Elenice Maria Sequetin Cunha

Antigenic typing of brazilian rabies virus samples isolated from animals and humans, 1989-2000

Animal and human rabies samples isolated between 1989 and 2000 were typified by means of a monoclonal antibody panel against the viral nucleoprotein. The panel had been previously established to study the molecular epidemiology of rabies virus in the Americas. Samples were isolated in the Diagnostic Laboratory of the Pasteur Institute and in other rabies diagnostic centers in Brazil. In addition to the fixed virus samples CVS-31/96-IP, preserved in mouse brain, and PV-BHK/97, preserved in cell culture, a total of 330 rabies virus samples were isolated from dogs, cats, cattle, horses, bats, sheep, goat, swine, foxes, marmosets, coati and humans. Six antigenic variants that were compatible with the pre-established monoclonal antibodies panel were defined: numbers 2 (dog), 3 (Desmodus rotundus), 4 (Tadarida brasiliensis), 5 (vampire bat from Venezuela), 6 (Lasiurus cinereus) and Lab (reacted to all used antibodies). Six unknown profiles, not compatible with the panel, were also found. Samples isolated from insectivore bats showed the greatest variability and the most commonly isolated variant was variant-3 (Desmodus rotundus). These findings may be related to the existence of multiple independent transmission cycles, involving different bat species.

Genetic diversity of bat rabies viruses in Brazil

SummaryThirty-three Brazilian bat rabies viruses (RVs) were studied by sequence analysis and were compared against sequences of bat-related RVs from other regions of the Americas. Phylogenetic analysis revealed that bat-related RVs formed several monophyletic lineages and that these were associated with bat species. Brazilian bat RVs were found to include nine major lineages, one of which grouped with RVs isolated from Lasiurus spp. from different regions of the Americas. These results suggest that there is considerable diversity among Brazilian bat RV variants and that some of these RV variants may be associated with bats from other countries.

Bat rabies in the north-northwestern regions of the state of São Paulo, Brazil: 1997-2002

OBJECTIVE Reports on bat rabies in Brazil are sporadic and isolated. This study aimed at describing the detection of rabies virus in bats in the state of São Paulo. METHODS A total of 7,393 bats from 235 municipalities of the north and northwestern areas of the state of São Paulo, Southeastern Brazil, were assessed according to their morphological and morphometric characteristics from 1997 to 2002. Fluorescent antibody test and mice inoculation were used for viral identification. RESULTS Of all samples examined, 1.3% was rabies virus positive, ranging from 0.2% in 1997 to 1.6% in 2001. There were found 98 bats infected, 87 in the urban area. Fluorescent antibody test was detected in 77 positive samples, whereas 92 produced rabies signs in mice; incubation period ranging from 4 to 23 days. In 43 cities at least one rabid bat was observed. The highest proportion (33.7%) of rabies virus was found in Artibeus lituratus. Eptesicus and Myotis were the most frequent positive species (24.5%) of the Vespertilionidae family. The species Molossus molossus and Molossus rufus showed 14.3% positive bats. There were no differences in the distribution of positive rabies between females (33; 48.5%) and males (35; 51.5%). CONCLUSIONS Rabies-infected bats were found in environments that pose a risk to both human and domestic animal population and there is a need for actions aiming at the control of these species and public education.

Rabies laboratory diagnosis: peculiar features of samples from equine origin

Rabies laboratory diagnosis is performed by using microscopic examination for Negri bodies (MEN), fluorescent-antibody test (FAT) and mouse inoculation test (MIT). In the majority of cases, when specimens are properly collected and conserved and the laboratory worker has good experience, agreement among employed techniques is verified. Comparing the sensitivity of these three diagnosis techniques in 3,713 samples (hippocampus and brain stem) received during 1981-1994 period, being 3,010 from bovine (983 positives) and 703 from equine (111 positives) species, it was observed that in equine rabid samples, this agreement was not maintained. For the latter specie, only in few opportunities the Negri bodies could be observed. With respect to FAT, the test detected a lower porcentage of positive equine samples compared to bovine species. Statistical analysis demonstrated that the difference was significative. Mouse inoculation test proved to be more sensitive. However, a significant difference in mice incubation period was observed for samples from both species. The absence of inclusion bodies and the longer incubation period for equine samples suggest that rabies pathogenesis studies for equine species have to be intensified.

West Nile virus surveillance, Brazil, 2008-2010

BACKGROUND West Nile virus (WNV) is an emergent pathogen that is widely distributed in North and Central America. The recent introduction in South America has focused attention on the spread of WNV across Southern American countries. The transmission network involves mosquitoes, birds, horses and humans. METHODS The serological evaluation of sera from 678 equids and 478 birds was performed using a WNV-specific blocking ELISA, and only the positive results were confirmed by plaque reduction neutralisation tests (PRNTs). Molecular analysis was performed on sera from 992 healthy equids and on 63 macerates of brains from equids that died of encephalitis and had previously tested negative for other pathogens. We also tested swabs from 928 birds. The samples analysed were collected in different biomes of Brazil. RESULTS We identified WNV antibodies by ELISA in thirteen equids and five birds, and PRNT90 confirmed WNV positivity in four equid samples collected in 2009 in an area between the Amazon and the Pantanal. None of the ELISA positive bird samples were confirmed by PRNT90, and all samples tested by RT-PCR were negative. CONCLUSION WNV circulation is confirmed by this large scale survey even in the absence of detection of clinical cases.

Rabies in southeast Brazil: a change in the epidemiological pattern

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.

Soroprevalência da anemia infecciosa eqüina, da arterite viral dos eqüinos e do aborto viral eqüino no município de Uruará, PA, Brasil

Equine infection anemia virus (EIAV), Equine arteritis virus (EAV) and Equine herpesvirus 1 (EHV 1) are the causal agents of diseases which may bring economical losses. The aim o of this study was to estimate the prevalence of herds and animals infected with EIA, EAV and EHV in Uruara municipal district, Para State-Brazil. Antibodies against EIAV were detected by the immunodiffusion test and those against EAV and HEV-1 by the serum neutralization test. Sample size was estimated from 2069 holder farms that raised Equidae and did not vaccinate against EAV and EHV. A 90% confidence level was adopted with 15% precision and 50% estimated prevalence. The herd was considered positive when it had at least one positive animal. The following prevalence of serum reactors animals were observed: VAIE: 17,71% (IC 10,67 - 26,83%), HVE-1: 17,71% (IC 10,67 - 26,83%) and VAVE: 0,00% (IC 0,00 - 3,77%). The following prevalence of positive herds were observed: VAIE: 53% (IC 38,12 - 68,12%), HVE-1: 40.62% (IC 25.96 - 56.65%) and VAVE: 0% (IC 0 - 6.94%).

Characterization of Aujeszky's disease virus isolates from South and Southeast Brazil by RFLP analysis

The genomic DNA of thirty strains of Aujeszkys disease virus (ADV) isolated in the South and Southeast regions of Brazil from 1982 to 1996 were characterized by restriction endonuclease analysis with BamHI. Twenty seven strains were isolated from pigs, 1 from cattle, 1 from cat and 1 from dog. Using a systematization previously described, the 30 ADV strains could be classified as genomic types I (n = 2) and II (n = 28). Genomic type III was not observed. In this first study of genomic type characterization of brazilian ADV strains, we could demonstrate the occurence in Brazil of the genomic types I and II, with a large predominance of genomic type II.

Diagnóstico laboratorial da raiva na região oeste do Estado de São Paulo

The Pólo da Alta Sorocabana laboratory, Presidente Prudente, SP, Brazil, and the Biological Institute in São Paulo State, performed an evaluation of rabies diagnosis from 1996 to 2003 in the west region of São Paulo State. For the tests, the laboratories used direct immunofluorescence and mice inoculation in 4,950 samples, that were sent for analysis involving dogs, cats, cattle, chiroptera (bats) and other animals. According to the results, the laboratories found 74 positive samples; of which 58 (78.4%) were non-hematophagous bats and 16 (21.6%) related to cattle. The present epidemiological study verified that in spite of the high positive index in chiroptera compared to the other species, there was not an outbreak of rabies in the species in the region of Presidente Prudente, from 1996 to 2003 but a rise in the positive index due to a marked increase in the number of chiroptera samples sent to the laboratories for virus rabies research.

One-step protocol for amplification of near full-length cDNA of the rabies virus genome

Full-length genome sequencing of the rabies virus is not a routine laboratory procedure. To understand fully the epidemiology, genetic variation and evolution of the rabies virus, full-length viral genomes need to be obtained. For rabies virus studies, cDNA synthesis is usually performed using nonspecific oligonucleotides followed by cloning. When specific primers are used, the cDNA obtained is only partial and is limited to the coding regions. Therefore, the development of methods for synthesizing long cDNA using rabies virus-specific primers is of fundamental importance. A new protocol for the synthesis of long cDNA and the development of 19 new primers are described in this study. This procedure allowed the efficient amplification of the full-length genome of the rabies virus variant maintained by hematophagous bat (Desmodus rotundus) populations following the synthesis of a complete long cDNA. Partial sequencing of the rabies virus genome was performed to confirm rabies-specific PCR amplification. Because degenerate primers were employed, this technique can be adapted easily to other variants. Importantly, this new method is faster and less expensive than cloning methods.