RESUMO: A leucose enzoótica bovina (LEB) é uma doença infecciosa natural dos bovinos com distribuição mundial causada pelo “bovine leukemia virus” (BLV). A imunodifusão em gel de ágar (IDGA) foi considerada por muitos anos o teste de eleição, porém ensaios imunoenzimáticos (ELISA) apresentam sensibilidade mais elevada e leitura mais rápida e objetiva. No entanto, a importação de kits de ELISA é um processo dispendioso e demorado, e atualmente não há kits de IDGA comercialmente disponíveis no Brasil. Desta forma, o objetivo deste trabalho foi padronizar um ELISA indireto (iELISA) para diagnóstico da LEB utilizando antígenos produzidos a partir do cultivo do BLV em linhagem celular Tadarida brasiliensis “lung” (Tb1Lu) livre de “bovine viral diarrhea virus” (BVDV), diferentemente do que acontece com as linhagens “fetal lamb kidney” (FLK) atualmente utilizadas na produção desses antígenos para uso em ensaios sorológicos. Após a padronização do iELISA, os resultados foram comparados com aqueles obtidos por IDGA e pelo ELISA comercial “Chekit Leucose-Serum”. Comparado ao IDGA, o iELISA apresentou 94,44% de sensibilidade, 75,68% de especificidade, valor preditivo positivo (VPP) de 79,1% e valor preditivo negativo (VPN) de 93,3%, com concordância entre os testes de 84% e o índice Kappa 0,699. Quando comparado ao ELISA “Chekit Leucose-Serum”, o iELISA apresentou sensibilidade de 92,6%, especificidade de 87,09%, VPP de 90,27% e VPN de 90%, com concordância de 90,27% e o índice Kappa 0,801. Portanto, devido à alta concordância com os testes tradicionais e ausência da ocorrência de reações inespecíficas com BVDV, o ensaio desenvolvido pode ser utilizado como ferramenta diagnóstica para o controle da LEB no Brasil.

ABSTRACT: Enzootic bovine leukosis (EBL) is an infectious disease caused by bovine leukemia virus (BLV) that affects cattle worldwide. Agar gel immunodiffusion (AGID) was the reference test for EBL diagnosis for many years, but enzyme-linked immunosorbent assay (ELISA) showed higher sensitivity, was faster to perform, and resulted in an objective reading. However, the importation of ELISA kits is lengthy and expensive, and currently, no AGID kits are available in Brazil. The aim of this work was to standardize an indirect ELISA (iELISA) for EBL diagnosis using BLV antigens produced in Tadarida brasiliensis lung (Tb1Lu) cells, which are Bovine viral diarrhea virus (BVDV) free, unlike fetal lamb kidney (FLK) cells, currently used for this purpose. Following standardization, iELISA results were compared with those obtained by AGID and the commercial Chekit Leucose-Serum ELISA. Compared to AGID, iELISA had 94,44% sensitivity, 75.68% specificity, 79.10% positive predictive value (PPV) and 93.30% negative predictive value (NPV), with 84% concordance and a Kappa index of 0.699. Compared to the Chekit Leucose-Serum ELISA, iELISA showed 92.60% sensitivity, 87.09% specificity, 90.27% PPV and 90,00% NPV, with 90.27% concordance and a Kappa index of 0.801. Taking into account the high agreement with the traditional tests and the absence of non-specific reactions with BVDV, the developed assay could be used as diagnostic method to control EBL in Brazil.

ABSTRACT Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct.