Rabies Tissue Culture Infection Test Research Articles

Evaluation of the effect of sample suspension concentration and viral load on the outcome of the rabies tissue culture infection test.

In this study, we examined various brain suspension concentrations and viral loads in Neuro-2a cell cultures using 20 rabies-positive bovine samples. The reproducibility of results varied: 65% showed consistent outcomes across all concentrations, while 35% disagreed in at least one. Viral titers ranged from less than 25 × 101 to 25 × 103.50 TCID50/mL, with 20% below 25 × 101 TCID50/mL. Concentrations between 5% and 20% yielded over 90% agreement in positive results, but at 30%, agreement dropped from 85% to 50%. Cell confluence was successfully maintained at 5%, 10%, and 20%, while concentrations of 30% and above led to confluence loss. Low viral loads also negatively impacted reproducibility. These results suggest that sample concentration has a direct influence on preservation of cell confluence and that low viral loads may influence the reproducibility of the rabies tissue culture infection test (RTCIT).

Vaccine-induced Rabies in a Red Fox in Poland.

Rabies as a zoonosis threatens public health worldwide. Several thousand people die each year of infections by the rabies virus (RABV). Oral rabies vaccination (ORV) of wildlife was successfully implemented in many European countries and led to rabies being brought under control there. In Poland, ORV was introduced in 1993 using vaccines containing an attenuated strain of the rabies virus. However, attenuated rabies viruses may have residual pathogenicity and cause the disease in target and non-target animals. A red fox carcass was tested as part of national rabies surveillance, and its brain was screened for RABV infection using two conjugates and a fluorescent antibody test (FAT). The rabies virus was isolated in mouse neuroblastoma cells by rabies tissue culture infection test (RTCIT), and viral RNA was detected by heminested reverse transcriptase PCR (hnRT-PCR) as well as by quantitative real-time RT-PCR (rtRT-qPCR). An amplicon of 600 bp was subjected to Sanger sequencing. To differentiate between vaccine and field RABV strains, PCR-restriction fragment length polymorphism (PCR-RFLP) using the Dra I, Msp I, Nla IV and Mbo II restriction endonucleases was performed. The rabies virus was detected in the fox's brain using FAT, RTCIT and molecular tests. The PCR-RFLP revealed of vaccine-induced rabies, and full-length genome analysis showed 100% nucleotide sequence identity of the isolate with the reference sequences of Street Alabama Dufferin Bern (SAD Bern) vaccine strains and other vaccine-induced rabies virus isolates detected in animals and deposited in GenBank. We detected vaccine-induced rabies for the first time in Poland in a fox during routine rabies surveillance.

Detection of rabies virus in cranial cavity lavage of naturally infected bats

The rabies virus (RABV) has been isolated in several bats species in the world, and among them, hematophagous, frugivorous and insectivorous species. Bats found in Brazil are small, which can lead to situations in which there are limitations in the collection of the central nervous system (CNS) and the amount of material may be insufficient to carry out laboratory diagnostic techniques for rabies. The objective of this work was to evaluate an alternative sample collection for the diagnosis of rabies in bats. A total of 92 bat samples, 82 positives and 10 negatives were selected. The cranial cavity was scraped with the aid of sterile tips and a virus diluent was added to create a suspension. All samples were submitted to Rabies Tissue Culture Infection Test (RTCIT) and reverse transcription polymerase chain reaction (RT-PCR). The diagnostic sensitivity and specificity of the RTCIT and RT-PCR using the cranial cavity lavage were calculated in comparison with the results of the laboratory routine (DFAT and RTCIT) performed with the CNS (considered gold standard). The results of the RTCIT show that the cranial cavity lavage is not an adequate sample for viral isolation, since the diagnostic sensitivity was low (37.8 %) when compared with the tests with the CNS. However, the RT-PCR of the cranial cavity lavage may be a tool to assist in the diagnosis, since it presented a sensitivity of 76.8 %. The results of this study suggest that cranial cavity lavage is an interesting alternative to enable the diagnosis of rabies in bats and increases the possibility of diagnosis contributing to rabies surveillance and control.

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.

Detection of rabies virus antigen by the indirect rapid immunohistochemistry test in equines and comparisons with other diagnostic techniques.

Laboratory diagnosis of rabies in equines is essential for distinguishing the disease from other sources of encephalitis. Diagnosis by conventional techniques such as a direct fluorescent antibody test (dFAT) or viral isolation in mice or cell culture can be difficult, and the application of molecular biological methods may be necessary. We performed an indirect rapid immunohistochemistry test (iRIT) for the detection of the rabies virus (RABV) antigen in the central nervous system (CNS) of equines and compared the results with those of other diagnostic techniques. We reviewed result records from the Rabies Diagnosis Laboratory at Instituto Pasteur, São Paulo, Brazil, of 174 samples of equine CNS from July 2014 to June 2016, which were investigated by dFAT, rabies tissue culture infection test (RTCIT), mouse inoculation test (MIT) and reverse transcription-polymerase chain reaction (RT-PCR) followed by genetic sequencing. These samples, 29 presented divergent results among techniques and were selected for the performed in the iRIT. The detected positivity rate was 4/29 (14%) by dFAT, 5/28 (18%) by RTCIT, 10/29 (35%) by MIT and 26/27 (96%) by RT-PCR. We analysed 29 samples through imprints of the cortex, hippocampus, cerebellum and brainstem in slides fixed in 10% buffered formaldehyde. Eighteen samples were identified as positive (62%) by iRIT assay, representing a greater number of positive cases than that detected by dFAT, MIT and RTCIT but not by RT-PCR. Among the brain regions, the brainstem presented the highest positivity (78%), followed by the hippocampus (69%), cerebellum (67%) and cortex (67%). Our results provide evidence that iRIT can contribute to a rapid diagnosis of rabies in equines and that complementary tests should be used to improve diagnostic accuracy in this species.

Analysis of rabies diagnosis in dogs and cats in the state of São Paulo, Brazil.

The genetic lineage of rabies virus (RABV) associated with dogs has not been found in the state of São Paulo since 1998, and all cases of rabies in domestic animals reported since then have involved the RABV lineage that circulates in bats. As there has been a change in the rabies transmission cycle in cats and dogs, we decided to analyze the tests used to diagnose rabies in these animals in the 15-year period from 2002 to 2016 in the state of São Paulo. During this period, 85,508 central nervous system (CNS) samples from dogs and cats were submitted to the Rabies Diagnosis Section at the Pasteur Institute of São Paulo for testing. All of the samples were tested by the fluorescent antibody test (FAT) and at least one of the following three tests: mouse inoculation test (MIT), rabies tissue culture infection test (RTCIT) and reverse transcription polymerase chain reaction (RT-PCR). Of all the samples tested, twenty were positive in at least one of these assays. Four other positive samples were identified at other institutions in the state of São Paulo. Of the twenty samples that tested positive at the Pasteur Institute of São Paulo, nine were tested by FAT, and the results were subsequently confirmed by other techniques; five gave inconclusive results, and therefore, other techniques had to be used as soon as possible in case the samples were positive; and six were negative by FAT and positive by one or more of the following tests: RTCIT, MIT and RT-PCR. Genetic typing of isolates from eighteen samples identified them as the lineage circulating in bats. In light of this finding, which indicates that genetic lineages associated with bats are circulating in domestic animals in the state of São Paulo, when the results of FAT carried out with samples from aggressive cats and dogs are inconclusive, complementary tests should be used. Decomposing samples and samples for which FAT was inconclusive should be tested using molecular techniques so that a definitive result can be obtained quickly and timely post-exposure prophylaxis can be administered to exposed individuals.

Disinfection protocols for necropsy equipment in rabies laboratories: Safety of personnel and diagnostic outcome

In the last decades, molecular techniques have gradually been adopted for the rapid confirmation of results obtained through gold standard methods. However, international organisations discourage their use in routine laboratory investigations for rabies post-mortem diagnosis, as they may lead to false positive results due to cross-contamination. Cleaning and disinfection are essential to prevent cross-contamination of samples in the laboratory environment. The present study evaluated the efficacy of selected disinfectants on rabies-contaminated necropsy equipment under organic challenge using a carrier-based test. The occurrence of detectable Rabies virus (RABV) antigen, viable virus and RNA was assessed through the gold standard Fluorescent Antibody Test, the Rabies Tissue Culture Infection Test and molecular techniques, respectively.None of the tested disinfectants proved to be effective under label conditions. Off label disinfection protocols were found effective for oxidizing agents and phenolic, only. Biguanide and quaternary ammonium compound were both ineffective under all tested conditions. Overall, discordant results were obtained when different diagnostic tests were compared, which means that in the presence of organic contamination common disinfectants may not be effective enough on viable RABV or RNA.Our results indicate that an effective disinfection protocol should be carefully validated to guarantee staff safety and reliability of results.

Real-Time RT-PCR for the Detection of Lyssavirus Species.

The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

Recurrence of Animal Rabies, Greece, 2012

Recurrence of Animal Rabies, Greece, 2012

International interlaboratory trials on rabies diagnosis: An overview of results and variation in reference diagnosis techniques (fluorescent antibody test, rabies tissue culture infection test, mouse inoculation test) and molecular biology techniques

Interlaboratory trials on rabies diagnosis were organised in 2009 and in 2010 by the European Union Reference Laboratory (EURL) for rabies. In 2009, two panels of virus samples were sent to participating laboratories to compare results on reference diagnosis techniques and on RT-PCR. A single panel was sent in 2010 to test FAT (fluorescent antibody test), RTCIT (rabies tissue culture infection test) and RT-PCR techniques. The virus panels included the RABV, EBLV-1, EBLV-2 and ABLV strains. Results revealed that laboratories produced the highest proportion of concordant results using RT-PCR (90.5%) and FAT (87.1%), followed by RTCIT (70.0%) and MIT (35.0%) in 2009 and in FAT (85.0%) and RT-PCR (80.6%) followed by RTCIT (77.3%) in 2010. Errors were only observed in bat strains (i.e. none in the RABV strain) for the RT-PCR or FAT techniques, highlighting the need to improve diagnosis most specifically in such strains. RT-PCR was the technique showing the lowest rate of false negative results in either trial year, while RTCIT and MIT (performed in 2009 only) were the techniques with the lowest proportion of false positive results. Nevertheless, the FAT technique represented a good compromise with both satisfactory sensitivity and specificity, as only a few false positive (1.6% in 2009, 5.8% in 2010) and false negative results (1.6% in both 2009 and 2010) were detected. The analysis of technical questionnaires describing the protocols used by participating laboratories revealed variation in the methods used that may induce inconsistencies in the results. In this study, the number of readers for FAT slide examination was identified as a factor affecting significantly the results of laboratories, suggesting that two independent readers are necessary for routine rabies diagnosis. Our findings highlight the need for all rabies diagnostic laboratories to improve harmonisation of procedures.

Rabies virus detection and phylogenetic analysis in samples from wild and domestic animals of Lithuania in 2007–2010

During the oral vaccination against rabies (ORV) in 2007–2010, phylogenetic analysis of rabies virus (RV) isolated from domestic and wild animals, was performed in the nucleoprotein (N) gene region. In total, 66 RV-positive brain samples were analysed: 52 samples from wild and 14 from domestic animals. The investigation was carried out at the National Food and Veterinary Risk Assessment Institute. For confirmation, RV identification in the analysed brain samples (n = 66) was performed by three methods: fluorescent antibody test (FAT), rabies tissue culture infection test (RTCIT) and reverse transcription polymerase chain reaction (RT-PCR). The results obtained by the three methods employed were similar: RV was identified in 66 samples of brain. The region of RT-PCR gene was amplified from (n = 66) positive brain samples and the obtained sequences of 362 bp nucleotides were compared with the selected other sequences of rabies virus from the GenBank. The phylogenetic analysis of N gene from wild and domestic animals showed a close genetic relationship between the different RV isolates irrespective of species or regional origin. The obtained results indicate that all RV strains in Lithuania belong to the first RV genotype of the North-East European group. Keywords: rabies virus, phylogenetic, wild, domestic

Quantification of the effectiveness of laboratory diagnostics of rabies using classical and molecular-genetic methods

In comparative experiments the diagnostic effectiveness of four methods of laboratory diagnostics of rabies – the mouse intracerebral inoculation test (MICIT, MIT), the rabies tissue culture infection test (RTCIT), the rapid rabies enzyme immune diagnosis test (RREID) and a molecular-genetic method, the nested reverse transcription polymerase chain reaction (nRT-PCR) – was quantified by the titration of serial dilutions of brain viral suspensions. The threshold value of the tests, i.e., the highest dilution of a specimen, which the method used is able to detect as a positive one, was determined. Further advantages and disadvantages of the tested methods were compared as well. Experimental optimization of procedures for RNA extraction was carried out and the optimum primer for RNA transcription to cDNA was selected. The RREID method was carried out in two variants: detection of the rabies antigen in a clarified (centrifugated) as well as in a non-clarified (noncentrifugated) brain suspension. In the experiments three autochthonous street isolates of rabies virus (in the form of primary isolates) were used; they had been isolated from naturally infected red foxes (Vulpes vulpes) and a lynx (Lynx lynx). The results of comparative experiments revealed a relative correlation of the diagnostic effectiveness of standard methods (MICIT and RTCIT), with standard MICIT being the more sensitive one, RTCIT however having several other advantages (among others the speed of performance) and thus being preferred. For quantitative comparison of diagnostic effectiveness two other methods (RREID and nRT-PCR) were examined in that street isolates of rabies virus, which revealed the highest titer after titration by MICIT and RTCIT. The sensitivity of the RREID method proved to be rather low. If used with noncentrifugated brain suspensions this method may yield nonspecific reactions. If compared particularly with RREID the nRT-PCR is characterized by a considerably higher diagnostic effectiveness. The sensitivity of nRT-PCR is not affected by preliminary clarification of the brain suspension. The reverse primer N12 seems to be more suitable for transcription of the extracted RNA to cDNA than random hexamers.