Evolution and divergence of the genetic lineage Desmodus rotundus/Artibeus lituratus of rabies virus in São Paulo State.

The last record of a rabies case caused by the dog-specific rabies virus (RABV) lineage in dogs or cats in São Paulo State was in 1998. From 2002 to 2021, 57 cases of rabies in these animals were reported, and the vast majority (51) were genetically characterized as belonging to the Desmodus rotundus/Artibeus lituratus RABV lineage. However, it is not currently possible to infer which of these bats is the source of infection by genome sequencing of RABV isolates. The aims of this study were (a) to characterize the Desmodus rotundus/Artibeus lituratus lineage to determine the relationships between the RABV lineages and each reservoir, (b) to assess the phylogeny and common ancestors of the RABV lineages found in D. rotundus and A. lituratus, and (c) to further understand the epidemiology and control of rabies. In this study, we genetically analyzed 70 RABV isolates from São Paulo State that were received by the Virology Laboratory of the Pasteur Institute of São Paulo between 2006 and 2015. Of these isolates, 33 were associated with the hematophagous bat D. rotundus and 37 with the fruit bat A. lituratus. A genomic approach using phylogenetic analysis and nucleotide sequence comparisons demonstrated that these isolates belonged to the same genetic lineage of RABV. We also found that, in São Paulo State, the D. rotundus/A. lituratus lineage could be subdivided into at least four phylogenetic sublineages: two associated with D. rotundus and two with A. lituratus. These results are of importance for the epidemiological surveillance of rabies in São Paulo.

Comparison of five different laboratory techniques for the rabies diagnosis in clinically suspected cattle in Brazil.

The direct-fluorescent antibody test (dFAT) is considered the "gold standard" assay to diagnose rabies. However, it is crucial to develop molecular techniques, such as RT-PCR and RT-qPCR, since many laboratories lack the needed supplies for performing complementary methods (viral isolation, for example). For this purpose, diagnostic techniques must be specific and sensitive to guarantee accuracy. This present investigation aimed to detect rabies virus (RABV) in 126 clinically suspected cattle in Brazil using different diagnostic tests [dFAT, mouse inoculation test (MIT), immunohistochemistry (IHC), RT-PCR and RT-qPCR] and to compare those results obtained under routine laboratory conditions. The results of the present investigation demonstrate that the molecular techniques are more sensitive and may detect low viral load, even though the non-homogeneous viral distribution caused a false-negative result in dFAT. We also observed a usual alteration in antigens distribution among regions of the central nervous system (CNS). By both dFAT and IHC assays, the most reliable CNS structures were thalamus and midbrain. Although this investigation demonstrated diagnostic sensitivity and specificity close to 100 % in all laboratory techniques employed, a dFAT auxiliary test is required for bovine specimens, such as molecular techniques, when there are poor sampling conditions (low viral load combined with unavailability of brainstem structures).

Characterization of the Th17 profile immune response in cases of human rabies transmitted by dogs and its interference in the disease pathogenesis.

The Th17 profile immune response is influenced by the presence of cytokines such as IL-1, IL-6, TGF-ß, IL-17, and IL-23. We sought to characterize the Th17 profile in CNS samples from human rabies cases transmitted by dogs and examine its possible influence on disease pathogenesis. We observed a high expression of TGF-ß, followed by IL-23, IL-17 and IL-6, and a low expression of IL-1ß and IFN-γ. Those results suggest the participation of Th17 in rabies virus neuroinfection transmitted by dogs. IL-23 probably plays a role in maintaining the Th17 profile, but it can also interfere with the establishment of the Th1 profile and viral clearance.

The Epidemiological Importance of Bats in the Transmission of Rabies to Dogs and Cats in the State of São Paulo, Brazil, Between 2005 and 2014.

In Brazil, rabies control in dogs and cats was pioneered by the state of São Paulo with the adoption of the Pan American Health Organization recommendations for prophylaxis and control, which led to a reduction in rabies cases from 1994 onwards. As a result of these measures, the rabies virus (RABV) genetic lineage associated with dogs has not been found in the state since 1998, and all the cases in domestic animals reported since then have been caused by bat-associated lineages of RABV. In the light of this, this study sought to investigate rabies cases in dogs and cats in the state of São Paulo between 2005 and 2014 and identify the associated transmission cycles by characterizing the RABV lineages responsible for these cases. Nine samples from dogs (n = 5) and from cats (n = 4) were collected between 2005 and 2014. The tenth animal, a rabid cat, was analysed by a different laboratory. The N gene nucleotide sequences obtained were analysed with the neighbor-joining algorithm and Kimura 2-parameter model using the MEGA 6 program. Phylogenetic analysis revealed that the genetic lineages identified in all the samples were those circulating in Brazilian bats. The findings of this study demonstrate that bats play an important role in the transmission of rabies to domestic animals in São Paulo state and that emphasis should be placed on the implementation of public policies to support surveillance of chiropterans for rabies.

Immunopathology of rabies infection in mice selected for high or low acute inflammatory reaction

Rabies is a severe and lethal disease that produces a slight inflammatory response during the infection process. We analyzed the immunopathological mechanisms that occur in the central nervous system (CNS) using mice genetically selected for maximal or minimal acute inflammatory reaction (AIRmax or AIRmin). As viral samples, we adopted the antigenic variant 3 (AgV3) of rabies virus from hematophagous bats and a fixed virus strain (PV1 43/3). Titration of specific antibodies was performed using enzyme-linked immunosorbent assay (ELISA). We observed a slight increase in IgG and IgG1 isotypes in infected AIRmax mice. Incubation period, determined by intracerebral inoculation with 100 LD50, was 6-7 days for PV1 43/4 strain and 9-10 days for AgV3. No difference in viral replication was noticed between AIRmax and AIRmin mice. Mortality was 100 percent with both viral strains. Histopathological analysis of brains and spinal cords showed inflammatory foci in all regions of the CNS. No differences were noticed in the number of neutrophils. Negri bodies were observed in practically all sites analyzed. Results suggested that inflammatory reaction is not a determining factor in the susceptibility to rabies infection.