Viral Genetics - Mutations and Recombination - PDF

**INTRODUCTION** Viral genetics fall into two general areas, which are; 1. Mutations and their effect on replication and pathogenesis. 2. The interaction of two genetically distinct viruses that infect the same cell. Viruses are simple entities, lacking an energy-generating system and...

**INTRODUCTION** Viral genetics fall into two general areas, which are; 1. Mutations and their effect on replication and pathogenesis. 2. The interaction of two genetically distinct viruses that infect the same cell. Viruses are simple entities, lacking an energy-generating system and having very limited biosynthetic capabilities. The smallest viruses have only a few genes; the largest viruses have as many as 200. Genetically, however, viruses have many features in common with cells. Viruses are subject to mutations, the genomes of different viruses can recombine to form novel progeny, the expression of the viral genome can be regulated, and viral gene products can interact. Viruses serve as vectors in gene therapy and in recombinant vaccines. By studying viruses, we can learn more about the mechanisms by which viruses and their host cells function. **Genetic Change in Viruses** The two principal mechanisms by which genetic changes occur in viruses are: mutation and recombination. Alterations in the genetic material of a virus may lead to changes in the function of viral proteins. Such changes may result in the creation of new viral serotypes or viruses of altered virulence. **MUTATIONS** Mutations in viral DNA and RNA occur by the same processes of base substitution, deletion, and frameshift probably the most important practical use of mutations is in the production of vaccines containing live, attenuated virus. These attenuated mutants have lost their pathogenicity but have retained their antigenicity---they therefore induce immunity without causing disease. There are two other kinds of mutants of interest. The first are antigenic variants such as those that occur frequently with influenza viruses, which have an altered surface protein and are therefore no longer inhibited by a person\'s preexisting antibody. The variant can thus cause disease, whereas the original strain cannot. The second are drug-resistant mutants, which are insensitive to an antiviral drug because the target of the drug, usually a viral enzyme, has been modified. Mutations arise by one of three mechanisms: 1. by the effects of physical mutagens (UV light, x-rays) on nucleic acids. 2. by the natural behavior of the bases that make up nucleic acids (resonance from keto to enol and from amino to imino forms). 3. through the fallibility of the enzymes that replicate the nucleic acids. The first two mechanisms act similarly in all viruses; hence, the effects of physical mutagens and the natural behavior of nucleotides are relatively constant. However, viruses differ markedly in their mutation rates, which is due primarily to differences in the fidelity with which their enzymes replicate their nucleic acids. Viruses with high-fidelity transcriptases have relatively low mutation rates and vice versa. **Mutation Rates and Outcomes** DNA viruses have mutation rates similar to those of eukaryotic cells because, like eukaryotic DNA polymerases, their replicatory enzymes have proof-reading functions. The error rate for DNA viruses has been calculated to be 10^-8^ to 10^-11^ errors per incorporated nucleotide. With this low mutation rate, replication of even the most complex DNA viruses, which have 2 × 10^-5^ to 3 × 10^-5^ nucleotide pairs per genome, will generate mutants rather rarely, perhaps once in several hundred to many thousand genome copies. The RNA viruses, however, lack a proofreading function in their replicatory enzymes, and some have mutation rates that are many orders of magnitude higher errors per incorporated nucleotide. Even the simplest RNA viruses, which have about 7,400 nucleotides per genome, will generate mutants frequently, perhaps as often as once per genome copy. Not all mutations that occur persist in the virus population. Mutations that interfere with the essential functions of attachment, penetration, uncoating, replication, assembly, and release do not permit misreplication and are rapidly lost from the population. However, because of the redundancy of the genetic code, many mutations are neutral, resulting either in no change in the viral protein or in replacement of an amino acid by a functionally similar amino acid. Only mutations that do not cripple essential viral functions can persist or become fixed in a virus population. **Phenotypic Variation by Mutations** Mutations that alter the viral phenotype but are not deleterious may be important. For example, mutation can create novel antigenic determinants. A mutation in the hemagglutinin gene of influenza A virus can give rise to a hemagglutinin molecule with an altered antigenic site (epitope) Provided the attachment function of the new hemagglutinin is intact, the mutant virus may be able to initiate an infection in an individual immune to viruses expressing the previous hemagglutinin. For example, from 1968 to 1979, mutations altered 10 percent of the amino acids in the influenza virus hemagglutinin serotype H3 molecule. This relatively modest mechanism of antigenic change through mutation, called antigenic drift, may allow a virus to outflank host defenses and cause disease in previously immune individuals. **Vaccine Strains from Mutations** Mutation has been a principal tool of virologists in developing attenuated live virus vaccines. For example, the Sabin vaccine strains of poliovirus were developed by growing polioviruses in monkey kidney cells. Mutation and selection produced variant polioviruses that were adapted for efficient replication in these cells. Some of the mutations in these variants affected the genes coding for the poliovirus coat proteins in such a way as to produce mutants unable to attach to human neural cells but still able to infect human intestinal cells. Infection of human intestinal cells does not produce paralytic disease but does induce immunity. Poliovirus vaccine strains 1 and 2 have multiple mutations in the coat proteins and are very stable. The type 3 vaccine strain is less stable and is subject to back-mutations (reversions) that restore neural virulence. This vaccine strain therefore causes paralytic disease in one out of every several million vaccinated individuals. Despite the possibility of back-mutations, the generation and selection of attenuated viral mutants remains an important mechanism for producing viral vaccines. **Recombination** Recombination is the exchange of genes between two chromosomes that is based on crossing over within regions of significant base sequence homology. Recombination can be readily demonstrated for viruses with double-stranded DNA as the genetic material and has been used to determine their genetic map. However, recombination by RNA viruses occurs at a very low frequency, if at all.Viral recombination occurs when viruses of two different parent strains co-infect the same host cell and interact during replication to generate virus progeny that have some genes from both parents. Recombination generally occurs between members of the same virus type (e.g., between two influenza viruses or between two herpes simplex viruses). Two mechanisms of recombination have been observed for viruses: independent assortment and incomplete linkage. Either mechanism can produce new viral serotypes or viruses with altered virulence. **Recombination by Independent Assortment** Independent assortment occurs when viruses that have multipartite (segmented) genomes trade segments during replication. These genes are unlinked and assort at random. Recombination by independent assortment has been reported, for example, for the influenza viruses and other orthomyxoviruses (8 segments of single-stranded RNA) and for the reoviruses (10 segments of double-stranded RNA). The frequency of recombination by independent assortment is 6 to 20 percent for orthomyxoviruses. Independent assortment between an animal and a human strain of influenza virus during a mixed infection can yield an antigenically novel influenza virus strain capable of infecting humans but carrying animal-strain hemagglutinin and/or neuraminidase surface molecules. This recombinant can infect individuals that are immune to the parent human virus. This mechanism results in an immediate, major antigenic change and is called antigenic shift. Antigenic shifts in influenza virus antigens can give rise to pandemics (worldwide epidemics) of influenza. Such antigenic shifts have occurred relatively frequently during recent history. Because the number of different serotypes of hemagglutinin and neuraminidase are limited, a given strain reappears from time to time. For example, the H1N1 influenza virus strain was responsible for the 1918 to 1919 influenza pandemic that caused 20 million deaths. The same virus also caused pandemics in 1934 and in 1947, then disappeared after 1958 and reappeared in 1977. The reappearance of virus strains after an absence is believed to be the result of recombinational events involving the independent assortment of genes from two variant viruses. **Recombination of Incompletely Linked Genes** Recombination also occurs between genes residing on the same piece of nucleic acid (Fig. 43-3). Genes that generally segregate together are called linked genes. If recombination occurs between them, the linkage is said to be incomplete. Recombination of incompletely linked genes occurs in all DNA viruses that have been studied and in several RNA viruses. Recombination by break-rejoin of incompletely linked genes.. The genetic interaction of DNA viruses can result in break-rejoin recombination, in which the two DNA molecules of different viruses break and then cross over. In DNA viruses, as in prokaryotic and eukaryotic cells, recombination between incompletely linked genes occurs by means of a break-rejoin mechanism. This mechanism involves the actual severing of the covalent bonds linking the bases of each of the two DNA strands in a DNA molecule. The severed DNA strands are then rejoined to the DNA strands of a different DNA molecule that has been broken in a similar site. Recombination rates for herpesviruses, which are DNA viruses that replicate in the nucleus of infected cells, approximate those expected for a eukaryotic genome of the size of the herpesvirus genome. Herpesviruses have an average recombination frequency of 10 to 20 percent for any two loci. However, the rate of recombination between a specific pair of genetic loci depends on the distance between them and varies from less than 1 percent to approximately 50 percent. Measurement of the recombination frequencies for different loci can be used to map the virus genome. In this type of genetic map, loci with high recombination frequencies are far apart and loci with low recombination frequencies are close together. Recombination has been shown to occur in several positive-sense single-stranded RNA virus groups: retroviruses, picornaviruses, and coronaviruses. That is initially surprising, as recombination between RNA molecules has not been observed in prokaryotic or eukaryotic cells. In retroviruses, recombination actually occurs at the point in replication when the retrovirus genome is in a DNA form and takes place by the same break-rejoin mechanism as in cells and DNA viruses. Recombination can occur both between two related retroviruses and between the retrovirus DNA and the host cell DNA. Recombination between two retroviruses gives rise to novel viral progeny with reassorted genes. Recombination between retroviruses and the host cell can give rise to novel viral progeny that carry nonviral genes. If these host genes code for growth factors, growth factor receptors, or a number of other specific cellular proteins, the recombinant retroviruses may be oncogenic. In picornaviruses and coronaviruses, recombination takes place at the level of the interaction of the viral RNA genomes and is not believed to occur by a break-rejoin mechanism. The mechanism is currently believed to be a copy-choice mechanism. Copy-choice may occur in these RNA viruses because the viral RNA polymerase binds to only a few bases of the template RNA at any one time. Such a weak interaction of the polymerase with the template RNA would permit the polymerase, carrying its RNA strand, to disassociate from the original template nucleic acid strand and then associate with a new template RNA strand. Recombination frequencies in the range of 0.2 to 0.4 percent have been reported. Therefore, the efficiency of this mechanism of recombination is low. **Phenotypic Variation from Recombination** As mentioned above, viral recombination is important because it can generate novel progeny viruses that express new antigenic and/or virulence characteristics. For example, the novel progeny viruses may have new surface proteins that permit them to infect previously resistant individuals; they may have altered virulence characteristics; they may have novel combinations of proteins that make them infective to new cells in the original host or to new hosts; or they may carry material of cellular origin that gives them oncogenic potential. **Vaccines and Gene Therapy through Recombination** Recombination is being used experimentally by virologists to create new vaccines. Vaccinia virus, a DNA virus of the poxvirus group, was used as a live vaccine in the eradication of smallpox. Recombinant vaccinia viruses are being developed that carry vaccinia virus DNA recombined with DNA from other sources (exogenous DNA). For example, vaccinia virus strains carrying DNA coding for bacterial and viral antigens have been produced. It is expected that after vaccination with the recombinant vaccinia virus, the bacterial or viral antigen (immunogen) will be produced. The presence of this immunogen will then stimulate specific antibody production by the host, resulting in protection of the host from the immunogen. Studies with these live, recombinant vaccinia viruses are currently under way to determine whether inoculation of the skin with the recombinant virus can induce a protective host antibody response to the bacterial or viral antigens. Other studies are investigating the use of live, recombinant adenoviruses containing bacterial or viral genes to infect the gastrointestinal tract and induce both mucosal and systemic immunity. Development of recombinant vaccinia virus for immunization against cholera toxin. Vaccinia virus genomic DNA is cut with an endonuclease. In a similar manner, recombinant viruses are also being developed that carry normal human genes. It is envisioned that such recombinant viruses could be useful for gene therapy. Target diseases for gene therapy span a wide range, including diabetes, cystic fibrosis, severe combined immunodeficiency syndrome, etc. Indeed, treatment of cystic fibrosis patients with replication deficient, recombinant adenoviruses bearing a normal copy of the cystic fibrosis transmembrane regulator gene has already been approved. If these studies give positive results, such directed generation of recombinant viruses may become an important tool in the development of vaccines and for gene therapy. **FUNGAL GENETICS** Fungi are eukaryotic achlorophyllous organisms with absorptive mode of nutrition. They are spore formers and are capable of both sexual and asexual modes of reproductions. They possess filament/ branched somatic structures and are surrounded by cell wall containing chitin as well as many other complex carbohydrates. Hence, Fungi genetics is the study of the mechanisms of Heritability information in fungi. Yeasts and filamentous fungi are extensively used as model organisms for eukaryotic genetic research, including cell cycle regulation, chromatin structure, genetic recombination and gene regulation. **Significance of Fungal Genetics**  Fungal cells are useful in the study of rare events (such as mutations and Recombination).  The concepts and techniques of fungal asexual and parasexual genetics have been applied to the genetic manipulation of cultured cells of higher eukaryotes such as humans and green plants. However, the techniques remain much easier to perform with fungi.  The fact that each enzyme is coded by its own specific gene was first recognized in fungi and was of paramount importance because it showed how the many Chemical reactions that take place in a living cell could be controlled by the genetic Apparatus. **Fungal Cell Contents** **1. The Cell Wall:** Except slime molds (Myxomycetes), the fungal cell consists of a rigid cell wall and cell organelles. Chemical analysis of cell wall reveals that it contains 80- 90% polysaccharides, and remaining proteins and lipids. Chitin (a polymer of N-acetyl glucosamine), cellulose (a polymer of Dglucose) or other glucans are present in cell walls in the form of fibrils forming layers. **2. Plasma Membrane (Cell Membrane):** In fungi too the cell wall is followed by plasma membrane that encloses the cytoplasm. It is semipermeable and, in structure and function, it is similar to that of prokaryotes. **3. Cytoplasm:** Cytoplasm is colorless in which sap-filled vacuoles are found. Except chloroplasts many of the familiar organelles and inclusions, characteristic of eukaryotes, are found in fungal cytoplasm. The cell organelles are endoplasmic reticulum, mitochondria, ribosomes, golgi bodies and vacuoles, Lomasomes are also present between plasma membrane and cell wall. **Fungal cell division** **Cell division** is an important characteristic of living cells is their ability to divide, so cell division is happened in all living organism. There is two kinds of division: mitosis and meiosis.  **Mitosis** is the process of one cell dividing into two daughters, such that each inherits a single and complete copy of the genome of their mother. This is achieved through the equal segregation of the sister chromatids between the daughter cells. In multicellular fungi, mitosis permits growth and repair of tissues while in unicellular fungi mitosis is a form of as asexual reproduction.  **Meiosis** produces specific types of reproductive cells. In animals, meiosis occurs in the gonads (ovaries/testes) and produces eggs or sperm. In plants and fungi, meiosis produces other reproductive cells called spores. Spores do not fuse; a spore is a single reproductive cell that simply begins to divide and grow on its own to become an offspring. **Reproduction** Fungi reproduce sexually and/or asexually. Perfect fungi reproduce both sexually and asexually, while imperfect fungi reproduce only asexually (by mitosis). In both sexual and asexual reproduction, fungi produce spores that disperse from the parent organism by either floating on the wind or hitching a ride on an animal. **Asexual reproduction in fungi:** **1. Fission:** In binary fission a mature cell elongates and its nucleus divides into two daughter nuclei. The daughter nuclei separate, cleaves cytoplasm centripetally in the middle till it divides parent protoplasm into two daughter protoplasm. A double cross wall is deposited in the middle to form two daughter cell. Ultimately the middle layer of double cross wall degenerates and daughter cells are separated. Examples: *Saccharomyces pombe*, *Psygosaccharomyces* 2. **Budding:** The cell wall bulges out and softens in the area probably by certain enzymes brought by vesicles. The protoplasm also bulges out in this region as small protuberance. The parent nucleus also divides into two, one of the daughter nucleus migrates into bud, the cytoplasm of bud and mother remain continuous for some time, As the bud enlarges, a septum is laid down at the joining of bud with mother cell. Then bud separates and leads independent life. **3. Fragmentation:** In some fungi, fragmentation or disjoining of hyphae occurs and each hyphae become a new organism. **4. formation spore of fungi:** Spore formation is the characteristic feature of fungi. Different fungi form different types of spore including;athrospores, chlamydospores, conidiospores, blastospores and sporangiospores. **Sexual reproduction in fungi:** Sexual reproduction is carried out by diffusion of compatible nuclei from two parent at a definite state in the life cycle of fungi. The process of sexual reproduction involves three phases:  Plasmogamy: fusion of protoplasm  Karyogamy: fusion of nucleus  Meiosis: reductional nuclear division Various methods by which compatible nuclei are brought together in plasmogamy. Some are: 1\. **Gametic copulation**: Fusion of two naked gametes, one or both of them are motile. 2\. **Gamete-gametangial copulation:** Male and female gametangia comes into contact but do not fuse. A fertilization tube formed from where male gametangium enters the female gametangium and male gamate passes through this tube. 3\. **Gametangial copulation**: Two gametangia or their protoplast fuse and give rise to zygospore 4\. **Somatic copulation:** Also known as somatogamy. In this process fusion of somatic cell occurs This sexual fusion of undifferentiated vegetative cells (monokaryons) results in dikaryotic hyphae, so the process is also called dikarotization. **5. Spermatization:** It is an union of special male structure called spermatia with a female receptive structure. Spermatium empties its content into receptive hyphae during plasmogamy **Parasexual Cycle** The parasexual cycle (or parasexuality) is a process peculiar to fungi and single-celled organisms. It is a nonsexual mechanism for transferring genetic material without meiosis or the development of sexual structures. The parasexual cycle resembles sexual reproduction. In Both cases, dissimilar hyphae (or modifications thereof) may fuse (plasmogamy) and their nuclei will occupy the same cell, the dissimilar nuclei then fuse (karyogamy) to form a diploid (zygote) nucleus. In contrast to the sexual cycle, in the parasexual cycle Recombination takes place during mitosis followed by Haploidization (but without meiosis), a type of recombination found in certain heterokaryotic Fungi, genetically distinct haploid nuclei fuse in the heterokaryon. The resulting diploid nuclei multiply by mitotic division, with some crossing over, and a diploid homokaryon develops from a diploid conidium.  Homokaryotic refers to multinucleate cells (multiple Nuclei share one common cytoplasm as it is found in Hyphal cells or mycelium of filamentous fungi) where All nuclei are genetically identical.  Heterokaryotic refers to cells where two or more Genetically different nuclei share one common cytoplasm. It is neither 1n or 2n. **Fungal Genome Structure** A genome is all the genetic information possessed by an organism. It includes; Chromosomal genes, Mitochondrial genes, Plasmids/mobile genetic elements and Virus genes. Each contributes to the overall phenotype of the fungus.  **Chromosomal genes:** Chromosomes are present in fungi cells. Most are haploid**,** Difficult to quantify **and** Very condensed**.** Number of chromosomes vary between 3 and 40 with most fungi having between 6 and 16**.** Genome size is small compared to other Eukaryotes, 3-8 times larger than E. coli, but 5-30 smaller than fruit flies or humans  **Mitochondrial genes:** Fungal mitochondrial genomes are small (19-121 kb in size) compared to plants (1 Mb), but larger than humans (6.6 kb). Both nuclear and mitochondrial genes are essential for function of the mitochondrial  **Plasmid genes:** Plasmids and transposable elements, they are self-replicating, Usually circular. Most common types include; "2 micron" plasmid found in the nucleus of *Saccharomyces cerevisiae* and many other plasmids found within mitochondria. There is no known function of any.  **Viruses' genes**: First discovered as a pathogen of mushrooms**.** Electron microscopy revealed isometric virus-like particles (VLPs) which are now found in numerous fungal species with few exceptions. VLPs are resident genetic elements, i.e., without a natural means of spreading, they don't cross species barriers**.** They can Can affect virulence of certain species **Genetic Transformation** This is a process that involves the insertion and expression of foreign genes in a genome of host organism, so the DNA becomes a permanent addition to the genome, so that it is inherited in subsequent generations. Transformation systems have been developed for only a handful of fungi that are pathogenic to humans, including several species of filamentous fungi, and for a lot of filamentous fungi pathogenic to plants. **Fungi Genetic Transformation Methods** The establishment of genetic transformation systems has enabled scientists to transform foreign DNA into filamentous fungi and thus obtained the desired strains for industrial purposes. We now can take full advantage of the superior secretary power of fungi and their excellent efficiency in manufacturing valuable metabolites. Metabolite: is an intermediate or end product of metabolism. Four techniques suitable for the genetic transformation of filamentous fungi: 1\. Protoplast-mediated transformation (PMT). 2\. *Agrobacterium* -mediated transformation (AMT). 3\. Electroporation. 4\. Biolistic methods. **Protoplast**-**mediated transformation (PMT)** PMT is the most commonly used fungal transformation method, which relies on a large number of competent fungal protoplasts. The principle is to use some commercially available enzymes to remove fungal complex cell wall components for generating protoplasts. Subsequently, some chemical reagents (such as Polyethylene glycol PEG) are used to promote the fusion of exogenous nucleic acids and protoplasts. **BIOINFORMATICS** Bioinformatics is an interdisciplinary field that combines biology, computer science, and information technology to analyze and interpret biological data. It involves the development and application of computational methods, algorithms, and software tools for understanding biological processes, organizing large datasets, and solving complex biological problems. Bioinformatics has become an indispensable tool in modern biological research, enabling scientists to extract meaningful insights from vast amounts of biological data. As technology continues to advance, bioinformatics will play an increasingly pivotal role in understanding the complexity of living systems, contributing to breakthroughs in medicine, agriculture, and environmental science. Embracing the interdisciplinary nature of bioinformatics is essential for researchers to harness its full potential and drive innovations in the life sciences. Bioinformatics implies an exclusive subject intended to comprehend the informatic framework of biosystems, and relevant pedagogy applies to learning synergistic details of biology, bioengineering, biotechnology, as well as the allied fields of medical science and health issues in the perspectives of information theoretics. **Importance/Objectives of Bioinformatics** Common uses of bioinformatics include the identification of candidate genes and single nucleotide polymorphisms (SNPs). Often, such identification is made with the aim of better understanding the genetic basis of disease, unique adaptations, desirable properties (especially in agricultural species), or differences between populations. Drug designing for newly occurring diseases. Phylogenetic analysis to understand the evolutionary pattern and development of particular protein/proteins, gene/genes or organism. Bioinformatics also tries to understand the organizational principles within nucleic acid and protein sequences. **History and Evolution of Bioinformatics** 1. **Historical Perspective:** Origins in the 1960s with the advent of computational biology and the need to manage and analyze the growing volume of biological data. 2. **Technological Advances:** Rapid progress in DNA sequencing, genomics, and other high-throughput technologies has fueled the expansion of bioinformatics. **Major Components of Bioinformatics** A. **Biological Databases** Biological databases are libraries of life sciences information, collected from scientific experiments, published literature, high-throughput experiment technology, and computational analysis. They contain information from research areas including genomics, proteomics, metabolomics, microarray gene expression, and phylogenetics. Information contained in biological databases includes gene function, structure, localization (both cellular and chromosomal), clinical effects of mutations as well as similarities of biological sequences and structures. Biological databases can be broadly classified into sequence, structure and functional databases. 1. **Sequence Databases:** Stores DNA, RNA, and protein sequences. 2. **Structural Databases:** Houses 3D structures of biological macromolecules. 3. **Functional Databases:** Annotates the function of genes and proteins. B. 1. **Sequence Alignment:** Identifying similarities and differences between biological sequences. 2. **Phylogenetic Analysis:** Inferring evolutionary relationships among species. 3. **Structural Bioinformatics:** Predicting and analyzing the three-dimensional structures of biomolecules. 4. **Functional Annotation:** Assigning biological functions to genes and proteins. C. 1. **Identifying Genes:** Locating and predicting the location of genes within a genome. 2. **Functional Annotation:** Associating biological functions with genes and their products. D. 1. **Network Analysis:** Studying the interactions between genes, proteins, and other molecules within biological systems. 2. **Pathway Analysis:** Understanding the interconnected pathways that regulate cellular processes. **Applications of Bioinformatics** 1. Determining the entire DNA sequence of an organism. 2. Studying similarities and differences between genomes to understand evolutionary processes. 3. Inferring the three-dimensional structure of proteins. 4. Identifying and characterizing interactions between proteins. **Challenges of Bioinformatics** 1. **Data Integration:** Managing and integrating diverse datasets from various biological sources. 2. **Data Privacy and Security:** Addressing ethical concerns related to the use and sharing of biological data. **OMICS** Informally refers to a field of study in biology ending in -omics. Purpose of omics aims at the collective characterization and quantification of pools of biological molecules that translate into the structure, function, and dynamics of an organism(s).