Zamudio Raphaela Mello, Garcia Jaíne Gonçalves, Barboza Camila Mosca, Rodrigues Adriana Candido, de Arruda Tamires Santos, Francisco Ana Lee Aparecida, de Oliveira Fahl Willian, Castilho Juliana Galera, da Silva Maria Cristina Carlan, de Carvalho Ruthner Batista Helena Beatriz
Federal University of ABC, Santo Andre, SP, Brazil; Rabies Diagnostic Sector, Pasteur Institute, Sao Paulo, SP, Brazil.
Federal University of ABC, Santo Andre, SP, Brazil; Rabies Diagnostic Sector, Pasteur Institute, Sao Paulo, SP, Brazil.
J Virol Methods. 2021 Aug;294:114195. doi: 10.1016/j.jviromet.2021.114195. Epub 2021 May 19.
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.
狂犬病在发展中国家是一个严重的公共卫生问题，由狂犬病病毒（RABV）引起，这是一种嗜神经性RNA病毒。狂犬病诊断的金标准检测方法是直接荧光抗体检测（DFAT）。然而，推荐采用一种确证方法，如狂犬病组织培养感染试验（RTCIT）。已经对几种细胞系进行了RTCIT测试，结果显示小鼠神经母细胞瘤（Neuro-2a）细胞系对感染的敏感性最高。人胚肾（HEK-293）细胞系由于能表达神经元蛋白且易于培养，最近被视为一种选择。在本研究中，我们评估了HEK-293细胞系与Neuro-2a细胞系相比对RTCIT的敏感性。我们总共使用了93个脑样本，其中48个为阴性，45个为阳性，这些样本之前已通过DFAT或RT-PCR以及在Neuro-2a细胞系中进行的RTCIT检测过是否感染狂犬病病毒。在阳性样本中，43个在使用Neuro-2a细胞系的传统RTCIT中呈阳性。我们测试了HEK-293细胞系用于RTCIT的两种方案（有和无病毒吸附），并设置了不同的孵育时间：24小时、48小时和72小时。HEK-293细胞系中阳性率最高的情况（41个阳性样本）是在72小时孵育期的吸附方案中出现的，相比之下，使用Neuro-2a细胞系的传统RTCIT有43个阳性样本。无论孵育时间如何，未吸附方案均未得到满意结果。尽管Neuro-2a细胞的敏感性略高，但使用HEK-293细胞仍有积极的方面，比如结果更快，与Neuro-2a细胞系相比具有生长快速且容易的优势。因此，我们的研究结果证实HEK-293细胞对狂犬病病毒敏感，可作为RTCIT的一种替代方法。
