jueves, 8 de noviembre de 2012


Viral Replication and Genetics D.J. Wise1 and G.R. Carter2 1Department of Biology, Concord University, Athens, West Virginia, USA.2Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, USA. Table of Contents Viral Replication Replication of DNA Viruses Replication of RNA Viruses Viral Genetics Mutation Antigenic Shift and Drift Interactions Between Two Viruses Complementation Recombination Genetic Reactivation Phenotypic Mixing Virus Applications: Gene Therapy and Recombinant Vaccines Gene Therapy Recombinant Vaccines Glossary Viral Replication Viral replication is a very complex and varied process. The mechanics of replication depends largely upon the type of nucleic acid and genomic organization of each particular virus. Despite the variation in replication strategies, there is commonality in several steps of viral replication. All viral replication schemes contain the following basic steps: attachment, penetration, uncoating (if necessary), protein synthesis (or gene expression), genome replication, assembly (morphogenesis), and release. •Attachment depends on the physical interaction between the virion and the surface of the host/target cell. Typically, the interaction is a receptor-ligand interaction. As a result, species specificity or specific cell type specificity is determined. Without attachment, viral infection cannot occur. On the other hand, not all attachments result in productive infection. In other words, attachment is required yet does not assure that replication will follow. •Penetration refers to the introduction of viral nucleic acid into the cell, the internalization of the nucleocapsid via receptor-mediated endocytosis or the fusion of the viral envelope with the plasma membrane. As an immediate result, the nucleic acid is either located in the cytosol or within an endocytic vesicle. •Uncoating of the nucleic acid from the nucleocapsid may require the participation of host proteins or other factors. Uncoating is a prerequisite for the genome to be expressed. Following uncoating, the viral nucleic acid either continues in the replication cycle or a copy of it becomes integrated within the host genome and is quiescent until triggered to become active (retroviruses). •Protein synthesis (gene expression)- mRNA is produced and translated into viral proteins. Regardless of whether the virus has a DNA or RNA genome, ss, ds, segmented or monopartite, it has to produce mRNAs that are recognized and translated by the host cell machinery. •As will be described for each of group of viruses, there is a unique mechanism whereby the host cell machinery becomes largely dedicated to synthesis of viral products and away from the synthesis of host cell products. •Genome replication: The mechanism of genome replication varies with the type of nucleic acid, its structure and topology. For the simplest viruses, it is a task of host cell enzymes; most viruses encode their own replication enzymes. •Maturation is the assembly of complete virus particles. Maturation of nonenveloped viruses is primarily the assembly of genome + capsid proteins forming the nucleocapsid. This occurs spontaneously through protein-protein and protein-nucleic acid interactions. In the maturation of enveloped virions, the nucleocapsid acquires an external envelope consisting of the host cell membranes (nuclear, Golgi, endoplasmic reticulum, or plasma membranes), which contains a lipid bilayer of cellular origin and viral encoded proteins. The envelope is acquired by a process known as budding. •Release of virions. For nonenveloped viruses, thousands of progeny virions are released by host cell death and lysis. For enveloped viruses, the progeny virions are released by budding out from the cell. Budding does not necessarily result in cell death, yet some enveloped viruses may be also released by cell lysis. Replication of DNA Viruses •In general, DNA viruses replicate within the nucleus. Exceptions are poxviruses and iridoviruses (viruses of insects and fish), which use cytoplasmic "factories". •Those DNA viruses that multiply within the nucleus use host DNA-dependent RNA polymerase for transcription. The majority of poxviruses and iridoviruses have virion-encoded transcriptases that allow them to replicate in the cytoplasm. •Replication of viral DNA is semiconservative and symmetrical with both strands being replicated. In dsDNA viruses, such as adenoviruses, the replication of both daughter strands does not necessarily follow the same mechanism. •Host DNA polymerases may be involved in replicating small- to moderate-sized genome viruses (papillomaviruses, polyomaviruses), whereas larger sized genome viruses usually code for their own polymerases (adenoviruses, herpesviruses, poxviruses). •Maturation of DNA viruses, with the exception of poxviruses and iridoviruses, occurs in the nucleus. •Structural proteins are transported from the cytoplasm to the nucleus, where they interact with each other and with the genome and are assembled into capsids that surround the nucleic acid. •Enveloped viruses complete maturation by budding through the nuclear membrane (iridoviruses) or the plasma membrane. Double-Stranded DNA Virus Replication These include the following animal-associated virus families: Asfarviridae, Poxviridae, Iridoviridae, Herpesviridae, Polyomaviridae, Papillomaviridae, and the Adenoviridae (Figure 3.1). •The genomes range in size from 5 - 8 kb (Polyomaviridae) to over 300 kb ( Poxviridae, and Iridoviridae). •In general, replication takes place in the nucleus by host enzymes (for small viruses such as polyoma and papillomaviruses) or by virus-encoded replicases (adenovirus, herpesvirus). Replication of poxviruses and some iridoviruses takes place in the cytoplasm resulting in the formation of inclusion bodies, which contain necessary enzymes of viral origin associated with replication, such as viral DNA-dependent DNA polymerases. •The dsDNA may be in the form of circular, linear, circularly permuted, or linear with covalently closed ends. •The small circular genomes replicate bidirectionally in a manner similar to plasmids. The replication of polyomavirus DNA (closed, circular and double-stranded) is postulated to be mediated by a "swivel mechanism" consisting of endonuclease and ligase. The endonuclease "nicks" one strand, allowing a short region to be replicated. The nick is then repaired by the ligase. Figure 3-1. General replication scheme of dsDNA viruses. To view click on figure . Single-Stranded DNA Virus Replication Includes the following animal-associated virus families: Circoviridae and Parvoviridae. •The genomes range in size from 3 kb to 6 kb. •The single-stranded circular DNA of circoviruses is thought to be replicated by a rolling circle mechanism. •Replication occurs in the nucleus and involves the generation of a - sense DNA strand to serve as a template for the + sense DNA genome for the progeny virions. This involves the production of a dsDNA intermediate, known as the replicative form. •Entry of the viral ssDNA into the nucleus stimulates its "repair" by host enzymes into the replicative form. In the case of the circular forms, the replicative form is associated with host histones and other nuclear proteins and thus "treated" as a host chromosome. Linear forms have derived mechanisms that allow the genome to be replicated without a loss of DNA following each replication. •The ssDNA may be in the forms of linear single-component (parvoviridae), circular single-component (circoviruses). Figure 3-2. General replication scheme of ssDNA viruses. To view click on figure . Double-Stranded DNA Viruses with Reverse Transcriptase •Includes the Hepadnaviridae. •Genome arrangement is a partially double-stranded non-covalently closed circular DNA, 3.2 kb in size. •Following attachment, penetration, and partial uncoating of the virion, the partially dsDNA enters the nucleus and is completed by viral polymerase and/or cellular enzymes. Once completed, the backbone is sealed by the action of host ligase. •In the nucleus, the viral DNA acts like a "mini-chromosome" following its association with host histones, etc. However, host DNA polymerase cannot replicate it. •In the replication cycle a large mRNA called the pg-RNA (pre-genomic RNA), which is longer than the DNA template from which it was transcribed due to the addition of a poly A tail is produced. It is this RNA intermediate that serves as the template for the virion DNA. Smaller mRNAs are also produced, leave the nucleus, and serve as template for translation, giving origin for viral polymerase and capsid protein. Partial assembly of capsids ensues. •Some of the pgRNA is encapsidated into these recently assembled, immature virions. Within the capsid, a cDNA copy of the pgRNA is made by encapsidated reverse transcriptase (viral polymerase). Following synthesis of the first complementary cDNA strand, the viral polymerase degrades the pgRNA template and begins synthesizing the second DNA strand. The virions are then released from the cells by budding, containing a DNA genome that is only partially double stranded. Replication of RNA Viruses •Replication of most RNA viruses occurs strictly within the cytoplasm of cells and is independent of nuclear machinery. Exceptions are orthomyxoviruses (bunyaviruses) that require factors from host DNA transcription and retroviruses that replicate via a DNA intermediate. •Attachment is an electrostatic interaction between the virions and specific cell receptors. •Viruses then enter the cell by receptor-mediated endocytosis or by fusing with the cell membrane or with the endocytic vesicle (enveloped viruses). •Uncoating occurs in the cytoplasm, or during passage (translocation) through the cell membrane, as appears to be the case for picornaviruses. The RNA of reoviruses, however, is never completely uncoated, but remains in viral cores during genome expression and replication. •The genome of some RNA viruses is a single molecule of RNA (monopartite); in others, the genome is segmented (multipartite). •The RNA of some animal viruses has mRNA function (+ sense) and can be directly translated, whereas the genome in others is antisense (- sense), and must first be transcribed into + RNAs by a viral-encoded RNA-dependent RNA polymerase (transcriptase). •Retroviruses have the enzyme reverse transcriptase (RNA-dependent DNA polymerase) permitting the formation of a dsDNA intermediate (provirus DNA), which becomes incorporated into the host genome, and is subsequently transcribed into mRNA by the host DNA-dependent RNA polymerase. •In general, replication of viral RNA is semiconservative and proceeds via a replicative intermediate (R1). The R1 consists of parental viral RNA that serves as a template for the transcription of several RNA strands, which eventually "peel off" and serve as templates for the synthesis of viral RNA. •Replication of double-stranded RNA of reoviruses is conservative and asymmetrical; only one strand is replicated, unlike double-stranded DNA. The replication processes requires RNA-dependent RNA polymerases (replicases) that are virus encoded. •Maturation occurs in the cytoplasm with the viral RNA becoming associated with the capsid proteins forming the nucleocapsid. Enveloped viruses complete maturation by budding through the endoplasmic reticulum, Golgi apparatus, or the plasma membrane. Double-Stranded RNA Viruses •Includes the following animal-associated virus families: Reoviridae and Birnaviridae. •The genomes of these viruses range in size from 4 kb to 20 - 27 kb in length. •Attachment is via receptor-mediated endocytosis. The virion is partially uncoated and the core particle remains in the endocytic vesicle. •Replication is by a conservative mechanism, the dsRNA serves as a template for the production of mRNA by a viral RNA-dependent RNA polymerase. Much of the remainder of the replication mechanism is poorly understood at this time. •Replication does not involve the formation of R1 intermediates. No free dsRNA is formed in the cytoplasm of the host cell. •All have segmented, linear genomes. Each segment corresponds to a monocistronic mRNA. •All of the genomes are linear, but may be two-component (Birnaviridae), or multi-component (reoviruses have 10 - 12 components). Figure 3-3. General replication scheme of dsRNA viruses. To view click on figure . Single-Stranded Positive Sense RNA Viruses •Includes the following animal-associated virus families: Caliciviridae, Picornaviridae, Astroviridae, HEV-like viruses, Nodaviridae, Flaviviruses, Coronaviridae, Togaviridae, and Arteriviridae. •Genome sizes range from less than 5 kb to 20 - 30 kb. •Attachment is via receptor-mediated endocytosis. There, the virion is uncoated and the ssRNA released to the cytoplasm. The viral genomes that are messenger-sense are totally or partially translated into proteins as the first step of virus replication. •Picornaviruses and Flaviviruses possess a positive sense RNA genome as genome, which behaves as a polycistronic mRNA. The genome is directly translated into one large polypeptide, which is co-translationally cleaved into a number of proteins by viral encoded or host cell proteases. •Coronaviruses have a complex transcription pattern, involving several rounds of translation in order to complete the replication cycle. •Linear forms of the following are possible: single-component with single open reading frames (ORFs) (picornaviruses), single-component with multiple ORFs (togaviruses and caliciviruses), and two-component with single ORF (nodaviruses). Figure 3-4. Replication scheme of positive-sense ssRNA viruses. To view click on figure . Single-Stranded Negative Sense RNA Viruses •Includes the following animal-associated virus families: Orthomyxoviridae, Rhabdoviridae, Paramyxoviridae, Bornaviridae, Filoviridae, Deltavirus, Arenaviridae, and Bunyaviridae. •Genomes range from 10 - 14 kb to 11 - 20 kb in size. As the genomes are negative-sense, they are not translated. Therefore, these viruses need to bring their replicases in the virion to proceed with transcription and replication of the genome. •Orthomyxoviruses have segmented genomes. The first step in the replication process is the transcription of - sense RNA by a virus encoded RNA-dependent-RNA polymerase. •Rhabdoviruses have nonsegmented genomes. Replication still requires the transcription via a viral RNA-dependent RNA polymerase. •In the case of the ambisense viruses, the transcriptase is encoded within the positive-sense portion that will eventually mediate the transcription of the negative-sense regions. •The following linear genome arrangements include single-component with multiple ORFs (filoviruses, paramyxoviruses, and rhabidoviruses), two-component ambisense (arenaviruses), three-component negative sense or ambisense (bunyaviruses), and six- to eight-component (orthomyxoviruses). Figure 3-5. Replication scheme of negative-sense ssRNA viruses. To view click on figure . Single-Stranded Positive Sense RNA Viruses with Reverse Transcriptase •Includes the vertebrate-associated viruses in the family Retroviridae. •This viral genome arrangement is comprised of diploid linear ssRNA held together by protein. It is 5'-capped and has a 3' poly A tail, and has four characteristic coding regions (gag-pro-pol-env). These regions are: gag (group specific antigen: matrix protein, nucleoprotein, capsid) genes; pro (protease) gene; pol (reverse transcriptase and RNase-H); and env (envelope, receptor binding) genes. •The conversion of RNA to ssDNA and then to dsDNA is mediated by the viral enzyme reverse transcriptase. The resulting dsDNA, called provirus DNA, is ultimately integrated into the host chromosome by the viral enzyme integrase. •Once integrated into the host genome, the viral dsDNA (or provirus) remains latent until "triggered" into active virion production. The provirus is then transcribed into mRNA by cellular RNA polymerase II. Viral Genetics Natural selection acting on viral genomes over the years has resulted in great genetic diversity for some viruses. Viral genomes are the key to understanding how viruses interact with the host cells they infect. The rapidly growing knowledge of viral genetics has led to many important applications and new fruitful techniques. Some of the important areas of interest are discussed below. Mutation A mutation is a change in the nucleic acid sequence of an organism. The organism possessing a mutation is referred to as a mutant. This change is based on comparison with the wild type (reference) virus. From this information, strains (wild types of the same virus), types (serological or biological), and variants (phenotypically different from wild type where genetic reason is unknown) can be identified. Mutations are neutral events that can be acted upon by natural selection. If the mutation enhances the survival (transmission and replication) of the organism, it has a selective advantage. If the mutation is detrimental to the growth and survival, the organism is eventually eliminated from the population. If the mutation does not alter the survivability or its phenotype, then the mutation may not be easily detected. Mutations occur by two different mechanisms, spontaneous mutation and induced mutation. •Spontaneous mutations are endogenous, being the result of DNA or RNA polymerase errors or the result of incorporation of naturally occurring tautomeric forms of nucleotides. DNA viruses are typically more genetically stable than RNA viruses; the spontaneous mutation rate is 10-8 to 10-11 per incorporated nucleotide. This is due to the fact that DNA polymerases often have some error correction ability. RNA viruses are considerably less genetically stable, with a spontaneous mutation rate of 10-3 to 10-4 per incorporated nucleotide. RNA polymerases typically lack error correction ability. In spite of this, some RNA viruses (e.g., poliovirus) are relatively genetically stable. It is thought that these viruses have the same high mutation rate as other RNA viruses, but are so precisely adapted for transmission and replication that minor changes result in their elimination. •Induced mutations are exogenous, the result of exposure to mutagens (either chemical or radiation) that significantly increase the mutation rate for a given organism. Chemical mutagens act either directly on the bases or indirectly by enhancing mispairing. Ultra violet radiation can induce the formation of pyrimidine dimers, ionizing radiation can damage DNA directly by breaking chemical bonds or indirectly by forming free radicals that in turn damage DNA. There are a variety of phenotypes that are generated as the result of mutation. Some of the more common ones include: Host Range Mutation Mutation allows for a change in the host range of a particular virus from the original one associated with the wild type virus. This type of change is believed to have occurred with feline parvovirus, which extended its host range and became capable of infecting dogs. Conditional Lethal Mutations This includes a series of mutations that replicate under a specific range of conditions, beyond this range wild type viruses are capable of replication but the conditional is not. Examples of conditional lethal mutants include temperature-sensitive mutants and cold-sensitive mutants. Temperature-sensitive mutants have been used in vaccine development and cold-sensitive mutants have been used in the analysis of viral replication cycles. Plaque Size Mutation As a result of mutation, these viruses produce plaques that deviate from those of the wild type. This information sometimes correlates with the infectivity of a particular virus strain. Nonsense (amber) Mutations Refers to point mutations that result in the formation of a translational stop codon at a position where an amino acid is incorporated in the wild type protein. As a result, the protein is truncated and frequently nonfunctional. The most common nonsense mutation is to the UAG codon, called amber. Deletion Mutations These are the result of a loss of nucleotides at some point in the genome, varying from a single nucleotide to whole sections of the genome. These can either occur in nature or be voluntarily produced in the lab and used in the development of viral vectors or to attenuate a virus for vaccine development. Antigenic Shift and Drift Antigenic shift refers to the change an antigen associated with a viral pathogen due to the acquisition of a novel entire gene or a change in an existing one. Typically antigenic shift is observed readily with those viruses that possess multipartite genomes, such orthomyxoviruses, arenaviruses and bunyaviruses. Coinfection of different strains in the same cell may result in packaging mixed genomes, containing some segments from one virus and other from the other. Antigenic drift is a result of accumulation of point mutations (single bases substitutions) has been identified as the mechanism associated with the antigenic variation observed with influenza viruses and may be the mechanism associated with the variability observed with rhinoviruses. Interactions Between Two Viruses Viral infections with two or more different viruses are known to occur in nature as well as in culture. These are referred to as mixed infections and can result in new viral combinations, and thus new variants of the virus. The following are some of the interactions that can occur during mixed infections. Complementation Complementation can occur during a mixed infection when one of the two viruses is deficient in a particular gene product. Without this protein, the virus is incapable of transmission and replication and is therefore a defective particle. In a mixed infection, if the second virus involved does make the product (thus complementing of the defect), the defective particle is capable of completing the transmission and replication processes. In nature, complementation occurs with the human Hepatitis D virusoid. The virusoid is defective in a surface antigen that is provided by Hepatitis B in mixed infections, allowing the replication of the Hepatitis D life cycle to be completed. Recombination Genetic recombination is the exchange of genetic material between two viral chromosomes in regions where a high level of genetic homology exists. As a result, the progeny are genetically distinct from the two "parental" viruses. Recombination is common in DNA viruses and those RNA viruses having a DNA phase (e.g., retroviruses). Currently, three mechanisms of recombination have been identified: Intramolecular Recombination Recombination that is mediated by cellular enzymes between two regions on a single dsDNA molecule, resulting in a looping-out of the intermediate region, yielding a shorter dsDNA molecule and a separate circular dsDNA molecule. The reverse of this reaction can also occur, resulting in the integration of a circular dsDNA molecule into another dsDNA molecule. This type of recombination is typically associated with monopartite DNA viruses. Copy-choice Recombination A genetic recombination in which the new nucleic acid molecule comes about by replicating selected parts of each parental molecule and by alternating between the two (maternal and paternal). This mechanism is poorly understood and occurs in monopartite RNA viruses. Reassortment This occurs in mixed infections with variant viruses having segmented genomes infecting a single cell. The progeny virions can contain some segments from one parent, some from the other. This is an efficient process observed with orthomyxoviruses, reoviruses, arenaviruses, and bunyaviruses. The mechanism is not well understood. Reassortment has been implicated in the appearance of new, highly virulent influenza virus strains throughout the 20th century. Genetic Reactivation Genetic reactivation is a special case recombination/reassortment that occurs in mixed infections when one or both of the viruses is noninfectious. The progeny, resulting from either recombination or reassortment, are now infectious and carry markers of both parents. If only one parent was noninfectious, the process is called cross-reactivation or marker rescue. If both parents were noninfectious, the process is called multiplicity reactivation. Phenotypic Mixing Phenotypic mixing is an example of non-genetic interactions between two viruses. As a result of mixed infection, the individual progeny possess structural proteins (envelope, capsid) from either or both parents. The genome of either parent virus can be encapsidated within any of the three types of capsids (envelopes), yielding six different types of progeny. Therefore, the genotype and phenotype of many of the progeny virions do not match. Virus Applications: Gene Therapy and Recombinant Vaccines Perhaps two of the most intriguing applications utilizing the knowledge of viral replication and genetics are gene therapy and development of recombinant vaccines. These techniques hold great promise for the development of novel ways to treat genetic diseases or afford protection against diseases in humans and animals. Gene Therapy Gene therapy is based on the premise of using viruses with no pathogenic properties, but retains their ability to selectively interact with and transmit their genes (plus any genetically-engineered genes) into specific host cells and tissues. Retroviruses are an excellent means for gene delivery into target host cells. The dsDNA of their genome is stable and readily integrated into the host genome. The viruses are engineered in such a manner that once the genome has been integrated into the genome they cannot replicate. Often this means using helper viruses to aid in the initial uptake of the engineered virions by the host cells by complementation. The limitation of this method of gene therapy is that in some cases the gene in question needs to be present in all cells of the host and not just a select group of cells or tissues. Retroviruses have been used in gene therapy for incorporation of the adenine deaminase (ADA) gene into the immune cells of patients with an ADA immunodeficiency. In addition to retroviruses, some other viruses currently being studied as potential vectors for gene therapy include the adenoviruses, adeno-associated viruses (parvoviruses) and herpesviruses. Recombinant Vaccines The three kinds of vaccines prepared with recombinant nucleic acid techniques are discussed in Chapter 6. Several of these recombinant vaccines are in current use to prevent animal and human viral diseases. Glossary Ambisense: Refers to an RNA genome containing sequence information that is both-positive sense (can be used directly as mRNA) and negative-sense (must be transcribed to form mRNA).Conservative Replication: Replication of dsDNA or dsRNA in such a manner that the original strands do not become a part of the newly formed progeny dsDNA or dsRNA.Endocytic Vesicle: A vesicle formed in the process of endocytosis, the "engulfment" of the virus, which can be mediated by surface receptors or cell membrane interactions.Golgi Membrane: A membrane associated with the Golgi apparatus of a eukaryotic cell. The Golgi apparatus receives newly synthesized lipids and proteins from the endoplasmic reticulum and chemically modifies and traffics them to the appropriate locations in the cell.Inclusion Bodies: These represent virus "factories" in which viral nucleic acid or protein is being synthesized.Ligase: A host enzyme that creates covalent bonds in nucleic acids associated with breaks in the sugar-phosphate backbone of the molecule.Monocistronic: Contains information for a single gene or gene product.Monopartite: A viral genome having a single segment.Multi-component Genomes: Genomes having more than one nucleic acid molecule making up its total genome.Mutagens: Chemical or physical agents that increase the mutation rate of the DNA of an organism.Negative-sense DNA: DNA whose transcription does not produce an RNA molecule that can be used directly as mRNA. It is the template for creation of negative-sense RNA genomes.Polycistronic: Contains information for several genes or gene products.Positive-sense DNA: DNA whose transcription produces the genome of positive-sense RNA genomes or can be used directly as mRNA.Reverse Transcriptase: A viral enzyme that uses a RNA template to synthesize DNA.Semiconservative Replication: Replication of dsDNA or dsRNA in such a manner that the original strands (one original, one newly synthesized) become a part of the newly formed progeny dsDNA or dsRNA.Single-component Genomes: Genomes having a single nucleic acid molecule making up its total genome.Tautomers: These are isomeric forms of organic compounds and when two of them exist in equilibrium it is referred to as tautomerism.Transcriptase: A viral enzyme capable of using an RNA molecule as a template for transcription.Wild Type: The natural virus; such viruses are used as reference strains for the comparisons of mutants and variants of a particular virus


Cultivo y caracterización de virus D.J. Wise1 and G.R. Carter2 1Department of Biology, Concord University, Athens, West Virginia, USA.2Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, USA. Traducido por: A. T. Pérez Méndez, Biotecnología Veterinaria de Puebla, S.A de C.V., Tehuacán, Puebla, Mexico. (21-Apr-2005). Indice Métodos de propagación viral Concentración y purificación de virus Infectividad y almacenamiento Visualización de virus Cuantificación directa de virus Cuantificación indirecta de virus Métodos misceláneos usados para caracterización Glosario Se han desarrollado métodos para el almacenamiento, visualización, cuantificación (directa e indirecta) y propagación de virus. También hay métodos para el diagnóstico de laboratorio de enfermedades virales, muchos de los cuales son métodos serológicos, basados en la detección de la respuesta del hospedero a la infección. Estos métodos diagnósticos serán discutidos en el capítulo 7. A lo largo del tiempo, fue posible observar que el contagio de ciertas enfermedades era capaz de pasar a través de filtros que las bacterias no podían pasar. Los filtrados obtenidos no eran capaces de crecer en medios de cultivos para bacterias, y eventualmente se demostró experimentalmente que eran infectivos y que contenían virus. A excepción de los virus de viruela, los virus no pueden ser observados con microscopio óptico. Eventualmente los virus fueron observados con microscopio electrónico. Algunos de los métodos importantes usados en los estudios básicos de virus se describen a continuación. Tal como se mencionó en el capítulo anterior, los virus animales presentan una considerable diversidad en sus características físicas. La característica que más refleja las propiedades del virión es la presencia o ausencia de la envoltura viral. Como se señala en la tabla 2.1, los virus no envueltos son, en general, sensibles a la radiación ultravioleta, relativamente termoestables y susceptibles al daño por los cristales de hielo. Debido principalmente a la presencia de la envoltura de membrana, los virus envueltos se inactivan con solventes lipídicos (como cloroformo y éter) y detergentes (como el desoxicolato), son sensibles a la radiación gama y ultravioleta, relativamente termolábiles y el daño que les produce la formación de cristales de hielo es más extenso que el que se produce en los virus no envueltos o desnudos. Tabla 2.1. Principales propiedades fisicoquímicas y biológicas de viriones envueltos y desnudos Características Virus desnudos Virus envueltos Radiación ultravioleta Sensible Sensible Radiación gama Sensible Sensible Termoestabilidad Termoestable Termolábil Susceptibilidad a daño por cristales de hielo Si Extenso Inactivación por solventes lipídicos y detergentes No Si Métodos de propagación viral Para aislar, caracterizar e identificar virus, así como para producir vacunas virales, se necesita una cantidad considerable de partículas virales. Esto se logra a través de diferentes métodos de propagación, los cuales se enlistan a continuación: Hospederos animales En el pasado, la propagación de virus en organismos hospederos susceptibles no infectados era la única manera de obtener grandes cantidades de virus. Actualmente el uso de animales experimentales como hospederos para propagación viral está limitado por razones éticas. La propagación viral en animales es más útil para aquellos virus que no crecen fácilmente en cultivos celulares. Por ejemplo: cepas vacunales del virus de la enteritis hemorrágica de pavo pueden ser propagadas tanto en aves vivas como en cultivos celulares. Sin embargo los productos propagados en bazo (de aves vivas) parecen ser más usados que los propagados en cultivo. Para fines diagnósticos la inoculación de animales es un medio para detectar virus en muestras clínicas, como el virus de la rabia en ratones lactantes. Huevos embrionados Antes del desarrollo de las técnicas de cultivo de células y de tejidos, el uso de huevo embrionado para propagación viral fue una de las primeras alternativas al uso de organismos animales hospederos. El huevo embrionado es aún el método preferido para la propagación de virus de influenza tipo A y para muchos otros virus aviares. El huevo embrionado también es útil en la diferenciación de algunos virus que producen lesiones similares, como los virus de la viruela de la vaca y los virus de la pseudo viruela de la vaca. Aunque el virus de la enfermedad de la lengua azul (BTV) es un virus de mamíferos, se replica bien en huevo embrionado, por lo que este sistema se usa para propagación viral con fines diagnósticos y de investigación. Cuando se usa huevo embrionado, uno debe considerar la posible presencia de anticuerpos maternos (IgY) en el saco vitelino del huevo. Por lo tanto, frecuentemente es preferible obtener huevo embrionado de parvadas libres de patógenos específicos (SPF). El dar pases en embrión de pollos es útil en la atenuación de ciertos virus para vacunas de virus vivo modificado. Cultivo de células/tejidos El cultivo de tejidos se refiere al crecimiento y mantenimiento de células de tejidos vivos in vitro. Hay básicamente dos tipos: cultivo de explantes y cultivo de células. Los explantes son pequeños fragmentos de tejidos del hospedero que se mantienen en cultivo, mientras que el cultivo de células se obtiene mediante de la disgregación de diferentes tejidos del hospedero en células individuales. La mayoría de los sistemas usados en virología son en realidad cultivos celulares y no cultivos de tejidos, aunque ambos términos se usan de manera indistinta. Los cultivos celulares se subdividen a su vez en cultivos primarios, cultivos semi-continuos y cultivos continuos. Figura 2-1. Cultivo celular normal. Cortesía de A. Wayne Roberts. Para ver oprima la figura . Cultivo de explantes Estos son cultivos de pequeños fragmentos de tejidos específicos, tomados directamente del hospedero animal. Los cultivos de explantes son útiles para el aislamiento viral y se requieren para el aislamiento de algunos coronavirus. La demostración de latencia de algunos alfa herpesvirus humanos y animales puede requerir explantes de ganglio nervioso sensitivo (por ejemplo, trigémino). Cultivos celulares primarios Estos se derivan de tejidos frescos que han sido digeridos enzimáticamente con tripsina u otras proteasas, para liberar células individuales. Como resultado, con frecuencia los cultivos primarios están compuestos de muchos tipos celulares diferentes. En condiciones in vitro las células de los cultivos primarios raramente pueden dividirse, o se dividen a una velocidad muy baja. Es por esto que tienen un tiempo de vida limitado, conocido como límite de Hayflick. A pesar del tiempo de vida limitado de los cultivos primarios, son ideales para el aislamiento de algunos virus. Los cultivos primarios raramente sobreviven más allá del vigésimo pase in vitro. Cultivos semi-continuos Son también conocidos como líneas celulares diploides, debido a que contienen el cromosoma diploide normal característico de la especie de la que ellos se derivan. Los cultivos semi-continuos son cultivos primarios que tienen algunas células que pueden ser cuidadas para sobrevivir más allá del límite de Hayflick. Los cultivos semi-continuos tienden a morir entre el 30° y el 50° pase in vitro. A pesar de esta limitante, los cultivos semi-continuos son útiles en la propagación de una gran variedad de virus. Los cultivos semi-continuos son por lo general de fibroblastos. Cultivos celulares continuos Se les conoce también como líneas celulares heterodiploides, debido a que poseen un número anormal de cromosomas. Estos cultivos se derivan de tejidos normales o neoplásicos y se caracterizan por su habilidad para ser propagados in vitro indefinidamente. De manera general, las líneas celulares continuas no son tan sensibles como las otras para propagación viral. Sin embargo, facilitan la propagación a gran escala de algunos virus para vacunas e investigación. Muchas líneas continuas están disponibles de proveedores como la Colección Americana de Cultivos Tipo (ATCC por sus siglas en inglés). La mayoría de los laboratorios de virología almacenan en congelación alícuotas iniciales de sus cultivos continuos, debido a que las líneas celulares que están continuamente en cultivo pueden sufrir cambios en sus características celulares. Las causas de estos cambios pueden ser infección por Micoplasma spp., o virus contaminantes (por ejemplo, circovirus porcino y virus de la diarrea viral bovina). Concentración y purificación de virus Una vez que un virus ha sido propagado adecuadamente, necesita ser recuperado de las células hospederas y los restos de éstas, y luego ser purificado. Esto se logra por varios procesos que involucran centrifugación diferencial (a diferentes velocidades), diálisis, precipitación, cromatografía y gradientes de densidad. El paso inicial de este proceso es la centrifugación diferencial; se usa una velocidad baja (~2,000 x g) para quitar restos celulares y a para concentrar se usa a continuación, en el caso de volúmenes pequeños, una velocidad alta de centrifugación (40K a 80K x g) y en el caso de volúmenes mayores, se usa diálisis y precipitación o precipitación con metanol frío (-70°C) o con polietilenglicol. La purificación se lleva a cabo mediante cromatografía o centrifugación usando gradientes de densidad. Los virus envueltos pueden ser purificados aprovechando su velocidad de sedimentación en gradientes de sacarosa. Los virus desnudos pueden ser purificados por centrifugación a través de gradientes de cloruro de cesio. Infectividad y almacenamiento Infectividad La infectividad es la habilidad de la partícula viral para infectar una célula hospedera. La temperatura en el exterior de la célula hospedera afecta fácilmente la capacidad del virus para conservar su infectividad, particularmente en el caso de los virus envueltos. Debido a que los virus no tienen actividad metabólica propia, la infectividad es el mejor medio para evaluar la integridad de la partícula viral después de que se ha expuesto a cierta temperatura. Las siguientes son consideraciones importantes al respecto: •A 60°C, la infectividad del virus disminuirá rápidamente, en segundos. •A 37°C, la infectividad disminuirá dramáticamente, en minutos. •A 20°C, la infectividad disminuirá, en cuestión de horas. •La infectividad en las temperaturas antes mencionadas influyen en la transmisión del virus por contacto directo (a 37°C) y por fomites (a 20°C). •A 4°C, la infectividad en tejidos se pierde en cuestión de días. Los clínicos deben tener esto en cuenta al considerar los especímenes clínicos. Con frecuencia se usan temperaturas abajo del punto de congelación para almacenamientos por periodos largos. Lo importante a considerar es mantener al mínimo la formación de cristales de hielo. Debe mantenerse en mente el hecho de que los virus presentan gran diversidad en su resistencia y labilidad Algunos son capaces de sobrevivir por horas, días, o incluso meses bajo condiciones ambientales, mientras que otros se inactivan en pocos minutos bajo las mismas condiciones. Los tres métodos principales para almacenar virus son: •Congelación a -70°C, con o sin criopreservante. •Para almacenamiento por largos periodos: congelamiento en nitrógeno líquido (-196°C). •Liofilización con almacenamiento en congelación o a temperatura ambiente. Visualización de los virus Los dos métodos principales usados para visualizar la estructura / morfología de los virus son: la microscopía electrónica y la microscopía de fuerza atómica. Otros tipos de microscopía se usan para observar cambios inducidos por la replicación viral en las células infectadas. Sin un medio para visualizar los virus es difícil obtener información acerca de la estructura o las interacciones célula-virus. Más aún, el visualizar las partículas virales le permite a uno estimar directamente el número de partículas (virales) presentes en una suspensión. Hay otros métodos que le permiten a uno estimar el número de virus indirectamente. En cualquier caso, la cuantificación directa o indirecta es siempre un estimado. Este estimado numérico es importante al preparar vacunas, al determinar el número mínimo de viriones necesarios para producir una enfermedad y en procedimientos virales de investigación. Microscopía óptica Si bien la microscopía óptica no es útil para la examinación directa de los virus (con excepción de los virus de la viruela), es útil para observar los efectos de la infección viral en la célula hospedera. El daño celular o la destrucción causada por el virus es conocida como efecto citopático (CPE por sus siglas en inglés). Los efectos citopáticos que pueden observarse incluyen: 1.Células redondeadas y agregados en forma de racimo de uvas, como se observa con adenovirus; 2.Células redondeadas, encogidas, lisadas, dejando gran cantidad de restos celulares, como se observa con los enterovirus; 3.Células agrandadas y redondeadas en áreas focales, como con herpesvirus; y 4.Fusión de células que se convierten en células multinucleadas (sinsicios) como en el caso de paramyxovirus. Además es posible observar cuerpos de inclusión, característicos de algunos virus. Figura 2-2. Efecto citopático del herpes virus "lento" equino. Cortesía de A. Wayne Roberts. Para ver oprima la figura . Microscopía de fluorescencia La microscopía de fluorescencia puede ser usada para visualizar células o tejidos infectados por virus, usando anticuerpos antígeno-específicos marcados con fluorocromos. El anticuerpo se une específicamente a los antígenos virales presentes dentro de las células o tejidos y los marca con la molécula fluorescente (generalmente fluoresceína). La marca fluorescente se observa posteriormente con un microscopio de luz ultravioleta que excita a la molécula del fluorocromo, lo que uno observa como una zona coloreada sobre un fondo relativamente oscuro. De manera alternativa, la visualización puede llevarse a cabo de forma indirecta usando anticuerpos sin marca (como los de sueros convalecientes) seguidos de la aplicación de anticuerpos marcados con fluoresceína que se unen al primer anticuerpo. Los ensayos basados en el uso de anticuerpos fluorescentes son usados comúnmente en el diagnóstico viral y la investigación. Microscopía electrónica La microscopía electrónica involucra la aceleración de electrones a un estado de alta energía y el enfoque magnético de los mismos hacia la muestra. Los electrones de alta energía tienen longitudes de onda muy cortas, proporcionando así una mejor resolución de estructuras muy pequeñas. La microscopía electrónica tiene suficiente poder de resolución para observar polímeros grandes como ADN y ARN, así como proteínas grandes. Para facilitar la visualización, las muestras pueden ser cubiertas con metales pesados como osmio, antes de la examinación con el microscopio electrónico. Los electrones impactan el metal pesado y posteriormente son visualizados en una pantalla fluorescente. La microscopía electrónica proporciona imágenes tridimensionales de viriones y de su localización (nuclear o citoplasmática) dentro de la célula hospedera en un momento dado durante la infección. La observación de viriones en células vivas no es posible, debido a que las muestras tienen que ser tratadas con metales pesados. Microscopía de fuerza atómica La microscopía de fuerza atómica trabaja a través de la medición de una propiedad local (tal como altura, absorción óptica, magnetismo, etc.) de una sonda puesta muy cerca de la muestra. Esto hace posible tomar mediciones en un área muy pequeña de la muestra. Los electrones son capaces de "atravesar por túnel" entre los átomos, provocando una fuerza pequeña pero cuantificable. El resultado de estas mediciones es un mapa detallado del contorno o la superficie de la estructura. La ventaja de la microscopía de fuerza atómica es que la preparación de la muestra es mínima y pueden usarse especímenes vivos. Este método ha sido útil para obtener imágenes detalladas de estructuras de cápsides y de interacciones virus-célula. Microscopía inmunoelectrónica Esta técnica permite la visualización de complejos antígeno / anticuerpo que son específicos para un virus en particular. En este método se obtienen cortes ultra finos (de la muestra) y se incuban con un anticuerpo específico para el virus. Después de un paso de lavado, el corte se incuba con Proteína A conjugada con partículas de oro (de un rango de 5 a 20 nm). Este conjugado se une a la porción Fc del anticuerpo y se detecta por microscopía electrónica. Enumeración directa de virus Estimar el número de virus tiene una cantidad importante de usos, incluyendo producción de vacunas e investigación. La microscopía electrónica se usa para cuantificar las partículas virales en una solución libre de células. Se examina un volumen conocido de la muestra y se cuenta el número de viriones. Este número se usa para calcular el número de virus. Una limitante es que las cápsides vacías, es decir, partículas no infectantes, también son contadas. En investigación, se compara el número de partículas infectantes y el número total, y se establece una relación de partículas totales / partículas infecciosas para un virus dado. Enumeración indirecta de virus Los métodos indirectos de cuantificación viral son aquellos que usan factores asociados con la infectividad (actividad biológica). Los tres métodos principales usados para determinar indirectamente concentraciones virales son: ensayos de hemoaglutinación, ensayos de formación de placas y el método de la dilución limitante. Hemoaglutinación Este ensayo se basa en la propiedad que tienen muchos virus envueltos para aglutinar glóbulos rojos (RBCs) o (GR). El ensayo se lleva a cabo en una microplaca, añadiendo glóbulos rojos a diluciones de la muestra que contiene virus, y observando posteriormente la hemoaglutinación. Son necesarias muchas partículas virales para cubrir los glóbulos rojos y producir hemoaglutinación. Por ejemplo: se necesitan aproximadamente 104 viriones de influenza por unidad hemoaglutinante (1 unidad de HA). Una unidad de HA se define como la máxima dilución de la muestra viral que causa hemoaglutinación completa. La hemoaglutinación es útil en la concentración y purificación de algunos virus, y como prueba presuntiva rápida para la presencia de este tipo de virus en fluidos de cultivos celulares infectados y de embriones de pollo. Es especialmente útil para probar actividad viral en cultivos celulares infectados con virus hemoaglutinantes que producen un efecto citopático (CPE) mínimo o no detectable. También pueden ser examinados directamente especímenes clínicos como heces, para buscar actividad hemoaglutinante de partículas virales (se discutirá posteriormente en el capítulo 7). Otros ensayos del mismo tipo, en los que se prueba una actividad enzimática de un virus en particular (como aquellos que producen transcriptasa reversa), pueden ser llevados a cabo de manera similar. Ensayo de formación de placas Este ensayo involucra la inoculación de células hospederas susceptibles con un virus, y usar su actividad biológica para estimar el número de viriones presentes. En este procedimiento se usan diluciones decimales seriadas del virus para inocular monocapas de células hospederas. Después de un periodo de incubación en el que se permite la adsorción del virus a la superficie de las células hospederas, la monocapa es cubierta por una capa de un gel compuesto por medio de cultivo para células hospederas y agarosa. La presencia del agar evita la diseminación a gran escala del virus en el cultivo celular, pero permite la diseminación localizada de célula a célula. Con los virus citopáticos la destrucción de las células lleva a la formación de zonas desocupadas llamadas placas, que pueden ser vistas entre las 24 y 72 horas de incubación. Un cálculo que involucra el número de placas observadas, el factor de dilución de la muestra y el volumen de muestra diluida utilizada, da como resultado las unidades formadoras de placa (PFU) por mililitro de muestra. El método de dilución limitante Este ensayo basado en titulación mide un efecto in vitro sobre las células, como el CPE, cuando éstas se expone a varias diluciones de la solución que contiene el virus. Si es posible, se usa una concentración conocida de un virus de referencia como control positivo. Dependiendo del virus, se hacen diluciones seriadas dobles o diluciones seriadas decimales del material viral y se ponen en contacto con las células. El título infectivo (el recíproco de la dilución más alta que provoca el 50% de CPE en el cultivo infectado) se expresa como TCID50/ml (dosis infectiva 50 cultivo celular). Este ensayo puede usarse con células en cultivo, embriones de pollo o incluso con animales de laboratorio. Métodos misceláneos usados para caracterización Hay algunos métodos en virología que son útiles en la identificación y clasificación de virus desconocidos. Algunas de esas técnicas serán mencionadas brevemente aquí, pero si se usan en el diagnóstico de laboratorio de un virus en particular, se explicarán con detalle posteriormente. Sensibilidad a solventes lipídicos La sensibilidad de los virus a solventes lipídicos como cloroformo y éter es útil en la taxonomía de ciertos virus. Cualquier virus que posea envoltura es susceptible a solventes lipídicos. Todos los virus envueltos de animales, excepto algunos virus de la viruela, son sensibles al éter. Identificación del tipo de ácido nucleico Esto se lleva a cabo examinando la síntesis de ácido nucleico en la célula, en presencia de inhibidores de la síntesis de ADN, como la 5-bromo-2-desoxiuridina (BRU). Si se inhibe la síntesis viral como consecuencia disminuirá la multiplicación viral. En el caso de que el crecimiento viral no sea inhibido se presume que al virus contiene ARN. Análisis con enzimas de restricción Las enzimas de restricción (RE) son endonucleasas que cortan el ADN de doble cadena en sitios de reconocimiento específicos que son secuencias palindrómicas que van desde cuatro hasta ocho pares de bases de longitud. El análisis con enzimas de restricción es particularmente útil en la clasificación de "subserotipos" virales, en la diferenciación de virus vivos modificados vacunales de virus virulentos y en el rastreo epidemiológico de brotes de enfermedades. Metodológicamente esta técnica consiste en tratar el ADN viral con una o varias enzimas de restricción y luego separar los fragmentos resultantes por medio de electroforesis en geles de poliacrilamida. Los virus de ARN pueden ser analizados de una manera similar haciendo primero el ADN complementario (ADNc) al ARN viral usando la enzima transcriptasa reversa, y luego amplificando este ADNc por el método de PCR descrito en el capítulo 7. Hemoadsorción Virus envueltos como los ortomixovirus y paramixovirus adquieren su envoltura externa por protrusión de yemas a través de la membrana celular. Antes de la protrusión, se incorporan a la membrana celular proteínas codificadas por el virus (hemoaglutininas). Esas células (aquellas a las se incorporaron hemoaglutininas a su membrana) adsorben eritrocitos a su superficie, lo que trae como consecuencia la formación de focos de hemoadsorción que pueden ser detectados microscópicamente. Métodos inmunológicos Los animales infectados con virus responden produciendo anticuerpos específicos. La detección y cuantificación de estos anticuerpos, que reflejan el estado de la enfermedad, son útiles en la planeación de programas de salud para el hato y estudios epidemiológicos de los brotes de enfermedades. Si bien la detección de anticuerpos es útil en el diagnóstico de enfermedades también, con frecuencia es un proceso tardado que requiere la comparación de los niveles de anticuerpos en sueros de la fase aguda de la enfermedad y de la convalecencia, sueros que se colectan con 10 a 14 días de diferencia unos de otros. Una estrategia más rápida es usar anticuerpos específicos anti-virus para detectar antígenos virales directamente en especímenes clínicos. Estos anticuerpos generalmente se obtienen por hiperinmunización de conejos o cabras con un virus específico. De manera alternativa pueden usarse anticuerpos monoclonales, si hay disponibles. Los anticuerpos monoclonales (mAbs) se preparan en ratones primero exponiendo al ratón al antígeno viral, que sensibiliza a las células B del bazo. Estas células se colectan y se fusionan químicamente con una línea celular de plasmocitoma de ratón que secreta IgG. Posteriormente estas células híbridas son clonadas y los hibridomas resultantes, que son derivados de una sola célula, se analizan para buscar secreción de la IgG antiviral específica. Las células del hibridoma seleccionado son inyectadas de regreso por vía intraperitoneal al ratón, donde las células se multiplican rápidamente y causan la acumulación de un fluido ascítico que contiene una alta concentración de anticuerpos monoclonales. La figura 2.1 muestra los pasos involucrados en la preparación de los anticuerpos monoclonales. Los anticuerpos monoclonales son especialmente útiles en la tipificación y subtipificación de virus. Cuando son acoplados a fluorocromos, los mAbs son muy usados para la detección de virus en tejidos. También son usados para la identificación de virus en muchos estuches diagnósticos comerciales de ELISAs. Las pruebas más comúnmente usadas en virología clínica o diagnóstica serán discutidas en el capítulo 7. Figure 2-3. Pasos asociados al desarrollo de anticuerpos monoclonales específicos. Para ver oprima la figura . Glosario Virus citopáticos:Son aquellos que alteran la apariencia microscópica de células en cultivo. Estos cambios pueden incluir redondeo de las células, fusión celular, desprendimiento celular, producción de cuerpos de inclusión, etc.Gradientes de densidad:Procedimiento para separar células o macromoléculas como proteínas y ácidos nucleicos, generalmente usando centrifugación a través de un gradiente de densidad. Este último consiste en una solución en la que hay un rango de densidades del soluto (generalmente sacarosa o cloruro de cesio), menos concentrado en la superficie y más concentrado en el fondo. Como resultado de la centrifugación las células o macromoléculas se desplazan a través del gradiente y forman una banda que se ubica donde su gravedad específica es igual a la densidad del medio.Palíndromos:Secuencias que se leen igual en ambas direcciones. La mayoría de los sitios de reconocimiento de las endonucleasas de restricción son palíndromos, por ejemplo la secuencia de reconocimiento de EcoR1 (E.coli) es: 5' GAATTC 3' 3' CTTAAG 5'

martes, 6 de noviembre de 2012


Developments in rabies vaccines. Hicks DJ, Fooks AR, Johnson N. Clin Exp Immunol. Volume 169, Issue 3, pages 199–204, September 2012 Summary The development of vaccines that prevent rabies has a long and distinguished history, with the earliest preceding modern understanding of viruses and the mechanisms of immune protection against disease. The correct application of inactivated tissue culture-derived vaccines is highly effective at preventing the development of rabies, and very few failures are recorded. Furthermore, oral and parenteral vaccination is possible for wildlife, companion animals and livestock, again using inactivated tissue culture-derived virus. However, rabies remains endemic in many regions of the world and causes thousands of human deaths annually. There also remain no means of prophylaxis for rabies once the virus enters the central nervous system (CNS). One reason for this is the poor immune response within the CNS to infection with rabies virus (RABV). New approaches to vaccination using modified rabies viruses that express components of the innate immune system are being applied to this problem. Preliminary reports suggest that direct inoculation of such viruses could trigger an effective anti-viral response and prevent a fatal outcome from RABV infection. Introduction Rabies virus (RABV) is the type species of the genus Lyssavirus within the family Rhabdoviridae. The virus has a single-stranded ribonucleic acid (RNA) genome in the negative sense orientation that encodes five proteins in the following order: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA polymerase (L) [1]. Infection with RABV causes in excess of 50 000 human deaths annually, the majority of which occur in Asia [2]. In addition to RABV, the lyssavirus genus contains a growing number of recognized and putative members (Table 1), many of which have been responsible for occasional human deaths [3]. RABV is a zoonotic virus and has a range of reservoir host species within the mammalian orders Carnivora and Chiroptera[4]. The most important of these reservoirs as a source of human disease is the domestic dog (Canis familiaris). In the Americas, a number of bat species are also responsible for transmission of rabies to humans, particularly the common vampire bat (Desmodus rotundus) in Latin America and a number of insectivorous bat species in North America [5]. This has emerged as a public health risk, as bites can occur without the victim realizing that an exposure has taken place, and many cases of bat-transmitted rabies have no recorded exposure to a bat prior to development of infection [6]. Table 1. The lyssavirus genus. Virus Reservoir or known host Causative agent of rabies in humans§ 1. *Bat species in the Americas only. †Bat species in Africa only. ‡Bat species in Australia only. §Rabies virus causes >50 000 human deaths annually; the other members of the genus Lyssavirus have caused occasional deaths (reviewed in [3]); no signifies no reported deaths. Rabies virus (RABV) Species of the orders Carnivora and Chiroptera* Yes Lagos bat virus (LBV) Chiroptera† No Mokola virus (MOKV) Not known Yes Duvenhage virus (DUVV) Chiroptera† Yes European bat lyssavirus type 1 (EBLV-1) Serotine bat (Eptesicus serotinus) Yes European bat lyssavirus type 2 (EBLV-2) Daubenton's bat (Myotis daubentonii) Yes Australian bat lyssavirus (ABLV) Chiroptera‡ Yes Aravan virus (ARAV) Lesser mouse-eared bat (Myotis blythi) No Khujand virus (KHUV) Whiskered bat (Myotis mystacinus) No Irkut virus (IRKV) Greater tube-nosed bat (Murina leucogaster) Yes West Caucasian bat virus (WCBV) Common bent-winged bat (Miniopterus schreibersii) No Shimoni bat virus (SHIBV) Commerson's leaf-nosed bat (Hipposideros commersoni) No Bokeloh bat virus (BBLV) Natterer's bat (Myotis nattereri) No Ikoma virus (IKOV) Civet (Civettictis civetta) No Transmission of RABV occurs following a bite from an infected host resulting in the deposition of virus-laden saliva into a wound. RABV is highly neurotrophic and following a highly variable period, often lasting months, virus infects a peripheral nerve and ascends to the dorsal root ganglion [7]. Once within the spinal cord, the virus spreads rapidly to the brain, resulting in an overwhelming encephalitis that eventually kills the host. Examination of infected brains by histopathological methods reveals few gross changes with the exception, in many cases, of the presence of distinctive inclusion or Negri bodies. Immunolabelling reveals numerous infected neurones with accompanying gliosis and the development of perivascular cuffs around the parenchymal vasculature. This is found predominantly in the hindbrain [8]. Once signs of infection develop there is no effective treatment and, uniquely among infectious diseases, it has a case fatality rate of almost 100%. However, current vaccines are highly effective at protecting against this outcome provided that vaccination is given before or shortly after exposure to a biting incident. The development of virus-neutralizing antibodies is critical to preventing infection [9], and rabies vaccines are efficient at inducing an anti-rabies antibody response. However, the late development of neutralizing antibodies during RABV infection [10] is unexplained, and may be a critical factor responsible for the high fatality rate associated with the disease. Furthermore, there are no effective anti-viral treatments for rabies despite extensive investigations [11]. This review will provide an overview of the past, present and possible future of rabies vaccination, particularly considering the potential of vaccination to treat disease. History of rabies vaccination A comprehensive review on the development of rabies vaccines has been published recently [12]. What follows is a brief overview of key developments. Louis Pasteur developed the earliest effective vaccine against rabies that was first used to treat a human bite victim on 6 July 1885 [13]. The method involved inoculation with homogenates of RABV-infected rabbit spinal cord that had been desiccated progressively in sterile air. Initially, the recipient received a subcutaneous injection of homogenate that was fully inactivated. This was followed by injection of material derived from infections of spinal cord desiccated for shorter periods that contained progressively more virulent preparations of virus. Pasteur's approach proved highly effective, and the methodology spread widely. Two problems were associated with the approach. The first was the consistency of inactivation, which in some cases led to recipients possibly developing rabies from the vaccination, and secondly, the ability to produce sufficient vaccine from rabbits to meet the demand for treatment. These problems were resolved by inactivation of infected sheep or goat brain with chemical agents such as phenol [14,15]. These vaccines also proved successful but, like the original Pasteur vaccine, contained high levels of myelin that caused sensitization in some vaccine recipients and, in extreme cases, fatal encephalitis. Alternatives to this approach included inactivation of infected chick embryos [16] or inactivation of infected suckling mouse brain that has a lower level of myelin compared to the adult brain [17]. However, even these approaches were not entirely free of autoimmune reactions, and the World Health Organization (WHO) does not advocate the use of vaccines containing nervous tissue, although they are still used in a number of countries. A new paradigm for rabies vaccines followed the development of cell culture for virus propagation. The first tissue culture vaccine was derived from virus grown in primary hamster kidney cells [18,19]. This was followed by growth of fixed RABV (see Box 1) in a human diploid cell line [21]. The lung-derived cell line WI-38 was used initially, but was switched subsequently to the MRC-5 cell line, which resulted in the development and licensing of a human diploid cell vaccine (HDCV) in the mid-1970s. An alternative to HDCVwas the use of purified chick embryo cells (PCEC) [22]. These vaccines are now used successfully worldwide. The concept of fixed virus Box 1. One of Louis Pasteur's achievements was to develop an animal model for the predictable passage of rabies virus (RABV). Pasteur solved this by infecting rabbits through introducing constant amounts of virus preparation onto the dura mater membrane after creating a hole in the skull (trepanation). After repeated passage by this method, in some instances more than 50 times, the incubation period from inoculation to the development of rabies became consistent at 7 days. The properties of the virus, which would now be termed a virus strain, were considered fixed. This has been applied to a large number of RABV strains that are now used commonly to develop rabies vaccines and in basic research on rabies pathogenesis. Examples include Pasteur virus (PV), Pitman Moore (PM), low egg passage (LEP) and Street Alabama Dufferin (SAD). The probable origins of these strains of RABV, many of which have been in existence for more than 50 years, were reviewed by Flamand and co-workers [20]. Administration of rabies vaccines Uniquely among vaccines, those for rabies can be given both pre- and post-exposure to virus. Pre-exposure vaccination is appropriate for travellers to RABV-endemic regions, veterinarians and researchers working with the virus. Post-exposure vaccination is possible because the exposure event, usually a bite, is easily identifiable and the incubation period is of sufficient length for vaccination to induce a protective immune response. This is principally through the development of neutralizing antibodies. Post-exposure vaccination is usually accompanied by injection of anti-rabies immunoglobulin of either human (HRIG) or equine (ERIG) origin, and is referred to collectively as post-exposure prophylaxis (PEP). Whether PEP is given can be decided by the level of exposure (Table 2) which, despite the extreme consequences of developing disease, is a factor in resource-poor areas of the world. Table 2. World Health Organization recommendations for vaccination in response to contact with a potentially rabid animal. Category Contact Action I Touch or normal animal husbandry such as feeding No treatment required II Contact leads to minor scratches or abrasions that do not result in bleeding Clean the wound with soap and water. Receive vaccination Biting that does not lead to breaks in the skin III Bites or scratches that lead to skin damage Clean wound with soap and water. Receive vaccination and anti-rabies immunoglobulin (human or equine origin) Exposure of any sort to bats Licks on broken skin/mucous membranes Pre-exposure vaccination consists of an intramuscular injection of 1 ml vaccine on days 0, 7, 21 and 28. Depending on the vaccine manufacturer, boosting is recommended at 3–5-year intervals. This has been borne out by recent cohort studies of UK bat workers who are required to be vaccinated against rabies prior to licensing to work with bats [23]. Post-exposure vaccination is given typically as an intramuscular injection on days 0, 3, 7, 14 and 30. HRIG is given on day 0, unless the recipient has received previous vaccination against rabies. One innovation has been the replacement of intramuscular inoculation with intradermal injection of vaccine [24]. This route of inoculation appears to require less vaccine to be effective, which reduces the cost of treatment. A disadvantage of this method is the increased difficulty in administering successful intradermal injections. Cell culture-derived vaccines can be used for the parenteral vaccination of companion animals and livestock, and have also been used to develop oral vaccines for wildlife immunization [25]. The combination of high titres of attenuated strains of RABV with an oral bait attractive to wildlife vectors such as the red fox (Vulpes vulpes) have been highly effective at eliminating rabies from western Europe and remain in use throughout eastern Europe and Turkey [26,27]. Parenteral vaccination with tissue culture-derived vaccines has been administered since the 1970s, and has been used extensively in all continents of the world. They have low levels of side effects, can be produced at low cost and have found application in both human and veterinary medicine. The major disadvantage for PEP is the need for compliance with repeated injections to ensure that treatment is successful. In practice, this requires multiple trips to a health clinic which, if not followed, can lead to vaccine failure. The cost of a full course of vaccination, particularly in parts of Asia and Africa, also remains a problem regarding the delivery of appropriate, effective treatment to those who require it. These two factors, along with ignorance of both the disease and the consequences of a dog bite, explain the continuing persistence of human deaths due to rabies in many areas of the world. A vaccine that could achieve protection against rabies, but with fewer injections, would be of great benefit in the treatment of rabies. Alternative development of rabies vaccines Despite the undoubted success of current commercial vaccines against rabies there have been numerous attempts to develop alternatives, all taking advantage of the genetic manipulation revolution. Antibodies have been shown to be critical for protection against the spread of RABV [9]. The key target for antibodies is virus glycoprotein. Glycoprotein is the only surface-exposed protein on the virion particle, and a number of antigenic sites to which neutralizing monoclonal antibodies bind have been identified on this protein [28,29]. The ability to clone the RABV glycoprotein into bacterial plasmids and then express the protein in a range of systems has led to a number of alternative approaches with the potential for new vaccines against rabies. In each case the recombinant protein, expressed in a range of vectors, has been shown to be protective in mouse models of vaccination and virus challenge. The following are examples of this: •  • RABV glycoprotein expressed on the surface of the vaccinia virus [30]. •  • RABV glycoprotein expressed on the surface of the canary pox virus [31]. •  • RABV glycoprotein expressed by canine adenovirus [32]. •  • A chimeric lyssavirus glycoprotein with segments derived from RABV and Mokola virus that provide immunization against more than one lyssavirus [33]. •  • DNA vaccination with RABV glycoprotein cloned into a plasmid vector [34]. Despite the ability to rapidly induce high titres of RABV neutralizing antibodies, effective at preventing infection in small animal models, they have been unable to challenge existing vaccines, principally on cost and acceptance for human use. Reverse genetics as a new paradigm in rabies vaccine development A new avenue for research on RABV biology and rabies vaccine development was opened with the ability to manipulate the viral genome. An attenuated, fixed strain of RABV, SAD B19, a European derivative of an American SAD strain (see Box 1), was recovered from a plasmid-encoded genome by Conzelmann and Schnell [35]. This has paved the way for a range of developments in vaccination biology through manipulation of the RABV genome. Table 3 provides a visualization of these modified genomes and the outcomes from their application to pathogenesis and vaccine development studies. Table 3. Examples of recombinant rabies virus (RABV). Schematic Comments Reference 1. HIV: human immunodeficiency virus; MIP1α: macrophage inflammatory protein 1α; GM-CSF: granulocyte–macrophage colony-stimulating factor. First demonstration of recovery of a recombinant RABV [35] Addition of a second glycoprotein coding sequenced increased expression, anti-glycoprotein antibody production and protection against subsequent infection with virulent RABV [45] Introduction of the HIV GAG protein produced a potential vaccine candidate for HIV. Further introduction of the interferon β gene attenuated the virus but enhanced the induction of activated CD8+ T cells [46] Addition of CCL3 (MIP1α) reduced virulence, increased recruitment of dendritic cells to the inoculation site and increased anti-RABV antibody production [47] Intracerebral inoculation with a recombinant RABV expressing GM-CSF prevented rabies in mice infected with a virulent strain of RABV [43] The persistent challenge in the field of RABV therapy is how to treat patients who have developed rabies beyond palliative care. A recent development and application of therapeutic coma [36] has met with a small number of successes, but also an increasing number of failures [10,37]. Experimental models suggest that the mammalian host produces a vigorous innate immune response to RABV infection in the brain [38–40]; however, there is strong evidence that this response is antagonized to some extent by the viral phosphoprotein [41,42]. This subversion may be the reason for the inability of the innate and adaptive immune response to control RABV replication in the central nervous system and leads ultimately to the death of the host. It is therefore encouraging that therapeutic treatment with modified rabies genomes appears to attenuate infection with a more virulent strain [43], and offers the future possibility that these may form the foundation of a future successful treatment to ameliorate the worst outcomes of RABV infection. Conclusions Rabies vaccines for humans are highly effective when given pre- or post-exposure to a bite from a potentially rabid animal. Recent alternatives have not (and are unlikely to in the near future) challenged current vaccines on the grounds of cost and acceptance. Indeed, it could be argued that there is no requirement for further development of vaccines for post-exposure treatment with current technologies, other than to ensure that the vaccine candidate is effective against the full spectrum of viruses within the genus Lyssavirus. However, the inability of current medicine to prevent patient death once RABV has entered the CNS remains a major challenge, particularly in the absence of effective anti-viral agents to RABV. Recent developments using reverse genetic approaches to modification of the RABV genome have generated potential tools for preventing rabies in murine models. Their use has been advocated for oral vaccination of dogs and wildlife [44]. These developments need to be explored further, but offer some hope for realizing the aim of preventing human deaths due to infection with RABV. Acknowledgements This work was funded by Department for Environment, Food and Rural Affairs (DEFRA) grant SV3500. 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