Indirect immunofluorescence antibody assays (IFA) were the first ones to be used for the detection of HHV-6 antibodies (Salahuddin et al., 1986; Linde et al., 1988;
Lopez et al., 1988) and remain still widely employed. In these tests, HHV-6-infected cells are fixed on a glass slide, a serum dilution is added and a fluorochrome-conjugated anti-immunoglobulin antibody is then applied to detect the binding of serum antibodies to specific antigens. When illuminated with ultraviolet light, the number of fluorescent foci as well as the characteristic pattern of cell staining observed with the microscope constitute the main parameters to be taken into account for the result. The staining of uninfected cells with a counterstain partly quenching non-specific fluorescence is also important, in particular, to check that the ratio of infected to uninfected cells is in agreement with the known characteristics of cell preparation. Infected cells consist of primary cells such as cord blood mononuclear cells or continuous cell lines such as HSB2 cells previously inoculated with a reference HHV-6 strain. These infected cells are used for the preparation of slides after a significant cytopathic effect (CPE) has been observed. A variation on conventional IFA is an anticomplement immunofluorescence assay (ACIF) in which human complement is applied to slides after the serum has been removed and before the conjugate is added; antigen-antibody-complement complex is detected by a fluorochrome-conjugated anti-C3 antibody (Lopez et al., 1988; Okuno et al., 1989; Robert et al., 1990). ACIF is considered to provide a lower background signal and a higher specific one than classical IFA. Although ACIF and IFA probably do not detect exactly the same HHV-6-specific antibodies, their results seem to be well correlated. As a general requirement, an extensive washing step after each exposure to a specific reagent (serum, complement, conjugate) is necessary to lower non-specific signal and reduce the rate of false-positive results. Nevertheless, this does not prevent the binding of cross-reactive antibodies, in particular those directed against other betaherpesviruses (Adler et al., 1993; Foa-Tomasi et al., 1994); as indicated below, more complex procedures are required to circumvent this phenomenon.
Enzyme-linked immunosorbent assays (ELISA) have been developed in recent years for the diagnosis of HHV-6 infection (Saxinger et al., 1988; Chou and Scott, 1990; Sloots et al., 1996). These assays generally use either crude lysate of infected cells or purified virus obtained from cell culture supernatant as antigens. The antigen preparation is coated on polystyrene plates or any other appropriate surface, the serum sample is then added after what an enzyme-conjugated anti-human immunoglobulin is applied; finally, a chromogenic reaction catalyzed by the enzyme permits to detect and, to some extent, quantify the complex formed by the reference antigens and corresponding serum antibodies. ELISA is usually known to be highly sensitive, simple, rather inexpensive and susceptible of being automated, some remarkable qualities which justify its extensive use for the diagnosis of many viral infections. In the case of HHV-6, the availability of commercialized ELISA kits has been less widespread than in other domains of clinical virology. As an overall consequence, ELISA is less used than IFA, despite its theoretical higher convenience. The specificity of HHV-6 ELISA has often been questioned and this test has not obtained better results than IFA on that point according to some authors (Dahl et al., 1990; Chokephaibulkit et al., 1997). In contrast, other authors have demonstrated that discrepancies between IFA and ELISA mainly corresponded to either false-positive or false-negative results in the IFA (Sloots et al., 1996).
Western blot and other immunoblot assays (IBA) permit the identification of antibodies to specific viral proteins. The specificity is higher than in ELISA but the sensitivity is usually lower. Viral antigens obtained either from infected cells or recombinant protein synthesis are denatured, separated by means of gel elect-rophoresis or isolated deposition, transferred to a membrane, and finally allowed to react with the serum specimen, basically using the same indicator system as in ELISA. So far, few attempts have been made to develop a HHV-6-specific IBA (Chen et al., 1992; LaCroix et al., 2000; Caselli et al., 2002; Zerr et al., 2005). The comparison between IFA, ELISA, and IBA in the case of human herpesvirus 7 (HHV-7), closely related to HHV-6, has confirmed that IBA was the most specific, exhibiting a sensitivity lower than ELISA and higher than IFA (Black et al., 1996b). Accordingly, the high specificity of an IBA designed to detect HHV-6-specific IgM antibodies was demonstrated in children below two years of age (LaCroix et al., 2000).
Serum samples are tested for neutralizing antibody by allowing serial dilutions of the serum to react with a standardized amount of infectious virus. The antibody titer is generally expressed as the highest serum dilution which blocks viral infec-tivity. Virus multiplication is read out from the observation of CPE (Suga et al., 1990), the counting of HHV-6-positive cells by means of IFA (Asada et al., 1989) or the measurement of viral antigen production by means of dot-blot assay (Tsukazaki et al., 1998). Neutralization assay (NTA) is generally considered to be highly specific, sensitive, and correlated with protective immunity. However, NTA is expensive and time consuming due to the requirements for cell culture and infectious virus production. It is often used as a reference method to validate other serologic assays or investigate complex immune responses in epidemiologic studies (Asano et al., 1990; Yoshikawa et al., 2001; Yoshida et al., 2002a,b).
The target antigens used in HHV-6 serologic testing encompass diverse proteins. IFA investigates the immune response against all the viral proteins expressed in infected cells. In contrast to that observed in the case of Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8), no distinction is done in HHV-6 IFA between the proteins corresponding either to lytic cycle or latency. This is also true for ELISA based on soluble antigen lysate from infected cells and, to a lesser extent, purified virions (Saxinger et al., 1988; Nielsen and Vestergaard, 1996; Choke-phaibulkit et al., 1997; Yoshida et al., 2002a). As a functional binding assay, NTA is assumed to reflect the interaction between neutralizing antibodies and glyco-proteins present on viral envelope. However, present knowledge about the target of neutralizing antibodies detected by NTA is poor.
A current trend is the identification of target proteins by means of immunoblot or immunoprecipitation assays combined with the use of monoclonal antibodies (Balachandran et al., 1989). Such approach has permitted to recognize the protein p100 (U11 gene product) as a major determinant of immune response (Yamamoto et al., 1990; Neipel et al., 1992). Similarly, a 101 kDa protein (101K) has been identified as an immunodominant virion protein for both IgG and IgM reactivity (LaCroix et al., 2000). The early antigen p41/38 (U27 gene product) had been chosen to develop a specific ELISA (Iyengar et al., 1991; Patnaik et al., 1995a). This early protein has been found to contain a divergent epitope which permits to differentiate HHV-6A from HHV-6B (Xu et al., 2001). However, further results obtained with recombinant p41 as the ELISA antigen have been rather disappointing both in terms of overall serum reactivity and variant specificity (Xu et al., 2002). The residues 4-10 of U24 gene product have been shown to be identical to the residues 96-102 of myelin basic protein (MBP). This core sequence with the flanking residues from either HHV-6 or MBP has been tested as an ELISA antigen: antibody titer for both peptides was increased in patients with multiple sclerosis (MS) as compared with healthy controls, suggesting a possible role of HHV-6 in the autoimmune reactivity to MBP and pathogenesis of myelin disease (Tejada-Simon et al., 2003). The recombinant protein REP (U94 gene product, expressed during virus latency) has been used to set up a novel ELISA which has permitted to observe a significant difference between MS and control patients, regarding both antibody prevalence and titer (Caselli et al., 2002). It is expected that future experiments will contribute to complete the list of relevant HHV-6 epitopes for se-rology. This would lead to define novel peptides or recombinant proteins that might be used in the design of serologic assays more specific than current ones.
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