Evaluation of LN34 Pan-Lyssavirus RT-qPCR assay for rabies diagnosis in Brazil.

Rabies, a fatal zoonotic viral disease affecting mammals, including humans, remains a significant global health concern, particularly in low-income countries. The disease, primarily transmitted through infected animal saliva, prompts urgent diagnosis for timely post-exposure prophylaxis (PEP). The gold standard diagnostic test, direct fluorescent antibody test (dFAT), while sensitive, suffers from limitations such as subjective interpretation and high costs. As a confirmatory technique, the LN34 Pan-Lyssavirus RT-qPCR assay has emerged as a promising tool for universal Lyssavirus detection. This study evaluated its performance using 130 rabies virus isolates representing eleven Brazilian variants and 303 clinical samples from surveillance operations. The LN34 assay demonstrated 100% sensitivity and 98% specificity compared to dFAT. Additionally, it detected all samples, including those missed by dFAT, indicating superior sensitivity. The assay's specificity was confirmed through Sanger nucleotide sequencing, with only a minimal false-positive rate. Comparative analysis revealed higher accuracy and concordance with dFAT than traditional rabies tissue culture infection tests (RTCIT). False-negative RTCIT results were attributed to low viral load or suboptimal sampling. These findings underscore the LN34 assay's utility as a confirmatory technique, enhancing rabies surveillance and control in Brazil. Its widespread adoption could significantly improve diagnostic sensitivity, crucial for effective PEP and public health interventions.

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.

Biotechnology advances: a perspective on the diagnosis and research of Rabies Virus.

Rabies is a widespread zoonotic disease responsible for approximately 55,000 human deaths/year. The direct fluorescent antibody test (DFAT) and the mouse inoculation test (MIT) used for rabies diagnosis, have high sensitivity and specificity, but are expensive and time-consuming. These disadvantages and the identification of new strains of the virus encourage the use of new techniques that are rapid, sensitive, specific and economical for the detection and research of the Rabies Virus (RABV). Real-time RT-PCR, phylogeographic analysis, proteomic assays and DNA recombinant technology have been used in research laboratories. Together, these techniques are effective on samples with low virus titers in the study of molecular epidemiology or in the identification of new disease markers, thus improving the performance of biological assays. In this context, modern advances in molecular technology are now beginning to complement more traditional approaches and promise to revolutionize the diagnosis of rabies. This brief review presents some of the recent molecular tools used for RABV analysis, with emphasis on rabies diagnosis and research.

Immunoperoxidase inhibition assay for rabies antibody detection.

An immunoperoxidase inhibition assay (IIA) for detection of rabies antibodies in human sera is described. Diluted test sera are added to microplates with paraformaldehyde-fixed, CER cells infected with rabies virus. Antibodies in test sera compete with a rabies polyclonal rabbit antiserum which was added subsequently. Next, an anti-rabbit IgG-peroxidase conjugate is added and the reaction developed by the addition of the substrate 3-amino-9-ethylcarbazole (AEC). The performance of the assay was compared to that of the "simplified fluorescence inhibition microtest" (SFIMT), an established virus neutralization assay, by testing 422 human sera. The IIA displayed 97.6% sensitivity, 98% specificity and 97.6% accuracy (Kappa correlation coefficient=0.9). The IIA results can be read by standard light microscopy, where the clearly identifiable specific staining is visible in antibody-negative sera, in contrast to the absence of staining in antibody-positive samples. The assay does not require monoclonal antibodies or production of large amounts of virus; furthermore, protein purification steps or specialized equipment are not necessary for its performance. The IIA was shown to be suitable for detection of rabies antibodies in human sera, with sensitivity, specificity and accuracy comparable to that of a neutralization-based assay. This assay may be advantageous over other similar methods designed to detect rabies-specific binding antibodies, in that it can be easily introduced into laboratories, provided basic cell culture facilities are available.

Efficacy of an inactivated, recombinant bovine herpesvirus type 5 (BoHV-5) vaccine.

Bovine herpesvirus type 5 (BoHV-5) is the causative agent of bovine herpetic encephalitis. In countries where BoHV-5 is prevalent, attempts to vaccinate cattle to prevent clinical signs from BoHV-5-induced disease have relied essentially on vaccination with BoHV-1 vaccines. However, such practice has been shown not to confer full protection to BoHV-5 challenge. In the present study, an inactivated, oil adjuvanted vaccine prepared with a recombinant BoHV-5 from which the genes coding for glycoprotein I (gI), glycoprotein E (gE) and membrane protein US9 were deleted (BoHV-5 gI/gE/US9(-)), was evaluated in cattle in a vaccination/challenge experiment. The vaccine was prepared from a virus suspension containing a pre-inactivation antigenic mass equivalent to 10(7.69) TCID(50)/dose. Three mL of the inactivated vaccine were administered subcutaneously to eight calves serologically negative for BoHV-5 (vaccinated group). Four other calves were mock-vaccinated with an equivalent preparation without viral antigens (control group). Both groups were boostered 28 days later. Neither clinical signs of disease nor adverse effects were observed during or after vaccination. A specific serological response, revealed by the development of neutralizing antibodies, was detected in all vaccinated animals after the first dose of vaccine, whereas control animals remained seronegative. Calves were subsequently challenged on day 77 post-vaccination (pv) with 10(9.25) TCID(50) of the wild-type BoHV-5 (parental strain EVI 88/95). After challenge, vaccinated cattle displayed mild signs of respiratory disease, whereas the control group developed respiratory disease and severe encephalitis, which led to culling of 2/4 calves. Searches for viral DNA in the central nervous system (CNS) of vaccinated calves indicated that wild-type BoHV-5 did not replicate, whereas in CNS tissues of calves on the control group, viral DNA was widely distributed. BoHV-5 shedding in nasal secretions was significantly lower in vaccinated calves than in the control group on days 2, 3, 4 and 6 post-challenge (pc). In addition, the duration of virus shedding was significantly shorter in the vaccinated (7 days) than in controls (12 days). Attempts to reactivate latent infection by administration of dexamethasone at 147 days pv led to recrudescence of mild signs of respiratory disease in both vaccinated and control groups. Infectious virus shedding in nasal secretions was detected at reactivation and was significantly lower in vaccinated cattle than in controls on days 11-13 post-reactivation (pr). It is concluded that the inactivated vaccine prepared with the BoHV-5 gI/gE/US9(-) recombinant was capable of conferring protection to encephalitis when vaccinated cattle were challenged with a large infectious dose of the parental wild type BoHV-5. However, it did not avoid the establishment of latency nor impeded dexamethasone-induced reactivation of the virus, despite a significant reduction in virus shedding after challenge and at reactivation on vaccinated calves.

A sandwich enzyme linked immunosorbent assay to detect rabies antibodiesin sera of different species

A sandwich ELISA (S-ELISA) was developed to detect antibodies to rabies virus in seraof different species. The test was performed as follows: ELISA plates were coated withpolyclonal mouse/anti-rabies antibodies for 2 hours at 37ºC. After adsorption, plateswere washed and non-specific binding blocked with 2% powdered milk. In a separateplate, serial threefold dilutions of test sera were incubated with inactivated rabies virusantigen. The mixtures were then placed on the rabies antibody-coated plates andincubated. These were then washed and incubated with polyclonal rabbit/anti-rabiesantibodies. Subsequently, a rabbit/IgG-peroxidase conjugate was added and platesincubated. After washing, the chromogen (ABTS with 0.15% H2O2) was added to platesand after incubation for 30 min were read in a spectrophotometer (OD405). To validatethe S-ELISA, 128 serum samples including humans, cattle, hematophagous and nonhaematophagous bats, mice, marmosets, ocelots - Leopardus pardalis, raccoons -Procyon lotor, jaguarondi - Herpailurus yaguarondi, fox - Cerdocyon thous and coati -Nasua nasua, were tested and compared to a standard fluorescent antibody virusneutralization test (FAVN). In comparison to FAVN, the S-ELISA showed highsensitivity (82.98%) and specificity (100%), with an accuracy of 87.5%. Subsequently,738 serum samples from different species were tested in the S-ELISA. Antibodies torabies were detected by S-ELISA in all species tested, with the exception of the threeserum samples from raccoons. The S-ELISA was shown to be a serological test of lowcost that can be easily implemented in diagnostic laboratories. In addition, no liveanimals, infectious virus, cell culture or fluorescence microscopy are required forperformance of the test. This is an additional advantage of the S-ELISA over othermethods of rabies antibody detection.

Herpesvírus bovinos (BoHV-1.1 e BoHV-1.2b) em forma infecciosa em encéfalos de bovinos submetidos ao diagnóstico de raiva no estado do Rio Grande do Sul / Bovine herpesviruses (BoHV-1 and BoHV-1.2b) in infectious form in brains of cattle submitted to rabies diagnosis in the State of Rio Grande do Sul, Brazil

Verificou-se a incidência de herpesvírus bovinos (BoHVs) em encéfalos de bovinos submetidos ao diagnóstico de raiva no estado do Rio Grande do Sul. Para tanto, amostras coletadas durante dois anos (n=70) foram submetidas ao isolamento viral em cultivos celulares. Os BoHVs foram isolados em dois (2,9 por cento) encéfalos. Após serem submetidas à caracterização antigênica e molecular, as amostras foram subtipadas como BoHV-1.1 e BoHV-1.2b. A BoHV-1.1 foi isolada de um encéfalo que foi também positivo para raiva. O vírus da raiva foi identificado em 11 amostras (15,7 por cento). Estes achados revelam que a incidência de BoHVs em forma infecciosa em bovinos com encefalite foi baixa, embora represente 16,7 por cento (2/12) dos encéfalos nos quais um agente viral foi identificado. Tal fato confirma a já reportada associação entre BoHV-1 e encefalites. Esse é o primeiro relato da ocorrência de BoHV-1.2b, um subtipo considerado menos patogênico, em um caso de doença neurológica em bovinos.

The incidence of bovine herpesviruses (BoHVs) was determined in brains of cattle submitted to rabies diagnosis in the State of Rio Grande do Sul, Brazil. Brain samples collected in a two-year interval (n=70) were submitted to virus isolation in cell culture. The BoHVs were isolated from two (2.9 percent) of the brains. After the antigenic and molecular characterization, the isolated strains were subtyped as BoHV-1.1 and BoHV-1.2b. The BoHV-1.1 isolate was recovered from a brain sample that was also positive for rabies. Rabies virus was identified in 11 (15.7 percent) samples. These findings reveal that the incidence of infectious BoHVs in brains of cattle with encephalitis was low, although these represented 16.7 percent (2/12) of samples from which at least one viral agent was identified. This confirms the previously reported link between BoHV-1 and bovine encephalitis. However, it is the first report on the association of BoHV-1.2b, a putatively less pathogenic BoHV subtype, with neurological disease in cattle.

Caracterização de amostras do vírus da raiva, isoladas nas regiões Norte e Centro-Oeste do Brasil, com anticorpos monoclonais antilissavírus / Antigenic characterization of Brazilian rabies virus isolate North and Central West regions of Brazil with anti-lyssavirus monoclonal antibodies

The occurrence of rabies virus antigenic variants in North and Central West regions of Brazil was studied using 61 rabies viruses isolated from different species: 30 from domestic dogs, 20 from cattle, four from horses, two from cats, one from a human and four from unidentified species. The isolates were submitted to antigenic analyses by indirect immunofluorescence with a panel of 12 monoclonal antibodies (Mabs) to lyssavirus antigens. Antigenic analyses revealed consistent differences between isolates whose natural hosts were dogs and those of haematophagous bats, often isolated from cattle. Three out of four isolates from horses and one from a domestic dog showed patterns of reactivity found only in viruses of insectivorous bats, indicating that non-haematophagous bats do play a unique role in the transmission of the virus to other species.

Phylogenetic comparison of the carboxy-terminal region of glycoprotein C (gC) of bovine herpesviruses (BoHV) 1.1, 1.2 and 5 from South America (SA).

Different types and subtypes of bovine herpesvirus 1 and 5 (BoHV-1 and BoHV-5) have been associated to different clinical conditions of cattle, in such a way that type/subtype differentiation has become an essential tool for understanding the pathogenesis and epidemiology of BoHV infections. In search for a genomic region that would allow a clear distinction between BoHV-1 and BoHV-5, the carboxy-terminal portion of glycoprotein C (gC), corresponding to residues 321-450 (BoHV-1) and 301-429 (BoHV-5) of 23 South American (SA) isolates (Brazil mostly) was amplified and sequenced. The nucleotide sequence alignments revealed levels of genomic similarity ranging from 98.7 to 99.8% among BoHV-1 isolates, 88.3 to 92% between BoHV-1/BoHV-5 and 96 to 99.7% among BoHV-5 isolates. At the amino acid level, sequence similarity varied ranging from 97.5 to 99.5% among BoHV-1, 77.5 to 84.4% between BoHV-1/BoHV-5 and 92.1 to 99.5% (BoHV-5/BoHV-5). The isolates could be clearly separated into BoHV-1.1, BoHV-1.2 and BoHV-5 after phylogenetic analysis. The results suggest that the phylogenetic analysis performed here can be used as a potential molecular epidemiological tool for herpesviruses.

Co-infections with bovine herpesvirus type 5 and bovine viral diarrhoea virus

During a series of experiments attempting to reproduce clinically apparent bovine herpesvirus type 5 (BoHV-5) infections, a group of calves was inadvertently infected with bovine viral diarrhoea virus (BVDV). Another group of calves was infected with BoHV-5 only. This paper reports the outcome of such infections. Two out of six calves solely infected with BoHV-5 displayed moderate to severe clinical signs. Three out of four calves of the group co-infected with BoHV-5 and BVDV developed severe clinical signs, two of them died. BoHV-5 virus was isolated to higher titres and for a longer period from the group of calves infected with both viruses. These results suggest that BVDV may enhance clinical signs induced by BoHV-5 and may also play a role in extending the period of BoHV-5 shedding.

Durante a realização de experimentos envolvendo inoculações experimentais com herpesvírus bovino tipo 5 (BoHV-5), um grupo de bovinos foi acidentalmente também inoculado com vírus da diarréia viral bovina (BVDV). Os dados obtidos nesta co-infecção foram então comparados a aqueles observados em animais inoculados exclusivamente com BoHV-5. No grupo infectado com BoHV-5 somente, dois dos seis animais inoculados mostraram sinais clínicos de moderados a graves. No grupo co-infectado com BoHV-5 e BVDV, três dos quatro animais desenvolveram doença grave, e dois deles morreram. BoHV-5 foi isolado em títulos maiores e por um período de tempo mais longo no grupo co-infectado. Os resultados sugerem que o BVDV pode exacerbar os sinais clínicos induzidos pelo BoHV-5 e, ainda, aumentou os níveis de excreção deste último.