Publications by authors named "Keila Iamamoto"

14 Publications

  • Page 1 of 1

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

Zoonoses Public Health 2020 09 14;67(6):651-657. Epub 2020 Jun 14.

Instituto Pasteur, São Paulo, Brazil.

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.
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http://dx.doi.org/10.1111/zph.12745DOI Listing
September 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

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

A rabies virus vampire bat variant shows increased neuroinvasiveness in mice when compared to a carnivore variant.

Arch Virol 2017 Dec 22;162(12):3671-3679. Epub 2017 Aug 22.

Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, São Paulo, SP, Brazil.

Rabies is one of the most important zoonotic diseases and is caused by several rabies virus (RABV) variants. These variants can exhibit differences in neurovirulence, and few studies have attempted to evaluate the neuroinvasiveness of variants derived from vampire bats and wild carnivores. The aim of this study was to evaluate the neuropathogenesis of infection with two Brazilian RABV street variants (variant 3 and crab-eating fox) in mice. BALB/c mice were inoculated with RABV through the footpad, with the 50% mouse lethal dose (LD) determined by intracranial inoculation. The morbidity of rabies in mice infected with variant 3 and the crab-eating fox strain was 100% and 50%, respectively, with an incubation period of 7 and 6 days post-inoculation (dpi), respectively. The clinical disease in mice was similar with both strains, and it was characterized initially by weight loss, ruffled fur, hunched posture, and hind limb paralysis progressing to quadriplegia and recumbency at 9 to 12 dpi. Histological lesions within the central nervous system (CNS) characterized by nonsuppurative encephalomyelitis with neuronal degeneration and necrosis were observed in mice infected with variant 3 and those infected with the crab-eating fox variant. However, lesions and the presence of RABV antigen, were more widespread within the CNS of variant-3-infected mice, whereas in crab-eating fox-variant-infected mice, RABV antigens were more restricted to caudal areas of the CNS, such as the spinal cord and brainstem. In conclusion, the results shown here demonstrate that the RABV vampire bat strain (variant 3) has a higher potential for neuroinvasiveness than the carnivore variant.
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http://dx.doi.org/10.1007/s00705-017-3530-yDOI Listing
December 2017

Group A rotavirus in Brazilian bats: description of novel T15 and H15 genotypes.

Arch Virol 2016 Nov 12;161(11):3225-30. Epub 2016 Aug 12.

Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine, University of São Paulo, Av. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, SP, 05508-270, Brazil.

This study aimed to survey for group A rotaviruses (RVA) in bats from Brazil and to perform phylogenetic inferences for VP4, VP7, NSP3, NSP4 and NSP5 genes. RVA was found in 9.18 % (28/305) of tested samples. The partial genotype constellation of a Molossus molossus RVA strain was G3-P[3]-Ix-Rx-Cx-Mx-Ax-Nx-T3-E3-H6, and that of a Glossophaga soricina RVA strain was G20-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-T15-Ex-H15. These findings demonstrate an important role of bats in RVA epidemiology and provide evidence of participation of bat RVA strains in interspecies transmission and reassortment events.
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http://dx.doi.org/10.1007/s00705-016-3010-9DOI Listing
November 2016

Erratum to: Alphacoronavirus in urban Molossidae and Phyllostomidae bats, Brazil.

Virol J 2016 07 7;13(1):124. Epub 2016 Jul 7.

Departament of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine, University of São Paulo, Av. Orlando Marques de Paiva, 87, São Paulo, CEP: 05508-270, Brazil.

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http://dx.doi.org/10.1186/s12985-016-0581-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937601PMC
July 2016

Alphacoronavirus in urban Molossidae and Phyllostomidae bats, Brazil.

Virol J 2016 06 24;13:110. Epub 2016 Jun 24.

Departament of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine, University of São Paulo, Av. Orlando Marques de Paiva, 87, CEP: 05508-270, São Paulo, Brazil.

Background: Bats have been implicated as the main reservoir of coronavirus (CoV). Thus the role of these hosts on the evolution and spread of CoVs currently deserve the attention of emerging diseases surveillance programs. On the view of the interest on and importance of CoVs in bats the occurrence and molecular characterization of CoV were conducted in bats from Brazil.

Findings: Three hundred five enteric contents of 29 bat species were tested using a panCoV nested RT-PCR. Nine specimens were positive and eight was suitable for RdRp gene sequencing. RdRp gene phylogeny showed that all CoVs strains from this study cluster in Alphacoronavirus genus, with one Molossidae and one Phlyllostomidae-CoV specific groups. Phylogenetic analyses of two S gene sequences showed a large diversity within the Alphacoronavirus genus.

Conclusions: This study indicated a CoV-to-host specificity and draws attention for CoV detection in Cynomops sp, a potential new reservoir. The phylogenetic analyses indicate that diversity of CoV in bats is higher than previously known.
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http://dx.doi.org/10.1186/s12985-016-0569-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920988PMC
June 2016

[Hematophagous bats as reservoirs of rabies].

Rev Peru Med Exp Salud Publica 2014 Apr;31(2):302-9

Instituto Pasteur, São Paulo, Brasil.

Rabies continues to be a challenge for public health authorities and a constraint to the livestock industry in Latin America. Wild and domestic canines and vampire bats are the main transmitter species and reservoirs of the disease. Currently, variations observed in the epidemiological profile of rabies, where the species of hematophagous bat Desmodus rotundus constitutes the main transmitting species. Over the years, knowledge has accumulated about the ecology, biology and behavior of this species and the natural history of rabies, which should lead to continuous development of methods of population control of d. Rotundus as well as prevention and diagnostic tools for rabies. Ecological relationships of this species with other hematophagous and non-hematophagous bats is unknown, and there is much room for improvement in reporting systems and surveillance, as well as creating greater awareness among the farming community. Understanding the impact of human-induced environmental changes on the rabies virus in bats should be cause for further investigation. This will require a combination of field studies with mathematical models and new diagnostic tools. This review aims to present the most relevant issues on the role of hematophagous bats as reservoirs and transmitters of the rabies virus.
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April 2014

Eastern equine encephalitis cases among horses in Brazil between 2005 and 2009.

Arch Virol 2014 Oct 27;159(10):2615-20. Epub 2014 May 27.

Seção de Diagnóstico da Raiva, Instituto Pasteur, Av. Paulista, 393-Cerqueira César, São Paulo, SP, CEP 01311-000, Brazil,

Eastern equine encephalitis is a viral zoonosis that exhibits complex distribution and epidemiology, and greater importance should be given to this disease by the public-health authorities. In Brazil, although eastern equine encephalitis virus (EEEV) has been identified in vectors and antibodies are sometimes detected in horses and humans, there have been no records of equine encephalitis in horses caused by this virus during the last 24 years. This study describes eighteen cases of eastern equine encephalomyelitis that occurred in six Brazilian states between 2005 and 2009. Viral RNA was identified using semi-nested RT-PCR to detect members of the genus Alphavirus, and by genetic sequencing. The gene encoding NSP1 was partially amplified, and after genetic sequencing, eighteen sequences were generated. All eighteen strains were classified as belonging to lineage III of American EEEV. These findings could be an indication of the importance of this virus in animal and human public health.
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http://dx.doi.org/10.1007/s00705-014-2121-4DOI Listing
October 2014

In vitro and in vivo inhibition of rabies virus replication by RNA interference.

Braz J Microbiol 2013 15;44(3):879-82. Epub 2013 Nov 15.

Faculdade de Medicina Veterinária, Universidade de São Paulo, São Paulo, SP, Brazil.

Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.
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http://dx.doi.org/10.1590/S1517-83822013005000050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910205PMC
February 2015

Desmodus rotundus and Artibeus spp. bats might present distinct rabies virus lineages.

Braz J Infect Dis 2012 Nov-Dec;16(6):545-51. Epub 2012 Nov 10.

Instituto Pasteur, São Paulo, SP, Brazil.

In Brazil, bats have been assigned an increasing importance in public health as they are important rabies reservoirs. Phylogenetic studies have shown that rabies virus (RABV) strains from frugivorous bats Artibeus spp. are closely associated to those from the vampire bat Desmodus rotundus, but little is known about the molecular diversity of RABV in Artibeus spp. The N and G genes of RABV isolated from Artibeus spp. and cattle infected by D. rotundus were sequenced, and phylogenetic trees were constructed. The N gene nucleotides tree showed three clusters: one for D. rotundus and two for Artibeus spp. Regarding putative N amino acid-trees, two clusters were formed, one for D. rotundus and another for Artibeus spp. RABV G gene phylogeny supported the distinction between D. rotundus and Artibeus spp. strains. These results show the intricate host relationship of RABV's evolutionary history, and are invaluable for the determination of RABV infection sources.
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http://dx.doi.org/10.1016/j.bjid.2012.07.002DOI Listing
June 2013

Phylogenetic analysis of partial RNA-polymerase blocks II and III of Rabies virus isolated from the main rabies reservoirs in Brazil.

Virus Genes 2012 Aug 18;45(1):76-83. Epub 2012 Apr 18.

Pasteur Institute, Av. Paulista 393, São Paulo, Brazil.

This study describes the results of the sequencing and analysis of segments of Blocks II and III of the RNA polymerase L gene of Rabies virus isolates from different reservoir species of Brazil. The phylogenetic relations of the virus were determined and a variety of species-specific nucleotides were found in the analyzed areas, but the majority of these mutations were found to be synonymous. However, an analysis of the putative amino acid sequences were shown to have some characteristic mutations between some reservoir species of Brazil, indicating that there was positive selection in the RNA polymerase L gene of Rabies virus. On comparing the putative viral sequences obtained from the Brazilian isolates and other Lyssavirus, it was determined that amino acid mutations occurred in low-restriction areas. This study of the L gene of Rabies virus is the first to be conducted with samples of virus isolates from Brazil, and the results obtained will help in the determination of the phylogenetic relations of the virus.
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http://dx.doi.org/10.1007/s11262-012-0743-8DOI Listing
August 2012

Rabies in southeast Brazil: a change in the epidemiological pattern.

Arch Virol 2012 Jan 28;157(1):93-105. Epub 2011 Oct 28.

Universidade Estadual Paulista, UNESP, Rua Clóvis Pestana, 793, Araçatuba, SP 16050-680, Brazil.

This epidemiological study was conducted using antigenic and genetic characterisation of rabies virus isolates obtained from different animal species in the southeast of Brazil from 1993 to 2007. An alteration in the epidemiological profile was observed. One hundred two samples were tested using a panel of eight monoclonal antibodies, and 94 were genetically characterised by sequencing the nucleoprotein gene. From 1993 to 1997, antigenic variant 2 (AgV-2), related to a rabies virus maintained in dog populations, was responsible for rabies cases in dogs, cats, cattle and horses. Antigenic variant 3 (AgV-3), associated with Desmodus rotundus, was detected in a few cattle samples from rural areas. From 1998 to 2007, rabies virus was detected in bats and urban pets, and four distinct variants were identified. A nucleotide similarity analysis resulted in two primary groups comprising the dog and bat antigenic variants and showing the distinct endemic cycles maintained in the different animal species in this region.
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http://dx.doi.org/10.1007/s00705-011-1146-1DOI Listing
January 2012

Outbreaks of Eastern equine encephalitis in northeastern Brazil.

J Vet Diagn Invest 2011 May;23(3):570-5

Veterinary Hospital, Federal University of Campina Grande (UFCG), Campus of Patos, 58700-000 Patos, Paraíba, Brazil.

Outbreaks of eastern equine encephalitis observed from May 2008 to August 2009 in the Brazilian states of Pernambuco, Ceará, and Paraíba are reported. The disease occurred in 93 farms affecting 229 equids with a case fatality rate of 72.92%. Main clinical signs were circling, depression or hyperexcitability, ataxia, and progressive paralysis with a clinical manifestation period of 3-15 days. Main histologic lesions were a diffuse lymphocytic encephalomyelitis with neuronal death, satellitosis, neuronophagia, and hemorrhages being more severe in the cerebral gray matter of the telencephalon, diencephalon, and mesencephalon. Some animals also had areas of malacia in the telencephalon, thalamus, and basal nuclei. From 1 case, the virus was isolated by mice inoculation, and in other 13 cases was identified as Eastern equine encephalitis virus by semi-nested reverse transcription polymerase chain reaction. After DNA sequencing, all samples were identified as eastern equine encephalitis through the BLASTn analysis, but samples from the Ceará and Paraíba states corresponded to the same cluster, while the sample from the state of Pernambuco corresponded to a different cluster.
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http://dx.doi.org/10.1177/1040638711403414DOI Listing
May 2011