between satellite RNA and viral genomic RNA (e. g., dsRNA satellites of fungus viruses) and it is very likely that other satellites, some with novel combinations of characters, remain to be discovered.
Defective interfering viruses are a special class of defective viruses that arise by recombination and rearrangement of viral genomes during replication. DIs are defective because they have lost essential functions required for replication. Thus, they require the simultaneous infection of a cell by a helper virus, which is normally the parental wild-type virus from which the DI arose. They interfere with the replication of the parental virus by competition for resources within the cell. These resources include the machinery that replicates the viral nucleic acid, which is in part encoded by the helper virus, and the proteins that encapsidate the viral genome to form virions.
DIs of many RNA viruses have been the best studied. Because DI RNAs must retain all cis-acting sequences required for the replication of the RNA and its encapsidation into progeny particles, sequencing of such DI RNAs can provide clues as to the identity of these sequences. Identification of cis-acting sequences is important for the construction of virus vectors used to express a particular gene of interest, whether in a laboratory experiment or for gene therapy.
The most highly evolved DI RNAs are often not translated and consist of deleted and rearranged versions of the parental genome. In the case of alphaviruses, whose genome is about 12kb (Chapter 3), DI RNAs have been described that are about 2kb in length. However, they have a sequence complexity of only 600 nucleotides, because sequences are repeated one or more times. The sequences of two such DI RNAs of Semliki Forest virus (SFV) are illustrated schematically in Fig. 9.1. From the sequences of these DIs as well as DIs of other alphaviruses, specific functions for the elements found in these DIs have been proposed. Other approaches have then been used to confirm the hypotheses derived from such sequence studies. Thus, the 3′ end of the parental RNA, which is retained in all alphavirus DI RNAs, forms a promoter for the initiation of minus-strand RNA synthesis from the plus-strand genome. The 5′ end of the RNA is also preserved in many DI RNAs, such as those illustrated in Fig. 9.1. Surprisingly, however, it has been replaced by a cellular tRNA in some DI RNAs. The complement of this sequence is present at the 3′ end of the minus strand, where it forms a promoter for initiation of genomic RNA synthesis. The finding that the DI RNAs with the tRNA as the 5′ terminus have a selective advantage over the parental genome during RNA replication suggests that this promoter is a structural element recognized by the viral replicase. It also suggests that the element present in the genomic RNA is suboptimal, perhaps because the genomic RNA must be translated as well as replicated. Finally, repeated sequences from two regions of the genome are present in all alphavirus DI RNAs. It is thought that one sequence (shown as red patterned blocks in is an enhancer element for RNA replication and the second (shown a UVs yellow and green patterned blocks) is a packaging signal. Repetition of these elements may increase the efficiency of replication and packaging of the DI RNA.