Funciones del cuento infantil

Detection of anti-KHV antibodies is often not feasible until later stages of KHV infection (Ronen et al., 2003; Perelberg et al., 2008; St-Hilaire et al., 2009; Matras et al., 2012), which may compromise the application of DIVA vaccination strategies as they require detection of anti-KHV antibodies. Additionally, anti-viral antibodies only indicate exposure to the virus, but do not confirm infection. Therefore accompanying methods for direct detection of the virus are required for its control, especially during the earliest stages of infection, so that appropriate measures can put in place.

Various methods have been used for detecting aquatic viruses within the host following infection, which have provided information not only on viral pathogenesis, but importantly, have also revealed the difficulties associated with the application of particular diagnostic methods with respect to specific stages of infection (Sano et al., 1991; 1992; 1994;

Lopez-Jimena et al., 2011; 2012).

The various in situ detection methods have their advantages and disadvantages (Adams et al., 2008). Compared to immunohistochemistry (IHC) and immunofluorescent antibody tests (IFAT), the use of In situ hybridisation (ISH) is less prone to false negative results that can occur in the former two methods due cross-linking of antigens, and subsequent masking of epitopes, in tissues following formalin fixation. However, viral DNA detection by ISH does not indicate whether or not viral replication is taking place, whereas IHC and IFAT can indicate the presence of viral structural proteins (e.g. γ; late proteins), and hence replication. Unlike IHC and ISH, IFAT does not suffer from problems with

non-specific staining due to the presence of endogenous peroxidases, but pathological lesions and host responses cannot be determined by IFAT as the tissue is not counterstained (as in IHC and ISH). IFAT is often the most ideal approach for virus detection compared to IHC because of its superior sensitivity (Adams and Thompson, 2006; Adams et al., 2008). In terms of histopathology, IHC is preferred to ISH as the tissue integrity is usually well preserved in IHC (Adams and Thompson, 2008). Proteinase K digestion is required during ISH to enable hybridisation of probes to DNA and this often leads to structural denaturation.

There are many reports relating to detection and analysis of KHV for both diagnostics and research, where the authors utilised polyclonal rabbit anti-sera for antigen detection (Hedrick et al., 2000; Pikarsky et al., 2004; Rosenkranz et al., 2008; Kempter et al., 2009;

Bergmann et al., 2010c), and only very few have used MAbs (Kempter et al., 2009;

Bergmann et al., 2010c; Aoki et al., 2011) or ISH for diagnostics (Bergmann et al., 2006;

2007; 2009b; 2010c; Kempter et al., 2009; Lee et al., 2012). No studies have been undertaken with these methods do determine their sensitivity for KHV. Application of such methods could also provide vital information with regards to pathogenesis, i.e. the expression of known antigens in infected carp tissues, the portal of entry and target tissues for viral replication, especially with respect to asymptomatic carriers.

DNA probes have been previously used to detect other aquatic herpesviruses, such as Channel catfish virus (CCV) in asymptomatic fish using Southern blotting (Wise et al., 1985;

Gou et al., 1991). Detection of viral nucleic acid, e.g. by expression ISH, does not always correlate with positive detection of viral protein by IHC, even within sections of the same tissue samples, as noted in turbot injected with a DNA vaccine against Nodavirus (Sommerset et al., 2005b). This can be associated with both assay sensitivity and time lag between transcription (detected by the ISH probe) and translation (detected by the IHC MAb) (Sommerset, et al., 2005b).

ISH has proved very sensitive for detecting viral DNA of eel herpesvirus (Herpesvirus anguillidae; HVA) at early stages (3 hpi) in infected cell cultures (Shih et al., 2003).

Conserved genomic regions have been targeted in ISH for the detection of fish viruses (Alonso et al., 2004; Huang et al., 2004) such as the major capsid protein gene of Iridovirus (Huang et al., 2004). Alonso et al. (2004) detected the virus in cell culture after 8 hpi with ISH compared to 24 hpi with IHC. However, specificity is key for KHV bearing in mind that it is closely related to carp herpesviruses, CyHV-1 (carp pox virus) and CyHV-2 (Goldfish herpesviral haematopoietic necrosis virus) (Waltzek et al., 2005).

Using IFAT on experimentally infected carp enabled the progressive systemic infection of another aquatic herpesvirus, carp pox, to be investigated in detail where antigens were detected in the gills and gastrointestinal tract after only 2-3 dpi then later in the skin (Sano et al., 1991). However, due to various sensitivities of detection methods, there has been much debate regarding the pathogenesis and portals of entry in fish. For example with IHNV the use of bioluminescence, IHC, TEM and virus infectivity titration resulted in conflicting results (Yamamoto and Clermont, 1990; Yamamoto et al., 1990; Harmache et al., 2006). In a study on the pathogenesis of Red Spotted Grouper Nervous Necrosis Virus (RGNNV) in European sea bass, ISH was unable to detect viral genome in some tissues that were positive by RT-qPCR during a time course of experimental infection (Lopez-Jimena et al., 2011).

Furthermore, IHC detected antigens in tissues where ISH was negative for genomic viral RNA in the same challenge (Lopez-Jimena et al., 2012). This, like other studies indicates the necessity to determine the most ideal target antigens of antibody-based methods as well as the target genes or sequences for ISH methods for in situ diagnostics.

Latency is a silent persistence of virus in the host (Pastoret et al., 1982), which results in less viral antigen within host tissues for detection by antibody based methods such as IHC and IFAT (Thiry et al., 1986). However, nucleic acid based detection methods such as ISH

have previously been successfully used to define mechanisms of herpesviruses during latent infections in mammals and fish (Teo and Griffin, 1990; Sano et al., 1994; Cardoso et al., 2012). Whereas viral antigens could not be detected during the period between acute infection and tumor formation in carp experimentally infected with carp pox, DNA could be detected by ISH (Sano et al., 1991; 1992; 1994).

It would useful to investigate the application of in situ diagnostic methods for detecting KHV infected carp during the early stages of infection, but it is vital to compare these methods with the most commonly, and reliably, used molecular detection methods from extracted DNA.