Crimean-Congo hemorrhagic fever virus is reported in many countries of Africa, Asia, the Middle East, and Eurasia [3, 4], and the geographical distribution has expanded in recent decades. An increasing number of epidemics and sporadic cases have been reported in Kosovo, Bulgaria, Albania, Greece, the Russian Federation, Tajikistan, Kazakhstan, Georgia, Iran, Pakistan, Afghanistan, Mauritania, Senegal, Kenya, and India [9, 10]. While serological evidence of CCHF has been found among humans since the 1970s, the first clinical CCHF infection was recognized in 2002. Currently, an exceptional outbreak of CCHF is occurring in Turkey with more than 7,000 cases reported creating a serious threat to public health [21–24].
Currently there are no specific treatments or licensed vaccines available for CCHFV. There have been few attempts to develop a vaccine because of the sporadic and limited numbers of cases, the lack of a suitable animal model to evaluate efficacy of vaccine candidates, and the high level biocontainment facilities required for working with the virus [25–27]. Recently, two research groups have found adult mice with defective interferon responses allowed to lethal CCHFV infection [28, 29]. These mouse models could provide invaluable information for further studies. Efforts to develop a vaccine against CCHFV are being made.
To determine the efficacy of vaccine candidates it is important to conduct serological studies that can accurately measure levels of protective antibodies. Reports on measuring neutralizing antibodies against CCHFV are limited, and the neutralizing antibody response is weak and difficult to demonstrate in CCHF infections [26, 30–32]. At present, neutralizing antibody titers to CCHF are most commonly measured using plaque reduction neutralization assay, which requires a monolayer of cells susceptible to the virus infection. However, the use of cell lines for CCHFV may produce little or no CPE, since the virus tends to develop a noncytopathic persistent infection, depending on the strain. An alternative method of measuring CCHF-neutralizing antibody response is fluorescent focus reduction neutralization assay (FFRNT). The two published reports on the detection of CCHF-neutralizing antibody with FFRNT are not recent [30, 31]. In the present study, we developed PPRNT, based on colorimetric immunoassay used for infectious particle assay and clonal isolation of adeno-associated virus . Fluorescent focus reduction neutralization assay was adapted for the measurement of neutralizing antibody responses against CCHF, and the results of FFRNT were regarded as the gold standard.
Parameters such as sensitivity, specificity, and reproducibility are important to evaluate when developing new or modified laboratory tests. In this study, the inter- and intra-assay variation of PPRNT revealed good reproducibility (Table 1). The PPRNT showed high sensitivity (98%), specificity (100%), and agreement (96.6%) in qualitative comparison with FFRNT (Table 2). The titers obtained by PPRNT and FFRNT were highly correlated (R2 = 0.92; Figure 2). In addition, we were able to define for both assays a positive cut-off (1:4) based on the geometric mean titers for the individual samples distributed. There was no significant difference between PPRNT and FFRNT with respect to determining CCHF-neutralizing antibody response. This is not surprising since both methods are based on the same fundamental principles and differ only in the surface area of the tissue culture plates and in the final visualization steps. However, a unique advantage of PPRNT is that the pseudo-plaques can be counted using the naked eye or by light microscopy. Moreover, the plates containing pseudo-plaques can be stored for an extended period of time.
Unlike the standard plaque reduction neutralization test, PPRNT does not require cell destruction or damage by the virus infection. It allows the use of virus strains that do not form plaques, such as CCHFV Turkey-Kelkit06, for the measurement of neutralizing antibodies. Therefore, results can be more rapidly obtained by PPRNT (3 days after infection) than with PRNT (usually 5 to 7 days after infection). Another potential advantage of the PPRNT compared to standard PRNT is that PPRNT is performed in 96-well plates, enabling investigation of a large number of samples.
In conclusion, the pseudo-plaque neutralization assay described here is a rapid, reproducible, and sensitive method for the measurement of CCHF neutralizing antibodies. This novel, high-throughput assay could serve as a useful tool for CCHF research in epidemiology, vaccine development, and other studies of immunity. It also provides an alternative to PRNT when viruses with no or poor CPE in cell culture.