Publications by authors named "Sandriana Ramos Silva"

10 Publications

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Rabies Virus Exposure in Wild Lowland Tapirs (Tapirus terrestris) from Three Brazilian Biomes.

J Wildl Dis 2021 Apr;57(2):443-446

Lowland Tapir Conservation Initiative, Institute for Ecological Research, Campo Grande, Mato Grosso do Sul 79046-150, Brazil.

We evaluated the presence of antibodies for rabies virus in 177 serum samples from 125 wild lowland tapirs (Tapirus terrestris) from three different Brazilian biomes. The rapid fluorescent focus inhibition test was performed. No antibody titers suggesting the circulation of the rabies virus in tapir habitat were detected.
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http://dx.doi.org/10.7589/JWD-D-20-00089DOI Listing
April 2021

Glycosylation is required for the neutralizing activity of human IgG1 antibodies against human rabies induced by pre-exposure prophylaxis.

Immunobiology 2021 Mar 23;226(2):152058. Epub 2021 Jan 23.

Instituto Pasteur, São Paulo, Brazil. Electronic address:

Rabies lyssavirus (RABV) neutralizing IgG antibodies confer protection after rabies vaccination, although how the RABV-specific antibodies neutralize the virus is still unknown. As changes in the antibody's carbohydrate chain can interfere with its effector functions, we compared the glycosylation patterns of both neutralizing and non-neutralizing IgG1 induced by pre-exposure prophylaxis to human rabies and analyzed their influence on in vitro antibody neutralizing activities. Specific IgG1 were purified from human serum using affinity chromatography. Purity and avidity were analyzed by SDS-PAGE and indirect ELISA using NHSCN respectively. The N-linked oligosaccharide chain of the purified IgG antibody was evaluated using a lectin-based ELISA assay with a panel of seven lectins. The activity of purified IgG1 and neutralizing IgG1 deglycosylated by PNGase F enzyme were analyzed using the rapid fluorescent focus inhibition test. The purified IgG1 showed an electrophoretic pattern compatible with human IgG. All of the antibodies recognized RABV, although neutralizing IgG1 had a higher avidity (RAI = 80%) than non-neutralizing IgG1 (RAI = 30%). The neutralizing IgG1 also showed higher binding to WFA, ECA, WGA, and ConA lectins, indicating possible different N-acetylgalactosamine, galactose, N-acetylglucosamine, and mannose contents. Non-neutralizing IgG1, on the other hand, showed strong binding at UEA-1 and SNA, which bind to fucose and sialic acid residues respectively. Different glycosylation profiles were also observed in Fab and Fc fragments from neutralizing and non-neutralizing IgG1, although the deglycosylated IgG1 lost its neutralizing activity. Our results suggest that antibody glycosylation is important for neutralizing RABV in vitro, since neutralizing IgG1 has a different glycosylation profile than non-neutralizing IgG1. Further research will be needed to better evaluate the differential glycosylation patterns between IgG1 antibodies following vaccination.
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http://dx.doi.org/10.1016/j.imbio.2021.152058DOI Listing
March 2021

Development of biotinylated polyclonal anti-ribonucleoprotein IgG for detection of rabies virus antigen by direct rapid immunohistochemical test.

Biologicals 2020 Nov 26;68:74-78. Epub 2020 Aug 26.

Instituto Pasteur, São Paulo, Brazil. Electronic address:

The direct rapid immunohistochemical test (dRIT) has been recommended for laboratorial diagnosis of rabies, especially in developing countries. The absence of commercial primary antibodies, however, still represents a major limitation to its wider use in testing. We describe here the development of a biotinylated polyclonal antibody against Rabies lyssavirus (RABV) ribonucleoprotein (RNP) and its use as a primary reagent in dRIT. Anti-RNP polyclonal horse IgG was purified by ionic exchange chromatography followed by immunoaffinity column chromatography, and its affinity, diagnostic sensitivity, and specificity were evaluated. CNS samples (120) of suspected rabies cases in different animal species were tested by dRIT, with the positive (n = 14) and negative (n = 106) results confirmed by direct fluorescence antibody testing (dFAT). Comparing the results of dRIT and dFAT, we found that the biotinylated anti-RNP IgG delivered 100% diagnostic specificity and sensibility for rabies diagnosis. Our findings show that the biotinylated anti-RNP polyclonal IgG can be produced with the quality required for application in dRIT. This work represents an important step in efforts to diagnose rabies in developing countries.
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http://dx.doi.org/10.1016/j.biologicals.2020.08.004DOI Listing
November 2020

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

J Virol Methods 2020 06 1;280:113879. Epub 2020 May 1.

Instituto Pasteur, São Paulo, Brazil. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jviromet.2020.113879DOI Listing
June 2020

Evaluation of polyclonal anti-RNP IgG antibody for rabies diagnosis by indirect rapid immunohistochemistry test.

Acta Trop 2020 Jun 21;206:105340. Epub 2020 Feb 21.

Instituto Pasteur, 393, Paulista Avenue, 01311-000, São Paulo, Brazil. Electronic address:

Rabies still represents a major public health threat and estimated to cause 60,000 human deaths annually, particularly in developing countries. Thus, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. The WHO and OIE recommended gold standard diagnostic technique for rabies is the direct immunofluorescence assay (dFAT). However, dFAT is expensive and requires a high level of expertise. As an alternative, the rapid immunohistochemistry technique is a promise to be a simple and cost effective diagnostic tool for rabies, and can be performed on field conditions prevalent in developing countries. However, no validated commercial conjugate antibody for rabies is available to meet the laboratory demand. Here, we evaluated the polyclonal anti-rabies virus ribonucleoprotein (RNP) IgG antibody for Rabies lyssavirus (RABV) detection by indirect rapid immunohistochemistry test (iRIT). We tested polyclonal anti-RNP IgG antibody against a batch of 100 brain specimens representing a wide phylogenetic origin in the State of São Paulo, Brazil. The purified IgG obtained 100% of diagnostic specificity and sensibility for RABV antigen detection in iRIT compared with the gold standard dFAT. In conclusion, our results demonstrate that the polyclonal anti-RNP IgG antibody may be used as a diagnostic reagent for rabies using iRIT, with the expectation of increase in availability and cost reduction of the epidemiological surveillance for developing countries.
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http://dx.doi.org/10.1016/j.actatropica.2020.105340DOI Listing
June 2020

Nyctinomops laticaudatus bat-associated Rabies virus causes disease with a shorter clinical period and has lower pathogenic potential than strains isolated from wild canids.

Arch Virol 2019 Oct 10;164(10):2469-2477. Epub 2019 Jul 10.

Pasteur Institute, Av. Paulista 393, São Paulo, SP, CEP 01311-000, Brazil.

Rabies is a lethal viral disease that can affect a wide range of mammals. Currently, Rabies virus (RABV) in some European and American countries is maintained primarily in wild species. The regulation of viral replication is one of the critical mechanisms involved in RABV pathogenesis. However, the relationship between replication and the pathogenesis of RABV isolated from wild animals remains poorly understood. In the present study, we evaluated the pathogenicity of the street viruses Nyctinomops laticaudatus bat-associated RABV (NYBRV) and Cerdocyon thous canid-associated RABV (CECRV). Infection of mice with NYBRV led to 33% mortality with rapid disease evolution and marked histopathological changes in the CNS. In contrast, infection with CECRV led to 67% mortality and caused mild neuropathological lesions. The proportion of RABV antigen was significantly higher in the cytoplasm of neuronal cells of the cerebral cortex and in the meninges of mice infected with CECRV and NYBRV, respectively. Moreover, the replication rate of NYBRV was significantly higher (p < 0.001) than that of CECRV in neuroblastoma cells. However, CECRV replicated to a significantly higher titer in epithelial cells. Our results indicate that NYBRV infection results in rapid disease progression accompanied by frequent and intense histopathological alterations in the CNS in mice, and in a high replication rate in neuroblastoma cells. Although, CECRV is more pathogenic in mice, it caused milder histopathological changes in the CNS and replicated more efficiently in epithelial cells. Our data point to a correlation between clinical aspects of disease and the replication of RABV in different cell lines.
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http://dx.doi.org/10.1007/s00705-019-04335-5DOI Listing
October 2019

Purification of IgG against ribonucleoprotein by a homemade immunoaffinity chromatography column for rabies diagnosis.

J Immunol Methods 2019 08 20;471:1-10. Epub 2019 Mar 20.

Instituto Pasteur, São Paulo, Brazil. Electronic address:

Polyclonal or monoclonal antibodies against rabies virus ribonucleoprotein (RNP) conjugated to fluorescein isothiocyanate (FITC) have been employed for Rabies virus (RABV) antigen detection by the direct fluorescent antibody test (DFA). To date, these biomolecules have been purified by traditional methods such as precipitation by ammonium sulfate or ion exchange chromatography followed by ammonium sulfate precipitation, which allows only for partial detection of the protein of interest. In this study, we aimed to purify anti-RNP polyclonal horse IgG antibodies by cation-exchange chromatography in combination with a homemade immunoaffinity chromatography on RNP immobilized (RNP-IAC). Furthermore, to evaluate the accuracy of the prepared anti-RNP IgG fluorescent antibody in diagnostic purposes, DFA was applied for RABV antigen detection in suspected brain samples of different animal species. The combination of these two techniques made it possible to obtain antibodies with high selectivity and purity. Compared with the performance of the traditional method, anti-RNP IgG antibodies purified by RNP-IAC can be obtained from a smaller volume of hyperimmune serum and with greater avidity. Furthermore, the results obtained by DFA analyses revealed that the prepared anti-RNP IgG fluorescent antibody achieved 100% diagnostic specificity and sensitivity for RABV antigen detection. Thus, two-technique chromatographic, including RNP-IAC technology could be appropriate methods for the purification of polyclonal anti-RNP IgG for the use as a diagnostic reagent for rabies.
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http://dx.doi.org/10.1016/j.jim.2019.03.007DOI Listing
August 2019

Street rabies virus strains associated with insectivorous bats are less pathogenic than strains isolated from other reservoirs.

Antiviral Res 2018 12 26;160:94-100. Epub 2018 Oct 26.

Laboratory of Diagnostic, Pasteur Institute, São Paulo, Brazil. Electronic address:

Rabies is a fatal and viral zoonosis that causes acute, progressive encephalitis and remains an important concern in public health. In the last few years, there has been a change in the epidemiological profile of rabies after implementing canine rabies control in the Americas, which has led to a significant increase in both human and pet cases of rabies associated with insectivorous bats. Thus, it is important to understand the pathogenesis caused by Rabies virus (RABV) isolates from insectivorous bats. Viral growth kinetics, cell-to-cell spread and virus uptake in vitro were analyzed for RABV isolates from Eptesicus furiralis and Myotis nigricans. For pathogenesis evaluation, mice were inoculated with RABV isolates from Eptesicus furiralis and Myotis nigricans, and clinical signs were observed for 40 days. We observed that the insectivorous bat strains showed a higher replication rate, faster cell-to-cell spread and delayed virus uptake in N2a cells. Furthermore, after the first sign of a clinical infection, mice infected with Myotis nigricans and Eptesicus furiralis isolates succumbed rapidly (6 ± 9 days) compared with RABV strains associated with other reservoirs. Our results show that the insectivorous bat RABV strains are less pathogenic for mice than strains associated with other reservoirs. In addition, this study also indicates that the differences in the biological characteristics of the RABV strains are important to their pathogenicity. An enhanced understanding of rabies pathogenesis may be important for the development of novel therapies for humans and in the implementation of rabies control strategies.
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http://dx.doi.org/10.1016/j.antiviral.2018.10.023DOI Listing
December 2018

Infection of neuroblastoma cells by rabies virus is modulated by the virus titer.

Antiviral Res 2018 01 6;149:89-94. Epub 2017 Nov 6.

Laboratory of Diagnostic, Pasteur Institute, São Paulo, Brazil. Electronic address:

Rabies is a lethal viral infection that can affect almost all mammals, including humans. To better understand the replication of Rabies lyssavirus, we investigated if the viral load in brains naturally infected with rabies influences viral internalization and viral growth kinetics in neuroblastoma cells, and if the viral load affects mortality in mice after intradermal infection. We noted that high initial viral loads in brains (group II) were unfavourable for increasing viral titers during serial passages in neuroblastoma cells when compared to low initial viral loads in brains (group I). In addition, group I strains showed higher viral growth and enhanced internalization efficiency in neuroblastoma cells than group II strains. However, we observed that the dominant virus subpopulation in group II promoted efficient viral infection in the central nervous system in the new host, providing a selective advantage to the virus. Our data indicate that rabies infection in animal models depends on not only the virus strain but also the amount of virus. This study may serve as a basis for understanding the biologic proprieties of Rabies lyssavirus strains with respect to the effects on viral replication and the impact on pathogenesis, improving virus yields for use in vaccine development.
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http://dx.doi.org/10.1016/j.antiviral.2017.11.003DOI Listing
January 2018

Immunological aspects of rabies: a literature review.

Arch Virol 2017 Nov 19;162(11):3251-3268. Epub 2017 Jul 19.

Instituto Pasteur, 393, Paulista Avenue, São Paulo, SP, 01311-000, Brazil.

Rabies is a lethal disease caused by the neurotropic virus rabies virus (RABV), and it remains an important public health problem globally. It is known that the host immune response is important for control of viral infection and promoting viral clearance. In this context, it is well documented that, in addition to RABV neutralizing antibody, interferons and cell-mediated immunity also have an important role in preventing the establishment of disease. On the other hand, RABV suppresses host immunity through different mechanisms, for example, direct inhibition of host gene expression, sequestration of pathogen-associated molecular patterns, or modification of cytokine signalling pathways, which hinder the protective host immune responses to RABV infection. Here, we review the immunological aspects of rabies, highlighting innate and adaptive immunity, as well as the host evasion immune mechanisms used by the virus. Finally, we briefly discuss how this knowledge can direct new research and be harnessed for future therapeutic strategies.
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http://dx.doi.org/10.1007/s00705-017-3484-0DOI Listing
November 2017