aav serotype 2. however, aav is highly prevalent in humans and other primates and several serotypes have been isolated from various tissue samples. serotypes 2, 3, 5, and 6 were discovered in human cells, aav serotypes 1, 4, and 7–11 in nonhuman primate samples. as of 2006 there have been 11 aav serotypes described, the 11th in 2004. aav capsid proteins contain 12 hypervariable surface regions, with most variability occurring in the threefold proximal peaks, but the parvovirus genome in general presents highly conserved replication and structural genes across serotypes. all of the known serotypes can infect cells from multiple diverse tissue types. tissue specificity is determined by the capsid serotype and pseudotyping of aav vectors to alter their tropism range will likely be important to their use in therapy. serotype 2 serotype 2 (aav2) has been the most extensively examined so far. aav2 presents natural tropism towards skeletal muscles, neurons, vascular smooth muscle cells and hepatocytes. three cell receptors have been described for aav2: heparan sulfate proteoglycan (hspg), avβ5 integrin and fibroblast growth factor receptor 1 (fgfr-1). the first functions as a primary receptor, while the latter two have a co-receptor activity and enable aav to enter the cell by receptor-mediated endocytosis. these study results have been disputed by qiu, handa, et al. hspg functions as the primary receptor, though its abundance in the extracellular matrix can scavenge aav particles and impair the infection efficiency. studies have shown that serotype 2 of the virus (aav-2) apparently kills cancer cells without harming healthy ones. "our results suggest that adeno-associated virus type 2, which infects the majority of the population but has no known ill effects, kills multiple types of cancer cells yet has no effect on healthy cells," said craig meyers, a professor of immunology and microbiology at the penn state college of medicine in pennsylvania in 2005. this could lead to a new anti-cancer agent. in march 2023, a series of nature papers linked infection of adeno-associated virus 2 (aav2) to a wave of childhood hepatitis. other serotypes although aav2 is the most popular serotype in various aav-based research, it has been shown that other serotypes can be more effective as gene delivery vectors. aav9 passes the blood-brain-barrier in humans, aav6 appears much better in infecting airway epithelial cells, aav7 presents very high transduction rate of murine skeletal muscle cells (similar to aav1 and aav5), aav8 transduce hepatocytes and aav1 and 5 were shown to be very efficient in gene delivery to vascular endothelial cells. in the brain, most aav serotypes show neuronal tropism, while aav5 also transduces astrocytes. aav6, a hybrid of aav1 and aav2, also shows lower immunogenicity than aav2. serotypes can differ with the respect to the receptors they are bound to. for example, aav4 and aav5 transduction can be inhibited by soluble sialic acids (of different form for each of these serotypes), and aav5 was shown to enter cells via the platelet-derived growth factor receptor. synthetic serotypes there have been many efforts to engineer and improve new aav variants for both clinical and research purposes. such modifications include new tropisms to target specific tissues, and modified surface residues to evade detection by the immune system. beyond opting for particular strains of recombinant aav (raav) to target particular cells, researchers have also explored aav pseudotyping, the practice of creating hybrids of certain aav strains to approach an even more refined target. the hybrid is created by taking a capsid from one strain and the genome from another strain. for example, research involving aav2/5, a hybrid with the genome of aav2 and the capsid of aav5, was able to achieve more accuracy and range in brain cells than aav2 would be able to achieve unhybridized. researchers have continued to experiment with pseudotyping by creating strains with hybrid capsids. aav-dj has a hybrid capsid from eight different strains of aav; as such, it can infect different cells throughout many areas of the body, a property which a single strain of aav with a limited tropism would not have. other efforts to engineer and improve new aav variants have involved the ancestral reconstruction of virus variants to generate new vectors with enhanced properties for clinical applications and the study of aav biology. immunology aav is of particular interest to gene therapists due to its apparent limited capacity to induce immune responses in humans, a factor which should positively influence vector transduction efficiency while reducing the risk of any immune-associated pathology. aav is not considered to have any known role in disease. however, host immune system response and immune tolerance reduce the efficacy of aav-mediated gene therapy. host immune response has been shown to respond to the aav vectors, the transduced cells, and the transduced proteins. the immune response can be subdivided into two categories: innate and adaptive, the latter of which is divided into humoral and cell-mediated. innate the innate immune response to the aav vectors has been characterised in animal models. intravenous administration in mice causes transient production of pro-inflammatory cytokines and some infiltration of neutrophils and other leukocytes into the liver, which seems to sequester a large percentage of the injected viral particles. both soluble factor levels and cell infiltration appear to return to baseline within six hours. by contrast, more aggressive viruses produce innate responses lasting 24 hours or longer. in-vivo studies indicate that aav vectors interact with the toll-like receptor (tlr)9- and tlr2-myd88 pathways to trigger the innate immune response by stimulating the production of interferons. it's shown that mice deficient in tlr9 are more receptive to aav treatment and demonstrate higher levels of transgene expression humoral due to previous natural infection, many people have preexisting neutralizing antibodies (nabs) against aav's, which can significantly hinder its application in gene therapy. even though aav's are highly variable among wild-type and synthetic variants, antibody recognition sites may be conserved evolutionarily. the virus is known to instigate robust humoral immunity in animal models and in the human population, where up to 80% of individuals are thought to be seropositive for aav2. antibodies are known to be neutralising, and for gene therapy applications these do impact vector transduction efficiency via some routes of administration. as well as persistent aav specific antibody levels, it appears from both prime-boost studies in animals and from clinical trials that the b-cell memory is also strong. in seropositive humans, circulating igg antibodies for aav2 appear to be primarily composed of the igg1 and igg2 subclasses, with little or no igg3 or igg4 present. cell-mediated the cell-mediated response to the virus and to vectors is poorly characterised, and has been largely ignored in the literature as recently as 2005. clinical trials using an aav2-based vector to treat haemophilia b seem to indicate that targeted destruction of transduced cells may be occurring. combined with data that shows that cd8+ t-cells can recognise elements of the aav capsid in vitro, it appears that there may be a cytotoxic t lymphocyte response to aav vectors. cytotoxic responses would imply the involvement of cd4+ t helper cells in the response to aav and in vitro data from human studies suggests that the virus may indeed induce such responses, including both th1 and th2 memory responses. a number of candidate t cell stimulating epitopes have been identified within the aav capsid protein vp1, which may be attractive targets for modification of the capsid if the virus is to be used as a vector for gene therapy. infection cycle there are several steps in the aav infection cycle, from infecting a cell to producing new infectious particles: attachment to the cell membrane receptor-mediated endocytosis endosomal trafficking escape from the late endosome or lysosome translocation to the nucleus uncoating formation of double-stranded dna replicative form of the aav genome expression of rep genes genome replication expression of cap genes, synthesis of progeny ssdna particles assembly of complete virions, and release from the infected cell. some of these steps may look different in various types of cells, which, in part, contributes to the defined and quite limited native tropism of aav. replication of the virus can also vary in one cell type, depending on the cell's current cell cycle phase. the characteristic feature of the adeno-associated virus is a deficiency in replication and thus its inability to multiply in unaffected cells. adeno-associated virus spreads by co-infecting a cell with a helper virus. the first helper virus that was described as providing successful generation of new aav particles, was the adenovirus, from which the aav name originated. it was then shown that aav replication can be facilitated by selected proteins derived from the adenovirus genome, by other viruses such as hsv or vaccinia, or by genotoxic agents, such as uv irradiation or hydroxyurea. depending on the presence or absence of a helper virus, the life cycle of aav follows either a lytic or lysogenic pathway, respectively. if there is a helper virus, aav's gene expression activates, allowing the virus to replicate using the host cell's polymerase. when the helper virus kills the host cell, the new aav virions are released. if there is not a helper virus present, aav exhibits lysogenic behavior. when aav infects a cell alone, its gene expression is repressed (aav does not replicate), and its genome is incorporated into the host genome (into human chromosome 19). in rare cases, lysis can occur without a helper virus, but usually aav can not replicate and kill a cell on its own. the minimal set of the adenoviral genes required for efficient generation, of progeny aav particles, was discovered by matsushita, ellinger et al. this discovery allowed for new production methods of recombinant aav, which do not require adenoviral co-infection of the aav-producing cells. in the absence of helper virus or genotoxic factors, aav dna can either integrate into the host genome or persist in episomal form. in the former case integration is mediated by rep78 and rep68 proteins and requires the presence of itrs flanking the region being integrated. in mice, the aav genome has been observed persisting for long periods of time in quiescent tissues, such as skeletal muscles, in episomal form (a circular head-to-tail conformation).