Discovery of a genome of a distant relative of chicken anemia virus reveals a new member of the genus Gyrovirus.

A 2.4-kb phi29 polymerase amplification product from serum of a diseased chicken was cloned and sequenced. The 2383-nucleotide sequence showed about 40% identity to a representative genome of chicken anemia virus (CAV), the only member of the genus Gyrovirus, family Circoviridae. The new genome had an organization similar to that of CAV: a putative 5' untranscribed region of about 400 nt followed by three partially overlapping open reading frames encoding VP1, VP2 and VP3 homologs. The amino acid identities between these homologs and those of CAV were 38.8%, 40.3%, and 32.2%, respectively. Based on these limited similarities, it is proposed that the newly identified virus is a member of a new species in the genus Gyrovirus. For this new species, the name Avian gyrovirus 2 (AGV2) is proposed.

Comparative evaluation of a competitive polymerase chain reaction (PCR) and a SYBR Green-based real-time PCR to quantify Porcine circovirus-2 DNA in swine tissue samples.

Porcine circovirus-2 (PCV-2) is considered the major etiological agent of post-weaning multisystemic wasting syndrome (PMWS) in pigs. The clinical manifestations of the disease are correlated with moderate to high amounts of PCV-2 DNA in biological samples of affected pigs. A threshold of 10(7) DNA copies/ml is suggested as the trigger factor for symptoms. A comparative study was conducted to determine which quantitative method would be more suitable to estimate the PCV-2 DNA load. Two polymerase chain reaction (PCR) assays were developed: a competitive PCR (cPCR) and a SYBR Green-based real-time PCR. The assays were compared for their capacity to detect PCV-2 in DNA samples extracted from liver, lung, spleen, mesenteric lymph nodes, and kidney of PMWS-affected (n = 23) or non-PMWS-affected pigs (n = 9). Both assays could successfully quantify PCV-2 DNA in all tissue samples and were able to detect significant differences between the numbers of PCV-2 DNA copies found in tissues of PMWS-affected and non-PMWS-affected pigs (≥ 10(2.5)). The highest mean viral loads were detected by the SYBR Green real-time PCR, up to 10(7.0 ± 1.5) copies/100 ng of total DNA sample, while the cPCR detected up to 10(4.8 ± 1.5). A mean difference of 10(1.8) was found between the amounts of PCV-2 DNA detected, using the SYBR Green real-time PCR and the cPCR, suggesting that the viral load threshold for PMWS should be determined for each particular assay.

Construction and characterization of a bovine herpesvirus 5 mutant with a deletion of the gI, gE and US9 genes

Bovine herpesvirus 5 (BoHV-5) is a important cause of viral encephalitis in cattle in South America. Within the framework of developing a differential vaccine against BoHV-5, a deletion mutant was constructed based on a Brazilian BoHV-5 isolate. The target of the deletions were genes that code proteins implicated in the neurovirulence of BoHV-5, the glycoprotein I (gI), glycoprotein E (gE) and membrane protein US9. To construct the deletion mutant of BoHV-5, the flanking regions of all three genes were cloned in a prokaryotic plasmid. This deletion fragment was co-transfected with the viral DNA into bovine cells. Identification of deletion mutants was performed by immunostaining with an anti-gE monoclonal antibody. One of the gE negative viral populations found was purified, amplified and further examined by restriction endonuclesase analysis of its genomic DNA. The plaque sizes and penetration kinetics of the deletion mutant and wild type viruses were compared. The plaque sizes of the deletion mutant were significantly smaller than those of the parental strain (p <= 0.05), but no statistical differences were observed in penetration kinetics. The results indicate that the gI/gE/US9 deletion mutant of BoHV-5 may have a reduced virulence in the host and is still viable enough to be a good candidate for the development of a BoHV-5 vaccine.

O herpesvírus bovino 5 (BoHV-5) é uma causa importante de encefalite viral em bovinos na América do Sul. Buscando o desenvolvimento de uma vacina diferencial contra o BoHV-5, um mutante deletado foi construído com base em um isolado brasileiro deste vírus. O alvo das deleções foram genes que codificam proteínas implicadas na neurovirulência do BoHV-5, a glicoproteína I (gI), a glicoproteína E (gE) e a proteína de membrana US9. Para construir o mutante deletado de BoHV-5, as regiões flanqueadoras dos três genes foram clonadas em um plasmídeo procarioto. Este fragmento de deleção foi co-transfectado com o DNA viral em células de bovinos. A identificação dos mutantes deletados foi feita por meio da técnica de imunoperoxidase com um anticorpo monoclonal anti-gE. Uma das populacões virais gE negativas encontradas foi purificada, amplificada e seu genoma foi examinado por análise de restrição enzimática. Os tamanhos de placas virais e taxas de penetração do vírus mutante foram determinados e comparados com os do vírus selvagem. As placas virais do vírus mutante deletado foram significativamente menores do que as do vírus selvagem (p <= 0,05), mas não foram encontradas diferenças significativas quando comparadas as taxas de penetração dos dois vírus. Estes resultados indicam que o vírus mutante deletado gI/gE/US9 de BoHV-5 pode apresentar virulência reduzida e é viável o suficiente para ser um bom candidato para o desenvolvimento de uma vacina contra o BoHV-5.