Role of cell death in the resolution of infections

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The innate immune response is the first line of host defense against pathogens. Phagocytes constitute a major cellular arm of innate immunity and, therefore, will be the focus of this review. In many organisms, including fish, macrophages and granulocytes are the two cell types specialized in the elimination of pathogen by phagocytosis. Macrophages are long-living, resident cells which are present in all compartments of the body, where they play a very important role in the initial stages of infection, as it is the first phagocyte to encounter invading microorganisms. The other major professional phagocyte type is the neutrophil, which is armed with powerful antimicrobial molecules and is rapidly recruited to infectious foci from the hemopoietic organs and blood. Macrophages have an efficient antibacterial phagocytic activity but they are also major scavenger cells and have the critical role of eliminating the potentially cytotoxic, moribund, apoptotic neutrophils and neutrophilic apoptotic bodies in infectious/inflammatory foci [1]. When this does not occur, apoptosing cells disintegrate through secondary necrosis [2] and [3], which is then responsible for triggering an inflammatory response and tissue damage [4], [5] and [6].

It has been demonstrated that apoptosis of the infected cells can also limit the spread of intracellular microorganisms by provoking inflammatory responses as a complementary mechanism for the removal of these cells through the recruitment of phagocytes [7]. Bacterial pathogens have developed different strategies to survive inside the host and to overcome their natural defenses, and thus cause disease. Destruction of host phagocytes by bacteria that proliferate extracellularly will deprive the infected host of the crucial defense mechanism represented by phagocytosis. So, the induction of host cell apoptosis by bacteria is considered an important mechanism for counteracting host immune defenses [8] and [9]. On the other hand, some obligate intracellular pathogens can exert an anti-apoptotic effect upon host cells, so that they can grow and multiple inside them [10], [11] and [12]. This is achieved by interfering with host cell apoptotic processes [13], [14], [15] S.K. Sukumaran, S.K. Selvaraj and N.V. Prasadarao, Inhibition of apoptosis by Escherichia coli K1 is accompanied by increased expression of BclXL and blockade of mitochondrial cytochrome c release in macrophages, Infect Immun 72 (2004), pp. 6012-6022. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (19)[15] and [16].

Although information about the role played by apoptosis in the resolution of bacterial infection in fish is scant, this section summarizes the few data available about these processes in some of the most important bacterial infectious diseases of fish.

1.1. Vibrio anguillarum

The Gram-negative bacterium V. anguillarum is an important pathogen that causes vibriosis in several species of freshwater and marine fish, particularly Salmoniformes (trouts and salmons) and Perciformes (sea bass, seabream, etc.). It has been shown that V. anguillarum is able to survive inside the phagocytes of the sea bass (Dicentrarchus labrax L.) disrupting the leukocyte respiratory burst, which is responsible for the generation of extremely toxic reactive oxygen intermediates [17]. In addition, V. anguillarum induces sea bass phagocyte survival through manipulation, for its own benefit, of the host apoptotic program. Thus, it is able to downregulate the expression of the apoptotic caspase-3 and caspase-9, thereby providing a safe haven for growth inside professional phagocytes [17]. Strikingly, formalin-killed bacteria slightly increase the respiratory burst of leukocytes and have no effect on the expression of caspase-3 and caspase-9. However, both live and formalin-killed bacteria induce similar cytokine and chemokine expression profiles in infected fish [17]. Collectively, these results support the idea that the pathogen actively interferes with the killing mechanisms and apoptotic program of host cells.

Unfortunately, the mechanisms involved in the regulation of apoptosis of sea bass leukocytes by V. anguillarum have not been investigated, but, taking into account the ability of mammalian bacterial pathogens to interfere with both pathways, interference with both the extrinsic and the intrinsic pathway of apoptosis are expected. Thus, interference with cytochrome c release from the mitochondria [15] and the activation of anti-apoptotic factors [14] are both well documented in mammals. In addition, a critical role for NO in regulating the initiation of macrophage apoptosis during infection has recently been described [16]. These authors [16] report that mice deficient in inducible NO synthase (iNOS−/−) show decreased rates of alveolar macrophage apoptosis in vivo and this is associated with a greater degree of inflammation in the lung. These results support the notion of a direct relationship between reactive nitrogen intermediates, apoptosis inhibition and inflammation.

1.2. Photobacterium damselae ssp. piscicida

The Gram-negative bacterium, P. damselae ssp. piscicida (Phdp), is an extracellular pathogen that causes fish pasteurellosis, a serious bacterial disease affecting several economically important marine fish species such as yellowtail, gilthead seabream, striped jack, sole and sea bass. A detailed immunocytochemical analysis on sea bass, seabream and sole infected with Phdp showed that the bacteria appeared almost exclusively as extracellular bacilli and were particularly abundant in the spleen and head kidney [18]. Phdp was also present in gut lamina propria, liver sinusoids, and blood, suggesting bacteremia and septicemia [18].

The infection of sea bass by intraperitoneal injection of Phdp results in apoptosis of neutrophils and macrophages in the peritoneal cavity and in the head kidney [19], the equivalent to mammalian bone marrow in terms of hematopoietic activity [20], [21] and [22]. A series of elegant studies by do Vale and co-workers has provided interesting insights into the mechanism involved in the induction of apoptosis of professional phagocytes by this bacterium. These authors used a wide array of techniques to shed light into these mechanisms, including, light and electron microscopic observation of peritoneal neutrophils and macrophages, and specific in situ detection on DNA fragmentation in peritoneal cells by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining and agarose gel electrophoresis of DNA extracted from peritoneal leukocytes. Notably, since macrophages are key cells in the elimination of both bacteria and apoptotic moribund cells and apoptotic bodies, the induction by Phdp of simultaneous macrophage and neutrophil apoptosis results, on the one hand, in the destruction of the two phagocytic cell types involved in the clearance of the bacteria and, on the other hand, in the progression of the apoptotic process towards secondary necrosis [23]. The lysis of neutrophils by secondary necrosis leads to the extensive release of their highly cytotoxic components and tissue damage. These two effects promote the survival and extracellular multiplication of Phdp [23].

It was also found that intraperitoneal injection of culture supernatants from virulent bacterial cultures results in extensive apoptosis of sea bass macrophages and neutrophils [19]. Since UV-killed virulent bacterial cells and avirulent strains do not trigger the apoptotic process and the fact that apoptogenic activity of culture supernatants is abolished by heat-treatment, secreted bacterial products emerge as candidates for the apoptogenic factor [19]. Analysis of mid-exponential culture supernatants from virulent and avirulent Phdp strains revealed different patterns of secreted proteins in both strains [24]. Subsequent biochemical work using concentrated and fractionated culture supernatants led to the identification of the protein AIP56 as the apoptogenic exotoxin secreted by Phdp [24]. Intraperitoneal injection of the fraction containing AIP56 as well of the recombinant protein expressed in Escherichia coli results in high numbers of apoptotic neutrophils and macrophages [24]. Importantly, passive immunization with anti-AIP56 antibodies neutralizes AIP56 activity and protects sea bass against Phdp infection [24]. As expected from these observations, an increase in caspase-3 and caspase-9 expression and activities were found in sea bass head kidney and the blood of Phdp-infected fish, in advanced stages of septicemia [18], [25] and [26]. Notably, this activity was accompanied by an increase in circulating active neutrophil elastase, which further supports the view that neutrophils undergo apoptotic secondary necrosis [27]. The increase of elastase activity in the blood of infected fish was significant in fish at 40 h post-infection and with circulating AIP56, but not in fish without toxaemia [18]. In addition, a high degree of correlation was found between the blood levels of caspase-3 and elastase in fish with toxaemia and advanced or terminal disease, suggesting that neutrophil elastase was released by lysis of apoptosing neutrophils rather than by degranulation of stimulated neutrophils [18]. The disintegration of phagocytes by lysis due to secondary necrosis explains the extensive occurrence of cell debris in the foci of cell destruction [18], since elastase is a highly tissue-damaging enzyme [28].

1.3. Streptococcus iniae

S. iniae is an endemic fish pathogen associated with bacterial meningitis of salmonids and other fish species [29] and [30]. S. iniae probably gains access to the bloodstream and maintains a high level of bacteremia, leading to the onset of a generalized disease and meningitis, as described for other diseases [31] and [32]. Similarly to Streptococcus pyogenes (group A streptococcus) infection in humans, where serotype replacement in a population [33] is most likely to be the result of the immune status of the individuals along with the introduction of a highly virulent organism [34], the propensity of S. iniae to cause an invasive disease in fish is probably related not only with the immune status of the fish but also with the pathogenic mechanism(s) of virulent strains [35]. One of the features that allows S. iniae to establish an infection is related to its ability to overcome the immune response of macrophages, which play a role in initial phagocytosis and eventual killing of streptococci [35]. In addition, salmonid leukocytes exposed in vitro to S. iniae showed DNA fragmentation, a characteristic feature of apoptotic cells [35]. Furthermore, apoptosis occurs without the release of cellular components. More importantly, in vitro assays with salmonid primary macrophages and the macrophage cell line RTS-11 demonstrated that non-invasive and invasive strains of S. iniae shared the mechanisms to overcome host immune defenses, but differed in their profiles of bacteremia, intracellular survival and induction of apoptosis, which are far pronounced in invasive strains [35]. Since apoptosis occurs without the release of cellular components, it reduces or suppresses inflammation. Therefore, in this case apoptosis may be advantageous for the pathogen, as it might avoid the triggering and recruitment of the non-specific host defense mechanism [35]. Furthermore, macrophage death could also contribute to delaying or hindering the development of an adaptive immune response [35]. In summary, generalized meningitis induced by S. iniae is a consequence of its capacity to survive in phagocytes and to induce their apoptosis. Consequently, the apoptotic phagocytes serve as a vector, which is loaded in the blood stream and unloaded after blood-brain barrier transcytosis in the central nervous system. These results highlight the importance of macrophages as "Trojan horses" in S. iniae infection [35].

1.4. Mycobacterium marinum

Tuberculosis is one of the most dangerous and lethal infectious diseases in the world, perhaps the most prevalent, responsible for 1.7 million people death per year and 9 million newly infected people caused by different species of mycobacteria belonging to Mycobacterium tuberculosis complex [36]. Although much has been learned about the biology and transmission of intracellular pathogen species, the mechanisms of their pathogenesis and host colonization remain largely unknown.

Thanks to the use of the zebrafish (Danio rerio) as a study model for some of these human infections, great advances have been made in our knowledge of the mechanisms triggered in the immune cells after infection, as well as in the host cells-pathogen relationships, particularly in the case of infections caused by Leptospira interrogans [37] and M. marinum [38], the fish counterpart of M. tuberculosis that naturally infects the zebrafish and produces a granulomatous infection similar to tuberculosis of mammals.

In the case of pathogenic mycobacteria, they are highly adapted intracellular pathogens that can survive for indefinite periods of time within their hosts. Infection results in the recruitment of host macrophages to the bacteria in the lung tissue, their phagocytosis, and the transit of infected macrophages into deeper tissues [38]. There, the infected macrophages recruit additional macrophages and other immune cells to form tightly aggregated immune structures called granulomas, pathological hallmarks of tuberculosis and considered host-protective structures formed to contain the infection [38]. During early stages of the infection, mycobacteria are eliminated by macrophages, so that the bacterial burden decreases in wild type zebrafish compared with those lacking macrophages [39]. However, phagocytic cells are required by mycobacteria for tissue dissemination after macrophage phagocytosis and cellular infection. During this step - an innate immune phase of infection - primary granulomas formed in deeper tissues contain infected macrophages that rapidly attract new uninfected macrophages, which phagocytose apoptotic infected macrophages, leading to rapid, iterative expansion of infected macrophages and hence bacterial numbers [39].

Tumor necrosis factor α (TNFα), a major pro-inflammatory cytokine, was found to be one of the first effector molecules essential to the host-protective response against tuberculosis [40]. Mice deficient either in TNFα or TNFα receptor 1 (TNFR1) have increased susceptibility to challenge by pathogenic mycobacteria [40], [41] and [42]. Furthermore, TNFα decisively influences infection control judging by the phenotypes of mice in which the TNFα gene has been deleted. The importance of TNFα signaling in protection against human tuberculosis is highlighted by the increasing use of TNF-neutralizing drugs in treating a variety of immune and inflammatory conditions such as rheumatoid arthritis [43] and [44]. Hence, patients receiving TNFα-neutralizing therapy have an increased rate of reactivation of latent tuberculosis [45].

Clay et al. [46] showed that upon intravenous M. marinum infection of zebrafish embryos, TNFR1-deficient zebrafish succumbed to infection significantly more quickly than their control counterparts. In addition, TNFR1-deficient zebrafish showed up to tenfold greater bacterial burdens compared with control embryos in the first six days after infection [46]. These results demonstrate that TNFα signaling is important for the modulation of mycobacterial infection from its early stages and does not require adaptive immunity for protective effects in vivo [46]. Furthermore, primary granuloma formation is accelerated in the absence of TNFα signaling in zebrafish [46].

In the same study, it has been shown that macrophage mycobactericidal and anti-apoptotic activities are highly dependent of TNFα signaling both in primary and secondary granulomas composed of infected and uninfected macrophages, lymphocytes and other immune cells [46]. In the absence of TNFα, apoptotic and necrotic infected macrophages increased in granulomas. Thus, a single apoptotic infected macrophage could be phagocytosed by several separate uninfected macrophages, indicating that the death of single infected host cells is capable of spreading bacteria to multiple uninfected macrophages [46]. Moreover, infected macrophage cell death could accelerate infection both by giving rise to extracellular bacteria as well as by spreading infection to new macrophages, since extracellular infecting mycobacteria were seen to accumulate in TNFR1-deficient zebrafish embryos [46].

Summarizing, apoptotic death of infected macrophages for the control of infection may be used by mycobacteria to favor their dissemination and proliferation. Therefore, TNFα seems to play an important role during mycobacterial infection by inducing an anti-apoptotic effect in infected macrophages, leading to bacteria isolation and elimination through increased macrophage mycobactericidal activity.

2. Role of cell death in the resolution of viral infections

In recent years, many viruses of different families have been found to induce apoptosis during their infection cycles. Host cells defend themselves from viral infection by apoptosis, but viruses have also developed a range of strategies to fight against the host immune response and apoptosis; they even make use of apoptosis to propagate. For some viruses, inhibition of apoptosis appears to be essential for the maintenance of viral latency. But for many other viruses, the careful induction of apoptosis during lytic infection may represent the basis for cytotoxity and be an important outlet for the dissemination of progeny virus.

Although information on the role played by host cell death in the onset of viral infection in fish is scarce, it seems that fish viruses use similar strategies and signaling pathways as their mammalian counterparts to interfere with host cell death. This study will continue by reviewing the major viral groups for which a substantial body of information is available concerning the mechanisms displayed by the fish viruses to interfere with host-programmed cell death.

2.1. Nodaviruses

Nodaviruses are small, non-enveloped, spherical viruses with bipartite positive-sense RNA genomes, which are capped but not polyadenylated. Two genera have been distinguished within the Nodaviridae family: the alphanodaviruses that predominantly infects insects and the betanodaviruses that infect fish [47].

Betanodavirus infection results in severe morbidity and mortality in fish which provokes important economical losses to the aquaculture industry. Infected fish suffer from encephalitis that derivates in abnormal swimming behavior and nervous necrosis [48] and [49]. Histopathological changes are characterized by extensive cellular vacuolations and neuronal degeneration in the central nervous system and retina [48] and [49]. The best characterized betanodaviruses are the greasy grouper (Epinephelus tauvina) nervous necrosis viruses (GGNNV) of the Singapore strain, and the red spotted grouper (Epinephelus akaara) nervous necrosis virus (RGNNV).

Infection of sea bass cells with GGNNV induced a typical cytopathic effect (CPE), with cytoplasmic vacuolation, thinning, rounding up, the detachment of infected cells from the cultured dish and, eventually, cell lysis and death. GGNNV infection induced apoptosis in sea bass cells [50]. The infected sea bass cells underwent DNA fragmentation and stained positive in TUNEL assay [50]. Furthermore, GGNNV-infected sea bass cells showed an increased activity of caspase-8-like and caspase-3-like proteases, whereas inhibitor of caspase-8 and caspase-3 reduced GGNNV-induced apoptosis [50]. Similarly, RGNNV induced the host apoptosis which precedes the onset of necrosis in a grouper liver cell line (GL-av) [51]. These processes seemed to be mediated through mitochondria disruption, but it was caspase-independent [51].

As stated above, these viruses contain two genomic RNA segments. The largest genomic sequence, RNA1, contains 3103 nt that encodes protein A, which is an RNA-dependent RNA polymerase [52]. The middle genomic segment RNA2 (1433 nt) encodes the capsid protein α [53]. In addition, nodaviruses also synthesize RNA3, a sub-genomic RNA species from the 3′ terminus of RNA1 [54]. RNA3 encodes two small proteins: B1 (111 amino acids) and B2 (75 amino acids) [50].

Recent studies have shown how several of these virus-encoded proteins could be involved in the survival and replication of nodavirus by regulating apoptosis in host cells. For example, RGNNV RNA 2-encoding protein α was cloned and transfected into tissue culture cells (GF-1) which then underwent apoptosis or post-apoptotic necrosis. Protein α induced the progressive loss of mitochondrial membrane potential (MMP) 24, 48, and 72 h post-transfection, which coincided with cytochrome c release, especially at 72 h post-transfection [55] and [56]. The mitochondrial permeability transition pore can be blocked by two anti-Bcl-2 family members from zebrafish, zfBcl-X(L) and zfMcl-1a [55] and [56]. In agreement with it, when RNA2-transfected cells were co-transfected with zfBcl-xL, loss of MMP was prevented at 24 and 48 h post-transfection, while initiator caspase-8 and effector caspase-3 activations were also blocked at 48 h post-transfection [57]. These data indicate, therefore, that RGNNV protein α induces apoptosis, followed by secondary necrotic cell death through a mitochondria-mediated death pathway and activation of caspase-8 and caspase-3, at least in GF-1 cells.

Protein B2 plays a dual-role in the viral replication process. On one hand, it is able to silence RNA interference (RNAi), which mediates the regulation of animal and plant innate immune responses [58], [59] and [60]. On the other hand, B2 could be implicated in host cell death, although this novel function is much less studied [58], [59] and [60]. In RGGNV-infected grouper liver (GL-av) cells, B2 was expressed 12 h post-infection (hpi), with increased expression between 24 and 72 hpi [50]. To further clarify its apoptotic role, B2 was transiently expressed in GL-av cells. Along with TUNEL positive cells 24 h post-transfection, the expression of the pro-apoptotic gene Bax, induced loss of MMP, but not mitochondrial cytochrome c release [50]. Using RNA interference to reduce B2 expression, both B2 and pro-apoptotic Bax expression were downregulated and RGNNV infected cells were rescued from secondary necrosis [60]. Furthermore, over-expression of anti-apoptotic Bcl-xL and Mcl-1 from zebrafish effectively prevented B2-induced mitochondria-mediated necrotic cell death [50], [56] and [57]. Taken together, these results suggest that B2 upregulates Bax and triggers mitochondria-mediated necrotic cell death, independently of cytochrome c release [60].

In sharp contrast, B1 has been shown to act as an early protein-in nodavirus infection, where it probably inhibits apoptosis. B1 showed a low level of expression in the early replication cycle being detected at 12 hpi, while its expression substantially increased at 24 hpi in RGNNV GL-av infected cells [61]. In gain-of-function studies, the over-expression of B1 was able to protect cells against necrotic cell death following RGNNV infection [61]. In loss-of-function studies, knockdown of B1 expression enhanced cell death [61]. However, the molecular mechanism underlying the role of B1 remains to be elucidated.

These results indicate that the expression of viral proteins (α, B1 and B2) is seemly coordinated. Viral B1 inhibitory-apoptosis protein was expressed in the early steps of infection allowing massive replication of the virus, while the pro-apoptotic viral proteins α and B2 were expressed in the late steps of infection, promoting cell necrosis and subsequently, virus dissemination. However, further in vivo studies need to be performed to confirm whether this coordinated expression of viral proteins reflects a viral-infection strategy. Also, deeper research will be needed to determine if initial apoptosis is due to host cell factors or by early viral gene expression.

In addition, it still remains unclear if betanodavirus-induced apoptosis requires cell caspase activation. GGNNV induced caspase-dependent apoptosis in sea bass cells [50]. Similarly, RGNNV induced caspase-dependent apoptosis in GF-1 cells, however in GL-av cells apoptosis was a caspase-independent process [50] and [56]. Collectively, these results indicate that betanodavirus may induce apoptosis via caspase-dependent or -independent processes depending on the cell type.

2.2. Birnaviruses

Infectious pancreatic necrosis virus (IPNV) is a fish pathogen and the prototype of the Birnaviridae virus family [62]. Birnaviruses possess a bi-segmented, double-stranded RNA genome contained within a medium-sized, unenveloped, icosahedral capsid. Viral gene expression involves the production of four unrelated major genes, which undergo various post-translational cleavage processes to generate three to five different structural proteins [63].

Several in vitro studies have shown that IPNV infection induces apoptosis in fish cell lines [64], [65] and [66]. Therefore, apoptosis might have an important role in the innate immune response in IPNV-infected fish. However, the role of apoptosis in IPNV infection in vivo remains controversial. In fact, apoptotic cells were observed in the head kidney, spleen, kidney and liver of IPNV-infected fish, but IPNV antigen-positive cells with an apoptotic nucleus were less frequent [67]. A plausible explanation for these paradoxical observations would be that the outcome for an IPNV-infected cell might depend upon the host cell ability to mount an apoptotic response to the virus infection [67]. Thus, if the virus has the upper hand, it will replicate at a high rate, resulting in the production of a high number of progeny, causing cell lysis and, finally, necrosis. When the balance is in favor of the host cell, it will enter into apoptosis at an early stage of the infection and limit the production and spread of virus progeny. Probably, interferon sensitizes nearby cells to the apoptosis-inducing effect of double-stranded RNA; consequently, the cells localized around the IPNV-infected cell would respond quicker to the viral infection than the cell initially infected [68].

In persistently infected fish, IPNV was detected in macrophages [69]. However, in another study of persistently infected rainbow trout, IPNV-positive cells in leukocyte smears from peripheral blood, head kidney and spleen did not have apoptotic nuclei [67]. Assuming the IPNV-positive cells to be macrophages, a possible explanation for the lack of apoptotic morphology in these cells is that differentiated macrophages are robust, long-lived cells and resistant to numerous death stimuli [70], [71] and [72]. An alternative explanation for this observation is that persistent infection of macrophages is associated with specific inhibition of host-induced apoptosis. Viral IPNV non-structural protein VP5 appeared as a good candidate for this function, since it contains Bcl-2 homologous domains and inhibits apoptosis when expressed in cell cultures. However, it was demonstrated that the induction of apoptosis in hepatocytes of IPNV-infected Atlantic salmon was independent of VP5 expression [73]. Therefore, the mechanism by which IPNV might inhibit host-induced apoptosis remains unclear.

Concerning the molecular events underlying IPNV-induced apoptosis, it seems that apoptosis requires new protein synthesis [66] and that activation of caspase-3 plays a major role [74]. In addition, activation of the transcription factor NF-κB via the tyrosine kinase pathway would be involved in the apoptosis induced by IPNV, since tyrosine kinase inhibitors block DNA fragmentation and enhanced cell viability in IPNV-infected salmon CHSE-214 cells [75]. Interestingly, zebrafish can be infected by IPNV [76], which represents a gain- and loss-of-function gene model for the study of IPNV-induced apoptosis.

2.3. Iridoviruses

The iridovirus family includes the genera Iridovirus, Chloriridovirus, Ranavirus and Lymphocystivirus. Some iridoviruses, such as epizootic hematopoietic necrosis virus (EHNV), frog virus 3 (FV 3), red sea bream iridovirus (RSIV), lymphocystis disease virus (LCDV), Chilo iridescent virus (CIV), Rana grylio virus (RGV) and grouper iridovirus (GIV), induce apoptosis in their host cells. The major morphological phenomenon observed when fish cells are infected with iridovirus is the typical CPE.

In RSIV-infected bluestriped grunt (Haemulon sciurus) fin (GF) cells, studies have been made to ascertain whether the CPEs need the activation of caspases. Although inhibitors of caspase-3 and caspase-6 were able to block cell enlargement and formation of apoptotic body-like vesicles in RSIV-infected GF, they failed to inhibit cell rounding [77]. In addition, LCDV studies provided evidence that induced apoptotic cell death occurred in vitro, as demonstrated by cell nucleus chromatin condensation, chromosomal DNA fragmentation and caspase activation in flounder gill cells [78]. Further, RGV infection of a fish cell line resulted in mitochondrial distribution changes [77]. Thus, a reduction in MMP provoked mitochondrial fragmentation and an increased activity of caspase-9 and caspase-3. Curiously, RGV caused NF-κB activation and intracellular Ca2+ increase during virus-induced apoptosis in fish cells [79]. Collectively, these data suggest that RGV triggers mitochondrion-mediated apoptosis.

The complete sequencing and analysis of the GIV genome revealed the presence of an open reading frame encoding an anti-apoptotic Bcl-2-like gene [66] and [80]. Moreover, a homolog of LPS-induced tumor necrosis factor (TNF)-α factor (LITAF) has been cloned from SGIV [81]. Both molecules could be involved in the apoptosis of SGIV-infected cells by regulating virus-host interactions. To date, it is known that LITAF from SGIV is able to associate with mitochondria and its over-expression induced apoptosis simultaneously, probably through disruption of MMP and activation of caspase-3, NF-κB and NFAT [81]. Therefore, current data suggest that apoptosis induced by LITAF from the SGIV, a protein encoded by the virus itself, may contribute to virus transmission during SGIV replication [81]. On the other hand, Bcl-2-like gene from this virus is an early expressed viral gene that localizes on the mitochondrial membrane and its over-expression was able to inhibit UV-irradiated apoptosis in grouper kidney cells [82].

Taking all these data together, it seems that RSIV induces caspase-dependent apoptosis during permissive replication in host cells. However, SGIV is capable of inhibiting the apoptosis process in its host cells at an early stage of virus infection, viral Bcl-2-like protein probably being involved in this early apoptosis inhibition process. In fact, a putative NF-κB binding site is located in the upstream promoter region of the SGIV-Bcl-2-like gene. Hypothetically, viral infection would cause translocation of NF-κB to the nucleus, where it would stimulate the expression of numerous genes involved in the immune response and apoptosis. Simultaneously, NF-κB could bind to the SGIV-Bcl-2-like promoter and induce the expression of this anti-apoptotic molecule. Further investigation is needed to verify whether the pro-apoptotic process initiated by host cells is counteracted by the Bcl2-like protein of the virus, which would lead to its replication within the cell. At a later stage, however, the virus would promote apoptosis via LITAF, facilitating cell rupture and viral dissemination. Moreover, the main targets of SGIV are the kidneys and spleen, two major lymphomyeloid organs of fish. This would definitely interfere with the host immune system; however, this, too, needs further investigations.

2.4. Rhabdoviruses

Spring viremia of carp virus (SVCV) is a tentative member of the Vesiculovirus genus within the Rhabdovirus family [83] and [84]. SVCV, induces lethal dropsy, hemorrhagic swim bladder inflammation and peritonitis in infected fish. The SVCV virion contains one molecule of linear, negative-sense, single-stranded RNA that encodes 5 structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and viral RNA-dependent RNA polymerase (L) in the order 3′-N-P-M-G-L-5′ [84]. Importantly, the G protein comprises the major antigenic determinant of the virus assembly and budding of the virus [84].

When epithelioma papulosum cyprini (EPC) cells were infected with SVCV and were observed by electron microscopy for evidence of apoptosis induction, the infected cells showed structural changes such as cell shrinkage, membrane blebbing, breakdown into apoptotic bodies, nuclear condensation and vacuolization of the cytoplasm [85]. The human endogenous acid cysteine proteinase inhibitor was capable of inhibiting, or at least delaying, DNA fragmentation and morphological changes associated with apoptosis during infection [85].

Unfortunately, no other studies focus on apoptosis in SVCV infections. As there is evidence supporting the effect of the M protein of vesicular stomatitis virus (VSV), a vesiculovirus that infect mammalian cells, on the caspase-8-dependent induction of apoptosis [86], [87] and [88], it could be interesting to determine whether the M protein of SVCV and other fish rhabdovirus could also induce apoptosis. Thus, there is one study showing that transfection of M gene from infectious hematopoietic necrosis virus (IHNV), another fish rhabdovirus responsible for important salmonid aquacultures losses [83], caused morphological changes of apoptosis, including nuclear fragmentation and cell shrinkage [89]. As zebrafish is susceptible to infection by SVCV under conditions that mimic a natural route of exposure [90] and [91], the SVCV-zebrafish model could be used to further investigate and elucidate the mechanism orchestrating the SVCV-induced apoptosis.

2.5. Orthomyxoviruses

Infectious salmon anemia virus (ISAV) is an aquatic orthomyxovirus with single-stranded RNA genome that is the causative agent of infectious salmon anemia (ISA), a disease of great importance in the Atlantic salmon farming industry. ISAV may infect and replicate in other fish as sea trout, brown trout, rainbow trout, eels, herring and Arctic char, resulting in asymptomatic fish, probably lifelong carriers of the virus [92].

ISAV infection causes a CPE in permissive cell lines from Atlantic salmon, including SHK-1, TO and ASK-2 cells, which are macrophage-like cell lines [93]. CPE in SHK-1 and ASK-2 cells is associated with apoptosis, but with necrosis and the release of high-mobility group 1 (HMGB1) protein in TO cells, so the mechanism of cell death during ISAV infection is cell type-specific [93]. This could explain clinical diseases and the pathology of this virus in vivo. In a natural infection, ISAV would destroy susceptible cells, such as leukocytes, by necrosis, leading to inflammatory reactions due to the release of HMGB1 protein and subsequent immune response [93]. However, ISAV is also capable of inducing apoptosis in less susceptible cells, such as the heart and hematopoietic portion of the kidney. This would not cause inflammatory reactions and, therefore, lead to subclinical disease and virus persistence during a natural infection [93].

There is evidence supporting the involvement of caspases in ISAV-induced apoptosis. For example, the use of the pan-caspase inhibitor, Z-VAD-fmk, inhibited apoptosis [93]. Others found that caspase-3 and caspase-7 activity increased during the course of infection in ASK and SHK-1 cells [94]. In addition, the protein encoded by the segment 7 ORF2, has the specific potential to bind caspase-8, which might have implications for ISAV-induced apoptosis [93]. So far, little is known about the mechanism through which ISAV evades immunological surveillance in fish. However, these preliminary data point to cell-induced apoptosis as a key for understanding the pathogenesis and persistence of ISAV in Atlantic salmon.

3. Conclusions

The choice between life and death is one of the major events in the regulation of the immune response and the resolution of infection. Pathogens have developed sophisticated mechanisms to interfere with programmed cell death of the host, allowing them to evade the immune response and to replicate within the host. While the induction of apoptosis would benefit the resolution of some infectious diseases, others require inhibition of the same. The use of genomics-based technologies has dramatically enhanced our knowledge of the mechanisms of pathogen virulence and of fundamental aspects of immunology and cell biology. A better understanding of the fish innate host defense, host cell death and viral and bacterial pathogenesis will shed light on the evolution of defense mechanisms against infectious agents and will likely pave the way for the development of vaccines, treatments and prophylactic agents designed to prevent and/or control infections in aquaculture.