Human embryonic kidney (HEK-293) cell line: An alternative for rabies virus diagnosis and research.

Rabies is a serious public health problem in developing countries and is caused by Rabies lyssavirus (RABV), a neurotropic RNA virus. The gold standard test for rabies diagnosis is the direct fluorescent antibody test (DFAT). Nevertheless, a confirmatory method is recommended, such as rabies tissue culture infection test (RTCIT). Several cell lines have been tested for RTCIT, and the murine neuroblastoma (Neuro-2a) cell line has been shown to be the most permissive for infection. The human embryonic kidney (HEK-293) cell line was recently thought as an option, due to neuronal protein expression and easy maintenance. In the present work, we evaluated the susceptibility of HEK-293 cell line to RTCIT compared to Neuro-2a. We used a total of 93 brain samples, 48 negatives and 45 positives for RABV previously tested by DFAT or RT-PCR and by RTCIT in Neuro-2a. Of the positive samples, 43 were positive in the traditional RTCIT using Neuro-2a. Two protocols of HEK-293 cell line to RTCIT were tested (with and without virus adsorption) with different incubations times: 24, 48 and 72 h. The highest positive rate in HEK-293 (41 positive samples) resulted from the adsorption protocol with 72 h incubation period, in contrast to 43 positive samples with the traditional RTCIT with Neuro-2a. No satisfactory results were observed using the protocol without adsorption, regardless of the incubation time. Despite the slightly higher sensitivity of Neuro-2a cells, the use of the HEK-293 cells still offers positive aspects, such as, more rapid results, with the advantage of fast and easy growth over Neuro-2a cell line. Therefore, our findings confirm that HEK-293 cells are susceptible to RABV and can be an alternative for RTCIT.

Performance evaluation of the polyclonal anti-rabies virus ribonucleoprotein IgG antibodies produced in-house for use in direct fluorescent antibody test.

Fluorescein isothiocyanate (FITC) labelled anti-rabies virus ribonucleoprotein (RNP) antibodies can be used as immunoreagents in direct fluorescent antibody testing (dFAT) for rabies diagnoses. While in-house products are occasionally used by laboratories, most conjugates are commercial reagents. Commercial anti-RNP antibodies are only available for research purposes in Brazil, however, which contributes to the increasing use of in-house produced antibodies. Considering that conjugate quality may influence the results obtained during rabies diagnosis, we sought to analyze the performance requirements of in-house produced polyclonal anti-RNP IgG-FITC for application in dFAT. To that end, their reproducibility, diagnostic sensitivity, and specificity were evaluated. The titer of polyclonal anti-RNP IgG-FITC was initially determined and evaluated by dFAT, using central nervous system (CNS) samples of different animal species (dogs, cats, bovines, equines, bats, and non-human primates). As our main result, the polyclonal anti-RNP IgG-FITC reached a titer of 1:30/1:40 in dFAT, with 100% of diagnostic sensitivity and specificity. In terms of reproducibility, the antibodies, regardless the production batch, presented the same performances. In conclusion, the in-house produced polyclonal anti-RNP IgG-FITC proved suitable for rabies virus antigen detection by dFAT.