Different linkages in the long and short regions of the genomes of duck enteritis virus Clone-03 and VAC Strains

<p>Abstract</p> <p>Background</p> <p>Duck enteritis virus (DEV) is an unassigned member in the family <it>Herpesviridae</it>. To demonstrate further the evolutionary position of DEV in the family <it>Herpesviridae</it>, we have described a 42,897-bp fragment. We demonstrated novel genomic organization at one end of the long (L) region and in the entire short (S) region in the Clone-03 strain of DEV.</p> <p>Results</p> <p>A 42,897-bp fragment located downstream of the <it>LOFR11 </it>gene was amplified from the Clone-03 strain of DEV by using 'targeted gene walking PCR'. Twenty-two open reading frames (ORFs) were predicted and determined in the following order: 5'<it>-LORF11-RLORF1</it>-<it>ORF1</it>-<it>ICP4</it>-<it>S1-S2-US1-US10-SORF3-US2-MDV091.5-like-US3-US4-US5-US6-US7-US8-ORFx-US1-S2-S1-ICP4 </it>-3'. This was different from that of the published VAC strain, both in the linkage of the L region and S region, and in the length of the US10 and US7 proteins. The <it>MDV091.5-like </it>gene, <it>ORFx </it>gene, <it>S1 </it>gene and <it>S2 </it>gene were first observed in the DEV genome. The lengths of DEV US10 and US7 were determined to be 311 and 371 amino acids, respectively, in the Clone-03 strain of DEV, and these were different from those of other strains. The comparison of genomic organization in the fragment studied herein with those of other herpesviruses showed that DEV possesses some unique characteristics, such as the duplicated US1 at each end of the US region, and the US5, which showed no homology with those of other herpesviruses. In addition, the results of phylogenetic analysis of ORFs in the represented fragment indicated that DEV is closest to its counterparts VZV (<it>Varicellovirus</it>) and other avian herpesviruses.</p> <p>Conclusion</p> <p>The molecular characteristics of the 42,897-bp fragment of Clone-03 have been found to be different from those of the VAC strain. The phylogenetic analysis of genes in this region showed that DEV should be a separate member of the subfamily <it>Alphaherpesvirinae</it>.</p

APP Processing Induced by Herpes Simplex Virus Type 1 (HSV-1) Yields Several APP Fragments in Human and Rat Neuronal Cells

Lifelong latent infections of the trigeminal ganglion by the neurotropic herpes simplex virus type 1 (HSV-1) are characterized by periodic reactivation. During these episodes, newly produced virions may also reach the central nervous system (CNS), causing productive but generally asymptomatic infections. Epidemiological and experimental findings suggest that HSV-1 might contribute to the pathogenesis of Alzheimer's disease (AD). This multifactorial neurodegenerative disorder is related to an overproduction of amyloid beta (Aβ) and other neurotoxic peptides, which occurs during amyloidogenic endoproteolytic processing of the transmembrane amyloid precursor protein (APP). The aim of our study was to identify the effects of productive HSV-1 infection on APP processing in neuronal cells. We found that infection of SH-SY5Y human neuroblastoma cells and rat cortical neurons is followed by multiple cleavages of APP, which result in the intra- and/or extra-cellular accumulation of various neurotoxic species. These include: i) APP fragments (APP-Fs) of 35 and 45 kDa (APP-F35 and APP-F45) that comprise portions of Aβ; ii) N-terminal APP-Fs that are secreted; iii) intracellular C-terminal APP-Fs; and iv) Aβ1-40 and Aβ1-42. Western blot analysis of infected-cell lysates treated with formic acid suggests that APP-F35 may be an Aβ oligomer. The multiple cleavages of APP that occur in infected cells are produced in part by known components of the amyloidogenic APP processing pathway, i.e., host-cell β-secretase, γ-secretase, and caspase-3-like enzymes. These findings demonstrate that HSV-1 infection of neuronal cells can generate multiple APP fragments with well-documented neurotoxic potentials. It is tempting to speculate that intra- and extracellular accumulation of these species in the CNS resulting from repeated HSV-1 reactivation could, in the presence of other risk factors, play a co-factorial role in the development of AD

Teste imunoenzimático com base em anticorpo monoclonal para a detecção de anticorpos contra os herpesvírus bovino tipos 1 e 5

Os herpesvírus bovino tipos 1 (BoHV-1) e 5 (BoHV-5) são agentes virais genética e antigenicamente relacionados, associados com diversas manifestações clínicas em bovinos, incluindo doença respiratória, genital, neurológica e abortos. Estudos epidemiológicos indicam que esses vírus estão amplamente disseminados no rebanho bovino brasileiro. O diagnóstico sorológico, que permite identificar animais portadores da infecção latente, se constitui em importante ferramenta para monitoramento individual e de rebanho. O presente artigo relata a padronização de um teste imunoenzimático do tipo ELISA, com base em anticorpo monoclonal (AcM), para a detecção de anticorpos séricos que reagem contra BoHV-1 e/ou BoHV-5. Inicialmente, determinou-se o AcM mais adequado para a sensibilização das placas, as diluições apropriadas do antígeno e dos soros-teste e o ponto de corte do ensaio. Após a padronização, o ensaio foi validado testando-se 506 amostras de soro bovino, previamente testadas para anticorpos neutralizantes contra BoHV-1 e/ou BoHV-5 pela técnica de soroneutralização (SN). Comparando-se com os resultados da SN frente a BoHV-1, o teste de ELISA apresentou sensibilidade e especificidade de 96,6% e 98,3%, respectivamente. Os valores preditivos positivo e negativo foram de 97,6%, a concordância foi de 97,6% e o índice de correlação kappa entre os testes foi de 0,95, o que indica uma excelente concordância. Comparando-se com os resultados da SN frente o BoHV-5, o ELISA apresentou 94,3% de sensibilidade; 97,9% de especificidade; 97,1% de valor preditivo positivo e 95,9% de valor preditivo negativo. Para BoHV-5, a concordância entre os testes foi de 96,4% e o índice de correlação foi de 0,92, também excelente. Esses resultados demonstram que o teste padronizado apresenta sensibilidade e especificidade adequados para o diagnóstico sorológico das infecções por BoHV-1 e BoHV-5 em nível individual e de rebanho. Dessa forma, o ensaio pode se constituir em alternativa para o teste de SN e para os kits de ELISA importados.Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are antigenic and genetically related viruses associated with different clinical syndromes in cattle, including respiratory, reproductive, neurological disease and abortion. Epidemiological studies indicate the widespread distribution of both viruses among Brazilian cattle. Serological diagnosis, that allows the identification of latently infected animals, represents an important tool for individual and herd monitoring. The present article describes the standardization of a monoclonal antibody (MAb)-based immunoenzymatic test (ELISA) for detection of antibodies to BoHV-1 and/or BoHV-5. The initial steps involved the determination of the most suitable MAb, the appropriate dilutions of viral antigen and serum samples, and the cut-off value of the assay. After standardization, the ELISA was validated by testing 506 cattle serum samples previously tested for neutralizing antibodies to BoHV-1 and BoHV-5 by virus neutralizing assay (VN). Comparing to the VN for BoHV-1 antibodies, the ELISA presented sensitivity and specificity of 96.6% and 98.3%, respectively. Positive and negative predictive values were 97.6%, the concordance between the tests was 97.6% and the coefficient of correlation k (kappa) was 0.95, demonstrating an excellent correlation. Comparing to the VN for BoHV-5 antibodies, the ELISA presented 94.3% of sensitivity, 97.9% of specificity, 97.1% of positive predictive value, 95.9% negative predictive value, concordance of 96.4% and kappa coefficient of 0.92. These results demonstrate that the ELISA presents suitable specificity and sensitivity to be used for individual and herd serological diagnosis of BoHV-1 and BoHV-5, thus, representing an alternative for VN assays and imported ELISA kits

Histopathological, immunohistochemical, and molecular study of BHV-5 infection in the central nervous system of experimentally infected calves

Bovine meningoencephalitis caused by BHV-5, a double-stranded DNA enveloped virus that belongs to the family Herpesviridae and subfamily Alphaherpesvirinae, is an important differential diagnosis of central nervous diseases. The aim of this study was to describe the histological changes in the central nervous system of calves experimentally infected with BHV-5 and compare these changes with the PCR and IHC results. Formalin-fixed paraffin-embedded central nervous system samples from calves previously inoculated with BHV-5 were microscopically evaluated and tested using IHC and PCR. All the animals presented with nonsuppurative meningoencephalitis. From 18 evaluated areas of each calf, 32.41% and 35.19% were positive by IHC and PCR, respectively. The telencephalon presented more accentuated lesions and positive areas in the PCR than other encephalic areas and was the best sampling area for diagnostic purposes. Positive areas in the IHC and PCR were more injured than IHC and PCR negative areas. The animal with neurological signs showed more PCR- and IHC-positive areas than the other animals

Ensaio imunoenzimático comercial no diagnóstico sorológico das infecções por herpesvírus bovino 1 A commercial enzyme immune assay in serodiagnosis of bovine herpesvirus 1 infections

Avaliou-se o desempenho de um ensaio imunoenzimático, obtido de fonte comercial, na identificação de anticorpos contra herpesvírus bovino tipo 1 (BHV-1), induzidos tanto por infecção natural quanto por vacinação, em 1000 amostras de soros sangüíneos de bovinos. A análise comparativa dos resultados obtidos no sistema avaliado e na técnica padrão de soroneutralização mostrou uma concordância de 97,05% (K=0,94) entre as duas metodologias de diagnóstico sorológico.<br>The performance of a commercial immune assay in the identification antibody of natural infection or vaccination against bovine herpesvirus type 1 (BHV-1) in 1000 samples of bovine serum was evaluated. The comparative analysis from the result of the evaluated system and standard serum neutralization technique showed a rate of agreement of 97.05% (K=0.94) between the two serologic diagnotic methods

Mutation of the Lck-Binding Motif of Tip Enhances Lymphoid Cell Activation by Herpesvirus Saimiri

The proline-rich SH3-binding (SH3B) motif of the tyrosine kinase-interacting protein (Tip) of herpesvirus saimiri (HVS) is required for binding to the cellular Src family kinase Lck. We constructed a mutant form of HVS in which prolines in the SH3B motif of Tip were altered to alanines. This mutant form of Tip was incapable of binding to Lck. The mutant virus, HVS/Tip mSH3B, retained its ability to immortalize common marmoset lymphocytes in culture. In fact, common marmoset lymphocytes immortalized by the HVS/Tip mSH3B mutant displayed increased expression of HLA-DR lymphocyte activation marker, an altered pattern of tyrosine phosphorylation, increased expression of the tyrosine kinase Lyn, and a shift in electrophoretic mobility of Lck compared to cells immortalized by wild-type HVS. Experimental infection of common marmosets resulted in fulminant lymphoma with both HVS/Tip mSH3B and wild-type HVS. However, HVS/Tip mSH3B produced greater infiltration of affected organs by proliferating lymphoid cells compared to wild-type HVS. These results demonstrate that Tip binding to Lck is not necessary for transformation and that abrogation of Tip binding to Lck alters the characteristics of transformed cells and the severity of the pathologic lesions